Recombinant immunotoxins containing ribotoxins or RNAses

Abstract

The present invention relates to binder-toxin fusion proteins comprising at least one protein binder selected from the group consisting of an antibody, an antibody fragment or derivative that retains target binding ability, or an antibody mimetic, a ribotoxin or a protoxin, and optionally a peptide linker connecting a) and b) and / or a cleavable domain contained in the protoxin.

Description

[Technical Field]

[0001] The present application relates to the field of binder-toxin fusion proteins. [Background technology]

[0002] Conjugates combining target binders and toxins were developed 40 years ago and now offer great hope for fighting cancer. These conjugates are primarily represented by the class of antibody-drug conjugates (ADCs), which consist of a monoclonal antibody chemically linked to a chemical cytotoxic agent via a linker. These drugs combine the specificity of monoclonal antibodies to target cancer cells with the high toxic potency of the payload, killing target cells while sparing healthy tissue.

[0003] There is still a need for such new entities to provide better treatments for various types of tumors. Accordingly, one object of the present invention is to provide such new entities.

[0004] A further object of the present invention is to provide alternative or even better treatment options for cancer patients.

[0005] These and other objects are achieved according to the invention by the methods and means set forth in the independent claims. The dependent claims relate to specific embodiments. Summary of the Invention

[0006] The methodology used to conceive and reduce the present invention is disclosed in PCT application PCT / EP2020 / 054263, the contents of which are incorporated herein by reference in their entirety. The definitions and embodiments disclosed therein form part of the present disclosure. For clarity, the text of PCT application PCT / EP2020 / 054263 is attached hereto and forms part of the disclosure thereof.

[0007] MODE FOR CARRYING OUT THE INVENTION Before describing the present invention in detail, it is to be understood that the present invention is not limited to the specific component parts of the described devices or to the process steps of the described methods, as such devices and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural references unless the context clearly dictates otherwise. Furthermore, when a range of parameters bounded by numerical values ​​is given, it should be understood that the range is intended to include those limits.

[0008] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate, unrelated embodiments. Features discussed in one embodiment are meant to be disclosed in relation to other embodiments shown herein. In one case, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, those skilled in the art will understand that this does not necessarily mean that the feature is not disclosed in the other embodiments. Those skilled in the art will understand that while it is the intent of the present application to disclose the feature in other embodiments as well, this has not been done merely for the sake of clarity and to keep the description manageable.

[0009] Furthermore, the contents of the prior art documents referred to herein are incorporated by reference. This refers in particular to prior art documents that disclose standard or conventional methods. In that case, incorporation by reference is intended to provide a fully enabling disclosure and to avoid lengthy repetition.

[0010] Embodiments of the invention are set forth in the claims.

[0011] According to one embodiment, there is provided a binder-toxin fusion protein comprising anisoprine or an active fragment thereof. Preferably, the binder-toxin fusion protein comprises a toxin sequence set forth in SEQ ID NO: 48 or 49, or a homolog thereof having at least 66% sequence identity to SEQ ID NO: 48 or 49.

[0012] According to one embodiment, there is provided a binder-toxin fusion protein comprising an anisopurine homolog or an active fragment thereof. Preferably, the binder-toxin fusion protein comprises a toxin sequence set forth in SEQ ID NO: 51, 52, or 53, or a homolog thereof having at least 66% sequence identity to SEQ ID NO: 51, 52, or 53.

[0013] Anisoprine is a fungal ribotoxin naturally produced by the entomopathogenic fungus Metarhizium anisopliae. M. anisopliae was first employed in the late 1800s for the biological control of wheat grain beetles. Since then, biocontrol agents using this fungus have evolved significantly. In recent years, as insecticide resistance has emerged and malaria control has become more difficult, M. anisopliae has emerged as a promising alternative for controlling adult malaria vectors, such as the Anopheles gambiae mosquito.

[0014] Anisoprine has not yet been reported in the context of antitumor therapy or as a toxin component of binder-toxin fusion proteins. The present inventors were the first to explore the potential of anisoprine in these settings and surprisingly discovered that this toxin possesses excellent properties that make it suitable for these applications.

[0015] In some embodiments, the toxin sequence has ≧67%;≧68%;≧69%;≧70%;≧71%;≧72%;≧73%;≧74%;≧75%;≧76%;≧77%;≧78%;≧79%;≧80%;≧81%;≧82%;≧83%;≧84%;≧85%;≧86%;≧87%;≧88%;≧89%;≧90%;≧91%;≧92%;≧93%;≧94%;≧95%;≧96%;≧97%;≧98%;≧99% sequence identity with SEQ ID NO: 48 or 49, and most preferably 100%.

[0016] According to one embodiment, the protein binder is selected from the group consisting of: ·antibody antibody fragments or derivatives that retain target binding ability, or · Antibody mimetics.

[0017] According to one embodiment, the binder-toxin fusion protein comprises a peptide linker connecting the binder or a domain thereof and the toxin or a cleavable domain contained in the toxin.

[0018] According to some embodiments of the binder-toxin fusion protein, the peptide linker or cleavable domain is specifically or non-specifically cleavable by an enzyme expressed by a mammalian cell or produced by a mammalian host; and / or the peptide linker or cleavable domain is not cleaved by an enzyme expressed by the plant cell or produced by the plant host; and / or The binder-toxin fusion protein is expressed in transfected plant cells or in whole transfected plants.

[0019] Those skilled in the art will appreciate that Peptide linker orThere are many standard methods at hand for determining whether a cleavable domain in a protoxin satisfies the condition that it is not cleaved by an enzyme expressed by a plant cell or produced by a plant host. See, for example, Wilbers et al. (2016). Those skilled in the art can also use standard methods to determine whether a peptide linker or cleavable domain is specifically or nonspecifically cleavable by an enzyme expressed by a mammalian cell or produced by a mammalian host.

[0020] According to one embodiment, the protein binder binds to human CD20 or human CD79B.

[0021] According to a further aspect of the invention, the binder-toxin fusion protein comprises at least: a) one protein binder selected from the group consisting of: ·antibody antibody fragments or derivatives that retain target binding ability, or Antibody mimetics, b) RNAse, ribotoxin or their respective protoxins, and c) Optionally, a peptide linker connecting the binder or a domain thereof to the cleavable domain contained in the toxin or protoxin.

[0022] According to one embodiment, such binder-toxin fusion proteins are in one of the formats selected from the group consisting of: (scFv-FC)-(linker)-toxin (dimer), a tetramer of two HC and two LC-(linker)-toxins, a tetramer of two LCs and two HC-(linker)-toxins, or a tetramer of two LC-(linker) toxins and two HC-(linker) toxins, Here, the linker is optional.

[0023] FIG. 1 illustrates some of the possible binder-toxin fusion protein formats. CH3 = heavy chain constant domain 3 CH2 = heavy chain constant domain 2 V L = light chain variable domain V H = heavy chain variable domain FC = antibody FC domain LC = light chain HC=heavy chain

[0024] According to a further aspect, · Peptide linker or Protoxin Medium the cleavable domain of is specifically or non-specifically cleavable by an enzyme expressed by a mammalian cell or produced by a mammalian host, and / or · Peptide linker or The cleavable domain in the protoxin cannot be cleaved by enzymes expressed by the plant cell or produced by the plant host.

[0025] According to one embodiment, such binder-toxin fusion proteins are expressed in transfected plant cells or in whole transfected plants.

[0026] According to one embodiment, the protein binder in such binder-toxin fusion protein binds to human CD20 or human CD79B.

[0027] CD79b (B-cell antigen receptor complex-associated protein β-chain) is a surface protein involved in humoral immune responses. CD79b is produced by B cells. It binds to CD79a via disulfide bridges. Two of these heterodimers bind to membrane-bound antibodies of the mIgM or mIgD subtype to form the antigen-binding B cell receptor (BCR). CD79b promotes the phosphorylation of CD79a. After antigen binding, the antigen-antibody BCR is endocytosed. CD79b is glycosylated. It contains ITAM motifs intracellularly, and upon BCR activation, it binds to and phosphorylates the protein kinases Syk and Lyn.

[0028] The complete sequence of CD79b was first published by Hashimoto et al. Immunogenetics. 1994;40(2):145-149. Protein binders to CD79b have been described in the art. The first mouse antibody against CD79b, called SN8, was published by Okazaki et al., Blood, 81:84-94 (1993). Polson et al., Blood. 2007;110(2):616-623, discusses the possibility of producing antibody-drug conjugates (ADCs) and recombinant immunotoxins against CD79b. The first humanized anti-CD79b antibody (polatuzumab) is disclosed in US8545850. This patent also discloses an ADC in which polatuzumab is linked to MMAE.

[0029] The B lymphocyte antigen CD20 is expressed on the surface of all B cells, beginning with the pro-B stage. In humans, CD20 is encoded by the MS4A1 gene. This protein has no known natural ligand, and its function is to enable optimal B cell immune responses, particularly to T-independent antigens. It is suspected to act as a calcium channel in the plasma membrane. CD20 is induced by CXCR4 / SDF1 (CXCL12) chemokine signaling during interactions with the microenvironment, and its molecular function has been linked to the signaling propensity of the B cell receptor (BCR).

[0030] CD20 is the target of the monoclonal antibodies rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, and ublituximab, all of which are effective agents for the treatment of B-cell lymphomas, leukemias, and B-cell autoimmune diseases. All of these antibodies, including their sequences, are well described in the prior art and are considered to be disclosed in terms of the present invention.

[0031] The term "ribotoxin" as used herein refers to a group of extracellular ribonucleases (RNases) secreted by fungi. Their most notable feature is their specificity. Ribosomes are inactivated by cleaving a single phosphodiester bond in rRNA at a universally conserved sequence. This cleavage triggers cell death by apoptosis. However, as extracellular proteins, they must first enter the target cell to exert their cytotoxic effect. This entry constitutes a rhythmic step in their activity.

[0032] All known ribotoxins are proteins of 130–150 amino acids that share at least two distinct secondary structural elements: a beta sheet containing the active center and a short alpha helix. This structural arrangement is very similar to that of other nontoxic extracellular fungal RNases, including RNase T1 from Aspergillus oryzae, which constitute a family of ribotoxins. For this reason, ribotoxins are considered to be representative of toxic RNases. Ribotoxins contain long, disordered, positively charged loops, whereas their nontoxic "relatives" are much shorter and negatively charged. Observation of their three-dimensional structures reveals their functional differences in toxicity. This binding of ribotoxins is responsible for recognizing both negatively charged acidic phospholipids, which facilitates cell entry, and specific ribosome-specific features, which cause inactivation.

[0033] Regardless of their toxicity, ribotoxins cleave RNA according to a general acid-base mechanism common to all extracellular fungal RNases characterized to date. Using dinucleosides such as GpA, cleavage of the 3'-5' phosphodiester bond of the substrate has been shown to occur via the formation of a cyclic intermediate that gives the corresponding derivative 3'-monophosphate as the final product of the reaction. This cyclic intermediate is then hydrolyzed following a transphosphorylation reaction. For this reason, these proteins are called cyclant RNases.

[0034] According to different embodiments, the ribotoxin is a toxin selected from the group consisting of: Sarusin Restrictocin Anisopurine Hirsutellin Clavin Mitogelin Ageritin, and Gigantin.

[0035] Ribotoxins have been detected in a variety of fungi, including insect pathogens and edible species, but the three-dimensional structures of only three have been elucidated: α-sarcin, restrictocin, and hirsuterin A (HtA). The first two are produced by Aspergillus giganteus and Aspergillus restrictus, respectively, and are nearly identical.

[0036] In one embodiment, the ribotoxin is .alpha.-sarcin, or an active fragment thereof.

[0037] Various variants of α-sarcin exist, examples of which are published under the UniProt identifiers P00655, Q7LVR0, O14446, O13323, O13324, O13322, O13325, and A0A0G2DUB2. While P00655 is used in some examples of the present application, other sarcin variants can be used as well. Those skilled in the art can find such variants in the respective databases with routine effort. An exemplary sequence of sarcin is shown in SEQ ID NO: 56, which represents a deimmunized variant thereof. SEQ ID NO: 55 represents the wild-type.

[0038] In one embodiment, the ribotoxin is hirsutellin A (HtA), or an active fragment thereof. HtA, produced by the entomopathogenic fungus Hirsutella thompsonii, is much smaller, exhibiting only 25% sequence identity with other large ribotoxins, yet inherits all the functional characteristics of the family. Various variants of hirsuterin A exist, examples of which are published under the UniProt identifiers N4VY63, P78696, A0A0B4HUA1, A0A0B4FSP6, T5AB58, A0A0B4EQU3, E9FCV0, A0A014PJJ6, A0A0B4GG41, L2G0X6, A0A063C0Y4, A0A179FJ94, A0A166WTA3, A1CDH8, I8AC84, A0A364MLV5, A0A4Q7JNA6, Q8NJP2, Q8NJP0, Q8NJP3, Q8NJP1, Q8NJN9, Q8NIC7, and E9E2C8. Some examples of the present application use N4VY63, but other hirsuterin A variants can be used as well. Those skilled in the art can find such variants in the respective databases with routine effort. One exemplary sequence of hirsuterin A is shown in SEQ ID NO:47.

[0039] In one embodiment, the ribotoxin is restrictocin (sometimes also called mitogenin), or an active fragment thereof (UniProt identifier: P67876). One exemplary sequence of restrictocin is shown in SEQ ID NO: 27.

[0040] Other variants may also be used, and those skilled in the art can find such variants in the respective databases with routine effort.

[0041] In one embodiment, the ribotoxin is clavin or an active fragment thereof. Different variants of clavin exist, examples of which are published under the UniProt identifiers P0CL70, P0CL71, E0YUC8, A0A4R8PRX1, A0A4R8T0U3, U4KU86, A0A4R8R208, and U4KUQ3. Other variants may also be used. Those skilled in the art can find such variants in the respective databases with routine effort.

[0042] In one embodiment, the ribotoxin is gigantin or an active fragment thereof (UniProt identifier P87063). Other variants may also be used. Those skilled in the art can find such variants in the respective databases with routine effort.

[0043] In one embodiment, the ribotoxin is anisopurine, or an active fragment thereof (see, e.g., SEQ ID NO: 48, and modified variants of SEQ ID NO: 49), which is produced by the fungus Metarhizium anisopliae, which is also an insect pathogen.

[0044] As used herein, the term "RNase" refers to a group of catalytic nucleases ("ribonucleases") that degrade RNA into smaller components. Ribonucleases are divided into endoribonucleases and exoribonucleases, and include several subclasses within the EC 2.7 (phosphorylating) and 3.1 (hydrolase) classes of enzymes.

[0045] The main types of endoribonucleases disclosed herein are: EC 3.1.27.5: RNase A is a commonly used RNase in research. RNase A (e.g., bovine pancreatic ribonuclease A: PDB:2AAS) is one of the most robust enzymes used in general laboratories. One method for isolating it is by boiling crude cell extracts until all enzymes except RNase A are denatured. It is specific for single-stranded RNA. It cleaves the 3'-terminus of unpaired C and U residues, ultimately forming the 3'-phosphorylated form via a 2',3'-cyclic monophosphate intermediate. It does not require cofactors for its activity.

[0046] EC 3.1.26.4: RNase H is a ribonuclease that cleaves the RNA in a DNA / RNA duplex to generate ssDNA. RNase H is a nonspecific endonuclease that catalyzes RNA cleavage by a hydrolytic mechanism with the aid of a divalent metal ion bound to the enzyme. RNase H leaves a 5'-phosphate.

[0047] EC 3.1.26.3: RNase III is a ribonuclease that cleaves rRNA (16s rRNA and 23s rRNA) from transcribed polycistronic RNA operons in prokaryotes. Also, the Dicer family of RNases, which digest double-stranded RNA (dsRNA), cleaves pre-mRNA (60-70 bp in length) at specific sites and converts them into miRNAs (22-30 bp) that are actively involved in regulating transcription and mRNA lifespan.

[0048] EC number 3.1.26: RNase L is an interferon-induced nuclease that, upon activation, destroys all RNA in the cell.

[0049] EC 3.1.26.5: RNase P is a type of ribonuclease characterized by its catalytic activity as a ribozyme, similar to an enzyme. One of its functions is to cleave the leader sequence from the 5' end of a single-stranded pre-tRNA. RNase P is known to be one of two naturally occurring multi-turnover ribozymes (the other being the ribosome). In bacteria, RNase P also carries out the catalytic activity of a holoenzyme, which combines with a cofactor to form an active enzyme system and consists of an apoenzyme that determines the substrate specificity of this system. Recently, a form of RNase P that is an RNA-free protein has been discovered.

[0050] EC number 3.1: RNase PhyM is sequence-specific for single-stranded RNA. It cleaves the 3' end of unpaired A and U residues.

[0051] EC 3.1.27.3: RNase T1 is sequence-specific for single-stranded RNA. It cleaves at the 3' end of unpaired G residues.

[0052] EC 3.1.27.1: RNase T2 is sequence-specific for single-stranded RNA. It cleaves the 3' end of all four residues, but preferentially cleaves the 3' end of As.

[0053] EC 3.1.27.4: RNase U2 is sequence-specific for single-stranded RNA. It cleaves at the 3' end of unpaired A residues.

[0054] EC 3.1.27.8: RNase V is specific for polyadenine and polyuridine RNA.

[0055] EC 3.1.26.12:RNaseE is a plant-derived ribonuclease that regulates the response to DNA damage stress by activating the SOS machinery through a RecA / LexA-dependent signaling pathway, transcriptionally repressing numerous genes that lead to the arrest of cell division and the initiation of DNA repair.

[0056] EC 3.1.26.-: RNase G is involved in processing the 16'-end of 5s rRNA. It is involved in chromosome segregation and cell division. It is thought to be one of the components of the cytoplasmic axial filament bundle. It is also thought to be able to regulate the formation of this structure. Main types of exoribonucleases

[0057] EC number EC 2.7.7.8: Polynucleotide phosphorylase (PNPase) functions not only as a nucleotide transferase but also as an exonuclease.

[0058] EC number EC 2.7.7.56: RNase PH functions as an exonuclease and a nucleotidyl transferase.

[0059] EC number 3.1.??RNase R is a close homolog of RNase II, but unlike RNase II, it can degrade RNA with secondary structure without the help of accessory factors.

[0060] EC number EC 3.1.13.5: RNase D is involved in the 3' to 5' processing of pre-tRNA.

[0061] EC number 3.1?RNase T is responsible for the 3' to 5' maturation of many stable RNAs.

[0062] EC 3.1.13.3: Oligoribonucleases degrade short oligonucleotides into mononucleotides.

[0063] EC 3.1.11.1: Exoribonuclease I degrades single-stranded RNA in the 5' to 3' direction and is found only in eukaryotes.

[0064] EC 3.1.13.1: Exoribonuclease II is a close homolog of exoribonuclease I. In some embodiments, the RNase is one selected from the following group:

[0065] Onconase: (Rampirinase, frog rnase): Different variants of onconase exist, examples of which are published under the UniProt identifiers Q8UVX5, Q9I8V8, Q6EUW9, Q6EUW8, Q6EUW7 or P22069. RNase 1: pancreatic ribonuclease (e.g., RNAse 1, e.g., Uniprot identifier P07998; see e.g., SEQ ID NO: 57) RNase 2: non-secretory ribonuclease (e.g., RNAse2, e.g., Uniprot identifier P10153) RNase 3: Eosinophil cationic protein (e.g., RNAse3 / Drosha, e.g., Uniprot identifier Q9NRR4 or P12724) RNase 4: Ribonuclease 4 (e.g., RNAse4, e.g., Uniprot identifier P34096) RNase 5: Angiogenin (e.g. RNAse 5, e.g. Uniprot identifier P03950, see e.g. SEQ ID NO: 50) RNase 6: ribonuclease K6 / ribonuclease T2 / ribonuclease K3 (e.g., RNAse6, e.g., Uniprot identifier Q93091) RNase 7: Ribonuclease 7 / Ribonuclease A E1 (e.g., RNAse7, e.g., Uniprot identifier Q9H1E1) RNase 8: Ribonuclease 8 (e.g., RNAse 8, e.g., Uniprot identifier Q8TDE3)

[0066] The above Uniprot identifiers are for illustrative purposes only. Other variants may also be used. Those skilled in the art can find such variants in the respective databases with routine effort.

[0067] In some embodiments, Peptide linker or Cleavable in protoxin Na The domain is specifically or non-specifically cleavable by an enzyme expressed by a mammalian cell or produced by a mammalian host, or is not cleavable by an enzyme expressed by a plant cell or produced by a plant host.

[0068] In one embodiment, the binder-toxin fusion protein is produced in a plant host or plant cells. As noted elsewhere, this provides the option of creating constructs with linkers that are cleavable by mammalian enzymes that are not present in the plant host. Thus, self-intoxication of the production system is avoided, and the cleavable linker allows for rapid release of the toxin in vivo.

[0069] Binder-Toxin Fusion Proteins In one embodiment, the binder-toxin fusion proteins are produced in mammalian cells, such as CHO cells.

[0070] In one embodiment, the plant host or plant cell is transiently modified with a vector that specifically encodes a binder-toxin fusion protein.

[0071] In one embodiment, the plant host or plant cell is permanently modified with a vector that specifically encodes a binder-toxin fusion protein.

[0072] Background on methods for transiently or permanently expressing binder-toxin fusion proteins in plant hosts or plant cells is provided in WO2020169620, the contents of which are incorporated herein by reference for enabling purposes.

[0073] In one embodiment, the plant host or plant cell is of the genus Nicotiana. Peptide linker or Cleavable in protoxin NaThe domain cannot be cleaved by enzymes expressed by the plant cell or produced by the plant host, thus protecting the producing plant cell or plant host from self-poisoning due to unwanted cleavage of the binder-toxin fusion protein.

[0074] In one embodiment, the plant or plant cell with which the nucleic acid construct is contacted is not a chloroplast or is not an algal chloroplast, particularly not a Chlamydomonas reinhardtii chloroplast. In another embodiment, the structure in the plant or plant cell with which the nucleic acid construct is contacted is not a chloroplast or is not an algal chloroplast, particularly not a Chlamydomonas reinhardtii chloroplast.

[0075] In another embodiment in which the protein binder comprises two or more strands, two nucleic acid constructs may be provided, the first nucleic acid construct comprising three polynucleotides encoding the first strand of the protein binder, a linker and a toxin, while the second nucleic acid construct comprises a polynucleotide encoding the second strand of the protein binder.

[0076] "Inducible promoters" allow for both transient and stable expression. These promoters selectively express operably linked DNA sequences following the presence of endogenous or exogenous stimuli or in response to chemical, environmental, hormonal, and / or developmental signals. These regulatory elements are sensitive to, but not limited to, ethanol, heat, light, stress, jasmone, salicylic acid, plant hormones, salt, flooding, or drought, as reviewed by Abdel-Ghany et al. (2015) and discussed in US 10,344,290 B2, both of which are incorporated herein by reference. Inducible promoters, including but not limited to, synthetic components, are discussed in Ali et al. (2019), the contents of which are incorporated herein by reference.

[0077] The genus Nicotiana includes tobacco plants. Tobacco plants or plant cells have already been tested to produce recombinant immunotherapeutic binder-toxin fusion proteins composed of small sFv fragments linked to protein toxins by stable linkers (Francisco et al. (1997) and US6140075A).

[0078] According to a further embodiment of the invention, the plant cell is at least one selected from the group consisting of: ·Nicotiana tabacum cv. BY2, Nicotiana tabacum NT-1, Arabidopsis thaliana, Daucus carota (wild carrot) and / or ·Oyrza sativa (rice).

[0079] Nicotiana tabacum cv. BY2, also known as Tobacco BY-2 cells, cv. Nicotiana tabacum 1 (NT-1, a sibling of BY-2), is a non-green, fast-growing plant cell line that can increase in number up to 100-fold within a week under appropriate culture conditions. This tobacco variety is cultivated as a cell culture, more specifically as a cell suspension culture (a specialized cell population grown in a liquid medium, cultivated by scientists to study specific biological properties of plant cells). In a cell suspension culture, each cell is independent, or at most, suspended in short chains in the culture medium. Each cell has similar properties to the other cells.

[0080] Model plant systems are comparable to HeLa cells for human research. Their relative simplicity and predictability facilitate the study of biological processes and can serve as an intermediate step toward understanding more complex organisms. They are used as model organisms by plant physiologists and molecular biologists, and also as model systems for higher plants due to their relatively high uniformity and rapid growth rate, characterizing the yet general behavior of plant cells. The diversity of cell types within any given part of a naturally grown plant (in vivo) makes it extremely difficult to investigate and understand some of the general biochemical phenomena of living plant cells. For example, solute transport into and out of cells is difficult to study because specialized cells in multicellular organisms behave differently. Cell suspension cultures, such as tobacco BY-2, provide a good model system for these studies at the level of single cells and their compartments, because tobacco BY-2 cells behave very similarly to each other. The influence of neighboring cell behavior is less significant in suspension than in intact plants. As a result, changes observed after a stimulus can be statistically correlated, allowing researchers to determine whether these changes represent a response to the stimulus or are merely coincidental. BY-2 and NT-1 cells are relatively well understood and are often used in studies involving the expression of heterologous proteins, particularly antibodies (Hellwig et al. (2004)). Such methods are disclosed in Hakkinen et al. (2018), the contents of which are incorporated herein by reference.

[0081] Torres (1989) discusses methods for establishing carrot cell suspension cultures (Daucus carota). Shaaltiel et al. (2007) discuss the production of enzymes using carrot cell-based expression systems, the contents of which are incorporated herein by reference. Daucus carota and Oryza sativa are also discussed as suitable plant cell-based expression systems in Santos et al. (2016), the contents of which are incorporated herein by reference. Production of recombinant proteins in Nicotiana tabacum, Arabidopsis thaliana, and Oryza sativa is disclosed in Plasson et al. (2009), the contents of which are incorporated herein by reference.

[0082] In general, the present invention can be practiced with any plant variety whose cells can be transformed with a DNA construct suitable for expression of a foreign polypeptide and cultured under standard plant cell culture conditions. Callus culture or other conventional plant cell culture methods may be used, although plant cell suspension or plant tissue culture is preferred.

[0083] According to another embodiment of the present invention, the plant is Nicotiana benthamiana. The production of antibodies in Nicotiana plants is disclosed, for example, in Daniell et al. (2001), the contents of which are incorporated herein by reference.

[0084] Other plants or plant cells that can be used in the context of the present invention include, but are not limited to, lettuce (Lactuca spp.), spinach (Spinacia oleracea), and Arabidopsis (Arabidopsis spp.).

[0085] In some embodiments, the cleavage site is selected from the group consisting of: a) Endosomal and / or lysosomal protease cleavage sites b) a cytosolic protease cleavage site, and / or c) Cleavage sites of cell surface proteases. Examples of such enzymes and their cleavage sites are shown in the table below (see also Choi et al. (2012)), the contents of which are incorporated herein by reference. The table also references the "Merops" database to access further information about each enzyme: https: / / www.ebi.ac.uk / merops / index.shtml. JPEG0007818834000001.jpg121170JPEG0007818834000002.jpg108170

[0086] The cleavage site is described from the cleavage point (represented by ↓). The letter x means all amino acids. If there are several preferred amino acids, they are separated by a slash ( / ).

[0087] Such enzymes are preferably proteases. In one embodiment, the peptide linker is not cleavable by plant enzymes.

[0088] Furin belongs to the subtilisin-like proprotein convertase family and is an enzyme that cleaves proteins at the C-terminus with the canonical basic amino acid sequence motif Arg-X-Arg / Lys-Arg (RX(R / K)R), where X can be any naturally occurring proteinogenic amino acid. Said motif is referred to herein as the furin cleavage site. ###

[0089] Preferably, the sequence is HRRRKRSLDTS (SEQ ID NO: 46, also referred to herein as Liop or FCS I ("Furin cleavage site 1")). Further cleavable linkers that can be used in the context of the present invention are TRHRQPRGWEQL (SEQ ID NO: 44, also referred to herein as Fpe or FCS II) and AGNRVRRSVG (SEQ ID NO: 45, also referred to herein as Fdt or FCS III).

[0090] Cathepsins are proteases found in all animals as well as other organisms. Most members are activated at the low pH found in lysosomes. Cathepsin B can cleave peptide sequences containing the dipeptide motif Val-Ala (VA). Said motif is referred to herein as the cathepsin B cleavage site. Those skilled in the art will find sufficient information regarding cathepsins and their cleavage sites in Turk et al (2012), the contents of which are incorporated herein by reference.

[0091] Caspases (cysteine-aspartic acid proteases, cysteine ​​aspartases, or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes that play an essential role in programmed cell death. More than 1,500 caspase substrates have been discovered in the human proteome. A common cleavage motif is DXXD-A / G / S / T, where X can be any naturally occurring proteinogenic amino acid. Those skilled in the art will find Kumar el al (2014) replete with information about caspases and their cleavage sites, the contents of which are incorporated herein by reference.

[0092] Matrix metalloproteinases (MMPs), also known as matrixins, are calcium-dependent, zinc-containing endopeptidases. Other family members are adamalysins, serralysins, and astacins. Collectively, these enzymes can degrade all types of extracellular matrix proteins, but can also process many bioactive molecules. Those skilled in the art will find ample information about matrix metalloproteinases and their cleavage sites in Eckard et al. (2016), the contents of which are incorporated herein by reference.

[0093] In general, those skilled in the art can, by routine consideration and literature reference, select specific cleavage sites consistent with the respective mammalian enzymes to control the target-specific release of protein toxins or protoxins. General guidelines for finding these cleavage sites are disclosed, for example, in Rawlings (2016).

[0094] According to one embodiment of the present invention, the protein toxin or protoxin is a deimmunized variant of a native protein toxin. Recombinant methods for deimmunizing protein toxins by sequence modification have been disclosed, for example, in Schmohl et al. (2015) or Grinberg and Benhar (2017), the contents of which are incorporated herein by reference.

[0095] In one embodiment, the protein toxin or protoxin is not toxic to plants or plant cells. Those skilled in the art have a range of routine methods at hand to check whether this condition is met. For an overview, see, for example, Klaine and Lewis (1995), the contents of which are incorporated herein by reference.

[0096] According to one embodiment of the present invention, the protein contains at least one plant-specific N-glycan. N-glycans are glycans attached to the amide group of asparagine (Asn) residues in proteins, often with the Asn-X-Thr or Asn-X-Ser (NXT or NXS) motif (where X is any amino acid except proline). Exemplary plant-specific N-glycans are described in Gomord et al. (2010) and are significantly different from mammalian N-glycan patterns. In this respect, it should be emphasized that the N-glycans produced by plants are significantly different from those produced by, for example, mammals. In particular, the N-glycans produced by tobacco plants have the following: A fucose residue bound to an N-acetylglucosamine residue via an α3 glycosidic bond (rather than an α6, as in mammals) A xylose residue linked to the proximal mannose residue via a β2 glycosidic bond Two distal N-acetyl-glucosamine residues (instead of neuraminic acid in mammals) carrying a fucose residue via an α3 glycosidic bond and a galactose residue via a β3 glycosidic bond, respectively

[0097] On the other hand, recombinantly expressed proteins in algae often lack glycosylation, but algae can express IgG antibodies and antibody fragments with one or more disulfide bridges.

[0098] The predominant plant-derived glycoform identified is the complex glycan (GnGn / GnGnXF). Other glycoforms (Man5-Man9, GnGnF, GnGnX, MMXF, Man5Gn, and GnM(X)(F)) are also detectable.

[0099] According to this nomenclature, MGnX means, for example: [ka] Background on methods for analyzing peptide glycoforms is provided in WO2020169620, the contents of which are incorporated herein by reference for enabling purposes.

[0100] According to another aspect of the present invention there is provided at least a binder-toxin fusion protein as described above, optionally comprising one or more pharmaceutically acceptable excipients.

[0101] According to another aspect of the present invention, there is provided a combination comprising (i) the binder-toxin fusion protein described above or the pharmaceutical composition described above, and (ii) one or more additional therapeutically active compounds.

[0102] According to another aspect of the present invention, binder-toxin fusion proteins, compositions or combinations are provided for use in the treatment of human or animal subjects suffering from, at risk of developing, and / or diagnosed with a neoplastic disease, or in the (manufacture of a medicament for) the prevention of such conditions.

[0103] According to another aspect of the present invention, there is provided a method for treating a human or animal subject suffering from, at risk of developing, and / or diagnosed with a neoplastic disease, or for preventing such a condition, said method comprising administering a therapeutically effective amount of a binder-toxin fusion protein, composition or combination as described above. [Brief explanation of the drawings]

[0104] [Figure 1] Some possible binder-toxin fusion protein formats are shown: CH3 = heavy chain constant domain 3; CH2 = heavy chain constant domain 2; VL = light chain variable domain; VH = heavy chain variable domain; FC = antibody FC domain; LC = light chain; HC = heavy chain. [Figure 2] See Table 3 [Figure 3] See Table 3 [Figure 4] See Table 3 [Figure 5] See Table 3 [Figure 6] See Table 3 [Figure 7] See Table 3 [Figure 8] See Table 3 [Figure 9] See Table 3 [Figure 10] See Table 3 [Figure 11] See Table 3 [Figure 12] See Table 3 [Figure 13] See Table 3 [Figure 14] See Table 3 [Figure 15] See Table 3 [Figure 16] See Table 3 [Figure 17] See Table 3 [Figure 18] See Table 3 [Figure 19] See Table 3 [Figure 20] See Table 3 [Figure 21] See Table 3 [Figure 22] See Table 3 [Figure 23] See Table 3 [Figure 24] See Table 3 [Figure 25] See Table 3 [Figure 26] See Table 3 [Figure 27] See Table 3 [Figure 28] See Table 3 [Figure 29] See Table 3 [Figure 30] See Table 3 [Figure 31] See Table 3 [Figure 32] See Table 3 [Figure 33] See Table 3 [Figure 34] See Table 3 [Figure 35] See Table 3 [Figure 36] See Table 3 [Figure 37] See Table 3 [Example]

[0105] While the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive, and the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0106] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0107] The examples are based on experiments performed with both HPRNAse and anisopurine, as well as other toxins, although the experimental protocols also apply to toxins in or closely related to these families.

[0108] The examples are based on experiments using furin-cleavable linkers, but the experimental protocols are applicable to other sequences susceptible to mammalian enzymes. Materials and Methods Gene construct binder-toxin fusion

[0109] The full-length rituximab HC and LC sequences have been used to develop mAb-based binder-toxin fusion proteins. The heavy and light chain variable regions of the rituximab sequence were assembled into a single-chain scFv and fused to a human IgG1 Fc region sequence. Next, the α-sarcin sequence was fused to the C-terminal portion of the LC or HC of full-length rituximab or to the C-terminal portion of the scFv-Fc using a human furin cleavage sequence, resulting in the HC+LC-FCS-α-sarcin, HC-FCS-α-sarcin+LC, and scFv-c-FCS-α-sarcin fusion protein sequences. Another binder-toxin fusion protein was realized by linking the scFv-Fc portion to α-sarcin without the cleavage site, resulting in scFv-Fc-α-sarcin. These sequences were generated by gene synthesis flanked by XbaI and IsceI. Genetic constructs containing antibodies

[0110] The full-length HC and LC antibody sequences were used to develop antibody-based binder-toxin fusion proteins. The unpublished heavy and light chain variable region sequences were assembled into a single-chain scFv and fused to a human IgG1 Fc region sequence. Next, using a human furin cleavage sequence, the human anisopurine sequence was fused to the LC or HC or both of the full-length undisclosed antibody or to the C-terminal end of the scFv-Fc, resulting in the following fusion protein sequences: HC+LC-FCS-anisopurine, HC-FCS-anisopurine+LC, and scFv-Fc-FCS-anisopurine or scFv-Fc-anisopurine. Additionally, the scFv-Fc, HC, and LC portions were linked to anisopurine without the cleavage site, resulting in the following binder-toxin fusion proteins: scFv-Fc-anisopurine, HC+LC-anisopurine, HC-anisopurine+LC, and LC-anisopurine+HC-anisopurine. These sequences were generated by gene synthesis flanked by XbaI and IsceI. Transient expression in Nicotiana benthamiana leaves

[0111] Nicotiana benthaminana was grown under a 16-hour light / 8-hour dark photocycle at 22±3°C. Leaves from 7-8 week-old plants were transiently transformed by syringe infiltration. The optical density at 600 nm (OD 600 Agrobacterium tumefaciens GV3101 (pMP90RK) carrying an unpublished plasmid containing a gene construct with an OD of 0.8–1.0 was harvested by centrifugation at 3500 g for 10 min. Finally, the bacteria were grown in infiltration buffer (10 mM MgCl2, 10 mM MES, 100 μM acetosyringone, pH 5.6) until the OD reached 0.8–1.0. 600 The pH was adjusted to 0.5 and infiltrated using a needless syringe. The infiltrated areas were harvested on days 4 and 6 after agroinfiltration. Whole leaves harvested on day 4 after agroinfiltration were used for protein A purification. Expression in N. tabacum cells

[0112] Nicotiana tabacum plant suspension cells were cultured in the plant culture medium described by Nagata et al. (1992), the contents of which are incorporated herein, at 130 rpm and 25°C for 5 days. The optical density at 600 nm (OD 600Agrobacterium tumefaciens LBA4404 (pBBR1MCS-5.virGN54D) harboring the pPZP-ATB binary plasmid, with a chromatin density of 0.8–1.0, was harvested by centrifugation at 2000 g for 5 minutes. Plant and bacterial cells were then co-cultured in co-culture medium for 30 minutes, followed by centrifugation at 2000 g for 5 minutes. After removing the supernatant, the cells were plated on solid co-culture medium for two days. For transient transformation, the cells were then harvested, washed three times, and cultured on plant growth medium containing cefotaxime and carbenicillin before being harvested for further analysis. For stable transformation, after two days of solid co-culture, the cells were washed and plated on plant medium containing selective antibiotics kanamycin, cefotaxime, and carbenicillin. After four weeks, calli were selected and subcultured on solid medium or in liquid suspension for further analysis. Protein analysis: ELISA, SDS-PAGE and Western blot

[0113] Harvested leaf tissue (120 mg) was ground in 400 μL of extraction buffer (250 mM sorbitol, 60 mM Tris, Na2EDTA, 0.6% Polyclar AT, pH 8.0). The homogenized tissue was centrifuged at 18,200 g for 40 minutes at 4°C. The supernatant was then collected, frozen in liquid nitrogen, and stored at -20°C.

[0114] Extracted tissues were analyzed by Western blotting. Proteins were boiled for 5 min in reducing or non-reducing SDS loading buffer (80 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue), centrifuged at 13,000 rpm for 5 min, and separated by SDS-PAGE (4-20% polyacrylamide gel electrophoresis). For Western blotting, proteins were electrophoresed onto a PVDF membrane (Biorad) using a semi-dry electrophoresis apparatus (Biorad Trans-Blot Turbo); the membrane was then blocked with 3% (w / v) nonfat dry milk in TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.5) for 1 h at room temperature and incubated (TBS-Tween 0.1% + 0.5% nonfat dry milk) at 1:10,000 dilution with an HRP-conjugated antibody against the Fc-specific region of human IgG (A0170; Sigma-Aldrich) or with an α-sarcin primary antibody (in-house reagent, anti-sarcin rabbit serum, Santa Cruz CAS 86243-64-3) at 1:10,000 dilution for 1 h at room temperature. After the anti-α-sarcin / HPRnase antibody, an HRP-conjugated anti-rabbit antibody (0545; Sigma) was added at a dilution of 1:10 000. Proteins were detected by enhanced chemiluminescence (Amersham Imager 600 / GE; GE Healthcare). Anti-CD79b ELISA

[0115] To analyze the specificity of CD79b-specific conjugates, purified binder-toxin fusion proteins containing CD79b-specific binders were analyzed in 96-well microplates (Greiner). Wells were coated with 50 μL of antigen CD79b (2.5 μg / mL) at 37°C for 1 h, followed by five washes with 250 μL of wash buffer (PBS Tween 0.1%). Blocking was then performed with 150 μL of hydrocasein (3.6%) in PBST for 30 min at RT and five washes. 50 μL of anti-antigen control antibody was loaded to create a standard curve ranging from 5 to 0 μg / mL. 50 μL of samples were loaded into the same 96-well plate for comparison at RT for 1 h and washed five times. 50 μL of 1 / 200,000 diluted detection antibody (goat anti-human HRPO, Bethyl) was loaded and incubated at RT for 1 h. Revelation was then carried out in 50 μL of TMB reaction buffer (Zentech) for 15 min and finally stopped with H3PO4 1M. Enzyme activity was then analyzed by spectrometry at 450 nm. The results are shown in Figure 4B. Protein A purification

[0116] Four days after agroinfiltration, leaves were harvested, weighed, and ground in a blender with 2 mL of extraction buffer (250 mM sorbitol, 60 mM Tris, Na2EDTA, 0.6% Polyclar AT, pH 8.0) per gram of fresh agroinfiltrated leaves. The mixture was then filtered through a double Miracloth (Millipore) layer. The filtrate was then centrifuged at 20,000 xg for 30 minutes at 4°C. The supernatant was loaded onto Protein A resin equilibrated with extraction buffer. The resin was then washed with 10 column volumes of 60 mM Tris, pH 8.0, and eluted with 100 mM glycine, pH 3.0, directly buffered with 10% Tris, pH 8.0. The concentrated protein fraction was then collected and frozen in liquid nitrogen. In vitro cytotoxicity assay

[0117] The effect of binder-toxin fusion proteins on the viability of cell lines expressing CD20 or CD79b was assessed using the Cell Titer Glo Assay (Promega, G9241), in which luciferase catalyzes the mono-oxygenation of luciferin in the presence of Mg 2+ and ATP. This reaction generates a luminescent signal proportional to the number of viable cells.

[0118] Depending on the cell line tested, cells were seeded into wells of a 96-well plate at a density of 2,000 or 5,000 cells / well in 50 μl of growth medium (RPMI 1640). Serial dilutions of binder-toxin fusions were prepared by adding 10 μl of binder-toxin fusion or buffer (PBS, Tween 0.02%) to 40 μl of growth medium. The mixtures were added to the cells and incubated at 37°C and 5% CO2 for 72 hours. Binder and toxin fusions were tested in duplicate. Buffer was used as a negative control, and medium and cells alone served as blank and untreated controls, respectively.

[0119] After 72 hours, the plate was equilibrated at room temperature for 30 minutes, and 100 μl of CellTiter Glo reagent was added to each well. The plate was then placed on a shaking platform for 2 minutes, and the signal was allowed to stabilize for 10 minutes in the dark at room temperature. Luminescence was then recorded.

[0120] Viability was measured by subtracting the mean luminescence signal of the blank (growth medium only) from each well, and the mean luminescence signal of untreated cells was set as 100% viability. The mean signal of treated cells was then normalized and plotted as a function of ATB concentration.

[0121] Anti-CD20-based binder-toxin fusion proteins were evaluated on target cells WSU-NHL (CD20+) and non-target cells K562 (CD20-).

[0122] Anti-CD79b-based binder-toxin fusion proteins were evaluated on target cells JEKO, OCY-LY3, BJAB, and WSU-DLCL2 (CD79+) and non-target cells K-562 (CD79-). In vivo assay: acute toxicity

[0123] To demonstrate the safety of the binder-toxin fusion in animals, an acute toxicity study was conducted using 20g female NOG mice (Taconic). The undisclosed antibody sc-Fv-Fc-α-sarcin (125), α-sarcin alone, and sc-Fv-Fc (86 as a control) were intravenously injected at 20mg / kg, 4.9mg / kg, and 15mg / kg, respectively. Body weights were measured daily for 8 consecutive days after injection. This study was conducted by EPO Experimentelle Pharmakologie & Onkologie Berlin-Buch GmbH, based on materials provided by ATB Therapeutics. Peptide glycoform analysis Background on methods for analyzing peptide glycoforms is provided in WO2020169620, the contents of which are incorporated herein by reference for enabling purposes. Cleavage assay

[0124] It has been demonstrated in vitro that the addition of recombinant furin to purified scFv-Fc-FCS-α-sarcin, scFv-Fc-α-sarcin, or HPRNAse (binder-toxin fusion proteins) releases the toxin via cleavage. One microgram of binder-toxin fusion protein was added to 15 μl of 25 units / ml furin (NEB P8077S) and 15 μl of cleavage buffer (1M sodium acetate, pH 5.5, + 10 mM CaCl2) and incubated at 37°C for 4 hours. Cleavage was visualized on an SDS-PAGE Coomassie Blue gel (4-20% polyacrylamide). CHO transient expression

[0125] For control purposes, several constructs, 414, 301, 452, 221, and 125, were also expressed in CHO cells. This was done using conventional (non-PCR) cloning techniques with a vector system developed by Evitria. Evitria vector plasmids were gene synthesized. Plasmid DNA was prepared under low-endotoxin conditions using anion-exchange chromatography. DNA concentration was determined by measuring absorbance at 260 nm. Sequence accuracy was confirmed by Sanger sequencing (up to two sequencing reactions per plasmid were performed, depending on the size of the cDNA).

[0126] For production, we used suspension-adapted CHO K1 cells (originally received from ATCC and adapted to serum-free culture at Evitria). Seeds were grown in eviGrow medium, a chemically defined, animal-component-free, serum-free medium. Cells were transfected with eviFect, a custom-made transfection reagent developed exclusively by Evitria, and the transfected cells were cultured in eviMake2, an animal-component-free, serum-free medium.

[0127] The supernatant was collected by centrifugation followed by filtration (0.2 pm filter).

[0128] Antibodies were purified using MabSelect SuRe (Cytivia). result

[0129] Several recombinant binder-toxin fusion proteins based on the scFv-Fc format have been constructed: scFv-Fc-FCS-anisoprine, scFv-Fc-anisoprine, scFv-Fc-FCS-HPRNAse, scFv-Fc-HPRNAse, scFv-Fc-FCS-α-sarcin, scFv-Fc-α-sarcin, scFv-Fc-FCS-α-sarcin homolog, scFv-Fc-α-sarcin homolog, scFv-Fc-FCS-HPRNAse homolog, and scFv-Fc-HPRNAse homolog. Full-length mAb-based binder-toxin fusion proteins were constructed with anisoprine, HPRNAse, and α-sarcin: HC+LC-anisoprine, HC-anisoprine or LC, HC-anisoprine+LCanisoprine, LC+HC-FCS-anisoprine or HPRNAse or α-sarcin. As a control, an unconjugated mAb was also constructed. Cell viability measurement method

[0130] The purified binder-toxin fusions were evaluated for cytotoxicity in cancer cell lines. All binder-toxin fusions were shown to impair the viability of positive cell lines. Furthermore, they demonstrated superiority over commercially available ADCs targeting the same antigen, including Polivy®, the first humanized anti-CD79b antibody conjugated to MMAE (polatuzumab), disclosed in US8545850 (see 425 and 507). Furthermore, binder-toxin fusion proteins showed very low efficacy against negative cell lines (see 425 and 125).

[0131] The binder-toxin fusion protein is harmless to target-negative primary cells such as HUVEC and HEP2, but is highly effective against cancer cells. acute toxicity

[0132] Ribotoxin-α-sarcin was well tolerated in mice at a high dose (4.91 mg / kg). Ribotoxin-based binder-toxin fusions exhibited greater activity against cancer cells than ribotoxin alone, and were well tolerated in animal models, with no acute toxicity observed even after intravenous injection of 20 mg / kg of binder-α-sarcin fusions. CHO transient expression

[0133] As a result, it was found that each of the constructs 414, 301, 452, 221, and 125 could actually be produced in CHO cells, although the yield was significantly low.

[0134] For example, the yield of construct 452 was 14-15 times lower in the CHO experiments compared to the Nicotinia experiments. Construct 452 has a G4S linker between the antibody and the toxin, which is not cleaved by mammalian proteases. However, without being bound by theory, it is still possible that one reason for the lower yield in CHO is that spontaneous cleavage or partial autointoxication, which occurs in CHO, does not occur in plants such as Nicotinia.

[0135] Construct 22 has a furin-cleavable linker (Fpe) and similarly has reduced expression in CHO compared to Nicotinia. [Table 2]

[0136] Nevertheless, construct 221 (with a furin-cleavable linker, Fpe) produced in CHO had an IC of 0.2595 nM. 50 Construct 125, which has a non-cleavable G4S linker, reduced cell viability in a dose-dependent manner in the positive cell lines, reaching IC 50 was 0.7792 nM. Summary of experimental results

[0137] The experimental results are summarized in the table below. [Table 3] JPEG0007818834000006.jpg204170JPEG0007818834000007.jpg223170JPEG0007818834000008.jpg47170 definition

[0138] As used herein, "percentage of sequence identity" is determined by comparing two optimally aligned biological sequences (amino acid sequences or polynucleotide sequences) over a comparison window, where the corresponding portions of the sequences in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence that does not contain additions or deletions due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0139] The terms "identical" or percent "identity," with respect to two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequence. Two sequences are "substantially identical" if they have a specified percentage of the same amino acid residues or nucleotides (i.e., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity over a specified region, or, if not specified, the entire reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region, measured using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection.

[0140] The present disclosure provides polypeptides that are substantially identical to the polypeptides exemplified herein.With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, in some cases at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, in some cases at least about 150, 200 or 250 amino acids in length, or over the entire length of the reference sequence.With respect to shorter amino acid sequences, for example, sequences of 20 or less amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted according to the conservative substitutions defined herein.

[0141] The terms "protein toxin" or "protein protoxin" do not exclusively refer to toxins that are, by their chemical nature, proteins (i.e., peptides having a length of 50 amino acid residues or more) or polypeptides (i.e., peptides having a length of 10 to 50 amino acid residues or more). Protoxins in the sense of the present invention are precursors of toxins, also called latent toxins, which must be activated, for example, by cleaving an inhibitory amino acid sequence or by undergoing a conformational change. The terms "protoxin" and " protein The terms "protoxin" and "protoxin" are used interchangeably herein and refer to the same entity.

[0142] As used herein, the term "fusion protein" means a protein having a peptide component operably linked to at least one additional component and which differs from the native protein in its domain composition and / or composition.

[0143] As used herein, the term "operably linked," when referring to two or more polynucleotides, refers to a situation in which different polynucleotides are placed in a functional relationship to each other. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Similarly, a signal peptide coding sequence is operably linked to a polypeptide coding sequence if the signal peptide affects the extracellular secretion of that polypeptide. According to one embodiment of the present invention, when each polynucleotide encodes a different peptide, "operably linked" means that the respective polynucleotides are contiguous and, where necessary to join two protein-coding regions, the open reading frames are aligned.

[0144] As used herein, the term "cleavable peptide linker" refers to an internal amino acid sequence within a fusion protein that contains residues linking the binder moiety and the toxin protein so as to render the toxin protein unable to exert its toxic effects outside the target cell or limit its ability to inhibit cell growth (cytostasis) or cause cell death (cytotoxicity). In this way, the protein toxin remains inactive in plasma until it reaches the target cell, where the cytotoxic payload will be selectively released and / or activated (Grawunder & Stein, 2017). Inside the target cell, the cleavable linker sequence is cleaved, rendering the toxin protein active or toxic. The fusion proteins of the present invention are composed of a cell-specific binder moiety and a protein toxin moiety linked by specific amino acid residues or amino acid sequences that have cleavage recognition sites for specific proteases, including, but not limited to, cancer-specific proteases, and / or are cleavable under specific conditions, such as, but not limited to, acid and / or reducing conditions. Sequences encoding cleavage recognition sites for specific proteases can be identified among known ubiquitous human proteases and / or by examining the expression of cancer-associated proteases. Also, the linker sequence should not interfere with the role of the binder moiety in cell binding and internalization into lysosomes.

[0145] The term "cleavable domain" of a protoxin refers to a sequence that, once cleaved by hydrolysis or enzymatic cleavage, activates the toxin moiety of the protoxin. Many protoxins possess an amino acid domain that is specifically cleaved by enzymatic or pH-dependent hydrolysis (e.g., after endocytosis in an endosome) to release the active toxin moiety into the cytoplasm. Such a cleavable domain doubles as a "naturally occurring" cleavable peptide linker (or "intrinsic cleavage site"), as opposed to a cleavable peptide linker that must be used when a toxin does not contain a cleavable domain for activation, e.g., because it does not contain a protoxin.

[0146] Thus, although cleavable linkers offer clear advantages over stable linkers in terms of activity profile, their use complicates the production of the respective binding protein-toxin conjugates in mammalian, insect, and yeast cells, as linker cleavage leads to autointoxication of the production system. However, this is not the case in plant-based production systems, because (i) they do not cleave the linker (due to the lack of the respective protease or the reducing and hydrolytic conditions), and / or (ii) Each protein toxin that is toxic to mammals or mammalian cells is not toxic to plants or plant cells.

[0147] The term antibody as used herein refers to an antibody composition having a homogeneous antibody population, ie, a homogeneous population of whole immunoglobulins or fragments or derivatives thereof that retain target binding ability.

[0148] Particularly preferably, such antibodies are IgG antibodies, or fragments or derivatives thereof that retain target binding ability. Immunoglobulin G (IgG) is a type of antibody. IgG, accounting for approximately 75% of human serum antibodies, is the most common type of antibody present in the blood. IgG molecules are produced and released by plasma B cells. Each IgG has two antigen-binding sites.

[0149] IgG antibodies are large molecules with a molecular weight of approximately 150 kDa, consisting of four peptide chains. They contain two identical class γ heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, resulting in a tetrameric structure. The two heavy chains are linked to each other and to the light chains by disulfide bonds. As a result, the tetramer has two identical halves that together form a Y-shape. Both ends of the fork contain identical antigen-binding sites. The Fc region of IgG contains a highly conserved N-glycosylation site. N-glycans bound to this site are primarily complex-type, core-fucosylated diantennary structures. Furthermore, a small proportion of these N-glycans have been observed to contain a bisecting GlcNAc and α-2,6-linked sialic acid residues.

[0150] In humans, there are four IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum (IgG1 being the most abundant). As used herein, the term "antibody fragment" refers to a fragment of such an antibody that retains target binding ability. For example, CDRs (complementarity determining regions), hypervariable regions, variable domain (Fv), IgG heavy chain (consisting of VH, CH1, hinge, CH2 and CH3 regions); IgG light chain (consisting of VL and CL regions), and / or Fab and / or F(ab)2.

[0151] As used herein, the term "derivative" refers to protein constructs that are structurally distinct but have some structural relationship to the common antibody concept, e.g., scFv, scFv-FC, Fab and / or F(ab)2, as well as bivalent, trivalent or higher specific antibody constructs or monovalent antibodies, and yet retain target binding ability, all of which are described below.

[0152] Other antibody derivatives known to those skilled in the art include diabodies, camelid antibodies, nanobodies, domain antibodies, bivalent homodimers consisting of two chains of scFvs, IgAs (two IgG structures linked by a J chain and secretory component), shark antibodies, antibodies consisting of a New World primate framework and non-New World primate CDRs, dimeric constructs containing CH3+VL+VH, and antibody conjugates (e.g., antibodies or fragments or derivatives linked to toxins, cytokines, radioisotopes, or labels). These types are well described in the literature and can be used by those skilled in the art based on the present disclosure to further enhance the activity of the invention.

[0153] Methods for producing hybridoma cells have been previously described (see Kohler and Milstein 1975, the contents of which are incorporated herein by reference). Essentially, for example, a mouse is immunized with human soluble guanylate cyclase (sGC) protein, followed by isolation of B cells from the mouse, and fusing the isolated B cells with myeloma cells.

[0154] Methods for producing and / or selecting chimeric mAb or humanized mAb are known in the art. Essentially, for example, the protein sequences from mouse anti-sGC antibody that are not involved in target binding are replaced with corresponding human sequences. For example, US6331415 by Genentech describes the production of chimeric antibodies, while US6548640 by Medical Research Council describes CDR grafting technology, and US5859205 by Celltech describes the production of humanized antibodies. All of these disclosures are incorporated herein by reference.

[0155] Methods for producing and / or selecting fully human mAbs are known in the art, including the use of transgenic animals immunized with human sGC or suitable display technologies such as yeast display, phage display, B cell display, or ribosome display, in which antibodies from a library are screened against human sGC in stationary phase.

[0156] In vitro antibody libraries are disclosed, inter alia, by MorphoSys in US6300064 and by MRC / Scripps / Stratagene in US6248516. Phage display technology is disclosed, for example, by Dyax in US5223409. Transgenic mammalian platforms are described, for example, in EP1480515A2 by TaconicArtemis, all of whose disclosures are incorporated herein by reference.

[0157] IgG, scFv, scFv-FC, Fab and / or F(ab)2 are antibody formats well known to those skilled in the art. The relevant validation techniques are available from the respective textbooks. As used herein, the term "Fab" refers to an IgG fragment containing the antigen-binding region, said fragment consisting of one constant and one variable domain from each of the heavy and light chains of the antibody.

[0158] The term "F(ab)2" as used herein refers to an IgG fragment consisting of two Fab fragments linked together by one or more disulfide bonds.

[0159] As used herein, the term "scFv" refers to a single-chain variable region fragment that is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, connected by a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker peptide.

[0160] As used herein, the term "scFv-FC" refers to a specific antibody format. This format is particularly stable and can be expressed in plant cells or plants with high yields. scFv-FC constructs are disclosed, for example, in Bujak et al. (2014), the contents of which are incorporated herein by reference. scFv-Fc constructs are, for example, dimeric constructs consisting of two chains linked to each other by one or more disulfide bonds, each chain having the following structure (N->C orientation): VL-linker-VH-linker-FC, or VH-linker-VL-linker-FC VL is the variable domain of the antibody light chain, VH is the variable domain of the antibody heavy chain, and FC is the constant domain of the antibody.

[0161] The use of full-length IgG antibodies or scFv-Fc binding domains confers a longer half-life to the conjugate. Furthermore, the Fc portion of the antibody may be most important when CDC (complement-dependent cytotoxicity) or ADCC (antibody-dependent cellular cytotoxicity) activation is required.

[0162] Modified antibody formats include, for example, bispecific or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, etc. These types are well described in the literature and can be used by those skilled in the art based on the present disclosure, further enhancing the activity of the invention. Furthermore, monovalent antibodies have also been previously described in US2004 / 0033561 A1 (referred to herein as monobodies) or WO2007048037, both of which are incorporated herein by reference.

[0163] Antibody mimetics are organic compounds—most often recombinant proteins or peptides—that can specifically bind to antigens in the same way as antibodies, but are structurally unrelated to antibodies. Their general advantages over antibodies include better solubility, tissue transport, thermal and enzymatic stability, and relatively low production costs. Antibody mimetics have been developed as therapeutic and diagnostic agents, and include, among others, affibody molecules, affilins, ubiquitins, affimas, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and nanoCLAMPs. Antibody mimetics are discussed in great detail, particularly in Gebauer and Skerra (2009), the contents of which are incorporated herein by reference.

[0164] Generally, the protein binder can consist of a single chain, such as when the protein binder is an scFv antibody, or scFv-FC, in which case the entire protein binder may be encoded on a single polynucleotide.

[0165] In another embodiment, the protein binder may comprise more than one chain, such as in a full-size IgG or F(ab)2 fragment, with the proviso that in such cases the nucleic acid construct may comprise two or more polynucleotides encoding different chains or domains for the protein binder.

[0166] As used herein, the term "plant" (including cells derived therefrom) relates to land plants (embryonic plants) including algae (including Chlorophyta and Charophyta / Streptophyta), as well as Mesostigmatophyceae, Chlorokybophyceae and Spirotaenia, and gymnosperms and angiosperms, including monocotyledons and dicotyledons.

[0167] The term "transient expression" as used herein relates to the temporary expression of a gene that is expressed for a short period of time after a nucleic acid, most often a plasmid DNA encoding an expression cassette, has been introduced into a host cell or plant.

[0168] As used herein, the term "stable expression" refers to the expression of a gene that is continuously expressed over time after a nucleic acid, most often a plasmid DNA encoding an expression cassette, has been introduced into the genome of a host cell (nuclear or plastid integration). In stably transfected cells, the foreign gene becomes part of the genome and is therefore replicated. References JPEG0007818834000009.jpg225170JPEG0007818834000010.jpg81170Array

[0169] The following sequences form part of the disclosure of this application. A WIPO ST 25 compatible electronic sequence listing is also provided with this application. For the avoidance of doubt, in the event of any discrepancy between a sequence in the table below and a sequence in the electronic sequence listing, the sequence in the table shall be deemed correct. Also, note that in some embodiments, each amino acid sequence may or may not have a signal peptide / lead peptide. All embodiments are considered to be disclosed with and without a signal peptide / lead peptide.

[0170] Also, note that in some embodiments, the amino acid sequence of each of the toxins represents a deimmunized version thereof, and all embodiments are considered to be disclosed using either the wild-type toxin sequence or the deimmunized variant. JPEG0007818834000011.jpg134170JPEG0007818834000012.jpg188170JPEG0007818834000013.jpg148170JPEG0007818834000014.jpg162170JPEG0007818834000015.jpg162170JPEG0007818834000016.jpg135170JPEG0007818834000017.jpg134170JPEG0007818834000018.jpg134170JPEG0007818834000019.jpg157170JPEG0007818834000020.jpg159170JPEG0007818834000021.jpg172170JPEG0007818834000022.jpg146170JPEG0007818834000023.jpg131170JPEG0007818834000024.jpg128170JPEG0007818834000025.jpg181170JPEG0007818834000026.jpg113170JPEG0007818834000027.jpg193170JPEG0007818834000028.jpg73170

Claims

1. A binder-toxin fusion protein comprising at least the following a) and b): a) one protein binder selected from the group consisting of an antibody, an antibody fragment and an antibody mimetic that retains target binding ability; and b) Anisoprine or an active fragment thereof comprising the sequence set forth in SEQ ID NO: 48 or 49 or a homolog thereof having at least 90% sequence identity thereto.

2. A binder-toxin fusion protein as described in claim 1, wherein the fusion protein includes a peptide linker connecting the binder or a domain thereof to the toxin or a cleavable domain contained in the toxin.

3. A binder-toxin fusion protein as described in claim 2, wherein the peptide linker is a non-cleavable linker and / or the peptide linker is a G4S linker.

4. A binder-toxin fusion protein as described in claim 2, wherein the peptide linker or cleavable domain is specifically or non-specifically cleavable by an enzyme expressed by a mammalian cell or produced by a mammalian host.

5. A binder-toxin fusion protein described in claim 3 or 4, wherein the peptide linker or cleavable domain is not cleaved by an enzyme expressed by a plant cell or produced by a plant host.

6. A binder-toxin fusion protein described in any one of claims 1 to 5, wherein the protein binder binds to human CD20 or human CD79B.

7. 7. The binder-toxin fusion protein of any one of claims 1 to 6, wherein the binder-toxin fusion protein is in one of any formats selected from the group consisting of: (scFv-FC)-(linker)-toxin (dimer), - a tetramer of two HC and two LC-(linker)-toxins, a tetramer of two LC and two HC-(linker)-toxins, or a tetramer of two LC-(linker)-toxins and two HC-(linker)-toxins; Here, the linker is optional.

8. 8. The binder-toxin fusion protein of any one of claims 1 to 7, wherein the cleavable linker or the cleavable domain in the protoxin comprises at least one cleavage site selected from the group consisting of: a) an endosomal and / or lysosomal protease cleavage site; b) a cytosolic protease cleavage site, and / or c) Cleavage sites of cell surface proteases.

9. comprising a binder-toxin fusion protein according to any one of claims 1 to 8, or A pharmaceutical composition comprising the binder-toxin fusion protein of any one of claims 1 to 8, further comprising one or more pharmaceutically acceptable excipients.

10. A combination comprising (i) a binder-toxin fusion protein according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9, and (ii) one or more further therapeutically active compounds.

11. A binder-toxin fusion protein according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9, or a combination according to claim 10, for use in the treatment of a human or animal subject suffering from, at risk of developing and / or diagnosed with a neoplastic disease, or for the prevention of such a condition.

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