Anti-gangliosides immunoglobulins with increased activity

Abstract

The present invention relates to antibodies directed against ganglioside tumor antigens which can be obtained in Phaeodactylum tricornutum, said antibodies presenting an increased FcRn affinity and an increased direct cytotoxicity activity; their uses as medicament and for cancer treatment more specifically.

Description

[0001] ANTI-GANGLIOSIDES IMMUNOGLOBULINS WITH INCREASED ACTIVITY

[0002] Field of the invention

[0003] [1] The present invention relates to immunoglobulins directed against tumor antigens corresponding to gangliosides and having increased cytotoxicity and a potential increased plasma half-life, to a method for producing such immunoglobulins, and to their uses, in particular for therapeutic purposes.

[0004] Background of the invention

[0005] [2] Protein therapeutic agents are today widely used in clinical practice and are rapidly replacing non-specific, low-molecular-weight therapeutic agents. Indeed, these protein therapeutic agents have the advantage of very high specificity, more limited side effects, and lower toxicity.

[0006] [3] Among them, monoclonal antibodies (mAbs) and their derivatives (immunoconjugates and Fc fusions) represent the majority of therapeutic proteins currently under development due to a blood half-life that may exceed 3 weeks. The immunotherapy market already amounted to more than 200 billion US dollars (USD) in 2023. However, this large amount corresponds only to a relatively limited number of therapeutic antibodies and should therefore continue to grow considerably in the coming years. Among these therapeutic antibodies, mention may in particular be made of RITUXAN, AVASTIN, HERCEPTIN, and REMICADE, each of which achieves sales of more than one billion dollars.

[0007] [4] Among the antibodies targets, gangliosides are acidic glyco sphingolipids (GSL) carrying one or more sialic acid residues on their carbohydrate moieties that are mainly located in glycolipid-enriched domains, also called lipid rafts, on the outer leaflet of the plasma membrane bilayer. Raft domains are composed of cholesterol, phospholipids, and glyco sphingolipids and enriched in specific proteins. They engage in major cellular pathways and are involved in cell biological properties under physiological conditions. The carbohydrate part of gangliosides is constituted by glucose, galactose, N- acetylgalactosamine and sialic acid residues, which could exhibit numerous structural modifications such as O-acetylation, A-acetylation or sulfation. Irrespective of the elongation status of their core structure (Gaipi-3GalNAcpi-4Gaipi-4Glcpi-lCer), gangliosides are classified in four series (0-, a-,b- and c-series depending on the number of sialic acid residues (from 0 to 3) linked to lacto sylceramide.

[0008] [5] Cancer development is generally associated with glycosylation changes of glycolipids and glycoproteins expressed at the cell surface, and thus can imply gangliosides. These modified carbohydrate epitopes are tumor antigens defined as tumor associated carbohydrate antigens (TACA), such as GD2 or GD3 ganglioside in neuroectodermal derived (ND) cancers.

[0009] [6] The ganglioside GD2 is a disialylated glycolipid prominently expressed on the surface of various tumor cells, including neuroblastoma, melanoma, small cell lung cancer, and certain sarcomas. Moreover, the expression of GD2 in malignant cells is associated with tumor progression, metastasis, and immune evasion, as it can modulate cell signaling pathways that promote survival and proliferation. Additionally, GD2 contributes to the suppression of immune responses by interacting with inhibitory receptors on immune cells, thereby creating a microenvironment conducive to tumor growth.

[0010] [7] Now, the GD2 expression on normal tissues is restricted — primarily limited to the central nervous system, peripheral nerves, and skin melanocytes — . Moreover, this expression level of ganglioside GD2 in normal cells of the peripheral nervous system is very low, even undetectable by conventional methods such as fluorescence microscopy. Quantitative studies show that GD2 expression in peripheral nerves (such as the phrenic nerve or vagus nerve) is significantly lower than in tumor cells (DORONIN et al., BMC Cancer, vol.14, p:295, 2014). Furthermore, GD2 represents only approximately 1 to 2% of total gangliosides on the surface of normal cells, whereas its expression in neuroblastomas can reach up to 10 million molecules per cell, a level 3 to 8 times higher than that of other tumor- associated gangliosides. [8] Accordingly, GD2 is an attractive target for cancer immunotherapy, particularly in neuroblastoma, where GD2 abundance and homogeneity of expression are exceptional. These characteristics have led to the development of several therapeutic strategies, most notably monoclonal antibodies such as dinutuximab and naxitamab.

[0011] [9] Dinutuximab (Chl4.18, tradename UNITUXIN) and dinutuximab beta (tradename QARZIBA) are monoclonal antibodies used as a second-line treatment for children with high-risk neuroblastoma. Each antibody is made of both mouse and human components and targets glycolipid GD2, expressed on neuroblastoma cells and on normal cells of neuroectodermal origin, including the central nervous system and peripheral nerves. They differ in that dinutuximab is manufactured using mouse cells, and dinutuximab beta is manufactured using hamster ovary cells.

[0012]

[0010] Therapies using these two antibodies are based on successive treatment cycles (5 in general), each cycle corresponding to the administration of a total antibody dose of 100 mg / m2over several days.

[0013]

[0011] While these therapies have demonstrated efficacy, challenges such as neurotoxicity and pain due to GD2 expression in normal nerve tissues remain significant hurdles in optimizing treatment outcomes. In fact, if lower doses cause fewer serious adverse effects (neuropathic pain, capillary leak syndrome, hypersensitivity reactions), this reduction impact the treatment anticancer efficacy. Thus, in relapsed or refractory neuroblastoma, doses below 100 mg / m2have shown a partial response in only 20 to 30% of patients, compared to 35 to 40% with the standard dose. Accordingly, and only in case of a severe reaction, a 50% dose reduction (i.e., 50 mg / m2) can be applied to the patient, but this is associated with a decrease in the immune response and antibody-dependent cytotoxicity (ADCC).

[0014] Summary of the invention

[0015]

[0012] In the context of evaluating the use of the microalga Phaeodactylum tricornutum for the production of therapeutic proteins, this microalga was used experimentally for the production of recombinant human antibodies directed against the Marburg virus (HEMPEL et al., Microb. Cell Fact., vol.16, p.131 , 2017) and against antigenic epitopes present on the surface of the hepatitis B virus (HEMPEL et al., PLoS One, vol.6(12), p.e28424, 2011; HEMPEL & MAIER, Microb. Cell Fact., vol.l l, p.126, 2012; VANIER et al., PLoS One, vol.l0(10), p.e0139282, 2015; and VANIER et al., Biotechnol. J., vol.13, p.1700496, 2018). These various studies have demonstrated that a monoclonal antibody manufactured by P. tricomutum is capable of binding to human Fey receptors, in particular FcyRI and FcyRIIIa, which suggests that it could be used effectively in human immunotherapy to induce phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC) (VANIER et al., cited above, 2018). Moreover, it has been shown that P. tricomutum is capable of glycosylating the Fc portions of monoclonal antibodies with oligomannosides bearing 5 to 9 mannose residues (VANIER et al., cited above, 2015), which are structurally identical to those of mammals (DUMONTIER et al., Carbohydr. Polym., vol.259, p.117660, 2021).

[0016]

[0013] In the context of a better characterization of the secreted antibodies produced by P. tricomutum, the inventors demonstrated that the obtained dinutuximab has the same 3D structure as compared to the QARZIBA -i.e., dinutuximab produced in CHO-. However, the inventors have unexpectedly demonstrated that the dinutuximab s obtained from P. tricomutum exhibit interesting distinctive properties including a higher binding affinity for human FcRn as compared to QARZIBA -i.e., the same antibody produced in CHO. Knowing that the antibody structure is the same, whether produced in CHO or in P. tricomutum, these properties therefore result in all likelihood from their specific glycosylation profile.

[0017]

[0014] The function of the FcRn receptor (neonatal Fc receptor) is based on its ability to handle IgG (and also albumin) through pH-dependent binding. The importance of this IgG-FcRn interaction in the half-life of antibodies was thus demonstrated in a mouse study, in which the half-life of mouse IgGl antibody was only 1.4 days in FcRn-deficient mice (mFcRn- / -), compared with a half-life of 9 days in mFcRn- / + and mFcRn+ / + mice. It is also now known that the clinical failure of murine monoclonal antibodies was due to their very short half-life in humans, which resulted from the low affinity of human FcRn for mouse IgG.

[0015] Following a better understanding of the associated mechanisms, it is now known that FcRn ensures functions of:

[0018]

[0016] - recycling allowing IgG (and albumin as well) to escape endothelial catabolism,

[0019]

[0017] - transcytosis promoting the biodistribution of IgG in the organism, and

[0020]

[0018] - cooperation during phagocytosis.

[0021]

[0019] The recycling function of IgG consists in extracting IgG from the pathway of endothelial catabolism of plasma proteins in order to restore them intact to the circulation. IgG share this property exclusively with albumin, which also binds to FcRn and benefits from recycling. In mice, it has been calculated that FcRn protects from degradation half of the internalized albumin molecules and four- fifths of IgG, which may be a mechanism energetically more economical for the organism than the de novo synthesis of these molecules. This recycling explains their long half-life (three weeks for IgG), while allowing the maintenance of high plasma concentrations since IgG and albumin represent 70% of plasma proteins. The catabolism of IgG and albumin increases when their blood concentrations are too high, due to the saturation of FcRn which cannot recycle more of them. It has been demonstrated that a variant M428L / N434S in bevacizumab and cetuximab results in a 11 higher affinity for FcRn. Such higher affinity results in an increase in the plasma half-life of the corresponding antibodies by a factor of 4.3 and 4.8 respectively in transgenic mouse expressing human FcRn, and an increased in their plasma half-life by a factor of 3.2 and 3.1 respectively in a monkey model (ZALEVSKY et al., Nat. Biotechnol., vol.28(2), p:157-159, 2010).

[0022]

[0020] On this basis, the 14-fold FcRn affinity increase of dinutuximab produced in P. tricornutum anticipates a 5- to 4-fold serum half-life increase of dinutuximab -i.e., 32 days instead of 8 days-.

[0023]

[0021] The transcytosis of IgG from one side to the other of epithelia or endothelia is the second major function of FcRn, making it possible to ensure their biodistribution in the organism. More specifically, FcRn is expressed on the endothelial cells of blood vessels, where it facilitates the bidirectional transport of IgG across the endothelial barrier. Their binding to FcRn is therefore a critical parameter of the tissue or solid tumor penetration of specific therapeutic antibodies (e.g., monoclonal antibodies such as trastuzumab or rituximab). Mouse models of neuroblastoma have shown that dinutuximab has limited penetration into tumors (especially tumors > 5 mm), which is mainly limited to their periphery.

[0024]

[0022] Also, the improved binding to FcRn of dinutuximab produced in P. tricornutum anticipates an improved tissue and solid tumor penetration compared to QARZIBA.

[0025]

[0023] Finally, a third major function of FcRn would be to cooperate with conventional FcyR in the functions of phagocytosis and presentation of immune complexes, perhaps taking over from FcyR for the binding to IgG in acidic endosomal compartments. This still poorly understood function of FcRn probably explains why it is expressed in antigen- presenting cells and neutrophil polymorphonuclear cells.

[0026]

[0024] Now, the improved binding to FcRn of dinutuximab produced in P. tricornutum also anticipates an improved presentation by antigen presenting cells (APCs) compared to QARZIBA.

[0027]

[0025] The CDC activity of antibodies can be important in their therapeutic efficacy. It is known that QARZIBA, while exhibiting ADCC activity, also exhibits moderate to low CDC activity.

[0028]

[0026] Now, it is known that a high mannose glycosylation profile of antibodies reduces this activity (as does the absence of galactose), as it affects their binding to the Clq protein. Now, the inventors have determined that dinutuximab produced in P. tricornutum exhibits a high mannose glycosylation profile. Therefore, it is very likely that dinutuximab produced in P. tricornutum exhibits lower CDC activity than QARZIBA.

[0029]

[0027] Nevertheless, while the contributing role of the complement-dependent cytotoxicity (CDC) in QARZIBA therapeutic effects is quite modest, its binding to the complement proteins is responsible for one of the main side effects associated with the use of GD2- specific monoclonal antibodies, namely antibody-induced allodynia (SORKIN et al., Pain, vol: 149, p: 135-142, 2010).

[0028] Thus, the reduced CDC activity of dinutuximab produced in P. tricornutum anticipates a decreased of the antibody-induced allodynia observed with QARZIBA.

[0030]

[0029] Now, the ADCC activity is critical for the therapeutic potential of QARZIBA. Unfortunately, the inventors have demonstrated that the binding of dinutuximab to the FCyRIIIa and FcyRI receptors is reduced (from 2-fold to 4-fold) compared to QARZIBA. These two receptors are involved in the ADCC response and, for FCyRI, in the ADCP response.

[0031]

[0030] Thus, the decreased binding of dinutuximab produced in P. tricornutum to FCyRIIIa and FcyRI also anticipates a decreased ADCC activity compared to QARZIBA. Simultaneously, the decreased binding of dinutuximab produced in P. tricornutum to FcyRI anticipates a decreased ADCP activity compared to QARZIBA.

[0032]

[0031] This decreased ADCC activity of dinutuximab produced in P. tricornutum as compared to QARZIBA was confirmed by the inventors in an in vitro assay on IMR-32 and LAN-1 cell lines both derived from human neuroblastoma.

[0033]

[0032] Now, as part of the characterization of their antibody, the inventors had already initiated in vivo experiments in which the test antibody (or QARZIBA) was co-injected with tumor cells (LAN1 and IMR32) into a two-day-old zebrafish embryo. The evolution of tumor size and the number of metastases was then determined three days after this coinjection.

[0034]

[0033] These experiments aimed to more precisely determine the therapeutic potential of an antibody, with the zebrafish embryo model constituting a first in vivo model allowing a more precise approach to the therapeutic potential of an antibody.

[0035]

[0034] FcRn is not functionally expressed in the zebrafish embryo, or its expression is very limited. Thus, its expression is confined to certain tissues such as the yolk sac or primitive intestine but is absent in the vessels or tissues relevant to the evaluation of injected therapeutic antibodies.

[0036]

[0035] Similarly, the two-day-old zebrafish embryo does not allow for the observation of a CDC response, the components of which are absent at this stage of development.

[0036] Finally, the zebrafish embryo model primarily allows for the assessment of an antibody's ADCC activity, even if all the components of this response are not yet present, and also the potential ADCP activity of the same antibody. It should be noted that this system also allows for the observation of potential direct effects of certain antibodies (cytotoxicity) when they exist.

[0037]

[0037] In this zebrafish model, the results unexpectedly showed a comparable response of dinutuximab produced in P. tricomutum with QARZIBA (dinutuximab in CHO) at the highest dose (3 mg / mL). Furthermore, the results have shown a much superior response (in fact much more gradual) of dinutuximab produced in P. tricomutum compared to QARZIBA (dinutuximab in CHO) at the lower doses (0.03 and 0.3 mg / mL).

[0038]

[0038] In view of the previous results on ADCC and ADCP, the results observed in zebrafish are therefore the opposite of what was expected, whether for the maximum dose or for the intermediate doses.

[0039]

[0039] The only coherent explanation for these results is that dinutuximab produced in P. tricomutum possesses an activity other than ADCP and ADCC, which cannot be CDC or FcRn-mediated, and which is much superior to that of QARZIBA. It is this complementary activity that would explain why dinutuximab produced in P. tricomutum has an activity similar to QARZIBA at 3 mg / ml (despite its much-reduced ADCC activity) and much superior to it at lower concentrations, whether at 0.3 or even 0.03 mg / ml.

[0040]

[0040] In the context of this "other" activity, it has been disclosed that the antitumor effects of GD2-specific antibodies -i.e., not only dinutuximab- are certainly based on antibody-dependent cellular cytotoxicity (ADCC), but also on direct induction of cell death [for a review, See PEREZ HORTA et al., Immunotherapy., vol:8, p: 1097-1117, 2016). This cell death induction is not classical and combined features of both apoptosis and necrosis in GD2-positive tumor cells, which include alteration of mitochondrial membrane potential, induction of apoptotic volume decreases and cell membrane permeability. Moreover, this cytotoxic effect was mediated exclusively by specific binding of anti-GD2 antibodies with ganglioside GD2, since it is not observed in GD2- negative tumors. Finally, the level of GD2 expression correlated with susceptibility of tumor cell lines to cytotoxic effect of anti-GD2 antibodies.

[0041]

[0041] Accordingly, the direct cell death induction activity of dinutuximab produced in P. tricornutum is anticipated to be far higher to the one of QARZIBA. This increased activity is determined by the inventors directly on cell lines expressing GD2 (IMR32 and LAN1) as compared to control cell lines.

[0042]

[0042] Since this direct cytotoxicity is disclosed for different anti-GD2 antibodies, an improved direct cell death induction activity of GD produced in P. tricornutum is anticipated to be far higher to the one of QARZIBA. This increased activity is determined by the inventors directly on cell lines expressing GD2 (IMR32 and LAN1) as compared to control cell lines not expressing GD2. Now, this direct cytotoxicity was not observed only with GD2 antibodies, but also with antibodies against various tumor- associated gangliosides including GM2 (RETTER et al., Cancer Res, vol.65(14), p:6425-6434, 2005), GM3 (DORVIGNIT et al., Immunobiology, vol.220(12), p:1343- 1350, 2015), O-acetyl GD2 (COCHONNEAU et al., Cancer Lett., vol.333(2), p:194- 204, 2013), or GD3 (KRISTAL et al., J. Biol. Chem., vol.274, p:23169-75, 1999).

[0043]

[0043] Ultimately, dinutuximab produced in P. tricornutum is positioned as an antibody with a therapeutic potential similar to the one of QARZIBA, while exhibiting a half-life, presentation to APCs and penetration into tumor masses much higher, and simultaneously lower side effects.

[0044]

[0044] Moreover, the inventors’ discovery makes it possible to anticipate the further obtaining antibodies directed against gangliosides with increased direct cytotoxicity activity.

[0045]

[0045] The inventors have further developed an antibody derived from dinutuximab with increased ADCC activity, -i.e., AB2S-. The inventors demonstrate that, the ADCC activity of this derived dinutuximab, when produced in P. tricornutum, is similar to the one of QARZIBA.

[0046]

[0046] Therefore, this derived dinutuximab is positioned as an antibody with a therapeutic potential greater to the one of QARZIBA, while exhibiting a tumor cell killing activity -i.e., with the addition of ADCC and direct cell death induction activities-, a halflife, presentation to APCs and penetration into tumor masses much higher, and simultaneously lower side effects. It should be noted that the use of this dinutuximab derivative at effective concentrations much lower than QARZIBA in all likelihood makes it possible to further reduce the side effects linked to CDC activity, which is of course dependent on antibody concentration.

[0047]

[0047] Consequently, a first subject of the invention relates to a method for producing antibodies having an increased plasma half-life, comprising the steps of:

[0048]

[0048] - producing the antibody in a cell system;

[0049]

[0049] - purifying said antibody; and

[0050]

[0050] - determining the binding affinity of said antibody for the FcRn receptor, preferably the human FcRn receptor;

[0051]

[0051] characterized in that said antibodies are directed against a ganglioside tumor antigen and said cell system is a culture of Phaeodactylum tricomutum.

[0052]

[0052] A second subject of the invention relates to an antibody obtained or which can be obtained by the method as described above.

[0053]

[0053] A third subject relates to a composition, used as a medicament, comprising such an antibody.

[0054]

[0054] A fourth subject relates to a composition comprising an antibody as described above, which antibody is directed against a ganglioside tumor antigen, for use in treating a subject suffering from cancer.

[0055]

[0055] Advantageously, said antibody as defined is administered at a frequency at least twice lower, preferably at least three times lower, and more preferably at least four times lower than the frequency with which the same antibody produced in mammalian cells, preferably in CHO, is administered.

[0056]

[0056] Still advantageously, said antibody as defined is administered at a dose which is at least 25% lower, preferably at least two-fold lower, as an example at least three-fold lower, and more preferably at least four-fold lower than the recommended dose at with which the same antibody but produced in mammalian cells, preferably in CHO, is administered.

[0057] Description of the figures

[0058]

[0057] Figure 1 shows the superposed FITR spectrum of dinutuximab produced in CHO cells (QARZIBA) or in Phaeodactylum tricornutum (AB IS) and the difference spectrum (black spectrum, between FTIR spectra of both samples).

[0059]

[0058] Figure 2 shows the FITR spectrum of dinutuximab produced in CHO cells (QARZIBA) or in Phaeodactylum tricornutum (AB IS), with a zoom on the spectral region related to the glycan absorption.

[0060]

[0059] Figure 3 shows the mean preprocessed FITR spectrum of dinutuximab produced in CHO cells (QARZIBA) or in Phaeodactylum tricornutum (AB IS), with a zoom on the spectral region related to the glycan absorption. A statistical pairwise comparison between FTIR spectra of both samples was also performed to evidence spectral changes.

[0061]

[0060] Figure 4 shows the mass spectrometry analysis of the A-glycosylation of the antibody produced in CHO (QARZIBA) allowing the identification of the A-gly can’s structures attached to the EEQYNSTYR (SEQ ID N°16) peptide present on the Fc part.

[0062]

[0061] Figure 5 shows the mass spectrometry analysis of the A-glycosylation of the AB IS antibody allowing the identification of the A-gly can’s structures attached to the EEQYNSTYR (SEQ ID N°16) peptide present on the Fc part.

[0063]

[0062] Figure 6 shows ADCC activity (Relative Unit: RLU) on LAN-1 of dinutuximab produced in CHO cells (QARZIBA) or in Phaeodactylum tricornutum (AB IS) and its derivative produced in Phaeodactylum tricornutum (AB2S).

[0064]

[0063] Figure 7 shows the binding affinity (Relative Unit: RLU) for human FcRn of dinutuximab produced in CHO cells (QARZIBA) or in Phaeodactylum tricornutum (AB IS) compared with the positive control (IgG Control).

[0064] Figure 8 shows the binding affinity (Relative Unit: RLU) for human FcRn of dinutuximab produced in CHO cells (QARZIBA) or in Phaeodactylum tricornutum (AB IS) together with dinutuximab derivative (AB2S-batch 1 and AB2S-batch 2) compared with the positive control (IgG Control).

[0065]

[0065] The figure 9 shows the normalized tumors size for zebrafish co-injected or not with the dinutuximab produced in P. tricornutum (AB IS) or in CHO (CHO-Ref for the three tested conditions against the vehicle control (no treatment - PBS only).

[0066] Detailed description of the invention

[0067]

[0066] Phaeodactylum tricornutum is a marine diatom. It is the only species of the genus Phaeodactylum and one of the model species widely used for the study and characterization of diatom metabolism. Although being a coastal species, it is found in many ecosystems throughout the world ranging from Finland to Ecuador. It can adopt different morphotypes (fusiform, oval, tri-radiate) and is the only diatom known to date capable of surviving and multiplying without a supply of silica. It has the real advantage of being cultivable and transformable by different methods (biolistic transformation, electroporation and bacterial conjugation), which makes it a particularly relevant production tool.

[0068]

[0067] Strains of Phaeodactylum tricornutum are available from CCAP (Culture Collection of Algae and Protozoa), BIGELOW (National Center for Marine Algae and Microbiota), or URX (Culture Collection of Algae - The University of Texas at Austin).

[0069]

[0068] The step of producing an antibody by Phaeodactylum tricornutum can be carried out using methods well known to the person skilled in the art. Such a production step typically includes the preparation of at least one vector allowing the expression of an antibody within Phaeodactylum tricornutum. To this end, the nucleotide sequence encoding the antibody of interest (its heavy chain and its light chain) is optimized to allow better expression by Phaeodactylum tricornutum (so as to use the codons preferentially used by this microalga), before being cloned into said at least one vector, which vector integrates the regulatory sequences necessary for the expression of said antibody within this microalga. Such regulatory sequences include in particular at least one promoter and at least one terminator associated with each open reading frame (ORF) (the heavy chain and the light chain of the antibody). However, these vectors are preferably associated with selection markers usable in Phaeodactylum tricornutum (example: zeocin, nourseothricin or blasticidin) allowing the screening of microalgae having integrated said vector. Advantageously, the two ORFs may be located on the same vector and are then separated by a “linker”. The transformation of Phaeodactylum tricornutum by said at least one vector is then carried out according to techniques well known such as those described in HU & PAN (Electroporation Transformation Protocol for Phaeodactylum tricornutum, Methods Mol. Biol., vol.2050, pp.163-167, 2020). After transformation, the selection of the clones producing the antibody is carried out using the selection marker of the vector before scaling up the culture for the specifically selected clone with a culture in a photobioreactor (1 L, 10 L, 200 L and 2000 L) allowing the production of the antibody. By way of example of such a production step, mention may be made of the methods described in detail (with their vector, promoter, terminator sequence, etc.) in international application WO 2023 / 208883.

[0070]

[0069] Advantageously, the production of the antibody in step (i) uses at least one signal peptide (secretion) functional in Phaeodactylum tricornutum, which allows the secretion of said antibody into the culture medium (e.g., signal peptide E and other alternatives described in international application WO 2023 / 208883), or a DDEL sequence (SEQ ID N°: 1) for retention in the endoplasmic reticulum (e.g., HEMPEL et al., cited above, 2011; or alternatively the HDEL sequence (SEQ ID N°:2) or the KDEL DDEL sequence (SEQ ID N°:3)), allowing the retention of said antibody in the endoplasmic reticulum of Phaeodactylum tricornutum.

[0071]

[0070] Preferably, the production of the antibody in step (i) uses at least one signal peptide allowing the secretion of said antibody into the culture medium.

[0072]

[0071] An antibody is an immunoglobulin molecule corresponding to a tetramer comprising four polypeptide chains, of which two are identical heavy chains (H) (about 50 to 70 kDa in full length) and two are identical light chains (L) (about 25 kDa in full length), interconnected by disulfide bonds. The light chains (L) are classified into kappa and lambda types. The heavy chains (H) are classified into five subtypes (y, p, a, a or 5). The nature of the heavy chain subtype determines the type of immunoglobulin (IgG: y, IgA: a, IgM: p, IgD: 5, IgE: a).

[0073]

[0072] Each light chain consists of one constant domain (CL) and one variable domain (VL). The heavy chains are composed of an N-terminal variable domain (VH) and a constant region composed of three or four constant domains (CHI, CH2, CH3 and CH4) depending on the isotype (CHI, CH2 and CH3 for IgG).

[0074]

[0073] The constant domains are characterized by an amino acid sequence very similar from one antibody to another, which is characteristic of the species and the isotype. They are not involved in antigen recognition as such but are involved in the activation of the complement system as well as in the elimination of immune complexes (antibody bound to its antigen) by immune cells possessing receptors for constant fragments (Fc receptors). The binding of an IgG thus involves a region overlapping the CH2 and CH3 domains of the IgG.

[0075]

[0074] The variable domains (VL and VH) can be subdivided into hypervariable regions (called complementarity determining regions (CDR)) which are interspersed with more conserved regions (called framework regions (FR)). Each VH and VL consists of three CDR and four FR, arranged from the amino-terminal end to the carboxy-terminal end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0076]

[0075] The attribution of amino acids to each domain follows well-known conventions, in particular with respect to their numbering (IMGT, The International Immunogenetics Information System).

[0077]

[0076] Ultimately, the functional capacity of an antibody to bind to a particular antigen depends on the variable domains of each of its light / heavy chain pairs and is largely determined by the CDR.

[0078]

[0077] For the purposes of the present invention, the term “antibody” refers to a monoclonal antibody per se.

[0079]

[0078] Advantageously, such an antibody is directed against a tumor antigen.

[0079] As used herein, a “tumor antigen” means an antigenic substance produced by tumor cells.

[0080]

[0080] The tumor antigen is a ganglioside.

[0081]

[0081] Monosialotetrahexosylganglioside (GM1) is a ganglioside that contains a sialic acid residue. GM1 has important physiological properties and impacts neuronal plasticity and repair mechanisms, as well as the release of neurotrophins in the brain. Antibodies directed against GM1 can be readily obtained by the person skilled in the art.

[0082]

[0082] GM2 is a glycosphingolipid expressed on the surface of various cancer cells, including melanoma, neuroblastoma, and some breast and lung cancers. Antibodies directed against GM2 include BIW-8962 (BAZ et al., Oncol. Ther., vol. 4(2), p:287-30, 2016) and KM966 (PARAJULI et al., Cancer Lett., vol.165(2), p: 179-84, 2001).

[0083]

[0083] GM3 is a sialylated glyco sphingolipid ubiquitously expressed in cell membranes, but its overexpression is frequently observed in various cancers, including melanoma, breast cancer, and gliomas. Among the monoclonal antibodies (mAbs) targeting GM3, 14F7hT is a humanized IgGl antibody specific for GM3(Neu5Gc), a tumor-associated variant of GM3 ((DORVIGNIT et al., abovementioned, 2015),).

[0084]

[0084] GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, and whose expression in normal tissues is very limited, mainly in the cerebellum and peripheral nerves. Antibodies directed against GD2 can be readily obtained by the person skilled in the art and include dinutuximab and naxitamab.

[0085]

[0085] O-acetyl-GD2 is a GD2 ganglioside whose terminal sialic acid is modified by the addition of an O-acetyl group. O-acetyl-GD2 is expressed, like GD2, in tumors of neuroectodermal origin, but is not expressed, unlike GD2, in normal tissues. Antibodies directed against O-acetyl-GD2 can be readily obtained by the person skilled in the art and include antibody 8B6 (WO 2008 / 043777).

[0086]

[0086] The ganglioside GD3 is a disialylated glyco sphingolipid highly expressed in several malignant tumors, including melanoma, neuroblastoma, sarcoma, and some brain cancers. GD3 antibodies include R24 (HOUGHTON et al., Proc Natl Acad Sci U S A , vol.82(4), p: 1242-6., 1985) and KM8138 (NAKAMURA et al., Cancer Immunol Immunother,, vol.50(5), p:275-84, 2001).

[0087]

[0087] Preferably, the ganglioside tumor antigen is GM2, GM3, GD2, O-acetyl GD2 or GD3.

[0088]

[0088] Alternatively, the ganglioside tumor antigen is GM1, GD2, and O-acetyl-GD2.

[0089]

[0089] Still preferably, the ganglioside tumor antigen is GD2 or O-acetyl-GD2.

[0090]

[0090] The invention is also directed to antibody targeting tumor antigen inducing direct cytotoxicity activity.

[0091]

[0091] Accordingly, the invention is directed to antibodies directed to EGFR or MUC1.

[0092]

[0092] ErbB receptors are expressed in various tissues of epithelial, mesenchymal, and neuronal origin. Under normal conditions, the activation of ErbB receptors is controlled by the spatial and temporal expression of their ligands, which are members of the EGF growth factor family. The binding of the ligand to ErbB receptors induces the formation of receptor homodimers and heterodimers and the activation of the intrinsic kinase domain, leading to phosphorylation on specific tyrosine kinase residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for various proteins whose recruitment leads to the activation of intracellular signaling pathways. Among the ErbB receptors, EGFR is known to play an essential role in the regulation of cell proliferation and differentiation. Antibodies directed against EGFR can be readily obtained by the person skilled in the art and include matuzumab (US 5,558,864; EP 0531 472), cetuximab (US 7,060,808), and panitumumab (US 6,235,883).

[0093]

[0093] The short variant SI of mucin (MUC1), also called polymorphic epithelial mucin (PEM) or epithelial membrane antigen (EMA), is a glycoprotein exhibiting extensive O- glycosylation of its extracellular domain. In the healthy organism, mucins line the apical surface of epithelial cells of the lungs, stomach, intestines, eyes, and several other organs to provide protection. By binding pathogens to the oligosaccharides of its extracellular domain, they prevent said pathogens from reaching the cell surface. MUC1 also has a signaling capacity. However, the overexpression of MUC1 is often associated with cancers of the colon, breast, ovary, lung, and pancreas. Antibodies directed against MUC1 can be readily obtained by the person skilled in the art and include clivatuzumab and gatipotuzumab.

[0094]

[0094] Preferably, the antibody is an IgG.

[0095]

[0095] Preferably also, said antibody is a human antibody or one of its derivatives.

[0096]

[0096] As used herein, the expression “derivative” refers to a chimeric or humanized antibody.

[0097]

[0097] By “derivative antibody” is also meant an antibody comprising one or more point mutations making it possible to improve its properties (e.g., ADCC). In this respect, such a derivative antibody may comprise one or more mutations (2, 3, 4, 5, etc.) in its constant regions (relative to the wild-type sequences of the human antibody) so as to increase its binding affinity for the FcRn receptor. By way of example of such mutations, mention may be made of patents EP 1817340 Bl, EP 2164873 Bl, EP 2552955 Bl and EP 3181581 Bl.

[0098]

[0098] Advantageously, said derivative antibody comprises at most three mutations in its constant regions making it possible to increase its binding affinity for the FcRn receptor, preferably at most two mutations, and more particularly preferably at most one mutation in its constant regions.

[0099]

[0099] According to a particular embodiment, said derivative antibody comprises no mutation making it possible to increase its binding affinity for the FcRn receptor.

[0100]

[0100] By “chimeric antibody” is meant an antibody composed of the variable domains of a non-human antibody (generally murine) and of constant domains. In this respect, such a chimeric antibody also comprises at least the light chain constant domain (CL) and the heavy chain constant domains (CHI, CH2, CH3 and optionally CH4) of a human antibody.

[0101]

[0101] By “humanized antibody” is meant an antibody that is partially or totally composed of amino acid sequences derived from a human antibody germline lineage by modifying the sequence of an antibody having non-human complementarity-determining regions (CDR). In this respect, such a humanized antibody also comprises at least the light chain constant domain (CL) and the heavy chain constant domains (CHI, CH2, CH3 and optionally CH4) of a human antibody. The humanization of the variable region of the antibody and optionally of the CDR is carried out by techniques that are now well known in the art. By way of example of such humanization techniques, mention may be made of British patent application GB 2188638A or US patent US 5,585,089, which describe recombinant antibodies produced comprising a substitution in only the complementaritydetermining regions or “CDR.”

[0102]

[0102] Step (ii) of antibody purification may be carried out by techniques well known to the person skilled in the art (e.g., OSSIPOW & FISCHER, Monoclonal Antibodies: Methods and Protocols; Methods in Molecular Biology Book 1131, Humana Press, Springer Protocols). Such a step typically includes harvesting the culture medium by filtration and concentrating the supernatant thus recovered. The antibody is then purified from this supernatant by affinity chromatography, typically on a protein A Sepharose column. By way of example of methods for carrying out this purification step, mention may be made of the methods described in international application WO 2023 / 208883.

[0103]

[0103] Step (iii) of determining the binding of the antibody to the FcRn receptor may be carried out by techniques well known to the person skilled in the art. Typically, such a step consists of performing a competition assay in the absence or presence of increasing concentrations of the antibody to be tested. The determination of the inhibition (IC50) by the antibody of the binding of the labeled ligand to the FcRn receptor makes it possible to deduce the binding affinity of this antibody for the FcRn receptor. Such a step is typically carried out using one of the kits available on the market. By way of example of such kits, mention may be made of the LUMIT FcRn BINDING IMMUNOASSAY kit (PROMEGA), the HTRF FcRn BINDING KIT (REVVITY), the HUMAN FcRn BINDING KIT (ACROBIOSYSTEMS), and the 1-0 FcRn BINDING ASSAY KIT (CREATIVE BIOLABS).

[0104]

[0104] According to a preferred embodiment, the method according to the invention comprises an additional step (iv) of comparing the binding affinity for FcRn of the same antibody (that is to say, the same polypeptide sequence), but produced in mammalian cells. Monoclonal antibodies produced today are mainly produced by hamster cells, namely CHO (Chinese Hamster Ovary) cells and BHK (Baby Hamster Kidney) cells, and to a lesser extent by murine cells (NSO cells). It should be noted that cells of human origin are increasingly used, including, inter alia, retinal cells (PER.C6 cells), cervical cancer cells (HeLa), T lymphocytes (Jurkat), and kidney cells (HEK 293, Human Embryonic Kidney).

[0105]

[0105] Preferably, the method according to the invention comprises an additional step (iv) of comparing the binding affinity for FcRn of the same antibody but produced in mammalian cells, preferably the same antibody produced in the CHO cell line.

[0106]

[0106] According to another preferred embodiment, the method according to the invention comprises an additional step (v) of selecting the antibody produced in Phaeodactylum tricomutum when its binding affinity for the FcRn receptor is at least 3 times higher, preferably at least 5 times higher, and more particularly preferably at least 10 times higher, than that of the same antibody (that is to say, the same polypeptide sequence) but produced in mammalian cells, preferably in CHO cells.

[0107]

[0107] The present invention also relates to an antibody obtained or which can obtained by the method described above.

[0108]

[0108] Because of its production Phaeodactylum tricomutum, such antibody has a specific A-glycosylation profile corresponding to oligomannosides -i.e., Man 5 to Man 9- . This specific N-glycosylation profile is devoid of galactose and of fucose.

[0109]

[0109] Advantageously, such an antibody is not dinutuximab.

[0110]

[0110] Preferably, said antibody is a dinutuximab derivative comprising one or more- point mutations making it possible to improve its properties, preferably its ADCC activity.

[0111] [I l l] In relation to point mutations for improving antibodies properties, they are well known from the skilled person and can include one or more of the mutations disclosed in DAMELANG et al. (Frontiers in Immunology, vol.14, 2024). Now and in relation to point mutations for improving the ADDCC activity of a specific antibody specifically , they are well known from the skilled person and can include one or more of the mutations in the heavy chain antibody sequences disclosed in SAXENA & WU (Frontiers in Immunology, vol.7, article 580, 2016), VAN DER HORST et al. (Cancers, vol.12, 3041, 2020), DIXON et al. (Cancers, vol: 13, 312, 2021) or DAMELANG et al. (Frontiers in Immunology, vol.14, 2024).

[0112]

[0112] Still preferably, said antibody one or more-point mutations related to ADCC activity are located in antibody heavy chain sequence and are selected in the group comprising G236A, S239D, F243L, P247I, R292P, S298A, Y300L, V305I, A330L,I332E, E333A, K334A, A339Q, and P396L, and more preferably are selected among S239D, A330L, and I332E.

[0113]

[0113] Still preferably, said dinutuximab derivative is AB2S, whose light and heavy chains have the sequences SED ID N°:16 and SED ID N°:17 respectively.

[0114]

[0114] Again advantageously, such an antibody is not naxitamab.

[0115]

[0115] The present invention further relates to a composition comprising such an antibody as a medicament.

[0116]

[0116] As used in the present application, the term “subject” in connection with the use of such a medicament refers to a mammal such as a rodent, a feline, a canine, a primate, or a human, preferably, said subject is a human.

[0117]

[0117] According to a preferred embodiment, the composition comprises a pharmaceutically acceptable carrier. By way of example of a pharmaceutically acceptable carrier, the composition may comprise emulsions, microemulsions, oil-in-water emulsions, anhydrous lipids, and water-in-oil emulsions, or other types of emulsions.

[0118]

[0118] Such a composition may then comprise one or more additives such as diluents, excipients, stabilizers, and preservatives. Such additives are well known to the person skilled in the art and are described in particular in Ullmann’s Encyclopedia of Industrial Chemistry, 6th Ed. (various editors, 1989-1998, Marcel Dekker) and in Pharmaceutical Dosage Forms and Drug Delivery Systems (ANSEL et al., 1994, WILLIAMS & WILKINS).

[0119]

[0119] The antibody may be solubilized in a buffer, in water, or incorporated into emulsions or microemulsions. By way of example of usable buffers, mention may be made of phosphate-buffered saline (PBS), artificial serum (150 mM NaCl in water), and Tris buffers.

[0120]

[0120] There are numerous causes of instability or degradation of antibodies, such as hydrolysis and denaturation, which may lead to a decrease in the induction of the humoral or cellular response. Stabilizers may be added to reduce or prevent such problems.

[0121]

[0121] By way of example of stabilizers, mention may be made of monosaccharides, disaccharides, polysaccharides, ionic and non-ionic detergents, alkali metal salts, phospholipids, fatty acids, polyols, and stabilizing peptides such as bovine serum albumin.

[0122]

[0122] The composition according to the invention may then be in a form suitable for parenteral administration, preferably for intravenous administration.

[0123]

[0123] The present invention finally relates to a composition comprising an antibody as described above, which antibody is directed against a tumor antigen, for use in treating a subject suffering from cancer.

[0124]

[0124] Advantageously, said antibody is administered at a frequency at least twice lower (that is to say, with a time interval at least twice longer), preferably at least three times lower, and more preferably at least four times lower than the frequency with which the same antibody produced in mammalian cells, preferably in CHO, is administered.

[0125]

[0125] As an example, the time interval between two cycles of administration of dinutuximab produced in CHO is 21 days. Accordingly, the time interval between two cycles of administration of dinutuximab produced in P. tricornutum is at least 42 days, preferably at least 63 days, and more preferably at least 84 days.

[0126]

[0126] Still advantageously, said antibody as defined is administered at a dose which is at least 25% lower, preferably at least two-fold lower, as an example at least three-fold lower, and more preferably at least four-fold lower than the recommended dose at with which the same antibody but produced in mammalian cells, preferably in CHO, is administered.

[0127]

[0127] As used herein, the term “recommend dose” correspond to the dose indicated in the New Drug Application (ND A) approval for the antibody produced in mammalian cells or which can be anticipated from its properties.

[0128]

[0128] As an example, and in relation to dinutuximab or its derivatives, the dinutuximab or derivative thereof produced in P. tricomutum is administrated at a total antibody dose lower than 75 mg / m2(antibody mass related to subject skin surface), preferably lower than 50 mg / m2, and most preferably than 25 mg / m2.

[0129]

[0129] Preferably, the antibody targets the ganglioside GM1, GD2 or O-acetyl-GD2, and most preferably GD2 or O-acetyl-GD2.

[0130]

[0130] Advantageously, the antibody is dinutuximab or a derivative thereof.

[0131]

[0131] Because of its reduced CDC activity, said antibody may result in decreased neurotoxicity or antibody-induced allodynia, that can be therapeutically used without any strong analgesic drugs co-administration unlike QARZIBA.

[0132]

[0132] Accordingly, said antibody as defined is administered, without any coadministration of at least one analgesic selected among opioids (e.g., Morphine) or gabapentinoids (e.g., Gabapentin) analgesics, preferably any co-administration of at least one opioid analgesic.

[0133]

[0133] As used herein, “co-administration” refers to administration before, simultaneously and or following the antibody administration.

[0134]

[0134] The following examples are provided for illustrative purposes and should not be construed as limiting the scope of the present invention.

[0135] Examples

[0135] Cloning of antibody sequences into expression vectors

[0136]

[0136] In order to precisely determine the production capacities of antibodies secreted by Phaeodactylum tricomutum and the properties of the antibodies obtained in this production system, the optimized nucleotide sequences encoding the antibodies listed in Table 1 are cloned into an expression vector (and with a secretion peptide) such as those described in international application WO 2023 / 208883.

[0137]

[0137] [Table 1]

[0138]

[0138] Culture of microalgal cells

[0139]

[0139] One of the strains used is the Phaeodactylum tricomutum strain CCAP1055 / 6 (Culture Collection of Algae & Protozoa). The strain is maintained in seawater medium

[0140] 33.3 g / L (reconstituted from INSTANT OCEAN®), supplemented with 1 mL / L of CONWAY solution (Table 2; KHATOON et al., Desalination and Water Treatment, vol.57(60), pp.29295-29302, 2016).

[0141]

[0140] [Table 2]

[0142]

[0141] The culture of microalgae is carried out in a climatic chamber at 19°C, with a day / night alternation and under agitation.

[0143]

[0142] Transformation

[0144]

[0143] The transformation of Phaeodactylum tricomutum is then carried out according to the protocol described in HU & PAN (Electroporation Transformation Protocol for

[0145] Phaeodactylum tricomutum, Methods Mol. Biol., vol.2050, pp.163-167, 2020).

[0146]

[0144] Briefly, the cell culture is washed with sorbitol to remove salts. The cells are brought into contact with the linearized vector and salmon sperm DNA, on ice, for 30 min. Then an electric current of 0.5 kV is applied to the cells. They are then diluted in their culture medium and left to rest for 24 h in the dark.

[0147]

[0145] Finally, the cells are plated on agar containing a selection agent appropriate for the vector used, namely zeocin (75 pg / mL), nourseothricin (100 pg / mL), or blasticidin S (8 pg / mL). For this selection step, the cells are cultured for several weeks.

[0148]

[0146] Selection and culture of clones expressing the antibody of interest

[0147] After 4 to 8 weeks of culture on the initial selection agars, colonies appear. These first colonies are subcultured onto a new selection agar, in a well-individualized manner, as soon as they reach a size of 1 mm. The agars are kept in culture until clone screening (at least one month later). For each antibody to be produced, between 80 and 140 clones were tested.

[0149]

[0148] Screening of clones by ELISA

[0150]

[0149] For each clone to be tested, 100 pL of culture medium from a liquid culture of the clone is collected (7-8 days of culture). The presence of antibodies in this volume is analyzed by ELISA using the “Human IgG ELISA” kit (MABTECH) according to the manufacturer’s instructions.

[0151]

[0150] Bioreactor culture

[0152]

[0151] For each antibody to be tested, the clone producing the highest amount of antibody is selected from among the 80 to 140 clones isolated and tested by ELISA. The selected clone is then cultured in a photobioreactor of 1 L, 10 L, or 200 L using the culture conditions described above.

[0153]

[0152] After 7 days of culture, the microalgal clone culture is harvested and clarified. The supernatant is collected before being concentrated and diafiltered by tangential flow filtration. The antibody is then purified by affinity chromatography using a protein A- Sepharose column (Sepharose CL-4B (CYTIVA); MABSELECT PRISMA (CYTIVA); or TOYOPEARL AF-RProtein A-650F (TOSOH)) following the associated manufacturer’s instructions.

[0154]

[0153] After elution of the antibody, the latter is concentrated using filtration units and then quantified with a microvolume spectrophotometer before being used in the functionality tests described below.

[0155]

[0154] Proteomic analysis

[0156]

[0155] The purified anti-GD2 antibodies produced respectively from microalgae cells (AB IS) and from CHO cells (QARZIBA) were analyzed using a proteomic approach combined to liquid Chromatography-ElectroSpray Mass spectrometry (LC-ESI MS).

[0156] In addition, analyses were run through the software PEAKS that consist in the comparison of the experimental MS and MS-MS data with the theoretical MS and MS- MS data deducted from the theoretical protein sequences of the heavy and light chains (HC and LC respectively) of the antibodies.

[0157]

[0157] Such analyses of the different mass spectra gave a protein coverage percentage of 95% of coverage for the Heavy Chains of both QARZIBA and AB IS and 96% of protein coverage for the Light Chains demonstrating that both antibodies are identical.

[0158]

[0158] A specific focus was made in order to analyze the N- and C-terminal extremities of the HC as they can be affected by a mis -cleavage of the signal peptide and a cleavage of the C-terminal lysine due to carboxypeptidase activities that are well none in mammalian cells like CHO.

[0159]

[0159] Therefore, the targeted analysis of the MS -MS spectra corresponding to the AB IS HC demonstrated that the signal peptide is cleaved as expected.

[0160]

[0160] The targeted analysis of the MS -MS analysis corresponding to the C-terminal end of the HC of AB IS shown that the C-terminal lysine is still present, therefore being non affected by carboxypeptidase activities.

[0161]

[0161] Secondary structure analysis

[0162]

[0162] In order to analyze further the structure of AB IS and compare it to the structure of QARZIBA, FTIR spectroscopy was used as it represents an interesting tool to analyze the higher-order structure, especially secondary structure. Indeed, vibrations of the amide bonds in proteins result in the largest bands in FTIR spectra of proteins.

[0163]

[0163] Nine characteristic amide bands have been identified but two of them are most commonly used to investigate higher-order structure of proteins. The exact frequencies of Amide I (mostly C=O stretching, 1695-1615 cm-1) and Amide II (mostly N-H bending, 1550-1520 cm-1) are influenced by the strength of hydrogen bonds involving amide C=O and N-H groups as well as the geometry of the polypeptide chain. Each type of secondary structure is associated with specific frequencies at which amides I and II occur.

[0164] Therefore, we have compared the AB IS antibody with QARZIBA in terms of secondary structure using FTIR. To assess the secondary structure, normalization for equal area was applied between 1740 and 1478 cm'1. This allows for comparison of FTIR spectra considering the same quantity of proteins.

[0164]

[0165] The Figure 1 presents the mean preprocessed spectra for each sample, with a zoom on the spectral region related to protein absorption. A statistical pairwise comparison between FTIR spectra of both samples was also performed to evidence spectral changes. The difference spectrum (black spectrum) presented in Figure 3 corresponds to the difference between the mean spectra of each sample. The black stars refer to significant differences defined by a Student’s t-test at each wavenumber.

[0165]

[0166] The results show that the mean spectra completely overlap leading to a flat difference spectrum with almost no significant variations (i.e. black stars). The shape of these difference spectra does not indicate a shift of the Amide I band position, which is the most sensitive amide band to structural changes.

[0166]

[0167] Multivariate analyses can also be applied to interpret the FTIR data in terms of secondary structure. Principal component analysis (PCA) is a powerful tool for high- dimension data to identify patterns and to express the data in such a way as to highlight their similarities and differences. This tool reduces the number of variables without much loss of information, taking into account most of the variance. It is an unsupervised analysis: the classification obtained does not suppose any a priori condition on grouping obtained.

[0167]

[0168] The estimation of the secondary structure moieties was realized using three wavenumbers in the Amide I and II bands. The wavenumbers used for this secondary structure determination are the following:

[0168]

[0169] • a-helix: 1545, 1655 and 1613 cm-1.

[0169]

[0170] • P-sheet: 1656, 1635 and 1692 cm-1.

[0170]

[0171] • Turn: 1678, 1528 and 1600 cm-1.

[0172] • Random: 1544, 1627 and 1692 cm-1.

[0171]

[0173] The Table 3 presents the results of this prediction for both samples. The mean and the standard deviation for the eight spectra recorded for each sample is shown in this table.

[0172]

[0174] [Table 3]

[0173]

[0175] Altogether, these results demonstrated that no difference has been evidenced in terms of secondary structure for AB IS produced in microalgae and the same antibody produced in mammalian cells (QARZIBA).

[0174]

[0176] Analysis of the A-glycosylation

[0175]

[0177] After demonstrating that the protein sequences and the secondary structures were identical for dinutuximab produced in P. tricomutum (AB IS) and in CHO (QARZIBA), the A-glycosylation sites of both antibodies were analyzed by two approaches that are FTIR and mass spectrometry as it is well established that the A-glycosylation of antibodies can impact the activities of the antibodies.

[0176]

[0178] 1- FTIR

[0177]

[0179] Carbohydrates also called glycans associated with proteins are responsible for absorption in other areas of the FTIR spectrum of these glycoproteins. The most interesting area for studying glycans is between 1200 and 950 cm'1. Recently, it has been evidenced that FTIR spectra of glycoproteins and especially mAbs provide a global but accurate fingerprint of the glycosylation profile. This fingerprint is not only sensitive to large differences such as the presence or absence of several monosaccharides but also to smaller modifications of the glycan and monosaccharide content.

[0178]

[0180] In order to compare the samples in terms of total glycan content (i.e. absorption band intensity), the normalization for equal area was applied between 1740 and 1478 cm- 1, i.e. on the protein specific bands. It allows a comparison of samples considering the same quantity of proteins. Using this normalization, spectral differences observed in the region of glycans must be assigned to differences in the mass ratio between glycans and proteins. It can thus evidence variations in the global glycosylation level.

[0179]

[0181] The results have shown that some spectral variations can be noted between both samples (figure 2). The spectra have been integrated between 1181 and 1000 cm-1 and between 1740 and 1478 cm-1 to obtain the ratios between the area of the peak corresponding to glycan absorption and the area of the Amide I & II bands corresponding to protein absorption. The peak area ratio reflects the mass ratio between carbohydrates and proteins. A ratio of 0.035 was obtained for the CHO-Ref antibody (QARZIBA) whereas a ratio of 0.029 for the antibody produced in microalgae (AB IS), evidencing a lower global glycan content for the antibody produced in microalgae.

[0180]

[0182] In order to compare the samples in terms of glycan composition, the FTIR spectra were analyzed after the scaling of the spectra. The normalization for equal area was applied between 1181 and 1000 cm-1. It allows a comparison of samples considering the same quantity of carbohydrates. It removes spectral variations due to variations in mass ratio between glycosylation and protein. Using this normalization, the spectral differences observed in the region of glycans are only due to differences in the glycan composition.

[0181]

[0183] Figure 3 presents the mean preprocessed spectra for each sample, with a zoom on the spectral region related to the glycan absorption. A statistical pairwise comparison between FTIR spectra of both samples was also performed to evidence spectral changes.

[0182]

[0184] The results have shown that significant spectral variations appear on the difference spectra which reveal some modifications in the glycan composition (Figure 3). Multivariate analyses have been applied to interpret the FTIR data in terms glycan composition.

[0183]

[0185] Now, these FTIR analyses established that the N-glycosylation of both antibodies (AB IS and QARZIBA) are different. Accordingly, a glycoproteomic combined to mass spectrometry analyses was performed to decipher the N-glycan structure of both antibodies.

[0186] 2- GLYCOPROTEOMIC COMBINED WITH MASS SPECTROMETRY

[0184]

[0187] The analysis of glycopeptides in the ESI LC-MS data was based on the search for fragment ions at m / z 204 and 366 characteristic of the respective presence of N- acetylglucosamine (GlcNAc) and of the disaccharide mannosyl-A-acetylglucosamine (Man-GlcNAc).

[0185]

[0188] The figures 4 and 5 shows the mass spectrometry analysis of the N-glycosylation of the CHO-Ref antibody (QARZIBA) and AB1-S respectively allowing the identification of the N-gly can’s structures attached to the EEQYNSTYR (SEQ ID N°16) peptide present on their Fc part.

[0186]

[0189] The results have shown that a set of three glycopeptides were detected and identified as A-glycans GOF, GIF, and G2F linked to the EEQYNSTYR peptide (SEQ ID N°16) for QARZIBA (Figure 4). Ions consisting of the EEQYNSTYR peptide (SEQ ID N°16) linked to carbohydrate fragments enable this identification (figure 4). The results have shown a different glycosylation profile for the AB IS with oligomannosides linked to the EEQYNSTYR peptide (SEQ ID N°16). The same glycosylation profile was observed for AB2S (data not shown).

[0187]

[0190] Altogether, these results demonstrated that AB IS and QARZIBA are bearing different N-glycans that are reflecting the N-glycosylation pathway of the production system used : microalgae and CHO.

[0188]

[0191] Antigen binding capacity

[0189]

[0192] The GD2 binding capacity for AB IS and QARZIBA was tested on cell lines expressing GD2 (LAN-1 and IMR-32). As a control, a GD2 non expressing cell lines was used (HEK-293).

[0190]

[0193] Disialoganglioside GD2 expressing cells (IMR32 or LAN- 1) were detached using Trypsin / EDTA and washed in cold PBS + BSA. Primary unconjugated antibody (AB IS, or AB2S, or QARZIBA) was incubated with the cells (diluted in PBS+ BSA). After washes cells were incubated with FITC-conjugated secondary antibody (diluted in PBS+BSA) in the dark. Following additional washes, cells were resuspended in PBS +BSA for acquisition by flow cytometry

[0194] The results have shown that the binding affinity of the antibodies tested for the different cell lines was quite similar, no matter the antibody was produced in Phaeodactylum tricomutum or in mammalian cells (data not shown).

[0191]

[0195] The affinity constants of the antibodies for the antigen were then determined specifically by SPR on a T200 by KIMIALYS.

[0192]

[0196] For the antigen affinity measurement, the antibody is immobilized on the chip at an optimal surface density and the antigen is injected at increasing concentrations. Sensograms were generated and kinetic constants are measured following the 1:1 Langmuir model.

[0193]

[0197] The results have shown that the binding affinity of the antibodies tested for the GD2 antigen (AB IS, AB2S and QARZIBA) were quite similar (data not shown), no matter the antibody was produced in Phaeodactylum tricomutum (AB IS or AB2S) or in mammalian cells (QARZIBA).

[0194]

[0198] Binding capacity to FcyR

[0195]

[0199] The binding capacity of each antibody to the different FcyR is measured using the various LUMIT™ FcyR Binding Immunoassay kits (PROMEGA) according to the manufacturer’s instructions. As a control, the binding affinity of each antibody produced in Phaeodactylum tricomutum (AB IS and AB2S) is compared with the affinity of the same antibody produced in mammalian cells (QARZIBA produced in a CHO cell line).

[0196]

[0200] The results showed that the binding affinity of the antibodies tested for the different FcyR was quite similar (whether produced in Phaeodactylum tricomutum or in mammalian cells), with values also comparable to those previously observed in VANIER et al. (cited above, 2018).

[0197]

[0201] The affinity constants of the antibodies for the antigen were then determined specifically by SPR on a T200 by KIMIALYS for FcyRI and FcyRIIIa. For these affinity measurements, the receptors FcyRI and FcyRIIIa were immobilized respectively, and each antibody was injected at increasing concentrations. For the receptors, complete characterization was performed at a constant pH (7.4). Kinetic constants are measured following the 1:1 Langmuir model for all Fc receptors.

[0198]

[0202] The results have shown that the FcyRI binding affinity of the antibody produced in Phaeodactylum tricornutum (AB IS or AB2S) is 10-fold less compared to the CHO produced antibody (QARZIBA).

[0199]

[0203] The results have further shown that the FcyRIIIa binding affinity of the antibody produced in Phaeodactylum tricornutum is 4-fold less compared to the CHO produced antibody.

[0200]

[0204] ADCC activity assay

[0201]

[0205] The ADCC activity of the different antibodies is measured using the ADCC REPORTER BIO ASSAY kit (PROMEGA) according to the manufacturer’s instructions.

[0202]

[0206] The LAN-1 and IMR-32 cell lines (culture medium: DMEM high glucose, 10% FCS) were used with this kit for testing the dinutuximab (anti-GD2) at different dilutions, whether it is produced in P. tricornutum (AB IS and AB2s) or in CHO (QARZIBA).

[0203]

[0207] The figure 6 shows the ADCC activity (Luminescence, Relative Unit: RLU) observed for LAN-1 incubated with dinutuximab produced in CHO cells (QARZIBA) or in Phaeodactylum tricornutum (AB IS) and the dinutuximab derivative produced in Phaeodactylum tricornutum (AB2S).

[0204]

[0208] The results have shown that the ADCC activity is far reduced for the dinutuximab produced in Phaeodactylum tricornutum (AB IS) as compared to the one produced in CHO cells (QARZIBA) with a nearly ten-fold reduction in EC50 (nearly 10'7M instead of 10'8M). Now, this difference in ADCC activities is corrected by the mutations in the dinutuximab derivative (AB2S).

[0205]

[0209] CPC activity assay

[0206]

[0210] The ADCC activity of the different antibodies is measured using the CYTOTOX- GLO™ CYTOTOXICITY ASSAY kit (PROMEGA) according to the manufacturer’s instructions.

[0211] The LAN-1 and IMR-32 cell lines (culture medium: DMEM high glucose, 10% FCS) are used with this kit for testing the dinutuximab (anti-GD2) at different dilutions, whether it is produced in P. tricomutum (AB IS and AB2s) or in CHO (QARZIBA).

[0207]

[0212] A decrease CDC activity is anticipated in antibodies produced in Phaeodactylum tricomutum (AB IS and AB2S) as compared to the one produced in CHO cells (QARZIBA), because of their glycosylation profile -i.e., with oligomannosides and devoid of galactose-.

[0208]

[0213] Binding capacity to FcRn

[0209]

[0214] To complete the characterization of the antibodies produced in Phaeodactylum tricomutum, the binding capacity of each antibody to the human FcRn receptor is measured using the LUMIT™ FcRn Binding Immunoassay kit (PROMEGA) according to the manufacturer’s instructions. Here again, each antibody is tested simultaneously with its reference produced in Chinese hamster ovary (CHO) cells.

[0210]

[0215] Figure 7 shows the binding affinity for the FcRn receptor of dinutuximab produced in CHO cells (QARZIBA, Reference) or in Phaeodactylum tricomutum (AB1S_CS58) compared with the positive control for FcRn binding (IgG Control).

[0211]

[0216] Figure 8 shows the binding affinity for the FcRn receptor of dinutuximab produced in CHO cells (QARZIBA, Reference) or in Phaeodactylum tricomutum (AB IS) together with dinutuximab derivative (AB2S-batch 1 and AB2S-batch 2) compared with the positive control for FcRn binding (IgG Control).

[0212]

[0217] Unexpectedly, the results show that the binding affinity of an antibody produced in Phaeodactylum tricomutum is much higher than that of the same antibody produced in CHO. Thus, it is observed that the binding affinity of dinutuximab produced in Phaeodactylum tricomutum (IC50 = 68 nM) is nearly 14 times higher than that of the same antibody produced in mammalian cells (IC50 = 950 nM; see Figure 1). In light of ZALEVSKY et al. (cited above, 2010) in particular, it is thus possible to predict that such an increase in FcRn binding affinity results in an increase in the plasma half-life of dinutuximab by a factor of 5 (when switching from production in mammalian cells (CHO) to production in Phaeodactylum tricornutum). These results were confirmed in second series of experiments on dinutuximab, but also on its derivative AB2S (See figure 2).

[0213]

[0218] In Vivo evaluation Zebrafish model

[0214]

[0219] To complete the characterization of the antibodies produced in Phaeodactylum tricornutum, the efficacy evaluation of dinutuximab was carried in zebrafish embryo according to the protocol of the service provider BIOREPERIA. Here again, the antibody was tested simultaneously with its reference QARZIBA produced in Chinese hamster ovary (CHO) cells. For these experiments, LAN-1 or IMR-32 cell xenografts were injected into the zebrafish embryo. The antibody to test was co-injected at different concentrations (0-0.03-0.3 and 3 mg / mL). The tumor volume was measured for each concentration after injection and at 96 hours. The study is carried out in parallel with the antibody.

[0215]

[0220] The figure 9 shows the normalized tumors size for zebrafish co-injected or not with the dinutuximab produced in P. tricornutum (AB IS) or in CHO (CHO-Ref for the three tested conditions against the vehicle control (no treatment - PBS only).

[0216]

[0221] The results show that there was no statistical difference in the mean tumor size between the two antibodies at a concentration of 3 mg / ml (figure 9). This suggests that the therapeutic effect is the same between both antibodies for this concentration (3mg / mL). The results further show that the AB IS antibody has a shifted dose responsecurve towards a more favorable profile compared with the CHO-Ref at the lowest concentrations of 0.03 and 0.3 mg / ml. Simultaneously, no significant reduction in the number of metastases in any of the concentrations of either antibody was observed suggesting the same mechanism of action for both antibodies (data not shown).

[0217]

[0222] Now, these results are far different from the expected ones. In fact, on the basis of the ADCC activity previously determined for AB IS and QARZIBA, the tumor growth inhibition expected with the P. tricornutum produced dinutuximab at 0.3 mg / ml was expected to be at the level observed with the ten-fold lower concentration (0.03 mg / ml) of dinutuximab produced in CHO (QARZIBA) and therefore null -i.e., 100% of normalized tumor size-. Similarly, the tumor growth inhibition expected with the P. tricomutum produced dinutuximab at 3 mg / ml was expected to be at the level observed with the ten-fold lower concentration (0.3 mg / ml) of QARZIBA and therefore around 30% i.e., 100% of normalized tumor size-.

[0218]

[0223] Accordingly, these results demonstrate that dinutuximab produced by P. tricomutum exhibits a tumor growth inhibition activity, which is distinct from an ADCC activity, and accounting for approximately 20% at 0.03 mg / ml -i.e., 80% of normalized tumor size-, for approximately 60% at 0.3 mg / ml -i.e., 40% of normalized tumor size-, and for at least 50% at 3 mg / ml (given that the normalized tumor size does not fall below 20%).

[0219]

[0224] Now, this activity other than ADCC cannot be ADCP, since the ADCP from dinutuximab produced by P. tricomutum is anticipated to be far lower than the one of dinutuximab produced by CHO because of its lower affinity for FcyRI. This activity is also distinct from CDC or FcRn-mediated, since these are absent from these injected two days zebrafish embryos.

[0220]

[0225] Accordingly, this “other” activity is certainly a direct antibody mediated cellular cytotoxicity, as already disclosed for anti-GD2 antibodies and other antibodies directed against gangliosides.

[0221]

[0226] In ovo evaluation of antibody efficacy and toxicity

[0222]

[0227] Evaluation of antibody efficacy and toxicity were performed according to the protocol of the service provider INOVOTION.

[0223]

[0228] LAN-1 cells are grafted into the chorioallantoic membrane of a hen’s egg on the 9thday of development.

[0224]

[0229] The antibody produced in Phaeodactylum tricomutum was administered 5 times at a concentration, and tumor volume was measured on the 18thday of development (9 days after xenografting). The study was conducted in parallel with the antibody and its reference produced in CHO.

[0225]

[0230] The mortality rate was calculated.

[0231] Antibody mediated cellular cytotoxicity

[0226]

[0232] Following the previous results in zebrafish, the antibody-mediated cellular cytotoxicity is determined for the antibodies produced in Phaeodactylum tricornutum - i.e. AB IS and AB2S - as compared to the one known from dinutuximab produced in CHO cells (QARZIBA, Reference) by colorimetric MTT (3-[[4,5]-dimethylthiazol-2- yl]-2,5-diphenyltetrazolium bromide as disclosed in DORONIN et al. (abovementioned, 2014).

[0227]

[0233] For these experiments, LAN-1 or IMR-32 cell lines expressing GD2 antigen and control cells (HEK) not expressing GD2 antigen are incubated with serial dilutions of the different antibodies for 72 h under standard culture conditions.

[0228]

[0234] Following this After incubation, the MTT solution (250 pg / ml final concentration) is added to each sample for 4 h. The optical density (OD) is read in a microplate reader at a test wavelength of 540 nm. Cell viability was measured as ratio of OD540 of cells treatment with antibodies to OD540 of control cells not incubated with any antibodies.

[0229]

[0235] A strongest antibody mediated cellular cytotoxicity is anticipated for the antibodies produced in Phaeodactylum tricornutum -i.e. AB IS and AB2S - as compared to the one known from dinutuximab produced in CHO cells (QARZIBA, Reference).

[0230]

[0236] The evolution of antibody mediated cellular cytotoxicity for antibodies directed against tumor antigens among GM1, GM2, GM3, O-acetyl GD2 or GD3 is determined depending on their method of production -i.e., produced in mammalian cells or in a culture of P. tricornutum-..

[0231]

[0237] Pharmacokinetics in mice

[0232]

[0238] In light of the particularly promising results obtained regarding FcRn receptor binding, a more precise determination of the plasma half-life of the antibodies produced in Phaeodactylum tricornutum is sought.

[0239] To this end, a pharmacokinetic study is carried out according to the protocol of the service provider NOV ALIX.

[0233]

[0240] The study uses the humanized mouse line Jackson Mouse SN 014565 F (B6.Cg- FcgrttmlDcr Tg(FCGRT)32Dcr / DcrJ). After administration of the antibody at 30 mg / kg by retro-orbital sinus route, the concentration of the antibody in the plasma is measured by ELISA at 0.25-0.5-1-2-4-6-24-48-7211 and 96h after the injection. The study is carried out in parallel with the antibody and its reference produced in CHO (QARZIBA).

Claims

Claims1. An antibody directed against a ganglioside tumor antigen, which can be obtained by a method comprising the steps of :(i) producing an antibody in a culture of Phaeodactylum tricornutum-,(ii) purifying said antibody; and(iii) determining the binding affinity of said antibody for the FcRn receptor, preferably for the human FcRn receptor.

2. The antibody according to the claim 1, characterized in that it is not dinutuximab or naxitamab.

3. The antibody according to the claim 1, characterized in that it said antibody has a specific A- glycosylation profile corresponding to oligomannosides -i.e., Man 5 to Man 9-, preferably this specific A-glycosylation profile being devoid of galactose and of fucose.

4. The antibody according to the claim 2, characterized in that the ganglioside tumor antigen is GM2, GM3, GD2, O-acetyl GD2 or GD3.

5. The antibody according to the claim 2, characterized in that the ganglioside tumor antigen is GM1, GD2, and O-acetyl-GD2.

6. The antibody according to any one of claim 4 or 5, characterized in that the ganglioside tumor antigen is GD2 or O-acetyl-GD2.

7. The antibody according to any one of claim 4 or 5, characterized in that said antibody is a dinutuximab derivative comprising one or more-point mutations making it possible to improve its properties, preferably its ADCC activity.

8. The antibody according to claim 7, characterized in that said one or more-point mutations related to ADCC activity are located in antibody heavy chain sequence and are selected in the group comprising G236A, S239D, F243L, P247I, R292P, S298A,Y300L, V305I, A330L, I332E, E333A, K334A, A339Q, and P396L, and more preferably are selected among S239D, A330L, and I332E.

9. The antibody according to claim 7, characterized in that said dinutuximab derivative is AB2S, whose light and heavy chains have the sequences SED ID N°: 16 and SED ID N°:17 respectively.

10. The antibody according to claim 7, characterized in that said dinutuximab derivative is AB2S, whose light and heavy chains have the sequences SED ID N°:16 and SED ID N°:17 respectively.

11. A composition comprising at least one antibody as defined in any one of claims 1 to 10, as a medicament.

12. A composition comprising an antibody as defined in any one of claims 1 to 10 for use in treating a subject suffering from cancer.

13. The composition according to claim 12, characterized in that said antibody is administered at a frequency at least twice lower, preferably at least three times lower, and more preferably at least four times lower than the frequency with which the same antibody but produced in mammalian cells is administered.

14. The composition of claim 13, wherein said antibody is dinutuximab and the time interval between two cycles of administration of said antibody is at least 42 days, preferably at least 63 days, and more preferably at least 84 days.

15. The composition according to claim 12, characterized in that said antibody is administered at a dose which is at least 25% lower, preferably at least two-fold lower, as an example at least three-fold lower, and more preferably at least four-fold lower than the recommended dose at with which the same antibody but produced in mammalian cells, preferably in CHO, is administered.

16. The composition of claim 15, wherein said antibody is dinutuximab or a derivative thereof, and said antibody is administrated at a total antibody dose lower than75 mg / m2(antibody mass related to subject skin surface), preferably lower than 50 mg / m2, and most preferably than 25 mg / m2.

17. The composition according to claim 12, characterized in that said antibody is dinutuximab or a derivative thereof, and said antibody is administered without any coadministration of at least one analgesic selected among opioids (e.g., Morphine) or gabapentinoids (e.g., Gabapentin) analgesics, preferably any co-administration of at least one opioid analgesic.

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