WO2020104479A1 - Methods and compositions for treating cancers and resistant cancers with anti transferrin receptor 1 antibodies - Google Patents

Methods and compositions for treating cancers and resistant cancers with anti transferrin receptor 1 antibodies

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Publication number
WO2020104479A1
WO2020104479A1 PCT/EP2019/081845 EP2019081845W WO2020104479A1 WO 2020104479 A1 WO2020104479 A1 WO 2020104479A1 EP 2019081845 W EP2019081845 W EP 2019081845W WO 2020104479 A1 WO2020104479 A1 WO 2020104479A1
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WIPO (PCT)
Prior art keywords
antibody
seq
amino acid
acid sequence
cancer
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PCT/EP2019/081845
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French (fr)
Inventor
Marie-Alix Poul
Ronan CREPIN
Rana MELHEM
Madeline NEIVEYANS
Original Assignee
INSERM (Institut National de la Santé et de la Recherche Médicale)
Université De Montpellier
Institut Régional Du Cancer De Montpellier
Université Paris-Sud
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Publication of WO2020104479A1 publication Critical patent/WO2020104479A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention is in the field of cancerology. More particularly, the invention relates to an antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of cancers and resistant cancers.
  • TfRl antibody anti- transferrin receptor 1
  • Transferrin receptor 1 (TfRl) is the main receptor responsible for the cell iron supply through receptor-mediated internalization of serum Fe3+- loaded transferrin (holo-Tf). Within the cell, Fe3+ is released, reduced, excluded from the early endosome by divalent metal ion transporter 1 (DMT1), and used for cell metabolism. Fe3+ excess is stored in ferritin, while TfRl is recycled at the cell surface together with iron- free transferrin (apo-Tf) 5 .
  • DMT1 divalent metal ion transporter 1
  • Cancer drug resistance has been and unfortunately still is a major problem in cancer therapy. Unfortunately there is a large group of patients that will either not respond to the applied therapy or will become resistant during therapy. Most of the patients ultimately become unresponsive, and relapse within 2-3 years.
  • the invention relates to an antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of cancers and resistant cancers.
  • TfRl transferrin receptor 1
  • the invention is defined by the claims.
  • H7 which is a fully human internalizing IgGl antibody. It works by a special mechanism upregulating TfRl levels which is not observed by other anti-TfRl antibodies which usually block Tf binding and internalization via blocking the TfRl on the cell surface or directing it for lysosomal degradation resulting in decreased TfRl levels.
  • inventors have demonstrated for the first time with a naked anti-TfRl antibody a complete inhibition of cell viability at less than 10 iig/ml of the antibody on a solid colon cancer cell line HCT116 at a low IC50 of 0.78 iig/ml.
  • variability on cell viability was observed when H7 was tested on a panel of colon cancer cell lines ranging between 50-80% of maximal inhibition at 10 iig/m 1 of H7 after 5 days of treatment.
  • TfRl is expressed by pancreatic cancer stem cells (CSCs) (HP AC and PanPec cells) grown in a specialized CSC medium and that the expression was higher in the PancPec cells grown in 3D compared to those in 2D.
  • CSCs pancreatic cancer stem cells
  • H7 was also found to decrease the growth of cells in 3D culture in CSC medium indicating that PDAC cancer stem cells (CSCs) are also sensitive to H7.
  • H7 decreased the tumor growth of the PDAC patient derived xenograft (PDX) model and improved the gemcitabine efficiency on the BxPC3 xenograft mice model.
  • PDX PDAC patient derived xenograft
  • Im9 B-cell lymphoma cell line which is resistant to apoptosis induced by rituximab (anti-CD20 antibody), was sensitive to one of the antibody anti- TfRl(e.g. H7).
  • Bp2 and Im9 B-cell lines (sensitive and resistant to rituximab-induced apoptosis, respectively) were incubated with H7 or rituximab. H7 strongly induced apoptosis in both cell lines.
  • H7 antibody has the characteristics and qualifications that allow it to be tried in clinical trials. It is fully human, highly efficient in blocking cell iron supply from transferrin, and induces ADCC.
  • the invention relates to an antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of cancers.
  • TfRl transferrin receptor 1
  • the terms “treating” or“treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject 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.
  • 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.
  • loading regimen 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., pain, disease manifestation, etc.]).
  • continuous therapy e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.
  • intermittent therapy e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • transferrin receptor refers to a membrane transport protein which is involved in iron uptake in vertebrates. It imports iron by internalizing the transferrin- iron complex through receptor-mediated endocytosis.
  • transferrin receptor protein 1 transferrin receptor protein 1
  • TfR2 Transferrin receptor 2
  • TfRl transferrin receptor protein 1
  • CD71 Cluster of Differentiation 71
  • TfRl is a transmembrane glycoprotein composed of two disulfide- linked monomers joined by two disulfide bonds.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity of TfRl . Typically, such antibody inhibits the iron transport in the tumour cells.
  • the term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"
  • Antibodies can be fragmented using conventional techniques. For example, F(ab’)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab’)2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al, 2006; Holliger & Hudson, 2005; Fe Gall et al, 2004; Reflf & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments.
  • the antibody is a“chimeric” antibody as described in U.S. Pat. No. 4,816,567.
  • the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409.
  • the antibody is a human antibody.
  • A“human antibody” such as described in US 6,075,181 and 6,150,584.
  • the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388.
  • the antibody is a monoclonal antibody.
  • Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.
  • the antibody anti-TfRl is conjugated to the drugs.
  • Said antibody is called as antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • such antibody is combined with the potency of chemotherapeutic agents.
  • the antibody anti-TfRl is able to induce cytotoxicity, also known as the antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies.
  • variable region Three highly divergent stretches within the variable region of the heavy and light chains are referred to as“hypervariable regions” which are interposed between more conserved flanking stretches known as“framework region” or“FRs”.
  • FR refers to amino acid sequences which are naturally found between and adjacent to hypervariable region.
  • three hypervariable regions of a light chain and three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen binding surface. This surface mediates recognition and binding of the target antigen.
  • the three hypervariable regions of each of the heavy and light chain are referred to as “complementary determining regions” or“CDRs” and are characterized, for example by Rabat et al.
  • 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).
  • the CDR of the antibodies of the invention are in the Kabat nomenclature.
  • the antibody for use according to the invention wherein said antibody is an anti-TfRl antibody chosen among H7, FI 2, C32, F2, H9, G9.
  • the antibody for use according to the invention wherein said antibody is H7 (SEQ ID NO: l).
  • the H7 antibody has the following sequences as described below:
  • the antibody for use according to the invention wherein said antibody is H7 antibody comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:3, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:5; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:7.
  • said antibody for use according to the invention wherein said antibody is F12 antibody (SEQ ID NO: 10).
  • the F12 antibody has the following sequences as described below:
  • the antibody for use according to the invention wherein said antibody is F12 antibody comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: l 1 , (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16.
  • F12 antibody comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: l 1 , (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, (d) a light chain CDR1 compris
  • the antibody for use according to the invention wherein said antibody is C32 antibody (SEQ ID NO: 19).
  • the C32 antibody has the following sequences as described below:
  • the antibody for use according to the invention wherein said antibody is C32 antibody comprising: a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:20, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:21, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:22, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:23; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:24, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:25.
  • the antibody for use according to the invention wherein said antibody is F2 antibody (SEQ ID NO: 28).
  • the F2 antibody has the following sequences as described below:
  • the antibody for use according to the invention wherein said antibody is F2 antibody comprising a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:29, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:30, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:31, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:32; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:33, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:34.
  • said antibody for use according to the invention wherein said antibody is H9 antibody (SEQ ID NO: 37).
  • the H9 antibody has the following sequences as described below:
  • the antibody for use according to the invention wherein said antibody is H9 antibody comprising a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:38, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:39, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:40, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:41 ; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:42, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:43.
  • the antibody for use according to the invention wherein said antibody is G9 antibody (SEQ ID NO: 46).
  • the H9 antibody has the following sequences as described below:
  • the antibody for use according to the invention wherein said antibody is G9 antibody comprising a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:47, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:48, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:49, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:50; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:51, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:52.
  • G9 antibody comprising a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:47, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:48, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:49, (d) a light chain CDR1 comprising
  • the antibody anti-transferrin receptor 1 (TfRl) for use according to the invention is conjugated with Monomethyl Auristatin F (MMAF) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization using a non-cleavable linker.
  • MMAF Monomethyl Auristatin F
  • the antibody anti-transferrin receptor 1 (TfRl) for use according to the invention is conjugated with Monomethyl Auristatin E (MMAE) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization.
  • MMAE Monomethyl Auristatin E
  • the term“cancer” refers to an abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. In the context of the invention, the term“cancers” relates to cancers which absorb iron. More particularly, iron uptake by transferrin receptor is the most important way for cancer cells to absorb iron which in required for their survival.
  • the cancer is selected from the following group but not limited to: pancreatic cancer, neuroblastoma, solid cancer, leukemia, lymphoma, glioblastoma, breast cancer, cancer related cachexia, gastrointestinal cancer such as colorectal cancer, cholangiocarcinoma, carcinoma of the oral cavity, gastric cancer, lung cancer such as small cell lung cancer, lung adenocarcinoma, melanoma, multiple myeloma, ovarian cancer, prostate cancer, renal cancer, hepatocarcinoma.
  • the cancer is selected from the group consisting of pancreatic cancer, neuroblastoma, solid cancer, leukemia, lymphoma, glioblastoma, breast cancer, cancer related cachexia, gastrointestinal cancer such as colorectal cancer, cholangiocarcinoma, carcinoma of the oral cavity, gastric cancer, lung cancer such as small cell lung cancer, lung adenocarcinoma, melanoma, multiple myeloma, ovarian cancer, prostate cancer, renal cancer, hepatocarcinoma.
  • Im9 B-cell lymphoma cell line which is resistant to apoptosis induced by rituximab (anti-CD20 antibody) was sensitive to the antibody H7. They also shown that H7 decreased the tumor growth of the PD AC patient derived xenograft (PDX) model and improved the gemcitabine efficiency on the BxPC3 xenograft mice model.
  • PDX PD AC patient derived xenograft
  • the invention relates to an antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of resistant cancers.
  • TfRl antibody anti- transferrin receptor 1
  • the term“resistant cancers” refers to a cancer that does not respond to treatment notably to convention therapies (chemotherapy, radiotherapy etc).
  • the cancer is resistant at the beginning of treatment.
  • the cancer become resistant during treatment, such cancer is called refractory cancer.
  • the resistant cancer is selected from the group consisting of pancreatic cancer, neuroblastoma, leukemia, lymphoma, glioblastoma, colon cancer.
  • the cancer is resistant to rituximab.
  • Rituximab is a chimeric monoclonal antibody against the protein CD20.
  • the antibody anti- transferrin receptor 1 is used in combination with a second agent for treatment of a disease or disorder.
  • an antibody anti- transferrin receptor 1 (TfRl) may be used in combination with conventional cancer therapies such as, e.g., surgery, radiotherapy, chemotherapy, or combinations thereof.
  • the present invention also provides for therapeutic applications where the antibody anti transferrin receptor 1 (TfRl) is used in combination with at least one further therapeutic agent, e.g. for treating cancers and metastatic cancers.
  • TfRl antibody anti transferrin receptor 1
  • 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.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolo melamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozeles).
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33 : 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyr
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the antibody anti- transferrin receptor 1 (TfRl) is used in combination with gemcitabine.
  • the antibody anti-transferrin receptor 1 (TfRl) is used in combination with an ATR inhibitor.
  • ATR also known as“ataxia telangiectasia and Rad3-related protein” has its general meaning in the art and refers to a serine/threonine-specific protein kinase that is involved in sensing DNA damage and activating the DNA damage checkpoint, leading to cell cycle arrest.
  • ATR inhibitor refers to chemical compounds that are effective in inhibiting ATR kinase.
  • ATR inhibitors include: ceralasertib; berzosertib (also known as VX- 970, VE822 or M6620); KU-55933; AZD0156; VE-821; BAY-1895344; AZ20; CGK733; ETP-46464; and AZD6738.
  • the antibody anti-transferrin receptor 1 (TfRl) is used in combination with Berzosertib.
  • the antibody anti-transferrin receptor 1 (TfRl) is used in combination with Auristatin.
  • Auristatin has its general meaning in the art and refers to a family of synthetic antineoplastic agent. Auristatins lead to the arrest of cancer cells in the mitosis stage and eventually, apoptosis.
  • the antibody anti- transferrin receptor 1 (TfRl) is used in combination with Monomethyl Auristatin F (MMAF) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization using a non-cleavable linker.
  • MMAF Monomethyl Auristatin F
  • the antibody anti-transferrin receptor 1 (TfRl) is used in combination with Monomethyl Auristatin E (MMAE) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization.
  • MMAE Monomethyl Auristatin E
  • the cancer is resistant to immune check point inhibitor.
  • immune checkpoint inhibitor refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins.
  • 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 stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS.
  • inhibitory checkpoint molecules examples include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA.
  • A2AR Adenosine A2A receptor
  • B7-H4 also called VTCN1
  • B and T Lymphocyte Attenuator (BTLA) and also called CD272 has HVEM (Herpesvirus Entry Mediator) as its ligand.
  • HVEM Herpesvirus Entry Mediator
  • Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA.
  • CTLA-4 Cytotoxic T-Lymphocyte- Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation.
  • IDO Indoleamine 2,3-dioxygenase
  • TDO tryptophan catabolic enzyme
  • Another important molecule is TDO, tryptophan 2,3-dioxygenase.
  • IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis.
  • KIR Killer-cell Immunoglobulin-like Receptor
  • LAG3, Lymphocyte Activation Gene-3 works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells.
  • PD- 1 Programmed Death 1 (PD-1) receptor
  • PD-L1 and PD-L2 This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014.
  • An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment.
  • TIM-3 short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines.
  • TIM-3 acts as a negative regulator of Thl /Tel function by triggering cell death upon interaction with its ligand, galectin-9.
  • VISTA Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response.
  • an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade.
  • the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.
  • the immune checkpoint inhibitor is an antibody.
  • antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
  • the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302.
  • anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).
  • the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897.
  • anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).
  • the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.
  • the immune checkpoint inhibitor inhibits Tim-3 or its ligand.
  • the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and WO2013006490.
  • the immune checkpoint inhibitor is a small organic molecule.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
  • small organic molecules interfere with transduction pathway of PD-1 and Tim-3.
  • they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.
  • the small organic molecules interfere with Indoleamine- pyrrole 2,3-dioxygenase (IDO) inhibitor.
  • IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677.
  • IDO inhibitors include without limitation 1 -methyl-tryptophan (IMT), b- (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-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5-bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydan
  • the IDO inhibitor is selected from 1 -methyl-tryptophan, b-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and b-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.
  • the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4- fluorophenyl)-N'-hydroxy-4- ⁇ [2-(sulfamoylamino)-ethyl]amino ⁇ -l,2,5-oxadiazole-3 carboximidamide :
  • the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-1, 2, 4-Triazole-3, 5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:
  • the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V- domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015).
  • PD-170 or AUPM-170
  • VISTA V- domain Ig suppressor of T cell activation
  • the immune checkpoint inhibitor is an aptamer.
  • the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
  • aptamers are DNA aptamers such as described in Prodeus et al 2015.
  • a major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration.
  • aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the aptamer is an anti-PD-1 aptamer.
  • the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.
  • the invention relates to a method for treating resistant cancer in a subject in need thereof comprising a step of administering said subject with a therapeutically amount of an antibody anti- transferrin receptor 1 (TfRl).
  • TfRl antibody anti- transferrin receptor 1
  • the term“subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • the subject according to the invention is a human. More particularly, the subject according to the invention has or susceptible to have resistant cancer. In a particular embodiment, the subject has or is susceptible to have resistance to rituximab.
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an antibody anti-TfRl) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • a “therapeutically effective amount” is meant a sufficient amount of an antibody anti- TfRl for use in a method for the treatment of melanoma at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • an effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the invention relates to an i) antibody anti- transferrin receptor 1 (TfRl) and ii) an immune check point inhibitor, for use in the treatment of resistant cancer, as a combined preparation.
  • the invention also relates to an i) antibody anti-transferrin receptor 1 (TfRl) and ii) an ATR inhibitors, for use in the treatment of cancer and resistant cancer.
  • TfRl antibody anti-transferrin receptor 1
  • ATR inhibitors for use in the treatment of cancer and resistant cancer.
  • the terms“combined treatment”, “combined therapy” or“therapy combination” refer to a treatment that uses more than one medication.
  • the combined therapy may be dual therapy or bi-therapy.
  • the antibody-anti TfRl according to the invention and an immune check point inhibitor for simultaneous, separate or sequential use in the method for treating resistant cancer.
  • the term“simultaneous use” refers to an administration of 2 active ingredients by the same route and at the same time or at substantially the same time.
  • the term“separate use” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes.
  • the invention relates to a pharmaceutical composition comprising an antibody anti- transferrin receptor 1 (TfRl) according to the invention.
  • TfRl antibody anti- transferrin receptor 1
  • the pharmaceutical composition according to the invention for use in the treatment of cancer.
  • the pharmaceutical composition according to the invention for use in the treatment of resistant cancer.
  • the anti-TfRl antibody as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxysulfonate, a pharmaceutically acceptable.
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intrap eritoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • 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 are a diagrammatic representation of FIGURES.
  • Figure 1 Effect of H7 on holo-Tf uptake and LIP,
  • A Non confluent cells were stained with 500 nM holo-Tf conjugated to Alexa 488 and either left untreated or treated with increasing concentrations of H7 for 4 hrs at 37°C. Internalized fluorescence was measured by FACS.
  • BxPC3 and CFPAC cell lines were labeled with 0.25 M of the intracellular iron chelating dye calcein-AM for 5 minutes. Cells were washed then either left non treated or treated as indicated for 8 hrs at 37°C. Fluorescence of calcein was measured by FACS. Results are expressed as the % of change in the fluorescence relative to the NT cells.
  • FIG. 2 H7 effect on cell viability of PDAC cells.
  • A, B, C BxPC3, CFPAC, and HP AC were treated for 5 days with H7 at the indicated concentrations (B) medium was complemented or not ferric ammonium citrate (FAC) at 0.25 mM.
  • A, B Cell viability was assessed by MTS assay Results are expressed as % of viable cells compared to non treated cells (C) Apoptosis was detected by by FACS using conjugated Annexin V-FITC and 7AAD.
  • the percentage of early apoptotic cells which are Annexin V positive 7AAD negative (An+) are shown in blue and late apoptotic cells which are Annexin V positive and 7AAD positive (An+ 7AAD+) are shown in white.
  • FIG. 3 Effect of H7 on BxPC3 xenograft and PDX mouse models.
  • BxPC3 cells (A) or PDX6 (B) were grafted in nude mice.
  • Established tumors were either treated with NaCl, H7 at 10 mg/kg, gemcitabine (gem) 100 mg/kg for BxPC3 or 50 mg/kg for PDX model, or the combinations H7 and gemcitabine for 4 weeks twice per week.
  • Tumor volume upper panels
  • the survival curve lower panels
  • Mice were sacrificed when their tumor reached 1500 mm3 for the BxPC3 model and 1400 mm3 for the PDX model. Arrows represent the treatments of the mice.
  • Figure 4 H7 and gemcitabine combination effect on viability of BxPC3 cell line.
  • Cells were plated, next day treated with either H7 alone at increasing concentrations (from 0.03 pg/ml to 2 pg/ml), gemcitabine alone at either 0.002 pg/ml (A) or 0.004 pg/ml (B), or gemcitabine fixed at the indicated concentrations with H7 varying with the range indicated above. 5 days later MTS reagent was added and cell viability was assessed.
  • Figure 5 A. TfRl expression by HP AC and PanPec cancer cells and CSCs. Cells were plated in IMDM N2 medium at 500 cells/well in 96 ultra-low attachment well plates.
  • FIG. 1 DFO and H7 Dose response on HCT116, DLD1, and DK04 colon cancer cells. Cells were plated, 24hrs later DFO (Fig A) or H7 (Fig B) were added with increasing concentrations. After 5 days cell viability was assessed using MTS assay (cellTiter 96, Promega).
  • Figure 7 Functional properties of anti-TfRl H7 scFv2-Fc on Im9 B-cell lymphoma.
  • Treatment with the anti-CD20 rituximab (RX, hlgGl) was tested. After the treatment, cells were collected and stained with a mix of Annexin conjugated to FITC and 7- AAD and analyzed by FACS. Results are expressed as the % of cells Annexin+/7AAD- that represent early apoptotic cells compared to non-treated.
  • Figure 8 Effect of H7 conjugated to Auristatin (ADC-H7) on cell viability of the BxPC3 cell line.
  • Cells were plated and next day either left non treated (NT), or treated with RX (negative control antibody), H7, or ADC-7 at different concentrations (0.05, 0.5, or 5 gg/ml). 5 days after treatment cell viability is measured by MTS (cell Titer, 96).
  • Figure 9 Comparative effect of the anti-TfRl H7 and the anti-HER2 trastuzumab as ADC conjugated to monomethyl-auristatin-E (MMAE) on the SKBR3 cancer cell line.
  • H7 was conjugated to MMAE (Drug antibody ratio of 3.5).
  • the anti-HER2 mAh trastuzumab (TZ) and the anti-CD20 rituximab (RX) were conjugated the same way.
  • 4000 cells of the SKBR3 human breast cancer cell line in 200 pF, DMEM, 10%FCS
  • Figure 10 Comparative effect of the anti-TfRl H7 and the anti-CD30 Adcetris as ADC conjugated to monomethyl-auristatin-E (MMAE).
  • MMAE monomethyl-auristatin-E
  • FIG. 11 Iron deprivation with an anti-TfRl H7 induces activation of the ATR / Chkl pathway indicating DNA damage.
  • A Upregulation of the ATR-ChklDNA damage checkpoint by anti-TfRl treatment.
  • BxPC3 and CFPAC cells either non treated (NT) or treated with cetuximab (Cx) as a control antibody at 10 pg/ml or H7 10 pg/ml or ironchelator deferoxamine for 48 hours and were analyzed for pThr ATR (1989), or pChkl (Ser 345) protein expression by Western blot.
  • B Synergistic effect of inhibition of DNA damage pathway and iron deprivation with H7 anti-TfRl antibody.
  • BxPC3 and CFPAC cells were treated with the ATR- inhibitor VE822 (Active Biochem; Ref VX-970, dissolved in DMSO) at the determined IC10 or IC10 (concentration inducing 10% of inhibition), i.e. 0.5 mM and 0.1 mM for BxPC3 and CFPAC, respectively, and with H7 2 pg/mF, or with with a VE/H7 combined treatment. Cell viability was determined after 5 days of treatment.
  • VE822 Active Biochem
  • Ref VX-970 dissolved in DMSO
  • Example 1 use of the antibody anti-TfRl (H7) in Pancreatic Cancer in pancreatic cells lines and in mouse models
  • DFO was obtained from Santa Cruz Biotechnology (sc-203331). Cetuximab antibody was obtained from Merck. Ferric ammonium citrate was purchased from Sigma Aldrich (F 5879). Gemcitabine (Hospira) was obtained from Val d’Aurelle hospital (Montpellier).
  • anti-human p21CIPl/WAFl 2946S
  • anti-human pchKl Ser345 2348T
  • anti-human NDRG1 5196S
  • anti-human pNDRGl Ser330 3506S
  • anti-human pNDRGl Thr346 3217S
  • the anti human TfRl (1360800) antibody was from Invitrogen, anti-human pH2AX Serl39 (05-636), and anti-human pThrl989 ATR (GTX128145) antibodies were obtained from Millipore and Genetex respectively and anti-human ferritin (ab75973) antibody was purchased from Abeam.
  • the anti-glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore (Billerica, MA) and tubulin from sigma aldrich (T4026). H7 in the IgGl format was produced in CHO cell line by EVITRIA (Switzerland) from the heavy and light variable domain sequences.
  • the PD AC cell lines: BxPC3, CFPAC-1, and HP AC were obtained from ATCC (Rockville, MD).
  • BxPC3 and HP AC are both epithelial cells that were derived from pancreatic adenocarcinomas.
  • the CFPAC-1 cells are epithelial cells derived from liver metastasis of pancreatic adenocarcinoma.
  • the BxPC3 and HP AC cell lines were cultured in RPMI 1640 medium while CFPAC in IMDM (Iscove’s Modified Dulbecco’s Medium).
  • the culture media was supplemented as recommended by ATCC with 10% FCS and penicillin, streptomycin antibiotics. All culture media were purchased from Fife Technologies, Inc (Gibco BRF, Gaithersburg, MD). Cells were grown at 37°C in a humidified atmosphere of 5% C02 and the medium was changed twice a week.
  • TFR1 on the cell surface of the PD AC cells was done as follows: non confluent cells recently plated (2 days) were trypsinated, washed with phosphate buffer saline (PBS) and incubated with APC-conjugated anti-human CD71 (BD pharmingen, 55134) for 1 hour at 4°C. The cells were then washed with cold PBS twice before FACS analysis.
  • PBS phosphate buffer saline
  • the effect of the anti-TfRl antibody on cell proliferation was done on the basis of the incorporation of 5-ethynyl-2’-deoxyuridine (EdU) into cells during DNA synthesis.
  • EdU 5-ethynyl-2’-deoxyuridine
  • the click- iT EdU Flow Cytometry Assay system (Invitrogen, Cl 0420) was used following the manufacturer’s protocol.
  • Cells were plated in medium complemented with 5% FCS at the following cell densities: BxPC3 50,000, CFPAC 15000, and HP AC 40,000 cells per well in 6 well plates. Next day they were either left untreated or treated with antibodies at 10 pg/ml at 10 pg/ml (H7 or Cetuximab as negative control), or DFO at 5 pM (positive control for iron deprivation) for 5 days.
  • Intracellular free iron levels were measured using the fluorescent probe calcein.
  • This probe binds iron rapidly and stochiometrically in a reversible manner while forming fluorescence-quenched calcein-iron complexes.
  • Once inside the cells calcein is cleaved by intracellular esterases to yield green fluorescent calcein, the fluorescence of which is quenched upon binding to iron.
  • PD AC cells were harvested, resuspended in medium without FCS, and incubated with 250 nM calcein- AM (Invitrogen) for 5 minutes at 37°C. They were washed and resuspended in prewarmed culture medium complemented with 1% FCS.
  • PKH26 staining Sigma Aldrich, Mini26-1 KT was used following the manufacturer’s instructions. Briefly, cells were harvested and stained with PKH26 fluorescent dye for 3 minutes at room temperature, washed and seeded in 6 well plates. 24 hours later they were treated with H7 at 10 iig/m 1 for 5 days. The Fluorescence intensity of PKH was then evaluated by FACS.
  • PI Propidium iodide obtained from sigma Aldrich, P4864 was used to do the cell cycle assay.
  • Cells were treated with H7 at 2 or 10 iig/ml in medium complemented with 5% FCS at 37°C for 3 days. They were then permeabilized with ethanol 70%, treated with RNase A (sigma Aldrich, R6513) at 100 pg/ml, and stained with PI at 40 pg/ml before being analyzed by FACS.
  • the Annexin V and 7AAD labeling kit (Beckman Coulter, IM3614) was used to examine apoptosis induced in response to H7 treatment.
  • Cells were treated with H7 at 2 or 10 pg/ml in medium supplemented with 5% FCS at 37°C. After 5 days of treatment, cells were harvested, washed with PBS and stained with 10 m ⁇ annexin V-FITC and 20 m ⁇ of 7AAD in 100 m ⁇ PBS. After 15 minutes of incubation in the dark at 4°C, 400 m ⁇ of PBS is added and cell apoptosis is examined by FACS.
  • Kook buffer was used for extracting proteins from cell lines. Cells were treated for 2 days with H7 at 10 pg/ml in 5% FCS medium in 6 well plates. Medium was then removed and the cells were washed three times with PBS and then 50 m ⁇ of Kook buffer (Tris-HCl PH 7.5 10 mM, SDS 20%) was added to cells which were then scraped and sonicated 4 times 5 seconds at amplitude 25%.
  • Tumors were homogenized for 30 minutes at 4°C with buffer containing 20 mM Tris-HCl (PH 7.5), 150 mM NaCl, 1.5 mM MgC12, 1 mM EDTA, 1% Triton, 0.1 mM phenylmethylsulfonyl fluoride, 100 mM sodium fluoride, 1 mM sodium orthovanadate (Sigma Aldrich), and one tablet of complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN). The mixture was then centrifuged at 13,400 rpm for 30 minutes at 4°C and the supernatant was collected. Proteins were then dosed and lameli 5X was added.
  • the extract was then heat denatured at 95°C for 5 minutes.
  • 70 pg of protein was loaded on 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) which was then saturated with 5% non-fat dry milk diluted in PBS-Tween 0.1 % for 1 hour at 25°C.
  • PVDF polyvinylidene difluoride membrane
  • Membranes were then incubated with the proper primary antibodies overnight at 4°C. After washing the membrane, the appropriate peroxidase-conjugated rabbit or mouse antibodies (Sigma- Aldrich) were added in PBS-Tween 0.1 %, 5% non-fat dry milk for 1 hour at 25°C.
  • the membrane was then washed and the blots were visualized using a chemiluminescent substrate (Western Lightning Plus-ECL; PerkinElmer).
  • ADCC Antibody Dependent Cell Cytotoxicity
  • BxPC3, CFPAC, and HP AC cells were stained with the PKH dye as described above in the cell division assay.
  • target cells 50 pF, 50,000 cells per well
  • the antibodies Rituximab (anti-CD20, negative control), Cetuximab (anti-EGFRl, positive control), or H7 (50 pF, 0.5 pg/ml) for 30 minutes at 37°C.
  • Peripheral Blood Mononuclear cells (PBMCs) 100 pL
  • 7AAD is added to the cells for 15 minutes at 4°C.
  • the % of 7AAD positive target cells (PKH positive) representing PD AC cells killed by ADCC was then quantified by FACS.
  • mice purchased from Harlan (Fe Malcourlet, France) were injected subcutaneously with BxPC3 cells (4x106 in 150 pF).
  • BxPC3 cells 4x106 in 150 pF.
  • mice were injected i.p. with either NaCl, H7 alone at 10 mg/kg, gemcitabine alone at 50 or 100 mg/kg, or combination of H7 plus gemcitabine. The treatment was done twice per week for 4 weeks. Tumor volumes were calculated by using the formula: DlxD2XD3/2. For survival curves mice were sacrificed when tumors reached a volume of 1400 or 1500 mm3.
  • the level of the TfRl was directly correlated to holo-Tf uptake in such a way that the CFPAC cell line expressing the highest TfRl level takes up transferrin the most (data not shown), which indicates that these cell lines are using the transferrin receptor to take up iron bound Tf
  • H7 decreases intracellular LIP by blocking holo-Tf uptake
  • the available anti-TfRl scFv H7 antibody binds to the TfRl at the binding site of holo- Tf (3D model structure of the scFv binding to TfRl not shown, collaboration Thomas Bourquard, BIOS, INRA Tours) and thus we expected that the full length reformated IgGl H7 may block the internalization of holo-Tf by cells.
  • holo- Tf 3D model structure of the scFv binding to TfRl not shown, collaboration Thomas Bourquard, BIOS, INRA Tours
  • H7 effectively blocked the uptake of holo-Tf whereby, at approximately 1/10 equimolar concentrations of H7 (45 nM) compared to Tf- Alexa 488 (500 nM), H7 decreases the holo-Tf uptake more than 50 % for all cell lines (Fig 1 A).
  • Holo-Tf blockade by H7 decreased intracellular LIP in a dose dependent fashion after 8 hrs. of H7 treatment shown with the increase in the calcein fluorescence (see methods).
  • DFO (10 or 100 mM) increased the fluorescence up to 29% and H7 up to 18% (H7 10 pg/mL) and 37% (H7 100 pg/ml) for the BxPC3 cell line.
  • the decrease in LIP is lower, such that with DFO (100 mM) the increase was 8% while with H7 whether 10 or 100 pg/ml there was an increase of 5% but remains significant (Fig 1 B).
  • H7 decreases cell viability and induces apoptosis of PD AC cell lines
  • Fig 2A shows a dose dependent decrease in cell viability upon H7 treatment with a maximal inhibition of 40% reached with 10 pg/rnl of antibody after 5 days of treatment. Increasing the antibody concentration to 100 pg/rnl did not lead to more reduction in cell viability.
  • culture medium was complemented with ferric ammonium citrate (0.25 mM)
  • ferric ammonium citrate 0.25 mM
  • H7 treatment induced apoptosis on the 3 cell lines (Fig 2C) as detected by Annexin V staining.
  • H7 reduces cell proliferation by blocking cells in S phase and inducing DNA damage
  • H7 effect on cell proliferation was tested by EdU incorporation in the newly replicating DNA of cells treated with H7 at 10 pg/ml for 5 days.
  • H7 decreased cell proliferation by 34 % on the BxPC3 cell line and 70% on the CFPAC and HP AC cell lines (data not shown).
  • DFO treatment (5 mM) also inhibited proliferation between 85 and 95% depending on the cell line tested.
  • PKH a fluorescent dye that binds to the cell membrane and allows cell tracking.
  • H7 has been recently described that iron deprivation using iron chelaror induce the expression of the tumor suppressor, NDRG1.
  • H7 at 10 pg/ml upregulates NDRG1 expression on the 3 cell lines tested.
  • the importance of NDRG1 phosphorylation in its antitumor functions has been recently revealed [20]
  • H7 also increased the phosphorylation of NDRG1 on Ser-330 and Thr-346. Therefore, we demonstrate for the first time the induction of NDRG1 by an anti-TfRl antibody taking into consideration that all the previous studies that reported NDRG1 induction were with iron chelators.
  • ADCC Antibody Dependent Cell Cytoxicity
  • H7 has been isolated in an antibody phage display screen for breast cancer specific internalizing antibodies. Interestingly, nevertheless, despite its internalizing properties, H7 treatment is up regulating its target TfRl on the cell surface.
  • PD AC cells were stained with the fluorescent dye PKH26 and incubated with H7 and PBMC as a source of NK cells.
  • H7 induced ADCC on both the BxPC3 and CFPAC cell lines (data not shown) in comparison to the negative control antibody Rituximab (anti-CD20) while the anti-EGFR Cetuximab was superior to H7 for ADCC induction. This indicates that in vivo H7 could recruit NK cells highlighting another mechanism of action of H7 antibody aside from its intrinsic killing effect through iron deprivation.
  • H7 inhibits tumor growth in PD AC mice models
  • H7 H7 + gemcitabine combination treatment at the same concentrations or vehicle (NaCl).
  • H7 alone decreased slighty tumor growth but not significantly compared to mice treated with NaCl while gemcitabine, as expected was efficient (p ⁇ 0,001) (Fig 3 A).
  • Fig 3 A Yet the combination of H7 with gemcitabine showed significantly improved effects on tumor growth as compared to gemcitabine alone (p ⁇ 0.05).
  • Gemcitabine is the standard treatment used in pancreatic cancer, it is a nucleoside analogue which when incorporated in DNA blocks further insertion of nucleotides and inhibits DNA replication (Shore et ah, 2003).
  • H7 can enhance the efficiency of gemcitabine.
  • a dose response treatment of gemcitabine on the BxPC3 cell line we found that the cells are extremely sensitive to gemcitabine (data not shown).
  • N2 contains growth factors including transferrin 10 mM, EGF for epidermal growth factor (20 ng / ml), bFGF for basic fibroblast growth factor (20 ng / ml) and insulin (5 g/ml).
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • insulin 5 g/ml
  • Cells are plated in 96 ultra-low attachment plates (500 cells/well). After that TfRl level was measured by FACs.
  • TfRl level was measured by FACs.
  • Example 2 use of the antibody anti-TfRl (H7) in colon cancer cells lines
  • Colon cancer cells are differentially sensitive to H7
  • HCT116 and DK04 cells harbor an activating mutation in the RAS oncogene.
  • DFD1 cell line has been generated from DK04 by targeted disruption of the mutated RAS leading to its inactivation.
  • Figure 6A demonstrates the sensitivity of these cells to DFO treatment, showing a dose dependent decrease in cell viability after 5 days of treatment.
  • H7 upregulates TfRl and downregulates ferritin levels of the colon cancer cell lines
  • H7 induced the upregulation of TfRl and the downregulation of ferritin levels on a variety of colon cancer cell lines.
  • HCT116, DK04, and DLD1 we observed the effect on 3 cell lines HCT116, DK04, and DLD1 (data not shown).
  • Treatment of these cell lines with 10 iig/rnl of H7 for 2 days induced an increase in the TfRl protein expression as shown in the western blot as well as a decrease in ferritin levels (data not shown) indicating that intracellular iron deprivation occurred in these cells after H7 treatment.
  • HCT116 we wanted to verify if H7 treatment induces apoptosis of the colon cancer cells and if it affects their progression in the different phases of the cell cycle, so we chose one cell line which is HCT116.
  • HCT 116 cells were arrested in S phase (an increase from 38 % in S phase with non-treated cells to 64% with H7 treated cells) so did DFO at 10 mM but to a lower extent (46 % of cells in S phase) (data not shown).
  • Apoptosis was confirmed both by an increase in the subGl phase of the cell cycle after 2 days of treatment (from 0.8% with non-treated cells to 1.4 % with H7 and 1.4 % with DFO treated cells) as demonstrated in our results (data not shown) and by an increase in the % of apoptosis detected using annexin V-FITC and 7AAD staining after 4 days of treatment (data not shown).
  • HCT116 is highly sensitive to H7 treatment which causes an arrest of cells in the cell cycle, death by apoptosis, and induction of NDRG1 protein expression. These features are similar to what was observed with the PD AC cell lines highlighting a therapeutic potential of H7 in colorectal cancer.
  • Example 3 use of the antibody anti-TfRl (H7) in rituximab-resistant B-cell lymphoma cells lines
  • Anti-TfRl antibody intrinsic cytotoxic activity After confirming that the ERY-1 and Raji cancer cell lines are sensitive to the iron chelator deferoxamine (DFO) (data not shown), these cell lines were used to test the effect of the H7 and Bal20 (another anti-TfRl antibody that induced the degradation of TfRl unlike H7, not shown) antibodies on cell growth. After 5 days of incubation, H7-Fc and H7-IgGl strongly decreased the viability of both cell lines (IC50 in the range of 0.1 pg/mL) (data not shown). Conversely, Bal20 had a limited effect, in agreement with its lower competition with holo-Tf (data not shown).
  • DFO iron chelator deferoxamine
  • H7-IgGl reduced rapidly (4h) the levels of the intracellular labile iron pool (FIP) in Raji and ERY-1 cells, while Bal20 had a more limited effect, especially at the lowest concentration used (1.5 pg/mL) (data not shown).
  • FIP intracellular labile iron pool
  • Bal20 had a more limited effect, especially at the lowest concentration used (1.5 pg/mL) (data not shown).
  • ERY-1 cells apoptosis could be detected already after 1 day of incubation with H7-Fc or H7-IgGE After 3 days, the percentage of apoptotic cells was higher than 50% using 5nM of H7-Fc or H7-IgGl (corresponding to 0.5 pg/mL and 0.75 pg/mL, respectively).
  • apoptosis was more limited with Bal20, even when used at high concentration (500 nM corresponding to 75 pg/mL) (data not shown).
  • Apoptosis upon H7 treatment was also detected in Raji cells, with the same kinetics than in ERY-1 cells, but to a lesser extent, consistent with this cell line displaying autophagic, but not apoptotic cell death features upon iron deprivation.
  • H7 Bal20 induced apoptosis in both cell lines, but was less efficient than H7 (Figure 1). H7 also induced early moderate free iron level decrease in both Bp3 and Im9 cell lines (data not shown). Altogether, these in vitro data indicate that the holo-Tf uptake blockade by H7 induces apoptosis in leukemia and lymphoma cell lines, including those resistant to rituximab. Accordingly, as shown in figure 7 inventors by in vitro methods have shown that H7 had a strong inhibitory effect in the rituximab-resistant B-cell lymphoma cell line Im9. H7 drastically reduced cell viability of Raji and ERY-1 cells (IC50 in the range of 0.1 pg/mL) and induced apoptosis in ERY-1, Bp3 and Im9 cells.
  • Example 4 an anti-TfRl conjugated to Auristatin
  • H7 was conjugated to Monomethyl Auristatin F (MMAF) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization using a non-cleavable linker.
  • MMAF Monomethyl Auristatin F
  • the drug antibody ratio obtained was estimated to be 2.5.
  • ADC-H7 or ADC-H7-MMAF.
  • Figure 8 reports the results on BxPC3 cell line obtained with 3 different concentrations of H7 conjugated or not to MMAF and as a negative control the anti-CD20 Rituximab (RX) conjugated with the same procedure to MMAF.
  • the figure show that ADC-H7 is very powerful compared to unconjugated H7. A clear inhibition is obtained for a concentration of H7 of 0.5 iig/mL and almost total inhibition is obtained with 5 iig/mL.
  • the H7 was also conjugated to Monomethyl Auristatin E (MMAE) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization.
  • MMAE Monomethyl Auristatin E
  • ADC-H7-MMAE The effect of the anti-TfRl H7 and the anti-HER2 trastuzumab (TZ) as ADC conjugated to MMAE on SKBR3 breast cancer cell lines were compared.
  • the ADC H7-MMAE has a similar IC50 compared to the ADC TZ-MMAE and decreases the viability to zero at similar concentrations.
  • the naked antibody alone has a cytostatic effect and decrease the viability of 40%. Therefore, the anti-TfRl -ADC could be used to treat HER2 amplified breast cancer resistant to trastuzumab or trastuzumab-Emtansin (Figure 9).
  • the ADC RX-MMAE is only active on the Raji cell line
  • the brentuximab vedotin is only active on the karpas 299 cell line.
  • the ADC H7-MMAE and MMAF have similar IC50 (in the range of 10 pM) on both cell lines and are 30 to 700 time more active than the naked antibody H7 probably due to the combined effect of iron deprivation and microtubule inhibition (Figure 10B).
  • the anti-TfRl ADC are 1 log less active than the potent clinically used brentixumab vedotin (anti-CD30) by display however strong activity (IC50 of 20 nM) ( Figure 10B). Therefore, the anti-TfRl ADC could be used on lymphoma patients resistant to brentuximab vedotin.
  • Example 5 Iron deprivation with an anti-TfRl H7 induces activation of the ATR /Chkl pathway indicating DNA damage.
  • the anti-human pThrl989 ATR (GTX128145) antibodies was obtained from Genetex and anti-human ferritin (ab75973) antibody was purchased from Abeam.
  • the anti- glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore (Billerica, MA) and tubulin from sigma aldrich (T4026). For extracting proteins, Kook buffer was used. Cells were treated for 2 days with H7 at 10 pg/ml in 5% FCS medium in 6 well plates.
  • PVDF polyvinylidene difluoride membrane
  • BxPC3 and CFPAC cells were treated with the ATR-inhibitor VE822 (Active Biochem; Ref VX-970, dissolved in DMSO) at the determined IC10 or IC10 (concentration inducing 10% of inhibition), i.e. 0.5 mM and 0.1 mM for BxPC3 and CFPAC, respectively, and with H7 2 pg/mL, or with with a VE/H7 combined treatment. Cell viability was determined after 5 days of treatment.
  • ATR-inhibitor VE822 Active Biochem
  • Ref VX-970 dissolved in DMSO
  • Cells were seeded at 5 x 103 (BxPC3), 1x103 (CFPAC) cells per well in 96-well plates with 200 pL of 5% FCS culture medium (RPMI for BxPC3 and .IMDM for CFPAC cells) 24 hours later cells they were treated and after 5 days of treatment 20 pL of MTS/PMS reagent was added in each well (CellTiter 96 AQueous One Solution Cell Proliferation Assay kit; Promega, G5430) for 2 hours. The absorbance at 490 nm was measured using a microplate reader (Multiskan) on 100 pL of supernatant.
  • FCS culture medium RPMI for BxPC3 and .IMDM for CFPAC cells
  • anti-TfRl with an ATR inhibitor (such as VE822) is synergistic for inhibit cell viability on pancreatic cancer lines BxPC3 and CFPAC.
  • the prensent invention shows that inventors have found an antibody anti transferrin receptor 1 (TfRl) for treating cancers and resistant cancers.
  • TfRl transferrin receptor 1
  • Torti SV Torti FM. Iron and cancer: more ore to be mined. Nat Rev Cancer 2013; 13:342-55.
  • Crepin R Goenaga AL, Jullienne B, Bougherara H, Legay C, Benihoud K, et al. Development of human single-chain antibodies to the transferrin receptor that effectively antagonize the growth of leukemias and lymphomas. Cancer research 2010; 70:5497-506.
  • Lepelletier Y Camara-Clayette V, Jin H, Hermant A, Coulon S, Dussiot M, et al. Prevention of mantle lymphoma tumor establishment by routing transferrin receptor toward lysosomal compartments. Cancer research 2007; 67: 1145-54.
  • TfR Transferrin receptor
  • Cymer F Beck H, Rohde A, Reusch D. Therapeutic monoclonal antibody N- glycosylation - Structure, function and therapeutic potential. Biologicals 2017. Abdel-Wahab O, Levine RL. Metabolism and the leukemic stem cell. The Journal of experimental medicine 2010; 207:677-80.
  • Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells. Mol Biol Cell 2008; 19:5490-505.

Abstract

Inventors have used an antibody (H7) which works by a special mechanism upregulating TfR1 levels which is not observed by other anti-TfR1 antibodies which usually block Tfbinding and internalization via blocking the TfR1 on the cell surface or directing it for lysosomal degradation resulting in decreased TfR1 levels. Interestingly, inventors have demonstrated for the first time with a naked anti-TfR1 antibody a complete inhibition of cell viability at less than 10 μg/ml of the antibody on a solid colon cancer cell line HCT116 at a low IC50 of 0.78 μg/ml. Inventors have shown that in vivo, H7 decreased the tumor growth of the PDAC patient derived xenograft (PDX) model and improved the gemcitabine efficiency on the BxPC3 xenograft mice model. Accordingly, inventors have shown that H7 antibody has the characteristics and qualifications that allow it to be tried in clinical trials. It is fully human, highly efficient in blocking iron supply from transferrin, and induces ADCC.

Description

METHODS AND COMPOSITIONS FOR TREATING CANCERS AND RESISTANT CANCERS WITH
ANTI
TRANSFERRIN RECEPTOR 1 ANTIBODIES
FIELD OF THE INVENTION:
The invention is in the field of cancerology. More particularly, the invention relates to an antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of cancers and resistant cancers.
BACKGROUND OF THE INVENTION:
Iron deprivation is an emerging strategy in cancer therapeutics. Tumors have high iron content and rely on iron for their growth and progression1. Cancer stem cells also require iron for their survival 2’ 3. Iron levels in cells can be reduced with iron chelators4 which are already used in the clinic for iron overload disorders. Transferrin receptor 1 (TfRl) is the main receptor responsible for the cell iron supply through receptor-mediated internalization of serum Fe3+- loaded transferrin (holo-Tf). Within the cell, Fe3+ is released, reduced, excluded from the early endosome by divalent metal ion transporter 1 (DMT1), and used for cell metabolism. Fe3+ excess is stored in ferritin, while TfRl is recycled at the cell surface together with iron- free transferrin (apo-Tf)5.
Cancer drug resistance has been and unfortunately still is a major problem in cancer therapy. Unfortunately there is a large group of patients that will either not respond to the applied therapy or will become resistant during therapy. Most of the patients ultimately become unresponsive, and relapse within 2-3 years.
Therefore, there is a need for efficient treatments of cancers and resistant cancers.
SUMMARY OF THE INVENTION:
The invention relates to an antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of cancers and resistant cancers. In particular, the invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION:
Inventors have used an antibody (H7) which is a fully human internalizing IgGl antibody. It works by a special mechanism upregulating TfRl levels which is not observed by other anti-TfRl antibodies which usually block Tf binding and internalization via blocking the TfRl on the cell surface or directing it for lysosomal degradation resulting in decreased TfRl levels. Interestingly, inventors have demonstrated for the first time with a naked anti-TfRl antibody a complete inhibition of cell viability at less than 10 iig/ml of the antibody on a solid colon cancer cell line HCT116 at a low IC50 of 0.78 iig/ml. In addition, variability on cell viability was observed when H7 was tested on a panel of colon cancer cell lines ranging between 50-80% of maximal inhibition at 10 iig/m 1 of H7 after 5 days of treatment.
Inventors have also found that TfRl is expressed by pancreatic cancer stem cells (CSCs) (HP AC and PanPec cells) grown in a specialized CSC medium and that the expression was higher in the PancPec cells grown in 3D compared to those in 2D. H7 was also found to decrease the growth of cells in 3D culture in CSC medium indicating that PDAC cancer stem cells (CSCs) are also sensitive to H7.
Inventors have shown that in vivo, H7 decreased the tumor growth of the PDAC patient derived xenograft (PDX) model and improved the gemcitabine efficiency on the BxPC3 xenograft mice model.
Inventors have also found that the Im9 B-cell lymphoma cell line, which is resistant to apoptosis induced by rituximab (anti-CD20 antibody), was sensitive to one of the antibody anti- TfRl(e.g. H7). Bp2 and Im9 B-cell lines (sensitive and resistant to rituximab-induced apoptosis, respectively) were incubated with H7 or rituximab. H7 strongly induced apoptosis in both cell lines. In Bp3 cells (rituximab- sensitive), the apoptotic rate was higher upon incubation with H7 than with rituximab (RX), although H7 effect was delayed compared with rituximab. Bal20 induced apoptosis in both cell lines, but was less efficient than H7. Altogether, these in vitro data indicate that holo-Tf uptake blockade by H7 rapidly reduces the LIP and induces apoptosis in leukemia and lymphoma cell lines, including those resistant to rituximab.
Accordingly, inventors have shown that H7 antibody has the characteristics and qualifications that allow it to be tried in clinical trials. It is fully human, highly efficient in blocking cell iron supply from transferrin, and induces ADCC.
Accordingly, in a first aspect, the invention relates to an antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of cancers. As used herein, the terms “treating” or“treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject 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., pain, disease manifestation, etc.]).
As used herein, the term“transferrin receptor” refers to a membrane transport protein which is involved in iron uptake in vertebrates. It imports iron by internalizing the transferrin- iron complex through receptor-mediated endocytosis. There are two types of transferrin receptors: transferrin receptor protein 1 (TfRl) and Transferrin receptor 2 (TfR2). In the context of the invention, the transferrin receptor is transferrin receptor protein 1 (TfRl) also known as Cluster of Differentiation 71 (CD71). TfRl is a transmembrane glycoprotein composed of two disulfide- linked monomers joined by two disulfide bonds. As used herein, the term“antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity of TfRl . Typically, such antibody inhibits the iron transport in the tumour cells.
The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Rabat et ak, 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab’)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab’)2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al, 2006; Holliger & Hudson, 2005; Fe Gall et al, 2004; Reflf & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a“chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A“human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388.
In a particular embodiment, the antibody is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.
In a particular embodiment, the antibody anti-TfRl is conjugated to the drugs. Said antibody is called as antibody drug conjugate (ADC). In a particular embodiment, such antibody is combined with the potency of chemotherapeutic agents. The technology associated with the development of monoclonal antibodies to tumor associated target molecules, the use of more effective cytotoxic agents, and the design of chemical linkers to covalently bind these components, has progressed rapidly in recent years (Ducry L, et a/. Bioconjugate Chemistry, 21 :5-13, 2010).
In a particular embodiment, the antibody anti-TfRl is able to induce cytotoxicity, also known as the antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies.
Three highly divergent stretches within the variable region of the heavy and light chains are referred to as“hypervariable regions” which are interposed between more conserved flanking stretches known as“framework region” or“FRs”. Thus the term“FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable region. In an antibody molecule, three hypervariable regions of a light chain and three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen binding surface. This surface mediates recognition and binding of the target antigen. The three hypervariable regions of each of the heavy and light chain are referred to as “complementary determining regions” or“CDRs” and are characterized, for example by Rabat et al. Typically, 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). According to the invention, the CDR of the antibodies of the invention are in the Kabat nomenclature.
In the context of the invention, the antibody for use according to the invention, wherein said antibody is an anti-TfRl antibody chosen among H7, FI 2, C32, F2, H9, G9.
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is H7 (SEQ ID NO: l).
In a particular embodiment, the H7 antibody has the following sequences as described below:
Figure imgf000008_0001
Table 1 : sequences of H7 antibody
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is H7 antibody comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:3, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:5; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:7. In a particular embodiment, the antibody for use according to the invention, wherein said antibody is F12 antibody (SEQ ID NO: 10).
In a particular embodiment, the F12 antibody has the following sequences as described below:
Figure imgf000010_0001
Table 2: sequences of F12 antibody
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is F12 antibody comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: l 1 , (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16.
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is C32 antibody (SEQ ID NO: 19).
In a particular embodiment, the C32 antibody has the following sequences as described below:
Figure imgf000011_0001
Table 3: sequences of C32 antibody
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is C32 antibody comprising: a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:20, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:21, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:22, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:23; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:24, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:25.
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is F2 antibody (SEQ ID NO: 28).
In a particular embodiment, the F2 antibody has the following sequences as described below:
Figure imgf000012_0001
Table 4: sequences of F2 antibody
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is F2 antibody comprising a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:29, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:30, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:31, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:32; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:33, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:34. In a particular embodiment, the antibody for use according to the invention, wherein said antibody is H9 antibody (SEQ ID NO: 37).
In a particular embodiment, the H9 antibody has the following sequences as described below:
Figure imgf000014_0001
Table 5 : sequences of H9 antibody
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is H9 antibody comprising a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:38, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:39, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:40, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:41 ; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:42, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:43.
The antibody for use according to the invention, wherein said antibody is G9 antibody (SEQ ID NO: 46). In a particular embodiment, the H9 antibody has the following sequences as described below:
Figure imgf000015_0001
Table 6: sequences of G9 antibody
In a particular embodiment, the antibody for use according to the invention, wherein said antibody is G9 antibody comprising a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:47, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:48, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:49, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:50; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:51, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:52.
In some embodiment, the antibody anti-transferrin receptor 1 (TfRl) for use according to the invention is conjugated with Monomethyl Auristatin F (MMAF) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization using a non-cleavable linker. In some embodiment, the antibody anti-transferrin receptor 1 (TfRl) for use according to the invention is conjugated with Monomethyl Auristatin E (MMAE) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization.
As used herein, the term“cancer” refers to an abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. In the context of the invention, the term“cancers” relates to cancers which absorb iron. More particularly, iron uptake by transferrin receptor is the most important way for cancer cells to absorb iron which in required for their survival. Typically, the cancer is selected from the following group but not limited to: pancreatic cancer, neuroblastoma, solid cancer, leukemia, lymphoma, glioblastoma, breast cancer, cancer related cachexia, gastrointestinal cancer such as colorectal cancer, cholangiocarcinoma, carcinoma of the oral cavity, gastric cancer, lung cancer such as small cell lung cancer, lung adenocarcinoma, melanoma, multiple myeloma, ovarian cancer, prostate cancer, renal cancer, hepatocarcinoma.
In some embodiment, the cancer is selected from the group consisting of pancreatic cancer, neuroblastoma, solid cancer, leukemia, lymphoma, glioblastoma, breast cancer, cancer related cachexia, gastrointestinal cancer such as colorectal cancer, cholangiocarcinoma, carcinoma of the oral cavity, gastric cancer, lung cancer such as small cell lung cancer, lung adenocarcinoma, melanoma, multiple myeloma, ovarian cancer, prostate cancer, renal cancer, hepatocarcinoma.
Inventors have also shown that Im9 B-cell lymphoma cell line, which is resistant to apoptosis induced by rituximab (anti-CD20 antibody), was sensitive to the antibody H7. They also shown that H7 decreased the tumor growth of the PD AC patient derived xenograft (PDX) model and improved the gemcitabine efficiency on the BxPC3 xenograft mice model.
Accordingly, in a second aspect, the invention relates to an antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of resistant cancers.
As used herein, the term“resistant cancers” refers to a cancer that does not respond to treatment notably to convention therapies (chemotherapy, radiotherapy etc). In a particular embodiment, the cancer is resistant at the beginning of treatment. In another embodiment, the cancer become resistant during treatment, such cancer is called refractory cancer. In a particular embodiment, the resistant cancer is selected from the group consisting of pancreatic cancer, neuroblastoma, leukemia, lymphoma, glioblastoma, colon cancer.
In another embodiment, the cancer is resistant to rituximab. Rituximab is a chimeric monoclonal antibody against the protein CD20.
In certain embodiments, the antibody anti- transferrin receptor 1 (TfRl) is used in combination with a second agent for treatment of a disease or disorder. When used for treating cancer, an antibody anti- transferrin receptor 1 (TfRl) may be used in combination with conventional cancer therapies such as, e.g., surgery, radiotherapy, chemotherapy, or combinations thereof.
The present invention also provides for therapeutic applications where the antibody anti transferrin receptor 1 (TfRl) is used in combination with at least one further therapeutic agent, e.g. for treating cancers and metastatic cancers. 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. The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolo melamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33 : 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In a particular embodiment, the antibody anti- transferrin receptor 1 (TfRl) is used in combination with gemcitabine.
In a particular embodiment, the antibody anti-transferrin receptor 1 (TfRl) is used in combination with an ATR inhibitor.
As used herein, the term“ATR”, also known as“ataxia telangiectasia and Rad3-related protein” has its general meaning in the art and refers to a serine/threonine-specific protein kinase that is involved in sensing DNA damage and activating the DNA damage checkpoint, leading to cell cycle arrest.
The term "ATR inhibitor" refers to chemical compounds that are effective in inhibiting ATR kinase. Examples of ATR inhibitors include: ceralasertib; berzosertib (also known as VX- 970, VE822 or M6620); KU-55933; AZD0156; VE-821; BAY-1895344; AZ20; CGK733; ETP-46464; and AZD6738.
In a particular embodiment, the antibody anti-transferrin receptor 1 (TfRl) is used in combination with Berzosertib.
In a particular embodiment, the antibody anti-transferrin receptor 1 (TfRl) is used in combination with Auristatin.
As used herein, the term“Auristatin” has its general meaning in the art and refers to a family of synthetic antineoplastic agent. Auristatins lead to the arrest of cancer cells in the mitosis stage and eventually, apoptosis.
In a particular embodiment, the antibody anti- transferrin receptor 1 (TfRl) is used in combination with Monomethyl Auristatin F (MMAF) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization using a non-cleavable linker. In a particular embodiment, the antibody anti-transferrin receptor 1 (TfRl) is used in combination with Monomethyl Auristatin E (MMAE) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization.
In another embodiment, the cancer is resistant to immune check point inhibitor. As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins.
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 stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. 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. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte- Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD- 1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl /Tel function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response.
In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.
In a particular embodiment, the immune checkpoint inhibitor is an antibody.
Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).
In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).
In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.
In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand.
In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and WO2013006490.
In some embodiments, the immune checkpoint inhibitor is a small organic molecule.
The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.
In a particular embodiment, the small organic molecules interfere with Indoleamine- pyrrole 2,3-dioxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1 -methyl-tryptophan (IMT), b- (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-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5-bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a b- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1 -methyl-tryptophan, b-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and b-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.
In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4- fluorophenyl)-N'-hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carboximidamide :
In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-1, 2, 4-Triazole-3, 5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:
In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V- domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.
In some embodiments, the immune checkpoint inhibitor is an aptamer.
Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.
The invention relates to a method for treating resistant cancer in a subject in need thereof comprising a step of administering said subject with a therapeutically amount of an antibody anti- transferrin receptor 1 (TfRl).
As used herein, the term“subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. More particularly, the subject according to the invention has or susceptible to have resistant cancer. In a particular embodiment, the subject has or is susceptible to have resistance to rituximab.
As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an antibody anti-TfRl) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.
By a "therapeutically effective amount" is meant a sufficient amount of an antibody anti- TfRl for use in a method for the treatment of melanoma at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. In a second aspect, the invention relates to an i) antibody anti- transferrin receptor 1 (TfRl) and ii) an immune check point inhibitor, for use in the treatment of resistant cancer, as a combined preparation.
The invention also relates to an i) antibody anti-transferrin receptor 1 (TfRl) and ii) an ATR inhibitors, for use in the treatment of cancer and resistant cancer.
As used herein, the terms“combined treatment”, “combined therapy” or“therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.
Typically, the antibody-anti TfRl according to the invention and an immune check point inhibitor for simultaneous, separate or sequential use in the method for treating resistant cancer. As used herein, the term“simultaneous use” refers to an administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term“separate use” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes.
The term“sequential use” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
In a third aspect, the invention relates to a pharmaceutical composition comprising an antibody anti- transferrin receptor 1 (TfRl) according to the invention.
In a particular embodiment, the pharmaceutical composition according to the invention for use in the treatment of cancer.
In a particular embodiment, the pharmaceutical composition according to the invention for use in the treatment of resistant cancer.
The anti-TfRl antibody as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intrap eritoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. 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: Effect of H7 on holo-Tf uptake and LIP, (A) Non confluent cells were stained with 500 nM holo-Tf conjugated to Alexa 488 and either left untreated or treated with increasing concentrations of H7 for 4 hrs at 37°C. Internalized fluorescence was measured by FACS. (B) BxPC3 and CFPAC cell lines were labeled with 0.25 M of the intracellular iron chelating dye calcein-AM for 5 minutes. Cells were washed then either left non treated or treated as indicated for 8 hrs at 37°C. Fluorescence of calcein was measured by FACS. Results are expressed as the % of change in the fluorescence relative to the NT cells.
Figure 2: H7 effect on cell viability of PDAC cells. (A, B, C) BxPC3, CFPAC, and HP AC were treated for 5 days with H7 at the indicated concentrations (B) medium was complemented or not ferric ammonium citrate (FAC) at 0.25 mM. (A, B) Cell viability was assessed by MTS assay Results are expressed as % of viable cells compared to non treated cells (C) Apoptosis was detected by by FACS using conjugated Annexin V-FITC and 7AAD. The percentage of early apoptotic cells which are Annexin V positive 7AAD negative (An+) are shown in blue and late apoptotic cells which are Annexin V positive and 7AAD positive (An+ 7AAD+) are shown in white.
Figure 3: Effect of H7 on BxPC3 xenograft and PDX mouse models. BxPC3 cells (A) or PDX6 (B) were grafted in nude mice. Established tumors were either treated with NaCl, H7 at 10 mg/kg, gemcitabine (gem) 100 mg/kg for BxPC3 or 50 mg/kg for PDX model, or the combinations H7 and gemcitabine for 4 weeks twice per week. Tumor volume (upper panels) and the survival curve (lower panels) were plotted. Mice were sacrificed when their tumor reached 1500 mm3 for the BxPC3 model and 1400 mm3 for the PDX model. Arrows represent the treatments of the mice. * p<0.05, **P<0.01, *** P<0.001, ns: not significant.
Figure 4: H7 and gemcitabine combination effect on viability of BxPC3 cell line. Cells were plated, next day treated with either H7 alone at increasing concentrations (from 0.03 pg/ml to 2 pg/ml), gemcitabine alone at either 0.002 pg/ml (A) or 0.004 pg/ml (B), or gemcitabine fixed at the indicated concentrations with H7 varying with the range indicated above. 5 days later MTS reagent was added and cell viability was assessed. Figure 5: A. TfRl expression by HP AC and PanPec cancer cells and CSCs. Cells were plated in IMDM N2 medium at 500 cells/well in 96 ultra-low attachment well plates. Surface TfRl is then measured by FACs after 2 weeks of plating. B. Sensitivity of HP AC CSCs to H7 treatment. Cells were plated at 500 cells/well and next day treated with either Rituximab or H7 at 10 gg/ml and the number of spheres are counted after 5 days of treatment.
Figure 6. DFO and H7 Dose response on HCT116, DLD1, and DK04 colon cancer cells. Cells were plated, 24hrs later DFO (Fig A) or H7 (Fig B) were added with increasing concentrations. After 5 days cell viability was assessed using MTS assay (cellTiter 96, Promega).
Figure 7: Functional properties of anti-TfRl H7 scFv2-Fc on Im9 B-cell lymphoma. Treatment with the anti-CD20 rituximab (RX, hlgGl) was tested. After the treatment, cells were collected and stained with a mix of Annexin conjugated to FITC and 7- AAD and analyzed by FACS. Results are expressed as the % of cells Annexin+/7AAD- that represent early apoptotic cells compared to non-treated.
Figure 8: Effect of H7 conjugated to Auristatin (ADC-H7) on cell viability of the BxPC3 cell line. Cells were plated and next day either left non treated (NT), or treated with RX (negative control antibody), H7, or ADC-7 at different concentrations (0.05, 0.5, or 5 gg/ml). 5 days after treatment cell viability is measured by MTS (cell Titer, 96).
Figure 9: Comparative effect of the anti-TfRl H7 and the anti-HER2 trastuzumab as ADC conjugated to monomethyl-auristatin-E (MMAE) on the SKBR3 cancer cell line. H7 was conjugated to MMAE (Drug antibody ratio of 3.5). The anti-HER2 mAh trastuzumab (TZ) and the anti-CD20 rituximab (RX) were conjugated the same way. 4000 cells of the SKBR3 human breast cancer cell line (in 200 pF, DMEM, 10%FCS) were incubated with various concentrations of the antibody-drug-conjugates of with the naked anti-TfRl H7 antibody. After 5 days, cell viability was assessed by a MTS assay. The concentration giving 50% of the maximum inhibition is indicated (in nM).
Figure 10: Comparative effect of the anti-TfRl H7 and the anti-CD30 Adcetris as ADC conjugated to monomethyl-auristatin-E (MMAE). A. Expression of TfRl, CD20 and CD30 on the Raji B lymphoma cell line and the Karpas 299 cell line (non-Hodgkin's lymphoma). Cell are stained with the naked anti-TfRl H7 and the anti-CD20 rituximab or the anti-CD30 ADC brentuximab vedotin (10 pg/mF in FACS buffer (PBS-1% foetal calf serum- FCS)) followed by an anti-human IgG conjugated with-FITC. Incubations and washes are performed at 4°C. Cells are analysed by FACS and MFI is represented auto, autofluorescence, 2nd, secondary antibody alone; iso, isotype control. MFI values: Raji, 13022, 75252, 432 and Karpas 299, 14159, 298, and 24878, for TfRl, CD20 and CD30 respectively. B. Comparative effect of the anti-TfRl H7-MMAE and H7-MMAF ADCs and the anti-CD30-MMAE (brentuximab vedotin). H7 was conjugated to MMAE or MMAF (Drug antibody ratio of 3.5). The anti-CD20 rituximab (RX) was conjugated the same way with MMAF. 4000 of Karpas 299 or Raji cells (in 200 mE) were incubated with various concentrations of the antibody-drug- conjugates of with the naked anti-TfRl H7 antibody, or with brentuximab vedotin. After 5 days, cell viability was assessed by a MTS assay. The concentration giving 50% of the maximum effect is indicated (in nM).
Figure 11. Iron deprivation with an anti-TfRl H7 induces activation of the ATR / Chkl pathway indicating DNA damage. A. Upregulation of the ATR-ChklDNA damage checkpoint by anti-TfRl treatment. BxPC3 and CFPAC cells either non treated (NT) or treated with cetuximab (Cx) as a control antibody at 10 pg/ml or H7 10 pg/ml or ironchelator deferoxamine for 48 hours and were analyzed for pThr ATR (1989), or pChkl (Ser 345) protein expression by Western blot. B. Synergistic effect of inhibition of DNA damage pathway and iron deprivation with H7 anti-TfRl antibody. BxPC3 and CFPAC cells were treated with the ATR- inhibitor VE822 (Active Biochem; Ref VX-970, dissolved in DMSO) at the determined IC10 or IC10 (concentration inducing 10% of inhibition), i.e. 0.5 mM and 0.1 mM for BxPC3 and CFPAC, respectively, and with H7 2 pg/mF, or with with a VE/H7 combined treatment. Cell viability was determined after 5 days of treatment.
EXAMPLES:
Example 1: use of the antibody anti-TfRl (H7) in Pancreatic Cancer in pancreatic cells lines and in mouse models
Material & Methods
Reagents used
DFO was obtained from Santa Cruz Biotechnology (sc-203331). Cetuximab antibody was obtained from Merck. Ferric ammonium citrate was purchased from Sigma Aldrich (F 5879). Gemcitabine (Hospira) was obtained from Val d’Aurelle hospital (Montpellier). For Western blots analysis, anti-human p21CIPl/WAFl (2946S), anti-human pchKl Ser345 (2348T), anti-human NDRG1 (5196S), anti-human pNDRGl Ser330 (3506S), anti-human pNDRGl Thr346 (3217S), primary antibodies were all obtained from Cell Signaling. The anti human TfRl (1360800) antibody was from Invitrogen, anti-human pH2AX Serl39 (05-636), and anti-human pThrl989 ATR (GTX128145) antibodies were obtained from Millipore and Genetex respectively and anti-human ferritin (ab75973) antibody was purchased from Abeam. The anti-glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore (Billerica, MA) and tubulin from sigma aldrich (T4026). H7 in the IgGl format was produced in CHO cell line by EVITRIA (Switzerland) from the heavy and light variable domain sequences.
Cell Culture
The PD AC cell lines: BxPC3, CFPAC-1, and HP AC were obtained from ATCC (Rockville, MD). BxPC3 and HP AC are both epithelial cells that were derived from pancreatic adenocarcinomas. The CFPAC-1 cells are epithelial cells derived from liver metastasis of pancreatic adenocarcinoma. The BxPC3 and HP AC cell lines were cultured in RPMI 1640 medium while CFPAC in IMDM (Iscove’s Modified Dulbecco’s Medium). The culture media was supplemented as recommended by ATCC with 10% FCS and penicillin, streptomycin antibiotics. All culture media were purchased from Fife Technologies, Inc (Gibco BRF, Gaithersburg, MD). Cells were grown at 37°C in a humidified atmosphere of 5% C02 and the medium was changed twice a week.
TfRl Measurement by FACS
The expression of TFR1 on the cell surface of the PD AC cells was done as follows: non confluent cells recently plated (2 days) were trypsinated, washed with phosphate buffer saline (PBS) and incubated with APC-conjugated anti-human CD71 (BD pharmingen, 551374) for 1 hour at 4°C. The cells were then washed with cold PBS twice before FACS analysis.
Holo-Tf uptake measurement
Cells were harvested and resuspended in medium supplemented with 1 % FCS and 500 nM Transferrin- Alexa 488 (Invitrogen, T13342). They were either left untreated or were treated with H7 (4.5, 45, 450 nM) for 4 hours at 37°C. Cells were then washed with cold PBS and the fluorescence of cell associated holo-Tf conjugated to Alexa 488 was measured by FACS (Gallios machine). Preliminary experiments with an additionnal stripping of the cells 10 min at 4°C with 50mM Glycine pH 2,8 + 500mM NaCl before FACS analysis showed that the fluorescence measured was more than 95% intracellular so this step was omitted to limit the steps before analysis. The mean fluorescence intensity of Alexa488 was calculated using Flow Jo Version 10.1r7 software.
Cell Viability Assays The3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetho xyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) and the electron coupling reagent phenazine methosulfate (PMS) reagents were used to study cell viability. Cells were seeded at 5 x 103 (BxPC3), 1x103 (CFPAC) or 25 x 102 (HP AC) cells per well in 96-well plates with 200 pF of 5% FCS culture medium. 24 hours later cells they were treated and after 5 days of treatment 20 pF of MTS/PMS reagent was added in each well (CellTiter 96 AQueous One Solution Cell Proliferation Assay kit; Promega, G5430) for 2 hours. The absorbance at 490 nm was measured using a microplate reader (Multiskan) on 100 pL of supernatant.
Cell Proliferation Assays
The effect of the anti-TfRl antibody on cell proliferation was done on the basis of the incorporation of 5-ethynyl-2’-deoxyuridine (EdU) into cells during DNA synthesis. The click- iT EdU Flow Cytometry Assay system (Invitrogen, Cl 0420) was used following the manufacturer’s protocol. Cells were plated in medium complemented with 5% FCS at the following cell densities: BxPC3 50,000, CFPAC 15000, and HP AC 40,000 cells per well in 6 well plates. Next day they were either left untreated or treated with antibodies at 10 pg/ml at 10 pg/ml (H7 or Cetuximab as negative control), or DFO at 5 pM (positive control for iron deprivation) for 5 days.
24 hours before processing cells for FACS analysis EdU at 5 pM was added. Cells were then permeabilized with ethanol 75% and incubated with Alexa 488 dye azide for 30 minutes, washed, and the fluorescence intensity of Alexa 488 was measured by FACS.
Intracellular free Iron detection assay
Intracellular free iron levels (labile iron pool, FIP) were measured using the fluorescent probe calcein. This probe binds iron rapidly and stochiometrically in a reversible manner while forming fluorescence-quenched calcein-iron complexes. Once inside the cells calcein is cleaved by intracellular esterases to yield green fluorescent calcein, the fluorescence of which is quenched upon binding to iron. PD AC cells were harvested, resuspended in medium without FCS, and incubated with 250 nM calcein- AM (Invitrogen) for 5 minutes at 37°C. They were washed and resuspended in prewarmed culture medium complemented with 1% FCS. Cells were then left untreated, treated with H7 (10, 100 pg/ml) or DFO (10, 100 pM) for 4 or 8 hrs. Intracellular calcein fluorescence was measured by flow cytometry and the % increase in calcein fluorescence relative to untreated control was calculated.
Cell-division assays The PKH26 staining (sigma Aldrich, Mini26-1 KT) assay was used following the manufacturer’s instructions. Briefly, cells were harvested and stained with PKH26 fluorescent dye for 3 minutes at room temperature, washed and seeded in 6 well plates. 24 hours later they were treated with H7 at 10 iig/m 1 for 5 days. The Fluorescence intensity of PKH was then evaluated by FACS.
Cell Cycle assay
Propidium iodide (PI) obtained from sigma Aldrich, P4864 was used to do the cell cycle assay. Cells were treated with H7 at 2 or 10 iig/ml in medium complemented with 5% FCS at 37°C for 3 days. They were then permeabilized with ethanol 70%, treated with RNase A (sigma Aldrich, R6513) at 100 pg/ml, and stained with PI at 40 pg/ml before being analyzed by FACS.
Apoptosis assays
The Annexin V and 7AAD labeling kit (Beckman Coulter, IM3614) was used to examine apoptosis induced in response to H7 treatment. Cells were treated with H7 at 2 or 10 pg/ml in medium supplemented with 5% FCS at 37°C. After 5 days of treatment, cells were harvested, washed with PBS and stained with 10 mΐ annexin V-FITC and 20 mΐ of 7AAD in 100 mΐ PBS. After 15 minutes of incubation in the dark at 4°C, 400 mΐ of PBS is added and cell apoptosis is examined by FACS.
Western Blot Analysis
Two extraction methods were used for protein extraction. For extracting proteins from cell lines (in vitro experiments), Kook buffer was used. Cells were treated for 2 days with H7 at 10 pg/ml in 5% FCS medium in 6 well plates. Medium was then removed and the cells were washed three times with PBS and then 50 mΐ of Kook buffer (Tris-HCl PH 7.5 10 mM, SDS 20%) was added to cells which were then scraped and sonicated 4 times 5 seconds at amplitude 25%. For protein extraction from tumors: Tumors were homogenized for 30 minutes at 4°C with buffer containing 20 mM Tris-HCl (PH 7.5), 150 mM NaCl, 1.5 mM MgC12, 1 mM EDTA, 1% Triton, 0.1 mM phenylmethylsulfonyl fluoride, 100 mM sodium fluoride, 1 mM sodium orthovanadate (Sigma Aldrich), and one tablet of complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN). The mixture was then centrifuged at 13,400 rpm for 30 minutes at 4°C and the supernatant was collected. Proteins were then dosed and lameli 5X was added. The extract was then heat denatured at 95°C for 5 minutes. 70 pg of protein was loaded on 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) which was then saturated with 5% non-fat dry milk diluted in PBS-Tween 0.1 % for 1 hour at 25°C. Membranes were then incubated with the proper primary antibodies overnight at 4°C. After washing the membrane, the appropriate peroxidase-conjugated rabbit or mouse antibodies (Sigma- Aldrich) were added in PBS-Tween 0.1 %, 5% non-fat dry milk for 1 hour at 25°C. The membrane was then washed and the blots were visualized using a chemiluminescent substrate (Western Lightning Plus-ECL; PerkinElmer).
Antibody Dependent Cell Cytotoxicity (ADCC) assays
BxPC3, CFPAC, and HP AC cells were stained with the PKH dye as described above in the cell division assay. In 96 wells plate (round bottom), target cells (50 pF, 50,000 cells per well) were mixed with the antibodies: Rituximab (anti-CD20, negative control), Cetuximab (anti-EGFRl, positive control), or H7 (50 pF, 0.5 pg/ml) for 30 minutes at 37°C. Peripheral Blood Mononuclear cells (PBMCs) (100 pL), isolated from normal human blood using the Ficoll reagent, were added to the mixture with the ratio of target cells to effector cells being equal to 50. After 3 hours of incubation at 37°C, 7AAD is added to the cells for 15 minutes at 4°C. The % of 7AAD positive target cells (PKH positive) representing PD AC cells killed by ADCC was then quantified by FACS.
In vivo experiments
All in vivo experiments were performed in compliance with the French regulations and ethical guidelines for experimental animal studies in an accredited establishment (Agreement No. C34-172-27). Athymic mice purchased from Harlan (Fe Malcourlet, France) were injected subcutaneously with BxPC3 cells (4x106 in 150 pF). For PDX models, approximately 60 mm3 of PDX6 tumor, developed in our laboratory were transplanted in the interscapular fat pads of Swiss nude female mice (Charles River Faboratories). When tumors reached 100 mm3, mice were injected i.p. with either NaCl, H7 alone at 10 mg/kg, gemcitabine alone at 50 or 100 mg/kg, or combination of H7 plus gemcitabine. The treatment was done twice per week for 4 weeks. Tumor volumes were calculated by using the formula: DlxD2XD3/2. For survival curves mice were sacrificed when tumors reached a volume of 1400 or 1500 mm3.
Results:
Iron requirement by the 3 PD AC cell lines (BXPC3, CFPAC, and HP AC)
Before treating the pancreatic cancer cells with the anti-TfRl antibody, we checked for their sensitivity to iron deprivation induced by the iron chelator Deferoxamine (DFO). Increasing concentrations of DFO decreased cell viability in a dose dependent manner on the 3 PD AC cell lines. The cells exhibit close IC50 values with BxPC3 and HP AC (IC50 2.91 pM and 2.16 pM respectively) being more sensitive to DFO than the CFPAC cell line (IC50 4.08 pM) (data not shown). As detected by FACS, PD AC cell lines expressed TfRl on their surface, with CFPAC showing the highest TfRl level while BxPC3 and HPAC show lower and approximately the same levels (data not shown). The level of the TfRl was directly correlated to holo-Tf uptake in such a way that the CFPAC cell line expressing the highest TfRl level takes up transferrin the most (data not shown), which indicates that these cell lines are using the transferrin receptor to take up iron bound Tf These results indicate that the PD AC cells need iron for their growth and that blocking holo-Tf uptake should affect their normal cellular functioning.
H7 decreases intracellular LIP by blocking holo-Tf uptake
The available anti-TfRl scFv H7 antibody binds to the TfRl at the binding site of holo- Tf (3D model structure of the scFv binding to TfRl not shown, collaboration Thomas Bourquard, BIOS, INRA Tours) and thus we expected that the full length reformated IgGl H7 may block the internalization of holo-Tf by cells. To check the antibody efficiency in blocking holo-Tf uptake, we incubated cells with 500 nM Alexa 488 conjugated Tf in the presence of increasing H7 concentrations for 4 hrs at 37°C and measured levels of internalized holo-Tf by FACS. H7 effectively blocked the uptake of holo-Tf whereby, at approximately 1/10 equimolar concentrations of H7 (45 nM) compared to Tf- Alexa 488 (500 nM), H7 decreases the holo-Tf uptake more than 50 % for all cell lines (Fig 1 A). Holo-Tf blockade by H7 decreased intracellular LIP in a dose dependent fashion after 8 hrs. of H7 treatment shown with the increase in the calcein fluorescence (see methods). DFO (10 or 100 mM) increased the fluorescence up to 29% and H7 up to 18% (H7 10 pg/mL) and 37% (H7 100 pg/ml) for the BxPC3 cell line. For the CFPAC cell line the decrease in LIP is lower, such that with DFO (100 mM) the increase was 8% while with H7 whether 10 or 100 pg/ml there was an increase of 5% but remains significant (Fig 1 B).
An indirect way of proving that LIP decreases upon treatment with H7 was used by measuring changes in TfRl and the iron storage protein ferritin expression that are regulated by the IRP/IRE system. By Western blot and FACS analysis, we show that TfRl level increases upon treatment with 10 pg/ml H7 on BxPC3 and CFPAC cells while ferritin levels decrease (data not shown). By FACS analysis we observe also an increase in surface TfRl expression upon H7 treatment on these 2 cell lines (data not shown). Consistently, we also observed upon H7 treatment of BxPC3 and HPAC cell lines an increase in Hypoxia inducible factor HIF1□, (data not shown) whose degradation has been described to be positively regulated by the iron dependent-prolyl hydroxylase PDH. Interestingly, TfRl increase was due to newly synthetized TfRl since it was blocked by cycloheximide and was not limited by protesomal degradation induced by H7 treatment since proteasome inhibitor bortezomib had no influence (data not shown). Altogether this data show that the reformatting of the scFv into a full length IgGl conserved blocking properties of H7 in the scFv format, with H7 IgGl inhibiting holoTf uptake leading to a decrease in both LIP and total iron storage without inducing TfRl degradation.
H7 decreases cell viability and induces apoptosis of PD AC cell lines
To evaluate the consequences of iron deprivation on the PDAC cells, we tested the effect of H7 on cell viability, proliferation, and apoptosis. Fig 2A shows a dose dependent decrease in cell viability upon H7 treatment with a maximal inhibition of 40% reached with 10 pg/rnl of antibody after 5 days of treatment. Increasing the antibody concentration to 100 pg/rnl did not lead to more reduction in cell viability. When culture medium was complemented with ferric ammonium citrate (0.25 mM), a source of soluble iron, sensitivity to H7 was reversed on the HP AC cell line indicating that decrease in cell viability upon H7 treatment was due to sustained iron deprivation (Fig 2B). Interestingly, H7 treatment induced apoptosis on the 3 cell lines (Fig 2C) as detected by Annexin V staining. These data show that H7 treatment decreases cell viability partly due to cell death.
H7 reduces cell proliferation by blocking cells in S phase and inducing DNA damage
Direct H7 effect on cell proliferation was tested by EdU incorporation in the newly replicating DNA of cells treated with H7 at 10 pg/ml for 5 days. At day 5 of treatment, H7 decreased cell proliferation by 34 % on the BxPC3 cell line and 70% on the CFPAC and HP AC cell lines (data not shown). DFO treatment (5 mM) also inhibited proliferation between 85 and 95% depending on the cell line tested. Next, to evaluate H7 effect on the rate of cell division, cells were labeled with PKH, a fluorescent dye that binds to the cell membrane and allows cell tracking. Treatment of the 3 PDAC cell lines with H7 at 10 pg/ml for 5 days resulted in a higher PKH26 fluorescence compared to untreated cells indicating a slower rate of cell division (data not shown).. .The analysis of the repartition of cells in the different cell cycle phases, after 3 days of treatment, showed that H7 caused an increase of the proportion of cells in S phase in the 3 cell lines tested (data not shown). Associated with this S-phase block was an induction of cyclin-dependent kinase inhibitor p21 in CFPAC cell line but not in BxPC3 cell line (data not shown). In HP AC cell line, depending on the experiment, p21 increased or not. We also noticed an increase in phosphorylation of histone H2AX ( y H2AX) in BxPC3 and HP AC cell lines indicating the occurrence of double strand breaks in DNA. In those 2 cell lines, phosphorylation of the ATR and Chkl kinase also occurred indicating replicative stress y H2AX increase was not observed in the CFPAC cell line nor ATR and Chkl phosphorylation (data not shown). The increase in p21 protein expression could be responsible of the block in S phase observed in CFPAC cells while replicative stress could be responsible for the block in S-phase observed in BxPC3 and HP AC cell lines. These findings indicate that although the overall effects accompanied with H7 treatment are the same on the different cell lines (the decrease in cellular proliferation and apoptosis induction) differential proteins and mechanisms might be implicated in reaching these effects depending on each cell line.
H7 upregulates (NDRG1) and its phosphorylation
It has been recently described that iron deprivation using iron chelaror induce the expression of the tumor suppressor, NDRG1. We therefore examined the effect of H7 on NDRG1 expression. H7 at 10 pg/ml upregulates NDRG1 expression on the 3 cell lines tested. The importance of NDRG1 phosphorylation in its antitumor functions has been recently revealed [20] H7 also increased the phosphorylation of NDRG1 on Ser-330 and Thr-346. Therefore, we demonstrate for the first time the induction of NDRG1 by an anti-TfRl antibody taking into consideration that all the previous studies that reported NDRG1 induction were with iron chelators.
H7 mediates Antibody Dependent Cell Cytoxicity (ADCC)
H7 has been isolated in an antibody phage display screen for breast cancer specific internalizing antibodies. Interestingly, nevertheless, despite its internalizing properties, H7 treatment is up regulating its target TfRl on the cell surface. To address the potential of H7 to recruit NK cells through its FcD l region, PD AC cells were stained with the fluorescent dye PKH26 and incubated with H7 and PBMC as a source of NK cells. In 3 hrs. H7 induced ADCC on both the BxPC3 and CFPAC cell lines (data not shown) in comparison to the negative control antibody Rituximab (anti-CD20) while the anti-EGFR Cetuximab was superior to H7 for ADCC induction. This indicates that in vivo H7 could recruit NK cells highlighting another mechanism of action of H7 antibody aside from its intrinsic killing effect through iron deprivation.
H7 inhibits tumor growth in PD AC mice models
We next evaluated the efficacy of H7 in vivo in two models of PD AC. First PD AC tumors were established in nude mice by s.c. injection of BxPC3 cells. Once tumors reached 100 mm3, mice received twice per week for 4 weeks i.p. injections of H7 antibody (10 mg/kg), gemcitabine (100 mg/kg), H7 + gemcitabine combination treatment at the same concentrations or vehicle (NaCl). H7 alone decreased slighty tumor growth but not significantly compared to mice treated with NaCl while gemcitabine, as expected was efficient (p<0,001) (Fig 3 A). Yet the combination of H7 with gemcitabine showed significantly improved effects on tumor growth as compared to gemcitabine alone (p< 0.05). In the second model, with the aim of using a more relevant preclinical model, we also tested H7 on a PD AC PDX generated in our institute. From the 6 PD AC PDX analyzed 4 expressed TfRl at detectable levels and 3 expressed NDRG- 1. PDX6 was chosen since it did not express NDRG1, indicating a more advanced tumor. Mice were treated the same way than in the first model except for gemcitabine dose (50 mg/kg). H7 treatment decreased slightly tumor growth (p<0,05) and gemcitabine alone (50 mg/kg) reduced drastically tumor size in this model. Interestingly, after the end of the treatment, combined treated mice had significant relapse delay compared to gemcitabine alone treated mice. (Fig 3B). Therefore H7 treatment alone has limited effects but improves gemcitabine therapeutic effects in these 2 models.
Combination treatment of gemcitabine and H7 in vitro
Gemcitabine is the standard treatment used in pancreatic cancer, it is a nucleoside analogue which when incorporated in DNA blocks further insertion of nucleotides and inhibits DNA replication (Shore et ah, 2003). We wanted to check in vitro if H7 can enhance the efficiency of gemcitabine. Before trying the combination we did a dose response treatment of gemcitabine on the BxPC3 cell line and we found that the cells are extremely sensitive to gemcitabine (data not shown). We tried different concentrations of gemcitabine with H7 and we show two of these combinations in Figure 4. To do the combinations we fixed the gemcitabine concentration at 0.002 pg/ml (0.00006 mM) (Figure 4A) or 0.004 pg/ml (Figure 4B) and varied H7 concentration from 0.03 iig/ml to 2 pg/ml (maximal inhibitory concentration of H7). Our results show that there is no combinatory effect in vitro for H7 and gemcitabine in contrast to the in vivo results that we obtained.
Because PD AC cell cultured in 2D were very sensitive to gemcitabine (IC50 = 0.00006 mM) which does not reflect the plasma concentration of 20 mM reached in patients (Tempera et al, 2003), we also tested the effect of gemcitabine in combination with H7 in 3D culture using low attachment 96-well plates. Cell viability was assessed by measurement of cellular ATP (Cell titer glow). We found that in those conditions, BxPC3 cells were less sensitive to gemcitabine (IC50 = 0.1 mM ), but not sensitive to H7 (5 days of culture, maximal concentration tested 10 pg/mL), H7 did not potentialize gemcitabine toxicity.
Effect of H7 on PD AC CSCs Based on studies that showed higher TfRl and LIP levels in CSCs compared to cancer cells and their sensitivity to iron chelation (Rychtarcikova et ah, 2017). We decided to determine if PD AC CSCs express TfRl and if they are sensitive to H7 treatment. We used two PDAC cell lines that can form spheres easily in those conditions, HP AC and PanPec. These cells were grown in medium without serum and specialized for cancer stem cells called IMDM N2 medium. N2 contains growth factors including transferrin 10 mM, EGF for epidermal growth factor (20 ng / ml), bFGF for basic fibroblast growth factor (20 ng / ml) and insulin (5 g/ml). Cells are plated in 96 ultra-low attachment plates (500 cells/well). After that TfRl level was measured by FACs. We found that both HP AC and PanPec CSCs cells express TfRl (Figure 5 A). In the case of HP AC CSCs the level was lower than that of HP AC cancer cells. While for PanPec cells showed higher TfRl levels when grown in 3D culture than the 2D culture. These results indicate that CSCs might be vulnerable to H7 treatment and to prove that we treated HP AC cells which we have already tested in 2D culture with either a negative control anti-CD20 antibody (Rituximab) or H7 at 10 iig/ml (Figure 5B). We found that indeed HP AC CSCs are sensitive to H7 treatment indicating that with H7 we can target both quiescent CSCs and rapidly proliferating cancer cells.
This study highlights the therapeutic potential of this fully human IgGl anti-TfRl mAh as a promising molecule in combination with chemotherapy in PDAC.
Example 2: use of the antibody anti-TfRl (H7) in colon cancer cells lines
Colon cancer cells are differentially sensitive to H7
We wanted to try H7 effect on other solid tumor cell lines, and so we tested its efficiency on a panel of colon cancer cell lines but we show the results on only 3 cell lines with different mutational statuses. HCT116 and DK04 cells harbor an activating mutation in the RAS oncogene. DFD1 cell line has been generated from DK04 by targeted disruption of the mutated RAS leading to its inactivation. Figure 6A demonstrates the sensitivity of these cells to DFO treatment, showing a dose dependent decrease in cell viability after 5 days of treatment. The most sensitive cell line was HCT 116 with a total inhibition of cell viability at 100 mM DFO concentration (IC50= 6.5 mM), at this same concentration DFD1 showed a 70% inhibition (IC50= 5.8 mM) while DK04 85% (IC50= 2.8 mM) inhibition.
These cell lines are also differentially sensitive to H7 in a similar manner to DFO. An almost complete inhibition of cell viability is detected with the HCT116 cell line with a very low IC50 of 0.78 pg/ml, an effect that we did not see on pancreatic cancer cell lines, to a maximal inhibition of 60% in viability with DFD1 (IC50= 0.79 pg/ml) and 75% on DK04 cell line (IC50= 0.51 pg/ml) at 10 pg/ml after 5 days of treatment (Figure 6B). These results show that the RAS mutation does not affect the efficiency of H7 as RAS mutation have been found to be associated with higher TfRl and LIP levels in other models (Yang and Stockwell, 2008; Dixon et a , 2012) (see discussion for commentaries on these data). Meaning that whether RAS is mutated or not colon cancer cells are sensitive to H7 treatment.
H7 upregulates TfRl and downregulates ferritin levels of the colon cancer cell lines
H7 induced the upregulation of TfRl and the downregulation of ferritin levels on a variety of colon cancer cell lines. We observed the effect on 3 cell lines HCT116, DK04, and DLD1 (data not shown). Treatment of these cell lines with 10 iig/rnl of H7 for 2 days induced an increase in the TfRl protein expression as shown in the western blot as well as a decrease in ferritin levels (data not shown) indicating that intracellular iron deprivation occurred in these cells after H7 treatment.
An S phase cell cycle arrest, Apoptosis, and NDRG1 induction with H7 treatment on the HCT116 cell line
We wanted to verify if H7 treatment induces apoptosis of the colon cancer cells and if it affects their progression in the different phases of the cell cycle, so we chose one cell line which is HCT116. We found that after 2 days of H7 treatment at 10 iig/rnl, HCT 116 cells were arrested in S phase (an increase from 38 % in S phase with non-treated cells to 64% with H7 treated cells) so did DFO at 10 mM but to a lower extent (46 % of cells in S phase) (data not shown). Apoptosis was confirmed both by an increase in the subGl phase of the cell cycle after 2 days of treatment (from 0.8% with non-treated cells to 1.4 % with H7 and 1.4 % with DFO treated cells) as demonstrated in our results (data not shown) and by an increase in the % of apoptosis detected using annexin V-FITC and 7AAD staining after 4 days of treatment (data not shown). We show that H7 at 10 iig/rn 1 induced NDRG1 expression in the HCT116 cell line compared to the negative control cetuximab and this induction is even more than that of the positive control DFO at 10 mM. These results indicate that HCT116 is highly sensitive to H7 treatment which causes an arrest of cells in the cell cycle, death by apoptosis, and induction of NDRG1 protein expression. These features are similar to what was observed with the PD AC cell lines highlighting a therapeutic potential of H7 in colorectal cancer.
Example 3: use of the antibody anti-TfRl (H7) in rituximab-resistant B-cell lymphoma cells lines
Anti-TfRl antibody intrinsic cytotoxic activity After confirming that the ERY-1 and Raji cancer cell lines are sensitive to the iron chelator deferoxamine (DFO) (data not shown), these cell lines were used to test the effect of the H7 and Bal20 (another anti-TfRl antibody that induced the degradation of TfRl unlike H7, not shown) antibodies on cell growth. After 5 days of incubation, H7-Fc and H7-IgGl strongly decreased the viability of both cell lines (IC50 in the range of 0.1 pg/mL) (data not shown). Conversely, Bal20 had a limited effect, in agreement with its lower competition with holo-Tf (data not shown). Moreover, H7-IgGl reduced rapidly (4h) the levels of the intracellular labile iron pool (FIP) in Raji and ERY-1 cells, while Bal20 had a more limited effect, especially at the lowest concentration used (1.5 pg/mL) (data not shown). In ERY-1 cells, apoptosis could be detected already after 1 day of incubation with H7-Fc or H7-IgGE After 3 days, the percentage of apoptotic cells was higher than 50% using 5nM of H7-Fc or H7-IgGl (corresponding to 0.5 pg/mL and 0.75 pg/mL, respectively). Conversely, apoptosis was more limited with Bal20, even when used at high concentration (500 nM corresponding to 75 pg/mL) (data not shown). Apoptosis upon H7 treatment was also detected in Raji cells, with the same kinetics than in ERY-1 cells, but to a lesser extent, consistent with this cell line displaying autophagic, but not apoptotic cell death features upon iron deprivation. Then, to compare apoptosis induced by rituximab (anti-CD20 antibody) and by H7, the Bp3 and Im9 B-cell lymphoma cell lines (sensitive and resistant to rituximab-induced apoptosis, respectively) were incubated with H7 or rituximab. H7 strongly induced apoptosis in both cell lines, (Figure 1). In Bp3 cells (rituximab- sensitive), the apoptotic rate was higher upon incubation with H7 than with rituximab (RX), although H7 effect was delayed compared with rituximab. Bal20 induced apoptosis in both cell lines, but was less efficient than H7 (Figure 1). H7 also induced early moderate free iron level decrease in both Bp3 and Im9 cell lines (data not shown). Altogether, these in vitro data indicate that the holo-Tf uptake blockade by H7 induces apoptosis in leukemia and lymphoma cell lines, including those resistant to rituximab. Accordingly, as shown in figure 7 inventors by in vitro methods have shown that H7 had a strong inhibitory effect in the rituximab-resistant B-cell lymphoma cell line Im9. H7 drastically reduced cell viability of Raji and ERY-1 cells (IC50 in the range of 0.1 pg/mL) and induced apoptosis in ERY-1, Bp3 and Im9 cells.
Example 4: an anti-TfRl conjugated to Auristatin
Through a collaboration with Dr. Nicolas Joubert (UMR7292 GICC CNRS - Universite de Tours), H7 was conjugated to Monomethyl Auristatin F (MMAF) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization using a non-cleavable linker. The drug antibody ratio obtained was estimated to be 2.5. This new ADC was named ADC-H7 (or ADC-H7-MMAF).
Figure 8 reports the results on BxPC3 cell line obtained with 3 different concentrations of H7 conjugated or not to MMAF and as a negative control the anti-CD20 Rituximab (RX) conjugated with the same procedure to MMAF. The figure show that ADC-H7 is very powerful compared to unconjugated H7. A clear inhibition is obtained for a concentration of H7 of 0.5 iig/mL and almost total inhibition is obtained with 5 iig/mL.
In the same way, The H7 was also conjugated to Monomethyl Auristatin E (MMAE) which is a synthetic antineoplastic agent and an inhibitor of microtubule polymerization. This new ADC was named ADC-H7-MMAE. The effect of the anti-TfRl H7 and the anti-HER2 trastuzumab (TZ) as ADC conjugated to MMAE on SKBR3 breast cancer cell lines were compared. The ADC H7-MMAE has a similar IC50 compared to the ADC TZ-MMAE and decreases the viability to zero at similar concentrations. The naked antibody alone has a cytostatic effect and decrease the viability of 40%. Therefore, the anti-TfRl -ADC could be used to treat HER2 amplified breast cancer resistant to trastuzumab or trastuzumab-Emtansin (Figure 9).
The effect of anti-Trfl H7, anti-CD20 (rituximab, RX) and anti-CD30 (Adcetris, brentuzumab vedotin) as ADC conjugated to monomethyl-auristatin-E (MMAE) or monomethyl-auristatin F (MMAF) on Raji B lymphoma cell line and the Karpas 299 cell line (non-Hodgkin’s lymphoma) were also compared. Both cell lines express similar levels of TfRl . CD20 is exclusively expressed by Raji cells, CD30 is exclusively expressed by Karpas 299 cells (Figure 10A). As expected, due to the distribution of their respective targets (Figure 10A), the ADC RX-MMAE is only active on the Raji cell line, the brentuximab vedotin is only active on the karpas 299 cell line. The ADC H7-MMAE and MMAF have similar IC50 (in the range of 10 pM) on both cell lines and are 30 to 700 time more active than the naked antibody H7 probably due to the combined effect of iron deprivation and microtubule inhibition (Figure 10B). On Karpas cells, the anti-TfRl ADC are 1 log less active than the potent clinically used brentixumab vedotin (anti-CD30) by display however strong activity (IC50 of 20 nM) (Figure 10B). Therefore, the anti-TfRl ADC could be used on lymphoma patients resistant to brentuximab vedotin.
Example 5: Iron deprivation with an anti-TfRl H7 induces activation of the ATR /Chkl pathway indicating DNA damage. Material&Methods:
The anti-human pThrl989 ATR (GTX128145) antibodies was obtained from Genetex and anti-human ferritin (ab75973) antibody was purchased from Abeam. The anti- glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore (Billerica, MA) and tubulin from sigma aldrich (T4026). For extracting proteins, Kook buffer was used. Cells were treated for 2 days with H7 at 10 pg/ml in 5% FCS medium in 6 well plates. Medium was then removed and the cells were washed three times with PBS and then 50 mΐ of Kook buffer (Tris-HCl PH 7.5 10 mM, SDS 20%) was added to cells which were then scraped and sonicated 4 times 5 seconds at amplitude 25%. The mixture was then centrifuged at 13,400 rpm for 30 minutes at 4°C and the supernatant was collected. Proteins were then dosed and lameli 5X was added. The extract was then heat denatured at 95°C for 5 minutes. 70 pg of protein was loaded on 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) which was then saturated with 5% non-fat dry milk diluted in PBS-Tween 0.1 % for 1 hour at 25°C. Membranes were then incubated with the proper primary antibodies overnight at 4°C. After washing the membrane, the appropriate peroxidase-conjugated rabbit or mouse antibodies (Sigma- Aldrich) were added in PBS-Tween 0.1 %, 5% non-fat dry milk for 1 hour at 25°C. The membrane was then washed and the blots were visualized using a chemiluminescent substrate (Western Lightning Plus-ECL; PerkinElmer).
BxPC3 and CFPAC cells were treated with the ATR-inhibitor VE822 (Active Biochem; Ref VX-970, dissolved in DMSO) at the determined IC10 or IC10 (concentration inducing 10% of inhibition), i.e. 0.5 mM and 0.1 mM for BxPC3 and CFPAC, respectively, and with H7 2 pg/mL, or with with a VE/H7 combined treatment. Cell viability was determined after 5 days of treatment. Cells were seeded at 5 x 103 (BxPC3), 1x103 (CFPAC) cells per well in 96-well plates with 200 pL of 5% FCS culture medium (RPMI for BxPC3 and .IMDM for CFPAC cells) 24 hours later cells they were treated and after 5 days of treatment 20 pL of MTS/PMS reagent was added in each well (CellTiter 96 AQueous One Solution Cell Proliferation Assay kit; Promega, G5430) for 2 hours. The absorbance at 490 nm was measured using a microplate reader (Multiskan) on 100 pL of supernatant.
Results:
BxPC3 and CFPAC treated with the iron deprivating H7 anti-TfRl antibody increase ATR and Chkl phosphorylation indicating a replicative stress. It can be hypothetize that combination of H7 treatment with an ATR inhibitor could enhance the effect of H7 (Figure 11 A). Iron deprivation and inhibition of ATR increase synergize to inhibit BxPC3 and CFPAC pancreatic cancer cell growth (Figure 1 IB).
Thus, the combination of anti-TfRl with an ATR inhibitor (such as VE822) is synergistic for inhibit cell viability on pancreatic cancer lines BxPC3 and CFPAC.
In conclusion, the prensent invention shows that inventors have found an antibody anti transferrin receptor 1 (TfRl) for treating cancers and resistant cancers.
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Claims

CLAIMS:
1. An antibody anti- transferrin receptor 1 (TfRl) for use in the treatment of cancers and resistant cancers.
2. The antibody for use according to claims 1, wherein said antibody is H7 antibody comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:3, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:5; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:7.
3. The antibody for use according to claim 1, wherein said antibody is F12 antibody comprising: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO : 11 , (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO : 12, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16.
4. The antibody for use according to claim 1, wherein said antibody is C32 antibody comprising: a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:20, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:21, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:22, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:23; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:24, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:25.
5. The antibody for use according to claim 1, wherein said antibody is F2 antibody comprising: a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:29, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:30, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:31, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:32; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:33, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:34.
6. The antibody for use according to claim 1, wherein said antibody is H9 antibody comprising: a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:38, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:39,
(c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:40, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:41; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:42, (f) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:43.
7. The antibody for use according to claim 1, wherein said antibody is G9 antibody comprising: a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:47, (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:48, (c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:49, (d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:50; (e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:51, (f) a light chain
CDR3 comprising the amino acid sequence of SEQ ID NO:52.
8. The antibody for use according to claims 1 to 7, wherein the cancer is selected from the group consisting of pancreatic cancer, neuroblastoma, solid cancer, leukemia, lymphoma, glioblastoma, breast cancer, cancer related cachexia, gastrointestinal cancer such as colorectal cancer, cholangiocarcinoma, carcinoma of the oral cavity, gastric cancer, Lung cancer such as small cell lung cancer, lung adenocarcinoma, Melanoma, Multiple myeloma, ovarian cancer, prostate cancer, renal cancer, hepatocarcinoma.
9. The antibody for use according to claims 1 to 7, wherein the resistant cancer is selected from the group consisting of pancreatic cancer, neuroblastoma, colon cancer, liver cancer, leukemia (including erythroleukemia), lymphoma, glioblastoma.
10. The antibody for use according to claims 1 to 9, wherein the cancer is resistant to rituximab.
11. The antibody for use according to claims 1 to 9, wherein the cancer is resistant to immune check point inhibitor.
12. An i) antibody anti- transferrin receptor 1 (TfRl) and ii) immune check point inhibitor, for use in the treatment of resistant cancer, as a combined preparation.
13. An i) antibody anti-transferrin receptor 1 (TfRl) and ii) ATR inhibitor, for use in the treatment of cancers and resistant cancers.
14. A pharmaceutical composition comprising an antibody anti- transferrin receptor 1
(TfRl) according to claims 1 to 13.
15. The pharmaceutical composition according to claim 14 is for use in the treatment of resistant cancer.
16. A method for treating cancer and resistant cancers in a subject in need thereof comprising a step of administering said subject with a therapeutically amount of an antibody anti- transferrin receptor 1 (TfRl) alone and/or in combination with an immune check point inhibitor.
PCT/EP2019/081845 2018-11-20 2019-11-19 Methods and compositions for treating cancers and resistant cancers with anti transferrin receptor 1 antibodies WO2020104479A1 (en)

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