Novel Anti-tfr1 (CD71) antibodies for cancer treatment

Abstract

The present invention relates to an antibody targeting transferrin receptor 1 (TfR1), which binds a transferrin-sensitive conformational active form of TfR1. It also relates to the use of said antibody for cancer treatment.

Description

[0001] Novel anti-TfR1 (CD71) antibodies for cancer treatment

[0002] The present invention concerns novel antibodies and antibody-drug conjugates targeting transferrin receptor 1 (TfR1) for the treatment of cancer.

[0003] Transferrin receptor 1 (TfR1 or CD71) is involved in the control of iron supply to the cell through the binding of transferrin, the major iron-carrier protein. TfR1 is overexpressed in several primary and metastatic cancer cells. Since cancer cells exhibit an increased dependence on iron than normal cells, TfR1 is an attractive target for the treatment of malignancies. However, given the broad expression of TfR1 on normal cells, anti-TfR1 antibodies might have a universal anti-proliferative activity that is not adequate for cancer treatment. A phase I study has been recently reported in advanced solid tumors with CX- 2029, a probody-drug conjugate targeting TfR1 , which shows dose-dependent hematologic toxicities (anemia, neutropenia and leukopenia) that required transfusion (Johnson et al. Clin Cancer Res. 27:4521-4530, 2021).

[0004] Therefore, there is a current need to optimize these antibodies with an increased tumor-specificity and minimum cross-reaction with normal cells for use for the treatment of cancer.

[0005] The present invention meets these needs.

[0006] SUMMARY OF THE INVENTION

[0007] The present invention relates to an antibody targeting transferrin receptor 1 (TfR1) comprising:

[0008] (a) a heavy chain variable (VH) domain, wherein CDR1 , CDR2 and CDR3 have at least 70% sequence identity to, respectively, SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3; and

[0009] (b) a light chain variable (VL) domain, wherein CDR1 , CDR2 and CDR3 have at least 70% sequence identity to, respectively, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0010] The invention also relates to an antibody targeting TfR1 having a VH domain, which has at least 70% sequence identity to SEQ ID NO: 7, and a VL domain, which has at least 70% sequence identity to SEQ ID NO: 8. The invention also relates to an antibody-drug conjugate (ADC) comprising an antibody targeting TfR1 and an anti-tumor agent.

[0011] Said antibody or ADC is especially for use as a medicament. Said antibody or ADC is especially for use in the treatment of cancer. Preferably, said antibody or ADC is used for the treatment of triple negative breast cancer (TNBC). Preferably, the antibody or ADC of the invention recognizes a transferrin-sensitive conformational active form of TfR1. In particular, the antibody or ADC of the invention recognizes a tumor-specific epitope of TfR1.

[0012] The invention also relates to a pharmaceutical composition comprising the antibody or the antibody-drug conjugate targeting TfR1 according to the invention, formulated with a pharmaceutically acceptable carrier.

[0013] The present invention further relates to a nucleic acid encoding the antibody or the antibody-drug conjugate targeting TfR1 according to the invention.

[0014] DETAILED DESCRIPTION OF THE INVENTION

[0015] Surprisingly, as shown in the examples, the inventors have demonstrated that the anti- TfR1 antibody according to the invention recognizes a transferrin (Tf)-sensitive conformational active form of TfR1 , which is elevated in tumoral tissues. The reactivity of said antibody is increased in cancer cells compared to normal cells, which make this antibody attractive for clinical development. Despite its large expression in tumors, TfR1 has not been considered a target for antibodies and antibody-drug conjugates (ADC) due to its broad expression on normal cells.

[0016] The antibody or ADC according to the invention overcomes this problem thanks to its limited off-target toxicity as it specifically binds a conformational tumor epitope of TfR1. Said antibody or ADC internalizes upon binding to TfR1 , allowing to efficiently deliver cytotoxic payloads into cancer cells, which is particularly suitable for the treatment of highly resistant cancers, such as triple negative breast cancer (TNBC).

[0017] The present invention thus relates to an antibody targeting transferrin receptor 1 (TfR1) comprising:

[0018] (a) a heavy chain variable (VH) domain, wherein CDR1 , CDR2 and CDR3 have at least 70% sequence identity to, respectively, SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3; and (b) a light chain variable (VL) domain, wherein CDR1 , CDR2 and CDR3 have at least 70% sequence identity to, respectively, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0019] Antibody

[0020] An antibody (or “immunoglobulin”) consists of a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (or domain) (abbreviated herein as VH) and a light chain variable region (or domain) (abbreviated herein as VL). According to the invention, the VL and the VH of the antibody form an antigen-binding site to TfR1 (also known as CD71).

[0021] As used herein, the term "antibody" encompasses not only full-length polyclonal or monoclonal antibodies, but also any polypeptide or protein comprising an antibody antigenbinding site. Preferably, the antibody of the invention is a monoclonal antibody. An antigenbinding site (paratope) is the part of an antibody that binds to and is complementary to the epitope of its target antigen (herein TfR1). The antigen-binding site is a polypeptide or domain that comprises one or more complementary determining regions (CDRs) of an antibody and is capable of binding the antigen.

[0022] Preferably, the anti-TfR1 antibody comprises a VH domain with the following combination of three CDRs:

[0023] H-CDR1 : GYTFTNYG (SEQ ID NO: 1)

[0024] H-CDR2: INTNTGEP (SEQ ID NO: 2)

[0025] H-CDR3: VYDGSFGFAY (SEQ ID NO: 3).

[0026] Preferably, the anti-TfR1 antibody comprises a VL domain with the following combination of three CDRs:

[0027] L-CDR1 : SSVSY (SEQ ID NO: 4)

[0028] L-CDR2: STS (SEQ ID NO: 5)

[0029] L-CDR3: QQRSNYPLT (SEQ ID NO: 6).

[0030] Preferably, H-CDR1 , H-CDR2 and H-CDR3 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98, at least 99% sequence identity to, respectively, SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3. Preferably, L-CDR1 , L-CDR2 and L-CDR3 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98, at least 99% sequence identity to, respectively, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0031] Preferably, the antibody targeting TfR1 of the invention comprises:

[0032] (a) a heavy chain variable (VH) domain, wherein CDR1 , CDR2 and CDR3 have sequences identical to, respectively, SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3; and

[0033] (b) a light chain variable (VL) domain, wherein CDR1 , CDR2 and CDR3 have sequences identical to, respectively, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0034] Preferably, the anti-TfR1 antibody comprises the following VH and VL domains:

[0035] - VH domain: MAWVWNLLFLMAAAQSAQAQIQLVQSGPELRKPGETVKISCKASGY TFTNYGMNWVKQAPGKGLEWMGWINTNTGEPAYAGDFKGRFAFSLETSASTAY LQINNLRNEDTATYFCVYDGSFGFAYWGQGTLVTVSA (SEQ ID NO: 7); and

[0036] - VL domain: MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSAFPGEKVTITCSAS SSVSYMHWFQQKPGTSPKVWIYSTSTLASGVPSRFSGSGSGTSYSLTISRMEAE DAATYYCQQRSNYPLTFGAGTRLELK (SEQ ID NO: 8).

[0037] Preferably, the VH domain and VL domain have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98, at least 99% sequence identity to, respectively, SEQ ID NO:7 and SEQ ID NO: 8.

[0038] Preferably, the antibody targeting TfR1 of the invention comprises a VH domain which has at least 70% sequence identity to SEQ ID NO: 7, and a VL domain which has at least 70% sequence identity to SEQ ID NO: 8. Preferably, the antibody targeting TfR1 of the invention comprises a VH domain identical to SEQ ID NO: 7, and a VL domain identical to SEQ ID NO: 8.

[0039] Preferably, the antibody targeting TfR1 of the invention comprises a VH domain identical to SEQ ID NO: 7, and a VL domain identical to SEQ ID NO: 8, and said antibody is the one of the examples, called 10G6.3.

[0040] The percentage of sequence identity is defined as the percentage of amino acid or nucleotide in a compared sequence that are identical to the reference sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity. Alignment for purposes of determining percent amino acid sequence or nucleotide identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403). When using such software, the default parameters, e.g., for gap penalty and extension penalty, are preferably used. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10. The percent identity is determined on at least 20 consecutive amino acid residues of the reference sequence.

[0041] The antibody targeting TfR1 of the invention may be chimeric, humanized or fully human.

[0042] The sequences used in the present invention are detailed in the following table:

[0043] Transferrin receptor 1

[0044] Transferrin receptor 1 (abbreviated herein as TfR1), also known as CD71 , is a type II transmembrane glycoprotein that is linked by two disulfide bonds to form a homodimeric protein. TfR1 is a key regulator of cellular iron homeostasis and proliferation. This receptor interacts with iron-bound transferrin (also known as holo-Tf or differic Tf) to import iron into the cell. The extracellular domain (ECD) of the homodimer can bind up to two molecules of transferrin. The holo-Tf-TfR1 complex is internalized through constitutive clathrin-mediated endocytosis. Due to the decrease in pH in intracellular vesicles, iron is released from Tf and the iron-free transferrin (apo-Tf)-TfR1 complex is recycled back to the cell surface where apo-Tf dissociates from the receptor. Recycling of TfR can occur hundreds of times during the lifetime of a single receptor. At any given time, a cell can express hundreds of thousands of copies of TfR1 with only a small percentage present at the cell surface. Depending on the position of the targeted TfR1 site (epitope), antibodies can be neutralizing, meaning that they inhibit TfR1 ligands binding, or they can be non-neutralizing. Said TfR1 ligands can be selected from holo-Tf, ferritin and HFE. Neutralizing antibodies inhibit Tf binding and subsequent TfR1 internalization, or disrupt receptor cycling due to its intracellular sequestration and degradation. All of which ultimately interfere with iron uptake leading to lethal iron starvation. Non-neutralizing antibodies may be internalized into cells. This strategy is particularly adapted for the delivery of a variety of agents including chemotherapeutic drugs, toxins, genes, oligonucleotides, small inhibitory RNA (siRNA), enzymes, viral vectors and nanodrugs

[0045] In some embodiments, the anti-TfR1 antibody of the invention is a non-neutralizing antibody. In some embodiments, the anti-TfR1 antibody of the invention does not block the binding of TfR1 ligands. Preferably, said antibody does not inhibit holo-Tf binding. In a preferred embodiment, the anti-TfR1 antibody of the invention is internalized into cells after binding to TfR1.

[0046] By “epitope”, it is meant the portion of the antigen that is recognized by an antibody. Epitopes may be defined as linear or conformational. Linear epitopes consist of continuous amino acid residues on a protein sequence, which directly contribute to the affinity of the interaction. Conformational epitopes consist of residues that are discontinuous in the protein sequence yet come within close proximity to form an antigenic surface on the protein's three-dimensional structure. Conformational epitopes are more difficult to identify than linear epitopes. Epitopes may also include determinants that are chemically active, surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. In some embodiments, the anti-TfR1 antibody of the invention recognizes a loop in the apical domain of TfR1 comprising an N-glycosylation site, preferably the loop aa 275-338 comprises the N- glycosylation site Asn317. By “loop aa 275-338”, it is meant a secondary structure of TfR1 comprising amino acid 275 to 338 of TfR1 sequence. In particular, the anti-TfR1 antibody of the invention recognizes an epitope, which is not directly involved in holo-Tf binding. Preferably, the anti-TfR1 antibody of the invention recognizes a tumor specific epitope of TfR1 , preferably a conformational epitope containing the N-glycosylation site Asn317.

[0047] The ECD of TfR1 contains three subdomains, all required for Tf binding: a helical, protease-like, and apical domain. Binding of holo-Tf to TfR1 induces conformational change in TfR1 , in particular in the apical and protease-like domains of TfR1 , which allows for the internalisation of TfR1. In some embodiments, the anti-TfR1 antibody of the invention recognizes a conformational epitope on TfR1. In a preferred embodiment, the anti-TfR1 antibody of the invention recognizes a Tf-sensitive conformational active form of TfR1. By “Tf-sensitive conformational active form of TfR1”, it is meant a three-dimensional structure of TfR1 that only occurs after binding to Tf, preferably holo-Tf.

[0048] Inhibition of TfR1

[0049] Functional activity of the antibody may be tested in vitro or in vivo. Suitable assays include enzyme-linked immunosorbent assays (ELISA) to control the binding characteristics of the antibody, cytotoxicity assays, in vitro internalization studies and in vivo studies of tumor growth in mice.

[0050] In some embodiments, the anti-TfR1 antibody of the invention has an affinity for TfR1 that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% higher in the presence of holo-Tf than without holo- Tf. Preferably, the anti-TfR1 antibody of the invention has an affinity for TfR1 that is at least 20%, more preferably at least 30% higher in the presence of holo-Tf than without holo-Tf. In some embodiments, the anti-TfR1 antibody of the invention has an affinity for the Tf- sensitive conformational active form of TfR1 that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% higher than for the non-activated form of TfR1. Preferably, the anti-TfR1 antibody of the invention has an affinity for the Tf-sensitive conformational active form of TfR1 that is at least 20%, more preferably at least 30% higher than for the non-activated form of TfR1 .

[0051] In some embodiments, the affinity of the antibody of the invention for TfR1 is between 0.01 nM to 100 nM, preferably between 0.05 nM to 50 nM, preferably between 0.1 nM to 20 nM, preferably between 0.5 nM to 20 nM, preferably between 1 nM to 20 nM, preferably between 5 nM to 15 nM, preferably between 8 nM to 12 nM. Preferably, the affinity of the antibody of the invention for TfR1 is between 8 to 12 nM.

[0052] In some embodiments, the affinity of the antibody of the invention for the Tf-sensitive conformational active form TfR1 is between 0.01 nM to 100 nM, preferably between 0.05 nM to 50 nM, preferably between 0.1 nM to 20 nM, preferably between 0.5 nM to 20 nM, preferably between 1 nM to 20 nM, preferably between 5 nM to 15 nM, preferably between 8 nM to 15 nM, preferably between 10 nM to 12 nM. Preferably, the affinity of the antibody of the invention for the Tf-sensitive conformational active form TfR1 is between 10 nM to 12 nM. The antibody affinity for an antigen may be expressed in half-maximal effective concentration (ECso, herein expressed in nM) or the equilibrium dissociation constant (KD), which is the ratio Ka / Kd of the association or on-rate (Ka) and the dissociation or off-rate (kd) of the binding interaction. Affinity (KD) is a measure of how strong the interaction of the antibody with its antigen is. Association rate (ka) shows how fast antigen is recognised. Dissociation rate (ka) is a measure of stability of binding. Taken together, kinetic data provide valuable information with implications for biological activity, pharmacokinetics and dosing regimen.

[0053] In some embodiments, the anti-TfR1 antibody of the invention has a higher efficacy in the presence of holo-Tf. In some embodiments, the efficacy of the anti-TfR1 antibody of the invention is at least twice, at least three times, at least four times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times higher in the presence of holo-Tf than without holo-Tf. Preferably, the efficacy of the anti-TfR1 antibody of the invention is six times higher in the presence of holo- Tf than without holo-Tf. By “efficacy”, it is meant the amount of antibody (herein concentration) required to obtain a desired effect, in the sense of this invention a 50% decrease of cell viability, preferably cancer cell viability.

[0054] Although TfR1 is ubiquitously expressed at a low to medium level in a broad variety of normal tissues, it is expressed at higher levels in rapidly proliferating cells. Said rapidly proliferating cells, include malignant cells, as well as normal cells such as precursors of blood cells in the bone marrow, hepatocytes in the liver, keratinocytes in the epidermis and enterocytes in crypts of intestinal epithelium. In some embodiments, the anti-TfR1 antibody of the invention does not bind to mature peripheral blood cells such as peripheral blood mononuclear cell (PBMC).

[0055] In some embodiments, the anti-TfR1 antibody of the invention has a low or limited reactivity on normal cells or tissues. By “normal”, it is meant healthy, preferably non- cancerous. Antibody reactivity to a target antigen can be assessed by any well-known methods in the art such as immunohistochemistry (IHC), enzyme linked immunoabsorbant assay (ELISA), flow cytometry or Western blotting. Preferably, said antibody reactivity is assessed by IHC. In some embodiments, the anti-TfR1 antibody of the invention has a high reactivity on cancer cells or tissues. In a preferred embodiment, the anti-TfR1 antibody of the invention has a high reactivity on breast cancer cells, preferably TNBC cells. In some embodiments, the anti-TfR1 antibody of the invention has a reduced off-target toxicity. By “reduced off-target toxicity”, it is meant that the antibody has a higher reactivity for target cells than non-target cells. Preferably, the target cell is a cancer cell. More preferably, the target cell is a TNBC cell.

[0056] In some embodiments, the anti-TfR1 antibody of the invention recognizes a tumorspecific epitope of TfR1. In some embodiments, the anti-TfR1 antibody of the invention recognizes a conformational tumor-specific epitope of TfR1. In some embodiments, the conformational tumor-specific epitope is only available in the presence of holo-Tf. In some embodiments, the anti-TfR1 antibody of the invention is internalized into cancer cells after binding to the conformational tumor-specific epitope. Preferably, the tumor-specific epitope of TfR1 comprises the glycosylation site Asn317. Tumor-specific epitope may be tumor antigen bearing cancer-specific mutations or cancer-specific epitopes. Said tumor-specific epitopes can be characterized through screening and identification of antibodies presenting differential binding specificity between healthy tissues and tumors.

[0057] Antibody-drug conjugate

[0058] The invention also relates to an antibody-drug conjugate (ADC) comprising the anti- TfR1 antibody according to the invention and an anti-tumor agent.

[0059] All the embodiments relative to the anti-TfR1 antibody according to the invention also apply to the ADC according to the invention

[0060] By “antibody-drug conjugate” (herein abbreviated ADC), it is meant an antibody covalently attached to a cytotoxic payload via a chemical linker. Said ADC combines both the advantages of highly specific targeting ability and highly potent killing effect to achieve accurate and efficient elimination of cancer cells. The ability of TfR1 to internalize make this receptor an attractive target for ADC therapy, preferably ADC cancer therapy. In some embodiments, the ADC of the invention is internalized upon binding to TfR1 so that the cytotoxic payload is released inside the target cell. Preferably, the target cell is a cancer cell. More preferably, the target cell is a TNBC cell. In some embodiments, the ADC of the invention has a high affinity for a tumor-specific epitope, which results in more rapid internalization into cancer cells and release of the cytotoxic payload.

[0061] The drug, or cytotoxic payload, can be conjugated to the antibody of the invention using any known methods for attaching agents to antibodies including, but not limited to, attachment to the carbohydrate moieties, to sulfhydryl groups, to amino groups, or to carboxylate groups. This can include any chemical reaction that will bind the two molecules as long as the antibody and the other agent retain their respective activities. Such chemical mechanisms can be covalent binding, affinity binding, intercalation, coordinate binding and complexation.

[0062] In some embodiments, the drug is conjugated to the antibody via a linker. Linker may include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. The linker may be cleavable or not. Preferably, the linker is a cleavable linker. By “cleavable linker”, it is meant a peptide linker that is susceptible to cleavage following exposure to acidic or reducing environments and / or proteolytic enzymes. The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cells are among the key factors determining the efficacy of the ADC. In some embodiments, the agent is linked to the antibody using a non-cleavable linker. In some embodiments, the agent is linked to the antibody using a cleavable valine-citrulline linker. Cleavage of the ADCs linker components by certain tumor-associated enzymes (i.e. matrix metalloproteinases) or by lower pH encountered in the tumour microenvironment results in the release of the active component. These non-internalizing ADC do not increase drug selectivity and, as a result, do not reduce toxicity considerably. Preferably, the ADC of the invention is internalized into cancer cells. Said internalized ADC allows the intracellular delivery of the cytotoxic payload directly into cancer cells, avoiding the immune system inactivation and solubility issues, while enhancing the therapeutic activity of the cytotoxic payload.

[0063] The present invention thus relates to an antibody-drug conjugate comprising the anti- TfR1 antibody according to the invention and an anti-cancer agent (or anti-tumor agent). The anti-cancer agent can be selected from an anthracycline, an antitumor antibiotic, an alkylating agent, an antimetabolite, an alkaloid, a topoisomerase inhibitor, an anti-mitotic agent, a DNA-intercalating agent, a taxane, a platin-based component, a specific kinase inhibitor, an androgen receptor antagonist, an hormone, a cytokine, an antiangiogenic agent, an antibody, in particular a monoclonal antibody, a modulator of the immunity system, an oncolytic virus and a TLR (Toll-Like Receptor)-3 ligand.

[0064] Anthracyclins include for example doxorubicin, daunorubicin, epirubicin, pirarubicin, idarubicin, zorubicin, aclarubicin, nemorubicin, sabarubicin or valrubicin. Antitumor antibiotics include for example Bleomycin, hydroxyurea, Mitomycin C or Mitoxantrone.

[0065] Alkylating agents include for example dacarbazine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, melphalan, mechlorethamine, oxaliplatin, uramustine or temozolomide.

[0066] Examples of antimetabolites are Azathioprine, Capecitabine, Cytarabine, Floxuridine, Fludarabine, Fluorouracil, Gemcitabine, Methotrexate, Fluorouracil (5-Fll) or Pemetrexed.

[0067] Alkaloids include for example vinblastine, or vincristine (Vinorelbine); Topoisomerase inhibitors include, for example Irinotecan, Topotecan or Etoposide; Antimitotic agents are for example selected from Vinblastine, Vincristine, Vinorelbine, and dolastatins and derivatives thereof;

[0068] Taxanes are for example selected from docetaxel, larotaxel, cabazitaxel, paclitaxel (PG-paclitaxel and D HA- paclitaxel), ortataxel, tesetaxel, and taxoprexin.

[0069] Examples of platin-based components are CDDP and OXP.

[0070] Examples of specific kinase inhibitors are for example BRAF kinase inhibitors such as vemurafenib and dabrafenib, or MEK inhibitors such as trametinib, or Plk1 inhibitors such as volasertib.

[0071] Androgen receptor antagonists are for example bicalutamide or enzalutamide.

[0072] Tamoxifen and anti-aromatase drugs are typically used in the context of hormonotherapy.

[0073] Examples of cytokines usable in the context of an immunotherapy are IL-2 (lnterleukine-2) and IFN (Interferon) alpha (IFNa).

[0074] Antiangiogenic agents are for example VEGF inhibitors such as itraconazole, bevacizumab or ranibizumab.

[0075] Anti-CD20 (pan B-Cell antigen) and anti-Her2 / Neu (Human Epidermal Growth Factor Receptor-2 / NEU) are examples of monoclonal antibodies. Monoclonal antibodies also include anti-immune checkpoint antibodies, such as anti-PD1 , anti-PDL1 , anti-CTLA4, anti-OX40L, anti-PDL2, anti-CD73, anti-CD80, anti-CD86, anti-TIGIT, anti-Galactin-3 or anti-HVEM antibodies.

[0076] Anti-PD1 antibodies include pembrolizumab or nivolumab.

[0077] Immunity system modulators are for example I DO1 , IDO2 or TDO2 inhibitors, A2a antagonists or STING agonists.

[0078] Oncolytic viruses are for exemple Talimogene laherparepvec. In some embodiments, the anticancer agent is a dolastatin or a derivative thereof, preferably an auristatin E. In some embodiments, the anticancer agent is monomethyl auristatin E (MMAE). In some embodiments, the ADC of the invention is linked to MMAE using a cleavable valine-citrulline linker (herein described by 10G6.3-vcMMAE).

[0079] Cancer treatment

[0080] The present invention further relates to the antibody or the ADC according to the invention for use for the treatment of cancer. As used herein, the terms "treatment" or "treat" refer to both prophylactic or curative treatment of cancer. A curative treatment is defined as a treatment that completely treat (cure) or partially treat (induces tumor growth stabilization, retardation or regression) cancer. By “treatment of cancer”, it is meant increasing the sensitivity of a cancer to a chemotherapeutic agent, and / or decreasing the resistance of a cancer with respect to a chemotherapeutic drug.

[0081] Cancer refers to tumors. The tumors to be treated include primary tumors and metastatic tumors, as well as refractory tumors.

[0082] Examples of cancers that may be treated by the anti-TfR1 antibody of the invention include, but are not limited to, cancer cells from the breast, bladder, blood, bone, bone marrow, brain, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangio sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non- Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. Preferably, the cancer is a breast cancer. Preferably, the cancer is a highly resistant breast cancer, preferably a triple-negative breast cancer. The high specificity of the anti- TfR1 antibody of the invention for a tumor epitope makes it suitable for targeted cancer therapy of resistant cancers. For a targeted therapy to be successful, it must exert strong action predominantly on the target cell while sparing the bystander cells and minimizing unwanted side effects.

[0083] Pharmaceutical composition

[0084] The present invention also relates to a pharmaceutical composition comprising the antibody or the ADC according to the invention, formulated with a pharmaceutically acceptable carrier.

[0085] "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. In 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 syringeability 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 bispecific protein complex 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 intraperitoneal 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 NaCI 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.

[0086] Compositions may contain suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. In some embodiments, the composition of the invention a lyophilized formulation.

[0087] In some embodiments, the composition of the invention is administered to a subject, in need thereof via any suitable route, such as administration by intravenous, intramuscular, intraperitoneal, intracerebrospinal, subeutaneous, intracutaneous, intraarticular, intrasynovial, intrathecal, intradermal, intratumoral, intranodal, intramedulla, oral, inhalation or topical routes; or it may be administered orally, by inhalation spray, topically, rectally, nasally, buccally or vaginally. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the treatment goal and the cancer site.

[0088] The term “subject” refers to any subject and typically designates a patient, in particular a subject undergoing a treatment of cancer such as chemotherapy and / or radiotherapy, or a subject at risk, or suspected to be at risk, of developing a cancer. The subject is preferably a mammal, even more preferably a human being, for example a human being suffering of a cancer and resistant to chemotherapy. The subject is typically a cancer patient, preferably a patient whose tumor cells are sensitive to iron deficiency.

[0089] In some embodiments, the composition of the invention is used for the treatment of cancer by administering to a subject in need thereof with an effective amount of the anti- TfR1 antibody of the invention. As used herein, “a therapeutically effective amount or dose” refers to an amount of the compound of the invention which removes, slows down the cancer or reduces or delays one or several symptoms or disorders caused by or associated with said disease in the subject, preferably a human being. The effective amount, and more generally the dosage regimen, of the compound of the invention and pharmaceutical compositions thereof may be determined and adapted by the one skilled in the art. An effective dose can be determined by the use of conventional techniques and by observing results obtained under analogous circumstances. The therapeutically effective dose of the compound of the invention will vary depending on the disease to be treated or prevented, its gravity, the route of administration, any co-therapy involved, the patient's age, weight, general medical condition, medical history, etc.

[0090] In some embodiments, one or more doses of the composition of the invention is administered. In some cases, a single dose may be effective to achieve a long-term benefit. Alternatively, multiple doses may be administered, usually sequentially and separated by a period of days, weeks or months.

[0091] Nucleic acids

[0092] The antibody of the invention can be recombinantly prepared using genetic engineering techniques. A DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody. Recombinant techniques are well-known and commonly used by the person in the art.

[0093] Isolated nucleic acid may be provided, encoding antibodies according to the present invention. Nucleic acid may be DNA and / or RNA. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode an antibody. Nucleic acid encoding an antibody can be introduced into a host cell via any suitable vector such as a plasmid, a phage or a viral vector. The introduction may be followed by expressing the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene, then optionally isolating or purifying the binder polypeptide, e.g., antibody. Vectors may contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

[0094] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is E. coli. Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production.

[0095] The present invention thus relates to a nucleic acid encoding the anti-TfR1 antibody of the invention. Preferably, the nucleic acid encodes the VH domain of the antibody targeting TfR1. Preferably, the nucleic acid encodes the VL domain of the antibody targeting TfR1. Preferably, the nucleic acid has the the nucleotide sequence of SEQ ID NO: 9. Preferably, the nucleic acid has the the nucleotide sequence of SEQ ID NO: 10.

[0096] FIGURES

[0097] Figure 1 : Experimental procedure for the identification of cancer specific antigen epitopes.

[0098] Figure 2: Comparative reactivity of anti-Tfr1 antibody and commercial anti-CD71 antibody on healthy tissues.

[0099] Figure 3: Reactivity of the anti-Tfr1 antibody-FITC and anti-CD71 FITC on CD3 + CD28 activating T-cells by FACS analysis.

[0100] Figure 4: Pattern of reactivity by FACS of the anti-Tfr1 antibody in normal cells vs breast cancer cells at the time of screening.

[0101] Figure 5: Characterization of the antibody reactivity by IHC.

[0102] Figure 6: H-score distribution of anti-Tfr1 antibody binding to primary TNBC by IHC.

[0103] Figure 7: Characterization of the antibody target.

[0104] Figure 8: Validation of the antibody target.

[0105] Figure 9: Biological properties of the antibody.

[0106] Figure 10: Activity of the ADC in vivo on xenografts.

[0107] Figure 11 : Holo-Tf increases antibody binding to TfR1.

[0108] Figure 12: X-Ray diffraction analysis of human transferrin receptor dimer and its glycosylation sites.

[0109] EXAMPLES

[0110] Material and methods

[0111] Cells and cell lines.

[0112] The SUM 190 PT cell line was purchased from Biolvt (Germany) and was cultured in Ham's F12 medium with 2% Fetal Bovine Serum (FBS), non-essential amino acids, ITS- X 1% (Gibco), 6.7ng / ml Triiodo-L-tyrosine, 1 .g / mL streptomycin and 2 mM glutamine. The culture supplement (human insulin, human holo-transferrin, selenium, ethanolamine (ITS-X (Gibco)) allow to reduce the FBS concentration. (Holo-transferrin concentration is 68.75 nM final). The T47D cell line was from ATCC and was cultured in RPMI-1640 medium supplemented with 10% FBS, 10 .g / mL insulin, 1 .g / mL streptomycin and 2 mM glutamine. The SLIM149 cell line was cultured in RPMI-1640 supplemented with 10% FBS, 1% non- essential amino acid, 0.1M HEPES Buffer, 10 .g / mL insulin, 2,5pg / mL hydrocortisone and 1 .g / mL streptomycin. The HCC38 cell line was cultured in RPMI-1640 supplemented with 10% FBS, 1% non-essential amino-acid, 1mM sodium pyruvate, 0,5% glucose and 1 .g / mL streptomycin. Absence of mycoplasma contamination was regularly controlled by PCR (Eurofins Genomics, Germany), and MycoAlertTMPLUS test (Lonza). Human Epidermal Keratinocytes (NHEK) (Lonza) were cultured in KBM medium from Lonza supplemented with Singlequots products as recommended by the manufacturer. Frozen Human Bone Marrow Mononuclear Cells were purchased from Bioivt (HUMANBMMC-0111546) and thawed in RPMI 10% FBS. PBMC donors (N=4) were from EFS Marseille.

[0113] Production of anti-breast cancer (BC) mAbs.

[0114] NMRI mice (Charles River, France) were immunized via intraperitoneal injection with human breast cancer cell lines (SUM149, SKBR3, HCC38 and SUM190PT). Lymphocytes from spleen were fused with the X63-Ag.853 murine myeloma cell line with PEG 1500 (Sigma Aldrich, France). To identify hybridoma clones producing mAbs that reacted with BC cells not with normal cells, normal cells (HME-1 , NHEK and PBMC) were first incubated with hybridoma supernatants, subsequently incubated with PE-conjugated anti-mouse IgG antibody (eBioscience), and finally analyzed through flow cytometry. Hybridoma clones producing mAbs that did not react with normal were tested on series of BC cell lines (SUM149, SKBR3, HCC38, SUM190PT and T47D). Sixteen clones were selected at the end of the screen.

[0115] IHC was carried on 5-pm sections from frozen tissue. Sections were fixed in acetone for 10 min, air-dried for 10 min and rehydrated in TBST. Staining was done with 20pg / ml 10G6.3 for 3h at 37°C. Secondary antibody OmnipMap anti-Ms HRP (Multimer HRP, Roche) was incubated for 15 min. Counterstaining was then done with Hematoxylin II and bluing reagent (Roche). Results were scored (H-score) as follow: H-score = (0 x %negatif) + (1 x %weak positive cells) + (2 x %moderate positive cells) + (3 x %strong positive cells). Overall score ranging from 0 (negative) to 300 (100% cells with strong staining).

[0116] Lysates from placenta and normal breast epithelial cells were lyzed in buffer containing 150mM NaCI + 1% NP40 + 50mM Tris pH8.0. Lysates (100pg) were biotinylated with 2mg / mL NHS-LC-Biotin (ThermoFisher Ref :21336) for 30min at RT. After removing excess biotin with Zeba spin desalting column, biotinylated lysates were incubated with the antibody of interest at 5pg / mL in lysis buffer for 1 H at 4°C. Mix were then incubated with G- protein sepharose beads for 1 H at 4°C. Beads were washed with lysis buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich Ref: P8340). The beads were denaturated in 4X Laemmli buffer (Bio-Rad Ref: 161-0747) supplemented with 0.25% [3-mercaptoethanol by heating at 95°C for 5min.

[0117] Mass spectrometry analysis.

[0118] The immunoprecipitated proteins were loaded on NuPAGE™ 4-12% Bis-tris acrylamide gels according to the manufacturer’s instructions (Invitrogen, Life Technologies). Running of samples was stopped after a short time as soon as proteins stacked as a single band. Protein containing bands were stained with Thermo Scientific Imperial Blue, cut from the gel, and following thiols reduction with 10 mM DTT and cystein alkylation with 55 mM iodoacetamide, digested with high sequencing grade trypsin (Promega, Madison, Wl, USA). Extracted peptides were concentrated before mass spectrometry analysis under speed-vacuum. Samples were reconstituted with 0.1 % trifluoroacetic acid in 2% acetonitrile and analyzed by liquid chromatography (LC)-tandem MS (MS / MS) using a Q Exactive Plus Hybrid Quadrupole-Orbitrap online with a nanoLC Ultimate 3000 chromatography system (Thermo Fisher Scientific™, San Jose, CA). For each biological sample, 3 microliters corresponding to 20 % of digested sample were injected in triplicate on the system. After pre-concentration and washing of the sample on a Acclaim PepMap 100 column (C18, 2 cm x 100 pm i.d. 100 A pore size, 5 pm particle size), peptides were separated on a LC EASY-Spray column (C18, 50 cm x 75 pm i.d., 100 A, 2 pm, 100A particle size) at a flow rate of 300 nL / min with a two steps linear gradient (2-22% acetonitrile / H20; 0.1 % formic acid for 100 min and 22-32% acetonitrile / H20; 0.1 % formic acid for 20 min). For peptides ionization in the EASYSpray source, spray voltage was set at 1.9 kV and the capillary temperature at 250 °C. All samples were measured in a data dependent acquisition mode. Each run was preceded by a blank MS run in order to monitor system background. The peptide masses were measured in a survey full scan (scan range 375-1500 m / z, with 70 K FWHM resolution at m / z=400, target AGO value of 3.00x106 and maximum injection time of 100 ms). Following the high-resolution full scan in the Orbitrap, the 10 most intense data-dependent precursor ions were successively fragmented in HCD cell and measured in Orbitrap (normalized collision energy of 25%, activation time of 10 ms, target AGO value of 1.00x105, intensity threshold 1.00x104 maximum injection time 100 ms, isolation window 2 m / z, 17.5 K FWHM resolution, scan range 200 to 2000 m / z). Dynamic exclusion was implemented with a repeat count of 1 and exclusion duration of 20s.

[0119] Data Processing Protocol.

[0120] Relative intensity-based label-free quantification (LFQ) was processed using the MaxLFQ algorithm from the freely available MaxQuant computational proteomics platform, version 1.6.3.4. Analysis was done on three replicates, corresponding to three injections on mass spectrometers. The acquired raw LC Orbitrap MS data were first processed using the integrated Andromeda search engine. Spectra were searched against the Human database extracted from UniProt on the 1stof February 2022 and containing 20395 entries. The following parameters were used for searches: (i) trypsin allowing cleavage before proline; (ii) one missed cleavages were allowed; (iii) cysteine carbamidomethylation (+57.02146) as a fixed modification and methionine oxidation (+15.99491) and N-terminal acetylation (+42.0106) as variable modifications. The false discovery rate (FDR) at the peptide and protein levels were set to 1 % and determined by searching a reverse database. For protein grouping, all proteins that cannot be distinguished based on their identified peptides were assembled into a single entry according to the MaxQuant rules. The statistical analysis was done with Perseus program (version 1.6.15) from the MaxQuant environment (www.maxquant.org). Quantifiable proteins were defined as those detected in above 70% of samples in one condition or more. Protein LFQ normalized intensities were base 2 logarithmized to obtain a normal distribution. Missing values were replaced using data imputation by randomly selecting from a normal distribution centred on the lower edge of the intensity values that simulates signals of low abundant proteins using default parameters (a downshift of 1.8 standard deviation and a width of 0.3 of the original distribution). To determine whether a given detected protein was specifically differential, a two-sample t-test was done using permutation-based FDR-controlled at 5 and employing 250 permutations. The p value was adjusted using a scaling factor sO with a value of 1 .

[0121] Transfection experiments.

[0122] COS-1 cells grown to 50-80% confluency were transfected with the appropriate cDNA expression plasmids by using the FuGENE™6 reagent method. The cells were cultivated for 1 day, and the medium was replaced. Cells were directly processed in the case of transient expression assays. Vector used in this study is the TFRC_QHuO6795D_pcDNA3.1 + / C -(K)-DYK. ELISA.

[0123] A sandwich enzyme-linked immunosorbent (ELISA) assay was used to control the binding characteritics of the 10G6.3 mAb.

[0124] Protocol 1 : TfR1 ELISA: 96-well plates (Nunc Maxisorp, Invitrogen) are coated with anti-CD71 antibody (Miltenyi ref: 130-108-043) at 33nM overnight at 4°C. Wells are then saturated with 1X BSA (R&D Systems, ref: DY995) for 2H, then recombinant human TfR1 (sequence Cys89-Phe760) (R&D Systems, ref: 2474-TR) or human Holo-Tf (sequence Cys89-Phe760) (negative control) (Sigma Aldrich ref: T0665) proteins are incubated at 20nM for 2H at room temperature. HRP-coupled 10G6.3 antibody is incubated at 15pg / mL for 2H. ABTS substrate (Thermo Scientific ref: 37615) is added for 25 minutes. The reaction is stopped by 100pL of 1% SDS before reading on the Optima plate reader (BMG Labtech) at 405nm.

[0125] Protocol 2: TfR1 + / - holo-Tf ELISA: 96-well plates (Nunc Maxisorp, Invitrogen) are coated with anti-CD71 antibody (Miltenyi ref: 130-108-043) at 33nM overnight at 4°C. Wells are then saturated with 1X BSA (R&D Systems, ref: DY995) for 2H, then recombinant human TfR1 protein (sequence Cys89-Phe760) (R&D Systems, ref: 2474-TR) is incubated at 10nM for 2H at room temperature. The ligands, holo-Tf, ferritin (recombinant Human Ferritin Heavy Chain protein, Novus Biologicals, ref: NBC1-18548) and HFE (Recombinant Human HFE His protein, Novus Biologicals, ref: NBP2-23110) are incubated in 5-fold dilution from 10nM to 0.08nM for 2H. HRP-coupled antibody 10G6.3 is incubated at 10nM for 2H. ABTS substrate (Thermo Scientific ref: 37615) is added for 25 minutes. The reaction is stopped by 100 pL of 1% SDS before reading on the Optima plate reader (BMG Labtech) at 405nm

[0126] Protocol 3: After coating with anti-CD71 antibody (Miltenyi ref: 130-108-043) at 33nM and saturation with 1X BSA, wells are incubated with TfR1 in 5-fold dilution from 60nM to 2.2nM in the presence of 20nM holo-Tf or ferritin.

[0127] Production of ADC.

[0128] ADC was produced from purified 10G6.3 monoclonal antibody as recommended by the manufacturer (Cell Mosaic, MA, USA). The linker used was the MC-Val-Cit-PAB-PNP (Maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate) covalently conjugated to monomethyl auristatin-E (MMAE). The drug-to-antibody ratio was 7.3.

[0129] Cytotoxicity / cell growth measurement. Experiments were done by incubating 3,000 cells / well in duplicate with serial dilutions of ADC at day 0 in 96-well plates. To evaluate the effect of ADC, cell growth / cytotoxicity was measured using the CellTiter-Blue® Reagent staining procedure as recommended (Promega, Wl, USA). The spectral properties of CellTiter-Blue® Reagent change upon reduction of resazurin to resorufin. Resazurin is dark blue in color and has little intrinsic fluorescence until it is reduced to resorufin by mitochondrial oxidation, which is pink and highly fluorescent (579 nm Excitation / 584 nm Emission). Hence, the intensity of the fluorescent signal reflects cell viability. Fluorescence was analyzed on the BMG LABTECH microplate reader ClarioStar Plus at 560Ex / 590Em.

[0130] Flow cytometry.

[0131] FACS analysis in PBS supplemented with 5% FBS. Cells were then stained with Alexa Fluor 488-conjugated (A488) goat anti-mouse antibody (Jackson Immunoresearch ref: 115-546-003) or Alexa Fluor 647-conjugated (A647) goat anti-mouse (Jackson Immunoresearch). Resting PBMC and activated T lymphocytes were stained with the 10G6.3-FITC and anti-CD71-FITC (Miltenyi, 130-115-028) in the presence of Human TruStain FcX™ (FcR block) (Biolegend, 422302). Human Bone Marrow Mononuclear Cells were stained with 10 pg / ml 10G6.3-FITC or anti-CD71-FITC (Miltenyi, 130-115-028) is association with anti-Glycophorin A+ / CD235a-APC (Miltenyi, 130-118-493) in the presence of Human TruStain FcX™ (FcR block). Cells were analysed with the Fortessa cytofluorimeter (Becton Dickinson).

[0132] Internalization studies.

[0133] Thiol-conjugation of monoclonal antibody was performed as recommended (Promega, Ref G9835). Briefly, the antibody was reduced with 2.5 mM DTT for 1h at 25°C. DTT was removed with Zeba spin desalting column. Thiol-conjugation was done with the pHdye reagent dissolved in 50% DMSO, for 3 minutes at 25°C under gentle agitation. Free dye was removed with Zeba spin desalting column. Concentration and Dye to Antibody Ratio were calculated at A280 and A532 as recommended. The 10G6-pHdye Dye to Antibody Ratio was 5.7. Internalization experiments were done in 96 well plates using 0.016 to 10 .g / mL of 10G6.3-pHdye for 24h at 37°C under 5% CO2. Cells were washed three times with 150 iL PBS and fluorescence quantified on the BMG Labtech Clariostar Plus spectrofluorimeter using Cy3 setting (530-20 nm 1 580-30 nm). in vivo studies in mice.

[0134] All experiments were done in agreement with the French Guidelines for animal handling and approved by the local ethics committee (Agreement n° 01152-01). The study, the care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Female NOD / SCID / yc null (NSG) and male NMRI-nude mice were obtained from Charles River laboratories. Mice were housed under sterile conditions with sterilized food and water provided ad libitum and maintained on a 12-h light and 12-h dark cycle. In NSG mice, cells and PDX were inoculated in both flanks in the mammary fat pads with 0.5 x 106cells suspended in 50% phenol red-free Matrigel (Becton-Dickinson Bioscience). Mice were treated when tumor reached average volume of 100-200 mm3. Mice were treated intravenously with ADCs at indicated concentration. Tumor growth was monitored by measuring with a digital caliper and by calculating the tumor volume (length x width2x TT / 6). All animals were randomly assigned into treatment groups, such that the mean tumor volume for each group was 100 to 200 mm3. Animal weight was monitored every 3 days in order to evaluate the toxicity of the different treatments. Mouse weight loss >20%, tumor volume >1500 mm3, ruffled coat and hunched back, weakness, and reduced motility were monitored and considered as endpoints.

[0135] Statistical analyses.

[0136] Prism software (Graphpad) was used for statistical analyses. To compare tumor sizes between treatment groups, 2-way ANOVA test and Bonferroni multiple comparison tests were used.

[0137] Results

[0138] Identification of 10G6.3 mAb

[0139] The inventors performed screening as presented in Figure 1. The inventors produced 554 hybridoma producing monoclonal antibodies (mAbs), among them 16 were selected after negative and positive selection on normal and breast tumor cells respectively. Pattern of reactivity of the 10G6.3 mAb contrasts with TfR1 expression described in healthy tissues (Figure 2). Increased expression is observed in cells that are constantly renewed, such as precursors of blood cells in the bone marrow, hepatocytes in the liver, keratinocytes in the epidermis and enterocytes in crypts of intestinal epithelium. The inventors found that 10G6.3 reacted mostly and markedly with placental cytotrophoblasts and not other adult tissues tested. In hematopoietic cells, 10G6.3 did not bind to mature peripheral blood cells. Reactivity was found to be low on activated T cells compared TfR1 expression seen by a reference TfR1 mAb. However, reactivity was rather similar to a reference TfR1 mAb on erythroid precursor cells of bone marrow (Figure 3). The reactivity of 10G6.3 mAb is detected on primary TNBC samples to higher levels than those described in healthy tissues (Figure 4). MAb 10G6.3 poorly reacts with HME-1 , normal human keratinocytes and PBMC (including T, B lymphocytes, NK cells, monocytes et neutrophils). Reactivity was detected on different breast tumor cells like HCC38 (TNBC), SLIM190 (HER2+), and T47D (luminal). The inventors measured apparent affinity of this mAb (10nM) (Figure 5) and proceeded to extensive analysis of its reactivity on healthy tissues. No or faint reactivity was detected in 19 out of 19 healthy adult tissues tested. Reactivity was strong on syncytiotrophoblast in placenta. The inventors analysed the reactivity in a panel of triple negative breast cancers. As seen on Figure 6, 10G6.3 stained TNBC at various levels. Staining is membranous and cytoplasmic ranging from H-score 0 to 180 (n=16). Staining was also detected on 3 selected PDX models of TNBC ranging from H-score 130 to 270. Together, these results showed that the antibody of the invention is highly specific for cancer, in particular for breast cancer, as it reacts with TNBC and poorly with healthy tissues.

[0140] 10G6.3 specifically recognizes the transferrin receptor- 1

[0141] Reactivity of 10G6.3 on cancer cells has been found to be as expected and the inventors proceeded to the identification of the target by immunoprecipitation followed by mass spectrometry procedures as described in material and methods. The inventors selected placental tissue that presents strong mAb reactivity by IHC and normal breast epithelial cells as negative control (Figure 7). After precipitation with 10G6.3, three major bands (between 90 kDa and 170 kDa) were detected by western-blot in placenta but not breast sample (Figure 7). Mass spectrometry analysis identified the transferrin receptor-1 (TfR1) as the unique membrane associated protein present in placenta and absent in normal breast epithelial cells (Figure 7). To confirm that 10G6.3 binds to TfR1 , the inventors performed RNA interference experiments and showed that 10G6.3 binding was strongly reduced as found with a commercial anti-TfR1 mAb (Miltenyi Biotech, Germany) (Figure 8). The mAb binds to Cos cells transiently transfected with human TfR1 cDNA expression vector as observed with a control anti-TfR1 mAb (Figure 8). Finally, the inventors showed specific and direct binding of 10G6.3 on recombinant soluble TfR1 by ELISA (Figure 8).

[0142] Antitumor activity in vitro

[0143] The ability of TfR1 to internalize make this receptor an attractive target for antibodydrug conjugate (ADC) therapy. To go further in the characterization of 10G6.3 as a valuable antibody for ADC targeting, the inventors quantified its internalization on SUM190PT breast tumor cells. MAb was conjugated with a pH sensor dye (Promega, Wl, USA) that have very low fluorescence level at pH>7 and a dramatic increase fluorescence when pH decreases in endosomes / lysosomes during internalization processes. Internalization of 10G6.3 is detected at a concentration of 0.4|jg / ml and is maximal at 10pg / ml (Figure 9). The inventors produced ADC format of 10G6.3 by conjugation to monomethyl auristatin-E (MMAE) via a cleavable valine-citrulline linker (10G6-vcMMAE). The inventors compared the in vitro efficacy of 10G6-MMAE with an irrelevant control CD30-vcMMAE / Adcetris on SUM 190 (positive control) and SKBR3 (negative control). Cytotoxicity was found with an IC50 value of 135 ng / ml (0.9nM) (Figure 9).

[0144] Antitumor activity in vivo

[0145] Activity of the ADC of the invention was tested in models of TNBC developed in immunocompromised NSG mice treated with one intravenous dose of 10G6.3-vcMMAE (10mg / kg). The inventors first analysed the cross-binding reactivity of 10G6.3 on murine TfR1. 10G6.3 did not react on murine placenta. The 10G6.3-vcMMAE induced a marked antitumor effect in CDX SUM190PT mice at 10 mg / kg and 7.5 mg / kg, with 99% of tumor mass regression that lasted for 57 days (Figure 10). These data showed a marked efficiency of the 10G6.3-vcMMAE ADC with complete and durable eradication of xenografted tumors. No pain or distress were noted in the absence of weight loss. The inventors also controlled red blood cells count and found no difference with control ADC brentuximab (Figure 10). Together these results indicated a marked efficacy on TNBC cancer of the 10G6.3-vcMMAE ADC of the invention and absence of toxicity.

[0146] Holo-Transferrin increase 10G6.3 affinity

[0147] Our data suggests that 10G6.3 recognizes a particular epitope that is different between healthy tissues and tumors. As many anti-TfR1 mAbs block diferric transferrin (holo-Tf) binding, the inventors measured ability of 10G6.3 to block holo-Tf binding. They performed ELISA in the presence of holo-Tf, ferritin and HFE, which are three known ligands for TfR1. As expected, 10G6.3 binds TfR1 (10nM) in the absence of ligands (0 nM and no TfR1 conditions). Binding to TfR1 is unchanged in the presence of increased concentration of ferritin and HFE ranging from 0.08 nM to 10 nM. Unexpectedly, the inventors observed that holo-Tf increased the binding of 10G6.3 to TfR1 by 2.7 fold, in a dose-dependent manner (Figure 11). To calculate the apparent affinity of the interaction, they performed an ELISA by varying the concentration of TfR1 from 2.2 nM to 20 nM with and without 20 nM holo-transferrin. The apparent affinity of 10G6.3 in the presence of holo-Tf increased in the presence of holo-Tf (from 11.7 nM to 8.8 nM). The maximum binding value also increased (from OD value 0.43 to 0.80) (Figure 11). In order to evaluate the effect of holo-Tf on 10G6.3-vcMMAE ADC efficacy, the inventors performed cytotoxicity experiments on SUM190PT cells. These cells usually grown in the presence of human holo-Tf (68.75nM). The inventors thus grown SUM190PT cells without holo-Tf and performed a cytotoxic assay comparing IC50 in absence of holo-Tf and after supplementation with 20 nM human holo- Tf (Figure 11). This concentration was defined from the previous ELISA experiment (Figure 11). In the absence of holo-Tf, IC50 value of 3.6 pg / ml shifted to 0.60 pg / ml in the presence of 20 nM holo-Tf, close to the IC50 presented in Figure 9B (experiment done in the presence of 68.75 nM holo-Tf). Thus, adding holo-Tf to culture medium results in a 6-fold increase of the 10G6.3-vcMMAE ADC efficacy. These data demonstrated that holo-Tf increase 10G6.3 binding and ADC efficacy. This may also indicate that 10G6.3 antibody of the invention recognizes a conformational epitope on TfR1.

[0148] Conclusion

[0149] The reactivity of the anti-TfR1 antibody of the invention is detected on primary TNBC samples to higher levels than those described in healthy tissues which make this antibody attractive for cancer treatment, in particular for resistant cancers such as TNBC. The Tf- dependent affinity of the 10G6.3 antibody could explain its differential reactivity between healthy tissues that contain less holo-Tf than tumors that are enrich in holo-Tf. The anti- TfR1 antibody affinity is indeed increased in the presence of holo-Tf, resulting in a 6 fold increased efficacy of the 10G6.3-vcMMAE ADC in vitro. These results also indicate that the 10G6.3 epitope is a conformational tumor-specific epitope. Together, the antibody or ADC of the invention represents a new alternative with reduced toxicity for the treatment of cancer.

Claims

CLAIMS1. An antibody targeting transferrin receptor 1 (TfR1) comprising:(a) a heavy chain variable (VH) domain, wherein CDR1 , CDR2 and CDR3 have at least 70% sequence identity to, respectively, SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3; and(b) a light chain variable (VL) domain, wherein CDR1 , CDR2 and CDR3 have at least 70% sequence identity to, respectively, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

2. The antibody targeting TfR1 according to claim 1 , wherein CDR1 to CDR3 of the VH domain have sequences identical to, respectively, SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3; and wherein CDR1 to CDR3 of the VL domain have sequences identical to, respectively, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

3. The antibody targeting TfR1 according to any one of the preceding claims having a VH domain which has at least 70% sequence identity to SEQ ID NO: 7, and a VL domain which has at least 70% sequence identity to SEQ ID NO: 8.

4. The antibody targeting TfR1 according to claim 3, wherein the VH domain is identical to SEQ ID NO: 7, and the VL domain is identical to SEQ ID NO: 8.

5. An antibody-drug conjugate comprising the antibody targeting TfR1 according to any one of the preceding claims, and wherein the drug is an anti-tumor agent.

6. The antibody targeting TfR1 according to any one of claims 1-4 or the antibodydrug conjugate according to claim 5, for use for the treatment of cancer.

7. The antibody targeting TfR1 or the antibody-drug conjugate according to claim 6, wherein the cancer is a triple negative breast cancer (TNBC).

8. The antibody targeting TfR1 according to any one of claims 1-4 or 6-7, or the antibody-drug conjugate according to any one of claims 5-7, wherein the antibody recognizes a Tf-sensitive conformational active form of TfR1 .

9. The antibody targeting TfR1 according to any one of claims 1-4 or 6-8, or the antibody-drug conjugate according to any one of claims 5-8, wherein the antibody recognizes a tumor specific epitope of TfR1 , preferably a conformational epitope containing the glycosylation site Asn317.

10. A pharmaceutical composition comprising an antibody targeting TfR1 according to any one of claims 1-4 or the antibody-drug conjugate according to claim 5, formulated with a pharmaceutically acceptable carrier.

11. A nucleic acid encoding the VH domain of the antibody targeting TfR1 according to any one of claims 1-4 or the antibody-drug conjugate according to claim 5.

12. A nucleic acid having the nucleotide sequence of SEQ ID NO: 9.

13. A nucleic acid encoding the VL domain of the antibody targeting TfR1 according to any one of claims 1-4 or the antibody-drug conjugate according to claim 5.

14. A nucleic acid having the nucleotide sequence of SEQ ID NO: 10.

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