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WO2025073856A1 - Association d'anticorps ou de fragments de liaison à l'antigène associés pour le traitement de cancers - Google Patents

Association d'anticorps ou de fragments de liaison à l'antigène associés pour le traitement de cancers Download PDF

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WO2025073856A1
WO2025073856A1 PCT/EP2024/077883 EP2024077883W WO2025073856A1 WO 2025073856 A1 WO2025073856 A1 WO 2025073856A1 EP 2024077883 W EP2024077883 W EP 2024077883W WO 2025073856 A1 WO2025073856 A1 WO 2025073856A1
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cancer
combination
antigen
antibodies
antibody
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Firas BASSISSI
Carine CIRON
Odile DUVAUX
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Xenothera
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Xenothera
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification

Definitions

  • the present invention relates to the field of treatment of cancers.
  • the present invention relates to the field of using polyclonal antibodies or combinations of antibodies directed against specific antigens and their implementation in treatment of cancers.
  • Cancer is the second leading cause of death in the World and is responsible for an estimated 9.6 million deaths in 2018. Globally, about 1 in 6 deaths worldwide is due to cancer. Inaccessible or non- sufficiently active treatment are common in this filed. There is thus a well- recognized need to develop new and improved therapies for treating cancers.
  • PTCL Peripheral T-cell Lymphoma
  • peripheral T-cell lymphoma not otherwise specified
  • angioimmunoblastic T-cell lymphoma extranodal NK/T-cell lymphoma, nasal type
  • ALK+ systemic anaplastic large T- cell lymphoma ALK- systemic anaplastic large T-cell lymphoma
  • mycosis fungoides Sezary syndrome, among others.
  • the intricate pathological classification system reflects the wide variability within this group of diseases.
  • PTCL non-Hodgkin's lymphoma
  • Immunotherapies represent a real hope in the treatment of a great number of cancers, in particular in the treatment of cancers not currently efficiently cured by conventional therapies.
  • This treatment mainly consists in the administration of monoclonal antibodies directed against the tumoral cells or directed against activators or inhibitors of a checkpoint of the immune response against cancers.
  • monoclonal antibodies mAb
  • ADC antibody drug conjugate
  • CAR-T chimeric antigen receptor T cells
  • bispecific antibodies are being actively explored and successfully introduced as innovative weapon in cancer treatment arsenal.
  • Antibodies directed against tumors can act through two complementary ways:
  • CDC cytotoxicity complement dependent
  • ADCC dependent from killer cells
  • ADCP dependent from phagocytes
  • PTCL PTCL
  • monoclonal antibodies which are being studied for the treatment of these cancers, including many new drugs being studied in clinical trials for the treatment of PTCL, including Alisertib, Bendamustine (Treanda), Bortezomib (Velcade), Lenalidomide (Revlimid), Nivolumab (Opdivo), Panobinostat (Farydak) and Pembrolizumab (Keytruda).
  • the escaping mechanisms comprise immuno selection mechanisms, i.e. the capacity of the tumoral cell to lose the antigen recognized by the immune system and immunosubversion mechanisms (induction of a specific tolerance).
  • immuno selection mechanisms i.e. the capacity of the tumoral cell to lose the antigen recognized by the immune system and immunosubversion mechanisms (induction of a specific tolerance).
  • immunosubversion mechanisms induction of a specific tolerance.
  • the appearance of less immunogenic tumoral variants can be particularly deleterious within treatments based on the use of monoclonal antibodies (which are specific to a unique epitope).
  • other PTCL subtypes have a 5- year survival rate ranging from 14% to 32%
  • a treatment based on the use of combinations of antibodies targeting different antigens on the tumoral cells might allow minimizing the escaping mechanisms.
  • polyclonality and polytargeting of a combination of antibodies can prevent the pitfalls encountered by various monoisotopic or mono target approaches of monoclonal antibodies.
  • cytotoxicity complement dependent CDC
  • ADCC cytotoxicity complement dependent from killer cells
  • ADCP cytotoxicity complement dependent from phagocytes
  • an antitumoral activity consisting of CDC, ADCC, apoptotic activity and ADCP.
  • the invention has for purpose to meet the above-mentioned needs.
  • the present invention in particular relates to the following items:
  • CD99 Cluster of differentiation 99
  • GPI Glucose-6-phosphate isomerase
  • SLC3A2 solute carrier family 3 member 2
  • GART glycosinamide ribonucleotide formyltransferase
  • CKAP4 Cytoskeleton-associated protein 4
  • Item 2 The combination for use according to item 1, further comprising at least a fourth antibody, or an antigen binding fragment thereof, specifically binding to an antigen selected from the group consisting of GPI, SLC3A2, GART, and CKAP4, in particular consisting of GPI and GART, said fourth antibody, or antigen binding fragment thereof, being different from the third antibody, or antigen-binding fragment thereof.
  • Item 3 The combination for use according to item 1 or 2, comprising at least:
  • Item 5 The combination for use according to any one of items 1 to 4, wherein the cancer is selected from the group consisting of myeloma; melanoma; skin cancer; breast cancer; brain tumors, bladder cancer, cervical cancer, prostate cancer; primary and metastatic colorectal cancer, in particular a colon cancer; mesothelioma; lung cancer, in particular non small cell lung cancer; liver cancer, in particular an hepatocarcinoma or a cholangiocarcinoma; primary and metastatic pancreas cancer; renal cancer; soft tissue sarcoma; thyroid cancer; lymphoma including Hodgkin and Non-Hodgkin lymphoma, in particular B-cell or T-cell lymphoma, more particularly T-cell lymphoma; gastric cancer; head and neck cancer; ovarian cancer; sarcoma; acute or chronic leukemia in particular T-cell or myeloid leukemia; osteosarcoma; anal cancer; testicular cancer; uterus cancer; thyroid cancer;
  • lymphoma in particular selected from the group consisting of:
  • Non-Hodgkin lymphomas selected from the group consisting of:
  • T- T-cell lymphomas in particular selected from the group consisting of: precursor T-lymphoblastic lymphoma (or precursor T-lymphoblastic leukaemia), Peripheral T- cell lymphoma and Cutaneous T-cell lymphoma; and (ii) Hodgkin lymphomas selected from the group consisting of nodular sclerosis, mixed cellularity, lymphocyte-depleted Hodgkin lymphoma and lymphocyte-rich Hodgkin lymphoma; or
  • a leukemia in particular selected from the group consisting of Acute Meyloid Leukemia (AML), Chronic Meyloid Leukemia (CML) and Histiocytic Leukemia.
  • T-ALL T-cell acute lymphoblastic leukemia
  • CTCL Cutaneous T-cell lymphoma
  • Item 9 The combination for use according to any one of items 1 to 8, wherein at least one of the antibodies of the combination, and in particular all the antibodies of the combination, is/are devoid of at least one antigenic determinant selected from (i) N- glycolylneuraminic acid (Neu5Gc) and (ii) a-l,3-galactose, and in particular is/are devoid of the two antigenic determinants N-glycolylneuraminic acid (Neu5Gc) and a-l,3-galactose.
  • N- glycolylneuraminic acid Ne- glycolylneuraminic acid
  • a-l,3-galactose a-l,3-galactose
  • Item 11 The combination for use according to item 10, wherein the polyclonal antibody composition is obtainable by immunization of an animal with CD3+, CD8+, TCR gamma/delta- and CD34- human tumor T-cells.
  • Item 13 The combination for use according to any one of items 1 to 12, wherein the combination is included in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • Item 14 The combination for use according to item 13, wherein the pharmaceutical composition further comprises at least one additional anticancer drug different from the antibodies of the combination of antibodies as defined in any one of items 1 to 4 and 8 to 11.
  • Item 16 The combination for use according to any one of items 13 to 15, wherein the pharmaceutical composition further comprises at least one chemotherapy treatment, in particular a chemotherapy treatment selected from the group consisting of a chemotherapy regimen consisting of Cyclophosphamide, Hydroxydaunorubicin, Oncovin and Prednisone (CHOP); a chemotherapy regimen consisting of Cyclophosphamide, Hydroxydaunorubicin, Oncovin, Etoposide and Prednisone (CHOEP); HD AC inhibitors; Ibrutinib, acalabrutininb, zanubrutinib, copanlisib and venetoclax.
  • a chemotherapy treatment selected from the group consisting of a chemotherapy regimen consisting of Cyclophosphamide, Hydroxydaunorubicin, Oncovin and Prednisone (CHOP); a chemotherapy regimen consisting of Cyclophosphamide, Hydroxydaunorubicin, Oncovin, Etoposide and Predn
  • Figure 1 describes the cytotoxicity activity of a combination of antibodies on different cell lines.
  • the figure represents the % of cytotoxicity of PAI (at a concentration of 100 pg/mL) for each of the following cell lines, from left to right: PBMC, HPB-ALL, Jurkat, T1301, HUT78 and OCI-LY-12.
  • Figure 3 describes the cytotoxicity activity of a combination of antibodies and of Alemtuzumab (Anti-CD52 monoclonal antibody) on different cell lines.
  • the figure represents de % of cytotoxicity of PAI (at a concentration of 100 pg/mL) (left column) and of Alemtuzumab (at a concentration of 100 pg/mL) (right column) for each of the following cell lines, from left to right: PBMC, HPB-ALL, Jurkat, and T1301.
  • Figure 12 describes the size of the tumor of Jurkat xenograft mouse models after 40 days of treatment with a combination of antibodies.
  • the figure represents the mean tumor size (in mm3) at Day 40 in the presence of PAI (right column) or in the absence of treatment (left column).
  • N 10 per group. * p ⁇ 0.05 - Mann- Whitney test.
  • Figure 16 describes the variation of the tumor size (in mm 3 ) over the time of the treatment (in days of treatment) for each of the following groups: Control group (•), PAI group ( ⁇ ) and CHOP group (A).
  • the arrows under the graph represent the days at which PAI treatment was administered (One way Anova - post Hoc test Fischer - *p ⁇ 0.05, **p ⁇ 0.01).
  • Figure 17 describes the Mean Tumor Volume (in mm 3 ) for each of the groups at D28, 28 days after the start of treatment. From left to right, the groups are: Control group (very dark grey), CHOP Group (light grey) and PAI Group (grey) (One way Anova - post Hoc test Fischer - *p ⁇ 0.05).
  • Figure 18 describes the percentage of cell death (in %) according to the PAI concentration (in pg/ml) in different cell lines: HPB-ALL (•), T-1301 ( ⁇ ), Jurkat ( A), HUT78 ( ⁇ ) and KARPASS 299 ( ⁇ ).
  • the words “/zave” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
  • the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements.
  • the term “consisting of’ implies the inclusion of the stated element(s), to the exclusion of any additional elements.
  • “pharmaceutically acceptable carriers” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible.
  • suitable carriers, diluents and/or excipients include one or more of water, amino acids, saline, phosphate buffered saline, buffer phosphate, acetate, citrate, succinate; amino acids and derivates such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts NaCl, calcium chloride; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as Polysorbate 80, polysorbate 20, poloxamer 188; and the like, as well as combination thereof.
  • isotonic agents such as sugars, polyalcohols, or sodium chloride
  • formulation may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.
  • antioxidant such as tryptamine
  • stabilizing agent such as Tween 20
  • the light chain includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH).
  • VL variable domain
  • VH variable domain
  • CH constant domain
  • “Fragments” of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody.
  • antibody fragments include Fv, Fab, F(ab’)2, Fab’, dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments.
  • a fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.
  • Fab denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of the heavy chain and the entire light chain are bound together through a disulfide bond. It is usually obtained among fragments by treating IgG with a protease, papain.
  • BsAb denotes an antibody which combines the antigen-binding sites of two antibodies within a single molecule. Thus, BsAbs are able to bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2 050764 Al.
  • multispecific antibody denotes an antibody which combines the antigenbinding sites of two or more antibodies within a single molecule.
  • Antibodies of compositions according to the invention may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.
  • the antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow et al., Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2 nd ed. (1988)).
  • polyclonal antibodies By “polyclonal antibodies” , “polyclonal antibody” or “polyclonal antibody composition” as used herein in an interchangeable manner is meant a mixture of antibodies recognizing different epitopes of a given antigen, or even different epitopes of different antigens expresses by a given cell or group of cells. Polyclonal antibodies encompass those which are contained in, or alternatively which are derived from, body fluids, especially serum or plasma from a non-human mammal organism, in particular from a pig. A polyclonal antibody composition of the invention is different from a composition containing more than one monoclonal antibodies.
  • monoclonal antibody refers to an antibody molecule of a single amino acid sequence, which is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method.
  • a monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e., produced by protein engineering.
  • humanized antibody refers to an antibody which is wholly or partially of non-human origin, and which has been modified to replace certain amino acids, in particular in the framework regions of the VH and VL domains, in order to avoid or minimize an immune response in humans.
  • the constant domains of a humanized antibody are most of the time human CH and CL domains.
  • recombinant as applied to an antibody, or an antigen-binding fragment thereof, a nucleic acid sequence, an expression vector or a host cell means that those are the products of various combinations of in vitro cloning, restriction, ligation steps, and other genetic engineering procedures.
  • the term “antibody” in particular encompasses an antibody from a pig, and more particularly an antibody from a pig, the said antibody being devoid of at least one antigenic determinant selected from (i) N- glycolylneuraminic acid (Neu5Gc) and (ii) a-l,3-galactose, and in particular is devoid of the two antigenic determinants N-glycolylneuraminic acid (Neu5Gc) and a-l,3-galactose; and which further preferably comprises at least one sugar moiety distinct from the antigenic determinants (i) N-glycolylneuraminic acid (Neu5Gc) and/or (ii) a-l,3-galactose.
  • a combination of antibodies for use according to the invention, and more particularly a polyclonal antibody composition for use according to the invention, as understood herein, is in particular a polyclonal antibody composition that is obtainable by immunization of an animal with CD3+, CD8+, TCR gamma/delta- and CD34- human tumor T-cells.
  • CD3+, CD8+, TCR gamma/delta- and CD34- it is understood that the human tumor T-cells which express CD3 and CD8 but that do not express TCR gamma/delta or CD34.
  • affinity means the strength of the binding of an antibody to an epitope presented on an antigen.
  • the affinity of an antibody is given by the dissociation constant KD, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody- antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • KD dissociation constant
  • Ka is defined by 1/Kd.
  • immunoglobulins comprise the classes or isotypes IgM, IgD, IgG, IgE and IgA antibodies and the IgG isotype comprise 11 subclasses (Butler et al., Developmental and Comparative Immunology 30 (2006) 199-221; Butler et al., Developmental and Comparative Immunology 33 (2009) 321-333).
  • Full-length IgGs consist of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, Cyl (also called CHI), Cy2 (also called CH2), and Cy3 (also called CH3).
  • antigenic determinant (or epitope), as applied herein to pig antibodies, as used herein is meant a structural component of an antigenic molecule, which includes an antigenic protein and an antigenic carbohydrate, responsible for its specific interaction with antibody molecules elicited by the same or related antigen.
  • antigenic determinant as applied herein to pig antibodies is also used collectively herein for an antigenic molecule comprising a plurality of epitopes, including conformational motives in which the sugar moiety is needed but represent only part of the epitope, susceptible to be recognized by antibody molecules elicited by the same or related antigen.
  • the antigenic molecule N-glycolylneuraminic acid may be called herein an “antigenic determinant”, although the said antigenic molecule may exhibit more than one epitope recognized by antibodies elicited with Neu5Gc or with Neu5Gc containing molecules.
  • cancer is herein used in its traditional sense, and refers to a cell or group of cells displaying uncontrolled growth, invasion upon adjacent tissues.
  • Cancers may in particular refer herein to a cancer selected from the group consisting of myeloma; melanoma; skin cancer; breast cancer; brain tumors, bladder cancer, cervical cancer, prostate cancer; primary and metastatic colorectal cancer, in particular a colon cancer; mesothelioma; lung cancer, in particular non small cell lung cancer; liver cancer, in particular an hepatocarcinoma or a cholangiocarcinoma; primary and metastatic pancreas cancer; renal cancer; soft tissue sarcoma; thyroid cancer; lymphoma including Hodgkin and Non-Hodgkin lymphoma, in particular B-cell or T-cell lymphoma, more particularly T-cell lymphoma; gastric cancer; head and neck cancer; ovarian cancer; sarcoma; acute or chronic leukemia in particular T-cell or myeloid leukemia; osteosarcoma; anal cancer; testicular cancer; uterus
  • the cancer may be selected from the group consisting of myeloma; melanoma; skin cancer; breast cancer; brain tumors, bladder cancer, cervical cancer, prostate cancer; primary and metastatic colorectal cancer, in particular a colon cancer; mesothelioma; lung cancer, in particular non small cell lung cancer; liver cancer, in particular an hepatocarcinoma or a cholangiocarcinoma; primary and metastatic pancreas cancer; renal cancer; soft tissue sarcoma; thyroid cancer; lymphoma including Hodgkin and Non-Hodgkin lymphoma, in particular B-cell or T-cell lymphoma, more particularly T-cell lymphoma; gastric cancer; head and neck cancer; ovarian cancer; sarcoma; acute or chronic leukemia in particular T-cell or myeloid leukemia; osteosarcoma; anal cancer; testicular cancer; uterus cancer; thyroid cancer; cancer of the central nervous system; gastrointestinal stromal
  • treatment refers to administering an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (i.e., a cancer), the symptoms of the cancer, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a disease, or otherwise arrest or inhibit further development of the cancer.
  • a condition i.e., a cancer
  • treating a cancer includes providing an antitumoral activity.
  • wild type animal or “WT animal” come herein in opposition with a genetically altered animal.
  • wild type pig is meant a pig which is not lacking at least one gene selected in a group comprising (i) a gene encoding a functional cytidine-5'- monophosphate N-acetyl neuraminic acid hydrolase (CMAH) and (ii) a gene encoding a functional a-(l,3)-galactosyltransferase.
  • CMAH cytidine-5'- monophosphate N-acetyl neuraminic acid hydrolase
  • polyclonal antibodies which may be present in antibody combinations of implemented according to the present invention, may notably be cited the method of fractionated precipitation with ethanol, with ammonium sulfate, with rivanol, with polyethylene glycol or with caprylic acid, the method by passage through ion exchange columns; other methods can involve affinity columns on protein A or G.
  • the antibodies obtained can be then subjected to conventional treatments for their intravenous administration, for example by enzymatic cleavage treatments plasmin, papain or pepsin.
  • Such antibodies can for example be generated through immunization of a nonhuman animal, in particular a non-human mammal, according to methods well known to the man skilled in the art.
  • the non-human mammal may be selected from the group consisting of rodents, such as mice, rats, guinea pigs and hamsters; lagomorphs, such as rabbits; ferrets; felines, such as cats; canines, such as dogs; goats; sheep; bovines, such as cows; swines, such as pigs and hogs; camelids; horses; and non-human primates.
  • the non-human mammal may more particularly be a pig.
  • anti-cancer pAb from pig origin for example, it is meant that the polyclonal antibodies have been obtained through immunization of a pig with a combination of proteins of interest or with a cell naturally expressing, or genetically altered in order to express on its surface, the proteins of interest against which it is desired to obtained polyclonal antibodies. See Reynard et al. pLoS One. 2016; 11(6): e0156775; Schieferdecker et al., Oncotarget. 2016 Oct 11; 7(41): 67061-67070 and Zhang et al. 2014 (DOI: 10.1038/srep04984).
  • polypeptide and protein are used interchangeably herein to refer to a sequence of amino acid residues.
  • the terms apply to amino acid sequences in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid sequences and non-naturally occurring amino acid sequence.
  • the present invention relates to the implementation of a combination of antibodies, or antigen-binding fragments thereof, for use in the treatment of a cancer in a human subject in need thereof.
  • Such combination comprises:
  • CD99 Cluster of differentiation 99
  • GPI Glucose-6-phosphate isomerase
  • SLC3A2 solute carrier family 3 member 2
  • GART glycosinamide ribonucleotide formyltransferase
  • CKAP4 Cytoskeleton-associated protein 4
  • the combination of antibodies for use according to the invention may comprise antibodies targeting more than three different proteins among the group consisting of FASN, CD99, GPI, SLC3A2, GART, and CKAP4.
  • the antibodies present in a combination of antibodies for use according to the invention may accordingly target at least 3 proteins, target 4 proteins, target at least 4 proteins, target 5 proteins, target at least 5 proteins, target 6 proteins, target at least 6 proteins, target 7 proteins, target at least 7 proteins, target 8 proteins, target at least 8 proteins, target 9 proteins or target at least 9 proteins from the group consisting of FASN, CD99, GPI, SLC3A2, GART, and CKAP4.
  • a combination of antibodies for use according to the invention comprises antibodies targeting 3, 4 or 5, more particularly 4 or 5, proteins selected from the group consisting of FASN, CD99, GPI, SLC3A2, GART, and CKAP4.
  • Some or all of the antibodies of an antibody combination for its use according to the invention may be devoid of at least one antigenic determinant selected from (i) N- glycolylneuraminic acid (Neu5Gc) and (ii) a-l,3-galactose. Some or all of the said antibodies for use according to the invention may in particular be devoid of the two antigenic determinants N-glycolylneuraminic acid (Neu5Gc) and a-l,3-galactose.
  • a method allowing to identify or characterize such antibodies falls within the general knowledge of a man skilled in the art.
  • a method that may be used by the one skilled in the art for identifying or characterizing antibodies according to the invention includes an Enzyme-linked immunosorbent assay (ELISA) wherein, for example, anti-Neu5Gc antibodies and anti-Gal antibodies are used as detection molecules.
  • ELISA Enzyme-linked immunosorbent assay
  • the antibodies from the antibody combination implemented according to the invention may be immunoglobulin G antibodies.
  • the relative composition of the IgG isotypes in pig DKO poly Ab is estimated to be >80% pig IgGla/b, of 11% IgG2a/b, of 5.5% IgG3, of 3% IgG4a/b, the remaining fraction being other isotypes (IgG5- 6). No detectable IgM or IgA isotype is present in pig DKO IgG polyclonal antibody preparation after purification using Protein A chromatography.
  • Protein FASN (Fatty acid synthase) is a multi-enzyme protein that serves as the key regulator in lipid metabolism, especially fatty acid synthesis.
  • Protein CD99 Cluster of differentiation 99 is a glycosylated transmembrane protein involved in many essential cellular functions such as cell adhesion and migration, cell death and differentiation, intracellular protein trafficking, endocytosis and exocytosis.
  • Protein GPI Glucose-6-phosphate isomerase
  • G6P glucose-6-phosphate
  • F6P fructose-6-phosphate
  • the amino acid sequence of GPI has for reference UniProt P06744.
  • Antibodies able to specifically target GPI are well known in the art. Mention can be made of the monoclonal antibody MABN691 (commercialized by Merck Millipore). Application W02000064469 may also be referred to concerning such antibodies.
  • SLC3a2 forms a dimer with SLC7a5 for its functionality and is involved in the delivery of amino acids together.
  • CD98 is a transmembrane protein and is found on the cell surface. Therefore, it can be easily targeted with drugs.
  • Antibodies able to specifically target SLC3A2 are well known in the art. Can for example be mentioned the monoclonal antibodies sc-390154 (commercialized by Santa Cruz Biotechnology), MA5-29573 (commercialized by ThermoFischer Scientific or the polyclonal antibody 15193-1-AP (commercialized by Proteintech Group Inc).
  • the application WO20172114458 also describes several antibodies directed against SLC3A2 and is incorporated by reference.
  • the amino acid sequence of GART has for reference UniProt Q71VH3.
  • Antibodies able to specifically target GART are well known in the art. Reference can be made to Joe Dotzlaf et al. (Hybridoma (Larchmt). 2006; 25:139-44).
  • the amino acid sequence of CKAP4 has for reference NCBI Reference Sequence :
  • Antibodies able to specifically target CKAP4 are well known in the art. Can for example be mentioned the polyclonal antibody 16686-1-AP (commercialized by Proteintech Group Inc), the monoclonal antibody MOB-3287z (commercialized by Creative Biolabs) or the monoclonal antibody sc-393544 (commercialized by Santa Cruz Biotechnology).
  • the application WO2019065747 also describes several antibodies directed against CKAP4 and is incorporated by reference.
  • a combination of antibodies, or antigen-binding fragments thereof, for use according to the invention may comprise:
  • CD99 Cluster of differentiation 99
  • GPI Glucose-6-phosphate isomerase
  • SLC3A2 solute carrier family 3 member 2
  • GART glycosinamide ribonucleotide formyltransferase
  • CKAP4 Cytoskeleton-associated protein 4
  • a fourth antibody or an antigen-binding fragment thereof, specifically binding to an antigen selected from the group consisting of GPI (Glucose-6-phosphate isomerase), SLC3A2 (solute carrier family 3 member 2), GART (glycinamide ribonucleotide formyltransferase), and CKAP4 (Cytoskeleton-associated protein 4), in particular consisting of GPI and GART, said fourth antibody, or antigen binding fragment thereof, being different from the third antibody, or antigen-binding fragment thereof.
  • GPI Glucose-6-phosphate isomerase
  • SLC3A2 solute carrier family 3 member 2
  • GART glycosinamide ribonucleotide formyltransferase
  • CKAP4 Cytoskeleton-associated protein 4
  • the combination of antibodies for use according to the invention comprises: - at least a first antibody, or an antigen-binding fragment thereof, specifically binding to FASN,
  • the antibodies of the combinations may, independently from the others, be devoid of at least one antigenic determinant selected from (i) N- glycolylneuraminic acid (Neu5Gc) and (ii) a-l,3-galactose, and may more particularly be devoid of the two antigenic determinants N-glycolylneuraminic acid (Neu5Gc) and a- 1,3- galactose.
  • the antibodies of the combinations are devoid of at least one antigenic determinant selected from (i) N-glycolylneuraminic acid (Neu5Gc) and (ii) a- 1,3-galactose, and are more particularly devoid of the two antigenic determinants N- glycolylneuraminic acid (Neu5Gc) and a-l,3-galactose.
  • the antibodies of a combination for use according to the invention may be of any origin, such as from a non-human mammal or synthetic, monoclonal or polyclonal.
  • non-human mammal may be selected from the group consisting of rodents, such as mice, rats, guinea pigs and hamsters; lagomorphs, such as rabbits; ferrets; felines, such as cats; canines, such as dogs; goats; sheep; bovines, such as cows; swines, such as pigs and hogs; camelids; horses; and non-human primates.
  • the antibodies of a combination for use according to the invention are preferably from a pig, and in particular from a pig lacking at least one gene selected in a group comprising (i) a gene encoding a functional cytidine-5'-monophosphate N-acetyl neuraminic acid hydrolase (CMAH) and (ii) a gene encoding a functional a-(l,3)-galactosyltransferase, and more particularly lacking both (i) a gene encoding a functional cytidine-5'-monophosphate N-acetyl neuraminic acid hydrolase (CMAH) and (ii) a gene encoding a functional a-( 1 ,3)-galactosyl transferase.
  • CMAH cytidine-5'-monophosphate N-acetyl neuraminic acid hydrolase
  • CMAH cytidine-5'-monophosphate N-acetyl neuraminic acid
  • the combination for use according to the present invention may be a combination of monoclonal antibodies or a polyclonal antibody composition and is preferably a polyclonal antibody composition.
  • the pharmaceutical composition may be in a solid form, which includes a lyophilized form.
  • the pharmaceutical composition may be formulated according to standard methods such as those described in Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins; Twenty first Edition, 2005).
  • the pharmaceutical composition may further comprise at least one additional anticancer drug different from the antibodies of the combination of antibodies.
  • the additional anticancer drug may for example be selected from the group consisting of monoclonal antibodies, in particular selected from the group consisting of anti- CD19, anti-CD20, anti-CD30, anti-CD137, anti-CTLA4, anti-TIM-3, anti-B7-H3, anti-CD123, anti-CD134, anti-CD154, anti-LAG-3, anti-CD227, anti-BTNA3, anti-CD39, anti-CD73, anti- CD115, anti-CD47, anti-SIRP alpha, anti-SIRP gamma, anti-CD28, anti-NCR, anti-NKp46, anti-NKp30, anti-NKp44, anti-NKG2D, anti-PDl, anti-PDLl, mogamulizumab, obinutuzumab, polatuzumab vedotin, Yttrium Y 90-ibritumomab tiuxetan, mosunetuzumab and anti-DNAM-1
  • the pharmaceutical composition may further comprise at least one chemotherapy treatment.
  • Chemotherapy treatments may be selected among those known in the art.
  • the chemotherapy treatment is selected from the group consisting of a chemotherapy regimen consisting of Cyclophosphamide, Hydroxy daunorubicin, Oncovin and Prednisone (CHOP); a chemotherapy regimen consisting of Cyclophosphamide, Hydroxydaunorubicin, Oncovin, Etoposide and Prednisone (CHOEP); HD AC inhibitors; Ibrutinib, acalabrutininb, zanubrutinib, copanlisib and venetoclax.
  • any injection of a combination for use according to the invention may be followed by any usual procedure to prevent and/or avoid anaphylactic reaction.
  • a combination or composition implemented according to the invention may be performed through a large peripheral access or, if possible, through a central catheter.
  • Administration of the combination or composition for use of the invention may be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, parenterally, intranasally, intrarespiratory (such as nebulization or intra-tracheal spray), intraortically, intraocularly, rectally, vaginally, transdermally, topically (e.g., gels), intraperitoneally, intramuscularly, intrapulmonary or intrathecally.
  • a combination or composition according to the invention may in particular be in a form suitable for administration by intravenous route.
  • the cancer may in particular be selected from the group consisting of myeloma; melanoma; skin cancer; breast cancer; brain tumors, bladder cancer, cervical cancer, prostate cancer; primary and metastatic colorectal cancer, in particular a colon cancer; mesothelioma; lung cancer, in particular non small cell lung cancer; liver cancer, in particular an hepatocarcinoma or a cholangiocarcinoma; primary and metastatic pancreas cancer; renal cancer; soft tissue sarcoma; thyroid cancer; lymphoma including Hodgkin and Non-Hodgkin lymphoma, in particular B-cell or T-cell lymphoma, more particularly T-cell lymphoma; gastric cancer; head and neck cancer; ovarian cancer; sarcoma; acute or chronic leukemia in particular T-cell or myeloid leukemia; osteosarcoma; anal cancer; testicular cancer; uterus cancer; thyroid cancer; cancer of the central nervous system; gastrointestinal stromal cancer; epi
  • a cancer according to the invention is a cancer expressing at least one antigen selected from the group consisting of FASN, CD99, GPI, SLC3A2, GART, and CKAP4, in particular consisting of GPI and GART.
  • the cancer may be selected from the group consisting of myeloma; melanoma; skin cancer; breast cancer; brain tumors, bladder cancer, cervical cancer, prostate cancer; primary and metastatic colorectal cancer, in particular a colon cancer; mesothelioma; lung cancer, in particular non small cell lung cancer; liver cancer, in particular an hepatocarcinoma or a cholangiocarcinoma; primary and metastatic pancreas cancer; renal cancer; soft tissue sarcoma; thyroid cancer; lymphoma including Hodgkin and Non-Hodgkin lymphoma, in particular B-cell or T-cell lymphoma, more particularly T-cell lymphoma; gastric cancer; head and neck cancer; ovarian cancer; sarcoma; acute or chronic leukemia in particular T-cell or myeloid leukemia; osteosarcoma; anal cancer; testicular cancer; uterus cancer; thyroid cancer; cancer of the central nervous system; gastrointestinal stromal
  • a combination for use according to the invention is such that:
  • the cancer is a lymphoma.
  • the cancer is:
  • lymphoma selected from the group consisting of:
  • Non-Hodgkin lymphomas selected from the group consisting of:
  • B- B-cell lymphomas in particular selected from the group consisting of: Diffuse large B-cell lymphoma, Follicular lymphoma, Small lymphocytic lymphoma (or chronic lymphocytic leukaemia), Mantle cell lymphoma, Marginal zone B-cell lymphoma, Burkitt lymphoma, Lymphoplasmacytic lymphoma (or Waldenstrom macroglobulinaemia), Hairy cell leukaemia and Primary central nervous system lymphoma; and
  • T- T-cell lymphomas in particular selected from the group consisting of: precursor T-lymphoblastic lymphoma (or precursor T-lymphoblastic leukaemia), Peripheral T- cell lymphoma and Cutaneous T-cell lymphoma; and
  • Hodgkin lymphomas selected from the group consisting of nodular sclerosis, mixed cellularity, lymphocyte-depleted Hodgkin lymphoma and lymphocyte-rich Hodgkin lymphoma; or - a leukemia, in particular selected from the group consisting of Acute Meyloid Leukemia (AML), Chronic Meyloid Leukemia (CML) and Histiocytic Leukemia.
  • AML Acute Meyloid Leukemia
  • CML Chronic Meyloid Leukemia
  • Histiocytic Leukemia Histiocytic Leukemia
  • the cancer is selected from the group consisting of:
  • T-ALL T-cell acute lymphoblastic leukemia
  • PTCL Peripheral T-cell lymphoma
  • PTCL-NOS PTCL-NOT OTHERWISE SPECIFIED
  • EATL Enteropathy associated T- cell lymphoma
  • MEITL Monomorphic epitheliotropic intestinal T-cell lymphoma
  • ACL Anaplastic large cell lymphoma
  • AITL Angioimmunoblastic T-cell lymphoma
  • Extranodal NK/T-cell lymphoma nasal type
  • ENKL Hepatosplenic gamma delta T-cell lymphoma
  • HSGDTCL Hepatosplenic gamma delta T-cell lymphoma
  • ICL Intestinal T-cell lymphoma
  • mycosis fungoides and Sezary syndrome
  • CTCL Cutaneous T-cell lymphoma
  • T-LBL T-cell lymphoblastic lymphoma
  • the present text further provides a method for treating cancer in an individual in need thereof, comprising at least the step of administering, to said individual, a combination of antibodies, or antigen-binding fragments thereof, comprising:
  • An individual in need thereof is an individual who suffers from cancer.
  • a cancer as described above.
  • an individual in need thereof is an individual who suffers from a lymphoma, in particular who suffers from a PTCL.
  • a method of treating cancer in an individual in need thereof comprising: immunizing an animal with CD3+, CD8+, TCR gamma/delta- and CD34- human tumor T-cells; isolating a combination of antibodies, or antigen -binding fragments thereof, from the immunized animal; and administering a therapeutically effective amount of a composition comprising the isolated combination of antibodies, or antigen -binding fragments thereof, to the individual, wherein the combination of antibodies, or antigen-binding fragments thereof, comprises:
  • compositions for treatment of cancers comprising a combination of antibodies, or antigen-binding fragments thereof, said combination of antibodies, or antigen-binding fragments thereof, comprising:
  • the combination of antibodies, or antigen-binding fragments thereof and the cancer may be as defined above.
  • Example 1 In vitro cytotoxicity study of a combination of antibodies of the invention in different human cell lines (CPC assay)
  • - HUT-78 cell line is representative of cutaneous T Cell lymphoma (Sezary Syndrome).
  • PBMCs Peripheral blood mononuclear cells isolated from 3 different healthy donors were exposed to the same concentration of PAI in presence of rabbit complement (dilution 1/3 final) during 30 min at 37°C. Cell viability was done using NucleoCounter®NC- 3000TM Advanced Image Cytometer (Chemometec, Denmark).
  • results show that PAI induced a strong cytotoxicity against T-ALL cell lines of between 62% and 92%, and around 40% for Cutaneous T Cell lymphoma and PTCL NOS cell lines (see Figure 1).
  • PAI targeted and killed specifically tumoral cells and spared healthy PBMC. No cross reactivity on healthy PBMC was observed.
  • Example 2 In vitro anti-tumor activity of a combination of antibodies of the invention in PTCL-NOS human cell line resistant to CHOP (CDC assay)
  • Example 3 Comparison between the cytotoxic activity of a combination of antibodies of the invention and Alemtuzumab in different human cell lines (CDC assay)
  • Complement depend cytotoxicity (CDC) of PAI a polyclonal antibody composition of the invention, in liquid cancer cell lines was compared to the CDC of Alemtuzumab, a monoclonal antibody anti-CD52.
  • Cytotoxic activity was evaluated on three T-ALL cell lines (HPB-ALL, Jurkat and T1301) and on a cutaneous T lymphoma cell line (HUT-78).
  • the polyclonal antibody composition (PAI) and the monoclonal antibody (Alemtuzumab) were incubated for 30 minutes at 37°C at an identical concentration of 100 pg/ml on these different cell lines in the presence of rabbit complement diluted to final 1/3 (100 000 tumoral cells per well). After 30min of incubation at 37°C, cell viability was measured using NucleoCounter®NC-3000TM Advanced Image Cytometer (Chemometec, Denmark).
  • PBMCs Peripheral blood mononuclear cells isolated from 3 different healthy donors was exposed to the same concentration of PAI and Alemtuzumab in presence of rabbit complement (dilution 1/3 final) during 30 min at 37°C. Cell viability was also measured using NucleoCounter®NC-3000TM Advanced Image Cytometer (Chemometec, Denmark).
  • Alemtuzumab showed low cytotoxicity against the tumour cell lines tested, but high cytotoxicity against PBMCs from healthy donors (see Figure 3).
  • Example 4 Apoptotic activity of a combination of antibodies of the invention in different human cell lines
  • T-ALL cells lines Jurkat, T1301 and HPB-ALL
  • a PTCL-NOS cell line 300 000 cells per well
  • serial concentrations from lOpg/mL to 300pg/mL
  • T-ALL cell lines Jurkat, T1301 and HPB-ALL
  • a cutaneous T cell lymphoma cell line HUT78
  • a multiple myeloma cell line 300 000 cells per well
  • Annexin V-CF488A conjugated and Hoechst 33342 (final concentrations: 10 pg/mL) were added and incubated for 15 min at 37°C. After washes, cell pellets were suspended in 100 pl Annexin V binding buffer supplemented with lOpg/mL Propidium iodure. Apoptotic cells were analyzed using NucleoCounter®NC- 3000TM Advanced Image Cytometer (Chemometec, Denmark)
  • Example 5 Comparison between the apoptosis activity of a combination of antibodies of the invention and Alemtuzumab in different cell lines
  • PAI polyclonal antibody composition PAI in different cell lines was compared to that of Alemtuzumab (monoclonal antibody anti-CD52).
  • Apoptotic assay was evaluated on three T-ALL cell lines (HPB-ALL, Jurkat and T1301).
  • the polyclonal antibody composition PAI and the monoclonal antibody (Alemtuzumab) were incubated for 24 hours at 37°C at an identical concentration of 30pg/ml on these three different cell lines (300 000 tumoral cells per well).
  • Example 6 Antibody-dependent cellular cytotoxicity (ADCC) of a combination of antibodies of the invention
  • NK cells were isolated from PBMC of a healthy donor according to the MojoSortTM Human NK Cell Isolation Kit protocol (Biolegend, San Diego, California, United States). The day of the assay, a T-ALL cell line (HPB-ALL) was labelled with Carboxyfluorescein succimidyl ester (CFSE) (Invitrogen, Waltham, Massachusetts, United States), incubated with a polyclonal antibody composition PAI at a concentration of lOpg/mL, 30pg/mL or lOOpg/mL and cocultured with human NK (ratio E:T 4:1) in RPMI 10% FCS for 16-24h.
  • CFSE Carboxyfluorescein succimidyl ester
  • Example 7 Antibody-dependent cellular Phagocytosis (ADCP) of a combination of antibodies of the invention
  • a THP-1 cell line was activated with PMA (phorbol myristate acetate (20 ng/ml final) during 30h to be differentiated in macrophages and then labelled with a human anti-CD68 tagged with alexa fluor 647.
  • PMA phorbol myristate acetate (20 ng/ml final) during 30h to be differentiated in macrophages and then labelled with a human anti-CD68 tagged with alexa fluor 647.
  • CFSE Carboxyfluorescein succimidyl ester
  • Example 8 Apoptotic pathways engaged by exposure to a combination of antibodies of the invention
  • caspases 8 and 9 were activated, suggesting an activation both the extrinsic and the intrinsic pathways by PAI (see Figure 8).
  • Example 9 In vivo anti-tumor activity of a combination of antibodies of the invention on T1301 xenograft
  • a xenograft in vivo mouse model of T-ALL is obtained by the subcutaneous injection of 3 000 000 T1301 cell line at day 0.
  • Treatment was initiated from the beginning of tumour growth for a total duration of 28 days.
  • Treatment consisted in the intraperitoneal injection of PAI at 35 mg/kg twice a week.
  • PAI showed to be effective against tumor growth in vivo.
  • D35 a 50% reduction in tumor size is observed in the group treated with PAI compared to vehicle (see Figure 10).
  • Example 10 In vivo anti-tumor activity of a combination of antibodies of the invention on
  • a xenograft in vivo mouse model of T-ALL was obtained by the subcutaneous injection of 3 000 000 Jurkat cell line at day 0 in 50% Matrigel.
  • PAI recognized 84.6% of NK/T PTCL biopsies, 81.8% of AITL biopsies; 93.3% of EATL biopsies, 73.9% of NOS biopsies and 83.3% of ALCL biopsies, as shown in table 5 below:
  • PAI induced a strong cytotoxicity activity against human solid tumor cell lines (ranging from 45,2% to 90,7%) (see Figure 13).
  • the solid cancer cell line tested are as follows:
  • Example 14 Global cytotoxicity of a combination of antibodies of the invention in different human solid tumor cell lines after 24 hours incubation
  • the solid cancer cell line tested are as follows:
  • PAI induces a strong cytotoxicity against the human solid tumor cell lines (ranging from about 50 % to 95 %) (see Figure 14).
  • Example 15 Targets of a combination of antibodies of the invention
  • HuProtTM array (CDI Labs, USA) was used for the assay of the sample : a polyclonal antibody composition of the invention named PAI. After blocking, the array was probed with PAI (Ipg/ml) at room temperature for 1 hour. Then the array was washed three times with TBST for 10 min and probed with Alexa647-anti- swine IgG secondary antibody under conditions optimized by CDI Labs for signal detection.
  • PAI polyclonal antibody composition of the invention
  • Z score is the average Z score of the duplicate spots of a given protein (each protein is printed in duplicate on a HuProtTM array).
  • L635(avg) and L635(std) are the average and standard deviation of the L635 values of all spots on the array, respectively.
  • Example 16 In vitro cytotoxicity assay
  • liquid cancer cell lines tested are as follows:
  • CellTiter-Glo® Cell Viability Assay Viability of cells was quantified by the CellTiter-Glo® One Solution cell viability assay (Promega G8462). After incubation of the cells, the CellTiter-Glo® One Solution Assay reagent was brought to ambient temperature. Next, 100 pl of CellTiter-Glo® One Solution Assay reagent were added to each well. Plates were shaken for 2 minutes to induce cell lysis and incubated for 20 minutes prior to reading luminescence (LU) by using the GloMax plate reader (Promega).
  • LU luminescence
  • Example 17 In vivo efficacy in a rat T cell lymphoma/leukemia model vs CHOP
  • SRG immunodeficient rats were injected with 15xl0 6 T1301 cells in 50% matrigel (final volume 500 pl) subcutaneously in the left flank. Tumor growth was monitored twice weekly by caliper measurement. Treatment started as soon as the tumor reached 500-1000 mm3 and consisted of one cycle of CHOP chemotherapy or PAI treatment twice a week, according to the protocol described above.
  • Group 1 control group without treatment (Control group),
  • Group 2 PAI treatment twice a week: 40 mg/kg (PAI group), and
  • CHOP treatment (1 cycle): 1 cycle at DO Cyclophosphamide 37.5 mg/kg, Doxorubicin 2.5 mg/kg, and vincristine 0.07 mg/kg, and at DO, DI, D2, D3, D4, D5 prednisolone 1.47 mg/kg (CHOP group).
  • PAI showed to be highly effective and well tolerated in SRG T1301 xenograft whereas only a modest and no persistent effect was observed with the standard of care (CHOP) treatment (see Figures 16 and 17).

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Abstract

La présente invention concerne une association d'anticorps ou de fragments de liaison à l'antigène associés destinée à être utilisée dans le traitement d'un cancer chez un patient humain en ayant besoin, l'association comprenant au moins un premier anticorps ou un fragment de liaison à l'antigène associé se liant spécifiquement à la synthase d'acides gras, au moins un deuxième anticorps ou un fragment de liaison à l'antigène associé se liant spécifiquement à CD99, ainsi qu'au moins un troisième anticorps ou un fragment de liaison à l'antigène associé se liant spécifiquement à un antigène choisi dans le groupe constitué par GPI, SLC3A2, GART et CKAP4 en particulier constitué de GPI et de GART. La présente invention concerne en outre une composition pharmaceutique destinée à être utilisée dans le traitement d'un cancer chez un patient humain en ayant besoin comprenant cette association d'anticorps ou de fragments de liaison à l'antigène de ceux-ci et un excipient pharmaceutiquement acceptable.
PCT/EP2024/077883 2023-10-04 2024-10-03 Association d'anticorps ou de fragments de liaison à l'antigène associés pour le traitement de cancers Pending WO2025073856A1 (fr)

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