Targeting fusion proteins

Abstract

This invention relates to targeting fusion proteins based on molecules which bind serum proteins, such as serum protein receptors. The invention also relates to amphipathic structures linked to the targeting fusion proteins. The invention also relates to methods of targeting and therapeutic methods using the fusion proteins and / or amphipathic structures.

Description

[0001] TARGETING FUSION PROTEINS

[0002] TECHNICAL FIELD

[0003] This invention relates to targeting fusion proteins based on molecules which bind serum proteins, such as serum protein receptors. The invention also relates to amphipathic structures linked to the targeting fusion proteins. The invention also relates to methods of targeting and therapeutic methods using the fusion proteins and / or amphipathic structures.

[0004] BACKGROUND

[0005] Therapies using amphipathic structures, such as engineered cells, vesicles, micelles, liposomes, lipid nanoparticles, or exosomes, are becoming increasingly common in the treatment of a variety of diseases, including cancer. In particular, immunotherapy using chimeric antigen receptor (CAR)-engineered T-cells has shown considerable potential in the treatment of B-cell malignancy and multiple myeloma. Other immunotherapeutics of increasing interest include the use of amphipathic structures as the delivery mechanism for a variety of therapeutic molecules, including gene therapies.

[0006] However, these efforts are frustrated by the inefficient targeting of the amphipathic structures in vivo. First, this means insufficient amphipathic structures arrive at the site of the disease or disorder and treat the disease or disorder effectively. This is especially important in the case of solid tumours, where sufficient targeting is required to allow a therapeutically effective number of active molecules or cells to reach the tumour site. Second, inefficient targeting has the potential to result in off-target effects as the amphipathic structures affect normal, healthy cells, as well as cells associated with the disease or disorder.

[0007] There remains a need for improved targeting of amphipathic structures. The present invention seeks to address one or more of these issues.

[0008] SUMMARY OF THE INVENTION

[0009] The inventors have surprisingly shown that novel targeting fusion proteins based on molecules which bind serum proteins, such as serum protein receptors (e.g., apolipoprotein receptors), are capable of efficiently targeting cells expressing a target molecule of interest ("target cells"). The inventors have specifically shown amphipathic structures, such as lipid nanoparticles (LNPs), coated with these novel fusion proteins are not only capable of targeting and delivering a cargo to target cells but are also capable of actively blocking uptake by and delivery to non-target cells. Use of the fusion proteins and amphipathic structures of the invention actively increase the amount and rate of endocytosis of the amphipathic structures by target cells. This allows improved delivery of amphipathic structures and their cargos with increased uptake by target cells and reduced uptake by non-target cells. The invention provides a fusion protein comprising (1) a first region comprising a peptide or polypeptide that specifically binds to at least a part of a receptor binding site of a membrane-binding serum protein and (2) a second region comprising a targeting peptide or polypeptide.

[0010] The invention also provides:

[0011] - a conjugate comprising (a) a fusion protein of the invention and (b) an antibody light chain or the variable region of an antibody light chain (VL); a construct comprising two conjugates of the invention; one or more polynucleotides encoding a fusion protein of the invention, a conjugate of the invention or a construct of the invention; a vector comprising one or more polynucleotides of the invention; an amphipathic structure linked to a fusion protein of the invention, a conjugate of the invention or a construct of the invention; an amphipathic structure of the invention, wherein the amphipathic structure is a vesicle, micelle, liposome, lipid nanoparticle, or exosome; a composition comprising an amphipathic structure of the invention, a polyethylene glycol (PEG)-lipid conjugate and a sterol, wherein the molar ratio of the PEG-lipid conjugate to the lipids in the amphipathic structure and the sterol is about 1.2% or less; a pharmaceutical composition comprising a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention or a composition of the invention and a pharmaceutically acceptable diluent and / or carrier;

[0012] - a method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a composition of the invention or a pharmaceutical composition of the invention; a method of producing an amphipathic structure of the invention comprising linking the amphipathic structure to the fusion protein, conjugate, or the construct; and - a method of improving the targeting of an amphipathic structure to a target cell comprising linking the amphipathic structure to a fusion protein of the invention, a conjugate of the invention or a construct of the invention, wherein the targeting peptide or polypeptide specifically binds a target antigen on the target cell.

[0013] DESCRIPTION OF THE FIGURES

[0014] Figure 1 shows CD3e binding by NanoPilot constructs using an indirect ELISA.

[0015] Figure 2 shows NanoPilot blocking non-specific transfection of LNPs by CD3-ve cells but maintaining transfection of the target CD3+ve cells.

[0016] Figure 3 shows NanoPilot containing a CDR that is not cross reactive with human CD3 blocks transfection via its Apo-E binding activity but does not target CD3+ve cells.

[0017] Figure 4 shows NanoPilot actively blocking non-specific uptake by CD3-ve cells.

[0018] Figure 5 shows the optimisation of NanoPilot concentrations.

[0019] Figure 6 shows the optimisation of NanoPilot incubation conditions.

[0020] Figure 7 shows LNPs incubated in the presence of NanoPilot at room temperature for up to 24 hours retained >90% of their transfectivity.

[0021] Figure 8 shows NanoPilot selectivity and MFI enhancement with two different Anti-CD3 sequences.

[0022] Figure 9 shows NanoPilot selectivity and MFI enhancement improvements when using LNPs formulated with DOPE as the helper lipid rather than DSPC. (A) LNPs containing DOPE helper lipids in place of DSPC give higher NanoPilot CD3+ve cell targeting ratios. (B) MFI enhancement from NanoPilot on DOPE LNPs can rise to as high as 200% and MFIs are retained at a higher level at high NanoPilot concentrations.

[0023] Figure 10 shows a PAI-1 NanoPilot construct binding to vitronectin. Changes in 280 nm absorbance as the Vitronectin Corona is established around the LNP, followed by sequential addition of PAI-1 NanoPilot.

[0024] Figure 11 shows NanoPilot (CYT344) is a more potent inhibitor of off-target LNP uptake than a recombinant human LDLR extracellular domain (rhLDLR-ED), as determined by IC50 values from a DiD-based blocking assay.

[0025] Figure 12 shows a comparison of LNP targeting using an LDLR-anchored NanoPilot versus an ApoE-anchored construct, demonstrating superior off-target blocking with the LDLR- anchored system. Figure 13 shows the modularity of the NanoPilot platform using an anti-CD117 construct (CYT391) to selectively target Kasumi-1 cells in a co-culture with off-target liver cell line HepG2.

[0026] Figure 14 shows that reducing the PEG-lipid concentration in the LNP formulation from 1.5 mol% to 0.75 mol% and 0.375 mol% increases on-target transfection and improves the targeting ratio a further 5-10 fold.

[0027] Figure 15 shows that the DiD signal from a membrane labelled LNP correlates with the GFP signal from the mRNA payload, confirming NanoPilot acts at the level of cellular uptake.

[0028] Figure 16 shows the adaptation of the NanoPilot platform to target primary murine T cells using anti-mCD3 (CYT346) and anti-mCD8 (CYT435) constructs.

[0029] Figure 17 shows targeted delivery of NanoPilot-coated LNPs to circulating T cells in Balb / c mice 24 hours post-injection, as measured by GFP and DiD signals in peripheral blood.

[0030] Figure 18 shows a significant reduction in LNP accumulation in the livers of mice treated with NanoPilot-coated LNPs compared to untargeted LNPs at 48 hours post-injection.

[0031] DETAILED DESCRIPTION

[0032] General disclosure

[0033] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0034] The present invention is described with respect to particular embodiments and with reference to certain Figures but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying Figures. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "an / one embodiment", "some embodiments" or a "preferred embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment(s) is included in at least one embodiment of the present invention. Thus, appearances of the embodiment phrases in various places throughout this specification are not necessarily all referring to the same embodiment, but may do so. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.

[0035] In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a part" includes two or more parts, "a binding site" includes two or more sites, "a region" includes two or more regions, "an antibody" includes two or more antibodies, "a receptor" includes two or more receptors, "a fragment" includes two or more fragments, "a fusion protein" includes two or more fusion proteins, reference to "a conjugate" includes two or more such conjugates, reference to "a construct" includes two or more such constructs, reference to "a polynucleotide" includes two or more polynucleotides, reference to "an amphipathic structure" refers to two or more amphipathic structures, reference to "a method" includes two or more methods and the like.

[0036] In all of the discussion herein, the standard one letter codes for amino acids are used. These are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0037] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0038] "About" as used herein when referring to a measurable value such as a percentage or an amount and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Any statement herein including the term "about" includes the same feature without the term. For instance, at least "about" 90% identity includes at least 90% identity.

[0039] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers, or steps. In all instances herein the terms "comprising" and "comprises" are interchangeable with "consisting essentially of" and "consists essentially of" and "consisting of" and "consists of".

[0040] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and Figures, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0041] Fusion proteins

[0042] The invention provides a fusion protein. The fusion protein may also be called a targeting fusion protein.

[0043] The fusion protein comprises two regions. The fusion protein may comprise at least two regions or two or more regions. The fusion protein may comprise any number of at least two regions or two or more regions, such as two, three, four, five, six, seven, eight, nine, ten or more regions. The fusion protein preferably comprises three regions, wherein the first region and second region are as defined above and wherein the third region comprises or consists of a linker. Linkers are discussed in more detail below.

[0044] The fusion protein may comprise any number of first regions. The fusion protein may comprise one first region. The fusion protein may comprise two or more first regions, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more first regions. The first regions may be the same or different. The fusion protein preferably comprises two identical first regions.

[0045] The fusion protein may comprise any number of second regions. The fusion protein may comprise one second region. The fusion protein may comprise two or more second regions, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more second regions. The second regions may be the same or different. The fusion protein preferably comprises two identical second regions.

[0046] First region

[0047] The first region comprises or consists of a peptide or polypeptide that specifically binds to at least a part of a receptor binding site of a membrane-binding serum protein.

[0048] The peptide or polypeptide may have any length. The peptide or polypeptide preferably has a length from about 10 amino acids to about 5000 amino acids, such as from about 20 to about 2000, from about 30 to about 1000, from about 40 to about 750 or from about 50 to about 500 amino acids in length. The peptide or polypeptide preferably has a length of at least about 10 amino acids, such as at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 200, at least about 300 or at least about 400 amino acids in length.

[0049] The peptide or polypeptide is preferably a protein, a receptor, an antibody or a fragment thereof, or an enzyme. The skilled person is capable of designing proteins, receptors antibodies, antibody fragments, or enzymes that function according to the invention.

[0050] The term "specifically binds to" means binding that is measurably different from a nonspecific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. Such methods are routine in the art. All instances herein of the term "specifically binds to" is interchangeable with "specifically interacts with," "specific for," "selectively binds to" "selectively interacts with" and "selective for".

[0051] The peptide or polypeptide specifically binds to at least a part of a receptor binding site of a membrane-binding serum protein if it binds to the part with preferential or high affinity, but does not bind or binds with only low affinity to other or different parts or to other or different molecules, such as other or different peptides, other or different polypeptides or proteins (including other membrane-binding serum proteins) or other or different polynucleotides. Preferably, the peptide or polypeptide binds to the at least a part of a receptor binding site of a membrane-binding serum protein with an affinity that is at least about 10 times, such as at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1000 or at least about 10,000 times, greater than its affinity for other molecules.

[0052] The term "Kassoc" or "Kon", as used herein, is intended to refer to the association rate of a particular binding molecule-target, whereas the term "Kdis" or "Koff," as used herein, is intended to refer to the dissociation rate of a particular binding molecule-target interaction. The term "Kd", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kon to Koff (i.e. Kon / Koff) and is expressed as a molar concentration (M).

[0053] The peptide or polypeptide has affinity for the at least a part of a receptor binding site of a membrane-binding serum protein. The receptor preferably has high affinity for the at least a part of a receptor binding site of a membrane-binding serum protein. The receptor has high affinity if it binds with a Kd of about 1 x IO-6M or less, such as about 1 x IO-7M or less, about 5 x IO-8M or less, about 1 x IO-8M or less, or about 5 x IO-9M or less. The peptide or polypeptide preferably has a Kd for the at least a part of a receptor binding site of a membrane-binding serum protein of about 100 nM or less, such as about 50 nM or less, about 20 nM or less or about 10 nM or less. A molecule or group binds with low affinity if it binds with a Kd of about 1 x IO-6M or more, about 1 x IO-5M or more, about 1 x 10-4M or more, about 1 x IO-3M or more, or about 1 x IO-2M or more.

[0054] Affinity can be measured using known binding assays, such as those that make use of fluorescence and radioisotopes. Competitive binding assays are also known in the art. The strength of binding between peptides or proteins and polynucleotides can be measured using nanopore force spectroscopy as described in Hornblower et al., Nature Methods. 4: 315-317. (2007) or Isothermal Titration Calorimetry (ITC), which is a label-free quantification technique used in studies of a wide variety of biomolecular interactions. ITC works by directly measuring the heat that is either released or absorbed during a biomolecular binding event. Kd values for antibodies can be determined using surface plasmon resonance, such as a Biacore® system, or solution equilibrium titration (SET) (see Friguet, et al., (1985) J. Immunol. Methods, 77(2):305-319, and Hanel et al., (2005) Anal. Biochem., 339(1): 182-184).

[0055] The membrane-binding serum protein is typically present in serum, such as mammalian serum. The membrane-binding serum protein is typically present in human serum.

[0056] The membrane-binding serum protein is capable of binding at least one membrane. The membrane-binding serum protein typically binds a membrane as part of its normal function in serum. The membrane is preferably a cell membrane in the organism from which the membrane-binding serum protein is derived. The membrane is preferably a mammalian cell membrane, such as a human cell membrane. The membrane is preferably present in any of the amphipathic structures described below. Binding of the membrane-binding serum protein to a membrane can be measured using routine techniques, including those described below and in the Examples. The membrane-binding serum protein is preferably present in one or more ultra low-density lipoproteins (ULDLs), one or more very low density-lipoproteins (VLDLs), one or more intermediate density-lipoproteins (IDLs), one or more low density-lipoproteins (LDLs), or one or more high density-lipoproteins (HDLs). The skilled person understands the types of proteins present in these lipoproteins.

[0057] The membrane-binding serum protein may be an apolipoprotein, a complement protein, an immunoglobulin or subunit thereof, fibrinogen chain, fibronectin, thrombin, globin, bradykinin, albumin or vitronectin.

[0058] The membrane-binding serum protein is preferably an apolipoprotein. The apolipoprotein is preferably selected from Apolipoprotein-A (Apo-A), Apo-B, Apo-C, Apo-D, Apo-E, Apo-F, Apo-H, Apo-L, Apo-M, Apo-N and Apo-(a). The Apo-A is preferably selected from Apo-AI, Apo-A2, Apo-A4 and Apo-A5. The Apo-B is preferably selected from Apo-B48 and Apo-B- 100. The Apo-C is preferably selected from Apo-C-I, Apo-C-II, Apo-C-III, and Apo-C-IV. The membrane-binding serum protein is preferably Apo-E. The Apo-E is preferably selected from Apo-Cl, Apo Apo-E2, Apo-E3, and Apo-E4.

[0059] The membrane-binding serum protein is preferably selected from albumin, Apo-E, fibrinogen beta chain, complement Clq subcomponent subunit B, fibrinogen gamma chain, immunoglobulin heavy constant mu, fibrinogen alpha chain, Apo-AI, fibronectin, complement Clq subcomponent subunit C, ceruloplasmin, immunoglobulin heavy constant alpha (fragment), Apo B-100, prothrombin, beta-globin, Apo-A4, complement C3, Apo-C-I, H-2 class I histocompatibility antigen, Q10 alpha chain, serotransferrin, interalpha-trypsin inhibitor heavy chain H3, pregnancy zone protein, carboxylesterase 1C, immunoglobulin heavy constant alpha (fragment), immunoglobulin kappa constant, vitronectin, immunoglobulin kappa variable 17-121 (fragment), interalpha-trypsin inhibitor heavy chain H2, fibrinogen beta chain, protein AMBP, alpha globin 1, interalpha-trypsin inhibitor heavy chain Hl, immunoglobulin heavy constant gamma 2C (fragment), alpha-l-antitrypsin 1-4 and bradykinin.

[0060] The peptide or polypeptide specifically binds to at least a part of a receptor binding site of a membrane-binding serum protein. The membrane serum-binding protein may comprise any number of receptor binding sites, such as 1, 2, 3, 4, 5 or more. The peptide or polypeptide may specifically bind to any number of these binding sites, preferably all of the binding sites. The peptide or polypeptide may specifically bind to any amount of a receptor binding site. The peptide or polypeptide may specifically bind to at least about 5% of the receptor binding site, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% of a receptor binding site. %s are usually calculated on the basis on the number of amino acids to which the peptide or polypeptide binds and the number of amino acids in the binding site. The peptide or polypeptide may specifically bind to at least about 3 amino acids in a receptor binding site of a membranebinding serum protein, such as at least about 4 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 110 amino acids, at least about 120 amino acids, at least about 130 amino acids, at least about 140 amino acids or at least about 150 amino acids.

[0061] The specific binding of the peptide or polypeptide typically reduces the ability of the receptor to bind to the membrane-binding serum protein. The specific binding of the peptide or polypeptide may reduce the binding of the receptor to the membrane-binding serum protein by any amount. It may reduce the binding by at least about 5%, such as by least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, as least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. Binding may be measured using any of the methods described above.

[0062] A receptor binding site is a set of amino acid residues on a protein wherein each residue either directly or indirectly interacts with a receptor. The amino acids forming the binding site are not required to be contiguous. The interaction may occur through hydrogen bonds, salt-bridges, electrostatic interactions, or any other non-covalent or covalent bonds. A receptor may be said to bind to a receptor binding site if it either interacts with one or more amino acid residues that are identified to be within the set of the receptor binding site amino acid residues, or if it binds in a way that causes steric hinderance of the interaction between the receptor and the amino acid residues within the set of the receptor binding site amino acid residues. A membrane-binding serum protein may have more than one receptor binding site.

[0063] A receptor binding site on a membrane-binding serum protein may be identified by mutating or modifying amino acid residues on the membrane-binding serum protein and determining which residues lead to changes in the level of interaction between the membrane-binding serum protein and a receptor. Binding may be measured using any of the methods described above.

[0064] Where a 3D structure has been solved for the membrane-binding serum protein bound to a receptor, a receptor binding site of the membrane-binding serum protein can be readily identified through identifying amino acid residues that form hydrogen bonds, salt-bridges, electrostatic interactions, or any other non-covalent or covalent bond between the membrane-binding serum protein and a receptor. The receptor binding site identification may be performed using software such as PDBePISA. Where independent 3D structures have been solved for each of the membrane-binding serum protein and a receptor but not together in a bound state, a structure of the membrane-binding serum protein bound to a receptor can be predicted using modelling software such as AlphaFold, PSIPRED or RoseTTAFold, and the output analysed for interacting amino acid residues.

[0065] The following table shows the amino acids positions in various membrane-binding serum proteins which correspond to a receptor binding site.

[0066] The first region may comprise an antibody or a fragment thereof that specifically binds to at least a part of a receptor binding site of a membrane-binding serum protein. Antibodies and antibody fragments are discussed below. The antibody or fragment thereof in the first region may be any of the types discussed below. In some embodiments, the fusion protein is a bispecific antibody which specifically binds to at least a part of a receptor binding site of a membrane-binding serum protein and specifically binds a target molecule. The target molecules may be any of those discussed below.

[0067] The first region preferably comprises or consists of a receptor for a membrane-binding serum protein or a functional fragment thereof. The invention provides a fusion protein comprising (1) a first region comprising a receptor for a membrane-binding serum protein or a functional fragment thereof and (2) a second region comprising a targeting peptide or polypeptide. The receptor may be for any membrane-binding serum protein. The receptor may be for any of the membrane-binding serum proteins discussed above.

[0068] Receptors for the membrane-binding serum proteins discussed above are known in the art. Receptors typically "specifically bind" the corresponding membrane-binding serum protein. The term "specifically binds to" is defined above. Any discussion above with reference to specific binding, including relating to affinity, also applies to the receptor for a membranebinding serum protein. In such discussion above, the term "the peptide or polypeptide" can be replaced with "the receptor for a membrane-binding serum protein" and term "at least a part of a receptor binding site of a membrane-binding serum protein" can be replaced with "the membrane-binding serum protein".

[0069] The receptor for a membrane-binding serum protein is preferably selected from a low- density lipoprotein receptor (LDLR), very low-density lipoprotein receptor (VLDLR), Apo-E receptor 2 (Apo-E-R2), LDLR-related protein 1 (LRP1), LRP1B, LRP4, LRP5, LRP6, lipoprotein receptor 11 (LR11, SorLA or SORL1), Megalin, Integrin-alpha-5, Integrin-beta-5, Lymphocyte antigen 75, Transferrin receptor protein 1, Macrophage scavenger receptor types I and II, Macrophage mannose receptor 1, C-type lectin domain family 9 member A, Stabilin-2, Sortilin-related receptor, Growth hormone receptor, C-type lectin domain family 4 member M, C-type mannose receptor 2, Low-density lipoprotein receptor-related protein

[0070] 12, Low-density lipoprotein receptor-related protein 10, Sortilin, Low-density lipoprotein receptor-related protein 3, Secretory phospholipase A2 receptor, Protein LMBR1L, Cubilin, Asialoglycoprotein receptor 1, Asialoglycoprotein receptor 2, CD209 antigen, and C-type lectin domain family 4 member F. The receptor for a membrane-binding serum protein is preferably a low-density lipoprotein receptor (LDLR).

[0071] Suitable receptors for use in the invention and their accession numbers are shown in the table below. All of the sequences at the accession numbers are incorporated herein in their entireties.

[0072] The first region may comprise or consist of a functional fragment of a receptor for a membrane-binding serum protein. The receptor may be any of those described above. The fragment is functional because it retains binding to the membrane-binding serum protein. The fragment preferably specifically binds to the membrane-binding serum protein. Specific binding is discussed in more detail above and any of the embodiments discussed above equally apply to the functional fragments. Binding or specific binding to the membranebinding serum protein can be measured using known assays including any of those discussed above.

[0073] The functional fragment may be any length as long as it is functional. The functional fragment typically comprises the region responsible for binding or specific binding to the membrane-binding serum protein. The fragment may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the receptor. The functional fragment may be from about 10 to about 150 amino acids in length, such as from about 20 to about 140, from about 30 to about 120, or from about 40 to about 110 amino acids in length. The functional fragment is preferably at least about 10 amino acids in length, such as at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 in length. The receptor for a membrane-binding serum protein or the functional fragment thereof may have structural similarity to the wild-type or native receptor or fragment. The receptor for a membrane-binding serum protein or the functional fragment thereof may be a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with wild-type or native receptor or fragment.

[0074] The receptor for a membrane-binding serum protein or the functional fragment thereof may comprise a variant sequence having at least about 90% homology or identity to the wildtype or native receptor or fragment. In preferred embodiments, the variant sequence comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the wild-type or native receptor or fragment. Homology and / or identity is / are typically measured over the entire length of the reference sequence, typically the wild-type or native receptor or fragment.

[0075] In all instances herein, the term "structural variant" encompasses a structure which is a naturally occurring polymorphic form of the reference structure as well as synthetic variants. The term "structural variant" is interchangeable with "functional structural variant". In all instances herein, the term "variant sequence" encompasses a sequence which is a naturally occurring polymorphic form of the reference sequence as well as synthetic variant sequences in which one or more amino acids are inserted, deleted, and / or substituted. The term "variant sequence" is interchangeable with "functional variant sequence". A functional variant is a structure or sequence which differs from the reference structure or sequence, typically because one or more amino acids are inserted, deleted and / or substituted, and retains at least some functional activity of the reference structure sequence. The functional activity of the variant may be substantially the same or about the same as the reference structure or sequence. The functional activity of the variant may be increased. The functional activity may be increased by any amount, such as by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 90%, at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more. The functional activity of the variant may be decreased. The functional activity may be decreased by any amount, such as by less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2% or less than about 1%. The functional activity is typically specifically binding at least a part of a receptor binding site of a membrane-binding serum protein, acting as receptor for, such as binding or specifically binding, a membrane-binding serum protein, binding or specifically binding a target molecule (as discussed below in relation to the second region), or the targeting activity of a fusion protein of the invention as defined herein. These activities can be measured using any of the methods described herein.

[0076] Standard methods in the art can be used to determine structural similarity, including RMSD. Suitable methods include, but are not limited to, TM-Align, US-Align or FATCAT.

[0077] Standard methods in the art may be used to determine homology or identity. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology or identity, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology and identity or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0078] The first region preferably comprises or consists of a low-density lipoprotein receptor (LDLR) polypeptide or a functional fragment thereof. The functional fragment preferably comprises or consists of at least LDLR A domain 5 (A5) or at least LDLR A domain 4 and A domain 5 (A4-5). The LDLR polypeptide or functional fragment thereof is preferably a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 5 or 18. This can be calculated as described above. The LDLR polypeptide or functional fragment thereof preferably comprises or consists of a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 5 or 18. This allows 10 amino acid differences with the sequence shown in SEQ ID NO: 5, which is 107 amino acids long. This allows 8 amino acid differences with the sequence shown in SEQ ID NO: 18, which is 89 amino acids long. In preferred embodiments, the LDLR polypeptide or functional fragment thereof comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 5 or 18. In preferred embodiments, the LDLR polypeptide or functional fragment thereof comprises or consists of a variant sequence having about 90.65%, about 91.59%, about 92.52%, at least about 93.46%, about 94.39%, about 95.33%, about 96.26%, about 97.20%, about 98.13%, or at least about 99.07% homology or identity to the sequence shown in SEQ ID NO: 5. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 5 or 18. Homology and / or identity can be measured as described above. The LDLR polypeptide or functional fragment thereof preferably comprises or consists of the sequence shown in SEQ ID NO: 5 or 18. In this paragraph, SEQ ID NO: 5 and all related embodiments are preferred over SEQ ID NO: 18 and its embodiments.

[0079] Second region

[0080] The second region comprises or consists of a targeting peptide or polypeptide. The targeting peptide or polypeptide may have any length. The targeting peptide or polypeptide preferably has a length from about 10 amino acids to about 5000 amino acids, such as from about 20 to about 2000, from about 30 to about 1000, from about 40 to about 750 or from about 50 to about 500 amino acids in length. The targeting peptide or polypeptide preferably has a length of at least about 10 amino acids, such as at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 200, at least about 300 or at least about 400 amino acids in length.

[0081] The targeting peptide or polypeptide is typically capable of targeting the fusion protein to a specific location. The targeting peptide or polypeptide is typically capable of targeting the fusion protein to a target cell or target cells. The target cell or target cells may be in vitro or ex vivo. The target cell or target cells may be in vivo. The target cell or target cells may be present in a subject. The subject may be any of those discussed below. The target cell or target cells may be associated with a disease or disorder. The disease or disorder may be any of those discussed below. The target cell or target cells preferably express(es) a target molecule to which the targeting peptide or polypeptide specifically binds.

[0082] The target cell or target cells may be derived from the ectoderm, endoderm, or mesoderm. The target cell or target cells be stem cells, such as embryonic stem cells, induced pluripotent stem cells or mesenchymal stem cells, bone cells, such as osteoclasts, osteoblasts or osteocytes, tendon cells, such as tenoblasts or tenocytes, chondrocytes, synovial cells, vascular cells, blood cells, such as red blood cells, immune cells, platelet, neutrophils or basophils, muscle cells, such as skeletal muscle cells, cardiac muscle cells or smooth muscle cells, reproductive cells, such as sperm, oocytes, duct cells or epididymal cells, secretory cells, adipocytes, liver lipocytes, pancreatic cells, pancreatic beta cells, epithelial cells, odontoblasts, cementoblasts, hormone-secreting cells, barrier cells, exocrine secretory epithelial cells, nerve cells, astrocytes, oligodendrocytes, or neurons.

[0083] The immune cells may be neutrophil granulocyte and precursors, such as myeloblasts, promyelocytes, myelocytes, or metamyelocytes, eosinophil granulocyte and precursors, basophil granulocyte and precursors, mast cells, leukocytes, lymphocytes, helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, B cells, macrophages, dendritic cells, plasma cells, neutrophils, or monocytes. Additional target cells are discussed below. The targeting peptide or polypeptide preferably specifically binds to a target molecule. The target molecule may be any type of molecule. The target molecule may be a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, a monosaccharide, an oligosaccharide, a polysaccharide, or a metabolite. The target molecule is preferably a target peptide or protein. The target molecule may be a target antigen.

[0084] The target molecule is preferably expressed by or is present on a target cell or on target cells. The target molecule is preferably expressed on the surface of a target cell or target cells. The target molecule may be intracellular or may be secreted by the target cell or target cells. The target molecule is preferably internalised by the target cell. This allows the fusion protein, conjugate, construct or amphipathic structure of the invention to be internalised by the target cell. In this manner, any cargo in the amphipathic structure can be delivered to the inside of the target cell.

[0085] The target molecule may be selected from AADAT, AAK1, ABCB1, ABCC3, ACTR6, ADA, ADH1A, ADH1B, ADH1C, AFAP1L2, AHNAK2, AKAP10, AKAP12, ALDH3A1, ANK2, ANXA1, ANXA3, APOLD1, AQP3, AQP4, ARHGAP19, ARHGAP29, ARHGAP36, ARHGEF15, ASH2L, ATP6V1H, ATP8B1, BCR, BFSP1, BLVRB, BMX, BVES, C19orf33, Clorf35, Clorf74, CA9, CALD1, CALN 1, CAPS, CCDC15, CCNYL1, CCR2, CD248, CD38, CD3E, CD44, CD93, CDC42EP1, CDH1, CDH5, CDH6, CDH8, CFL2, CHRM3, CLCN6, CLDN18, CLDN19, CMTM7, COL17A1, CORIN, CPPED1, CRYAB, CRYBG3, CSAD, CSPG4, CST6, CTSV, DAGLB, DCT, DDR2, DLL3, DNAJB13, DOK3, DYSF, EEF2, ENKD1, ENO1, ENO2, ENO3, ENSG00000288684, EPB41L1, EPHB2, EPN1, ERMN, ETNK1, EZR, F2RL1, FAM186A, FAM210B, FBP2, FCGR2A, FCGR2C, FCHSD2, FILIP1, FKBP2, FLRT3, FLT1, FYN, GABRA1, GABRA2, GABRA3, GABRA5, GAP43, GAPDH, GAS7, GDI1, GDI2, GFI1B, GHR, GLRA1, GLRA2, GLRA3, GPR148, GPR160, GPR50, GRAP2, GRID2IP, GRIK5, GRPR, GTSE1, HFE, HMGCS1, HPS3, HS6ST3, HYI, ICAM1, IFRD1, IL10RA, IL7R, INVS, IRS1, ITGA3, ITGB7, JAK2, JAML, KCNK17, KIT, KLHL41, KLK7, KLRC1, KLRC2, L1CAM, LCP1, LEMD1, LIPI, LOXHD1, LY9, LZTS2, MAP3K7, MCEMP1, MEGF10, MISP, MLIP, MLKL, MMP10, MPP1, MS4A1, MYH9, MYO1G, MYO3A, NCAM2, NCOA1, NDRG4, NECTIN4, NFAM1, NKTR, NRP2, NRXN1, OTOP2, PAIP1, PALLD, PALM2AKAP2, PARD3, PARP6, PCDHA12, PCDHGB2, PDE3A, PDE4A, PDPN, PHGDH, PIGW, PIK3AP1, PLEKHA6, PPL, PPP3R1, PPP3R2, PRAME, PRKCZ, PYGL, RAD51C, RASA4, RASA4B, RASGRP2, RDX, RET, RGR, RHEX, RHOBTB2, RHOXF2, RHOXF2B, RIF1, RIGI, RIMS2, RNF146, ROBO2, ROM1, RPIA, SAMSN1, SC5D, SCEL, SCIN, SCN8A, SCN9A, SGCE, SHKBP1, SHROOM2, SIPA1L3, SLC13A5, SLC16A1, SLC1A6, SLC2A1, SLC2A2, SLC3A2, SLC5A6, SLC9A3, SLCO1A2, SLCO1B3, SLCO4C1, SMPX, SPN, SPRYD3, SRC, SRD5A3, SUMF2, SYNPO, SYT4, TACR2, TACSTD2, TASP1, TDP1, TEK, TEX9, TGFBR2, TGM2, TMEM101, TNFRSF21, TNFRSF8, TNKS1BP1, TNNI2, TOGARAM2, TRAT1, TRIBI, TRPM7, TSHZ3, TTLL5, TXNDC11, TYW3, UAP1, UBA3, UBE2C, USP2, VCPIP1, VTCN1, WDPCP, ZDHHC5, ZNF536AADAT, AAK1, ABCB1, ABCC3, ACTR6, ADA, ADH1A, ADH1B, ADH1C, AFAP1L2, AHNAK2, AKAP10, AKAP12, ALDH3A1, ANK2, ANXA1, ANXA3, APOLD1, AQP3, AQP4, ARHGAP19, ARHGAP29, ARHGAP36, ARHGEF15, ASH2L, ATP6V1H, ATP8B1, BCR, BFSP1, BLVRB, BMX, BVES, C19orf33, Clorf35, Clorf74, CA9, CALD1, CALN1, CAPS, CCDC15, CCNYL1, CCR2, CD248, CD38, CD3E, CD44, CD93, CDC42EP1, CDH1, CDH5, CDH6, CDH8, CFL2, CHRM3, CLCN6, CLDN18, CLDN19, CMTM7, COL17A1, CORIN, CPPED1, CRYAB, CRYBG3, CSAD, CSPG4, CST6, CTSV, DAGLB, DCT, DDR2, DLL3, DNAJB13, DOK3, DYSF, EEF2, ENKD1, ENO1, ENO2, ENO3, ENSG00000288684, EPB41L1, EPHB2, EPN1, ERMN, ETNK1, EZR, F2RL1, FAM186A, FAM210B, FBP2, FCGR2A, FCGR2C, FCHSD2, FILIP1, FKBP2, FLRT3, FLT1, FYN, GABRA1, GABRA2, GABRA3, GABRA5, GAP43, GAPDH, GAS7, GDI1, GDI2, GFI1B, GHR, GLRA1, GLRA2, GLRA3, GPR148, GPR160, GPR50, GRAP2, GRID2IP, GRIK5, GRPR, GTSE1, HFE, HMGCS1, HPS3, HS6ST3, HYI, ICAM1, IFRD1, IL10RA, IL7R, INVS, IRS1, ITGA3, ITGB7, JAK2, JAML, KCNK17, KIT, KLHL41, KLK7, KLRC1, KLRC2, L1CAM, LCP1, LEMD1, LIPI, LOXHD1, LY9, LZTS2, MAP3K7, MCEMP1, MEGF10, MISP, MLIP, MLKL, MMP10, MPP1, MS4A1, MYH9, MYO1G, MYO3A, NCAM2, NCOA1, NDRG4, NECTIN4, NFAM1, NKTR, NRP2, NRXN 1, OTOP2, PAIP1, PALLD, PALM2AKAP2, PARD3, PARP6, PCDHA12, PCDHGB2, PDE3A, PDE4A, PDPN, PHGDH, PIGW, PIK3AP1, PLEKHA6, PPL, PPP3R1, PPP3R2, PRAME, PRKCZ, PYGL, RAD51C, RASA4, RASA4B, RASGRP2, RDX, RET, RGR, RHEX, RHOBTB2, RHOXF2, RHOXF2B, RIF1, RIGI, RIMS2, RNF146, ROBO2, ROM1, RPIA, SAMSN1, SC5D, SCEL, SCIN, SCN8A, SCN9A, SGCE, SHKBP1, SHROOM2, SIPA1L3, SLC13A5, SLC16A1, SLC1A6, SLC2A1, SLC2A2, SLC3A2, SLC5A6, SLC9A3, SLCO1A2, SLCO1B3, SLCO4C1, SMPX, SPN, SPRYD3, SRC, SRD5A3, SUMF2, SYNPO, SYT4, TACR2, TACSTD2, TASP1, TDP1, TEK, TEX9, TGFBR2, TGM2, TMEM101, TNFRSF21, TNFRSF8, TNKS1BP1, TNNI2, TOGARAM2, TRAT1, TRIBI, TRPM7, TSHZ3, TTLL5, TXNDC11, TYW3, UAP1, UBA3, UBE2C, USP2, VCPIP1, VTCN1, WDPCP, ZDHHC5, and ZNF536.

[0086] The target molecule is preferably expressed by immune cells. The target molecule is preferably expressed by leukocytes, The target molecule is preferably selected from CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD47, CD147, CD117, CD38, integrin 7.

[0087] The targeting molecule is preferably selected from CD8, TCR alpha, TCR beta, CD10, CD33, CD34, CD68, CD19, CD62L, CD25, CXCR3, CCR2, CCR3, CCR4, CCR5, CCR6, or CCR7.

[0088] The target molecule is preferably expressed by B cells. The target molecule is preferably selected from CD38, CD138, CD10, CD19, CD38 and CD20.

[0089] The target molecule is preferably expressed by T cells. The target molecule is preferably selected from CCR5, CXCR3B, CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR)p, TCR-a, TCR-a / p, TCR-y / 5, PD1 , CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CDI la, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor The target molecule is preferably expressed by monocytes and macrophages. The target molecule is preferably selected from Cl lb, CDl lc, CD14 and CD206.

[0090] The target molecule is preferably expressed by granulocytes. The target molecule is preferably selected from CD44, CD107a, CD23, integrin alpha-4, CDl lb, CDl lc and CD14.

[0091] The target molecule is preferably expressed by dendritic cells. The target molecule is preferably selected from CD21, CD23, DEC205 and clusterin.

[0092] The target molecule is preferably expressed by mast cells. The target molecule is preferably selected from CD9, CD25, CCD44, integrin alpha-4, integrin beta-1 and CD71.

[0093] The target molecule is preferably expressed by megakaryocytes. The target molecule is preferably selected from CD36, CD41, CD42b, integrin beta-3, and P-selectin.

[0094] The target molecule is preferably expressed by natural killer (NK) cells. The target molecule is preferably selected from CDl lc, CD16 and CD56.

[0095] The target molecule is preferably expressed by Myeloid-Derived Suppressor Cells (MDSCs). The target molecule is preferably selected from CDllb, CDllb, CD14, CD33 and VEGFR-1.

[0096] The target molecule is preferably expressed by hematopoietic stem cells (HSCs). The target molecule is preferably selected from CD45, CD90 and CD117.

[0097] The target molecule is preferably associated with a disease or disorder. The disease or disorder may be any of those discussed below.

[0098] The target molecule is preferably associated with cancer. The cancer may be any of those discussed below. The target molecule or target antigen may be selected from a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, Wilms' tumour gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), CD19, p53, cyclin (DI), CD38, B-cell maturation antigen (BCMA, BCM), B-cell activating factor receptor (BAFFR, BR3), transmembrane activator and CAML interactor (TACI) or a related protein thereof, CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, alpha folate receptor, 5T4, DvD6 integrin, B7-H3, B7-H6, CAIX, CD20, CD22, CD33, CD44, CD44v6,

[0099] CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, Claudin 6, Claudin 18.2, CSPG4, CMV, CSF1R, EBV, EGFR, EGFR family including ErbB2 (HER2), ErbB family homo and heterodimers, EGFRvIII, EGP2, EGP40, EPCAM, EphA2, FAP, foetal AchR, GD2, GD3, Glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, HPV, IL-llR.alpha., IL-13R.alpha.2, Lambda, Lewis-Y, Kappa, Mesothelin, NOAM, PRAME, PSCA, ROR1, SSX, TAG72, TEMs, UPAR, VEGFR2, TSHR, CS-1, CLL-1, Tn Ag, FLT3, KIT, IL-13Ra2, IL-llRa, PRSS21, CD24, PDGFR- beta, SSEA-4, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, LMP2, gplOO, bcr-abl, tyrosinase, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, LAGE-la, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, prostein, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, and an NKG2D ligand. The NKG2D ligand may be selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0100] The target molecule is preferably associated with diabetes and / or obesity. The target molecule is preferably selected from GIPR, GLP1R, and ZnT8.

[0101] The target molecule may be CD38.

[0102] The targeting peptide or polypeptide typically specifically binds to the target molecule. The targeting peptide or polypeptide may specifically bind to the whole or part of the target molecule. The targeting peptide or polypeptide preferably specifically binds to a target epitope on a target molecule or target antigen.

[0103] The term "specifically binds to" is defined above. Any discussion above with reference to specific binding, including relating to affinity, also applies to the targeting peptide or polypeptide. In such discussion above, the term "the peptide or polypeptide" can be replaced with "the targeting peptide or polypeptide" and term "at least a part of a receptor binding site of a membrane-binding serum protein" can be replaced with "the target molecule / epitope / antigen". a target molecule or a target epitope on a target molecule means binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. Such methods are routine in the art. All instances herein of the term "specifically binds to" is interchangeable with "specifically interacts with," "specific for," "selectively binds to" "selectively interacts with" and "selective for". The targeting peptide or polypeptide specifically binds to target molecule if it binds to the target molecule with preferential or high affinity, but does not bind or binds with only low affinity to other or different molecules, such as other or different peptides, other or different polypeptides or proteins or other or different polynucleotides. Preferably, the targeting peptide or polypeptide binds to the target molecule with an affinity that is at least about 10 times, such as at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1000 or at least about 10,000 times, greater than its affinity for other molecules.

[0104] The targeting peptide or polypeptide has affinity for the target molecule. The targeting peptide or polypeptide preferably has high affinity for the target molecule. The targeting peptide or polypeptide has high affinity for the target molecule if it binds with a Kd of about 1 x IO-6M or less, such as about 1 x IO-7M or less, about 5 x IO-8M or less, about 1 x IO-8M or less, or about 5 x IO-9M or less. The targeting peptide or polypeptide preferably has a Kd for the target molecule of about 100 nM or less, such as about 50 nM or less, about 20 nM or less or about 10 nM or less.

[0105] Affinity can be measured using any of the assays discussed above. Kd values for antibodies can be determined using surface plasmon resonance, such as a Biacore® system, or solution equilibrium titration (SET) (see Friguet, et al., (1985) J. Immunol. Methods, 77(2):305-319, and Hanel et al., (2005) Anal. Biochem., 339(1): 182-184).

[0106] The targeting peptide or polypeptide is preferably a protein, an antibody or a fragment thereof, or an enzyme. These may be known as a targeting protein, targeting antibody, or targeting enzyme. The skilled person is capable of designing targeting proteins, targeting antibodies, or targeting enzymes that function according to the invention.

[0107] The targeting protein preferably binds or specifically binds a target molecule. The targeting protein may be a receptor or fragments thereof. Receptors and fragments are discussed above with reference to the first region. The receptor may be for any of the target molecules discussed above.

[0108] The receptor may be a cellular adhesion molecule (CAM). The CAM may be an member of the immunoglobulin super family of cell adhesion molecules (IgCAMs), a cadherin, an integrins, or a member of the superfamily of C-type of lectin-like domains proteins (CTLDs).

[0109] The IgCAM may be selected from a Neural Cell Adhesion Molecule (NCAM), Intercellular Cell Adhesion Molecule (ICAM-1), Vascular Cell Adhesion Molecule (VCAM-1), Platelet-endothelial Cell Adhesion Molecule (PECAM-1), Mucosal Vascular Addressin Cell Adhesion Molecule (MAdCAM-1), the LI family including LI (protein), CHL1, Neurofascin and NrCAM, the SIGLEC family including Myelin-associated glycoprotein (MAG, SIGLEC-4), CD22 and CD83, the CTX family including CTX, Junctional adhesion molecule (JAM), BT-IgSF, Coxsackie virus and adenovirus receptor (CAR), VSIG and ESAM, Nectins and related proteins including CADM1 and other Synaptic Cell Adhesion Molecules, CD2, CD48, HEPACAM, HEPACAM2 and Down syndrome cell adhesion molecule (DSCAM).

[0110] The cadherin may be selected from CDH1 (E-cadherin), CDH2 (N-cadherin), CDH12 (cadherin 12, type 2 (N-cadherin 2)), CDH3 (P-cadherin), Desmoglein 1 (DSG1), DSG2, DSG3, DSG4, Desmocollin 1 (DSC1), DSC2, DSC3, PCDH1, PCDH7, PCDH8, PCDH9, PCDH10, PCDH11X / 11Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20, PCDHA1, PCDHA2, PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHAC1, PCDHAC2, PCDHB1, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, PCDHB8, PCDHB9, PCDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB17, PCDHB18, PCDHGA1, PCDHGA2, PCDHGA3, PCDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGB1, PCDHGB2, PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2, FAT4, CDH4 (R-cadherin), CDH5 (VE- cadherin), CDH6 (K-cadherin), CDH7 (cadherin 7, type 2), CDH8 (cadherin 8, type 2), CDH9 (cadherin 9, type 2 (Tl-cadherin)), CDH10 (cadherin 10, type 2 (T2-cadherin)), CDH11 (OB-cadherin (osteoblast)), CDH13 (T-cadherin), CDH15 (M-cadherin), CDH16 (KSP- cadherin, CDH17 (LI cadherin), CDH18 (cadherin 18, type 2), CDH19 (cadherin 19, type 2), CDH20 (cadherin 20, type 2), CDH23 (cadherin 23), CDH22, CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2,, LOC389118, PCLKC, RESDA1, and RET.

[0111] The alpha subunits of the integrin may be selected from CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD11D, CD103, CDlla, CDllb, CD51, CD41 and CDl lc. The beta subunits in the integrin may be selected from CD29, CD18, CD61, CD104, ITGB5, ITGB6, ITGB7 and ITGB8. The integrin may be selected from Oipi, O2pi, O3P1, 04P1, O4P7, O5P1, O6P1, o7|31, oL|32, oM|32, ollb|33, avpi, aV|33, aV|35, aVP6, aVP8 and a6[34.

[0112] The CTLD may be selected from the mannose receptor (MR), the classic asialoglycoprotein receptor macrophage galactose-type lectin (MGL), DC- SIGN (CLEC4L), Langerin (CLEC4K), Myeloid DAP12-associating lectin (MDL)-l (CLEC5A), DC-associated C-type lectin 1 (Dectin 1) subfamily including dectin 1 / CLEC7A, DNGR1 / CLEC9A, Myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (also called CLEC1B) and CLEC12B, and the DC immunoreceptor (DCIR) subfamily, including DCIR / CLEC4A, Dectin 2 / CLEC6A, Blood DC antigen 2 (BDCA2) (CLEC4C), and macrophage-inducible C-type lectin (Mincle or CLEC4E).

[0113] The antibody may specifically bind any target antigen. Suitable target antigens may be any of those discussed above. The term "antibody" as referred to herein includes whole antibodies. Naturally occurring antibodies typically comprise a tetramer which is usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, usually comprised of three domains (CHI, CH2 and CH3). Heavy chains can be of any isotype, including IgG (IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (IgAl and IgA2 subtypes), IgM and IgE. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Light chain includes kappa (K) chains and lambda (A) chains. The heavy and light chain variable region is typically responsible for antigen recognition, whilst the heavy and light chain constant region may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0114] The term "fragment thereof" with reference to an antibody refers to any fragment of an intact antibody. Such fragments include, but are not limited to, an antibody heavy chain, the variable region of an antibody heavy chain (VH), an antibody light chain and the variable region of an antibody light chain (VL). The fragment retains the ability to specifically bind to a target antigen, such as CD38, although in some instances an accessory protein is required for specific binding. For instance, if the fragment is an antibody heavy chain or the variable region of an antibody heavy chain (VH), an antibody light chain or the variable region of an antibody light chain (VL) is needed for specific binding. The antibody light chain or the variable region of an antibody light chain (VL) need not form part of the fusion protein of the invention and can be supplied or expressed separately so that the fusion protein forms an antibody or functional fragment thereof capable of specific binding to a target antigen.

[0115] The fragment is preferably a functional fragment. The second region preferably comprises a functional antibody fragment. The term "functional fragment" with reference to an antibody, including a "functional antibody fragment", refers to a fragment of an intact antibody that retains the ability to specifically bind to a target antigen, such as CD38. Such fragments include, but are not limited to, Fab fragments, Fab' fragments, monovalent fragments consisting of the VL, VH, CL and CHI domains; F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragment consisting of the VH and CHI domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 : 544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). 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); see e.g. Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "functional fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0116] The antibody, fragment thereof or functional fragment thereof typically comprises one or more, such as 6 or 12, complementarity determining regions (CDRs). CDRs are defined according to the Kabat definition unless specified that the CDR are defined according to another definition. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al- Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme). For example, for classic formats, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to "Kabat"). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0117] By convention, the CDR regions in the heavy chain are typically referred to as HCDR1, HCDR2 and HCDR3 and in the light chain as LCDR1, LCDR2 and LCDR3. They are numbered sequentially in the direction from the amino terminus to the carboxy terminus. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. The targeting peptide or polypeptide may be a monoclonal antibody, a fragment thereof or a functional fragment thereof.

[0118] The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g. human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik, et al., (2000) J Mol Biol; 296:57-86). The targeting peptide or polypeptide may be a human antibody, a fragment thereof or a functional fragment thereof.

[0119] The human antibody may include amino acid residues not encoded by human sequences (e.g. mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0120] An antibody can be prepared using an antibody having one or more of the VH and / or VL sequences shown herein as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and / or VL), for example within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

[0121] One type of variable region engineering that can be performed is antibody binding region / paratope or CDR grafting. Because paratope sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR / paratope sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522- 525; Queen, C. et al., 1989 Proc. Natl. Acad. See. U.S.A. 86: 10029-10033; US5,225,539, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370). Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc- cpe.cam.ac.uk / vbase), as well as in 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; Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. J Immunol. 24:827-836.

[0122] An example of framework sequences for use in the antibodies are those that are structurally similar to the framework sequences used by selected antibodies of the disclosure, e.g., consensus sequences and / or framework sequences used by monoclonal antibodies of the disclosure. The VHCDR1, 2 and 3 sequences, and the VLCDR1, 2 and 3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0123] Another type of variable region modification is to mutate amino acid residues within the VHand / or VLCDR1, CDR2 and / or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation." Site- directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. Conservative modifications (as discussed above) can be introduced. The mutations may be amino acid substitutions, additions, or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

[0124] A wide variety of antibody / immunoglobulin frameworks or scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which specifically binds to NPR1. Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof (such as those disclosed elsewhere herein), and include immunoglobulins of other animal species, preferably having humanized aspects. Single heavy-chain antibodies such as those identified in camelids are of particular interest in this regard. Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art. Antibody proteins obtained from members of the camel and dromedary {Camelus bactrianus and Camelus dromaderius) family including new world members such as llama species {Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals, see WO94 / 04678.

[0125] A region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "camelid nanobody". See US5,759,808; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256- 1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be "humanized". Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.

[0126] The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

[0127] The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitate drug transport across the blood brain barrier, see US2004 / 0161738. These features combined with the low antigenicity to humans indicate great therapeutic potential. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins with bacteriophage and are functional.

[0128] The targeting peptide or polypeptide may be a camelid antibody or nanobody, a fragment thereof or a functional fragment thereof. In one embodiment, the camelid antibody, nanobody, fragment thereof or a functional fragment thereof is obtained by grafting the CDRs sequences of the heavy or light chain of a human antibody into nanobody or single domain antibody framework sequences, as described for example in WO94 / 04678.

[0129] The antibody, fragment thereof or functional fragment thereof may comprise nonimmunoglobulin frameworks. Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, Adnectins (fibronectin) (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA), now part of GSK) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA, now Emergent BioSolutions), maxybodies (Avidia, Inc. (Mountain View, CA)), Protein A (Affibody AB, Sweden) and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

[0130] The adnectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (US6,818,418).

[0131] These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprises the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the disclosure using standard cloning techniques.

[0132] Ankyrin technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel o- helices and a 0-turn. Binding of the variable regions is mostly optimized by using ribosome display.

[0133] Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in humans over 250 proteins are structurally based on A-domains. Avimers consist of a number of different "A- domain" monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, US20040175756 US20050053973 US20050048512 and US20060008844.

[0134] Affibody® affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants (See e.g., US5,831,012). Affibody® molecules mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of Affibody® molecules is similar to that of an antibody.

[0135] Anticalins® are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids.

[0136] The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being just marginally bigger than a single immunoglobulin domain.

[0137] The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity.

[0138] One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing "anticalins" is WO1999 / 16873.

[0139] Affilin™ molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New Affilin™ molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin™ molecules do not show any structural homology to immunoglobulin proteins. Scil Proteins employs two Affilin™ scaffolds, one of which is gamma crystalline, a human structural eye lens protein and the other is "ubiquitin" superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of gamma crystalline derived proteins are described in W02001 / 04144 and examples of "ubiquitin-like" proteins are described in W02004 / 106368.

[0140] PEM are medium-sized, cyclic, peptide-like molecules (MW l-2kDa) mimicking beta-hairpin secondary structures of proteins, the major secondary structure involved in protein-protein interactions. More generally, any polypeptide that mimicks the 3D structure of the epitope of the disclosed antibodies are part of the present disclosure.

[0141] These polypeptides may further be engineered to increase half-life or improve solubility. Especially, fusion constructs of these polypeptides with serum proteins, such as Fc fragments of IgG or human serum albumin can be generated to increase half-life, similarly to Fc engineering described in the following paragraph for antibody fragment molecules of the disclosure.

[0142] Engineered antibodies include those in which modifications have been made to framework residues within VH and / or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site- directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the disclosure.

[0143] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in US2003 / 0153043.

[0144] In addition or alternative to modifications made within the framework or CDR regions, antibodies may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0145] In one embodiment, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in US5,677,425. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0146] In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in US6, 165,745.

[0147] In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in US5,869,046 and US6,121,022.

[0148] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigenbinding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in US5,624,821 and US5,648,260. In particular, residues 234 and / or 235 may be mutated, optionally to alanine. Thus, in one embodiment, an antibody according to the disclosure has a mutation in the Fc region at one or both of amino acids 234 and 235. Substitution of both amino acids 234 and 235 results in reduced ADCC activity.

[0149] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in US6,194,551. In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in WO94 / 29351.

[0150] In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids. This approach is described further in WO00 / 42072. Moreover, the binding sites on human IgGl for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chen. 276:6591-6604).

[0151] In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen". Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in US5,714,350 and US6,350,861.

[0152] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fuco sy I residues or an antibody having increased bisecting GIcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. WO03 / 035835 describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). WO99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GIcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17: 176-180). Another modification of the antibodies herein that is contemplated by the disclosure is pegylation. An antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody.

[0153] Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See for example, EP0154316 and EP0401384.

[0154] The antibody may have any of the Kd values described above for the targeting peptide or polypeptide. The antibody preferably has a Kd of about 100 x 10-9M (lOOnM) or less, such as about 10 x 10-9M (lOnM) or less. Antibody Kd may be measured as described above.

[0155] The antibody fragment is preferably a single chain antibody. The second region preferably comprises a single chain antibody. The single chain antibody may be any of those described above, including a single-chain variable fragment (scFv). The scFv is typically a fusion protein comprising the variable regions of an antibody heavy chain (VH) and light chain (VL). The VH and VL are typically fused by a linker of about 10 to about 25 amino acids. The single chain antibody preferably binds or specifically binds a target molecule. The target molecule may be any of those discussed above. This embodiment allows the fusion protein of the invention itself to target, i.e., is capable of targeting to a specific location without any accessory molecule.

[0156] The antibody fragment is preferably an antibody heavy chain or the variable region of an antibody heavy chain (VH). The second region preferably comprises an antibody heavy chain or a VH. The heavy chain or VH may be any of those discussed above. The antibody heavy chain or the VH is preferably capable of binding or specifically binding a target molecule. The target molecule may be any of those discussed above. The fusion proteins in this embodiment are typically used in conjunction with an antibody light chain or a variable region of an antibody light chain (VL). The antibody heavy chain in the fusion protein and the antibody light chain together form half an intact antibody. The VH in the fusion protein and the VL together form a Fab fragment. The antibody light chain or the VL may be provided or expressed separately from the fusion protein comprising an antibody heavy chain or VH. The antibody is preferably a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with any one of SEQ ID NOs: 1-4, 16 or 17, preferably any one of SEQ ID NOs: 1, 3 and 16. This can be calculated as described above.

[0157] The antibody is preferably a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with any one of SEQ ID NOs: 1-4, 16, 17, 36 and 37. This can be calculated as described above.

[0158] The antibody preferably comprises or consists of the sequence shown in any one of SEQ ID NOs: 1-4, 16 or 17, preferably any one of SEQ ID NOs: 1, 3 and 16, or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 1-4, 16 or 17, preferably any one of SEQ ID NOs: 1, 3 and 16, such as at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 1-4, 16 or 17, preferably any one of SEQ ID NOs: 1, 3 and 16. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 1-4, 16 or 17, preferably any one of SEQ ID NOs: 1, 3 and 16. Homology and / or identity may be measured as described above.

[0159] The antibody preferably comprises or consists of the sequence shown in any one of SEQ ID NOs: 1-4, 16, 17, 36 and 37, preferably any one of SEQ ID NOs: 1, 3 and 16, or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 1-4, 16, 17, 36 or 37, preferably any one of SEQ ID NOs: 1, 3 and 16, such as at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 1-4, 16, 17, 36 or 37, preferably any one of SEQ ID NOs: 1, 3 and 16. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 1-4, 16, 17, 36 or 37, preferably any one of SEQ ID NOs: 1, 3 and 16. Homology and / or identity may be measured as described above.

[0160] The embodiments based on SEQ ID NO: 1 and 3 are most preferred.

[0161] Fusion of regions

[0162] The first region and the second region may be fused in any order. The fusion protein may comprise from N to C terminus (1) the first region and (2) the second region. The fusion protein may comprise from N to C terminus (2) the second region and (1) the first region. The first region and second region may be fused in any way. The first region and the second region are preferably genetically fused. This means the fusion protein is expressed from a single polynucleotide expressing the fusion protein. Polynucleotides and their expression are described in more detail below.

[0163] The first region and second region may be fused by a linker. The linker is preferably an amino acid sequence. Suitable amino acid linkers, such as peptide linkers, are known in the art. The length, flexibility and hydrophilicity of the amino acid or peptide linker are typically designed such each region in the fusion protein can perform its function. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and / or glycine amino acids. More preferred flexible linkers include (SG)i, (SG)2, (SG)3, (SG)4, (SG)5, (SG)8, (SG)io, (SG)i5or (SG)2O wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigid linkers include (P)i2wherein P is proline.

[0164] The linker is preferably a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 14 or 15. This can be calculated as described above.

[0165] The linker preferably comprises or consists of the sequence shown in SEQ ID NO: 14 or 15 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 14 or 15, such as at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 14 or 15. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 14 or 15. Homology and / or identity may be measured as described above.

[0166] Preferred fusion protein

[0167] The fusion protein is preferably a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 7. This can be calculated as described above.

[0168] The fusion protein preferably comprises or consists of the sequence shown in SEQ ID NO: 7 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 7. In preferred embodiments, the fusion protein comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 7. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 7. Homology and / or identity can be measured as described above. The fusion protein preferably comprises or consists of the sequence shown in SEQ ID NO: 7. Fusion proteins based on the sequence shown in SEQ ID NO: 7 are typically used in constructs comprising the sequence shown in SEQ ID NO: 9 or variant sequences thereof.

[0169] The fusion protein is preferably a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 11. This can be calculated as described above.

[0170] The fusion protein preferably comprises or consists of the sequence shown in SEQ ID NO: 11 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 11. In preferred embodiments, the fusion protein comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 11. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 11. Homology and / or identity can be measured as described above. The fusion protein preferably comprises or consists of the sequence shown in SEQ ID NO: 11. Fusion proteins based on the sequence shown in SEQ ID NO: 11 are typically used in constructs comprising the sequence shown in SEQ ID NO: 13 or variant sequences thereof.

[0171] The fusion protein is preferably a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 20, 24 or 28. This can be calculated as described above.

[0172] The fusion protein preferably comprises or consists of the sequence shown in SEQ ID NO: 20, 24 or 28 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 20, 24 or 28. In preferred embodiments, the fusion protein comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 20, 24 or 28. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 20, 24 or 28. Homology and / or identity can be measured as described above. The fusion protein preferably comprises or consists of the sequence shown in SEQ ID NO: 20, 24 or 28. Fusion proteins based on the sequence shown in SEQ ID NO: 20 or 24 are typically used in constructs comprising the sequence shown in SEQ ID NO: 22 or 26 or variant sequences thereof.

[0173] The fusion protein is preferably a structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 39. This can be calculated as described above.

[0174] The fusion protein preferably comprises or consists of the sequence shown in SEQ ID NO: 39 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 39. In preferred embodiments, the fusion protein comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 39. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 39. Homology and / or identity can be measured as described above. The fusion protein preferably comprises or consists of the sequence shown in SEQ ID NO: 39. Fusion proteins based on the sequence shown in SEQ ID NO: 39 are typically used in constructs comprising the sequence shown in SEQ ID NO: 41 or variant sequences thereof.

[0175] Production of fusion proteins

[0176] The fusion protein of the invention may be modified in any way. Methods for introducing or substituting non-naturally occurring amino acids in proteins are also well known in the art. The fusion protein may also be produced using D-amino acids or a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.

[0177] Any fusion protein of the invention may be chemically modified. The fusion protein can be chemically modified in any way and at any site. The fusion protein may be chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art. The protein may be chemically modified by the attachment of any molecule, such as a dye or a fluorophore. Any of the fusion proteins may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of a signal sequence to promote their secretion from a cell where the protein does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the protein. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the protein.

[0178] Any of the fusion proteins may be labelled with a revealing label. The revealing label may be any suitable label which allows the fusion protein to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g., 1251, 35S, enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin.

[0179] The fusion protein may also contain other non-specific modifications as long as they do not interfere with the function of the protein. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the protein(s). Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidation with methylacetimidate or acylation with acetic anhydride.

[0180] Any of the fusion proteins can be produced using standard methods known in the art. Polynucleotide sequences encoding a protein may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a protein may be expressed in a bacterial host cell using standard techniques in the art. The protein may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0181] Proteins may be produced in large scale following purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system, and the Gilson HPLC system.

[0182] Conjugates

[0183] The invention also provides a conjugate comprising (a) a fusion protein of the invention, wherein the second region comprises an antibody heavy chain or the variable region of an antibody heavy chain (VH) and (b) an antibody light chain or a variable region of an antibody light chain (VL). The conjugate preferably comprises (a) a fusion protein of the invention, wherein the second region comprises an antibody heavy chain and (b) an antibody light chain. The conjugate preferably comprises (a) a fusion protein of the invention, wherein the second region comprises a VH and (b) a VL.

[0184] The antibody heavy chain and antibody light chain together form half an intact antibody. The VH and VL together form a Fab fragment. Any of the embodiments discussed above with reference to the fusion protein of the invention equally apply to the conjugates of the invention.

[0185] Components (a) and (b) in the conjugate may be separate or may be linked. Methods for linking sections of antibodies are known in the art. Components (a) and (b) in the conjugate may be expressed from a single polynucleotide or separate polynucleotides. This is discussed in more detail below.

[0186] The conjugate of the invention preferably comprises (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 7 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 9.

[0187] The conjugate of the invention preferably comprises (a) a fusion protein comprising the sequence shown in SEQ ID NO: 7 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 7 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 9 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 9. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 7 and / or 9. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 7 and / or 9.

[0188] The conjugate of the invention preferably comprises (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 11 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 13. The conjugate of the invention preferably comprises (a) a fusion protein comprising the sequence shown in SEQ ID NO: 11 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 11 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 13 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 13. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 11 and / or 13. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 11 and / or 13.

[0189] The conjugate of the invention preferably comprises (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 20 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 22.

[0190] The conjugate of the invention preferably comprises (a) a fusion protein comprising the sequence shown in SEQ ID NO: 20 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 20 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 22 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 22. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 20 and / or 22. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 20 and / or 22.

[0191] The conjugate of the invention preferably comprises (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 24 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 26. The conjugate of the invention preferably comprises (a) a fusion protein comprising the sequence shown in SEQ ID NO: 24 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 24 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 26 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 26. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 24 and / or 26. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 24 and / or 26.

[0192] The conjugate of the invention preferably comprises (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 39 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 41.

[0193] The conjugate of the invention preferably comprises (a) a fusion protein comprising the sequence shown in SEQ ID NO: 39 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 39 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 41 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 41. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 39 and / or 41. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 39 and / or 41.

[0194] Constructs

[0195] The invention also provides a construct comprising two conjugates of the invention. The construct comprises two fusion proteins each comprising an antibody heavy chain or the variable region of an antibody heavy chain (VH). The construct also comprises two antibody light chains or two variable regions of an antibody light chain (VLs). The construct preferably comprises two conjugates of the invention each comprising a fusion protein of the invention, wherein the second region comprises an antibody heavy chain, and an antibody light chain. The construct preferably comprises two conjugates of the invention each comprising a fusion protein of the invention, wherein the second region comprises a VH, and a VL.

[0196] The two antibody heavy chains form a complete antibody together with the two antibody light chains. The two VHs form a F(ab')2 fragment together with two VLs. The two conjugates may be the same or different.

[0197] The construct of the invention preferably two conjugates each comprising (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 7 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 9.

[0198] The construct of the invention preferably comprises two conjugates each comprising (a) a fusion protein comprising the sequence shown in SEQ ID NO: 7 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 7 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 9 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 9. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 7 and / or 9. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 7 and / or 9. The two conjugates in the construct may be the same or may be different.

[0199] The construct of the invention preferably two conjugates each comprising (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 11 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 13.

[0200] The construct of the invention preferably comprises two conjugates each comprising (a) a fusion protein comprising the sequence shown in SEQ ID NO: 11 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 11 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 13 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 13. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 11 and / or 13. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 11 and / or 13. The two conjugates in the construct may be the same or may be different.

[0201] The construct of the invention preferably two conjugates each comprising (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 20 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 22.

[0202] The construct of the invention preferably comprises two conjugates each comprising (a) a fusion protein comprising the sequence shown in SEQ ID NO: 20 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 20 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 22 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 22. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 20 and / or 22. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 20 and / or 22. The two conjugates in the construct may be the same or may be different.

[0203] The construct of the invention preferably two conjugates each comprising (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 24 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 26.

[0204] The construct of the invention preferably comprises two conjugates each comprising (a) a fusion protein comprising the sequence shown in SEQ ID NO: 24 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 24 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 26 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 26. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 24 and / or 26. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 24 and / or 26. The two conjugates in the construct may be the same or may be different.

[0205] The construct of the invention preferably two conjugates each comprising (a) a fusion protein structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 39 and (b) an antibody light chain structural variant having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with SEQ ID NO: 41.

[0206] The construct of the invention preferably comprises two conjugates each comprising (a) a fusion protein comprising the sequence shown in SEQ ID NO: 39 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 39 and (b) an antibody light chain comprising the sequence shown in SEQ ID NO: 41 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 41. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 39 and / or 41. The variant sequence is typically a functional variant sequence retaining the function of the reference sequence. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 39 and / or 41. The two conjugates in the construct may be the same or may be different.

[0207] Polynucleotides

[0208] The invention also provides one or more polynucleotides encoding a fusion protein of the invention, a conjugate of the invention, or a construct of the invention. The invention also provides a polynucleotide encoding a fusion protein of the invention, a conjugate of the invention, or a construct of the invention. The invention also provides two polynucleotides encoding a fusion protein of the invention, a conjugate of the invention, or a construct of the invention, wherein one polynucleotide encodes component (a) in the conjugate and the other polynucleotide encodes component (b) in the conjugate.

[0209] The one or more polynucleotides preferably comprise the sequence shown in SEQ ID NO: 6 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 6. The variant sequence may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 6. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 6.

[0210] The one or more polynucleotides preferably comprises (a) the sequence shown in SEQ ID NO: 6 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 6 and (b) the sequence shown in SEQ ID NO: 8 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 8. The one or more polynucleotides preferably comprise (a) one polynucleotide comprising the sequence shown in SEQ ID NO: 6 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 6 and (b) another polynucleotide comprising the sequence shown in SEQ ID NO: 8 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 8. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 6 and / or 8. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 6 and / or 8.

[0211] The one or more polynucleotides preferably comprise the sequence shown in SEQ ID NO: 10 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 10. The variant sequence may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 10. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 10.

[0212] The one or more polynucleotides preferably comprises (a) the sequence shown in SEQ ID NO: 10 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 10 and (b) the sequence shown in SEQ ID NO: 12 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 12. The one or more polynucleotides preferably comprise (a) one polynucleotide comprising the sequence shown in SEQ ID NO: 10 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 10 and (b) another polynucleotide comprising the sequence shown in SEQ ID NO: 12 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 12. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 10 and / or 12. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 10 and / or 12.

[0213] The one or more polynucleotides preferably comprise the sequence shown in SEQ ID NO: 19, 23 or 27 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 19, 23 or 27. The variant sequence may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 19, 23 or 27. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 19, 23 or 27.

[0214] The one or more polynucleotides preferably comprises (a) the sequence shown in SEQ ID NO: 19 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 19 and (b) the sequence shown in SEQ ID NO: 21 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 21. The one or more polynucleotides preferably comprise (a) one polynucleotide comprising the sequence shown in SEQ ID NO: 19 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 19 and (b) another polynucleotide comprising the sequence shown in SEQ ID NO: 21 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 21.

[0215] The one or more polynucleotides preferably comprises (a) the sequence shown in SEQ ID NO: 23 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 23 and (b) the sequence shown in SEQ ID NO: 25 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 25. The one or more polynucleotides preferably comprise (a) one polynucleotide comprising the sequence shown in SEQ ID NO: 23 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 23 and (b) another polynucleotide comprising the sequence shown in SEQ ID NO: 25 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 25. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the reference sequence. Homology and / or identity is / are typically measured over the entire length of the reference sequence.

[0216] The one or more polynucleotides preferably comprise the sequence shown in SEQ ID NO: 38 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 38. The variant sequence may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 38. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 38.

[0217] The one or more polynucleotides preferably comprises (a) the sequence shown in SEQ ID NO: 38 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 38 and (b) the sequence shown in SEQ ID NO: 40 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 40. The one or more polynucleotides preferably comprise (a) one polynucleotide comprising the sequence shown in SEQ ID NO: 38 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 38 and (b) another polynucleotide comprising the sequence shown in SEQ ID NO: 40 or a variant sequence having at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 4. The variant sequence(s) may have at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in SEQ ID NO: 38 and / or 40. Homology and / or identity is / are typically measured over the entire length of SEQ ID NO: 38 and / or 40.

[0218] A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The nucleotides can be naturally occurring or artificial.

[0219] A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'O-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and the nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The nucleosides may be adenosine, guanosine, uridine and cytidine.

[0220] The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides may be deoxyribonucleotides. The nucleotides typically contain a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.

[0221] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5- hydroxy methylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5- hydroxy methylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate and 5-hydroxymethyl-2'- deoxycytidine triphosphate. The nucleotides may be selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP. In some embodiments, the nucleotides are selected from dAMP, dTMP, dGMP or dCMP.

[0222] The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'-amino cytidine and 2'-amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro pyrimidines (such as 2'- fluorocytidine and 2'fluoro uridine), hydroxyl pyrimidines (such as 5'-o-P-borano uridine), 2'-O-methyl nucleotides (such as 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O- methyl cytidine and 2'-O-methyl uridine), 4'-thio pyrimidines (such as 4'-thio uridine and 4'- thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2'- deoxy uridine, 5-(3-aminopropyl)-uridine and l,6-diaminohexyl-N-5-carbamoylmethyl uridine).

[0223] One or more nucleotides in the polynucleotides may be modified, for instance with a label or a tag. The label may be any suitable label which allows the polynucleotides to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g.125I,35S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin.

[0224] The nucleotides in the polynucleotides may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups. The nucleotides may be connected via their nucleobases as in pyrimidine dimers. The polynucleotide may comprise a deoxyribonucleic acid (DNA) or a ribonucleic acid (RIMA). The polynucleotide may be any synthetic polynucleotide known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. The polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.

[0225] Unless otherwise indicated, a particular polynucleotide also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a different base, mixed- base and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0226] Substitutions may be used for the practices of codon optimisation and codon wobble, both of which are known to those skilled in the art. Thus, it will be appreciated that codon- optimised and codon-wobbled polynucleotides are also envisaged. In an embodiment, the polynucleotides are codon-optimised for human expression.

[0227] The polynucleotides can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence. Direct chemical synthesis of polynucleotides can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68: 109; the diethylphosphoramidite method of Beaucage et al., 1981, Tetra. Lett., 22: 1859; and the solid support method of U.S. Pat. No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1: 17.

[0228] Vectors

[0229] The invention also provides a vector comprising the one or more polynucleotides of the invention. The vector is typically an expression vector. Various expression vectors can be employed. Both viral-based and non-viral expression vectors can be used in the invention. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors useful for expression in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C, (Invitrogen, San Diego, Calif.), MPS V vectors, and numerous other vectors known in the art for expressing other proteins and / or nucleotide sequences. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68: 143.

[0230] Preferably, the vector is a retroviral, lentiviral, adenoviral, or adeno-associated viral vector. The vector is preferably a retroviral or lentiviral vector. The vector is preferably a lentiviral vector.

[0231] Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell typespecific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, the SSFV promoter, the EFl alpha promoter, the phosphoglycerate kinase (PGK) promoter and promoter-enhancer combinations known in the art.

[0232] Amphipathic structure

[0233] The invention also provides an amphipathic structure linked to a fusion protein of the invention, a conjugate of the invention, or a construct of the invention. The fusion protein, conjugate, or construct is capable of targeting the amphipathic structure. The fusion protein, conjugate, or construct may be capable of targeting the amphipathic structure in any of the ways discussed above.

[0234] The amphipathic structure is preferably linked to the fusion protein, conjugate, or construct via a membrane membrane-binding serum protein. The membrane-binding serum protein may be any of those discussed above. The membrane-binding serum protein is typically present on the surface of the amphipathic structure. The membrane-binding serum protein is typically present in the outer membrane of the amphipathic structure. The fusion protein, conjugate, or construct typically comprises a receptor for the membrane-binding serum protein. The receptor may be any of those described above. The amphipathic structure is preferably linked to the fusion protein, conjugate, or construct via an LDL. The LDL may be any of those described above. The LDL is typically present on the surface of the amphipathic structure. The LDL is typically present in the outer membrane of the amphipathic structure. The fusion protein, conjugate, or construct typically comprises a LDL receptor for the LDL. The LDL receptor may be any of those described above. The LDL is Apolipoprotein-E (Apo-E).

[0235] An amphipathic structure is a structure formed of molecules comprising polar and non-polar regions. Typically, the amphipathic structure will comprise lipids, optionally phospholipids. The amphipathic structure may be a vesicle, micelle, liposome, lipid nanoparticle (LNP), or exosome. Other amphipathic structures are known in the art.

[0236] In addition to the fusion protein, conjugate, or construct of the invention, the amphipathic structure may comprise other proteins, polynucleotides and / or polysaccharides, such as oligosaccharides. Suitable proteins, polynucleotides and / or polysaccharides include, but are not limited to, those described in WO 2023 / 144089 (incorporated herein by reference in its entirety).

[0237] The amphipathic structure preferably comprises a therapeutic molecule and / or a genetic cargo. The therapeutic molecule can be any molecule intended to treat a disease or disorder. The disease or disorder may be any of those discussed below. The skilled person is capable of identifying therapeutic molecules that may be delivered using the amphipathic structure. Examples include, but are not limited to, small molecules, chemotherapeutic agents, proteins, enzymes, antibodies, antisense oligonucleotides (ASOs), such as gapmers, small interfering RNAs (siRNAs), messenger RNA (mRNA), mRNA vaccines, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems, including CRISPR / Cas9, bridge RNAs, exonucleases, and homing endonucleases or meganucleases.

[0238] A genetic cargo is a cargo which encodes a molecule or system of interest. The genetic cargo typically comprises a polynucleotide which encodes the molecule or system of interest and an expression vector and / or promoter. The genetic cargo may comprise any of the expression vectors and / or promoters discussed above with reference to the vectors of the invention. The genetic cargo is typically expressed in the target cell.

[0239] The genetic cargo may encode any of the therapeutic molecules listed above. The genetic cargo preferably encodes a gene editing system. The gene editing system may be selected from a zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) system, such as CRISPR / Cas9, bridge RNAs, and a homing endonuclease or meganuclease. The genetic cargo preferably encodes a chimeric antigen receptor (CAR). Suitable CARs are known to those skilled in the art. In particular, the CAR may comprise or consist of a first, second, third, or fourth generation CAR.

[0240] First-generation CARs comprise or consist of an extracellular binding domain, a transmembrane domain, and one or more intracellular signalling domains. The extracellular binding domain may comprise a single-chain variable fragment (scFv) from a monoclonal antibody. In particular, the antibody may be specific for a tumour antigen. A first-generation CAR typically comprises the CD3 chain domain or a modified derivative thereof as the intracellular signalling domain, which is the primary transmitter of signals.

[0241] In addition to the components specified for first-generation CARs, second-generation CARs also contain a co-stimulatory domain, such as CD28 and / or 4-1BB. The inclusion of an intracellular co-stimulatory domain improves T-cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence. The co-stimulatory domain of a second- generation CAR is typically in cis with and upstream of the one or more intracellular signalling domains.

[0242] Third-generation CARs combine multiple co-stimulatory domains in cis with one or more intracellular signalling domains, to augment T-cell activity. For example, a third-generation CAR may comprise co-stimulatory domains derived from CD28 and 41BB, together with an intracellular signalling domain derived from CD3z. Other third-generation CARs may comprise co-stimulatory domains derived from CD28 and 0X40, together with an intracellular signalling domain derived from CD3z.

[0243] Fourth-generation CARs (also known as TRUCKS or armoured CARs), combine the features of a second-generation CAR with further factors to enhance anti-tumour activity (e.g., cytokines, co-stimulatory ligands, chemokines receptors or further chimeric receptors of immune regulatory or cytokine receptors). The factors may be in trans or in cis with the CAR, typically in trans with the CAR.

[0244] The genetic cargo may encode a molecular marker. Suitable molecular markers include, but are not limited to, lacZ (b-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (betaglucuronidase), GFP (green fluorescent protein) and other fluorescent proteins, bla (betalactamase) and other antibiotic resistance markers, and heavy metals resistance genes.

[0245] The invention also provides a population of amphipathic structures of the invention. The population may comprise or consist of vesicles, micelles, liposomes, lipid nanoparticles (LNPs), or exosomes of the invention or a combination or mixture thereof. For instance, the population may comprise a combination of liposomes and LNPs, or a combination of LNPs and exosomes. Other combinations can be envisaged by the skilled person. The amphipathic structures in the population may comprise the same fusion protein of the invention or different fusion proteins of the invention.

[0246] The population may comprise or consist of any number of amphipathic structures of the invention, such as at least about 1.00 x 103, at least about 1.00 x 104, at least about 1.00 x 105, at least about 1.00 x 106, at least about 1.00 x 107, at least about 1.00 x 108, at least about 1.00 x 109, or at least about 1.00 x 1010. The population may comprise or consist of even more amphipathic structures of the invention, such as at least about 1011or at least about 1012.

[0247] Compositions of the invention

[0248] In some embodiments, the amphipathic structure or the population is present in a composition further comprising a polyethylene glycol (PEG)-lipid conjugate and a sterol and the molar ratio of the PEG-lipid conjugate to the lipids in the amphipathic structure and the sterol is about 1.2% or less. The invention also provides a composition comprising an amphipathic structure of the invention or a population of the invention, a PEG-lipid conjugate and a sterol, wherein the molar ratio of the PEG-lipid conjugate to the lipids in the amphipathic structure and the sterol is about 1.2% or less. The amphipathic structure or the population may be any of those described above. The amphipathic structure or the population comprises lipids. The amphipathic structure may be a vesicle, micelle, liposome, lipid nanoparticle (LNP), or exosome. The amphipathic structure is preferably a liposome or LNP.

[0249] PEG is defined above. The PEG in the conjugate may be any PEG. Suitable PEGs include, but are not limited to, PEG200, PEG350, PEG400, PEG600, PEG1000, PEG1500, PEG2000 and PEG4000. The PEG is preferably PEG2000.

[0250] The lipid in the conjugate may be any lipid. The lipid may be myristoyl diglyceride (DMG) or l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0251] The PEG and the lipid may be conjugated in any way known in the art. The lipid is typically PEGylated to form the conjugate.

[0252] The lipid conjugate is preferably DMG-PEG2000.

[0253] The sterol may be cholesterol, p-sitosterol, campesterol, stigmasterol or ergosterol. The sterol may be cholesterol or p-sitosterol. The sterol is preferably cholesterol.

[0254] The composition may comprise two or more sterols. The two or more sterols may be selected from the ones described above. In such embodiments, the molar ratio of the PEG- lipid conjugate to the lipids in the amphipathic structure and the two or more sterols is about 1.2% or less.

[0255] The molar ratio of the PEG-lipid conjugate to the lipids in the amphipathic structure and the sterol(s) is about 1.2% or less. The molar ratio may be about 1.1% or less, about 1.0% or less, about 0.9% or less, about 0.8% or less, about 0.75% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.375% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less, The molar ratio is preferably about 0.75% or less or about 0.75%. The molar ratio is preferably about 0.375% or less or about 0.375%. The skilled person is capable of calculating molar ratios using standard techniques.

[0256] The skilled person is capable of designing suitable compositions. The composition may form part of the a pharmaceutical composition of the invention discussed below.

[0257] Making amphipathic structures

[0258] The invention also provides a method of producing an amphipathic structure of the invention. The method comprises linking the amphipathic structure to the fusion protein, the conjugate, or the construct. The method typically comprises linking the amphipathic structure to the fusion protein, the conjugate, or the construct by incubating the amphipathic structure with the fusion protein, the conjugate, or the construct. This method equally applies to making populations of amphipathic structures of the invention.

[0259] Incubation may be for at least about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours or about 4 hours. Incubation may be for longer, for example up to about 12 hours, about 16 hours, about 24 hours, about 48 hours or about 72 hours.

[0260] The total incubation may be for at least about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 12 hours, about 16 hours, about 24 hours, about 48 hours or about 72 hours.

[0261] Incubation may be at a temperature of at least about 5°C, about 10°C, about 15°C, about 30°C, about 35°C, about 37°C, about 40°C, or at least about 45°C. Incubation may be at a temperature of about 37°C.

[0262] Incubation is typically in a buffered aqueous solution, for example a salt solution such as phosphate buffered saline (PBS). Incubation may be at a pH of about pH 6-9, for example about pH 6, 6.5, 7, 7.5, 8, 8.5 or about 9. In some embodiments, incubation is at a pH of from about pH 5 to about pH 9. In some embodiments, incubation is at a pH of about 7. Any of the embodiments discussed above with reference to the fusion protein, conjugate, construct, amphipathic structure, or population of the invention equally apply to this method.

[0263] Improving targeting

[0264] The invention also provides a method of improving the targeting of an amphipathic structure to a target cell or target cells comprising linking the amphipathic structure to a fusion protein, conjugate, or construct of the invention, wherein the targeting peptide or polypeptide specifically binds a target antigen on the target cell or target cells. Any of the embodiments discussed above with reference to the fusion proteins, conjugates, constructs, and amphipathic structures of the invention equally apply to this method.

[0265] The method preferably increases the amount and / or rate of endocytosis of the amphipathic structure by the target cell or target cells. The method preferably also decreases the rate and / or amount of endocytosis of the amphipathic structure by non-target cells. This can be measured using routine techniques including those described in the Examples. The target cell or target cells may be associated with a disease or disorder. The target cell or target cells may be any of those discussed above. The target cell or target cells may be a cancer cell or cancer cells. The target cell or target cells is / are preferably a cancer cell or cancer cells and the non-target cells are preferably corresponding, healthy cells. For instance, if the target cells are pancreatic cancer cells, the non-target cells are preferably healthy pancreatic cells. The target cell or target cells is / are preferably in a patient.

[0266] Pharmaceutical compositions

[0267] The invention also provides a pharmaceutical composition comprising a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention or a composition of the invention. The pharmaceutical composition also comprises a pharmaceutically acceptable diluent and / or carrier.

[0268] The invention also provides a pharmaceutical composition comprising a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention or an amphipathic structure of the invention. The pharmaceutical composition also comprises a pharmaceutically acceptable diluent and / or carrier.

[0269] The invention also provides a pharmaceutical composition comprising a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention or a population of the invention. The pharmaceutical composition also comprises a pharmaceutically acceptable diluent and / or carrier.

[0270] The carrier and / or diluent is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Typically, these carriers and / or diluents include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.

[0271] Suitable further agents for inclusion in the pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulphite, or sodium hydrogensulphite), buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), colouring, flavouring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, excipients and / or pharmaceutical adjuvants.

[0272] The carrier and / or diluent may be a parenteral, optionally intravenous vehicle. Suitable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases, one might include agents to adjust tonicity of the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. The precise formulation will depend on the route of administration. Additional relevant principles, methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22ndEdition).

[0273] A pharmaceutical composition of the invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled person, the route and / or mode of administration will vary depending upon the desired results. Routes of administration for pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrapleural and intra-sternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intratumourally. In other embodiments, administration is intrapleural or intraperitoneal. When parenteral administration is contemplated, the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved. The pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake.

[0274] Alternatively, the pharmaceutical composition of the invention can be administered by a nonparenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0275] In some embodiments, the pharmaceutical composition is for subcutaneous administration. Typically, the pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids, such as methionine, and or saccharides such as sucrose), buffering agents and tonicifying agents.

[0276] Alternatively, the pharmaceutical composition may be for intravenous administration.

[0277] Therapeutic methods

[0278] The invention also provides a method of treating or preventing a disease or disorder in a subject. The subject is typically in need thereof. The subject may be a mammal. Optionally, the subject is a human, horse, dog, or cat. The subject is preferably human.

[0279] The method comprises administering to the subject a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention, a composition of the invention or a pharmaceutical composition of the invention.

[0280] The method comprises administering to the subject a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention or a pharmaceutical composition of the invention.

[0281] The method comprises administering to the subject a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention or a pharmaceutical composition of the invention.

[0282] The invention also provides a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention, a composition of the invention or a pharmaceutical composition of the invention for use in therapy. The invention also provides a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention, a composition of the invention or a pharmaceutical composition of the invention for use in a method of treating or preventing a disease or disorder in a subject

[0283] The invention also provides a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention or a pharmaceutical composition of the invention for use in therapy. The invention also provides a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention or a pharmaceutical composition of the invention for use in a method of treating or preventing a disease or disorder in a subject.

[0284] The invention also provides a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention or a pharmaceutical composition of the invention for use in therapy. The invention also provides a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention or a pharmaceutical composition of the invention for use in a method of treating or preventing a disease or disorder in a subject.

[0285] The invention also provides the use of a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention, a composition of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating or preventing a disease or disorder.

[0286] The invention also provides the use of a fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating or preventing a disease or disorder.

[0287] The invention may be used to treat or prevent any disease or disorder. The disease or disorder may be an infection, cancer, an immune disease or disorder, or a genetic disease or disorder.

[0288] The infection may be caused by any pathogenic agent. The pathogenic agent may be a bacterium, an archaeon, a fungus, or a virus.

[0289] The bacterium may be Gram negative or Gram positive. The Gram-positive bacterium is preferably from the genus Bacillus, Clostridium, Enterococcus, Mycobacterium, Staphylococcus or Streptococcus. The Gram-positive bacterium may be from the genus Pasteurella or Nocardia.

[0290] The Gram-negative bacterium is preferably from the genus Aggregatibacter, Bacteroides, Bartonella, Brucella, Campylobacter, Chylamidia, Enterbacter, Francisella, Haemophilus, Heliobacter, Klebsiella, Legionella, Moraxella, Neisseria, Porphyromonas, Pseudomonas, Salmonella, Serratia, Stenotrophomonas, Vibrio or Yersinia. The Gram-negative bacterium may be from the genus Escherichia or Pseudomonas.

[0291] The bacterium may be from the genus Borrelia, Chlamydophila, Listeria, Mycoplasma, Proteus, or Treponema. The bacterium is preferably Aggregatibacter actinomycetemcomitans, Bacillus anthracis, Bacillus licheniformis, Bacteroides fragilis, Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Campylobacter jejuni, Chlamydia trachomatis, Chlamydophila pneumoniae, Clostridium difficile, Clostridium perfringens, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycoplasma genitalium, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Porphyromonas gingivalis, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Stenotrophomonas maltophilia, Streptococcus mutans, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Treponema pallidum, Vibrio cholera, Vibrio parahaemolyticus or Yersinia enterocolitica.

[0292] Other specific examples of bacteria include, but are not limited, to Mycobacterium tuberculosis, Mycobacterium intracellilare, Mycobacterium kansaii, Mycobacterium gordonae, Streptococcus agalactiae, Streptococcus viridans group, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, Corynebacterium diptheriae, Erysipelothrix rhusiopathie, Clostridium tetani, Klebsiella pneumoniae, Pasteurella multocida, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pertenue and Actinomyces israelii.

[0293] The fungus is preferably from the genus Absidia, Acremonium, Aspergillus, Aureobasidium, Basidiobolus, Blastomyces, Blastoschizomyces, Candida, Cladosporium, Coccidioides, Cryptococcus, Cunninghamella, Curvularia, Debaryomyces, Exophiala, Exserohilum, Fonsecea, Fusarium, Geotrichum, Histoplasma, Issatchenkia, Kluyveromyces, Malezzesia, Mucor, Paracoccidioides, Paecilomyces, Penicillium, Pichia, Pneumocystis, Rhizomucor, Rhizopus, Rhodotorula, Saccharomyces, Scedosporium, Schizophyllum, Scopulariopsis, Sporothrix, Trichoderma, Trichophyton or Trichosporon. The fungus is preferably Aspergillus fumigatus, Aspergillus flavus, Aspergillus lentulus, Aspergillus terreus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus niger, Candida albicans, Candida caribbica ( Candida fermentati), Candida dubliniensis, Candida famata (Debaryomyces hansenii), Candida fukuyamaensis (Candida xestobii or Candida carpophila), Candida guilliermondii, Candida kefyr (Kluyveromyces marxianus), Candida krusei (Issatchenkia orientalis), Candida metapsilosis, Candida orthopsilosis, Candida parapsilosis, Candida parapsilosis, Candida pelliculosa, Candida psychrophila, Candida rugosa, Candida smithsonii, Candida tropicalis, Candida utilis, Coccidioides immitis , Cryptococcus bacillisporus, Cryptococcus gattii, Cryptococcus grubii, Cryptococcus neoformans, Debaryomyces coudertii, Debaryomyces maramus, Debaryomyces nepalensis, Debaryomyces prosopidis, Debaryomyces robertsiae, Debaryomyces udenii, Histoplasma capsulatum, Kluyveromyces lactis, Pichia cecembensis, Rhodotorula araucariae, Rhodotorula babjevae, Rhodotorula dairensis, Rhodotorula diobovatum, Rhodotorula glutinis, Rhodotorula kratochvilovae, Rhodotorula paludigenum, Rhodotorula sphaerocarpum, Rhodotorula toruloides, Rhodotorula mucliaginosa, Saccharomyces 'sensu stricto', Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces cariocanus, Saccharomyces kudiavzevii, Saccharomyces mikatae, Saccharomyces paradioxus, Saccharomyces pastorianus, Saccharomyces uvarum, Saccharomyces cerevisiae or Tsuchiyaea wingfieldii. The virus may belong to the family Retroviridae, such as human deficiency viruses, such as HIV-I (also referred to as HTLV- III), HIV-II, LAC, IDLV-III / LAV, HIV-III or other isolates such as HIV-LP, the family Picornaviridae, such as poliovirus, hepatitis A, enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses, the family Calciviridae, such as viruses that cause gastroenteritis, the family Togaviridae, such as equine encephalitis viruses and rubella viruses, the family Flaviviridae, such as dengue viruses, encephalitis viruses and yellow fever viruses, the family Coronaviridae, such as coronaviruses, including SARS-Cov-2 (COVID-19), the family Rhabdoviridae, such as vesicular stomata viruses and rabies viruses, the family Filoviridae, such as Ebola viruses, the family Paramyxoviridae, such as parainfluenza viruses, mumps viruses, measles virus and respiratory syncytial virus, the family Orthomyxoviridae, such as influenza viruses, the family Bungaviridae, such as Hataan viruses, bunga viruses, phleoboviruses and Nairo viruses, the family Arena viridae, such as hemorrhagic fever viruses, the family Reoviridae, such as reoviruses, orbiviruses and rotaviruses, the family Bimaviridae, the family Hepadnaviridae, such as hepatitis B virus, the family Parvoviridae, such as parvoviruses, the Papovaviridae, such as papilloma viruses and polyoma viruses, the family Adenoviridae, such as adenoviruses, the family Herpesviridae, such as herpes simplex virus (HSV) I and II, varicella zoster virus and pox viruses, or the family Iridoviridae, such as African swine fever virus). The virus may be an unclassified virus, such as the etiologic agents of Spongiform encephalopathies, the agent of delta hepatitis, the agents of non-A, non-B hepatitis (class 1 enterally transmitted; class 2 parenterally transmitted such as Hepatitis C), Norwalk and related viruses and astroviruses.

[0294] The cancer may include, but not necessarily be limited to, a solid tumour cancer, a soft tissue tumour, a metastatic lesion, and a haematological cancer. For example, the cancer can be liver cancer, lung cancer, breast cancer, prostate cancer, lymphoid cancer, colon cancer, renal cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, such as squamous cell carcinoma of the head and neck (SCCHN), cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the oesophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukaemias including acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, solid tumours of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, myelodysplastic syndrome (MDS), chronic myelogenous leukaemia-chronic phase (CMLCP), diffuse large B-cell lymphoma (DLBCL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), hepatocellular carcinoma (HCC), gastrointestinal stromal tumours (GIST), non-small cell lung carcinoma (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), environmentally induced cancers including those induced by asbestos, and combinations of said cancers. In embodiments, the cancer is selected from the above group.

[0295] The cancer may be a solid tumour cancer.

[0296] In some embodiments, the cancer is selected from the group consisting of cancer of the head and / or neck, ovarian cancer, malignant mesothelioma, breast cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, prostate cancer, oesophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, cancer of the central nervous system or renal cell carcinoma.

[0297] In some embodiments, the cancer is selected from ovarian cancer, breast cancer, optionally triple-negative breast cancer, pancreatic cancer, malignant mesothelioma, and combinations of said cancers.

[0298] In some embodiments the cancer is breast cancer, ovarian cancer, melanoma or mesothelioma.

[0299] In some embodiments the cancer is ovarian cancer, breast cancer or combinations thereof.

[0300] The breast cancer may be triple negative breast cancer.

[0301] The subject may have been pre-treated with a chemotherapeutic agent.

[0302] The genetic disorder may be lp36 deletion syndrome, lq21.1 deletion syndrome, 2q37 deletion syndrome, 5,10-methenyltetrahydrofolate synthetase deficiency, 5q deletion syndrome, 7p22.1 microduplication syndrome, 17ql2 microdeletion syndrome, 17ql2 microduplication syndrome, 18p deletion syndrome, 21-hydroxylase deficiency, 47, XXX (triple X syndrome), AAA syndrome (achalasia-addisonianism-alacrima syndrome), Aarskog-Scott syndrome, ABCD syndrome, Absence deformity of leg-cataract syndrome, Aceruloplasminemia, Acheiropodia, Achondrogenesis type II, Achondroplasia, Acute intermittent porphyria, Adenylosuccinate lyase deficiency, Adrenoleukodystrophy, ADULT syndrome, Aicardi-Goutieres syndrome, Alagille syndrome, Albinism, Alexander disease, Alfi's syndrome, Alkaptonuria, Alpha 1-antitrypsin deficiency, Alport syndrome, Alstrbm syndrome, Alternating hemiplegia of childhood, Alzheimer's disease, Amelogenesis imperfecta, Aminolevulinic acid dehydratase deficiency porphyria, Amish lethal microcephaly, Amyotrophic lateral sclerosis - Frontotemporal dementia, Androgen insensitivity syndrome, Angel-shaped phalango-epiphyseal dysplasia, Angelman syndrome, Aortic arch anomaly - peculiar facies - intellectual disability, Apert syndrome, Aphalangy- syndactyly-microcephaly syndrome, Arthrogryposis-renal dysfunction-cholestasis syndrome, Ataxia telangiectasia, Axenfeld syndrome, Bainbridge-Ropers syndrome, Beare- Stevenson cutis gyrata syndrome, Beckwith-Wiedemann syndrome, Benjamin syndrome, biotinidase deficiency, Birt-Hogg-Dube syndrome, Bjbrnstad syndrome, Blepharophimosis intellectual disability syndromes, Bloom syndrome, Brody myopathy, Brunner syndrome, CADASIL syndrome, Campomelic dysplasia, Camptodactyly-taurinuria syndrome, Canavan disease, Carpenter syndrome, Cat eye syndrome, CATSHL syndrome, CDKL5 deficiency disorder, Cerebral dysgenesis-neuropathy-ichthyosis-keratoderma syndrome (CEDNIK), Charcot-Marie-Tooth disease, CHARGE syndrome, Chediak-Higashi syndrome, Chondrodysplasia, Grebe type, Chronic granulomatous disorder, Cleft palate short stature vertebral anomalies syndrome, Cleidocranial dysostosis, Cockayne syndrome, Coffin-Lowry syndrome, Cohen syndrome, Collagenopathy, types II and XI, Combined malonic and methylmalonic aciduria (CMAMMA), Combined malonic and methylmalonic aciduria (CMAMMA), Congenital insensitivity to pain with anhidrosis (CIPA), Congenital muscular dystrophy, Congenital muscular dystrophy-infantile cataract-hypogonadism syndrome, Corneal dystrophy-perceptive deafness syndrome, Cornelia de Lange syndrome (CDLS), Cowden syndrome, CPO deficiency (coproporphyria), Cranio-lenticulo-sutural dysplasia, CRASIL syndrome, Cri du chat, Crohn's disease, Crouzon syndrome, Crouzonodermoskeletal syndrome (Crouzon syndrome with acanthosis nigricans), Currarino syndrome, Cystic fibrosis, Darier's disease, De Grouchy syndrome, Dent's disease (Genetic hypercalciuria), Denys-Drash syndrome, DiGeorge syndrome, Distal hereditary motor neuropathies, multiple types, Distal muscular dystrophy, Dolichonychia, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, Ectrodactyly-polydactyly syndrome, Edwards syndrome, Ehlers-Danlos syndrome, Emanuel syndrome, Emery-Dreifuss syndrome, Epidermolysis bullosa, Erythropoietic protoporphyria, Fabry disease, Factor V Leiden thrombophilia, Familial adenomatous polyposis, Familial Creutzfeld-Jakob disease, Familial dysautonomia, Familial episodic pain syndrome, Familial thoracic aortic aneurysm and aortic dissection, Fanconi anemia (FA), Fatal familial insomnia, FBXW7 neurodevelopmental syndrome, Feingold syndrome, FG syndrome, Fibular aplasia-ectrodactyly syndrome, Fine- Lubinsky syndrome, Fragile X syndrome, Friedreich's ataxia, G6PD deficiency, Galactosemia, Gaucher disease, Gerstmann-Straussler-Scheinker syndrome, Gillespie syndrome, Glutaric aciduria, type I and type 2, GRACILE syndrome, GRIN2B-related neurodevelopmental disorder, Griscelli syndrome, Gustavson syndrome, Haemochromatosis type 3, Hailey- Hailey disease, Harlequin type ichthyosis, Hemochromatosis type 1, Hemochromatosis type 2A, Hemochromatosis type 2B, Hemochromatosis type 4, Hemochromatosis type 5, Hemophilia, Hepatoerythropoietic porphyria, Hereditary coproporphyria, Hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu syndrome), Hereditary inclusion body myopathy, Hereditary multiple exostoses, Hereditary neuropathy with liability to pressure palsies (HNPP), Hereditary spastic paraplegia (infantile-onset ascending hereditary spastic paralysis), Hermansky-Pudlak syndrome, Heterotaxy, Homocystinuria, Hunter syndrome, Huntington's disease, Hurler syndrome, Hutchinson-Gilford progeria syndrome, Hyperlysinemia, Hyperoxaluria, primary, Hyperphenylalaninemia, Hypoalphalipoproteinemia (Tangier disease), Hypochondrogenesis, Hypochondroplasia, Immunodeficiency-centromeric instability-facial anomalies syndrome (ICF syndrome), Incontinentia pigmenti, Infantile cerebral and cerebellar atrophy with postnatal progressive microcephaly, Ischiopatellar dysplasia, Isodicentric 15, Jackson-Weiss syndrome, Jacobsen syndrome, Joubert syndrome, Juvenile primary lateral sclerosis (JPLS), Juvenile-onset dystonia, Keloid disorder, KIFIA-Associated neurological disorder, Kleefstra syndrome, Kniest dysplasia, Kosaki overgrowth syndrome, Krabbe disease, Kufor-Rakeb syndrome, LCAT deficiency, Lesch-Nyhan syndrome, Li-Fraumeni syndrome, Limb-Girdle Muscular Dystrophy, lipoprotein lipase deficiency, Lynch syndrome, Malignant hyperthermia, Maple syrup urine disease, Marfan syndrome, Maroteaux-Lamy syndrome, McCune-Albright syndrome, McLeod syndrome, Mediterranean fever, familial, MEDNIK syndrome, Menkes disease, Methemoglobinemia, Methylmalonic acidemia, Micro syndrome, Microcephaly, Miller-Dieker syndrome, Morquio syndrome, Mowat-Wilson syndrome, Muenke syndrome, Multiple endocrine neoplasia type 1 (Wermer's syndrome), Multiple endocrine neoplasia type 2, Muscular dystrophy, Muscular dystrophy, Duchenne and Becker type, Myostatin-related muscle hypertrophy, Myotonic dystrophy, Natowicz syndrome, Neurofibromatosis type I, Neurofibromatosis type II, Niemann-Pick disease, Nonketotic hyperglycinemia, Nonsyndromic deafness, Noonan syndrome, Norman-Roberts syndrome, Ogden syndrome, Omenn syndrome, Osteogenesis imperfecta, Ostravik-Lindemann-Solberg syndrome, Pantothenate kinase-associated neurodegeneration, Patau syndrome (Trisomy 13), PCC deficiency (propionic acidemia), Pendred syndrome, Peutz-Jeghers syndrome, Pfeiffer syndrome, Phelan-McDermid syndrome, Phenylketonuria, Pipecolic acidemia, Pitt-Hopkins syndrome, Polycystic kidney disease, Polycystic ovary syndrome (PCOS), Porphyria, Porphyria cutanea tarda (PCT), Prader-Willi syndrome, PRICKLEl-related progressive myoclonus epilepsy with ataxia, Primary ciliary dyskinesia (PCD), Primary pulmonary hypertension, Protein C deficiency, Protein S deficiency, Proximal 18q deletion syndrome, Pseudo-Gaucher disease, Pseudoxanthoma elasticum, Retinitis pigmentosa, Rett syndrome, Roberts syndrome, Rubinstein-Taybi syndrome (RSTS), Sandhoff disease, Sanfilippo syndrome, Scheuermann's disease, Schwartz-Jampel syndrome, Shprintzen-Goldberg syndrome, Sickle cell anemia, Siderius X-linked mental retardation syndrome, Sideroblastic anemia, Sjogren-Larsson syndrome, Skin fragility-woolly hair-palmoplantar keratoderma syndrome, Sly syndrome, Smith-Lemli-Opitz syndrome, Smith-Magenis syndrome, Snyder- Robinson syndrome, Spinal muscular atrophy, Spinocerebellar ataxia (types 1-29), Split hand split foot-nystagmus syndrome, Spondyloepiphyseal dysplasia congenita (SED), SSB syndrome (SADDAN), Stargardt disease (macular degeneration), Stickler syndrome (multiple forms), Strudwick syndrome (spondyloepimetaphyseal dysplasia, Strudwick type), Tay-Sachs disease, Tetrahydrobiopterin deficiency, Thanatophoric dysplasia, Thickened earlobes-conductive deafness syndrome, Treacher Collins syndrome, Tuberous sclerosis complex (TSC), Turner syndrome, Usher syndrome, Variegate porphyria, Viljoen-Kallis- Voges syndrome, von Hippel-Lindau disease, von Willebrand disease, Waardenburg syndrome, Warkany syndrome 2, Weissenbacher-Zweymuller syndrome, Weyer's ulnar ray / oligodactyly syndrome, Williams syndrome, Wilson disease, Wolf-Hirschhorn syndrome, Woodhouse-Sakati syndrome, X-linked intellectual disability and macroorchidism (fragile X syndrome), X-linked severe combined immunodeficiency (X-SCID), X-linked sideroblastic anemia (XLSA), X-linked spinal-bulbar muscle atrophy (spinal and bulbar muscular atrophy), Xeroderma pigmentosum, Xpll.2 duplication syndrome, XXXX syndrome (48, XXXX), XXXXX syndrome (49,XXXXX), XXXXY syndrome (49,XXXXY), XXXY syndrome (48,XXXY), XXYY syndrome (48,XXYY), XYY syndrome (47,XYY), XYYY syndrome (48,XYYY), XYYYY syndrome (49,XYYYY), or Zellweger syndrome.

[0303] The genetic disease or disorder may be Angelman syndrome, Canavan disease, Charcot- Marie-Tooth disease, Color blindness, Cri du chat syndrome, Cystic fibrosis (CF), DiGeorge syndrome, Down syndrome, Duchenne muscular dystrophy, Familial hypercholesterolemia, Haemochromatosis type 1, Haemophilia, Klinefelter syndrome, Neurofibromatosis, Phenylketonuria, Polycystic kidney disease, Prader-Willi syndrome, Scheuermann's disease, Sickle cell disease, Spinal muscular atrophy, Tay-Sachs disease, Turner syndrome, congenital adrenal hyperplasia, thalassemia, severe combined immune deficiency (SCID), Huntington's disease, or retinitis pigmentosa. For instance, the invention may be used to deliver gene editing systems capable of correcting globin gene mutations associated with sickle cell disease or thalassemia, correcting mutations in the adenosine deaminase gene associated with SCID, reducing the expression of HTT, the disease-causing gene of Huntington's disease, or correcting mutations in the rhodopsin gene for the treatment of retinitis pigmentosa.

[0304] One particular embodiment of the invention relates to in vivo CAR therapy. The method of preventing cancer preferably comprises administering to the subject an amphipathic structure of the invention, a population of the invention or a pharmaceutical composition of the invention and thereby delivering a genetic cargo encoding a chimeric antigen receptor (CAR) to one or more immune cells in the subject. The CAR may be any of those described above. The one or more immune cells may be one or more T-cells, one or more natural killer (NK) cells, one or more neutrophils, one or more B-cells or subsets or derivatives thereof. Optionally, the one or more immune cells are one or more T-cells, one or more B- cells or NK cells. The one or more immune cells may be one or more CD4+T-cells, one or more CD8+T-cells, one or more NK cells, or any combination thereof. The one or more immune cells may be one or more CD4+ T-cells and / or one or more CD8+T-cells. The one or more immune cells may be one or more o[3 T-cells and / or one or more y6 T-cells. The one or more immune cells typically express the CAR.

[0305] The CAR may be delivered to any number of immune cells in the subject. Any number of immune cells in the subject may express the CAR. Suitable numbers for treating or preventing a disease or disorder are from about 1 x 106to about 1 x 1011cells. In one embodiment, from about 1 x 107to about 1 x 1010cells, or from about 1 x 108to about 1 x 109cells in the subject express the CAR.

[0306] General therapeutic methods

[0307] The administration of fusion protein of the invention, a conjugate of the invention, a construct of the invention, the one or more polynucleotides of the invention, a vector of the invention, an amphipathic structure of the invention, a population of the invention or a pharmaceutical composition of the invention may reduce symptoms by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated subject. The administration may result in a decrease in tumour size of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated tumour.

[0308] The amount and / or number of the fusion protein, conjugate, construct, polynucleotide, vector, amphipathic structure, population, or pharmaceutical composition administered to the subject should take into account the route of administration, the disease or disorder being treated, the weight of the subject and / or the age of the subject. From about 1 mg / ml to about 100 mg / ml may be administered to the subject. In some embodiments, from about 1 mg / ml to about 10 mg / ml are administered to the subject. For amphipathic structures, from about 1 x 106to about 1 x 1011amphipathic structures are administered to the subject. In one embodiment, from about 1 x 107to about 1 x 1010cells, or from about 1 x 108to about 1 x 109amphipathic structures are administered to the subject.

[0309] The administration route may be any of those discussed above. In preferred embodiments, administration is conducted by intravenous injection.

[0310] The therapy or method typically comprises administering a therapeutically effective amount or a prophylactically effective amount of the fusion protein, conjugate, construct, polynucleotide, vector, amphipathic structure, population, or pharmaceutical composition. A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease or disorder and / or abolishes one or more symptoms, such as all the symptoms, of the disease or disorder. The therapeutically effective amount preferably cures the disease or disorder. A prophylactically effective amount is an amount which prevents the onset of the disease or disorder and / or prevents the onset of one or more symptoms, such as all the symptoms, of the disease or disorder. The prophylactically effective amount preferably prevents the subject from developing the disease or disorder. Suitable amounts are discussed in more detail below.

[0311] Any of the fusion proteins, conjugates, constructs, polynucleotides, vectors, amphipathic structures, populations, or pharmaceutical compositions may be administered to a subject that displays symptoms of disease or disorder. Any of the populations or the pharmaceutical composition may be administered to a subject that is asymptomatic, i.e., does not display symptoms of disease or disorder. Any of the populations or the pharmaceutical composition may be administered when the subject's disease status is unknown, or the subject is expected not to have a disease or disorder. Any of the populations or the pharmaceutical composition may be administered to a subject that is predisposed, such as genetically predisposed, to developing the disease or disorder.

[0312] The method or therapy may comprise one or more diagnostic tests to establish the presence or absence of the disease or disorder and / or for use in determining treatment options. Examples of suitable diagnostic tests include detection of specific mutations in cancer cells (e.g., specific mutation in EGFR, HER.2, and the like), detection of specific mutations associated with particular diseases (e.g., trinucleotide repeats, mutations in p-globin associated with sickle cell disease, specific SNPs, etc.), detection of hepatitis, detection of viruses (e.g., SARS-Cov-2), and so forth.

[0313] In embodiments where the subject is human, the subject may be a human adult or child. In the context of the present invention, an adult will be understood to be an at least 18-year- old human. A child will be understood to be a human less than 18 years old. In some embodiments, the adult is at least 60 years old.

[0314] The invention may be used in combination with other means of, and substances for, treating disease or disorder. In some cases, any of the fusion proteins, conjugates, constructs, polynucleotides, vectors, amphipathic structures, populations, or pharmaceutical compositions may be administered simultaneously, sequentially, or separately with other substances which are intended for treating the disease or disorder or ameliorating the symptoms of the disease or disorder, or for providing pain relief. Any of the fusion proteins, conjugates, constructs, polynucleotides, vectors, amphipathic structures, populations, or pharmaceutical compositions may be used in combination with existing treatments for disease or disorder and may, for example, be simply mixed with such treatments. Thus the invention may be used to increase the efficacy of existing treatments for disease.

[0315] All of the embodiments in this section equally apply to the composition of the invention. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0316] SEQUENCE LISTING

[0317] SEQ ID NO: 1 (Anti-CD3e_Cibisatamab_VH)

[0318] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKG RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0319] SEQ ID NO: 2 (Anti-CD3e_Cibisatamab_VL)

[0320] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLL GGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSS

[0321] SEQ ID NO: 3 (Anti-CD3e_OKT3_VH)

[0322] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKA TLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

[0323] SEQ ID NO: 4 (Anti-CD3e_OKT3_VL)

[0324] DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSG TSYSLTISSM EAEDAATYYCQQWSSN PLTFG AGTKLELK

[0325] SEQ ID NO: 5 (LDLR_LA4-LA5)

[0326] GSEAAAKEAAAKEAAAKGSVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQG DSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCA

[0327] Cibisatamab NanoPilot sequences (CYT344)

[0328] SEQ ID NO: 6 (CYT341_HC_ORF)

[0329] GCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAGG TTCAACTTCTAGAGTCTGGGGGTGGGCTAGTTCAGCCCGGAGGGAGTCTTCGGCTTTCCTGCGCCG CGTCCGGCTTTACATTTTCAACATATGCTATGAATTGGGTGAGACAGGCTCCGGGTAAGGGTCTGGA GTGGGTCAGCCGCATTCGCAGCAAGTACAACAACTACGCAACCTACTACGCCGACTCAGTCAAGGG ACGATTTACAATCAGCAGGGATGACTCTAAAAACACACTGTACCTCCAGATGAACTCCCTCCGTGCC GAAGACACCGCAGTCTATTACTGTGTGCGACACGGCAATTTCGGAAATTCCTATGTAAGTTGGTTCG CCTATTGGGGACAGGGTACCTTGGTGACTGTTAGCTCCGCTTCCACCAAGGGCCCCTCTGTTTTCCC ATTGGCTCCTTCATCAAAGTCTACAAGCGGCGGCACCGCTGCTCTAGGATGTCTCGTCAAAGACTAC TTCCCTGAACCTGTTACAGTGAGCTGGAACAGTGGCGCGCTAACGTCTGGAGTGCACACATTCCCAG CTGTGCTGCAGAGTTCTGGCCTGTATTCTCTGTCTAGCGTGGTGACAGTGCCTTCCAGTTCCCTCGG TACGCAGACTTACATTTGCAACGTAAACCACAAACCCTCAAACACAAAAGTGGATAAGAAGGTTGAG

[0330] CCTAAATCTTGCGATAAGACACACACATGCCCCCCATGCCCAGCTCCTGAGCTGCTAGGGGGGCCAT CCGTCTTTCTGTTTCCTCCTAAACCAAAAGACACACTCATGATCTCCCGGACCCCAGAGGTAACTTGC GTGGTGGTGGACGTGTCTCATGAAGACCCAGAGGTGAAGTTCAACTGGTATGTCGATGGAGTCGAG GTACATAACGCAAAGACTAAACCCAGGGAAGAGCAGTACAACAGCACCTACCGTGTTGTGTCTGTGC TGACTGTGCTCCACCAGGACTGGCTCAACGGCAAGGAATATAAATGCAAAGTCTCCAATAAGGCACT

[0331] ACCCGCGCCCATTGAGAAAACCATCTCTAAGGCAAAAGGGCAACCACGCGAGCCTCAGGTTTACACT CTGCCCCCCAGCAGGGAAGAGATGACTAAGAATCAGGTTTCCCTGACATGTCTCGTGAAGGGCTTTT ACCCATCGGATATTGCCGTCGAATGGGAGAGTAACGGACAGCCCGAGAATAATTACAAGACTACAC CCCCCGTACTCGACTCCGATGGAAGCTTTTTCCTGTACTCCAAGCTGACCGTTGACAAGAGCCGCTG GCAACAAGGCAATGTGTTCTCTTGCAGTGTAATGCACGAGGCCCTCCATAATCACTACACCCAGAAG

[0332] TCACTGAGTTTATCCCCCGGTTCAGAAGCTGCAGCTAAGGAAGCCGCTGCAAAAGAAGCTGCTGCTA AGGGTTCTGTCCTGACATGCGGCCCCGCCAGTTTTCAATGCAATTCATCTACCTGTATTCCCCAGCTC TGGGCATGCGACAACGATCCAGATTGCGAGGATGGTAGCGACGAATGGCCTCAGAGGTGTAGAGG ACTGTACGTGTTTCAGGGGGACAGCTCCCCATGTTCTGCTTTCGAGTTCCATTGTTTGAGTGGAGAG TGCATCCACTCATCGTGGCGGTGCGACGGGGGCCCTGATTGTAAGGATAAATCCGACGAAGAGAAC

[0333] TGTGCCTAGT

[0334] SEQ ID NO: 7 (CYT341_HC_translation)

[0335] MGWSCIILFLVATATGVHSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

[0336] KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEAAAKEA AAKEAAAKGSVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFE FHCLSGECIHSSWRCDGGPDCKDKSDEENCA*

[0337] SEQ ID NO: 8 (CYT341_LC_ORF)

[0338] GCCACCATGGAGACCGACACTCTGCTGCTTTGGGTTCTGCTGCTGTGGGTACCAGGCAGTACAGGG GATCAAGCAGTCGTGACTCAGGAGCCTTCCCTGACCGTGTCGCCTGGGGGTACCGTGACTCTGACT TGCGGCTCTAGCACCGGGGCGGTGACAACTTCTAACTATGCCAACTGGGTACAGGAAAAACCAGGC CAGGCCTTCAGGGGATTAATCGGTGGGACCAATAAGAGAGCTCCAGGCACCCCCGCTCGGTTTAGT GGTTCTCTCCTGGGTGGCAAAGCTGCCTTGACGTTGTCCGGCGCACAGCCCGAAGATGAAGCAGAG TATTACTGTGCTCTGTGGTACTCGAATCTGTGGGTGTTCGGCGGCGGAACGAAGCTGACAGTCCTCG

[0339] GCCAGCCCAAGGCAGCCCCATCCGTTACCCTGTTTCCCCCATCCTCCGAAGAATTGCAGGCAAATAA

[0340] AGCTACCCTGGTGTGCCTTATCTCCGATTTTTACCCCGGGGCCGTGACTGTGGCATGGAAGGCAGAT

[0341] TCTTCGCCTGTGAAGGCTGGTGTTGAAACAACCACACCCTCCAAGCAAAGCAACAACAAGTACGCAG

[0342] CCAGCTCCTATCTGTCCCTGACCCCGGAACAATGGAAGTCTCATAGGTCCTACAGCTGTCAGGTCAC

[0343] ACATGAGGGCTCCACAGTGGAGAAAACAGTGGCTCCAACAGAGTGTAGTTAG

[0344] SEQ ID NO: 9 (CYT341_LC_translation)

[0345] ATMETDTLLLWVLLLWVPGSTGDQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQA

[0346] FRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKA

[0347] APSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS*

[0348] 0KT3 NanoPilot sequences (CYT344)

[0349] SEQ ID NO: 10 (CYT344_HC_ORF)

[0350] GCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGATAT

[0351] TAAGTTGCAACAGAGCGGAGCAGAACTTGCTCGGCCAGGGGCCTCCGTCAAAATGTCATGCAAGAC

[0352] CAGTGGGTACACATTCACACGGTACACCATGCACTGGGTTAAACAGAGACCTGGACAAGGGCTGGA

[0353] ATGGATCGGTTATATTAACCCGAGCAGGGGCTATACTAACTATAACCAGAAGTTTAAGGACAAAGCC

[0354] ACTCTAACCACAGATAAGTCTTCAAGCACAGCATACATGCAGCTCAGCTCTCTGACCAGTGAAGATA

[0355] GTGCTGTTTACTACTGCGCCAGGTATTACGATGATCATTACTGTCTGGACTATTGGGGTCAGGGTAC

[0356] CACCCTCACCGTGTCCTCTGCTTCCACCAAGGGCCCCTCTGTTTTCCCATTGGCTCCTTCATCAAAGT

[0357] CTACAAGCGGCGGCACCGCTGCTCTAGGATGTCTCGTCAAAGACTACTTCCCTGAACCTGTTACAGT

[0358] GAGCTGGAACAGTGGCGCGCTAACGTCTGGAGTGCACACATTCCCAGCTGTGCTGCAGAGTTCTGG

[0359] CCTGTATTCTCTGTCTAGCGTGGTGACAGTGCCTTCCAGTTCCCTCGGTACGCAGACTTACATTTGCA

[0360] ACGTAAACCACAAACCCTCAAACACAAAAGTGGATAAGAAGGTTGAGCCTAAATCTTGCGATAAGAC

[0361] ACACACATGCCCCCCATGCCCAGCTCCTGAGCTGCTAGGGGGGCCATCCGTCTTTCTGTTTCCTCCT

[0362] AAACCAAAAGACACACTCATGATCTCCCGGACCCCAGAGGTAACTTGCGTGGTGGTGGACGTGTCTC

[0363] ATGAAGACCCAGAGGTGAAGTTCAACTGGTATGTCGATGGAGTCGAGGTACATAACGCAAAGACTA

[0364] AACCCAGGGAAGAGCAGTACAACAGCACCTACCGTGTTGTGTCTGTGCTGACTGTGCTCCACCAGG

[0365] ACTGGCTCAACGGCAAGGAATATAAATGCAAAGTCTCCAATAAGGCACTACCCGCGCCCATTGAGAA

[0366] AACCATCTCTAAGGCAAAAGGGCAACCACGCGAGCCTCAGGTTTACACTCTGCCCCCCAGCAGGGA

[0367] AGAGATGACTAAGAATCAGGTTTCCCTGACATGTCTCGTGAAGGGCTTTTACCCATCGGATATTGCC

[0368] GTCGAATGGGAGAGTAACGGACAGCCCGAGAATAATTACAAGACTACACCCCCCGTACTCGACTCC

[0369] GATGGAAGCTTTTTCCTGTACTCCAAGCTGACCGTTGACAAGAGCCGCTGGCAACAAGGCAATGTGT

[0370] TCTCTTGCAGTGTAATGCACGAGGCCCTCCATAATCACTACACCCAGAAGTCACTGAGTTTATCCCCC

[0371] GGTTCAGAAGCTGCAGCTAAGGAAGCCGCTGCAAAAGAAGCTGCTGCTAAGGGTTCTGTCCTGACA

[0372] TGCGGCCCCGCCAGTTTTCAATGCAATTCATCTACCTGTATTCCCCAGCTCTGGGCATGCGACAACG ATCCAGATTGCGAGGATGGTAGCGACGAATGGCCTCAGAGGTGTAGAGGACTGTACGTGTTTCAGG GGGACAGCTCCCCATGTTCTGCTTTCGAGTTCCATTGTTTGAGTGGAGAGTGCATCCACTCATCGTG GCGGTGCGACGGGGGCCCTGATTGTAAGGATAAATCCGACGAAGAGAACTGTGCCTAG

[0373] SEQ ID NO: 11 (CYT344_HC_tranlsation)

[0374] MGWSCIILFLVATATGVHSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIG YINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV

[0375] SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEAAAKEAAAKEAAA KGSVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGE CIHSSWRCDGGPDCKDKSDEENCA

[0376] SEQ ID NO: 12 (CYT344_LC_ORF)

[0377] GCCACCATGGAGACCGACACTCTGCTGCTTTGGGTTCTGCTGCTGTGGGTACCAGGCAGTACAGGG GATGACATCCAGCTGACACAGAGCCCTGCTATCATGTCTGCTTCCCCCGGCGAGAAAGTGACTATGA CTTGTCGTGCTAGTTCTAGTGTGTCTTACATGAATTGGTATCAGCAGAAGTCGGGCACATCCCCCAA GCGCTGGATCTACGACACTAGTAAAGTGGCCTCCGGAGTCCCATATCGATTCTCAGGCTCTGGCTCA GGTACTTCATACTCTTTGACGATATCCAGCATGGAGGCAGAGGACGCCGCGACCTATTACTGCCAGC

[0378] AATGGAGCTCCAATCCCCTGACGTTTGGCGCCGGAACAAAGCTTGAGTTAAAGAGGACAGTTGCCG CCCCTAGTGTGTTCATCTTTCCGCCTTCAGACGAGCAGCTGAAGTCCGGTACAGCTTCCGTGGTGTG CCTTCTAAATAATTTCTATCCGAGGGAAGCGAAGGTGCAGTGGAAGGTCGATAATGCACTGCAAAGC GGCAACAGTCAGGAGTCCGTCACAGAACAGGATAGCAAAGACTCCACTTATAGTCTGTCCTCGACCC TTACACTATCCAAGGCCGACTACGAAAAACACAAAGTCTACGCTTGCGAGGTGACCCACCAGGGTCT

[0379] GAGCTCCCCTGTAACAAAGTCTTTCAACCGCGGAGAGTGCTAG

[0380] SEQ ID NO: 13 (CYT344_LC_translation)

[0381] ATMETDTLLLWVLLLWVPGSTGDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKR WIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0382] SEQ ID NO: 14

[0383] GSEAAAKEAAAKEAAAKGS

[0384] SEQ ID NO: 15 GGSGGSGGSGGSGG

[0385] SEQ ID NO: 16 (Anti-murine_CD3£_2Cl l_VH)

[0386] EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFT

[0387] VSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS

[0388] SEQ ID NO: 17 (Anti-murine_CD3£_2Cl l_VL)

[0389] DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGR

[0390] DSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK

[0391] SEQ ID NO: 18 (Murine_LDLR_A4-A5)

[0392] ATTCGPAHFRCNSSICIPSLWACDGDVDCVDGSDEWPQNCQGRDTASKGVSSPCSSLEFHCGSSECI

[0393] HRSWVCDGEADCKDKSDEEHCA

[0394] SEQ ID NO: 19 (CYT346_HC_ORF)

[0395] ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAGGTCCAGCT

[0396] GGTTGAATCAGGTGGCGGTTTGGTGCAGCCTGGCAAGTCTCTGAAGCTCTCTTGTGAGGCTAGCGG

[0397] GTTTACATTCAGCGGGTATGGCATGCACTGGGTGAGGCAAGCACCCGGACGAGGACTCGAATCCGT

[0398] GGCCTACATTACCTCTTCCAGCATCAACATCAAATACGCCGACGCTGTCAAAGGAAGATTCACTGTAT

[0399] CACGCGACAATGCCAAAAACCTACTGTTCTTACAGATGAATATACTTAAGAGTGAGGACACTGCGAT

[0400] GTATTATTGCGCACGGTTTGATTGGGATAAGAACTACTGGGGCCAGGGGACAATGGTGACCGTTAG

[0401] TTCCGCTTCCACCAAGGGCCCCTCTGTTTTCCCATTGGCTCCTTCATCAAAGTCTACAAGCGGCGGC

[0402] ACCGCTGCTCTAGGATGTCTCGTCAAAGACTACTTCCCTGAACCTGTTACAGTGAGCTGGAACAGTG

[0403] GCGCGCTAACGTCTGGAGTGCACACATTCCCAGCTGTGCTGCAGAGTTCTGGCCTGTATTCTCTGTC

[0404] TAGCGTGGTGACAGTGCCTTCCAGTTCCCTCGGTACGCAGACTTACATTTGCAACGTAAACCACAAA

[0405] CCCTCAAACACAAAAGTGGATAAGAAGGTTGAGCCTAAATCTTGCGATAAGACACACACATGCCCCC

[0406] CATGCCCAGCTCCTGAGGCCGCTGGGGGGCCATCCGTCTTTCTGTTTCCTCCTAAACCAAAAGACAC

[0407] ACTCATGATCTCCCGGACCCCAGAGGTAACTTGCGTGGTGGTGGACGTGTCTCATGAAGACCCAGA

[0408] GGTGAAGTTCAACTGGTATGTCGATGGAGTCGAGGTACATAACGCAAAGACTAAACCCAGGGAAGA

[0409] GCAGTACAACAGCACCTACCGTGTTGTGTCTGTGCTGACTGTGCTCCACCAGGACTGGCTCAACGGC

[0410] AAGGAATATAAATGCAAAGTCTCCAATAAGGCACTAGGCGCGCCCATTGAGAAAACCATCTCTAAGG

[0411] CAAAAGGGCAACCACGCGAGCCTCAGGTTTACACTCTGCCCCCCAGCAGGGAAGAGATGACTAAGA

[0412] ATCAGGTTTCCCTGACATGTCTCGTGAAGGGCTTTTACCCATCGGATATTGCCGTCGAATGGGAGAG

[0413] TAACGGACAGCCCGAGAATAATTACAAGACTACACCCCCCGTACTCGACTCCGATGGAAGCTTTTTC

[0414] CTGTACTCCAAGCTGACCGTTGACAAGAGCCGCTGGCAACAAGGCAATGTGTTCTCTTGCAGTGTAA

[0415] TGCACGAGGCCCTCCATAATCACTACACCCAGAAGTCACTGAGTTTATCCCCCGGTTCAGAAGCTGC

[0416] AGCTAAGGAAGCCGCTGCAAAAGAAGCTGCTGCTAAGGGTTCTGCCACTACCTGTGGGCCTGCTCA

[0417] CTTTCGGTGCAACAGCTCTATCTGTATCCCCAGCCTCTGGGCTTGCGATGGTGATGTGGACTGTGTT GATGGAAGTGATGAGTGGCCGCAGAATTGTCAGGGGAGGGACACAGCAAGTAAGGGCGTCTCTTC CCCATGCTCTAGCCTGGAGTTCCATTGTGGATCATCCGAGTGTATTCACAGATCCTGGGTGTGCGAT GGCGAAGCGGACTGCAAGGACAAATCAGACGAAGAGCATTGCGCCTAG

[0418] SEQ ID NO: 20 (CYT346_HC_translation)

[0419] MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVA

[0420] YITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

[0421] NKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEAAAKEAAAKEAAAK GSATTCGPAHFRCNSSICIPSLWACDGDVDCVDGSDEWPQNCQGRDTASKGVSSPCSSLEFHCGSSE CIHRSWVCDGEADCKDKSDEEHCA*

[0422] SEQ ID NO: 21 (CYT346_LC_ORF)

[0423] ATGGAGACCGACACTCTGCTGCTTTGGGTTCTGCTGCTGTGGGTACCAGGCAGTACAGGGGATGAC

[0424] ATACAGATGACACAGTCGCCCTCCTCCCTACCTGCCTCCCTTGGTGATCGGGTGACCATCAACTGCC AAGCCTCTCAGGACATCAGCAACTACCTGAATTGGTATCAGCAAAAACCAGGAAAGGCACCTAAACT CCTCATCTACTACACAAACAAGCTGGCTGATGGGGTCCCCTCCAGATTTTCTGGTAGCGGCAGCGGA AGGGATAGCTCTTTCACCATTTCAAGTTTGGAGAGTGAAGACATCGGCTCATACTATTGTCAGCAGT

[0425] ATTATAATTACCCGTGGACTTTCGGGCCAGGCACTAAGCTGGAGATTAAGAGGACAGTTGCCGCCCC

[0426] TAGTGTGTTCATCTTTCCGCCTTCAGACGAGCAGCTGAAGTCCGGTACAGCTTCCGTGGTGTGCCTT CTAAATAATTTCTATCCGAGGGAAGCGAAGGTGCAGTGGAAGGTCGATAATGCACTGCAAAGCGGC AACAGTCAGGAGTCCGTCACAGAACAGGATAGCAAAGACTCCACTTATAGTCTGTCCTCGACCCTTA CACTATCCAAGGCCGACTACGAAAAACACAAAGTCTACGCTTGCGAGGTGACCCACCAGGGTCTGA

[0427] GCTCCCCTGTAACAAAGTCTTTCAACCGCGGAGAGTGCTAG

[0428] SEQ ID NO: 22 (CYT346_LC_translation)

[0429] METDTLLLWVLLLWVPGSTGDDIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLI

[0430] YYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC*

[0431] SEQ ID NO: 23 (CYT349_HC_ORF)

[0432] ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAGGTCCAGCT GGTTGAATCAGGTGGCGGTTTGGTGCAGCCTGGCAAGTCTCTGAAGCTCTCTTGTGAGGCTAGCGG GTTTACATTCAGCGGGTATGGCATGCACTGGGTGAGGCAAGCACCCGGACGAGGACTCGAATCCGT GGCCTACATTACCTCTTCCAGCATCAACATCAAATACGCCGACGCTGTCAAAGGAAGATTCACTGTAT

[0433] CACGCGACAATGCCAAAAACCTACTGTTCTTACAGATGAATATACTTAAGAGTGAGGACACTGCGAT

[0434] GTATTATTGCGCACGGTTTGATTGGGATAAGAACTACTGGGGCCAGGGGACAATGGTGACCGTTAG

[0435] TTCCGCTAAAACGACACCCCCATCGGTCTATCCACTGGCCCCAGGATCTGCTGCACAAACTAACTCC

[0436] ATGGTGACCCTGGGGTGCCTTGTTAAGGGCTATTTTCCTGAGCCAGTGACAGTGACCTGGAACTCTG

[0437] GATCCCTGTCCAGCGGTGTGCATACCTTCCCAGCTGTCTTGCAATCTGACCTATACACTTTAAGTAGT

[0438] TCAGTGACCGTCCCTTCCTCAACCTGGCCCTCAGAGACCGTCACATGCAACGTTGCCCACCCGGCCA

[0439] GCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGT

[0440] CCCAGAAGTATCATCTGTTTTTATCTTCCCCCCAAAACCTAAGGATGTGCTCACCATTACACTGACTC

[0441] CTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCGGAGGTCCAGTTTAGCTGGTTTGT

[0442] AGATGATGTGGAAGTGCACACGGCCCAGACCCAACCCCGGGAGGAGCAGTTCAACTCCACTTTCCG

[0443] CTCAGTTAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTTAAATGCCGAGTT

[0444] AATAGTGCAGCTTTTCCTGCCCCCATCGAAAAAACAATCTCCAAAACAAAAGGCAGACCTAAGGCTC

[0445] CCCAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTAAGTCTGACCTGCAT

[0446] GATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAA

[0447] CTACAAGAACACTCAGCCCATCATGGACACAGACGGGTCTTATTTCGTCTACAGCAAGCTCAATGTG

[0448] CAGAAGTCGAACTGGGAGGCAGGAAATACTTTTACCTGTTCTGTGTTGCATGAAGGCCTGCATAATC

[0449] ACCATACTGAAAAGAGCCTATCCCACTCTCCTGGTAAAGGTTCAGAAGCTGCAGCTAAGGAAGCCGC

[0450] TGCAAAAGAAGCTGCTGCTAAGGGTTCTGCCACTACCTGTGGGCCTGCTCACTTTCGGTGCAACAGC

[0451] TCTATCTGTATCCCCAGCCTCTGGGCTTGCGATGGTGATGTGGACTGTGTTGATGGAAGTGATGAGT

[0452] GGCCGCAGAATTGTCAGGGGAGGGACACAGCAAGTAAGGGCGTCTCTTCCCCATGCTCTAGCCTGG

[0453] AGTTCCATTGTGGATCATCCGAGTGTATTCACAGATCCTGGGTGTGCGATGGCGAAGCGGACTGCA

[0454] AGGACAAATCAGACGAAGAGCATTGCGCCTAG

[0455] SEQ ID NO: 24 (CYT349_HC_translation)

[0456] MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVA

[0457] YITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSA

[0458] KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVP

[0459] SSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVD

[0460] ISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK

[0461] TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGS

[0462] YFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGSEAAAKEAAAKEAAAKGSATTCG

[0463] PAHFRCNSSICIPSLWACDGDVDCVDGSDEWPQNCQGRDTASKGVSSPCSSLEFHCGSSECIHRSWV

[0464] CDGEADCKDKSDEEHCA*

[0465] SEQ ID NO: 25 (CYT349_LC_ORF)

[0466] ATGGAGACCGACACTCTGCTGCTTTGGGTTCTGCTGCTGTGGGTACCAGGCAGTACAGGGGATGAC

[0467] ATACAGATGACACAGTCGCCCTCCTCCCTACCTGCCTCCCTTGGTGATCGGGTGACCATCAACTGCC

[0468] AAGCCTCTCAGGACATCAGCAACTACCTGAATTGGTATCAGCAAAAACCAGGAAAGGCACCTAAACT CCTCATCTACTACACAAACAAGCTGGCTGATGGGGTCCCCTCCAGATTTTCTGGTAGCGGCAGCGGA

[0469] AGGGATAGCTCTTTCACCATTTCAAGTTTGGAGAGTGAAGACATCGGCTCATACTATTGTCAGCAGT

[0470] ATTATAATTACCCGTGGACTTTCGGGCCAGGCACTAAGCTGGAGATTAAGAGAGCAGATGCTGCTCC

[0471] CACAGTATCCATCTTTCCACCTTCCAGTGAGCAGCTTACATCTGGAGGGGCCTCAGTGGTGTGCTTT

[0472] TTGAACAATTTCTACCCCAAAGACATCAATGTTAAGTGGAAGATAGATGGTTCTGAACGGCAAAATG

[0473] GCGTGCTGAACAGTTGGACTGATCAGGACTCGAAAGACTCTACCTACAGCATGAGCTCCACCCTCAC

[0474] GCTGACCAAGGACGAGTATGAAAGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACT

[0475] TCCCCTATTGTCAAAAGCTTCAACAGGAATGAGTGCTGA

[0476] SEQ ID NO: 26 (CYT349_LC_translation)

[0477] METDTLLLWVLLLWVPGSTGDDIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLI

[0478] YYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKRADAAPTVSIF PPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY ERHNSYTCEATHKTSTSPIVKSFNRNEC*

[0479] SEQ ID NO: 27 (CYT350_HC_ORF)

[0480] ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAGGTCCAGCT

[0481] GGTTGAATCAGGTGGCGGTTTGGTGCAGCCTGGCAAGTCTCTGAAGCTCTCTTGTGAGGCTAGCGG

[0482] GTTTACATTCAGCGGGTATGGCATGCACTGGGTGAGGCAAGCACCCGGACGAGGACTCGAATCCGT

[0483] GGCCTACATTACCTCTTCCAGCATCAACATCAAATACGCCGACGCTGTCAAAGGAAGATTCACTGTAT

[0484] CACGCGACAATGCCAAAAACCTACTGTTCTTACAGATGAATATACTTAAGAGTGAGGACACTGCGAT

[0485] GTATTATTGCGCACGGTTTGATTGGGATAAGAACTACTGGGGCCAGGGGACAATGGTGACCGTTAG

[0486] TTCCGCTTCCACCAAGGGCCCCTCTGTTTTCCCATTGGCTCCTTCATCAAAGTCTACAAGCGGCGGC

[0487] ACCGCTGCTCTAGGATGTCTCGTCAAAGACTACTTCCCTGAACCTGTTACAGTGAGCTGGAACAGTG

[0488] GCGCGCTAACGTCTGGAGTGCACACATTCCCAGCTGTGCTGCAGAGTTCTGGCCTGTATTCTCTGTC

[0489] TAGCGTGGTGACAGTGCCTTCCAGTTCCCTCGGTACGCAGACTTACATTTGCAACGTAAACCACAAA

[0490] CCCTCAAACACAAAAGTGGATAAGAAGGTTGAGCCTAAATCTTGCGATAAGACACACACATGCCCCC

[0491] CATGCCCAGCTCCTGAGGCCGCTGGGGGGCCATCCGTCTTTCTGTTTCCTCCTAAACCAAAAGACAC

[0492] ACTCATGATCTCCCGGACCCCAGAGGTAACTTGCGTGGTGGTGGACGTGTCTCATGAAGACCCAGA

[0493] GGTGAAGTTCAACTGGTATGTCGATGGAGTCGAGGTACATAACGCAAAGACTAAACCCAGGGAAGA

[0494] GCAGTACAACAGCACCTACCGTGTTGTGTCTGTGCTGACTGTGCTCCACCAGGACTGGCTCAACGGC

[0495] AAGGAATATAAATGCAAAGTCTCCAATAAGGCACTAGGCGCGCCCATTGAGAAAACCATCTCTAAGG

[0496] CAAAAGGGCAACCACGCGAGCCTCAGGTTTACACTCTGCCCCCCAGCAGGGAAGAGATGACTAAGA

[0497] ATCAGGTTTCCCTGACATGTCTCGTGAAGGGCTTTTACCCATCGGATATTGCCGTCGAATGGGAGAG

[0498] TAACGGACAGCCCGAGAATAATTACAAGACTACACCCCCCGTACTCGACTCCGATGGAAGCTTTTTC

[0499] CTGTACTCCAAGCTGACCGTTGACAAGAGCCGCTGGCAACAAGGCAATGTGTTCTCTTGCAGTGTAA

[0500] TGCACGAGGCCCTCCATAATCACTACACCCAGAAGTCACTGAGTTTATCCCCCGGAGGCAGCGGGG

[0501] GTAGTGGGGGCTCTGGCGGATCCGGTGGAGCCACTACCTGTGGGCCTGCTCACTTTCGGTGCAACA

[0502] GCTCTATCTGTATCCCCAGCCTCTGGGCTTGCGATGGTGATGTGGACTGTGTTGATGGAAGTGATGA GTGGCCGCAGAATTGTCAGGGGAGGGACACAGCAAGTAAGGGCGTCTCTTCCCCATGCTCTAGCCT

[0503] GGAGTTCCATTGTGGATCATCCGAGTGTATTCACAGATCCTGGGTGTGCGATGGCGAAGCGGACTG

[0504] CAAGGACAAATCAGACGAAGAGCATTGCGCCTAG

[0505] SEQ ID NO: 28 (CYT350_HC_translation)

[0506] MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVA YITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP

[0507] EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSGGSGGSGGAT TCGPAHFRCNSSICIPSLWACDGDVDCVDGSDEWPQNCQGRDTASKGVSSPCSSLEFHCGSSECIHR

[0508] SWVCDGEADCKDKSDEEHCA*

[0509] SEQ ID NO: 29 (CYT354_PAI-l_sfGFP)

[0510] VHHPPSYVAHLASDFGVRVFQQVAQASKDRNVVFSPYGVASVLAMLQLTTGGETQQQIQAAMGFKIDD KGMAPALRHLYKELMGPWNKDEISTTDAIFVQRDLKLVQGFMPHFFRLFRSTVKQVDFSEVERARFIIND WVKTHTKGMISNLLGKGAVDQLTRLVLVNALYFNGQWKTPFPDSSTHRRLFHKSDGSTVSVPMMAQT NKFNYTEFTTPDGHYYDILELPYHGDTLSMFIAAPYEKEVPLSALTNILSAQLISHWKGNMTRLPRLLVLP

[0511] KFSLETEVDLRKPLENLGMTDMFRQFQADFTSLSDQEPLHVAQALQKVKIEVNESGTVASSSTAVIVSA RMAPEEIIMDRPFLFVVRHNPTGTVLFMGQVMEPGSEAAAKEAAAKEAAAKGSGMSKGEELFTGVVPIL VELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFF KSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITA

[0512] DKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE FVTAAGITHGMDELYKGS

[0513] CYT435 (Anti-murine CD8a clone YTS-105.18 on human IgGl with PG-LALA silencing mutations fused to murine LDL-R (LA4-LA5) fragment)

[0514] SEQ ID NO: 30 (YTS-105.18_VH)

[0515] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSNSVHWVRQPPGKGLEWMGGIWGDGDTDYNSALKSRL SISRDTSKNQVFLKMNSLQTDDTAIYFCTPLIGSWYFDFWGPGTMVTASS

[0516] SEQ ID NO: 31 (YTS-105.18_VL)

[0517] DIVMTQSPSSLAVSAGERVTLNCKASQNVRNNIAWYQQKPGQSPKLLIYYASYRYTGVPDRFTGDGFGT DFTLAINSVQADDAAFYYCQRIYNSPYTFGAGTKLELI

[0518] SEQ ID NO: 32 (CYT435_HC_ORF) GCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATCAAGTCCA GTTGAAGGAATCTGGACCTGGACTGGTGCAGCCCTCCCAGACATTGAGCCTTACCTGTACAGTATCA GGGTTTAGTCTCACTAGTAATTCGGTGCACTGGGTCCGGCAACCACCAGGGAAAGGACTAGAGTGG ATGGGTGGCATATGGGGCGACGGCGATACTGATTACAACAGCGCTTTAAAATCTAGACTGAGCATCT

[0519] CCAGGGATACTTCCAAGAACCAGGTGTTCCTGAAGATGAATTCTCTCCAGACCGACGACACCGCCAT

[0520] TTACTTCTGCACGCCCCTGATCGGGTCATGGTATTTCGACTTTTGGGGTCCTGGCACAATGGTTACC GCATCTTCCGCTTCCACCAAGGGCCCCTCTGTTTTCCCATTGGCTCCTTCATCAAAGTCTACAAGCGG CGGCACCGCTGCTCTAGGATGTCTCGTCAAAGACTACTTCCCTGAACCTGTTACAGTGAGCTGGAAC AGTGGCGCGCTAACGTCTGGAGTGCACACATTCCCAGCTGTGCTGCAGAGTTCTGGCCTGTATTCTC

[0521] TGTCTAGCGTGGTGACAGTGCCTTCCAGTTCCCTCGGTACGCAGACTTACATTTGCAACGTAAACCA CAAACCCTCAAACACAAAAGTGGATAAGAAGGTTGAGCCTAAATCTTGCGATAAGACACACACATGC CCCCCATGCCCAGCTCCTGAGGCCGCTGGGGGGCCATCCGTCTTTCTGTTTCCTCCTAAACCAAAAG ACACACTCATGATCTCCCGGACCCCAGAGGTAACTTGCGTGGTGGTGGACGTGTCTCATGAAGACC

[0522] CAGAGGTGAAGTTCAACTGGTATGTCGATGGAGTCGAGGTACATAACGCAAAGACTAAACCCAGGG AAGAGCAGTACAACAGCACCTACCGTGTTGTGTCTGTGCTGACTGTGCTCCACCAGGACTGGCTCAA CGGCAAGGAATATAAATGCAAAGTCTCCAATAAGGCACTAGGCGCGCCCATTGAGAAAACCATCTCT AAGGCAAAAGGGCAACCACGCGAGCCTCAGGTTTACACTCTGCCCCCCAGCAGGGAAGAGATGACT

[0523] AAGAATCAGGTTTCCCTGACATGTCTCGTGAAGGGCTTTTACCCATCGGATATTGCCGTCGAATGGG AGAGTAACGGACAGCCCGAGAATAATTACAAGACTACACCCCCCGTACTCGACTCCGATGGAAGCTT TTTCCTGTACTCCAAGCTGACCGTTGACAAGAGCCGCTGGCAACAAGGCAATGTGTTCTCTTGCAGT GTAATGCACGAGGCCCTCCATAATCACTACACCCAGAAGTCACTGAGTTTATCCCCCGGTTCAGAAG

[0524] CTGCAGCTAAGGAAGCCGCTGCAAAAGAAGCTGCTGCTAAGGGTTCTGCCACTACCTGTGGGCCTG CTCACTTTCGGTGCAACAGCTCTATCTGTATCCCCAGCCTCTGGGCTTGCGATGGTGATGTGGACTG TGTTGATGGAAGTGATGAGTGGCCGCAGAATTGTCAGGGGAGGGACACAGCAAGTAAGGGCGTCT CTTCCCCATGCTCTAGCCTGGAGTTCCATTGTGGATCATCCGAGTGTATTCACAGATCCTGGGTGTG

[0525] CGATGGCGAAGCGGACTGCAAGGACAAATCAGACGAAGAGCATTGCGCCTAG

[0526] SEQ ID NO: 33 (CYT435_HC_translation)

[0527] MGWSCIILFLVATATGVHQVQLKESGPGLVQPSQTLSLTCTVSGFSLTSNSVHWVRQPPGKGLEWMGG

[0528] IWGDGDTDYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTAIYFCTPLIGSWYFDFWGPGTMVTASSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

[0529] NKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEAAAKEAAAKEAAAK GSATTCGPAHFRCNSSICIPSLWACDGDVDCVDGSDEWPQNCQGRDTASKGVSSPCSSLEFHCGSSE CIHRSWVCDGEADCKDKSDEEHCA*

[0530] SEQ ID NO: 34 (CYT435_LC_ORF) GCCACCATGGAGACCGACACTCTGCTGCTTTGGGTTCTGCTGCTGTGGGTACCAGGCAGTACAGGG GATGACATTGTGATGACTCAGTCCCCTTCATCTTTGGCAGTGTCCGCCGGGGAACGAGTGACCCTTA ACTGTAAGGCCTCTCAAAACGTACGGAACAATATCGCCTGGTATCAGCAGAAGCCCGGCCAAAGCC CTAAACTGCTGATCTATTATGCTAGTTACAGGTATACGGGCGTTCCAGATCGCTTTACCGGAGATGG

[0531] ATTCGGCACAGACTTCACACTAGCTATCAATAGCGTCCAGGCCGATGACGCGGCATTCTACTACTGC

[0532] CAGAGAATATACAATTCGCCCTACACCTTTGGTGCTGGGACTAAACTGGAGCTCATTAGGACAGTTG CCGCCCCTAGTGTGTTCATCTTTCCGCCTTCAGACGAGCAGCTGAAGTCCGGTACAGCTTCCGTGGT GTGCCTTCTAAATAATTTCTATCCGAGGGAAGCGAAGGTGCAGTGGAAGGTCGATAATGCACTGCAA AGCGGCAACAGTCAGGAGTCCGTCACAGAACAGGATAGCAAAGACTCCACTTATAGTCTGTCCTCGA CCCTTACACTATCCAAGGCCGACTACGAAAAACACAAAGTCTACGCTTGCGAGGTGACCCACCAGGG TCTGAGCTCCCCTGTAACAAAGTCTTTCAACCGCGGAGAGTGCTAG

[0533] SEQ ID NO: 35 (CYT435_LC_translation)

[0534] METDTLLLWVLLLWVPGSTGDDIVMTQSPSSLAVSAGERVTLNCKASQNVRNNIAWYQQKPGQSPKLL IYYASYRYTGVPDRFTGDGFGTDFTLAINSVQADDAAFYYCQRIYNSPYTFGAGTKLELIRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC*

[0535] CYT391 (Anti-human CD117 clone 9P3 on human IgGl with PG-LALA silencing mutations fused to murine LDLR (LA4-LA5) fragment)

[0536] SEQ ID NO: 36 (9P3_VH)

[0537] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKGLEWVANINYDGSSTYYLDSVKGR FTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSS

[0538] SEQ ID NO: 37 (9P3_VL)

[0539] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQGKKLWSFGGGTKVEIK

[0540] SEQ ID NO: 38 (CYT391_HC_ORF)

[0541] GCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAAGT

[0542] CCAGCTCGTGGAGAGCGGGGGCGGACTGGTGCAGCCTGGGGGTTCTTTGCGCCTCTCATGCGCCG

[0543] CTAGTGGTTTCACATTCTCCGACTACTACATGGCCTGGGTCCGTCAGGCGCCCGGCAAGGGCCTTG AATGGGTGGCAAATATTAACTACGACGGCTCATCTACATACTATCTGGATTCCGTAAAGGGAAGATTT ACCATCAGCCGGGACAACGCTAAAAACTCCTTATACCTACAAATGAATAGCCTGCGAGCCGAGGACA CAGCTGTTTACTATTGTGCAAGGGGAGATTATTACGGTACTACTTATTGGTATTTTGATGTGTGGGGC

[0544] CAGGGGACCACCGTGACGGTTAGTTCTGCTTCCACCAAGGGCCCCTCTGTTTTCCCATTGGCTCCTT CATCAAAGTCTACAAGCGGCGGCACCGCTGCTCTAGGATGTCTCGTCAAAGACTACTTCCCTGAACC TGTTACAGTGAGCTGGAACAGTGGCGCGCTAACGTCTGGAGTGCACACATTCCCAGCTGTGCTGCA GAGTTCTGGCCTGTATTCTCTGTCTAGCGTGGTGACAGTGCCTTCCAGTTCCCTCGGTACGCAGACT

[0545] TACATTTGCAACGTAAACCACAAACCCTCAAACACAAAAGTGGATAAGAAGGTTGAGCCTAAATCTTG

[0546] CGATAAGACACACACATGCCCCCCATGCCCAGCTCCTGAGGCCGCTGGGGGGCCATCCGTCTTTCT

[0547] GTTTCCTCCTAAACCAAAAGACACACTCATGATCTCCCGGACCCCAGAGGTAACTTGCGTGGTGGTG

[0548] GACGTGTCTCATGAAGACCCAGAGGTGAAGTTCAACTGGTATGTCGATGGAGTCGAGGTACATAAC

[0549] GCAAAGACTAAACCCAGGGAAGAGCAGTACAACAGCACCTACCGTGTTGTGTCTGTGCTGACTGTG

[0550] CTCCACCAGGACTGGCTCAACGGCAAGGAATATAAATGCAAAGTCTCCAATAAGGCACTAGGCGCG

[0551] CCCATTGAGAAAACCATCTCTAAGGCAAAAGGGCAACCACGCGAGCCTCAGGTTTACACTCTGCCCC

[0552] CCAGCAGGGAAGAGATGACTAAGAATCAGGTTTCCCTGACATGTCTCGTGAAGGGCTTTTACCCATC

[0553] GGATATTGCCGTCGAATGGGAGAGTAACGGACAGCCCGAGAATAATTACAAGACTACACCCCCCGT

[0554] ACTCGACTCCGATGGAAGCTTTTTCCTGTACTCCAAGCTGACCGTTGACAAGAGCCGCTGGCAACAA

[0555] GGCAATGTGTTCTCTTGCAGTGTAATGCACGAGGCCCTCCATAATCACTACACCCAGAAGTCACTGA

[0556] GTTTATCCCCCGGTTCAGAAGCTGCAGCTAAGGAAGCCGCTGCAAAAGAAGCTGCTGCTAAGGGTT

[0557] CTGTCCTGACATGCGGCCCCGCCAGTTTTCAATGCAATTCATCTACCTGTATTCCCCAGCTCTGGGCA

[0558] TGCGACAACGATCCAGATTGCGAGGATGGTAGCGACGAATGGCCTCAGAGGTGTAGAGGACTGTAC

[0559] GTGTTTCAGGGGGACAGCTCCCCATGTTCTGCTTTCGAGTTCCATTGTTTGAGTGGAGAGTGCATCC

[0560] ACTCATCGTGGCGGTGCGACGGGGGCCCTGATTGTAAGGATAAATCCGACGAAGAGAACTGTGCCT AG

[0561] SEQ ID NO: 39 (CYT391_HC_translation)

[0562] MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKGLEWVA

[0563] NINYDGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTT

[0564] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

[0565] LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL

[0566] MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0567] KCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE

[0568] NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEAAAKEAAA

[0569] KEAAAKGSVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFH CLSGECIHSSWRCDGGPDCKDKSDEENCA*

[0570] SEQ ID NO: 40 (CYT391_LC_ORF)

[0571] GCCACCATGGAGACCGACACTCTGCTGCTTTGGGTTCTGCTGCTGTGGGTACCAGGCAGTACAGGG

[0572] GATGACATCCAGATGACCCAGAGCCCAAGTAGCTTGTCAGCATCGGTTGGCGATCGGGTGACCATC

[0573] ACATGCCGCGCTTCACAGAGTATTTCCTCTTATCTGAACTGGTACCAGCAGAAGCCTGGCAAAGCCC

[0574] CTAAGCTGCTAATATACTACACCTCCAGACTCCAATCCGGAGTGCCCTCCAGGTTTAGCGGGTCTGG

[0575] TAGTGGAACAGATTACACGCTGACAATCAGCTCTCTCCAGCCCGAAGACTTCGCCACTTATTATTGTC

[0576] AACAGGGAAAGAAACTTTGGTCTTTCGGCGGGGGTACTAAGGTCGAGATTAAAAGGACAGTTGCCG

[0577] CCCCTAGTGTGTTCATCTTTCCGCCTTCAGACGAGCAGCTGAAGTCCGGTACAGCTTCCGTGGTGTG

[0578] CCTTCTAAATAATTTCTATCCGAGGGAAGCGAAGGTGCAGTGGAAGGTCGATAATGCACTGCAAAGC GGCAACAGTCAGGAGTCCGTCACAGAACAGGATAGCAAAGACTCCACTTATAGTCTGTCCTCGACCC

[0579] TTACACTATCCAAGGCCGACTACGAAAAACACAAAGTCTACGCTTGCGAGGTGACCCACCAGGGTCT

[0580] GAGCTCCCCTGTAACAAAGTCTTTCAACCGCGGAGAGTGCTAG

[0581] SEQ ID NO: 41 (CYT391_LC_translation)

[0582] METDTLLLWVLLLWVPGSTGDDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI

[0583] YYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGKKLWSFGGGTKVEIKRTVAAPSVFIFP

[0584] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC*

[0585] CYT420 (Anti-human CD3e clone OKT3 on human IgGl fused to full length human ApoE3)

[0586] SEQ ID NO: 42 (ApoE3 AA sequence ( a. a. 19-317))

[0587] KVEQAVETEPEPELRQTEWSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMK

[0588] ELKAYKSELEEQLTPVAEETRARLSKELAAARLGADMEDVCGRLVYRGEVAMLGSTEELRVRLASHLRKL

[0589] RKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGE

[0590] RLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGL VEKVQAAVGTSAAPVPSDNH

[0591] SEQ ID NO: 43 (CYT420_HC_ORF)

[0592] GCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGATAT

[0593] TAAGTTGCAACAGAGCGGAGCAGAACTTGCTCGGCCAGGGGCCTCCGTCAAAATGTCATGCAAGAC

[0594] CAGTGGGTACACATTCACACGGTACACCATGCACTGGGTTAAACAGAGACCTGGACAAGGGCTGGA

[0595] ATGGATCGGTTATATTAACCCGAGCAGGGGCTATACTAACTATAACCAGAAGTTTAAGGACAAAGCC

[0596] ACTCTAACCACAGATAAGTCTTCAAGCACAGCATACATGCAGCTCAGCTCTCTGACCAGTGAAGATA

[0597] GTGCTGTTTACTACTGCGCCAGGTATTACGATGATCATTACTGTCTGGACTATTGGGGTCAGGGTAC

[0598] CACCCTCACCGTGTCCTCTGCTTCCACCAAGGGCCCCTCTGTTTTCCCATTGGCTCCTTCATCAAAGT

[0599] CTACAAGCGGCGGCACCGCTGCTCTAGGATGTCTCGTCAAAGACTACTTCCCTGAACCTGTTACAGT

[0600] GAGCTGGAACAGTGGCGCGCTAACGTCTGGAGTGCACACATTCCCAGCTGTGCTGCAGAGTTCTGG

[0601] CCTGTATTCTCTGTCTAGCGTGGTGACAGTGCCTTCCAGTTCCCTCGGTACGCAGACTTACATTTGCA

[0602] ACGTAAACCACAAACCCTCAAACACAAAAGTGGATAAGAAGGTTGAGCCTAAATCTTGCGATAAGAC

[0603] ACACACATGCCCCCCATGCCCAGCTCCTGAGCTGCTAGGGGGGCCATCCGTCTTTCTGTTTCCTCCT

[0604] AAACCAAAAGACACACTCATGATCTCCCGGACCCCAGAGGTAACTTGCGTGGTGGTGGACGTGTCTC

[0605] ATGAAGACCCAGAGGTGAAGTTCAACTGGTATGTCGATGGAGTCGAGGTACATAACGCAAAGACTA

[0606] AACCCAGGGAAGAGCAGTACAACAGCACCTACCGTGTTGTGTCTGTGCTGACTGTGCTCCACCAGG

[0607] ACTGGCTCAACGGCAAGGAATATAAATGCAAAGTCTCCAATAAGGCACTACCCGCGCCCATTGAGAA

[0608] AACCATCTCTAAGGCAAAAGGGCAACCACGCGAGCCTCAGGTTTACACTCTGCCCCCCAGCAGGGA

[0609] AGAGATGACTAAGAATCAGGTTTCCCTGACATGTCTCGTGAAGGGCTTTTACCCATCGGATATTGCC

[0610] GTCGAATGGGAGAGTAACGGACAGCCCGAGAATAATTACAAGACTACACCCCCCGTACTCGACTCC GATGGAAGCTTTTTCCTGTACTCCAAGCTGACCGTTGACAAGAGCCGCTGGCAACAAGGCAATGTGT TCTCTTGCAGTGTAATGCACGAGGCCCTCCATAATCACTACACCCAGAAGTCACTGAGTTTATCCCCC GGTAAGGGGGGCGGTGGATCTAAGGTGGAGCAGGCGGTCGAGACCGAACCAGAGCCAGAGCTGA GACAAACCGAATGGAGCGGCCAGAGGTGGGAATTGGCCCTCGGAAGGTTTTGGGATTATCTGAGGT GGGTTCAGACTTTGTCGGAGCAAGTGCAGGAGGAGCTGCTGTCATCTCAAGTGACTCAGGAACTCC GTGCCCTCATGGACGAAACTATGAAAGAGCTGAAAGCATACAAGAGTGAACTTGAGGAGCAGTTGA CACCGGTGGCAGAGGAAACAAGAGCCCGGCTTAGCAAGGAGTTAGCCGCCGCACGGCTCGGTGCT GATATGGAAGATGTCTGCGGACGGTTGGTATATCGAGGTGAGGTCGCTATGCTGGGATCCACCGAG

[0611] GAGCTGCGAGTGAGACTCGCTTCTCACTTACGAAAGCTTAGGAAGAGACTTCTGAGGGACGCAGAC GACCTGCAAAAGCGGCTCGCAGTATACCAAGCCGGCGCAAGAGAAGGCGCCGAAAGAGGACTAAG CGCTATCCGTGAGCGGCTGGGTCCCTTGGTGGAGCAGGGCAGGGTCCGGGCAGCGACGGTTGGGT CCCTGGCTGGCCAACCCCTCCAGGAGCGAGCCCAGGCTTGGGGGGAACGCCTTCGCGCTCGCATG GAGGAGATGGGGAGTCGCACCCGTGACCGTCTGGATGAAGTTAAAGAGCAGGTAGCCGAAGTGCG AGCGAAACTGGAAGAACAGGCCCAGCAGATTCGCCTACAGGCTGAGGCTTTCCAGGCCCGGCTAAA GTCCTGGTTCGAACCCCTGGTCGAGGACATGCAGCGCCAGTGGGCCGGCCTGGTTGAGAAAGTGC AGGCTGCAGTGGGGACATCAGCCGCTCCTGTGCCTTCTGATAACCATTAG

[0612] SEQ ID NO: 44 (CYT420_HC_translation)

[0613] MGWSCIILFLVATATGVHSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIG YINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSKVEQAVETE PEPELRQTEWSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELE EQLTPVAEETRARLSKELAAARLGADMEDVCGRLVYRGEVAMLGSTEELRVRLASHLRKLRKRLLRDAD

[0614] DLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEM GSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVG TSAAPVPSDNH*

[0615] EXAMPLES

[0616] Example 1

[0617] In this Example, two NanoPilot fusion proteins were produced and tested. CYT341 is formed by fusing variable light and heavy chains derived from the CD3e binder of Cibisatamab, and CYT344 is formed by fusing light and heavy chains derived from the CD3e binder of OKT3, each with the LDLR LA4-LA5 repeat fragment (a. a. V124-A211) with an N-terminal rigid linker. CYT346 contains a CDR that is not cross reactive with human CD3 (CYT346- Anti murine CD3 clone 2011). Materials and Methods

[0618] NanoPilot Cloning

[0619] Variable light and heavy chains derived from the CD3e binders of Cibisatamab and OKT3 were codon optimised for production in CHO and produced by gene synthesis (Twist Bioscience). Using Golden Gate DNA assembly these fragments were then inserted into human IgGl backbones (in a pTwist CMV BG WPRE Neo Vector) to obtain the full length IgGl heavy and IgG light chain constructs bearing the Anti-CD3e CDRs. The coding region for the LDLR LA4-LA5 repeat fragment (a. a. V124-A211) with an N-terminal rigid linker was similarly gene synthesised and inserted onto the C-terminus of the IgGl heavy chain to generate the heavy chain of the NanoPilot molecule.

[0620] NanoPilot Expression

[0621] ExpiCHO-S Cells were grown to a density of 6 x 10 6 cells / ml in ExpiCHO Expression Medium (Gibco) in Optimum Growth Flasks with a 0.2 pm vented cap (Thomson) in a humidified incubator at 37 °C, 8% CO2, with shaking at 120 RPM (19 mm orbit diameter). For transfection, DNA for the IgGl heavy-LDLR fusions and IgG light chains were complexed at a 1 : 1 mass ratio using ExpiFectamine CHO reagent and OptiPRO serum free medium (Gibco) according to the ExpiCHO transfection protocol. After complexation DNA was added to the cells at a DNA concentration of 1 pg DNA / ml of cell culture. After 18 hrs post transfection, cells were fed with ExpiCHO feed according to the ExpiCHO protocol and left to express for a further 3-4 days.

[0622] NanoPilot Purification

[0623] Cell cultures were harvested by centrifugation at 600g for 10 mins at 4°C to pellet the cells. Supernatants were then filtered to 0.2 pm (Disposable PES 50mm membrane filter units, Fisherbrand) before being applied to a 1 ml HiTrap Protein A HP column pre equilibrated in Protein A binding Buffer (25 mM NaPO4 pH 7.0, 150 mM NaCI, 0.5 mM CaCI2) (Cytiva) at 1.5ml / min. After a 15 column volume wash with Protein A binding buffer, bound material was eluted via a 20 CV gradient into 100% Protein A elution buffer (100 mM Citric acid pH 2.5, 150 mM NaCI, 0.5 mM CaCI2). Elution fractions were then supplemented with IM TRIS pH 9.0 (200 pl per ml of elution) to neutralise the acid. For further polishing, pooled protein-containing fractions were diluted ~7X with IEX A buffer (50 mM TRIS pH 8.0, 0.5 mM CaCI2) prior to loading onto a 5 ml HiTrap Q HP column (Cytiva). After a 10CV wash with 3 % IEX-B (50 mM TRIS pH 8.0, 30 mM NaCI, 0.5 mM CaCI2), bound proteins were eluted via a 10 CV gradient to 100% IEX-B buffer (50 mM TRIS pH 8.0, 30 mM NaCI, 0.5 mM CaCI2). Protein-containing anion exchange elution fractions were then pooled and concentrated using a Amicon Ultra 15 mL centrifugal device with a 10,000 Dalton molecular weight cutoff (Millipore) before buffer exchanging into DPBS + 0.5 mM CaCI2. Protein concentration was determined via UV spectroscopy at A280 nm and purity and homogeneity assessed by SDS-PAGE and analytical size exclusion using a 10 / 300 Superdex 200 column (Cytiva).

[0624] LNP production

[0625] For production of the lipid organic phase, lipids (dissolved to a final concentration of 10 mM in 100% Ethanol) were mixed at the following molar ratios: 50% Dlin-MC3-DMA (MedChemExpress), 38.5% Cholesterol (Plant Cholesterol, Merck), 10% DSPC (Merck), and 1.5% DMG-PEG2000 (Merck). LNPs were assembled via microfluidic mixing using a NanoAssemblr SparkTM device (Precision NanoSystems). For the aqueous phase, 20 pg of CleanCap EGFP mRNA (TriLink) was diluted to a final concentration of 0.3125 pg / pl RNA in 100 mM Sodium acetate pH 4.5 (final). 64 pl of the aqueous phase was then added to a Spark Cartridge along with 32 pl of the premixed organic phase and 96 pl of RNase free DPBS as dilution buffer. After microfluidic mixing, assembled LNPs were dialysed against 2 L of DPBS using a Slide-A-Lyzer MINI Dialysis Device with a 10K MWCO (Thermo Scientific) at 4 °C overnight. mRNA concentration and encapsulation efficiency was determined using a modified Quant-it Ribogreen Assay (ThermoFisher Scientific) as described in the GenVoy- ILM User Guide from Precision NanoSystems.

[0626] LNP Size Characterisation

[0627] For characterization, the diameter and concentration of the generated mRNA-LNPs were determined via Nanoparticle Tracking Analysis on a Zetaview PMX-120 (Particle Metrix) using the following parameters: measurement type: size; laser wavelength: 488 nm; filter types: scatter or 510 nm (for fluorescence readings); dispersant: PBS; temperature: 21°C; positions measured: 11; range of average counted particles per frame: 100-200.

[0628] Jurkat cell culture and transfection

[0629] The JRT3-T3.5 (TCR0-ve) cell line which does not express the TCRo[3 heterodimer or CD3 on the surface was obtained from ATCC. The CD3+ve JRT3-T3.5 cell line (transduced with a MEL5 TCR0 chain as well as CD8o[3 chains) was obtained from Lea Knezevic (University of Bristol). Jurkat cell lines were grown to a density of 0.5-1.5x10^6 cells / ml in R10 media (RPMI-1640 medium modified with sodium bicarbonate (Sigma), IX GlutaMAX (Gibco), 10% heat inactivated Fetal Bovine Serum (Gibco), and 10 units / ml penicillin and 100 pg / ml streptomycin) in a humidified incubator at 37 °C, 5% CO2. To test the effects of NanoPilot on LNP transfection, CD3+ve and CD3-ve Jurkat cells were pelleted by centrifugation at 600g for 5 mins before being washed and resuspended in ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies) supplemented with 1 pg / ml human Apo-E3 (Merck), 10 units / ml penicillin, 100 pg / ml streptomycin, and 100 pg / ml Kanamycin. CD3+ve and CD3- ve cells were then mixed at the desired ratio before plating a total of 62,500 cells in a 100 pl volume per well of a flat bottomed 96 Well TC-Treated Microplate (Corning). LNPs are diluted to an encapsulated mRNA concentration of 20 ng / pl in DPBS + 0.5 mM CaCI2before adding 2 pg / ml human Apo-E3 and incubated at room temperature for 5 minutes. NanoPilot serial dilutions (made in sterile DPBS + 0.5 mM CaCI2) are then added at a 1: 1 volume ratio bringing the final encapsulated mRNA concentration to 10 ng / pl. After a further 5 minute incubation, 12.5 pl of the LNP-NanoPilot complexes were added to the cells for a total encapsulated mRNA dose of 125 ng. Cells were incubated for 18 hrs in a humidified incubator at 37 °C, 5% CO2.

[0630] Staining and FACS analysis

[0631] For analysis cells were transferred to a U bottomed 96 Well TC-Treated Microplate (Corning) plate prior to centrifugation at 500 g for 5 mins to pellet the cells. Media was removed and cells resuspended in 50 pl of DPBS supplemented with 0.5 % W / V BSA and 0.25 pg of Human TruStain FcX (BioLegend) to block Fc receptors for 20 mins at 4°C. Cells were then stained with 50 pl of DPBS supplemented with 0.5 % W / V BSA and 0.5 pl of Human CD8 Alexa Fluor 350 MAb (Clone RPA-T8, Bio-Techne Ltd) for a further 20 mins at 4°C. The plate was then centrifuged at 500g for 5 mins and stain removed before resuspension of the cells in 200 pl of PBS + 0.5 % W / V BSA. GFP expression, CD3e surface markers and cell viability were determined using flow cytometry on a BD FACSCanto II with a HTS 96 well plate loader (BD Biosciences). Data was evaluated in FlowJo Software (version 10.1, BD Biosciences).

[0632] ELISA analysis

[0633] To test CD3E binding by NanoPilot constructs an indirect ELISA was performed by first immobilising biotinylated mouse or human CD3e6 heterodimer proteins (CDD-M82W5 and CDD-H82W6 respectively from ACROBiosystems) at 1 pg / ml in a volume of 100 pl per well of a pre-coated and blocked Streptavidin Microplate (Pierce, 15124). After a 2 hour incubation at room temperature, wells were washed 3X in 200 pl of ELISA wash buffer (130 mM NaCI, 20 mM TRIS PH 7.5, 0.05% Tween-20, 0.1% BSA) before applying serial dilutions of NanoPilot constructs for incubation for 1 hour at room temperature. Unbound material was removed by a further 3X washes before applying Anti IgG-HRP conjugate antibodies (Goat Anti-Mouse IgGl-HRP AB97240 or Mouse Anti-Human IgG Fc-HRP AB99759 from Abeam) for incubation for 30 minutes at room temperature. For quantification, unbound HRP conjugates were first removed with 6X washes before addition of 100 pl of TMB substrate solution (ThermoFisher Scientific) to start the reaction. After a 10 min incubation at room temperature the reaction was stopped with 100 pl of ELISA Stop Solution (SS04, Invitrogen) and colorimetric change determined by measuring absorbance at 450 nm with wavelength subtraction at 570 nm using a plate reader (Cytation 1 Imager, BioTek). Curves were fitted with the Agonist vs. response variable slope (four parameter) model within GraphPad Prism (Version 10.2.1) to obtain EC50 values.

[0634] Results

[0635] CD3 epsilon binding by NanoPilot

[0636] To test CD3E binding by NanoPilot constructs, an indirect ELISA was performed by first immobilising biotinylated mouse or human CD3e6 heterodimer proteins (0.1 pg / well) before applying serial dilutions of NanoPilot constructs. Bound NanoPilots were quantified by applying Anti IgG-HRP conjugate antibodies before addition of TMB substrate solution and acidification with stop solution for measurement of colorimetric change by absorbance at 440 nm using a plate reader (Figure 1).

[0637] Target cell selection by NanoPilot

[0638] NanoPilot blocked nonspecific transfection of LNPs by CD3-ve cells but maintains transfection of the target CD3+ve Jurkats when added to a 1 : 1 ratio of CD3+ve and CD3-ve Jurkats (Figure 2A). This effect also remained when the CD3+ve target cells were dropped to just 10% of the cell mixture (Figure 2B). %GFP+ve cell was the percentage of cells identified as singlets that fluoresce at wavelengths within the GFP filter bandwidths above that of the untransfected control cells. The targeting ratio was defined as the ratio of GFP+ve and CD3+ve to GFP+ve and CD3-ve cells.

[0639] When NanoPilot was designed with a CDR that was not cross reactive with human CD3 (CYT346- Anti murine CD3 clone 2C11), it blocked transfection via its Apo-E binding activity but does not redirect to the CD3+ve cells and thus does not lead to selection (Figure 3). An IC50 of 24.7 nM + / - 4.5 nM was obtained for CYT346 blocking of Jurkat transfection by fitting the dose response curve using a Variable slope (four parameters) non linear fit (GraphPad Prism Ver. 10).

[0640] Active off target blocking by NanoPilot

[0641] CYT341 was shown to be actively blocking non-specific uptake through a monoculture study with CD3 -ve cells, which showed almost complete abrogation of transfection for CYT341 coated LNPs (Figure 4). This is important because there are a far greater amount of nonspecific targets in vivo, so active blocking is preferential to just redirection to target cells.

[0642] MFI Enhancement by NanoPilot

[0643] Optimisation of NanoPilot purity and LNP incubation conditions revealed that NanoPilot can enhance the transfection of target cells (GFP mean fluorescence intensity) while also blocking off-target (i.e. CD3-ve) transfection (Figures 5 and 6). These data show that CYT341 was able to simultaneously block Apo-E mediated uptake of LNPs by off target cells while still directing and even enhancing transfection of target cells. At higher concentrations the Apo-E binding sites on the LNPs likely became saturated leading to excess free NanoPilot and in turn blocking of the target CD3 receptors.

[0644] Stabilisation of LNPs by NanoPilot

[0645] LNPs incubated in the presence of NanoPilot at room temperature for up to 24 hours retained >90% of their transfectivity, whereas the uncoated LNPs retained <70% (Figure 7). We are investigating the LNP-stabilising effects of our AMBPs further.

[0646] NanoPilot activity differs with targeting sequence used

[0647] NanoPilot was produced with two different Anti-CD3e targeting sequences derived from the CD3e binder of Cibisatamab (CYT341) and clone OKT3 (CYT344). LNPs were coated with titrations of either CYT341 or CYT344 before testing their effects on LNP targeting on a mixture of CD3+ve and CD3-ve Jurkat cells. Transfection of LNPs coated with CYT344 led to a higher proportion of GFP+ve / CD3+ve cells at NanoPilot concentrations above 100 nM than LNPs coated with CYT341 (Figure 8A). CYT344 also gave higher MFI enhancements at optimal concentrations vs CYT341 (Figure 8B).

[0648] NanoPilot targeting is enhanced using LNP formulations containing DOPE

[0649] Previous studies have shown that LNPs containing the helper lipid l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) are capable of binding more ApoE protein. We therefore hypothesised that this could affect the targeting activity of NanoPilot. Two LNP organic phase formulations were produced, one containing 10% DOPE and another with 10% DSPC keeping the molar ratios of the other lipid components constant (50% Dlin-MC3-DMA , 38.5% Cholesterol, 1.5% DMG-PEG2000). CYT344 was titrated into both LNP formulations prior to testing its effects on LNP targeting using a mixture of CD3-ve and CD3+ve Jurkat cells. LNPs containing 10% DOPE gave significantly higher levels of transfection and in turn a better ratio of CD3+ve targeting (Figure 9A) and MFI enhancement (Figure 9B).

[0650] PAI-1 anchored NanoPilot binds vitronectin

[0651] Vitronectin is a membrane-binding serum protein that preferentially binds to certain LNP formulations. We therefore produced a NanoPilot with a PAI-1 fragment that binds vitronectin, fused to sfGFP as a reporter (SEQ ID NO:28). LNPs incubated with vitronectin were able to bind the recombinantly expressed PAI-l_sfGFP. When analysed using size exclusion chromatography, a decrease in UV absorption in each of the expected elution peaks of the vitronectin and PAI-l_sfGFP were observed. This was coupled with an increase in the peak corresponding to the LNP. The composition of the elution peaks was then confirmed using SDS-PAGE and fluorescence spectroscopy.

[0652] Example 2

[0653] Materials and Methods

[0654] NanoPilot Cloning

[0655] As described above in Example 1, variable light and heavy chains derived from the CD3e binders of Anti- murine CD3e 2C11, and Anti human-CD117 clone 9P3 were codon optimised and then inserted into human IgGl backbones (in a pTwist CMV BG WPRE Neo Vector) to obtain the full length IgGl heavy and IgG light chain constructs bearing the CDRs of interest. The coding region for the human LDLR LA4-LA5 repeat fragment (a. a. V124-A211) and murine LDLR LA4-LA5 fragment (a. a. A124-A212) with an N-terminal rigid linker was similarly gene synthesised and inserted onto the C-terminus of the IgGl heavy chain to generate the heavy chain of the NanoPilot molecule. For Fc silencing of NanoPilot constructs, variable regions and LDLR fragments were cloned onto an IgGl backbone sequence synthesised with PG-LALA silencing mutations L234A, L235A, and P329G.

[0656] NanoPilot expression and purification were conducted as described in Example 1.

[0657] LNP production

[0658] This was conducted as described above for Example 1.

[0659] For optimized formulations, DSPC was replaced with DOPE (Merck) and / or DMG-PEG2000 was reduced to 0.75%. For LNP tracking, a 0.25% molar ratio of DiD'; (DiIC18(5) solid, Invitrogen) was added to the lipid organic phase prior to LNP formulation. The size of LNPs was characterized as described in Example 1 above.

[0660] Preparation of NanoPilot-Coated Lipid Nanoparticles (LNPs) for in vitro transfection

[0661] LNPs encapsulating eGFP mRNA were diluted to 2X the desired final dose concentration (typically 20-10 pg / ml) in DPBS + 0.5 mM CaCI2. The LNP stock was incubated with 2 pg / mL of human (Preprotech 350-02-500 pg) or murine (Cambridge Bioscience APO- MM102) ApoE3 for 5 minutes at room temperature. NanoPilots were serially diluted in DPBS containing 0.5 mM CaCI2to create a 2X working stock. For transfection, the serially diluted NanoPilots were mixed 1: 1 with the LNP-ApoE mixture and incubated for 5 minutes at room temperature. The LNP-NanoPilot complexes were then added to the cells at a 1: 10 LNP: cell culture ratio for a final dose of 100 ng mRNA-LNP per 100,000 cells. Cells were incubated for 18 hrs in a humidified incubator at 37 °C, 5% CO2 prior to FACS analysis. Titration concentrations presented in all graphs are the concentration of NanoPilot incubated with the LNP, not the final concentration after addition to cells. Jurkat cell culture and transfection

[0662] This was conducted largely as described in Example 1 above. CD3+ve and CD3-ve cells were then mixed at the desired ratio before plating a total of 62,500 cells in a 100 pl volume per well of a round bottomed 96 Well TC-Treated Microplate (Corning). The coculture was then treated with 100 ng per well of LNP complexes with or without the Anti- CD3 NanoPilots (CYT341 or CYT344). Optimised DOPE and DOPE 0.75% PEG formulations were dosed at a lower 50 ng dose per well.

[0663] For FACS analysis, Jurkat cells were pelleted in a U bottomed 96 Well TC-Treated Microplate (Corning) plate via centrifugation at 500 g. Media was removed and cells resuspended in 50 pl of DPBS supplemented with 0.5 % W / V BSA and 0.25 pg of Human TruStain FcX (BioLegend) to block Fc receptors for 20 mins at 4°C. For labelling of the CD3+ve Jurkats, cells were then stained with 50 pl of DPBS supplemented with 0.5 % W / V BSA and 0.5 pl of Human CD8 Alexa Fluor 350 MAb (Clone RPA-T8, Bio-Techne) for a further 20 mins at 4°C. The plate was then centrifuged at 500g for 5 mins and stain removed before resuspension of the cells in 200 pl of PBS + 0.5 % W / V BSA. GFP expression, surface markers and cell viability were determined using flow cytometry on a BD FACSCanto II with a HTS 96 well plate loader (BD Biosciences). Data was evaluated in FlowJo Software (version 10.1, BD Biosciences). The targeting ratio was determined by dividing the median GFP fluorescence intensity (MFI) of the target cells (e.g., CD3+) by the MFI of the non-target cells (e.g., CD3- )■

[0664] ELISA analysis was conducted as described in Example 1.

[0665] HepG2-Kasumi-l co-culture and Anti-CD117 NanoPilot targeting assay

[0666] The human hepatocellular carcinoma cell line, HepG2 (ATCC HB-8065), was cultured in R10 medium. Cells were maintained in T-75 flasks in a humidified incubator at 37°C with 5% CO2. The medium was replaced every 2-3 days. Upon reaching 70% confluency, cells were sub-cultured. The monolayer was washed with phosphate-buffered saline (PBS) and detached using TrypLE Express trypsin enzyme (Gibco). The trypsin was neutralized with R10 medium, and the cell suspension was centrifuged at lOOx g for 5 minutes. The cell pellet was resuspended in fresh medium, and viable cell concentration was determined by Trypan Blue exclusion. For transfections assays, HepG2 cells were re-seeded at a density of 45,000 cells per well in flat bottom TC treated microplates (Corning) and incubated for 24hrs prior to transfection. Kasumi-1 cells (ATCC-CRL-2724) were cultured in R10 medium and split every 3-5 days. For the co-culture, the medium was removed from the adherent HepG2 cells, and 35,000 stained Kasumi-1 cells, suspended in 100 pL of ImmunoCult-XF T Cell Expansion Medium (supplemented with 1 pg / mL human ApoE3, 1% penicillinstreptomycin, and 100 pg / mL Kanamycin), were plated on top. The co-culture was then treated with 100 ng per well of LNP complexes with or without the Anti-CD117 NanoPilot (CYT391).

[0667] The mixed cell population (post TrypLE Express digest) was transferred to a U-bottom 96- well plate and pelleted at 500 x g for 5 minutes. The supernatant was removed, and the cells were washed and resuspended in 200 pL of staining buffer (DPBS + 0.5% w / v BSA). The two cell populations were distinguished based on the pre-staining of Kasumi-1 cells with CellTrace Far Red. LNP-mediated mRNA delivery and translation were quantified by measuring GFP expression (FITC channel). Data were acquired on a BD FACSCanto II and analysed using FlowJo software.

[0668] Murine Splenocyte Pan T Cell Isolation and Transfection

[0669] Pan T cells were isolated from Balb / C mouse splenocyte suspension (Tebu-bio, IQB- MSP102) by negative selection using a Pan T Cell Isolation Kit (Miltenyi 130-095-130). All steps were performed at 2-8°C. Briefly, splenocytes were resuspended in MACS buffer (PBS pH 7.2, 0.5% BSA, 2 mM EDTA). The cell suspension was incubated with a Biotin-Antibody Cocktail for 5 minutes, followed by a 10-minute incubation with Anti-Biotin MicroBeads (Miltenyi Biotec). The labeled cell suspension was applied to an LS Column (Miltenyi Biotec) within a MACS Separator. The flow-through, containing the enriched, unlabeled pan T cells, was collected. The purity of the isolated T cells was assessed by flow cytometry. All further cell culture was performed in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, 1% L-glutamine, 40 U / mL murine IL-2 (Miltenyi, 130-120-662), and 0.01 mM 2-Mercaptoethanol.

[0670] Isolated, unactivated pan T cells were seeded into a 96-well plate at 60,000 cells (100 pL) per well. Ten microliters of the diluted LNP-NanoPilot complexes were added to the cells. The plate was incubated overnight (16-20 hours) at 37°C and 5% CO2. Post-transfection, cells were analysed for LNP uptake and T cell subset identification. Cells were first incubated with TruStain FcX™ PLUS (anti-mouse CD16 / 32, BioLegend) to block Fc receptors.

[0671] Subsequently, cells were stained with fluorophore-conjugated antibodies against mouse CD4 (PE / Cyanine7, BioLegend, clone RM4-5) and CD8a (APC / Cyanine7, BioLegend, clone 53- 6.7). LNP uptake was assessed by measuring the fluorescence of a DiD-labeled lipid within the LNP formulation (APC channel) and translation of the mRNA via GFP fluorescence (FITC channel). Data were acquired on a MACSQuant Analyser 10 flow cytometer.

[0672] LNP-ApoE Blocking Assay

[0673] To compare the blocking potential of NanoPilot and a recombinant human LDLR extracellular domain (rhLDLR-ED, Bio-Techne, 2148-LD / CF), a inhibition assay was performed. Two different lipid nanoparticle (LNP) formulations were used: one containing 10% DSPC helper lipid and another containing 10% DOPE, both encapsulating luciferase-encoding mRNA (Trilink) labelled with 0.25% DiIC18 lipid dye. The rhLDLR-ED was reconstituted in PBS with 0.5 mM CaCI2to a concentration of approximately 3 pM. The NanoPilot (CYT344) and rhLDLR-ED were then serially diluted in PBS with 0.5 mM CaCI2from a starting concentration of 3 pM. LNP stocks were incubated with human ApoE for 5 minutes at room temperature. Subsequently the LNP-ApoE complexes were mixed with the serially diluted NanoPilot or rhLDLR-ED solutions and incubated for 5 minutes. This mixture was then added to CD3-ve human T cells for transfection. After 18 hours, DiD median Fis of the CD3-ve cells were determined via FACS on a MACSQuant Analyser 10 flow cytometer. The half-maximal inhibitory concentration (IC50) values for DiIC18 MFI (LNP uptake) were calculated by fitting the data to a four-parameter logistic curve using GraphPad Prism.

[0674] Comparison of LDLR vs. ApoE Anchoring

[0675] An alternative LNP anchoring strategy was evaluated by generating an ApoE-anchored construct (CYT420). This construct was created by fusing full-length human ApoE to the C- terminus of the anti-human CD3e heavy chain (OKT3 construct CYT344), replacing the LDLR fragment. For a direct comparison, the standard LDLR-anchored NanoPilot (CYT344) was complexed with LNPs pre-coated with ApoE3. In parallel, the ApoE-anchored NanoPilot (CYT420) was complexed with identical LNPs that had not been pre-coated with ApoE3. Both complexes were prepared using a DiD-labeled, 0.75% PEG DOPE LNP formulation and were evaluated in the Jurkat co-culture assay. LNP uptake (DiD) and mRNA translation (GFP) were subsequently quantified by flow cytometry using a MACSQuant Analyser 10 flow cytometer.

[0676] Animal Studies

[0677] All animal procedures were performed in collaboration with the Medicines Discovery Catapult (MDC) and conducted in accordance with institutional guidelines. For in vivo administration, LNP formulations were first concentrated to 200 pg / mL mRNA using 100 kDa MWCO Amicon filters and subsequently sterile-filtered. Final dosing solutions were prepared by mixing the LNPs 1: 1 (v / v) with the Anti-murine CD3 NanoPilot (CYT346), resulting in a final mRNA concentration of 100 pg / mL. Balb / c mice received a single tail vein injection containing 10 pg of total mRNA in a 100 pl volume. Experimental groups were dosed with LNPs complexed with murine ApoE and either 2 pM or 0.5 pM of CYT346 NanoPilot. Control groups received either untargeted LNPs (complexed with murine ApoE only) or the vehicle control (DPBS with 0.5 mM CaCI2). Each group contained a total of six mice. At 24 hr peripheral blood was collected by micro-bleeds for flow cytometric analysis using a NovoCyte 3000 cytometer.

[0678] IVIS Organ Imaging and Analysis At 48 hours post-injection, mice were euthanised, and major organs were harvested for ex vivo fluorescence imaging. Organs were imaged using an IVIS Lumina III in vivo imaging system (Perkin Elmer). Fluorescence from the DiD lipid tracer was imaged using an excitation filter of 644 nm and an emission filter of 665 nm. GFP fluorescence from the translated mRNA payload was measured using an excitation filter of 488 nm and an emission filter of 510 nm. For quantitative analysis, regions of interest (ROIs) were drawn around each organ, and the average radiant efficiency ([p / s / cm2 / sr] / [pW / cm2]) was calculated using Living Image software (Perkin Elmer).

[0679] Statistical Analysis

[0680] Data are presented as mean ± standard deviation unless otherwise noted. Statistical significance was determined with an unpaired t-test using GraphPad Prism software. A p- value of less than 0.05 was considered statistically significant.

[0681] Results

[0682] NanoPilot Inhibition of LNP Uptake

[0683] A blocking experiment was performed using either the NanoPilot (CYT344) or a soluble, recombinant human LDLR extracellular domain (rhLDLR-ED, Bio-Techne) as a competitor to inhibit LNP delivery. The inhibition of LNP uptake was measured by DiD fluorescence via flow cytometry. For LNPs formulated with DSPC, the NanoPilot exhibited an IC50 of 41 nM, while the rhLDLR-ED had an IC50 of 188 nM. For DOPE-formulated LNPs, the NanoPilot had an IC50 of 40 nM compared to 313 nM for the rhLDLR-ED. The results are shown in Figure 11.

[0684] Comparison of LDLR-Anchored vs. ApoE-Anchored Constructs

[0685] An anti-CD3 NanoPilot construct comprising a full-length human ApoE fused to the C- terminus of the heavy chain (CYT420) was generated. The performance of the LDLR- anchored (CYT344, with ApoE-coated LNPs) and ApoE-anchored (CYT420, with uncoated LNPs) constructs was evaluated in a Jurkat co-culture system. Both constructs resulted in preferential GFP expression in CD3+ve cells (Figure 12a). Off target CD3-ve inhibition of LNP uptake was more potent with the LDLR-anchored system resulting in a targeting ratio more than five-fold higher than that achieved with the ApoE-anchored system (Figure 12a, b).

[0686] Modular Targeting Specificity of the NanoPilot Platform

[0687] A construct was engineered with an antigen-binding domain specific for human CD117 (c- Kit), derived from antibody clone 9P3 (CYT391). In a monoculture of CD117-positive Kasumi-1 cells, CYT391-coated LNPs resulted in a dose-dependent increase in GFP expression, with a 200% increase in median fluorescence intensity observed between 30- 120 nM (Figure 13a). In a co-culture of Kasumi-1 cells and CD117-negative HepG2 cells, the CYT391 NanoPilot resulted in enhanced GFP expression in Kasumi-1 cells and a reduction in LNP uptake of up to 88% in HepG2 cells (Figure 13b).

[0688] Effect of Reduced PEG-Lipid Content on Targeting

[0689] The DMG-PEG2000 content in a DOPE-LNP formulation was reduced from 1.5 mol% to 0.75 mol% and 0.375 mol%. The performance of NanoPilot with these formulations was evaluated in the Jurkat co-culture system. The lower PEG content resulted in an increase in on-target transfection in CD3+ve cells, while blocking of off-target cells was maintained (Figure 14a). This resulted in a 4- to 9-fold increase in CD3+ve target MFIs compared to the 1.5 mol% PEG DOPE-LNP (Figure 14b), achieving a targeting specificity of over 500-fold (Figure 14c).

[0690] NanoPilot blocks physical uptake of LNPs

[0691] LNPs were co-labeled with the lipophilic dye DiD to track particle location independent of GFP expression. In a Jurkat co-culture system, the DiD signal correlated with the GFP signal across all concentrations of the CYT344 NanoPilot (Figure 15). With increasing NanoPilot concentration, the DiD signal was progressively excluded from off-target CD3-ve cells and accumulated in on-target CD3+ve cells, mirroring the GFP expression pattern.

[0692] Adaptation of NanoPilot for Murine T Cells

[0693] The NanoPilot platform was adapted for murine targets by replacing the human-specific OKT3 targeting domain with the 2C11 antibody sequence (anti-mCD3, CYT346) or the YTS105.18 sequence (anti-mCD8, CYT435). In primary murine splenocytes, the anti-mCD3 NanoPilot (CYT346) delivered LNPs to both CD4+ and CD8+ T cells. The anti-mCD8 NanoPilot (CYT435) resulted in GFP expression and DiD uptake only in the CD8+ T cell population (Figure 16a, b,c). The DiD signal was higher for the anti-mCD8 construct, which also exhibited greater potency, with peak percent GFP-positive cells observed at 125-250 nM, compared to 500 nM for the anti-mCD3 construct (Figure 16a, c). However, the overall GFP fluorescence intensity was higher following delivery with the anti-mCD3 NanoPilot than with the anti-mCD8 construct (Figure 16b).

[0694] Targeted Delivery to Circulating T Cells In Vivo

[0695] An optimised LNP formulation (DOPE, 0.75% PEG) co-loaded with EGFP mRNA and DiD was complexed with the anti-murine CD3 NanoPilot (2 pM and 0.5 pM) and administered to Balb / c mice. Control groups received untargeted LNPs or a vehicle control. At 24 hours postinjection, peripheral blood was analysed by flow cytometry. In mice treated with the anti- mCD3 NanoPilot, a significant population of circulating T cells was positive for both GFP and DiD. Negligible transfection was observed in animals that received untargeted LNPs (Figure 17a, b).

[0696] Reduction of LNP Accumulation in the Liver In Vivo At 48 hours post-injection, livers were harvested for ex vivo fluorescence imaging. Livers from mice that received NanoPilot-coated LNPs exhibited a statistically significant reduction in both DiD and GFP fluorescence compared to the untargeted LNP group (Figure 18a, b).

Claims

CLAIMS1. A fusion protein comprising (1) a first region comprising a peptide or polypeptide that specifically binds to at least a part of a receptor binding site of a membrane-binding serum protein and (2) a second region comprising a targeting peptide or polypeptide.

2. A fusion protein according to claim 1, wherein the first region comprises (i) a receptor for a membrane-binding serum protein or a functional fragment thereof or (ii) an antibody or a fragment thereof that specifically binds to at least a part of a receptor binding site of a membrane-binding serum protein.

3. A fusion protein according to claim 2, wherein the first region comprises a low-density lipoprotein receptor (LDLR) polypeptide or a functional fragment thereof4. A fusion protein according to claim 3, wherein the functional fragment comprises at least LDLR A domain 5 (A5) or at least LDLR A domain 4 and A domain 5 (A4-5).

5. A fusion protein according to claim 3 or 4, wherein the LDLR polypeptide or the functional fragment comprises a variant sequence having at least about 90% identity to the sequence shown in SEQ ID NO: 5 or 18.

6. A fusion protein according to any one of the preceding claims, wherein the targeting peptide or polypeptide is a protein, an antibody or fragment thereof, or an enzyme.

7. A fusion protein according to any one of the preceding claims, wherein the first and second regions are genetically fused and / or are fused via a linker.

8. A fusion protein according to claim 7, wherein linker comprises or consists of a variant sequence having at least about 90% identity to the sequence shown in SEQ ID NO: 14 or 15.

9. A fusion protein according to any one of the preceding claims, wherein the second region comprises a single-chain antibody.

10. A fusion protein according to any one of claims 1-8, wherein the second region comprises an antibody heavy chain or the variable region of an antibody heavy chain (VH).

11. A fusion protein according to claim 10, wherein the fusion protein comprises the sequence shown in SEQ ID NO: 7, 11, 20, 24 or 28 or a variant sequence having at least about 90% identity to the sequence shown in SEQ ID NO: 7, 11, 20, 24 or 28.9412. A fusion protein according to claim 10, wherein the fusion protein comprises the sequence shown in SEQ ID NO: 39 or a variant sequence having at least about 90% identity to the sequence shown in SEQ ID NO: 39.

13. A conjugate comprising (a) a fusion protein according to claim 10, 11 or 12 and (b) an antibody light chain or the variable region of an antibody light chain (VL).

14. A construct comprising two conjugates according to claim 13.

15. One or more polynucleotides encoding a fusion protein according to any one of claims 1-12, a conjugate according to claim 13 or a construct according to claim 14.

16. A vector comprising one or more polynucleotides according to claim 15.

17. An amphipathic structure linked to a fusion protein according to any one of claims 1-12, a conjugate according to claim 13 or a construct according to claim 14.

18. An amphipathic structure according to claim 17, wherein the amphipathic structure is a vesicle, micelle, liposome, lipid nanoparticle, or exosome.

19. An amphipathic structure according to claim 17 or 18, wherein the amphipathic structure is linked to the fusion protein, conjugate, or construct via an LDL.

20. An amphipathic structure according to claim 19, wherein the LDL is Apolipoprotein-E (Apo-E).

21. An amphipathic structure according to any one of claims 17-20, wherein the amphipathic structure comprises a therapeutic molecule and / or a genetic cargo.

22. An amphipathic structure according to any one of claims 17-21, wherein the amphipathic structure is present in a composition further comprising a polyethylene glycol (PEG)-lipid conjugate and a sterol and wherein the molar ratio of the PEG-lipid conjugate to the lipids in the amphipathic structure and the sterol is about 1.2% or less.

23. A composition comprising an amphipathic structure according to any one of claims 17- 21, a PEG-lipid conjugate and a sterol, wherein the molar ratio of the PEG-lipid conjugate to the lipids in the amphipathic structure and the sterol is about 1.2% or less.

24. A pharmaceutical composition comprising a fusion protein according to any one of claims 1-12, a conjugate according to claim 13, a construct according to claim 14, the one or more polynucleotides according to claim 15, a vector according to claim 16, an amphipathic structure according to any one of claims 17-21, or a composition according to claim 23 and a pharmaceutically acceptable diluent and / or carrier.9525. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a fusion protein according to any one of claims 1-12, a conjugate according to claim 13, a construct according to claim 14, the one or more polynucleotides according to claim 15, a vector according to claim 16, an amphipathic structure according to any one of claims 17-21, a composition according to claim 23 or a pharmaceutical composition according to claim 24.

26. A method of producing an amphipathic structure according to any one of claims 17-21 comprising linking the amphipathic structure to the fusion protein, conjugate, or the construct.

27. A method of improving the targeting of an amphipathic structure to a target cell comprising linking the amphipathic structure to a fusion protein according to any one of claims 1-12, a conjugate according to claim 13 or a construct according to claim 14, wherein the targeting peptide or polypeptide specifically binds a target antigen on the target cell.

28. A method according to claim l , wherein the method increases endocytosis of the amphipathic structure by the target cell and / or method decreases endocytosis of the amphipathic structure by non-target cells.96

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