WO2024251979A1

WO2024251979A1 - Backpacked-antibody constructs and their uses

Info

Publication number: WO2024251979A1
Application number: PCT/EP2024/065791
Authority: WO
Inventors: Edurne RUJAS DIEZ; José Luis NIEVA ESCANDÓN; Sara INSAUSTI GONZÁLEZ; Izaskun MORILLO MELERO
Original assignee: FUNDACION BIOFISICA BIZKAIA; Euskal Herriko Unibertsitatea
Current assignee: FUNDACION BIOFISICA BIZKAIA; Euskal Herriko Unibertsitatea
Priority date: 2023-06-07
Filing date: 2024-06-07
Publication date: 2024-12-12
Anticipated expiration: 2025-12-07

Abstract

The present invention is comprised within the fields of molecular biology and biotechnology. It specifically relates to polypeptides for the generation of Backpacked-antibody (B-ab) molecules for a variety of uses, in particular, for the specific delivery of drugs to tumor cells.

Description

BACKPACKED-ANTIBODY CONSTRUCTS AND THEIR USES

The present invention is comprised within the fields of molecular biology and biotechnology. It specifically relates to polypeptides for the generation of Backpacked- antibody (B-ab) molecules for a variety of uses, in particular, for the specific delivery of drugs to tumor cells.

BACKGROUND ART

Nanolipoprotein particles (NLPs), commonly referred to as nanodiscs (NDs), are nanometer-sized particles usually comprised of an amphipathic lipid bilayer and an apolipoprotein (Apo). NLPs have been used for various biotechnology applications, such as membrane protein stabilization/solubilization, drug delivery, and in particular vaccine delivery, and diagnostic imaging.

In some instances, NLPs can self-assemble under appropriate conditions into nanoscale amphipathic apolipoprotein-stabilized lipid bilayer particles possibly comprising additional molecules, such as one or more integral membrane proteins or other proteins and molecules attached to the amphipathic component of the NLP.

Nanodiscs can function as drug-delivery vehicles for a wide range of payloads. Their hydrophobic core can stably incorporate water-insoluble chemotherapeutic agents, while the surface can be used to adsorb molecules such as small interfering RNA (siRNA) through electrostatic interactions with the lipid polar head groups or to load hydrophilic drugs through chemical conjugation with lipids.

Despite the advancement of this technology, providing NLPs delivery platforms, in particular, stable compositions and systems, for delivering of drugs, therapeutic agents or diagnostic agents can be challenging.

SUMMARY OF THE INVENTION

To overcome the disadvantages of the current strategies, the authors of the present invention have engineered an IgG scaffold to generate antibody-like particles that incorporate a lipid cage mimicking a “backpack” for loading and delivery of therapeutically effective concentrations of cytotoxic drugs to tumor cells. These antibodylike particles are capable of efficiently delivering cytotoxic drugs in a more efficient manner and with higher payload than antibody-drug conjugates. In addition, they show an increased serum half-life in comparison with the lipid cages which do not contain the IgG scaffold. Moreover, their serum half-life is not affected by the incorporation of therapeutic compounds into the lipid cage, which contrasts with the situation of other types of antibody-based delivery agents such as antibody-drug conjugates, which suffer a decrease in their serum half-life with respect to the non-conjugated form as a result of the coupling of the therapeutic compound.

The innovative design of these molecules harnesses the strengths of lipidic nanocarriers and targeted therapy to facilitate more efficient delivery of chemotherapeutic drugs. Its modular nature allows exchanging the different components within the molecule in a strategic fashion to rapidly produce efficient therapies against a wide range of cancers and to continuously integrate new discoveries in the field of therapeutic antibodies and antitumor agents. Overall, it will result in the development of a novel antibody-based platform for cancer treatment with improved therapeutic properties in comparison to conventional approaches.

In view of the aforementioned, a first aspect of the present invention relates to a fusion protein, hereinafter “the fusion protein of the invention” comprising:

(i) a single-chain antigen-binding fragment (scFab) or a fragment of an immunoglobulin chain containing a variable region;

(ii) a membrane scaffold protein (MSP); and

(iii) a crystallisable domain fragment (Fc).

In another aspect, the present invention relates to a polynucleotide, hereinafter “the polynucleotide of the invention” encoding the fusion protein of the invention.

In another aspect, the present invention relates to a vector, hereinafter “the vector of the invention” comprising the polynucleotide of the invention.

In another aspect, the present invention relates to a host cell comprising the vector of the invention.

In another aspect, the present invention relates to a lipid nanodisc, hereinafter “the lipid nanodisc of the invention” characterize in that it comprises multiple copies of the fusion protein of the invention and a membrane-forming lipid. In another aspect, the present invention relates to a pharmaceutical composition, hereinafter “the pharmaceutical composition of the invention” comprising the lipid nanodisc of the invention, and at least one pharmaceutically acceptable excipient.

In another aspect, the present invention relates to the lipid nanodisc of the invention or the pharmaceutical composition of the invention for use in medicine.

In another aspect, the present invention relates to the lipid nanodisc of the invention or the pharmaceutical composition of the invention for use in drug administration.

In another aspect, the present invention relates to the lipid nanodisc of the invention or the pharmaceutical composition of the invention for use in the treatment of a cancer characterized in that it contains cells expressing on their surface the protein to which the scFab forming part of the fusion protein specifically binds or the protein to which the reconstituted antigen-binding site formed by the fragment of the immunoglobulin chain and a second immunoglobulin chain binds.

In another aspect, the present invention relates to a method to obtain the fusion protein of the invention, wherein the method comprises:

(i) growing a cell comprising the polynucleotide of the invention in conditions suitable for allowing the expression of the fusion protein from the polynucleotide; and

(ii) recovering the fusion protein from the culture.

In another aspect, the present invention relates to a method to obtain the lipid nanodisc of the invention, wherein the method comprises:

(i) providing the fusion protein of the invention; and

(ii) contacting the fusion protein with a membrane-forming lipid under conditions adequate to provide a lipid bilayer formed by the membrane forming lipid stabilized by the fusion protein.

In another aspect, the present invention relates to an in vitro method for the delivery of a compound of interest to a cell population or to a tissue comprising contacting the lipid nanodisc of the invention comprising the compound of interest with the cell population or tissue, wherein cells in the tissue or cells of the cell population express on their surface the protein to which the scFab forming part of the fusion protein specifically binds or the protein to which the reconstituted antigen-binding site formed by the fragment of the immunoglobulin chain and a second immunoglobulin chain.

DESCRIPTION OF THE FIGURES

Figure 1 . Diagrammatic representation of a Backpacked Antibody (B-ab) in comparison to conventional mAbs and ADCs. B) Diagrammatic representation of Fc homodimerization in the B-ab.

Figure 2. A) size exclusion chromatography profile of a properly assembled B-ab. Inset: Rhodamine spectrum of the main peak to follow lipid incorporation. B) SDS-PAGE of MSP constructs: MSP2N2, scFab-MSP2N2 and scFab-MSP2N2-Fc. C) Hydrodynamic radius of MSP2N2 nanodiscs and B-ab in comparison with natural IgG and IgM molecules. D) Negative stain electron micrograph of B-abs. E) Thermostability profile of scFab-MSP2N2 and scFab-MSP2N2-Fc and the corresponding IgG. Black arrows indicate the melting temperature (T_m) observed in the different samples.

Figure 3. A) Concentration-response curves for the binding of B-Ab to CD19, the target/epitope of denintuzumab, included as Fab in the B-ab. The parental IgG is added for comparison. B) SDS-PAGE of the B-Ab under reducing and no reducing conditions. C) Concentration-response curves for the binding of B-Ab containing wild-type I gG 1 Fc or the Fc lgG1 Leu234Ala, Leu235Ala (LALA) mutations to the human FcyR.

Figure 4. Stability of the B-Ab to undergo a lyophilization process. Side-by-side comparison of a three-month lyophilized B-Ab and a B-Ab stored at -80 measured by SDS-PAGE (A), DLS (B) and ELISA (C).

Figure 5. A) Binding and internalization (arrows) of B-Abs containing denintuzumab Fabs, MSP2N2 nanodiscs and parental IgGs to CD19+ Daudi cells and CD19- Jurkat cells. B) 2 h incubation of B-Abs containing rituximab Fabs and parental IgGs to CD20+ Daudi cells. Scale bar= 10 pm.

Figure 6. Cell binding and internalization of B-Abs containing denintuzumab and rituximab Fabs double labeled with AF488 thiol reactive dyes to label the Fc fragment and SR-DPPE to label the lipid compartment. Scale bar= 10 pm.

Figure 7. Colocalization of internalized B-Abs and IgGs with lysosomes labeled with LysoTracker DND-26 Green. Scale bar= 10 pm.

Figure 8. Loading of the B-Abs with the cytotoxic drug MMAE: A) Chemical conjugation of the MMAE drug to the head group of lipid 16:0 Ptd Thioethanol. B) Mass spectrometry calibration curves of 16:0_Ptd_thioEtOH_MMAE_conjugate (matrix spiked, 5nM to 5000pM). Retention times are shown above each detected peak. Intensity values of each peak are represented on the calibration curve in the inset. C) Quantification of the amount of lipid-drug conjugate incorporated into the B-Abs upon folding. The relative amount of the conjugate can be inferred from the intensity of the detected peak. The inset provides evidence of the conjugate's identity (theoretical molecular weight of 1619.0543).

Figure 9. Cytotoxicity of human cancer cells lines Daudi and Raji induced by the B-Ab containing polatuzumab Fabs loaded with MMAE-Ptd (40% of the lipid content).

Figure 10. Evaluation of MMAE-loaded ADC and B-Abs on Daudi B cell division capacity. A) Quantification of the DNA content of Daudi tumor cells in the presence (right) or absence (left) of 0.1 pM of the MMAE drug. B) Comparison of the effect on the cell division capacity of Daudi B cells after incubation with different concentrations of ADC and B-Abs loaded with MMAE. Antibodies without the drug and non-functionalized nanodiscs loaded with the same amount of drug at 20 pg/mL were used as controls in the assay.

Figure 11. Ability of the platform to swap the different components of the B-Ab: the MSP rendering molecules of different sizes (A) and the Fab specificity allowing recognition of different targets (binding at 1.5 pg/mL) (B).

Figure 12. Cell binding and internalization comparison between conventional IgGs and corresponding B-Abs of different specificities. SR thiol reactive dye was used to label both type of molecules. Scale bar= 10 pm.

Figure 13. Plasticity of the platform to combine multiple antibody specificities in tandem. Schematic representation (A) and functional assembly (B) of a bispecific B-Ab.

Figure 14. Bioavailability of the B-Abs. A) Concentration-response curves for pH- dependent binding to human FcRn by B-Ab, and corresponding IgG (at endosomal pH (top) and physiological pH (bottom)). Gray lines represent raw data and black lines represent global fit. B) FcRn binding affinity constant (KD) to a B-Ab, and the corresponding IgG at pH 5.6. The values for two independent measurements are shown. C) Five SCID mice per group were used to assess the serum concentration of a nonfunctionalized nanodisc (NanoD), a B-Ab (Fc-modified, LALAP mutation), and the parental IgG after intraperitoneal (IP) administration of 5 mg/kg.

Figure 15. Bioavailability of loaded B-Abs. Comparison of the serum concentration of A) IgG and the corresponding ADC loaded with the cytotoxic drug MMAE (DAR 3-6), B) B- Ab (Fc-modified, LALAP mutation) empty and loaded with MMAE through covalent conjugation to the lipids (DAR of 160), and C) ADC and loaded B-Ab in A and B.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have engineered an IgG scaffold to generate antibody-like particles that incorporate a lipid cage mimicking a “backpack” for loading and delivery of therapeutically effective concentrations of cytotoxic drugs to tumor cells. These backpacked antibodies incorporate three key elements: a lipid nanocage to adsorb a high number of drugs with poor solubility and high cytotoxicity, Fab fragments for the recognition of tumor antigens and to trigger drug internalization and an Fc domain to confer long serum circulation time.

Fusion protein of the invention

Thus, in a first aspect, the invention relates to a fusion protein, hereinafter “the fusion protein of the invention” comprising:

(ii) a membrane scaffold protein (MSP); and

(iii) a crystallisable domain fragment (Fc).

The term “fusion protein” is well known in the art, referring to a single polypeptide chain artificially designed which comprises two or more sequences from different origins, natural and/or artificial. The fusion protein, per definition, is never found in nature as such.

The term “fused to”, as used herein, and interchangeably used herein as “connected to”, “conjugated to”, “ligated to” refers, in particular, to “genetic fusion”, e.g., by recombinant DNA technology, as well as to “chemical and/or enzymatic conjugation” resulting in a stable covalent link.

The fusion protein of the invention comprises a single-chain binding fragment or a fragment of an immunoglobulin chain containing a variable region.

The term “antigen-binding fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Such "fragments" are, for example between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably about 8 to about 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. The antigen binding fragment contain one constant and one variable domain of each of the heavy and the light chains. Only the variable regions of the heavy and light chains are fused together to form a single-chain variable fragment (scFv), which is half the size of the Fab fragment. The term “Fab” refers to the VH and CH1 domain of the heavy chain and the VL and CL domain of the light chain, i.e. a single chain antigen binding fragment is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1-linker- VL-CL, b) VL-CL-linker-VH-CH1 , c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL, and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CH1 domain.

The term “antigen-binding fragment” also refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Such "fragments" are, for example between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably about 8 to about 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. The antigen binding fragment contain one constant and one variable domain of each of the heavy and the light chains. Only the variable regions of the heavy and light chains are fused together to form a single-chain variable fragment (scFv), which is half the size of the Fab fragment. The term “Fab” refers to the VH and CH1 domain of the heavy chain and the VL and CL domain of the light chain, i.e. a single chain antigen binding fragment is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1-linker- VL-CL, b) VL-CL-linker-VH-CH1 , c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL, and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CH1 domain. As mentioned above, the fusion protein of the invention comprises a scFab or a fragment of an immunoglobulin chain containing a variable region. If the fusion protein of the invention comprises a fragment of an immunoglobulin chain containing a variable region, then the variable region can be a heavy chain ora light chain. In a particular embodiment, the fragment of an immunoglobulin containing a variable region is a heavy chain. In another particular embodiment, the fragment of an immunoglobulin containing a variable region is a light chain. Such "fragment" is, for example between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably about 8 to about 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length.

In another particular embodiment, the antigen binding fragment include an Fv fragment consisting of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv).

The fusion protein of the invention comprises a membrane scaffold protein (MSP). The term membrane scaffold protein (MSP) refers to any amphipathic protein that is capable of self-assembly with an amphipathic lipid in an aqueous environment, organizing the amphipathic lipid into a bilayer, and comprise apolipoproteins, lipophorins, derivatives thereof (such as truncated and tandemly arrayed sequences) and fragments thereof (e.g. peptides) which maintains the amphipathic nature and capability of self-assembly. In general scaffold protein have an alpha helical secondary structure in which a plurality of hydrophobic amino acids form an hydrophobic face and a plurality of hydrophilic amino acids form an opposing hydrophilic face.

The term “apolipoprotein” as used herein indicates an amphipathic protein that binds lipids to form lipoproteins. The term “amphipathic” pertains to a molecule containing both hydrophilic and hydrophobic properties. Exemplary amphipathic molecules comprise, a molecule having hydrophobic and hydrophilic regions/portions in its structure. Examples of biomolecules which are amphipathic include but not limited to phospholipids, cholesterol, glycolipids, fatty acids, bile acids, saponins, and additional lipids identifiable by a skilled person. A “lipoprotein” as used herein indicates a biomolecule assembly that contains both proteins and lipids. In particular, in lipoproteins, the protein component surrounds or solubilizes the lipid molecules enabling particle formation. Exemplary lipoproteins include the plasma lipoprotein particles classified under high-density (HDL) and low-density (LDL) lipoproteins, which enable fats to be carried in the blood stream, the transmembrane proteins of the mitochondrion and the chloroplast, and bacterial lipoproteins. In particular, the lipid components of lipoproteins are insoluble in water, but because of their amphipathic properties, apolipoproteins such as certain Apolipoproteins A and Apolipoproteins B and other amphipathic protein molecules can surround the lipids, creating the lipoprotein particle that is itself water-soluble, and can thus be carried through water-based circulation (e.g. blood, lymph in vivo or in vitro). Apolipoproteins known to provide the protein components of the lipoproteins can be divided into six classes and several sub-classes, based on the different structures and functions. Exemplary apolipoprotein known to be able to form lipoproteins comprise Apolipoproteins A (apo A-l, apo A-l I , apo A-IV, and apo A-V), Apolipoproteins B (apo B48 and apo B100), Apolipoproteins C (apo C-1 , apo C-ll, apo C-lll and apo C-IV), Apolipoproteins D, Apolipoproteins E (ApoE2, ApoE3 and ApoE4), Apolipoproteins F and Apolipoproteins H.

The MSPs of the present invention must be amphipathic, with one part of its structure more or less hydrophilic and facing the aqueous solvent and another part more or less hydrophobic and facing the center of the hydrophobic bilayer that is to be stabilized. Examination of the basic biochemical literature reveals two candidate protein structures that can have this required amphipathic character: the helix and the pleated sheet. Each MSP has an amino acid sequence which forms amphipathic helices with more hydrophobic residues (such as A, C, F, G, I, L, M, V, W or Y) predominantly on one face of the helix and more polar or charged residues (such as D, E, N, Q, S, T, H, K or R) on the other face of the helix. In addition, the helical structure is punctuated with residues such as proline (P) or glycine (G) periodically, which can introduce flexibility into the overall structure by interrupting the general topology of the helix. In one embodiment, these punctuations occur about every 20-25 amino acids to form “kinks” or to initiate turns to facilitate the “wrapping” of the MSP around the edge of a discoidal phospholipid bilayer.

In an alternative embodiment, the engineered amphiphilic MSP contain regions of secondary structure such as parallel or antiparallel pleated sheets, with spacer regions of appropriate length to allow association of hydrophobic regions with a hydrophobic target molecule which is protected from the aqueous milieu, and thus stabilized and solubilized. The fusion protein of the invention comprises a membrane scaffold protein (MSP). In a particular embodiment, the MSP is selected from the group consisting of: Apo Al, Apo E, MSP1 D1 , MSP1 D1 D73C, MSP1 D1 (-), MSP1 E1 D1 , MSP1 E1 D1 D73C, MSP1 E2D1 , MSP1 E2D1 D73C, MSP1 E3D1 , MSP1 E3D1 D73C, MSP1 FC, MSP1 FN, MSP2N2, MSP1 D1 biotin labelled or MSP1 D1 DHS.

In a more particular embodiment, the membrane scaffold protein of the fusion protein of the invention is the membrane scaffold protein 2N2 (MSP2N2).

In some embodiments, the MSP sequence is selected from the group consisting of: SEQ ID NO: 1 (MSP1 E1), SEQ ID NO: 2 (MSP1 E2), SEQ ID NO: 3 (MSP1 E3), SEQ ID NO: 4 (MSP1 D1), SEQ ID NO: 5 (MSP1 E3D1), SEQ ID NO: 6 (MSP2N2), SEQ ID NO: 7 (SAPOSIN-A), SEQ ID NO: 8 (MSP1 FN), SEQ ID NO: 9 (MSP1 E1 D1), SEQ ID NO: 10 (ApoA1), SEQ ID NO: 11 (ApoE) or a sequence with at least 70% identity thereto. In some embodiments, the MSP comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.

The fusion protein of the invention comprises an immunoglobulin Fc fragment. An "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, an Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). An immunoglobulin Fc domain may include, e.g., immunoglobulin CH2 and CH3 domains. An immunoglobulin Fc domain may include, e.g., immunoglobulin CH2 and CH3 domains and an immunoglobulin hinge region. For example, as used herein, an “Fc” or “Fc domain” can refer to a polypeptide comprising a CH2 domain, a CH3 domain, and optionally a hinge or a portion thereof. This polypeptide can bind (e.g., dimerize) to another polypeptide comprising a CH2 domain, a CH3 domain, and optionally a hinge or a portion thereof, wherein the dimer is capable of binding to an Fc receptor. The Fc can be provided in the fusion protein as a scFc, comprising two Fc chains linked through a flexible linker. Boundaries between immunoglobulin hinge regions, CH2, and CH3 domains are well known in the art, and can be found, e.g., in the PROSITE database (available on the World Wide Web at prosite.expasy.org).

In IgG, IgA and IgD antibody isotypes, the Fc region comprises two identical protein fragments, derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position C226 or P230 (or amino acid between these two amino acids) to the carboxy-terminus of the heavy chain, wherein the numbering is according to the Ell index as in Kabat. The CH2 domain of a human IgG Fc region extends from about amino acid 231 to about amino acid 340, whereas the CH3 domain is positioned on C-terminal side of a CH2 domain in an Fc region, i.e. , it extends from about amino acid 341 to about amino acid 447 of an IgG.

In a particular embodiment, the Fc region may be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc). A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human lgG1 Fc region; native sequence human lgG2 Fc region; native sequence human lgG3 Fc region; and native sequence human lgG4 Fc region as well as naturally occurring variants thereof.

A "hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to the domain of a heavy chain constant region that joins the CH1 domain to the CH2 domain and includes the upper, middle, and lower portions of the hinge (Roux et al. J. Immunol. 1998 161 :4083). The hinge provides varying levels of flexibility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions.

The term "CH1 domain" refers to the heavy chain constant region linking the variable domain to the hinge in a heavy chain constant domain. The term "CH1 domain" includes wildtype CH1 domains as well as variants thereof (e.g., non-naturally-occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes wildtype CH1 domains and variants having 1 , 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions. Exemplary CH1 domains include CH1 domains with mutations that modify a biological activity of an antibody, such as ADCC, CDC or half-life.

The term "CH2 domain" refers to the heavy chain constant region linking the hinge to the CH3 domain in a heavy chain constant domain. The term "CH2 domain" includes wildtype CH2 domains, as well as variants thereof (e.g., non-naturally-occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wildtype CH2 domains and variants having 1 , 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions. Exemplary CH2 domains include CH2 domains with mutations that modify a biological activity of an antibody, such as ADCC, CDC or half-life.

The term "CH3 domain" refers to the heavy chain constant region that is C-terminal to the CH2 domain in a heavy chain constant domain. The term "CH3 domain" includes wildtype CH3 domains, as well as variants thereof (e.g., non-naturally-occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wildtype CH3 domains and variants having 1 , 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions. Exemplary CH3 domains include CH3 domains with mutations that modify a biological activity of an antibody, such as ADCC, CDC or half-life.

Exemplary Fc domains include Fc domains with mutations that promote the heterodimerization such as but not limited to: the knobs-into-hole (KiH) (T369W, T366S/L368A/Y407V), electrostatic steering (E356K/D399K, K392D/K409D) or the Zymeworks mutations (T350V/L351Y/F405A/Y407V, T350V/T366L/K392L/T394W).

In certain embodiments, the immunoglobulin Fc domain is derived from a human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, and IgM Fc domain.

The fusion protein of the invention comprises an Fc domain. In an embodiment, the Fc domain has the same sequence or 99 percent or greater sequence similarity with a human lgG1 Fc domain. In an embodiment, the Fc domain has the same sequence or 99 percent or greater sequence similarity with a human lgG2 Fc domain. In an embodiment, the Fc domain has the same sequence or 99 percent or greater sequence similarity with a human lgG3 Fc domain. In an embodiment, the Fc domain has the same sequence or 99 percent or greater sequence similarity with a human lgG4 Fc domain. In some embodiments, the Fc domain is selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or a sequence with at least 70% identity thereto. In some embodiments, the Fc domain comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identical to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18.

In addition, it is understood that the immunoglobulin Fc domain can be modified to prevent the glycosylation of the Fc domain. For example, in certain constructs, the immunoglobulin Fc domain is derived from a human IgGI Fc domain and comprises a mutation to prevent glycosylation, for example, a mutation at position N297, for example, an N297A or N297G mutation (residue numbers according to Ell numbering, Kabat, et al., E.A., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242).

In a particular embodiment, the single-chain antigen-binding fragment (scFab) is N- terminal to the MSP.

In another particular embodiment, the immunoglobulin Fc fragment is C-terminal to the MSP.

In another particular embodiment, the single-chain antigen-binding fragment comprises a light chain (LC) and a heavy chain (HC) and the LC and the HC are connected by a linker sequence.

In another particular embodiment, the single-chain antigen-binding fragment is a VHH.

The term “VHH” refers to a single-chain antigen binding fragment which consist solely of a variable domain (VHH) of the heavy chain of an antibody. The VHH domains have four framework regions (FR) and three hypervariable regions (HV) or CDR complementarity determining regions. VHH has four amino acid substitutions in the FR2 region, at positions 37, 44, 45, and 47. FR2 of conventional VH does not have these substitutions and is involved in the formation of the hydrophobic interface with the VL domain. Conventional VH has a highly conserved hydrophobic tryptophan residue at position 103 that interacts with the VL, however, VHH usually has a hydrophilic arginine residue, which is determinant in its single domain nature.

In another particular embodiment, the single-chain antigen binding fragment is VNAR. The term “VNAR” refers to the variable domain of the immunoglobulin new antigen receptors (IgNARs), which is a class of natural heavy-chain only antibody. IgNAR is a unique and unconventional antibody which has been identified in several different types of shark. Each chain in IgNAR consists of five constant domains followed by one variable domain. The variable domain of IgNAR, or referred to as VNAR, contains only two complementarity-determining regions (CDRs), as known as CDR1 and CDR3. Unlike conventional IgG, shark VNAR has been undergone evolution by compensating CDR2.

As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences and corresponds to an antibody region having a structure that is complimentary to its target antigen or epitope. Other portions of variable domains, not interacting with antigen, are referred to as "framework regions" (FRs). A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, which are known as "framework regions" ("FR"). VH and VL domains have four framework regions (FRs) each positioned before, after, and between CDR regions. VH framework regions are referred to herein as FRH1 , FRH2, FRH3, and FRH4 and VL framework regions are referred to herein as FRL1 , FRL2, FRL3, and FRL4. FRs and CDRs of VH domains are typically in the order of FRH1-CDRH1-FRH2-CDRH2- FRH3- CDRH3-FRH4, from N- to C-terminus. FRs and CDRs of VL domains are typically in the order of FRL 1 -CDRL 1 -FRL2-CDRL2-FRL3 -CDRL3 -FRL4, from N- to C-terminus. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and/or (e.g., and) the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242; IMGT(R), the international ImMunoGeneTics information system(R) www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209- 212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res., 31 :307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol.196:901-917, Al-lazikani et al (1997) J. Molec. Biol.273:927-948; and Almagro, J. Mol. Recognit.17: 132-143 (2004). See also bioinf.org.uk/abs. As used herein, a CDR may refer to the CDR defined by any method known in the art.

In another particular embodiment, the single-chain antigen-binding fragment or the fragment of the immunoglobulin chain is connected to the MSP by a linker sequence.

In another particular embodiment, the immunoglobulin Fc fragment is connected to the MSP by a linker sequence.

The term “linker” means a suitable peptide that allows for two or more functional domains joined together in a fusion protein. Linkers can be flexible or rigid linkers. In a preferred embodiment the linker is a flexible linker. “Flexible linker” as it is used herein means that the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavourable interaction between the linker and the protein moieties.

In certain embodiment, the linker comprises from about 1 to about 100 amino acid residues, e.g., about 1 to about 70, about 2 to about 70, about 1 to about 30, or about 2 to about 30 amino acid residues. In some embodiments, the linker comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid residues.

Exemplary linkers include glycine and serine-rich linkers, e. g. a (G_nS)_m sequence (e.g., GGS, GGGS (SEQ ID NO: 19), or GGGGS (SEQ ID NO: 20) sequence) that is present in at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, or at least 14 copies within the linker. The single-chain antigen-binding fragment (scFab) or the fragment of an immunoglobulin chain may derive from different antibodies. In a particular embodiment, the single-chain antigen-binding fragment (scFab) or the fragment of an immunoglobulin chain are derived from denintuzumab, rituximab, pinatuzumab, polatuzumab, epratuzumab trastuzumab oratezolizumab. In a more particular embodiment, the single-chain antigenbinding fragment (scFab) or the fragment of an immunoglobulin chain derive from denintuzumab or polatuzumab.

In another particular embodiment, the single-chain antigen-binding fragment comprises more than one antigen-binding region.

In another particular embodiment, the fusion protein of the invention further comprises a tag. As it is used herein, the term “tag” means a polypeptide useful for making the detection, isolation and/or purification of a protein easier. Generally, said labelling sequence is located in a part of the protein of interest that does not adversely affect the functionality thereof. In a more particular embodiment, the detection tag is selected from the group consisting of: His tag, Strep tag, Avi tag, Ha tag and myc tag.

Polynucleotide of the invention

The terms “nucleic acid”, “polynucleotide” and “nucleotide sequence”, as used interchangeably herein, relate to any polymeric form of nucleotides of any length and composed of ribonucleotides or deoxyribonucleotides. The terms include both singlestranded and double-stranded polynucleotides, as well as modified polynucleotides (e.g., methylated, protected). Typically, the nucleic acid is a “coding sequence” which, as used herein, refers to a DNA sequence that is transcribed and translated into a polypeptide in a host cell when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence. It will be understood that, in order for the polynucleotide of the invention to be expressed in the host of cell of interest, the polynucleotide can be provided in an operably linked manner with a regulatory region. The person skilled in the art will understand that suitable regulatory regions can be used based on the host cell in which the polynucleotide can be expressed. The nature of the regulatory region is not particularly limitative in the present invention.

Vector of the invention

In another aspect, the present invention relates to a vector, hereinafter “the vector of the invention” comprising the polynucleotide of the invention. As it is used herein, the term “vector” or “expression vector” refers to a replicative DNA construct used for expressing the polynucleotide of the invention in a cell. The choice of expression vector will depend upon the choice of host. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Esherichia coli, including pCR 1 , pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and filamentous single-stranded DNA phages.

Host cell of the invention

The polynucleotide and vector according to the invention may be provided within host cells. In another aspect, the present invention relates to a host cell, hereinafter “the host cell of the invention”, comprising the polynucleotide of the invention encoding the fusion protein of the invention, as well as a host cell comprising the vector of the invention comprising a polynucleotide as described above.

The term “host cell”, as used herein, refers to a cell into which a nucleic acid of the invention, such as a polynucleotide or a vector according to the invention, has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The term “host cell” includes stable cell lines. Once the polynucleotide has been introduced into the host cell, i.e. after transfection, the cells begin to express the fusion protein for a transient period before ceasing production altogether. However, a small subpopulation maintains its ability to express the fusion protein for long periods of time due to the integration of the foreign DNA into its genome. These cells are known as stable transfected cells.

According to the invention, the host cell can be a bacterium, wherein said bacterium can be a gram-negative bacterial cell, this term intended to include all facultative anaerobic Gram-negative cells of the family Enterobacteriaceace such as Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafhia, Edwardsiella, Providencia, Proteus and Yersinia. In another particular embodiment, the bacterium is a gram-positive bacterial cell of the genus Mycobacteriaceae such as M. bovis-BCG, M. leprae, M. marinum, M. smegmatis and M. tuberculosis.

In another particular embodiment, the host cell can be a human derived cell line such as but not limited to HEK 293F and expiCHO cells.

Lipid nanodisc of the invention

In another aspect, the present invention relates to lipid nanodisc, hereinafter the lipid nanodisc of the invention, characterized in that it comprises multiple copies of the fusion protein of the invention and a membrane-forming lipid.

‘Nanodiscs’ are described herein, wherein the belt contains amphipathic proteins or amphipathic peptides, preferably at each hydrophobic edge, aligning in a double belt. Nanodiscs are structurally very similar to discoidal high-density lipoproteins (HDL), and their belt proteins involve helix-rich membrane scaffold proteins (MSPs), which are known in the art and concern artificially designed proteins comprising truncated forms of apolipoprotein (apo) A-l, wherein several helix elements are repeated or shuffled or engineered further, as to create diverse options for wrapping around the patch of a lipid bilayer to form a disc-like particle.

Nanodiscs are widely applied to reconstitute (detergent-)solubilized membrane proteins in an artificial environment resembling the native membrane, thereby stabilizing membrane proteins to study binding of ligands, agonists or antagonists. Additional apolipoprotein-based nanoparticle systems with varying diameters of the nanodiscs, depending on the MSP variant used to constitute the nanodics, have been developed and are included herein as nanodisc systems.

The lipid nanodisc of the invention comprises multiple copies of the fusion protein of the invention. In a particular embodiment, the lipid nanodisc comprises at least 2 copies of the fusion protein of the invention. In another particular embodiment, the lipid nanodisc comprises between 2 and 4 copies of the fusion protein of the invention.

In a particular embodiment, if the component (i) of the fusion protein of the invention is a fragment of an immunoglobulin chain, then the lipid nanodisc further comprises a second fragment of an immunoglobulin chain containing its variable region, wherein said second fragment is associated with the fragment of the immunoglobulin chain of component (i) so that the antigen binding site is reconstituted by the variable regions of the immunoglobulin chain fragment of component (i) and the second fragment of an immunoglobulin chain.

In another particular embodiment, the component (i) is a fragment of an immunoglobulin light chain and the second fragment of the immunoglobulin chain is from an immunoglobulin heavy chain or wherein component (i) is a fragment of an immunoglobulin heavy chain and the second fragment of the immunoglobulin chain is from an immunoglobulin light chain.

The term “membrane-forming lipid” as used herein refers to a group of compounds (structurally similar to fats and oils) which form the double-layered surface of all cells (lipid bilayer). The three major classes of membrane lipids are phospholipids, glycolipids, and cholesterol.

In a particular embodiment, the membrane-forming lipid is selected from the group consisting of phospholipids, sphingolipids, glycolipids, sterols and alkylphosphocholines.

In a more particular embodiment, the membrane-forming lipid is selected from the group consisting of: phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylserine (PS), dimyristoylphosphatidylcholine (DMPC) dioleoylphosphoethanolamine (DOPE) dioleoylphosphatidylcholine (DOPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3- phospho-L-serine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dipalmitoylphosphatidylcholine (DPPC), cholesterol (CHOL), sphingomyelin, Ptd thioethanol (1 ,2-Dipalmitoyl-sn- Glycero-3-Phosphothioethanol), Ptd ethylene Glycol (2-dipalmitoyl-sn-glycero-3- phospho(ethyleneglycol), 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1 ,2- dioleyloxy-3-dimethylaminopropane (DODMA), 1 ,2-dioleoyl-3-dimethylammonium- propane (DODAP), D-Lin-MC3-DMA and D-Lin-KC2-DMA.

In another particular embodiment, the lipid nanodisc comprises a molar ratio protein: lipid of 1 :500, preferably 1 :300, more preferably 1 :200. In some embodiments, the lipid nanodisc comprises a molar ratio protein: lipid of 1 : 150, 1 : 100, 1 :50 or 1 :20. The molar ratio protein:lipid will vary depending of the MSP that is comprised in the fusion protein of the invention, i.e., the higher the MSP, the more lipids can be used.

In another particular embodiment, the lipid nanodisc of the invention further comprises a detection tag. In a more particular embodiment, the detection tag is intercalated in the lipid fraction.

In a particular embodiment, the detection tag is a fluorescent lipid. In a more particular embodiment, the detection tag is selected from the group consisting of: Rho-PE, NBD- PE, StarRed-PE, Fluorescein-PE, Cy5-PE, Cy5.5-PE, Cy7-PE, TopFluor-PE, TopFluor- PC, ATTOO647N-SM, TF-CHOL.

In another particular embodiment, the detection tag is adsorbed to the acyl chain of the lipids without the need to be conjugated. In a more particular embodiment, the detection tag is selected from the group consisting of: DiD, DiA, Dil, DiO, Laurdan, BODIPY.

In another particular embodiment, the lipid nanodisc of the invention further comprises one or a combination of compounds of interest.

In a more particular embodiment, the compounds of interest is selected from the group consisting of: cytotoxic agent such as MMAE, MMAF, DM1 , Doxorubicin, SN-38, Docetaxel, anticancer agent, siRNA or shRNA, anti-sense oligonucleotides (ASO), microARNs, TLR-agonists such as E104, cytotoxic peptides such as melittin, Magainin 2, Cathelicidins LL37 hCAP18 or Cecropin B. In some embodiments, the lipid nanodisc of the invention comprises a combination of compounds of interest. In some embodiments, the lipid nanodisc comprises two lipid- drug conjugates of the same type, e.g. cytotoxic or with different anti-tumour mechanism. For example one cytotoxic and one immune co-stimulatory (TLR7-stimulating E104). In other embodiment, the lipid nanodisc of the invention comprises cytotoxic drugs in combination with siRNAs to reach synergic effects for combating tumour heterogenicity.

In some embodiments, the anticancer compound of interest is adsorbed to the acyl chain of the lipids without the need to be conjugated. In other embodiments, the compound of interest is conjugated by covalent bonding with the polar heads of the nanodisc. In other embodiments, the compound of interest is conjugated by a linker that is sensitive to degradation by proteases present in the tumour environment, in lysosomes or pH- sensitive. If the linker between the agent and the lipid is degradable in the tumour microenvironment, the drug could enter neighbouring cells.

In a particular embodiment, the compound of interest is a cytotoxic agent. Cytotoxicity is the quality of being toxic to cells. Cytotoxicity assays are known by an expert in the art and are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components. Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components. Cytotoxicity can also be monitored using the 3-(4, 5-Dimethyl- 2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) or with 2,3-bis-(2-methoxy-4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), which yields a water-soluble product, or the MTS assay. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. Cytotoxicity can also be measured by the sulforhodamine B (SRB) assay, WST assay and clonogenic assay. In another particular embodiment, the compound of interest is an anticancer agent.

The term “anticancer agent” refers to an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term.

Several anti-cancer agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), anti-microtubule agents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), anthracyclines, vinca alkaloids, or epipodophyllotoxins.

Additional examples of anti-cancer agents include without limitation Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Bortezomib (VELCADE); Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin (a platinum- containing regimen); Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin (a platinum-containing regimen); Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquone; Docetaxel (TAXOTERE); Doxorubicin; Droloxifene; Dromostanolone; Duazomycin; Edatrexate; Eflornithine; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin; Erbulozole; Erlotinib (TARCEVA), Esorubicin; Estramustine; Etanidazole; Etoposide; Etoprine; Fadrozole; Fazarabine; Fenretinide; Floxuridine; Fludarabine; 5-Fluorouracil; Flurocitabine; Fosquidone; Fostriecin; Gefitinib (IRESSA), Gemcitabine; Hydroxyurea; Idarubicin; Ifosfamide; llmofosine; Imatinib mesylate (GLEEVAC); Interferon alpha-2a; Interferon alpha-2b; Interferon alpha-nl; Interferon alpha-n3; Interferon beta-l a; Interferon gamma-l b; Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLLMID, REVIMID); Letrozole; Leuprolide; Liarozole; Lometrexol; Lomustine; Losoxantrone; Masoprocol; Maytansine; Mechlorethamine; Megestrol; Melengestrol; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed (ALIMTA), Pegaspargase; Peliomycin; Pentamustine; Pentomone; Peplomycin; Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate; Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin; Prednimustine; Procarbazine; Puromycin; Pyrazofurin; Riboprine; Rogletimide; Safingol; Semustine; Simtrazene; Sitogluside; Sparfosate; Sparsomycin; Spirogermanium; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tamsulosin; Taxol; Taxotere; Tecogalan; Tegafur; Teloxantrone; Temoporfin; Temozolomide (TEMODAR); Teniposide; Teroxirone; Testolactone; Thalidomide (THALOMID) and derivatives thereof; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan; Toremifene; Trestolone; Triciribine; Trimetrexate; Triptorelin; Tubulozole; Uracil; Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine; Vincristine; Vindesine; Vinepidine; Vinglycinate; Vinleurosine; Vinorelbine; Vinrosidine; Vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin.

The anti-cancer agent may be an enzyme inhibitor including without limitation tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor. The tyrosine kinase inhibitor may be without limitation Genistein (4', 5, 7- trihydroxyisoflavone), Tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]- propanedinitrile, Herbimycin A, Daidzein (4',7-dihydroxyisoflavone), AG-126, trans-1- (3'- carboxy-4'-hydroxyphenyl)-2-(2",5"-dihydroxy-phenyl)ethane, or HDBA (2- Hydroxy5- (2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid. The CDK inhibitor may be without limitation p21 , p27, p57, pl5, pl6, pl8, or pl9. The MAP kinase inhibitor may be without limitation KY12420 (C23H24O8), CNI-1493, PD98059, or 4-(4- Fluorophenyl)-2-(4- methylsulfinyl phenyl)-5-(4-pyridyl) IH-imidazole. The EGFR inhibitor may be without limitation erlotinib (TARCEVA), gefitinib (IRESSA), WHI- P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.

The anti-cancer agent may be a VEGF inhibitor including without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin. The anti-cancer agent may be an antibody or an antibody fragment including without limitation an antibody or an antibody fragment including but not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER-2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma Rl)), oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory conditions such as RSV infection), ibritumomab tiuxetan (ZEVALIN, indicated for Non-Hodgkin's lymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72), I0R- C5, 10R-T6 (anti-CD 1), IOR EGF/R3, celogovab (ONCOSCINT OV 103), epratuzumab (LYMPHOCIDE), pemtumomab (THERAGYN), and Gliomab-H (indicated for brain cancer, melanoma).

It is contemplated that in certain embodiments of the invention a protein that acts as an angiogenesis inhibitor is targeted to a tumor. These agents include, in addition to the anti-angiogenic polypeptides mentioned above, Marimastat; AG3340; COL-3, BMS- 275291 , Thalidomide, Endostatin, SU5416, SU6668, EMD121974, 2-methoxyoestradiol, carboxiamidotriazole, CMIOI, pentosan polysulphate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM- 1470, platelet factor 4 or minocycline.

Other suitable active agents are DNA cleaving agents. Examples of DNA cleaving agents suitable for inclusion as the cell toxin in the conjugates used in practicing the methods include, but are not limited to, anthraquinone-oligopyrrol-carboxamide, benzimidazole, leinamycin; dynemycin A; enediyne; as well as biologically active analogs or derivatives thereof (i.e., those having a substantially equivalent biological activity). Known analogs and derivatives are disclosed, for examples in Islam et al., J. Med. Chem. 34 2954-61 , 1991 ; Skibo et al., J. Med. Chem. 37:78-92, 1994; Behroozi et al., Biochemistry 35:1568- 74, 1996; Helissey et al., Anticancer Drug Res. 11 :527-51 , 1996; Unno et al., Chem. Pharm. Bull. 45:125-33, 1997; Unno et al., Bioorg. Med. Chem., 5:903-19, 1997; Unno et al., Bioorg. Med. Chem., 5: 883-901 , 1997; and Xu et al., Biochemistry 37:1890-7, 1998). Other examples include, but are not limited to, endiyne quinone imines (U.S. Pat. No. 5,622,958); 2,2r-bis (2-aminoethyl)-4-4'-bithiazole [Lee et al., Biochem. Mol. Biol. Int. 40:151-7, 1996]; epilliticine-salen. copper conjugates [Routier et al., Bioconjug. Chem., 8: 789-92, 1997],

Some of the aforementioned chemotherapy agents can be grouped together under a common category as antimetabolites. “Antimetabolite” as used herein, refers to the compounds which inhibit the use of a metabolite that is part of normal metabolism. Antimetabolites are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid. Non-limiting examples of antimetabolites include the following compounds: bleomycin, busulfan, capecitabine, carmustine, carboplatin, chlorodeoxyadenosine, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate mitomycin, mitoxantrone, oxaliplatin, paclitaxel, procarbazine, SN-38, thioguanine, thiotepa, teniposide vinblastine, vincristine, and vinorelbine.

In a particular embodiment, the cytotoxic agent is monomethyl auristatin E (MMAE).

In another more particular embodiment, the compound of interest is a RNA inhibitor, such as small interfering RNA (siRNA) or a short hairpin RNA or a small hairpin RNA (shRNA).

Small interference RNA or siRNA are agents which are capable of inhibiting the expression of a target gene by means of RNA interference. A siRNA can be chemically synthesized, can be obtained by means of in vitro transcription or can be synthesized in vivo in the target cell. Typically, the siRNA consists of a double stranded RNA between 15 and 40 nucleotide long and may contain a 3' and/or 5' protruding region of 1 to 6 nucleotides. The length of the protruding region is independent of the total length of the siRNA molecule. The siRNA acts by means of degrading or silencing the target messenger after transcription. The siRNAs that can be used in the present invention has to be substantially homologous to the mRNA of the gene sequence which encodes a protein of interest. "Substantially homologous" is understood as having a sequence which is sufficiently complementary or similar to the target mRNA such that the siRNA is capable of degrading the latter through RNA interference. The siRNA suitable for causing said interference include siRNA formed by RNA, as well as siRNA containing different chemical modifications such as: (i) siRNA in which the bonds between the nucleotides are different than those appear in nature, such as phosphorothionate bonds; (ii) conjugates of the RNA strand with a functional reagent, such as a fluorophore; (iii) modifications of the ends of the RNA strands, particularly of the 3' end by means of the modification with different hydroxyl functional groups in 2' position; (iv) nucleotides with modified sugars such as 0-alkylated residues on 2' position like 2'-O-methylribose or 2'- O-fluororibose; or (v) nucleotides with modified bases such as halogenated bases (for example 5-bromouracil and 5-iodouracil), alkylated bases (for example 7- methylguanosine).

Vectors suitable for expressing siRNA are those in which the two DNA regions encoding the two strands of siRNA are arranged in tandem in one and the same DNA strand separated by a spacer region which, upon transcription, forms a loop and wherein a single promoter directs the transcription of the DNA molecule giving rise to the so-called short hairpin RNA (shRNA).

The siRNA and shRNA that can be used in the lipid nanodisc of the invention can be obtained using a series of techniques known by the person skilled in the art. The region of the nucleotide sequence taken as a basis for designing the siRNA is not limiting and it may contain a region of the coding sequence (between the start codon and the end codon) or it may alternatively contain sequences of the non-translated 5' or 3' region preferably between 25 and 50 nucleotides long and in any position in 3' direction position with respect to the start codon.

In another particular embodiment, the compound of interest is an antisense oligonucleotide (ASO). The antisense nucleic acids can be bound to the target protein by means of conventional base complementarity or, for example, in the case of binding to double stranded DNA through specific interaction in the large groove of the double helix. An antisense oligonucleotide can be distributed, for example, as an expression plasmid which, when it is transcribed in cell, produces RNA complementary to at least one unique part of the cellular mRNA encoding the protein of interest. Alternatively, the antisense oligonucleotide is an oligonucleotide probe generated ex vivo which, when introduced into the cell, produces inhibition of gene expression hybridizing with the mRNA and/or gene sequences of a target nucleic acid. Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, for example, exonucleases and/or endonucleases and are therefore stable in vivo. Examples of nucleic acid molecules for use thereof as antisense oligonucleotides are DNA analogs of phosphoramidate, phosphothionate and methylphosphonate. With respect to the antisense oligonucleotide, the oligodeoxyribonucleotide regions derived from the starting site of the translation, for example, between -10 and +10 of the target gene are preferred. The antisense approximations involve the oligonucleotide design (either DNA or RNA) that are complementary to the mRNA encoding the target polypeptide. The antisense oligonucleotide will be bound to the transcribed mRNA and translation will be prevented.

The oligonucleotides which are complementary to the 5' end of the mRNA, for example the non-translated 5' sequence up to and including the start codon AUG must function in the most efficient manner to inhibit translation. Nevertheless, it has been shown that the sequences complementary to the non-translated 3' sequences of the mRNA are also efficient for inhibiting mRNA translation.

The antisense oligonucleotides may comprise at least one modified base. The antisense oligonucleotide may also comprise at least a modified sugar group selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose. The antisense oligonucleotide may also contain a backbone similar to a neutral peptide. Such molecules are known as peptide nucleic acid (PNA) oligomers. In a particular embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone. In another embodiment, the antisense oligonucleotide is an alpha-anomeric oligonucleotide.

While antisense oligonucleotides complementary to the coding region of the target mRNA sequence can be used, those complementary to the transcribed non translated region can also be used.

In another particular embodiment, the compound of interest is a microRNA. MicroRNAs are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of microRNA action involves sequence specific hybridization of the microRNA molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of microRNA molecules preferably includes one or more sequences complementary to a target mRNA, and the well-known catalytic sequence responsible for mRNA cleavage or a functionally equivalent sequence.

In another particular embodiment, the lipid nanodisc has a hydrodynamic radius (Rh) between 2 nm and 20 nm, preferably between 5 and 20 nm. Pharmaceutical composition of the invention

In another aspect, the invention relates to a pharmaceutical composition, hereinafter the pharmaceutical composition of the invention, comprising the lipid nanodisc of the invention and at least one acceptable excipient.

The term "excipient" refers to a substance that aids the absorption of any of the components or compounds of the pharmaceutical composition of the invention, or stabilises the components or compounds and/or aids the preparation of the pharmaceutical composition in the sense of giving it consistency or flavours to make it more palatable. Thus, excipients may have the function, by way of example, but not limited to, binding the components (e.g. starches, sugars or cellulose), sweetening, colouring, protecting the active substance (e.g. to insulate it from air and/or moisture), filling a pill, capsule or any other presentation or a disintegrating function to facilitate the dissolution of the components, not excluding other excipients not listed in this paragraph. The term 'excipient' is therefore defined as a material which, included in the dosage forms, is added to the active substances or their associations to enable their preparation and stability, to modify their organoleptic properties or to determine the physical and chemical properties of the pharmaceutical composition and their bioavailability.

The expression “pharmaceutically acceptable excipient”, as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible with the lipid nanodisc of the invention.

The “dosage form” is the configuration to which the active ingredients and excipients are adapted to provide a pharmaceutical composition or medicinal product. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered.

The lipid nanodisc of the invention or the pharmaceutical composition of the invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (a) oral administration, such as drenches (aqueous or nonaqueous solutions or suspensions), tablets, boluses, powders, granules, pastes, mouthwash or hydrogels, (b) parenteral administration, for instance, by subcutaneous, intramuscular or intravenous injection of, for example, a sterile solution or suspension, (c) intracavity administration (e.g. intraperitoneal instillation), intravesical (i.e. urinary bladder) instillation, (d) intraorgan administration (e.g. intraprostatical administration), (e) topical application (i.e. cream, ointment or spray applied to the skin), (f) intravaginal or intrarectal administration (e.g. as a pessary, cream, foam, enema or suppository) or (g) aerosol (e.g. as an aqueous aerosol, liposomal preparation or solid particles containing the agent (s)).

In a preferred embodiment, the lipid nanodisc of the invention or the pharmaceutical composition of the invention may be specially formulated for intravenous administration, intramuscular administration and/or intraperitoneal administration.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

In addition to what is described above, the present invention also covers the possibility that the pharmaceutical composition of the invention may be administered to a subject together with other components or compounds, even if these do not form part of the pharmaceutical composition of the invention.

In a particular embodiment, the pharmaceutical composition of the invention is lyophilised. The term “lyophilised” refers to high vacuum freeze-dried pharmaceutical composition. Freeze drying, also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product and lowering pressure, removing the ice by sublimation.

In another aspect, the present invention relates to the lipid nanodisc of the invention or the pharmaceutical composition of the invention for use in the treatment of a cancer characterized in that it contains cells expressing on their surface the protein to which the scFab forming part of the fusion protein specifically binds or the protein to which the reconstituted antigen-binding site formed by the fragment of the immunoglobulin chain and a second immunoglobulin chain.

As used herein, the term "treating" (or "treat" or "treatment") refers to processes involving a slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related symptoms, conditions, or disorders. The treatment of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The "treatment" of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease. Accordingly, the "treatment" of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a broad range of responses to a treatment.

The term “subject”, as used herein, refers to an individual, plant or animal, such as a human, a nonhuman primate (e.g., chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs. The term does not denote a particular age or sex. In a preferred embodiment of the invention, the subject is a human.

The term "cancer", as used herein, refers to a disease characterized by uncontrolled cell division (or by an increase of survival or apoptosis resistance) and by the ability of said cells to invade other neighbouring tissues (invasion) and spread to other areas of the body where the cells are not normally located (metastasis) through the lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body. Depending on whether or not they can spread by invasion and metastasis, tumors are classified as being either benign or malignant: benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they only grow locally; whereas malignant tumors are tumors that are capable of spreading by invasion and metastasis. Biological processes known to be related to cancer include angiogenesis, immune cell infiltration, cell migration and metastasis. As used herein, the term cancer includes, but is not limited to, the following types of cancer: breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; T- cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer; testicular cancer including germinal tumours such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumours, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor.

Methods of the invention

In another aspect, the present invention relates to a method, hereinafter the first method of the invention, to obtain the fusion protein of the invention, wherein the method comprises:

(ii) recovering the fusion protein from the culture.

The first method of the invention comprises a first step of growing a cell comprising the polynucleotide of the invention. The polynucleotide of the invention which is comprised in the vector of the invention may be introduced into the cell by means of well-known techniques such as, transfection, electroporation, via particle bombardment and transformation using the vector of the invention that has been isolated. In a preferred embodiment the vector is introduced by transformation or electroporation. The transformed cell may be recovered on a solid nutrient media or in liquid media. In addition, the first method of the invention comprises growing said cell in conditions suitable for allowing the expression of the fusion protein from the polynucleotide of the invention. Culture conditions suitable for the growth of the cell and for the expression of the fusion protein may be different for each type of cell. However, those conditions are well known in the art and are readily determined.

The second step of the first method of the invention comprises the recovery of the fusion protein from the culture. The fusion protein can be recovered from the cells or from the supernatant of the culture medium. In the first case, the step of recovering requires the separation of the cells from the culture medium by any method known by the persons skilled in the art (trypsinization and centrifugation or filtration, trypsinization step is necessary for adherent cells, if the cells are in suspension, trypsinization is not necessary) and the rupture of said cells in an inert solution (freezing/thawing cycles, homogenization, sonication, cavitation, use of detergents and the like). Subsequently, it is possible to recover the fusion protein from the cell homogenate or from the supernatant of the culture medium using well known methods such as density gradient ultracentrifugation, or protein purification columns, such as affinity chromatography.

In another aspect, the present invention relates to a method, hereinafter the second method of the invention, to obtain the lipid nanodisc of the invention, wherein the method comprises:

(i) providing the fusion protein of the invention; and

Conditions suitable to provide a lipid bilayer formed by the membrane forming lipid are well known in the art and are readily determined.

In a particular embodiment, when the component (i) of the fusion protein is a fragment of an immunoglobulin chain, then the fusion protein is provided in association with a second fragment of an immunoglobulin chain containing its variable region, said fusion protein associated with the second fragment of an immunoglobulin chain contains an antigen binding site formed by the variable regions of the immunoglobulin chain fragment of component (i) and the second fragment of an immunoglobulin chain.

In another particular embodiment, the fusion protein of component (i) is a fragment of an immunoglobulin light chain and is provided in association with a second fragment of an immunoglobulin chain which is from an immunoglobulin heavy chain or wherein the fusion protein of component (i) is a fragment of an immunoglobulin heavy chain and is provided in association with a second fragment of an immunoglobulin chain which is from an immunoglobulin light chain component.

In another particular embodiment, the second method of the invention further comprising the step of lyophilizing the lipid nanodisc. The term “lyophilised” or “lyophilizing” has been defined or explained above, and this definition is applicable to the second method of the invention.

The term “cell population” refers to the number of cells in a given area. In the present invention this area can be the tumor microenvironment. In some embodiments, the compound of interest is conjugated to the lipid by a linker that is sensitive to degradation by proteases present in the tumour environment, in lysosomes or pH-sensitive. As this linker is degradable in the tumour microenvironment, the compound of interest could enter in the cell population or in the tissue.

EXAMPLES

The following examples illustrate the invention and must not be considered as limiting the scope thereof.

Material and methods

Protein expression and purification.

Genes encoding scFab-MSP-Fc fusions, IgGs were synthesized and cloned by GeneArt (Life Technologies) into the pcDNA3.4 expression vector while the receptors were cloned into de pHLsec expression vector. In addition, expression of the glycoproteins CD79b and HER2 was promoted through their genetic fusion to a monomeric variant of Venus. 200 ml of HEK 293F cells (Thermo Fisher Scientific) were seeded at a density of 0.8x10⁶ cells/mL in Freestyle expression media and incubated with 135 rpm oscillation at 37° C, 8% CO2, and 70% humidity. Within 24 h of seeding, cells were transiently transfected using 50 pg of filtered DNA. In the case of IgGs 90 pg of DNA at a 1 :2 LC:HC ratio. The DNA was preincubated for 10 min at room temperature (RT) with the transfection reagent PEIpro (VWR) at a 1 :3 ratio. After 6-7 days, cell suspensions were harvested by centrifugation at 6000 xg for 10 min and the supernatants filtered through a 0.22 pm Steritop filter (EMD Millipore). The scFab-MSP-Fc fusions and the IgGs particles were purified by affinity chromatography using a Protein A column and eluted using 20mM glycine pH 2.2. The eluted protein was neutralized using 1M Tris pH 9.0 and fractions containing protein were pooled, concentrated and loaded onto a Superose 6 10/300 GL size exclusion column (GE Heathcare) and in a Superdex200 10/300 GL size exclusion column (GE Heathcare), respectively in 20 mM sodium phosphate, 150 mM NaCI, 10% glycerol, pH 8.0. In the case of the recombinant receptors, they contain a C-terminal His6x tag for downstream purification. Hence, filtered supernatants were passed through a HisTrap Ni-NTA column (GE Healthcare) at 4 ml min-1. After washing the column with 20 mM Tris pH 9.0, 150 mM NaCI, 5 mM imidazole, the receptors were eluted with an increasing gradient of imidazole (up to 500 mM). Fractions containing protein were pooled, concentrated and loaded onto a Superdex 200 Increase size exclusion column (GE Heathcare) in 20 mM Tris pH 8.0, 150 mM NaCI buffer, 10% glycerol. The proteins were stored at -80° C until further use.

Assembly of Backpacked-antibodies.

The desired amount of lipid (stocks in chloroform, Avanti Polar Lipids) was dispensed into a glass tube. The solvent was dried up using a stream of nitrogen obtaining a lipid thin film and then the tube was placed in a vacuum desiccator under high vacuum to further remove residual solvent for at least 1 hour. The lipid was solubilized adding a buffer containing sodium cholate (12 - 40 mM final concentration). Then, the B-Ab was added to a final molar ratio of 1 :200 (protein: lipid). The mixture was vortexed and incubated for 15 minutes at RT in a roller. After the incubation and to allow detergent removal, the mixture was subjected to sequential incubations of 15 min, 30 min, 12h and additional 15 min with 50 - 70 mg of BioBeads SM-2 Adsorbents (Bio-Rad) each time. After the incubations, the assembled material was loaded into a Superose 6 10/300 GL size exclusion column (GE Heathcare) in 20 mM TRIS, 150 mM NaCI, 10% glycerol, pH 8.0. The protein fractions were polled, concentrated and stored at -80° C until further use. 1 % of Rhodamine phosphatidylethanolamine (Rho-PE) was added to the lipid mixture in order to monitor the lipid component within the B-Abs. Rhodamine emission spectra was obtained by fluorescence spectroscopy using an excitation wavelength of 550 nm. For microscopy experiments, Abberior STAR RED (SR) probe (Abberior GmbH) or Alexa Fluor (AF) 488 was used to labelled the IgG and B-Ab antibodies via thiol chemistry according to the dye manufacturer’s instructions. Alternatively, 1% of SR- DPPE lipid was used to label the lipid compartment of the B-Abs.

Negative-stain electron microscopy.

Eight microliters of B-Ab at a concentration approximately of 0.01 mg/mL was placed on the surface of parafilm and carbon-coated copper grid that had previously been glow- discharged in air for 45 sec was placed on the drop, allowed to adsorb for 2 min, and then blotted. The grid was placed again in a drop of eight microliters of sample buffer, allowed to wash for 30 sec and blotted. Then, the grid was placed in a drop of eight microliters of uranyl acetate 2% for 45 sec. The stain was removed immediately from the grid using filter paper. Grids were imaged with a JEOL JEM 1400 Plus electron microscope operating at 120 kV and equipped with an sCMOS camera.

Dynamic light scattering.

The Rh of the B-Abs was determined by dynamic light scattering (DLS) using a Zetasizer Nano - ZS (Malvern Panalytical). About 50 pL of the B-Abs at a concentration of 1 mg/mL was added to a UVette 220 nm - 1600 nm (Eppendorf) and measured at a fixed temperature of 25 °C. Particle size determination and polydispersity were obtained from the accumulation of 10 reads using the Zetesizer software.

Differential Scanning Calorimetry (DSC).

The thermal stability of the B-Ab and the corresponding IgG (1 mg/mL) was measured using a Nano Differential Scanning Calorimeter (TA Instruments). Nanodisc containing molecules were generated with POPC. All samples were preparated in PBS buffer (137 mM NaCI, 2.7 mM KCI, 10 mM Na₂HPO₄, 1 ,8 mM KH2PO4, pH 7.4). Heating curves were recorded from 20 °C to 100 °C with a rate of 1 °C/min and analyzed with NanoAnalyze software (TA Instruments) using a Gaussian model.

Enzyme-linked Immunosorbent Assay (ELISA).

96-well plates (C96 MAXISORP Nunc-lnmuno plate, Thermo Scientific) were coated overnight at 4° C with 0.5 pg/mL of the assembled B-Ab. After a 2-h well blocking with 3% (w/v) bovine serum albumin (BSA), 1 to 4 serial dilutions of the receptors (starting at 5 pg/mL) were incubated 1 h at RT. The bound protein was detected with an HRP- StrepTag II immunoglobulin (Sigma-Aldrich). The reaction was measured by absorbance at a wavelength of 492 nm in a Synergy HT microplate reader. Biolayer interferometry

Binding kinetics were measured using an Octet R8 Biolayer Interferometer (Sartorius ForteBio, Freemont, CA). B-Ab and IgG samples were loaded onto FAB2G biosensors (Sartorius ForteBio, Freemont, CA) to reach a 0.8 nm signal response. Association rates were measured by transferring the loaded sensors to wells containing two-fold serial dilutions (20 nM to 0.62 nM) of the recombinantly expressed FcRn for 180 s. Dissociation rates were measured by dipping the sensors into buffer-containing wells for another 180 s. FcRn pH-dependent binding was measured using two different bindings buffers (PBS containing 137mM NaCI, 2.7mM KCI, 10mM Na2HPO4 and 1.8mM KH2PO4 pH 7.4, 0.01% BSA and 0.0002% Tween-20 and 20mM NaAc, 150mM NaCI pH 5.6, 0.01 % BSA and 0.0002% Tween-20) at 25 °C. Analysis was performed using the Octet software, with a 1 :1 fit model.

Cell binding and internalization.

Glass bottom 8-well p-Slides (Ibidi) were coated with 0.01 % (v/v) poly-L-lysine (Sigma) for 10 minutes, washed three times with water and let dry. 300,000 cells were added in 250 pL RPMI + 10% FBS and incubated for 15 minutes at 37° C, 5% CO2 to adhere. Afterwards, antibodies, B-Abs or nanodiscs were added to a final concentration of 10 pg/mL. In the case of LysoTracker DND-26 Green imaging, it was added 5 min prior to imaging to a final concentration of 50 nM as recommended by the manufacturer. Live cell imaging was performed in an Abberior STEDYCON confocal system connected to an Olympus IX83 inverted microscope equipped with a 60x 1 .2 NA Olympus UPlanSApo water immersion objective lens. AlexaFluor 488 and STAR RED were excited with 488 nm and 640 nm laser lines subsequently. Laser power was adjusted to be 20 pW at the sample plane. Pixel size was set to 100 nm and pixel dwell time to 10 ps. Each image line was scanned two times and the signal integrated.

Cell Viability Assay.

Daudi and Raji B cell lines were grown in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (FBS). 10,000 cells/well of each cancer cell line in 100 pl media was co-cultured with 100 pl of 3-fold serial dilutions of Ptd-MMAE loaded B-Polatuzumab at 37 °C. After 72 h incubation, cell viability was monitored using CellTiter Gio 2.0 kit (Promega) following the manufacturer's instructions. Luminescence in relative light units (RLUs) was measured using 96-well white plates (Sigma-Aldrich) in a Synergy HT microplate reader (Biotek Instruments). Cell cycle analysis by flow cytometry

The B-Ab, ADC and control samples were incubated with T 10⁶ Daudi cells in a 24-well plate. After 1 hour, cells were washed by centrifugation and fresh media was added to each well. Cells were harvested, washed with PBS and counted after 72h. 5 10⁵ cells were fixed with 70% cold ethanol at -20 °C for at least 1 h, and incubated with 0.5% Triton X-100 and 25 pg/mL RNase A in PBS for 30 min at room temperature. Then, DNA was stained with 25 ng/mL propidium iodide (Molecular Probes) for 15 min and samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Co., Miami, FL, USA).

Pharmacokinetic study

In vivo studies were performed using 8- week-old C.B-17/lcrHan®Hsd-Prkdc^scid mice purchased from Innotiv (Envigo), housed in individually-vented cages under 12 h light/dark cycle at a temperature of 21-23 °C and a humidity of 40-55%. All animal handling and protocols were approved by the animal care ethic committee of Biogipuzkoa Institute and were conducted in conformity with the EU guidelines and regulations for animal experimentation. For pharmacokinetic studies, a single injection of 5 mg/kg of Backpacked Antibodies (B-Ab) or control samples (IgG, ADC and naked nanodisc) were intraperitoneally injected. Blood samples were collected at multiple time points from the saphenous vein and serum samples were assessed for levels of circulating antibodies by ELISA using standard curves. Briefly, 96-well Pierce Nickel Coated Plates (Thermo Fisher) were coated with 50 pL at 5 pg/ml of the His6x-tagged antigen hCD79b. 1 :100 sera dilution was incubated for 1 h at RT and further develop using as secondary antibody the Goat anti-Human IgG F(ab')2 - AP (ThermoFisher Scientific) (dilution 1 :2000). The optical density was measured using the halo led 96 dynamic ELISA Reader at 405nm. For naked nanodisc containing serum samples, Goat anti-Human IgG Fc - HRP (ThermoFisher Scientific) (dilution 1 :3000) was used as secondary antibody, and the optical density was measured at 450 nm.

Example 1. Molecule design and production.

To assess the capacity of nanodiscs to self-assemble in the presence of antibody fragments (Fabs and Fc), the authors of the present invention have used the membrane scaffold protein MSP2N2 and the antibody fragments (Fab and Fc) derived from denintuzumab, a CD19-targeting lgG1 mAb. The Fab fragment is a hetero-dimer consisting of a light and a heavy chain. In order to use a single polypeptide for the generation of the B-Ab, the inventors have created a single-chain Fab (scFab) through genetic fusion of the LC and HC using a 70 amino acid flexible linker [(GGGGS)x14], The scFab was fused at the N-terminus of MSP2N2 while the C-terminus of this membrane scaffold protein was fused to the Fc chain. The Fc fragment is a homodimer and its dimerization occurs upon assembly of the lipid nanocage, which requires the presence of two MSP. Like in mAbs the Fc:Fab ratio will be 1 :2 but in the case of the B- Ab, the molecule carries a lipid nanocage to load hydrophobic drugs (Fig. 1A-B).

Example 2. Functional and biophysical characterization.

Denintuzumab derived B-Abs (B-Denin) self-assemble into homogeneous (Fig. 2A-B) disc-shaped particles with protruding Fabs and Fc and have a hydrodynamic radius (Rh) of 10 nm, which is within the range of molecules found in nature such as IgG and IgM (Fig. 2C-D). Incorporation of lipids in the B-Ab was monitored using fluorescence spectroscopy. For that, B-Denin molecules were assembled in the presence of 1% of Rhodamine phosphatidylethanolamine (Rho-PE) (Fig. 2A). The resulting B-Ab particles showed similar thermodynamic stability to the parental IgG molecules. In both samples, two melting temperatures (T_m1 and T_m2) corresponding to the Fab and Fc region was observed, respectively. Deletion of the Fc fragment in the B-Ab yielded a particle with a single T_m1 (Fig. 2E). Functional assembly of the antibody fragments within the B-Ab was assessed using enzyme-linked immunosorbent assays (ELISA) (Fig. 3). A dosedependent titration curve to the recombinant CD19 showed similar binding affinities between the scFab that constitutes the B-Ab and the Fab fragment of the parental IgG (Fig. 3A). Dimerization of the Fc was confirmed by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) from a shift in its molecular weight under reducing conditions (Fig. 3B). Functional binding of the Fc fragment to the human Fc gamma receptor I (FcyRI) was confirmed by ELISA (Fig. 30). In addition, similar to what has been observed in the case of IgG molecules, the double mutations LALA (Leu234Ala, Leu235Ala) abolished binding of the B-Ab to FcyRI (Fig. 3C). Importantly, lyophilization of the B-Abs does not alter their biophysical and functional properties offering a pathway to further increase the thermostability of these molecules for a prolonged period of time (Fig. 4). No signs of degradation (Fig. 4A), aggregation (Fig. 4B) or lost in binding affinities to their targeted receptors (Fig. 40) have been observed upon lyophilization and storage of the B-Ab for three months.

Example 3. Specific tumor cell targeting.

B-Denin molecules were labelled with fluorescent dyes and their cell targeting and internalization to Daudi B, which express the targeted receptor CD19 was monitored using confocal microscopy. B-Ab labeling was employed with the thiol reactive fluorophores STAR RED (SR) or Alexa Fluor (AF) 488 for conjugation of the Fc fragment to label the protein or though the addition of a SR conjugated lipid (SR-DPPE) to label the lipid compartment. Specific tumor cell targeting of the labelled B-Abs was confirmed from the ability of B-Denin to bind CD19+ Daudi cells but not the CD19- Jurkat cells lacking the receptor (Fig. 5A). This process was strictly dependent on the presence of the Fab since the lipid nanocage made only with the MSP2N2 scaffold protein was unable to target Daudi cells (Fig. 5A). Upon receptor binding, B-Abs undergo a rapid cell internalization similar to that observed for the parental IgG (Fig. 5A). In order to confirm that the observed endocytosis was receptor-mediated and not triggered by the nanodisc, inventors replaced the scFab specificity in the B-Ab by that of rituximab, an anti-CD20 mAb that is not internalized upon antibody binding. As expected, incubation of the rituximab derived B-Ab (B-Rtx) with Daudi cells resulted in accumulation of the molecule at the membrane surface following the trend of the parental IgG (Fig. 5B). Importantly, when the protein and the lipid compartment of the B-Ab was simultaneously labelled with two different fluorophores, inventors observed a co-localization of the two dyes confirming that, as expected, the B-Ab components stayed assembled during the whole process (Fig. 6). Finally, the inventors have confirmed that the particles undergo lysosomal trafficking upon cell uptake since inventors observed co-localization of the labelled B-Abs with the Lysotracker probe, which specifically labels the lysosomal compartment (Fig. 7). Overall, these data demonstrate the ability of these novel molecules to specifically deliver cargo inside tumor cells.

Example 4. In vitro tumor cell cytotoxicity

In order to test the efficiency of the B-Ab molecules to induce cell death of tumor cells, inventors loaded the lipid nanodisc with a lipid-drug conjugate generated using a reactive linker and the cytotoxic drug MMAE (Fig. 8A). A dry lipid film of 40% of the lipid-drug conjugate and 60% of POPO was generated for assembly of a B-Ab containing polatuzumab specificity. The final amount of incorporated drug was determined through mass spectrometry (LC-MS) using a matrix calibration curve constructed by mixing the assembled B-Ab in the absence of the lipid-drug conjugate and adding serial dilutions of the pure conjugate (Figure 8B). The calculated ratio of POPO to 16:0_Ptd_thioEtOH_MMAE_conjugate in the B-Abs is 1 :1.21. Hence, since each nanodisc is composed of approximately 350 lipid molecules, the B-Abs incorporate up to 160 drugs per particle (Figure 8B-C). Consistent with an efficient internalization (Fig. 5A), incubation of the loaded B-Pola with Daudi and Raji tumor cells resulted in potent cell death with half inhibitory concentrations (IC50) of 0.046 pg/mL and 0.061 pg/mL, respectively. Incubation with unloaded B-Pola molecules did not alter the viability of the cells suggesting that the anti-tumor cytotoxic activity observed was due to the delivered drugs (Fig. 9). In addition, Incorporating the antineoplastic agent MMAE into the B-Ab nanodiscs provides these molecules with the ability to inhibit cell growth (cell cycle arrest at G2/M) with a potency much greater than that of ADCs (Fig. 10). As expected, the potency of B-Abs is more than 1 order of magnitude higher, consistent with the number of drugs these new molecules can deliver to cancer cells, which is more than 10 times greater (Fig. 10B).

Example 5. Plug-and-play nature of the platform.

Consistent with the development of a modular platform, the inventors have shown that the different components of the B-Ab can be easily altered. First, the size of the membrane scaffold protein can be modified resulting in the generation of B-Abs with different hydrodynamic radius (Rh) (Fig. 11 A). In addition, the inventors were able to generate B-Abs with different target specificities by easily swapping the scFab in the particles. All the resulting molecules were able to recognize their recombinant receptors (Fig. 11 B) and to specifically internalize their loaded cargo inside the cell (Fig. 12). In addition, this design is compatible with the generation of bispecific molecules through tandem fusions of single chain variable fragments (Fig. 13A). As shown in Fig. 13B, the two specificities built in the bispecific B-Ab were able to bind their targeted epitope.

Example 6. Pharmacokinetic study in immunodeficient mice.

The presence of the Fc allowed the B-Abs to interact with the FcRn in vitro in a pH- dependent manner, showing a binding affinity similar to IgG (Fig 14A, 14B). This led to prolonged plasma circulation times for empty B-Abs compared to nanodiscs, aligning with IgG ranges (Fig 14C). While cytotoxic drugs negatively impact the ADC's pharmacokinetic profile, causing faster decay (Fig. 15A), the behavior of the B-Ab remains consistent regardless of drug loading (Fig. 15B). Importantly, pharmacokinetic profiles of the ADC and loaded B-Ab demonstrate similarity (Fig. 15C), despite the B-Ab carrying over 10 times more MMAE molecules than the IgG. Hence, lipid nanodiscs serve to conceal drugs from the surrounding media, safeguarding the antibody's half-life against their hydrophobic nature.

Claims

1 . A fusion protein comprising:

(ii) a membrane scaffold protein (MSP); and

(iii) an immunoglobulin Fc fragment.

2. The fusion protein according to claim 1 , wherein the membrane scaffold protein is selected from the group consisting of: Apo Al, Apo E, MSP1 D1 , MSP1 D1 D73C, MSP1 D1 (-), MSP1 E1 D1, MSP1 E1 D1 D73C, MSP1 E2D1 , MSP1 E2D1 D73C, MSP1 E3D1 , MSP1 E3D1 D73C, MSP1 FC, MSP1 FN, MSP2N2, MSP1 D1 biotin labelled or MSP1 D1 DHS.

3. The fusion protein according to claim 2, wherein the membrane scaffold protein is the membrane scaffold protein 2N2 (MSP2N2).

4. The fusion protein according to any of claims 1 to 3, wherein the single-chain antigen-binding fragment (scFab) is N-terminal to the MSP.

5. The fusion protein according to any of claims 1 to 4, wherein the immunoglobulin Fc fragment is C-terminal to the MSP.

6. The fusion protein according to any of claims 1 to 5, wherein the immunoglobulin chain containing the variable region is a heavy chain or a light chain.

7. The fusion protein according to any of claims 1 to 6, wherein the single-chain antigen-binding fragment comprises a light chain (LC) and a heavy chain (HC) and wherein the LC and the HC are connected by a linker sequence or wherein the single-chain antigen-binding fragment is a VHH, VNAR or a tandem fusion of the above.

8. The fusion protein according to any of claims 1 to 7, wherein the single-chain antigen-binding fragment or the fragment of the immunoglobulin chain is connected to the MSP by a linker sequence.

9. The fusion protein according to any of claims 1 to 8, wherein the immunoglobulin Fc fragment is connected to the MSP by a linker sequence.

10. The fusion protein according to any of claims 1 to 9, wherein the single-chain antigen-binding fragment (scFab) or wherein the fragment of an immunoglobulin chain derived from denintuzumab rituximab, pinatuzumab, polatuzumab, epratuzumab trastuzumab or atezolizumab.

11. The fusion protein according to any of claims 1 to 10, wherein the single-chain antigen-binding fragment comprises more than one antigen-binding region.

12. The fusion protein according to any of claims 1 to 11 , wherein the fusion protein further comprises a tag.

13. A polynucleotide encoding the fusion protein according to any of claims 1 to 12.

14. A vector comprising the polynucleotide according to claim 13.

15. A host cell comprising the vector according to claim 14.

16. A lipid nanodisc characterized in that it comprises multiple copies of the fusion protein according to any of claims 1 to 12 and a membrane-forming lipid.

17. The lipid nanodisc according to claim 16 wherein, when component (i) of the fusion protein is a fragment of an immunoglobulin chain, then the lipid nanodisc further comprises a second fragment of an immunoglobulin chain containing its variable region, wherein said second fragment is associated with the fragment of the immunoglobulin chain of component (i) so that the antigen binding site is reconstituted by the variable regions of the immunoglobulin chain fragment of component (i) and the second fragment of an immunoglobulin chain.

18. The lipid nanodisc according to claim 17, wherein component (i) is a fragment of an immunoglobulin light chain and the second fragment of the immunoglobulin chain is from an immunoglobulin heavy chain or wherein component (i) is a fragment of an immunoglobulin heavy chain and the second fragment of the immunoglobulin chain is from an immunoglobulin light chain.

19. The lipid nanodisc according to any of claims 16 to 18, wherein the membraneforming lipid is selected from the group consisting of phospholipids, sphingolipids, glycolipids, sterols and alkylphosphocholines.

20. The lipid nanodisc according to claim 19, wherein the membrane-forming lipid is a phospholipid selected from the group consisting of: phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylserine (PS).

21 . The lipid nanodisc according to any of claims 16 to 20, wherein the lipid nanodisc comprises a molar ratio protein: lipid of 1 :200.

22. The lipid nanodisc according to any of claims 16 to 21 , wherein the lipid nanodisc further comprises a detection tag.

23. The lipid nanodisc according to claim 22, wherein the detection tag is intercalated in the lipid fraction.

24. The lipid nanodisc according to any of claims 16 to 23, wherein the lipid nanodisc further comprises one or a combination of compounds of interest.

25. The lipid nanodisc according to claim 24, wherein the compound of interest is an anticancer agent.

26. The lipid nanodisc according to any of claims 16 to 25, wherein the lipid nanodisc has a hydrodynamic radius (Rh) between 5 nm and 20 nm.

27. A pharmaceutical composition comprising the lipid nanodisc according to any of claims 16 to 26, and at least one pharmaceutically acceptable excipient.

28. The pharmaceutical composition according to claim 27, wherein the composition is lyophilised.

29. The lipid nanodisc according to any of claims 16 to 26 or the pharmaceutical composition according to any of claims 27 or 28 for use in medicine.

30. The lipid nanodisc according to any of claims 16 to 26 or the pharmaceutical composition according to any of claims 27 or 28 for use in drug administration.

31. The lipid nanodisc according to any of claims 16 to 26 or the pharmaceutical composition according to any of claims 27 or 28 for use in the treatment of a cancer characterized in that it contains cells expressing on their surface the protein to which the scFab forming part of the fusion protein specifically binds or the protein to which the reconstituted antigen-binding site formed by the fragment of the immunoglobulin chain and a second immunoglobulin chain.

32. A method to obtain the fusion protein according to any of claims 1 to 12, wherein the method comprises:

(i) growing a cell comprising a polynucleotide according to claim 13 in conditions suitable for allowing the expression of the fusion protein from the polynucleotide; and

(ii) recovering the fusion protein from the culture.

33. A method to obtain the lipid nanodisc according to any of claims 16 to 26, wherein the method comprises:

(i) providing a fusion protein according to any of claims 1 to 12; and

34. The method according to claim 33 wherein, when the component (i) of the fusion protein is a fragment of an immunoglobulin chain, then the fusion protein is provided in association with a second fragment of an immunoglobulin chain containing its variable region, said fusion protein associated with the second fragment of an immunoglobulin chain contains an antigen binding site formed by the variable regions of the immunoglobulin chain fragment of component (i) and the second fragment of an immunoglobulin chain.

35. The method according to claim 34, wherein the fusion protein of component (i) is a fragment of an immunoglobulin light chain and is provided in association with a second fragment of an immunoglobulin chain which is from an immunoglobulin heavy chain or wherein the fusion protein of component (i) is a fragment of an immunoglobulin heavy chain and is provided in association with a second fragment of an immunoglobulin chain which is from an immunoglobulin light chain component.

36. The method according to any of claims 33 to 35, further comprising the step of lyophilizing the lipid nanodisc.

37. An in vitro method for the delivery of a compound of interest to a cell population or to a tissue comprising contacting the lipid nanodisc according to any of claims 16 to 26 comprising the compound of interest with the cell population or tissue, wherein cells in the tissue or cells of the cell population express on their surface the protein to which the scFab forming part of the fusion protein specifically binds or the protein to which the reconstituted antigen-binding site formed by the fragment of the immunoglobulin chain and a second immunoglobulin chain.