Recombinant immunotoxins containing ribosome-inactivating proteins

Abstract

The present invention relates to a binder-toxin fusion protein comprising at least a protein binder and a ribosome-inactivating protein (RIP) type 1 or an active fragment thereof.

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 represent a great hope in fighting cancer. These conjugates are mainly represented by the class of antibody-drug-conjugates (ADCs), which consist of a monoclonal antibody chemically bound 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 the target cells while sparing healthy tissues. Summary of the Invention [Problem to be solved by the invention]

[0003] There is still a need for such new entities in order to provide better treatments for various types of tumors.It is therefore an object of the present invention to provide such new entities. A further object of the present invention is to provide alternative or even better treatment options for cancer patients.

[0004] 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.

[0005] The methodology used to conceive and put into practice 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 to this application and forms part of the disclosure. [Brief description of the drawings]

[0006] [Figure 1] A selection of possible formats of binder-toxin fusion proteins. All formats shown contain an antibody Fc domain. [Figure 2A] Alignment of different RIP types I (ribosome-inactivating proteins type I). Some highly conserved residues (Tyr21, Phe24, Arg29, Tyr80, Tyr123, Gly140, Ala165, Glu177, Ala178, Arg180, Glu208, Asn209 and Trp211) are shown in bold. [Figure 2B] Further alignment and homology matrix of different RIP types I. [Figure 2C] Further alignment and homology matrix of different RIP types I. [Diagram 3] Cell killing activity of binder-toxin proteins containing RIP I bryodin or the α domain of ricin (RIP II) against target positive cell lines. The antibody is anti-CD79B. [Figure 4] Cell killing activity of RIP I trichosanthin-containing binder-toxin proteins against target-positive and target-negative cell lines. The antibody is anti-CD79B. [Diagram 5] Cell killing activity of binder-toxin proteins, including RIP I momordin, against target-positive cell lines. [Figure 6] Evaluation of transient expression of IgG-G4S-RIP type I toxin in Nicotinia benthamiana. All binder-toxin fusion proteins are well expressed (>200mg / Kg fresh leaves), which is often considered the threshold for industrialization. [Figure 7] Cell killing activity of binder-toxin fusion proteins containing different RIP type I against target positive cell lines. The antibody is anti-CD79B (polatuzumab). [Figure 8]Figure 2 shows the cell killing activity of binder-toxin fusion proteins containing wild-type or aglycosylated RIP I bryodin against target positive cell lines. The antibody is anti-CD79B (polatuzumab). It can be seen that deglycosylation of the toxin does not affect efficacy. [Figure 9] Cell killing activity of binder-toxin fusion proteins containing RIP I bryodin with G4S linker (ATB 678) and the same construct with Liopt linker (ATB 679) in target positive cell lines (mammalian). ATB 673 is a naked antibody. The antibody is anti-CD22 inotuzumab. Liopt is a furin-cleavable linker, which is cleaved in mammalian cells, whereas G4S is non-cleavable. The cleavability of the linker does not yet affect the potency of the binder-toxin fusion protein in this cellular assay. [Figure 10] Cell killing activity of binder-toxin fusion proteins containing wild-type RIP I bryodin and the liopt linker against target-negative cell lines. The antibody is anti-CD22 inotuzumab. [Figure 11] Cell killing activity of binder-toxin fusion proteins containing RIP I momordin and a G4S linker against target-positive cancer cells. The antibody binds to an antigen present on the surface of the cancer cell. [Figure 12] Inhibition of protein synthesis in HELA cell lysates. Target-independent activity of intact binder-toxin fusion proteins. In HELA cell lysates, all binder-toxin fusion proteins tested showed similar activity. Deglycosylation of MOM and BD1 by mutating glycosylation sites does not affect activity. [Figure 13] Internalization of constructs containing anti-CD79B antibody (polatuzumab) into CD79 positive cells. Binder-toxin fusion proteins containing BD1 (ATB 639) are internalized much better than naked antibody (ATB 372). [Figure 14]Differences in pharmacokinetics between binder-toxin fusion proteins containing glycosylated toxins (ATB 639, ATB 657) and binder-toxin fusion proteins containing deglycosylated toxins (ATB 752, ATB 662). As shown in Figures 8 and 12, deglycosylation does not adversely affect activity but increases serum half-life. [Figure 15] Embodiments of binder-toxin fusion proteins according to the present invention Each combination of features between toxins, binders, linkers and fusion sites is considered to be disclosed as an individual embodiment without list selection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Before describing the invention in detail, it is to be understood that the invention is not limited to the specific component parts of the devices described, or to the process steps of the methods described, 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 is to 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 parameter range bounded by numerical values ​​is given, it is to be understood that the range is deemed to include these limits.

[0008] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate embodiments unrelated to each other. 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, a person skilled in the art will understand that this does not necessarily mean that the feature is not meant to be disclosed in the other embodiment. A person skilled in the art will understand that it is the intent of the present application to disclose the features in other embodiments as well, but this has not been done merely for the sake of clarity and to keep the present specification to a manageable volume.

[0009] Embodiments of the invention are set forth in the claims.

[0010] According to a first aspect of the present invention, at least a) a protein binder, and b) ribosome-inactivating protein (RIP) type 1 or an active fragment thereof A binder-toxin fusion protein comprising:

[0011] Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate eukaryotic and prokaryotic rRNA, thereby halting protein synthesis during translation. RIPs are widely present in various plant species and tissues. These proteins are known to play an important role in defense against pathogens and have been suggested to confer disease resistance. Plant-derived RIPs have been found in more than 50 species from 14 families, including Cucurbitaceae, Euphorbiaceae, Poaceae, and Caryophyllaceae. RIPs have been found not only in plants, but also in bacteria, fungi, algae, and even mosquitoes.

[0012] RIPs constitute a large family of proteins that can be classified according to their structural composition into RIP type I and type II.

[0013] Type I RIPs are low molecular weight proteins of around 30 KD and occur as single-chain proteins. The single chain of type I RIPs consists of an enzymatically active domain (A domain or α domain) that exerts N-glycosidase activity.

[0014] The amino acid sequences of type I RIPs have special sequence characteristics: in addition to several highly conserved hydrophobic amino acids, 11 absolutely conserved residues in particular are present in almost all type I RIPs: Tyr21, Phe24, Arg29, Tyr80, Tyr123, Gly140, Ala165, Glu177, Ala178, Arg180, Glu208, Asn209 and Trp211. These residues are marked in the alignment in Figure 2.

[0015] Vivanco et al. (1999) have shown that the plant type 1 RIPs, ME1 and ME2, exhibit antibacterial activity against Agrobacterium tumefaciens and Agrobacterium radiobacter.

[0016] It is therefore surprising that, independent of the suspected antibacterial activity of RIP I toxins, we were able to produce RIP I-based binder-toxin fusion proteins in Agrobacterium-transfected plant cells and plant hosts.

[0017] (2002) showed that the RIP type I trichosanthin expressed in transgenic tobacco plants confers acquired resistance to pathogens such as tobacco mosaic virus. The authors proposed three possible explanations for the extent of phenotypic abnormalities and low expression levels of RIPs in transgenic plants: (1) the mode of expression (constitutive or tissue-specific), (2) the target sequence (or lack thereof) within the coding gene, and (3) the diverse toxicity of different RIPs and their effects on host plant ribosomes. RIP proteins vary widely in their enzymatic and rRNA N-glycosidase activities, as well as their activities on prokaryotic and / or eukaryotic ribosomes.

[0018] Krishnan et al. reported that while it has been very difficult to express most type I RIPs in transgenic plants, they have succeeded in expressing cereal seed ribosome-inactivating proteins (RIPs) at high levels in transgenic tobacco and rice cultures. Cereal seed RIPs are intracellular proteins that are not cleaved post-translationally and do not exhibit depurination activity on plant ribosomes either in vitro or in vivo.

[0019] It was therefore surprising to find that type I RIPs could indeed be produced in transgenic plants when coupled to protein binders, even though the fusion construct as a whole retained ribosome-inactivating activity, as we show in Figure 12, where a binder-toxin fusion protein IgG with bryodin fused to the heavy chain still possesses ribosome-inactivating activity on lysates of antibody target-negative HeLa cells.

[0020] Type II RIPs are large proteins of 50-65 kDa, characterized by an enzymatically active A chain and a somewhat larger B chain (or β chain, lectin subunit) bearing a galactose-like sugar chain.

[0021] In addition to RIP types I and II, a third class, type III, has been described, with a small number of members that have an N-terminal domain that correlates with the A domain of RIPs and are fused to an unknown functional C-terminal domain that appears to serve as a protective function against self-inactivation. It has only been found in barley and maize.

[0022] Interestingly, type I RIPs are less cytotoxic than type II RIPs. The lower cytotoxicity of type I RIPs is due to the absence of the cell-binding B chain (β chain). It is therefore surprising that we found that binder-toxin fusion proteins containing type I RIPs are extremely potent (see, in particular, FIG. 3). According to some embodiments, the ribosome-inactivating protein (RIP) type 1 in the binder-toxin fusion protein is at least one selected from the group consisting of: Momordin (MOM), Bryodin I (BD1), Cucurmocin (CUC), Bryodin II (BD2), Trichosanthin (TRI), Karasurin (KAR), ·MOMC, ME1, and / or ME2.

[0023] Below we provide more detailed information and examples of these ribosome-inactivating proteins.

[0024] [Table 1]

[0025] The sequences and characteristics of ME1 and ME2 are disclosed in Vivanco et al. (1999).

[0026] According to some embodiments, the ribosome-inactivating protein (RIP) comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, or a homolog thereof having at least 66% sequence identity thereto.

[0027] In some embodiments, the toxin sequence has 67% or more; 68% or more; 69% or more; 70% or more; 71% or more; 72% or more; 73% or more; 74% or more; 75% or more; 76% or more; 77% or more; 78% or more; 79% or more; 80% or more; 81% or more; 82% or more; 83% or more; 84% or more; 85% or more; 86% or more; 87% or more; 88% or more; 89% or more; 90% or more; 91% or more; 92% or more; 93% or more; 94% or more; 95% or more; 96% or more; 97% or more; 98% or more; 99% or more sequence identity, and is most preferably 100% identical to any one of SEQ ID NOs: 1-8.

[0028] In particular, mutants of these toxins that retain toxic function are also included. Such mutants can include, for example, mutations / substitutions of N-glycosylation motifs (e.g., NXS or NXT, where X cannot be P) to produce deglycosylated mutants of the toxins, or mutations / substitutions to deimmunize the respective toxins (see, for example, Zinsli et al. 2020, the contents of which are incorporated herein by reference for operability).

[0029] Deglycosylated variants of BD1 can include, for example, one or more of the following substitutions: N192S, A228V, S229D, S229G, R230G, A230S, and / or R231D. Deglycosylated variants of MOM can include, for example, one or more of the following substitutions: T252G, T252D, S253A, K254G, K254D, D1A, and / or D1S. Deglycosylated variants of CUC can include, for example, one or more of the following substitutions: N189S, T227G.

[0030] The inventors surprisingly showed that deglycosylated variants could improve pharmacokinetics and extend serum half-life, and without being bound by theory, this may be due to reduced hepatic clearance due to underglycosylation.

[0031] Interestingly, while a number of currently approved protein pharmaceuticals require proper glycosylation for optimal therapeutic efficacy (as glycosylation can affect various physiological processes (e.g., protein-protein binding, protein molecular stability) at both the cellular and protein levels), such effects do not appear to play a role in the toxin moiety of the binder-toxin protein according to the present invention. The present inventors have realized this fact and therefore established a feasible route to improve pharmacokinetics and serum half-life without compromising efficacy.

[0032] According to one embodiment, the ribosome-inactivating protein (RIP) type 1 in the binder-toxin fusion protein is momordin or a deimmunized, deglycosylated variant thereof or a variant thereof having sequence identity to momordin as defined above.

[0033] According to one embodiment, the ribosome-inactivating protein (RIP) type 1 in the binder-toxin fusion protein is bryodin 1 or a deimmunized, deglycosylated variant thereof or a variant thereof having sequence identity to momordin as defined above.

[0034] According to one embodiment, the protein binder is selected from the group consisting of: ·antibody, an antibody fragment or derivative that retains target binding ability, or Antibody mimetics.

[0035] 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. 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 a transfected plant cell or a transfected plant host.

[0036] Those skilled in the art have at hand a number of routine methods to determine whether a peptide linker or cleavable domain in a toxin 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 routinely determine whether a 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. Transfection of the plant cell or plant host can be transient or stable.

[0037] According to one embodiment, the binder-toxin fusion protein comprises a non-cleavable peptide linker connecting the binder or a domain thereof and the toxin.

[0038] According to one embodiment, the protein binder binds to human CD20, human CD22 or human CD79B.

[0039] CD79b (B-cell antigen receptor complex-associated protein β-chain) is a surface protein that is involved in humoral immune responses.

[0040] CD79b is produced by B cells. It binds to CD79a and is linked by 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 is phosphorylated by the protein kinases Syk and Lyn.

[0041] The complete nucleotide sequence of CD79b was first published in 1994.

[0042] Protein binders against CD79B have been described in the art. The first antibody against CD79b (mouse) was called SN8 and was published by Okazaki et al. (1993).

[0043] Polson et al. (2007) discuss the possibility of generating antibody-drug conjugates (ADCs) and recombinant immunotoxins directed against CD79b.

[0044] The first humanized anti-CD79b antibody (Polatuzumab) is disclosed in US8545850, which also discloses an ADC in which polatuzumab is linked to MMAE.

[0045] The B lymphocyte antigen CD20 or CD20 is expressed on the surface of all B cells from the pro-B stage (CD45R+, CD117+) and gradually increases in concentration until maturation. In humans, CD20 is encoded by the MS4A1 gene. This gene encodes a member of the transmembrane 4A gene family. Members of this nascent protein family are characterized by common structural features and similar intron / exon splice boundaries and display unique expression patterns among hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule and plays a role in B cell development and differentiation into plasma cells. This family is localized to 11q12 and is within a cluster of family members. Alternative splicing of this gene results in two transcript variants that code for the same protein. This protein has no known natural ligand and its function is to enable optimal B cell immune responses, especially against T-independent antigens. It is suspected to act as a calcium channel in the cell membrane. CD20 is induced by CXCR4 / SDF1 (CXCL12) chemokine signals during interactions with the microenvironment, and in this respect, the molecular function of CD20 has been linked to the signaling propensity of the B cell receptor (BCR).

[0046] CD20 was discovered in 1980 by Lee Nadler of the Dana-Farber Cancer Institute. Protein binders to CD20 have been described in the art. Over the last 30 years, considerable progress has been made in understanding the structure and function of the CD20 molecule and in the development of artificial anti-CD20 mAbs. Information regarding anti-CD20 antibodies can be found, for example, in Lim et al. (2010), the contents of which are incorporated herein by reference. The following table shows some anti-CD20 antibodies known to those skilled in the art: [Table 2]

[0047] The contents of the mentioned references are hereby incorporated by reference in the context of enabling functionality.

[0048] CD22 (cluster of differentiation 22) is a lectin molecule belonging to the SIGLEC family. It is present on the surface of mature B cells and to some extent on immature B cells. CD22 is a transmembrane protein with a molecular mass of 140 kDa. The extracellular portion of CD22 consists of seven immunoglobulin domains, and the intracellular portion is formed by a cytoplasmic tail of 141 amino acids.

[0049] Because CD22 is restricted to B cells, it has been considered as a target for immunotherapy of B cell malignancies, including monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, radioimmunoconjugates, and CAR-T cells. The following table shows some anti-CD22 antibodies known to those skilled in the art: [Table 3]

[0050] The contents of the mentioned references are hereby incorporated by reference in the context of enabling functionality.

[0051] According to one embodiment, such binder-toxin fusion protein is in one of the formats selected from the group consisting of: (scFv-Fc)-(linker)-toxin (dimer), Toxin-(linker)-Fc-VH / VL, A tetramer of two HC and two LC-(linker)-toxins (IgG format), A tetramer of two LC and two HC-(linker)-toxins (IgG format), or A tetramer of two LC-(linker)-toxins and two HC-(linker)-toxins (IgG format), Here, the linker is optional.

[0052] FIG. 1 shows a selection of embodiments of binder-toxin fusion protein formats according to the present invention. CH 3 = Heavy chain constant domain 3 CH 2 = 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

[0053] As can be seen, all of the embodiments depicted in Figure 1 include an Fc domain. Such a domain is lacking in antibody fragments, such as, for example, scFv, Fab, (Fab)2. Thus, in one embodiment of the binder-toxin fusion protein, an antibody or fragment thereof includes at least one Fc domain.

[0054] In addition to those shown in FIG. 1, toxin molecules can be fused to both the heavy and light chains simultaneously.

[0055] Protein binders may also be bispecific. In one such embodiment, an antibody can be designed such that one VF / VL pair binds target 1 and the other VF / VL pair binds target 2. Such an approach is called "quadroma" or further developed as "knobs into holes". In another such embodiment, two different VH domains and two different VL domains can be chimerized to generate dual variable domain antibodies (called "DVD-Ig"). See, e.g., Brinkmann and Kontermann (2017), the contents of which are incorporated herein by reference for enablement.

[0056] Additionally, a binder toxin fusion can include two different toxins fused to, for example, two chains of an antibody.

[0057] In another embodiment where the protein binder comprises two or more strands, two nucleic acid constructs may be provided, a first nucleic acid construct comprising three polynucleotides encoding the first strand of the protein binder, a linker and a toxin, while a second nucleic acid construct comprises a polynucleotide encoding the second strand of the protein binder.

[0058] "Inducible promoters" could induce 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 10344290 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.

[0059] According to one embodiment of the invention, the plant or plant cell is of the genus Nicotiana.

[0060] The genus Nicotiana includes the tobacco plant. 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).

[0061] According to a further embodiment of the invention, the plant cell is at least one selected from the group consisting of: Nicotiana tabacum (Tobacco) cv. BY2, Nicotiana tabacum NT-1, Arabidopsis thaliana, Daucus carota, and / or · Oryza sativa (rice).

[0062] Nicotiana tabacum cv. BY2, also known as Tobacco BY-2 cells, cv.Nicotiana tabacum 1 (NT-1, sibling of BY-2), is a non-green, fast-growing plant cell that can increase its number up to 100-fold within a week under suitable medium and good culture conditions. This tobacco variety is bred as a cell culture, more specifically as a cell suspension culture (a specialized population of cells growing in liquid medium, grown by scientists to study specific biological properties of plant cells). In cell suspension cultures, each cell is independent or at most suspended in short chains in the culture medium. Each cell has similar properties to the other cells.

[0063] Model plant systems are comparable to HeLa cells for human research. The organisms are relatively simple and predictable, which facilitates the study of biological processes and can be an intermediate step to 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 high growth rate, which characterize the still general behavior of plant cells. Due to the diversity of cell types within any part of a naturally grown plant (in vivo), it is very difficult to investigate and understand some of the general biochemical phenomena of living plant cells. For example, the transport of solutes in 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 the behavior of neighboring cells is less important in suspension than it is in intact plants. As a result, changes observed after a stimulus is applied can be statistically correlated to determine whether these changes are a response to the stimulus or simply 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.

[0064] 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. The 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.

[0065] In general, the 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. Although callus culture or other conventional plant cell culture methods may be used, plant cell suspension or plant tissue culture is preferred.

[0066] 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.

[0067] 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.).

[0068] 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.

[0069] 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. For more detailed information on each enzyme in this table, please refer to the "Merops" database: https: / / www.ebi.ac.uk / merops / index.shtml. [Table 4] JPEG2025508742000006.jpg30170

[0070] The cleavage site is described from the cleavage site point (represented by ↓). The letter x stands for all amino acids. If there are several preferred amino acids, they are separated by a slash ( / ).

[0071] Such an enzyme is preferably a protease. In one embodiment, the peptide linker is not cleavable by a plant enzyme.

[0072] Furin belongs to the subtilisin Cathepsins-like proprotein convertase family and is an enzyme that cleaves proteins C-termini at 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. Preferably, the sequence is HRRRKRSLDTS (also referred to herein as "liopt").

[0073] 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 that contain 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 the information regarding cathepsins and their cleavage sites in Turk et al. (2012) fully enabling, the contents of which are incorporated herein by reference.

[0074] 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 1500 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 et al. (2014) fully enabling information about caspases and their cleavage sites, the contents of which are incorporated herein by reference.

[0075] Matrix metalloproteinases (MMPs), also known as matrixins, are calcium-dependent zinc-containing endopeptidases. Other family members are adamalysin, serralysin, and astacin. Taken together, these enzymes can degrade all kinds of extracellular matrix proteins, but can also process many bioactive molecules. Those skilled in the art will find ample potential information on matrix metalloproteinases and their cleavage sites in Eckard et al. (2016), the contents of which are incorporated herein by reference.

[0076] In general, the skilled artisan can, by routine consideration and literature reference, select specific cleavage sites that correspond to the respective mammalian enzymes in order 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).

[0077] According to one embodiment of the 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.

[0078] In one embodiment, the protein toxin or protoxin is not toxic to plants or plant cells. The skilled artisan has a set 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.

[0079] According to one embodiment of the present invention, the protein comprises 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 motif Asn-X-Thr or Asn-X-Ser (NXT or NXS), where X is any amino acid except proline. Exemplary plant-specific N-glycans are disclosed in Gomord et al. (2010) and are significantly different from mammalian N-glycan patterns.

[0080] 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: Fucose residues linked to N-acetylglucosamine residues by α3 glycosidic bonds (rather than α6 as in mammals) A xylose residue linked to the proximal mannose residue via a β2 glycosidic bond · Two distal N-acetyl-glucosamine residues (as a replacement for neuraminic acid in mammals) bearing a fucose residue via an α3 glycosidic bond and a galactose residue via a β3 glycosidic bond, respectively.

[0081] In contrast, recombinantly expressed proteins in algae often lack glycosylation, but algae can express IgG antibodies and antibody fragments with one or more disulfide bridges.

[0082] The predominant plant-derived glycoform identified is the complex glycan (GnGn / GnGnXF). Other glycoforms (Man5-Man9, GnGnF, GnGnX, MMXF, Man5Gn, GnM(X)(F)) are also detectable.

[0083] According to this nomenclature, MGnX means, for example: [ka]

[0084] Background on methods for analyzing peptide glycoforms is provided in WO2020169620, the contents of which are incorporated herein by reference for enabling purposes.

[0085] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising at least a binder-toxin fusion protein according to the above description, optionally comprising one or more pharma- ceutically acceptable excipients.

[0086] According to another aspect of the invention there is provided a combination comprising (i) a binder-toxin fusion protein or a pharmaceutical composition according to the above description, and (ii) one or more further therapeutically active compounds.

[0087] According to another aspect of the invention, a binder-toxin fusion protein, composition or combination as described above is provided for use (for the manufacture of a medicament for) 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.

[0088] This language is to be regarded as embracing both the Swiss-type claim language accepted in some countries (in which case the brackets are to be regarded as not being enclosed) and the EPC 2000 language (in which case the brackets and any content within them are to be regarded as not being enclosed).

[0089] 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 the prevention of such a condition, said method comprising administration of a therapeutically effective amount of a binder-toxin fusion protein, composition or combination as described above.

[0090] definition As used herein, the "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 sequence portions 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 at which identical nucleic acid bases or amino acid residues occur 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.

[0091] The term "identical" or percent "identity" refers to two or more sequences or subsequences that are the same sequence, with respect to two or more nucleic acid or polypeptide sequences. 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 specified region, using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection. 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. For shorter amino acid sequences, for example sequences of 20 or fewer amino acids, substantial identity exists if one or two amino acid residues are conservatively substituted in accordance with conservative substitutions as defined herein.

[0092] The term "protein toxin" or "protein protoxin" does not refer exclusively 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, that need to be activated, for example, by cleaving an inhibitory amino acid sequence or by undergoing a conformational change. The terms "protoxin" and "protein protoxin" are used interchangeably herein and refer to the same subject matter.

[0093] As used herein, the term "fusion protein" refers to 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.

[0094] As used herein, the term "operably linked" refers to a situation in which different polynucleotides are placed in a functional relationship with each other when referring to two or more polynucleotides. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription of the coding sequence. Similarly, a coding sequence for a signal peptide is operably linked to a coding sequence for a polypeptide if the signal peptide affects the extracellular secretion of the polypeptide. According to one embodiment of the present invention, when each polynucleotide encodes a different peptide, "operably linked" refers to the fact that each polynucleotide is contiguous and, where necessary to link two protein coding regions, the open reading frames are aligned.

[0095] The term "cleavable peptide linker" as used herein refers to an internal amino acid sequence within the fusion protein that includes residues that link the binder moiety and the toxin protein so as to render the toxin protein unable to exert its toxic effect outside the target cell or limit the toxin protein's ability to inhibit cell growth (cytostasis) or cause cell death (cytotoxicity). In this way, the protein toxin will remain inactive as long as it is in the 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, causing the toxin protein to become activated 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 a cleavage recognition site for a specific protease, including but not limited to a cancer-specific protease, and / or are cleavable under specific conditions, including 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 testing the expression of cancer-associated proteases, and the linker sequence should not interfere with the role of the binder moiety in cell binding and internalization into lysosomes.

[0096] The term "cleavable domain" of a toxin refers to a sequence that, once cleaved by hydrolysis or enzymatic cleavage, activates the toxin moiety of the toxin. Many toxins have amino acid domains that are specifically cleaved by enzymes or by pH-dependent hydrolysis (e.g., after endocytosis in endosomes) to release the active toxin moiety into the cytoplasm. Such cleavable domains function dually as "naturally occurring" cleavable peptide linkers (or "intrinsic cleavage sites"), as opposed to the cleavable peptide linkers that must be used if the toxin does not contain a cleavable domain for activation.

[0097] Thus, although cleavable linkers may, under certain circumstances, offer 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 cleavage of the linker results in release of the toxin from the protein conjugate, resulting in autointoxication of the production system. (i) they do not cleave the linker (due to lack of a protease or reducing / hydrolysis conditions, respectively) and / or (ii) each protein toxin that is toxic to mammals or mammalian cells is not toxic to plants or plant cells; This is not the case for plant-based production systems.

[0098] However, the inventors have experienced that even binder-toxin fusion proteins that are not cleaved in mammalian cells can be problematic when testing their production in mammalian cells. Without wishing to be bound by theory, this may be because immediately after intracellular protein expression, the ribosome-inactivating protein moiety, even though bound to an antibody, exerts a toxic effect on the ribosomes of the expressing cell. Such adverse effects were not observed when using non-mammalian expression systems, such as plants or plant cells (e.g., Nicotiana, as disclosed elsewhere herein), which, without wishing to be bound by theory, may be due to differences in the ribosome structure of plant and mammalian cells.

[0099] Eukaryotic cytoplasmic ribosomes consist of a 60S large subunit (LSU) and a 40S small subunit (SSU). The latter encodes the mRNA, while the former catalyzes the peptidyl transferase reaction that leads to the formation of peptide bonds in newly synthesized proteins. The subunits consist of rRNA and accessory ribosomal proteins (RPs). The large subunit consists of 5S, 5.8S, and 25S rRNA, and is between 25S and 26S in plant cells, but is 28S in mammalian cells.

[0100] The term "antibody" as used herein refers to an antibody composition having a homogenous antibody population, ie, a homogenous population of whole immunoglobulins or fragments or derivatives thereof that retain target binding ability.

[0101] 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, which accounts for about 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.

[0102] IgG antibodies are large molecules with a molecular weight of about 150 kDa consisting of four peptide chains. They contain two identical class γ heavy chains of about 50 kDa and two identical light chains of about 25 kDa, and thus have a tetrameric structure. The two heavy chains are linked to each other and to the light chains, respectively, by disulfide bonds. As a result, the tetramer has two identical halves that together form a Y-shaped shape. Both ends of the fork contain identical antigen-binding sites. The Fc region of IgG has a highly conserved N-glycosylation site. The N-glycans that bind to this site are mainly complex-type core-fucosylated diantennary structures. In addition, a small amount of these N-glycans have been identified that have bisecting GlcNAc and α-2,6-linked sialic acid residues.

[0103] In humans, there are four IgG subclasses (IgG1, 2, 3, 4), named in order of their abundance in serum (IgG1 being the most abundant).

[0104] As used herein, the term "antibody fragment" is intended to refer to a fragment of such an antibody which retains target binding ability and, in one embodiment, still contains only the Fc domain, or the CH2 or CH3 domain.

[0105] The term "derivative" as used herein refers to protein constructs that are structurally distinct but have some structural relatedness to the common antibody concept, such as 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.

[0106] Other antibody derivatives known to those skilled in the art are IgA (two IgG structures linked by a J chain and a secretory component), shark antibodies, antibodies consisting of New World primate frameworks and non-New World primate CDRs, dimerized constructs containing CH3+VL+VH.

[0107] Methods for producing hybridoma cells have been described previously (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.

[0108] Methods for producing and / or selecting chimeric or humanized mAbs are known in the art. Essentially, for example, the protein sequences from mouse anti-sGC antibodies that are not involved in target binding are replaced by 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.

[0109] Methods for the production and / or selection of fully human mAbs are known in the art, including the use of transgenic animals immunized with human sGC or the use of suitable display technologies such as yeast display, phage display, B cell display or ribosome display, where antibodies from a library are screened against human sGC in stationary phase.

[0110] 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.

[0111] IgG, scFv-Fc, Fc-VH / VL are antibody formats well known to those skilled in the art. The relevant validation techniques are available from the respective textbooks.

[0112] As used herein, the term "scFv-Fc" refers to a specific antibody format. This format is particularly stable and can be expressed in high yields in plant cells and plants. scFv-Fc constructs are disclosed, for example, in Bujak et al. (2014), the contents of which are incorporated herein by reference. The scFv-Fc construct is, for example, a dimeric construct comprising two chains linked together by one or more disulfide bonds, each chain consisting of the following structure (NC orientation): VL-linker-VH-linker-Fc, or VH-linker-VL-linker-Fc, VL is the variable domain of the light chain of an antibody, VH is the variable domain of the heavy chain of an antibody, and Fc is the constant domain of an antibody.

[0113] As used herein, the terms "Fc-VH-VL" and "Fc-VH-VL" refer to a specific antibody format in which a VH domain is fused to one arm (CH2 and CH3) of an Fc domain and a VH domain is fused to the other arm of the Fc domain.

[0114] The use of full-length IgG antibodies, or scFv-Fc binding domains, or other formats that include the Fc domain or CH2 and / or CH3, increases the half-life of the conjugate. Furthermore, the Fc portion of the antibody may be of primary importance when CDC (complement-dependent cytotoxicity) or ADCC (antibody-dependent cellular cytotoxicity) activation is required.

[0115] Modified antibody formats are, 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 this disclosure, further adding the activity of the invention.In addition, monovalent antibodies have also been previously described in US 2004 / 0033561 A1 (herein referred to as monobody) or WO2007048037, both of which are incorporated herein by reference.

[0116] Antibody mimetics are organic compounds - most often recombinant proteins or peptides - that can specifically bind antigens in the same way as antibodies, but are not structurally related to antibodies. Their general advantages over antibodies are better solubility, tissue delivery, heat 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 have been discussed in great detail, particularly in Gebauer and Skerra (2009), the contents of which are incorporated herein by reference.

[0117] Generally, the protein binder can consist of a single chain, for example when the protein binder is an scFv antibody, or an scFv-Fc, in which case the entire protein binder can be encoded on a single polynucleotide.

[0118] 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.

[0119] As used herein, the term "plants" (including cells derived therefrom) relates to land plants (embryophytes) including algae (including Chlorophyta and Charophyta / Streptophyta), as well as gymnosperms and angiosperms including Mesostigmatophyceae, Chlorokybophyceae and Spirotaenia, and monocotyledonous and dicotyledonous plants.

[0120] 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 frequently a plasmid DNA encoding an expression cassette, is introduced into a host cell or plant.

[0121] As used herein, the term "stable expression" refers to 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. EXAMPLES

[0122] While the present invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered as illustrative or exemplary and not restrictive, and the present 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.

[0123] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'-3'.

[0124] This example is based on experiments with bryodin and other toxins from the ribosome-inactivating protein family, but the experimental protocol is applicable to other RIP type I toxins and other constructs. [Table 5] JPEG2025508742000009.jpg67170

[0125] Materials and Methods Genetic construct binder toxin fusion 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 sequences of the Rituximab sequence were assembled into a single-chain scFv and fused to a human IgG1 Fc sequence. The bryodin sequence was then 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 HC+LC-FCS-bryodin, HC-FCS-bryodin+LC, and scFv-c-FCS-bryodin fusion protein sequences. These sequences were generated by gene synthesis flanked by XbaI and IsceI.

[0126] A non-public plasmid was used to insert the ORF encoding the binder-toxin fusion.

[0127] Genetic constructs containing antibodies 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 sequences were assembled into a single-chain scFv and fused to a human IgG1 Fc sequence. Human bryodin sequences were then fused to the LC or HC or both of the full-length undisclosed antibody or to the C-terminal portion of the scFv-Fc using human furin cleavage sequences, respectively, to obtain HC+LC-FCS-bryodin, HC-FCS-Bryodin+LC, and scFv-Fc-FCS-bryodin or scFv-Fc-bryodin fusion protein sequences. Another binder-toxin fusion protein was realized in which the scFv-Fc, HC and LC portions were linked to bryodin without cleavage sites, to obtain scFv-Fc-bryodin, HC+LC-bryodin, HC-bryodin+LC, LC-bryodin+HC-bryodin. These sequences were generated by gene synthesis flanked by XbaI and IsceI.

[0128] Transient expression in Nicotiana benthamiana leaves 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. Optical density at 600 nm (OD 600 Agrobacterium tumefaciens GV3101 (pMP90) 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 OD of 0.8–1.0. 600 The pH was adjusted to 0.5 and infiltrated using a needless syringe. The infiltrated areas were harvested on the 4th and 6th days after agroinfiltration. Whole leaves harvested on the 4th day after agroinfiltration were used for protein A purification.

[0129] Expression in N. tabacum cells 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 by reference, at 130 rpm and 25° C. for 5 days. The optical density at 600 nm (OD 600 Agrobacterium tumefaciens LBA4404 (pBBR1MCS-5.virGN54D) carrying the pPZP-ATB binary plasmid with a chromatin ratio of 0.8–1.0 was harvested by centrifugation at 2000 g for 5 min. Then, plant and bacterial cells were co-cultured in co-culture medium for 30 min, followed by centrifugation at 2000 g for 5 min. After removing the supernatant, the cells were plated on solid co-culture medium for 2 days. For transient transformation, the cells were then harvested, washed three times, and cultured on plant growth medium containing cefotaxime and carbenicillin, and then harvested for further analysis. For stable transformation, after 2 days of solid co-culture, the cells were washed and plated on plant medium containing selective kanamycin and cefotaxime and carbenicillin antibiotics. After 4 weeks, calli were selected and subcultured on solid medium or liquid suspension culture for subsequent analysis.

[0130] Protein analysis: ELISA, SDS-PAGE and Western blot The collected leaf tissue (5 g 120 mg) was ground in 400 10 μL of extraction buffer (Tris 0.1 M, NaCl 460 mM, EDTA 5 mM, sodium metabisulfite 5 mM, pH 7.5, 250 mM Sorbitol, 60 mM Tris, Na2EDTA, 0.6% Polyclar AT, pH 8.0). The homogenized tissue was centrifuged at 18,200 g for 540 min at 4°C. The supernatant was then collected and analyzed by AmMag. TM Binder-toxin fusion proteins were purified using protein A magnetic beads (L00695; genescript), frozen in liquid nitrogen, and stored at -20°C. Elution was performed with 0.1 M glycine, 460 mM NaCl, pH 3.0, and neutralized with 10% Tris 1 M, pH 8.0. Purified binder-toxin proteins were visualized by SDS-PAGE.

[0131] 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). 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) with either an HRP-conjugated antibody against anti-human IgG Fc-specific region (A0170; Sigma-Aldrich) at a dilution of 1:10.000 or a bryodin primary antibody from Santa Cruz at a dilution of 1:10.000 for 1 h at room temperature. Following the anti-bryodin antibody, an HRP-conjugated anti-rabbit antibody (0545; Sigma) was used at a dilution of 1:10,000. Proteins were detected by enhanced chemiluminescence (Amersham Imager 600 / GE; GE Healthcare).

[0132] Anti-CD20ELISA For anti-CD20 conjugate specificity analysis, plant extracts were analyzed in 96-well microplates (Greiner). 96-well microplates were coated with 100 μL of 5 μg / mL CD20 (AcroBiosystems) for 2 h at 37 °C and washed 5 times with washing buffer (TBS Tween 0,1%). Blocking was then performed with 200 μL of BSA 1% in TBS pH 8.0 for 30 min at RT, followed by 5 washes. 100 μL of anti-CD20 control antibody was loaded to provide a standard curve from 5 to 0 μg / mL, and 100 μL of samples were loaded into the same 96-well plate for comparison for 2 h at RT, followed by 5 washes. 100 μL of 1 / 150.000 diluted detection antibody (goat anti-human HRPO, Bethyl) was added and incubated for 1 h at RT. This was followed by 30 min of reaction in 100 μL of TMB reaction buffer (Zentech), followed by HO. 3 PO 4 The reaction was stopped at 1 M. The enzyme activity was then analyzed by spectrometry at 450 nm.

[0133] Further ELISAs For specificity analysis of binders specific for an undisclosed antigen (a structure expressed on the human cell surface and overexpressed in some cancers, referred to herein as antigen X), purified binder-toxin fusion proteins containing binders against X were analyzed in 96-well microplates (Greiner). Wells were coated with 50 μl of antigen X (2,5 μg / mL) for 1 h at 37 °C and then washed 5 times with 250 μL of wash buffer (PBS Tween 0,1%). Then, blocking was performed for 30 min at RT with 150 μL of hydrocasein (3,6%) in PBST and washed 5 times. A standard curve from 5 to 0 μg / mL was generated by loading 50 μL of anti-antigen control antibody and 50 μL of samples were loaded in the same 96-well plate for comparison for 1 h at RT and washed 5 times. 50 μL of 1 / 200,000 diluted detection antibody (goat anti-human HRPO, Bethyl) was loaded and incubated for 1 h at RT. Then, revelation was performed for 15 min with 50 μL of TMB reaction buffer (Zentech), and finally, HO was added. 3 PO4 The reaction was stopped at 1 M. The enzyme activity was then analyzed by spectrometry at 450 nm. The results are shown in Figure 4B.

[0134] Protein A purification Four days after agroinfiltration, the leaves were collected, weighed, and ground in a mixer with 2 mL of extraction buffer (TRIS 0.1 M, NaCl 460 mM, EDTA 5 mM, Sodium metabisulfite 5 mM pH 7.5) per gram of fresh agroinfiltrated leaves. The mixture was then filtered through a double Miracloth (Millipore) layer. The filtrate was centrifuged at 40.000 g for 10 min at 4 °C. The supernatant was then loaded onto Protein A resin pre-equilibrated with wash buffer. The resin was then washed with 10 column volumes of 60 mM TRIS 25 mM, 460 mM NaCl pH 7.5 and elution was performed with 100 mM glycine, 460 mM NaCl pH 3.0 directly buffered with 10% Tris 1 M pH 8.0. The concentrated protein fraction was collected, dialyzed, and frozen in liquid nitrogen.

[0135] In vitro cytotoxicity test The effect of binder-toxin fusion proteins on the viability of cell lines expressing CD20, CD22 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 Mg2+ and ATP, generating a luminescent signal proportional to the number of viable cells.

[0136] Depending on the cell line tested, cells were seeded into wells of 96-well plates at a density of 2000 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, 5% CO. 2The cells were incubated at 4°C for 72 h. Binder-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.

[0137] After 72 hours, plates were equilibrated at room temperature for 30 minutes and 100 μl of CellTiter Glo reagent was added to each well. Plates were 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.

[0138] 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 taken as 100% viability. The mean signal of treated cells was then normalized and plotted as a function of ATB concentration.

[0139] Anti-CD20-based binder-toxin fusion proteins were evaluated on target cells (CD20+) and non-target cells K562 (CD20-).

[0140] Anti-CD22-based binder-toxin fusion proteins were evaluated on target cells (CD22+) and non-target cells K562 (CD22-).

[0141] Anti-CD79b-based binder-toxin fusion proteins were evaluated on target cells (CD79+) and non-target cells K562 and LOUCY (CD79-).

[0142] Internalization and Caspase Assays: IncuCyte To visualize internalization, caspase activation, and cell proliferation in ATB-treated cells, 10,000 cells were seeded in 100 μl of growth medium (RPMI1640) into wells of poly-L-ornithine-coated plates (96 wells). The Incucyte® Caspase-3 / 7 Dye for Apoptosis Green reagent contains an oligopeptide cleavage sequence (DEVD) conjugated to a DNA-binding dye. The green reagent labels apoptotic cells when the sequence is cleaved by caspase-3 / 7. Immediately after seeding, the cells were added with the Incucyte® Caspase-3 / 7 Dye for Apoptosis Green reagent at a final concentration of 5 μM.

[0143] To visualize ATB internalization, ATB (Fc portion) needs to be labeled with Incucyte® Human Fabfluor-pH Antibody Labeling Dye for Antibody Internalization at a molar ratio of 1:3. After conjugation, the labeled antibody is added to the cells, and the Fab-Ab complex is taken up into the cells via acidic lysosomes and endosomes, processed, and a red fluorescent signal is quickly observed. ATB was diluted with RPMI in the presence of Incucyte® Human Fabfluor-pH antibody labeling dye (1:3 molar ratio) and allowed to bind at 37°C for 15 minutes in the dark. After binding, 100 μl of bound ATB was added to the wells (final ATB concentration: 30 nM) and immediately placed in an incubator. Cell proliferation was measured by phase contrast. Four images per well were taken in the green channel every 10–15 min for 4–8 h, then every 4 h for 72 h. After subtraction of background signal, red and green signals were quantified.

[0144] 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.

[0145] Cleavage assay Addition of recombinant Furin to purified HC-FCS-Bryodin+LC (binder-toxin fusion protein) demonstrated in vitro cleavage that allowed release of the toxin. 1 μl of 25 units / ml Furin (NEB P8077S) was added to 15 μl of cleavage buffer (1M sodium acetate pH 5.5 + 10 mM CaCl2) per microgram of binder-toxin fusion protein, followed by incubation at 37°C for 30 minutes, 2 hours, and 5 hours. Cleavage was visualized on SDS-Page Coomassie Blue gels (4-20% polyacrylamide). Sequence similarity

[0146] Bryodin I (BD1) is a RIP (type I) with N-glycosidase activity. The protein has two N-glycosylation sites. The percentage of sequence identity with trichosanthin (TRI), karasurin (KAR), momordin I (MOM), MOMC and cucurmocin (CUC) was assessed (Figure 2A-2C). Trichosanthin, the natural non-glycosylated homolog, showed the highest identity with BD1 (86,23%). Karasurin has a very close sequence to trichosanthin (97,57% identity) and is also non-glycosylated. Momordin has 67,48% identity with BD1 and one N-glycan site. WT CUC has two N-glycan sites, and the modified sequence CUC (N189S / T227G) has 59,84% identity with BD1 as directly assessed.

[0147] Assay Binder-toxin fusion proteins based on full-length mAbs were constructed using bryodin, momordin, trichosanthin: HC+LC--bryodin, HC-bryodin or LC, HC-bryodin+LC bryodin, LC+HC-FCS-bryodin or momordin or trichosanthin, and unconjugated mAbs alone were constructed as controls.

[0148] Cell viability measurement method The purified binder-toxin fusions were evaluated for cytotoxicity in cancer cell lines. All binder-toxin fusions have been shown to impair the viability of positive cell lines. Furthermore, the inventors demonstrated that type I-based binder-toxin fusions were superior to RIP type II-based binder-toxin fusions targeting the same antigen. In addition, the binder-toxin fusion proteins described above were highly ineffective against target-negative cell lines.

[0149] The binder fusion toxin is harmless to primary cells such as HUVEC and HEPG2, but is highly effective against cancer cells.

[0150] Assay for measuring inhibition of protein synthesis in cell lysates The Thermo Scientific 1-Step Human Coupled IVT Kit from Thermo Fisher was used. Briefly, all components of the reaction (Hela lysate, reaction mix, pCFE-GFP-DNA) and ATB or vehicle are added. 25 μL of the reaction mix is ​​transferred to a 384-well plate and fluorescence is measured at 480ex / 520em for 90 min at 37°C. GFP fluorescence values ​​are plotted on a graph and converted to a slope. The slope is normalized to the reaction mixture containing vehicle (100% protein synthesis) and to the reaction mixture without pCFE-GFP-DNA and vehicle (0% protein synthesis).

[0151] RIP type I expression assay Five-week-old plants of N. benthamiana were infiltrated with a suspension of A. tumefaciens. After 5 days, the plants were harvested and homogenized in a ratio of 2:1 extraction buffer volume / leaf weight. The extract was clarified by centrifugation at 40,000 g for 5 min, and SPR (Biacore) measurements were performed to evaluate the content of expressed proteins in the crude extract.

[0152] PK evaluation in mouse models Pharmacokinetics were evaluated in immunocompromised female C57BL / 6 mice, 7-12 weeks old, weighing 20-25 g. The protein-toxin fusion protein was injected intravenously (tail vein) at a dose of 20 mg / kg. Blood samples (20 μL) were collected at each sampling time point: before administration, 30 min, 4 h, 8 h, 24 h (day 1), 48 h (day 2), 72 h (day 3), and 96 h (day 4). Blood was collected in tubes containing K2-EDTA and plasma was separated from the blood by centrifugation at 1,500 × g for 10 min. Plasma was then transferred into sterile cryovials, aliquoted, and stored at -80 °C until analysis.

[0153] The concentration of the protein-toxin fusion protein was measured by sandwich ELISA, an ELISA in which the target protein (CD79b) is coated onto a microplate. The protein-toxin fusion protein was diluted with mouse plasma and detected with a rabbit-IgG-Fc HRP-conjugated secondary antibody. Sandwich ELISA is commonly used for the PK evaluation of biologics in animal models and is well described in the review by Stephanie D. (2013).

[0154] The experimental results are summarized in the table below. [Table 6] JPEG2025508742000011.jpg170170

[0155] References The contents of the prior art documents referred to in this specification 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. [Table A-1] [Table A-2] [Table A-3]

[0156] array The following sequences form part of the disclosure of this application. A WIPO ST 26 compatible Electronic Sequence Listing is also provided with this application. For the avoidance of doubt, in case of any discrepancy between the sequences in the table below and the sequences in the Electronic Sequence Listing, the sequences 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.

[0157] Sequence Listing [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5]

Claims

1. A binder-toxin fusion protein comprising at least: a) a protein binder, and b) Ribosome-inactivating protein (RIP) type 1 or an active fragment thereof.

2. Ribosome-inactivating proteins (RIPs) Momordin, Bryodin I, - Cucurmosin, ・Bryodin II, - Trichosanthin, ・ Karasurin, ・MOMC, ME1, and / or ・ME2 2. The binder-toxin fusion protein of claim 1, which is at least one selected from the group consisting of:

3. 2. The binder-toxin fusion protein of claim 1, wherein the ribosome-inactivating protein (RIP) comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, or a homolog thereof having at least 66% sequence identity thereto.

4. Protein binders ·antibody, - an antibody fragment or derivative that retains target binding ability, or ・Antibody mimetics 2. The binder-toxin fusion protein of claim 1, selected from the group consisting of:

5. 10. The binder-toxin fusion protein of claim 1, wherein the fusion protein comprises a peptide linker connecting the binder or a fragment thereof to the toxin or a cleavable domain contained in the toxin.

6. 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, or The binder-toxin fusion protein is expressed in a transfected plant cell or a transfected plant host; The binder-toxin fusion protein of claim 1.

7. 2. The binder-toxin fusion protein of claim 1, wherein the protein binder binds to human CD20 or human CD79B.

8. 2. The binder-toxin fusion protein of claim 1, wherein the binder-toxin fusion protein is in any format selected from the group consisting of: (scFv-Fc)-(linker)-toxin (dimer), Toxin-(linker)-Fc-VH / VL, - a tetramer of two HC and two LC-(linker)-toxins (IgG format), a tetramer of two LC and two HC-(linker)-toxins (IgG format), or a tetramer of two LC-(linker)-toxins and two HC-(linker)-toxins (IgG format); Here, the linker is optional.

9. 10. The binder-toxin fusion protein of claim 1, wherein the plant or plant cell is of the genus Nicotiana.

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

11. 10. The binder-toxin fusion protein of claim 1, wherein the protein comprises at least one plant-specific N-glycan.

12. A pharmaceutical composition comprising a binder-toxin fusion protein according to any one of claims 1 to 11, and optionally one or more pharmaceutically acceptable additives.

13. A combination product comprising (i) a binder-toxin fusion protein according to any one of claims 1 to 11, and (ii) one or more further therapeutically active compounds.

14. 12. A binder-toxin fusion protein according to any one of claims 1 to 11 for use in the treatment of a human or animal subject suffering from and / or diagnosed with a neoplastic disease or in the prevention of such a condition.

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