Dimeric protein complexes and uses thereof

Abstract

Dimeric protein complexes and uses thereof The present invention is located in the field of multimers used in targeted therapy. More specifically, the present invention provides a dimeric protein complex comprising a first polypeptide comprising a first functional component and a C-terminal fragment of C4bp β chain, the first functional component being linked to the C-terminus of the C-terminal fragment of C4bp β chain; and a second polypeptide comprising a second functional component and a C-terminal fragment of C4bp β chain, the second functional component being linked to the C-terminus of the C-terminal fragment of C4bp β chain, the first and second polypeptides being the same or different, the first and second functional components being proteins or polypeptides. Further provided are nucleic acids encoding the first or second polypeptide of the dimeric protein complex, and pharmaceutical compositions comprising the dimeric protein complex. The present invention also relates to the use of the dimeric protein complex in immunotherapy.

Description

[Technical field]

[0001] The present invention is located in the field of multimeric protein complexes and their use in targeted therapy. More specifically, the present invention relates to dimeric protein complexes that exhibit two or more functional components and include a scaffold. [Background technology]

[0002] Recombinant DNA technology offers the possibility of large-scale production of biologically active proteins for use in several therapeutic applications. Many of the products produced from recombinant DNA are already in clinical use or are currently in development, including large proteins, small peptides, and antibody fragments. Multimerization of protein and peptide molecules has been shown to increase the half-life of these molecules in vivo, allowing them to exert their activity for a longer period of time. Furthermore, multimeric protein complexes offer the opportunity to combine multiple functional components (e.g., components with therapeutic and targeting functionality) into one molecule, often enabling targeted therapies with increased efficacy and reduced unwanted side effects. Furthermore, co-administration of different therapeutic components can lead to synergistic therapeutic effects. Additionally or alternatively, the combination of different targeting components can increase binding specificity and affinity or bring two target cells into close proximity with each other. Summary of the Invention [Problem to be solved by the invention]

[0003] Although very efficient, such multimeric protein complexes have drawbacks. First, their chemical formulation is not precisely defined. Second, the number of functional moieties attached to the polymer molecule is often random and uncontrollable. This is especially problematic when functional moieties are combined and loaded onto such polymers. Moreover, in most cases, the functional moieties are attached to a scaffold or framework. Such scaffolds or frameworks are often large molecules, which may result in poor tissue penetration, poor binding to areas of the molecular surface accessible to smaller sized molecules, and / or poor production yields. Therefore, there is an urgent need to improve multimeric protein complexes. [Means for solving the problem]

[0004] C4b-binding protein (C4bp) is a normal plasma protein and therefore not immunogenic. C4bp is a potent soluble negative regulator of the classical complement pathway (CP), but is also involved in the regulation of the coagulation process through the binding of protein S to the first two short consensus repeats (SCRs) (SCR1-SCR2) of the C4bp β chain. C4bp has a quaternary structure consisting of seven identical α chains and one β chain (7α1β) as the major molecular species (70%). Minor molecular species showing 6α, 7α or 6α1β without β have also been found. Each α chain consists of eight short consensus repeats (SCRs, SCR1-SCR8) and a non-SCR C-terminus (C4bpα) of about 58 amino acids containing two cysteines. The first three SCRs (SCR1-SCR3) of the C4bp α chain bind soluble or membrane-bound C4b and either prevent the formation of the classical pathway C3 convertase (C4b2a) on the target membrane surface or bind the formed C4b3a C3 convertase and attenuate its current biological activity. A single β chain is composed of three SCRs and a non-SCR C-terminus (C4bpβ) of 59 amino acids that contains two cysteines (C202, C216) that form disulfide bonds with the two adjacent α chains. Thus, the seven α and β chains are covalently linked by the formation of interchain disulfide bridges.

[0005] Surprisingly, the inventors have found that the C-terminal portion of the C4bp β-chain (C4bpb) can be used as a dimerization scaffold, and that functional components can be introduced C-terminally (i.e., downstream) of said C-terminal portion of the C4bp β-chain. Such dimeric protein complexes may be referred to herein as "boro" or "β (beta)-boro". Unexpectedly, it has been found that the presence of functional components downstream of the C4bp β-chain, a non-natural location in an endoplasmic reticulum / Golgi molecule that transports intracellular organelles, does not prevent spontaneous dimerization of C4bp in eukaryotic cells. Attaching functional components to the C-terminal side of the C-terminal fragment of the C4bp β-chain has many advantages. Attaching functional components that are naturally located at the C-terminus of a polypeptide, such as the Fc fragment of an antibody, upstream of the C-terminal portion of the C4bp β chain may impair the biological activity of such functional components due to steric hindrance issues with the proximal dimerization scaffold. When these polypeptides are introduced C-terminal to the C-terminal fragment of the C4bp β chain, these C-terminal functional components are able to exert their biological functions, such as the "IgG mimics" described herein.

[0006] In addition, e.g., scFv or V H It has been surprisingly found that, even for proteins or polypeptides that have a biological function / activity, such as a binding domain, at their N-terminus, such as the H polypeptide, cloning said protein or polypeptide C-terminally to the dimerization scaffold of C4bpβ does not reduce or eliminate the biological activity of said protein or polypeptide (e.g., reduce binding ability), for example for some of the "tetraboro" as described herein. The advantage is that scFv or V HSuch proteins or polypeptides, such as H, can be included at both the N-terminus and C-terminus of the dimerization scaffold of C4bp β, resulting in a dimeric protein complex with high valency (e.g., up to 4 valency) of said protein or polypeptide. The inventors have also found that it is possible to obtain multifunctional (e.g., bifunctional) dimeric protein complexes from a single expression vector containing at least one functional component upstream and at least one functional component downstream of the C-terminal fragment of the C4bp β chain. When a single expression vector containing at least two functional components is transfected into a cell, the cell releases a single multifunctional dimeric molecular species into the cell culture supernatant.

[0007] In contrast to using the C4bp α chain as a scaffold, by using the C-terminal fragment of the C4bp β chain as a scaffold, it is possible to obtain dimeric protein complexes with relatively small sizes (e.g., around 70-240 kDa, e.g., 100-240 kDa). Small protein complexes are more physiological than large protein complexes, have improved biodistribution space, and faster renal excretion, making them suitable as therapeutic agents. For example, by using a C-terminal fragment of the C4bp β-chain as a scaffold, it is possible to produce molecules with molecular weights and therefore extravascular diffusion space in the same range as monomeric IgG. Since the dimeric protein complexes of the invention can be composed of more than two valencies, such as trivalent or tetravalent, the binding activity of the dimeric protein complexes of the invention, such as monofunctional "homotetraboro" as described herein, can be superior to that of IgG. More specifically, such "homotetraboro" may have better binding capacity, greater antigen capture capacity, and / or better neutralization capacity than IgG.

[0008] The dimeric protein complexes of the present invention can also selectively modulate cellular functions, for example, by generating dimeric protein complexes that bring tumor cells or pathogens into physical proximity with immune effector cells, such as NK cells, and force synapse formation to activate the immune effector cells toward the tumor cells or pathogens.

[0009] Furthermore, because the C-terminal portion of the C4bp β chain is derived from a natural circulating molecule and the C-terminal portion of the C4bp β chain has no known biological function, the potential immunogenicity of the dimeric protein complex of the present invention is limited.

[0010] Furthermore, dimeric protein complexes of the present invention comprising identical first and second polypeptides can be prepared using a single construct that produces a single molecular species (as can be determined using SDS-PAGE followed by SYPRO ruby ​​staining or Western blotting under non-reducing or reducing conditions), which greatly simplifies the production and purification design and processes.

[0011] Thus, in a first aspect, a dimeric protein complex is provided comprising two polypeptides, each of which comprises a functional component and a sequence corresponding to a C-terminal fragment of the C4bp β-chain. The term "functional component" is defined in more detail herein, but generally refers to an amino acid sequence having a given function. In a particular embodiment, the first aspect comprises: a first polypeptide comprising a first functional component and a C-terminal fragment of a C4b binding protein (C4bp) β chain, the first functional component being linked to the C-terminus of the C-terminal fragment of the C4bp β chain; and a second polypeptide comprising a second functional component and a C-terminal fragment of a C4bp β chain, said second functional component being linked to the C-terminus of said C-terminal fragment of a C4bp β chain. A dimeric protein complex comprising: The first and second polypeptides are the same or different, preferably the first and second functional components are peptides, proteins or polypeptides. It may be described as relating to a dimeric protein complex.

[0012] In certain embodiments, the first polypeptide further comprises a third functional component linked to the N-terminus of the C-terminal fragment of the C4bp β chain; the second polypeptide further comprises a fourth functional component linked to the N-terminus of the C-terminal fragment of the C4bp β chain; The first, second, third and fourth functional components may be the same or different.

[0013] In certain embodiments, the first, second, third and / or fourth functional components are proteins or polypeptides, preferably proteins or polypeptides selected from the group consisting of a binding domain, a recombinant viral structural protein, an oncolytic agent, a cytotoxic agent, a cytokine, a receptor binding peptide, or a monomeric Fc, more preferably proteins or polypeptides selected from the group consisting of a binding domain (e.g., an antigen recognition domain), a recombinant viral structural protein, a cytotoxic agent, a cytokine, a receptor binding peptide, or a monomeric Fc.

[0014] In certain embodiments, the binding domain (e.g., antigen recognition domain) is a single chain variable fragment (scFv) of an antibody specific for an antigen or a single domain variable fragment (VFv) of a heavy chain antibody. H H), an antibody-like scaffold, an extracellular domain of a viral envelope protein, a cognate extracellular domain of a receptor or ligand for an antigen, or an antigen-binding portion of said receptor or ligand or a soluble or synthetic receptor. In certain embodiments, the binding domain (e.g., an antigen recognition domain) specifically binds to a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), a bacterial antigen, a viral antigen or a virus-associated antigen, a fungal antigen, an activating NK cell receptor, a cytokine, a toxin, or a contaminant.

[0015] In certain embodiments, two of the first, second, third, or fourth functional components comprise a binding domain (e.g., an antigen recognition domain) that specifically binds to an activating NK cell receptor, and binding of the binding domain (e.g., the antigen recognition domain) to an activating NK cell receptor can activate an NK cell, and two of the first, second, third, or fourth functional components comprise a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA, a TAA, a bacterial antigen, a viral antigen, a virus-associated antigen, or a fungal antigen.

[0016] The inventors have also found that inclusion of a monomeric Fc, preferably an IgG Fc comprising an IgG hinge, CH2 domain and CH3 domain, downstream (C-terminal) of the C-terminal fragment of the C4bp β chain of both the first and second polypeptides of the dimeric protein complex results in unexpectedly enhanced but regulated Fc functions, such as Fc receptor (FcR) binding properties, complement activation (as indicated by complement deposition), cell lysis and / or cell activation, upon antigen binding, as a result of the double hinge formed (i.e., the presence of an Fc hinge and a C4bp β chain hinge) compared to conventional antibodies (e.g. enhanced complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC) / antibody dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDCC) / complement dependent cell-mediated phagocytosis (CDCP)). For example, such dimeric protein complexes result in potent activation of host complement, such as through (i) Fc-CD16 [Fc(γ)RIIIA] interactions and (ii) C3b degradation products (i.e., iC3b, C3d,g, C3d)-CD11b interactions, enhanced NK activation, and enhanced phagocytosis of target cells by macrophages. Examples of such dimeric protein complexes comprising monomeric Fc of IgG are also referred to herein as "pseudo-IgG". Furthermore, without being bound by theory, the aforementioned properties of pseudo-IgG may result from the inert nature of the C-terminal fragment of the C4bp β-chain and / or the flexibility of the monomeric Fc located at the C-terminal portion of the pseudo-IgG.

[0017] It should be noted that, since binding of such dimeric protein complexes to cells or antigens is required to result in complement activation, complement activation does not occur when a dimeric protein complex as taught herein, comprising a monomeric Fc, such as a pseudo-IgG as referred to herein, downstream (C-terminal) of the C-terminal fragment of the C4bp β-chain of both the first and second polypeptides of the dimeric protein complex, is in the liquid phase (i.e., unbound). Thus, in certain embodiments, the first and second functional components are monomeric Fc, preferably monomeric IgG Fc comprising an IgG hinge, CH2 domain and CH3 domain; the hinge region of the monomeric Fc in the first polypeptide is connected to the hinge region of the monomeric Fc in the second polypeptide by at least two disulfide bonds; The third and / or fourth functional components include a binding domain (eg, an antigen recognition domain).

[0018] A further aspect is encoding a first polypeptide of a dimeric protein complex comprising a first functional component and a C-terminal fragment of a C4bp β chain, said first functional component being linked to the C-terminus of said C-terminal fragment of a C4bp β chain as defined herein; A second polypeptide encoding a second functional component and a C-terminal fragment of a C4bp β chain, the second functional component being linked to the C-terminus of the C-terminal fragment of a C4bp β chain as defined herein. A nucleic acid is provided.

[0019] A further aspect provides an expression cassette comprising a nucleic acid as taught herein.

[0020] A further aspect provides an expression vector comprising a nucleic acid as taught herein or an expression cassette as taught herein.

[0021] A further aspect provides an expression vector comprising a nucleic acid encoding a C-terminal fragment of a C4bp β chain, an insertion site for a nucleic acid encoding a functional component located immediately 3' to the nucleic acid encoding the C-terminal fragment of a C4bp β chain; and, optionally, an insertion site for a nucleic acid encoding a functional component located immediately 5' to the nucleic acid encoding the C-terminal fragment of a C4bp β chain.

[0022] A further aspect provides a pharmaceutical composition comprising a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, or an expression vector as taught herein, and a pharma- ceutically acceptable carrier.

[0023] A further aspect provides a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, an expression vector as taught herein, or a pharmaceutical composition as taught herein for use as a medicament, preferably in immunotherapy.

[0024] A further aspect provides a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, an expression vector as taught herein, or a pharmaceutical composition as taught herein for use in the treatment of a neoplastic disease or an infectious disease.

[0025] Further aspects provide a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, or an expression vector as taught herein for use in a method of molecular imaging of a living body.

[0026] These and further aspects and preferred embodiments of the present invention are set out in the following paragraphs and in the appended claims, the subject matter of which is specifically incorporated into this specification. [Brief description of the drawings]

[0027] [Figure 1]1 shows the "boro" technology taught herein based on the inclusion of a functional component C-terminal to the C-terminal portion of the C4bp β chain, which serves as a scaffold for dimerization of a therapeutic biologic. (A) Exemplary schematic of expression cassettes for expression of "diboro" in which the first and second polypeptides of the dimeric protein complex contain a functional component C-terminal to the C-terminal portion of the C4bp β chain and no functional component N-terminal to the C-terminal portion of the C4bp β chain. Transfection of one of the exemplary expression cassettes can form homodiboro containing either functional component 1 or 2 (i.e., monofunctional). Co-transfection of two different expression cassettes for diboro (one for functional component 1 and one for functional component 2) results in the formation of a bifunctional heterodiboro. (B-C) Exemplary schematic diagrams of expression cassettes for the expression of "tetrabolo", in which the first and second polypeptides of the dimeric protein complex consist of a functional component C-terminal to the C-terminal portion of the C4bp β-chain and a functional component N-terminal to the C-terminal portion of the C4bp β-chain. Transfection of one of the illustrated expression cassettes allows the formation of a bifunctional tetrabolo. Co-transfection of two different expression cassettes for tetrabolo results in the formation of a trifunctional (B) or tetrafunctional (C) tetrabolo. (D-E) Exemplary schematic diagrams of expression cassettes for the expression of scFv (D) or VHH (E), in which the scFv or VHH is cloned either upstream (homodimer) or downstream (homodimer) of the C4bp C-terminal β-chain. An scFv is the result of combining two N-terminal variable domains of a human immunoglobulin light and heavy chain, whereas a VHH is the N-terminal single variable domain of the heavy chain of a camelid immunoglobulin (e.g. Bactrian camel, dromedary, llama, alpaca, vicuña and guanaco). When the scFv or VHH is located at the N-terminal end of the construct and thus linked at the C-terminus, such as upstream of the C4bp C-terminal beta strand (top right in D and E), they are in the "natural position" and are referred to herein as homodimers.In contrast, when scFv or VHH are located C-terminal to the construct and linked at the N-terminus, such as downstream of the C-terminal β-strand of C4bp (bottom right of D and E), they are located in the "non-natural" inverted position, also referred to herein as the "boro" position, and such multimers are referred to herein as "homodibolo". Homodibolo is depicted with the recognition domain (white semicircle) in the top position and the attachment linker in close proximity. In the first situation, the His-tag is located just downstream of the C4bp C-terminal β-strand, and in the second situation, it is located downstream of the boro-scFv or VHH. (F-G) Exemplary schematics of expression cassettes for expression of scFv homotetrabolo (F) and VHH homotetrabolo (G). The scFv or VHH downstream of the C4bp C-terminal β-strand is depicted upside down with the recognition domain on top and the attachment close to the recognition domain. (H-I) Exemplary schematic diagrams of expression cassettes for the expression of bifunctional VHH-scFv(H) or scFv-VHH(I) heterotetraboro. [Diagram 2] A two-step purification strategy for selecting multifunctional diboro- or tetraboro-proteins is shown. By introducing tags such as HIS-tag or FLAG-tag, the desired multifunctional diboro- or tetraboro-proteins can be selected, for example, by HIS-TRAP purification (step 1) followed by FLAG affinity chromatography (step 2). FuCo: functional component, SP: signal peptide, DS: C-terminal C4bp β-strand, HIS: 8x His-tag, FLAG: FLAG-tag. [Diagram 3] (A-B) Expression of bifunctional heterotetraboro sIL-15Rα.C4bpβ.scFv-anti-NKG2A. This construct is either single-transfected (A) or co-transfected (B) with human recombinant IL-15 plasmid. The scFv anti-NKG2A expressed C-terminal to the C4bp C-terminal β chain is in the "reverse" position. [Figure 4]Figure 1 shows that purified heterotetrabolo specifically binds to natural killer (NK) cells. His-Trap™ Excel-IMAC purified heterotetrabolo sIL-15Rα.C4bpβ.scFv.Anti-NKG2A.His8x expressed from HEK293F cells with or without cotransfection with human IL-15 was incubated with peripheral blood mononuclear cells (PBMC) (A and B) or natural killer cells NK92MI (C and D). Cells were then stained for NK markers, IL-15, and His. [Diagram 5] Functional effects of heterotetrabolo on NK cell degranulation and cytotoxic capacity. His-Trap™ Excel-IMAC purified heterotetrabolo sIL-15Rα.C4bpβ.scFv.Anti-NKG2A.His8x expressed from HEK293F cells cotransfected or not with human IL-15 (huIL-15) were incubated with PBMCs for 48 hours (A, C, D) or 4 hours (B). Cells were then stimulated with Raji target cells, coincubated with anti-CD107a for 5 hours (A), permeabilized, and stained with interferon gamma (IFN-γ) for 5 hours (B). In (C), NK cells were stimulated with HIV-1 infected ACH2 cells, coincubated with anti-CD107a for 5 hours, and in (D) permeabilized and stained for IFN-γ intracellular expression for 5 hours. [Figure 6] Functional effect of heterotetrabolo on NK cell cytotoxicity. His-Trap™ Excel-IMAC purified heterotetrabolo sIL-15Rα.C4bpβ.scFv.Anti-NKG2A.His8x expressed from HEK293F cells cotransfected with or without recombinant huIL-15 plasmid was incubated with PBMC for 48 h. Cells were then incubated with Raji target cells (stained with Cell Tracer Violet) (A) or ACH2 cells (B) for 24 h and stained for L / D. [Figure 7]Details of the constructs are shown: Construct A' (NKG2D / Psl): MS scFv [(RTX VL 10-first amino acid / MS (VL-VH)] anti-NKG2D.C4bpβ.scFv (VL-VH) anti-Psl.His8x and Construct B' (SLAMF7 / Psl): ELO scFv [(RTX VL 10-first amino acid / ELO (VL-VH)] anti-SLAMF7.C4bpβ.scFv (VL-VH) anti-Psl.FLAG. Transfection of A' or B' alone results in expression of BiKE (NKG2D / Psl or SLAMF7 / Psl), whereas cotransfection of A' and B' constructs results in expression of TriKE (NKG2D / SLAMF7 / Psl). [Figure 8]Binding of BiKE and TriKE bearing scFv-anti-Psl to P. aeruginosa using ELISA with unfixed coated bacteria. (A) Sketch of the BiKE and TriKE biologics used. The top two panels are the BiKE and the bottom panel is the TriKE biologic. (B) Results of binding of molecules to bacteria. Ten clinical Pseudomonas isolates were used. (C) Double staining violet+ / PKH26+ (crosslinked Pseudomonas / NK92MI) induced by BiKE anti-SLAMF7 / anti-Psl (1.85%), tTriKE anti-NKG2D / anti-SLAMF7 / anti-Psl (0.9%) and to a lesser extent BiKE anti-NKG2D / anti-Psl (0.43%). Negative controls showed double staining of 0.29% and 0.31%. Of note, NKG2D was much less expressed in NK92MI than in SLAMF7 (data not shown), which may explain why NKG2D staining was weaker in MK92MI. (D) ELISA with unfixed coated P. aeruginosa and exposure with anti-HIS or anti-Flag antibodies indicates that BiKEs NKG2D / Psl, BiKEs SLAMF7 / Psl, and TriKE NKG2D / SLAMF7 / Psl express the Psl scFv as well as their respective NK receptor targeting sites. BiKE NKG2D / Psl (A') was purified on a HIS-TRAP column, BiKEs SLAMF7 / Psl (B') was purified using FLAG affinity chromatography, and the His and FLAG tags in the structure of TriKEs (A'B') were purified using a two-step method including first HIS affinity chromatography and then FLAG affinity chromatography to demonstrate that the selected TriKE clones carry both anti-NKG2D and anti-SLAMF-7 scFvs. In (E) NK cell-induced killing of luminescent Pseudomonas strain PAO1-lux after binding with 3 and 6 μg of TriKE NKG2D / SLAMF7 is shown. 2.105 NK92-CD16 cells were plated with Pseudomonas strain PAO1-lux in 200 μl of complete RPMI medium without antibiotics at a final E / T ratio of 1 / 3 and cultured at 37°C.Molecules were added at T0 and bacterial growth was measured in triplicate over time in a luminescence microplate reader for 9 h. [Figure 9] A sketch showing the structure of an exemplary double hinge "long neck" antibody-like glycoprotein, also referred to herein as "pseudo-IgG". The "double hinge" state means that (i) the dimerization scaffold of the C4bp C-terminal β-strand exhibiting two cysteines is present in parallel with the subsequent (ii) hinge of the IgG1-Fc exhibiting two cysteines, thus providing high stability of the dimer and providing a "long neck" domain separating the targeting moiety from the CH2-CH3 Fc IgG1 domain. The length of the linker between the targeting moiety and the C4bp C-terminal β-strand is variable. [Figure 10] Expression cassettes for pseudo-IgG are shown: (A) Two constructs are depicted, both showing functional component 1 as IgG1 Fc (hinge+CH2+CH3) downstream of C4bp β and functional components 3 and 4 upstream of C4bp β, respectively. A single transfection results in the expression of monospecific pseudo-IgG with recognition function 3 or 4. Co-transfection of the two constructs resulted in the expression of bifunctional 3 and 4 pseudo-IgG along with their monospecific 3 and 4 counterparts. (B) Combining the charge repulsion induced heterodimeric Fc platform (also known as the "knob-into-hole" technology) with the boro technology allows 100% expression of bispecific pseudo-IgG, eliminating the need to purify the bispecific pseudo-IgG from its other two monospecific counterparts. Alternative knob-into-hole techniques can also be applied (as described in John BB Ridgway, Leonard G Presta & Paul Carter. Protein Engineering, 1996; 9; 7; pp. 617-621). [Figure 11] Comparative testing of fluid-phase complement activation using the CH50 assay is shown. Importantly, pseudo-IgG behaves similarly to conventional therapeutic antibodies in that it has a reduced ability to activate soluble phase plasma. [Figure 12]Flow cytometric analysis of CDC (A) and C3b deposition (B) on Daudi cells coated with saturating concentrations (20 μg / ml) of (i) RTX scFv.C4bpβ.Fc (mock RTX), (ii) RTX (MabThera) or (iii) control (no molecule) followed by incubation (37°C, 30 min) with 25% normal human serum (NHS) in gelatin veronal buffer (GVB) supplemented with Ca++ and Mg++ (GVB++; 141 mM NaCl, 0. 3 mM CaCl2, 1 mM MgCl2, 0.1% gelatin, 1.8 mM sodium barbiturate and 3.1 mM barbituric acid, pH 7.3-7.4). Cells were then stained with mouse anti-human C3b mAb (clone 7C12), secondary goat anti-mouse IgG Ab AF647 labelled and live / dead UV. Pseudo-IgG induces superior C3b deposition (B) and subsequent increased CDC in Daudi cells (A) compared to the reference rituximab (RTX). [Figure 13] Comparative flow cytometric analysis of C3b deposition versus Fc density on Daudi cells using RTX and mock RTX. (A) Details of the molecules used. (B) Daudi cells were incubated with two-fold serial dilutions of the molecules (starting concentration 20 μg / ml for 30 min at 4°C). Molecules-coated Daudi cells were incubated with 25% normal human serum (NHS) in 50 μl of GVB++ for 30 min at 37°C. Cells were stained with mouse anti-human C3b mab (7C12), goat anti-mouse IgG pAb PE conjugate, goat anti-human Fc pAb AF647 conjugate and UV live / dead. Fc concentration and mean fluorescence intensity (MFI) of C3b deposition are shown on the X-axis and Y-axis, respectively. At comparable concentrations, mock RTX deposited 5.6-fold more C3b than RTX. [Figure 14]Flow cytometric analysis of NK activation (percentage of CD107 positive NK) and complement activation (MFI anti-C3b) on BT474 target cells. (A) BT474 were stained with CFSE and incubated with 5-fold serial dilutions (20, 4, 0.8 μg / ml) of bispecific pseudo-IgG (Z199 / 2D3 or Z199 / Trastu), or trastuzumab, or no molecule. BT474 cells were incubated with GVB++ (30 min at 37 °C / 5% CO2) or complete RPMI medium, followed by co-culture with PBMC for 5 h at 37 °C / 5% CO2. Cells were stained with anti-human CD107 / BV421 (after 1 h co-incubation), anti-human IgG / AF647 pAb, and anti-C3b / PE, anti-CD3 / BUV496, CD14 / PE-Cy5, CD16 / BUV737, CD19 / PE-Cy5, CD56 / BV786 mAbs. The gating strategy was CD3- / CD14-CD19- / CD16+ / CD56+ to access NK staining on Fc, knowing that BT474 cells are CSFE positive. (B-C) NK cells were analyzed for CD107 degranulation (B) and C3b deposition (C). Bispecific pseudo-IgG shows similar efficacy for NK activation compared to trastuzumab, but pseudo-IgG is a potent complement activator in contrast to trastuzumab. Using a trastuzumab-derived scFv cloned into a pseudo-IgG scaffold, a “pseudo-trastuzumab” with strong complement-activating activity was generated. [Figure 15]Analysis of the percentage of phagocytic macrophages using Andor spinning disk confocal microscopy shows the results of antibody-dependent cell-mediated phagocytosis of Daudi cells (involving ADCP, IFNγRIIIA / Fc interactions when using decomplemented ΔC5-HS) ​​or ADCP AND complement-dependent cell-mediated phagocytosis (involving CDCP, both IFNγRIIIA / Fc and CD11b / iC3b interactions when using ΔC5-HS). Mock RTX is more than two-fold better than RTX in phagocytosis of Daudi cells by macrophages in the presence of complement and approximately four-fold better than RTX in phagocytosis by macrophages in the absence of complement. With regard to Fc-mediated phagocytosis of Daudi by macrophages, compared to RTX, mock RTX has a more balanced dual Fc function through both IFNRIIIA / F and CD11b / iC3b interactions. [Figure 16] Design of cassettes and expression vectors, and results of pseudo-IgG expressed after single (monospecific) or double (bispecific) transfection in HEK293T cells. The knob-into-hole technology combined with pseudo-IgG allows the expression of 100% bispecific pseudo-IgG. [Figure 17](A) Direct bactericidal effect of Psl pseudo-IgG against PAO1-luciferase P. aeruginosa strain. 3 μg of Psl pseudo-IgG is applied to 1.5.105 bacteria / well in 50% NHS or ΔNHS (in GVB++ veronal buffer) for 5 h. Bacterial growth is measured at different time points (luciferase signal in RLU). Psl pseudo-IgG has a strong killing effect in the presence of 50% NHS, preventing bacterial growth. (B) Mechanism of action of pseudo-IgG showing its strong ability to recruit C1q, involved in the activation of the classical complement pathway. The terminal complement complex (membrane attack complex) is finally formed, leading to pore formation, membrane disruption and lysis (CDC). (C) Dose-dependent binding (using ELISA with non-fixed coated bacteria) of two purified Psl and pano pseudo-IgG against three different P. aeruginosa strains: PAO1 reference strain, serotype O11 (ATCC 33358) and clinical isolate IPP6247290 (tracheal secretions from a tracheotomy patient). Psl pseudo-IgG has strong binding affinity for PAO1 and IPP6247290, but low affinity for O11. Conversely, pano pseudo-IgG shows good affinity only for the O11 serotype, which is the main serotype recognized by the original panobacumab. [Figure 18] Dose-dependent pano scFv (VL-VH).C4bpβ.Fc pseudo-IgG and anti-Psl scFv (VL-VH) C4bpβ.Fc pseudo-IgG-mediated C3b deposition of P. aeruginosa reference strain PAO1 (left), ATCC serotype O11 (middle), and clinical isolate IPP6247290 (right) are shown. The results are consistent with the dose-response binding and affinity results of the two pseudo-IgGs in Figure 17C. [Figure 19] Dose-dependent pano scFv (VL-VH).C4bpβ.Fc pseudo-IgG and anti-Psl scFv (VL-VH) C4bpβ.Fc pseudo-IgG mediated C5b9 (MAC) deposition against the P. aeruginosa reference strain PAO1 (left), ATCC serotype O11 (middle), and clinical isolate IPP6247290 (right). The results are consistent with the dose-response binding and affinity of the two pseudo-IgGs and C3b deposition results shown in Figures 17C and 18, respectively. [Figure 20]Panobacumab scFv (VL-VH).C4bpβ.Fc pseudo-IgG and anti-Psl scFv (VL-VH) C4bpβ.Fc pseudo-IgG molecules were shown to enhance the complement killing capacity of human serum by 35% (strain PAO1), 26% (strain O11) and 31% (clinical isolate IPP6247290). [Figure 21] Exemplary amino acid and nucleic acid sequences for knob-into-hole technology are shown. (A) Codon-optimized sequence for expression in human cells (e.g., HEK293T cells) encoding a linker between BspE1 (T / CCGGA) and the stop codon TGA.C4bpβ.Fc, followed by a multiple cloning site terminating with NotI (GC / GGCCGC), whose CH3 Fc IgG1 exhibits three mutations (S354C, T366W, K409A), also known as Fc[knob]. (B) Codon-optimized sequence encoding a linker between BspE1 and the stop codon TGA.C4bpβ, followed by a multiple cloning site terminating with NotI, whose CH3 Fc IgG1 exhibits five mutations (Y349C, T366S, L368A, F405K and Y407V), also known as Fc[hole]. SEQ ID NO: 45 provides the amino acid sequence of a codon-optimized sequence encoding a linker between BspE1 and the stop codon TGA, .C4bpbeta.Fc, followed by a multiple cloning site terminating in NotI. [Figure 22]We present a complement-mediated strategy for bacterial target destruction using bifunctional heterotetraboro. We used a fusion protein consisting of (i) Psl scFv (VL-VH) and (ii) the last three short consensus repeats (SCR3-5) of factor H-related protein 1 (FHR1) cloned upstream and downstream of the C4bp C-terminal β-chain, respectively. (A) Exemplary schematic of an expression cassette for the expression of Psl / FHR1(SCR3-5) heterotetraboro, where the first and second polypeptides of the dimeric protein complex comprise FHR1 SCR35 C-terminal to the C-terminal portion of the C4bp β-chain and Psl scFv (VL-VH) N-terminal to the C-terminal portion of the C4bp β-chain. Transfection of one of the exemplary expression cassettes allows the formation of the Psl / FHR1(SCR3-5) heterotetraboro. (B) A sketch illustrating the mechanism of action of the Psl / FHR1(SCR3-5) construct is shown. Psl / FHR1(SCR3-5) constructs bind to P. aeruginosa via the Psl binding site. On the target bacterial surface, the heterotetraboro FHR1(SCR3-5) C-terminal effector moiety competed with hijacked bound factor H (FH) for C3b and regulated FH-mediated complement breakdown. As a result, dysregulation of FH locally leads to activation of the alternative complement pathway (AP) without engaging the classical complement pathway (CP) / lectin pathway (LP). Final MAC formation disrupts the bacterial membrane, leading to bacterial lysis. (C) Dose-dependent binding of Psl / FHR1(SCR3-5) molecules to PAO1 P. aeruginosa cells. 1.5.105 bacteria / well were immobilized on MaxiSorpTM 96-well flat-bottom polystyrene 96-well ELISA plates. After blocking, serial dilutions of Psl scFv / FHR1(SCR3-5) molecules were added. Bound molecules were revealed with 100 ng / well of mouse anti-HIS HRP-conjugated detection monoclonal antibody (SIGMA). (D) Effect of Psl scFv / FHR1(SCR3-5) on C3b deposition and complement activation. 1.5.105 bacteria / well were immobilized and incubated with serial dilutions of Psl scFv / FHR1(SCR3-5) molecules. 0.5% normal human serum (NHS) or decomplemented human serum (ΔNHS) was added for 30 min.C3b deposition was measured using mouse anti-human C3 / C3b / iC3b mAb (clone 7C12) followed by goat anti-mouse IgG HRP-conjugated revealing antibody. Addition of Psl scFv / FHR1(SCR3-5) molecules increased C3b deposition and promoted complement activation of PAO1 P. aeruginosa cells. (E) Direct bactericidal effect of Psl / FHR1(SCR3-5) purified construct against PAO1-luciferase P. aeruginosa strain. 3 μg of Psl / FHR1(SCR3-5) molecules are applied to 1.5.105 bacteria / well in 50% NHS or decomplemented NHS (ΔNHS) (in GVB++ veronal buffer) for 5 h. Bacterial growth is measured at different time points (luciferase signal in RLU). The Psl scFv / FHR1(SCR3-5) molecule exhibits potent killing effects and inhibits bacterial growth in the presence of 50% NHS. The last three SCRs of FHR1 (SCR3-5), which contain the binding domain for C3b that activates the alternative complement pathway, are sufficient to induce efficient local regulation of FH and kill bacteria. [Diagram 23]Pseudo-IgGs displaying the first two N-terminal "complement control proteins" (CCP1-2) or "short consensus repeats (SCR1-2)" of the C4bp α chain are shown. (A) Instead of classical antibody fragments (e.g., scFv, VHH), we used CCP1-2 cloned into a pseudo-IgG scaffold as an anchor site (CCP1-2.C4bpβ.Fc or CCP1-2 pseudo-IgG). Purified CCP1-2 pseudo-IgGs were analyzed using SDS-PAGE (4-15% acrylamide gradient gels) under non-reducing (NR) and reducing (R) conditions, followed by SYPRO RUBY staining. The apparent molecular weight of CCP1-2 pseudo-IgG was 120 kDa and 60 kDa under NR and R conditions, respectively. (B) The binding ability of CCP1-2 pseudo-IgGs to N. gonorrhoeae was tested using FACS analysis. Bacteria coated with 10 μl of CCP1-2 pseudo-IgG (or control bacteria) were incubated with secondary goat anti-human IgG / AF648 pAb. The graph shows specific binding to N. gonorrhoeae. (C) FACS analysis of CCP1-2 pseudo-IgG-mediated MAC formation in the presence of 10% NHS or ΔNHS. The results show robust MAC formation via CCP1-2 pseudo-IgG. MAC formation was completely abolished using ΔNHS in the presence of the same concentration of CCP1-2 pseudo-IgG. (D) FACS analysis of CCP1-2 pseudo-IgG-mediated bacterial killing in the presence of 10% NHS for 30 or 90 min. A 50% growth inhibition was observed after 30 min incubation at 2 μl / ml. Prolonged incubation with factor 3 completely abolished bacterial growth. At 10 μl / ml, bacterial growth was completely abolished after 30 min. No activity was observed when serum was decomplemented. The use of NHS alone does not affect bacterial growth. This experiment shows that this new pseudo-IgG, displaying CCP1-2 from the N-terminal α-chain of C4bp, has a strong bactericidal effect associated with selective, localized, directed complement-mediated killing. In contrast to mostly single-target antibodies, this CCP1-2 pseudo-IgG could potentially target all pathogens that evade complement-mediated killing by employing the C4bp soluble complement control protein. Thus, we have created a multi-target pseudo-IgG. [Figure 24](A) Homodibolo (homoB2C-scFvLi7) and homotetrabolo (homoB4-scFvLi7) of scFv anti-sphingomyelinase D (SMaseD) from Loxosceles intermedia (Li) scFvLi7 (VH-VL) were generated in HEK293 cells. Homodibolo "homoB2C": the gene encoding scFvLi7 was cloned downstream of the C-terminal C4bp β-strand in the unnatural "reverse" position. In this product, no gene was present upstream of the C-terminal C4bp β-strand. Homodibolo showed a valence of two scFvLi7. Homotetrabolo "homoB4": the gene encoding scFvLi7 was cloned upstream and downstream of the C-terminal C4bp β-strand in the natural and reverse positions, respectively. Homotetrabolo showed a valence of four scFvLi7. To increase protein expression, two mutants of HomoB4 (herein referred to as HomoB4 bis and ter) were generated. For HomoB4 bis, the original scFvLi7 (VH-VL) upstream of the C-terminal C4bp β-chain dimerization scaffold was reverted to VL-VH and the first eight amino acids of VLscFvLi7 (DIVMTQSP (SEQ ID NO: 46)) were replaced with those of Rituximab VL (QIVLSQSP (SEQ ID NO: 5)). For HomoB4 ter, a (AASGGGGSSGGGGSSGGGGS: SEQ ID NO: 47) linker was introduced between the signal peptide and the beginning of the original scFvLi7 (VH-VL). (B) HomoB2C, HomoB4, HomoB4 bis and HomoB4 ter were analyzed using SDS-PAGE under non-reducing conditions, followed by Western blotting with rabbit anti-HIS pAb, secondary goat anti-rabbit IgG AF488 conjugate. Western blots were revealed using a Typhoon Imager. The size of homodiboro (HomoB2C) is 75 kDa, and that of homotetraboro (HomoB4) is 150 kDa. The expression yield was increased by introducing a short linker after the peptide signal. (C) Specific binding of purified HomoB2C to SmaseD was analyzed by SDS-PAGE and WB (non-reducing conditions) of Loxosceles intermedia venom (5 μg / well).Revelation with (i) horse anti-SmaseD Li serum (α-lox) (positive control), (ii) purified scFv Li7-HomoB2C (10 μg / ml), or (iii) irrelevant mAb (negative control). ECL (20 sec exposure), DAB and nitrocellulose membranes stained with Rouge Ponceau. A band with an apparent molecular mass of 32-35 kDa was revealed by both HomoB2C and the immune horse serum, which corresponds to the molecular mass of SmaseD from a different loxosceles species (31-34 kDa) (GJ Binford et al. Mol Biol Evol. 2009, 26(3), 547-566). Conclusion: scFv cloned downstream of the C-terminal C4bp β-strand functionally binds the target even in the non-native "upside-down" position. (D) Dose-response binding of HomoB4 and HomoB2C with Li venom coated on an ELISA plate. ELISA plates were blocked and then incubated with serial dilutions of Homo B2C, Homo B4, Homo B4 bis and Homo B4 ter (concentrations expressed in ng / ml). ELISA plates were revealed with rabbit anti-His pAb and goat anti-rabbit IgG HRP-conjugated pAb. ELISA plates were revealed with OPD / H2O2 chromogenic substrate for HRP. Molecule concentrations in Dulbecco's modified Eagle's medium (DMEM) complete medium were measured using HIS-HIS enzyme-linked immunosorbent assay (ELISA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] As used herein, the singular forms "a," "an," and "the" include both singular and plural references unless the context clearly dictates otherwise. As used herein, the terms "comprise," "comprising," and "consisting of" are synonymous with "including" or "containing" and are inclusive or open-ended terms and do not exclude additional, unrecited members, elements, or method steps. The terms also encompass "consisting of" and "consisting essentially of," which have their established meanings as patent terms. The recitation of numerical ranges by endpoints includes all numerical values ​​and subranges subsumed within each range, as well as the recited endpoints. This applies whether the numerical range is recited "from to" or "between" or other expressions. The term "about" as used herein when referring to a measurable value such as a parameter, amount, duration, etc., means to include, as appropriate for the disclosed invention, a variation from / of the specified value, for example, a variation of ±10% or less, preferably ±5% or less, more preferably ±1% or less, and even more preferably ±0.1% or less from / of the specified value. It is to be understood that a value with the modifier "about" is itself specifically and preferably disclosed. The term "one or more" or "at least one," such as one or more or at least one member of a group of members, is clear in itself, but by way of further example, the term specifically encompasses reference to any one of said members, or any two or more of said members, up to all of said members, such as any >= 3, 4, 5, 6 or 7 of said members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0029] This specification includes a reference to the background of the invention to explain the context of the invention. This reference should not be construed as an admission that any of the referenced material was published, known, or part of the general knowledge in any country as of the priority date of any claim. Throughout this disclosure, various publications, patents, and published patent specifications are referenced by way of specific reference. All documents cited herein are incorporated by reference in their entirety. Specifically, the teachings or passages of documents specifically mentioned herein are incorporated by reference.

[0030] Unless otherwise specified, all terms used in the disclosure of the present invention, including technical and scientific terms, have the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. As a further guide, definitions of terms are included to better understand the teachings of the present invention. When a particular term is defined in relation to a particular aspect or embodiment of the present invention, the implication or meaning is meant to apply throughout the specification, i.e., in the context of other aspects or embodiments of the present invention, unless otherwise stated.

[0031] In the following, different aspects or embodiments of the invention are described in more detail. Each described aspect or embodiment may be combined with any other aspect or embodiment, unless expressly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with one or more of any other features indicated as preferred or advantageous.

[0032] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, although some embodiments described herein include some features and do not include other features included in other embodiments, it will be understood by one of ordinary skill in the art that combinations of features of different embodiments are within the scope of the invention and constitute different embodiments. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0033] Recombinant biomolecules are often used in biotechnology, for example to produce vaccines, diagnostic kits, therapeutic molecules, etc. Such recombinant biomolecules include antigens, antibodies or antibody fragments (scFv, V H H), enzymes, hormones, cytokines, growth factors, agonist or antagonist ligands for regulating immune responses, or checkpoint inhibitors. Many of these biological molecules require assembly to be functional or to exhibit optimal biological activity. In order to produce therapeutic dimeric molecules, it is necessary to use methods that allow the production of dimeric recombinant therapeutics where the assembly occurs naturally, without the need for an additional dimerization step that requires the use of immunogenic molecules.

[0034] The inventors have found that the C-terminal portion of the C4bp β chain can be used as a scaffold for dimerization and that functional components can be introduced C-terminally (i.e. downstream) and optionally N-terminally to said C-terminal portion of the C4bp β chain. The present invention allows for the natural expression of a dimeric protein complex in which (i) the association of dimers occurs in the eukaryotic export machinery, (ii) the associated dimers are secreted into the cell culture supernatant, and (iii) the dimerization scaffold (i.e., the C-terminal fragment of the C4bp β chain) is derived from a native circulating human molecule. In the present invention, a gene encoding a protein or polypeptide of interest can be cloned into a non-native location downstream of the C-terminal dimerization scaffold of the C4bp β chain, since the native C4bp β chain does not have a protein or polypeptide at its C-terminus. Introducing a protein or polypeptide of interest into a non-native location in the C-terminal portion of the C4bp β chain unexpectedly led to spontaneous dimerization of the fusion protein, similar to a protein or polypeptide of interest located in the native N-terminal portion of the C-terminal portion of the C4bp β chain. Cloning a gene encoding a protein or polypeptide of interest into this location is particularly advantageous when the biological activity of the protein or polypeptide encoded by the gene is located at its C-terminus, such as a monomeric Fc. Cloning such a gene upstream of the C4bp β-chain would likely inhibit or reduce the activity of the resulting dimeric protein complex due to the close proximity of the dimerization scaffold to the biological function of the protein or polypeptide of interest. In contrast, when such a protein encoded by a gene of interest is cloned downstream of a C-terminal fragment of the C4bp β-chain, as in the present invention, its biological function is distal to the dimerization scaffold and thus maintained.

[0035] Furthermore, the inventors were surprised to find that, for example, scFv or V H Even if a gene encoding a target protein or polypeptide having a naturally occurring (wild type) domain that exerts a biological function / activity, such as a binding domain located at its N-terminus, such as the H polypeptide, is cloned downstream (i.e., on the C-terminal side) of the C-terminal fragment of the C4bp β-chain as in the present invention, the biological function / activity of the protein or polypeptide is not reduced or eliminated. Moreover, the dimerization of the dimerization scaffold is maintained. The advantage is that it is possible to use scFv or V HSuch proteins or polypeptides, such as H, can be included both at the N-terminus and C-terminus of the dimerization scaffold of C4bp β, resulting in a dimeric protein complex with high valency (e.g., up to 4 valency) of said protein or polypeptide. The present invention encompasses multifunctional dimeric protein complexes that can be produced by co-transfecting cells with two expression vectors encoding different functional components fused at the C-terminus to the C-terminal portion of the C4bp β chain, without a functional component fused at the N-terminus to the N-terminal portion of the C4bp β chain (e.g., first plasmid: N-terminus-C4bpβ.functional component AC terminus; second plasmid: N-terminus-C4bpβ.functional component BC terminus). In this case, three molecular species are released from the cells into the cell culture supernatant: AA "homodibolo", BB "homodibolo" and AB bifunctional "heterodibolo". The dimeric protein complexes can then be purified (Figures 1 and 2). The present invention also encompasses dimeric protein complexes that contain functional components both C-terminal and N-terminal to the C-terminal portion of the C4bp β chain, also referred to herein as homotetrabolo or heterotetrabolo, the former being mono- or bifunctional (which can be generated by a single transfection) and the latter being tri- or tetrafunctional (which can be generated by co-transfection of two plasmids) (Figures 1 and 2). Furthermore, the present invention encompasses dimeric protein complexes that contain a monomeric Fc as a functional component C-terminal to the C-terminal portion of the C4bp β chain. The molecular weight - and therefore the extravascular distribution space - of such dimeric protein complexes is in the same range as IgG, but homotetrabolo can exhibit tetravalency, resulting in a functional binding activity that is much greater than that of antibodies.

[0036] In conclusion, the dimeric protein complex of the present invention is particularly interesting for use as a therapeutic agent since it has approximately the size of an antibody, is robust as a result of a covalent interchain bond between the two C-terminal parts of the C4bp β-chain, and can be easily cloned with high expression yields.

[0037] Thus, the first aspect is a first polypeptide comprising a first functional component and a C-terminal fragment of a C4bp β chain, said first functional component being linked to the C-terminus of said C-terminal fragment of a C4bp β chain; and a second polypeptide comprising a second functional component and a C-terminal fragment of a C4bp β chain, said second functional component being linked to the C-terminus of said C-terminal fragment of a C4bp β chain. A dimeric protein complex comprising: The first and second polypeptides are the same or different. A dimeric protein complex is provided.

[0038] In certain embodiments, the dimeric protein complex is non-naturally occurring, meaning that the dimeric protein complex is recombinantly produced.

[0039] The term "protein" as used herein generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term encompasses natural, recombinant, semi-synthetically or synthetically produced proteins. The term also encompasses proteins having one or more co-expressed or post-expressed modifications of the polypeptide chain, such as, but not limited to, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of a proenzyme or prehormone to its active form, and the like. The term further encompasses protein variants or mutants having changes in amino acid sequence relative to the corresponding native protein, such as, for example, deletion, addition and / or substitution of amino acids. The term contemplates both full-length proteins and portions or fragments of proteins, e.g., naturally occurring protein portions resulting from processing of such full-length proteins.

[0040] The term "polypeptide" as used throughout this specification generally includes a polymeric chain of amino acid residues linked by peptide bonds. Thus, in particular where a protein is composed of only a single polypeptide chain, the terms "protein" and "polypeptide" may be used interchangeably herein to refer to such a protein. The term is not limited to the minimum length of the polypeptide chain. The term includes natural, recombinant, semi-synthetically or synthetically produced polypeptides. The term also includes polypeptides having one or more co-expressed or post-expressed modifications of the polypeptide chain, including, but not limited to, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of a proenzyme or prehormone to its active form. The term further includes polypeptide variants or mutants having amino acid sequence changes relative to the corresponding native polypeptide, including, for example, deletions, additions and / or substitutions of amino acids. The term contemplates both full-length polypeptides and polypeptide portions or fragments, such as naturally occurring polypeptide portions resulting from processing of such full-length polypeptides.

[0041] The term "peptide" as used throughout the present specification preferably refers to a polypeptide as used herein that consists essentially of 50 amino acids or less, such as 45 amino acids or less, preferably 40 amino acids or less, such as 35 amino acids or less, more preferably 30 amino acids or less, such as 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less or 5 amino acids or less.

[0042] Reference to any peptide, polypeptide, protein or nucleic acid may specifically include a peptide, polypeptide, protein or nucleic acid having a native sequence, i.e., one whose primary sequence is identical to the sequence of a peptide, polypeptide, protein or nucleic acid found or derived from nature. Those skilled in the art understand that native sequences may differ due to genetic divergence between different species. Furthermore, native sequences may differ between or within different individuals of the same species due to normal genetic variation (mutation) within a given species. Native sequences may also differ between or within individuals of the same species due to somatic mutations or post-transcriptional or post-translational modifications. Any such variants or isoforms of peptides, polypeptides, proteins or nucleic acids are contemplated herein. Thus, any peptide, polypeptide, protein or nucleic acid sequence that exists in or is derived from nature is considered "native".

[0043] In certain embodiments, the peptides, polypeptides, proteins or nucleic acids may be human, i.e., their primary sequences may be identical to the corresponding primary sequences of naturally occurring human peptides, polypeptides, proteins or nucleic acids, or may be identical to the corresponding primary sequences present in naturally occurring human peptides, polypeptides, proteins or nucleic acids. In certain embodiments, the modifier "human" refers to the primary sequence of the respective peptide, polypeptide, protein or nucleic acid, and not its origin or source. For example, such peptides, polypeptides, proteins or nucleic acids are present in or isolated from a sample of a human subject, or obtained by other means (e.g., recombinant expression, cell-free transcription or translation, or non-biological nucleic acid or peptide synthesis).

[0044] In certain embodiments, a peptide, polypeptide, protein, or nucleic acid may be wild-type. Although most native peptides, polypeptides, proteins, or nucleic acids may be considered wild-type, those with naturally occurring mutations leading to partial or complete loss of function that may contribute to a disease phenotype or cause a disease are generally excluded from the scope of the term "wild-type." Reference to any peptide, polypeptide, protein or nucleic acid may also include variants or fragments of such peptide, polypeptide, protein or nucleic acid, particularly naturally occurring, native or wild-type forms.

[0045] The term "variant" of a protein, polypeptide, peptide or nucleic acid generally refers to a protein, polypeptide, peptide or nucleic acid whose amino acid sequence or whose nucleotide sequence is substantially identical (i.e., largely but not completely identical) to the sequence of the protein, polypeptide, peptide or nucleic acid, e.g., at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, even more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the referenced protein, polypeptide, peptide or nucleic acid. Preferably, the variant may exhibit such a degree of identity to the referenced protein, polypeptide, peptide or nucleic acid when the entire base sequence of the referenced protein, polypeptide, peptide or nucleic acid is referenced in a sequence alignment (i.e., overall sequence identity). Sequence identity can be determined using sequence alignment and, of course, an appropriate algorithm for carrying out the determination of sequence identity.Exemplary, but non-limiting, algorithms include those based on the Basic Local Alignment Search Tool (BLAST) described by Altschul et al. 1990 (J Mol Biol 215: 403-10), e.g., the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174:247-250), using, for example, published default settings or other suitable settings (e.g., for the BLASTN algorithm: cost to open a gap=5, cost to extend a gap=2, penalty for mismatch=-2, reward for match=1, gap x dropoff=50, expectation=10.0, word size=28; or for the BLASTP algorithm: matrix=Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89:10915-10919), cost to open a gap = 11, cost to expand a gap = 1, expectation = 10.0, word size = 3).

[0046] An example of a procedure for determining the percent identity between a particular amino acid sequence and an amino acid sequence of a query polypeptide involves aligning the two amino acid sequences using the Blast 2 sequences (Bl2seq) algorithm available as a web application or as a standalone executable program (BLAST version 2.2.31+) on the NCBI website (www.ncbi.nlm.nih.gov) with appropriate algorithm parameters. Examples of suitable algorithm parameters are: matrix=Blosum62, cost to open a gap=11, cost to extend a gap=1, expectation=10.0, word size=3). If the two compared sequences share homology, the output shows the regions of homology as aligned sequences. If the two compared sequences do not share homology, the output does not show aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where identical amino acid residues are displayed in both sequences. The percentage of identity is determined by dividing the number of matches by the length of the query polypeptide and then multiplying the resulting value by 100. Percent identity values ​​may, but need not, be rounded to the nearest tenth (0.001) place. For example, 78.11, 78.12, 78.13, and 78.14 would be rounded down to 78.1, and 78.15, 78.16, 78.17, 78.18, and 78.19 would be rounded up to 78.2. Furthermore, the detailed display for each segment of the alignment produced by Bl2seq already includes the percentage of identity for convenience.

[0047] A variant of a protein, polypeptide, peptide, or nucleic acid may be a homolog (e.g., an ortholog or paralog) of that protein, polypeptide, peptide, or nucleic acid. As used herein, the term "homology" generally refers to the structural similarity between two macromolecules from the same or different taxa, said similarity resulting from a common ancestry. A variant of a protein, polypeptide, or peptide may include an addition, deletion, or substitution of one or more amino acids, such as a conservative amino acid substitution or a non-conservative amino acid substitution, relative to (i.e., compared to) the corresponding protein or polypeptide. A variant of a nucleic acid may include an addition, deletion, or substitution of one or more nucleotides relative to (i.e., compared to) the corresponding nucleic acid.

[0048] As described elsewhere herein, C4bp is a normal plasma protein with an octopus-like structure consisting of seven branched α chains and one central β chain. Each chain contains short consensus repeats (SCRs). Each SCR, consisting of approximately 60 amino acids, contains two intrachain disulfide bonds. Both the C-terminal portions of the C4bp α chain and β chain lack biological functions, the former being responsible for polymerization of the molecule in the cytoplasm of C4bp-producing cells, and the latter being responsible for covalently linking one β chain to the heptameric core of the C4bp glycoprotein by forming two disulfide bonds with two cysteines at the C-terminus of the C4bp α chain. The β chain subunits are not required for oligomerization of the α chain.

[0049] The C-terminal fragment of the C4bp β chain can spontaneously dimerize into a dimeric protein complex in the absence of the C4bp α chain. Thus, one skilled in the art will understand that the first and second polypeptides referred to herein refer to two components of a dimeric protein complex that are expressed as separate polypeptides and spontaneously associate after expression by a covalent or intermolecular bond. The term homodimer is used when the first and second polypeptides are identical. The term heterodimer is used when the first and second polypeptides are not identical.

[0050] In certain embodiments, a C-terminal fragment of a C4bp β chain formed by a first polypeptide has the ability to dimerize with a C-terminal fragment of a C4bp β chain formed by a second polypeptide. The C-terminal fragment of a C4bp β chain formed by a first polypeptide and the C-terminal fragment of a C4bp β chain formed by a second polypeptide are preferably dimerized by a covalent bond.

[0051] In certain embodiments, the C-terminal fragment of the C4bp β chain contains at least one, and preferably at least two, cysteine ​​residues, for example, one of the cysteines of the C-terminal fragment of the C4bp β chain may be mutated.

[0052] In a particular embodiment, the C-terminal fragment of the C4bp β chain is the 194-252 fragment of the C4bp β chain, or a functional variant that at least preserves the ability to form a dimeric protein.

[0053] In a particular embodiment, a functional variant of a C-terminal fragment of the C4bp β chain, preferably a 194-252 fragment of the C4bp β chain, is: a modified sequence of the 194-252 fragment of the C4bp β chain, in which less than 25%, preferably less than 10%, of the amino acids of the 194-252 fragment have been truncated or substituted, while the cysteines at positions 202 and 216 and at least three amino acids upstream and downstream of each cysteine ​​are conserved; a modified sequence of the 194-252 fragment of the C4bp beta chain, in which the cysteine ​​involved in dimerization is replaced by an amino acid selected from alanine, valine, phenylalanine, proline, methionine, isoleucine, leucine and tryptophan, and another amino acid of the fragment is replaced by a cysteine; the sequence of the 194-252 fragment of the C4bp β chain modified by the insertion of a sequence heterologous to the C4bp β chain between the cysteines involved in dimerization; or This is the sequence of the 194-252 fragment of the C4bp β chain modified by truncating the amino acids between the cysteines involved in dimerization.

[0054] In certain embodiments, the C-terminal fragment of the C4bp β chain is a C-terminal fragment of a human C4bp β chain. By way of example, the human C4bp β chain may comprise the amino acid sequence as annotated in NCBI Genbank under accession number NP_001017367.1 (isoform 1 precursor) and in Uniprot (www.uniprot.org) under accession number P20851.1.

[0055] In a particular embodiment, the C-terminal fragment of the C4bp β chain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 1 (IQEAPKPECEKALLAFQESKNLCEAMENFMQQLKESGMEELKYSLELKKAELKAKLL); or an amino acid sequence having at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, or a functional fragment thereof (e.g. a fragment that allows dimerization of the C-terminal fragment of the C4bp β chain). The amino acid sequence set forth in SEQ ID NO: 1 corresponds to amino acids 194 to 252 of the amino acid sequence of the human C4bp β chain annotated under NCBI Genbank accession number NP_001017367.1 (isoform 1 precursor).

[0056] In certain embodiments, a C-terminal fragment of a C4bp beta chain comprising, consisting essentially of, or consisting of an amino acid sequence having at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1 is a functional variant of the C-terminal fragment of a C4bp beta chain.

[0057] In certain embodiments, the dimeric protein complex, the first polypeptide and / or the second polypeptide do not comprise the C4bp alpha chain or a fragment thereof. For example, the first and second polypeptides do not comprise the C-terminal fragment of the C4bp alpha chain. By way of example, the precursor of the human C4bp alpha chain comprises the amino acid sequence annotated under NCBI Genbank accession number NP_000706.1 and Uniprot (www.uniprot.org) accession number P04003.2.

[0058] The C-terminal fragment of the C4bp β chain is sufficient to achieve spontaneous dimerization, preferably covalent dimerization, e.g., the C-terminal fragment of the C4bp β chain contains at least two cysteines, allowing spontaneous interchain covalent dimerization of two C-terminal fragments of the C4bp β chain in the cellular export apparatus. In order to obtain a dimeric protein complex of the invention with the smallest possible size, fragments of the C4bp β chain that are not necessary to achieve natural dimerization may be deleted. Thus, in a particular embodiment, the first and second polypeptides do not include an N-terminal fragment of the C4bp β chain. In a more particular embodiment, the first and second polypeptides do not include the amino acid sequence set forth in SEQ ID NO: 2 (MFFWCACCLMVAWRVSASDAEHCPELPPVDNSIFVAKEVEGQILGTYVCIKGYHLVGKKTLFCNASKEWDNTTTECRLGHCPDPVLVNGEFSSSGPVNVSDKITFMCNDHYILKGSNRSQCLEDHTWAPPFPICKSRDCDPPGNPVHGYFEGNNFTLGSTISYYCEDRYYLVGVQEQQCVDGEWSSALPVCKL), or a fragment thereof.

[0059] In certain embodiments, the dimeric protein complex is a homodimer. In other words, in certain embodiments, the first and second polypeptides of the dimeric protein complex are identical.

[0060] In certain embodiments, the dimeric protein complex is a heterodimer. Or, in other words, in certain embodiments, the first and second polypeptides of the dimeric protein complex are not identical (i.e., different from each other).

[0061] In certain embodiments, the first and second polypeptides of the dimeric protein complex are identical.

[0062] In certain embodiments, the first and second polypeptides of the dimeric protein complex are not identical (ie, are different from each other). In the context of the present invention, the term "connected" as used herein is synonymous with "coupled," "bonded," "fused," and "joined," and refers to a physical link between at least two elements or components.

[0063] In certain embodiments, the first or second functional component is attached to the C-terminus of the C-terminal fragment of the C4bp β chain by a peptide bond. Coupling of the two proteins can be achieved by any method known in the art, such as expressing a nucleic acid comprising a nucleic acid encoding a C-terminal fragment of the C4bp β chain and a nucleic acid encoding a first or second functional component, where the nucleic acid encoding the first or second functional component is located 3' to the nucleic acid encoding the C-terminal fragment of the C4bp β chain, as described elsewhere herein.

[0064] In certain embodiments, the first functional component is located immediately C-terminal to the C-terminal fragment of the C4bp β chain of a first polypeptide of the dimeric protein complex, and / or the second functional component is located immediately C-terminal to the C-terminal fragment of the C4bp β chain of a second polypeptide of the dimeric protein complex. Alternatively, a linker, such as a peptide linker, can be included between the C-terminus of the C-terminal fragment of the C4bp β chain and the N-terminus of the first or second functional component. The term "linker" as used herein refers to a linking element that serves to link other elements. A linker may be a rigid linker (also referred to herein as a spacer) or a flexible linker. In certain embodiments, the linker is a covalent linker that achieves a covalent bond. The term "covalent" or "covalent bond" refers to a chemical bond that involves the sharing of one or more electron pairs between two atoms. In many molecules, the sharing of electrons allows each atom to obtain the equivalent of an outermost electron shell, which corresponds to a stable electron configuration. Covalent bonds include various types of interactions, including σ-bonds, π-bonds, metal-metal bonds, agostic interactions, bent bonds, and three-center two-electron bonds.

[0065] In certain embodiments, the linker is a (poly)peptide linker or a non-peptide linker, e.g. a non-peptide polymer, such as a non-biological polymer. Preferably, the link between the C-terminal fragment of the C4bp β-chain and the functional component may be a hydrolytically stable linkage, i.e. a linkage that is substantially stable in water for extended periods of time, e.g. several days, especially at useful pH values, including under physiological conditions. In certain embodiments, the linker is a peptide linker of one or more amino acids. More specifically, the peptide linker may be 1 to 50 or 2 to 50 amino acids long, or 1 to 45 or 2 to 45 amino acids long, preferably 1 to 40 or 2 to 40 or 1 to 35 or 2 to 35 amino acids long, more preferably 1 to 30 or 2 to 30 amino acids long. More preferably, the linker is 5 to 25 or 5 to 20 amino acids long. Particularly preferably, the linker is 5 to 15 or 7 to 15 amino acids long. Thus, in certain embodiments, the linker may be 1, 2, 3, or 4 amino acids long. In other embodiments, the linker may be 5, 6, 7, 8, or 9 amino acids long. In further embodiments, the linker may be 10, 11, 12, 13, or 14 amino acids long. In still other embodiments, the linker may be 15, 16, 17, 18, or 19 amino acids long. In further embodiments, the linker may be 20, 21, 22, 23, 24, or 25 amino acids long. In certain embodiments, the linker is 4-10, or 5-9, or 6-8, or 7 amino acids in length, in other embodiments the linker is 12-18, or 13-17, or 14-16, or 15 amino acids in length. The nature of the amino acids constituting the linker is not particularly important, so long as the biological activity of the polypeptide segments linked thereby is not substantially impaired and the linker provides the intended spatial separation of the C-terminal fragment of the C4bp β chain and the functional component. Preferred linkers are essentially non-immunogenic and / or not susceptible to proteolytic cleavage.

[0066] In certain preferred embodiments, the peptide linker may comprise, consist essentially of, or consist of amino acids selected from the group consisting of glycine, serine, alanine, threonine, and combinations thereof. In further preferred embodiments, the linker may comprise, consist essentially of, or consist of amino acids selected from the group consisting of glycine, serine, and combinations thereof. Such linkers provide particularly good flexibility. In certain embodiments, the linker may consist only of glycine residues. In certain embodiments, the linker may consist only of serine residues.

[0067] In certain embodiments, the linker has the amino acid sequence (SGGGGS) n (SEQ ID NO: 7), where n is an integer between 1 and 11, preferably between 2 and 7, more preferably between 3 and 7, such as 3, 5 or 7, even more preferably 5 or 7. In certain embodiments, the linker is a flexible linker comprising, consisting essentially of or consisting of the amino acid sequence 3x(SGGGGS) (SEQ ID NO: 3), 5x(SGGGGS) (SEQ ID NO: 48), or 7x(SGGGGS) (SEQ ID NO: 49). One skilled in the art will appreciate that the flexible linker and its size may be optimized depending on the functional moiety or moieties to be linked to the C-terminal portion of the C4bp β-chain.

[0068] In a particular embodiment, the linker is a spacer that comprises, consists essentially of, or consists of the amino acid sequence RDCDPPGNPVHGYFEGNNFTLGSTISYYCEDRYYLVGVQEQQCVDGEWSSALPVCKL (SEQ ID NO: 4). Such a spacer corresponds to SCR3 of the C4bp β chain and exhibits four cysteine ​​residues folded over by the presence of two internal disulfide bridges. SCR3 has no biological function and serves as a natural spacer of the C4bp β chain, separating the first two SCRs from each other and providing a scaffold for dimerization.

[0069] The inventors have found that inclusion of the peptide QIVLSQSP (SEQ ID NO:5) C-terminal to the signal peptide (preferably immediately 3' to the signal peptide) and N-terminal to the C-terminal fragment of the C4bp β-chain, and optionally N-terminal to the third or fourth functional component, results in increased expression of the first or second polypeptide described herein. The peptide defined in SEQ ID NO:5 is derived from rituximab (RTX). The peptide defined in SEQ ID NO:5 is located N-terminal to the rituximab hypervariable domain of the light chain (RTX VL). This sequence is located N-terminal to framework 1 (FR1) of the VL of RTX, away from (or away from) the "complementarity determining region 1 (CDR1)" downstream of the VL (involved in the recognition portion of the paratope), with the FR1 region located upstream of the CDR1. Thus, in certain embodiments, the first polypeptide comprises a C-terminal side of a signal peptide and an N-terminal side of a C-terminal fragment of the C4bp β chain, and, if a third functional component is present, comprises the peptide QIVLSQSP (SEQ ID NO:5) N-terminal to said third functional component. In certain embodiments, the second polypeptide comprises a C-terminal side of a signal peptide and an N-terminal side of a C-terminal fragment of the C4bp β chain, and, if a fourth functional component is present, comprises the peptide QIVLSQSP (SEQ ID NO:5) N-terminal to said fourth functional component. Preferably, the peptide defined in SEQ ID NO:5 is used in combination with a functional component that is a scFv, such as panobacumab. The scFv may have the structure [VH-linker-VL] or the inverse structure [VL-linker-VH]. The N-terminal portion (e.g., the first 8 amino acids) of the scFv VL is preferably replaced with the peptide defined in SEQ ID NO:5.

[0070] In a particular embodiment, the peptide defined by SEQ ID NO:5 is located immediately C-terminal to the signal peptide. Preferably, the signal peptide ends in a Bgl2 restriction site, more preferably in the amino acid sequence RS. Thus, the first and second polypeptides may comprise the sequence "signal peptide-RS-QIVLSQSP (SEQ ID NO:6)" at their N-terminus. Such a sequence has been found by the inventors to result in high expression yields of the dimeric protein complex.

[0071] In certain embodiments, the first polypeptide does not include a functional component that is not a tag on the N-terminal side of the C-terminal fragment of C4bp β chain, and / or the second polypeptide does not include a functional component that is not a tag on the N-terminal side of the C-terminal fragment of C4bp β chain. Such a dimeric protein complex may be referred to herein as a "diboro." Such a diboro may be used to test whether a functional component fused to the C-terminal side of the C-terminal fragment of C4bp β chain functions effectively before establishing a dimeric protein complex that also includes a functional component on the N-terminal side of the C-terminal fragment of C4bp β chain, as described elsewhere herein. A diboro in which the first and second functional components are identical may be referred to herein as a homodiboro, and a diboro in which the first and second functional components are different may be referred to as a heterodiboro.

[0072] In certain embodiments, the first and / or second polypeptide comprises one or more, such as two or three, functional components N-terminal and / or C-terminal to the C-terminal fragment of C4bp β chain. Thus, in certain embodiments, the first and / or second polypeptide comprises at least one, such as at least two or at least three, functional components N-terminal and / or C-terminal to the C-terminal fragment of C4bp β chain.

[0073] In certain embodiments, the first polypeptide further comprises a third functional component attached to the N-terminus of the C-terminal fragment of C4bp β chain; and / or the second polypeptide further comprises a fourth functional component attached to the N-terminus of the C-terminal fragment of C4bp β chain, wherein the first, second, third and / or fourth functional components are identical or different. A dimeric protein complex comprising first, second, third and fourth functional components is also referred to herein as a "tetraboro."

[0074] In certain embodiments, the third functional component is located immediately N-terminal to the C-terminal fragment of the C4bp β chain of a first polypeptide of the dimeric protein complex, and / or the fourth functional component is located immediately N-terminal to the C-terminal fragment of the C4bp β chain of a second polypeptide of the dimeric protein complex. Alternatively, a linker, such as a peptide linker, may be included between the N-terminus of the C-terminal fragment of the C4bp β chain and the C-terminus of the third or fourth functional component, as described elsewhere herein.

[0075] In certain embodiments, said first polypeptide of the dimeric protein complex comprises: A linker on the N-terminal side of the C-terminal fragment of the C4bp β chain and on the C-terminal side of the first functional component; and A linker on the C-terminal side of the C-terminal fragment of the C4bp β chain and on the N-terminal side of the third functional component, preferably the linker is a flexible linker, for example a flexible linker comprising, consisting essentially of or consisting of the amino acid sequence SGGGGS as defined by SEQ ID NO:7, or a spacer, such as a spacer comprising, consisting essentially of or consisting of the amino acid sequence RDCDPPGNPVHGYFEGNNFTLGSTISYYCEDRYYLVGVQEQQCVDGEWSSALPVCKL as defined by SEQ ID NO:4.

[0076] In certain embodiments, said second polypeptide of the dimeric protein complex comprises: a linker N-terminal to the C-terminal fragment of the C4bp β chain and C-terminal to the second functional component; and A linker between the C-terminal side of the C-terminal fragment of the C4bp β chain and the N-terminal side of the fourth functional component, preferably the linker is a flexible linker, for example a flexible linker comprising, consisting essentially of or consisting of an amino acid sequence as defined by SEQ ID NO:7, or a spacer, such as a spacer comprising, consisting essentially of or consisting of an amino acid sequence as defined by SEQ ID NO:4.

[0077] A dimeric protein complex as taught herein may be monofunctional, bifunctional, trifunctional, or tetrafunctional, depending on the amounts of different functional components present in the dimeric protein complex.

[0078] In certain embodiments, the first, second, third and fourth functional components are identical. For example, the first, second, third and fourth functional components can be functional component A. Thus, in such embodiments, the first and second polypeptides of the dimeric protein complex taught herein each comprise functional component AA. Such dimeric protein complexes are sometimes referred to herein as monofunctional homotetraboro.

[0079] In certain embodiments, the first and second functional components are identical, the third and fourth functional components are identical, and the first and third components are not identical (or different). For example, the first and second functional components may be functional components A, and the third and fourth functional components may be functional components B, where A and B represent different functional components. Thus, in such embodiments, the first and second polypeptides of the dimeric protein complex taught herein each comprise functional components AB. Such dimeric protein complexes may be referred to herein as bifunctional homotetrabolo. Examples of such bifunctional homotetrabolo include dimeric protein complexes that comprise complementary scFvs against a given strain of Pseudomonas. The term "different" when referring to functional components includes differences not only in their primary amino acid sequence but also in post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulfonation, glutathionylation, acetylation, oxidation of methionine to methionine sulfoxide or methionine sulfone, and the like.

[0080] In certain embodiments, at least three of the first, second, third and fourth functional components are different (meaning that two of the first, second, third and fourth functional components may be the same). For example, the first and second functional components may each be functional component A, the third functional component may be functional component B, and the fourth functional component may be functional component C, where A, B and C represent different functional components. For example, the third and fourth functional components may each be functional component A, the first functional component may be functional component B, and the second functional component may be functional component C, where A, B and C are different functional components. Such dimeric protein complexes may be referred to herein as trifunctional heterotetraboro (Figure 1).

[0081] In certain embodiments, the first, second, third and fourth functional components are all different. For example, the first functional component may be functional component A, the first functional component may be functional component B, the third functional component may be functional component C, and the fourth functional component may be functional component D, where A, B, C and D represent different functional components. Such dimeric protein complexes are sometimes referred to herein as tetrafunctional heterotetraboro.

[0082] In certain embodiments, the first, second, third and / or fourth functional components can be selected independently of each other. The term "functional component" as used herein refers to an element, more particularly an amino acid sequence, capable of exerting a given function. In the present context, three main functions are distinguished: effector function, targeting function, and tracking function, but the functional components used in the present invention are not limited thereto. The skilled person will select a functional component that functions for the desired application of the dimeric protein complex taught herein. For example, a functional component may be an enzyme (e.g., SmaseD from the venom from Loxoceres intermedia (Li)), an enzyme activity regulator, a receptor ligand, a hapten, an antigen, an antibody, an antibody fragment (e.g., scFv or V). HH), a prophylactic or therapeutic agent (e.g., an immune response regulator (e.g., FHR1 (i.e., FHR-1) or a fragment thereof, e.g., SCR3, 4 and 5 derived from FHR1), an immune checkpoint inhibitor, an immunotoxin, an antioxidant, an antibiotic, a growth factor, a hormone, a cytokine (e.g., IL-2, IL-15, INFγ or TNFα), an oncolytic or cytotoxic agent such as a toxin or a protoxin, a neuromediator, an antimicrobial agent such as an antiviral, antibacterial, or antiparasitic agent, an anti-neoplastic agent, or any other therapeutic or prophylactic agent for any purpose), a metal ion of a heavy metal, a lanthanide, a radioactive nucleotide, a chemiluminescent molecule, a fluorescent molecule, or a receptor such as a monochain receptor or a proteinaceous drug receptor. In certain embodiments, the first, second, third and fourth functional components are not tags, as described elsewhere herein.

[0083] In certain embodiments, at least one of the first, second, third and / or fourth functional components is a protein or polypeptide. For example, the first, second, third and / or fourth functional components are an enzyme, an enzyme activity modulator, a receptor ligand, a hapten, an antigen, an antibody, an antibody fragment (e.g., scFv or VFv). H H), prophylactic or therapeutic agents (such as immune response regulators, immune checkpoint inhibitors, immunotoxins, antioxidants, antibiotics, growth factors, hormones, cytokines, oncolytic and cytotoxic agents such as toxins and protoxins, neuromediators, antimicrobial agents such as antivirals, antibacterials, antiparasitics, antitumor agents, or any other therapeutic or prophylactic agent for any other purpose) or receptors such as monochain receptors or proteinaceous drug receptors. The receptor may be a soluble receptor or a fragment thereof. In certain embodiments, the first, second, third and / or fourth functional components, preferably the first and second functional components, more preferably the first, second, third and fourth functional components, are proteins or polypeptides, e.g., functional proteins or polypeptides.

[0084] In certain embodiments, the first, second, third and fourth functional components are proteins or polypeptides, preferably selected from the group consisting of a binding domain (e.g., an antigen recognition domain), a receptor (e.g., a soluble receptor), a recombinant viral structural protein (such as a recombinant viral structural protein for a virus-like particle (VLP)), an oncolytic agent, a cytotoxic agent, a cytokine, a receptor-binding peptide (e.g., a ligand), or a monomeric Fc, and more preferably selected from the group consisting of a binding domain (e.g., an antigen recognition domain), a receptor (e.g., a soluble receptor), a recombinant viral structural protein (such as a recombinant viral structural protein for a virus-like particle (VLP)), an oncolytic agent, a cytotoxic agent, a cytokine, a receptor-binding peptide (e.g., a ligand), or a monomeric Fc. More preferably, the binding domain is a protein or polypeptide selected from the group consisting of a binding domain (e.g., an antigen recognition domain), a recombinant viral structural protein (such as a recombinant viral structural protein for a virus-like particle (VLP)), a cytotoxic agent, a cytokine, a receptor-binding peptide (e.g., a ligand), or a monomeric Fc, and even more preferably, the binding domain is a protein or polypeptide selected from the group consisting of a binding domain (e.g., an antigen recognition domain), a recombinant viral structural protein (such as a recombinant viral structural protein for a virus-like particle (VLP)), a cytotoxic agent, a cytokine, a receptor-binding peptide (e.g., a ligand), or a monomeric Fc.

[0085] The inventors have found that SCRs 3-5 of factor H related protein 1 (FHR1) are sufficient to compete with factor H (FH), as described elsewhere herein. Thus, in a particular embodiment, the first, second, third or fourth functional component, preferably said third and said fourth functional component, comprises, consists essentially of or consists of short consensus repeats 3, 4 and 5 (SCRs 3, 4 and 5) from factor H related protein 1 (FHR1), preferably SCRs 3, 4 and 5 from human FHR1.

[0086] In a particular embodiment, the first, second, third or fourth functional component, preferably said third and said fourth functional component, has an amino acid sequence having at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 50 (human SCR3), at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90% or 100% sequence identity to SEQ ID NO: 51 (human SCR4). and an amino acid sequence having at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52 (human SCR5). In certain embodiments, the first, second, third or fourth functional component, preferably said third and said fourth functional component, comprises, consists essentially of or consists of an amino acid sequence having at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 53 (human SCR3-5).

[0087] FHR1 (SCR3-5) competes with hijacked and bound factor H (FH) for C3b and controls FH-mediated complement breakdown. As a result, dysregulation of FH locally triggers the alternative complement pathway (AP) without activating the classical complement pathway. Ultimate MAC formation destroys target cells to which the dimeric protein complex is directed, such as pathogenic cells like bacteria, resulting in cell lysis. Thus, in certain embodiments, when the third and fourth functional components of the dimeric protein complex taught herein comprise, consist essentially of, or consist of SCRs 3, 4 and 5 of FHR1, the first and second functional components of said dimeric protein complex preferably comprise functional components with a targeting function, more preferably a binding domain (e.g., an antigen recognition domain) that binds a pathogen or tumor cell, preferably a binding domain (e.g., an antigen recognition domain) that binds a pathogen that evades human complement attack, such as scFv PsI that binds Pseudomonas aeruginosa as described elsewhere herein.

[0088] In certain embodiments, two of the first, second, third or fourth functional components comprise SCR3, 4 and 5 from FHR1, wherein two of the first, second, third or fourth functional components comprise a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA, a TAA, a bacterial antigen, a viral antigen, a virus-associated antigen or a fungal antigen.

[0089] In a particular embodiment, the first, second, third or fourth functional component, preferably said third and said fourth functional component, does not comprise short consensus repeats 1 and 2 (SCR1 and 2) derived from Factor H related protein 1 (FHR1), preferably from human FHR1.

[0090] In certain embodiments, the third and / or fourth functional component (i.e., a functional component located N-terminal to the C-terminal fragment of the C4bp β chain) is a protein or peptide with a biological function / activity located at its N-terminus, such as an active receptor capable of capturing a ligand (e.g., an enzyme), or an antibody fragment, preferably an scFv (e.g., in a VH-VL or VL-VH orientation) or a VFv. H H, and more preferably scFv.

[0091] In certain embodiments, the third and / or fourth functional component (i.e., a functional component located C-terminal to the C-terminal fragment of the C4bp β chain) is a protein or peptide with a biological function / activity located at its N-terminus, such as an active receptor capable of capturing a ligand (e.g., an enzyme), or an antibody fragment, preferably an scFv (e.g., in a VH-VL or VL-VH orientation) or a VFv. H More preferably, the protein or peptide is a C-terminal fragment of the C4bp β-chain, and more preferably an scFv, wherein the protein or peptide is linked at its N-terminus to the C-terminus of the C-terminal fragment of the C4bp β-chain. Thus, in such an embodiment, the C-terminus of the protein or peptide having a biological function / activity located at its N-terminus is located at the C-terminus of the first and / or second polypeptide of the dimeric protein complex taught herein. (SGGGGS) n A linker such as (SEQ ID NO: 7), where n is an integer between 1 and 11, may be present between the C-terminus of the C-terminal fragment of the C4bp β chain and the N-terminus of a protein or peptide having a biological function / activity located at its N-terminus. Thus, in such an embodiment, the N-terminus of the protein or peptide having a biological function / activity located at its N-terminus is freely accessible in its "native", unbound form, but is now fused to another protein or polypeptide.

[0092] In certain embodiments, the first, second, third and fourth functional components are not themselves multimeric complexes, but may complex with each other to form a multimeric complex.

[0093] In certain embodiments, the first, second, third and fourth functional components may be encoded by tandem associated genes separated by a linker or spacer.

[0094] In certain embodiments, the functional components, such as the first and second functional components, comprise, consist essentially of, or consist of at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids.

[0095] In certain embodiments, the first and second polypeptides comprise a protein or polypeptide (e.g., comprising one or more functional components) having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 amino acids C-terminal to the C-terminal fragment of the C4bp β chain.

[0096] In certain embodiments, the functional components, such as the first and second functional components, do not comprise, do not consist essentially of or do not consist of a tag, preferably do not consist of a tag. In certain embodiments, the functional components, such as the first and second functional components, do not comprise, do not consist essentially of or do not consist of a protein purification tag, such as a FLAG tag, a His tag, an HA tag or a Myc tag, preferably do not consist of a protein purification tag.

[0097] In certain embodiments, the first, second, third and / or fourth functional components, preferably the first and second functional components, are not the C4bp α or β chain or fragments thereof.

[0098] In a particular embodiment, the first, second, third and / or fourth functional component, preferably the first and second functional components, are polypeptides or proteins that are heterologous to the C-terminal fragment of the C4bp β chain. By a functional component being heterologous to the C4bp β chain, it is meant that it is not naturally associated with the C-terminal fragment of the C4bp β chain to which it is bound.

[0099] In certain embodiments, the first, second, third and / or fourth functional components comprise, consist essentially of or consist of an enzyme, an enzyme activity modulator, a receptor ligand, a hapten, an antigen, an antibody, an antibody fragment, a prophylactic or therapeutic agent, an oncolytic agent, a cytotoxic agent, a cytokine, a recombinant viral structural protein, an antibody-like scaffold, an extracellular domain of a viral envelope protein, a cognate extracellular domain of a receptor or ligand for an antigen (e.g. a signaling molecule) or an antigen-binding portion of said receptor or ligand, a soluble receptor, or a synthetic receptor, preferably an enzyme, an enzyme activity modulator, a receptor ligand, a hapten, an antigen, an antibody, an antibody fragment, a prophylactic or therapeutic agent, a cytotoxic agent, a cytokine, a recombinant viral structural protein, an antibody-like scaffold, an extracellular domain of a viral envelope protein, a cognate extracellular domain of a receptor or ligand for an antigen (e.g. a signaling molecule) or an antigen-binding portion of said receptor or ligand, a soluble receptor, or a synthetic receptor.

[0100] Unless otherwise clear from the context, reference herein to a peptide or polypeptide or protein may generally also include fragments and / or variants of that peptide, polypeptide or protein. When the present specification refers to or includes fragments and / or variants of proteins, polypeptides or peptides, this preferably refers to variants and / or fragments that are "functional", i.e. that at least partially retain the biological activity or intended functionality (e.g. antigen recognition properties) of the respective protein, polypeptide or peptide. Preferably, functional fragments and / or variants may retain at least about 20%, such as at least about 30%, or at least about 40%, or at least about 50%, such as at least about 60%, more preferably at least about 70%, such as at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95%, or even about 100% or more of the intended biological activity or functionality compared to the corresponding protein, polypeptide or peptide. For example, the extracellular domain of a viral envelope protein may also be a fragment thereof, a soluble receptor may also be a fragment thereof, or a synthetic receptor may also be a fragment thereof.

[0101] In certain embodiments, the first, second, third and / or fourth functional components comprise, consist essentially of or consist of a binding domain (e.g., an antigen recognition domain). The term "antigen" or "Ag" as used herein is defined as a molecule capable of binding to an antigen recognition domain, such as capable of binding to an antibody or a receptor (e.g., a T cell receptor). Antigens are found on the cell surface of target cells, such as pathogens or tumor cells. The term "antigen recognition domain" or "binding domain" or "antigen-specific binding domain" as used herein refers to a domain of a functional component that binds to a specific target molecule. An antigen recognition domain may bind to a single target molecule or to multiple target molecules. Binding domains include naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partners for a target molecule. Binding domains are not limited to antibodies or fragments thereof, but should be considered to include other molecules such as receptors. The present inventors have demonstrated that the C4bp α-chain, preferably the short consensus repeats 1 and 2 (SCR1-2) from the human C4bp α-chain (also known as complement control proteins 1 and 2 (CCP1-2)) can be used as a binding domain (e.g., antigen recognition domain) to bind to bacterial pathogens (e.g., Bordetella pertussis & burgdorferi, Haemophilus influenza, Moraxella catarrhalis, Neisseria gonorrhoeae & meningitidis, Streptococcus pneumoniae & pyogenes, Yersinia enterocolitica, Staphylococcus aureus) as well as fungi (Candida dubliensis, Aspergillus fumigatus & We have found that the dimeric protein complexes taught herein can be used to target a wide variety of pathogens, including those that evade complement-mediated killing by employing C4bp soluble complement control proteins, including, but not limited to, ...

[0102] In certain embodiments, the binding domain (e.g., antigen recognition domain) comprises, consists essentially of, or consists of SCR1 and SCR2 (also known as CCP1 and CCP2) from the C4bp alpha chain, preferably the human C4bp alpha chain (e.g., the human C4bp alpha chain having Uniprot accession number P04003.2).

[0103] In certain embodiments, the binding domain (e.g., antigen recognition domain) comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 54 (SCR1) and an amino acid sequence having at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55 (SCR1). In certain embodiments, the binding domain (e.g., antigen recognition domain) comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:56 (SCR1-2).

[0104] In certain embodiments, the binding domain (e.g., the antigen recognition domain) does not include CCP3, CCP4, CCP5, CCP6, CCP7, and CCP8, ... of the C4bp α chain, preferably the human C4bp α chain (e.g., the human C4bp α chain having Uniprot accession number P04003.2).

[0105] In certain embodiments, the antigen recognition domain (e.g., an extracellular antigen recognition domain) is derived from an antibody or an antibody fragment. The term "antibody" is used herein in the broadest sense and generally refers to any immunological binding agent, such as a whole antibody, including but not limited to a chimeric antibody, a humanized antibody, a human antibody, a recombinant antibody, a transgenic antibody, a grafted antibody, a single chain antibody, or the like, or any fusion protein, conjugate, fragment, or derivative thereof, that contains one or more domains that selectively bind to an antigen of interest. Thus, the term antibody includes whole immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, or any immunologically effective fragment thereof. Thus, the term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., bivalent, trivalent or higher) and / or multispecific antibodies (e.g., bivalent or higher specific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity (e.g., the ability to specifically bind to an antigen of interest), as well as multivalent and / or multispecific complexes of such fragments. The term "antibody" includes not only antibodies produced by methods including immunization, but also any polypeptide, e.g., recombinantly expressed polypeptide, that has been engineered to include at least one complementarity determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Thus, the term applies to such molecules, whether produced in vitro, in cell culture, or in vivo. The term "antibody fragment" or "antigen-binding site" includes a portion or region of a full-length antibody, typically the antigen-binding or variable domain thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, Fv, scFv fragments, V, and VFv fragments. H Domain, V L Domain, V HThese include single domain (sd) Fvs, such as H domains, diabodies, linear antibodies, single chain antibody molecules, particularly heavy chain antibodies, and multivalent and / or multispecific antibodies formed from antibody fragments, such as dibodies, tribodies, and multibodies. The designations Fab, Fab', F(ab')2, Fv, scFv, etc. above are intended to have their established meanings in the art. Naturally occurring full-length antibodies are immunoglobulin molecules that contain two heavy (H) chains and two light (L) chains linked by disulfide bonds. The amino-terminal portion of each chain contains a variable region of about 100-110 amino acids that is primarily responsible for antigen recognition via the complementarity-determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector functions.

[0106] The CDRs are interspersed with more conserved regions called framework regions (FRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are called "LCDR1, LCDR2, and LCDR3" and the three CDRs of the heavy chain are called "HCDR1, HCDR2, and HCDR3". The CDRs contain most of the residues that form specific interactions with the antigen. The numbering and positioning of the CDR amino acid residues within the LCVR and HCVR regions follows the well-known Kabat numbering convention. This refers to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Ann. NYAcad. Sci. 190:382-93 (1971); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). The positioning of the CDRs in the variable regions of an antibody follows the Kabat numbering, or simply "Kabat".

[0107] Light chains are classified as kappa or lambda and are characterized by a particular constant region as known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies are further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4. Each heavy chain type is characterized by a particular constant region whose sequence is well known in the art. In certain embodiments, the antibody may be of any of the IgA, IgD, IgE, IgG and IgM classes, preferably of the IgG class.

[0108] In certain embodiments, the antigen recognition domain (eg, an extracellular antigen recognition domain) comprises, consists essentially of, or consists of the antigen binding region of an antibody or antibody fragment.

[0109] An antibody or antibody fragment not only recognizes a specific target, such as a specific receptor, enzyme, or toxin, but is also able to activate (ie, agonize) or inhibit (ie, antagonize) that specific target. For example, if one or more of the functional components comprises, consists essentially of, or consists of an scFv anti-SmaseD from the venom of Loxoceres intermedia (Li), the dimeric protein complex may bind to SmaseD and inhibit its activity. For example, when one or more of the functional components comprises, consists essentially of, or consists of SmaseD from the venom of Loxosceles intermedia (Li), and one or more of the functional components comprises an antigen-binding moiety (e.g., scFv anti-HER2) that targets cancer cells, the dimeric protein complex binds to the target cells and kills them by accumulating SmaseD on their cell surface, thus exerting tumor suppressor activity. SmaseD converts sphingomyelin to ceramide-1-phosphate. In situ generation of ceramide-1-phosphate by SmaseD enzymatic activity alters the lateral structure and morphology of the target membrane, which is considered a tumor suppressor lipid.

[0110] The term "antigen-binding portion" or "antigen-binding region" refers to one or more fragments of an antibody, such as a particular site, portion, domain or stretch of amino acid residues that retains the ability to specifically bind to an antigen of interest. It has been shown that the antigen-binding function of an antibody may be carried by fragments of a full-length antibody. These specifically bind to two or more different antigens in a bispecific, dual specific or multispecific format. Examples of binding fragments encompassed by the term "antigen-binding site" of an antibody include (i) Fab fragments, VL , V H , C L and CHI domains; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) V H and an Fd fragment consisting of a CHI domain; (iv) a V of a single arm of an antibody. L and V H (v) a dAb fragment comprising a single variable domain (Ward et al., Nature, 341: 544-546 (1989); PCT publication WO 90 / 05144); and (vi) an isolated complementarity determining region (CDR). In addition, the two domains of the Fv fragment, V L Although VH and VH are encoded by separate genes, they can be synthesized using recombinant methods. L and V H The domains can be linked by a synthetic linker that allows them to be made into a single protein chain that pairs to form a monovalent molecule (known as single-chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are defined as those consisting of a V H Domain and V LBivalent, bispecific antibodies in which the domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to pair with complementary domains on another chain, forming two antigen-binding sites (Holliger, et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). Such antibody-binding moieties are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5). Also, the term "sequence" as used herein (e.g., "variable domain sequence," "V H A reference to a sequence (such as an amino acid sequence) or a nucleic acid sequence (such as a "protein sequence") should generally be understood to include both the related amino acid sequence as well as the nucleic acid or nucleotide sequence encoding it, unless the context requires a more restrictive interpretation.

[0111] In certain embodiments, the antigen-binding region of the antibody or antibody fragment specifically binds to an antigen of interest, such as a TSA or TAA, as described elsewhere herein. The term "specifically binds" means that an agent (also referred to herein as "binding agent" or "specific binding agent") binds to one or more desired targets (e.g., peptides, polypeptides, proteins, nucleic acids, or cells) to the substantial exclusion of other random or unrelated entities, and optionally to the substantial exclusion of other structurally related molecules. The term "specifically binds" does not necessarily require that an agent binds only to its intended target. For example, an agent may have at least about 2-fold, preferably at least about 5-fold, more preferably at least about 10-fold, even more preferably at least about 25-fold, even more preferably at least about 50-fold, even more preferably at least about 100-fold, or at least about 10 ... 4 times, or at least about 10 5 times, or at least about 10 6 A substance can be said to specifically bind to a target of interest if its affinity for the substance is greater than or equal to its affinity for a non-target by 2-fold or more.

[0112] The bond or interaction between an agent and its intended target may be covalent (i.e., mediated by one or more chemical bonds involving the sharing of electron pairs between atoms) or, more typically, non-covalent (i.e., mediated by non-covalent forces such as, for example, hydrogen bridges, dipole interactions, van der Waals interactions, etc.). Preferably, the agent is A ≧ 1×10 6 M -1 , more preferably K A ≧ 1×10 7 M -1 , more preferably K A ≧ 1×10 8 M -1 , more preferably K A ≧ 1×10 9 M -1 , and even more preferably, K A ≧ 1×10 10 M -1 Or K A ≧ 1×10 11 M-1 , the affinity constant of such a bond (K A ) which binds or interacts with its intended target, where K A = [A_T] / [A][T], where A represents the drug and T represents the intended target. A Determination of can be performed by methods known in the art, for example, using equilibrium dialysis and Scatchard plot analysis. In certain embodiments, the antigen recognition domain (e.g., the extracellular antigen recognition domain) is an scFv (e.g., V H -V L Or V L -V H Orientation) or V H H, preferably an antibody fragment such as an scFv, comprising, consisting essentially of, or consisting of a protein or peptide whose biological function / activity is located at its N-terminus.

[0113] In a preferred embodiment, the antigen recognition domain (eg, an extracellular antigen recognition domain) comprises, consists essentially of, or consists of a single chain variable fragment (scFv) of an antibody.

[0114] In further embodiments, the antigen recognition domain (e.g., an extracellular antigen recognition domain) can comprise, consist essentially of, or consist of a bivalent scFv. In a dimeric protein complex as taught herein that includes di-scFvs, the two scFvs specific for each antigen are represented by two V H Area and two V L The domains are linked together to produce a single peptide chain resulting in a tandem scFv as described in Xiong, CY et al., 2006, Protein Engineering Design and Selection 19 (8): 359-367; Kufer, P. et al., 2004, Trends in Biotechnology 22 (5): 238-244. The scFv may be obtained using standard recombinant DNA techniques. For example, the scFv can be prepared by isolating the coding sequence from an antibody producing hybridoma, identifying the V chain type, and designing the nucleic acid encoding the scFv, as described in Koksal H. et al., 2019, Antibody Therapeutics, 2(2):56-63. The scFv can be prepared by isolating the coding sequence from an antibody producing hybridoma, identifying the V chain type, and designing the nucleic acid encoding the scFv. L Sequence, linker peptide, and V H Contains the sequence V L The sequence, the linker peptide, and the V H or the V L The sequence, the linker peptide, and the V H Array, where V L The sequence is located on the N-terminal side of the linker peptide, and the linker peptide is V H Located at the N-terminus of the sequence. For example, V L and V H Different scFv designs are possible in which the positions of the sequences are swapped.

[0115] In certain embodiments, the functional component may comprise, consist essentially of, or consist of an scFv, such as: scFv specifically binding to NKG2D. The scFv specifically binding to NKG2D is an RTX hybrid (V) derived from a whole IgG4 as described in patent US 9.127.064-B2, which is incorporated herein by reference. L -Linker-V H ) codon-optimized human MS anti-NKG2D scFv spanning between the Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG) restriction sites. Preferably, the scFv that specifically binds to NKG2D comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO:8 and / or the nucleic acid sequence defined by SEQ ID NO:9. scFv that specifically binds to NKG2A. The scFv that specifically binds to NKG2A is the Z199 codon-optimized (V199) derived from the Z199 humanized anti-human NKG2A monoclonal antibody described in International Patent Application WO2009092805, which is incorporated herein by reference. H -Linker-V L ) scFv anti-human NKG2A, which is a non-competitive antagonist of the CD94 / NKG2A receptor spanning between the Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG) restriction sites. Preferably, the scFv that specifically binds to NKG2A comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO: 10 and / or the nucleic acid sequence defined by SEQ ID NO: 11. KIR-specific scFv. KIR-specific scFv is a codon-optimized (V H -Linker-V L ) scFv, spanning between the restriction sites of Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG). Preferably, the scFv that specifically binds to KIR comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO: 12 and / or the nucleic acid sequence defined by SEQ ID NO: 13. An scFv that specifically binds to SLAMF7. The scFv that specifically binds to SLAM7 may be an scFv derived from elotuzumab:RTX-hybrid inversion (V L -Linker-V H) The codon-optimized humanized IgG1-derived SLAMF7 (anti-CD319) scFv spans between the Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG) restriction sites. Preferably, the scFv that specifically binds to SLAMF7 comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO: 14 and / or the nucleic acid sequence defined by SEQ ID NO: 15. The scFv that specifically binds to the CD20 antigen is a codon-optimized (V) fragment derived from rituximab (RTX) that specifically binds to the CD20 antigen and spans the Bgl2 (A / GATCT, amino acid RS) and BspE1 (T / CCGGA, amino acid SG) restriction sites. L -Linker-V H The scFv that specifically binds to CD20 may be a codon-optimized (V) scFv derived from ofatumumab (OFA) scFv pseudo-IgG that specifically binds to CD20 spanning between the Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG) restriction sites. H -Linker-V L ) scFv that specifically binds to CD20. Preferably, the scFv comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO: 16 or 18 and / or the nucleic acid sequence defined by SEQ ID NO: 17 or 19. HER2-specific scFv. The HER2-specific scFv is a codon-optimized fragment (V) derived from trastuzumab that spans the restriction sites Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG). H -Linker-V LThe scFv that specifically binds to HER2 can be a codon-optimized 2D3 V scFv that specifically binds to HER2, spanning the restriction sites Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG), as described in International Patent Application WO2009068625-A2, which is incorporated herein by reference. H The scFv that specifically binds to HER2 can be the codon-optimized 47D5 V scFv that specifically binds to HER2, spanning between the Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG) restriction sites, as described in International Patent Application WO2009068625-A2, which is incorporated herein by reference. H H. Preferably, the scFv that specifically binds to HER2 comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO: 20, 22, 24 and / or the nucleic acid sequence defined by SEQ ID NO: 21, 23, or 25. The scFv that specifically binds to P. aeruginosa Psl is a codon-optimized scFv that specifically binds to P. aeruginosa Psl (V L -Linker-V H ) scFv, comprising the sequence as defined in SEQ ID NO: 9 of International Patent Application WO2017095744 A1, which is incorporated herein by reference, and spanning between the Bgl2 (A / GATCT, amino acids RS) and the BspE1 (T / CCGGA, amino acids SG) restriction sites. Preferably, the scFv that specifically binds to Psl of P. aeruginosa comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO: 26 and / or the nucleic acid sequence defined by SEQ ID NO: 27. scFv that specifically binds to P. aeruginosa PcrV. The scFv that specifically binds to P. aeruginosa PcrV is a codon-optimized (V) scFv that specifically binds to P. aeruginosa PcrV spanning the Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG) restriction sites, as described in International Patent Application WO2017095744 A1, which is incorporated herein by reference. L -Linker-V H ) scFv or a codon-optimized RTX hybrid inversion (V) derived from panobacumab (Aridis Pharmaceuticals, AR-101) that specifically binds to Pseudomonas aeruginosa and spans between the Bgl2 (A / GATCT, amino acid RS) and BspE1 (T / CCGGA, amino acid SG) restriction sites. L -Linker-V H ) scFv. Preferably, the scFv that specifically binds to PcrV of P. aeruginosa comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO: 28 or 30 and / or the nucleic acid sequence defined by SEQ ID NO: 29 or 31. As described in Karim-Silva, S., et al., Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential. Toxins (Basel), 2020. 12(4) or Karim-Silva, S., et al., Generation of recombinant antibody fragments with toxin-neutralizing potential in loxoscelism. Immunol Lett, 2016. 176: p. 90-6, the scFv that specifically binds to SMaseD is SMaseD (scFvLi7) (V H -Linker-V L) may be an scFv that specifically binds to SMaseD. As described in Karim-Silva, S., et al., Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential. Toxins (Basel), 2020. 12(4) or Karim-Silva, S., et al., Generation of recombinant antibody fragments with toxin-neutralizing potential in loxoscelism. Immunol Lett, 2016. 176: p. 90-6, the scFv that specifically binds to SMaseD may be an scFv anti-SMaseD (scFvLi7) (V L -Linker-V H ), spanning between the Bgl2 (A / GATCT, amino acid sequence RS) and BspE1 (T / CCGGA, amino acid sequence SG) restriction sites and containing the amino acid sequence QIVLSQSP (SEQ ID NO: 5) derived from RTX immediately C-terminal to the signal peptide. Preferably, the scFv that specifically binds SmaseD comprises, consists essentially of, or consists of the amino acid sequence defined by SEQ ID NO: 58 (codon-optimized mouse scFv-Li7(VH-linker-VL) anti-SmaseD from Loxoceles intermedia (Li)) and / or the nucleic acid sequence defined by SEQ ID NO: 57 (codon-optimized mouse scFv-Li7(VH-linker-VL) anti-SmaseD from Loxoceles intermedia (Li)).

[0116] It is known in the art that NK activation is improved when an NK activating component (e.g., an antigen recognition domain that specifically binds and antagonizes an activating NK cell receptor) is present as a dimer. For example, the NKG2D receptor consists of a homodimer of two disulfide-linked transmembrane proteins. Each NKG2D monomer interacts with a different surface of the ligand MIC-A. A dimeric protein complex containing multiple, e.g., two anti-NKGD2 scFvs, binds to the NKG2D dimeric receptor with much higher efficacy than a dimeric protein complex containing only one anti-NKGD2 scFv, resulting in greater NK activation. Alternatively, a dimeric protein complex containing two different scFvs that recognize two different NK receptors (e.g., NKG2D and SLAMF7) may be used, and these scFvs can bind to two NK receptors simultaneously and activate NK cells more efficiently.

[0117] In certain embodiments, the first and second functional components comprise, consist essentially of, or consist of a binding domain (e.g., an antigen recognition domain) that specifically binds to an activating NK cell receptor, such as natural cytotoxicity receptor (NCR), natural killer group 2, member D (NKG2D), NKG2A, killer immunoglobulin-like receptor (KIR), or SLAMF7, and the third and fourth functional components comprise, consist essentially of, or consist of sIL15Rα. In a more particular embodiment, the first and second functional components comprise, consist essentially of, or consist of scFv anti-NKG2A or scFv anti-KIR, and the third and fourth functional components comprise, consist essentially of, or consist of sIL15Rα. Preferably, the scFv anti-NKG2A is derived from the specific monoclonal antibody (mAb) Z199. As described in Carretero et al., The CD94 and NKG2-A C-type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules. Eur J Immunol 1997 27:563, Z199 is a non-competitive agonist of the human CD94 / NKG2A receptor. Such dimeric protein complexes allow for a dual approach to activating NK cells: activation by the IL15 pathway and activation by NK checkpoint inhibitors presented by two scFvs against NKG2A or KIR. Thus, such dimeric protein complexes act as NK superagonists. Here, an approach equivalent to that of checkpoint inhibitors for CD8 T cells is used for dimeric protein complexes for NK cells. Through the scFv anti-NKG2A site, the dimeric protein complexes inhibit the NKG2A pathway, which is a pathway for NK cell exhaustion. The IL15 portion exhibits selective NK activation.

[0118] The dimeric protein complex may be used to crosslink NK cells with unwanted cells (e.g., tumor cells) or pathogenic microorganisms, such as bacteria (e.g., Pseudomonas aeruginosa), viruses (e.g., SARS-CoV2) or fungi (e.g., Aspergillus) to kill the unwanted cells or pathogenic microorganisms. For such purposes, the dimeric protein complex may exhibit (i) a binding domain (e.g., an antigen recognition domain) that specifically binds to an activating NK cell receptor, such as an scFv against NKG2D or SLAM7, and (ii) a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA, TAA, or an antigen of a pathogenic microorganism (e.g., a bacterial antigen, a viral antigen, a virus-associated antigen, or a fungal antigen). For example, the dimeric protein complex may be used to cross-link NK cells and P. aeruginosa and kill P. aeruginosa. For such purpose, the dimeric protein complex may present (i) two different monomeric NK-activating scFvs against two NK natural cytotoxicity receptors (NCRs), NKG2D (CD314) and SLAMF7 (CS1, CD319, CRACC), and (ii) a dimeric PcrV or Psl scFv anti-P. aeruginosa as a therapeutic to activate NK cells against P. aeruginosa. The scFv anti-NKG2D acts as an agonist of the NKG2D / DAP10 / Grb2 / VAv1 / PI3K / Akt pathway, and the scFv anti-SLAMF7 derived from elotuzumab acts as an agonist of the SLAMF7 / ITSM / PLC / Ca pathway. ++ / ERK pathway agonists, both of which activate NK cells and enhance ADCC.

[0119] Thus, in a particular embodiment, the first and second functional components comprise, consist essentially of or consist of a scFv anti-PcrV of P. aeruginosa or a scFv anti-Psl of P. aeruginosa, the third functional component comprises, consist essentially of or consist of a scFv anti-NKG2D and the fourth functional component comprises, consist essentially of or consist of a scFv anti-SLAM7, as described in International Patent Application WO2017095744 A1. Such a dimeric protein complex includes a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA, a TAA, a bacterial antigen, a viral antigen or a virus-related antigen, or a fungal antigen, and further includes two scFvs (e.g., V H -V L Or V L -V H Orientation) or V H Any dimeric protein complex containing H, each having a different specificity for an activating NK cell receptor, is referred to herein as a "dimeric protein complex." Tri -specific K iller E The NK activation pathway is also sometimes referred to as "TriKE," which stands for "NK activators." By utilizing two distinct pathways for NK activation, the overall NK activation capacity of TriKE is dramatically improved.

[0120] In addition, the antibody may further comprise a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA, a TAA, a bacterial antigen, a viral antigen or a virus-related antigen, or a fungal antigen, and further comprises one scFv (e.g., a V H -V L Or V L -V H V with specificity for activating NK cell receptors H A dimeric protein complex further comprising H is referred to herein as " Bi -specific K iller EThey are also sometimes called "BiKE" as "ngagers". When all functional components in a dimeric protein complex are scFvs, the dimeric protein complex taught herein may contain tetravalents of said scFvs, resulting in blocking scFvs with high affinity. An example of such a dimeric protein complex includes a dimeric protein complex in which the first and second functional components, or the first, second, third and fourth functional components, comprise the scFv anti-sphingomyelinase D (SMase D) from the spider venom Loxosceles intermedia (scFvLi7).

[0121] In a preferred embodiment, the antigen recognition domain (e.g., an extracellular antigen recognition domain) is a heavy chain antibody (V H H) comprises, consists essentially of or consists of a single domain variable fragment of said single domain variable fragment. The term "Nanobody" or "Nanobodies" is a trademark of Ablynx NV (Belgium). The term "Nanobody" is well known in the art and, as used herein in its broadest sense, refers to: (1) a V-type antibody of a naturally occurring heavy chain antibody, preferably a heavy chain antibody of camelid origin; H (2) by isolating the naturally occurring V H (3) by expression of a nucleotide sequence encoding a naturally occurring V H By "humanization" of the H domain or by such a humanized V H (4) by expression of a nucleic acid encoding a H domain; (5) by expression of a naturally occurring V domain from any animal species, particularly a mammalian species, such as a human; H "Camelization" of a domain, or such camelization V H(5) by "camelization" of a "domain antibody" or "dAb" as described in the art, or by expression of a nucleic acid encoding such a camelized dAb, for the term "dAb" see, e.g., Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490, and Domantis Ltd, e.g. WO 06 / 030220, WO 06 / 003388 and other published patent applications; single domain antibodies or single variable domains can be derived from certain species of sharks (e.g., the so-called "IgNAR domains", see, e.g., International Patent Application WO (6) by using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other known amino acid sequences; (7) by preparing a nucleic acid encoding a Nanobody using known nucleic acid synthesis techniques and then expressing the nucleic acid so obtained; and / or (8) by any combination of one or more of the foregoing. As used herein, "camelids" includes Old World camelids (Camelus bactrianus and Camelus dromaderius) and New World camelids (e.g., Lama paccos, Lama glama, and Lama vicugna).

[0122] The amino acid sequence and structure of a Nanobody can be considered to be composed of, but not limited to, four framework regions or "FR", which are referred to in the art and herein as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively; these framework regions are interrupted by three complementarity determining regions or "CDRs", which are referred to in the art as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. The total number of amino acid residues in a Nanobody can be a region of 110-120, preferably 112-115. It should be noted, however, that Nanobody parts, fragments, analogs or derivatives are not particularly limited in terms of their length and / or size, so long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are preferably suitable for the purposes described herein.

[0123] The heavy chain variable domain (referred to herein as "V") present in a conventional four-chain antibody H domain) present in conventional four-chain antibodies (herein referred to as the “V L To distinguish them from the variable domains found in naturally occurring heavy chain antibodies, the variable domains are referred to as "V H Also called the H domain. H The H domain is isolated V H H domains (as well as naturally occurring V H H domains share these structural and functional properties and have many unique structural and functional properties that make them highly advantageous for use as functional antigen-binding domains or proteins (Nanobodies based thereon, which share these structural and functional properties with H domains). HH domains (which are "designed" by nature to functionally bind antigens in the absence of and without interactions with light chain variable domains) and Nanobodies can function as single, relatively small, functional antigen-binding structural units, domains or proteins. H The H domain is the V domain of a conventional four-chain antibody. H Domain and V L These are distinct from domains, which by themselves are generally not useful as single antigen-binding proteins or domains, but must be somehow linked to provide a functional antigen-binding unit (e.g., as in conventional antibody fragments such as Fab fragments; VL domains covalently linked to VL domains, as described elsewhere herein). H (such as an ScFv fragment consisting of domains). For example, the functional component may be a V H H, e.g. 2D3 V H H or 47D5 V H H, for example, 2D3 V as described in International Patent Application WO2009068625 A2 H H or 47D5 V H Contains H or the V H H, or H It can consist of H.

[0124] In a further embodiment, the antigen-binding region is obtained from a multispecific antibody or antibody fragment (such as a bispecific antibody, trispecific antibody, etc.) that comprises at least two (2, 3, etc.) binding sites, each directed against a different antigen or antigenic determinant. The antigen-binding region may be derived from an antibody or antibody fragment derived from one or more moieties from any animal species, preferably a vertebrate species, including, for example, birds and mammals. The antibody may be, but is not limited to, chicken, turkey, goose, duck, guinea fowl, quail, or pheasant. The antibody may also be, but is not limited to, human, murine (e.g., mouse, rat, etc.), porcine, donkey, rabbit, goat, sheep, guinea pig, monkey (e.g., chinese monkey), camel (e.g., Camelus bactrianus and Camelus dromaderius) containing camel heavy chain antibodies, llama (e.g., Lama paccos, Lama glama, or Lama vicugna) containing camel heavy chain antibodies, or horse.

[0125] In certain embodiments, the antigen-binding region may be derived from a chimeric antibody or chimeric antibody fragment, such as a chimeric antibody or chimeric antibody fragment derived from at least two animal species. More specifically, the term "chimeric antibody" refers to an antibody that comprises heavy and light chain variable region sequences of one species and constant region sequences of another species, such as an antibody having murine heavy and light chain variable regions linked to human, non-human primate, canine, equine, or feline constant regions. Chimeric antibodies are antibodies that contain portions of the heavy and / or light chains that are identical or homologous to corresponding sequences from antibodies from a particular species or class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences from antibodies from another species or class or subclass, and fragments of such antibodies, that exhibit the desired biological activity (see, e.g., US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies are produced by combining DNA encoding a portion of a monoclonal antibody, such as the Fv region of a monoclonal antibody from one species, e.g., mouse or monkey, with antibody-producing DNA from another species, e.g., human.

[0126] In certain embodiments, the antigen-binding region is derived from a fully human antibody or antibody fragment. As used herein, the term "fully human antibody" refers to an antibody whose encoding genetic information is derived from a human. Thus, the term "fully human antibody" refers to an antibody having variable and constant regions derived exclusively from human germline immunoglobulin sequences. Thus, the term "fully human antibody" does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mouse, have been grafted onto human framework sequences.

[0127] In certain embodiments, the antigen-binding region is obtained from a humanized antibody or antibody fragment. More specifically, the term "humanized antibody" refers to antibodies that include heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but with a V H and / or V L Humanized antibodies refer to antibodies in which at least a portion of the sequence has been altered to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is the CDR-grafted antibody, in which non-human CDR sequences have been substituted for human V H and V L sequence and replace the corresponding human CDR sequences. Those skilled in the art will understand that the antigen-binding region derived from an antibody or antibody fragment may contain one or more amino acid deletions, additions and / or substitutions (e.g., conservative substitutions), so long as such modifications maintain binding to the respective antigen. In certain embodiments, the functional component comprises, consists essentially of, or consists of an antibody-like scaffold, an extracellular domain of a viral envelope protein, a cognate extracellular domain of a receptor or ligand for an antigen or an antigen-binding portion of said receptor or ligand, a soluble receptor, or a synthetic receptor.

[0128] In certain embodiments, the functional component comprises one or more antibody-like scaffolds. The term "antibody-like scaffold" as used herein refers to a synthetic or natural binding molecule that has a stable scaffold that holds the molecules together and variable arms that bind to specific targets, thereby mimicking the general structure and function of an antibody. Non-limiting examples of antibody-like scaffolds include designed ankyrin repeat proteins (DARPins), affimers, monobodies, etc. For example, the functional component may include a DARPin directed against HER-2 having the amino acid sequence defined by SEQ ID NO: 2294-2295, or an affibody directed against HER-2 having the amino acid sequence defined by SEQ ID NO: 2297, as described in WO2017172981A2, which is incorporated herein by reference.

[0129] In certain embodiments, the functional component comprises the cognate extracellular domain of a receptor or ligand for an antigen, or an antigen-binding portion of said receptor or ligand. The term "cognate" as used herein with respect to a receptor or ligand refers to a receptor or ligand with which a target molecule preferentially interacts under physiological conditions, or under in vitro conditions that substantially approximate physiological conditions. As used herein, the term "preferentially interacts" is synonymous with "preferentially binds" and refers to an interaction that is statistically significantly greater than a control.

[0130] As used herein for antibodies, the term "antigen-binding portion" or "antigen-binding region" refers to one or more fragments of a receptor or ligand that retain the ability to specifically bind to an antigen.

[0131] The receptor may be a soluble receptor, e.g., a soluble receptor capable of binding a pathogenic microorganism or a toxin. For example, the functional component may comprise a soluble IL-15 receptor alpha chain (sIL15Ralpha) capable of binding IL-15, or a complement receptor type 1 (CR1, CD35). For example, a soluble IL-15 receptor alpha chain having an amino acid sequence as defined by SEQ ID NO: 32 and a nucleic acid sequence as defined by SEQ ID NO: 33, spanning between the Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG) restriction sites of a codon-optimized soluble version of the human IL15 receptor alpha chain that binds human interleukin-15 [UniProtKB - Q13261.1 (I15RA_HUMAN) - domain used: residues 31-96].

[0132] CR1 is a membrane-anchored complement regulatory protein (mCRP). CR1 is the most versatile mCRP. CR1 reduces the deposition of C3b and C4b and inactivates bound C3b / C4b to C3d / C4d. Loss of CR1 expression in podocytes may contribute to complement-mediated injury in the kidney. A dimeric protein complex can be engineered by cloning a gene encoding soluble CR1 upstream of the C-terminal fragment of the C4bp β-chain and a gene encoding a targeting moiety downstream of the C-terminal fragment of the C4bp β-chain. This complex generates a soluble complement inhibitor that selectively accumulates on the target membrane surface, which can locally induce complement activation. Such a complex is also called the "complement switch-off strategy" or protective cell targeting approach. The ligand may be a cytokine, a growth factor, or a ligand that acts as an agonist or antagonist of a receptor that can modulate an immune response in vivo.

[0133] In certain embodiments, the functional component comprises, consists essentially of, or consists of the extracellular domain of a viral envelope protein.

[0134] In certain embodiments, the functional component comprises a synthetic receptor. The term "synthetic receptor" or "recombinant receptor" refers to a receptor that does not exist in nature and is artificially created. For example, the sequence of a polypeptide can be intentionally altered by a human in a laboratory. The functional component is a single chain variable fragment (scFv) of an antibody specific for an antigen or a single domain variable fragment (VFv) of a heavy chain antibody. H H), antibody-like scaffolds, cognate receptors or ligands for the antigen, or antigen-binding portions of said receptors or ligands, or synthetic receptors known to specifically bind to the antigen of interest, the ability of such functional components to bind to an antigen of interest, such as TSAs or TAAs, viral antigens, or virus-associated antigens, can be determined by the binding of scFv or VFv specific for the antigen. H H., antibody-like scaffold, cognate receptor or ligand for the antigen or an antigen-binding portion of said receptor or ligand, soluble receptor, or synthetic receptor.

[0135] In certain embodiments, the binding domain (e.g., antigen recognition domain) specifically binds to a tumor antigen, a pathogen antigen, an activating NK cell receptor, a cytokine, a toxin, or a contaminant. In certain embodiments, the binding domain (e.g., antigen recognition domain) specifically binds to a tumor specific antigen (TSA), a tumor associated antigen (TAA), a bacterial antigen, a viral antigen or a virus associated antigen, a fungal antigen, an activating NK cell receptor, a cytokine, a toxin, or a contaminant.

[0136] As used herein, the term "tumor antigen" refers to an antigenic substance produced by tumor cells that can elicit an immune response in the host. The term "tumor specific antigen" refers to an antigen that is present on tumor cells and not present on other cells. The term "tumor associated antigen" refers to an antigen that is present on tumor cells and normal cells. Non-limiting examples of TSAs and / or TAAs include CD19, CD319 / CS1, ROR1, CD20, CD5, CD7, CD22, CD70, CD30, BCMA, CD25, NKG2D ligand, MICA / MICB, carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), CA-125, MUC-1, CO17-1A, melanoma associated antigen (MAGE), mutant p 53, mutant ras, HER2 / Neu, ERBB2, folate binding protein, GD2, CD123, CD33, CD37, CD30, CD56, c-Met, mesothelin (MSLN), GD3, HERV-K, IL-11Rα, CSPG4, WT-1, EGFRvIII, TRAIL / DR4, VEGFR2, glycoprotein 100 (gp100 / Pme117), NY-BR-1, NY-CO-58 , NY-ESO-1, MART1, 5T4, αvβ6 integrin, B7-H3, B7-H6, CAIX, CD20, CD44, CD44v6, CD44v7 / 8, CD79a, CD79b, CD1 38, CD171, CEA, CSPG4, EGFR, EGFR family, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, glypican-3 (GPC3), HL A-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-1 Examples include 3Rα2, Lambda, Lewis-Y, Kappa, mesothelin, Muc16, NCAM, PRAME, PSCA, PSMA, SSX, Survivin, TAG72, or TEM.

[0137] Protein descriptions for the protein abbreviations used herein can be found in the U.S. government's National Center for Biotechnology Information (NCBI) protein database (http: / / www.ncbi.nlm.nih.gov / ) or the UniProt homepage (https: / / www.uniprot.org / ).

[0138] In a particular embodiment, the TSA or TAA is human epidermal growth factor receptor 2 (HER2). By way of example, the HER2 is human HER2 annotated in Uniprot (www.uniprot.org) under accession number P04626.1.

[0139] In a specific embodiment, the bacterial antigen is PcrV or Psl of Pseudomonas aeruginosa.

[0140] In certain embodiments, the viral antigen or virus-associated antigen is selected from the group consisting of human cytomegalovirus (HCMV) antigen, gp160, hepatitis C virus (HCV) antigen, human papillomavirus (HPV) antigen, lysozyme (LYZ), pp50, Tat, VSV8, Epstein-Barr virus (EBV) antigen, gp33, hepatitis delta virus (HDV), influenza virus, nef, pp65, tuberculosis antigen, gag, hepatitis B virus (HBV) antigen, human immunodeficiency virus (HIV) antigen, LMP2, p21 protein, severe acute respiratory syndrome (SARS)-CoV antigen, and vpr.

[0141] In certain embodiments, the fungal antigen is an Aspergillus antigen.

[0142] In certain embodiments, the activating NK cell receptor is natural cytotoxicity receptor (NCR), NKG2D, NKG2A, killer immunoglobulin-like receptor (KIR), or SLAMF7 (CRACC). As examples, human NCR1 is as annotated in Uniprot (www.uniprot.org) under accession number O76036.1, human NKG2D is as annotated in Uniprot (www.uniprot.org) under accession number P26718.1, human NKG2A is as annotated in Uniprot (www.uniprot.org) under accession number P26715.2, human KIR is as annotated in Uniprot (www.uniprot.org) under accession numbers Q99706.3, Q8N743.2 or P43628.1, and human SLAMF7 is as annotated in Uniprot (www.uniprot.org) under accession number Q9NQ25.1.

[0143] In certain embodiments, the binding domain (e.g., antigen recognition domain) is capable of antagonizing (i.e., inhibiting) an activating NK cell receptor. Dimeric protein complexes as taught herein that comprise, consist essentially of, or contain a functional component that consists of a binding domain (e.g., antigen recognition domain) that specifically binds to and antagonizes an activating NK cell receptor may be used to activate NK cells.

[0144] In a specific embodiment, the cytokine is IL-15.

[0145] In certain embodiments, the toxin is sphingomyelinase-D (SmaseD) from Loxoceres intermedia or brown recluse spider. Dimeric protein complexes as taught herein that contain, consist essentially of, or contain a functional component that consists of a binding domain (e.g., an antigen recognition domain) that specifically binds to the toxin can be used to neutralize the toxic activity of this toxin.

[0146] In certain embodiments, the first, second, third and / or fourth functional components comprise, consist essentially of or consist of recombinant viral structural proteins, such as recombinant viral structural proteins for a virus-like particle (VLP).

[0147] In certain embodiments, the first, second, third and / or fourth functional components comprise a cytotoxic agent such as sphingomyelinase-D (SmaseD) from Loxoceles intermedia or brown recluse spider. A dimeric protein complex comprising SmaseD from Loxoceles intermedia as a functional component and further comprising a functional component comprising a binding domain (e.g., an antigen recognition domain) as a further functional component can be used to kill a target cell as a result of accumulation of the toxin on the cell surface of the target cell. In a particular embodiment, one of the first, second, third or fourth functional components comprises a binding domain (e.g., an antigen recognition domain) that specifically binds to an activating NK cell receptor, and binding of the binding domain (e.g., the antigen recognition domain) to an activating NK cell receptor is capable of activating an NK cell, and wherein at least two, e.g., two or three, of the first, second, third or fourth functional components comprise a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA, a TAA, a bacterial antigen, a viral antigen, a virus-associated antigen or a fungal antigen. Such a dimeric protein complex is referred to herein as "BiKE."

[0148] In a particular embodiment, two of the first, second, third or fourth functional components comprise a binding domain (e.g., an antigen recognition domain) that specifically binds to an activating NK cell receptor, and the binding of the binding domain (e.g., the antigen recognition domain) to the activating NK cell receptor can activate an NK cell, and two of the first, second, third or fourth functional components comprise a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA, a TAA, a bacterial antigen, a viral antigen, a virus-associated antigen or a fungal antigen. Preferably, the two binding domains (e.g., antigen recognition domains) that specifically bind to an activating NK cell receptor are different, and more preferably, each specifically binds to a different activating NK cell receptor. Such a dimeric protein complex is referred to herein as "TriKE."

[0149] In certain embodiments, three of the first, second, third, or fourth functional components comprise a binding domain (e.g., an antigen recognition domain) that specifically binds to an activating NK cell receptor, and binding of the binding domain (e.g., the antigen recognition domain) to the activating NK cell receptor is capable of activating an NK cell, and wherein one of the first, second, third, or fourth functional components comprises a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA, a TAA, a bacterial antigen, a viral antigen, a virus-associated antigen, or a fungal antigen. Preferably, each of the three binding domains (e.g., antigen recognition domains) that specifically bind to an activating NK cell receptor are different, and more preferably, each specifically binds to a different activating NK cell receptor. The at least two, two or three binding domains (e.g., antigen recognition domains) that specifically bind to a TSA, TAA, bacterial antigen, viral antigen, virus-associated antigen or fungal antigen may each recognize different epitopes of the same ligand / receptor present on the surface of the same pathogen or tumor, different ligands / receptors on the same pathogen or tumor, or different ligands / receptors on different pathogens or tumors.

[0150] In certain embodiments, the first and / or second functional components of the dimeric protein complex taught herein comprise, consist essentially of, or consist of a molecule or a fragment thereof, preferably a peptide, whose main biologically active domain is located at its C-terminus, such as a monomeric Fc. Preferably, the first and / or second functional components of the dimeric protein complex taught herein do not comprise, consist essentially of, or consist of a molecule or a fragment thereof, preferably a peptide, whose main biologically active domain is located at its N-terminus, such as a glycoprotein. Alternatively, a flexible linker or a rigid spacer can be introduced between the molecule whose main biologically active domain is located at its N-terminus and the C-terminal portion of the C4bp β chain in order to distance the biologically active domain of the molecule from the C-terminal portion of the C4bp β chain. As described elsewhere herein, the inventors have also found that the inclusion of a monomeric Fc, preferably an IgG monomeric Fc including an IgG hinge, CH2 domain, and CH3 domain, downstream (C-terminal) of the C-terminal fragment of the C4bp β-chain of both the first and second polypeptides of the dimeric protein complex results in an unexpected enhanced ability of the dimeric Fc formed by the two monomeric Fcs to (i) activate the complement system and C3b deposition on targets, (ii) activate NK cells and antibody-dependent cell-mediated cytotoxicity (ADCC) / complement-dependent cytotoxicity (CDCC), and (iii) activate macrophages and macrophage-mediated phagocytosis of target cells compared to conventional therapeutic antibodies. These enhanced abilities are the result of the double hinge (i.e., the presence of an Fc hinge and a C4bp β-chain hinge N-terminal to the Fc hinge) created in the dimeric protein complex in which the first and second functional components are monomeric Fcs, as taught herein. Moreover, such dimeric protein complexes can be produced in a simpler manner than antibodies, which typically consist of two separate constructs: (i) one encoding the light chain (V L, C L ) and (ii) those encoding the heavy chain (V H For dimeric protein complexes in which the first and second functional components are monomeric Fc, as taught herein, a single construct encoding a polypeptide comprising the C-terminal portion of the C4bp β-chain can be used, and thus only a single transfection into the host cell is required. Also, as taught herein, dimeric protein complexes in which the first and second functional components are monomeric Fc have an overall size smaller than a conventional IgG1 antibody (e.g., about 130 kDa, whereas the MW of an IgG1 antibody is typically about 150 kDa).

[0151] In certain embodiments, the first, second, third and / or fourth functional components comprise, consist essentially of, or consist of a monomeric Fc. In certain embodiments, the first and second functional components comprise, consist essentially of, or consist of a monomeric Fc (also referred to herein as an Fc domain); a hinge region of the monomeric Fc in the first polypeptide is connected to a hinge region of the monomeric Fc in the second polypeptide by at least two disulfide bonds; The third and / or fourth functional components include a binding domain (eg, an antigen recognition domain).

[0152] In certain embodiments, the first and second functional components comprise, consist essentially of, or consist of the same monomeric Fc.

[0153] In certain embodiments, the third and / or fourth functional components do not inhibit a biological function of the monomeric Fc.

[0154] In certain embodiments, the monomeric Fc in the first and second polypeptides is linked immediately C-terminal to the C-terminal portion of the C4bp β chain, meaning that there is no linker between the monomeric Fc and the C-terminal portion of the C4bp β chain. The monomeric Fc may be derived from a known antibody or fragment thereof, such as a known therapeutic antibody or fragment thereof. Thus, in certain embodiments, the dimeric protein complex as taught herein allows for improving the biological function of the Fc of an antibody or fragment thereof without modifying the sequence of the Fc. In certain embodiments, the monomeric Fc comprises a hinge (also referred to herein as a hinge region or hinge domain), a CH2 domain, and a CH3 domain. In certain embodiments, the monomeric Fc is a monomeric Fc of IgG comprising a hinge (also referred to herein as a hinge region or hinge domain), a CH2 domain, and a CH3 domain of IgG. In a preferred embodiment, the monomeric Fc is a monomeric Fc of IgG1 comprising, or consisting essentially of, a hinge (i.e., hinge region), a CH2 domain, and a CH3 domain of IgG1. For example, a monomeric Fc has the amino acid sequence EPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA It may comprise, consist essentially of, or consist of the amino acid sequence: KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW (SEQ ID NO: 34).

[0155] In certain embodiments, the knob-into-hole technology as described in International Patent Application WO2013097430, incorporated herein by reference, may be applied to a monomeric Fc, e.g. as shown in Figure 21. For example, a CH3 Fc IgG1 constituted by a first polypeptide of a dimeric protein complex may exhibit three mutations (S354C, T366W and K409A), also known as Fc[knob], and a CH3 Fc IgG1 constituted by a second polypeptide of a dimeric protein complex may exhibit five mutations (Y349C, T366S, L368A, F405K and Y407V). In certain embodiments, the third functional component comprises, consists essentially of, or consists of a binding domain (e.g., an antigen recognition domain) and the fourth functional component comprises, consists essentially of, or consists of a binding domain (e.g., an antigen recognition domain), and the binding domains (e.g., antigen recognition domains) are different, so that they may be referred to as bispecific pseudo-IgG. Bispecific pseudo-IgGs are capable of cross-linking two different types of cells, such as target cells and effector cells.

[0156] In a particular embodiment, the first and second functional components comprise, consist essentially of, or consist of a monomeric Fc (also referred to herein as an Fc domain), wherein a hinge region of the monomeric Fc in the first polypeptide is linked to a hinge region of the monomeric Fc in the second polypeptide by at least two disulfide bonds, and the third and / or fourth functional components comprise scFv that specifically binds to the CD20 antigen (e.g., scFv derived from rituximab or ofatumumab), scFv that specifically binds to HER2, or V H H (e.g., 2D3 V H H, 47D5 V H scFv or VFv as described elsewhere herein, such as scFv derived from VH anti-HER, or trastuzumab. HH, an scFv that specifically binds to Pseudomonas aeruginosa (e.g., an scFv anti-PcrV or scFv anti-Psl derived from panobacumab), an scFv that specifically binds to NKG2A, an scFv that specifically binds to SLAMF7, or an scFv that specifically binds to NKG2D, or H H, or the scFv or the V H H. As shown in Table 1 below, numerous combinations are possible, which can create bispecific pseudo-IgGs. [Table 1]

[0157] For example, the first and second polypeptides of the dimeric protein complexes encompassed herein may comprise a codon-optimized RTX scFv pseudo-IgG that specifically binds to CD20, and has the amino acid sequence: [ka] (SEQ ID NO:35); and / or the nucleic acid sequence: [ka] (SEQ ID NO:36).

[0158] The signal peptide (first bold sequence of the sequence defined in SEQ ID NO:36) is cloned between the EcoRI (GAATTC or NS) and Bgl2 (AGATCT or RS) restriction sites and is the signal sequence from tumor necrosis factor receptor superfamily member 16 (TNFR16) (UniProt number P08138.1). AGATCT or RS ) and BspE1( T.C.G.A. or SG) restriction sites. The sequence encoding the linker is between the BspE1 restriction site and the start of the C4bp C-terminal β-strand (the second bolded sequence in the sequence defined in SEQ ID NO: 36). The sequence encoding linker.C4bp β.Fc is between the BspE1 and the stop codon TGA (.) between NotI( GCGGCCGC ), followed by a multiple cloning sequence ending with . The sequence encoding the C-terminal portion or fragment of the C4bp β-chain dimerization scaffold is the first underlined sequence of the sequence defined in SEQ ID NO:36. The sequence encoding the hinge of Fc IgG1 is shown in italics. The sequence encoding CH2 Fc IgG1 is the second underlined sequence of the sequence defined in SEQ ID NO:36. The sequence encoding CH3 Fc IgG1 is the third bolded sequence of the sequence defined in SEQ ID NO:36.

[0159] Preferably, the "pseudo-IgG" taught herein is encoded by a nucleic acid sequence that includes a cassette encoding a monomeric Fc spanning between EcoRI (G / AATTC) and NotI. Genes encoding other functional components located N-terminal to the C-terminal portion of the C4bp β-chain of such exemplary pseudo-IgG are interchangeable and are preferably located between the restriction sites Bgl2 (A / GATCT) and BspE1, with the restriction sites and genes of interest in the same open reading frame as depicted in the sequence above.

[0160] The inventors have also found that the previously described "knob-into-hole technology" as described in European Patent EP2235064B1 or International Patent Application WO2013097430 (which are incorporated herein by reference) can be applied to pseudo-IgG as described herein. The "knob-into-hole technology" allows for the production of 100% of a single molecular species consisting of a dimeric protein complex as taught herein, without the use of purification steps to separate multiple different molecular species of the dimeric protein complex. For example, the knob-into-hole technology comprises: (i) a first polypeptide encoded by a nucleic acid sequence comprising a codon-optimized sequence encoding a linker .C4bpβ.Fc between BspE1 (T / CCGGA) and a stop codon TGA, optionally followed by a multiple cloning site terminating in NotI (GC / GGCCGC), whose encoded CH3 Fc IgG1 exhibits three amino acid mutations (S354C, T366W and K409A), also known as Fc[knob], comprising, consisting essentially of or consisting of a nucleic acid sequence encoding an amino acid sequence as defined in SEQ ID NO: 38 and / or as defined in SEQ ID NO: 37; and (ii) a first polypeptide encoded by a nucleic acid sequence comprising a codon-optimized sequence encoding a linker .C4bpβ.Fc between BspE1 and a stop codon TGA, optionally followed by a multiple cloning site terminating in NotI, whose encoded CH3 Fc IgG1 exhibits three amino acid mutations (S354C, T366W and K409A), also known as Fc[knob], comprising, consisting essentially of or consisting of a nucleic acid sequence encoding an amino acid sequence as defined in SEQ ID NO: 38 and / or as defined in SEQ ID NO: 37. IgG1 exhibits five amino acid mutations (Y349C, T366S, L368A, F405K and Y407V) and is also known as Fc[hole] and can be used to generate a dimeric protein complex ( FIG. 21 ) comprising a second polypeptide as described in International Patent Application WO2013097430, which second polypeptide comprises, consists essentially of or consists of a nucleic acid sequence encoding an amino acid sequence as defined in SEQ ID NO: 40 and / or as defined in SEQ ID NO: 39.

[0161] In certain embodiments, the functional component may be a tag.

[0162] In certain embodiments, the first and / or second polypeptide comprises a fifth and / or sixth functional component, said fifth and / or sixth functional component being a tag. In certain embodiments, when the first and / or second polypeptide comprises two functional components N-terminal and / or C-terminal to the C-terminal fragment of the C4bp β chain, one of these two functional components may be a tag.

[0163] In certain embodiments, the tag may be located immediately after the signal peptide, immediately N-terminal or C-terminal to the C-terminal fragment of the C4bp β chain, or C-terminal to the first and / or second polypeptide. Tags can be added to proteins for a variety of purposes, such as purification (e.g., poly(His) tag), aiding in proper folding of the protein (e.g., thioredoxin), separation techniques (e.g., FLAG tag), enzymatic or chemical modification (e.g., biotin ligase tag, FIAsH), or detection (e.g., tracking or visualization). Detection tags can be visualized directly or indirectly, usually through detection with a labeled antibody or other protein or molecule that binds to or interacts with the tag. Examples of such tags include, but are not limited to, Avi tag, calmodulin tag, polyglutamic acid tag, E tag, EE tag, EPEA / C tag, FLAG tag, HA tag, His tag, Myc tag (e.g., c-myc tag), HSV epitope, HAT, S tag, SBP tag, Sof tag1, Sof tag3, Strep tag, TC tag, V5 tag, VSV tag, Xpress tag, Isopep tag, Spy tag, biotin carboxyl carrier protein, glutathione-S-transferase (GST) tag, green fluorescent protein tag, Halo tag, maltose binding protein (MSB) tag, Nus tag, thioredoxin tag, or Fc tag. As used herein, the term "tag" refers to any of a variety of proteins, including fluorescent proteins (eGFP, eRFP, Cherry), magnetic beads, biotin for staining with labeled avidin or streptavidin conjugates, enzymes, substrates, coenzymes, chemiluminescent groups (e.g., nanoluciferase), chromogenic agents, colorimetric labels, molecular imaging probes (e.g., 18 F, 11 C, or 64 Cu, 99m TC, iron oxide nanoparticles, or luciferase). Preferably, the tag is a peptide, protein or polypeptide. In certain embodiments, the tag is a peptide, protein or polypeptide having an amino acid sequence of at most 10 amino acids. In certain embodiments, the tag is a protein purification tag or a protein separation tag. More preferably, the tag is a FLAG tag, a His tag, an HA tag or a Myc tag.

[0164] In certain embodiments, the first and / or second polypeptide comprises a tag C-terminal to the second and / or second functional component. For therapeutic uses in humans where the presence of a fluorescent moiety is undesirable, a proteolytic cleavage site can be introduced N- or C-terminal to the tag, depending on the location of the tag in the first or second polypeptide, to remove the tag after purification of the dimeric protein complex.

[0165] In certain embodiments, a proteolytic cleavage site is included immediately N-terminal to the tag, such that the skilled artisan will understand that the proteolytic cleavage site is located between the first or second functional component and the tag. Examples of such cleavage sites are well known in the art and include, for example, a Tobacco Etch Virus (TEV) protease cleavable site, such as one that comprises the amino acid sequence ENLYFQ / G (SEQ ID NO: 41), or a Human Rhinovirus (HRV) 3C protease cleavable site, such as one that comprises the amino acid sequence LEVLFQ / GP (SEQ ID NO: 42), where " / " represents the peptide bond to be cleaved, and methods for introducing them into constructs of the invention or using them to release protein moieties. Preferably, the proteolytic cleavage site is a TEF protease cleavable site, such as comprising the amino acid sequence ENLYFQ / G (SEQ ID NO: 41), or an HRV 3C protease cleavable site, such as comprising the amino acid sequence LEVLFQ / GP (SEQ ID NO: 42). A further aspect provides a nucleic acid encoding a first polypeptide of a dimeric protein complex comprising a first functional component and a C-terminal fragment of a C4bp β chain, said first functional component being linked to the C-terminus of said C-terminal fragment of a C4bp β chain, as defined herein.

[0166] A further aspect provides a nucleic acid encoding a second polypeptide comprising a second functional component and a C-terminal fragment of a C4bp beta chain, said second functional component being linked to the C-terminus of said C-terminal fragment of a C4bp beta chain, as defined herein. In certain embodiments, the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are in separate cistrons or are in a multicistronic construct.

[0167] Thus, a further aspect provides a nucleic acid encoding a first polypeptide of a dimeric protein complex comprising a first functional component and a C-terminal fragment of a C4bp β chain, wherein said first functional component is linked to the C-terminus of the C-terminal fragment of a C4bp β chain, and encoding a second polypeptide comprising a second functional component and a C-terminal fragment of a C4bp β chain, wherein said second functional component is linked to the C-terminus of said C-terminal fragment of a C4bp β chain, as defined herein. By "encodes" it is meant that a nucleic acid sequence, or a portion thereof, corresponds to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides, or to another nucleic acid sequence in the context of a template transcription product (e.g., an RNA or RNA analog).

[0168] In certain embodiments, the nucleic acids encoding the first, second, third and / or fourth functional components are codon optimized.

[0169] In certain embodiments, the nucleic acid encoding the first and second functional components, such as a nucleic acid encoding an scFv, spans between the Bgl2 (A / GATCT, amino acids RS) and BspE1 (T / CCGGA, amino acids SG) restriction sites.

[0170] In certain embodiments, the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide comprise a nucleic acid encoding a signal peptide.

[0171] In certain embodiments, the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide comprise a nucleic acid encoding a signal peptide 5' (or upstream) of the nucleic acid encoding the C-terminal fragment of the C4bp β chain. One skilled in the art will understand that when the nucleic acid encoding the first polypeptide comprises a nucleic acid encoding a third functional component located 5' of the nucleic acid encoding the C-terminal fragment of the C4bp β chain, the nucleic acid encoding the signal peptide is located 5' of the nucleic acid encoding the third functional component. Similarly, when the nucleic acid encoding the second polypeptide comprises a nucleic acid encoding a fourth functional component located 5' of the nucleic acid encoding the C-terminal fragment of the C4bp β chain, the nucleic acid encoding the signal peptide is located 5' of the nucleic acid encoding the fourth functional component.

[0172] Furthermore, one skilled in the art will understand that when the nucleic acid encoding the first polypeptide comprises a nucleic acid encoding a tag 5' of the nucleic acid encoding a third functional component and / or 5' of the nucleic acid encoding the C-terminal fragment of the C4bp beta chain, the nucleic acid encoding the signal peptide is located 5' of said nucleic acid encoding said tag. Similarly, when the nucleic acid encoding the second polypeptide comprises a nucleic acid encoding a tag 5' of the nucleic acid encoding a fourth functional component and / or 5' of the nucleic acid encoding the C-terminal fragment of the C4bp beta chain, the nucleic acid encoding the signal peptide is located 5' of said nucleic acid encoding said tag. The signal peptide may be a homologous or heterologous signal peptide, depending on the host cell used to produce the agent as taught herein. Preferably, the nucleic acid encodes a signal peptide having the amino acid sequence LNGFR (SEQ ID NO: 42).

[0173] After expression of the first and / or second polypeptides of the dimeric protein complexes taught herein, the signal peptide is typically cleaved after fulfilling its function, typically resulting in a "mature" form of the first and / or second polypeptide. The first and / or second polypeptides including the signal peptide may be referred to as precursors of the first and / or second polypeptides, respectively. The first and / or second polypeptides of the dimeric protein complex taught herein may comprise one or more restriction sites between the signal peptide and the C-terminal fragment of the C4bp β chain. To allow expression of a nucleic acid encoding a first and / or second polypeptide as defined herein, the nucleic acid may be inserted into a nucleic acid expression cassette and / or vector, as is well known in the art.

[0174] A further aspect provides an expression cassette comprising a nucleic acid encoding a first polypeptide of a dimeric protein complex taught herein or a second polypeptide of a dimeric protein complex, preferably wherein the nucleic acid encoding the first polypeptide or the nucleic acid encoding the second polypeptide is operably linked to a promoter and / or transcriptional and translational regulatory signals.

[0175] The term "nucleic acid expression cassette" or "expression cassette" as used herein refers to a nucleic acid molecule, typically DNA, into which a nucleic acid fragment, preferably a recombinant nucleic acid molecule as defined herein, can be inserted so as to be expressed, said nucleic acid molecule comprising one or more nucleic acid sequences that control the expression of the nucleic acid fragment. Non-limiting examples of such more nucleic acid sequences that control the expression of the nucleic acid fragment include promoter sequences, open reading frames and transcription terminators. Preferably, a nucleic acid expression cassette may contain one or more open reading frames (ORFs) that code for one or more polypeptides of interest. An "open reading frame" or "ORF" refers to a sequence of coding nucleotide triplicates (codons) beginning with a translation initiation codon, ending with a translation termination codon known per se, and containing no in-frame translation termination codons, which may code for a protein, polypeptide, or peptide. Thus, this term may be synonymous with "coding sequence" as used in the art.

[0176] "Operable linkage" refers to a linkage in which a control sequence and a sequence to be expressed are linked in such a way as to allow said expression. For example, sequences such as a promoter and an ORF may be said to be operably linked if the nature of the linkage between said sequences does not: (1) result in the introduction of a frameshift mutation; (2) inhibit the ability of the promoter to direct the transcription of the ORF; or (3) inhibit the ability of the ORF to be transcribed from the promoter sequence. Thus, "operably linked" means that an expression control sequence, such as a promoter, is incorporated into a genetic construct so as to effectively control the transcription / expression of the sequence of interest.

[0177] The precise nature of transcriptional and translational regulatory sequences or elements required for expression may vary depending on the expression environment, but typically include transcription terminators and, optionally, enhancers. Reference to a "promoter" is taken in its broadest context and includes transcriptional regulatory sequences necessary for accurate transcription initiation and, where applicable, precise spatial and / or temporal control of gene expression or, for example, in response to internal or external (e.g., exogenous) stimuli. More specifically, a "promoter" may refer to a region on a nucleic acid molecule, preferably a DNA molecule, to which RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, located upstream, i.e., 5', of the sequence that controls its transcription. Typically, in prokaryotes, a promoter region may include both the promoter itself and a sequence that, when transcribed into RNA, signals the initiation of protein synthesis (e.g., Shine-Dalgarno sequence). A promoter sequence may also include an "enhancer region," which is one or more regions of DNA that can bind proteins (i.e., trans-acting factors) to increase the level of transcription of genes in a gene cluster. Enhancers are typically present at the 5' end of a coding region, but can also be present separately from the promoter sequence, for example, within an intron region of a gene or 3' of a coding region of a gene.

[0178] In embodiments, promoters contemplated herein may be constitutive or inducible. A constitutive promoter is understood to be a promoter whose expression is constant under standard culture conditions. An inducible promoter is a promoter that responds to one or more induction cues. For example, an inducible promoter may be chemically regulated (e.g., a promoter whose transcriptional activity is controlled by the presence or absence of a chemical inducer such as alcohol, tetracycline, steroids, metals, or other small molecules) or physically regulated (e.g., a promoter whose transcriptional activity is controlled by the presence or absence of a physical inducer such as light or high or low temperature). An inducible promoter may also be indirectly regulated by one or more transcription factors that are themselves directly regulated by chemical or physical cues. Non-limiting examples of promoters include T7, U6, H1, retroviral ras sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. The term "terminator" or "transcription terminator" generally refers to a sequence element at the end of a transcription unit that signals the end of transcription. For example, a terminator is usually located downstream, i.e., 3', of an ORF encoding a polypeptide of interest. For example, when a recombinant nucleic acid contains two or more ORFs, e.g., arranged contiguously, forming together a multicistronic transcription unit, a transcription terminator may be advantageously located 3' of the most downstream ORF.

[0179] In certain embodiments, the expression cassette comprises a nucleic acid encoding a first or second polypeptide as disclosed herein operably linked to one or more promoters, enhancers, ORFs and / or transcription terminators. A further aspect provides an expression vector comprising a nucleic acid as taught herein (i.e., a nucleic acid encoding a first and / or second polypeptide of a dimeric protein complex) or an expression cassette as taught herein.

[0180] The term "expression vector" or "vector" as used herein refers to a nucleic acid molecule, typically DNA, into which a nucleic acid fragment, preferably a recombinant nucleic acid molecule as defined herein, can be inserted and cloned, i.e. propagated. Thus, vectors usually contain one or more unique restriction sites and may be capable of autonomous replication in a defined cell or vehicle organism, so that the cloned sequence is reproducible. Vectors also preferably contain a selection marker, such as an antibiotic resistance gene, to allow the selection of recipient cells containing the vector. Suitable vectors include, but are not limited to, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PACs, BACs, linear nucleic acids, e.g., linear DNA, transposons, viral vectors, etc. (see, e.g., Sambrook et al., 1989; Ausubel 1992). Viral vectors include, in particular, retroviral, lentiviral, adenoviral, or adeno-associated viral vectors, such as vectors based on HIV, SV40, EBV, HSV, or BPV. Expression vectors are generally constructed to allow and / or result in the expression of a nucleic acid or open reading frame introduced therein in a desired expression system, e.g., in vitro, in a cell, an organ and / or an organism. For example, expression vectors may advantageously contain appropriate regulatory sequences. Important factors in selecting a particular vector include, among others, the choice of recipient cell, the ease of recognizing and selecting vector-containing recipient cells from vector-free recipient cells, the copy number of the vector desired in a particular recipient cell, whether it is desired for the vector to be integrated chromosomally in the recipient cell or to remain extrachromosomal, and whether it is desired to be able to "shuttle" the vector between recipient cells of different species.

[0181] Expression vectors can be autonomous or integrative. Nucleic acids can be introduced into cells in the form of expression vectors, such as plasmids, phages, transposons, cosmids, or viral particles. Recombinant nucleic acids can be maintained extrachromosomally or can be integrated into the chromosomal DNA of the cell. Expression vectors can contain a selectable marker gene encoding a protein required for cell survival under selected conditions (e.g., URA3 encoding an enzyme required for uracil biosynthesis, or LEU2 encoding an enzyme required for leucine biosynthesis, or TRP1 encoding an enzyme required for tryptophan biosynthesis) to allow detection and / or selection of cells transformed with the desired nucleic acid. Expression vectors can also contain an autonomously replicating sequence (ARS). The ARS can include a centromere (CEN) and an origin of replication (ORI). For example, the ARS can be ARS18 or ARS68. Prior to introducing the vector into the cells of interest, the vector can be propagated (e.g., amplified) in bacterial cells such as Escherichia coli. Vector DNA can be isolated from the bacterial cells by any method known in the art that will purify the vector DNA from the bacterial environment. Purified vector DNA can be extensively extracted with phenol, chloroform, and ether to confirm the absence of E. coli proteins in the plasmid DNA preparation. A further embodiment provides an expression vector comprising a nucleic acid encoding a C-terminal fragment of a C4bp β chain, a first insertion site for a nucleic acid located immediately 3' to the nucleic acid encoding the C-terminal fragment of a C4bp β chain, and optionally a second insertion site for a nucleic acid located immediately 5' to the nucleic acid encoding the C-terminal fragment of a C4bp β chain.

[0182] In certain embodiments, the expression vector comprises a nucleic acid encoding a C-terminal fragment of a C4bp β chain, a first insertion site for a nucleic acid encoding a functional component located immediately 3' to the nucleic acid encoding the C-terminal fragment C4bp β chain, and optionally, a second insertion site for a nucleic acid encoding a functional component located immediately 5' to the nucleic acid encoding the C-terminal fragment C4bp β chain. The insertion site allows a nucleic acid, such as a DNA fragment, to be inserted into the region. The insertion site may include one or more restriction sites for one or more restriction enzymes. Preferably, the restriction site is a unique restriction site. That is, the restriction site occurs only once in a particular vector. In certain embodiments, the restriction site of the first insertion site is different from the restriction site of the second insertion site. The insertion site allows a nucleic acid, such as a DNA fragment, to be inserted into the region. For example, the insertion site may be a multiple cloning site (MSC), as known in the art, which is a short nucleotide segment that includes multiple (such as up to 20) restriction sites. The dimeric protein complex taught herein can be prepared by a simple cloning method.Furthermore, the method for preparing the dimeric protein complex taught herein allows the production of the dimeric protein complex with a high expression yield, such as 20 mg / L or more of the dimeric protein complex. A further aspect provides a method of preparing a dimeric protein complex as taught herein.

[0183] In certain embodiments, when the first and second functional components are proteins or polypeptides, the method of preparing a dimeric protein complex taught herein comprises: - providing, in an expressible manner, one or more nucleic acids encoding the first and / or second polypeptides of the dimeric protein complex taught herein; - introducing said one or more nucleic acids encoding said first and / or second polypeptides into a host cell; and - recovering the dimeric protein complex from the host cell or its supernatant; and - optionally purifying the recovered dimeric protein complex. Includes.

[0184] In certain embodiments, a method for preparing a dimeric protein complex as taught herein comprises culturing a host cell into which one or more nucleic acids encoding a first and / or second polypeptide have been introduced under conditions suitable for expression of the one or more nucleic acids and suitable for dimerization, preferably covalent binding, of the first and second polypeptides.

[0185] In certain embodiments, when the first and second polypeptides of the dimeric protein complex are identical, the method of preparing the dimeric protein complex taught herein involves only introducing into a host cell one nucleic acid encoding either the first or the second polypeptide (which are identical in the case of homodimers) in an expressible format.

[0186] In certain embodiments, when the first and second polypeptides of the dimeric protein complex are not identical, the method of preparing a dimeric protein complex taught herein comprises introducing one nucleic acid encoding the first polypeptide and one nucleic acid encoding the second polypeptide into a host cell, such as by co-transfection, in an expressible manner. Typically, a dimeric protein complex (or simply a dimer) is formed by random association of a first and / or second polypeptide comprising the C-terminal portion of the C4bp β-chain, which may or may not comprise additional features such as one or more components. Thus, a host cell into which a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide (wherein the first and second polypeptides are different) have been introduced produces a dimeric protein complex consisting of two or more molecular specifications. For example, if the first polypeptide comprises a functional component A and the second polypeptide comprises a functional component B, the host cell produces dimeric protein complexes of three different molecular species, i.e., A:A, A:B, and B:B dimeric protein complexes. Thus, following production and collection of the dimeric protein complex, the dimeric protein complex can be purified to obtain a population of dimeric protein complexes that consists substantially (e.g., at least 95%; preferably at least 99%, more preferably at least 99.9%) of a single molecular species dimeric protein complex of interest, such as an A:A, A:B or B:B dimeric protein complex.

[0187] In certain embodiments, purification of dimeric protein complexes may be performed by utilizing one or more tags incorporated into one or more nucleic acids encoding the first and / or second polypeptides. For example, in the case of a heterodiboro or heterotetraboro where the first polypeptide comprises a HIS tag and the second polypeptide comprises a FLAG tag, a two-step purification may be performed, including a first purification by HIS-TRAP purification, which first captures all dimers comprising the first polypeptide comprising a HIS tag, and then a second purification by FLAG affinity chromatography, which captures all dimers comprising the first polypeptide comprising a HIS tag and the second polypeptide comprising a FLAG tag (see, e.g., FIG. 2).

[0188] The term "host cell" as used herein refers to a cell into which one or more nucleotides, preferably DNA, have been introduced, such as by transfection. The host cell may be a eukaryotic cell, such as a yeast cell, such as S cerevisiae, a filamentous fungal cell, such as Aspergillus sp, an insect cell, such as Drosophila S2 cell or Spodoptera sf9, a mammalian cell or a plant cell. The most commonly used host cells for protein production are mammalian immortalized cell lines, such as CHO cells or HEK 293 cells, preferably HEK 293 cells, which are easily transfected and have a high growth rate, thus facilitating protein production and increasing yield. Large-scale production of protein complexes usually requires host cells capable of growing in suspension, such as suspension-adapted CHO cells or HEK293 cells, preferably suspension-adapted HEK293 cells.

[0189] Methods for introducing nucleic acids into living cells are known to those skilled in the art and may include calcium phosphate co-precipitation, electroporation, microinjection, protoplast fusion, lipofection, exosome-mediated transfection, transfection using polyamine transfection reagents, bombardment of cells with nucleic acid-coated tungsten microprojectiles, viral particle delivery, and the like. Such introduction is also referred to as delivery, transfection, or transformation. Cell-penetrating peptides (CPPs) may be used to deliver polypeptides or nucleic acids into cells. CPPs include, but are not limited to, Penetratin, Tat(48-60), Transportan, (R-AhX-R4), and the like.

[0190] In certain embodiments, the resulting dimeric protein complex may be concentrated.

[0191] In certain embodiments, when the first, second, third and / or fourth functional components to be included in the dimeric protein complex are not proteins or polypeptides, the method of preparing a dimeric protein complex taught herein comprises: - preparing a protein complex comprising a dimerized C-terminal fragment of the C4bp β-chain and a first, second, third and / or fourth functional component which is a protein or polypeptide; - adding a first, second, third and / or fourth functional component which is not a protein or polypeptide to the prepared protein complex to obtain the desired dimeric protein complex. Includes.

[0192] Unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, as will be apparent to those skilled in the art. A further aspect provides a eukaryotic cell engineered to produce a dimeric protein complex as taught herein, the cell comprising, in an expressible manner, one or more nucleic acids encoding the first and / or second polypeptides of the dimeric protein complex as taught herein. The eukaryotic cell is not a human embryo. Depending on the type of functional components contained in the dimeric protein complexes as taught herein, the dimeric protein complexes as taught herein may be used for multiple applications, including but not limited to therapeutic applications, in general healthcare (e.g., molecular imaging), environmental purposes, purification applications, and the like.

[0193] A further aspect provides a pharmaceutical composition comprising a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, or an expression vector as taught herein, and a pharma- ceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein, consistent with the art, means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof. As used herein, "carriers" or "excipients" include any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate buffered saline, etc.), solubilizers, colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione, etc.), amino acids (e.g., glycine, etc.), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavors, fragrances, thickeners, agents for achieving depot effect, coating agents, antifungal agents, preservatives, antioxidants, tonics, absorption retardants, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as a conventional media or agent is incompatible with the active substance, its use in the therapeutic compositions is contemplated. Exemplary, non-limiting carriers for use in formulating pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic co-solvents suitable for intravenous use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to those of skill in the art. Pharmaceutical compositions as contemplated herein can be formulated for essentially any route of administration, including, but not limited to, oral administration (e.g., oral ingestion or inhalation), nasal administration (e.g., nasal inhalation or nasal mucosal application), parenteral administration (e.g., subcutaneous, intravenous (IV), intramuscular, intraperitoneal or intrathoracic injection or infusion), transdermal or transmucosal (e.g., oral, sublingual, nasal, etc.) administration, topical administration, rectal, vaginal or intratracheal injection, etc. In this manner, the therapeutic effect achievable by the present methods and compositions can be tailored to the particular needs of a given application, e.g., systemic, local, tissue-specific, etc.

[0194] In a particular embodiment, the pharmaceutical composition is a neutralizing antiserum.Preferably, the pharmaceutical composition is a neutralizing antiserum against SmaseD from Li, and comprises a dimeric protein complex comprising a first and a second functional component, the first and the second functional component comprising, essentially consisting of, or consisting of scFv anti-SmaseD from venom from Loxoceles intermedia (Li), or comprises a dimeric protein complex comprising a first, a second, a third and a fourth functional component, the first, the second, the third and the fourth functional component comprising, essentially consisting of, or consisting of scFv anti-SmaseD from venom from Li.

[0195] A further aspect provides a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, an expression vector as taught herein, or a pharmaceutical composition as taught herein for use as a medicament, preferably in immunotherapy.

[0196] Further aspects provide a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, an expression vector as taught herein, or a pharmaceutical composition as taught herein for use in the treatment of a neoplastic or infectious disease. That is, provided herein is a method of treating a neoplastic or infectious disease in a subject in need of such treatment, comprising administering a therapeutically effective amount of a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, an expression vector as taught herein, or a pharmaceutical composition as taught herein. Also provided is the use of a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, an expression vector as taught herein, or a pharmaceutical composition as taught herein for the manufacture of a medicament for the treatment of a neoplastic disease or an infectious disease. Also provided are dimeric protein complexes as taught herein, nucleic acids as taught herein, expression cassettes as taught herein, expression vectors as taught herein, or pharmaceutical compositions as taught herein for use in treating venom bites, for example from insects, spiders, or snakes. For example, as described elsewhere herein, when one or more of the functional components comprises, consists essentially of, or consists of scFv anti-SmaseD from venom from Loxoceles intermedia (Li), the dimeric protein complexes can bind to and inhibit the activity of SmaseD, and thus treat venom bites from Loxoceles intermedia.

[0197] In certain embodiments, a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, an expression vector as taught herein, or a pharmaceutical composition as taught herein is used to treat a skin necrotic event following varicositis (i.e., the process where venom is injected by the spider bite). The term "therapy" or "treatment" encompasses both therapeutic treatment of an already established disease or condition, such as treatment of an already established neoplastic or infectious disease, and prophylactic measures aimed at preventing or reducing the likelihood of an undesired disease occurring, such as preventing the onset, onset and progression of a neoplastic or infectious disease. Beneficial or desired clinical results may include, but are not limited to, alleviation of one or more symptoms or one or more biological markers, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliative of the disease state, and the like. "Treatment" may also mean prolonging survival compared to expected survival if not receiving treatment. Except where noted, the terms "subject" or "patient" may be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, even more preferably primates, including in particular human patients, non-human mammals and primates. A preferred subject is a human subject. The term "subject" or "patient" includes subjects in need of treatment, more particularly subjects who would benefit from treatment of a given condition, in particular a central nervous system (CNS) disorder, including a neurovascular disorder or neurovascular dysfunction. Such subjects may include, but are not limited to, those diagnosed with the condition, those susceptible to developing the condition, and / or those in whom the condition is to be prevented.

[0198] The products and methods taught herein allow for the administration of therapeutically and / or prophylactically effective amounts of the dimeric protein complexes taught herein, the nucleic acids taught herein, the expression cassettes taught herein, the expression vectors taught herein, or the pharmaceutical compositions taught herein to subjects with neoplastic or infectious diseases who would benefit from such treatment. As used herein, the term "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical agent that induces in a subject the biological or pharmaceutical response desired by a surgeon, researcher, veterinarian, medical doctor, or other clinician, including, among other things, the alleviation of symptoms of the disease or condition being treated. The term "prophylactically effective amount" refers to the amount of an active compound or pharmaceutical agent that inhibits or delays the onset of a disorder in a subject as desired by a researcher, veterinarian, medical doctor, or other clinician. Methods for determining therapeutically and / or prophylactically effective doses of the dimeric protein complexes taught herein, the nucleic acids taught herein, the expression cassettes taught herein, the expression vectors taught herein, or the pharmaceutical compositions taught herein are known in the art. The term "therapeutically effective amount," as used herein, refers to an amount of a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, an expression vector as taught herein, or a pharmaceutical composition as taught herein, that, when administered, results in a positive therapeutic response for the treatment of a patient having a neoplastic or infectious disease.

[0199] An appropriate therapeutically effective amount of a dimeric protein complex taught herein, a nucleic acid taught herein, an expression cassette taught herein, an expression vector taught herein, or a pharmaceutical composition taught herein can be determined by a qualified physician taking into account the nature of the disease state and its severity, as well as the age, size and condition of the patient.

[0200] The term "neoplastic disease" generally refers to any disease or disorder characterized by the growth and proliferation of neoplastic cells, whether benign (does not invade surrounding normal tissues and does not form metastases), premalignant (pre-cancerous), or malignant (can invade adjacent tissues and give rise to metastases). The term neoplastic disease generally includes all transformed cells and tissues, as well as all cancerous cells and tissues. Neoplastic diseases or disorders include, but are not limited to, abnormal cell proliferation, benign tumors, pre-malignant or pre-cancerous lesions, malignant tumors, and cancer. Examples of neoplastic diseases or disorders include benign, pre-malignant, or malignant neoplasms in any tissue or organ, such as the prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, breast, or genitourinary tract. As used herein, the term "tumor" or "tumor tissue" refers to an abnormal mass of tissue resulting from excessive cell division. A tumor or tumor tissue includes tumor cells that have abnormal growth characteristics and no useful bodily function. Tumors, tumor tissues, and tumor cells can be benign, pre-malignant, malignant, or lesions with no potential for cancer. A tumor or tumor tissue may also include non-tumor cells associated with the tumor, such as vascular cells that form blood vessels that supply blood to the tumor or tumor tissue. Non-tumor cells may be induced to replicate or grow by tumor cells, such as by induction of angiogenesis in the tumor or tumor tissue. As used herein, the term "cancer" refers to a malignant neoplasm characterized by dysregulated or unregulated cell proliferation. The term "cancer" includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignant tumor or tumor) and secondary malignant cells or tumors (e.g., those resulting from metastasis, i.e., the migration of malignant or tumor cells to a secondary site different from the site of the original tumor). The term "metastatic" or "metastasis" generally refers to the spread of cancer from one organ or tissue to another non-adjacent organ or tissue. The occurrence of neoplastic disease in other non-adjacent organs or tissues is referred to as metastasis. Examples of cancer include, but are not limited to, carcinoma, lymphoma, germinoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancer include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, and lung large cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and central nervous system cancer, melanoma, head and neck cancer, bone cancer, bone marrow cancer, duodenal cancer, esophageal cancer, thyroid cancer, or blood cancer.

[0201] In certain embodiments, the tumor is a solid tumor. Solid tumors include any tumor that forms a tumor mass, but usually does not contain cysts or fluid. Solid tumors can be benign, pre-malignant, or malignant. Examples of solid tumors include carcinomas, sarcomas, melanomas, and lymphomas.

[0202] In certain embodiments, the neoplastic disease or cancer is one that comprises cells expressing one or more antigens that are targeted by the dimeric protein complexes taught herein.

[0203] In certain embodiments, when the disease to be treated is a neoplastic disease, the dimeric protein complex taught herein comprises a functional component in which at least one of the first, second, third and fourth functional components is a binding domain (e.g., an antigen recognition domain) that specifically binds to a TSA or TAA. Thus, one skilled in the art will understand that when the dimeric protein complex taught herein comprises a functional component that comprises, consists essentially of, or consists of a binding domain (e.g., an antigen recognition domain) that specifically binds to HER2, the neoplastic disease may be HER2-positive breast cancer.

[0204] In certain embodiments, the infection may be any infection known in the art, such as an infection caused by a pathogen, such as a bacterium, a virus, or a fungus, including, but not limited to, Pseudomonas aeruginosa infection, Bordetella pertussis infection, Borrelia burgdorferi infection, Borrelia recurrentis infection, Haemophilus influenza infection, Moraxella catarrhalis infection, Neisseria gonorrhoeae infection, Neisseria meningitidis infection, Streptococcus pneumoniae infection, Yersinia enterocolitica and Staphylococcus aureus infection, Candida dubliensis infection, Escherichia coli infection, Pasteurella pneumoniae infection, Aspergillus fumigatus terreus infection, human cytomegalovirus (HCMV) infection, hepatitis C virus (HCV) infection, human papillomavirus (HPV) infection, Epstein-Barr virus (EBV) infection, hepatitis delta virus (HDV) infection, influenza virus infection, tuberculosis infection, hepatitis B virus (HBV) infection, human immunodeficiency virus (HIV) infection, severe acute respiratory syndrome (SARS)-CoV infection, Candida albicans infection, Aspergillus fumigatus infection, and Toxoplasma gondii infection.

[0205] In certain embodiments, when the disease to be treated is an infectious disease, the dimeric protein complex taught herein comprises at least one of the first, second, third and fourth functional components which is a binding domain (e.g., an antigen recognition domain) that specifically binds to a viral antigen, a virus-associated antigen, a bacterial antigen or a fungal antigen.

[0206] In certain embodiments, when the disease to be treated is an infectious disease caused by a pathogen that evades human complement attack, the dimeric protein complex as taught herein comprises at least one of the first, second, third and fourth functional components comprising, essentially consisting of, or consisting of SCRs 3, 4 and 5 from FHR1, as described elsewhere herein.

[0207] In certain embodiments, where the disease to be treated is an infectious disease caused by a pathogen that evades complement-mediated killing by employing C4bp soluble complement regulatory proteins, the dimeric protein complex taught herein comprises at least one of the first, second, third, and fourth functional components comprising, consisting essentially of, or consisting of CCP1 and CCP2 from the C4bp N-terminal alpha chain as described elsewhere herein. Non-limiting examples of pathogens that hijack C4bp via CCP1-2 binding include Streptococcus pneumoniae and pyogenes, Staphylococcus aureus, Escherichia coli, Candida albicans, Aspregillus fumigatus, Toxoplasma gondii.

[0208] In certain embodiments, the disease to be treated is an infection caused by an antibiotic-resistant pathogen, such as Pseudomonas aeruginosa.

[0209] Further aspects provide a dimeric protein complex as taught herein, a nucleic acid as taught herein, an expression cassette as taught herein, or an expression vector as taught herein for use in methods of molecular imaging of a living body. In certain embodiments, when the dimeric protein complex taught herein is used in a method for molecular imaging of a living organism, the dimeric protein complex comprises at least one functional component comprising a binding domain (e.g., an antigen recognition domain) and further comprises at least one molecular imaging probe. Non-limiting examples of molecular imaging probes include: 18 F, 11 C, or 64 Cu (for use in positron emission tomography (PET)); 99m Tc (for use in single photon emission computed tomography (SPECT)), magnetically active elements such as iron oxide nanoparticles, and luciferase (for use in diagnostic molecular imaging).

[0210] The present application also provides aspects and embodiments as defined in the following statements: Statement 1) a first polypeptide comprising a first functional component and a C-terminal fragment of a C4bp β chain, the first functional component being linked to the C-terminus of the C-terminal fragment of the C4bp β chain; and a second polypeptide comprising a second functional component and a C-terminal fragment of a C4bp β chain, said second functional component being linked to the C-terminus of said C-terminal fragment of a C4bp β chain. A dimeric protein complex comprising: the first and second polypeptides are the same or different; the first and second functional components are proteins or polypeptides; Dimeric protein complex. statement 2) the first polypeptide further comprises a third functional component linked to the N-terminus of the C-terminal fragment of the C4bp β chain; the second polypeptide further comprises a fourth functional component linked to the N-terminus of the C-terminal fragment of the C4bp β chain; the first, second, third and fourth functional components are the same or different; 1. A dimeric protein complex according to statement 1. Statement 3) The dimeric protein complex according to statement 2, wherein the first, second, third and fourth functional components are proteins or polypeptides, preferably proteins or polypeptides selected from the group consisting of an antigen recognition domain, a recombinant viral structural protein, an oncolytic agent, a cytotoxic agent, a cytokine, a receptor-binding peptide or a monomeric Fc. Statement 4) The antigen recognition domain is an antigen-specific single chain variable fragment (scFv) of an antibody or a single domain variable fragment (VFv) of a heavy chain antibody. H H), an antibody-like scaffold, an extracellular domain of a viral envelope protein, a cognate extracellular domain of a receptor or ligand of an antigen, or an antigen-binding portion of said receptor or ligand, a soluble receptor, or a synthetic receptor. Statement 5) The dimeric protein complex according to statement 3 or 4, wherein the antigen recognition domain specifically binds to a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), a bacterial antigen, a viral antigen or a virus-associated antigen, a fungal antigen, an activating NK cell receptor, a cytokine, a toxin or a contaminant. Statement 6) The dimeric protein complex according to Statements 2 to 5, wherein two of the first, second, third or fourth functional components comprise an antigen recognition domain that specifically binds to an activating NK cell receptor, and binding of the antigen recognition domain to the activating NK cell receptor is capable of activating an NK cell, and two of the first, second, third or fourth functional components comprise an antigen recognition domain that specifically binds to a TSA, a TAA, a bacterial antigen, a viral antigen, a virus-related antigen or a fungal antigen. Statement 7) the first and second functional components are monomeric Fc, preferably monomeric IgG Fc comprising an IgG hinge, CH2 domain, and CH3 domain; the hinge region of the monomeric Fc in the first polypeptide is connected to the hinge region of the monomeric Fc in the second polypeptide by at least two disulfide bonds; The third and / or fourth functional components comprise an antigen recognition domain; 7. A dimeric protein complex according to any one of statements 2 to 6. Statement 8) encoding a first polypeptide comprising a first functional component and a C-terminal fragment of a C4bp β chain, the first functional component being linked to the C-terminus of the C-terminal fragment of a C4bp β chain as defined in any one of statements 1 to 7; and / or encoding a second polypeptide comprising a second functional component and a C-terminal fragment of a C4bp β chain, said second functional component being linked to the C-terminus of said C-terminal fragment of a C4bp β chain as defined in any one of statements 1 to 7; Nucleic acid. Statement 9) An expression cassette comprising the nucleic acid according to statement 8. Statement 10) An expression vector comprising the nucleic acid according to statement 8 or the expression cassette according to statement 9. Statement 11) An expression vector comprising a nucleic acid encoding a C-terminal fragment of the C4bp β chain, an insertion site for a nucleic acid encoding a functional component located immediately 3' to the nucleic acid encoding the C-terminal fragment of the C4bp β chain; and, optionally, an insertion site for a nucleic acid encoding a functional component located immediately 5' to the nucleic acid encoding the C-terminal fragment of the C4bp β chain. Statement 12) A pharmaceutical composition comprising a dimeric protein complex according to any one of statements 1 to 7, a nucleic acid according to statement 8, an expression cassette according to statement 9, or an expression vector according to statement 10 or 11, and a pharma- ceutical acceptable carrier. Statement 13) A dimeric protein complex according to any one of statements 1 to 7, a nucleic acid according to statement 8, an expression cassette according to statement 9, an expression vector according to statement 10 or 11 or a pharmaceutical composition according to statement 12 for use as a medicament, preferably for use in immunotherapy. Statement 14) A dimeric protein complex according to any one of statements 1 to 7, a nucleic acid according to statement 8, an expression cassette according to statement 9, an expression vector according to statement 10 or 11, or a pharmaceutical composition according to statement 12 for use in the treatment of a neoplastic disease or an infectious disease. Statement 15) A dimeric protein complex according to any one of statements 1 to 7, a nucleic acid according to statement 8, an expression cassette according to statement 9, or an expression vector according to statement 10 or 11, for use in a method for molecular imaging of a living organism.

[0211] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations within the spirit and scope of the appended claims. Aspects and embodiments of the invention disclosed herein are further supported by the following non-limiting examples. EXAMPLES

[0212] Example 1. Bifunctional heterodimer (tetraboro) capable of activating NK cells Introduction NKG2A (CD159a) is a type II transmembrane protein that is constantly co-expressed with CD94 forming a disulfide-linked NKG2A / CD94 heterodimeric complex. NKG2A mediates Ca ++It is a member of the dependence (C-type) lectin family. The CD94 / NKG2A dimer is an inhibitory NK receptor for HLA class I histocompatibility antigen, alpha chain E (HLA-E). The cytoplasmic domain of NKG2A contains two characteristic ITIM motifs (immunoreceptor tyrosine based inhibition motifs) involved in the transmission of inhibitory signals. HLA-E plays a very specific role in the recognition of self and non-self cells by NK cells. HLA-E molecules are expressed at low levels in most tissues, but are commonly overexpressed on the surface of various cancers. Thus, high HLA-E + Tumors send strong inhibitory signals upon NKG2A / CD94 / HLA-E interaction, inhibiting the function of NK cells. Z199 is a mouse monoclonal antibody (IgG2b) that is a non-competitive antagonist of the human NKG2A (CD159a) receptor. The present inventors have generated a scFv derived from Z199 mAb as a blocker of the human NK inhibitory pathway, comprising the amino acid sequence defined in SEQ ID NO: 10 and the nucleic acid sequence defined in SEQ ID NO: 11, and may be referred to herein as "anti-NKG2A scFv". IL-15 is a regulatory cytokine that belongs to the common γc-chain family that includes IL-2, IL-4, IL-7, IL-9, and IL-21. IL-15 uses the cytokine-specific receptor α-chain IL-15Rα (CD215) in a heterotrimeric receptor with IL-2. IL-2 and IL-15 stimulate T cell proliferation, generation of cytotoxic lymphocytes, and proliferation of natural killer (NK) cells. In contrast to IL-2, IL-15 suppresses IL-2-mediated activation-induced cell death and does not activate functional Tregs. IL-15 acts primarily as a cell surface molecule, delivering IL-15 in trans to mononuclear cells such as NK cells and CD8 memory cells as part of the immunological synapse with IL-15Rα on antigen-presenting cells. IL-15 is very difficult to produce alone due to its short half-life, low solubility, and tight transcriptomic regulation. The IL15 / sIL15Rα soluble complex is much more stimulatory than soluble IL15 alone (Rubinstein, Kovar et al. 2006). This complex inhibits IL15 internalization and inhibits βγ c IL15Rα may also protect IL15 from degradation when complexed with the IL15Rα soluble receptor. In this example, we aimed to develop a bifunctional heterodimer that uses a 58 amino acid C4bp C-terminal β-chain dimerization scaffold (C4bpβ) to associate (i) the IL-15 / IL-15Rα soluble complex with (ii) Z199 scFv anti-NKG2A to activate NK cells by delivering IL-15 at the surface of the NK cells. The gene encoding the soluble IL-15 receptor α-chain (sIL-15Rα) is located upstream of C4bpβ, while the gene encoding the Z199 scFv is located downstream of C4bpβ, as shown in Figure 3A. The two functions are dimers, respectively, and the heterotetraboro "IL-15 / sIL-15Rα.C4bpβ.scFv anti-NKG2A.His8x" simultaneously activates two different NK pathways via blocking the IL-15 / sIL-15Rα complex and the NKG2A receptor.

[0213] 1.2. Genes used and cassette design Genes used: - Signal peptide (SP) of tumor necrosis factor receptor superfamily member 16 (NGFR): UniProtKB - P08138.1 TNR16_HUMAN, amino acids 1-28; - soluble recombinant human IL-15 receptor α chain (sIL-15Rα): UniProt n° Q13261.1, amino acids 31-205); - Human C4b binding protein C-terminal beta chain (C4bpβ) dimerization scaffold: UniProtKB - P20851.1 C4BPB_HUMAN, amino acids 194-252. 3x(SGGGGS) (SEQ ID NO:3) linkers are cloned on both ends of the C-terminal part of C4bpβ; - Z199(V), as described in International Patent Publication WO2009 / 092805, which is incorporated herein by reference. H -V L ) anti-NKG2A scFv; - Human Interleukin 15 (hIL-15): UniProtKB - P40933.1 IL-15_HUMAN, amino acids 49-162. IL-15 is preceded by a 35 amino acid tPA signal peptide (MDAMKRGLCCVLLCGAVFVSPSQEIHARFRRGAR (SEQ ID NO: 44)) optimized for secretory expression in eukaryotic cells from Proteogenix. The IL-15 gene was cloned into pcDNA3.1 (containing a G418 resistance gene for stable expression) by Proteogenix; - pEF-IRESpac is a bicistronic expression recipient vector driven by the human polypeptide chain elongation factor 1α promoter and is used to generate stable mammalian cell lines expressing high levels of recombinant proteins. The plasmid contains the puromycin resistance gene (pac) for stable expression of recombinant proteins. Heterotetraboro cassette: · SP-sIL-15Rα-[3x(SGGGGS)]-C4bpβ-[3x(SGGGGS)]-Z199 scFv anti-NKG2A-His8x cloned into pEF-IRESpac · tPA SP.hIL-15, cloned into pcDNA3.1. The cDNA encoding the cassette (sIL-15Rα.C4bpβ.scFv anti-NKG2A), spanning Bgl2 and NotI restriction sites, was codon-optimized for expression in human cells, synthesized and cloned into pEF-IRESp by Proteogenix (Illkirsch, Strasbourg). Figure 3 shows the steps of transfection of the bifunctional heterotetraboro (Figure 3A, 3B).

[0214] 1.3. Transfection in HEK293F cells, cloning / screening, production, scale-up culture, purification using immobilized metal affinity chromatography (IMAC) technique using 1 ml or 5 ml Ni-Sepharose Excel columns, FPLC One day before transfection, 1.3 x 10 6HEK293F cells were plated in 6-well cell culture plates and cultured in DMEM complete medium. Two hours prior to transfection, complete DMEM was replaced with pre-warmed optiMEM. HEK293F cells were transfected with the heterotetraboro-encoding expression vector alone or co-transfected with pcDNA3.1-hIL-15 using Lipofectamine 3000 according to the manufacturer's protocol (Thermo Fisher, catalogue no. L3000001). 4 μg of DNA was transfected with 5 μl of Lipofectamine and 4 μL of reagent. A DNA ratio of 1:1 was used for co-transfection. 24 hours after transfection, 1 ml of complete DMEM medium was added. After 48 hours, the cells were transferred to 10 cm cell culture dishes and cultured in complete DMEM medium supplemented with 5–20 μg / ml puromycin for single transfections, or complete DMEM medium supplemented with 5–20 μg / ml puromycin and 100–500 μg / ml geneticin (G418) for cotransfections. After approximately 2 weeks, single clones that had grown to antibiotic resistance were individually transferred to 96-well plates. Approximately 1 week later, when the clones reached a certain density and the culture medium turned yellowish, the supernatants from the isolated clones were screened for heterotetrabolo expression using anti-IL-15 / anti-HIS and anti-HIS / anti-HIS sandwich ELISAs to identify the highest bifunctional heterotetrabolo expression levels. The cell clones showing the best protein expression were then selected and expanded. The cells were then amplified in large culture flasks, transferred to 5-chamber cell stacks, and left in 500 ml of complete DMEM medium for 24 hours. The next day, the DMEM was replaced with 500 ml of optiMEM and left for 48 hours as the first production run. This was followed by a 24 hour incubation in complete DMEM, followed by a second production run of 48 hours in 500 ml of fresh optiMEM. The optiMEM was then loaded onto a His-Trap column: the two preparations were pooled (to make 1 L) and diluted in 1000 ml of Nalgene TM Rapid-Flow TMThe supernatant was filtered through a vacuum filter unit. The supernatant was passed through a 5 ml Excel column in a closed loop for 5 days at a flow rate of 2 ml / min to immobilize the bifunctional heterotetraboro. The column was then connected to an FPLC. After washing, the molecules were eluted using a stepwise imidazole gradient. The eluate was concentrated using an Amicon® Ultra-15 centrifugal filter unit with a molecular weight cutoff (MWCO) of 30 kDa. The imidazole was then removed by dialysis against PBS.

[0215] 1.4. Western blot analysis of bifunctional heterotetraboro The following purified molecules were analyzed using Western blotting (WB) under non-reducing or reducing conditions: 1. Bifunctional heterotetraboro: sIL-15Rα.C4bpβ.scFv anti-NKG2A.His8x + huIL-15 2. Bifunctional heterotetraboro: sIL-15Rα.C4bpβ.scFv Anti-NKG2A.His8x 1 μg of heterotetraboro purified from transient transfections or 1 μg of molecules purified from stable transfections were mixed in 4X Laemmli sample buffer without or with 10% (v / v) β2-mercaptoethanol (reducing conditions) and diluted with 4-15% Mini-Protean® Tris-Glycin eXtended (TGX) buffer. TM ) precast protein gels (Bio-Rad) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using XT MES running buffer (Bio-Rad). After electrophoresis, the gels were electroblotted onto low-fluorescence background PVDF membranes (activated with methanol before use) using TTB (25 mM Tris, 192 mM glycine, 0.01% (v / v) SDS, 20% (v / v) methanol, pH 8.8) blotting buffer. Protein transfer was performed using a Bio-Rad Mini Trans-Blot (R)Electrophoretic transfer was performed using a transfer cell. After blocking with PBS-5% (w / v) low-fat milk, the membrane was incubated for 1 h in the same buffer with 1 μg of rabbit anti-His 6x-tag pAb. After three washes with TBS-Tween buffer (pH 7.2), the membrane was incubated for 1 h with goat anti-rabbit IgG AF647-labeled antibody in PBS-1% (w / v) low-fat milk. After three washes, the membrane was dried and transferred to an Amersham Fluorescence Imaging System (FMS) using an adapted filter for the AF647 (APCC) fluorescent dye (Cy5). TM Typhoon TM The samples were analyzed with a biomolecular imager. Scanned images were processed using the open source image processing program "Image J". WB showed that under non-reducing conditions, the heterotetraboro exists as a dimer with a size of approximately 120 kDa (data not shown). Under reducing conditions, the dimer is reduced to a monomer with a size of 60 kDa (data not shown).

[0216] 1.5. Characterization of purified heterotetraboro using anti-HIS or anti-IL-15 ELISA To assess the presence of the molecules in the crude supernatant or after purification, different ELISA setups were used. For this purpose, either the purified molecules, mouse anti-human IL-15 (clone CT2NU; Invitrogen) or 6-His Tag rabbit anti-His polyclonal (Bethyl, A190-114-P) antibodies were injected into MaxiSorp TM96-well flat-bottom polystyrene 96-well ELISA plates were coated for 12 hours (when molecules were directly coated) or 72 hours when antibodies (100 ng / 100 uL PBS / well) were coated. All ELISA washes were performed 5 times with 1% PBS / BSA and all incubations were performed at 4°C for 1 hour. After the first wash, plates were blocked with 5% PBS / BSA, washed again and incubated with 100-200 ul of supernatant, purified molecule or rhuIL-15. After further washing, the His tag was detected using 100 ng per well of mouse anti-HIS HRP-conjugated detection monoclonal antibody (SIGMA). Detection of IL-15 was performed in two steps: first, with 100 ng per well of mouse anti-IL-15, then with 100 ng per well of HRP (Sigma; whole molecule)-conjugated rabbit anti-mouse. Color development was achieved using the chromogenic substrates OPD and HRP. 2 O 2 The reaction was carried out in 1x citrate buffer containing 0.5NH 2 SO 4 The absorbance was read at 492 and 630 nm on a Polarstar plate reader.

[0217] 1.5.1. Demonstration of rhIL-15 / sIL-15Rα Complex Formation in Purified sIL-15Rα.C4bpβ.scFv Anti-NKG2A.His8x Heterotetraboro Protein / anti-His ELISA demonstrated that purified heterotetraboro with or without IL-15 had similar dose-response binding patterns to ELISA plates. Protein / anti-IL-15 ELISA demonstrated that only sIL-15Rα.C4bpβ.scFv anti-NKG2A.His8x+hIL-15 showed a dose-response signal specific to the presence of IL-15 complexed to the molecule (data not shown).

[0218] 1.5.2. Demonstration of hIL-15 / sIL-15Rα complexes formed on heterotetraboro after purification To determine whether huIL-15 remained bound to the molecule after multiple steps of purification and dialysis, a sandwich ELISA was performed, coated with anti-IL-15 and revealed with anti-His. It was demonstrated that proper complex formation between hIL-15 on the heterotetraboro and its co-receptor IL-15Rα occurred (data not shown). sIL-15Rα.C4bpβ.scFv.anti-NKG2A.His8x without hIL-15 showed only background signal, indicating the absence of IL-15 in this construct (data not shown).

[0219] 1.5.3. Demonstration of saturation of hIL-15 within sIL-15Rα.C4bpβ.scFv.Anti-NKG2A.His8x + hIL-15 heterotetraboro ELISAs were set up to (i) demonstrate specific binding of rhuIL-15 to sIL-15R.C4bpβ.scFv anti-NKG2A bifunctional heterotetrabolo and (ii) evaluate the rhuIL-15 saturation level of His-Trap purified sIL-15R.C674bp.scFv anti-NKG2A bifunctional heterotetrabolo expressed from HEK293T cells cotransfected with rhuIL-15 expression vector [sIL-15R.C4bp.scFv anti-NKG2A+rhuIL-15]. As a control molecule, the His-Trap purified sIL-15Rα.C4bpβ.scFv anti-NKG2A corresponding bifunctional heterotetrabolo expressed from HEK293T cells not cotransfected with rhuIL15 expression vector [sIL-15R.C4bpβ.scFv anti-NKG2A] was used. ELISA plates were coated with 6 μg / 100 μl / well of [sIL-15Rα.C4bpβ.scFv NKG2A+rhuIL-15] or [sIL-15Rα.C4bpβ.scFv anti-NKG2A]. After washing, plates were incubated with serial dilutions of commercial rhuIL-15 (starting concentration 20 μg / ml) and revealed with mouse anti-human IL-15 detection pAb followed by rabbit anti-mouse IgG HRP conjugate. After washing, HRP and HRP were added to the plates. 2 O 2ELISA was performed in 1x citrate buffer containing 0.5NH OPD colorimetric substrate. 2 SO 4 The absorbance was read at 492 nm and 630 nm in a spectrophotometer. For sIL-15Rα.C4bpβ.scFv anti-NKG2A bifunctional heterotetraboro, the OD varies from 0.05 to 2. ELISA results show that the 50% rhuIL-15 saturation of sIL-15Rα.C4bpβ.scFv anti-NKG2A bifunctional heterotetraboro is 0.15 ng (OD=1). Full saturation is reached between 10-100 ng rhuIL-15 (OD=2). For the sIL-15Rα.C4bpβ.scFv anti-NKG2A+rhuIL-15 bifunctional heterotetraboro, the OD varies from 1.55 to 1.95. The saturation of rhuIL-15 without the addition of commercial rhuIL-15 can be considered to be about 75-80%. With the addition of rhuIL-15, it becomes fully saturated. This experiment shows that sIL-15Rα.C4bpβ.scFv anti-NKG2A+rhuIL-15, when co-expressed with an rhuIL-15 expression vector, is already saturated with at least 75% of rhuIL-15, and the remaining unoccupied valencies without rhuIL-15 can still capture additional rhu-IL-15 until fully saturated.

[0220] 1.5.4. Demonstration of Heterotetraboroyl Binding to the PBMC-Derived NK92MI Cell Line or to NK Cells To demonstrate proper binding of the heterotetraboro to target NK cells, the molecule was incubated with either PBMC or the NK92MI cell line and stained for IL-15 and His. In Figure 4, both heterotetraboro showed anti-His signals on PBMC-derived NK cells and NK92MI cells, indicating specific binding of the molecule to the target cells. In contrast, only cells incubated with sIL-15Rα.C4bpβ.scFv anti-NKG2A.His8x + hIL-15 stained positive for anti-IL-15. These results indicate that scFv anti-NKG2A cloned in a non-natural location downstream of the C4bp C-terminal β-chain dimerization scaffold can bind to PBMC-derived NK cells and the NK92MI cell line expressing NKG2A. Furthermore, the heterotetraboro formed sIL-15Rα / IL-15 complexes, which were shown to be robust and unaffected during the purification process.

[0221] 1.5.5. CD107a / IFN expression profile in NK cells preincubated with heterotetraboro or rhuIL-15 and stimulated with RAJI target cells or latently HIV-1 infected ACH2 cells We investigated the biological function and ability of the heterotetraboro to activate NK cells derived from PBMCs in the presence of RAJI target cells (a human B-lymphoblastoid cell line derived from a patient with Burkitt's lymphoma) and ACH-2 target cells (an acute lymphoblastic leukemia T-cell line A3.01 infected with HIV-1 strain LAI). The Raji cell line is known to be resistant to NK cells by expressing multiple major histocompatibility complexes, including the NKG2A ligand HLA-E. The latent ACH-2 cell line is a model of chronic HIV-1 infection that harbors a single integrated copy of HIV-1. We investigated the profile of CD107a-positive NK cells, a marker reflecting the degranulation state of NK cells, and intracellular IFN-γ expression, reflecting cytolytic activity. A total of 1.5 x 10 6PBMCs were incubated with 3 μg / ml of each molecule in RPMI medium in 24-well plates for 4 or 48 hours. PBMCs were further incubated with Raji and ACH-2 cells in the presence of anti-CD107a-BV421 at an E:T ratio of 10:1. After 1 hour of incubation, GolgiStop TM and GolgiPlug TM (BD Biosciences) was added and incubated for an additional 4 h, then washed and stained for extracellular markers (anti-CD3 / BUV496, CD14 / PE-Cy5, CD16 / BUV737, CD19 / PE-Cy5, CD56 / BV786, CD8 / BV711, and L / D). Cells were permeabilized for 20 min according to the cytoperm / cytofix manufacturer's protocol (BD Biosciences, ref. 554714) and stained for intracellular IFN-γ / FITC. After 48 h preincubation with sIL-15Rα.C4bpβ.scFv.anti-NKG2A.His8x+hIL-15 or commercial rhIL-15, CD107a positive NK cells were significantly increased compared to incubation with medium alone. This data clearly indicates that the dimeric sIL-15Rα / hIL-15 complex has the same effect on NK cells as rhIL-15 on CD107a-mediated degranulation after stimulation with Raji cells, but NK cell degranulation after stimulation with latently HIV-1-infected ACH2 cells is significantly enhanced compared to rhIL-15. The increase in CD107a positive NK cells after incubation with heterotetraboro without hIL-15 was not significant compared to medium control when stimulated with Raji cells, but was significant when stimulated with latently HIV-1-infected ACH-2 cells. This indicates that blocking the NKG2A receptor with scFv has only a marginal effect on NK cell degranulation. Furthermore, heterotetraboro+hIL-15 induced 60% IFNγ-positive NK cells after 4 h preincubation with Raji cells and 25% IFNγ-positive NK cells with ACH-2 cells, whereas heterotetraboro without hIL-15 and commercial rhIL-15 induced only 2% IFNγ-positive NK cells with Raji cells and 10% with ACH-2 cells. With regard to IFNγ expression by NK cells, the bivalent sIL-15Rα / huIL-15 complex was more efficient than rhIL-15 alone (Figure 5).

[0222] 1.5.6. Cytotoxic activity of NK cells against Raji cells after incubation with heterotetraboro or heterotetraboro+rhuIL-15 To examine the ability of heterotetraboro stimulated NK cells to lyse target cells, a cytotoxicity experiment was performed. 6PBMCs were incubated with 3 μg of each molecule in 1 ml RPMI medium in 24-well plates for 48 h. Raji and ACH-2 cells were stained with cell tracer violet (ThermoFisher, Ref. C34557) according to the manufacturer's protocol and added at an E:T ratio of 10:1 for an additional 24 h. Cells were stained with live / dead (ThermoFisher, Ref. L34975) and analyzed by flow cytometry. As negative controls, Raji and ACH-2 cells incubated without NK cells were used. Cytotoxicity was calculated as follows: ([control viable cells %]-[co-culture viable cells %]) / (control viable cells %). FIG. 6 shows that sIL-15Rα.C4bpβ.scFv.anti-NKG2A.His8x+huIL-15 was able to significantly increase the cytotoxic activity of NK cells against resistant Raji and ACH-2 cells, whereas sIL-15Rα.C4bpβ.scFv.anti-NKG2A.His8x and commercial rhuIL-15 had no effect compared to media control when stimulated with Raji cells or showed significantly less effect than sIL-15Rα.C4bpβ.scFv.anti-NKG2A.His8x + huIL-15 when stimulated with ACH-2 cells.

[0223] Example 2. Redirected optimized NK-mediated killing of Pseudomonas aeruginosa using trifunctional heterotetraboro as a trispecific killer engager ("TriKE"). Introduction NK cells are innate lymphoid cells that have the following functions: · Cytokine production and cytotoxic responses via perforin / enzyme release via granule exocytosis in the extracellular environment. NK cells patrol the circulation and tissues, detecting and distinguishing between normal and abnormal (infected or tumor) cells via activating and inhibitory receptors. ·NK cells play a key role in immunity against bacteria and tumors through (i) cytokine production and (ii) release of cytotoxic granules. NK cells sense the environment through inhibitory or activating receptors. Thus, NK cell responses depend on the balance of activating and inhibitory signals, and as a result, NK cell cytotoxic responses are driven by an imbalance of inhibitory and activating ligands. P. aeruginosa infection alters the membrane expression of activating receptors on NK cells. NKG2D is involved in antitumor, antiviral, and antibacterial immunity. NKG2D has been reported to be involved in bacterial clearance in a mouse model of P. aeruginosa pneumonia. NKG2D expression was significantly decreased after P. aeruginosa infection. As a result, reduced NKG2D expression could explain the change in NK cell cytotoxicity after P. aeruginosa infection. SLAMF7 is a member of the SLAM family. Like most SLAM receptors, SLAMF7 is an autoligand, i.e., it recognizes another SLAMF7 molecule on other cells as a ligand. SLAMF7 is present on natural killer (NK) cells, activated T cells, most B cells, and myeloid cells. In NK cells, SLAMF7 is a positive regulator of NK cell activation. This activity requires expression of Ewing's sarcoma-associated transcript 2 (EAT-2), an adaptor for the SAP family. The SLAMF7 receptor is a key regulator of IFN-α-induced innate immune responses. Resistance to multiple antibiotics is an increasingly urgent problem in healthcare worldwide. However, the pharmaceutical industry does not seem to be focusing on the development of new antibiotics. In contrast to new immunotherapies for cancer patients, the development of new antibiotics is costly and less profitable. Therefore, the risk that healthcare workers face infections caused by bacteria resistant to all known antibiotics is steadily increasing, with dramatic consequences for the prognosis of patients. Therefore, new antibacterial therapeutic avenues are essential if science and medicine do not want to lose the battle against these pathogens. Some pathogens, such as Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, are considered particularly dangerous by the World Health Organization (WHO). In the field of cancer, various immunotherapies have emerged in the past decade and are considered the new best hope in the fight against tumors. Among them, the “trispecific killer engager (TriKE)”, first developed by Jeffrey Miller at the University of Minnesota, USA, is a promising approach. These allow the survival and activation of NK cells by combining three immunoglobulin (antibody)-derived single-chain variable fragments (scFv) against an activating receptor on NK cells (mostly CD16) and two antigens on cancer cells, or by replacing one of the tumor antigens with human interleukin-15 (IL-15). The inventors have used the TriKes principle (Felices M et al. Methods Mol Biol, 2016, 1441:333-346) to generate dimeric protein complexes for use in the treatment of bacterial infections, such as P. aeruginosa infections. Such dimeric protein complexes contain two scFv fragments against the NK cell activating receptors NKG2D (CD314) and SLAMF7 (or CD319). This is based on the knowledge that NK cells are generally optimally stimulated when two activating receptors are engaged, rather than one. The dimeric protein complexes further contain a third functional component, which is an scFv derived from an anti-bacterial antibody directed against a surface structure of P. aeruginosa, such as, for example, a dimeric PcrV or Psl scFv anti-P. aeruginosa (scFv anti-PcrV or scFv anti-Psl as described in International Patent Application WO2017095744-A1, which is incorporated herein by reference). We hypothesized that NK cell degranulation would occur through cross-linking of NK cells with bacteria via a TriKE-based dimeric protein complex (also referred to herein as "TriKE"). Human cytotoxic lymphocytes are known to contain cytolytic granules filled with apoptosis inducers (perforin, granzymes) and the direct bactericidal molecule granulysin. After degranulation, bacteria are killed whether or not they are resistant to antibiotics. Thus, our approach is new and original, since the TriKE dimeric protein complex acts not as an antibiotic by inhibiting bacterial metabolism, but as a direct inducer of cell death. Autologous NK cells in infected tissues and organs are activated in situ and degranulate there.

[0224] 2.2. Genes used and cassette design (Figure 7) - Construct A': MS scFv [(RTX V L 10-First Amino Acid / MS (V L -V H )]Anti-NKG2D.C4bpβ.scFv (V L -V H ) Anti-Psl.His8x The MS anti-NKG2D scFv (disclosed in European Patent Application EP2769993-A1, incorporated herein by reference) is an agonist of the NKG2D / DAP10 / Grb2 / VAv1 / PI3K / Akt pathway, whereas the ELO scFv anti-SLAMF7 (derived from elotuzumab) is an agonist of the SLAMF7 / ITSM / PLC / Ca pathway. ++ / erk pathway agonists, and both pathways activate NK cells. Psl scFv anti-Pseudomonas aeruginosa is the scFv anti-Psl described in International Patent Application WO2017095744-A1. - Construct B':ELO scFv [(RTX V L 10-First amino acid / ELO (V L -V H )] anti-SLAMF7.C4bpβ.scFv (V L -V H ) anti-Psl.FLAG The anti-NKG2D and anti-SLAMF7 scFvs are located upstream of the C-terminal part of the C4bp β chain, while the scFv anti-Psl is located downstream (C-terminal side) of the C-terminal part of the C4bp β chain. This cassette was cloned into the pEF-IRESpac expression vector. Three clones were selected for further experiments: A' (BiKE), B' (BiKE) and A'B' (TriKE) (see FIG. 8A for a schematic representation), where BiKE is a dimeric protein complex as taught herein in which the first and second polypeptides are identical, and TriKE is a dimeric protein complex as taught herein in which the first and second polypeptides are different from each other. Purified BiKEs A' and TriKEs A'B' were confirmed using SDS-PAGE and SYPRO analysis under non-reducing conditions (data not shown). Stepwise purification using increasing concentrations of imidazole allowed the isolation of dimers while simultaneously removing low (monomer) or high (tetramer) valency species.

[0225] 3.2. Binding of BiKE and TriKE expressing scFv anti-PslI to Pseudomonas aeruginosa using ELISA with coated bacteria Bacteria were grown overnight in TBS medium to an optical density of 1.0 measured at 600 nm. One OD unit is 2 x 10 8 CFU / ml are expressed. Bacteria were centrifuged at 2500 rpm for 10 min. The pellet was resuspended in 10 ml of PBS. After centrifugation, bacteria were resuspended in 4 ml of PBS containing 1% (v / v) formaldehyde and left at room temperature for 2 h. Bacteria were diluted 10-fold in PBS (10-fold: 2 x 10 7 CFU / ml). Bacteria were soaked in NUNC MaxiSorp TM Immobilized overnight at 4°C on a 96-well flat-bottom polystyrene ELISA plate (200 μl / well = 4 x 10 6 CFU / well). After washing with PBS-1% (w / v) BSA, the plates were blocked with 100 μl of PBS-5% (w / v) BSA for 1 h at 4°C. The wells were incubated with 100 μl of BiKE- and TriKE-containing crude cell culture supernatant for 1 h at 4°C. After washing five times with PBS-1% BSA, the wells were incubated with goat anti-FLAG / HRP-conjugated pAb (Bethyl) or mouse anti-HIS HRP-conjugated mAb (SIGMA) for 1 h at 4°C. After five washes, the ELISA plates were incubated with o-phenylenediamine dihydrochloride (ODP) / H 2 O 2 or tetramethylbenzidine (TMB) / H 2 O 2 When the color was suitable for revealing with a chromogenic substrate and reading at 450 / 492 nm in a spectrophotometer, 0.5NH 2 SO 4 The reaction was stopped with. Data from the binding of BiKEs and TriKEs heterotetraboro to P. aeruginosa showed that scFv anti-PslI bound to 1 of 4 strains from patients with mucous cysts and 5 of 5 strains from secretions from patients with endotracheal tubes. scFv anti-PslI also bound to strain RP73. This example shows that the scFv anti-Pseudomonas located C-terminal to the C-terminal fragment of the C4bp β-chain is functional and able to bind to the target (Figure 8B).

[0226] 3.3. ELISA and FACS analysis of binding and cross-linking of BiKEs and TriKEs with Pseudomonas / NK92MI, and bacterial killing NK92MI and P. aeruginosa (strain RP73) were first stained with violet cell tracer and PKH26, respectively. BiKEs anti-NKG2D / anti-Psl (A'), anti-SLAMF7 / anti-PSL (B') and TriKEs anti-NKG2D / anti-SLAMF7 / anti-Psl (A'B') were incubated with NK92MI alone, P. aeruginosa alone, or both NK92MI and P. aeruginosa for 1 h at 4°C. As negative controls, cells / bacteria were incubated with either DMEM supernatant of HEK293 cells or PBS / 10% FBS. Cells were analyzed using flow cytometry and the percentage of NK92MI / P. aeruginosa colocalization was determined for all conditions (see Table 2 and Figure 8C for results of FACS analysis). [Table 2] Cells were stained with violet cell tracer and PKH26 with BiKE anti-SLAMF7 / anti-Psl (B') and TriKE anti-NKG2D / anti-SLAMF7 / anti-Psl (A'B') demonstrating that the two tetraboro scFvs were functional and capable of binding and cross-linking NK cells to target P. aeruginosa . The expression of different scFvs on BiKE and TriKE was confirmed by ELISA using anti-HIS or / and anti-Flag antibodies. BiKE NKG2D (A') was purified on a HIS-TRAP column and BiKE SLAMF7 (B') was purified using FLAG affinity chromatography, while the His and FLAG tags in the structure of TriKE (A'B') allowed a two-step purification method to isolate clones with both anti-NKG2D and anti-SLAMF-7 scFvs. As shown in Figure 8D, each BiKE and TriKE expresses scFvs against their respective targets NKG2D and / or SLAMF-7. In Figure 8E, we used the luminescent Pseudomonas PAO1-lux strain to evaluate bacterial killing induced by NK cells after binding to BiKE and TriKE. 2.10 5NK92-CD16 cells were plated with Pseudomonas strain PAO1-lux in 200 μl of complete RPMI medium without antibiotics at a final E / T ratio of 1 / 3 and cultured at 37°C. 0 Molecules were added at 100 µg / mL and bacterial growth was measured in triplicate over a 9-h time course in a luminescence microplate reader. TriKE anti-NKG2D / anti-SLAMF7 / anti-PSl (A'B') reduced bacterial growth of PAO1-lux by 2-fold at a concentration of 3 µg and completely blocked growth after 9 h of incubation with NK cells at a concentration of 6 µg. BiKE anti-NKG2A / anti-Psl (A') and BiKE anti-SLAMF7 / anti-Psl (B') had no significant effect on bacterial growth compared to NK cells incubated with PAO1-lux without the molecules.

[0227] Example 3. "Two-hinge long-neck pseudo-immunoglobulin G (pseudo-IgG)" utilizing the C-terminal β-chain dimerization scaffold of C4bp and Fc (hinge + CH2 + CH3) from IgG downstream of the dimerization scaffold to enhance Fc biological function 3.1. Design and characterization of pseudo-IgG We have constructed a two-hinge long-neck pseudo-immunoglobulin G1 (LNPIgG1) as a "pseudo-IgG" (Figure 9). Like known scFv-Fc antibody-like molecules, the pseudo-IgG is produced as a single cassette cloned into an expression vector for eukaryotic cells. The pEF-IRESpac bicistronic expression vector is a vector suitable for stable expression of recombinant therapeutic glycoproteins in mammalian cells. pEF-IRESpac contains an "Internal Ribosome Entry Site" (IRES) downstream of a multiple cloning site (MCS) followed by a puromycin resistance gene (pac). Protein expression is driven by the strong human polypeptide chain elongation factor 1α promoter, resulting in high protein expression. The signal peptide of tumor necrosis factor receptor superfamily member 16 (TNFR16, UniProt number P08138.1) was cloned between the restriction sites ECoRI and Bgl2. Pseudo-IgG (scFv or V HA cDNA encoding H.Linker.C4bpβ.Fc) spanning between the Bgl2 and NotI restriction sites, codon-optimized for human cells, was synthesized and cloned into pEF-IRESp by Proteogenix (Illkirsch, Strasbourg). The C-terminal β-strand of C4bp (UniProt no. P20851.1, aa: 137-252) contains a (3x SGGGGS (SEQ ID NO: 3)) linker at the upstream end starting with a BspE1 restriction site, followed by the hinge, CH2 and CH3 of Fc from human IgG1, and ending with a stop codon and a NotI restriction site (Figure 10). MabThera or Rituximab (RTX) derived (V L -V H ) Pseudo-IgG containing scFv typically has a molecular weight of about 140 kDa under non-reducing conditions. The pseudo-IgG is a dimeric molecule that presents an N-terminal recognition domain upstream of the C-terminal part of the C4bp β-chain dimerization scaffold with a 3x(SGGGGS) (SEQ ID NO: 3) linker between them. The targeting moiety can be an antibody fragment (scFv, Nanobody) but also a soluble recombinant receptor or ligand. Downstream of the dimerization scaffold is the Fc of IgG1, consisting of (i) the hinge, (ii) CH2, and (iii) CH3. There is no linker between the dimerization scaffold and the Fc. While C4bpβ is responsible for the covalent dimerization of the two chains, the hinge of the Fc allows dimeric Fc binding, leading to a fully functional Fc dimer. Transfection of a single monocistronic construct containing either a third or fourth functional component N-terminal to the C-terminal fragment of the C4bp β-chain and a monomeric Fc of IgG1 C-terminal to the C-terminal fragment of the C4bp β-chain into eukaryotic cells such as HEK293T cells results in the secretion of a single molecular species, a monospecific pseudo-IgG, into the cell culture supernatant (Figure 10A). Co-transfection of two constructs, where one construct contains a third functional component N-terminal to the C-terminal fragment of C4bp β chain and a monomeric Fc of IgG1 at the C-terminal side of the C-terminal fragment of C4bp β chain and a second construct contains a fourth functional component N-terminal to the C-terminal fragment of C4bp β chain and a monomeric Fc of IgG1 at the C-terminal side of the C-terminal fragment of C4bp β chain, where the third and fourth functional components are different, results in the generation of a bispecific 3,4 pseudo-IgG along with two monospecific 3 and 4 counterparts (Figure 10A). Combining the known "knob-into-hole" technology, as described in International Patent Application WO2013097430, incorporated herein by reference, or by Ridgway JB and P Carter LGP, 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Eng., 1996, Jul 9 (7): 617-21, with the pseudo-IgG technology as taught herein allows the expression of 100% bispecific pseudo-IgG (Figure 10B). The pseudo-IgG features a double-hinge "long-neck" Fc, which has particularly enhanced biological properties for activating the complement system on target surfaces compared to conventional antibodies. The ability to activate FcγR is also enhanced, leading to enhanced NK activation as well as phagocytic activity of macrophages against targets. Our in vitro data suggest that the long-neck pseudo-IgG with two hinges has improved accessibility to C1q, and this configuration also appears to favor C1q binding and subsequent complement activation and complement-mediated cytotoxicity (CDC), as well as simultaneous engagement of Fc for complement-dependent cell-mediated cytotoxicity / phagocytosis (CDCC / CDCP) and Fc engagement to FcγR of immune effector cells (ADCC / ADCP) over conventional IgG1.

[0228] 3.2. Cloning and screening of pseudo-IgG-containing single clone crude supernatants using ELISA or flow cytometry analysis 3.2.1. Screening of monospecific and "knob-into-hole" bispecific pseudo-IgG-containing supernatants using a symmetric Fc / Fc ELISA HEK293T cells were used to express the different molecules. The day before transfection, HEK293T cells were plated in a 6-well cell culture plate with complete DMEM medium at 1.3x10 6 The cells were plated at a density of 1000 cells / well. The next day, the complete DMEM medium was replaced with optiMEM medium. The cells were transfected with the expression vectors using a Lipofectamine 3000 kit according to the manufacturer's instructions (ThermoFisher, catalog no. L3000001). 48 hours after transfection, the cells were transferred to 10 cm cell culture dishes and supplemented with DMEM medium supplemented with Pen / Strep antibiotics (P / S), glutamine (Gln), 10% (v / v) fetal bovine serum (FBS), and 5–20 μg / ml puromycin (PURO20) selection antibiotic (depending on the construct). After 2 weeks, the grown clones that were resistant to puromycin were manually picked one by one and transferred to 96-well plates. After about 1 week, the supernatants of the isolated clones were screened for expression levels of tetraboro / pseudo-IgGs using symmetric anti-HIS or anti-human IgG1 ELISA. Screening of individual clone supernatants was established using a home-made Fc ELISA. Goat anti-human IgG pAb (ABCAM ab97221) was coated onto NUNC MaxiSorp 96-well ELISA plates at 1 μg / ml in PBS (100 ng / well) for 48 h at 4°C. After blocking with PBS-5% BSA for 1 h at 4°C, the plates were incubated with 200 μl of crude supernatant of individual cell clones for 1 h at 4°C. The dilution of the supernatant needs to be adjusted so that the signal is not saturated. Usually, a dilution of 1 / 200 is required. Afterwards, the plates were revealed with goat anti-human Fc pAb HRP conjugate (ABCAM ab997225) diluted 1 / 1000. The ELISA was then performed using OPD / H.2 O 2 The clones were revealed with HRP chromogenic substrate and read at 492 nm and 620 nm. The best clones (i.e., those expressing the most pseudo-IgG) were selected and expanded for scale-up production.

[0229] 3.2.2. Double screening of bispecific pseudo-IgG-containing supernatants using FACS analysis and two target cells This is RTX scFv / anti-HER2 V from individually picked cell clones. H This is an example of screening of H-bispecific pseudo-IgG-containing supernatants using target cells and flow cytometry analysis. The supernatants were tested in parallel on HER2-positive BT474 cells and CD20-positive DAUDI cells. BT474 and DAUDI cells (1,5.10 5 Cells / well) were incubated with 150 μl of crude supernatant from each transfected clone for 30 min at 4° C. After washing (PBS / 1% FBS), cells were incubated with goat anti-human IgG AF647 conjugated pAb. After fixation with 1% paraformaldehyde in PBS, cells were analyzed by flow cytometry. Cells whose supernatants showed the strongest staining for both cell types (i.e. clones D1, D12 and E1) (data not shown) were expanded for scale-up production.

[0230] 3.3. Large-scale production, protein G purification and characterization of pseudo-IgG 3.3.1. Large-scale production As a result of screening, the clone with the highest pseudo-IgG expression level was selected and cultured 25 cm 2 After reaching confluence, cells were first transferred to a 75 cm 2 (T75) flask and then 175 cm 2The cells from seven confluent T175 flasks were transferred to a polystyrene CellSTACK® in a 5-chamber format in complete medium (DMEM with 10% (v / v) FBS, penicillin / streptomycin, L-glutamine). After 24 hours, the complete DMEM medium was replaced with OptiMEM supplemented with penicillin / streptomycin and L-glutamine. After 48 hours, the OptiMEM medium was harvested and the process was repeated (24 hours complete DMEM followed by 48 hours OptiMEM). The harvested OptiMEM medium (1 liter) was centrifuged (20 minutes, 4000 rpm) in a 50 ml Falcon tube and filtered through a 0.22 μm PVDF 1 L Millipore vacuum filter unit.

[0231] 3.3.2. G-protein-based purification of pseudo-IgG The filtered OptiMEM medium was incubated with 1 ml of Protein G Sepharose® 4 Fast Flow (GE healthcare, GE17-0618-01) for 48 h at 4°C with agitation. Protein G beads were collected by centrifugation and transferred to an empty 1 ml disposable column (Qiagen) connected to a peristaltic pump. After washing the beads with 50 ml of PBS, pseudo-IgG was eluted using 20 ml of elution buffer (phosphate citrate buffer, pH 2.7, supplemented with 10% (v / v) glycerol). The collected fraction was immediately neutralized with neutralization buffer (bicarbonate buffer pH 9 supplemented with 10% (v / v) glycerol) in a ratio of elution buffer 4 / 5 + neutralization buffer 1 / 5 to a final pH of 7.2. The eluate was then concentrated using an Amicon(c) 30 kDa MWCO centrifugal filter device. The concentration of the purified molecules was measured using a NanoDrop TM The contents were measured using a microspectrophotometer, dispensed, and then stored frozen.

[0232] 3.3.3. Molecular pattern analysis by SDS-PAGE electrophoresis and SYPRO Ruby staining 1 μg of purified pseudo-IgG was mixed with 4 μl of 4X Laemmli sample buffer without (non-reducing conditions) or with (reducing conditions) 10% (v / v) β2-mercaptoethanol. Samples were diluted with 4-15% Mini-Protean® Tris-Glycin eXtended (TGX TM ) precast protein gels (Bio-Rad) and XT MES running buffer (Bio-Rad) were loaded and electrophoresed. The gels were then fixed for 30 min with 100 ml of 2x 50% (v / v) methanol + 7% (v / v) acetic acid. After fixing, the gels were incubated overnight at 4°C with 30 ml of SYPRO Ruby gel stain. The next day, the gels were washed for 30 min with 100 ml of 10% (v / v) methanol + 7% (v / v) acetic acid. Finally, the gels were stained with Amersham TM Typhoon TM The gel was analyzed using a biomolecular imager with a Cy5 filter. RTX anti-CD20 pseudo-IgG, 47D5 V H Molecular pattern analysis of the H anti-HER2 pseudo-IgG and bispecific CD20 / HER2 pseudo-IgG showed that bispecific pseudo-IgG was present in 100% of the knob-into-hole generation. H A size difference was observed between H and 47D5 V H The apparent molecular weight of H is 2D3 V H It was smaller than H.

[0233] 3.3.4. FACS analysis of binding of RTX scFv.C4bpβ.Fc pseudo-IgG, RTX scFv.C4bpα.Fc and MabThera (Rituximab) to Daudi cells Binding of purified RTX scFv.C4bpα.Fc (Multi-Di-Fc), RTX scFv.C4bpβ.Fc (mimetic IgG or mono-Di-Fc), MabThera (RTX) or no molecule (negative control) to Daudi cells was analyzed by flow cytometry. Daudi cells (1.5 x 10 5 / well) were incubated with a saturating concentration of the molecule (18 μg / ml) for 30 min at 4° C. After washing, the cells were incubated with goat anti-human IgG / AF647 or Protein A / AF647 for 30 min at 4° C. The RTX scFv was demonstrated to function on both scaffolds [C4bpα.Fc and C4bpβ.Fc] and showed binding patterns similar to those observed with both revelation systems (anti-human IgG or protein A) (data not shown).

[0234] 3.4. CH 50 Comparison of fluid complement activation of RTX scFv.C4bpα.Fc, RTX scFv.C4bpβ.Fc pseudo-IgG and MabThera (RTX) using an assay We compared the liquid-phase complement activation mediated by the RTX scFv.C4bpβ.Fc pseudo-IgG molecule with MabThera. 50 The assay was used. 50 The assay verifies the functional ability of classical pathway complement components in normal human serum (NHS) to lyse sheep red blood cells (SRBCs) presensitized with rabbit anti-SRBC antibodies (hemolysins). Hemolysis is mediated by the formation of the membrane attack complex (MAC). CH 50 To calibrate the assay, we first established a serial dilution of normal human serum (NHS): 80 μl of serum was added to 20 μl of PBS to obtain the working serum solution. The serial dilutions of the working serum solution were incubated with sensitized SRBCs (30 min at 37° C.). The SRBCs were then centrifuged and the amount of hemoglobin released in the supernatant was measured at 418 nm using a spectrophotometer. A hemolysis calibration curve was thus established. The serum dilution that induced approximately 75% hemolysis, which corresponds to the top of the linear hemolysis curve before reaching a plateau, was used as the final serum dilution for the assay. The three molecules were serially diluted in PBS. 20 μl of the serially diluted molecules were mixed with 80 μl of NHS and incubated at 37°C for 1 h. Afterwards, a hemolysis test was performed (30 min at 37°C) with sensitized SRBCs using serum dilutions set during calibration. If the molecule consumes the liquid complement of NHS, the hemolytic ability of NHS will decrease. Figure 11 shows CH 50 Assay data are shown. CH represents the molecular concentration that inhibits 50% hemolysis. 50 teeth: 41 mg / l for RTX scFv.C4bp(beta).Fc - MabThera (RX) 4 mg / l. These data show that RTX-mimetic IgG (or "mimetic RTX") - which represents a single dimeric Fc - is 50 At a concentration of around 4 mg / l, the antibody showed similar characteristics to MabThera in terms of liquid-phase complement activation. In contrast, the CH of the two multi-Di-Fc antibodies 50 The concentration is 4 times lower. The hemolysis curve started to decline at a concentration of 28 mg / l for RTX pseudo-IgG and 12 mg / l for MabThera. The results of this assay show that up to 45 mg / l, the liquid-phase complement consumption by pseudo-IgG is lower than that by conventional antibodies (RTX). Conventional antibodies and pseudo-IgG start to consume liquid-phase complement at concentrations of 12 mg / l and 28 mg / l, respectively. In conclusion, pseudo-IgG is more reliable than Multi-Di-Fc and its liquid-phase complement activation properties are similar to conventional therapeutic antibodies. Most antibodies must be injected in high doses to achieve serum concentrations of 10-100 mg / l to show therapeutic effects. From our assay, pseudo-IgG can be used more safely than conventional IgG up to 45 mg / l.

[0235] 3.5. Flow cytometric analysis of bispecific anti-CD20 / anti-HER2 mimetic IgG-mediated cross-linking of Daudi lymphoma and BT474 breast tumor cells We report the bispecific 47D5 anti-HER2 V HThe H.C4bpβ.Fc / RTX anti-CD20 scFv.C4bpβ.Fc pseudo-IgG was tested for (i) its ability to bind to CD20-positive Daudi lymphoma cells and HER2-positive BT474 breast cancer cells, and (ii) its ability to cross-link the two cell types. As a control, the 47D5 anti-HER2 and RTX anti-CD20 monospecific corresponding pseudo-IgG was used. Daudi cells were stained with CFSE and BT474 cells with KPH26 cell tracer. Daudi and BT474 cells were mixed at a ratio of Daudi:BT474 = 2:1. Cells were stained with (i) purified bispecific anti-HER2 / anti-CD20 pseudoIgG, (ii) monospecific 47D5 V H H anti-HER2 pseudo-IgG, (iii) a saturating concentration of monospecific RTX scFv anti-CD20 pseudo-IgG, or no molecule as a control. After washing, the cells were incubated with goat anti-human IgG / AF647 pAb. After washing, the cells were analyzed by flow cytometry. The bispecific pseudo-IgG was shown to bind to the two cell types, leading to their cross-linking (Table 3). In contrast, 47D5 pseudo-IgG was shown to bind only to BT474 cells, whereas RTX pseudo-IgG was shown to bind only to Daudi cells, neither of which was able to cross-link the two cell types. This experiment demonstrated the binding of scFv anti-CD20 and V in bispecific pseudo-IgG. H This study validates the functionality of both anti-HER2 antibodies. [Table 3] In the second experiment, bispecific [knob-into-hole] RTX anti-CD20 scFv.C4bpβ.Fc[knob] / 47D5 anti-HER2 V HH.C4bpβ.Fc[hole] pseudo-IgG was compared with RTX, trastuzumab, or the two conjugated mAbs for binding and cross-linking Daudi and BT474. We clearly demonstrated that neither RTX, trastuzumab, nor the conjugated mAbs, even together, were able to cross-link both cell types. In contrast, the bispecific [knob-into-hole] pseudo-IgG is able to (i) bind to each cell type and (ii) cross-link the two cell types. The double-positive population Violet / PKH26 also shows a strong anti-Fc signal (Table 4). Thus, we verified that the two relevant recognition sites of the bispecific pseudo-IgG are functional in the [knob-into-hole] bispecific construct. [Table 4] Taken together these two experiments validate the bispecific pseudo-IgG with and without knob-into-hole technology.

[0236] 3.6. Flow cytometric analysis of CDC and C3b deposition on Daudi cells after incubation with serial dilutions of pseudo-RTX (RTX scFv.C4bpβ.Fc) or MabThera reference mAb in the presence of 25% normal human serum In this experiment, Daudi cells were incubated with (i) two-fold serial dilutions of scFv.C4bpβ.Fc (RTX pseudo-IgG), MabThera, or no molecule (starting concentration 20 μg / ml). The cells were then incubated with GVB ++The cells were incubated with 25% normal human serum (NHS) in PBS for 30 min at 37 °C. After washing, the cells were stained with mouse anti-human C3b mAb (clone 7C12 from Cedarlane) for 30 min at 4 °C, followed by goat anti-mouse IgG AF647-conjugated secondary pAb, live / dead. Cells were fixed with 1% (v / v) paraformaldehyde in PBS and analyzed by flow cytometry. Figure 12A shows that RTX-mimetic IgG induces higher cytotoxicity compared to RTX-mab-thera for all concentrations used: at a molecular weight of 5 μg / ml, the percentage of dead cells was 1.7-fold higher for mock-RTX (38%) than for RTX (22%). Only at the highest concentration used (20 μg / ml), RTX showed a similar effect to mock-RTX. In Figure 12B, C3b deposition via mimetic RTX is 3.5-fold, 2.2-fold, 2.14-fold, and 1.55-fold higher at concentrations of 2.5, 5, 10, and 20 μg / ml, respectively, compared to RTX. RTX is a therapeutic antibody whose biological activity is dominated by CDC. Expression of RTX scFv on a mimetic IgG scaffold enhances the Fc function of RTX.

[0237] 3.7. Design of pseudo-RTX and comparison of the efficacy of RTX and pseudo-RTX in C3b deposition on Fc density on DAUDI cells FIG. 13A shows the structural differences of RTX mimetic IgG (or mimetic RTX or RTX scFv.C4bpβ.Fc) compared to RTX: - RTX scFv.C4bpβ.Fc or pseudo-IgG or "pseudo-RTX" is on the left - RTX or MabThera is on the right Figure 13B shows the amount of C3b deposition versus Fc density on Daudi at different concentrations used. RTX-mimetic IgG rose more rapidly than RTX, demonstrating a higher C3b activation effect at the same Fc density. This is consistent with the comparative dose-response analysis of mimetic and RTX on cytotoxicity and C3b deposition reported in Figure 12. Figure 13B shows that for the same MFI anti-Fc (12,200), C3b deposition was 5.6-fold better for mimetic compared to RTX. This experiment demonstrated that the C4bpβ.Fc scaffold is effective for designing and expressing "mimetic IgG" with Fc that has enhanced complement activation and CDC activity on target surfaces.

[0238] 3.8. FACS analysis of mimic IgG-mediated complement activation and NK activation (percentage of CD107-positive NK) on BT474 We wanted to explore the biological functions of two bispecific "knob-into-hole" pseudo-IgGs: Z199 scFv anti-NKG2A.C4bpβ.Fc[knob] / 2D3 V H H Anti-HER2.C4bpβ.Fc[hole]; abbreviation: Z199 / 2D3 Z199 scFv anti-NKG2A.C4bpβ.Fc[knob] / Trastu scFv anti-HER2.C4bpβ.Fc[hole]; Abbreviation: Z199 / T (i) NK activation from PBMCs, (ii) complement activation of BT474 cells upon incubation with 20% C5-deficient human serum as a complement source. The experimental protocol is briefly detailed in Figure 14A. BT474 were stained with CFSE and incubated with 5-fold serial dilutions (20, 4, 0.8 μg / ml) of bispecific pseudo-IgG (Z199 / 2D3 or Z199 / Trastu), or trastuzumab, or no molecule. ++ (37℃ / 5% CO 2 30 min at 4°C) or complete RPMI medium, then incubated with PBMCs at 37°C / 5% CO 2The cells were co-cultured with BT474 for 5 h. Cells were stained with anti-human CD107 / BV421 (after 1 h of co-incubation) and anti-human IgG / AF647 pAb, as well as anti-C3b / PE, anti-CD3 / BUV496, CD14 / PE-Cy5, CD16 / BUV737, CD19 / PE-Cy5, and CD56 / BV786 mAbs. The gating strategy was set up to access NK staining of Fc, CD3, and BT474 cells, knowing that BT474 cells were CSFE positive. - / CD14 - CD19 - / CD16 + / CD56 + It was. Figure 14B shows that Z199 / 2D3 bispecific pseudo-IgG activated NK cells slightly more than trastuzumab in the presence of complement and showed similar activity to trastuzumab in the absence of complement. Z199 / T activity was slightly weaker than trastuzumab with or without complement, but the presence of complement on BT474 cells slightly enhanced Z199 / T-mediated NK activation. Bispecific pseudo-IgG, but not trastuzumab, clearly strongly induces complement to target cells. Z199 / T enhances NK activation in the presence of complement. NK activation occurs through two mechanisms: (i) interaction of FcγRIIIA (CD16) with Fc, and (ii) interaction of complement degradation products (iC3b, C3dg, C3d, etc.) with CD11b (CR3) and CD11c (CR4). Trastuzumab has no complement activation effect. Figure 14C shows C3b deposition only with bispecific pseudo-IgG, but not with trastuzumab. Z199 / T activated complement more than Z199 / 2D3. For both pseudo-IgGs coated on BT474, the additional presence of complement tended to enhance their ability to activate NK cells, but seemed to have a negative effect on trastuzumab. These data demonstrate that introduction of trastuzumab-derived scFvs into the C4bpβ.Fc pseudo-IgG scaffold generates pseudo-IgGs with dramatically enhanced complement activation biological activity compared to original trastuzumab. The Te Z199 / 2D3 bispecific pseudo-IgG was shown to crosslink NK92MI and BT474, as was the bispecific CD20 / HER2, which was able to crosslink Daudi and BT474 cells (data not shown). We validated this bispecific pseudo-IgG in an NK / tumor cell model.

[0239] 3.9. Comparative analysis of pseudo-IgG- and RTX-mediated phagocytosis of Daudi cells by mature monocyte-derived macrophages using Andor spinning disk confocal microscopy Experimental setup. Daudi were stained with CFSE and macrophages with KPH26. Daudi cells were then incubated with saturating concentrations (20 μg / ml) of the following: · MabThera (RTX) RTX scFv.C4bpβ.Fc (pseudo-RTX) No molecules After washing, Daudi cells were cultured in GVB. ++ Incubate with 15% C5-deficient human serum (C5-HS) ​​in a 37°C / 5% CO 2 The cells were co-cultured for 18 h at 100°C for 18 h. Afterwards, the cells were fixed with 1% (v / v) formaldehyde, stained with DAPI, and analyzed by confocal microscopy (magnification 40x). NIKON NIH-elements software was used for analysis. 50 fields were photographed with three colors, DAPI (blue), CFSE (green), and PKH26 (red), and bright light, and large pictures were assembled. A minimum of three large pictures were analyzed for each condition to establish statistical analysis (mean and SD). After thresholding, macrophages and Daudi were automatically counted with high accuracy, and automatic colocalization was performed using the Pearson coefficient. Figure 15 is a histogram summarizing the analysis. Percentage of phagocytic macrophages in the presence (left) or absence (right) of complement. The presence of ΔC5-HS allows deposition of complement without subsequent CDC. Using Δ□5-HS evaluates ADCP+CDCP, whereas using decomplemented ΔC5-HS evaluates ADCP only. The percentage of phagocytic macrophages with / without complement on Daudi was as follows: MabThera (RTX): 24.1% / 8.6%, coefficient 2.8 decrease RTX scFv.C4bpβ.Fc (pseudo-RTX): 52% / 34.1%, reduced by 1.52 No molecules: 5% / 1.8% In the absence of complement, the loss in the percentage of phagocytic macrophages was 33% for pseudo-IgG and 66% for RTX, compared to the phagocytosis results obtained in the presence of complement. The cytotoxic activity mediated by RTX is more towards complement-mediated cytotoxicity (CDC) compared to pseudo-RTX. A possible explanation is that in pseudo-IgG, the binding of C1q to Fc, which leads to further engagement of the CD11b / iC3b axis, has less effect on the simultaneous engagement of Fc with the FcγRIIIA receptor on the macrophage surface. Pseudo-RTX outperforms RTX by 2.16- and 4-fold in the presence and absence of complement, respectively, in inducing macrophage phagocytosis of lymphoma Daudi targets. This experiment validates the pseudo-IgG scaffold and shows that the generation of pseudo-IgG with scFv of RTX dramatically improves the biological activity of pseudo-RTX compared to the gold standard of RTX. The biological activity of RTX is more dependent on complement (CDCP) than RTX pseudo-IgG. Although phagocytosis is reduced by 1.52-fold and 2.8-fold in the absence of complement for pseudo-RTX and RTX, the overall activity of pseudo-RTX is higher than that of RTX in the absence of complement. Thus, the combination of ADCP and CDCP is more efficient for pseudo-RTX than for RTX. This can be explained by the fact that the binding of C1q to Fc (responsible for activation of the classical complement pathway and subsequent CDCC / CDCP) has less effect on the ability of Fc to subsequently bind to CD16 (and thus participate in ADCC / ADCP) in the case of pseudo-RTX compared to RTX. Thus, pseudo-IgG technology may improve the overall therapeutic activity of current therapeutic mAbs, especially the concomitant ADCC / CDCC and ADCP / CDCP activities.

[0240] Example 4. Preparation of anti-Pseudomonas aeruginosa pseudo-IgG 4.1. Cassette / expression vector design (Figure 16) The recombinant cDNA construct for the expression of pseudo-IgG was synthesized by ProteoGenix SAS, Schiltigheim: - scFv KBPA-101 (anti-O11 scFv V derived from panobacumab, described in International Patent Application WO2006 / 084758A1) L -V H orientation)_hu C4bp C-terminal β chain (UniProt nr. P20851.1, aa 137-252)_hu IgG H chain constant γ (UniProt nr. P01857.1) -scFv anti-Psl (derived from MEDI3902, described in international patent application WO2017095744A1)_hu C4bp C-terminal β chain (UniProt nr.P20851.1, aa 137-252)_hu IgG H chain constant γ (UniProt nr.P01857.1) -(V L -V H ) scFv panobacumab.(3xSGGGGS).C4bp(beta).hinge.CH2.CH3 -(V L -V H ) scFv anti-Psl.(3xSGGGGS).C4bp(beta).hinge.CH2.CH3 -(V L -V H ) scFv anti-PcrV.(3xSGGGGS).C4bp(beta).hinge.CH2.CH3 -(V L -V H ) scFv panobacumab.(3xSGGGGS).hinge.CH2.CH3 -(V L -V H ) scFv panobacumab.(3xSGGGGS).C4bp(beta).hinge.CH2.CH3[knob] / (V L -V H ) scFv anti-Psl.(3xSGGGGS).C4bpβ.hinge.CH2.CH3[hole] The expression cassette was cloned between the restriction enzymes BglII and BspEI in the multiple cloning site of the bicistronic pEF-IRESpac expression vector.

[0241] 4.2. Transfection & Clone Selection Panobacumab scFv (V L -V H The production of .C4bpβ.Fc pseudo-IgG and anti-Psl scFv.C4bpβ.Fc pseudo-IgG molecules was performed in a similar manner as in Example 3 above. Briefly, HEK293T cells were transfected with recombinant plasmid DNA using Lipofectamine 3000 kit. Two days after transfection, cells were trypsinized and transferred to 10 cm cell culture dishes in complete medium (DMEM supplemented with 10% (v / v) FBS, penicillin / streptomycin, L-glutamine) supplemented with selection antibiotic (40 μg / ml puromycin). Successfully transfected cells were transferred to a single chamber of a 96-well plate when the clones reached a diameter of 0.5-1 mm. Protein concentrations in the supernatants of isolated clones were analyzed using a symmetric anti-Fc ELISA.

[0242] 4.3. Determination of protein concentration using symmetric anti-Fc ELISA Goat anti-human IgG Fc (Abcam, ab97221) was diluted in PBS and incubated at 1 μg / ml in NUNC MaxiSorp TM96-well flat-bottom polystyrene plates were coated with 100 ng / well in 100 μl PBS. After 48 h incubation at 4°C, the plates were washed 5 times with 150 μl PBS-1% BSA and blocked with 100 μl PBS-5% BSA for 1 h at 4°C. After the washing steps, 2 μl cell culture supernatant + 198 μl PBS was added (100-fold dilution). The plates were incubated for 1 h at 4°C. Goat anti-human IgG Fc(HRP) (Abcam, ab97225) was added at the same concentration as the coating antibody (100 ng / well) and incubated for 1 h at 4°C. After the final washing step, the plates were washed with 50 μl TMB / H2O. 2 O 2 The staining reaction was revealed with a chromogenic substrate and 0.5NH 2 SO 4 The plate was read in a spectrophotometer at 450 nm.

[0243] 4.4. Large-scale production The clone with the highest absorbance (anti-Fc ELISA) was selected and plated 25 cm from the 96-well cell culture plate. 2 After reaching confluence, cells were first transferred to a 75 cm 2 (T75) flask and then 175 cm 2 Cells from seven confluent T175 flasks were transferred to a polystyrene CellSTACK® in a 5-chamber format in complete medium (DMEM with 10% FBS, penicillin / streptomycin, and L-glutamine). After 24 hours, the complete DMEM medium was replaced with OptiMEM supplemented with penicillin / streptomycin and L-glutamine. After 48 hours, the OptiMEM medium was harvested and the process was repeated (24 hours complete DMEM and 48 hours OptiMEM). The harvested OptiMEM medium (1 liter) was centrifuged (20 minutes, 4000 rpm) in a 50 ml Falcon tube and filtered through a 0.22 μm PVDF 1L vacuum filter unit.

[0244] 4.5. Protein Purification by Protein G Affinity Chromatography The filtered OptiMEM medium was incubated with 1 ml of Protein G Sepharose® 4 Fast Flow (GE healthcare, GE17-0618-01) for 48 h at 4°C with agitation. Protein G beads were collected by centrifugation and transferred using a peristaltic pump into an empty 1 ml disposable column (Qiagen) previously washed with 25% ethanol and PBS. After washing the beads with 50 ml of PBS, elution buffer (phosphate citrate buffer, pH 2.7) was added. 1600 μl-s of eluate was collected in a 2 ml Eppendorf tube already containing 400 μl-s of neutralization buffer (bicarbonate buffer pH 9). Fifteen 2 ml tubes were filled with eluate and mixed in a 50 ml Falcon. The eluate was then concentrated using an Amicon(c) 30 kDa MWCO centrifugal filter device. The concentration of the purified molecules was determined using a NanoDrop TM Measurements were made using a microspectrophotometer.

[0245] 4.6. Molecular pattern analysis by SDS-PAGE electrophoresis and SYPRO Ruby staining 1 μg of purified panobacumab scFv (V L -V H The C4bpβ.Fc pseudo-IgG molecules were mixed with 4 μl-s of 4X Laemmli sample buffer without (non-reducing conditions) or with (reducing conditions) 10% β2-mercaptoethanol. Samples were diluted with 4-15% Mini-Protean® Tris-Glycin eXtended (TGX TM ) precast protein gels (Bio-Rad) and XT MES running buffer (Bio-Rad) were loaded and electrophoresed. The gels were then fixed for 30 min with 100 ml of 2x 50% methanol + 7% acetic acid. After fixing, the gels were incubated overnight at 4°C with 30 ml of SYPRO Ruby gel stain. The next day, the gels were washed for 30 min with 100 ml of 10% methanol + 7% acetic acid. Finally, the gels were stained with AmershamTM Typhoon TM The gels were analyzed using a biomolecular imager with a Cy5 filter. Panobacumab scFv (V L -V H The three purified products of C4bpβ.Fc pseudo-IgG were analyzed and shown to have a MW of approximately 140 kDa in the non-reduced form and approximately 70 kDa in the reduced form (data not shown).

[0246] 4.7. Panobacumab scFv (V L -V H Dose-dependent binding of C4bpβ.Fc pseudo-IgG and anti-Psl scFv.C4bpβ.Fc pseudo-IgG to Pseudomonas aeruginosa - Whole cell ELISA Bacterial cells (reference strains PAO1 and O11, clinical isolate IPP6247290) were grown overnight in 4 ml of TBS medium until the optical density at 600 nm reached 1.0. The cells were transferred to a 15 ml Falcon tube and centrifuged at 2500 rpm for 10 min. The supernatant was discarded and the pellet was suspended in 10 ml of PBS. After centrifugation, the bacteria were resuspended (fixed) in 1% (v / v) PFA-PBS solution. Then, 4 x 10 6 CFU / well NUNC MaxiSorp TM Immobilization was performed overnight on a 96-well flat-bottom polystyrene ELISA plate. After washing five times with PBS-1% BSA, the plate was blocked with 100 μl of PBS-5% BSA solution at 4°C for 1 h. Different concentrations of purified pseudo-IgG molecules [Pano scFv.C4bpβ.Fc or Psl scFv.C4bpβ.Fc] were added for 1 h at 4°C (three-fold serial dilution starting from 1000 ng / well). After washing, goat anti-human IgG Fc (HRP) was added at a concentration of 1 μg / ml (100 ng / well) and incubated for 1 h at 4°C. The plate was blocked with 100 μl of OPD / H. 2 O 2 The staining reaction was carried out with 0.5NH 2 SO 4 The incubation was stopped at 40° C. Plates were read in a spectrophotometer at 492 nm. Data are means ± SD from triplicate experiments. The reference strain PAO1 expresses anti-Psl scFv (EC 50 = 6 ng / well) but not by Pano scFv.C4bpβ.Fc (Figure 17C, left). ATCC33358 serotype O11: Pano scFv.C4bpβ.Fc pseudo-IgG specifically binds to Pseudomonas aeruginosa International Antigenic Typing System (IATS) serotype O11 and recognizes the O-polysaccharide portion of lipopolysaccharide (LPS) (EC 50 =110 ng / well), whereas recognition by Psl scFv.C4bpβ.Fc was much weaker (EC 50 The recognition by C4bpβ.Fc was much weaker (EC 50 (Figure 17C, center). Serotype O11 accounts for ~20% of P. aeruginosa infections. The clinical isolate (IPP6247290) was isolated from a tracheotomy patient and expressed pano scFv.C4bpβ.Fc (EC 50 =5 ng / well) and Psl scFv.C4bpβ.Fc (EC 50 =110 ng / well), with pano scFv.C4bpβ.Fc having 22-fold better affinity than Psl scFv.C4bpβ.Fc (Figure 17C, right). As a negative control, we used coated bacteria but no mock IgG, and a secondary anti-human IgG pAb revelation system. Figure 17A depicts the direct killing activity of Psl scFv.C4bpβ.Fc pseudo-IgG via complement activation against the reference strain PAO1-luciferase, preventing bacterial growth and resulting in attenuation of the luciferase signal over a period of 4-5 h. In the presence of Psl scFv.C4bpβ.Fc and decomplemented human serum, there is no effect on bacterial growth compared to the control (25% NHS non-complemented or decomplemented). This experiment shows evidence of a complement-dependent bacterial killing mediated by pseudo-IgG. Figure 17B shows the mechanism of action of Pano & Psl pseudo-IgG, which upon binding to Pseudomonas recruits C1q and is involved in activation of the classical complement pathway, leading to deposition of C3b, formation of the membrane attack complex, and ultimately bacterial killing through pore formation and membrane disruption.

[0247] 4.8. Pano scFv.C4bpβ.Fc and Psl scFv.C4bp(beta).Fc pseudo-IgG mediate Pseudomonas aeruginosa complement activation (C3b deposition) in a dose-responsive manner using ELISA with coated bacteria Bacterial cells (PAO1, O11 reference strain or clinical isolate IPP6247290) were cultured in NUNC MaxiSorp™ as described in section 4.7. TM 96-well flat-bottom polystyrene ELISA plates were coated with IgG [Pano scFv.C4bpβ.Fc or Psl scFv.C4bpβ.Fc] serially diluted 3-fold in PBS / 1% BSA (starting concentration 100 ng / well) and incubated for 1 h. As a control, no IgG was added and normal human serum (NHS) was then applied to measure background C3b deposition. After washing, the plates were incubated with 100 ng / well of GVB ++ Plates were incubated for 30 min at 37°C with 0.5% decomplemented or decomplemented human serum (NHS vs. ΔNHS, respectively) diluted in buffer to a final volume of 100 μl. After washing, plates were incubated with mouse anti-human C3 / C3b / iC3b mAb (clone 7C12) (Cedarlane, CL7636AP) for 1 h at 4°C. Plates were then revealed with 100 ng / well goat anti-mouse IgG HRP-conjugated pAb (Biolegend, 405306). Finally, plates were stained with 100 μl of OPD / H. 2 O 2 The plates were revealed with a chromogenic substrate. The staining reaction was 2 SO 4 The mixture was stopped using 500 mM NaCl. The OD was read at 492 nm and 605 nm using a spectrophotometer. The data for pseudo-IgG-mediated C3b deposition on bacteria depicted in FIG. 18 are consistent with the data for pseudo-IgG binding observed in FIG. 17C: Psl pseudo-IgG, but not Pano pseudo-IgG, was detected by the PAO1 reference strain (EC 50 =111 ng / well) induces strong C3b deposition (Figure 18, left). Pano pseudo-IgG was detected by the O11 ATCC33358 reference strain (EC 50 =300 ng / well) induced strong C3b deposition, and Psl pseudo-IgG (EC 50 The OD was 3 vs. 0.7 for the first and second plates, respectively, at the same mock IgG concentration (1000 ng / well). Pano pseudo-IgG and Psl pseudo-IgG were isolated from the clinical isolate IPP627290 (both from EC 50 = 111 ng / well). The weaker binding of Pano pseudo-IgG compared to Psl pseudo-IgG does not affect the dose-response of the most rapid C3b deposition effect. In the presence of 0.5% NHS, no C3b deposition occurs even in the absence of pseudo-IgG.

[0248] 4.9. Pano scFv.C4bpβ.Fc and Psl scFv.C4bpβ.Fc pseudo-IgG mediate complement activation (C5b9 membrane attack complex formation) of Pseudomonas aeruginosa in a dose-responsive manner in ELISA using coated bacteria Strain PAO1 (Figure 19, left), ATCC reference strain O11 (Figure 19, center), or clinical isolate IPP6247290 (Figure 19, right) were solubilized with NUNC MaxiSorp™ as described in section 4.7. TM 96-well flat-bottom polystyrene ELISA plates were coated with IgG. Uncoated wells were used as negative controls. After blocking with 100 μl PBS-5% BSA, plates were incubated for 1 h at 4° C. with 3-fold serial dilutions (starting at 1000 ng / well) of either Pano scFv.C4bpβ.Fc or Psl scFv.C4bpβ.Fc pseudo-IgG. After washing five times with 150 μl PBS-1% BSA, plates were incubated with IgG from GVB ++Plates were incubated for 30 min at 37°C with 2% normal human serum (NHS) or decomplemented normal human serum (ΔNHS) diluted in buffer to a final volume of 100 μl. After washing, plates were incubated with mouse anti-human C5b9 mAb (clone aE11) (Abcam, ab66768) for 1 h at 4°C. Plates were incubated with goat anti-mouse IgG HRP-conjugated pAb (Biolegend, 405306) 100 ng / well. Finally, 100 μl of OPD / H were added to the wells. 2 O 2 The plates were revealed with a chromogenic substrate. The staining reaction was 2 SO 4 The mixture was stopped with 0.5% CO. The OD was read at 492 nm. Data are means ± SD from three experiments. The data for pseudo-IgG-mediated C5b9 MAC formation on bacteria depicted in FIG. 19 are consistent with the data for pseudo-IgG binding observed in FIG. 17C and the data for pseudo-IgG-mediated C3b deposition observed in FIG. 18: Psl-mocked IgG, but not Pano-mocked IgG, induces MAC formation on the PAO1 reference strain (FIG. 19, left). At the highest mocked IgG concentration (1000 ng / well), the measured OD was 2.5 and 1 for Psl-mocked IgG and Pano-mocked IgG, respectively. The background OD in the absence of mocked IgG was 0.5. Pano pseudo-IgG induced MAC formation in the O11 ATCC33358 reference strain, as did Psl pseudo-IgG to a lesser extent. The OD measured at the highest pseudo-IgG concentration (1000 ng / well) was 1.5 for Pano pseudo-IgG and 0.9 for Psl pseudo-IgG. Pano- and Psl-mocked IgG induced strong MAC formation against the clinical isolate IPP627290 (Psl-mocked IgG and Pano-mocked IgG, respectively, EC 50 = 12 and 37 ng / well). The presence of 2% NHS does not lead to MAC formation in the absence of pseudo-IgG. Overall, Figures 17-19 show that the two pseudomonas IgG anti-P. aeruginosa are potent complement activators. Pano pseudomonas IgG recognized 9% and 26% of the cystic fibrosis and tracheostomy isolates, respectively, whereas Psl pseudomonas IgG recognized 45% and 100% of the cystic fibrosis isolates (11 strains) and tracheostomy isolates (15 strains), respectively (data not shown). Thus, Psl pseudomonas IgG appears to have a broader Pseudomonas recognition spectrum compared to Pano pseudomonas IgG. We further investigated whether C3b deposition leads to the formation of terminal complement complexes (TCCs), also called membrane attack complexes (MACs), which not only cause extensive damage to membranes and form pores that are the origin of complement-dependent cytotoxicity (CDC), leading not only to direct lysis but also to tag targets for recognition by immune effector cells, phagocytosis, and facilitating the activation of cytotoxic cells.

[0249] 4.10. Complement-dependent killing assay Bacterial cells (SPAO1, reference strain O11, clinical isolate IPP6247290) were cultured in tryptic soy broth (TSB) medium at 37°C until the optical density at 600 nm reached 1.0. The cells were washed and then diluted with 10 6 Dilute in PBS to a concentration of 5 x 10 cells / ml per well. 3 Add cells (5 μl of bacterial suspension + 45 μl of GVB ++ The plates were incubated with or without buffer, pseudo-IgG molecules (5 μg / well) for 2 h at 4°C. Then, 50 μl-s of 10% (v / v) normal human serum (NHS) or heat-inactivated human serum (ΔNHS) was added for 30 min at 37°C. The mixtures were then diluted 10-fold and 100-fold with PBS, incubated overnight, and plated on Trypric Soy Agar (TSA) plates to quantify CFU. Data are means ± SD from triplicate experiments. Panobacumab scFv(V L -V HThe direct killing capacity of the .C4bpβ.Fc pseudo-IgG and anti-Psl scFv.C4bpβ.Fc pseudo-IgG molecules was evaluated by a complement-dependent killing assay. Bacterial cells (PAO1, reference strain O11, clinical isolate IPP6247290) were incubated with activated (NHS) or decomplemented human serum (ΔNHS) in the presence or absence of the pseudo-IgG molecules and after overnight incubation plated on TSA agar plates to quantify CFUs. Figure 20 shows that when bound, both pseudo-IgG-s promoted complement activation and bacterial killing, resulting in a reduced number of colony forming units (CFU) / plate compared to NHS. Anti-Psl pseudo-IgG reduced the CFU count of PAO1 by 35% and IPP6247290 by 31%. Pano pseudo-IgG reduced the CFU count of ATCC33358 O11 by 26%. This experiment demonstrated that Pano and Ps1 pseudo-IgG-mediated C3b and C5b9 deposition had a bactericidal effect on P. aeruginosa. The PAO1-luciferase Pseudomonas reference strain, however, was much simpler to demonstrate the pseudo-IgG-mediated complement-directed killing effect and further inhibition of bacterial growth (Figure 17A).

[0250] NK92 humCD16 Flow cytometric analysis of panobacumab scFv.C4bpβ.Fc pseudo-IgG-mediated cross-linking of cell lines with Pseudomonas aeruginosa The present inventors have demonstrated that panobacumab (V L -V H ) The ability of scFv.C4bpβ.Fc pseudo-IgG to cross-link P. aeruginosa and NK cells was examined. humCD16 The cell lines were stained with violet cell tracer, and the P. aeruginosa O11 ATCC strain was stained with PKH26. 1 μg of mock IgG (or no molecule as a control) was added to (i) violet-NK92 humCD16 (ii) PKH26-O11 alone, or (iii) with both cells and bacteria (1:5 ratio) were incubated for 1 h at 4° C. After washing, cells, bacteria, or cells / bacteria were incubated with goat anti-human IgG AF647 conjugate. Cells were analyzed using flow cytometry (Quanteon). NK92humCD16 Flow cytometric analysis of Pano scFv pseudo-IgG-mediated capture of P. aeruginosa by cell lines revealed that pseudo-IgG bound to P. aeruginosa and that pseudo-IgG suppressed NK92 humCD16 In the absence of pseudo-IgG, P. aeruginosa and NK92 were not bound to each other (data not shown). humCD16 No cross-linking between the NK92 and Fc-positive populations was observed (data not shown). Only in the presence of pseudo-IgG was a violet-positive and Fc-positive population present, and only in the presence of pseudo-IgG was a PKH26-positive and Fc-positive population present (data not shown). Furthermore, only in the presence of pseudo-IgG was a cell violet tracer and PKH26 double-positive population present (data not shown). In conclusion, pseudo-IgG-P. aeruginosa complexes promote NK92 cell violet binding through CD16-Fc interactions. humCD16 In contrast, free pseudo-IgG can be captured by NK92 cells. humCD16 This experiment demonstrates that the pseudoIgG is fully functional and recruits NK effector cells upon binding to bacterial targets.

[0251] Example 5. Bifunctional heterodimer (tetraboro) capable of killing Pseudomonas aeruginosa Introduction The opportunistic pathogen Pseudomonas aeruginosa causes life-threatening infections in humans and is a major cause of hospital-acquired infections, especially in immunocompromised patients. The complement system plays a key role in the early clearance of bacterial infections. Upon contact with body fluids, P. aeruginosa evades human complement attack via human factor H (FH), the major inhibitor of the alternative complement pathway. The FH protein family includes FH, the major alternative pathway regulator, its splice variant FH-like protein 1 (FHL-1), and five FH-related proteins (FHRs). Recent evidence indicates a role for FHR-1 in the control or regulation of complement activation. FHR-1 is composed of five CCPs that are homologous to CCPs 6 and 7 and CCPs 18-20 of FH, respectively, which are responsible for host surface recognition and contain important C3b-binding sites. More specifically, the C-terminal SCR3-5 domain of FHR-1 is 100% / 97%, 100% and 98% identical to FH / SCR18-20, respectively, and binds C3b and C3d. FH, which controls the amplification reaction at the level of C3, can bind bacterial cells via complement control protein (CCP) domains 6-7 and 19-20. FHR1 competes with FH and acts as a positive regulator of complement attack. Importantly, FHR-1 is recruited to the microbial surface and subsequently outcompetes factor H as the concentration of FHR-1 increases, resulting in downregulation of C3 convertase and enhanced complement activation. Activation of the alternative complement pathway amplifies the recruitment, priming and activation of neutrophils and monocytes, ultimately creating a self-amplifying inflammatory loop that destructively targets cells and bacteria. The inventors have generated bifunctional heterotetraboro antibodies using scFv anti-psl derived from anti-bacterial antibodies directed against the surface structure of P. aeruginosa, such as dimeric PcrV or Psl scFv anti-P. aeruginosa (scFv anti-PcrV or scFv anti-PstI, as described in International Patent Application WO2017095744-A1, incorporated herein by reference), targeting the last three Short Consensus Repeats (SCR3-5) of the exopolysaccharide psl and (FHR1) of P. aeruginosa. The inventors have: (i) Psl scFv (V L -VH ) and (ii) a fusion protein consisting of SCR3-SCR5 from FHR1 cloned upstream and downstream of the C4bp C-terminal β-strand, respectively (Figure 22A). The Psl / FHR1(SCR3-5) construct binds to P. aeruginosa via the Psl-binding site. Thus, on the targeted bacterial surface, the heterotetraboro FHR1(SCR3-5) C-terminal effector moiety competes with the hijacked binding factor H (FH) for C3b, modulating FH-mediated complement breakdown and locally leading to activation of the alternative complement pathway (AP) and subsequent bacterial lysis, as sketched in Figure 22B.

[0252] 5.2. Genes used and cassette design C4bpβ-(SGGGGS) 5 -MluI-FHR1(SCR3-5)-His8x-Stop-NotI-BspE1-(SGGGGS) 5 -C-terminal part Genes used: - Psl scFv anti-Pseudomonas aeruginosa is the scFv anti-PstI described in international patent application WO2017095744-A1. - SCR3-SCR5 of complement factor H-related protein 1 (CFHR1): UniProtKB - Q03591 (FHR1_HUMAN) - All other ingredients are listed elsewhere in Example 1.

[0253] 5.3. Demonstration of heterotetraboro Psl / FHR1 (SCR3-5) binding and complement activation in Pseudomonas aeruginosa Dose-dependent binding of heterotetraboro Psl / FHR1 (SCR3-5) to P. aeruginosa PAO1 cells was observed by ELISA using 1.5.10 immobilized on 96-well ELISA plates. 5The results were confirmed by ELISA using 1000 cells / well. Bound molecules were revealed using mouse anti-HIS HRP-conjugated mAb (Figure 22C). To measure C3b deposition on bacteria, we used the same ELISA in the presence of 0.5% normal human serum (NHS) or decomplemented human serum (ΔNHS) for 30 min. C3b was detected with mouse anti-human C3 / C3b / iC3b mAb, followed by goat anti-mouse IgG HRP-conjugated revealing antibody. Addition of Psl scFv / FHR1 (SCR3-5) heterotetraboro increased C3b deposition only in the presence of non-complemented serum, thereby activating complement on the surface of PAO1 P. aeruginosa cells, whereas ΔNHS completely abolished C3b deposition (Figure 22D).

[0254] 5.4. Demonstration of killing of Pseudomonas aeruginosa by heterotetraboro Psl / FHR1 (SCR3-5) Direct killing of Psl / FHR1(SCR3-5) was demonstrated using PAO1-luciferase P. aeruginosa strain after 5 h incubation with heterotetraboro. Bacterial growth was measured (luciferase signal in RLU) at different time points (5 h). Psl scFv / FHR1(SCR3-5) heterotetraboro induced direct killing of bacteria in the presence of 50% NHS and blocked bacterial growth throughout the experimental period (5 h). The last three SCRs of FHR1 (SCR3-5), which contain the binding domain of C3b that activates the alternative complement pathway, are sufficient to trigger efficient FH regulation locally. NHS alone has only a weak bactericidal effect (background). This innovative construct selectively and locally induces activation of the alternative complement pathway (AP) at targeted membrane surfaces and represents a new approach for destructive cell targeting.

[0255] Example 6. Preparation of multi-target CCP1-2 mimetic IgG Introduction The complement system is a complex innate immune surveillance system responsible for defense against invading pathogens, inflammation, and host homeostasis. It can distinguish between self, modified self (such as senescent, apoptotic, or necrotic cells), and non-self, and tag the last two types of cells / pathogens, but not the first type, as danger signals, which (i) triggers direct complement-mediated cytotoxicity (CDC) or (ii) recruits and activates immune effector cells (such as NK cells, macrophages) for complement-mediated cytotoxicity (CDCC) or phagocytosis (CDCP). The complement system is the first line of defense against invading microorganisms and is responsible for eliminating the majority of bacterial infections. Selective pattern recognition molecules such as mannan-binding lectin (MBL), C1q, properdin, and antibodies are circulating molecules responsible for this self / modified self / non-self discrimination. Four mem...

Claims

1. A first polypeptide comprising a first functional component and a C-terminal fragment of the C4b-binding protein (C4bp) β-chain, wherein the first functional component is bound to the C-terminus of the C-terminal fragment of the C4bp β-chain; and A second polypeptide comprising a second functional component and a C-terminal fragment of the C4bp β-chain, wherein the second functional component is bound to the C-terminus of the C-terminal fragment of the C4bp β-chain A dimeric protein complex comprising, The first and second functional components are monomeric Fc; the hinge region of the monomeric Fc in the first polypeptide is connected to the hinge region of the monomeric Fc in the second polypeptide by at least two disulfide bonds; The first polypeptide further comprises a third functional component bound to the N-terminus of the C-terminal fragment of the C4bp β-chain; The second polypeptide further comprises a fourth functional component bound to the N-terminus of the C-terminal fragment of the C4bp β-chain; The third and / or fourth functional components comprise a binding domain; The first and second polypeptides are the same or different, A dimeric protein complex.

2. The dimeric protein complex according to claim 1, wherein the binding domain is a single-chain variable fragment (scFv) of an antibody specific for an antigen or a single-domain variable fragment (VHH) of a heavy-chain antibody, an antibody-like scaffold, an extracellular domain of a viral envelope protein, a homologous extracellular domain of an antigen receptor or ligand, or an antigen-binding portion of the receptor or ligand or a soluble receptor or synthetic receptor.

3. The dimeric protein complex according to claim 1, wherein the binding domain specifically binds to a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), a bacterial antigen, a viral antigen or a virus-related antigen, a fungal antigen, an activated NK cell receptor, a cytokine, a toxin or a contaminant.

4. The dimeric protein complex according to claim 3, wherein the third and fourth functional components comprise complement control proteins (CCP) 1 and 2 of the C4bp α-chain.

5. The dimeric protein complex according to claim 1, wherein the first and second functional components are monomeric Fc of IgG comprising the hinge, CH2 domain and CH3 domain of IgG.

6. A nucleic acid encoding the first polypeptide of the dimeric protein complex according to any one of claims 1 to 5, and / or the second polypeptide of the dimeric protein complex according to any one of claims 1 to 5.

7. An expression cassette comprising the nucleic acid according to claim 6.

8. An expression vector comprising the nucleic acid according to claim 6 or the expression cassette according to claim 7.

9. A pharmaceutical composition comprising the dimeric protein complex according to any one of claims 1 to 5, the nucleic acid according to claim 6, the expression cassette according to claim 7, or the expression vector according to claim 8, and a pharmaceutically acceptable carrier.

10. The dimeric protein complex according to any one of claims 1 to 5, the nucleic acid according to claim 6, the expression cassette according to claim 7, the expression vector according to claim 8, or the pharmaceutical composition according to claim 9 for use as a medicament, preferably for use in immunotherapy.

11. The dimeric protein complex according to any one of claims 1 to 5, the nucleic acid according to claim 6, the expression cassette according to claim 7, the expression vector according to claim 8, or the pharmaceutical composition according to claim 9 for use in the treatment of a neoplastic disease or an infectious disease.

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