M3c65 antibody and derivatives thereof for targeting inflammation

Abstract

The present invention relates to the use of an M3C65 antibody or derivatives thereof, and constructs comprising M3C65 or derivatives thereof, for targeting inflammatory tissues and / or treating inflammatory diseases. In particular, the present invention relates to a chimeric construct or conjugate comprising an M3C65 antibody or a derivative thereof, and to a cell that has been modified to express an M3C65 antibody or a derivative thereof, for targeting inflammatory tissues and / or treating inflammatory diseases.

Description

[0001] M3C65 ANTIBODY AND DERIVATIVES THEREOF

[0002] FOR TARGETING INFLAMMATION

[0003] The present invention relates to the use of an M3C65 antibody or derivatives thereof, and constructs comprising M3C65 or derivatives thereof, for targeting inflammatory tissues and / or treating inflammatory diseases. In particular, the present invention relates to a chimeric construct or conjugate comprising an M3C65 antibody or a derivative thereof, and to a cell that has been modified to express an M3C65 antibody or a derivative thereof, for targeting inflammatory tissues and / or treating inflammatory diseases.

[0004] BACKGROUND OF THE INVENTION

[0005] Cells produce reactive oxygen species (ROS) whose biological effects depend on the amount produced. At low concentrations, ROS are involved in proliferation, differentiation and cell metabolism, while at high concentrations, they are involved in the formation of neutrophil extracellular traps (NETs) that promote microbial elimination. In response to certain exogenous and endogenous stimuli, such as inflammation, this balance can be disrupted and lead to the accumulation of ROS that participate in oxidative stress, causing irreversible alteration of DNA, RNA, proteins and lipids. A major consequence of oxidative stress is lipid peroxidation, which generates a number of highly reactive breakdown products, that in turn react with lipids, apoproteins and proteins, thereby forming stable covalent adduct and creating “oxidationspecific epitopes” (OSE).

[0006] Oxidation-specific epitopes (OSE) are present on oxidized proteins or oxidized lipids that are themselves involved in or trigger inflammation, such as sterile inflammation. Sterile inflammation is an inflammation which occurs in the absence of any microorganisms, and that is elicited in response to damage-associated molecular patterns (DAMPs), which are released locally in response to tissue damage. Sterile inflammation is triggered by the activation of the innate immune response through the recognition of DAMPs by pattern recognition receptors (PRRs), resulting in the enhanced secretion of cytokines and chemokines. Increased cytokine and chemokine secretion at the site of initial damage ultimately results in an enhanced recruitment of immune cells, such as neutrophils and macrophages. Unresolved sterile inflammation is implicated in the development of several medical conditions, such asautoimmune diseases, gout, Alzheimer disease, and atherosclerosis. For example, oxidized phospholipids (OxPL) are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes. Some studies therefore suggest that therapies targeting and / or inactivating OxPL may be beneficial for reducing inflammation (Que, X., Hung, MY., Yeang, C. et al. Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice. Nature 558, 301-306 (2018)).

[0007] Due to their accumulation in inflammatory conditions, OSE-modified proteins or lipids, such as oxidized phospholipids, can be used as targets. In this regard, recent developments have focused on chimeric IL2 constructs designed to selectively bind to and accumulate in inflammatory tissues. In particular, recent research has demonstrated the efficacy of chimeric constructs comprising IL2 fused to an OSE-binding targeting moiety, such as an E06 antibody (WO23057588). This strategy was able to enhance the efficacy of IL2 therapy by increasing IL2 levels specifically in inflammatory tissues.

[0008] Nevertheless, there is still a need for strategies to target inflammatory tissues, in particular to treat inflammatory diseases.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention relates to the use of an M3C65 antibody or derivatives thereof, and constructs comprising M3C65 or derivatives thereof, for targeting inflammatory tissue and / or treating inflammatory diseases. In particular, the present invention relates to a chimeric construct or conjugate comprising M3C65 antibody or a derivative thereof, to a cell that has been modified to express M3C65 antibody or a derivative thereof, for targeting inflammatory tissue and / or treating inflammatory diseases.

[0011] The M3C65 antibody is known as an anti-Phosphocholine (PC)-epitope. However, some previous studies have concluded that it has poor properties in vivo, rendering it unsuitable for therapeutic or targeting use. For example, P.X Shaw et al. showed that M3C65 fails to stain atherosclerotic plaques in vivo, which are however well stained by other anti-PC antibodies such as PG14 or EO6 antibody (see Figure 5 of Peter X. Shaw, et al., The Autoreactivity of Anti-Phosphorylcholine Antibodies for Atherosclerosis-Associated Neo-Antigens and Apoptotic Cells. J Immunol 15 June 2003; 170 (12): 6151-6157.). In view of the fact thatM3C65 antibody showed very weak binding in vivo, it was previously concluded that M3C65 was less able to target inflammatory regions containing PC and PC-derived epitopes.

[0012] The inventors unexpectedly showed that M3C65 actually outperforms other anti-PC antibodies (such as the E06 antibody) for targeting protein or cells to various sites of inflammation. In particular, the inventors showed that M3C65 has a 20-fold higher avidity for PC than EO6. This is important because it allows a stronger binding to OSE than E06.

[0013] Thus, according to a first aspect, the invention relates to an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, for use in treating an inflammatory disease. In particular, the invention relates to an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, for use in treating inflammation, preferably inflammation in a patient with an inflammatory disease and / or an auto-immune disease.

[0014] Another aspect of the invention relates to an M3C65 chimeric construct. Specifically, the chimeric construct of the invention comprises :

[0015] (i) a targeting moiety, which is an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, that is fused to

[0016] (ii) a payload that is a heterologous peptide or protein.

[0017] Preferably, the targeting moiety is an M3C65 scFv.

[0018] In a particular embodiment, the payload is a cytokine.

[0019] In a preferred embodiment, the payload is interleukin 2 (IL-2). In a particular embodiment, said IL-2 is human IL-2 or homologous variant thereof, wherein the variant has at least 80% amino acid identity with human wild-type IL-2. In a preferred embodiment, the variant is an active analogue of human IL-2 which has at least 85% amino acid identity with human wild-type IL-2. In a preferred embodiment, the variant is an active analogue of human IL-2 which has at least 90% amino acid identity with human wild-type IL-2. In a particular embodiment, said IL-2 is an IL2 mutein that comprises a substitution at position N88 of SEQ ID NO: 12, still preferably substitution N88R or N88D. In a particular embodiment, said IL-2 has the amino acid sequence of SEQ ID NO: 12 or has at least 85%, 90% or 95% amino acid identity with SEQ ID NO: 12. In another particular embodiment, said IL-2 has the amino acid sequence of SEQ ID NO:30 orhas at least 85%, 90% or 95% amino acid identity with SEQ ID NO:30. In another particular embodiment, said IL-2 has the amino acid sequence of SEQ ID NO:31 or has at least 85%, 90% or 95% amino acid identity with SEQ ID NO:31.

[0020] In a particular embodiment, said chimeric construct further comprising a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof that is capable of forming a dimeric protein. In a preferred embodiment, the fragment of C4BPP comprises, or consists of, amino acid residues 194 to 252 of C4BPP or a longer fragment of C4BPP that extends at the N-term up to at most amino acid 135. In a particular embodiment, said chimeric construct comprises IL-2 fused at the N-terminus of C4BPP or said fragment thereof, wherein the C-terminus of C4BPP or said fragment thereof is preferably linked to the targeting moiety.

[0021] Another aspect of the invention relates to a nucleic acid encoding said chimeric construct or to a vector comprising said nucleic acid. Another aspect of the invention relates to a host cell expressing said nucleic acid or vector.

[0022] Another aspect of the invention relates to said chimeric construct, wherein the payload is interleukin 2 (IL-2), for use in treating an auto-immune disease or an inflammatory disease.

[0023] Another aspect of the invention relates to a conjugate comprising :

[0024] (i) a targeting moiety, which is an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, that is conjugated, preferably by a linker, to

[0025] (ii) a payload that is a drug or an imaging moiety.

[0026] Another aspect of the invention relates to a cell, preferably an immune cell, that has been modified to express an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, wherein the M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof is preferably expressed on the cell surface. According to a particular embodiment, the C-terminus said M3C65 antibody, functional variant or fragment thereof, is fused to a transmembrane domain of a cell surface protein or a GPI anchor.

[0027] In a preferred embodiment, the cell is a regulatory T (Treg) cell, such as a conventional Treg cell or a converted Treg cell made by expressing Foxp3 in a conventional T cell. In a particular embodiment, the cell, preferably a Treg cell, has been further modified to express a proteinimproving its functionality, such as IL-2. In a particular embodiment, the cell is autologous, allogeneic or xenogeneic.

[0028] In a particular embodiment, the cell has been modified to express a CAR based on said M3C65 antibody, functional variant, or fragment thereof. According to a particular embodiment, the C-terminus of M3C65 antibody is preferably fused to a transmembrane domain. According to a particular embodiment, the transmembrane domain is further fused to an intracellular domain permitting the cell activation, such as CD3^, CD28, and / or 4-1BB.

[0029] According to a particular embodiment, said chimeric construct, conjugate or cell comprises an M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, which comprises :

[0030] - a heavy chain variable region (VH) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:32, a CDR2 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:33, and a CDR3 having the amino acid sequence of SEQ ID NO:3 ; and / or - a light chain variable region (VL) comprising a CDR1 having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:34, a CDR2 having the amino acid sequence of SEQ ID NO:5 (GTK) or SEQ ID NO:35, and a CDR3 having the amino acid sequence of SEQ ID NO:6.

[0031] According to a particular embodiment, said chimeric construct, conjugate or cell comprises an M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, which comprises :

[0032] - a heavy chain variable region (VH) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO:2, and a CDR3 having the amino acid sequence of SEQ ID NO:3 ; and / or

[0033] - a light chain variable region (VL) comprising a CDR1 having the amino acid sequence of SEQ ID NO:4, a CDR2 having the amino acid sequence of GTK (SEQ ID NO:5), and a CDR3 having the amino acid sequence of SEQ ID NO:6.

[0034] In a particular embodiment, the M3C65 antibody or functional variant thereof, preferably an M3C65 scFv, comprises :

[0035] - a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:7 ; and- a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 8.

[0036] In a particular embodiment, the M3C65 antibody or functional variant thereof is a humanized antibody or a fragment thereof, preferably a humanized M3C65 scFv. In a particular embodiment, said humanized M3C65 antibody or fragment thereof, in particular said humanized M3C65 scFv, comprises :

[0037] - a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9 ; and

[0038] - a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10.

[0039] Another aspect of the invention relates to the chimeric construct, conjugate or cell according to the present invention, for use as a medicament.

[0040] Another aspect of the invention relates to the chimeric construct, conjugate or cell according to the present invention, for use in treating an inflammatory disease.

[0041] DESCRIPTION OF THE FIGURES

[0042] Figure 1: Assessment of avidity and affinity interaction between E06 or M3C65 fusion proteins and PC-BSA.

[0043] A) PC-BSA (1 pg / ml) was coated on a 96-well plate and incubated overnight at 4°C. After washing and blocking, 5-fold dilutions of E06 or M3C65 were incubated for 2 hours at 37°C in triplicate. After a subsequent washing, bound fusion proteins were exposed to sodium thiocyanate (NaSCN; 0.5M, 1.5M) or left untreated (control). Residual binding was quantified by absorbance (Optical Density measurement) at 450 nm, with NaSCN resistance indicating avidity.B) Affinity was determined by surface plasmon resonance (SPR) using a BIACORE 3000. Anti-BSA IgG was covalently immobilized onto a sensor chip and infused with the same concentration of PC-BSA. A dose response of E06 or M3C65 fusion proteins was serially infused to evaluated binding of the proteins. Binding kinetics, i.e. the association (kon) and dissociation (koff) rate constants, were measured during 5-minute association / dissociation phases, and the dissociation constant (KD) was calculated using a 1 : 1 Langmuir binding model.

[0044] Figure 2: Therapeutic effects of the targeted fusion proteins in colitis inflammatory experimental model.

[0045] A) The therapeutic efficacy of IL-2, E06, and M3C65 fusion proteins was evaluated in an 8-week-old female C57B1 / 6 mouse model of DSS-induced colitis (n=5). Colitis was induced by administering 3% DSS in drinking water for 6 consecutive days, starting on day 0. One day before the addition of 3% dextran sulfate sodium (DSS) to drinking water, mice began a 5-day course of daily intraperitoneal injections (50,000 IU of IL-2, E06, or M3C65 fusion proteins, or PBS for the control group). Additionally, a second 5-day course started on day 6.

[0046] Mice were monitored daily for clinical symptoms, including body weight loss (B), expressed as a percentage of initial weight; scored 0-4, stool consistency (scored 0-4), and blood presence in feces (scored 0-4). These parameters were used to calculate a Disease Activity Index (DAI) (C) ranging from 0 (normal) to 12 (severe disease).

[0047] Figure 3: In vivo targeting of IL-2C4bpP-M3C65 fusion protein

[0048] To evaluate the in vivo targeting of the IL-2C4bpP-M3C65 fusion protein, eight- week-old female C56B16 / J mice received IL-2C4bpP-M3C65 by the mean of injection of 1011vg of AAV-IL-2C4bpP-M3C65, or PBS. Ten days after injection, psoriasis-like inflammation was induced by applying 62.5 mg of 5% imiquimod cream to the right ear daily and Vaseline to the left ear as a control. After six days, the mice were euthanized, their ears were collected and fixed, and they were paraffin-embedded for future sections (figure 3A). The ears were sectioned and stained directly with an anti-HisTag-Cy5 antibody to detect the presence of IL-2C4bpP-M3C65 and counterstained with DAPI to visualize the nuclei (figure 3B).

[0049] Figure 4: Direct binding of IL-2C4bpP-M3C65 fusion protein to inflammatory tissues The direct binding of the IL-2-C4bpP-M3C65 fusion protein to inflamed ears was evaluated. Eight-week-old female C57B16 / J mice were induced with psoriasis-like inflammation by applying 62.5 mg of 5% imiquimod cream to the right ear daily with vaseline applied to the leftear as a control. After six days, the mice were euthanized and their ears were collected, fixed, and paraffin-embedded for future sections (Figure 4A). The ears were sectioned and stained with IL-2-C4bpP-M3C65, followed by an anti-HisTag-Cy5 antibody, in order to evaluate the protein’s direct binding. The cells were also counterstained with DAPI to visualize nuclei. Controls consisted of anti-HisTag-Cy5 antibody staining that had not been pre-incubated with the IL-2-C4bpP-M3C65 (Figure 4B).

[0050] Figure 5: In Vivo Targeting of CAR-M3C65 T-Cells

[0051] A) To evaluate the in vivo targeting of CAR-M3C65 T-cells, 10-week-old female NSG mice were injected intravenously with 2x10sCAR-M3C65 T-cells expressing a luciferase transgene (CAR-M3C65-LUC). Control mice received non-transduced T-cells.

[0052] B) On day 7 (D7), bioluminescence imaging was performed using the IVIS Spectrum system. Mice were administered 0.295M Viviren, a substrate specific for Renilla luciferase. Images were acquired for 2 minutes, 5 minutes after administration of said substrate. Following imaging, all mice received a subcutaneous injection of PC-BSA (20 pg) complexed and adjuvanted with Alum and Complete Freund’s Adjuvant (CFA, 1 mg / ml) in the upper back. C) On day 9 (D9), bioluminescence imaging was repeated under the same conditions to evaluate CAR-M3C65-LUC T-cell recirculation and accumulation at the PC-BSA injection site.

[0053] Figure 6: Flow Cytometry Analysis of CAR-M3C65-Tregs proliferation

[0054] CAR-M3C65-Tregs (Tregs transduced with the full CAR-M3C65) or CAR-M3C65ATS-Tregs (Tregs transduced with CAR-M3C65 lacking the intracellular transducing signal domain) were cultured for 72 hours with either coated PC-BSA or plain BSA as control. The cells were stained with CellTrace (CTV) prior to culturing to determine Treg proliferation by flow cytometry analysis.

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056] Definitions

[0057] The “subject” or “patient” to be treated may be any mammal, preferably a human being. The human subject may be a child, an adult or an elder.The term “treating” or “treatment” means any improvement in the disease. It includes alleviating at least one symptom, or reducing the severity or the development of the disease. When the disease is an inflammatory and / or autoimmune disorder, the term more particularly includes reducing the risk, occurrence or severity of acute episodes (flares). The term “treating” or “treatment” encompasses reducing the progression of the disease. In particular the invention encompasses preventing or slowing down the progression of the disease. The term “treating” or “treatment” further encompasses prophylactic treatment, by reducing the risk or delaying the onset of the disease, especially in a subject who is asymptomatic but has been diagnosed as being “at risk”.

[0058] “Regulatory T cells” or “Tregs” are T lymphocytes having immunosuppressive activity. Natural Tregs are characterized as CD4+CD25+Foxp3+ cells. Tregs play a major role in the control of inflammatory diseases, although their mode of action in such disease is not well understood. In fact, in most inflammatory diseases, Treg depletion exacerbates disease while Treg addition decreases it. Most Tregs are CD4+ cells, although there also exists a rare population of CD8+ Foxp3+ T lymphocytes with a suppressive activity.

[0059] An antibody “specifically binds” to a target antigen if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. “Specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of this disclosure.

[0060] “Antibody fragments" comprise only a portion of an antibody, wherein the portion typically retains at least one, more commonly most or all, of the functions normally associated with that portion when present in the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules or single-domain antibodies; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises an antigen binding site of the original antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in the original antibody, such as FcRn binding, antibody half-life modulation, ADCC function and complement binding.The antigen-binding regions or antigen-binding fragments correspond to the arms of the Y-shaped structure of the antibody, which consist each of the complete light chain paired with the VH and CHI domains of the heavy chain, and are called the “Fab fragments” (for Fragment antigen binding). Fab fragments were first generated from native immunoglobulin molecules by papain digestion which cleaves the antibody molecule in the hinge region, on the aminoterminal side of the interchains disulfide bonds, thus releasing two identical antigen-binding arms. Other proteases such as pepsin, also cleave the antibody molecule in the hinge region, but on the carboxy -terminal side of the interchains disulfide bonds, releasing fragments consisting of two identical Fab fragments and remaining linked through disulfide bonds; reduction of disulfide bonds in the F(ab')2 fragments generates Fab' fragments.

[0061] The part of the antigen binding region corresponding to the VH and VL domains is called the Fv fragment (for Fragment variable); it contains the CDRs (complementarity determining regions), which form the antigen-binding site (also termed paratope).

[0062] The effector region of the antibody which is responsible of its binding to effector molecules or cells, corresponds to the stem of the Y-shaped structure, and contains the paired CH2 and CH3 domains of the heavy chain (or the CH2, CH3 and CH4 domains, depending on the class of antibody), and is called the Fc region (for Fragment crystallisable region).

[0063] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This scFv fragment retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.

[0064] As used herein, a “single-domain antibody” (sdAb) also known as nanobody (Nb) refers to a single-variable domain, derived from a heavy-chain only antibody, which is able to bind an antigen, an epitope or a ligand alone, that is to say, without the requirement of another binding domain. A single domain antibody may derive from, or consists in, a VHH that refers to a single variable domain found in heavy-chain antibodies of Camelidae.As used herein, the term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g. chimeric antibodies that contain minimal sequence derived from a non-human antibody). A “humanized form” of an antibody, e.g., a non-human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. The donor antibody can be any suitable non-human antibody, such as a mouse antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by framework region residues from the donor antibody. Alternatively, selected framework region residues of the donor antibody are replaced by framework region residues from a human or humanized antibody. Additional framework region modifications may be made within the human framework sequences. Humanized antibodies thus may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such amino acid modifications may be made to further refine antibody function and / or increased the humanization process.

[0065] The sequence listing shows the following sequences:

[0066] SEQ ID NO:1 is GFSLTGYG: the amino acid sequence of heavy chain CDR1 (H-CDR1) of M3C65.

[0067] SEQ ID NO:2 is IWGDGST: the amino acid sequence of heavy chain CDR2 (H-CDR2) of M3C65.

[0068] SEQ ID NO:3 is VRDYGPY: the amino acid sequence of heavy chain CDR3 (H-CDR3) of M3C65.

[0069] SEQ ID NO:4 is TGAVTTSNY: the amino acid sequence of light chain CDR1 (L-CDR1) of M3C65.

[0070] SEQ ID NO:5 is GTK: the amino acid sequence of light chain CDR2 (L-CDR2) of M3C65. SEQ ID NO:6 is ALWYSNHWV: the amino acid sequence of light chain CDR3 (L-CDR3) of M3C65.

[0071] SEQ ID NO:7 is the amino acid sequence of M3C65 variable heavy chain.

[0072] SEQ ID NO: 8 is the amino acid sequence of M3C65 variable light chain.

[0073] SEQ ID NO:9 is the amino acid sequence of humanized M3C65 variable heavy chain.SEQ ID NO: 10 is the amino acid sequence of humanized M3C65 variable light chain.

[0074] SEQ ID NO: 11 is wild-type human IL2 (253 amino acids, including the signal peptide) SEQ ID NO: 12 is mature wild-type human IL2 (233 amino acids, without the signal peptide) SEQ ID NO: 13 is C4BP beta chain (1-252)

[0075] SEQ ID NO: 14 is fragment 194-252 of C4BP beta chain

[0076] SEQ ID NO: 15 is fragment 137-252 of C4BP beta chain

[0077] SEQ ID NO: 16 is the GGGGS pattern (linker)

[0078] SEQ ID NO: 17 is linker GGGGS GGGGS GGGGS

[0079] SEQ ID NO: 18 is the GGGS pattern (linker)

[0080] SEQ ID NO: 19 is linker GGGSGGGSGGGS

[0081] SEQ ID NO:20 is the amino acid sequence of M3C65 scFv

[0082] SEQ ID NO:21 is the amino acid sequence of humanized M3C65 scFV

[0083] SEQ ID NO:22 is the amino acid sequence of hIL-2 fused to C4BPB

[0084] SEQ ID NO:23 is the amino acid sequence of IL-2 (N88R) fused to C4BPB

[0085] SEQ ID NO:24 is the amino acid sequence of hIL-2 fused to M3C65

[0086] SEQ ID NO:25 is the amino acid sequence of hIL-2 fused to C4BPB fused to M3C65 SEQ ID NO:26 is the amino acid sequence of IL-2 (N88R) fused to M3C65

[0087] SEQ ID NO:27 is the amino acid sequence of IL-2 (N88R) fused to C4BPB fused to M3C65 SEQ ID NO:28 is the amino acid sequence of IL-2 (Aldel+C125S) fused to M3C65

[0088] SEQ ID NO:29 is the amino acid sequence of IL-2 (Aldel+C125S) fused to C4BPB fused to M3C65

[0089] SEQ ID NO:30 is IL-2 variant (N88R)

[0090] SEQ ID NO:31 is IL-2 variant (Aldel+C125S)

[0091] SEQ ID NO:32 is the extended sequence of H-CDR1 of M3C65

[0092] SEQ ID NO:33 is the extended sequence of H-CDR2 of M3C65

[0093] SEQ ID NO:34 is the extended sequence of L-CDR1 of M3C65

[0094] SEQ ID NO:35 is the extended sequence of L-CDR2 of M3C65

[0095] SEQ ID NO:36 is the amino acid sequence of humanized M3C65 variable heavy chain (with extended CDRs)

[0096] SEQ ID NO:37 is the amino acid sequence of humanized M3C65 variable light chain (with extended CDRs)

[0097] SEQ ID NO:38 is the amino acid sequence of humanized M3C65 scFV (with extended CDRs) SEQ ID NO:39 is the PPPPK pattern (linker)1- M3C65 chimeric construct

[0098] The chimeric construct of the present invention comprises (i) a targeting moiety that is fused to (ii) a payload.

[0099] Tarsetins moiety

[0100] In the context of the present invention, the targeting moiety of the chimeric construct refers to an M3C65 antibody or a fragment thereof, or to a functional variant of M3C65 antibody or a fragment thereof.

[0101] As used herein, the terms “M3C65” and “M3C65 antibody” are used interchangeably to designate a hybridoma antibody composed of y2b heavy chains and XI light chains and produced in the secondary immune response to phosphocholine (PC)-protein antigens. M3C65 antibody is known in the state of the art as exhibiting high affinity for PC-protein conjugates. The enhanced properties of M3C65 are attributed to somatic mutations, particularly in the CDR2 region of the XI light chain, with key mutations at positions 52, 53, and 55. However, the potential of M3C65 as afunctional anti-oxidation-specific epitope (anti-OSE) antibody has been assessed and found to be suboptimal for therapeutic or targeting applications. Notably, M3C65 does not effectively stain atherosclerotic plaques, in contrast to other anti-OSE antibodies, such as PG14 or EO6, which exhibit robust staining capabilities (Shaw, P. X. et al. (2003).

[0102] Surprisingly, the present inventors observed that chimeric constructs comprising M3C65 antibody were actually more effective at targeting proteins or cells at different sites of inflammation than constructs comprising E06 antibody (which has the same specificity to PC antigen).

[0103] It is therefore an object of the present invention to provide a chimeric construct comprising a targeting moiety which is an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof.In a particular embodiment, the targeting moiety is an M3C65 antibody or an M3C65 antibody fragment such as a Fab, Fab', F(ab')2, Fv or scFv fragment of M3C65 antibody, or functional variants thereof.

[0104] In a particular embodiment, the targeting moiety is a functional variant of M3C65 antibody or an M3C65 antibody fragment such as a Fab, Fab', F(ab')2, Fv or scFv fragment of M3C65 antibody, or functional variants thereof.

[0105] In a particular embodiment, the targeting moiety is a fragment of M3C65 antibody, or a fragment of a functional variant of M3C65 antibody.

[0106] In a preferred embodiment, the targeting moiety is a scFv fragment of M3C65 antibody or a scFv fragment of a functional variant of M3C65 antibody. In a particular embodiment, the targeting moiety is a scFv fragment of M3C65 antibody. Indeed, a scFv fragment lacks a Fc domain and therefore has a silenced effector function.

[0107] The terms “functional variant”, “derivative”, “mutated derivative”, or “mutant” designate a sequence that differs from the parent sequence to which it refers by deletion, substitution or insertion of one or several amino acids, without substantially impacting the function of the antibody or the fragment thereof. Preferably, the functional variant shows at least 60%, 65% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity with the native sequence of the M3C65 antibody. In a particular embodiment, the mutations do not substantially impact the function of the antibody. A functional variant of an antibody or a fragment thereof possesses similar antigen-binding affinity relative to the reference antibodies (e.g., having a KD less than 1 x 10-7 M, 10-8 M, preferably less than 1 x 10-9 or 1 x 10-10 M). The affinity of the binding is defined by the terms ka (associate rate constant), kd (dissociation rate constant), or KD (equilibrium dissociation). Typically, specifically binding when used with respect to an antibody refers to an antibody that specifically binds to (“recognizes”) its target(s) with an affinity (KD) value less than 10-7 M, preferably less than 10-8 M, e.g., less than 10-9 M or 10-10 M. A lower KD value represents a higher binding affinity (i.e., stronger binding) so that a KD value of 10-9 indicates a higher binding affinity than a KD value of 10-8. In one embodiment, the equilibrium KD value of the M3C656 antibody or functional variants thereof is less than 5x10-8 M.Mutated derivatives, or functional variants, can comprise a VH chain that comprises an amino acid sequence at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any of the reference sequences recited herein, a VL chain that has an amino acid sequence at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any of the reference sequences recited herein, or both. These variants are capable of binding to Phosphocholine (PC). In some examples, the variants possess similar antigen-binding affinity relative to the reference antibodies described above (e.g., having a Kd less than 1 x 10-8 M).

[0108] A functional variant comprises mutations (deletions, substitutions or insertions) that do not adversely impact the binding function (e.g., affinity, specificity, or both) and bioactivity of the antibody (such as reducing the binding affinity by more than 50% as compared to the original antibody). In some embodiments, the modified antibody has the same binding specificity and has at least 50%, such as at least 60%, at least 70% or at least 80% or at least 90% of the affinity of the original antibody. The present disclosure also encompasses M3C65 antibody variants with improved biological properties of the antibody, such as higher binding affinity, or with altered ADCC properties, or with improved effects on Phosphocholine (PC)-expressing cells.

[0109] In a particular embodiment, the functional variants described herein can contain one or more mutations (e.g., conservative substitutions) which preferably do not occur at residues which are predicted to interact with one or more of the CDRs of the M3C65 antibody. In a particular embodiment, the functional variants described herein do not comprise mutation (such as deletions, substitutions or insertions) in the CDR regions.

[0110] Conservative substitutions will produce molecules having functional and chemical characteristics similar to those of the molecule from which such modifications are made. For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with another residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art. For example, amino acid substitutions can be used to identify important residues of the molecule sequence, or to increase or decrease the affinity of the molecules described herein. Variants comprising one or more conservative amino acid substitutions can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J.Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0111] Amino acid sequence variants of the antibody can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or via peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to achieve the final construct, provided that the final construct possesses the desired characteristics. Nucleic acid molecules encoding amino acid sequence variants of the antibody can be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant (natural) version of the antibody.

[0112] Any of the M3C65 antibodies, functional variants or fragments thereof described herein can be examined to determine their properties, such as antigen-binding activity, antigen-binding specificity, and biological functions, following routine methods. The binding affinity may be determined using techniques known in the art, such as ELISA or biospecific interaction analysis, or other techniques known in the art.

[0113] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0114] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:1 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 1; - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:2 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:2; and- H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with respect to SEQ ID NO:3 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:3.

[0115] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises extended variants of H-CDR1, H-CDR2 and / or H-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said H-CDR1, H-CDR2 and / or H-CDR3.

[0116] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0117] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or an extended variant thereof having the amino acid sequence of SEQ ID NO:32;

[0118] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or an extended variant thereof having the amino acid sequence of SEQ ID NO:33; and

[0119] - H-CDR3 having the amino acid sequence of SEQ ID NO:3.

[0120] Preferably, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following light chain complementarity-determining regions (L-CDRs):

[0121] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:4;

[0122] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO:6 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6.

[0123] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises extended variants of L-CDR1, L-CDR2 and / or L-CDR3, i.e.variants having additional amino acid residues at the N-terminus or C-terminus of said L-CDR1 , L-CDR2 and / or L-CDR3.

[0124] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following light chain complementarity-determining regions (L-CDRs):

[0125] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or an extended variant thereof having the amino acid sequence of SEQ ID NO:34;

[0126] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or an extended variant thereof having the amino acid sequence of SEQ ID NO:35; and

[0127] - L-CDR3 having the amino acid sequence of SEQ ID NO:6.

[0128] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0129] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:32, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:1 or SEQ ID NO:32;

[0130] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:33 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:2 or SEQ ID NO:33;

[0131] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0132] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0133] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:34 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:4 or SEQ ID NO:34;

[0134] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:35 a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:5 or SEQ ID NO:35; and

[0135] - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.In a preferred embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0136] - H-CDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 1;

[0137] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 2;

[0138] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0139] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0140] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 4;

[0141] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0142] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, comprises :

[0143] - a heavy chain variable region (VH) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO:2, and a CDR3 having the amino acid sequence of SEQ ID NO:3 ; and / or

[0144] - a light chain variable region (VL) comprising a CDR1 having the amino acid sequence of SEQ ID NO:4, a CDR2 having the amino acid sequence of SEQ ID NO:5, and a CDR3 having the amino acid sequence of SEQ ID NO:6.

[0145] In a particular embodiment, the M3C65 antibody or functional variant thereof, preferably an M3C65 scFv, comprises :

[0146] - a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:7 ; and

[0147] - a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 8.In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, comprises :

[0148] - a variable heavy chain (VH) domain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 7 ; and

[0149] - a variable light chain (VL) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 8.

[0150] In a particular embodiment, the targeting moiety is a scFv fragment of M3C65 antibody, which comprises :

[0151] - a variable heavy chain (VH) domain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 7 ; and

[0152] - a variable light chain (VL) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 8.

[0153] In a particular embodiment, the VH domain and the VL domain of the M3C65 scFv are fused through an amino acid linker. The term “linker” refers to a (poly)peptide comprising 5 to 80 amino acids, preferably 5 to 30, still preferably 10 to 20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises GGGS or GGGGS repeats. Linkers composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility, and allows for mobility of the connecting functional domains. Rigid peptide linkers can also be employed in the design of fusion proteins and antibody fragments, such as the (PPPPK)xN linker. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17 or SEQ ID NO: 19. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17.

[0154] In a particular embodiment, the M3C65 scFv fragment comprises or consists of the amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:20.In a particular embodiment, the M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, is a humanized form of the reference M3C65 antibody, which, in its original form, is an antibody with heavy and light chain variable regions of murine origin.

[0155] In a particular embodiment, the M3C65 antibody or functional variant thereof as described herein is a humanized M3C65 antibody or a fragment thereof.

[0156] In a particular embodiment, the targeting moiety is a humanized M3C65 antibody or a fragment thereof such as a Fab, Fab', F(ab')2, Fv or scFv fragment of humanized M3C65 antibody.

[0157] In a preferred embodiment, the targeting moiety is a scFv fragment of a humanized M3C65 antibody.

[0158] In the context of the present invention, the term "humanized antibody” refers to an antibody in which the constant and variable framework region derived from one or more human immunoglobulins, e.g. the human anti-HER2 monoclonal antibody (4D5), is fused with the binding region, e.g. the CDRs, of an animal immunoglobulin, e.g. a murine (mouse) antibody M3C65. The humanized antibodies are designed to maintain the binding specificity of the nonhuman antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered" to produce specific human antibodies in response to antigenic challenge (see, e.g., Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun 6:579, the entire teachings of which are herein incorporated by reference). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275). In a humanization approach, complementarity determining regions (CDRs) and certain other amino acids from donor mouse variable regions are grafted into human variable acceptor regions and then joined to human constant regions. See, e.g. Riechmann et al., Nature 332:323-327 (1988); U.S. Pat. No. 5,225,539.11

[0159] Such humanized antibody typically comprises one or more variable domains in which the antigen binding domains are derived from the non-human antibody, and framework regions derived from human or humanized antibody sequences.

[0160] Additional framework region modifications may be made within the human framework sequences. Such amino acid modifications may be made to further refine antibody function and / or increased the humanization process.

[0161] In a particular embodiment, the humanized antibody of the present invention or fragment thereof, is generated by CDR-grafting, preferably by grafting the complementarity determining regions from the murine M3C65 antibody into the framework regions of a human monoclonal antibody, preferably of a human anti-HER2 monoclonal antibody, preferably 4D5 antibody.

[0162] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0163] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:1 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 1; - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:2 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:2; and

[0164] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with respect to SEQ ID NO:3 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:3.

[0165] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises extended variants ofH-CDRl, H-CDR2 and / or H-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said H-CDR1, H-CDR2 and / or H-CDR3.

[0166] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises the following heavy chain complementarity-determining regions (H-CDRs):- H-CDR1 having the amino acid sequence of SEQ ID NO:1 or an extended variant thereof having the amino acid sequence of SEQ ID NO:32;

[0167] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or an extended variant thereof having the amino acid sequence of SEQ ID NO:33; and

[0168] - H-CDR3 having the amino acid sequence of SEQ ID NO:3.

[0169] Preferably, the M3C65 humanized antibody or fragment thereof further comprises the following light chain complementarity-determining regions (L-CDRs):

[0170] L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:4;

[0171] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO:6 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6.

[0172] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises extended variants of L-CDR1, L-CDR2 and / or L-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said L-CDR1, L-CDR2 and / or L-CDR3.

[0173] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises the following light chain complementarity-determining regions (H-CDRs):

[0174] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or an extended variant thereof having the amino acid sequence of SEQ ID NO:34;

[0175] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or an extended variant thereof having the amino acid sequence of SEQ ID NO:35; and

[0176] - L-CDR3 having the amino acid sequence of SEQ ID NO:6.

[0177] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises the following heavy chain complementarity-determining regions (H-CDRs):- H-CDR1 having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:32, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:1 or SEQ ID NO:32;

[0178] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:33, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:2 or SEQ ID NO:33;

[0179] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0180] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0181] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:34, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:4 or SEQ ID NO:34;

[0182] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:35, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:5 or SEQ ID NO:35; and

[0183] - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0184] The following table shows the sequences of SEQ ID NO:1 to 6, as identified in the patent application :

[0185]

[0186] In a preferred embodiment, the M3C65 humanized antibody or fragment thereof comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0187] - H-CDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 1;- H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 2;

[0188] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0189] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0190] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 4;

[0191] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0192] In a preferred embodiment, the M3C65 humanized antibody or fragment thereof comprises :

[0193] - the heavy chain complementarity-determining regions (H-CDRs) of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3,

[0194] - the light chain complementarity-determining regions (L-CDRs) of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and

[0195] - framework regions from a human monoclonal antibody, preferably from a human anti- HER2 monoclonal antibody, preferably 4D5 antibody.

[0196] In a particular embodiment, the humanized antibody or fragment thereof, preferably a humanized M3C65 scFv, comprises :

[0197] - a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9 ; and

[0198] - a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10.

[0199] In a particular embodiment, the humanized antibody or fragment thereof, preferably a humanized M3C65 scFv, comprises :- a variable heavy chain (VH) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9; and

[0200] - a variable light chain (VL) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10.

[0201] In a particular embodiment, the VH domain and the VL domain of the humanized M3C65 scFv are fused through an amino acid linker. The term “linker” refers to a (poly)peptide comprising 5 to 80 amino acids, preferably 5 to 30, still preferably 10 to 20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises GGGS or GGGGS repeats. Linkers composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility, and allows for mobility of the connecting functional domains. Rigid peptide linkers can also be employed in the design of fusion proteins and antibody fragments, such as the (PPPPK)xN linker. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 19. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17.

[0202] In a particular embodiment, the scFv fragment of humanized M3C65 antibody comprises or consists of the amino acid sequence as shown in SEQ ID NO:21 or SEQ ID NO:38, preferably SEQ ID NO:21 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:21 or SEQ ID NO:38, preferably SEQ ID NO:21.

[0203] Payload of the chimeric construct

[0204] The chimeric construct of the present invention also comprises a payload, which is fused to the targeting moiety (i.e. the M3C65 antibody, functional variant or fragment thereof as described above).

[0205] As used herein, the term “payload” refers to a moiety, in particular a peptide moiety, that is distinct from the targeting moiety itself (i.e. that is distinct from the M3C65 antibody, functionalvariant or fragment thereof as described above). A "distinct moiety" is a moiety that has a structure / activity different from that of the M3C56 antibody.

[0206] According to a particular embodiment, the payload is an heterologous peptide or protein. According to a particular embodiment, the payload is a therapeutic peptide or protein, preferably a peptide or protein having therapeutic activity on inflammatory diseases. According to a preferred embodiment, the payload is a peptide or protein having anti-inflammatory activity.

[0207] In a particular embodiment, the pay load is an antibody, which is fused to the M3C65 antibody, functional variant or fragment thereof as described above. According to a particular embodiment, the payload is a monoclonal antibody that either neutralizes inflammatory cytokine(s) or their receptor(s). For example, the payload can be an anti-TNF-a antibody or an anti-IL-1 antibody.

[0208] In a particular embodiment, the payload is a cytokine. In a particular embodiment, the cytokine is selected from the group consisting of: interleukin 2 (IL-2), interleukin 10 (IL- 10) and Transforming growth factor beta (TGFb).

[0209] According to a preferred embodiment, the payload is Interleukin 2 (IL-2).

[0210] According to this particular embodiment, the chimeric construct of the invention comprises :

[0211] (i) a targeting moiety, which is an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof as described above, that is fused to

[0212] (ii) IL-2.

[0213] As used herein, Interleukin-2 (IL-2) encompasses mammal wild type Interleukin-2, and variants thereof. Preferably, IL-2 is human IL-2 or an homologous variant thereof.

[0214] Active variants of IL-2 have been disclosed in the literature. Variants of the native IL-2 can be fragments, analogues, and derivatives thereof. By "fragment" is intended a polypeptide comprising only a part of the polypeptide sequence. An "analogue" designates a polypeptide comprising the native polypeptide sequence with one or more amino acid substitutions, insertions, or deletions. Muteins and pseudopeptides are specific examples of analogues."Derivatives" include any modified native IL-2 polypeptide or fragment or analogue thereof, such as glycosylated, phosphorylated, fused to another polypeptide or molecule, polymerized, etc., or through chemical or enzymatic modification or addition to improve the properties of IL-2 (e.g., stability, specificity, etc.). The IL-2 active variants generally have at least 75%, preferably at least 80%, 85%, more preferably at least 90% or at least 95% amino acid sequence identity to the amino acid sequence of the reference IL-2 polypeptide, for instance mature wild type human IL-2.

[0215] As used herein, "wild type IL-2" means IL-2, whether native or recombinant, comprising the 133 normally occurring amino acid sequence of native human IL-2, whose amino acid sequence is described in Fujita, et. al., PNAS USA, 80,7437-7441 (1983). SEQ ID NO: 12 (133 amino acids) is the human IL-2 sequence less the signal peptide, consisting of an additional 20 N-terminal amino acids. SEQ ID NO: 11 (153 amino acids) is the human IL-2 sequence including the signal peptide.

[0216] In a particular embodiment, the payload is a human IL-2 or variant thereof, wherein the variant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 98%, or at least 99% amino acid identity with human wild-type IL-2. In a particular embodiment, the payload is a human IL-2 or variant thereof, wherein the variant has at least 80% amino acid identity with human wild-type IL-2. In a particular embodiment, the payload is a human IL-2 or variant thereof, wherein the variant has at least 85% amino acid identity with human wildtype IL-2. In a particular embodiment, the payload is a human IL-2 or variant thereof, wherein the variant has at least 90% amino acid identity with human wild-type IL-2.

[0217] In another embodiment, the IL-2 is an IL2 mutein.

[0218] As used herein, "IL-2 mutein" means a polypeptide in which specific amino acid substitutions to the human mature interleukin-2 protein have been made. All numbering of the amino acids is made with respect to human mature interleukin-2 protein of SEQ ID NO: 12, unless otherwise indicated.

[0219] In some embodiments, the cysteine at position 125 is replaced with a neutral amino acid such as serine (C125S), alanine (Cl 25 A), threonine (C125T) or valine (Cl 25V).For example, elimination of the O-glycosylation site results in a more homogenous product when active variant is expressed in mammalian cells such as CHO or HEK cells.

[0220] In certain embodiments active variant comprises an additional amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2. In one embodiment said additional amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2 is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a specific embodiment, said additional amino acid mutation is the amino acid substitution T3A.

[0221] In a particular embodiment, the IL-2 is an IL2 mutein that comprises at least one amino acid substitution at position N88 of SEQ ID NO: 12. In a preferred embodiment, said amino acid substitution is selected from the group consisting of N88R and N88D.

[0222] In a particular embodiment, the payload is IL-2 or an IL-2 variant, which is further fused to a molecule or peptide in order to improve its therapeutic potential or its pharmacokinetic properties.

[0223] Active IL-2 variants that selectively promote T-reg cell proliferation, survival, activation and / or function are particularly useful in treating inflammatory disorders.

[0224] By "selectively promote", it is meant that the active variant promotes the activity in T-reg cells but has limited or lacks the ability to promote the activity in non-regulatory T cells. Further described herein are assays to screen for active variants that selectively promote T-reg cell proliferation, survival, activation and / or function. Methods for determining whether a variant IL-2 polypeptide is active are available in the art. See e.g. WO2016 / 014428. An active variant is defined as a variant that shows an ability to stimulate Tregs, including variants with an improved ability, or a similar ability, or even a reduced ability to stimulate Tregs when compared to wild-type IL-2 or aldesleukin (as defined below), to the extent it does not stimulate Teffs more than it stimulates Tregs. Methods for testing whether a candidate molecule stimulate T cells, Tregs in particular, or NK cells are well-known. Variants may be tested for their ability to stimulate effector T cells (such as CD8+ T cells), CD4+Foxp3+ Tregs, or NK cells. In apreferred embodiment, the active variant shows a reduced ability to stimulate NK cells, compared to wild type IL2 or aldesleukin. Monitoring STAT5 phosphorylation is a simple way of assessing variants for their ability to preferentially stimulate Tregs over Teff, as described in Yu et al, Diabetes 2015;64:2172-2183. In a particular embodiment, a variant is particularly useful when a given level of STAT5 phosphorylation is achieved with doses at least 10 times inferior for Tregs than for other immune cells, including Teffs.

[0225] Said active variants induce signaling events that preferentially induce survival, proliferation, activation and / or function of Treg cells. In certain embodiments, the IL-2 variant retains the capacity to stimulate, in Treg cells, STAT5 phosphorylation and / or phosphorylation of one or more of signaling molecules downstream of the IL-2R, e.g., p38, ERK, SYK and LCK. In other embodiments, the IL-2 variant retains the capacity to stimulate, in Treg cells, transcription or protein expression of genes or proteins, such as FOXP3, Bcl-2, CD25 or IL-10, that are important for Treg cell survival, proliferation, activation and / or function. In other embodiments, the IL-2 variant exhibits a reduced capacity to stimulate endocytosis of IL-2 / IL-2R complexes on the surface of CD25+ T cells. In other embodiments, the IL-2 variant demonstrates inefficient, reduced, or absence of stimulation of PI3 -kinase signaling, such as inefficient, reduced or absent phosphorylation of AKT and / or mTOR (mammalian target of rapamycin). In yet other embodiments, the IL-2 variant retains the ability of wild type IL-2 to stimulate STAT5 phosphorylation and / or phosphorylation of one or more of signaling molecules downstream of the IL-2R in Treg cells, yet demonstrates inefficient, reduced, or absent phosphorylation of STAT5, AKT and / or mTOR or other signaling molecules downstream of the IL-2R in FOXP3-CD4+ or CD8+ T cells or NK cells. In other embodiments, the IL-2 variant is inefficient or incapable of stimulating survival, growth, activation and / or function ofFOXP3- CD4+ or CD8+ T cells or NK cells.

[0226] In all cases, these variants have the capacity to stimulate cell lines such as CTLL-2 or HT-2 which can be universally used to determine their biological activity. For instance, the biological activity of IL-2 may be determined by a cell-based assay performed on HT-2 cell line (clone A5E, ATCC® CRL-1841™) whose growth is dependent on IL-2. Cell growth in the presence of a range of test interleukin-2 product is compared with the growth recorded with IL-2 international standard (WHO 2nd International Standard for INTERLEUKIN 2 (Human, rDNA derived) NIBSC code: 86 / 500). Cell growth is measured after addition and transformation of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(inner salt, MTS) into formazan by active viable cells. Formazan concentration is then measured by spectrophotometry at 490 nm.

[0227] Examples of IL-2 variants are disclosed, for instance, in EP109748, EP136489, US4,752,585; EP200280, EP118617, WO99 / 60128, EP2288372, US9,616,105, US9,580,486, W02010 / 085495, WO2016 / 164937.

[0228] For instance, certain mutations may result in a reduced affinity for the signaling chains of the IL-2 receptor (IL-2RP / CD122 and / or IL-2Ry / CD132) and / or a reduced capacity to induce a signaling event from one or both subunits of the IL-2 receptor. Other mutations may confer higher affinity for CD25 (IL-2Ra). In both cases, those mutations define active variants that preferentially induce survival, proliferation, activation and / or function of Treg. This property may be monitored using surface plasmon resonance.

[0229] Particular examples of useful variants include IL-2 muteins which show at least one amino acid substitution at position D20, N30, Y31, K35, V69, Q74, N88, V91, or Q126, numbered in accordance with wild type IL-2, meaning that the chosen amino acid is identified with reference to the position at which that amino acid normally occurs in the mature sequence of wild type IL-2 ofSEQ ID NO: 12.

[0230] Preferred IL-2 muteins comprise at least one substitution at position D20H, D20I, D20Y, N30S, Y31H, K35R, V69AP, Q74, N88R, N88D, N88G, N88I, V91K, or Q126L.

[0231] In some embodiments, the IL-2 mutein molecule comprises a V91K substitution. In some embodiments, the IL-2 mutein molecule comprises aN88D substitution. In some embodiments, the IL-2 mutein molecule comprises a N88R substitution. In some embodiments, the IL-2 mutein molecule comprises a substitution of H16E, D84K, V91N, N88D, V91K, or V91R, any combinations thereof. In some embodiments, these IL-2 mutein molecules also comprise a substitution at position 125 as described herein. In some embodiments, the IL-2 mutein molecule comprises one or more substitutions selected from the group consisting of: T3N, T3 A, L12G, L12K, L12Q, L 12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20H, D20I, D20Y, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81 S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q D84R, D84S, D84T, S87R,N88A, N88D, N88E, N88I, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91 S, I92K, I92R, E95G, and Q126. In some embodiments, the amino acid sequence of the IL-2 mutein molecule differs from the amino acid sequence set forth in mature IL-2 sequence with a Cl 25 A or Cl 25 S substitution and with one substitution selected from T3N, T3A, L12G, L12K, L12Q L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81 S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88I, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91 S, I92K, I92R, E95G, Q126I, Q126L, and Q126F. In some embodiments, the IL-2 mutein molecule differs from the amino acid sequence set forth in mature IL-2 sequence with a Cl 25 A or C125S substitution and with one substitution selected from D20H, D20I, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R, H16T, H16V, I92K, I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D, V91G, V91K, and V91S. In some embodiments, the IL-2 mutein comprises N88R and / or D20H mutations.

[0232] These substitutions can be used alone or in combination with one another. In some embodiments, the mutein comprises each of these substitutions. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations.

[0233] In some embodiments, the IL-2 mutein comprises a N88R or a N88D mutation, preferably N88R. In some embodiments, the IL-2 mutein comprises a C125A or C125S mutation. These substitutions can be used alone or in combination with one another. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises each of these substitutions.

[0234] In a particular embodiment, the IL-2 is aldesleukin. Aldesleukin is the active ingredient of Proleukin®. Aldesleukin is a variant of mature human IL-2 comprising two amino acid modifications as compared to the sequence of mature human IL-2 (SEQ ID NO: 12): the deletion of the first amino acid (alanine) and the substitution of cysteine at position 125 by serine. Conservative modifications and substitutions at other positions of IL-2 (i. e., those that have a minimal effect on the secondary or tertiary structure of the mutein) are encompassed. Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978), and by Argos in EMBO J., 8: 779-785 (1989). For example, amino acidsbelonging to one of the following groups represent conservative changes: -ala, pro, gly, gin, asn, ser, thr; -cys, ser, tyr, thr; -val, ile, leu, met, ala, phe; -lys, arg, his; -phe, tyr, trp, his ; and -asp, glu.

[0235] Variants with mutations which disrupt the binding to the a subunit of IL-2R are not preferred, as those mutants may have a reduced capacity to stimulate Tregs.

[0236] Active IL-2 variants that promote Teff cell proliferation, survival, activation and / or function may be useful in treating cancers.

[0237] Such active variants of IL-2 comprise at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor (CD25) and preserves affinity of the mutant IL-2 polypeptide to the intermediate-affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide. This property may be monitored using surface plasmon resonance.

[0238] Preferred active variants include IL-2 mutein comprising F42A, K43N, Y45A, and / or E62A substitution(s).

[0239] Active variants such as mutants of human IL-2 (hIL-2) with decreased affinity to CD25 may for example be generated by amino acid substitution at amino acid position 35, 38, 42, 43, 45, 62 or 72 or combinations thereof (numbering relative to the human IL-2 sequence SEQ ID NO: 12). Exemplary amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, E62G, E62A, E62S, E62T, E62Q, E62E, E62N, E62D, E62R, E62K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Particular active variants useful in the chimeric construct for the present invention comprise an amino acid mutation at an amino acid position corresponding to residue 42, 45, or 72 of human IL-2, or a combination thereof. In one embodiment said amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, more specifically an amino acid substitution selected from the group of F42A, Y45A and L72G. These active variants exhibit substantially similar binding affinityto the intermediate-affinity IL-2 receptor, and have substantially reduced affinity to the a-subunit of the IL-2 receptor and the high-affinity IL-2 receptor (IL2RaPy) compared to a wildtype form of the IL-2 mutant.

[0240] Other characteristics of useful active variants may include the ability to induce proliferation of IL-2 receptor-bearing T and / or NK cells, the ability to induce IL-2 signaling in IL-2 receptorbearing T and / or NK cells, the ability to generate interferon (IFN)-y as a secondary cytokine by NK cells, a reduced ability to induce elaboration of secondary cytokines - particularly IL- 10 and TNF-a - by peripheral blood mononuclear cells (PBMCs), a reduced ability to activate regulatory T cells, a reduced ability to induce apoptosis in T cells, and a reduced toxicity profile in vivo.

[0241] Particular active variants comprise three amino acid mutations that abolish or reduce affinity of the active variants to the a-subunit of the IL-2 receptor but preserve affinity of the active variant to the intermediate affinity IL-2 receptor. In one embodiment said three amino acid mutations are at positions corresponding to residue 42, 45 and 72 of human IL-2. In one embodiment said three amino acid mutations are amino acid substitutions. In one embodiment said three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a specific embodiment said three amino acid mutations are amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 12). In certain embodiments said amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor by at least 5-fold, specifically at least 10-fold, more specifically at least 25 -fold. In embodiments where there is more than one amino acid mutation that reduces the affinity of the active variant to the a-subunit of the IL-2 receptor, the combination of these amino acid mutations may reduce the affinity of the active variant to the a-subunit of the IL-2 receptor by at least 30-fold, at least 50-fold, or even at least 100-fold. In one embodiment said amino acid mutation or combination of amino acid mutations abolishes the affinity of the active variant to the a-subunit of the IL-2 receptor so that no binding is detectable by surface plasmon resonance.Substantially similar binding to the intermediate-affinity receptor, i.e. preservation of the affinity of the mutant IL-2 polypeptide to said receptor, is achieved when the active variant exhibits greater than about 70 percent of the affinity of a wild-type form of the IL-2 mutant to the intermediate-affinity IL-2 receptor. Active variants useful in the invention may exhibit greater than about 80 percent and even greater than about 90 percent of such affinity.

[0242] Reduction of the affinity of IL-2 for the a-subunit of the IL-2 receptor in combination with elimination of the O-glycosylation of IL-2 results in an IL-2 protein with improved properties. In a specific embodiment, the active variant can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer (NK) cell, cytotoxic activity in a NK cell, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and NK / lymphocyte activated killer (LAK) antitumor cytotoxicity.

[0243] In some embodiments, these active variants also comprise a substitution at position 125 as described herein.

[0244] Optional moiety having dimerization properties

[0245] The chimeric construct of the invention, which comprises a targeting moiety (i.e. an M3C65 antibody, variant thereof or fragment thereof) and a payload (such as IL-2), may optionally further comprise a moiety having multimerization properties, i.e. a fragment or moiety that is able to form multimeric proteins.

[0246] Preferably, the chimeric construct of the invention, which comprises a targeting moiety (i.e. an M3C65 antibody, variant thereof or fragment thereof) and a payload (such as IL-2), may optionally further comprise a moiety having dimerization properties, i.e. a fragment or moiety that is able to form dimeric proteins.

[0247] For example, the chimeric construct may further comprise a Fc fragment of an IgG, or a functional variant thereof which has the capacity to form at least one dimer, for example ahomodimer or a heterodimer, a trimer, a tetramer or any multimer containing a different number of chimeric constructs.

[0248] In a particular embodiment, the chimeric construct of the invention, which comprises a targeting moiety (i.e. an M3C65 antibody, variant thereof or fragment thereof) and a payload (such as IL-2), may optionally comprise a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof that is capable of forming a dimeric protein.

[0249] The C4BP / 3 or C4BP / 3 fragment

[0250] The C4BP protein is involved in coagulation and the complement system. The major form of C4BP is composed of 7 identical 75 kD alpha chains and one 45 kD beta chain. The alpha and beta chains respectively contain 8 and 3 SCR (short consensus repeat) domains, those motifs being found in many complement-regulating proteins and constituted by 50-70 amino acids organized into beta sheets. The amino acid sequence of the beta chain of human C4BP is shown as SEQ ID NO: 13.

[0251] A nucleic acid sequence corresponding to this polypeptide sequence has also been described by Hillarp and Dahlback (1990, PNAS, vol 87, pp 1183-1187). The role of the alpha chain in polymerizing the C4BP protein has been studied by Kask et al (Biochemistry 2002, 41, 9349-9357). Those authors have shown that the C-terminal portion of the alpha chain, in particular its helical structure and the presence of two cysteines, is necessary for polymerization of the C4BP protein when the alpha chain is expressed in a heterologous system.

[0252] European patent application 2 227 030 describes the production of heteromultimeric recombinant proteins by using C-terminal fragments of the alpha and beta chains of the C4BP protein in fusion with polypeptides of interest. US patent 7,884,190 describes the use of the beta chain of the C4BP protein, independently of its use in association with the alpha chain of the C4BP protein for the production of dimeric proteins.

[0253] The C4BP protein used to carry out the invention is advantageously the human C4BP protein. In a preferred embodiment, the chimeric construct comprises a fragment of the C4BPP chain that comprises or consists of at least amino acids 194 to 252 (SEQ ID NO: 14).Sequences coding for longer fragments of the beta chain, or even the whole beta chain, may also be used. For certain applications, it is preferable to avoid using a sequence coding for a beta chain which is capable of binding the S protein participating in coagulation. If the selected sequence codes for a fragment containing the two first SCR motifs of the beta chain, these will preferably by versions mutated by addition, deletion or substitution of amino acids to cut out with the possibility of interaction with the S protein. SCR motifs and / or [GS] domains may be added with the aim of modifying, for example increasing, the flexibility of the fusion polypeptide obtained or to allow the chimeric protein to adopt a suitable conformation to form multimers, particularly dimers.

[0254] A longer fragment of C4BPP that extends at the N-term up to at most amino acid 135 may be used.

[0255] In a particular embodiment, the fragment of the C4BPP chain may comprise or consist of at least amino acids 185 to 252, 180 to 252, 175 to 252, 170 to 252, 165 to 252, 160 to 252, 155 to 252, 150 to 252, 145 to 252, 140 to 252, or 135 to 252 (with respect to SEQ ID NO: 13).

[0256] In a particular embodiment, the fragment of the C4BPP chain comprises or consists of at least amino acids 137 to 252 (SEQ ID NO: 15).

[0257] A functional variant of C4BPP may be used. The functional variant has maintained the capacity to form at least one dimer, for example a homodimer or a heterodimer, a trimer, a tetramer or any multimer containing a different number of chimeric proteins.

[0258] Within the context of the invention, the term "functional variant of a fragment of the C4BPP chain" means a polypeptide sequence modified with respect to the sequence of fragment of the beta chain by deletion, substitution or addition of one or more amino acids, said modified sequence retaining, however, the capacity to form at least dimer proteins using the method of the invention. More precisely, the production of dimer proteins using a sequence coding for a functional variant of the fragment may be at least 80% equal to that obtained with a native sequence coding for the fragment (SEQ ID NO: 13, or a fragment thereof), preferably at least 90%, still preferably 95%) in an identical expression system. Preferably, the variant is such that more than 80% of the fusion polypeptides which it contains are produced in the form of dimers in a eukaryotic expression system in accordance with the invention.In a particular embodiment, a variant of the fragment of the beta chain is encoded by a nucleic acid that is capable of hybridizing under stringent conditions with the wildtype sequence coding for the fragment, as described by Hillarp and Dahlback (1990, PNAS, Vol. 87, pp 1183-1187). The term "stringent conditions" means conditions which allow specific hybridization of two single strand DNA sequences at about 65°C., for example, in a solution of 6*SSC, 0.5% SDS, 5 * Denhardf s solution and 100 pg of non specific DNA or any other solution with an equivalent ionic strength and after washing at 65°C., for example in a solution of at most 0.2*SSC and 0.1% SDS or any other solution with an equivalent ionic strength.

[0259] Preferably, the nucleotide sequence coding for a functional variant of said wildtype fragment and hybridizing under stringent conditions with the sequence coding for said fragment has, in the portion which hybridizations, a length of at least 50%, preferably at least 80%, of the length of the sequence coding for the fragment. In a particular implementation, the nucleotide sequence coding for a functional variant of said fragment and hybridizing under stringent conditions with the sequence coding for said fragment has, in the portion which hybridizations, substantially the same length as the sequence coding for said fragment.

[0260] In a further implementation, a functional variant is a modified sequence of the wildtype fragment one or more amino acids of which, not essential to the dimerization function, have been removed or substituted and / or one or more amino acids essential to dimerization have been replaced by amino acids with equivalent functional groups (conservative substitution). It is particularly recommended that the two cysteines, located at positions 201 and 215, and the peptide structure around these cysteines be conserved to allow the formation of disulfide bridges which are necessary for dimerization, for example by conservation of at least 3 amino acids upstream and downstream of each cysteine. In particular, a functional variant may also be obtained by inserting a heterologous sequence of the beta chain, and in particular domains of the alpha chain of C4BP, between the cysteines responsible for dimerization or, in contrast, by doing away with certain amino acids present between those same cysteines. Alternatively, a functional variant may be produced by point modification of certain amino acids, in particular substitution of a cysteine responsible for dimerization by a neutral amino acid as regards implication in the dimerization process (for example the amino acids A, V, F, P, M, I, L and W) and at the same time substituting another amino acid by a cysteine to conserve the capacity to form intracatenary and / or intercatenary disulfide bridges between the cysteines. Thesemodifications thus result in a variation in the distance between the various cysteines involved in the multimerization process, in particular dimerization.

[0261] Preferably, less than 50% of the amino acids of the 194 to 252 fragment are done away with or replaced, preferably less than 25% or even less than 10% (for example 5 amino acids or fewer) or less than 5% (e.g. 1 or 2 amino acids).

[0262] In a particular embodiment, the functional variant comprises or consists of

[0263] a) a modified sequence of the fragment (preferably the 194 - 252 fragment) of C4BPP, wherein less than 25 percent of the amino acids of the fragment (preferably the 194 - 252 fragment), preferably less than 10 percent, have been cut out or replaced, in which the cysteines located in positions 202 and 216 (numbered with respect to SEQ ID NO: 13) as well as at least 3 amino acids upstream and downstream of each cysteine have been conserved; or

[0264] b) a modified sequence of the fragment (preferably the 194 - 252 fragment) of the C4BPP, wherein a cysteine responsible for dimerization is substituted with an amino acid, preferably selected from alanine, valine, phenylalanine, proline, methionine, isoleucine, leucine and tryptophan, and another amino acid of the fragment is substituted with a cysteine; or c) a sequence of the fragment (preferably the 194 - 252 fragment) of C4BPP modified by insertion of a sequence which is heterologous to the beta chain, between the cysteines responsible for dimerization; or

[0265] d) a sequence of the fragment (preferably the 194- 252 fragment) of C4BPP modified by cutting out amino acids between the cysteines responsible for dimerization.

[0266] The M3C65 chimeric construct

[0267] Preferably, the targeting moiety (i.e. an M3C65 antibody, variant thereof or fragment thereof) and the payload (such as IL-2), of the chimeric construct are fused to each other.

[0268] The payload may be fused to the N-terminus or to the C-terminus of the M3C65 antibody, functional variant, or fragment thereof. In a preferred embodiment, the payload is fused to the N-terminus of the M3C65 antibody, functional variant, or fragment thereof. Preferably, the C-terminus of the payload is fused to the N-terminus of the M3C65 antibody, functional variant, or fragment thereof.According to a particular embodiment, the payload is IL-2 or IL-2 variant as described above, and is fused to the N-terminus or to the C-terminus of the M3C65 antibody, functional variant, or fragment thereof. In a preferred embodiment, said IL-2 or IL-2 variant is fused to the N-terminus of the M3C65 antibody, functional variant, or fragment thereof. Preferably, the C-terminus of IL-2 or IL-2 variant is fused to the N-terminus of the M3C65 antibody, functional variant, or fragment thereof.

[0269] The targeting moiety (i.e. an M3C65 antibody, variant thereof or fragment thereof) and the payload (such as IL-2), may be fused in frame (directly) or through an amino acid linker, preferably a polyG linker. In the context of the present invention, the term “linker” refers to a (poly)peptide comprising 5 to 80 amino acids, preferably 5 to 30, still preferably 10 to 20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises GGGS or GGGGS repeats, although an artisan skilled in the art will recognize that other sequences following the general recommendations (Argos, 1990, J Mol Biol. 20;211(4):943-58; George R, Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. 2002;15:871-879) can also be used. Linkers composed of small, nonpolar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility, and allows for mobility of the connecting functional domains. Rigid peptide linkers can also be employed in the design of fusion proteins and antibody fragments, such as the (PPPPK)xN linker. In a preferred embodiment, the payload and the targeting moiety are liked through the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17 or SEQ ID NO:19.

[0270] In a preferred embodiment, said IL-2 or IL-2 variant and the M3C65 antibody, variant thereof or fragment thereof are liked through the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17.

[0271] In a particular embodiment, the chimeric construct further comprises a moiety having dimerization properties, such as a of C4BPP or a functional fragment thereof, as described above. Such chimeric construct preferably forms a homodimer, or may be used to produce a heterodimer, as described below. In a particular embodiment, (i) the targeting moiety, (ii) thepayload and (iii) the moiety having dimerization properties are fused to each other, in frame (directly) or through amino acid linker(s).

[0272] In a preferred embodiment the payload, preferably IL-2, is fused at the N-terminus of C4BPP or said functional fragment thereof. In a particular embodiment, the payload, preferably IL-2, is fused at the N-terminus of C4BPP or said functional fragment thereof, through an amino acid linker, preferably a polyG linker. In some embodiments, the IL-2 is fused to the C4BPP moiety via a linker comprising GGGS or GGGGS repeats, preferably via the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17 or SEQ ID NO: 19, preferably via the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:17.

[0273] In a particular embodiment, the amino acid sequence corresponding to the IL-2 fused to the C4BPP moiety (“IL2-C4BPP”), comprises or consists of SEQ ID NO: 22 or SEQ ID NO: 23.

[0274] In a preferred embodiment the C-terminus of the IL-2 is fused at the N-terminus of C4BPP or said functional fragment thereof, wherein the C-terminus of C4BPP or said fragment thereof is linked to the N-terminus of the targeting moiety.

[0275] In a particular embodiment, the chimeric construct comprises from N-terminus to C-terminus :

[0276] - The payload, preferably IL-2 or a functional variant thereof,

[0277] optionally a linker; and

[0278] - the targeting moiety, which is an M3C65 antibody, a functional variant thereof or a fragment thereof.

[0279] In a preferred embodiment, the chimeric construct comprises from N-terminus to C-terminus :

[0280] - the payload, preferably IL-2 or a functional variant thereof ;

[0281] optionally a linker ;

[0282] - the moiety having dimerization properties, such as a of C4BPP or a functional fragment thereof ;

[0283] optionally a linker ; andthe targeting moiety, which is an M3C65 antibody, a functional variant thereof or a fragment thereof.

[0284] In a preferred embodiment, the chimeric construct comprises from N-terminus to C-terminus :

[0285] - IL-2 or a functional variant thereof ;

[0286] optionally a linker ;

[0287] C4BPP or a functional fragment thereof ;

[0288] optionally a linker ; and

[0289] - the targeting moiety, which is an M3C65 antibody, a functional variant thereof or a fragment thereof.

[0290] In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29 or comprises or consists of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.

[0291] In a particular embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28 or comprises or consists of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28.

[0292] In a particular embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29 or comprises or consists of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29.

[0293] In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:24, or comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:24.

[0294] In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:25, or comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:25.In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:26, or comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:26.

[0295] In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:27, or comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:27.

[0296] In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:28, or comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:28.

[0297] In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO:29, or comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:29.

[0298] In another particular embodiment, the chimeric construct comprises from N-terminus to C-terminus :

[0299] - the targeting moiety, which is an M3C65 antibody, a functional variant thereof or a fragment thereof ;

[0300] optionally a linker ; and

[0301] a payload, preferably IL-2 or a functional variant thereof.

[0302] In a preferred embodiment, the chimeric construct comprises from N-terminus to C-terminus :

[0303] - the targeting moiety, which is an M3C65 antibody, a functional variant thereof or a fragment thereof.

[0304] optionally a linker ;

[0305] - the moiety having dimerization properties, such as a of C4BPP or a functional fragment thereof ;

[0306] optionally a linker ; and

[0307] - the payload, preferably IL-2 or a functional variant thereof.

[0308] Homodimer and heterodimer constructsIt is herein described a method for producing a recombinant dimer protein comprising: a) transfecting host cells with a vector allowing expression of a nucleotide sequence coding for a chimeric construct that is a fusion polypeptide comprising i) at least one payload, such as interleukin 2 (IL2), ii) a moiety that is capable of forming a dimeric protein, such as a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof, and iii) at least one targeting moiety as described above ;

[0309] b) culturing transfected cells under conditions which are suitable for expressing the nucleotide sequence coding for the fusion polypeptide and the covalent association of two fusion polypeptides in vivo to form a dimeric protein;

[0310] c) recovering, and preferably purifying, the dimeric proteins formed.

[0311] The transfected cells preferably do not contain any nucleic acid allowing expression of a nucleotide sequence coding for the C-terminal fragment of the alpha chain of the C4BP protein involved in polymerization of the C4BP protein.

[0312] In a particular embodiment, it is herein described a method for producing heterodimers, said method comprising:

[0313] a. transfecting host cells with one or more vectors to allow the expression of one or more nucleotide sequences coding for:

[0314] i. a first fusion polypeptide comprising i) at least one payload, such as interleukin 2 (IL2), and ii) a moiety that is capable of forming a dimeric protein, such as a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof ; and

[0315] ii. a second fusion polypeptide, comprising i) at least one targeting moiety as described above, such as an M3C65 scFv or a functional variant thereof, and ii) a moiety that is capable of forming a dimeric protein, such as a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof ;

[0316] b. culturing transfected cells under conditions appropriate for expressing the nucleotide sequence or sequences coding for the first and second fusion polypeptides and association of two fusion polypeptides in vivo to form a heterodimeric protein;

[0317] c. recovering, and preferably purifying, the heterodimeric proteins formed.

[0318] In a particular embodiment, it is herein described a method for producing heterodimers, said method comprising:a. transfecting host cells with one or more vectors to allow the expression of one or more nucleotide sequences coding for:

[0319] i. a first fusion polypeptide comprising i) at least one payload, such as interleukin 2 (IL2), ii) a moiety that is capable of forming a dimeric protein, such as a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof, and iii) at least one targeting moiety as described above such as an M3C65 scFv or a functional variant thereof; and ii. a second fusion polypeptide, comprising i) at least one heterologous polypeptide wherein the heterologous polypeptide is defined as being different from the payload of the first fusion polypeptide, ii) a moiety that is capable of forming a dimeric protein, such as a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof, and iii) optionally at least one targeting moiety as described above such as an M3C65 scFv or a functional variant thereof;

[0320] b. culturing transfected cells under conditions appropriate for expressing the nucleotide sequence or sequences coding for the first and second fusion polypeptides and association of two fusion polypeptides in vivo to form a heterodimeric protein;

[0321] c. recovering, and preferably purifying, the heterodimeric proteins formed.

[0322] Preferably, in the second fusion polypeptide, C4BPP or said fragment is fused to the C-terminal end of the heterologous polypeptide.

[0323] The term "different" when referring to the heterologous polypeptide means a polypeptide which has a primary amino acid sequence that is different by at least one amino acid from the primary sequence of the payload, such as interleukin 2 (moiety) of the first fusion polypeptide. Alternatively, the term "different" also covers heterologous polypeptides having the same primary sequence but having different post-translational modifications, for example in terms of acetylation, amidation, biotinylation, carboxylation, hydroxylation, methylation, phosphorylation or sulfatation, or by adding lipids (isoprenylation, palmitoylation and myristoylation), glucides (glycosylation) or polypeptides (ubiquitination).

[0324] Such heterodimer proteins are also part of the invention.

[0325] In a particular embodiment, the host cell allows co-expression of the two fusion polypeptides, a first fusion polypeptide A comprising i) at least one payload, such as interleukin 2 (IL2), ii) a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variantthereof that is capable of forming a dimeric protein and iii) at least one targeting moiety as described above such as an M3C65 scFv or a functional variant thereof ; and a second fusion polypeptide A, comprising i) at least one payload, such as interleukin 2 (IL2), ii) a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof that is capable of forming a dimeric protein, and iii) at least one targeting moiety as described above such as an M3C65 scFv or a functional variant thereof. In this particular embodiment, coexpression of the two fusion polypeptides can also allow the production of homodimers A- A.

[0326] In a particular embodiment, the host cell allows co-expression of the two fusion polypeptides, a first fusion polypeptide A comprising i) at least one payload, such as interleukin 2 (IL2), ii) a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof that is capable of forming a dimeric protein and iii) at least one targeting moiety as described above such as an M3C65 scFv or a functional variant thereof ; and a second fusion polypeptide B, comprising i) at least one heterologous polypeptide, ii) a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof that is capable of forming a dimeric protein, and iii) at least one targeting moiety as described above such as an M3C65 scFv or a functional variant thereof ; wherein the heterologous polypeptide is defined as being different from the payload of the first fusion polypeptide. In this particular embodiment, co-expression of the two fusion polypeptides can also allow the production of homodimers A-A and B-B and the production of heterodimers A-B.

[0327] It is also provided a recombinant eukaryotic cell allowing synthesis of a dimer or heterodimer protein as defined above, and obtainable by carrying out step a) of the production method defined above. Greater details for the production in host cells are described below.

[0328] Production methods of the chimeric construct

[0329] The chimeric construct, which is in the form of a fusion protein, and the homo- or heterodimers can be produced by DNA recombinant technique in a suitable expression vector or by a RNA molecule.

[0330] The expression vector is selected as a function of the host cell into which the construct is introduced. Preferably, the expression vector is selected from vectors that allow expression in eukaryotic cells, especially from chromosomal vectors or episomal vectors or virus derivatives,in particular vectors derived from plasmids, yeast chromosomes, or from viruses such as baculovirus, papovirus or SV40, retroviruses, Adenoviruses, Adeno-associated Viruses, retroviral vectors such as Gamma-retroviral vectors or lentiviral vectors, or combinations thereof, in particular phagemids and cosmids. In a particular embodiment, it is a vector allowing the expression of baculovirus, capable of infecting insect cells.

[0331] If necessary, the sequence coding for the fusion polypeptide also comprises, preferably in its 5' portion, a sequence coding for a signal peptide for the secretion of fusion polypeptide. Conventionally, the sequence of a signal peptide is a sequence of 15 to 20 amino acids, rich in hydrophobic amino acids (Phe, Leu, He, Met and Vai).

[0332] The vector comprises all of the sequences necessary for the expression of the sequence coding for the fusion polypeptide. In particular, it comprises a suitable promoter, selected as a function of the host cell into which the construct is to be introduced.

[0333] Within the context of the invention, the term "host cell" means a cell capable of expressing a gene carried by a nucleic acid which is heterologous to the cell and which has been introduced into the genome of that cell by a transfection method.

[0334] Preferably, a host cell is a eukaryotic cell. A eukaryotic host cell is in particular selected from yeast cells such as S cerevisiae, filamentous fungus cells such as Aspergillus sp, insect cells such as the S2 cells of Drosophila or sf9 of Spodoptera, mammalian cells and plant cells. Mammalian cells which may in particular be cited are mammalian cell lines such as CHO, COS, HeLa, C127, 3T3, HepG2 or L(TK-) cells. In a preferred implementation, said host cells are selected from eukaryotic cell lines, preferably Sf9 insect cells. Methods for preparing recombinant dimeric proteins in sf9 insect cells are described in US patent 7,884,190. Any transfection method known to the skilled person for the production of cells expressing a heterologous nucleic acid may be used to carry out step a) of the method. Transfection methods are, for example, described in Sambrook et al, 2001, "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0335] Alternatively, the chimeric construct can be produced by chemical peptide synthesis. For instance, the protein can be produced by the parallel synthesis of shorter peptides that are subsequently assembled to yield the complete sequence of the protein with the correct disulfidebridge. A synthesis of IL-2 is illustrated for instance in Asahina et al., Angewandte Chemie International Edition, 2015, Vol.54, Issue 28, 8226-8230, the disclosure of which being incorporated by reference herein.

[0336] In another embodiment, the chimeric protein may be expressed in vivo, after administering the subject with a nucleic acid encoding said chimeric protein. In a preferred embodiment, the nucleic acid is carried by an RNA or a viral vector, such as an adeno-virus associated virus (AAV).

[0337] The invention also relates to a nucleic acid encoding the chimeric construct as described above, wherein said chimeric construct preferably comprises an M3C65 antibody, a variant thereof or a fragment thereof, fused to a payload that is IL-2 or a variant thereof.

[0338] The invention also relates to a vector comprising said nucleic acid.

[0339] The invention also relates to a host cell expressing said nucleic acid or said vector.

[0340] 2- M3C65 conjugate

[0341] Another aspect of the invention relates to a conjugate comprising a targeting moiety, which is an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof as described above. More particularly, the targeting moiety as described above is conjugated to a payload.

[0342] As used herein, the terms “conjugate” and “conjugated” are used interchangeably to specify that the targeting moiety and the payload are covalently linked.

[0343] The conjugate comprises a pay load that can be any compound or molecule, organic, peptide, protein, nucleic acid, carrier, adjuvant, that is operably linked to said M3C65 antibody, functional variant or fragment thereof.

[0344] In a particular embodiment, the payload is a drug or an imaging moiety.

[0345] As used herein, the term “drug” refers to any chemical entity, compound, or product capable of inducing a therapeutic effect when administered to a subject in need thereof. The drug may beselected from the group consisting of small molecule compounds, peptides, proteins, nucleic acids, antibodies, antibody fragments, and combinations thereof. The drug includes, but is not limited to, chemotherapeutic agents, cytotoxic agents, anti-inflammatory agents, immunomodulatory agents, enzyme inhibitors, receptor antagonists and receptor agonists. In particular, the drug can be any suitable drug for the treatment of an inflammatory disease. In a particular embodiment, the conjugate comprises an anti-inflammatory drug as a pay load.

[0346] In a particular embodiment, the anti-inflammatory drug is a steroidal anti-inflammatory drug or a non-steroidal anti-inflammatory drug. “Steroidal anti-inflammatory drugs”, also known as corticosteroids, work by mimicking the effects of hormones naturally produced in the adrenal glands. They have potent anti-inflammatory and immunosuppressive properties. “Non-steroidal anti-inflammatory drugs” (NSAIDs), on the other hand, primarily work by inhibiting cyclooxygenase enzymes, which are involved in the production of prostaglandins, key mediators of inflammation.

[0347] As used herein, the term "imaging moiety" refers to any chemical entity, molecule, or compound capable of generating a detectable signal suitable for imaging applications, in particular for diagnostic applications. Such imaging moiety may be selected from the group consisting of radioisotopes, fluorescent compounds, luminescent compounds, magnetic resonance imaging (MRI) contrast agents, positron emission tomography (PET) tracers, single-photon emission computed tomography (SPECT) agents, ultrasound contrast agents, X-ray contrast agents, computed tomography (CT) contrast agents, and combinations thereof. In particular, the imaging moiety can be selected from radioisotopes suitable for nuclear imaging.

[0348] For example, the term “imaging moiety” includes, but is not limited to, an enzyme, a radioisotope, a radionuclide, a colloidal metal, a luminescent compound, a biotinyl group and a predetermined polypeptide epitope recognized by a secondary reporter such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains or epitope tags.

[0349] In a particular embodiment, the imaging moiety is an enzyme. As used herein, the term “enzyme” include, but is not limited to, peroxidase (e.g., horse radish peroxidase), luciferase, alkaline phosphatase, b-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6 phosphate dehydrogenase, preferably luciferase. Luciferase is a generic term for the class of oxidative enzymes thatproduce bioluminescence, for example the firefly luciferase (EC 1.13.12.7) from the firefly Photinus pyralis.

[0350] In a particular embodiment, the imaging moiety may be biotin, digoxin-genin, or 5-bromo-desoxy uridine.

[0351] In a particular embodiment, the imaging moiety is a luminescent compound. For example, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, and a colorimetric compound may be used in the practice of the invention, more preferably a fluorescent compound.

[0352] In a particular embodiment, the imaging moiety is a fluorescent compound. Suitable fluorescent compound include, but are not limited to, rhodamine, lanthanide phosphors, fluorescein and its derivatives, fluorochromes, rhodamine and its derivatives, Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Yellow Fluorescent Protein (YFP), Cyan Fluorescent Protein (CFP), Blue Fluorescent Protein (BFP), dansyl, umbelliferone, and others. Additionally, commercially available fluorescent moiety including, but not limited to, fluorescent phosphoramidites such as FluorePrime, Fluoredite, FAM, hydroxycoumarin, aminocoumarin, methoxy coumarin, cascade Blue, pacific Blue, pacific Orange, lucifer yellow, R-phycoerythrin, PE-Cy5 conjugates, PE-Cy7 conjugates, red 613, perCP, truRed, HuorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X- Rhodamine, lissamine Rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugates can be used. The fluorescent compound can be made of a combination of fluorescent compounds listed above.

[0353] Other detectable imaging moieties may include radioactive labels such as iodine-123, iodine-125, iodine-126, iodine-133, iodine-131, bromine-77, technetium-99m, indium- 1 13m, gallium-67, gallium-68, ruthenium-95, ruthenium-97, ruthenium- 103, ruthenium- 106, mercury-203, scandium-47, tellurium-121 m, tellurium-128, thulium- 165, thulium-167, thulium- 168, fluorine- 18, yttrium-99, and zirconium-89.

[0354] In a particular embodiment, the targeting moiety and the payload, that is a drug or an imaging moiety, may be conjugated in frame (directly) or through an amino acid linker.In a preferred embodiment, the targeting moiety (i.e. M3C65 antibody, functional variant or a fragment thereof) is conjugated by a linker to the pay load, that is preferably a drug or an imaging moiety. Preferably, the targeting moiety and the payload, that is preferably a drug or an imaging moiety, are conjugated by the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17 or SEQ ID NO: 19. Preferably, the targeting moiety and the payload, that is preferably a drug or an imaging moiety, are conjugated by the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17.

[0355] In a particular embodiment, the conjugate comprises a targeting moiety which is an M3C65 antibody or an M3C65 antibody fragment such as a Fab, Fab', F(ab')2, Fv or scFv fragment of M3C65 antibody, or functional variants thereof.

[0356] In a preferred embodiment, the conjugate comprises atargeting moiety which is a scFv fragment of M3C65 antibody or a functional variant thereof.

[0357] In a particular embodiment, the conjugate comprises an M3C65 antibody or functional variant that is a humanized antibody or a fragment thereof, preferably a humanized M3C65 scFv.

[0358] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0359] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:1 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 1; - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:2 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:2; and

[0360] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with respect to SEQ ID NO:3 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:3.In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises extended variants of H-CDR1, H-CDR2 and / or H-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said H-CDR1, H-CDR2 and / or H-CDR3.

[0361] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0362] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or an extended variant thereof having the amino acid sequence of SEQ ID NO:32;

[0363] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or an extended variant thereof having the amino acid sequence of SEQ ID NO:33; and

[0364] - H-CDR3 having the amino acid sequence of SEQ ID NO:3.

[0365] Preferably, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following light chain complementarity-determining regions (L-CDRs):

[0366] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:4;

[0367] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO:6 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6.

[0368] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises extended variants of L-CDR1, L-CDR2 and / or L-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said L-CDR1 , L-CDR2 and / or L-CDR3.In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following light chain complementarity-determining regions (L-CDRs):

[0369] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or an extended variant thereof having the amino acid sequence of SEQ ID NO:34;

[0370] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or an extended variant thereof having the amino acid sequence of SEQ ID NO:35; and

[0371] - L-CDR3 having the amino acid sequence of SEQ ID NO:6.

[0372] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0373] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:32, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:1 or SEQ ID NO:32;

[0374] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:33 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:2 or SEQ ID NO:33;

[0375] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0376] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0377] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:34 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:4 or SEQ ID NO:34;

[0378] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:35 a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:5 or SEQ ID NO:35; and

[0379] - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0380] In a preferred embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, comprises the following heavy chain complementarity-determining regions (H-CDRs):- H-CDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 1;

[0381] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 2;

[0382] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0383] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0384] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 4;

[0385] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0386] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, comprises :

[0387] - a heavy chain variable region (VH) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO:2, and a CDR3 having the amino acid sequence of SEQ ID NO:3 ; and / or

[0388] - a light chain variable region (VL) comprising a CDR1 having the amino acid sequence of SEQ ID NO:4, a CDR2 having the amino acid sequence of SEQ ID NO:5, and a CDR3 having the amino acid sequence of SEQ ID NO:6.

[0389] In a particular embodiment, the M3C65 antibody or functional variant thereof, preferably an M3C65 scFv, comprises :

[0390] - a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:7 ; and

[0391] - a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 8.

[0392] In a particular embodiment, said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, comprises :- a variable heavy chain (VH) domain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 7 ; and

[0393] - a variable light chain (VL) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 8.

[0394] In a particular embodiment, the targeting moiety is a scFv fragment of M3C65 antibody, which comprises :

[0395] - a variable heavy chain (VH) domain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 7 ; and

[0396] - a variable light chain (VL) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 8.

[0397] In a particular embodiment, the VH domain and the VL domain of the M3C65 scFv are fused through an amino acid linker. The term “linker” refers to a (poly)peptide comprising 5 to 80 amino acids, preferably 5 to 30, still preferably 10 to 20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises GGGS or GGGGS repeats. Linkers composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility, and allows for mobility of the connecting functional domains. Rigid peptide linkers can also be employed in the design of fusion proteins and antibody fragments, such as the (PPPPK)xN linker. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17 or SEQ ID NO: 19. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17.

[0398] In a particular embodiment, the M3C65 scFv fragment comprises or consists of the amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:20.

[0399] In a particular embodiment, the M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, is a humanized form of the reference M3C65 antibody, which, in its original form, is an antibody with heavy and light chain variable regions of murine origin.In a particular embodiment, the M3C65 antibody or functional variant thereof as described herein is a humanized M3C65 antibody or a fragment thereof.

[0400] In a particular embodiment, the targeting moiety is a humanized M3C65 antibody or a fragment thereof such as a Fab, Fab', F(ab')2, Fv or scFv fragment of humanized M3C65 antibody.

[0401] In a preferred embodiment, the targeting moiety is a scFv fragment of a humanized M3C65 antibody.

[0402] In the context of the present invention, the term "humanized antibody” refers to an antibody in which the constant and variable framework region derived from one or more human immunoglobulins, e.g. the human anti-HER2 monoclonal antibody (4D5), is fused with the binding region, e.g. the CDRs, of an animal immunoglobulin, e.g. a murine (mouse) antibody M3C65. The humanized antibodies are designed to maintain the binding specificity of the nonhuman antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered" to produce specific human antibodies in response to antigenic challenge (see, e.g., Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun 6:579, the entire teachings of which are herein incorporated by reference). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275). In a humanization approach, complementarity determining regions (CDRs) and certain other amino acids from donor mouse variable regions are grafted into human variable acceptor regions and then joined to human constant regions. See, e.g. Riechmann et al., Nature 332:323-327 (1988); U.S. Pat. No. 5,225,539.

[0403] Such humanized antibody typically comprises one or more variable domains in which the antigen binding domains are derived from the non-human antibody, and framework regions derived from human or humanized antibody sequences.Additional framework region modifications may be made within the human framework sequences. Such amino acid modifications may be made to further refine antibody function and / or increased the humanization process.

[0404] In a particular embodiment, the humanized antibody of the present invention or fragment thereof, is generated by CDR-grafting, preferably by grafting the complementarity determining regions from the murine M3C65 antibody into the framework regions of a human monoclonal antibody, preferably of a human anti-HER2 monoclonal antibody, preferably 4D5 antibody.

[0405] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0406] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:1 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 1; - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:2 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:2; and

[0407] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with respect to SEQ ID NO:3 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:3.

[0408] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises extended variants ofH-CDRl, H-CDR2 and / or H-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said H-CDR1, H-CDR2 and / or H-CDR3.

[0409] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0410] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or an extended variant thereof having the amino acid sequence of SEQ ID NO:32;

[0411] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or an extended variant thereof having the amino acid sequence of SEQ ID NO:33; and

[0412] - H-CDR3 having the amino acid sequence of SEQ ID NO:3.Preferably, the M3C65 humanized antibody or fragment thereof further comprises the following light chain complementarity-determining regions (L-CDRs):

[0413] L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:4;

[0414] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO:6 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6.

[0415] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises extended variants of L-CDR1, L-CDR2 and / or L-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said L-CDR1, L-CDR2 and / or L-CDR3.

[0416] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises the following light chain complementarity-determining regions (H-CDRs):

[0417] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or an extended variant thereof having the amino acid sequence of SEQ ID NO:34;

[0418] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or an extended variant thereof having the amino acid sequence of SEQ ID NO:35; and

[0419] - L-CDR3 having the amino acid sequence of SEQ ID NO:6.

[0420] In a particular embodiment, the M3C65 humanized antibody or fragment thereof comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0421] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:32, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:1 or SEQ ID NO:32;

[0422] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:33, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:2 or SEQ ID NO:33;- H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0423] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0424] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:34, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:4 or SEQ ID NO:34;

[0425] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:35, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:5 or SEQ ID NO:35; and

[0426] - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0427] In a preferred embodiment, the M3C65 humanized antibody or fragment thereof comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0428] - H-CDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 1;

[0429] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 2;

[0430] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0431] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0432] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 4;

[0433] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0434] In a preferred embodiment, the M3C65 humanized antibody or fragment thereof comprises :

[0435] - the heavy chain complementarity-determining regions (H-CDRs) of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3,

[0436] - the light chain complementarity-determining regions (L-CDRs) of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and- framework regions from a human monoclonal antibody, preferably from a human anti- HER2 monoclonal antibody, preferably 4D5 antibody.

[0437] In a particular embodiment, the humanized antibody or fragment thereof, preferably a humanized M3C65 scFv, comprises :

[0438] - a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9; and

[0439] - a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10.

[0440] In a particular embodiment, the humanized antibody or fragment thereof, preferably a humanized M3C65 scFv, comprises :

[0441] - a variable heavy chain (VH) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NOV; and

[0442] - a variable light chain (VL) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10.

[0443] In a particular embodiment, the VH domain and the VL domain of the humanized M3C65 scFv are fused through an amino acid linker. The term “linker” refers to a (poly)peptide comprising 5 to 80 amino acids, preferably 5 to 30, still preferably 10 to 20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises GGGS or GGGGS repeats. Linkers composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility, and allows for mobility of the connecting functional domains. Rigid peptide linkers can also be employed in the design of fusion proteins and antibody fragments, such as the (PPPPK)xN linker. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17 or SEQ ID NO: 19. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17.In a particular embodiment, the scFv fragment of humanized M3C65 antibody comprises or consists of the amino acid sequence as shown in SEQ ID NO:21 or SEQ ID NO:38, preferably SEQ ID NO:21 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:21 or SEQ ID NO:38, preferably SEQ ID NO:21.

[0444] 3- Cell expressing M3C65

[0445] A further object of the present invention relates to a cell that has been modified to express an M3C65 antibody, a functional variant, or a fragment thereof as described above.

[0446] In a particular embodiment, the cell is an immune cell.

[0447] As used herein, the term "immune cell" includes any type of immune cells categorized as lymphocytes, neutrophils, and monocytes / macrophages, whether recombinant (engineered) or not. For example, suitable immune cells include, but are not limited to, T cell, chimeric antigen receptor (CAR)-T cell, T cell receptor (TCR)-transgenic T cell, tumor infiltrating lymphocyte (TIL), NK cell, NK-T cell, CAR-NK cell, CAR-NKT cell, TCR- transgenic NK cell, TCR-transgenic NK-T cell, dendritic cell, macrophage, CAR-macrophage or any synthetic tumor specific immune cells.

[0448] In a preferred embodiment, the cell is a regulatory T (Treg) cell, such as a conventional Treg cell or a converted Treg cell made by expressing Foxp3 in a conventional T cell.

[0449] In a particular embodiment, the cell has been optionally further modified to express a protein improving its functionality. In a particular embodiment, the cell, preferably a Treg cell, has been further modified to express IL-2.

[0450] In a particular embodiment, the immune cells can be autologous, allogeneic or xenogeneic. In a particular embodiment, the immune cells can be autologous or allogeneic.

[0451] As used herein, the term "autologous" refers to any material derived from the same individual to which it is later to be re-introduced.As used herein, the term "allogeneic" refers to any material derived from one individual which is then introduced to another individual of the same species, e.g., allogeneic T cell transplantation.

[0452] “Xenogeneic cells” are cells that originate from a different species than the recipient organism.

[0453] In a particular embodiment, the cell comprises or expresses the M3C65 antibody, functional variant, or fragment thereof, on its surface, i.e. the cell displays the M3C65 antibody, functional variant or fragment thereof on its outer membrane. According to a particular embodiment, the M3C65 antibody, functional variant, or fragment thereof is surface-expressed by the cell.

[0454] The M3C65 antibody, functional variant, a fragment thereof as described above can be expressed on the cell surface through several mechanisms. For example, the antibody or antibody fragment can be engineered to be expressed on the cell surface by fusing it to a transmembrane domain or an anchoring protein, such as Glycosylphosphatidylinositol (GPI) anchors. The antibody or antibody fragment can be chemically modified to allow covalent attachment to cell surface proteins.

[0455] In a particular embodiment, the cell expresses a CAR.

[0456] As used herein, the terms "Chimeric antigen receptor" and “CAR” are used interchangeably to refer to engineered receptors, which graft an antigen binding specificity onto immune cells (e.g. T cells or NK cells), thus combining the antigen binding properties of the antigen binding domain with the immunogenic activity of the immune cell, such as the lytic capacity and selfrenewal of T cells. Particularly, a CAR refers to a fused protein comprising an extracellular domain able to bind an antigen, a transmembrane domain, optionally a hinge domain and at least one intracellular domain.

[0457] The terms “extracellular domain able to bind an antigen”, “external domain”, “ectodomain” and “antigen binding domain” are used interchangeably herein and mean any oligopeptide or polypeptide that can bind to a targeted antigen, such as PC. Particularly, the term "antigen binding domain” or "antigen-specific targeting domain" as used herein refers to the region of the CAR which targets and binds to specific antigens, such as PC-antigen.When a CAR is expressed in a host cell, this domain forms the extracellular domain (ectodomain) of the receptor. The antigen binding domain of a CAR typically derives from an antibody, for example M3C65, and may consist of an antigen-binding domain of a single-chain antibody (scFv) or antigen-binding fragments (Fab), for example the antigen-binding domain of a single-chain antibody (scFv) or antigen-binding fragments (Fab) of M3C65 antibody.

[0458] The terms “intracellular domain”, “internal domain”, “cytoplasmic domain” and “intracellular signaling domain” are used interchangeably herein and mean any oligopeptide or polypeptide known to function as a domain that transmits a signal that causes activation or inhibition of a biological process in a cell. The intracellular signaling domain may generate a signal that promotes an immune effector function of the cell transduced with a nucleic acid sequence comprising a CAR, e.g. cytolytic activity and helper activity, including the secretion of cytokines. In particular, the intracellular domain as described herein is able to transmit a signal that causes activation of T cells. Suitable intracellular domains include, but are not limited to, intracellular signaling domains such as CD3^, and co-stimulatory domains such as CD28 and 4-1BB (CD137).

[0459] The CAR can be :

[0460] a first-generation CAR, which consists of an scFv antibody fragment, a transmembrane domain, and a single CD3^ intracellular signaling domain ;

[0461] a second-generation CAR, which includes an additional co-stimulatory domain (e.g., CD28 or 4- IBB) alongside CD3^ ;

[0462] a third-generation CAR, which contains two co-stimulatory domains (e.g., CD28 and 4- 1BB) in addition to CD3^.

[0463] The term “transmembrane domain” means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signaling domains. This may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. Typically, the transmembrane domain denotes a single transmembrane alpha helix of a transmembrane protein, also known as an integral protein.

[0464] A chimeric antigen receptor may optionally comprise a “hinge domain” which serves as a linker between the extracellular and transmembrane domains. As used herein the terms “hinge”, "spacer", or "linker" refers to an amino acid sequence of variable length typically encodedbetween two or more domains of a polypeptide construct to confer for example flexibility, improved spatial organization and / or proximity. As used herein, the term “cleavable linker” refers to a peptide chain of variable length that can be proteolytically cleaved or digested by proteases or enzymes or that self-cleaves. After cleavage of the peptide linker, its integrity is generally compromised and results to the separation of the domains located on either side of the cleavable linker (C-terminus and N-terminus). A “cleavable linker” generally comprises a cleavage site. As used herein, the term "cleavage site" refers to a specific sequence of amino acids that can be cleaved specifically by a cleavage agent, such as a protease, or that selfcleaves. The term "linker" as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and / or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.

[0465] A chimeric antigen receptor may optionally comprise a signal peptide. The terms "signal peptide" “targeting signal”, “localization signal”, “transit peptide” or “leader sequence” refer to a short peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. The core of the signal peptide may contain a long stretch of hydrophobic amino acids. The signal peptide may or may not be cleaved from the mature polypeptide.

[0466] In a particular embodiment, the cell expresses a CAR based on M3C65 antibody, a functional variant, or a fragment thereof, preferably an M3C65 scFv. In a particular embodiment, said M3C65 antibody, functional variant thereof, or fragment thereof is as described above. In a particular embodiment, the cell expresses a CAR comprising a scFv fragment of an M3C65 antibody or a functional variant thereof. In a particular embodiment, the cell expresses a CAR comprising a scFv fragment of a humanized M3C65 antibody.

[0467] In a particular embodiment, the cell expresses a CAR which comprises a scFv comprising the following heavy chain complementarity-determining regions (H-CDRs):

[0468] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:1 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 1; - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:2 and / orhaving a substitution of one, two or three amino acids with respect to SEQ ID NO:2; and

[0469] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with respect to SEQ ID NO:3 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:3.

[0470] In a particular embodiment, said M3C65 antibody, preferably said M3C65 scFv comprises extended variants ofH-CDRl, H-CDR2 and / or H-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said H-CDR1, H-CDR2 and / or H-CDR3.

[0471] In a particular embodiment, said M3C65 antibody, preferably said M3C65 scFv, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0472] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or an extended variant thereof having the amino acid sequence of SEQ ID NO:32;

[0473] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or an extended variant thereof having the amino acid sequence of SEQ ID NO:33; and

[0474] - H-CDR3 having the amino acid sequence of SEQ ID NO:3.

[0475] Preferably, the cell expresses a CAR which comprises a scFv comprising the following light chain complementarity-determining regions (L-CDRs):

[0476] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:4;

[0477] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO:6 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6.

[0478] In a particular embodiment, said M3C65 antibody, preferably said M3C65 scFv, comprises extended variants of L-CDR1, L-CDR2 and / or L-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said L-CDR1, L-CDR2 and / or L-CDR3.In a particular embodiment, said M3C65 antibody, preferably said M3C65 scFv, comprises the following light chain complementarity-determining regions (L-CDRs):

[0479] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or an extended variant thereof having the amino acid sequence of SEQ ID NO:34;

[0480] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or an extended variant thereof having the amino acid sequence of SEQ ID NO:35; and

[0481] - L-CDR3 having the amino acid sequence of SEQ ID NO:6.

[0482] In a particular embodiment, said M3C65 antibody, preferably said M3C65 scFv, comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0483] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:32, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:1 or SEQ ID NO:32;

[0484] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:33 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:2 or SEQ ID NO:33;

[0485] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0486] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0487] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:34 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:4 or SEQ ID NO:34;

[0488] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:35 a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:5 or SEQ ID NO:35; and

[0489] - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0490] In a preferred embodiment, the cell expresses a CAR which comprises a scFv comprising the following heavy chain complementarity-determining regions (H-CDRs):

[0491] - H-CDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 1;- H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 2;

[0492] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0493] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0494] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 4;

[0495] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0496] In a particular embodiment, the cell expresses a CAR which comprises a scFv comprising: - a heavy chain variable region (VH) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO:2, and a CDR3 having the amino acid sequence of SEQ ID NO:3 ; and / or

[0497] - a light chain variable region (VL) comprising a CDR1 having the amino acid sequence of SEQ ID NO:4, a CDR2 having the amino acid sequence of SEQ ID NO:5, and a CDR3 having the amino acid sequence of SEQ ID NO:6.

[0498] In a particular embodiment, the cell expresses a CAR which comprises a scFv comprising : - a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:7 ; and

[0499] - a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 8.

[0500] In a particular embodiment, the cell expresses a CAR which comprises a scFv comprising: - a variable heavy chain (VH) domain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 7 ; and

[0501] - a variable light chain (VL) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 8.In a particular embodiment, the VH domain and the VL domain of the M3C65 scFv are fused through an amino acid linker. The term “linker” refers to a (poly)peptide comprising 5 to 80 amino acids, preferably 5 to 30, still preferably 10 to 20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises GGGS or GGGGS repeats. Linkers composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility, and allows for mobility of the connecting functional domains. Rigid peptide linkers can also be employed in the design of fusion proteins and antibody fragments, such as the (PPPPK)xN linker. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17 or SEQ ID NO: 19. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17.

[0502] In a particular embodiment, the M3C65 scFv fragment comprises or consists of the amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:20.

[0503] In a particular embodiment, the cell expresses a CAR which comprises a scFv fragment of a humanized M3C65 antibody.

[0504] In a particular embodiment, the scFv fragment of a humanized M3C65 antibody is generated by CDR-grafting, preferably by grafting the complementarity determining regions from the murine M3C65 antibody into the framework regions of a human monoclonal antibody, preferably of a human anti-HER2 monoclonal antibody, preferably 4D5 antibody.

[0505] In a particular embodiment, the scFv of humanized M3C65 antibody comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0506] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:1 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 1; - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with SEQ ID NO:2 and / orhaving a substitution of one, two or three amino acids with respect to SEQ ID NO:2; and

[0507] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof, said variant having at least 80%, 90% or 95% sequence identity with respect to SEQ ID NO:3 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:3.

[0508] In a particular embodiment, the scFv of humanized M3C65 antibody comprises extended variants of H-CDR1, H-CDR2 and / or H-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said H-CDR1, H-CDR2 and / or H-CDR3.

[0509] In a particular embodiment, the scFv of humanized M3C65 antibody comprises the following heavy chain complementarity -determining regions (H-CDRs):

[0510] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or an extended variant thereof having the amino acid sequence of SEQ ID NO:32;

[0511] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or an extended variant thereof having the amino acid sequence of SEQ ID NO:33; and

[0512] - H-CDR3 having the amino acid sequence of SEQ ID NO:3.

[0513] Preferably, the scFv of humanized M3C65 antibody comprises the following light chain complementarity-determining regions (L-CDRs):

[0514] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:4;

[0515] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO:5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO:5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO:6 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6.

[0516] In a particular embodiment, the scFv of humanized M3C65 antibody comprises extended variants of L-CDR1, L-CDR2 and / or L-CDR3, i.e. variants having additional amino acid residues at the N-terminus or C-terminus of said L-CDR1, L-CDR2 and / or L-CDR3.O

[0517] In a particular embodiment, the scFv of humanized M3C65 antibody comprises the following light chain complementarity-determining regions (H-CDRs):

[0518] L-CDR1 having the amino acid sequence of SEQ ID NO:4 or an extended variant thereof having the amino acid sequence of SEQ ID NO:34;

[0519] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or an extended variant thereof having the amino acid sequence of SEQ ID NO:35; and

[0520] - L-CDR3 having the amino acid sequence of SEQ ID NO:6.

[0521] In a particular embodiment, the scFv of humanized M3C65 antibody comprises the following heavy chain complementarity-determining regions (H-CDRs):

[0522] - H-CDR1 having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:32, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:1 or SEQ ID NO:32;

[0523] - H-CDR2 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:33, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:2 or SEQ ID NO:33;

[0524] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0525] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0526] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:34, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:4 or SEQ ID NO:34;

[0527] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:35, or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:5 or SEQ ID NO:35; and

[0528] - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0529] In a preferred embodiment, the CAR comprises a scFv of humanized M3C65 antibody comprising the following heavy chain complementarity-determining regions (H-CDRs):

[0530] - H-CDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 1;- H-CDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 2;

[0531] - H-CDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 3 ;

[0532] and comprises the following light chain complementarity-determining regions (L-CDRs):

[0533] - L-CDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 4;

[0534] - L-CDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO: 5; and - L-CDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof having a mutation of one, two or three amino acids with respect to SEQ ID NO:6.

[0535] In a preferred embodiment, the CAR comprises a scFv of a humanized M3C65 antibody comprising :

[0536] - the heavy chain complementarity-determining regions (H-CDRs) of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3,

[0537] - the light chain complementarity-determining regions (L-CDRs) of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and

[0538] - framework regions from a human monoclonal antibody, preferably from a human anti- HER2 monoclonal antibody, preferably 4D5 antibody.

[0539] In a particular embodiment, the CAR comprises a scFv of a humanized M3C65 antibody comprising :

[0540] - a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9; and

[0541] - a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10.

[0542] In a particular embodiment, the CAR comprises a scFv of a humanized M3C65 antibody comprising :- a variable heavy chain (VH) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:36, preferably SEQ ID NO:9; and

[0543] - a variable light chain (VL) domain comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 10 or SEQ ID NO:37, preferably SEQ ID NO: 10.

[0544] In a particular embodiment, the VH domain and the VL domain of the humanized M3C65 scFv are fused through an amino acid linker. The term “linker” refers to a (poly)peptide comprising 5 to 80 amino acids, preferably 5 to 30, still preferably 10 to 20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises GGGS or GGGGS repeats. Linkers composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility, and allows for mobility of the connecting functional domains. Rigid peptide linkers can also be employed in the design of fusion proteins and antibody fragments, such as the (PPPPK)xN linker. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or SEQ ID NO: 19 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17 or SEQ ID NO: 19. In a preferred embodiment, the linker is the linker of SEQ ID NO: 17 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 17.

[0545] In a particular embodiment, the scFv fragment of humanized M3C65 antibody comprises or consists of the amino acid sequence as shown in SEQ ID NO:21 or SEQ ID NO:38, preferably SEQ ID NO:21 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:21 or SEQ ID NO:38, preferably SEQ ID NO:21.

[0546] In a particular embodiment, the cell has been modified to express a CAR based on said M3C65 antibody, functional variant, or fragment thereof, wherein the C-terminus of M3C65 antibody, preferably a scFv, is fused to a transmembrane domain of a surface protein or a GPI anchor, and wherein the transmembrane domain is preferably further fused to an intracellular domain permitting the cell activation, such as CD3^, CD28, and / or 4-1BB.

[0547] 4- Formulations and uses

[0548] Formulations and routes of administrationIt is also provided a pharmaceutical composition comprising an M3C65 antibody, functional variant or a fragment thereof as described above, preferably in association (e.g., in solution, suspension, or admixture) with a pharmaceutically acceptable vehicle, carrier or excipient.

[0549] It is also provided a pharmaceutical composition comprising a chimeric construct, a conjugate, or a cell as described above, preferably in association (e.g., in solution, suspension, or admixture) with a pharmaceutically acceptable vehicle, carrier or excipient.

[0550] Suitable excipients include any isotonic solution, saline solution, buffered solution, slow release formulation, etc. Liquid, lyophilized, or spray-dried compositions are known in the art and may be prepared as aqueous or nonaqueous solutions or suspensions. Preferably the pharmaceutical compositions comprise appropriate stabilizing agents, buffering agents, bulking agents, or combinations.

[0551] The pharmaceutical composition may further contain another active ingredient, or may be administered in combination with any other active ingredient.

[0552] The pharmaceutical composition may be administered using any convenient route, including parenteral, e.g. intradermal, subcutaneous, or intranasal route. The subcutaneous route is preferred. Oral, sublingual or buccal administrations are also encompassed.

[0553] An example of a formulation suitable for a subcutaneous injection is described in international patent application W02017 / 068031.

[0554] According to the present invention, the chimeric constructs, conjugates, cells, nucleic acids, vectors, host cells, and compositions as described herein have numerous utilities and applications.

[0555] More particularly, the conjugates of the present invention which may comprise an imaging moiety as a payload are useful as optical imaging agents of tissues and organs in various biomedical applications.In some embodiment, the conjugates as described herein may be used in a method of imaging, detecting or diagnosing a disease or a disorder in a subject in vivo, preferably an inflammatory disease.

[0556] In a particular embodiment, the methods for imaging include: (a) administering a conjugate to a subject, wherein the conjugate comprises (i) a targeting moiety, which is an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, that is conjugated, preferably by a linker, to (ii) a payload, that is an imaging moiety; and (b) detecting the presence of the conjugate in the subject in vivo by imaging.

[0557] In a particular embodiment, the presence of the conjugate is detected in real time.

[0558] In another particular embodiment, the presence of the conjugate is detected by a non-invasively and / or minimally invasively method.

[0559] Imaging can be performed by many procedures well-known to those having ordinary skill in the art. Particularly, imaging can be performed by any technique allowing the visualization of detection signals provided by the above-described imaging moieties. In particular, imaging moieties generally provide signals detectable by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like.

[0560] In a preferred embodiment, the conjugates can be used in a method of imaging, detecting or diagnosing an inflammatory disease or disorder in a subject. Examples of such inflammatory diseases are more detailed below.

[0561] Another embodiment of the invention relates to the conjugates as described herein, for use in a method of staining a biological sample from a subject. As used herein, a "biological sample" may be any sample, such as a tissue sample, that may be taken from a subject.

[0562] The conjugates as described herein can be detected in a biological sample by a variety of known methods. Examples of suitable methods include, but are not limited to, immunohistochemistry, immunofluorescence, Enzyme-linked Immunosorbent Assay (ELISA), fluorescence microscopy, confocal microscopy, multiphoton microscopy, electron microscopy,autoradiography and the likes. Any skilled person can easily select the appropriate methods depending on the biological sample to be analyzed.

[0563] In a particular embodiment, the biological sample is a tissue sample, preferably analyzed by an immunochemistry method.

[0564] In a particular embodiment, the biological sample is from a subject suffering from an inflammatory disease or disorder as detailed hereinafter.

[0565] In some embodiments, said inflammatory disease or disorder can be an autoimmune disease or disorder.

[0566] Treatment of auto-immune and / or inflammatory disorders

[0567] The present invention also relates to the use of the chimeric construct, nucleic acid, vector, conjugate, cell, M3C65 antibody, or to the pharmaceutical composition according to the present invention for the treatment of a disease or a disorder in a subject.

[0568] In some embodiments, the chimeric construct, nucleic acid, vector, conjugate, cell, M3C65 antibody, or the pharmaceutical composition according to the present invention may be used in the treatment of an inflammatory disease, an autoimmune disease, or a cancer. In a particular embodiment, the invention relates to the use of an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, for use in treating an inflammatory disease.

[0569] In some embodiments, the chimeric construct, nucleic acid, vector, conjugate, cell, M3C65 antibody, or the pharmaceutical composition according to the present invention are useful in methods for treating an inflammatory and / or autoimmune disorder, In some embodiments, the chimeric construct, nucleic acid, vector, conjugate, cell, M3C65 antibody, or the pharmaceutical composition according to the present invention are useful in methods for treating systemic lupus erythematous, type I diabetes, HCV-related vasculitis, uveitis, myositis, systemic vasculitis, psoriasis, allergy, asthma, Crohn’s disease, multiple sclerosis, rheumatoid arthritis, atherosclerosis, autoimmune thyroid disease, auto-inflammatory diseases, neuro-immune diseases, neuro-inflammatory diseases or neuro-degenerative diseases, including Alzheimer’s disease and amyotrophic lateral sclerosis, acute and chronic graft-versus-hostdisease, spontaneous abortion and allograft rejection; solid organ transplantation rejection, vasculitis, inflammatory bowel disease (IBD), and allergic asthma; spondyloarthritis or ankylosing Spondylitis; Sjogren’s syndrome, Systemic sclerosis, Alopecia aerate, or Ulcerative Colitis.

[0570] Herein, the term “inflammatory disease” and “inflammatory disorder” are used interchangeably and refer to any disease or disorder that encompasses an inflammatory component, and / or any disease or disorder in which inflammation is a prominent contributor to the clinical condition.

[0571] The term “inflammatory disease” encompasses conditions where inflammation plays a significant role in the pathophysiology, regardless of whether it is the primary cause or a secondary effect. Inflammatory diseases can affect various organs and systems in the body, including the digestive tract, immune system, circulatory system, respiratory system, metabolic system, and nervous system. These conditions can be acute (short-term) or chronic (long-term), and may result from infectious or non-infectious causes. Chronic inflammation can induce tissue damage, impair organ function, and contribute to various pathological conditions, including autoimmune disorders, metabolic syndrome, cardiovascular diseases, neurological diseases or cancers.

[0572] In a particular embodiment, the inflammatory disease may be an autoimmune disease. The term "autoimmune disease" is used to designate a condition that arises from an abnormal immune response of the body against substances and tissues normally present in the body. The disease may be restricted to certain organs or involve a particular tissue in different places.

[0573] In a preferred embodiment, the invention concerns a chimeric construct as described hereabove or a pharmaceutical composition comprising such, for use in treating an auto-immune and / or inflammatory disease, wherein said chimeric construct comprises a targeting moiety and a payload that is interleukin 2 (IL-2).

[0574] A chimeric construct as described hereabove comprising a targeting moiety and a payload that is interleukin 2 (IL-2) or a pharmaceutical composition comprising such, is also suitable in conditions where the activation of Tregs tissue regeneration properties are desired, such as in muscle diseases, neurodegeneration, cardiac or other tissues infarction.In a preferred embodiment, the invention concerns a chimeric construct as described hereabove or a pharmaceutical composition comprising such, for use in treating an auto-immune and / or inflammatory disease, wherein said chimeric construct comprises a targeting moiety and a payload that is interleukin 2 (IL-2), and wherein the dosage is less than 3.5 MIU / day.

[0575] In a particular embodiment, it is herein described a method of treatment of an auto-immune and / or inflammatory disorder, comprising administering the chimeric construct, the conjugate, cell, M3C65 antibody, or the pharmaceutical composition once or twice a week, or even once or twice a month, preferably by subcutaneous route.

[0576] In one embodiment, a chronic administration is implemented, e.g. comprising administration once every 3 days to once every three months. Such sequences of administration may be repeated if needed.

[0577] In certain aspects of the present invention, the chimeric construct, conjugate, cell, nucleic acid, vector, M3C65 antibody or pharmaceutical composition described herein are useful in methods for treating a cancer. In some embodiments, the subject is suffering from locally advanced or metastatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colon cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, breast cancer, kidney cancer, esophageal cancer, or prostate cancer.

[0578] The compositions can be administered once from one or more times per day to once or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject can include a single treatment or, can include a series of treatments.

[0579] Another aspect of the invention is the use of the chimeric construct, nucleic acid, vector, conjugate, cell, or M3C65 antibody according to the invention for the manufacture of a pharmaceutical composition. A further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising a chimeric construct, nucleic acid, vector, conjugate, cell, or M3C65 antibody, according to the invention.EXAMPLES

[0580] EXAMPLE 1 : Assessment of avidity and affinity interaction between IL-2-E06 or IL2-M3C65 fusion proteins and PC-BSA.

[0581] MATERIAL AND METHODS

[0582] Fusion proteins

[0583] The avidity / affinity of M3C65 antibody was compared to the avidity / affinity of E06 antibody which is another anti-PC antibody. Two fusion proteins were designed :

[0584] IL-2-C4BPB-M3C65 (SEQ ID NO:25), which comprises from N-ter to C-ter: hIL-2 (SEQ ID NO: 12), a C4BPB fragment (SEQ ID NO: 14) and M3C65 scFv (SEQ ID NO:20); and

[0585] IL-2-C4BPB-E06 which comprises from N-ter to C-ter: hIL-2, a C4BPB fragment and E06 scFv.

[0586] The sequence of the fusion protein was transcribed into a nucleotide sequence to which was added a 6xHis tag at the C-ter, codon-optimized for human cells and inserted into a lentiviral vector under the control of the SFFV promoter. Then, HEK 293T cells were transduced with the lentiviral vector, and cultured in serum-free medium for 48-72 hours at 37°C, 5% CO2. The cell culture media was then collected and purified using a Ni-NTA column to purify the HisTag proteins. After elution, the proteins were concentrated and buffer exchanged for PBSlx.The fusion proteins were then stored at 4°C and analyzed using an SDS-PAGE gel with Coomassie blue staining.

[0587] ELISA

[0588] Avidity assessment was performed by using a standard ELISA protocol.

[0589] Phosphocholine-BSA (PC-BSA) (1 pg / ml) was coated on a 96-well plate and incubated overnight at 4°C. After washing and blocking, 5-fold dilutions of E06 or M3C65 were incubated for 2 hours at 37°C in triplicate. A subsequent washing was then performed. Bound fusion proteins were exposed to various concentrations of sodium thiocyanate (NaSCN) (0.5M, 1.5M)or left untreated (control). Residual binding was quantified by absorbance (Optical Density measurement) at 450 nm, with NaSCN resistance indicating avidity.

[0590] Surface plasmon resonance (SPR)

[0591] Affinity measurement and dissociation constant (KD) estimation were performed by Surface plasmon resonance (SPR) using a BIACORE 3000 instrument (GE Healthcare Bio-Sciences AB, Sweden).

[0592] Anti-BSA IgG was covalently immobilized onto a BIACORE Sensor Chip CM5 (GE Healthcare Bio-Sciences, Sweden) and infused with the same concentration of PC-BSA. A dose response of IL-2 / E06 or IL-2 / M3C65 fusion proteins was serially infused to evaluated binding of the proteins. Binding kinetics, i.e. the association (kon) and dissociation (koff) rate constants, were measured during 5-minute association / dissociation phases, and the dissociation constant (KD) was calculated using a 1:1 Langmuir binding model.

[0593] RESULTS

[0594] Avidity Assay

[0595] As detailed above, E06 and M3C65 antibodies are known to be specific for oxidized phospholipids such as phosphocholine (PC).

[0596] To determine the strength of binding interactions between said antibodies and an antigen (PC-BSA), the inventors evaluated the resistance of antibody-antigen interactions to disruption by sodium thiocyanate (NaSCN), a chaotropic agent well-known to unbind low-avidity binding. More precisely, PC-BSA was coated on a 96-well plate before being incubated with either IL-2-C4BPB-E06 or IL-2-C4BPB-M3C65 fusion proteins.

[0597] In ELISA, whereas both IL-2-C4BPB-E06 and IL-2-C4BPB-M3C65 bind specifically precoated PC-BSA, addition of NaSCN at 0.5M and 1.5M, induced dramatic reduction of IL-2 -C4BPB-06 binding. Upon exposure to 0.5M NaSCN, IL-2-C4BPB-E06 exhibited a substantial decrease in binding capacity, maintaining only 35% of its original binding activity. When challenged with 1.5M NaSCN, IL-2-C4BPB-E06 binding was further reduced, retaining merely 15% of initial binding. In contrast, IL-2-C4BPB-M3C65 demonstrated superior bindingstability, maintaining approximately 90% and 80% of its original binding activity when exposed to 0.5M and 1.5M NaSCN, respectively (Figure 1A).

[0598] Affinity Assay

[0599] Besides assessing avidity of either IL-2 / E06 or IL-2 / M3C65 fusion proteins with PC-BSA, the inventors performed an additional experiment to assess the binding affinity of said proteins to the PC-BSA antigen.

[0600] To determine the binding affinity of these proteins, the inventors evaluated the kinetic parameters, i.e. the association (kon) and dissociation (koff) rate constants, to identify how quickly the proteins associate with or dissociate from the antigen. This experiment has been conducted as above-detailed by Surface plasmon resonance (SPR) using a BIACORE 3000 instrument.

[0601] As well as the avidity assay, while IL-2-C4BPB-E06 showed low association (kon) and high dissociation (koff), IL-2 / M3C65 fusion protein demonstrated higher association to PC-BSA with reduction of dissociation. These observations were confirmed by the calculation of KD, reaching 3.10’8M for IL-2-C4BPB-E06 and 1.8.10’9M for IL-2 / M3C65 for PC, showing a 20-fold increase in affinity compared to IL-2 / E06 (Figure IB).

[0602] EXAMPLE 2 : Therapeutic effects of the targeted fusion proteins in colitis inflammatory experimental model.

[0603] MATERIAL AND METHODS

[0604] Colitis inflammatory experimental model

[0605] 8-week-old female C57BL / 6 mice (n=5) were immunized by oral administration of 3% of Dextran Sulfate Sodium (DSS; Sigma) in drinking water for 6 days starting on day 0. One day before the addition of 3% dextran sulfate sodium (DSS) to drinking water, mice began a 5-day course of daily intraperitoneal injections (50,000 IU of IL-2, E06, or M3C65 fusion proteins, or PBS for the control group). Additionally, a second 5-day course started on day 6.Mice were monitored daily for clinical symptoms including body weight, stool consistency and presence of blood during 15 days. The following scoring system has been used to evaluate the severity:

[0606] Loss of body weight between 0-5% : 1 / 5-10% : 2 / 10-15% : 3 / and over 15% : 4. Stool consistency: normal stool : 0 / formed but soft stool : 1 / loose stool : 2 / mild diarrhea : 3 / watery diarrhea : 4.

[0607] Gross bleeding: absence : 0 / presence : 2 / gross bleeding : 4.

[0608] These parameters were used to calculate a Disease Activity Index (DAI) (C) ranging from 0 (normal) to 12 (severe disease).

[0609] RESULTS

[0610] The targeted fusion proteins (IL-2 fused to E06 antibody and IL-2 fused to M3 C65 antibody) were evaluated in a colitis inflammatory experimental model for their ability to control the severity of the disease. More precisely, the inventors evaluated both IL-2-C4BPB-E06 and IL-2-C4BPB-M3C65 constructs in comparison to native IL-2 and PBS (control group).

[0611] After six days of DSS administration, all mice developed clinical manifestations such as a significant reduction in their body weight (Figure 2A). However, while both native IL-2 and IL-2 / E06 demonstrated efficacy profiles substantially similar to untreated control subjects, the results depicted in Figure 2B demonstrate that administration of IL-2 / M3C65 resulted in significantly attenuated body weight loss compared to the other groups. Moreover, at D15, the body weight of mice treated with IL-2 / M3C65 had returned to a level that was either equivalent to or greater than the body weight recorded at DO.

[0612] Furthermore, IL-2 / M3C65 treatment corresponded with marked reduction in DAI scores (Figure 2B), indicating superior therapeutic efficacy in this inflammatory disease model characterized by the production and accumulation of various oxidation-specific epitopes (OSEs), notably phosphorylcholine (PC).

[0613] EXAMPLE 3: In vivo targeting of IL2-M3C65 fusion protein

[0614] To evaluate the in vivo targeting of the IL2-C4bpP-M3C65 fusion protein (SEQ ID NO:25), eight-week-old female C56B16 / J mice were given either 1011vg of AAV-IL-2C4bpP-M3C65 orPBS. Ten days after injection, psoriasis-like inflammation was induced by applying 62.5 mg of 5% imiquimod cream to the right ear daily and Vaseline to the left ear as a control. After six days, the mice were euthanized, their ears were collected and fixed, and they were paraffin-embedded for future sections. The ears were sectioned and stained directly with an anti-Histag-Cy5 antibody to detect the presence of IL2-C4bpP-M3C65 and counterstained with DAPI to visualize the nuclei (Figure 3A).

[0615] RESULTS

[0616] In mice that received IL2-C4bpP-M3C65, a strong HisTag-Cy5 signal was detected around the inflamed ear (white arrows) that had received imiquimod, whereas no signal was detected in the control ear of the same mice (Figure 3B). No staining was detected in the PBS group. These results confirm the in vivo targeting of IL2-C4bpP-M3C65 to inflammatory sites.

[0617] The direct binding of the IL-2-C4bpP-M3C65 fusion protein to inflamed ears was also evaluated. Eight-week-old female C57B16 / J mice were induced with psoriasis-like inflammation by applying 62.5 mg of 5% imiquimod cream to the right ear daily with vaseline applied to the left ear as a control. After six days, the mice were euthanized and their ears were collected, fixed, and paraffin-embedded for future sections (Figure 4A). The ears were sectioned and stained with IL-2-C4bpP-M3C65, followed by an anti-HisTag-Cy5 antibody, in order to evaluate the protein’s direct binding. The cells were also counterstained with DAPI to visualize nuclei. Controls consisted of anti-HisTag-Cy5 antibody staining that had not been preincubated with the IL-2-C4bpP-M3C65.

[0618] RESULTS

[0619] While we never detected a Cy5 signal on Vaseline-treated ears, a strong signal (indicated by the white arrows) similar to that observed with AAV in vivo was detected around the imiquimod-treated ears (i.e. those with inflammation). Thus, IL-2-C4bpP-M3C65 was able to target and bind to inflamed tissues specifically (see Figure 4B).

[0620] EXAMPLE 4: In Vivo Targeting of CAR-M3C65 T-Cells

[0621] MATERIAL AND METHODSProduction of CAR-M3C65 T-cells

[0622] The CAR comprises the M3C65 scFv sequence (SEQ ID NO:20) to which a 6xHistag has been added upstream of the scFv. This sequence was then fused to the extracellular domain of CD28, the transmembrane and intracytoplasmic domains of CD28 and CD3z for signal transduction. This sequence was transcribed into a nucleotide sequence, codon optimized and inserted into an MSCV retroviral vector under the control of the MSCV promoter. The resulting retroviral vector was then concentrated and used to transduce T cells, which had been cytometrically sorted and cultured for 5 days.

[0623] In vivo tarsetins models

[0624] On Day 0, 10-week-old female Immunodeficient NOD scid gamma (NSG) mice were injected intravenously with either 2x10sCAR-M3C65 T-cells expressing a luciferase transgene (CAR-M3C65-LUC) or non-transduced T-cells (control group).

[0625] Bioluminescence imasins

[0626] On day 7 (D7), bioluminescence imaging was performed using the IVIS Spectrum system (Caliper Life Sciences) according to the manufacturer's instructions. Mice were administered 0.295M ViviRen™ Live Cell Substrate (Promega Corp.; Madison, Wis., USA), a substrate specific for Renilla luciferase. Images were acquired for 2 minutes, 5 minutes after administration of said substrate. Following imaging, all mice received a subcutaneous injection of PC-BSA (20 pg) complexed and adjuvanted with Alum and Complete Freund’s Adjuvant (CFA, 1 mg / ml) in the upper back.

[0627] On day 9 (D9), bioluminescence imaging was repeated under the same conditions as abovedetailed to evaluate CAR-M3C65-LUC T-cell recirculation and accumulation at the PC-BSA injection site.

[0628] RESULTS

[0629] The inventors evaluated the in vivo targeting and trafficking of immune cells that have been modified to express a CAR based on said M3C65 antibody. More precisely the inventorsevaluated the migration and accumulation of engineered T-cells to co-expressing CAR-M3C65 and Renilla Luciferase (CAR-M3C65-LUC). At Day 0 (DO), NSG mice receiving intravenous administration of 2xl06CAR-M3C65-LUC T-cells, while control subjects received nontransduced T-cells (Figure 5A). Initial biodistribution analysis was conducted at day 7 postadministration (D7) utilizing an IVIS spectrum imaging system and bioluminescence was detected as detailed above.

[0630] As anticipated, minimal signal localization was observed, predominantly in hepatic regions with minor signal detection in periorbital regions corresponding to the administration site (Figure 5B).

[0631] Subsequently, all test subjects received subcutaneous administration of PC-BSA (20 pg) complexed and adjuvanted with Alum and Complete Freund’s Adjuvant (CFA, 1 mg / ml) in the upper back (denoted by circles in Figures 5B and 5C).

[0632] Follow-up imaging was performed at D9 to evaluate potential CAR-M3C65-LUC T-cell migration to the PC-BSA administration site. Notably, both mice tested with CAR-M3C65-LUC T-cells demonstrated a bioluminescence signal exactly at the site of injection of adjuvanted PC-BSA, demonstrating the in vivo targeting of T-cells bearing CAR-M3C65 (Figure 5C).

[0633] EXAMPLE 5: CAR-M3C65-mediated Tregs activation with PC-BSA

[0634] To evaluate the activation of CAR-mediated Tregs, CAR-M3C 65 -Tregs (Tregs transduced with the full CAR-M3C65 as described above) or CAR-M3C 65 ATS -Tregs (Tregs transduced with CAR-M3C65 lacking the intracellular transducing signal domain) were cultured for 72 hours with either BSA or PC-BSA. The cells were stained with CellTrace (CTV) prior to culturing to determine Treg proliferation.

[0635] RESULTS

[0636] CAR-M3C 65 -Tregs cell proliferation was assessed by tracking CellTrace Violet (CTV) by flow cytometry. Only the Tregs transduced with the full CAR-M3C65 proliferated in the presence of PC-BSA, demonstrating its specificity and functionality (Figure 6).CONCLUSION

[0637] From the results depicted in Figures 1-6, M3C65 antibody has been shown to be particularly effective in targeting proteins or cells in various inflammatory sites.

[0638] Unexpectedly, the inventors revealed that M3C65 exhibits substantially enhanced binding properties for oxidized phospholipids, such as phosphocholine (PC), when compared to the previously characterized E06 antibody, which was considered as the standard for high-affinity PC binding (Figure 1). This enhanced binding profile manifests in both improved affinity and avidity properties. Moreover, experiments conducted in vivo, including on relevant models of inflammatory diseases, have demonstrated the clear therapeutic benefit of chimeric constructs comprising M3C65 antibody. Particularly, these constructs have demonstrated remarkable efficacy in IL2 delivery to targeted inflammatory sites (Figures 2, 3 and 4). The superior targeting capabilities of M3C65 were further validated through biodistribution studies, which established its capacity to facilitate precise localization and proliferation of immune cells to inflammatory sites, thereby minimizing potential off-target effects and associated complications (Figures 4 and 5).

[0639] Collectively, these findings establish M3C65 as a highly specific targeting moiety, particularly in therapeutic applications requiring precise delivery to inflammatory sites.

Claims

86CLAIMS1. A chimeric construct comprising :(i) a targeting moiety, which is an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, that is fused to(ii) a payload that is a heterologous peptide or protein.

2. The chimeric construct of claim 1, wherein the targeting moiety is an M3C65 scFv.

3. The chimeric construct of claim 1 or 2, wherein the payload is a cytokine, preferably interleukin 2 (IL-2), optionally wherein said IL-2 is human IL-2 or homologous variant thereof, wherein the variant has at least 80% amino acid identity with human wild-type IL-2, preferably wherein the variant is an active analogue of human IL-2 which has at least 90% amino acid identity with human wild-type IL-2, wherein said IL-2 is preferably an IL2 mutein that comprises a substitution at position N88 of SEQ ID NO: 12, still preferably substitution N88R or N88D.

4. The chimeric construct of claim 3, said chimeric construct further comprising a beta chain of the C4b-binding protein (C4BPP) or at least one fragment or functional variant thereof that is capable of forming a dimeric protein, preferably wherein the fragment of C4BPP comprises, or consists of, amino acid residues 194 to 252 of C4BPP or a longer fragment of C4BPP that extends at the N-term up to at most amino acid 135,wherein said chimeric construct preferably comprises IL-2 fused at the N-terminus of C4BPP or said fragment thereof, wherein the C-terminus of C4BPP or said fragment thereof is preferably linked to the targeting moiety.

5. A conjugate comprising:(i) a targeting moiety, which is an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof, that is conjugated, preferably by a linker, to(ii) a payload that is a drug or an imaging moiety.

6. The conjugate of claim 5, wherein the targeting moiety is an M3C65 scFv.

877. A cell, preferably an immune cell, that has been modified to express an M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof on its surface,wherein said M3C65 antibody, functional variant or fragment thereof is preferably fused to a transmembrane domain of a surface protein or to a GPI anchor ,wherein the cell is more preferably a regulatory T (Treg) cell, such as a conventional Treg cell or a converted Treg cell made by expressing Foxp3 in a conventional T cell, wherein the cell has been optionally further modified to express a protein improving its functionality, and / or wherein the cell is preferably autologous, allogeneic or xenogeneic.

8. The cell of claim 7, that has been modified to express a CAR based on said M3C65 antibody, functional variant, or fragment thereof,wherein the C-terminus of M3C65 antibody, functional variant or fragment thereof, preferably a scFv, is preferably fused to a transmembrane domain ; andwherein the transmembrane domain is preferably further fused to an intracellular domain permitting the cell activation, such as CD3^, CD28, and / or 4-1BB.

9. The chimeric construct, conjugate or cell of claims 1-8, wherein said M3C65 antibody, functional variant of M3C65 antibody, or fragment thereof, preferably an M3C65 scFv, comprises:- a heavy chain variable region (VH) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO:2, and a CDR3 having the amino acid sequence of SEQ ID NO:3 ; and / or- a light chain variable region (VL) comprising a CDR1 having the amino acid sequence of SEQ ID NO:4, a CDR2 having the amino acid sequence of GTK (SEQ ID NO:5), and a CDR3 having the amino acid sequence of SEQ ID NO:6.

10. The chimeric construct, conjugate or cell of any of claims 1-9, wherein the M3C65 antibody or functional variant thereof, preferably an M3C65 scFv, comprises:- a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:7 ; and- a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 8.8811. The chimeric construct, conj ugate or cell of any of claims 1 -9, wherein the M3C65 antibody or functional variant thereof is a humanized antibody or a fragment thereof, preferably a humanized M3C65 scFv,wherein, the humanized antibody or fragment thereof, in particular a humanized M3C65 scFv, more preferably comprises:- a variable heavy chain (VH) domain comprising an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:36, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:9 or SEQ ID NO:36 ; and- a variable light chain (VL) domain comprising an amino acid sequence as shown in SEQ ID NOTO or SEQ ID NO:37, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NOTO or SEQ ID NO:37.

12. A nucleic acid encoding the chimeric construct of any of claims 1-4, or a vector comprising said nucleic acid.

13. A vector comprising the nucleic acid encoding the chimeric construct of any one of claims 1-4, wherein the vector is a viral vector, preferably a retroviral vector such as a lentiviral vector, or an Adeno-Associated Virus (AAV) vector.

14. A host cell expressing the nucleic acid or the vector of claim 12 or 13.

15. The chimeric construct, conjugate or cell according to any of claims 1 to 11, for use as a medicament.

16. The chimeric construct, conjugate or cell according to any of claims 1 to 11, for use in treating inflammation, preferably inflammation in a patient with an inflammatory disease and / or an auto-immune disease.

17. A chimeric construct of claim 3 or 4, wherein the payload is interleukin 2 (IL-2) or variants thereof, for use in treating an auto-immune disease or an inflammatory disease.8918. An M3C65 antibody, a functional variant of M3C65 antibody, or a fragment thereof such as an M3C65 scFv, for use in treating inflammation, preferably inflammation in a patient with an inflammatory disease and / or an auto-immune disease.

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