Antibody-peptide conjugates for immunotherapy of diseases such as cancer

Abstract

The invention relates to an antibody capable of binding a cell surface antigen, wherein said antibody is conjugated to a peptide comprising an epitope capable of being presented by a CD1 molecule on the cell surface. The invention further relates to a method for the treatment of a disease, comprising administration, to a subject, of the antibody-peptide conjugate of the invention.

Description

[0001] Antibody-peptide conjugates for immunotherapy of diseases such as cancer

[0002] Field of the invention

[0003] The present invention is in the field of immunology and describes antibody-peptide conjugates and their use in the treatment of diseases, such as tumors, expressing specific or associated cell surface antigens.

[0004] Background of the invention

[0005] The treatment of tumors remains a major unmet medical need despite recent introduction of immunotherapy based on administration of antibodies to suppressive molecules such as PD-1, PD-L1 or CTLA-4, CAR-T cells or antibody drug conjugates (ADCs).

[0006] An increasing number of antigens or neo antigens associated with tumors are, however, identified, which could serve as a base to mount specific immune responses for tumor eradication. Such antigens include tumor-specific antigens, expressed only on tumor cells, and tumor-associated antigens, expressed at higher concentrations on tumors than on normal cells.

[0007] Both of such antigens constitute a preferential target for either CAR-T cells or ADCs. Variable efficacy and cost related to the production of CAR-T cells have led to a profusion of ADCs towards increasing numbers of tumor antigens. ADCs are antibodies coupled to a payload which, after internalization of the antibody, is liberated and exert its tumoricidal activity. Payloads can be of different types, including toxins, chemotherapeutic agents and enzymes. A major risk of payloads is toxicity associated with their diffusion from dying cells to bystander or distant cells, or dissociation of payloads from antibodies in circulation, thereby limiting their usage.

[0008] Summary of the invention

[0009] The inventor proposes a therapeutic approach wherein the payload conjugated to an antibody is a sequence of amino acids carrying an epitope which binds and is presented by a class I MHC-like CD1 molecule at the target cell surface, thereby constituting a target for epitope-specific natural killer T (NKT) cells. The potent cytotoxic properties of NKT cells eliminate the target cell, which preferably is a tumor cell. Accordingly, in a first aspect, the invention relates to an antibody capable of binding a cell surface antigen, wherein said antibody is conjugated to a peptide comprising an epitope capable of being presented by a GDI molecule on the cell surface.

[0010] In further aspect, the invention relates to a method for the treatment of a disease, comprising administration, to a subject, of the antibody-peptide conjugate of the invention as described herein.

[0011] Similarly, the invention relates to the antibody-peptide conjugate of the invention as described herein for use in method for the treatment of a disease, such as cancer, wherein the treatment comprises administration of the antibody-peptide conjugate.

[0012] Considering the non-polymorphic nature of CD1 molecules, a single peptide containing the CD1 binding epitope may be used for all patients in need of a treatment. The method proposed here circumvents the difficulties associated with the development of class l-restricted cytotoxic CD8+ T cells: (1) the large variation in class I MHC molecules forcing the design of patient-tailored epitopes, and (2) the usual exhausted status of class l-restricted cytotoxic cells.

[0013] The antibody-peptide conjugate of the invention may be used in combination with a step wherein the subject is immunised with (i) a peptide antigen comprising the same epitope as comprised within the peptide conjugated to the antibody or (ii) a nucleic acid encoding such a peptide antigen. Alternatively, epitope-specific NKT cells may be administered passively. NKT cells specific for the epitope presented by target cells may be elicited by peripheral tissue immunisation. In case of patients with immunocompromised status, immunisation may be replaced by in vitro expanded NKT cells. The combination of an antibody-vehicle carrying a GDI-restricted epitope and the immunisation process (or passive administration of NKT cells) eliciting specific NKT cells carries the advantages of absence of toxic effects associated to commonly used cytotoxic drug payloads and of specifically targeting cells by a vaccine-induced population of cytotoxic cells.

[0014] Accordingly, in one embodiment, the invention relates to a method for the treatment of a disease, comprising administration, to a subject, of the antibody-peptide conjugate of the invention as described herein, wherein said method comprises a further step of eliciting or administering epitope-specific NKT cells in said subject. Detailed description of the invention

[0015] In a first aspect, the invention relates to an antibody capable of binding a cell surface antigen, wherein said antibody is conjugated to a peptide comprising an epitope capable of being presented by a GDI molecule on the cell surface. The antibody-peptide conjugate gets internalized into the cell upon binding of the antibody to the cell surface antigen. The peptide is subsequently released, binds a GDI molecule and is presented by GDI on the cell surface.

[0016] The inventor of the present application has previously described that GDI can present stretches of amino acids that include hydrophobic amino acids and that this presentation is followed by activation of non-invariant (non-type 1) NKT cells. Thus, activation of antigen-specific NKT cells has led to a series of therapeutic applications as described in, for instance, WO 2012 / 069572, WO 2012 / 069575, WO 2013 / 174805 and WO 2018 / 189405 (all hereby incorporated by reference).

[0017] The cell surface antigen to which the antibody-peptide conjugate of the invention binds is typically a tumor cell surface antigen, i.e. an antigen found on the surface of tumor cells. Such antigens may be antigens that are not expressed on somatic cells (tumor-specific antigens) or antigens that are preferentially expressed by tumor cells (tumor-associated antigens). The antibody-peptide conjugate is typically capable of being internalized, i.e. internalized into the target cell, upon binding to a cell expressing the cell surface antigen. Thus, the cell surface antigen is typically an internalizing antigen. In some embodiments, binding of the antibody-peptide induces internalization into the target cell.

[0018] The term "antibody" as used herein is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological and / or tumor-specific conditions with a half-life of significant periods of time. The structure of immunoglobulins has been well characterized (see for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Antibodies typically contain heavy chain and / or light chains which each contain constant and variable regions. The variable regions are typically responsible for antigen binding and may be further subdivided into regions of hypervariability, also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In the context of the present invention, the term "antibody" includes a full-length antibodies as well as an antibody fragments retaining the ability to specifically bind to the antigen. Furthermore, the term includes chimeric, humanized as well as human antibodies.

[0019] Antibodies to cell surface antigens, such as tumor cell surface antigens, can easily be produced for example in recombinantly modified host cells, hybridomas or systems that use cellular extracts supporting in vitro transcription and / or translation of nucleic acid sequences encoding the antibody. A number of antibodies is used in the clinic.

[0020] The conjugated antibody of the invention may have any format suitable for binding to the cell surface antigen and internalization into the target cell. Suitable formats include, but are not limited to, full-length antibodies, such as IgG antibodies, as well as antibody fragments containing the variable part (Fab'2 or Fab fragments), single-chain antibodies, scFv antibodies and singledomain antibodies, such as nanobodies, minibodies or extended variable domains.

[0021] Full-length antibodies may provide additional advantages associated to the presence of the heavy chain, including interaction with Natural Killer (NK) cells CD16 Fc receptor (thereby recruiting NK cells at cell surface), activation of the classical complement pathway (for lgG3 mainly, triggering the assembly of the cell lytic C5b-C9 complex) or macrophage activation. However, shorter versions of antibody may facilitate cell uptake and therefore antibody-payload internalization.

[0022] In some embodiments, the antibody is a multivalent antibody, such as a bivalent antibody. Bi- or multivalent binding may increase target oligomerization on the target cell and thus facilitate internalization.

[0023] In some embodiments, the antibody is a multispecific antibody, wherein said multispecific antibody further comprises an antigen-binding region that binds a different epitope of the same cell surface antigen or further comprises an antigen-binding region binds to a different cell surface antigen. Preferably, the multispecific antibody binds two or more cell surface antigens that are present on the surface of the same target cell. Multispecific binding may be used to increase the specificity of antibody binding to tumor cells, by choosing antigens that are only found in combination on tumor cells.

[0024] The antibody of the invention is conjugated to a peptide comprising an epitope capable of being presented by a CD1 molecule on the cell surface. In one embodiment, the peptide has a length of of at least 7 amino acids, such as 8, 9, 10, 11, 12, 13 ,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids, such as at least 11 amino acids, for example at least 15 amino acids, such as at least at least 20 amino acids, for example between 20 and 50 amino acids, for example between 20 and 40, amino acids, such as between 22 and 30 amino acids.

[0025] In one embodiment, the peptide as such is not cytotoxic to human cells.

[0026] In one embodiment, the peptide does not contain HLA-I epitopes.

[0027] In one embodiment, the peptide does not contain pathogen-derived sequences or pathogen- derived epitopes, such as viral sequences or viral epitopes.

[0028] In one embodiment, the peptide does not contain a sequence which can be cleaved extracellularly.

[0029] In one embodiment, the peptide does not contain a cleavage site for a metallo-proteinase.

[0030] In one embodiment, the peptide does not elicit peptide-specific CD8+ cytotoxic T cells.

[0031] In one embodiment, the peptide does not elicit peptide-specific MHC class l-restricted CD8+ cytotoxic T cells.

[0032] Peptides comprising an epitope capable of being presented by a CD1 molecule on the cell surface have been defined and described in WO 2012 / 069572, WO 2012 / 069575, WO 2013 / 174805 and WO 2018 / 189405 (all hereby incorporated by reference). Suitable peptides can further be identified via in silica analysis of peptide interactions with GDI molecules, e.g. CDld, based on the crystal structure of human CDld (Koch et al. (2005) Nature Immunology 6, 819) or based on the structure of CDld described in Gracia-Alles et al. (2006) EMBO J. 25:3684 for CDlb. The analysis may, for example, be performed as described in Example 2 herein. Suitable in silica platforms for such an analysis include, but are not limited to, those provided by Schrodinger (https: / / www.schrodinger.com), e.g. the PIPER platform.

[0033] In a preferred embodiment, the epitope comprises the sequence X1-X2-X3-X4-X5-X6-X7, wherein Xi and X7 independently are F (Phe), W (Trp), T (Thr), H (His) or Y (Tyr), wherein X2, X3, X5, and Xe may be any amino acid, including a non-natural amino acid, and wherein X4 is I (He), L (Leu), M (Met) or V (Vai).

[0034] In one embodiment, Xi and X7 are independently selected from F, W, T or H.

[0035] In one embodiment, Xi and X7 are independently selected from F, W, H or Y.

[0036] In one embodiment, Xi and X7 are independently F or W.

[0037] The peptide and / or epitope may be derived from a natural sequence, with or without amino acid substitutions, so as to increase epitope binding to a CD1 molecule, or be an artificial sequence not found in nature. The peptide and / or epitope may optionally contain non-natural amino acids, such as D-amino acids, or additional moieties such as short-length lipids, palmitoyl or myristoyl chains.

[0038] The CD1 molecule presenting the epitope may be a CDla, CDlb, CDlc or CDld molecule.

[0039] Conjugation of the antibody with the peptide-based payload can be carried out by various linkers and conjugation methods (linkage) methods. Several of such linkers and their use in antibodydrug conjugates have been described (see for instance Bargh JD, et al, Chem. Soc. Rev, 2019, 48: 4361-4374; Fu Z, et al, Signal Transduction and Targeted Therapy, 2022, 7:93).

[0040] The presence of a reducible disulfide bond between antibody and peptide payload preferentially leads to reduction in cytosol due to the high concentration of glutathione. Disulfide bonds can be created directly on the targeting antibody or via a linker. Some antibodies, such as IgGl, comprise four interchain disulfide bridges, which can be used to link a payload.

[0041] Suitable linkers, such as dipeptide linkers, have also been described, designed as to constitute a substrate for intracellular enzymes, preferentially in the acidic environment of endosomes or lysosomes. Thus, such linkers may be cleaved by cathepsins, such as cathepsin B / L, enzymes confined to lysosomes. Such linkers may contain motifs such as Phe-Arg, Val-Ala or Phe-Gly (not limitative). Alternatively, the peptide payload can be cleaved from the antibody by enzymes such as beta-glycosidase, beta-galactosidase, or phosphatase (Bargh JD, et al, Chem. Soc. Rev, 2019, 48: 4361-4374). In a preferred embodiment, a peptide linker is used which can be cleaved or can only be cleaved under acidic conditions. Such conditions prevail in the endosomal pathway and lysosomes in particular. The payload (i.e. the CDl-restricted epitope-containing peptide) may then either directly bind to a free GDI molecule and be directed to the cell surface, or exit into the cell cytosol, be taken up by the proteasome, the endoplasmic reticulum and the transGolgi for cell surface expression bound to a GDI molecule (so-called secretory pathway). Thus, suitable linkers for use in the present invention include chemical cleavage linkers (e.g. based on hydrazone bonds or disulfide bonds) and enzyme cleavage linkers (e.g. based on glucuronide bonds or and peptide bonds). Hydrazone is a typical acid-sensitive (pH sensitive) linker. Disulfide bond based linkers are other chemically sensitive cleavable linker that are sensitive to reductive glutathione (GSH). Peptide based linkers are typically sensitive to lysosomal proteases, such as cathepsin B. Specific peptide based linkers include valine- citrulline containing linkers such as those used in brentuximab vedotin. Beta-glucuronide linkers are another type of enzyme-sensitive linker commonly used in antibody conjugates. They can be cleaved for payloads release in cells by betaglucuronidase, the levels of which are often found higher in tumor environments. In some embodiments, the linker is not cleavable by extracellular proteases, such as matrix-metallo proteinases.

[0042] As mentioned above, in a further aspect, the invention relates to a method for the treatment of a disease, comprising administration, to a subject, of the antibody-peptide conjugate of the invention as described herein.

[0043] Similarly, the invention relates to the antibody-peptide conjugate of the invention as described herein for use in method for the treatment of a disease, such as cancer, wherein the treatment comprises administration of the antibody-peptide conjugate.

[0044] Passive administration of cell surface specific antibodies is followed by internalization and uptake by the endosomal pathway. Antibody is coupled to the peptide-based payload, a sequence of amino acids carrying a CDl-restricted epitope, which is liberated after antibody internalization and decoupling within the endosomal pathway, and loaded onto free GDI molecules for cell surface expression and recognition by NKT cells.

[0045] In addition to their intrinsic cytotoxic properties, NKT cell activation results in the recruitment of natural killer (NK) cells and of adaptive immunity including class I- and class Il-restricted CD8+ and CD4+ T cells, respectively. The synergic activation of an immune response leads to higher levels of tumor control, recruiting cytotoxic properties of NKT-, CD8+ T- and NK cells, resulting in a highly efficient method to eliminate tumor cells (see W02022008479).

[0046] In one embodiment, the method comprises a further step of eliciting or administering epitopespecific NKT cells in said subject.

[0047] Thus, in a further embodiment, the method comprises, or further comprises, immunisation of said subject with (i) a peptide antigen comprising the same epitope as comprised within the peptide conjugated to the antibody or (ii) a nucleic acid encoding such a peptide antigen.

[0048] The peptide used for the immunisation comprises the same GDI epitope as the peptide that forms part of the antibody-peptide conjugate, but may otherwise differ in sequence, e.g. in the sequences flanking the epitope.

[0049] Immunisation with peptide antigens containing GDI epitopes can be carried out using current strategies used to increase the stability of peptides and / or uptake by antigen-presenting cells, such as those described in W02022008479, including adsorption or coupling to nanoparticles, coupling to a carrier such as KLH, addition of stappling or cell penetrating amino acids. The peptide may be co-administered with an adjuvant, e.g. aluminum hydroxide or an innate activator such as Montanide. In embodiments wherein method comprises immunisation with a nucleic acid encoding such a peptide antigen, said nucleic acid may be in mRNA or DNA format.

[0050] Injections are preferably carried out close to a peripheral lymph node. In another embodiment, the method comprises, or further comprises, administration, to said subject, of epitope-specific NKT cells, which may be allogenic or autologous. In one embodiment, the epitope-specific NKT cells are ex vivo expanded or ex vivo loaded autologous epitope-specific NKT cells.

[0051] Thus, peptides can be loaded in vitro on antigen-presenting cells, such as dendritic cells, and the cells administered by the intravenous route, a strategy which could circumvent the immunocompromised status of many patients suffering from tumors. Alternatively, NKT cells can be prepared in vitro for passive administration to patients in need.

[0052] Accordingly, the present invention describes a combination therapy, including (a) an immunization by peripheral administration, using a natural or synthetic CDl-epitope containing peptide, so as to elicit a population of epitope-specific NKT cells, or passive administration of ex vivo generated epitope-specific NKT cells, and (b) rendering the target cells, for example tumor cells, exquisitely sensitive to such NKT cells by administration of an antibody specific for the cell surface receptor coupled by a cleavable bond to the peptide containing the CD1 epitope used for immunisation. In one embodiment, the immunization (a) is performed prior to administration of the antibody-peptide conjugate (b). In a further embodiment, two or more immunizations with the CDl-epitope containing peptide are performed prior to administration of the antibody- peptide conjugate. Preferably, there is a time interval of at least 2 weeks, such as 2-4 weeks, for example 3 weeks between the immunisation and the administration of the antibody-peptide conjugate. In an embodiment wherein the administration of the antibody-peptide conjugate is combined with passive administration of ex vivo generated epitope-specific NKT cells, such NKT cells are preferably administered at least 24 hours, such as 24-48 hours before administration of the antibody-peptide conjugate.

[0053] In one embodiment, the present invention relates to a therapy combining an immunisation made using a CDl-restricted protein epitope so as to elicit specific natural killer T (NKT) cells, followed by administration of an antibody towards a surface antigen specific or associated with a tumor cell, coupled via a linker to a payload made of the peptide used for immunisation, wherein:

[0054] • the said tumor cell is expressing said surface antigen, which preferably is a tumor-specific antigen or a tumor-associated antigen; • the said GDI epitope is a natural or artificial sequence of amino acids comprising the sequence X1-X2-X3-X4-X5-X6-X7, wherein Xi and X7 independently are F, W, T, H or Y, wherein X2, X3, X5, and Xe may be any amino acid, including a non-natural amino acid, and wherein X4 is I, L, M or V,

[0055] • said immunisation comprises administration of said epitope-containing peptide, preferably with an adjuvant, to a patient suffering from said tumor

[0056] • said GDI epitope-containing peptide is coupled to said antibody by a cleavable bond, which is cleavable under acidic conditions.

[0057] Sensitivity of tumors cells to NKT cell cytotoxicity may depend on the degree of expression of GDI molecules at the cell surface. In some embodiments, the method of the invention further comprises administration of a compound that increases the level of GDI molecules, such as CDld molecules, on the cell surface. The transcription of CDld is under control of epigenetic regulators, which can be the target for inhibitors of histone deacetylase, retinoic acid or rapamycin. Furthermore, CDld expression is increased by irradiation. Thus, in some embodiments, the method of the invention further comprises administration of irradiation or a compound that increases the level of said CD1 molecule, e.g. CDld, on the cell surface, wherein said compound is retinoic acid, rapamycin, an epigenomic regulator, or an inhibitor of methylation of the promoter of SIP1, which promotes CDld expression (see Wang et al. (2023) Front. Immunol. 14:1152228. doi: 10.3389 / fimmu.2023.1152228). In a further embodiment, the method of the invention further comprises administration of a cytokine, such as GM-CSF or IL-4, to increase the level of the CD1 molecule, in particular of CDlb, on the cell surface.

[0058] The present method provides several significant advantages:

[0059] • Taking into account that CD1 molecules are non-polymorphic, an identical CDl-epitope payload may be used for all individuals and be independent of the nature of the tumor or target cell considered. An immunisation step with the peptide containing the CD1 epitope leads to the activation of epitope-specific NKT cells, which specifically recognize and eliminate cells expressing the epitope. However, in the particular case of patients with a compromised immune system, passive administration of either antigen-presenting cells loaded in vitro with the GDI epitope of interest, or of NKT cells expanded in vitro, constitute valid alternatives. In vitro-expanded specific NKT cells may provide an "off-the-shelf" universal preparation ready for use. • Substituting toxin or enzyme payloads by a stretch of amino acids carrying a GDI epitope avoids off-target adverse effects due to toxin release in circulation or from dying cells or enzymatic interference exerted at distance of the target site, such as those observed with topoisomerase (cardiac toxicity). This eliminates the need to determine and maintain a subtle equilibrium in between efficacy and toxicity.

[0060] • The peripheral tissue immunisation step generates specific NKT cells showing characteristics of memory cells, opening the possibility of repeating passive administration of the specific antibodypayload complex as many times as requested for achieving meaningful clinical results.

[0061] As described, tumor cells can:

[0062] •express tumor-specific surface antigens, not expressed on somatic cells, such as the MAGE antigens or MART-1, or the B cell receptor (BCR) of myeloma cells or from a mature B cell leukemia, or the T cell receptor expressed by mature T cell leukemia, and / or

[0063] • express tumor-associated surface antigens expressed at much higher levels than in normal cells, such as the HER-2 / neu or HER-3 epidermal growth factor receptor (EGFR)

[0064] Each of these categories of antigens can serve as the target for an antibody-peptide conjugated of the invention.

[0065] Specific suitable cell surface antigens include, but are not limited to: CD30, CD79B, CD22, BCMA, CD19, CD33, CD20, ROR1, IL2RA, CD37, CD45, HER2, Nectin-4, Tissue Factor, TACSTD2, EGFR, F0LR1, C-MET, CEACAM5, HER3, Mesothelin, PSMA, CD71, B7-H3, MUC1, IGF1R, ADAM9, CEACAM6, CLDN18, AXL, ROR2, MUC18, SDPTP2B, MAGE, MART-1, tumor-associated B Cell Receptor, tumor-associated T Cell Receptor, TROP-2, DLL-3 and CD56. In one embodiment, the cell surface antigen is not EpCAM and is not CD20.

[0066] In one embodiment, the antibody-peptide conjugate includes anti-HER2 antibody, for example a humanized anti-HER2 antibody, such as trastuzumab, coupled to a peptide payload. HER-2 / neu is an epidermal growth factor receptor including an extracellular ligand-binding domain, a transmembrane region and an intracellular domain. In the presence of a ligand, homodimers are formed, which initiates activation of a network of cell signaling pathways regulating cell proliferation, differentiation, migration and survival. Though HER-2 / neu is expressed on normal healthy cells, its expression is increased by several-fold in cancer cells, making HER-2 / neu an exquisite target for the therapy of cancer, including breast, prostate, lung and pancreas cancers.

[0067] Myeloma is an incurable malignancy of postgerminal center B cells resulting from the translocation of genes such as C-Maf to the heavy chain encoding gene or chromosomal hyperploidy. Myeloma cells carry a unique B cell receptor (BCR) which can be targeted by specific antibodies. Myeloma cells in early development phase express high density of surface CDld, making them a preferred target for NKT cells. Myeloma cells can also be treated by recognition of BCMA, a major surface tumor-associated antigen present in a large proportion of myelomas, at levels significantly higher than on healthy cells.

[0068] Mature T cell leukemia and some T cell lymphomas carry a T cell receptor of the alpha-beta or gamma-delta type, which can similarly become a target for specific antibodies.

[0069] In one embodiment of the method of treatment or use according to the invention, the disease to be treated is cancer, such as a solid tumor cancer or a hematological cancer.

[0070] Furthermore, in one embodiment, the disease is leukemia or lymphoma and the cell surface antigen is the B cell receptor (BCR).

[0071] In one embodiment, the disease is breast cancer and the cell surface antigen is CD25, CD174, CD197, CD205, CD228, c-MET, CRIPTO, HER2, HER3, FL0R1, GloboH, GPNMB, IGF-1R, integrinp- 6, PTK7, nectin-4, ROR2, TROP-2 or SLC39A6.

[0072] In one embodiment, the disease is HER2+ breast cancer and the cell surface antigen is HER2.

[0073] In one embodiment, the disease is HER2- breast cancer and the cell surface antigen is TACSTD2.

[0074] In one embodiment, the disease is triple-negative breast cancer and the cell surface antigen is TACSTD2 or CEACAM5.

[0075] In one embodiment, the disease is ovarian cancer and the cell surface antigen is CA125, CD142, CD205, FL0R1, GloboH, mesothelin, PTK7 or TIM-1.

[0076] In one embodiment, the disease is prostate cancer and the cell surface antigen is CD46, PSMA, STEAP-1, SLC44A4, TENB2 or CD276(B7-H3). In one embodiment, the disease is prostate cancer, the cell surface antigen is CD276(B7-H3) and the tumor of the patient does not comprise p53 mutations. In one embodiment, the disease is lung cancer and the cell surface antigen is CD25, CD56, CD71, CD228, CD326, CRIPTO, EGFR, HER3, FAP, GloboH, GD2, IGF-1R, integrinp-6, mesothelin, PTK7, R0R2, SLC34A2, SLC39A6, Axl or avp6.

[0077] In one embodiment, the disease is pancreatic cancer and the cell surface antigen is CD25, CD71, CD74, CD227, CD228, GRP20, GCC, IGF-1R, integrinp-6, nectin-4, SLC34A2, SLC44A4, avp6, CLDN18, CEACAM5 or mesothelin.

[0078] In one embodiment, the disease is melanoma and the cell surface antigen is CD276, GD2, GPNMB, ED-B, PMEL17, MART-1, MUC18, MAGE or the endothelin B receptor.

[0079] In one embodiment, the disease is gastric cancer and the cell surface antigen is CD25, CD197(CCR7), CD228(P79, SEMF), FLORl(FRa), GloboH, GRP20, GCC, HER2, CEACAM5 or SLC39A6(LIV1AZIP6).

[0080] In one embodiment, the disease is colorectal cancer and the cell surface antigen is CD74, CD174, CD166, CD227, CD326, CEACAM5, CEACAM6, CRIPTO, FAP, ED-B or HER3.

[0081] In one embodiment, the disease is bladder cancer and the cell surface antigen is CD25, HER2 or CD205(Ly75).

[0082] In one embodiment, the disease is metastatic transitional (urothelial) tract cancer and the cell surface antigen is Nectin-4, TACSTD2 or HER2.

[0083] In one embodiment, the disease is ureter cancer and the cell surface antigen is HER2.

[0084] In one embodiment, the disease is muscle invasive bladder cancer cancer and the cell surface antigen is Nectin-4.

[0085] In one embodiment, the disease is liver cancer and the cell surface antigen is CD276(B7-H3) or c- MET.

[0086] In one embodiment, the disease is renal cancer and the cell surface antigen is AGS-16, EGFR, c- MET, CAIX, CD70, FL0R1 or TIM-1.

[0087] In one embodiment, the disease is multiple myeloma and the cell surface antigen is CD38, CD46, CD56, CD74, CD138, CD269 or the endothelin B receptor.

[0088] In one embodiment, the disease is refractory / relapsed multiple myeloma and the cell surface antigen is BCMA.

[0089] In one embodiment, the disease is head and neck cancer and the cell surface antigen is CD71 (transferrinR), CD197(CCR7), EGFR or SLC39A6 (LIV1AZIP6). In one embodiment, the disease is recurrent head and neck cancer and the cell surface antigen is EGFR.

[0090] In one embodiment, the disease is non-Hodgkin lymphoma and the cell surface antigen is CD19, CD20, CD21, CD22, CD25, CD30, CD37, CD70, CD71, CD72, CD79a / b, CD18, CD205 or R0R1.

[0091] In one embodiment, the disease is B-cell non-Hodgkin lymphoma and the cell surface antigen is CD20.

[0092] In one embodiment, the disease is follicular lymphoma and the cell surface antigen is CD20.

[0093] In one embodiment, the disease is B-cell Hodgkin's lymphoma and the cell surface antigen is CD30.

[0094] In one embodiment, the disease is Hodgkin's lymphoma and the cell surface antigen is CD25, CD30 or CD197.

[0095] In one embodiment, the disease is diffuse large B-cell lymphoma and the cell surface antigen is CD79B, RORl or CD19.

[0096] In one embodiment, the disease is mantle cell lymphoma and the cell surface antigen is R0R1.

[0097] In one embodiment, the disease is acute myeloid leukemia and the cell surface antigen is CD25, CD33, CD123 or FLT3.

[0098] In one embodiment, the disease is refractory or relapsed acute myeloid leukemia and the cell surface antigen is CD33.

[0099] In one embodiment, the disease is B-cell acute myeloid leukemia and the cell surface antigen is CD22.

[0100] In one embodiment, the disease is glioma and the cell surface antigen is CD25 or EGFR.

[0101] In one embodiment, the disease is mesothelioma and the cell surface antigen is mesothelin or CD228.

[0102] In one embodiment, the disease is small cell lung cancer and the cell surface antigen is DLL-3 or CEACAM5.

[0103] In one embodiment, the disease is bile duct cancer and the cell surface antigen is CEACAM5.

[0104] In one embodiment, the disease is endometrial cancer and the cell surface antigen is CEACAM5.

[0105] In one embodiment, the disease is non-small cell lung cancer and the cell surface antigen is MAGE, HER2, TACSTD2, CEACAM5 or c-Met.

[0106] In one embodiment, the disease is biliary cancer and the cell surface antigen is CLDN18.

[0107] In one embodiment, the disease is colon cancer and the cell surface antigen is CEACAM6.

[0108] In one embodiment, the disease a cancer of epithelial origin and the cell surface antigen is MUC1. In one embodiment, the disease is uterine cancer and the cell surface antigen is mesothelin.

[0109] In one embodiment, the disease is lung cancer and the cell surface antigen is mesothelin.

[0110] In one embodiment, the disease is metastatic breast cancer and the cell surface antigen is HER3 or CEACAM5.

[0111] In one embodiment, the disease is cervical cancer and the cell surface antigen is Tissue Factor or CEACAM5.

[0112] In one embodiment, the disease is epithelial ovarian cancer cancer and the cell surface antigen is Tissue Factor, CEACAM5 or F0LR1R.

[0113] In one embodiment, the disease is peritoneal cancer and the cell surface antigen is Tissue Factor or F0LR1R.

[0114] In one embodiment, the disease is fallopian tube cancer and the cell surface antigen is Tissue Facto or F0LR1R.

[0115] In one embodiment, the disease is adenocarcinoma of the gastroesophageal junction and the cell surface antigen is CEACAM5.

[0116] In one embodiment, the disease is bladder carcinoma and the cell surface antigen is CEACAM5.

[0117] In one embodiment, the disease is a solid tumor cancer and the cell surface antigen is CEACAM5. In one embodiment, the disease is transitional cell carcinoma (urothelial cell carcinoma) and the cell surface antigen is CEACAM5.

[0118] In one embodiment, the disease is pancreatic ductal adenocarcinoma and the cell surface antigen is CEACAM5.

[0119] In one embodiment, the disease is anaplastic large cell lymphoma and the cell surface antigen is CD30.

[0120] In one embodiment, the disease is cutaneous T-cell lymphoma and the cell surface antigen is CD30.

[0121] In one embodiment, the disease is mycosis fungoides and the cell surface antigen is CD30.

[0122] In one embodiment, the disease is peripheral T-cell lymphomas and the cell surface antigen is

[0123] CD30.

[0124] Example 1 General description of the procedure 1. Selection of tumor and production of specific antibodies

[0125] Peripheral blood obtained from a patient suffering from a mature lymphoma is obtained. Cells showing lymphoma transformation are identified by FACS analysis and cell sorting. The sequence of the surface receptor is then identified by gene amplification.

[0126] The corresponding protein, or its mRNA or DNA counterpart is then produced for generating specific antibodies either by animal immunization or by selection on libraries of phages displaying random Fab fragments. Antibodies are then humanized by replacement of constant regions with the human counterparts.

[0127] 2. Production of the antibody-CDld epitopes payload compound

[0128] Specific antibodies or their equivalent under shorter formats, including nanobodies or peptides containing antibody binding sites are coupled to a peptide comprising a CDld-restricted epitope by way of a linker made of a dipeptide containing an acid-sensitive cleavable bound. The construct made of the CDld epitope and the peptide payload is bound to antibody by a disulfide bond.

[0129] 3. CDld epitope presentation

[0130] To determine whether the CDld epitope is expressed on lymphoma cells after incubation with the antibody-payload complex, cells are maintained in culture in the presence of various concentrations of the antibody-CDld epitope conjugate, which is tagged by a fluorochrome. The presentation of the CDld epitope onto cell surface is then verified by FACS analysis.

[0131] 4. Lysis of tumor cells in vitro

[0132] Wildtype mice are immunized with the CDld epitope to obtain NKT cells. To note, the high homology in between human and mouse CDld allows mouse NKT cells to be activated by presentation of human epitopes by mouse CDld. NKT cells obtained from immunized wildtype mice are prepared by cell sorting and added at various concentrations to cell cultures of lymphoma cells treated by the antibody-CDld epitope conjugate. Lysis of tumors cells is then evaluated by the release of a fluorochrome or chromium.

[0133] 5. Lysis of tumor cells in vivo

[0134] To observe the effects of NKT cells directly in vivo, nude mice are grafted with the human lymphoma and the growth of the tumor is followed in the presence of specific NKT cells. One group of nude mice is grafted with lymphoma cells as a control. Anothergroup is grafted with the same tumor treated in cell culture with the antibody-epitope compound. Specific NKT cells obtained by immunisation with the CDld epitope in a healthy mouse are then administered intravenously to each one of the two groups of nude mice. It is shown that the growth of the tumor is kept under control only in nude mice having received lymphoma cells preincubated with the antibody-peptide conjugate, showing that recognition of tumor via NKT-CDld epitope recognition is sufficient for controlling tumor cell growth.

[0135] Example 2 Identification of peptide epitopes binding to CDld or CDlb class I MHC-like presentation molecules

[0136] The 3D structure of CDld and CDlb is obtained from publicly accessible resources, such as: RCSB PDB - 5FKP: Crystal structure of the mouse CDld in complex with the p99 peptide or RCSB PDB - 2H26: human CDlb in complex with endogenous phosphatidylcholine and spacer, respectively.

[0137] To identify stretches of amino acids that constitute epitopes binding to CDld or CDlb, the entire sequence of the starting protein is examined in stretches of 20 amino acids with an overlap of 19 amino acids. The free binding energy of each amino acid stretch on CDld or CDlb is calculated using the modification of the Fast Fourrier Transform correlation, modified as described in Kozakov et al (Proteins: Structure, function and bioinformatics. 2006. 65:392-406).

[0138] Amino acid sequences are then classified according to calculated binding energy and compared to values obtained with an artificial reference peptide epitope (Girardi, Enrico et al. "Structure of an a-Helical Peptide and Lipopeptide Bound to the Nonclassical Major Histocompatibility Complex (MHC) Class I Molecule CDld." The Journal of biological chemistry vol. 291,20 (2016): 10677-83. doi:10.1074 / jbc.M115.702118).

[0139] Sequences with a free energy binding score matching at least that of the standard reference peptide are produced by solid-phase chemistry.

[0140] To confirm that such selected sequences contain relevant epitopes, namely capable of inducing activation of CDld or b -restricted natural killer T cells, the following experiments are carried out:

[0141] In the mouse:

[0142] • selected in vitro identified CDld epitopes are incubated for 2 hours with B cell-depleted splenocytes and the population of CD3+ T cells is then characterized for markers of activation and expression of phenotypic and transcriptomic signatures of natural killer T cells.

[0143] • selected CDld epitopes are injected by the IP route and splenocytes are obtained 2 days after for the detection of activated natural killer T cells.

[0144] As natural killer T cells are effector cells, they react within less than 2 hours when in contact with a cognate antigen, by contrast with T cells from the adaptive immune system requiring time and several antigenic contacts before reaching a status of activation.

[0145] In the human:

[0146] • selected in vitro identified CDld or CDlb epitopes are incubated for 2 hours with B cell-depleted PBMC and the presence of activated natural killer T cells is identified as above, including the transcription of cytokines such as IL-4, IL-21 and GM-CSF, all implicated in the generation of adaptive immune responses.

[0147] Example 3 Production and use of antibody-peptide conjugates

[0148] 3.1. Identification of a NKT activating peptide and immunisation A sequence of amino acids is identified by use of a combination of algorithms as described in patents WO 2012 / 069572, WO 2012 / 069575, WO 2013 / 174805 and WO 2018 / 189405. Selected sequences are then evaluated in silica to identify those with higher free binding energy, corresponding to highest binding affinities to CDld. Such epitopes can optionally be mutated by substitution of amino acids anchoring the epitope into the CDld cleft so as to increase CDld binding.

[0149] Selected peptides are then produced by solid-phase synthesis and used in an immunisation protocol in several mouse strains, including C57BL / 6, BALB / c and FVB / N. Natural killer T (NKT) cells elicited by this immunization protocol are identified in regional lymph nodes and in the spleen by determination of surface phenotype showing co-expression of CD3 and CD56. The peptide specificity of such cells is verified by either reactivity with CDld tetramers loaded with the corresponding peptide or by incubation in the presence of antigen-presenting cells expressing CDld, such as JAWS II cells.

[0150] 3.2. Expression of CDld on cell lines

[0151] Available cell lines such as the NT2.5 expressing the rat HER-2 / neu oncogene are maintained in culture and tested for expression of CDld by use of a specific antibody. The binding of such antibody is detected by addition of an anti-mouse IgG antibodies coupled, for instance, to a fluorochrome. If required, cells can be first incubated with reagents such as retinoic acid or rapamycin, known to increase CDld expression at cell surface, or histone deacetylases or demethylases. Alternatively, cells can be transfected with a construct containing CDld.

[0152] 3.3. Production of an antibody to HER2 and coupling to a CDld epitope payload

[0153] Antibodies to HER2 are obtained by immunization with the soluble protein in adjuvant by methods known in the art.

[0154] The CDld epitope selected above is coupled to anti-HER2 antibody by a disulfide bond and a di- peptidic linker of the Phe-Gly type, which is susceptible to cleavage by cathepsin under the acidic conditions prevailing in the endosomal pathway. See for instance Bargh JD, et al. (Chem. Soc. Rev, 2019, 48: 4361-4374) for details on the procedure.

[0155] 3.4. Inoculation of FVB / N mice with the NT2.5 cell line

[0156] The NT2.5 cell line carried the rat HER-2 antigen. Its administration in FVB / N mice elicits a tumor after inoculation into the mammary glands. The growth of the tumors is followed over time. The same experiment is repeated including IV administration of the anti-HER2 specific antibody, or with such antibody coupled to the CDld epitope payload. Tumor growth is measured and compared in between control mice and mice receiving the anti-HER2 antibody with or without payload.

[0157] 3.5. Immunisation with the selected CDld epitope

[0158] FVB / N mice are immunized with the peptide containing the selected CDld epitope, so as to elicit a population of antigen-specific NKT cells as described above, followed by inoculation of the NT2.5 cell line. The growth of the tumor is followed over time and compared to growth obtained in the absence of NKT cells as a control.

[0159] In a parallel experiment, the antibody to HER2 is administered by the IV route just after tumor inoculation. In a third group, mice are treated by the anti-HER2 antibody coupled to the CDld epitope payload.

[0160] Tumor growth is compared between these three experimental conditions and shown to be minimally reduced by administration of the anti-HER2 antibody, but significantly reduced by administration of the anti-HER2 antibody containing the CDld epitope payload.

[0161] Example 4 Addition of a flanking sequence specifically recognizing the CD8 cofactor to boost the efficacy of administration of antibody-peptide payload complexes

[0162] By contrast to class I or class II MHC molecules, CDld does not contain a sequence specific for the binding of CD8 coactivator. In situations of tumors or chronic intracellular infections, eliciting NKT cells showing the highest cytotoxic activity, such as NKT cells carrying CD8+ coactivator, may be desirable.

[0163] The CDld-restricted epitope X1-X2-X3-X4-X5-X6-X7 defined above is modified by incorporating in its flanking regions an amino acid sequence with binding properties for CD8. Thus, the sequence DQTQDTE (SEQ ID NO:1), which corresponds to the CD8 binding domain of the alpha 3 chain of class I MHC, is added at the carboxy-terminal end of the X1-X2-X3-X4-X5-X6-X7 peptide. The resulting sequence is then coupled via a covalent bond cleavable under acidic conditions (as prevailing in late endosome) to a monoclonal antibody directed towards the HER2 surface antigen.

[0164] The antibody-peptide conjugate is administered by the IV route in a humanized mouse model grafted with a HER-2+ human tumor. The humanized mouse model obtained from irradiated C57BL / 6 mice reconstituted with human CD34+ progenitors purified from umbilical vein blood. Such mice are vaccinated by administration of the peptide X1-X2-X3-X4-X5-X6-X7 extended with the sequence of SEQ ID NO:1 twice in adjuvant at a week interval prior to administration of the antibody-peptide conjugate.

[0165] By comparison, mice first vaccinated with the peptide X1-X2-X3-X4-X5-X6-X7 extended with the sequence of SEQ ID NO:1 show a significant reduction of an HER2+ tumor, as compared to mice with no peptide vaccination and / or no antibody-peptide conjugate administration, indicating that the vaccination has induced specific NKT cells towards the peptide. Moreover, practically all specific NKT cells obtained by vaccination show a CD8+ NKT phenotype.

Claims

Claims1. An antibody capable of binding a cell surface antigen, wherein said antibody is conjugated to a peptide comprising an epitope capable of being presented by a CD1 molecule on the cell surface.

2. The antibody according to claim 1, wherein said cell surface antigen is a tumor cell surface antigen.

3. The antibody according to claim 1 or 2, wherein said antibody is capable of being internalized upon binding to a cell expressing the cell surface antigen.

4. The antibody according to any one of the preceding claims, wherein said cell surface antigen is selected from the group consisting of: CD30, CD79B, CD22, BCMA, CD19, CD33, CD20, ROR1, HER2, Nectin-4, Tissue Factor, TACSTD2, EGFR, F0LR1, C-MET, CEACAM5, HER3, Mesothelin, PSMA, CD71, B7-H3, MUC1, IGF1R, ADAM9, CEACAM6, CLDN18, AXL, ROR2, MUC18, SDPTP2B, MAGE, MART-1, tumor-associated B Cell Receptor, tumor-associated T Cell Receptor, TROP-2, DLL-3 and CD56.

5. The antibody according to any one of the preceding claims, wherein said antibody is an IgG antibody, such as a full-length IgGl antibody, a Fab fragment, a Fab'2 fragment, a single-chain antibody, such as an scFv antibody, or an antibody domain.

6. The antibody according to any one of the preceding claims, wherein said antibody is a multivalent antibody, such as a bivalent antibody.

7. The antibody according to any one of the preceding claims, wherein said antibody is a multispecific antibody, wherein said multispecific antibody further binds a different epitope of the same cell surface antigen or further binds to a different cell surface antigen.

8. The antibody according to claim 7, wherein said multispecific antibody binds two or more cell surface antigens that are present on the surface of the same target cell.

9. The antibody according to any one of the preceding claims, wherein said peptide has a length of at least 7 amino acids, such as at least 11 amino acids, for example at least 15 amino acids, such as at least 20 amino acids, for example between 20 and 50 amino acids, such as between22 and 30 amino acids.

10. The antibody according to any one of the preceding claims, wherein said epitope comprises the sequence X1-X2-X3-X4-X5-X6-X7, wherein Xi and X7 independently are F, W, T, H or Y, wherein X2, X3, X5, and Xe may be any amino acid, including a non-natural amino acid, and wherein X4 is I, L, M or V.

11. The antibody according to any one of the preceding claims, wherein said GDI molecule is CDla, CDlb, CDlc or CDld.

12. The antibody according to any one of the preceding claims, wherein said antibody is conjugated to said peptide via a linker, such as a covalent linker.

13. The antibody according to claim 12, wherein said linker is a self-immolative linker.

14. The antibody according to claim 12 or 13, wherein said linker is susceptible to cleavage by cathepsins, such as cathepsin D.

15. A method for the treatment of a disease, comprising administration, to a subject, of the antibody of any one of the preceding claims.

16. The method according to claim 15, wherein the method comprises a further step of eliciting or administering epitope-specific NKT cells in said subject.

17. The method according to claim 15 or 16, wherein the method comprises, or further comprises, immunisation of said subject with (i) a peptide antigen comprising the same epitope as comprised within the peptide conjugated to the antibody or (ii) a nucleic acid encoding such a peptide antigen.

18. The method according to claim 15 or 16, wherein the method comprises, or further comprises, administration of epitope-specific NKT cells, preferably ex vivo expanded, or ex vivo autologous antigen-presenting cells loaded with an NKT-specific epitope, to said subject.

19. The method according to any one of claims 15 to 18, further comprising administration of irradiation or a compound that increases the level of said CD1 molecule, such as a CDld molecule, on the cell surface.

20. The method according to claim 19, wherein said compound is retinoic acid, rapamycin, an epigenomic regulator.

21. The method according to any one of claims 15 to 20, wherein the antibody is administered more than once.

22. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 14 and a pharmaceutically-acceptable carrier.

23. A method for producing an antibody according to any one of claims 1 to 14 comprising the step of conjugating a peptide comprising an epitope capable of being presented by a CD1 molecule on the cell surface to an antibody capable of binding a cell surface antigen.

Images