IMMUNOGLOBULINS WITH INCREASED AFFINITY FOR FCRN

Abstract

The present invention relates to a method for producing antibodies with an increased plasma half-life 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.

Description

Title of the invention: IMMUNOGLOBULINS WITH INCREASED AFFINITY FOR FCRN Scope of the invention

[0001] The present invention relates to immunoglobulins with an increased plasma half-life, to a method for producing such immunoglobulins, and to their uses, particularly for therapeutic and prophylactic purposes. Technological background of the invention

[0002] Protein-based therapeutic agents are now widely used in clinical practice and are rapidly replacing non-specific, low-molecular-weight therapeutic agents. Indeed, these protein-based therapeutic agents offer the advantage of very high specificity, fewer side effects, and lower toxicity.

[0003] 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 can exceed 3 weeks. The immunotherapy market was already worth over $200 billion (USD) in 2023. This significant amount now corresponds to only a relatively limited number of therapeutic antibodies and is therefore expected to continue growing considerably in the coming years. Examples of these therapeutic antibodies include Rituxan, Avastin, Herceptin, and Remicade, each of which has sales exceeding $1 billion.

[0004] Now, therapeutic antibodies have very poor absorption from the gastrointestinal system (when administered orally) and their bioavailability is very low because they are easily denatured in the gastrointestinal system or readily degraded by proteolytic enzymes. As a result, these therapeutic agents must be administered by intravenous or subcutaneous injection, which must be frequent to achieve a noticeable therapeutic effect. In the case of a therapeutic antibody with good stability, it typically needs to be administered once every 2-3 weeks at a high dose (2-8 mg / kg body weight) to achieve an effective therapeutic effect.

[0005] Besides the inconvenience to the patient, which should be limited, this stability can vary considerably depending on the antibodies. Generally, the half-life (t / 2) of IgG1, IgG2, and IgG4 in humans is approximately 21 days, while that of other immunoglobulins (e.g., IgG3) is between 2.5 and 7 days. Now, the Monoclonal antibodies have highly variable half-lives ranging from 6 to 32 days.

[0006] It is clear that the half-life of a therapeutic antibody is a critical parameter since increasing it will allow for longer intervals between doses of the associated treatment. For example, nirsevimab, an anti-RSV monoclonal antibody, has a half-life of several months, allowing a single administration to provide passive immunization of infants during the epidemic period (i.e., nearly 4 months).

[0007] The pharmacokinetics of therapeutic antibodies are now better understood and are known to be closely associated with their ability to bind to the FcRn receptor (neonatal Fc receptor). The function of this receptor relies on its ability to bind IgG (and also albumin) through pH-dependent interactions. The importance of this IgG-FcRn interaction in the half-life of antibodies has thus been demonstrated in a mouse study, where the half-life of the mouse IgG antibody is only 1.4 days in FcRn-deficient mice (mFcRn- / -), compared to a half-life of 9 days in mFcRn- / + and mFcRn+ / + mice. It is now known that the clinical failure of murine monoclonal antibodies stemmed from their very short half-life in humans, which resulted from the low affinity of human FcRn for mouse IgG.

[0008] Following a better understanding of the associated mechanisms, it is now known that FcRn performs the following functions:

[0009] - recycling allowing IgG (and albumin as well) to escape from endothelial catabolism,

[0010] - transcytosis promoting the biodistribution of IgG in the body, and

[0011] - cooperation during phagocytosis.

[0012] The recycling function of IgG consists of extracting IgG from the endothelial catabolism pathway of plasma proteins and returning them intact to the circulation. IgG shares this property exclusively with albumin, which also binds to FcRn and benefits from recycling. In mice, it has been calculated that FcRn protects half of the internalized albumin molecules and four-fifths of the IgG molecules from degradation, which could be an energetically more economical mechanism for the body than the de novo synthesis of these molecules. This recycling explains their long half-life (three weeks for IgG), while also 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, following the saturation of FcRn, which can no longer recycle them.

[0013] Transcytosis of IgG from one pole to the other of epithelia or endothelia is the second major function of FcRn, ensuring their biodistribution in the body. As with recycling, FcRn is thus closely associated with vesicular trafficking and the accompanying pH variations. At neutral pH (extracellular compartment), the binding of IgG to FcRn is negligible, but the protonation of IgG histidine residues at slightly acidic pH allows for binding to FcRn.

[0014] Finally, a third major function of FcRn would be to cooperate with classical FcyRs in phagocytosis and immune complex presentation, perhaps taking over from FcyRs for IgG binding in acidic endosomal compartments. This still poorly understood function of FcRn probably explains why it is expressed in antigen-presenting cells and neutrophils.

[0015] As for the binding of IgG to FcRn, it is located in a region that overlaps the CH2 and CH3 domains of IgG and involves, in particular, residues His310 of the CH2 domain and His435 of the CH3 domain (replaced by an arginine on IgG3s of lower affinity). These two histidine residues are positively charged at pH below 6.5, forming salt bridges with the corresponding residues on FcRn. Consequently, when the pH rises above 7.0, the charge of these histidine residues disappears, leading to a decrease in binding to FcRn and the release of the antibody.

[0016] Based on this knowledge and in the search for an increase in the half-life of antibodies, many groups have sought to increase the binding affinity of antibodies to FcRn by integrating point mutations within their sequences.

[0017] It has thus been demonstrated that an M428L / N434S variant has an 11-fold greater binding affinity to FcRn than wild-type IgG at pH 6. The introduction of these mutations into two antibodies (bevacizumab and cetuximab) confirmed this increase in affinity, which led to an increase in the plasma half-life of the corresponding antibodies by a factor of 4.3 and 4.8 in a transgenic mouse expressing human FcRn, and an increase in their plasma half-life by a factor of 3.2 and 3.1 in a monkey model (ZALEVSKY et al., Nat. Biotechnol., vol. 28(2), p: 157-159, 2010).

[0018] Examples of such mutations include those described in patents EP 1817340 Bl, EP 2164873 Bl, EP 2552955 Bl, or EP 3181581 Bl.

[0019] Now, even though the use of these mutations can improve the plasma half-life of the associated antibodies, their number is still too limited.

[0020] Also, the search for new technologies to improve the plasma half-life of therapeutic antibodies is currently particularly dynamic, with the objective of the possible therapeutic use of monoclonal antibodies which have high potential but too short a lifespan. Summary of the invention

[0021] In the context of evaluating the use of the microalga Phaeodactylum tricomutum for the production of therapeutic proteins, the latter 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. 11, p: 126, 2012; VANIER et al., PLoS One, vol. 10(10), p: e0139282, 2015; and VANIER et al., Biotechnol. J., vol. 13, p:1700496, 2018). These different studies have demonstrated that a monoclonal antibody produced by P.P. tricomutum is capable of binding to human Fcy receptors, including FcyRI and FcyRIIIa, suggesting that it could be used effectively in human immunotherapy to induce phagocytosis and an antibody-dependent cell-mediated cytotoxicity (ADCC) response (VANIER et al., cited previously, 2018). Furthermore, P. tricomutum has been shown to glycosylate the Fc portions of monoclonal antibodies with oligomannosides bearing 5 to 9 mannose residues (VANIER et al., cited previously, 2015) that are structurally identical to those of mammals (DUMONTIER et al., Carbohydr. Polym., vol. 259, p: 117660, 2021).

[0022] In the context of a better characterization of the secreted antibodies produced by P. tricomutum, the inventors unexpectedly demonstrated that the antibodies obtained exhibit a binding affinity for human FcRn much higher than that of the same antibodies produced in CHO. The difference is so significant that it anticipates an increase in the plasma half-life of these antibodies, which could be by a factor of 5 in patients.

[0023] Although they represent only 2 to 3% of the mass of an IgG molecule, glycans are known to affect critical antibody functions, particularly binding to Fc receptors. For example, N-glycosylation of a conserved site in the CH2 domain of ItGg is known to be necessary for antibody binding to Fc receptors. Furthermore, the specific composition of the incorporated glycoform has been shown to modify certain activities of associated antibodies. For instance, afucosylated glycans confer enhanced binding to FcyRIIIa of IgG3. Patent EP 1896071 B1 thus uses the enzymatic treatment of an antibody with an α-(2,3)-sialytransferase to improve some of its properties, notably increasing its plasma half-life.

[0024] It therefore appears that the specific glycosylation profile of immunoglobulins produced in P. tricomutum confers upon them an improved binding affinity for FcRn, which affinity is the corollary of a much longer plasma half-life to that obtained for the same immunoglobulin produced in mammalian cells, particularly in CHO.

[0025] This discovery therefore provides access to a new technology for obtaining immunoglobulins with a considerably increased plasma half-life. In addition to the use of such a technology for therapeutic antibodies for the treatment of cancer or autoimmune diseases, this technology appears particularly relevant for the production of therapeutic antibodies with an increased plasma half-life, notably for the production of IgG3 to obtain antibodies usable in therapy, but also for the production of neutralizing antibodies for the prophylactic treatment of infectious diseases.

[0026] Consequently, a first object of the invention relates to a process for producing antibodies with an increased plasma half-life comprising the steps of:

[0027] - production of an antibody in a cellular system;

[0028] - purification of said antibody; and

[0029] - determination of the binding affinity of said antibody for the FcRn receptor, of preferentially the human FcRn receptor;

[0030] Characterized in that said cell system is a culture of Phaeodactylum tricomutum.

[0031] A second object of the invention relates to an antibody obtained or capable of being obtained by the process as described above.

[0032] A third object relates to a composition, used as a medicinal product, comprising such an antibody.

[0033] A fourth object relates to a composition comprising an antibody as described above, which antibody is directed against an infectious antigen, and preferably constitutes a neutralizing antibody, for use in preventing infection in a subject by the infectious agent associated with said infectious antigen.

[0034] A fifth object relates to a composition comprising an antibody as described above, which antibody is directed against an immunoregulatory antigen, for use in treating a subject suffering from an autoimmune disease, suffering from an allergy or undergoing transplantation.

[0035] A sixth object comprising an antibody as described above, which antibody is directed against an immunoregulatory antigen or against a tumor antigen, for use in treating a subject suffering from cancer. Description of figures

[0036] Figure 1 shows the binding affinity (Relative Unit: RLU) for human FcRn of dinutuximab produced in CHO cells (QARZIBA, Reference) or in Phaeodactylum tricornutum (ABlS_CS58) compared to the positive control (IgG Control). Detailed description of the invention

[0037] Phaeodactylum tricornutum is a marine diatom. It is the only species in the genus Phaeodactylum and one of the model species widely used for the study and characterization of diatom metabolism. Although a coastal species, it is found in many ecosystems worldwide, 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 silica. It has the significant advantage of being able to be cultured and processed by various methods (biological transformation, electroporation, and bacterial conjugation), making it a particularly relevant production tool.

[0038] 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).

[0039] The step of producing an antibody using Phaeodactylum tricornutum can be carried out using methods well known to those skilled in the art. Such a production step typically includes the preparation of at least one vector enabling the expression of an antibody within Phaeodactylum tricornutum. To this end, the nucleotide sequence encoding the antibody of interest (its heavy and light chains) 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 incorporates 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 and light chains of the antibody).These vectors are now preferably associated with selection markers usable in Phaeodactylum tricornutum (e.g., zeocin, nourseothricin, or blasticidin), allowing for the screening of microalgae that have integrated the vector. Advantageously, the two ORFs can be located on the same vector and are then separated by a linker. The transformation of Phaeodactylum tricornutum with at least one of these vectors is then carried out using well-known techniques 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 antibody-producing clones is performed using the marker. Vector selection precedes scaling up the culture for the specifically selected clone using photobioreactor culture (IL, 10L, 200L, and 2000L) to produce the antibody. An example of such a production step is the method described in detail (including its vector, promoter, termination sequence, etc.) in international application WO 2023 / 208883.

[0040] Advantageously, the production of the antibody in step i) uses at least one functional signal (secretory) peptide 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 NO:1) for retention in the endoplasmic reticulum (e.g. HEMPEL et al., previously cited, 2011; or possibly the HDEL (SEQ ID NO:2) or KDEL DDEL (SEQ ID NO:3) sequences), allowing the retention of said antibody in the endoplasmic reticulum of Phaeodactylum tricornutum.

[0041] 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.

[0042] An antibody is an immunoglobulin molecule corresponding to a tetramer comprising four polypeptide chains, including two identical heavy chains (H) (approximately 50 to 70 kDa along their entire length) and two identical light chains (L) (approximately 25 kDa along their entire length) interconnected by disulfide bonds. The light chains (L) are classified as kappa and lambda. The heavy chains (H) are classified into 5 subtypes (y, p, a, e, or β). The nature of the heavy chain subtype determines the immunoglobulin type (IgG:y, IgA:a, IgM:p, IgD:β, IgE:e).

[0043] Each light chain consists of a constant domain (CL) and a variable domain (VL). Heavy chains are composed of an N-terminal variable domain (VH) and a constant region composed of three or four constant domains (CH1, CH2, CH3 and CH4) depending on the isotype (CH1, CH2 and CH3 for IgG).

[0044] Constant domains are characterized by an amino acid sequence that is very similar from one antibody to another, and which is characteristic of the species and isotype. They are not involved in antigen recognition per se, but play a role in the activation of the complement system as well as in the elimination of immune complexes (antibodies bound to their 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.

[0045] The variable domains (VL and VH) can be subdivided into hypervariability regions (called complementarity-determining regions (CDRs)) which are interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino end terminal to the carboxy-terminal end in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0046] The allocation of amino acids to each domain conforms to well-known conventions, particularly with regard to their numbering (IMGT, The International Immunogenetics Information System).

[0047] Ultimately, the functional ability 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 CDRs.

[0048] For the purposes of the present invention, the term “antibody” refers to a monoclonal antibody per se.

[0049] Advantageously, such an antibody is directed against an immunoregulatory antigen, an infectious antigen or even a tumor antigen.

[0050] As used here, an “immunoregulatory antigen” means an antigen expressed by a molecule involved in the regulation of the immune system, which may in particular be expressed by activated or non-activated inducing immune cells, such as T and / or NK cells, or by activated or non-activated suppressor cells.

[0051] Such antibodies can be used to neutralize certain specific cell populations (to reduce the immune system response) in the treatment of subjects suffering from autoimmune diseases or allergies, or in subjects undergoing transplantation. Such antibodies can be easily identified by those skilled in the art and target, in particular, CD20,1TL-2R, or TNFα. Such antibodies directed against an immunoregulatory antigen can also be used to neutralize other specific cell populations (this time to increase the immune system response) in the treatment of subjects suffering from cancer. Here again, such antibodies can be easily identified by those skilled in the art and target, in particular, CTLA4, PD-1, or PD-L1.

[0052] Examples of immunoregulators include CTL-A4 (Cytotoxic T-lymphocyte-Associated protein 4, also known as CD 152), PD-1 (Programmed cell Death protein 7, also known as CD279), PDL1 (Programmed Death-Ligand 1, also known as CD274 or B7H1), BTLA (B- and T-Lymphocyte Attenuator, also known as CD272), KIRs (Killer cell Immunoglobulin-like Receptors), CAMPATH-1 (also known as CD52), 1TL2RA (alpha chain of the interleukin-2 receptor, also known as CD25), TNFα (Tumor necrosis factor alpha, also known as cachectin), IL-4Ra (alpha chain of the interleukin-4 receptor), 1TL-17A (interleukin-17A), 1TL23A (alpha subunit of interleukin 23, also known as IL-23pl9), 1TTGAL (InTeGrin Alpha L, also known as CD1 IA antigen) and CD20.

[0053] CTLA4 is a protein receptor located on the cell membrane of T lymphocytes (BRUNET et al., Nature, vol. 328, pp. 267-270, 1987). CTLA4 is constitutively expressed in regulatory T lymphocytes but only upon activation in conventional T lymphocytes. It acts as an inhibitor of lymphocyte activity when it comes into contact with CD80 or CD86 proteins on the surface of an antigen-presenting cell. Antibodies directed against CTLA4 can be easily produced by those skilled in the art and include ripilimumab or tremelimumab.

[0054] PD-1 is expressed on the surface of activated T lymphocytes. This protein plays a role in regulating the immune system's response to human body cells by lowering the immune reaction and promoting autotolerance by suppressing the inflammatory activity of T lymphocytes. Antibodies directed against PD-1 can be easily obtained by those skilled in the art and include nivolumab, pembrolizumab, dostarlimab, or toripalimab.

[0055] PD-L1 is a protein expressed on the surface of macrophages, particularly tumor-associated macrophages. This protein binds to the PDI receptor and modulates the activation or inhibition of immune system cells. PD-L1 is now also expressed on the surface of several cancer cells and prevents apoptosis of the carrier cells. Antibodies directed against PD-L1 can be easily obtained by those skilled in the art and include atezolizumab and durvalumab.

[0056] BTLA expression is induced upon T cell activation and exhibits both activating and inhibiting capabilities. Antibodies directed against BTLA can be easily obtained by those skilled in the art and include icatolimab and HFB200603.

[0057] Killer cell immunoglobulin-like receptors (KIRs) are a family of type I transmembrane glycoproteins expressed on the plasma membrane of natural killer (NK) cells and a minority of T cells. They regulate the lethal function of these cells by interacting with major histocompatibility molecules (MHC) class I, which are expressed on all types of nucleated cells. KIR receptors can distinguish allelic variants of MHC class I, enabling them to detect virus-infected or transformed cells. Antibodies directed against a KIR receptor can be easily obtained by those skilled in the art and include lirilumab and 1-7F9 (ROMAGNE et al., Blood, vol. 114(13), pp. 2667–2677, 2009).

[0058] The CAMPATH-1 antigen, also known as cluster of differentiation 52 (CD52), is a glycoprotein that, in humans, is encoded by the CD52 gene. CD52 is present on the surface of mature lymphocytes, but not on the stem cells from which these lymphocytes originate. It is also found on monocytes. and dendritic cells. Antibodies targeting CAMPATH-1 can be easily obtained by a person skilled in the art and include alemtuzumab.

[0059] The interleukin-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, which binds to and responds to a cytokine called IL-2. The alpha chain of the interleukin-2 receptor (IL2RA) is a protein involved in the assembly of the high-affinity interleukin-2 receptor, composed of the alpha (IL2RA), beta (IL2RB), and common gamma (IL2RG) chains. Antibodies directed against IL2RA can be easily obtained by those skilled in the art and include basiliximab or daclizumab.

[0060] TNFα is an important cytokine involved in systemic inflammation and the acute-phase response. TNFα is primarily produced by activated macrophages, but it can also be produced by many other cell types, such as helper T cells, NK cells, neutrophils, eosinophils, and mast cells. Antibodies against TNFα can be easily produced by those skilled in the art and include adalimumab, infliximab, and golimumab.

[0061] The interleukin-4 receptor (IL4R) is a type I cytokine receptor. It is a heterodimer, composed of the alpha chain of the interleukin-4 receptor (IL-4Ra) combined either with the common gamma chain (yc, forming the type I IL4 receptor) or with IL-13Ral (forming the type II IL4 receptor). This receptor is involved in the production of IgE in B cells. Antibodies directed against IL-4Ra can be easily obtained by those skilled in the art and include dupilumab.

[0062] Interleukin-17A is a pro-inflammatory cytokine produced by lymphocytes Activated T cells. Antibodies directed against 1TL17A can be easily obtained by a person skilled in the art and include ixekizumab.

[0063] The IL23A gene codes for one of the two subunits of interleukin 23: the alpha subunit. Interleukin 23 is a pro-inflammatory cytokine. Antibodies directed against 1TL23A can be easily obtained by those skilled in the art and include guselmab.

[0064] LTTGAL is a pro-inflammatory cytokine involved in cell adhesion between leukocytes and costimulatory signaling between lymphocytes. It is the target of the drug efalizumab. Antibodies directed against LTTGAL can be easily obtained by a person skilled in the art and include efalizumab.

[0065] CD20 is a surface molecule of B lymphocytes expressed from the pre-B phase that disappears at the end of differentiation. More specifically, the CD20 molecule regulates a step in the activation process necessary for cell cycle initiation and differentiation. Antibodies directed against CD20 can be obtained simply by a person skilled in the art and includes rituximab, ibritumomab, tiuxetan, tositumomab and ofatuumumab.

[0066] As used here, an "infectious antigen" means an antigen expressed by an infectious agent or by a cell infected by such an infectious agent.

[0067] As used here, "infectious agent" means a microorganism (bacteria, virus, parasite) capable of causing an infection.

[0068] Preferably, the antibody directed against an infectious antigen is a neutralizing antibody.

[0069] By "neutralizing antibody" is meant an antibody that binds specifically to a surface antigen of an infectious agent (referred to as an infectious particle), thereby preventing it from coming into contact with host cells that it could infect and potentially destroy. Those skilled in the art can easily identify antibodies directed against an infectious agent that exhibit such neutralizing capacity. An example of a neutralizing antibody is palivizumab, which is used prophylactically against the most common cause of bronchiolitis (RSV) in infants.

[0070] Preferably, said neutralizing antibody is different from the antibody directed against the Marburg virus described in HEMPEL et al. (previously described, 2017) and from the antibodies directed against epitopes of the hepatitis B virus described in HEMPEL et al. (previously described, 2011; HEMPEL & MAIER, previously described, 2012; VANIER et al., previously described, 2015 and VANIER et al., previously described, 2018).

[0071] Advantageously, such an antibody is directed against a parasite antigen. Examples of parasites against which such a neutralizing antibody is directed include parasites selected from the group comprising Plasmodium falciparum (malaria), schistosomes (bilharzia) or protozoa of the genus Leishmania (leishmaniasis).

[0072] Advantageously, such an antibody is directed against a bacterial antigen. Examples of bacteria against which such a neutralizing antibody is directed include bacteria selected from the group comprising Streptococcus pneumoniae, Haemophilus influenzae type B (meningitis), Bordetella pertussis (whooping cough), Clostridium tetani (tetanus), Corynebacterium diphtheriae (diphtheria), Mycobacterium tuberculosis (tuberculosis), Neisseria meningitidis (meningococcal C meningitis and septicemia).

[0073] Advantageously, such an antibody is always directed against a viral antigen. Examples of viruses against which such a neutralizing antibody is directed include viruses selected from the group comprising respiratory syncytial virus (Orthopneumovirus hominis), influenza A virus (Alphainfluenza virus), Influenza B virus (Betainfluenza virus), hepatitis B virus (HBV), measles virus (Orbillivirus hominis), rubella virus (Rubivirus rubellae), mumps virus (Myxovirus parotidis), polioviruses, Zika virus (Orthoflavivirus zikaense), and yellow fever virus (Orthoflavivirus flavi).

[0074] As used here, a "tumor antigen" means an antigenic substance produced by tumor cells.

[0075] In this respect, many tumor antigens are well known to those skilled in the art, and examples include, but are not limited to, CEA, EGFR, HER2, EPCAM, MUC1, PSMA, CD-19, CAIX, phospholipid antigens such as phosphatidylserine, and gangliosides such as GM1, GD2, and GD2-O-acetylated. It should also be noted that certain immunoregulatory antigens can also be antitumor antigens (e.g., CD20). Such antibodies can be used to target tumor cells in the treatment of cancer patients.

[0076] The CEA (CarcinoEmbryonic Antigen) glycoprotein is a tumor marker involved in cell adhesion. Antibodies directed against CEA can be easily obtained by those skilled in the art and include arcitumomab.

[0077] ErbB receptors are expressed in various tissues of epithelial, mesenchymal, and neuronal origin. Under normal conditions, ErbB receptor activation is controlled by the spatial and temporal expression of their ligands, which are members of the EGF growth factor family. Ligand binding to ErbB receptors induces the formation of receptor homodimers and heterodimers and the activation of the intrinsic kinase domain, leading to phosphorylation of 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 and HER2 are known to play an essential role in the regulation of cell proliferation and differentiation.They have a strong tendency to assemble with other HER receptors into homo- and / or heterodimers upon binding to extracellular growth factor, resulting in various forms of signal transduction pathway activation, leading to apoptosis, cell survival, or proliferation. Antibodies targeting EGFR can be easily produced by those 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). Regarding HER2, antibodies against this receptor can also be easily produced by those skilled in the art and include trastuzumab (US 5,821,337).

[0078] EPCAM (EPithelial Cell Adhesion Molecule) is expressed in a variety of human epithelial tissues and in stem and progenitor cells. This molecule is now also expressed by numerous carcinomas, but is not expressed in cancers of non-epithelial origin. EPCAM is a 30-40 kDa glycosylated type I membrane protein involved, in particular, in homotypic cell adhesion. Antibodies directed against EPCAM can be easily obtained by those skilled in the art and include edrecolomab, catumaxomab, and nofetumomab.

[0079] The short SI variant of mucin (MUC1), also known as epithelial polymorphic mucin (EMP) or epithelial membrane antigen (EMA), is a glycoprotein with extensive O-glycosylation of its extracellular domain. In a healthy body, mucins line the apical surface of epithelial cells in the lungs, stomach, intestines, eyes, and several other organs for protection. By binding pathogens to oligosaccharides in its extracellular domain, it prevents these pathogens from reaching the cell surface. MUC1 also exhibits signaling capabilities. Overexpression of MUC1 is now frequently associated with colon, breast, ovarian, lung, and pancreatic cancers. Antibodies against MUC1 can be easily obtained by those skilled in the art and include clivatuzumab and gatipotuzumab.

[0080] Prostate-specific membrane antigen (PSMA) is a membrane glycoprotein primarily expressed in the prostate epithelium, proximal tubules of the kidney, the jejunal brush border of the small intestine, and ganglia of the nervous system. PSMA is now highly expressed in the male prostate, with expression levels one hundred times greater than in other tissues. For some prostate cancers, PSMA is the second most overexpressed gene product. Antibodies directed against MUC1 can be easily obtained by those skilled in the art and include capromab.

[0081] Cluster of differentiation 19 (CD19, also known as CVID3) is a transmembrane protein expressed by all B-lineage cells. This protein participates in the membrane recruitment of cytoplasmic signaling proteins. This protein is now also expressed in B-lineage cells that have undergone neoplastic transformation. Therefore, CD19 is a target for immunotherapies targeting neoplastic lymphocytes. Antibodies directed against CD19 can be easily produced by those skilled in the art and include tafasitamab and loncastuximab.

[0082] Carbonic anhydrase IX (CA IX) is a transmembrane dimeric metalloenzyme with an extracellular active site that facilitates acid secretion in the gastrointestinal tract. CA IX is overexpressed in many types of cancer, including clear cell renal cell carcinoma (RCC) and other carcinomas. of the cervix, breast, and lung, where it promotes tumor growth by increasing tumor acidosis. Antibodies targeting CAIX can be easily obtained by a person skilled in the art and include girentuximab,

[0083] Phosphatidylserine (abbreviated Ptd-L-Ser or PS) is a phospholipid and a component of the cell membrane. It plays a key role in cell cycle signaling, particularly with regard to apoptosis. Antibodies directed against PS can be easily obtained by those skilled in the art and include bavituximab.

[0084] Monosialotetrahexosylganglioside (GM1) is a ganglioside containing a sialic acid residue. GM1 possesses important physiological properties and impacts neuronal plasticity and repair mechanisms, as well as neurotrophin release in the brain. Antibodies directed against GM1 can be easily obtained by those skilled in the art.

[0085] 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 easily obtained by those skilled in the art and include dinutuximab and naxitamab.

[0086] GD2-O-acetylated is a GD2 ganglioside in which the terminal sialic acid is modified by the addition of an O-acetyl group. GD2-O-acetylated is expressed like GD2 in neuroectodermal tumors, but unlike GD2, it is not expressed in normal tissues. Antibodies directed against GD2-O-acetylated can be easily obtained by those skilled in the art and include antibody 8B6 (WO2008 / 043777).

[0087] While CD20 is a molecule expressed by "normal" B cells (from the pre-B phase until the end of differentiation), it is also generally expressed, and at very high levels, on neoplastic ("tumor") B cells. Thus, the CD20 surface antigen has the potential to serve as a candidate for "targeting" B-cell lymphomas. Antibodies directed against CD20 can be easily obtained by those skilled in the art and include rituximab, ribritumomab, tiuxetan, tositumomab, and ofatuumumab.

[0088] Preferably, the antibody is an IgG.

[0089] According to a particular embodiment, said IgG is an IgG3.

[0090] The human subclass IgG3 is notably absent among the formats approved for monoclonal antibody therapies and Fc fusion protein-based biologics. This limited use is the result of concerns about its potential for rapid degradation and short plasma half-life that have apparently outweighed the potential benefits of IgG3, which include IgG3 exhibits a high affinity for Fcy receptor activation, efficient complement binding, and a long hinge that appears better suited to targets with low abundance. The mean plasma half-life of IgG3 is thus only 7 days, compared to 21 days for other IgGs (IgG1, IgG2, and IgG4). Consequently, only one IgG3 is currently undergoing clinical trials for the treatment of lung cancer (GT103, NCT04314089, GRID THERAPEUTICS), despite its significant therapeutic potential.

[0091] Preferably, said antibody is a human antibody or one of its derivatives.

[0092] As used here, the term "derived" refers to a chimeric or humanized antibody.

[0093] The term "derived" antibody also refers to an antibody comprising one or more point mutations that enhance its properties (e.g., ADCC). As such, a derived antibody may include one or more mutations (2, 3, 4, 5, etc.) in its constant regions (relative to the wild-type sequences of the human antibody) in order to increase its binding affinity for the FcRn receptor. Examples of such mutations include patents EP 1817340 Bl, EP 2164873 B1, EP 2552955 B1, and EP 3181581 Bl.

[0094] Advantageously, said derived antibody comprises at most three mutations in its constant regions allowing to increase its binding affinity for the FcRn receptor, preferably at most two mutations and, particularly preferably, at most one mutation in its regions relative.

[0095] According to a particular embodiment, said derived antibody does not include any mutation enabling an increase in its binding affinity for the FcRn receptor.

[0096] By "chimeric antibody" is meant an antibody that is composed of the variable domains of a non-human (generally murine) antibody and the constant domains. As such, such a chimeric antibody includes at a minimum also the constant light chain domain (CL) and the constant heavy chain domains (CH1, CH2, CH3 and possibly CH4) of a human antibody.

[0097] By "humanized antibody" is meant an antibody that is composed partially or entirely of amino acid sequences derived from a germline human antibody by modifying the sequence of an antibody having non-human complementarity-determining regions (CDRs). As such, such a humanized antibody includes, at a minimum, the constant light chain (CL) domain and the constant heavy chain domains (CH1, CH2, CH3, and possibly 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. As examples of such humanization techniques, one can cite British patent application GB 2188638A or US patent US 5,585,089, which describe recombinant antibodies produced comprising a substitution only at the level of the complementarity-determining regions or "CDR".

[0098] Step (ii) of antibody purification can be carried out using techniques well known to those skilled in the art (e.g., OSSIPOW & FISCHER, Monoclonal Antibodies: Methods and Protocols; Methods in Molecular Biology Book 1131, Humanapress, Springer Protocols). Such a step typically includes collecting the culture medium by filtration and concentrating the supernatant thus recovered. The antibody is then purified from this supernatant by affinity chromatography, conventionally on a Sepharose protein A column. As an example of methods for carrying out this purification step, the methods described in international patent application WO 2023 / 208883 may be cited.

[0099] Step (iii) of determining antibody binding to the FcRn receptor can be performed using techniques well known to those 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. Determining the inhibition (IC50) by the antibody of the binding of the labeled ligand to the FcRn receptor allows the binding affinity of this antibody for the FcRn receptor to be deduced. Such a step is typically performed using one of the commercially available kits. Examples of such kits include the LUMIT FcRn BINDING IMMUNOASSAY kit (PROMEGA), HTRF FcRn BINDING KIT (REVVITY), HUMAN FcRn BINDING KIT (ACROBIOSYSTEMS), and 1-0 FcRn BINDING ASSAY KIT (CREATIVE BIOLABS).

[0100] According to a preferred embodiment, the process according to the invention includes a complementary step (iv) of comparing the binding affinity for FcRn of the same antibody (i.e., the same polypeptide sequence), but produced in mammalian cells. Monoclonal antibodies produced today are mainly produced by hamster cells, namely CHO (Chinese Hamster Ovary) and BHK (Baby hamster kidney) cells, and to a lesser extent by murine cells (NSO cells). It should be noted that human cells are increasingly used, including, among others, retinal cells (PER.C6 cells), cervical cancer cells (HeLa), T lymphocytes (Jurkat), and kidney cells (HEK 293, Human Embryonic Kidney).

[0101] Preferably, the method according to the invention includes a complementary step (iv) of comparing the binding affinity for FcRn of the same antibody but produced in mammalian cells, preferably of the same antibody produced in the CHO cell line.

[0102] According to another preferred embodiment, the process according to the invention includes a supplementary step (v) of selecting the antibody produced in Phaeodactylum tricornutum provided that its binding affinity for the FcRn receptor is less than 3 times greater, preferably at least 5 times greater, and particularly preferably at least 10 times greater, than that of the same antibody (i.e. the same polypeptide sequence) but produced in mammalian cells, preferably in CHO cells.

[0103] The present invention also relates to an antibody obtained or capable of being obtained by the process described above.

[0104] Advantageously, such an antibody does not belong to the group consisting of rituximab, trastuzumab, adalimumab, bevacizumab, infliximab, cetuximab, motavizumab, palivizumab, alemtuzumab, dinutuximab, naxitamab, benralizumab, catumaxomab, daratumumab, elotuzumab, epratuzumab, farletuzumab, galiximab, gemtuzumab, ibritumomab, lumiliximab, necitumumab, nimotuzumab, ocrelizumab, ofatumumab, oregocomab, pertuzumab, tositumomab, zalutumumab, zanolimumab, the antibody directed against the Marburg virus described in HEMPEL et al. (described previously, 2017), antibodies directed against hepatitis B epitopes described in HEMPEL et al. described previously, 2011; HEMPEL & MAIER, described previously, 2012; VANIER et al., described previously, 2015 and VANIER et al., described previously, 2018).

[0105] The present invention further relates to a composition comprising such an antibody as a drug.

[0106] As used in this application, the term "subject" in connection with the use of such a drug refers to a mammal such as a rodent, a feline, a canine, a primate or a human, preferably said subject is a human.

[0107] 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.

[0108] Such a composition may then include one or more additives such as diluents, excipients, stabilizers and preservatives. Such additives are well known to those skilled in the art and are described in particular in "Ullmann's Encyclopedia of Industrial Chemistry, 6th Ed." (various publishers, 1989-1998, Marcel Dekker); and in "Pharmaceutical Dosage Forms and Drug Delivery System" (ANSEL et al., 1994, WILLIAMS & WILKINS).

[0109] The antibody can be solubilized in a buffer, in water, or incorporated into emulsions or microemulsions. Examples of usable buffers include: Examples include phosphate saline buffer (PBS), artificial serum (150 mM NaCl in water) and Tris buffers.

[0110] There are many causes of antibody instability or degradation, such as hydrolysis and denaturation, which can lead to a decrease in the induction of the humoral or cellular response. Stabilizers can be added to reduce or prevent such problems.

[0111] Examples of stabilizers include monosaccharides, disaccharides, polysaccharides, ionic and non-ionic detergents, alkali metal salts, phospholipids, fatty acids, polyols and stabilizing peptides such as bovine serum albumin.

[0112] The composition according to the invention may then be in a form administrable by parenteral route, preferably by intravenous route.

[0113] The present invention further relates to a composition comprising an antibody as described above, which antibody is directed against an infectious antigen, and preferably constitutes a neutralizing antibody, for use in preventing infection in a subject by the infectious agent associated with said infectious antigen.

[0114] Preferably, a single administration of said antibody to a subject prevents infection of the subject for a period of at least two months following said administration, preferably for a period of at least three months following said administration and, particularly preferably, for a period of at least three months following said administration.

[0115] Accordingly, no further administration of said antibody is performed during this period. Indeed, the plasma half-life resulting from the discovery made by the inventors is such that it allows for the expectation of obtaining antibodies with a half-life similar to or greater than that of palivizumab (approximately 68 days) starting from any conventional antibody (approximately 21 days) when it is produced in Phaeodactylum tricornutum. As a reminder, a single administration of palivizumab prevents RSV infection for more than 4 months.

[0116] Such use relates to a method of prophylactic treatment of a pathology associated with an infectious agent comprising the administration of a therapeutically effective amount of a composition comprising an antibody as described above and directed against an infectious antigen associated with said infectious agent, which antibody being preferentially a neutralizing antibody.

[0117] The term "therapeutically effective amount" means an amount sufficient to prevent infection by the infectious agent. A person skilled in the art will be able to determine the therapeutically effective amount based on their general knowledge and / or by means of simple routine experiments.

[0118] The present invention further relates to a composition comprising an antibody as described above, which antibody is directed against an immunoregulatory antigen, for use in treating a subject suffering from an autoimmune disease, suffering from an allergy or undergoing transplantation.

[0119] A person skilled in the art will be able to easily identify the immunoregulatory antigens that should be targeted for such treatments (e.g., CD20, IL2R, or TNFα).

[0120] Advantageously, said antibody as defined is administered with a periodicity at least twice greater (i.e. with a time interval at least twice greater), preferably at least three times greater and, particularly preferably, at least 4 times greater than that with which the same antibody produced in mammalian cells, preferably in CHO, is administered.

[0121] The present invention further relates to a composition comprising an antibody as described above, which antibody is directed against an immunoregulatory antigen, for use this time to treat a subject suffering from cancer.

[0122] Here again, a person skilled in the art will be able to easily identify the immunoregulatory antigens that should be targeted for such treatments (e.g. CTLA4, PDI, PD-L1, etc.).

[0123] Here again advantageously, said antibody as defined is administered with a periodicity at least twice greater (i.e. with a time interval at least twice greater), preferably at least three times greater and, particularly preferably at least 4 times greater than that with which the same antibody produced in mammalian cells, preferably in CHO, is administered.

[0124] 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.

[0125] Here again advantageously, said antibody as defined is administered with a periodicity at least twice greater (i.e. with a time interval at least twice greater), preferably at least three times greater and, particularly preferably at least 4 times greater than that with which the same antibody produced in mammalian cells, preferably in CHO, is administered.

[0126] Preferably, the antibody targets the GD2 ganglioside or the GD2-O-Acetylated.

[0127] Advantageously, the antibody is dinutuximab.

[0128] The following examples are provided for illustrative purposes only and shall not limit the scope of the present invention. Examples

[0129] Cloning of antibody sequences into expression vectors

[0130] To determine precisely the production capacities of antibodies secreted by Phaeodactylum tricornutum 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) like those described in international application WO 2023 / 208883.

[0131] [Tables 1] Antibody Light chain Heavy chain dinutuximab SEQ ID N°4 SEQ ID N°5 rituximab SEQ ID N°6 SEQ ID N°7 trastuzumab SEQ ID N°8 SEQ ID N°9 adalimumab SEQIDN°10 SEQIDN°11 bevacizumab SEQIDN°12 SEQIDN°13 Infliximab SEQIDN°14 SEQIDN°15

[0132] Culture of microalgae cells

[0133] One of the strains used is Phaeodactylum tricornutum CCAP1055 / 6 (Culture Collection of Algae & Protozoa). The strain is maintained in 33.3g / L seawater medium (reconstituted from Instant Ocean®), supplemented with ImL / L of Conway solution (Table 2; KHATOON et al., Desalination and Water Treatment, vol. 57(60), p: 29295-29302, 2016).

[0134] [Tables2] Compound Concentration (g / m³) kNO3 100 Na3PO4 20 Na2H2EDTA-2H2O 45 FeCl3-6H2O 1.3 ZnCl2 4.2 MnCl2-4H2O 0.36 CoCl2-6H2O 4.0 CuSO4-5H2O 4.0 (NH4)6Mo7O24-4H2O 1.8 h3bo3 33.4 Thiamine Hydrochloride 0.2 Cyanocobalamin 0.01

[0135] Microalgae culture is carried out in a climate chamber at 19°C, with a Day / Night alternation and under agitation.

[0136] Transformation

[0137] The transformation of Phaeodactylum tricomutum is then carried out according to the protocol described in HU & PAN (Electroporation Transformation Protocol for Phaeodactylum tricomutum, Methods Mol. Biol., vol.2050, p: 163-167, 2020).

[0138] Briefly, the cell culture is washed with sorbitol to remove salts. The cells are placed in contact with the linearized vector and salmon sperm DNA on ice for 30 minutes. 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 hours in the dark.

[0139] Finally, the cells are plated on agar containing a selection agent adapted to the vector used, namely zeocin (75 pg / mL), nurseothricin (100 pg / mL) or blasticidin S (8 µg / mL). For this selection step, the cells are cultured for a few weeks.

[0140] Selection and culture of clones expressing the antibody of interest

[0141] After 4 to 8 weeks of culture on the first selection agar plates, colonies colonies appear. These first colonies are subcultured onto a new selection agar plate, carefully separated into individual cells, as soon as they reach a size of 1 mm. The agar plates are then cultured until the clones are screened (at least one month later). For each antibody to be produced, between 80 and 140 clones were tested.

[0142] Targeting clones by ELISA

[0143] For each clone to be tested, 100 pL of culture medium from a liquid culture of the clone is taken (7-8 day culture). The presence of antibodies in this volume is analyzed by ELISA using the "Human IgG ELISA" kit (MABTECH) following the manufacturer's instructions.

[0144] Bioreactor Culture

[0145] For each antibody to be tested, the clone producing the most antibody is selected from among the 80 to 140 clones isolated and tested by ELISA. The selected clone is then cultured in a 10L or 200L IL photobioreactor using the culture conditions described above.

[0146] After 7 days of culture, the microalgae clone culture is harvested and then clarified. The supernatant is recovered 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-R Protein A-650F (TOSOH)) following the associated manufacturer's instructions.

[0147] After elution of the antibody, the latter is concentrated using filtration units and then measured by micro-volume spectrophotometer before being used in functionality tests described below.

[0148] FcyR Binding Capability

[0149] The binding capacity of each antibody to the different FcyRs is measured using the different LUMIT™ FcyR Binding Immunoassay (PROMEGA) kits, following the manufacturer's instructions. As a control, the binding affinity of each antibody produced in Phaeodactylum tricomutum is compared to the affinity of the same antibody produced in mammalian cells (typically in CHO cell lines).

[0150] The results showed that the binding affinity of the tested antibodies for the different FcyR receptors was quite similar (whether produced in Phae o dactylum tricomutum or in mammalian cells) with also values ​​comparable to those observed previously in VAUTIER et al. (previously cited, 2018).

[0151] ADCC Activity Test

[0152] The ADCC activity of the different antibodies is measured using the PROMEGA ADCC REPORTER BIOASSAY kit, following the manufacturer's instructions. Target cells are cultured under their usual conditions at 37°C, 5% CO2. The SkBr3 cell line (culture medium: DMEM Hi glucose 10% FCS) is used for Trastuzumab (anti-ErbB2). The Raji cell line (culture medium: RPMI 1640 10% FCS) is used for Rituximab (anti-CD20). The LAN-1 and IMR-32 cell lines (culture medium: DMEM Hi glucose 10% FCS) are used for dinutuximab (anti-GD2).

[0153] FcRn binding capability

[0154] To further characterize the antibodies produced in Phaeodactylum tricomutum, the binding capacity of each antibody for the human FcRn receptor is measured using the LUMIT™ FcRn Binding Immunoassay (PROMEGA) kit, following the manufacturer's instructions. Again, each antibody is tested simultaneously with its reference produced in Chinese hamster ovary (CHO) cells.

[0155] Fig. 1 shows the FcRn receptor binding affinity of dinutuximab produced in CHO cells (QARZIBA, Reference) or in Phaedactylum tricomutum (AB1S_CS58) compared to the positive control for FcRn binding (IgG Control).

[0156] Unexpectedly, the results show that the binding affinity of an antibody produced in Phaedactylum tricornutum is much higher than that of the same antibody produced in CHO. Specifically, the binding affinity of dinutuximab produced in Phaedactylum tricornutum (IC50 = 68 nM) is observed to be nearly 14 times higher than that of the same antibody produced in mammalian cells (IC50 = 950 nM; see [Fig. 1]). In light of Zalevsky et al. (previously cited, 2010), in particular, it is therefore possible to predict that such an increase in binding affinity for FcRn results in a fivefold increase in the plasma half-life of dinutuximab (when switching from production in mammalian cells (CHO) to production in Phaedactylum tricornutum).

[0157] Pharmacokinetics in mice

[0158] In light of the particularly exciting results obtained in connection with binding to the FcRn receptor, a more precise determination of the plasma half-life of antibodies produced in Phaedactylum tricornutum is sought.

[0159] To this end, a pharmacokinetic study is performed by expressing each antibody to be tested in the humanized B6.Cg-Fcgrt mouse line <tmldcr>Tg(FCGRT)32Dcr / DcrJ (Tg32 mouse; stock number 0014565), described in PROETZEL & ROOPENIAN; Methods, vol.65(l), p: 148-53, 2014) which expresses human FcRn according to the supplier's protocol (JACKSON LABORATORY).< / tmldcr>

Claims

Demands

1. A process for producing antibodies with an increased plasma half-life comprising the steps of: (i) producing an antibody in a cell system; (ii) purifying said antibody; (iii) determining the binding affinity of said antibody for the FcRn receptor, preferably for the human FcRn receptor; Characterized in that said cell system is a culture of Phaeodactylum tricomutum:

2. The method according to claim 1, characterized in that step i) of antibody production uses a functional signal (secretory) peptide from Phaeodactylum tricomutum or an endoplasmic reticulum retention DDEL sequence, preferably step i) of antibody production uses a functional signal (secretory) peptide from Phaeodactylum tricomutum enabling secretion of the antibody into the culture medium.

3. The method according to any one of the preceding claims, characterized in that it comprises a step (iv) of comparing the binding affinity for the FcRn receptor of the same antibody, but produced in mammalian cells, preferably of the same antibody produced in the CHO cell line.

4. The method according to any one of the preceding claims, characterized in that it comprises a step (v) of selecting the antibody produced in Phaeodactylum tricomutum provided that its binding affinity for the FcRn receptor is at least 3 times greater, preferably at least 5 times greater, and particularly preferably at least 10 times greater, than that of the same antibody (i.e. the same polypeptide sequence) but produced in mammalian cells, preferably in CHO cells.

5. An antibody obtained by the process according to any one of the preceding claims, preferably said antibody is an IgG.

6. The antibody according to claim 5, characterized in that it does not belong to the group consisting of rituximab, trastuzumab, adalimumab, bevacizumab, infliximab, cetuximab, motavizumab, palivizumab, alemtuzumab, dinutuximab, naxitamab, benralizumab, catumaxomab, daratumumab, elotuzumab, epratuzumab, farletuzumab, galiximab, the gemtuzumab, ribritumomab, lumiliximab, necitumumab, nimotuzumab, ocrelizumab, ofatumumab, oregovomab, pertuzumab, tositumomab, zalutumumab, and zanolimumab.

7. The antibody according to claim 5, characterized in that said antibody is an IgG3.

8. A composition comprising at least one antibody as defined in any one of claims 5 to 7 and directed against an immunoregulatory antigen, an infectious antigen or a tumor antigen as a drug.

9. A composition comprising an antibody as defined in any one of claims 5 to 7 and directed against an infectious antigen, preferably a neutralizing antibody, for use in preventing infection in a subject by the infectious agent associated with said infectious antigen.

10. A composition comprising an antibody as defined in any one of claims 5 to 7 and directed against an immunoregulatory antigen, for use in treating a subject suffering from an autoimmune disease, suffering from an allergy or undergoing transplantation.

11. A composition comprising an antibody as defined in any one of claims 5 to 7 and directed against an immunoregulatory antigen or against a tumor antigen, for use in treating a subject suffering from cancer.

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