WO2025073856A1

WO2025073856A1 - Combination of antibodies, or antigen-binding fragments thereof, for the treatment of cancers

Info

Publication number: WO2025073856A1
Application number: PCT/EP2024/077883
Authority: WO
Inventors: Firas BASSISSI; Carine CIRON; Odile DUVAUX
Original assignee: Xenothera
Current assignee: Xenothera
Priority date: 2023-10-04
Filing date: 2024-10-03
Publication date: 2025-04-10
Anticipated expiration: 2026-04-04

Abstract

The present invention relates to a combination of antibodies, or antigen-binding fragments thereof, for use in the treatment of a cancer in a human subject in need thereof, the combination comprising at least a first antibody, or an antigen-binding fragment thereof, specifically binding to FASN, at least a second antibody, or an antigen-binding fragment thereof, specifically binding to CD99, and at least a third 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. The present invention further relates to a pharmaceutical composition for use in the treatment of a cancer in a human subject in need thereof comprising this combination of antibodies, or antigen-binding fragments thereof and a pharmaceutically acceptable carrier.

Description

COMBINATION OF ANTIBODIES. OR ANTIGEN-BINDING FRAGMENTS THEREOF. FOR THE TREATMENT OF CANCERS

Field of the Invention

The present invention relates to the field of treatment of cancers. In particular, 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.

Description of Related Art

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.

Among cancers, PTCL, or Peripheral T-cell Lymphoma, constitutes a diverse group of lymphoproliferative tumors that originate from mature post-thymic T cells. This group exhibits considerable heterogeneity, with the majority of cases displaying aggressive invasiveness. PTCL can be categorized into four main groups, encompassing over 30 subtypes which include 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.

Geographically, there are variations in the incidence and distribution of PTCL subtypes. Asia has a slightly higher incidence rate compared to Europe and the United States. Notably, in China, PTCL accounts for approximately 25% to 35% of non-Hodgkin's lymphoma (NHL), which is significantly higher than the 10% to 15% observed in European and American countries.

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. In addition to these immunomodulating monoclonal antibodies (mAb), antibody drug conjugate (ADC), chimeric antigen receptor T cells (CAR-T) and more recently bispecific antibodies are being actively explored and successfully introduced as innovative weapon in cancer treatment arsenal. However, for many cancers, there is still a lack of effective treatments, especially treatments that can result in long-term cancer- free survival and lower metastatic and relapse risks.

Antibodies directed against tumors (passive immunotherapy) can act through two complementary ways:

- by cytotoxicity complement dependent (CDC) or dependent from killer cells (ADCC) or dependent from phagocytes (ADCP);

- by induction of an adaptative immune response. It has indeed been demonstrated that opsonization of a target and the local production of complement’s molecules (C3a, C5a) (see Strainic et al., Immunity, 2008 Mar;28(3):425-35) activates the T lymphocyte costimulation and increases its survival. Thus, the passive administration of antitumor antibodies can, as a first step, generate the discharge of factors from the complement that, in a second step, ease the T cells response.

Concerning more particularly PTCL, there are currently several 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).

However, 40% to 85% of the patients are resistant to treatments based on the use of monoclonal antibodies. 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). 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).

More particularly, the outlook for PTCL patients is not promising. The overall response rate to initial treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP-based regimens is relatively low, and PTCL tends to relapse, resulting in a poor prognosis. Despite the recent major advances in the understanding of PTCLs, including new laboratory methods for diagnosis and new therapeutic approaches, the prognosis of the majority of PTCLs remains poorer than with aggressive B-cell lymphoma. With the exception of ALK+ systemic anaplastic large T-cell lymphoma (ALCL), other PTCL subtypes have a 5- year survival rate ranging from 14% to 32% (Vose J et al. International peripheral T-cell and natural killer/T-cell lymphoma study: pathology findings and clinical outcomes. J Clin Oncol. 2008 Sep 1; 26(25): 4124-4130).

Accordingly, a treatment based on the use of combinations of antibodies targeting different antigens on the tumoral cells might allow minimizing the escaping mechanisms.

In particular, the polyclonality and polytargeting of a combination of antibodies can prevent the pitfalls encountered by various monoisotopic or mono target approaches of monoclonal antibodies.

However, despite their potential efficacy as observed by Richet more than 100 years ago, combinations of antibodies, such as polyclonal antibodies of animal origin, have rarely been used in the treatment of tumors due in particular to high toxic risks in patients. This toxicity is mainly linked to the expression of Neu5GC and alpha- 1,3-galactose carbohydrates on animal immunoglobulins which in human elicit potent anti-Neu5GC and anti-alpha- 1,3-galactose immune responses associated with allergy, serum sickness disease and formation of immune complexes. In order to mimic the polyclonal humoral immune response for cancer treatment, combinations of monoclonal have been considered. However, their clinical efficacy as monotherapy does not predicate their safety and clinical efficacy in combination (Berlin et al., Investigational News Drugs (2022) 40:586-595). The difficulty of such combination mainly lies in the association of antibodies with different PKs (which makes it more difficult to control toxicity), the stability of the product according to the isotypes and the manufacturing (see for example Larbouret et al., Cancers (Basel). 2021 Sep 15;13(18):4620).

There accordingly remains a need in the field for novel anti-cancer therapeutic agents, and in particular novel therapeutic agents efficient against a great variety of cancers.

There accordingly remains a need in the field for novel anti-cancer therapeutic agents, and in particular novel therapeutic agents efficient against a plurality of PTCL subtypes.

There also remains a need in the art for the provision of an effective and improved therapy for treating cancers and endowed with reduced or no adverse effects, and in particular with very low interaction with PBMC.

There also remains a need in the art for the provision of novel anti-cancer therapeutic agents having an anti-cancer activity against both solid and liquid cancers.

There also remains a need in the art for the provision of novel anti-cancer therapeutic agents efficient against PTCL subtypes resistant to the standard of care CHOP or CHOEP (CHOP + etoposide). There also remains a need in the art for the provision of novel anti-cancer therapeutic agents able to activate complement and to activate apoptotic mechanisms to kill a great variety of cancers, and in particular to activate both the intrinsic and extrinsic apoptotic pathways, and in particular through the induction of both caspase 8 and caspase 9.

There also remains a need in the art for the provision of novel anti-cancer therapeutic agents able to provide an antitumoral activity selected from the group consisting of comprises cytotoxicity complement dependent (CDC), cytotoxicity complement dependent from killer cells (ADCC), apoptotic activity and/or cytotoxicity complement dependent from phagocytes (ADCP), and in particular an antitumoral activity consisting of CDC, ADCC, apoptotic activity and ADCP.

The invention has for purpose to meet the above-mentioned needs.

Summary of the invention

The present invention in particular relates to the following items:

Item 1: A combination of antibodies, or antigen-binding fragments thereof, for use in the treatment of a cancer in a human subject in need thereof, the combination comprising:

- at least a first antibody, or an antigen-binding fragment thereof, specifically binding to FASN (FasN fragment),

- at least a second antibody, or an antigen-binding fragment thereof, specifically binding to CD99 (Cluster of differentiation 99), and

- at least a third 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.

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:

- at least a first antibody, or an antigen-binding fragment thereof, specifically binding to FASN, - at least a second antibody, or an antigen-binding fragment thereof, specifically binding to CD99,

- at least a third antibody, or an antigen-binding fragment thereof, specifically binding to GPI, and

- at least a fourth antibody or an antigen-binding fragment thereof, specifically binding to GART.

Item 4: The combination for use according to any one of items 1 to 3, wherein the antigen-binding fragments of the antibodies present in the combination are independently Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, or diabodies.

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; cancer of the central nervous system; gastrointestinal stromal cancer; epidermal carcinoma; and oesophageal cancer.

Item 6: The combination for use according to any one of items 1 to 5, wherein the cancer is:

- a lymphoma, in particular selected from the group consisting of:

(i) Non-Hodgkin lymphomas selected from the group consisting of:

- 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-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.

Item 7; The combination for use according to any one of items 1 to 6, wherein the cancer is selected from the group consisting of:

- T-cell acute lymphoblastic leukemia (T-ALL);

- Peripheral T-cell lymphoma (PTCL), in particular PTCLs selected from the group consisting of PTCL-NOT OTHERWISE SPECIFIED (PTCL-NOS), Enteropathy associated T- cell lymphoma (EATL), Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), Anaplastic large cell lymphoma (ALCL), and Angioimmunoblastic T-cell lymphoma (AITL), Extranodal NK/T-cell lymphoma, nasal type (ENKL), Hepatosplenic gamma delta T-cell lymphoma (HSGDTCL), Intestinal T-cell lymphoma (ITCL), mycosis fungoides, and Sezary syndrome;

- Cutaneous T-cell lymphoma (CTCL); and

- T-cell lymphoblastic lymphoma (T-LBL).

Item 8 : The combination for use according to any one of items 1 to 7, wherein the cancer expresses at least one antigen selected from the group consisting of FASN, CD99, GPI, SLC3A2, GART, and CKAP4, in particular consisting of GPI and GART.

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.

Item 10; The combination for use according to any one of items 1 to 9, wherein the combination is a combination of monoclonal antibodies or a polyclonal antibody composition.

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 12; The combination for use according to item 11, wherein the animal is a pig, and in particular is 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)-galactosyltransfcrasc

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 15; The combination for its use according to items 14, wherein the at least one additional anticancer drug is 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 monoclonal antibodies.

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.

Item 17; The combination for use according to any one of items 1 to 16, for use in providing an antitumoral activity selected from the group consisting of comprises cytotoxicity complement dependent (CDC), cytotoxicity complement dependent from killer cells (ADCC), apoptotic activity and/or cytotoxicity complement dependent from phagocytes (ADCP), and in particular an antitumoral activity consisting of cytotoxicity complement dependent (CDC), cytotoxicity complement dependent from killer cells (ADCC), apoptotic activity and cytotoxicity complement dependent from phagocytes (ADCP).

Brief description of the figures 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 2: describes the anti-tumor activity of a combination of antibodies on a CHOP resistant PTCL-NOS cell line. The figure represents the % of cell death at different PAI concentrations (in pg/mL).

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 4: describes the apoptotic activity of a combination of antibodies on different cell lines. The figure represents the % of apoptosis at different PAI concentrations (in pg/mL) for each of the following cell lines: PBMC (• curve), Jurkat (■ curve), T1301 (A curve), HPB-ALL (▼ curve) and OCI-LY-12 (♦ curve).

Figure 5: describes the apoptotic activity of a combination of antibodies and of Alemtuzumab on different cell lines. The figure represents de % of apoptotic activity of PAI (at a concentration of 30 pg/mL) (left column) and of Alemtuzumab (at a concentration of 30 pg/mL) (right column) for each of the following cell lines, from left to right: HPB-ALL, T1301 and Jurkat.

Figure 6: describes ADCC activity of a combination of antibodies on a HPB-ALL cell line. The figure represents the % cell death at a concentration of PAI of lOpg/mL (column on the left), of 30pg/mL (column in the middle), or of lOOpg/mL (column on the right).

Figure 7: describes ADCP activity of a combination of antibodies on a HPB-ALL cell line. The figure represents the % cell death at a concentration of PAI of lOpg/mL (column on the left), of 30pg/mL (column in the middle), or of lOOpg/mL (column on the right).

Figure 8: describes the activation of caspase 8 and caspase 9 by a combination of antibodies on different cell lines. The figure represents the % of positive cells for caspase 8 (first two columns on the left of each graph) or for caspase 9 (last two columns on the right of each graph) in each of the cell lines in the absence (CT column) or in the presence of PAI (PAI column). Paired T test, *: p<0.05; **: p<0.01; ***: p<0.001. Figure 9: describes the evolution of the tumor size of T1301 xenograft mouse models in the presence of a combination of antibodies. The figure represents the mean tumor size (in mm3) over time (in days) in the presence of PAI (■ curve) or in the absence of treatment (• curve). N=10 per group. * p<0.05 - Mann-Whitney test.

Figure 10: describes the size of the tumor of T1301 xenograft mouse models after 35 days of treatment with a combination of antibodies. The figure represents the mean tumor size (in mm3) at Day 35 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 11: describes the evolution of the tumor size of Jurkat xenograft mouse models in the presence of a combination of antibodies. The figure represents the mean tumor size (in mm3) over time (in days) in the presence of PAI (A curve) or in the absence of treatment (■ curve). N=10 per group. * p<0.05 - Mann-Whitney test.

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 13: describes the CDC of a combination of antibodies on solid cancers. The figure represents the % of cytotoxicity at different PAI concentrations (in pg/mL) for each of the following cell lines: A549 (•), HepG2 (■), HCT116 (A), MDA-MB231 (▼), LNCAP (♦) and SKMEL30 (o).

Figure 14: describes the global cytotoxicity of a combination of antibodies on solid cancers. The figure represents the % of cytotoxicity at different PAI concentrations (in pg/mE) for each of the following cell lines: A549 (•), HCT116 (A), LNCAP (▼) and SKMEL30 (♦).

Figure 15: describes the biding of a combination of antibodies to different protein targets. The figure represents the Z score of PAI for the following targets, from left to right: FasN, GPI, CD99, GART, SLC3A2 and CKAP4.

Figure 16: describes the variation of the tumor size (in mm³) 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 mm³) 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 (♦).

Detailed description of the invention

1. Definitions

Several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2^nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3^rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, may provide one of skill with a general dictionary of many of the terms used in this disclosure. In case of conflict, the present specification, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Units, prefixes, and symbols are denoted in their Systeme International des Unites (SI) accepted form. The headings provided herein are not limitations of the various aspects of the disclosure.

All publications and other references mentioned herein are incorporated by reference in their entirety.

It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody ” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more ” and “at least one” can be used interchangeably herein.

Throughout this specification and embodiments, 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. The term “consisting essentially of’ implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the basic characteristic(s) of the disclosure. It is understood that the different embodiments of the disclosure using the term “comprising'” or equivalent cover the embodiments where this term is replaced with “comprising only”, “consisting of’ or “consisting essentially o ”.

It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided.

Furthermore, “and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

“Pharmaceutically” or “pharmaceutically acceptable” refers 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.

As used herein, “pharmaceutically acceptable carriers” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of 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. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition, and formulation may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20. Suitable excipients, as well as pharmaceutical formulation requirements, are described in "Remington: The Science & Practice of Pharmacy", which is a reference work in the field.

As used herein the term "antibody" have the same meaning and will be used equally in the present description. The term "antibody" as used herein refers to isolated or recombinant immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that immunospecifically binds an antigen, such as proteins FASN, CD99, SLC3A2, GART or CKAP4. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulphide bonds and each heavy chain is linked to a light chain by a disulphide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Ec receptors (EcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, L- CDR3-L and CDR1-H CDR2-H, CDR3-H, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. The term “antibody” includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, camelid antibodies and chimeric antibodies. The antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).

In the context of the present disclosure, the term “antibody” specifically includes an antibody binding a FASN protein (also termed an anti-FASN antibody), and/or an antibody binding to a CD99 protein (also termed an anti-CD99 antibody), and/or an antibody binding to a GPI protein (also termed an anti-GPI antibody), and/or an antibody binding a SEC3A2 protein (also termed an anti-SEC3A2 antibody), and/or an antibody binding to a GART protein (also termed an anti-GART antibody), and/or an antibody binding a CKAP4 protein (also termed an anti-CKAP4 antibody).

It is intended that all references to the term “antibody” or “antigen-binding fragment thereof ’ as used herein, referred to the antibodies or the antigen-binding fragments thereof as described in the present disclosure.

“Fragments” of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody. Examples of 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.

The term “Fab” denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of 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.

The term “F(ab’)2” refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than 2 identical Fab fragments bound via a disulfide bond of the hinge region. It is usually obtained among fragments by treating IgG with a protease, pepsin.

The term “Fab’“ refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab’)2.

A single chain Fv (“ cFv”) polypeptide is a covalently linked VH::VE heterodimer which is usually expressed from a gene fusion including VH and VE encoding genes linked by a peptide-encoding linker. The human scFv fragment of the disclosure includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. “dsFv” is a VH::VL heterodimer stabilized by a disulphide bond. “(dsFv)2” denotes two dsFv coupled by a peptide linker.

The term “bispecific antibody” or “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.

The term “multispecific antibody” denotes an antibody which combines the antigenbinding sites of two or more antibodies within a single molecule.

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

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)).

A “combination of antibodies, or antigen-binding fragments thereof' may in particular be a polyclonal antibody composition or a combination of monoclonal antibodies, and may preferably be a polyclonal antibody composition.

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. Polyclonal rabbit antithymocyte globulins (ATGs) are FDA and EMA approved drugs, commonly used in organ transplantation as induction (Lacorcia et al., Transplantation, Apr 15;87(7):966-74, 2009). New generation of humanized polyclonal antibodies were also tested in humans for different indication and showed satisfactory tolerability (Poulakou et al., Front. Immunol. 2024 Apr 17:15:1330178).

The term “monoclonal antibody" or “mAb” as used herein 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.

The term “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.

The term “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.

In the context of the present invention, 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. By 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. In particular, the animal is a pig, and in particular is 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-(l,3)- galactosyltransferase.

The term “antigen” as used in the present disclosure refers to a molecule or a portion of a molecule capable of being bound by one or more antibodies. An antigen can have one or more than one epitope. For instance, in the context of the disclosure, an antigen is in particular selected from the group consisting of proteins FASN, CD99, GPI, SLC3A2, GART and CKAP4.

The term “affinity”, as used herein, 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. The affinity constant Ka is defined by 1/Kd. Methods for determining the affinity of Abs can be found in, for example, Harlow, et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988, Coligan et al., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), or Muller, Methods Enzymol. 1983;92:589-601, which references are entirely incorporated herein by reference. A preferred and standard method well known in the art for determining the affinity of mAbs is the measurement of surface plasmon resonance) by using the Biacore instruments (Laure et al. Curr Protoc Protein Sci. 2006 Sep;Chapter 19:Unit 19.13). For multimeric antigens, such as virus capsids, to which both antigen binding sites of an antibody can bind simultaneously, Ka and KD values determined by such standard methods measure the functional affinity, or avidity of the interaction. Illustratively, an antibody is considered to bind an antigen when a functional binding affinity (KD), preferably measured by surface plasmon resonance, is of 10-8 mol/1 (M) or less, preferably 10-9 M to 10-12 M.

By “IgG” as used herein is meant a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, IgG comprises the subclasses or isotypes IgGl, IgG2, IgG3, and IgG4. In mice, IgG comprises IgGl, IgG2a, IgG2b, IgG3. In pigs, 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).

By “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. By extension, the term “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. Illustratively, the antigenic molecule N-glycolylneuraminic acid (Neu5Gc) 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.

By “conventional polyclonal antibodies” , as used herein is meant polyclonal antibodies, and in particular pig or rabbit polyclonal antibodies, that are not devoid of the antigenic determinants N-glycolylneuraminic acid (Neu5Gc) and a-l,3-galactose. In this regard, it may be notably cited the products commercialized under the name Thymoglobulin, Grafalon, Atgam or p-ALG®.

The term “cancer” is herein used in its traditional sense, and refers to a cell or group of cells displaying uncontrolled growth, invasion upon adjacent tissues.

Examples of cancers more particularly considered in the present text are provided further. 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 cancer; thyroid cancer; cancer of the central nervous system; gastrointestinal stromal cancer; epidermal carcinoma; and oesophageal cancer. In a particular embodiment, 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.

In a particular embodiment, 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 cancer; epidermal carcinoma; and oesophageal cancer that express at least one antigen selected from the group consisting of FASN, CD99, GPI, SLC3A2, GART, and CKAP4, in particular consisting of GPI and GART.

By a “therapeutically effective amount” of a combination of antibodies used according to the invention is meant a sufficient amount of the antibodies to obtain the desired effect as presented in the present text, and in particular for treating a human subject affected with a cancer. It will be understood, however, that the total daily usage of the combination of antibodies implemented according to the invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the nature of the cancer being treated; the activity of the specific antibodies employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific antibodies employed; the duration of the treatment; drugs used in combination or coincidental with the specific antibodies employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The terms “treatment", “treating” or “therapy” 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. In particular, in the case of the present invention, treating a cancer includes providing an antitumoral activity. In particular, the antitumoral activity comprises cytotoxicity complement dependent (CDC), cytotoxicity complement dependent from killer cells (ADCC), apoptotic activity and/or cytotoxicity complement dependent from phagocytes (ADCP), more particularly an antitumoral activity consisting of cytotoxicity complement dependent (CDC), cytotoxicity complement dependent from killer cells (ADCC), apoptotic activity and cytotoxicity complement dependent from phagocytes (ADCP).

The terms “wild type animal” or “WT animal” come herein in opposition with a genetically altered animal. For example, by “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.

As an appropriate method for obtaining 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. In this regard, may be more particularly cited the protocol implemented in example 3 of EP 0 335 804, which implements an ion exchange chromatography on DEAE cellulose.

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. Accordingly, by “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).

The terms “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.

2. Antibody combinations implemented according to the invention

As mentioned above, 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:

Each one of the antibodies present in a combination for use according to the invention may, independently from the others, be a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof. As defined above, said antigen-binding fragment may independently be selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, or diabodies.

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.

In particular, 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.

The antibodies from the antibody combination implemented according to the invention may be immunoglobulin G antibodies.

Porcine IgG structure and genetics have for example been described in literature (Butler et al. 2009 - DOI: 10.1007/s00251-009-0356-0). Eleven isotypes called IgGla, IgGlb, IgG2a, IgG2b, IgG3, IgG4a, IgG4b, IgG5a, IgG5b, IgG6a, IgG6b have been proposed based on analysis of the genomic IgH locus sequences. According to the available knowledge on relative abundance of pig IgG subclasses and Fc domains affinity for Protein A (Butler et al. 2009 - DOI: 10.1007/s00251-009-0356-0), during a conventional purification, 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.

The amino acid sequence of FASN has for reference UNIPROT A0A0U1RQF0. Antibodies able to specifically target FASN (anti-FASN antibodies) are well known in the art. Can for example be mentioned the monoclonal antibody 15973814 (commercialized by Fischer Scientific), the polyclonal antibody LS-B3636 (commercialized by LifeSpan BioSciences) or the polyclonal antibody GTX109833 (commercialized by GeneTex).

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.

The amino acid sequence of CD99 has for reference: UniProt P14209.

Antibodies able to specifically target CD99 (anti-CD99 antibodies) are well known in the art. Mention can for example be made of the monoclonal antibody AA 23-122. Application WO2015161267 may also be referred to concerning such antibodies.

Several studies have demonstrated, with varying degrees of success, the potential of targeting CD99 in T-cell acute lymphoblastic leukemia owing to its high overexpression in T-ALL compared to normal T cells (Ebadi et al. Scientific Reports 2021, 11:24374; Kotemul et al. Exploration of Targeted Anti-tumor Therapy 2024, 5:96-107). However, targeting CD99 with a monoclonal antibody appears rather challenging, particularly because of the existence of different isoforms. Co-expression of CD99LF and CD99SF induces apoptosis, whereas CD99LF alone induces cell aggregation without apoptosis (Alberti et al. FASEB 2002, Dec; 16(14): 1946-8). In addition, numerous epitopes essential for apoptosis induction have been identified, with apoptosis occurring through either the Fas-dependent or-independent pathway, underscoring the intricate nature of CD99-mediated cell death mechanisms (Pettersen et al. J Immunol 2001, 166 (8): 4931-4942). Furthermore, effective apoptosis induction seems contingent upon CD99 clustering, mandating an antibody valence of three or more for optimal efficacy (Romero et al. J of Molecular Biology 2022, 167402)

Protein GPI (Glucose-6-phosphate isomerase) has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions such as as a glycolytic enzyme (glucose-6-phosphate isomerase) that interconverts glucose-6- phosphate (G6P) and fructose-6-phosphate (F6P), as a neurotrophic factor that promotes survival of skeletal motor neurons and sensory neurons, and as a lymphokine that induces immunoglobulin secretion.

The amino acid sequence of GPI has for reference UniProt P06744.

Antibodies able to specifically target GPI (anti-GPI antibodies) 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.

Protein SLC3A2 belongs to the SLC (solute carriers) carrier proteins consisting of genes that form a 65-membered superfamily. They are mainly involved in molecule/drug delivery and are a frequently referenced research resource in drug resistance studies. A subclassification is made into SLC families according to what type of molecule delivery they are responsible for, and these families include: SLC1 family, SLC2 family.... SLC65 family. In addition, every family has sub-members. For example, the SLC3 family has 2 members: Such as, SLC3al and SLC3a2. In general, in the operating mechanism, they act together with members of the SLC7 family, so most studies usually analyze both together. On the other hand, 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.

The amino acid sequence of SLC3A2 has for reference GenBank : KAI2560544.1.

Antibodies able to specifically target SLC3A2 (anti-SLC3A2 antibodies) 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.

Protein GART (glycinamide ribonucleotide formyltransferase) catalyzes the N- formylation of glycinamide ribonucleotide. GART is an essential step in the synthesis of purine nucleotides, and a target for blocking the proliferation of malignant cells.

The amino acid sequence of GART has for reference UniProt Q71VH3.

Antibodies able to specifically target GART (anti-GART antibodies) are well known in the art. Reference can be made to Joe Dotzlaf et al. (Hybridoma (Larchmt). 2006; 25:139-44).

Protein CKAP4 (cytoskeleton-associated protein 4) is a Dickkopfl (DKK1) receptor, DKK1 being a secretory protein that antagonizes oncogenic Wnt signaling by binding to the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) and may may also regulate its own signaling to promote cancer cell proliferation.

The amino acid sequence of CKAP4 has for reference NCBI Reference Sequence :

NP-006816.2. Antibodies able to specifically target CKAP4 (anti-CKAP4 antibodies) 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 for use according to the invention may comprise 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. Said antigen targeted by the said at least fourth antibody may in particular be different from the three antigens to which the first, second and third antibodies, or antigen-binding fragments thereof, bind to.

Accordingly, a combination of antibodies, or antigen-binding fragments thereof, for use according to the invention may comprise:

- at least a second antibody, or an antigen-binding fragment thereof, specifically binding to CD99 (Cluster of differentiation 99),

- at least a third 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, and

- at least 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.

In a particular embodiment, 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,

- at least a second antibody, or an antigen-binding fragment thereof, specifically binding to CD99,

An example of such a combination is illustrated in the examples of the present specification under the denomination PAI.

As indicated previously, in all of these embodiments, the antibodies of the combinations, in particular the polyclonal 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.

In a particular embodiment, in all of the above-mentioned embodiments, the antibodies of the combinations, in particular the polyclonal antibodies, 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, and in particular polyclonal antibodies for use according to the invention, may be of any origin, such as from a non-human mammal or synthetic, monoclonal or polyclonal. Such 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, and in particular polyclonal antibodies 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.

In particular, 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.

In a particular embodiment, the combination for use according to the present invention is included in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are defined further above.

The pharmaceutical composition may be in liquid form.

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 terms “combination of antibodies” are also used herein to designate “a combination of antibodies, or antigen-binding fragments thereof'.

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 monoclonal antibodies.

The pharmaceutical composition may further comprise at least one chemotherapy treatment.

Chemotherapy treatments may be selected among those known in the art. In particular, one skilled in the art will know to adapt the choice of the chemotherapy according to the cancer that is to be treated. In a particular embodiment, 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.

Dosages may range from 0.001 to 100 mg or more of the combination for use according to the invention as defined herein per kg of body weight (mg/kg) or greater, for example 0.1, 1.0, 10, or 50 mg/kg of body weight, with 1 to 20 mg/kg being preferred. The dosage and frequency of administration may be adapted as previously detailed.

Also, 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.

Besides, the injection of a combination or composition implemented according to the invention may be performed through a large peripheral access or, if possible, through a central catheter.

As is known in the art, adjustments for protein degradation, systemic versus localized delivery, as well as the age, body weight, general health, sex, diet, time of administration, possible allergy, drug interaction and the severity of the condition may be necessary, and is easily determined with routine experimentation by those skilled in the art.

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.

Administration of the combination or composition of the invention may be done by following the Besredka method.

A combination or composition according to the invention may in particular be in a form suitable for administration by intravenous route.

3. Implementation of a combination of antibodies, or antigen-binding fragments thereof, or a composition, according to the invention

A combination of antibodies, or antigen-binding fragments thereof, or a composition comprising it, implemented according to the invention is used in a therapeutically effective amount. As previously indicated, a combination of antibodies or composition thereof is for use in the treatment of a cancer in a human subject.

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; epidermal carcinoma; and oesophageal cancer.

In a particular embodiment, 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.

In a particular embodiment, a combination for use according to the invention is such that:

- at least a first antibody, or an antigen-binding fragment thereof, specifically binding to FASN, - at least a second antibody, or an antigen-binding fragment thereof, specifically binding to CD99, and

- at least a third 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; with the cancer being 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; epidermal carcinoma; and oesophageal cancer.

In a particular embodiment, the cancer is a lymphoma.

In particular, the cancer is:

- a lymphoma selected from the group consisting of:

(i) Non-Hodgkin lymphomas selected from the group consisting of:

- 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.

In a particular embodiment, the cancer is selected from the group consisting of:

- T-cell acute lymphoblastic leukemia (T-ALL);

- Peripheral T-cell lymphoma (PTCL), in particular PTCLs selected from the group consisting of PTCL-NOT OTHERWISE SPECIFIED (PTCL-NOS), Enteropathy associated T- cell lymphoma (EATL), Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), Anaplastic large cell lymphoma (ALCL), and Angioimmunoblastic T-cell lymphoma (AITL), Extranodal NK/T-cell lymphoma, nasal type (ENKL), Hepatosplenic gamma delta T-cell lymphoma (HSGDTCL), Intestinal T-cell lymphoma (ITCL), mycosis fungoides, and Sezary syndrome; and;

- Cutaneous T-cell lymphoma (CTCL); and

- T-cell lymphoblastic lymphoma (T-LBL).

It further provides the use of a combination of antibodies, or antigen-binding fragments thereof, prepared by immunizing an animal with CD3+, CD8+, TCR gamma/delta- and CD34- human tumor T-cells, in the manufacture of a medicament.

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:

- at least a first antibody, or an antigen-binding fragment thereof, specifically binding to FASN,

- at least a second antibody, or an antigen-binding fragment thereof, specifically binding to CD99, and

- at least a third 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.

An individual in need thereof is an individual who suffers from cancer. In particular a cancer as described above. In a particular embodiment, an individual in need thereof is an individual who suffers from a lymphoma, in particular who suffers from a PTCL. It is further described 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:

It is further described a method of preparation of a combination of antibodies, or antigen -binding fragments thereof, comprising at least the steps of: immunizing an animal with CD3+, CD8+, TCR gamma/delta- and CD34- human tumor T-cells; and isolating a combination of antibodies, or antigen -binding fragments thereof, from the immunized animal.

The present text also describes a pharmaceutical composition for treatment of cancers, comprising a combination of antibodies, or antigen-binding fragments thereof, said combination of antibodies, or antigen-binding fragments thereof, comprising:

- at least a second antibody, or an antigen-binding fragment thereof, specifically binding to CD99, and - at least a third 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.

The combination of antibodies, or antigen-binding fragments thereof and the cancer may be as defined above.

The present disclosure is further illustrated, without in any way being limited to, by the Examples hereafter.

EXAMPLES

Example 1: In vitro cytotoxicity study of a combination of antibodies of the invention in different human cell lines (CPC assay)

Different hematological cancer cell lines were exposed to a combination of antibodies of the invention, a polyclonal antibody composition named “PAI” at a concentration of 100 g/mL in presence of rabbit complement (dilution 1/3 final) (n=3).

The liquid cancer cell lines tested were as follows:

- HPB-ALL, Jurkat and T1301 cell lines are representative of T-ALL;

- HUT-78 cell line is representative of cutaneous T Cell lymphoma (Sezary Syndrome); and

- OCI-LY-12 cell line is representative of PTCL-NOS and described as resistant to CHOP (Magni et al. 2019).

After 30min of incubation at 37°C, cell viability was determined using NucleoCounter®NC-3000™ Advanced Image Cytometer (Chemometec, Denmark).

Peripheral blood mononuclear cells (PBMCs) 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- 3000™ Advanced Image Cytometer (Chemometec, Denmark).

Results: The 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).

In particular, it was shown that 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)

Serial dilutions of polyclonal antibody composition PAI (from 10 pg/ml to 300 pg/ml) were incubated for 24 hours at 37°C with OCI-LY-12 cells representative of PTCL- NOS cancers in the presence of rabbit complement (final dilution 1/3). The 24h incubation with rabbit complement was used to assess the overall anti-tumor activity of the PAI polyclonal antibody composition (Complement-dependent cytotoxicity (CDC) and apoptosis). It should be noted that the PTCL-NOS cell line is resistant to the standard CHOP treatment authorised for the treatment of PTCL-NOS cancers (Sibon et al, 2022; Deng et al. 2019, Ruan et al. 2023).

After 24 hours of incubation at 37 °C, cell viability was determined using NucleoCounter®NC-3000™ Advanced Image Cytometer (Chemometec, Denmark).

Results:

The results showed that PAI induced a strong anti-tumor activity against OCI-LY-

12 cell line, with 80% of cell death at 30 pg/ml (see Figure 2).

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-3000™ Advanced Image Cytometer (Chemometec, Denmark).

Peripheral blood mononuclear cells (PBMCs) 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-3000™ Advanced Image Cytometer (Chemometec, Denmark).

Results:

It was demonstrated that PAI induced a stronger cytotoxicity activity against all T-ALL and against cutaneous T cell lymphoma cell lines compared to Alemtuzumab.

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) and a PTCL-NOS cell line (300 000 cells per well) were exposed to serial concentrations (from lOpg/mL to 300pg/mL) of a polyclonal antibody composition PAI of the invention.

Further, T-ALL cell lines (Jurkat, T1301 and HPB-ALL), a cutaneous T cell lymphoma cell line (HUT78) and a multiple myeloma cell line (300 000 cells per well) were exposed to a concentration of 250 pg/mL of the polyclonal antibody composition PAL

After 20 hours of incubation at 37°C, 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- 3000™ Advanced Image Cytometer (Chemometec, Denmark)

Apoptotic activity was also assessed in PBMC from a healthy donor using the same experimental protocol. Results:

The results showed that PAI induced a strong apoptotic activity in all T-ALL and PTCL-NOS cell lines tested with a number of apoptotic cells ranging from 60% to 96% at a concentration of lOOpg/mL. It was shown that PAI targeted and killed specifically tumoral cells and spared healthy PBMC. No cross reactivity on healthy PBMC up to the highest tested concentration of 300pg/mL was observed (see Figure 4).

As demonstrated in table 1 below, PAI at a concentration of 250 pg/mL also induced a strong and significant induction of apoptosis of KMS-12-BM myeloma cells and HUT-78 cells.

Table 1

Example 5: Comparison between the apoptosis activity of a combination of antibodies of the invention and Alemtuzumab in different cell lines

Apoptotic assay of a 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).

After 24 hours of incubation at 37°C, Annexin V-CF488A conjugated and Hoechst 33342 (final concentration: lOpg/mL) were added and incubated for 15 min at 37°C. After washes, cell pellets were suspended in lOOpl Annexin V binding buffer supplemented with lOpg/mL Propidium iodure. Apoptotic cells were analyzed using NucleoCounterONC- 3000™ Advanced Image Cytometer (Chemometec, Denmark).

Results: Contrary to PAI, Alemtuzumab showed a weak apoptotic activity against HPB- ALL (less than 20%) and no apoptosis in T1301 and Jurkat cell lines (see Figure 5).

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 MojoSort™ 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. After 16-24h incubation, propidium iodine was added and cells were analyzed (CFSE+/IP+ cells) by the NucleoCounter® NC-3000™ Advanced Image Cytometer (ChemoMetec A/S, Allerod, Danemark).

Results:

The results showed that the ADCC process was partially involved in cancer cell death induced by PAI (see Figure 6).

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. 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 polyclonal antibody composition PAI at a concentration of lOpg/mL, 30pg/mL and lOOpg/mL and cocultured with human macrophages (ratio E:T 4:1) in RPMI 10% FCS for 16-24h. After 16-24h incubation, propidium iodine was added and cells were analyzed (CFSE+/IP+ cells) by the NucleoCounter® NC-3000™ Advanced Image Cytometer (ChemoMetec A/S, Allerod, Danemark).

Results: The results showed that ADCP process is involved in tumor cell death induced by PAI with concentration-response relationship (see Figure 7).

Example 8: Apoptotic pathways engaged by exposure to a combination of antibodies of the invention

To decipher the apoptotic pathways engaged by exposure to a combination of antibodies of the invention, activation of caspase 8 (initiating the extrinsic apoptosis pathway) and caspase 9 (involved in intrinsic apoptosis pathway) was investigated in tumor cell lines T1301, Jurkat, HUT-78, HPB-ALL and KMS-12-BM. The cell lines were treated with 250pg/ml of a polyclonal antibody composition PAI during 20h at 37°C.

Caspase 8 and 9 activity was determined after 20h with a caspase 8 or 9- specific fluorescent probe and fluorescent intensity is analyzed using NucleoCounterONC- 3000™ Advanced Image Cytometer (Chemometec, Denmark).

Results:

In all the tested cell lines, 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.

Two groups of mice were included in this study (n=10 per group): a control group without treatment and a treated group with a polyclonal antibody composition PAI . 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.

Results:

PAI showed to be effective against tumor growth in vivo. At D35, a 50% reduction in tumor size is observed in the group treated with PAI compared to vehicle (see Figure 10).

Further, table 2 below summarizes the EC20, EC50 and ECiuax obtained by in vitro CDC with PAI on the T1301 cell line according to a protocol as per examples 1, 2 and 3.

Table 2

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.

Two groups of mice were included in this study (n=10 per group): a control group without treatment and a treated group with a polyclonal antibody composition PAI . Treatment was initiated from the beginning of tumour growth and occurred for a total duration of 28 days. Treatment consisted in the intraperitoneal injection of PAI at 35 mg/kg twice a week.

Results:

PAI showed to be effective against tumor growth in vivo. At D40, a 72,3% reduction in tumor size is observed in the group treated with PAI compared to vehicle (see Figure 12).

Further, table 3 below summarizes the EC20, EC50 and ECiuax obtained by in vitro CDC with PAI on the Jurkat cell line according to a protocol as per examples 1, 2 and 3.

Table 3

Example 11: Binding of a combination of antibodies of the invention to PTCL tumors in patient biopsies To assess the targeting and recognition of a combination of antibodies according to the invention in PTCL, the inventors evaluated the immunolabelling of PAI, a polyclonal antibody composition of the invention, on patients’ biopsies. For this purpose, the inventors analysed a Tissue microarray (TMA) constituting of 119 cores of biopsies from various PTCL (Pantomics Inc. United States): 13 cores of biopsies from NK/T Cell lymphoma, 33 cores of biopsies from Angioimmunoblastic T Cell lymphoma (AITL), 15 cores of biopsies from enteropathy-associated T Cell lymphoma (EATL), 46 cores of biopsies from PTCL- NOS, and 12 cores of biopsies from anaplastic large Cell lymphoma (ALCL).

Briefly, after deparaffinization and antigen retrieval, the TMA was incubated with an optimum concentration of 2.5pg/mL of PAI, incubated with a secondary antibody-HRP conjugate and revealed with the ImmPACT VIP Peroxidase Substrate. An internal grading system, adapted from standard grading score used in Tissue cross reactivity studies, was applied to rate the intensity and the distribution of the staining using ImageJ software. The grading system is provided in table 4 below:

Table 4

Cases were considered positive if 10% or more of the tumor cells were stained by

PAI.

Results:

Surprisingly, 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:

Table 5

Among the PTCL patient' biopsies studied, the majority (>50%) were at score 2, which can be considered high.

Photomicrographs showed strong PAI staining in NK/T Cell lymphoma, in AITL, in EATL, and in NOS PTCL (all in score 2). Photomicrograph showed an unstained PTCL (score 0) of the same TMA.

Example 12: In vitro CPC study of a combination of antibodies of the invention in different human solid tumor cell lines

Different solid cancer cells line were exposed to the anti-tumor polyclonal antibody composition PAI at serial concentrations ranging from 12,5pg/mL to 800pg/mL in presence of rabbit complement (dilution 1/3 final) (n=3).

The solid cancer cell lines tested were as follow:

- A549 cell line representative of Non Small cell lung cancer,

- Hep-G2 cell line representative of hepatocellular carcinoma,

- HCT-116 cell line representative of colon cancer,

- MDA-MB-231 cell line representative of triple negative breast cancer,

- LNCAP cell line representative of prostate cancer,

- SK-MEL-30 cell line representative of melanoma.

After 1 hour of incubation at 37 °C, cell viability was measured using NucleoCounter®NC-3000™ Advanced Image Cytometer (Chemometec, Denmark).

Results:

PAI induced a strong cytotoxicity activity against human solid tumor cell lines (ranging from 45,2% to 90,7%) (see Figure 13).

Example 13: In vitro apoptotic activity of a combination of antibodies of the invention in different human solid tumor cell lines Different solid cancer cell lines (300 000 cells per well) were exposed to a concentration of 250 pg/mL of the polyclonal antibody composition PAI.

The solid cancer cell line tested are as follows:

- A549 cell line representative of Non small cell lung cancer

- Hep-G2 cell line representative of hepatocellular carcinoma

- HCT-116 cell line representative of colon cancer

- MDA-MB-231 cell line representative of triple negative breast cancer

- LNCaP cell line representative of prostate cancer

After 20 hours of incubation at 37°C, Annexin V-CF488A conjugated and Hoechst 33342 (final concentration: 10 pg/mL) were added and incubated for 15 min at 37°C. After washes, cell pellets were suspended in 100 pl of Annexin V binding buffer supplemented with lOpg/mL Propidium iodure. Apoptotic cells were analyzed using NucleoCounterONC- 3000™ Advanced Image Cytometer (Chemometec, Denmark).

Results:

PAI induced apoptosis of the tested cancer cell lines. The results for each line are provided in table 6 below:

Table 6

Example 14: Global cytotoxicity of a combination of antibodies of the invention in different human solid tumor cell lines after 24 hours incubation

Different solid cancer cell lines were exposed to the polyclonal antibody composition PAI at serial concentration ranging from 12,5pg/mL to 800pg/mL in presence of rabbit complement (dilution 1/3 final).

The solid cancer cell line tested are as follows:

- A549 cell line representative of Non small cell lung cancer

- Hep-G2 cell line representative of Hepatocellular carcinoma

- HCT-116 cell line representative of colon cancer

- MDA-MB-231 cell line representative of triple negative breast cancer - LNCAP cell line representative of prostate cancer

- SK-MEL-30 cell line representative of melanoma

After 24 hours of incubation at 37 °C, cell viability was measured using NucleoCounter®NC-3000™ Advanced Image Cytometer (Chemometec, Denmark).

Results:

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

HuProt™ 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.

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). The Z score of each spot on a given array is calculated according to the algorithm below: Z= [L635 - L635(avg)] / L635(std) L635(avg) and L635(std) are the average and standard deviation of the L635 values of all spots on the array, respectively.

Results:

Binding of the polyclonal antibody composition was observed for all six of the protein targets.

Example 16: In vitro cytotoxicity assay

Different liquid cancer cell lines were exposed to the polyclonal antibody composition PAI at serial concentrations ranging from 12,5 pg/mL to 800 pg/mL in presence of rabbit complement (dilution 1/3 final).

The liquid cancer cell lines tested are as follows:

- HPB-ALL, Jurkat, and T1301 cell line representative of T-ALL, - HUT-78 cell line representative of cutaneous T Cell lymphoma (Sezary Syndrome), and

- KARPAS 299 cell line representative of ALCL.

After 24 hour of incubation at 37°C, cell viability was studied using 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).

Results:

The results showed that PAI induces a strong cell death after 24 hour of incubation, even in HUT78 cell line which was described as a cell line that does not express CD99 membrane protein (see Figure 18).

Example 17 : In vivo efficacy in a rat T cell lymphoma/leukemia model vs CHOP

SRG immunodeficient rats were injected with 15xl0⁶ 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.

Design: 3 groups with n=7 rats per group

Group 1: control group without treatment (Control group),

Group 2: PAI treatment twice a week: 40 mg/kg (PAI group), and

Group 3: 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).

Results: The results showed that PAI administration completely blocked tumor growth for the duration of treatment. No tumor escape was observed with PAI treatment.

Five days after the end of the CHOP chemotherapy cycle, at DIO, tumor growth slowed in the CHOP group, with a 40% reduction observed at 21 days. After 21 days the tumor progressed and seemed similar to the progression the tumor growth of the control 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).

Claims

1. A combination of antibodies, or antigen-binding fragments thereof, for use in the treatment of a cancer in a human subject in need thereof, the combination comprising:

2. The combination for use according to claim 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.

3. The combination for use according to claim 1 or 2, comprising at least:

4. The combination for use according to any one of claims 1 to 3, wherein the antigen-binding fragments of the antibodies present in the combination are independently Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, or diabodies.

5. The combination for use according to any one of claims 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; cancer of the central nervous system; gastrointestinal stromal cancer; epidermal carcinoma; and oesophageal cancer.

6. The combination for use according to any one of claims 1 to 5, wherein the cancer is:

- a lymphoma, in particular selected from the group consisting of:

(i) Non-Hodgkin lymphomas selected from the group consisting of:

(ii) Hodgkin lymphomas selected from the group consisting of nodular sclerosis, mixed cellularity, lymphocyte-depleted Hodgkin lymphoma and lymphocyte-rich Hodgkin lymphoma, or

7. The combination for use according to any one of claims 1 to 6, wherein the cancer is selected from the group consisting of: - T-cell acute lymphoblastic leukemia (T-ALL);

- Cutaneous T-cell lymphoma (CTCL); and

- T-cell lymphoblastic lymphoma (T-LBL).

8. The combination for use according to any one of claims 1 to 7, wherein the cancer expresses at least one antigen selected from the group consisting of FASN, CD99, GPI, SLC3A2, GART, and CKAP4, in particular consisting of GPI and GART.

9. The combination for use according to any one of claims 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.

10. The combination for use according to any one of claims 1 to 9, wherein the combination is a combination of monoclonal antibodies or a polyclonal antibody composition.

11. The combination for use according to claim 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.

12. The combination for use according to claim 11, wherein the animal is a pig, and in particular is 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

13. The combination for use according to any one of claims 1 to 12, wherein the combination is included in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

14. The combination for use according to claim 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 claims 1 to 4 and 8 to 11.

15. The combination for its use according to claim 14, wherein the at least one additional anticancer drug is 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 monoclonal antibodies.

16. The combination for use according to any one of claims 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); HDAC inhibitors; Ibrutinib, acalabrutininb, zanubrutinib, copanlisib and venetoclax.

17. The combination for use according to any one of claims 1 to 16, for use in providing an antitumoral activity selected from the group consisting of comprises cytotoxicity complement dependent (CDC), cytotoxicity complement dependent from killer cells (ADCC), apoptotic activity and/or cytotoxicity complement dependent from phagocytes (ADCP), and in particular an antitumoral activity consisting of cytotoxicity complement dependent (CDC), cytotoxicity complement dependent from killer cells (ADCC), apoptotic activity and cytotoxicity complement dependent from phagocytes (ADCP).