WO2017053290A1 - Ribotoxin molecules derived from sarcin and other related fungal ribotoxins - Google Patents

Abstract

The present application relates to modified T cell epitopes derived from fungal ribotoxins, including α-sarcin, clavin, gigantin, mitogillin, and restrictocin, as well as modified ribotoxin molecules comprising the modified epitopes. The modified ribotoxin molecules inhibit protein synthesis, like the wild type ribotoxins, but exhibit reduced immunogenicity as compared to the corresponding wild type ribotoxin. Another aspect relates to a fusion protein which comprises a modified ribotoxin fused or conjugated or otherwise linked to a targeting molecule that is effective for binding a target of interest. Another aspect relates to the use of the modified ribotoxin or fusion protein for treating or managing a disease or condition.

Description

RIBOTOXIN MOLECULES DERIVED FROM SARCIN AND OTHER RELATED

FUNGAL RIBOTOXINS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and relies on the filing date of, U.S. provisional patent application number 62/222,405, filed 23 September 2015, the entire disclosure of which is herein incorporated by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 16, 2016, is named 0185_0003- PCT_SL.txt and is 24, 1 18 bytes in size.

BACKGROUND

[0003] a-Sarcin was one of the first ribotoxins to be discovered as a product of the mold Aspergillus giganteum MDH18894 in 1965. It was named because of its toxicity to certain sarcoma cell lines. This toxicity was determined later in the mid-1970s to be due to specific cleavage by the toxin of a certain segment of ribosomal RNA (the sarcin-ricin loop) conserved throughout the animal kingdom. Cleavage of that ribosomal RNA by the toxin inhibits protein production by the cell. It is highly toxic, killing cells through an apoptotic mechanism.

[0004] a-Sarcin is a 150 amino acid protein (Lacadena et al., 2007, FEMS Microbiol Rev 31 , 212-237). Much is known about the structure of a-sarcin. Tyr48, His50, Glu96, Arg121 , His137 and Leu145 are critical amino acids for the active site of the RNAse activity. The five-stranded beta sheet and single a-helix are important for the molecule's 3D structure. The protein contains two disulfide bonds. Most of the natural variation between a-sarcin and molecules from related organisms resides in the loops between these structural elements. Deletion of amino acids 7-22 does not appear to affect the protein's conformation. (It did however affect membrane interaction.) The molecule is highly negatively charged with a high isoelectric point. Amino acids 1 16— 139 may be involved in cell membrane interactions, such as crossing of the cell membrane. Asn54 may be involved in the binding pocket for the substrate. Arg121 may be critical for interaction with lipid membranes. The immunogenicity of sarcin has not been well studied.

[0005] Other fungal ribotoxins belong to the same family as a-sarcin and are produced by other Aspergillus species, including, for example, clavin, gigantin, mitogillin, and restrictocin The members of this family of ribotoxins share a high degree of amino acid identity, generally greater than 85%. (Lacadena et al., 2007, FEMS Microbiol Rev 31 , 212-237) and mediate toxicity through the same mechanism, i.e., by cleaving a phosphodiester bond in the conserved sarcin-ricin loop of ribosomal RNA. Clavin and gigantin are 150 amino acids in length, while restrictocin and mitogillin, which are variants of the same polypeptide isolated from A. restrictus, are 149 amino acids in length.

SUMMARY

[0006] Briefly, the present disclosure features modified ribotoxin epitopes of the fungal ribotoxins, including a-sarcin, clavin, gigantin, mitogillin, and restrictocin, e.g., "modified ribotoxin epitopes." Without intending to be bound by any theory or mechanism, it is believed that the modified ribotoxin epitopes disclosed in this application possess reduced binding to human MHC class II and/or elicit a reduced T cell response as compared to the corresponding wild type ribotoxin epitopes.

[0007] The present disclosure also features modified molecules based on the structure of the fungal ribotoxins, including a-sarcin, clavin, gigantin, mitogillin, and restrictocin, e.g., "modified ribotoxin molecules." Without intending to be bound by any theory or mechanism, it is believed that the modified ribotoxin molecules of the present invention are less immunogenic to humans as compared to the wild type ribotoxin. A molecule's efficacy may be limited by an unwanted immune response, particularly if the molecule is used in a therapeutic or prophylactic setting. Therefore, it may be desirable in certain instances to reduce the immunogenicity of a molecule.

[0008] In one embodiment, the modified sarcin polypeptide comprises at least two mutations as compared to a wild type α-sarcin polypeptide (SEQ ID NO: 1 ), wherein the at least two mutations comprise a first mutation within a first T cell epitope and a second mutation within a second T cell epitope of the wild type α-sarcin polypeptide, wherein the first T cell epitope consists of the amino acid sequence XKNPKTNKY (SEQ ID NO:42), wherein X is Q or DQ and the second T cell epitope consists of the amino acid sequence IIAHTKENQ (SEQ ID NO:4) and wherein the first mutation is D9T and the second mutation is Q142T. [0009] In another embodiment, the modified sarcin polypeptide comprises at least two mutations as compared to a wild type a-sarcin polypeptide (SEQ ID NO: 1 ), wherein the at least two mutations comprise a first mutation within a first T cell epitope and a second mutation within a second T cell epitope of the wild type a-sarcin polypeptide, wherein the first T cell epitope consists of the amino acid sequence XKNPKTNKY (SEQ ID NO:42), wherein X is Q or DQ and the second T cell epitope consists of the amino acid sequence IIAHTKENQ (SEQ ID NO:4) and wherein the first mutation is P13I and the second mutation is Q142T.

[0010] The present disclosure also features fusion proteins comprising modified ribotoxin molecules (e.g. , a-sarcin, clavin, gigantin, mitogillin, and restrictocin) and targeting molecules. Targeting molecules may include but are not limited to antibodies, Fab fragments, single chain variable fragments (scFvs), VH domains, engineered CH2 domains, peptides, cytokines, hormones, other protein scaffolds, etc. The fusion proteins may be used as therapeutic agents. For example, in some embodiments, the fusion proteins target an unwanted pathogen or a cancer cell. Thus, certain embodiments are directed to methods of using a fusion protein comprising a modified ribotoxin molecule to treat or manage a disease or condition.

[0011] Another aspect is directed to nucleic acid constructs encoding the modified ribotoxin molecules (e.g. , a-sarcin, clavin, gigantin, mitogillin, and restrictocin) or fusion proteins comprising the same. The nucleic acid constructs can be used, for example, in a method of producing the modified ribotoxin molecule or fusion protein by expressing the nucleic acid construct in a host cell and isolating the modified ribotoxin molecule or fusion protein.

[0012] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0013] The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate aspects of the invention and together with the description serve to explain the principles of the invention. In the drawings:

[0014] Figure 1A shows a plasm id map for a Herceptin® scFV/sarcin fusion protein with a furin cleavage linker sequence. Figure 1 B is a diagram of a representative Herceptin® scFV/sarcin fusion protein with a furin cleavage linker sequence.

[0015] Figure 2A shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with one of the following mutations: Q142T or Q10K. Figure 2B shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with one of the following mutations: N16R or K139E.

[0016] Figure 3 shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with multiple mutations as follows: Q10K/Q142T or N16R/K139E/Q142T.

[0017] Figure 4A shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with multiple mutations as follows: Q10K/K139E/Q142T. Figure 4B shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with the null mutation H137Q.

[0018] Figure 5A shows the results of stability assay, where Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence were incubated for 48 hours at 37°C at a concentration of 10 pg/ml in human serum, cell conditioned media, or PBS before assessing binding to BT-474 cells using an anti-HIS antibody for detection. Figures 5B and 5C show the results of stability assay, where Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence were incubated for 48 hours at 37°C at a concentration of 10 pg/ml in human serum before assessing binding to BT- 474 cells using an anti-HIS antibody (Figure 5B) or an anti-sarcin antibody for detection (Figure 5C).

[0019] Figure 6A shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with two mutations as follows: D9T/Q142T (pRCT06-07) and Q10A/Q142T (pRCT06-08). Figure 6B shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with two mutations as follows: P13I/Q142T (pRCT06-09) and T15G/Q142T (pRCT06-10). Figure 6C shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a furin cleavage linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with two mutations as follows: Y18K/Q142T (pRCT06-1 1 ) and Y18R/Q142T (pRCT06-12).

[0020] Figure 7A shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a GS linker sequence, where the sarcin portion of the fusion protein is a wild type (WT) sarcin (RCT04-22 or pRCT06-20). Figure 7B shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a GS linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin or a modified sarcin polypeptide with a single mutaton (Q142T) or two mutations (Q10K/Q142T).

[0021] Figure 8A (Experiment 1 ) and Figure 8B (Experiment 2) show the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a GS linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin (RCT06-20) or a modified sarcin polypeptide with two mutations as follows: D9T/Q142T and P13I/Q142T. Figure 8C shows the results of a cytotoxicity assay using BT-474 cells and Herceptin® scFV/sarcin fusion proteins with a GS or furin linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin (RCT06-20) or a modified sarcin polypeptide with two mutations as follows: Q10K/Q142T (GS linker) and P13I/Q142T (furin linker).

[0022] Figure 9A (Experiment 1 , Day 5) and Figure 9B (Experiment 2, Day 6) show the results of a cytotoxicity assay using NCI-N87 cells and Herceptin® scFV/sarcin fusion proteins with a GS linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin (RCT06-20) or a modified sarcin polypeptide with two mutations as follows: D9T/Q142T and P13I/Q142T. Figure 9C shows the results of a cytotoxicity assay using NCI-N87 cells and Herceptin® scFV/sarcin fusion proteins with a GS or furin linker sequence, where the sarcin portion of the fusion protein is either a wild type (WT) sarcin (RCT06-20) or a modified sarcin polypeptide with two mutations as follows: Q10K/Q142T (GS linker) and P13I/Q142T (furin linker).

[0023] Figure 10 shows the results of stability assay, where Herceptin® scFV/sarcin fusion proteins with a GS linker sequence were incubated for 48 hours at 37°C at a concentration of 10 pg/ml in human serum, BT-474 cell conditioned medium, or PBS (37°C or 4°C) before assessing binding to BT-474 cells with an anti-HIS antibody.

[0024] Figure 11 A is a box and whisker plot showing healthy donor T cell proliferation responses. Bars represent the 10-90 percentile. Figure 11 B is a box and whisker plot showing healthy donor T cell IL-2 ELISpot responses. Bars represent the 10-90 percentile.

[0025] Figure 12 shows a comparison of immunogenicity predicted using EpiScreen™ technology and immunogenicity observed in a clinical setting. Sixteen therapeutic proteins were tested for their relative risk of immunogenicity using EpiScreen™ technology. Results were plotted against the frequency of immunogenicity (anti-therapeutic antibody responses) observed for each protein when used in the clinic (data sourced from PubMed). The line of regression and the correlation coefficient is shown.

DEFINITIONS

[0026] In order to facilitate the review of the various embodiments of the invention, the following explanations of specific terms are provided:

[0027] Definitions of common terms in molecular biology, cell biology, and immunology may be found in Kuby Immunology, Thomas J. Kindt, Richard A. Goldsby, Barbara Anne Osborne, Janis Kuby, published by W. H. Freeman, 2007 (ISBN 14292021 14); and Genes IX, Benjamin Lewin, published by Jones & Bartlett Publishers, 2007 (ISBN-10: 0763740632).

[0028] Antibody: A protein (or complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The immunoglobulin genes may include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. Light chains may be classified as either kappa or lambda. Heavy chains may be classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0029] As used herein, the term "antibodies" includes intact immunoglobulins as well as fragments (e.g., having a molecular weight between about 10 kDa to 100 kDa). Antibody fragments may include: (1 ) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with the enzyme pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) scFv, single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making antibody fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999). Antibody fragments are not limited to the aforementioned examples, e.g., an antibody fragment may include a VH, a VL, etc.

[0030] Antibodies can be monoclonal or polyclonal. Monoclonal antibodies can be prepared from a variety of methods, e.g., methods involving phage display and human antibody libraries. Examples of procedures for monoclonal antibody production are described in Longberg and Huzar (lnt Rev Immunol., 1995, 13:65-93), Kellermann and Green (Curr Opin Biotechnol., 2002, 13:593-7, and Harlow and Lane (Using Antibodies: A Laboratory Manual, CSHL, New York, 1999). Classical methods of preparing murine hybridomas are discussed in Kohler and Milstein (Nature 256:495- 97, 1975).

[0031] A standard "humanized" immunoglobulin, such as a humanized antibody, is an immunoglobulin including a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, synthetic, etc.) immunoglobulin. A humanized antibody binds to the same or similar antigen as the donor antibody that provides the CDRs. The molecules can be constructed by means of genetic engineering (see, for example, U.S. Patent No. 5,585,089).

[0032] Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response, including compositions that are injected or absorbed. An antigen (Ag) reacts with the products of specific humoral or cellular immunity. In some embodiments, an antigen also may be the specific binding target of the modified sarcin molecule and/or ribotoxin fusion protein (e.g., binding moieties) whether or not such interaction could produce an immunological response.

[0033] Avidity: binding affinity (e.g., increased) as a result from bivalent or multivalent binding sites that may simultaneously bind to a multivalent target antigen or receptor that is either itself multimeric or is present on the surface of a cell or virus such that it can be organized into a multimeric form. For example, the two Fab arms of an immunoglobulin can provide such avidity increase for an antigen compared with the binding of a single Fab arm, since both sites must be unbound for the immunoglobulin to dissociate.

[0034] Binding affinity: The strength of binding between a binding site and a ligand (e.g., between a binding moiety, e.g., an antibody, and an antigen or epitope). The affinity of a binding site X for a ligand Y is represented by the dissociation constant (Kd), which is the concentration of Y that is required to occupy half of the binding sites of X present in a solution. A lower (Kd) indicates a stronger or higher- affinity interaction between X and Y and a lower concentration of ligand is needed to occupy the sites. In general, binding affinity can be affected by the alteration, modification and/or substitution of one or more amino acids in the epitope recognized by the paratope (portion of the molecule that recognizes the epitope). Binding affinity can also be affected by the alteration, modification and/or substitution of one or more amino acids in the paratope. Binding affinity can be the affinity of antibody binding an antigen.

[0035] In one example, binding affinity can be measured by end-point titration in an Ag- ELISA assay. Binding affinity can be substantially lowered (or measurably reduced) by the modification and/or substitution of one or more amino acids in the epitope recognized by the antibody paratope if the end-point titer of a specific antibody for the modified/substituted epitope differs by at least 4-fold, such as at least 10-fold, at least 100-fold or greater, as compared to the unaltered epitope.

[0036] CH2 or CH3 domain molecule: A polypeptide (or nucleic acid encoding a polypeptide) derived from an immunoglobulin CH2 or CH3 domain. Unless noted otherwise, the immunoglobulin can be IgG, IgA, IgD, IgE or IgM. The CH2 or CH3 molecule is composed of a number of parallel β-strands connected by loops of unstructured amino acid sequence. The CH2 or CH3 domain molecule can further comprise an additional amino acid sequence(s), such as a complete hypervariable loop. In some embodiments, the CH2 or CH3 domains comprise one or more mutations in a loop region of the molecule. In some embodiments, the CH2 or CH3 domains comprise one or more mutations in a scaffold region (e.g., for stabilization, etc.). A "loop region" of a CH2 or CH3 domain refers to the portion of the protein located between regions of β-sheet (for example, each CH2 domain comprises seven β-sheets, A to G, oriented from the N- to C-terminus). A CH2 domain comprises six loop regions: Loop 1 , Loop 2, Loop 3, Loop A-B, Loop C-D and Loop E-F. Loops A-B, C-D and E-F are located between β-sheets A and B, C and D, and E and F, respectively. Loops 1 , 2 and 3 are located between β-sheets B and C, D and E, and F and G, respectively. These loops in the natural CH2 domain are often referred to as structural loops. Non-limiting examples of CH2 domain molecules can be found in WO 2009/099961 .

[0037] Naturally occurring CH2 and CH3 domain molecules are small in size, usually less than 15 kD. Engineered CH2 and CH3 domain molecules can vary in size depending on the length of donor loops inserted in the loop regions, how many donor loops are inserted and whether another molecule (such as a binding moiety, an effector molecule, or a label) is conjugated or linked to the CH2 or CH3 domain. The CH2 domain may be from IgG, IgA or IgD. The CH2 domain may be from a CH3 domain from IgE or IgM, which is homologous to the CH2 domains of IgG, IgA or IgD.

[0038] CH2D: A CH2 or CH3 domain molecule. The CH2 or CH3 domain molecule may be engineered such that the molecule specifically binds an antigen. The CH2 and CH3 domain molecules engineered to bind antigen are among the smallest known antigen-specific binding antibody domain-based molecules that can retain Fc receptor binding.

[0039] Contacting: Placement in direct physical association, which includes both in solid and in liquid form.

[0040] Degenerate polynucleotide: As used herein, a "degenerate polynucleotide" is a polynucleotide encoding a protein (e.g., a modified sarcin molecule, a fusion protein) that includes a sequence that is degenerate as a result of redundancies in the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the protein (e.g., the modified sarcin molecule, fusion protein) encoded by the nucleotide sequence is unchanged.

[0041] Preferably, the codons are well expressed in the selected host organism. Use of the degenerate versions of the encoding nucleic acids may optimize expression ("codon optimization") in different expression systems. For example, E. coli expression systems may prefer one codon for an amino acid while a Pichia protein expression system may prefer a different codon for the same amino acid in that position of the protein.

[0042] Domain: A protein structure that retains its tertiary structure independently of the remainder of the protein. In some cases, domains have discrete functional properties and can be added, removed or transferred to another protein without a loss of function.

[0043] Effector molecule: A molecule, or the portion of a chimeric molecule, that is intended to have a desired effect on a cell to which the molecule or chimeric molecule is targeted. An effector molecule is also known as an effector moiety (EM), therapeutic agent, or diagnostic agent, or similar terms. Examples of effector molecules include, but are not limited to, a detectable label, biologically active protein, drug, cytotoxic molecule, or toxin (cytotoxic molecule).

[0044] Epitope: An antigenic determinant. These are particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody binds a particular antigenic epitope based on the three dimensional structure of the antibody and the matching (or cognate) epitope.

[0045] Expression: The translation of a nucleic acid sequence into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane, or be secreted into the extracellular matrix or medium.

[0046] Expression control sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (e.g., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0047] A promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see, for example, Bitter et al. (1987) Methods in Enzymology 153:516-544).

[0048] Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see, for example, Bitter et al. (1987) Methods in Enzymology 153:516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In some embodiments, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as the metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5 K promoter, etc.) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.

[0049] A polynucleotide can be inserted into an expression vector that contains a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

[0050] Expression system: A system for expressing a gene product, e.g., a protein. Expression systems may be cell-based or cell-free. Examples of expression systems include but are not limited to bacterial systems (e.g., E. coli, B. subtilis), yeast systems (e.g., Pichia, S. cerevisiae), an insect system, a eukaryotic system, viral systems (e.g., baculovirus, lambda, retrovirus), and the like.

[0051] Fc binding regions: The FcRn binding region of the CH2 region is known to comprise the amino acid residues M252, 1253, S254, T256, V259, V308, H310, Q31 1 (Kabat numbering of IgG). These amino acid residues have been identified from studies of the full IgG molecule and/or the Fc fragment to locate the residues of the CH2 domain that directly affect the interaction with FcRn. Three lines of investigation have been particularly illuminating: (a) crystallographic studies of the complexes of FcRn bound to Fc, (b) comparisons of the various human isotypes (lgG1 , lgG2, lgG3 and lgG4) with each other and with IgGs from other species that exhibit differences in FcRn binding and serum half-life, correlating the variation in properties to specific amino acid residue differences, and (c) mutation analysis, particularly the isolation of mutations that show enhanced binding to FcRn, yet retain the pH-dependence of FcRn interaction. All three approaches highlight the same regions of CH2 region as crucial to the interaction with FcRn. The CH3 domain of IgG also contributes to the interaction with FcRn, but the protonation/deprotonation of H310 is thought to be primarily responsible and sufficient for the pH dependence of the interaction. In the present invention, a ribotoxin fusion protein may optionally comprise a CH2 domain with a functional FcRn binding site (or additional binding sites) for enhanced half life of the fusion protein molecule.

[0052] Heterologous: A heterologous polypeptide or polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species.

[0053] Immune response: A response of a cell of the immune system, such as a B- cell, T cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen. An immune response can include any cell of the body involved in a host defense response for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response or inflammation.

[0054] Immunoconjugate: A covalent linkage of an effector molecule to a targeting molecule. The effector molecule can be a detectable label, biologically active protein, drug, cytotoxic molecule, or toxin (cytotoxic molecule).

[0055] Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, small molecule toxins, saporin, restrictocin or gelonin, sarcin, ricin, fragments thereof, or modified toxins thereof. Other cytotoxic agents may include auristatin, maytansinoids, and cytolytic peptides. Other immunoconjugates may be composed of a binding protein (e.g., a targeting molecule with a binding moiety) linked to drug molecules (ADC or "antibody drug conjugates"; Ducry and Stump, Bioconj Chem 21 : 5-13, 2010; Erikson et al., Bioconj Chem 21 : 84-92, 2010). These toxins/immunotoxins may directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as domain la of PE and the B chain of DT) and replacing it with a different targeting moiety. In some embodiments, a modified sarcin molecule or a fusion protein of the present invention is joined to an effector molecule (EM). Antibody drug conjugates (ADCs), which are drugs (e.g., cytotoxic agents) conjugated to antibodies (or fragments thereof), deliver therapeutic molecules to their conjugate binding partners. The effector molecule may be a small molecule drug or biologically active protein, such as erythropoietin. In some embodiments, the effector molecule may be an immunoglobulin domain, such as a VH or CH1 domain. In some embodiments, the modified sarcin molecule or the fusion protein joined to an effector molecule is further joined to a lipid or other molecule to a protein or peptide to increase its half-life. The linkage can be either by chemical or recombinant means. "Chemical means" refers to a reaction between the modified sarcin molecule or the fusion protein and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the modified sarcin molecule or the fusion protein and the effector molecule. Such a linker may be subject to proteolysis by an endogenous or exogenous linker to release the effector molecule at a desired site of action. Because immunoconjugates were originally prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as "chimeric molecules." The term "chimeric molecule," as used herein, therefore refers to a targeting moiety, such as a ligand, antibody or fragment or domain thereof, conjugated (coupled) to an effector molecule.

[0056] The terms "conjugating," "joining," "bonding" or "linking" refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionucleotide or other molecule to a polypeptide. In the specific context, the terms can in some embodiments refer to joining a ligand, such as an antibody moiety, to an effector molecule ("EM"). The terms "conjugating," "joining," "bonding" or "linking" may also refer to attaching a peptide to a toxin (e.g., sarcin, modified sarcin molecule, etc.).

[0057] Immunogen: A compound, composition, or substance that is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal.

[0058] The term "Immunogenicity" as used herein is the ability of an immunogen to elicit an immune response. The immune response can be both a humoral or cellular response. Preferably, the immune response is a T cell response. Measuring the activation of an immune response can be done by several methods well known in the art.

[0059] The term "reduced immunogenicity" as used herein means that the modified ribotoxin or modified ribotoxin fusion protein is less immunogenic than the corresponding non-modified ribotoxin or non-modified ribotoxin fusion protein. Preferably, the modified ribotoxin or modified ribotoxin fusion protein elicits a reduced T cell response as compared to the corresponding non-modified ribotoxin or non- modified ribotoxin fusion protein.

[0060] The term "reduced T cell response" as used herein means that the modified ribotoxin or modified ribotoxin fusion protein induces less T cell activation than the corresponding non-modified ribotoxin or non-modified ribotoxin fusion protein, as measured by an in vitro T cell proliferation (³{H}-thymidine incorporation) assay using CD8+ depleted, human peripheral blood mononuclear cells. In one embodiment, the stimulation index (SI) of the modified ribotoxin or modified ribotoxin fusion protein is less than 2.0, and more preferably less than 1.5. The term "stimulation index" as used herein refers to the ability of the modified ribotoxin or modified ribotoxin fusion protein to activate T cells. The SI is conventionally presented as the mean cpm per test samples/mean cpm per control samples (without any test peptide).

[0061] Isolated: An "isolated" biological component (such as a nucleic acid molecule or protein) that has been substantially separated or purified away from other biological components from which the component naturally occurs (for example, other biological components of a cell), such as other chromosomal and extra- chromosomal DNA and RNA and proteins, including other antibodies. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. An "isolated antibody" is an antibody that has been substantially separated or purified away from other proteins or biological components such that its antigen specificity is maintained. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins, or fragments thereof.

[0062] Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule (e.g., a modified sarcin molecule, a targeting molecule, a ribotoxin fusion protein, etc.) to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.

[0063] Ligand contact residue or Specificity Determining Residue (SDR): An amino acid residue within a molecule that participates in contacting a ligand or antigen. A ligand contact residue is also known as a specificity determining residue (SDR).

[0064] Linkers: covalent or very tight non-covalent linkages; chemical conjugation or direct gene fusions of various amino acid sequences, especially those rich in Glycine, Serine, Proline, Alanine, or variants of naturally occurring linking amino acid sequences that connect immunoglobulin domains, and/or carbohydrates including but not limited to polyethylene glycols (PEGs), e.g., discrete PEGs (dPEGs). Typical lengths may range from 2 up to 20 or more amino acids, however the present invention is not limited to these lengths (e.g., the linker may be a peptide between 1 and 20 amino acids). The optimal lengths may vary to match the spacing and orientation of the specific target antigen(s), minimizing entropy but allowing effective binding of multiple antigens.

[0065] Modification: changes to a protein sequence, structure, etc., or changes to a nucleic acid sequence, etc. As used herein, the term "modified" or "modification," can include one or more mutations, deletions, substitutions, physical alteration (e.g., cross-linking modification, covalent bonding of a component, post-translational modification, e.g., acetylation, glycosylation, the like, or a combination thereof), the like, or a combination thereof. Modification, e.g., mutation, is not limited to random modification (e.g., random mutagenesis) but includes rational design as well.

[0066] Multimerizing Domain. Many domains within proteins are known that form a very tight non-covalent dimer or multimer by associating with other protein domain(s). Some of the smallest examples are the so-called leucine zipper motifs, which are compact domains comprising heptad repeats that can either self-associate to form a homodimer (e.g. GCN4); alternatively, they may associate preferentially with another leucine zipper to form a heterodimer (e.g. myc/max dimers) or more complex tetramers (Chem Biol. 2008 Sep 22; 15(9):908-19. A heterospecific leucine zipper tetramer. Deng Y, Liu J, Zheng Q, Li Q, Kallenbach NR, Lu M.). Closely related domains that have isoleucine in place of leucine in the heptad repeats form trimeric "coiled coil" assemblies (e.g. HIV gp41 ). Substitution of isoleucine for leucine in the heptad repeats of a dimer can alter the favoured structure to a trimer. Small domains have advantages for manufacture and maintain a small size for the whole protein molecule, but larger domains can be useful for multimer formation. Any domains that form non-covalent multimers could be employed. For example, the CH3 domains of IgG form homodimers, while CH1 and CL domains of IgG form heterodimers.

[0067] Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non- naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-0-methyl ribonucleotides, peptide- nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes a complementary RNA sequence (i.e., A, U, G, C) in which "U" replaces "T. "

[0068] Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5'-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences;" sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0069] cDNA refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0070] Recombinant nucleic acid refers to a nucleic acid having nucleotide sequences that are not naturally joined together and can be made by artificially combining two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Recombinant nucleic acids include nucleic acid vectors comprising an amplified or assembled nucleic acid, which can be used to transform or transfect a suitable host cell. A host cell that comprises the recombinant nucleic acid is referred to as a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce a "recombinant polypeptide." A recombinant nucleic acid can also serve a non-coding function (for example, promoter, origin of replication, ribosome-binding site and the like).

[0071] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0072] Pharmaceutically acceptable vehicles: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure may be conventional but are not limited to conventional vehicles. For example, E. W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 15th Edition (1975) and D. B. Troy, ed. Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore MD and Philadelphia, PA, 21 ^st Edition (2006) describe compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more antibodies, and additional pharmaceutical agents.

[0073] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. As a non-limiting example, the formulation for injectable trastuzumab includes L-histidine HCI, L-histidine, trehalose dihydrate and polysorbate 20 as a dry powder in a glass vial that is reconstituted with sterile water prior to injection. Other formulations of antibodies and proteins for parenteral or subcutaneous use are well known in the art. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0074] Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are a- amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" may cover naturally occurring proteins, depending on the context, as well as those that are recombinantly or synthetically produced. The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0075] "Conservative" amino acid substitutions are those substitutions that do not substantially affect or decrease an activity or antigenicity of a polypeptide. For example, a polypeptide can include at most about 1 , at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and specifically bind an antibody that binds the original polypeptide. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Examples of conservative substitutions include: (i) Ala - Ser; (ii) Arg - Lys; (iii) Asn - Gin or His; (iv) Asp - Glu; (v) Cys - Ser; (vi) Gin - Asn; (vii) Glu - Asp; (viii) His - Asn or Gin; (ix) lie - Leu or Val; (x) Leu - lie or Val; (xi) Lys - Arg, Gin, or Glu; (xii) Met - Leu or lie; (xiii) Phe - Met, Leu, or Tyr; (xiv) Ser - Thr; (xv) Thr - Ser; (xvi) Trp - Tyr; (xvii) Tyr - Trp or Phe; (xviii) Val - lie or Leu.

[0076] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and/or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, serine or threonine, is substituted for (or by) a hydrophobic residue, for example, leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, for example, glutamate or aspartate; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

[0077] Preventing, treating, managing, or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Managing" refers to a therapeutic intervention that does not allow the signs or symptoms of a disease to worsen. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease.

[0078] Probes and primers: A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Primers are short nucleic acids, and can be DNA oligonucleotides 15 nucleotides or more in length, for example. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.

[0079] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified molecule is one that is isolated in whole or in part from naturally associated proteins and other contaminants in which the molecule is purified to a measurable degree relative to its naturally occurring state, for example, relative to its purity within a cell extract or biological fluid.

[0080] The term "purified" includes such desired products as analogs or mimetics or other biologically active compounds wherein additional compounds or moieties are bound to the molecule in order to allow for the attachment of other compounds and/or provide for formulations useful in therapeutic treatment or diagnostic procedures.

[0081] Generally, substantially purified molecules include more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the respective compound with additional ingredients in a complete pharmaceutical formulation for therapeutic administration. Additional ingredients can include a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other like co-ingredients. More typically, the molecule is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation may be essentially homogeneous, wherein other macromolecular species are less than 1 %. [0082] Recombinant protein: For a recombinant nucleic acid, see "Recombinant Nucleic Acid" above. A recombinant protein or polypeptide is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Recombinant proteins may be made in cells transduced, transfected, or transformed with genetic elements to direct the synthesis of the heterologous protein. They may also be made in cell-free systems. Host cells that are particularly useful include mammalian cells such as CHO and HEK 293, insect cells, yeast such as Pichia pastoris or Saccharomyces, or bacterial cells such as E. coli or Pseudomonas.

[0083] Sample: A portion, piece, or segment that is representative of a whole. This term encompasses any material, including for instance samples obtained from a subject.

[0084] A "biological sample" is a sample obtained from a subject including, but not limited to, cells, tissues and bodily fluids. Bodily fluids include, for example, saliva, sputum, spinal fluid, urine, blood and derivatives and fractions of blood, including serum and lymphocytes (such as B cells, T cells and subfractions thereof). Tissues include those from biopsies, autopsies and pathology specimens, as well as biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin.

[0085] In some embodiments, a biological sample is obtained from a subject, such as blood or serum. A biological sample is typically obtained from a mammal, such as a rat, mouse, cow, dog, guinea pig, rabbit, or primate. In some embodiments, the primate is macaque, chimpanzee, or a human.

[0086] Scaffold: A platform molecule often used for introduction of other domains, loops, mutations, and the like. As an example, a CH2 or CH3 domain scaffold is a CH2 or CH3 domain that can be used to introduce donor loops and/or mutations (such as into the loop regions) in order to confer antigen binding to the CH2 or CH3 domain. In some embodiments, a scaffold is altered to exhibit increased stability compared with the native molecule. For example, a scaffold may be mutated to introduce pairs of cysteine residues to allow formation of one or more non-native disulfide bonds. Scaffolds are not limited to these definitions. In another example a scaffold can be the fibronectin type III domain, Centryns, Affibodies, DARPINS, cyclic peptides, nanoantibodies (VHH domains from llamas), shark domains, etc.

[0087] Sequence identity: The similarity between nucleotide or amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants will possess a relatively high degree of sequence identity overall or in certain regions when aligned using standard methods.

[0088] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman and Wunsch, Journal of Molecular Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5: 151 -153, 1989; Corpet et al., Nucleic Acids Research 16: 10881 -10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genetics 6: 1 19-129, 1994.

[0089] The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., Journal of Molecular Biology 215:403-410, 1990.) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

[0090] Specific binding agent: An agent that binds substantially only to a defined target. Thus an antigen specific binding agent is an agent that binds substantially to an antigenic polypeptide or antigenic fragment thereof. In one embodiment, the specific binding agent is a monoclonal or polyclonal antibody or a peptide or a scaffold molecule that specifically binds the antigenic polypeptide or antigenic fragment thereof.

[0091] The term "specifically binds" refers to the preferential association of a binding agent or targeting moiety (such as hormones, peptides, peptide fragments, domains, cytokines, other ligands and receptors, scaffolds, etc.), in whole or part, with target (e.g., a cell or tissue bearing that target of that binding agent) and not to non-targets (e.g., cells or tissues lacking a detectable amount of that target). It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, specific binding may be distinguished as mediated through specific recognition of the antigen. A variety of immunoassay formats are appropriate for selecting molecules specifically reactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used.

[0092] Subject: Living multi-cellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.

[0093] Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, small molecules, recombinant viruses, or the like. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Alternatively, the molecule linked to a targeting moiety may be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art. See, for example, U.S. Patent No. 4,957,735; and Connor et al. 1985, Pharm. Ther. 28:341 -365. Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as Tc^99m, In¹¹¹, ³²P, ¹²⁵l, and ¹³¹1, fluorophores, chemiluminescent agents, and enzymes.

[0094] Therapeutically effective amount: A quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Such agents include the modified ribotoxin molecules (e.g., modified sarcin, clavin, gigantin, mitogillin, or restrictocin molecule) and fusion proteins described herein. For example, this may be the amount of a fusion protein comprising a modified sarcin molecule useful in preventing, treating or ameliorating a disease or condition, such as cancer. Ideally, a therapeutically effective amount of a modified ribotoxin molecule (e.g., modified sarcin, clavin, gigantin, mitogillin, or restrictocin molecule) or fusion protein is an amount sufficient to prevent, treat or ameliorate the condition or disease, in a subject without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent useful for preventing, ameliorating, and/or treating a subject will be dependent on the subject being treated, the type and severity of the affliction, and the manner of administration of the therapeutic composition.

[0095] Toxin: See Immunoconjugate

[0096] Transduced: A transduced cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transduction encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. Such cells are sometimes called transformed cells.

[0097] Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

DETAILED DESCRIPTION

[0098] The present disclosure provides modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, and restrictocin) molecules, wherein the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, and restrictocin) molecules are less or non- immunogenic compared to the corresponding wild type ribotoxin (e.g., wild type a- sarcin, clavin, gigantin, mitogillin, or restrictocin). The wild type ribotoxin (e.g., wild type a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is modified to create the "modified ribotoxin molecule," where the modification of the wild type ribotoxin molecule reduces its immunogenicity, e.g., reduces or eliminates the number of T cell epitopes (as described below). As used herein, the term "modified" can include one or more mutations, deletions, additions, substitutions, truncations, physical alteration (e.g., cross-linking modification, covalent bonding of a

component, post-translational modification, e.g., acetylation, glycosylation), and the like.

T CELL EPITOPES

[0099] When an antigen-presenting cell of the immune system takes up a protein, the protein is proteolytically digested ("processed") into peptides, some of which can bind to MHC class II molecules and be presented on the surface of antigen- presenting cells to T cells. The binding of peptides to MHC class II is believed to be due to interactions between amino acid side chains of the peptides and specific binding "pockets" within the MHC groove, e.g. , pocket positions p1 , p4, p6, p7 and p9 within the open-ended binding grooves of 34 human MHC class II alleles. The amino acids of the peptide that interact with the p1 , p4, p6, p7, and p9 pocket positions of the class II MHC molecule are called anchor residues (e.g. , P1 , P4, P6, P7, and P9 class II MHC anchor residues).

[00100] In situations where such presented peptides activate CD4+ (helper) T cells, these peptides are defined as CD4+ T cell epitopes, which arise where the complex of peptide and MHC class II is bound by a T cell receptor and, in conjunction with co- stimulatory signals, result in T cell activation. In such cases, these peptides bind within a groove within the MHC class II molecule and allotypic variations in MHC class II can influence the binding of such peptides and, in some cases, can restrict binding to a small number of allotypes ("allotype-restricted"). In other cases, peptides can bind broadly to different MHC allotypes - such non-restricted binding is referred to as "promiscuous" or "degenerate" binding.

MODIFIED SARCIN MOLECULES

[00101] Table 1 shows the sequence corresponding to wild type a-sarcin (SEQ ID NO: 1 ). The modified sarcin molecules of the present invention are derived from a "parent" a-sarcin, for example wild type α-sarcin or fragments of wild type a-sarcin.

[00102] TABLE 1

[00103] International Patent Application PCT/US2013/020035, published as WO 2014/158770, which is incorporated by reference in its entirety, describes in silico analysis of the wild type α-sarcin protein to identify potential T cell epitopes. Briefly, all overlapping 9mer peptides from the wild type α-sarcin sequence were threaded through a database of 34 human MHC class II DR allotypes and individually scored based on their fit and interactions with each of the MHC class II molecules.

[00104] The results of this work suggest that wild type α-sarcin contains at least three potential T cell epitopes comprising a single promiscuous high affinity MHC binding peptide with p1 anchor at residue 24 (L/leucine), and two promiscuous moderate affinity MHC binding peptides with p1 anchors at residues 122 (V/valine) and 134 (l/isoleucine) (see Table 2). Other potential low to very low immunogenic T cell epitopes were also identified.

[00105] TABLE 2

[00106] The wild type α-sarcin was further analyzed by the EpiScreen™ (Cambridge, UK) immunogenicity assay to identify the presence and potency of T cell epitopes within the wild type a-sarcin. Briefly, 46 15-mer peptides overlapping by 12 amino acids and spanning the wild type α-sarcin were tested for proliferation against 50 healthy PBMC donors selected to best represent the spread of HLA-DR alleles in the population. From this analysis, two T cell epitopes were identified within the wild type a-sarcin, as shown in Table 3.

[00107] TABLE 3

[00108] Sarcin Epitope 1 corresponds to amino acid residues 10-18 of the wild type a- sarcin within the N-terminal 22 amino acid region involved in membrane and interaction and binding of α-sarcin to the ribosome. Sarcin Epitope 1 can optionally include the immediately adjacent N-terminal amino acid (P-1 anchor residue) and, thus, comprise the amino acid sequence DQKNPKTNKY (SEQ ID NO:6) corresponding to amino acids 9-18 of the wild type a-sarcin. [00109] The Sarcin Epitope 1 can be modified to reduce or eliminate human MHC class II binding. In one embodiment, the modified Sarcin Epitope 1 has one or more mutations in one or more of the P-1 , P1 , P4, P6, P7, or P9 MHC class II anchor residues of Sarcin Epitope 1 , where the P-1 anchor residue corresponds to the amino acid (D) directly N-terminal to the Sarcin Epitope 1 in the wild type a-sarcin. In another embodiment, the modified Sarcin Epitope 1 has one or more of the following substitutions: P-1 at residue D9: D9T or D9A; P1 anchor at residue Q10: Q10K, Q10R, or Q10A; P4 anchor at residue P13: P13I; P6 anchor at residue T15: T15G, T15Q, or T15H; P7 anchor residue at N16: N16R, N16K, N16A; and/or P9 anchor at residue Y18: Y18H, Y18K, or Y18R. Put another way, the modified Sarcin Epitope 1 has the amino acid sequence of XiX2KNX3KX₄XsKX6, wherein Xi is D, A, or T; X2 is Q, K, R, or A; X₃ is P or I; X₄ is T, G, Q, or H; Xs is N, R, K or A; and Xe is Y, H, K, or R (SEQ ID NO:7).

[00110] In addition to modifying one or more anchor residues, it is also possible to modify one or more non-anchor residues in the Sarcin Epitope 1 provided the modified epitope retains reduced MHC class II binding as compared to wild type a-sarcin. An alignment of Sarcin Epitope 1 with the corresponding epitope in other related, fungal ribotoxins provides guidance as to possible non-anchor residue substitutions. One of ordinary skill in the art could readily identify other non-anchor residue substitutions using conventional methods and techniques.

[00111] Sarcin Epitope 2 corresponds to amino acid residues 134-142 of the wild type α-sarcin and, thus spans H137, which is part of the catalytic triad. The Sarcin Epitope 2 can be modified to reduce or eliminate human MHC class II binding. In one embodiment, the modified Sarcin Epitope 2 has one or more mutations in one or more of the P1 , P6, P7, or P9 MHC class II anchor residues of Sarcin Epitope 2. In another embodiment, the modified Sarcin Epitope 2 has one or more of the following substitutions: P1 anchor at residue 1134: I134A; P6 anchor at residue K139: K139D, K139E, K139G, K139Q, K139H, or K139N; P7 anchor residue at E140: E140D; and/or P9 anchor at residue Q142: Q142D, Q142N, Q142T, Q142E, Q142R, or Q142G. Put another way, the modified Sarcin Epitope 2 has the amino acid sequence of Xi lAHTX₂X₃NX₄, wherein Xi is I or A; X₂ is K, D, E, G, Q, H, or N; X₃ is E or D; and X₄ is Q, D, N, T, E, R, or G (SEQ ID NO:8).

[00112] In addition to modifying one or more anchor residues, it is also possible to modify one or more non-anchor residues in the Sarcin Epitope 2 provided the modified epitope retains reduced MHC class II binding as compared to wild type a-sarcin. An alignment of Sarcin Epitope 2 with the corresponding epitope in other related, fungal ribotoxins provides guidance as to possible non-anchor residue substitutions. One of ordinary skill in the art could readily identify other non-anchor residue substitutions using conventional methods and techniques.

[00113] Without intending to be bound by any theory or mechanism, it is believed that the mutations that reduce or eliminate human MHC class II binding as described herein may help reduce or eliminate the immunogenicity of wild type α-sarcin in humans (e.g. , via reducing the number and/or immunogenicity of T cell epitopes).

[00114] In certain embodiments, the modified sarcin molecule comprises a first and a second mutation compared with a "parent" α-sarcin (e.g. , a wild type a-sarcin, a fragment of wild type a-sarcin, etc.), wherein the first mutation comprises a D9T or P13I mutation and wherein the second mutation comprises a Q142T mutation. In one embodiment, the modified sarcin molecule comprises a first and a second mutation compared with a "parent" α-sarcin (e.g. , a wild type a-sarcin, a fragment of wild type a-sarcin, etc.), wherein the first mutation comprises a D9T mutation and wherein the second mutation comprises a Q142T mutation. In another embodiment, the modified sarcin molecule comprises a first and a second mutation compared with a "parent" a- sarcin (e.g. , a wild type a-sarcin, a fragment of wild type a-sarcin, etc.), wherein the first mutation comprises a P13I mutation and wherein the second mutation comprises a Q142T mutation.

[00115] In other embodiments, the modified sarcin molecule comprises three mutations compared with a "parent" α-sarcin (e.g. , a wild type a-sarcin, a fragment of wild type a-sarcin, etc.). For example, the modified sarcin molecule may comprise a first (D9T or P13I) and second mutation within Sarcin T Cell Epitope 1 (SEQ ID NO:5 or SEQ ID NO:6) and a third mutation (Q142T) within Sarcin T Cell Epitope 2 (SEQ ID NO:4). Alternatively, the modified sarcin molecule may comprise a first mutation (D9T or P13I) within Sarcin T Cell Epitope 1 (SEQ ID NO:5 or SEQ ID NO:6) and a second (Q142T) and third mutation within Sarcin T Cell Epitope 2 (SEQ ID NO:4).

[00116] Table 4 provides the amino acid sequences of the modified sarcin molecules. [00117] TABLE 4

[00118] Modification of the wild type a-sarcin may include an amino acid substitution as described above. In some embodiments, the amino acid substitution is a 2 amino acid substitution (e.g., D9T and Q142T), a 3 amino acid substitution, a 4 amino acid substitution, a 5 amino acid substitution, 6 amino acid substitution, a 7 amino acid substitution, an 8 amino acid substitution, a nine amino acid substitution, a 10 amino acid substitution, or a more than 10 amino acid substitution.

[00119] Modification of the wild type a-sarcin is not limited to an amino acid substitution. For example, the modification may further include an amino acid deletion or an amino acid addition. In some embodiments, the amino acid deletion is a 1 amino acid deletion, a 2 amino acid deletion, a 3 amino acid deletion, a 4 amino acid deletion, a 5 amino acid deletion, 6 amino acid deletion, a 7 amino acid deletion, an 8 amino acid deletion, a nine amino acid deletion, a 10 amino acid deletion, or a more than 10 amino acid deletion. In some embodiments, the amino acid addition is a 1 amino acid addition, a 2 amino acid addition, a 3 amino acid addition, a 4 amino acid addition, a

5 amino acid addition, 6 amino acid addition, a 7 amino acid addition, an 8 amino acid addition, a nine amino acid addition, a 10 amino acid addition, or a more than 10 amino acid addition. Deletions and/or additions may optionally correspond to deletions in regions of the molecule other than T cell epitope regions.

[00120] Wild type α-sarcin comprises two disulfide bonds (between amino acids Cys

6 and Cys 148 and between amino acids Cys 76 and Cys 132). In some embodiments, the modified sarcin molecule comprises an additional disulfide bond. In some embodiments, the additional disulfide bond can be added in sites adjacent to the wild type disulfide bond sites. In some embodiments, additional disulfide bonds are incorporated into the molecule by adding amino acids. In some embodiments, disulfide bonds are incorporated into the molecule by substituting amino acids. In some embodiments, the modified sarcin molecule has no disulfide bonds.

[00121] Modification of the wild type a-sarcin may include an amino acid substitution (as described above) and an additional modification, for example a deletion, an addition, a truncation (e.g., N-terminal truncation, C-terminal truncation), or a combination thereof.

OTHER MODIFIED FUNGAL RIBOTOXIN MOLECULES

[00122] As discussed above, in addition to a-sarcin, there are other related ribotoxin family members produced by other Aspergillus species, including clavin, gigantin, mitogillin, and restrictocin. Table 5 shows the sequences corresponding to wild type clavin (SEQ ID NO:24), gigantin (SEQ ID NO:25), mitogillin (SEQ ID NO:26), and restrictocin (SEQ ID NO:37). The modified clavin, gigantin, mitogillin, and restrictocin molecules of the present invention are derived from a "parent" clavin, gigantin, mitogillin, and restrictocin, respectively, for example wild type clavin, gigantin, mitogillin, or restrictocin, or fragments of wild type clavin, gigantin, mitogillin, or restrictocin.

[00123] TABLE 5

[00124] An example of a rapid method for analysis of the immunogenicity of a protein molecule involves the prediction of peptide binding to human MHC class II molecules. While only a proportion of peptides that bind to MHC class II will be actual T cell epitopes, the analysis of peptide binding to MHC class II can provide a rapid analysis of the potential for immunogenicity of a protein sequence because CD4+ T cell epitopes bind MHC class II. Furthermore, promiscuous high affinity MHC class II binding peptides have been shown to correlate with the presence of T cell epitopes (Hill et al., 2003, Arthritis Res Ther, 1 :R40-R48) and thus analysis of such promiscuous binding peptides provides a basis for analysis of "potential" T cell epitopes.

[00125] Computer methods have been developed to model such interactions, such as iTope (Perry et al., 2008, Drugs in R&D, 9(6) 385-396), which is based on Peptide Threading software (WO 02/069232, WO 98/59244). In iTope, overlapping 9mers from a sequence of interest are individually tested for interaction with 34 different human MHC class II DR allotypes and individually scored based on their fit and interactions with each of the MHC class II molecules. For each MHC allotype, the combined strength of interactions can provide a prediction of the strength of physical binding of each 9mer peptide and the designation of high affinity binding peptides. By collective analysis of the binding of a 9mer to all 34 MHC class II allotypes, the extent of promiscuous or restricted binding can be determined. This allows the identification of promiscuous high affinity MHC class II binding peptides that are thus considered to have high potential for having T cell epitope activities.

[00126] The wild type amino acid sequences of clavin, gigantin, mitogillin, and restrictocin were analyzed for non-self human MHC class II binders. All overlapping 9mers from the wild type ribotoxin sequences were threaded through a database of 34 human MHC class II DR allotypes and individually scored based on their fit and interactions with each of the MHC class II molecules. The predicted binding to MHC class II where the position of the first residue of a 9mer peptide binding to MHC class II allotype ("p1 anchor") has a binding score of 0.55-0.6 or a binding score was >0.6. Regions containing potentially immunogenic peptides are indicated as "Promiscuous High" and "Promiscuous Moderate." "Promiscuous High" MHC binding peptides are defined as both 50% of Total Alleles Binding and High Affinity alleles binding to MHC class II. "Promiscuous Moderate" MHC binding peptides are defined as 50% of Total Alleles Binding to MHC class II but <50% of High Affinity alleles binding to MHC class II.

[00127] The results of this work suggest that wild type clavin contains several potential T cell epitopes, including a promiscuous high affinity MHC binding peptide with p1 anchor at residue 134 (l/isoleucine), and three promiscuous moderate affinity MHC binding peptides with p1 anchors at residues 63 (L/leucine), 122 (V/valine), and 130 (V/valine) (see Table 6). Potential low to very low immunogenic T cell epitopes were also identified.

[00128] TABLE 6

[00129] In addition, the EpiScreen™ (Cambridge, UK) immunogenicity analysis of a- sarcin, suggests that clavin contains the following T cell epitope having a p1 anchor residue of Q10: QKNPKTNKY (SEQ ID NO:5).

[00130] The in silico work also suggests that wild type gigantin contains several potential T cell epitopes, including two promiscuous high affinity MHC binding peptides with p1 anchors at residue at residues 63 (L/leucine) and 122 (V/valine) (see Table 7). Potential low to very low immunogenic T cell epitopes were also identified.

[00131] TABLE 7

[00132] In addition, the EpiScreen™ (Cambridge, UK) immunogenicity analysis of a- sarcin, suggests that gigantin contains the following two T cell epitopes having p1 anchor residues of Q10 and 1134, respectively: QKNIKTNKY (SEQ ID NO:31 ) and IIAHTRENQ (SEQ ID NO:32).

[00133] The in silico work also suggests that wild type mitogillin and restrictocin, which are variants of the same protein isolated from Aspergillus restrictus, contain several potential T cell epitopes, including three promiscuous high affinity MHC binding peptides with p1 anchors at residue at residues 62 (l/lsoleucine), 129 (V/valine), and 133 (l/isoleucine) and a single promiscuous moderate affinity MHC binding peptide with a p1 anchor at residue 121 (V/valine) (see Table 8). Potential low to very low immunogenic T cell epitopes were also identified.

[00134] TABLE 8

[00135] In addition, the EpiScreen™ (Cambridge, UK) immunogenicity analysis of a- sarcin suggests that mitogillin and restrictocin contain the following T cell epitope having a p1 anchor residue of Q10: QLNPKTNKW (SEQ ID NO:36).

[00136] The above-identified clavin, gigantin, mitogillin, and restrictocin T cell epitopes can be modified to reduce or eliminate human MHC class II binding. In one embodiment, the modified clavin polypeptide comprises at least two mutations as compared to a wild type clavin polypeptide (SEQ ID NO:24), wherein the at least two mutations comprise a first mutation within a first T cell epitope and a second mutation within a second T cell epitope of the wild type a-clavin polypeptide, wherein the first T cell epitope consists of the amino acid sequence QKNPKTNKY (SEQ ID NO:5) and the second T cell epitope consists of the amino acid sequence IVAHTRENQ (SEQ ID NO:27) and wherein the first mutation is P13I and the second mutation is Q142T.

[00137] In another embodiment, the modified mitogillin polypeptide comprises at least two mutations as compared to a wild type mitogillin polypeptide (SEQ ID NO:26), wherein the at least two mutations comprise a first mutation within a first T cell epitope and a second mutation within a second T cell epitope of the wild type mitogillin polypeptide, wherein the first T cell epitope consists of the amino acid sequence QLNPKTNKW (SEQ ID NO:36) and the second T cell epitope consists of the amino acid sequence IVAHQRGNQ (SEQ ID NO:35) and wherein the first mutation is P13I and the second mutation is Q141 T.

[00138] In another embodiment, the modified restrictocin polypeptide comprises at least two mutations as compared to a wild type restrictocin polypeptide (SEQ ID NO:37), wherein the at least two mutations comprise a first mutation within a first T cell epitope and a second mutation within a second T cell epitope of the wild type restrictocin polypeptide, wherein the first T cell epitope consists of the amino acid sequence QLNPKTNKW (SEQ ID NO:36) and the second T cell epitope consists of the amino acid sequence IVAHQRGNQ (SEQ ID NO:35) and wherein the first mutation is P13I and the second mutation is Q141 T.

[00139] In another embodiment, the modified gigantin polypeptide comprises at least one mutation as compared to a wild type gigantin polypeptide (SEQ ID NO:25), wherein the wild type gigantin polypeptide comprises at least a first and a second T cell epitope, wherein the first T cell epitope consists of the amino acid sequence QKNIKTNKY (SEQ ID NO:31 ) and the second T cell epitope consists of the amino acid sequence IIAHTRENQ (SEQ ID NO:32) and wherein the at least one mutation is within the second T cell epitope and is Q142T.

[00140] In addition to modifying one or more anchor residues, it is also possible to modify one or more non-anchor residues in the above-identified clavin, gigantin, mitogillin, and restrictocin T cell epitopes, provided the modified epitope retains reduced MHC class II binding as compared to the corresponding wild type ribotoxin.

[00141] Modification of the wild type clavin, gigantin, mitogillin, or restrictocin may include an amino acid substitution as described above. In some embodiments, the amino acid substitution is a 2 amino acid substitution (e.g. , P13I and Q141 T or Q142T), a 3 amino acid substitution, a 4 amino acid substitution, a 5 amino acid substitution, 6 amino acid substitution, a 7 amino acid substitution, an 8 amino acid substitution, a nine amino acid substitution, a 10 amino acid substitution, or a more than 10 amino acid substitution.

[00142] Modification of the wild type clavin, gigantin, mitogillin, or restrictocin is not limited to an amino acid substitution. For example, the modification may include an amino acid deletion or an amino acid addition. In some embodiments, the amino acid deletion is a 1 amino acid deletion, a 2 amino acid deletion, a 3 amino acid deletion, a 4 amino acid deletion, a 5 amino acid deletion, 6 amino acid deletion, a 7 amino acid deletion, an 8 amino acid deletion, a nine amino acid deletion, a 10 amino acid deletion, or a more than 10 amino acid deletion. In some embodiments, the amino acid addition is a 1 amino acid addition, a 2 amino acid addition, a 3 amino acid addition, a 4 amino acid addition, a 5 amino acid addition, 6 amino acid addition, a 7 amino acid addition, an 8 amino acid addition, a nine amino acid addition, a 10 amino acid addition, or a more than 10 amino acid addition. Deletions and/or additions may optionally correspond to deletions in regions of the molecule other than T cell epitope regions. [00143] In certain embodiments, it is possible to substitute a wild type epitope 1 and/or epitope 2 sequence from a first Aspergillus ribotoxin with a deimmunized epitope 1 and/or epitope 2 sequence from a second Aspergillus ribotoxin, wherein the first and second Aspergillus ribotoxin are different. For example, in one embodiment, a wild type Aspergillus ribotoxin is modified by replacing the wild type epitope 1 and/or epitope 2 with a deimmunized epitope 1 and/or 2 from an a-sarcin, clavin, gigantin, mitogillin, or restrictocin ribotoxin.

[00144] In another embodiment, the wild type a-sarcin ribotoxin is modified by replacing the epitope 1 sequence (SEQ ID NO:5 or SEQ ID NO:6) and/or the epitope 2 sequence (SEQ ID NO:4) with a deimmunized epitope 1 and/or 2 from a different Aspergillus ribotoxin, such as clavin, gigantin, mitogillin, or restrictocin. In yet another embodiment, the wild type clavin ribotoxin is modified by replacing the epitope 1 sequence (SEQ ID NO:5) and/or the epitope 2 sequence (SEQ ID NO:27) with a deimmunized epitope 1 and/or 2 from a different Aspergillus ribotoxin, such as a- sarcin, gigantin, mitogillin, or restrictocin. In yet another embodiment, the wild type gigantin ribotoxin is modified by replacing the epitope 1 sequence (SEQ ID NO:31 ) and/or the epitope 2 sequence (SEQ ID NO:32) with a deimmunized epitope 1 and/or 2 from a different Aspergillus ribotoxin, such as a-sarcin, clavin, mitogillin, or restrictocin. In yet another embodiment, the wild type mitogillin or restrictocin ribotoxin is modified by replacing the epitope 1 sequence (SEQ ID NO:36) and/or the epitope 2 sequence (SEQ ID NO:35) with a deimmunized epitope 1 and/or 2 from a different Aspergillus ribotoxin, such as a-sarcin, clavin, or gigantin.

RIBOTOXICITY AND CYTOTOXICITY

[00145] The modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule may retain the cytotoxicity of the corresponding wild type ribotoxin. Cytotoxicity may refer to ribonucleolytic activity toward a specific substrate, e.g. , an oligonucleotide substrate (e.g. , the ribosome), ability to interfere with protein synthesis in a cell-based assay, or cell killing activity toward a particular cell type. For example, a cytotoxicity assay may measure the ability of the toxin to degrade the ribosome. Cytotoxicity is not limited to the aforementioned definitions.

[00146] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule may be as cytotoxic as the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is at least as cytotoxic as the corresponding wild type ribotoxin. It was surprisingly discovered that in certain embodiments, the modified sarcin molecule was more cytotoxic than wild type a-sarcin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is less cytotoxic than the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 10% less cytotoxic than the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 15% less cytotoxic than the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 20% less cytotoxic than the corresponding wild type ribotoxin.

[00147] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule retains the core ribotoxin structure of the corresponding wild type ribotoxin. As used herein, the term "core ribotoxin structure" refers to the arrangement of the alpha helix and beta sheet of wild type ribotoxin. For example, in some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has the same alpha helix arrangement as the corresponding wild type ribotoxin, e.g., the general structure of the alpha helix remains the same. In some embodiments, the amino acids of the alpha helix remain the same as the wild type ribotoxin. The alpha helix amino acids may refer to Glu27-Ala37 (Perez-Canadilas et al., J Mol Biol 2009, 299: 1061 -73) or Glu26-Ala36 for mitogillin or restrictocin. In some embodiments, one or more amino acids in the alpha helix may be modified but the alpha helix structure is still maintained. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has the same beta sheet structure as the corresponding wild type ribotoxin, e.g., the general structure of the beta sheet remains the same. In some embodiments, the amino acids of the beta sheet remain the same as wild type ribotoxin. In some embodiments, one or more amino acids in the alpha helix may be modified but the alpha helix structure is still maintained. The amino acids of the beta sheet may refer to His50-Phe52 and/or Leu94-Phe97 and/or Ala120-Tyr124 and/or Gly 133-Thr138 and/or Glu144-Leu146 (Perez-Canadilas et al., J Mol Biol 2009, 299:1061 -73) or His49-Phe51 and/or Leu93-Phe96 and/or Ala1 19-Tyr123 and/or Gly 132-Gln138 and/or Asp143-Leu146 in mitogillin or restrictocin. In some embodiments, one or more of the amino acids of the active site, e.g., His 50 and/or Glu 96 and/or Arg 121 and/or His137 (or His 49, Glu 95, Arg 120, and/or His 136 in mitogillin or rest ctocin) are not changed in the modified ribotoxin molecule. In some embodiments, one or more of the amino acids of the active site are modified.

[00148] The modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule may retain the ribotoxicity of the corresponding wild type ribotoxin. Ribotoxicity may refer to ribotoxic (e.g., nucleolytic) activity toward a specific substrate, e.g., oligonucleotide substrate (e.g., the ribosome) or ability to interfere with protein synthesis in a cell-based assay. Ribotoxicity is not limited to the aforementioned definitions.

[00149] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule may be as ribotoxic as the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is at least as ribotoxic as the corresponding wild type ribotoxin. It was surprisingly discovered that in certain embodiments, the modified sarcin molecule is more ribotoxic than wild type a-sarcin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is less ribotoxic than the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 10% less ribotoxic than the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 15% less ribotoxic than the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 20% less ribotoxic than the corresponding wild type ribotoxin.

[00150] Assays for ribotoxicity and cytotoxicity of sarcin are well known in the art and described in Carreras-Sangra et al., 2012, PEDS 25, 425-35. Conventional ribotoxicity and cytoxicity assays include the in vitro transcription translation (IVTT) assay described in the Examples of this application.

STABILITY AND SOLUBILITY

[00151] Stability of a protein may determine the ability of the protein to withstand storage or transport conditions. Stability may also affect the protein's half-life after administration (e.g., in serum). The melting temperature of the protein, or the temperature at which the protein loses it tertiary structure, are non-limiting examples of measurements of the physical stability of a protein.

[00152] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule retains the melting temperature of the

corresponding wild type ribotoxin. (The term "retains the melting temperature" may refer to plus or minus 2%, plus or minus 5%, plus or minus 10%). For example, a modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule retains the melting temperature of the corresponding wild type ribotoxin if its melting temperature is within plus or minus 5% of the melting temperature of the

corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a higher melting temperature than the corresponding wild type ribotoxin.

[00153] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a lower melting temperature than the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is no more than 2 degrees less than the melting temperature of the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is no more than 5 degrees less than the melting temperature of the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is no more than 10 degrees less than the melting temperature of the corresponding wild type ribotoxin.

[00154] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is at least 40°C. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is at least 50°C. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is at least 60°C. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is at least 65°C. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is at least 70°C. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a melting temperature that is at least 80°C. Protocols for determining melting temperature of such proteins are well known to one of ordinary skill in the art (e.g., see Gong et al., 2009, JBC 284:21 , pp 14203-14210, and WO 2009/099961 A2).

[00155] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule retains the solubility of the corresponding wild type ribotoxin. (The term "retains the solubility" may refer to plus or minus 2%, plus or minus 5%, plus or minus 10%). For example, a modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule retains the solubility of the corresponding wild type ribotoxin if its solubility is within plus or minus 5% of the solubility of wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a- sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a higher solubility than the corresponding wild type ribotoxin.

[00156] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a lower solubility than the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a solubility that is no more than 10% less than the solubility of the corresponding wild type ribotoxin. In some

embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a solubility that is no more than 15% less than the solubility of the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule has a solubility that is no more than 20% less than the solubility of the corresponding wild type ribotoxin.

[00157] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule or fusion protein comprises a tag. A tag may include but is not limited to a His tag, a flag tag, or the like.

[00158] Without intending to be bound by any theory or mechanism, it is believed that a-sarcin, clavin, gigantin, mitogillin, and restrictocin are not degraded by serum proteases. They are also believed to be relatively resistant to lysosomal and cytosolic proteases. In some embodiments, the modification(s) to the wild type ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule to create the modified ribotoxin molecule do not affect the protease resistant properties of wild type ribotoxin. For example, in some embodiments, the modification(s) do not add a protease cleavage site.

[00159] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule maintains the protease resistant property of the corresponding wild type ribotoxin (e.g., when subjected to serum proteases and/or lysosomal proteases and/or cytosolic proteases). In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 10% less protease resistant (e.g., when subjected to serum proteases and/or lysosomal proteases and/or cytosolic proteases) as compared to the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 20% less protease resistant (e.g., when subjected to serum proteases and/or lysosomal proteases and/or cytosolic proteases) as compared to the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 30% less protease resistant (e.g., when subjected to serum proteases and/or lysosomal proteases and/or cytosolic proteases) as compared to the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 40% less protease resistant (e.g., when subjected to serum proteases and/or lysosomal proteases and/or cytosolic proteases) as compared to the corresponding wild type ribotoxin. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is no more than 50% less protease resistant (e.g., when subjected to serum proteases and/or lysosomal proteases and/or cytosolic proteases) as compared to the corresponding wild type ribotoxin.

RIBOTOXIN FUSION PROTEINS

[00160] The present invention also features ribotoxin fusion proteins, e.g., ribotoxin fusion proteins comprising a modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule as described above. In some embodiments, the ribotoxin fusion protein comprises a modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule having reduced immunogenicity in humans as compared to the corresponding wild type ribotoxin and a targeting molecule effective for binding a target. [00161] The targeting molecule may be linked to the modified ribotoxin (e.g., a- sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the targeting molecule may be incorporated in the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule.

[00162] In some embodiments, the targeting molecule is linked to the N-terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the targeting molecule is linked to the C-terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is linked to the N-terminus of the targeting molecule. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is linked to the C-terminus of the targeting molecule. In some embodiments, the N-terminus of the targeting molecule is linked to the C-terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the N-terminus of the targeting molecule is linked to the N-terminus of the modified ribotoxin (e.g., a- sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the C-terminus of the targeting molecule is linked to the C-terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the C-terminus of the targeting molecule is linked to the N-terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule.

LINKERS

[00163] Linkers may optionally be used to link the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule and the targeting molecule together in a fusion protein. In some embodiments, the targeting molecule is linked to the C-terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule via a linker. In some embodiments, the targeting molecule is linked to the N-terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule via a linker. In some embodiments, the fusion protein is an oligomer of modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules and targeting molecules. For example, in some

embodiments, the fusion protein comprises two targeting molecules and one modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the fusion protein comprises two modified ribotoxin (e.g., a- sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules and one targeting molecule. One or more linkers may optionally be used to link fusion proteins together to form an oligomer or to link components within the fusion protein together.

[00164] Linkers may affect the overall structure of the fusion protein and the accessibility of functional regions of the components of the fusion protein. For example, proline residues are known to bend or kink the structure of a protein, and thus a linker comprising one more proline residues may bend or kink the structure of the fusion protein.

[00165] A linker, for example, may include but is not limited to a peptide of various amino acid lengths and/or sequences. In some embodiments, the linker is between 0 to 10 amino acids in length. In some embodiments, the linker is between 0 to 15 amino acids in length. In some embodiments, the linker is between 0 to 20 amino acids in length. In some embodiments, the linker is between 1 to 10 amino acids in length. In some embodiments, the linker is between 1 to 15 amino acids in length. In some embodiments, the linker is between 1 to 20 amino acids in length. In some embodiments, the linker is between 2 to 20 amino acids in length. In some

embodiments, the linker is between 3 to 20 amino acids in length. In some

embodiments, the linker is between 4 to 20 amino acids in length. In some

embodiments, the linker is between 5 to 10 amino acids in length. In some embodiments the linker is between 10 to 15 amino acids in length. In some embodiments, the linker is between 15 to 20 amino acids in length. In some embodiments, the linker is more than 20 amino acids in length. The optimal lengths may vary to match the spacing and orientation of the specific target (s).

[00166] The linker may be encoded in the gene that encodes the fusion protein. In some embodiments, the linker may be covalently bonded (e.g., cross-linked) to a portion of the fusion protein. The linkers may be covalent or very tight non-covalent linkages; chemical conjugation or direct gene fusions of various amino acid sequences, e.g., those (a) rich in Glycine, Serine, Proline, Alanine, or (b) variants of naturally occurring linking amino acid sequences that connect immunoglobulin domains. In some embodiments, the linker comprises the GGR sequence, as disclosed in Lacadena et al., 2007, FEMS Microbiol Rev 31 , 212-237, or the GSR sequence. In other embodiments, the linker comprises glycine and serine residues (i.e., a GS linker). In certain embodiments, the GS linker has 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 amino acid residues. For example, the GS linker may comprise the sequence GS, GGS, GSS, GSG, GSGS (SEQ ID NO: 15), GSGSGS (SEQ ID

NO: 16), GSGSGSGS (SEQ ID NO: 17), GSGSGSGSGS (SEQ ID NO: 18), GSGSG (SEQ ID NO: 19), GGGGS (SEQ ID NO:20), GSSSS (SEQ ID NO:21 ), GGSSSS (SEQ ID NO:22), or SSGGGG (SEQ ID NO:23). In other embodiments, the linker comprises a furin cleavage sequence, wherein the furin cleavage sequence comprises the sequence RXKR (SEQ ID NO:38) or RXRR (SEQ ID NO:39), where X is Q, N, S, or V. In one embodiment, the furin cleavage sequence comprises the sequence RSKR (SEQ ID NO: 1 1 ). It is also possible to include addition amino acid residues at either end of the furin cleavage sequence. For example, GS or GG can be used interchangeably at both sides of the furin cleavage sequence.

[00167] In some embodiments, the linker comprises a non-peptide component (e.g. , a sugar residue, a heavy metal ion, a chemical agent such as a therapeutic chemical agent, polyethylene glycols (PEGs), e.g. , discrete PEGs, etc.).

[00168] In some embodiments, the dPEG is linked to the modified ribotoxin (e.g. , a- sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule at either one of a serine, tyrosine, cysteine, or lysine of the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the dPEG is linked to a glycosylation site of the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the dPEG is linked to the targeting molecule at either one of a serine, tyrosine, cysteine, or lysine of the targeting molecule. In some embodiments, the dPEG is linked to a glycosylation site of the targeting molecule. In some embodiments, the dPEG is between about 200 to 10,000 daltons.

[00169] In some embodiments, the linker is a hinge component. For example, the targeting molecule may comprise a first half hinge component capable of binding a second half hinge component on the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the hinge components may comprise one or more multimerizing domains. The multimerizing domains may be configured such that they can be cleaved subsequently from the hinge components via proteolysis. Any protease might be used that exhibits sufficient specificity for its particular recognition sequence designed into the linker, but does not cleave any other sequence in the fusion protein. The cleavage may occur at the extreme end of the recognition motif, so that the final fusion protein molecule does not retain any additional amino acid residues that are part of the protease recognition site. The protease may be an enzyme that has little or no effect on a patient if trace amounts were carried over following purification (e.g., Factor X, thrombin).

[00170] An example of a cleavable linker (or adapter) can be found in Heisler et al., 2003, Int. J. Cancer 103 277-282 and Keller et al., 2001 , J Control Release 74, 259- 261. For example, the linker (adapter) comprises a cytosolic cleavable peptide (CCP), membrane transfer peptide (MTP) and endosomal cleavable peptide (ECP). Upon endocytosis of the fusion protein, enzymatic cleavage releases the ligand exposing the MTP, allowing translocation into the cytosol where the MTP is released from the toxin (e.g., sarcin, clavin, gigantin, mitogillin, or restrictocin) by an enzymatic cleavage of the CCP. The ribotoxin fusion proteins described herein may use a similar cleavable linker or various components of such a linker as described in the above references.

[00171] As previously discussed, the fusion protein may be an oligomer, e.g., the fusion protein may comprise a targeting molecule dimer (or multiple targeting molecules) linked to a modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the targeting molecule is a dimer. In some embodiments, the targeting molecule is a trimer. In some embodiments, the targeting molecule is a tetramer. In some embodiments, the targeting molecule is a pentamer. In some embodiments, the targeting molecule comprises more than five subunits. In some embodiments, the fusion protein may be an oligomer, e.g., the fusion protein may comprise a modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule dimer (or multiple modified ribotoxin molecules) linked to a targeting molecule. In some embodiments, the modified ribotoxin (e.g., a- sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is a dimer. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is a trimer. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is a tetramer. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is a pentamer. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule comprises more than five subunits. [00172] The two or multiple targeting molecules or the two or multiple modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules may be coupled by a linker, wherein the linker can be attached to the individual targeting molecules or modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules at any appropriate location. Examples of where a linker may attach onto the targeting molecules include: the C-terminus, the N-terminus. a cysteine preceding or following the C-terminus or N-terminus of the CH2 domain. In some embodiments, a linking of two or more targeting molecules or modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules (e.g., to form a dimer, a trimer, etc.) is driven by the formation of a disulfide bond between cysteines.

[00173] In some embodiments, a linker may be selected from the group consisting of 2-iminothiolane, N-succinimidyi-3-(2-pyridyldithio) propionate (SPDP), 4- succinimidyloxycarbonyl~a-(2~pyridyldithio)toluene (SMPT), m-maieimidobenzoyi-N- hydroxysuccinimide ester (MBS), N-succinimidyi (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4~(p-maleimidophenyl)but- yrate (SMPB), 1 -ethyI-3-(3- dimethylaminopropy!)carbodiimide (EDC), bis-diazobenzidine and glutaraldehyde. In some embodiments, a linker may be attached to an amino group, a carboxyiic group, a suifhydry! group or a hydroxy! group of an amino acid group. The amino group that a linker may attach to includes, for example, alanine, lysine, or proline. The carboxyiic group that a linker may be attached to may be, for example, aspartic acid, glutamic acid. The suifhydryl group that a linker may be attached to may be, for example, cysteine. The hydroxyl group that a linker may be attached to may be, for example, serine, threonine, or tyrosine. Any coupling chemistry known to those skilled in the art capable of chemically attaching targeting molecule to another targeting molecule (or a targeting molecule to a modified ribotoxin molecule) can be used.

TARGETING MOLECULE AND TARGETS

[00174] The fusion protein comprises targeting molecules effective for binding a target. In some embodiments, the targeting molecule comprises a peptide. In some embodiments, the targeting molecule comprises an antibody, an antibody fragment, a single chain variable fragment (scFv), a nanobody, an abdurin, a CH2 domain molecule, a CH2 domain fragment, a CH3 domain molecule, a CH3 domain fragment, a protein scaffold, a hormone, a receptor-binding peptide, the like, or a combination thereof. In some embodiments, the targeting molecule comprises a binding moiety, the binding moiety comprises a VH domain, a VL domain, a tenth type three domain of fibronectin, a designed ankyrin repeat protein, a centyrin scaffold, a peptide ligand, a protein ligand, a receptor, hormone, an enzyme, a cytokine, a small molecule, a fragment thereof, the like, or a combination thereof. The targeting molecule is not limited to the aforementioned examples.

[00175] In some embodiments, the targeting molecule comprises an antigen binding region. In some embodiments, the targeting molecule is a CH2 domain molecule having a molecular weight less than about 20 kDa. In some embodiments, the targeting molecule comprises at least one functional FcRn binding site. In some embodiments, the targeting molecule comprises multiple FcRn binding sites (e.g., for enhanced serum half life).

[00176] In some embodiments, the ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) fusion protein is a monospecific molecule, e.g., the ribotoxin fusion protein is specific for one target. In some embodiments, the ribotoxin fusion protein is a bispecific molecule, e.g., the ribotoxin fusion protein is specific for two targets. In some embodiments, the ribotoxin fusion protein is a trispecific molecule, e.g., the ribotoxin fusion protein is specific for three targets. In some embodiments, the ribotoxin fusion protein is specific for more than three targets.

[00177] In some embodiments, the targeting molecule comprises at least a first paratope specific for a first epitope. In some embodiments, the targeting molecule comprises at least two first paratopes each specific for a first epitope. In some embodiments, the targeting molecule comprises a first paratope specific for a first epitope and a second paratope specific for a second epitope.

[00178] As previously discussed, the ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) fusion protein may further comprise at least one additional targeting molecule. For example, in some embodiments, ribotoxin fusion protein further comprises a second targeting molecule, e.g., linked to either the targeting molecule or the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the ribotoxin fusion protein further comprises a third targeting molecule. In some embodiments, the ribotoxin fusion protein further comprises a fourth targeting molecule. [00179] In some embodiments, the second targeting molecule is linked to the N- terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule and the targeting molecule is linked to the C-terminus of the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the second targeting molecule is linked to the C-terminus of the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule and the targeting molecule is linked to the N-terminus of the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule.

[00180] In some embodiments, the second targeting molecule comprises a first paratope specific for the first epitope. In some embodiments, the second targeting molecule comprises a second paratope specific for a second epitope. In some embodiments, the targeting molecule comprises a third paratope specific for the first epitope or a fourth paratope specific for a third epitope.

[00181] As previously discussed, the ribotoxin fusion protein may further comprise at least one additional modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. For example, in some embodiments, ribotoxin fusion protein further comprises a second modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule. In some embodiments, the second modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is linked to the modified ribotoxin molecule. In some embodiments, the second modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule is linked to the targeting molecule.

[00182] The target may be any appropriate target. A target may include a cell, a tumor cell, an immune cell, a protein, a peptide, a molecule, a bacterium, a virus, a protist, a fungus, the like or a combination thereof. For example, in some

embodiments, a target is a receptor, e.g. , a cell surface receptor. Non-limiting examples of specific targets include Her2 receptor, PMSA, nucleolin, death receptors (e.g. , Fas receptor, tumor necrosis factor receptors, etc.), CD22, CD19, CD79b, DR5, ephA2, Mud , EGFR, VEGFRs, CTLA-4, bacterial and fungal cell surface receptors, CD80, and the like.

[00183] In some embodiments, the ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) fusion protein further comprises an imaging reagent, an isotope, a drug, an immunoconjugate, the like, or a combination thereof. The imaging reagent, isotope, drug, or immunoconjugate may be linked to the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule and/or the targeting molecule.

CELL PERMEABILITY AND RETENTION

[00184] It may be beneficial for the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule (e.g., of a ribotoxin fusion protein) to lack membrane permeability (or have reduced membrane permeability as compared to wild type ribotoxin). This may allow the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule to be administered more safely to patients. For example, if the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule (e.g., of a ribotoxin fusion protein) were to be cleaved from the targeting molecule, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule would not be taken up (or would be less likely to be taken up) by a cell that is not the intended target cell (according to the specificity of the targeting molecule of the ribotoxin fusion protein). In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule comprises a mutation in one or more amino acids important in membrane interaction. For example, in some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule comprises a mutation in amino acid R120 or R121. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule comprises the mutation R120Q or R121 Q. In some embodiments, the modified ribotoxin molecule comprises the mutation R120S or R121 S.

[00185] The membrane permeability mutation may not necessarily be coupled with a mutation in a T cell epitope site. However, in some embodiments, the membrane permeability mutation is coupled with one or multiple mutations in a T cell epitope site (mutations described above)

[00186] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule comprises a mutation that reduced its membrane permeability but does not reduce its cytotoxicity. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule comprises a mutation that reduced its membrane permeability but does not reduce its ribotoxicity (e.g., targeting and/or binding to the SRL site of the ribosome is not affected). [00187] In some embodiments, a molecule is bound to the N terminus of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule (e.g., of a ribotoxin fusion protein), wherein the molecule can be cleaved upon uptake of the modified sarcin molecule in a target cell.

[00188] The modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule having reduced membrane permeability is not limited to the R120Q, R120S, R121 Q, or R121 S mutations. For example, the first 22 amino acids of a-sarcin, gigantin, or clavin or the first 21 amino acids of restrictocin or mitogillin may be important for membrane interaction (and trafficking to the rRNA sarcin-rich loop target site). In some embodiments, one or more of the first 21 or 22 amino acids of the ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) are modified to alter membrane interaction. For example, in some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule comprises a deletion in the first 5 amino acids, a deletion in the first 10 amino acids, a deletion in the first 15 amino acids, a deletion in the first 20 amino acids, or a deletion in the first 22 amino acids. Alternatively, amino acids may be added to the N-terminus (e.g., a tag, etc.) to help eliminate (or reduce) membrane permeability.

[00189] The ribotoxin fusion protein may have enhanced properties (e.g., enhanced cell retention) as compared to the wild type ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) alone, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule alone, and/or the targeting molecule alone. For example, in some embodiments, the targeting molecule is modified to enhance its cell

permeability. In some embodiments, the ribotoxin is modified to reduce its cell permeability (as described above). In some embodiments, the targeting molecule is modified to enhance cell permeability and the ribotoxin is modified to reduce its cell permeability.

[00190] In some embodiments, the fusion protein has increased cell permeability as compared to the targeting molecule alone. In some embodiments, the fusion protein has increased cell permeability as compared to the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule alone. In some embodiments, the fusion protein is modified to increase cell permeability as compared to wild type ribotoxin. In some embodiments, the fusion protein is modified to increase cell permeability as compared to the targeting molecule alone. In some embodiments, the fusion protein is modified to increase cell permeability as compared to the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restnctocin) molecule alone. In some embodiments, the fusion protein has increased cell retention as compared to wild type ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin).

[00191] In some embodiments, the fusion protein has increased cell retention as compared to the targeting molecule alone. In some embodiments, the fusion protein has increased cell retention as compared to the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule alone. In some embodiments, the fusion protein is modified to increase cell retention as compared to wild type ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin). In some

embodiments, the fusion protein is modified to increase cell retention as compared to the targeting molecule alone. In some embodiments, the fusion protein is modified to increase cell retention as compared to the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule alone.

[00192] The ribotoxin fusion protein may comprise a means (e.g., a linker) of allowing it to escape from the endosomes. In some embodiments, the linker is designed to be cleaved in the cytosol. In some embodiments, the linker cannot be cleaved in the blood, e.g., serum.

EXPRESSION

[00193] The modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule and/or the ribotoxin fusion protein may be expressed in any appropriate expression system. For example, in some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule and/or the ribotoxin fusion protein is expressed in an E. coli expression system. In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule and/or the ribotoxin fusion protein is expressed in a Pichia pastoris expression system.

PHARMACEUTICAL COMPOSITIONS

[00194] In some embodiments, the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule comprises or is contained in a pharmaceutical composition. In some embodiments, the fusion protein comprises or is contained in a pharmaceutical composition. Examples of pharmaceutical compositions for antibodies and peptides are well known to one of ordinary skill in the art and are described below.

[00195] In some embodiments, the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule or the fusion protein is bound to a molecule (or molecules) that confers increased stability (e.g. , serum half-life). Dextrans, various polyethylene glycols (PEG), and albumin-binding peptides are extremely common scaffolds for this purpose (see, for example, Dennis et al. , 2002, Journal of Biological Chemistry 33:238390). The molecules may be conjugated to the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule or the fusion protein by a variety of mechanisms, for example via chemical treatments and/or modification of the protein structure, sequence, etc (see, for example, Ashkenazi et al. , 1997, Current Opinions in Immunology 9: 195-200; U.S. Patent No. 5,612,034; U.S. Patent No. 6, 103,233). The molecule (e.g. , dextran, PEG, etc.) may be bound to the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule or the fusion protein through a reactive sulfhydryl by incorporating a cysteine at the end of the protein opposite the binding loops. Such techniques are well known in the art. In another example, a modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule or a fusion protein may bind specifically to albumin to utilize the albumin in serum to increase circulating half-life.

[00196] Choosing pharmaceutical compositions that confer increased protein stability or binding of the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule or the fusion protein to scaffolds that confer increased protein stability are not the only ways in which the stability of the protein can be improved. In some embodiments, the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule or the fusion protein of the present invention may be modified to alter stability. The term "modified" or "modification" in this context can include one or more mutations, additions, deletions, substitutions, disulfide bond additions, physical alteration (e.g. , cross-linking modification, covalent bonding of a component, post-translational modification, e.g. , acetylation, glycosylation, pegylation, the like, or a combination thereof), the like, or a combination thereof. Gong et al. (2009, Journal of Biological Chemistry 284: 14203-14210) shows examples of modified proteins having increased stability. [00197] Due to the unstable nature of proteins, pharmaceutical compositions are often transported and stored via cold chains, which are temperature-controlled uninterrupted supply chains. For example, some pharmaceutical compositions may be stored and transported at a temperature between about 2 to 8 degrees Celsius. Cold chains dramatically increase the costs of such pharmaceutical compositions. Without intending to be bound by any theory or mechanism, it is believed that increasing the stability of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules or the fusion proteins of the present invention (e.g., via pharmaceutical compositions, etc.) may help reduce or eliminate the need to store and transport the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules or the fusion proteins via cold chains.

[00198] The pharmaceutical carrier (vehicles) may be a conventional but is not limited to a conventional carrier (vehicle). For example, E. W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 15th Edition (1975) and D. B. Troy, ed. Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore MD and Philadelphia, PA, 21 st Edition (2006) describe compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more antibodies, and additional pharmaceutical agents. U.S. Patent No. 7,648,702 features an aqueous pharmaceutical composition suitable for long-term storage of polypeptides containing an Fc domain of an immunoglobulin.

[00199] Pharmaceutical compositions may comprise buffers (e.g., sodium phosphate, histidine, potassium phosphate, sodium citrate, potassium citrate, maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris), acetate, diethanolamine, etc.), amino acids (e.g., arginine, cysteine, histidine, glycine, serine, lysine, alanine, glutamic acid, proline), sodium chloride, potassium chloride, sodium citrate, sucrose, glucose, mannitol, lactose, glycerol, xylitol, sorbitol, maltose, inositol, trehalose, bovine serum albumin (BSA), albumin (e.g., human serum albumin, recombinant albumin), dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), polyethylene glycol (PEG), ethylene glycol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), hydrochloride, sacrosine, gamma-aminobutyric acid, Tween-20, Tween-80, sodium dodecyl sulfate (SDS), polysorbate, polyoxyethylene copolymer, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, zinc ions, copper ions, calcium ions, manganese ions, magnesium ions, CHAPS, sucrose monolaurate, 2-O-beta-mannoglycerate, the like, or a combination thereof. The present invention is in no way limited to the pharmaceutical composition components disclosed herein, for example pharmaceutical compositions may comprise propellants (e.g., hydrofluoroalkane (HFA)) for aerosol delivery. U.S. Patent No. 5,192,743 describes a formulation that when reconstituted forms a gel which can improve stability of a protein of interest (e.g., for storage).

[00200] Pharmaceutical compositions may be appropriately constructed for some or all routes of administration, for example topical administration (including inhalation and nasal administration), oral or enteral administration, intravenous or parenteral administration, transdermal administration, epidural administration, and/or the like. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non- toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[00201] In some embodiments, a parenteral formulation may comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. As a non-limiting example, the formulation for injectable trastuzumab includes L-histidine HCI, L-histidine, trehalose dihydrate and polysorbate 20 as a dry powder in a glass vial that is reconstituted with sterile water prior to injection. Other formulations of antibodies and proteins for parenteral or subcutaneous use are well known in the art. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically- neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. The aforementioned pharmaceutical compositions and protein modifications to increase protein stability can be applied as described in U.S. Patent Application 2009/032692, which is hereby incorporated by reference in its entirety.

METHODS OF PRODUCING MODIFIED RIBOTOXIN MOLECULES AND FUSION PROTEINS

[00202] Methods for producing modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules and fusion proteins described herein are well known to one of ordinary skill in the art. For example, the modified ribotoxin (e.g. , a- sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules may be expressed in a bacterial system (e.g., including but not limited to Escherichia coli; Henze et al., Eur J Biochem 192: 127-131, 1990), a yeast system, a phage display system, an insect system, a mammalian system, a ribosomal display, a cis display system (Odegrip et al. , 2004, PNAS 101 , 2806-2810), the like, or a combination thereof. Construction of fusion proteins with sarcin in a P. pastoris expression system has been described in Carreras-Sangra et al. , 2012, PEDS 25, 425-35. The present invention is not limited to the methods (e.g. , protein expression and display systems) described herein. Briefly, as an example, the method may comprise obtaining a vector having a sequence for a modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule; producing a protein product of the sequence for the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule in an expression system; and at least partially purifying the protein product.

[00203] The present invention also features a modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule having reduced immunogenicity as compared to the corresponding wild type ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) produced from methods described herein (e.g. , see Examples below). As discussed above, the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) optionally has enhanced solubility and stability and/or reduced membrane permeability or enhanced cell retention as compared to the corresponding wild type ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) and can be produced from the methods described herein.

TREATING OR MANAGING DISEASES WITH RIBOTOXIN FUSION PROTEINS

[00204] The modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules of the present disclosure may be important tools for treating or managing diseases or conditions. The present disclosure also provides methods of treating or managing a disease or a condition (e.g., in a mammal, e.g. , a human). The methods may comprise obtaining a modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule (or fusion protein comprising the same) and introducing the modified ribotoxin molecule or fusion protein into a patient, wherein the modified ribotoxin molecule or fusion protein binds to a target and the binding functions to cause neutralization or destruction of the target.

[00205] Optionally, the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule (or fusion protein comprising the same) binds to a first or second target that causes either activation or inhibition of a signaling event through that target. The modified ribotoxin molecule or fusion protein comprising the same may comprise an agent (e.g. , chemical, peptide, toxin) that functions to neutralize or destroy the first target. In some embodiments, the agent is inert or has reduced activity when it is constructed as the modified ribotoxin molecule or fusion protein comprising the same and the agent may be activated or released upon uptake or recycling.

[00206] Binding of the modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule (or fusion protein comprising the same) fusion protein may function to cause the neutralization or destruction of the target. The target may be, for example, a cell, a tumor cell, an immune cell, a protein, a peptide, a molecule, a bacterium, a virus, a protist, a fungus, the like, or a combination thereof. The target is not limited to the aforementioned examples. As an example, destruction of a target cell (in this example a tumor) may be achieved by therapy using the following fusion protein: a modified ribotoxin (e.g. , a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecule and a targeting molecule comprising a CH2 domain molecule directed to a particular tumor surface antigen (such as an EGFR, IGFR, nucleolin, ROR1 , CD20, CD19, CD22, CD79a, stem cell markers).

[00207] In some embodiments, the fusion protein can bind to an immune effector cell surface antigen (for example, a T cell specific antigen like CD3, or an NK cell specific surface antigen, like FcyRllla).

[00208] Various methods may be used for detecting the binding of the fusion protein (e.g. , targeting molecule) to the target in the sample. Such methods are well known to one of ordinary skill in the art. DNA SEQUENCES AND CONSTRUCTS

[00209] While not explicitly described, the present invention also features isolated DNA sequences and recombinant constructs for production of the modified ribotoxin (e.g., a-sarcin, clavin, gigantin, mitogillin, or restrictocin) molecules and fusion proteins described herein. DNA sequences can be codon-optimized for the various expression hosts.

EXAMPLE 1 - CONSTRUCTION OF MODIFIED SARCIN/SCFV FUSION

PROTEINS WITH A FURIN CLEAVAGE LINKER

[00210] Fusion proteins comprising a modified sarcin polypeptide linked to a single chain variable fragment antibodies) scFV derived from the Herceptin® antibody were constructed and assayed to test for activity (cytotoxicity) and stability. Herceptin® binds to the Human Epidermal growth factor Receptor 2 (HER2), which is

overexpressed in many breast cancer patients. Initially, a linker comprising a furin cleavage sequence (RSKR) (SEQ ID NO: 1 1 ) was used to join the modified sarcin polypeptide and scFV. The fusion protein constructs also contained a 6X HIS tag (SEQ ID NO:40). See Figures 1 A and 1 B. The modified sarcin portion of the fusion proteins contained either a single mutation (Q10K, N16R, K139E, and Q142T), two mutations (Q10K/Q142T), or three mutations (Q10K/K139E/Q142T and

N16R/K139E/Q142T), as set forth in the table below. Control fusion proteins comprising the wild type sarcin and the sarcin H137 null mutant, which is not toxic, were also constructed.

[00211] TABLE 9

Plasmid Herceptin Furin Sarcin HiSe Epitope MW name scFv cleavage (Kda) pRCT06-06 Yes Yes N16R/K139E/Q142T Yes 1 and 2 -45

Table 9 discloses "Hise" as SEQ ID NO:40.

[00212] In order to express the fusion proteins, an E. coli BL21 strain SHuffle™ T7 Express (NEB, Hitchin, UK) derivative overexpressing the chaperonins GroEL/S was used. Bacteria were transformed with expression plasm ids encoding the fusion protesin and plated out. Single colonies were picked and grown in 2YT broth overnight at 37°C. All constructs were confirmed by sequencing. The following day, the overnight culture was diluted 1 :20 in 3 L 2YT broth and bacterial growth at 37°C was monitored by OD600 measurement. Protein expression was induced by adding IPTG and following cell lysis, proteins were purified using Ni-NTA agarose. Proteins of the expected MW were obtained, demonstrating that the scFv-sarcin fusion proteins are soluble and can be purified using Ni-NTA agarose.

[00213] Cytotoxicity assays were performed using a human breast cancer cell line (BT-474) that expresses HER2. Cell viability was assayed on day 7 using CellTiter- Glo®. All modified sarcin fusion proteins have improved cytotoxicity relative to the H137 null mutant-containing fusion protein, demonstrating that Herceptin scFV effectively targets sarcin to BT-474 cells. The fusion proteins containing a modified sarcin with a single mutation had higher cytotoxicity than the fusion proteins containing a modified sarcin with multiple mutations. Figures 2-4. The single mutation variant Q142T performed as well as the wild type sarcin. Figure 2A. The double mutation variant Q10K/Q142T performed better than the triple mutants.

Figure 3. Thus, an inverse correlation was observed between the number of mutations in the sarcin portion of the fusion protein and the cytotoxicity of the fusion protein, with the fusion protein containing the single Q142T mutation having the highest cytotoxicity.

[00214] Next the stability of these scFV/modified sarcin fusion proteins was tested. For the stability studies, the fusion proteins were incubated in human serum (50%), conditioned media (from a 4-day culture of BT-474 cells) or 1 X PBS at a final concentration of 10 pg/ml fusion protein for 48 hours at 37°C. Following the 48-hour incubation, the binding of the fusion proteins to BT-474 cells was assessed using an anti-HIS antibody that recognizes the His tag of the fusion protein. All of the fusion proteins appear stable in conditioned media, however, only the fusion proteins containing the single Q142T mutation or the wild type sarcin appear stable in human serum. Figure 5A.

[00215] The stability studies were repeated except that the binding of the fusion proteins to BT-474 cells was assessed using either an anti-HIS antibody or an anti- sarcin antibody. Again, only the fusion proteins containing the single Q142T mutation or the wild type sarcin appeared stable in human serum. Reducing binding of Q10K/K139E/Q142T and to a lesser extent Q10K/Q142T appears to correspond to a loss of sarcin, not the HIS tag. Figures 5B and 5C. Thus, results from the initial scFV/sarcin fusion proteins suggested that modified sarcin molecules with a single mutation would yield fusion proteins with the highest cytotoxicity and best stability.

[00216] Additional Herceptin® scFv fusion constructs containing different modified sarcin polypeptides were constructed as shown in the table below.

[00217] TABLE 10

Table 10 discloses "Hise" as SEQ ID NO:40.

[00218] The cytotoxicity of these fusion proteins were assayed using BT-474 cells as described above. The results are shown in Figures 6A-C. Unexpectedly, fusion proteins containing modified sarcin with two mutations (D9T/Q142T, P13I/Q142T, and Q10A/Q142T) exhibited a cytotoxicity profile comparable to the fusion protein containing the wild type sarcin.

EXAMPLE 2 - CONSTRUCTION OF MODIFIED SARCIN/SCFV FUSION

PROTEINS WITH A GS LINKER

[00219] Herceptin® scFv fusion constructs containing a GS linker instead of a furin cleavage sequence were constructed as shown in the table below.

[00220] TABLE 11

Table 1 1 discloses "Hise" as SEQ ID NO:40.

[00221] The cytotoxicity of these fusion proteins were assayed using BT-474 cells as described above. The results are shown in Figures 7A-B. The fusion protein containing the GS linker and the double mutation (Q10K/Q142T) appeared to retain higher cytotoxicity than the same fusion protein constructed with the furin cleavage linker sequence. Compare Figure 3 to Figures 7B.

[00222] Based on these results, additional Herceptin® scFv fusion constructs with the GS linker and the D9T/Q142T and P13I/Q142T mutations were constructed as shown in the table below.

[00223] TABLE 12

Table 12 discloses "Hise" as SEQ ID NO:40.

[00224] The cytotoxicity of these fusion proteins were assayed in two separate experiments using BT-474 cells as described above. The results are shown in Figures 8A-C. Surprisingly, the D9T/Q142T and P13I/Q142T fusion proteins had comparable cytotoxicity to the fusion protein containing the wild type sarcin. Figures 8A-B. This result was not expected given the initial findings where fusion proteins with multiple mutations in the sarcin portion had significantly reduced cytotoxicity compared with the control fusion protein comprising the wild type sarcin. See Figures 3 and 4A. The P13I/Q142T fusion protein with the GS linker also showed better cytotoxicity as compared to the Q10K/Q142T fusion protein with the GS linker. Figure 8C. [00225] These cytotoxicity assays were repeated in a second, HER2 positive cell line, NCI-N87. Cell viability was assayed on day 5 or 6 using CellTiter-Glo®. The results are shown in Figures 9A-C. Again, the D9T/Q142T and P13I/Q142T fusion proteins unexpectedly had comparable cytotoxicity to the fusion protein containing the wild type sarcin. Figures 9A and 9B. The P13I/Q142 fusion protein with the GS linker also showed better cytotoxicity as compared to the Q10K/Q142T fusion protein with the GS linker in the NCI-N87 cells. Figure 9C. Thus, the fusion proteins containing the double mutants D9T/Q142T and P13I/Q142T unexpectedly showed cytotoxicity comparable to the fusion protein with the wild type sarcin in two different cell lines.

[00226] Next the stability of the fusion proteins with the GS linkers was compared to the stability of the same fusion proteins with the furin cleavage linker sequence. For the stability studies, the fusion proteins were incubated in human serum (50%), conditioned media (from a 4-day culture of BT-474 cells) or 1 X PBS at a final concentration of 10 pg/ml fusion protein for 48 hours at 37°C. Stability in 1 X PBS at 4°C was also tested. Following the 48-hour incubation, the binding of the fusion proteins to BT-474 cells was assessed using an anti-HIS antibody that recognizes the 6X HIS tag (SEQ ID NO:40) of the fusion protein. The results are shown in Figure 10.

[00227] For all of the fusion proteins, the presence of the GS linker enhanced stability in human serum and cell conditioned media. The fusion proteins with the GS linker and the D9T/Q142T or P13I/Q142T double mutations are both stable in human serum, exhibiting a stability profile comparable to the fusion protein with the GS linker and wild type sarcin. A fusion protein with the GS linker and the triple mutation Q10K/K139E/Q142T exhibited enhanced stability in human serum and conditioned media as compared to the same constructs with the furin cleavage linker sequence, however, the stability was significantly less as compared to the other GS linker fusion proteins (i.e. , wild type, D9T/Q142T, and P13I/Q142T).

EXAMPLE 3 - IMMUNOGENICITY TESTING OF MODIFIED SARCIN

POLYPEPTIDES

[00228] The immunogenicity of modified sarcin polypeptides was evaluated using the EpiScreen™ DC:T cell assay. Dendritic cells (DC) derived from monocytes isolated from PBMC were loaded with the test samples and induced to mature in order to present T cell epitopes to autologous purified CD4⁺ T cells. CD4⁺ T cell responses to the test samples were assessed in ³H-Thymidine proliferation and IL-2 ELISpot assays.

[00229] EpiScreen™ donor selection

[00230] PBMC were isolated from healthy community donor buffy coats (from blood drawn within 24 hours) obtained from the UK National Blood Transfusion Service (Addenbrooke's Hospital, Cambridge, UK) and commercial suppliers, under informed consent. Cells were separated by Lymphoprep (Axis-shield, Dundee, UK) density centrifugation and donor HLA-DR and DQ haplotypes were identified by HISTO Spot SSO HLA typing (MC Diagnostics, St. Asaph, UK). T cell responses to the control antigen, Keyhole Limpet Haemocyanin (KLH, Sigma, Poole, UK) were also determined. PBMC were then frozen and stored in liquid nitrogen until required.

[00231] A cohort of 20 donors was selected to best represent the number and frequency of HLA-DR and DQ allotypes expressed in the European/North American and the world populations. Analysis of the allotypes expressed revealed that the cohort covered all major HLA-DR and DQ allotypes. Currently HLA-DP is not considered in the selection, due to its more limited polymorphism and likely low levels of expression (Edwards et al 1986).

[00232] Preparation of Test Samples

[00233] Test samples, as detailed in Table 13, were stored at 4°C until use and purity was assessed by denaturing SDS PAGE on a 4-12% silver stained (Silver Xpress Silver Staining Kit, Invitrogen, Paisley, UK) gradient gel. The protein was of the expected size and there were no contaminating protein bands and/or

degradation products present. Endotoxin levels in all test samples were measured using a chromogenic kinetic LAL assay kit according to the manufacturer's instructions (Charles River, Margate, UK) and found to be within the limit acceptable for the assay (<5.0 EU/mg) (Table 13). The test samples were diluted to 3 μΜ in AIM-V® culture medium (Invitrogen) just before use (final assay concentration: 0.3 μΜ). KLH was stored at -20°C at 10 mg/ml in dH2O. For the studies, an aliquot of KLH was thawed immediately before diluting to 3 μΜ in AIM-V® (final assay concentration 0.3 μΜ). Humanised anti-A33 antibody (humanised A33) was used as a clinical benchmark control (Scott et al 2005) and was stored at -80°C as a 1 .05 mg/ml stock solution and diluted in AIM-V® to 3 μΜ before use (final assay concentration: 0.3 μΜ). PHA (Sigma) was used as a positive control in the ELISpot assay and a 1 mg/ml stock was stored at -20°C before diluting to a concentration of 10 g/ml in AIM-V® (final assay concentration: 2.5 Mg/ml).

[00234] TABLE 13

[00235] Sample 3 is directed to the E96Q null mutant that is not cytotoxic. Samples 1 and 2 also contain the E96Q mutation, rendering these variants non-toxic. For the immunogenicity assay, it is necessary to include a null mutation to render the variant non-toxic. Otherwise, the sarcin polypeptide would kill the cells in the

immunogenicity assay.

[00236] Preparation of monocyte-derived DC and autologous CD4⁺ T cells

[00237] To prepare MoDC, PBMC from each donor were revived in AIM-V® culture medium and CD14+ cells (monocytes) were isolated using Miltenyi Pan Monocyte Isolation kits and LS columns (Miltenyi Biotech, Oxford, UK) according to the manufacturer's instructions. Monocytes were resuspended in DC culture media (AIM-V® supplemented with 1000 lU/ml IL-4 and 1000 lU/ml GM-CSF (Peprotech, London, UK)) and plated in low-bind 24 well plates (2 ml final culture volume). Cells were fed on day 2 by half volume DC culture media change. On day 3, antigens (test samples, KLH and humanised A33) were added to the cells in DC culture medium to a final concentration of 0.3 μΜ. In addition, an equivalent volume of DC culture medium was added to the untreated control wells. MoDC were incubated with antigen for 24 hours after which cells were washed three times, and

resuspended in DC culture medium containing 50 ng/ml TNF-a (Peprotech) in order to mature the cells. [00238] Cells were fed again on day 7 by a half volume medium change with DC culture medium containing 50 ng/ml TNF-a before harvesting on day 8. The harvested MoDC were counted and viability assessed using trypan blue (Sigma) dye exclusion. MoDC were then γ-irradiated (40 Gy) before use in the proliferation and ELISpot assays. Also on day 8, autologous CD4+ T cells were isolated by negative selection from PBMC using a CD4+ T Cell Isolation Kit and LS columns (Miltenyi Biotech) according to the manufacturer's instructions.

[00239] Proliferation assays

[00240] After counting and assessing cell viability, 1x10⁵ CD4⁺ T cells were co- cultured with 1x10⁴ irradiated MoDC in 96 well round bottom plates. All cultures were set up in sextuplicate. Following a 7 day co-culture, the cultures were pulsed with 1.0 pCi ³H-thymidine (Perkin Elmer, Buckinghamshire, UK) in 50 μΙ AIM-V® medium and incubated for a further 6 hours before harvesting onto filter mats using a TomTec Mach III cell harvester. Counts per minute (cpm) for each well were determined by Meltilex™ (Perkin Elmer) scintillation counting on a Microplate Beta Counter in paralux, low background counting.

[00241] ELISpot assays

[00242] ELISpot plates (Millipore, Watford, UK) were pre-wetted and coated overnight with 100 μΙ/well IL-2 capture antibody (R&D Systems, Abingdon, UK) in PBS. Plates were then washed 3 times in PBS, incubated overnight in blocking buffer (1 % BSA/PBS) and washed in AIM-V® medium. 50 μΙ autologous CD4⁺ T cells and 50 μΙ of irradiated MoDC or controls were added to the appropriate wells. Each sample was tested in sextuplicate cultures and, for each donor, a negative control (AIM-V® medium alone), no cells control and a mitogen positive control (PHA at 2.5 μg/ml - used as an internal test for ELISpot function and cell viability) were also included on each plate. After the 7-day incubation period, ELISpot plates were developed by sequential washing in dhteO and PBS prior to the addition of 100 μΙ filtered, biotinylated detection antibody (R&D Systems) in 1 % BSA/PBS. Following incubation at 37°C for 1 hour, plates were further washed in PBS and 100 μΙ filtered streptavidin-AP (R&D Systems) in 1 % BSA/PBS was added for 1 .5 hours (incubation at room temperature). Streptavidin-AP was discarded and plates were washed in PBS. 100 μΙ BCIP/NBT substrate (R&D Systems) was added to each well and incubated for 30 minutes at room temperature. Spot development was stopped by washing the wells 3 times with dh O. Plates were scanned on an Immunoscan® Analyser (CTL, Bonn, Germany) and spw were determined using Immunoscan®

Version 5 software.

[00243] Assessment of cell viability

[00244] Following MoDC harvest on day 8, MoDC were assessed for viability using trypan blue dye exclusion. Viability was expressed as a percentage of cells unstained with trypan blue out of the total number of cells.

[00245] EpiScreen™ data analysis

[00246] For proliferation and IL-2 ELISpot assays, an empirical threshold of a SI equal to or greater than 2 (SI > 2.00) has been previously established whereby samples inducing responses above this threshold are deemed positive (where included, borderline SI > 1 .90 are highlighted). Extensive assay development and previous studies have shown that this is the minimum signal to noise threshold allowing maximum sensitivity without detecting large numbers of false positive responses or omitting subtle immunogenic events. For both proliferation (n=6) and IL-2 ELISpot (n=6) data sets, positive responses were defined by statistical and empirical thresholds:

1 ) Significance (p < 0.05) of the response by comparing cpm or spw of test wells against medium control wells using unpaired two sample Student's t-test.

2) Stimulation index greater than 2 (SI > 2.00), where SI = mean of test wells (cpm or spw) / baseline (cpm or spw). Data presented in this way is indicated as SI > 2.00, p <0.05.

[00247] In addition, intra-assay variation was assessed by calculating the CV and SD of the raw data from replicate cultures.

[00248] Results and Discussion

[00249] The immunogenicity of three test samples was evaluated using PBMC isolated from a cohort of 20 HLA typed healthy donors in the EpiScreen™ DC:T cell assay. Briefly, MoDC were semi-matured and left untreated or loaded with the test samples or controls (KLH or humanised A33). Following the removal of the test samples and controls, MoDC were matured with the pro-inflammatory cytokine, TNF- a. Using the EpiScreen™ DC:T cell assay system, it has previously been shown that the treatment of MoDC with TNF-a induces DC maturation, up-regulating the surface expression of HLA class II, CD40 and the co-stimulatory molecules CD80 and CD86. In order to assess the immunogenic potential of the samples, two markers (IL-2 production and proliferation) were used to measure autologous T cell activation by the mature MoDC. While there is generally a good correlation between IL-2 production and proliferation after T cells have been activated, differences can sometimes still occur. The pharmacology of drugs that modulate the immune response may result in lower correlations between T cell proliferation and IL-2 production due to bias in the type of DC or CD4+ T cell induced. Differences can also be due to the kinetics of T cell responses in cultures where transient

proliferative responses can potentially be missed, particularly if the proliferation occurs during the very early stages of the autologous T cell culture (i.e. before day 7). Additional differences can also be due to activation of specific T cell subsets that undergo limited proliferation. Since the IL-2 ELISpot assay comprises a membrane pre-coated with capture antibody which binds secreted cytokine during the entire incubation time, both early and late responses will be detected. Proliferation and IL- 2 ELISpot assays have therefore been interpreted independently, and differences and similarities then highlighted between the respective assay data.

[00250] Assessment of cell viability

[00251] To exclude any direct toxic effects of the test samples on MoDC, the viability of cells four days after the removal of the test samples (day 8 of MoDC culture) was assessed by trypan blue dye exclusion. The null mutant sarcin test samples did not affect cell viability since the mean viability of MoDC treated with medium alone was similar to that of MoDC treated with test samples or control antigens (KLH and humanised A33), i.e. between 92-95% (data not shown).

[00252] Screening of test samples using EpiScreen™ DC:T cell assays

[00253] Table 14 summarizes the CD4+ T cell proliferation in response to the test samples. In Table 14, positive CD4⁺ T cell responses (SI > 2.00, significant p < 0.05) for proliferation ("P") and for IL-2 ELISpot ("E") are shown. Borderline responses (significant p < 0.05 with SI > 1.90) are also indicated (^*). The frequency of positive responses for proliferation and IL-2 ELISpot assays are shown as a percentage at the bottom of the columns. Correlation is expressed as the

percentage of proliferation responses also positive in the ELISpot assay.

[00254] TABLE14

Donor Sample 1 Sample 2 Sample 3 Humanised KLH

Donor 7

Donor 8 E

Donor 9

Donor 10

Donor 11 PE PE P PE

Donor 12 E

Donor 13 p* E

Donor 14 E

Donor 15 E PE

Donor 16 PE

Donor 17 PE

Donor 18 PE

Donor 19 PE* PE

Donor 20 PE PE

Proliferation % 0 10 20 20 50

ELISpot % 0 5 15 25 65

Proliferation and ELISpot % 0 5 15 15 45

Correlation % N/A 50 75 75 90

[00255] Both KLH and humanised A33 elicited positive donor responses (SI > 2.00, p < 0.05, including borderline) in 50% and 20% of the donor cohort respectively (Table 14). These rates are within the expected range across multiple EpiScreen™ DC:T cell assays. Test samples 2 and 3 induced positive CD4+ T cell proliferation with SI > 2.00 (p < 0.05) in 10% and 20% of the donor cohort respectively. No responses were detected against test sample 1 .

[00256] Analysis of the mean magnitude (SI) of positive CD4+ T cell proliferation in response to test samples 2 and 3 (SI > 2.00, p < 0.05) showed low mean Sis of 2.54 and 2.18 respectively (Table 15).

[00257] TABLE15

[00258] Variance analysis of the proliferation data set was used to determine the statistical significance of the magnitude of CD4+ T cell responses amongst test samples and the clinical benchmark control, humanised A33. The magnitude of the responses towards test samples 1 , 2 and 3 was not significantly different from the response to humanised A33. There was no significant difference between the test samples (Figure 1 1A). [00259] Based on the frequency of CD4+ T cell responses and the mean SI the relative ranking of immunogenicity potential was, from highest to lowest: Sample 3 > Sample 2 > Sample 1 (where ">" indicates a difference of more than one donor).

[00260] EpiScreen™ IL-2 ELI Spot assay

[00261] Table 14 also summarizes IL-2 secretion by CD4+ T cells in response to stimulation with test sample-loaded MoDC. Both KLH and humanised A33 elicited positive donor responses (SI > 2.00, p < 0.05, including borderline) in 65% and 25% of the donor cohort respectively (Table 14). These rates are within the expected range across multiple EpiScreen™ DC:T cell assays. The overall correlation between the proliferation and ELISpot assay for the positive control KLH was 90%. All PHA wells were positive for the presence of spots; however determination of an SI value for these wells was not possible, as after 7 days of culture, the majority of wells contained spots too numerous to count (data not shown).

[00262] As with the proliferation assay, test sample 1 did not elicit any positive donor responses. One donor responded positively to test sample 2 (5% of donor cohort) and test sample 3 induced positive responses in 15% of the donor cohort.

[00263] Assessment of the mean magnitude of positive (including borderline) T cell responses (SI > 2.00, p < 0.05) showed low mean Sis of 2.35 for test sample 2 and 2.18 for the test sample 3 (Table 16).

[00264] TABLE 16

[00265] Variance analysis of the IL-2 ELISpot data set was used to determine the statistical significance of the magnitude of CD4+ T cell responses amongst test samples and the clinical benchmark control, humanised A33. There was no significant difference between responses to the test samples or to humanised A33 (Figure 1 1 B).

[00266] Based on the frequency and magnitude of CD4+ IL-2 responses the relative ranking of immunogenicity potential was the same as the ranking for the proliferation assay, from highest to lowest: Sample 3 > Sample 2 > Sample 1 (where ">" indicates that the difference is a single donor response and ">" indicates a difference of more than one donor).

[00267] Interpretation of results

[00268] All donors produced a positive T cell response against PHA indicating that cells in the ex vivo cultures were functional (data not shown). The overall correlation between positive donor responses in the proliferation and IL-2 ELISpot assays was high (90% for KLH) and thus, as in previous studies, responding donors were defined as those that mounted a positive response to a test sample in both IL-2 ELISpot and proliferation assays.

[00269] Analysis of the combined datasets from these two assays showed that the overall frequency of T cell responses was highest for sample 3 (null mutant E96Q control) with 15% of the donor cohort responding positively. A positive response to sample 2 (P13I/Q142T) was detected in 5% of the donor cohort and no positive responses to sample 1 (D9T/Q142T) were detected in the proliferation assay or the IL-2 ELISpot assay. From these results test sample 3 was considered to have a moderate risk of clinical immunogenicity, whereas samples 1 and 2 were considered low risk.

[00270] Variance analysis of the proliferation data set and the IL-2 ELISpot data set was used to determine the statistical significance of the magnitude of CD4+ T cell responses amongst test samples and the clinical benchmark control, humanised A33. In the proliferation and ELISpot assays the magnitudes of the responses towards test samples 1 , 2 and 3 were not significantly different to the magnitude of responses to humanised A33, and there was no significant difference between the test samples (Figures 1 1 A and 1 1 B).

[00271] Conclusions

[00272] The EpiScreen™ DC:T cell assay was used to determine the potential for clinical immunogenicity of three related test samples, a null mutant of wild type sarcin (sample 3) and two deimmunised null mutants of sarcin (sample 1 and 2). The ability of mature MoDC, loaded with the test samples, to induce CD4+ T cell responses in a panel of 20 healthy HLA-typed donors was measured by proliferation and IL-2 production. The results showed that sample 3 (null mutant of wild type sarcin) presented the highest potential risk for clinical immunogenicity with 20% of the donor cohort responding positively in the proliferation assay. Sample 2 (null mutant sarcin P13I/Q142T mutant) showed a lower frequency of positive responses of 10% and sample 1 (null mutant sarcin D9T/Q142T mutant) did not induce any positive responses, indicating a low potential risk for clinical immunogenicity. Based on the empirical dataset (SI > 2.00, p < 0.05) in which the frequency of positive T cell responses was determined for each test sample, the relative risk of all the samples for immunogenicity (from highest to lowest) was: Null mutant of wild type sarcin> null mutant sarcin P13I/Q142T mutant > null mutant sarcin D9T/Q142T mutant.

[00273] Previous EpiScreen™ time course T cell assays with a range of biologies have shown a clear correlation between the percentage of donor T cell responses in the EpiScreen™ assay and the level of immunogenicity (anti-protein therapeutic antibody responses) observed in the clinic. Figure 12. High frequency donor responses were observed in EpiScreen™ assays for immunogenic antibodies such as Infliximab, whereas relatively low frequency donor responses were observed for non-immunogenic antibodies such as Xolair and Herceptin. In general, protein therapeutics that induce > 10% positive responses in the EpiScreen™ assay are associated with a significant risk of immunogenicity in the clinic, for example

Infliximab frequently induced anti-drug antibody responses in the clinic and in the EpiScreen™ assays induces responses in > 10% of donors. In comparison to other protein therapeutics tested in EpiScreen™ assays, the data from this study show that sample 3 (null mutant of wild type sarcin) falls into the same range as Humira and would be considered to have an increased risk of clinical immunogenicity.

Samples 1 and 2 (null mutant sarcin P13I/Q142T and D9T/Q142T mutants) fall into the same range as Herceptin and Avastin and therefore would be considered to have a low potential risk for clinical immunogenicity.

[00274] The disclosures of the following U.S. Patents are incorporated in their entirety by reference herein: U.S. Patent No. 7,750, 136; U.S. Patent No. 8,252,897; U.S. Patent Application No. 2007/0178082; U.S. Patent Application No.

2007/0135620. In addition, International Patent Application PCT/US2013/020035, published as WO 2014/158770, is hereby incorporated by reference in its entirety.

[00275] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[00276] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the invention.

[00277] ADDITIONAL DISCLOSURES:

[00278] THE BELOW DISCLOSURES ARE NOT CLAIMS.

[00279] ADDITIONAL EMBODIMENTS - MODIFIED FUNGAL RIBOTOXINS AND METHODS OF DEIMMUNIZING A Rl BOTOX IN

[00280] 1 . A modified sarcin molecule or fragment thereof comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 1 and comprising a first amino acid modification and a second amino acid modification, wherein the first amino acid modification is D9T or P13I and the second amino acid modification is Q142T, and wherein said modified sarcin molecule or fragment thereof inhibits protein synthesis and elicits a reduced T cell response as compared to wild type a-sarcin (SEQ ID NO: 1 ).

[00281] 2. The modified sarcin molecule of embodiment 1 , wherein the first amino acid modification is D9T.

[00282] 3. The modified sarcin molecule of embodiment 1 , wherein the first amino acid modification is P13I.

[00283] 4. A composition comprising the modified sarcin molecule of embodiment 1 and a pharmaceutically acceptable excipient or carrier.

[00284] 5. The modified sarcin molecule of embodiment 1 , further comprising at least one cell binding ligand.

[00285] 6. The modified sarcin molecule of embodiment 5, wherein the cell-binding ligand is an antibody or antigen-binding fragment thereof, a cytokine, a polypeptide, a hormone, a growth factor, or insulin.

[00286] 7. The modified sarcin molecule of embodiment 6, wherein the cytokine is IL-2 or IL-5.

[00287] 8. The modified sarcin molecule of embodiment 6, wherein the antibody is monoclonal, polyclonal, humanized, genetically engineered or grafted.

[00288] 9. The modified sarcin molecule of embodiment 6, wherein the antigen-binding fragment is a Fab, a Fab2, a F(ab')2, a ScFv, a (ScFv)2, a single chain binding polypeptide, a VH, or a VL.

[00289] 10. The modified sarcin molecule of embodiment 9, wherein the antigen-binding fragment is fused to the modified sarcin molecule or fragment thereof via a linker sequence.

[00290] 1 1 . A composition comprising the modified sarcin molecule of embodiment 5 and a pharmaceutically acceptable excipient or carrier.

[00291] 12. A modified sarcin polypeptide, wherein the modified sarcin polypeptide comprises at least a first mutation as compared to a wild type α-sarcin polypeptide (SEQ ID NO: 1 ), wherein the at least first mutation is within a first T cell epitope consisting of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, wherein the first T cell epitope is replaced with a deimmunized T cell epitope from a different Aspergillus ribotoxin having the at least first mutation, and wherein said modified sarcin polypeptide inhibits protein synthesis and elicits a reduced T cell response as compared to wild type α-sarcin (SEQ ID NO: 1 ).

[00292] 13. A modified sarcin polypeptide, wherein the modified sarcin polypeptide comprises at least a first mutation as compared to a wild type α-sarcin polypeptide (SEQ ID NO: 1 ), wherein the at least first mutation is within a first T cell epitope consisting of the amino acid sequence of SEQ ID NO:4, wherein the first T cell epitope is replaced with a deimmunized T cell epitope from a different Aspergillus ribotoxin having the at least first mutation, and wherein said modified sarcin polypeptide inhibits protein synthesis and elicits a reduced T cell response as compared to wild type α-sarcin (SEQ ID NO: 1 ).

[00293] 14. The modified sarcin polypeptide of embodiment 12, further comprising at least a second mutation, wherein the at least second mutation is within a second T cell epitope consisting of the sequence of SEQ ID NO:4, and wherein the second T cell epitope is replaced with a deimmunized T cell epitope from the different Aspergillus ribotoxin having the at least second mutation. [00294] 15. The modified sarcin polypeptide of any one of embodiments 12-14, wherein the different Aspergillus ribotoxin is selected from the group consisting of clavin, gigantin, mitogillin, and restrictocin.

[00295] 16. A modified clavin polypeptitde, wherein the modified clavin polypeptide comprises at least a first mutation as compared to a wild type clavin polypeptide (SEQ ID NO:24), wherein the at least first mutation is within a first T cell epitope consisting of the amino acid sequence of SEQ ID NO:5, wherein the first T cell epitope is replaced with a deimmunized T cell epitope from a different Aspergillus ribotoxin having the at least first mutation, and wherein said modified sarcin polypeptide inhibits protein synthesis and elicits a reduced T cell response as compared to wild type clavin (SEQ ID NO:24).

[00296] 17. A modified clavin polypeptide, wherein the modified clavin polypeptide comprises at least a first mutation as compared to a wild type clavin polypeptide (SEQ ID NO:24), wherein the at least first mutation is within a first T cell epitope consisting of the amino acid sequence of SEQ ID NO:27, wherein the first T cell epitope is replaced with a deimmunized T cell epitope from a different Aspergillus ribotoxin having the at least first mutation, and wherein said modified sarcin polypeptide inhibits protein synthesis and elicits a reduced T cell response as compared to wild type clavin (SEQ ID NO:24).

[00297] 18. The modified clavin polypeptide of embodiment 16, further comprising at least a second mutation, wherein the at least second mutation is within a second T cell epitope consisting of the sequence of SEQ ID NO:27, and wherein the second T cell epitope is replaced with a deimmunized T cell epitope from the different Aspergillus ribotoxin having the at least second mutation.

[00298] 19. The modified clavin polypeptide of any one of embodiment s 16-18, wherein the different Aspergillus ribotoxin is selected from the group consisting of sarcin, gigantin, mitogillin, and restrictocin.

[00299] 20. A modified gigantin polypeptide, wherein the modified gigantin polypeptide comprises at least a first mutation as compared to a wild type gigantin polypeptide (SEQ ID NO:25), wherein the at least first mutation is within a first T cell epitope consisting of the amino acid sequence of SEQ ID NO: 31 , wherein the first T cell epitope is replaced with a deimmunized T cell epitope from a different Aspergillus ribotoxin having the at least first mutation, and wherein said modified sarcin polypeptide inhibits protein synthesis and elicits a reduced T cell response as compared to wild type gigantin (SEQ ID NO:25).

[00300] 21 . A modified gigantin polypeptide, wherein the modified gigantin polypeptide comprises at least a first mutation as compared to a wild type gigantin polypeptide (SEQ ID NO:25), wherein the at least first mutation is within a first T cell epitope consisting of the amino acid sequence of SEQ ID NO:32, wherein the first T cell epitope is replaced with a deimmunized T cell epitope from a different Aspergillus ribotoxin having the at least first mutation, and wherein said modified sarcin polypeptide inhibits protein synthesis and elicits a reduced T cell response as compared to wild type gigantin (SEQ ID NO:25).

[00301] 22. The modified gigantin polypeptide of embodiment 20, further comprising at least a second mutation, wherein the at least second mutation is within a second T cell epitope consisting of the sequence of SEQ ID NO:32, and wherein the second T cell epitope is replaced with a deimmunized T cell epitope from the different Aspergillus ribotoxin having the at least second mutation.

[00302] 23. The modified gigantin polypeptide of any one of embodiments 20-22, wherein the different Aspergillus ribotoxin is selected from the group consisting of sarcin, clavin, mitogillin, and restrictocin.

[00303] 24. A modified mitogillin or restrictocin polypepitde, wherein the modified mitogillin or restrictocin polypeptide comprises at least a first mutation as compared to a wild type mitogillin or restrictocin polypeptide (SEQ ID NO:26 or SEQ ID NO:37), wherein the at least first mutation is within a first T cell epitope consisting of the amino acid sequence of SEQ ID NO: 36, wherein the first T cell epitope is replaced with a deimmunized T cell epitope from a different Aspergillus ribotoxin having the at least first mutation, and wherein said modified sarcin polypeptide inhibits protein synthesis and elicits a reduced T cell response as compared to wild type mitogillin or restrictocin (SEQ ID NO:26 or SEQ ID NO:37).

[00304] 25. A modified mitogillin or restrictocin polypeptide, wherein the modified mitogillin or restrictocin polypeptide comprises at least a first mutation as compared to a wild type mitogillin or restrictocin polypeptide (SEQ ID NO:26 or SEQ ID NO:37), wherein the at least first mutation is within a first T cell epitope consisting of the amino acid sequence of SEQ ID NO: 35, wherein the first T cell epitope is replaced with a deimmunized T cell epitope from a different Aspergillus ribotoxin having the at least first mutation, and wherein said modified sarcin polypeptide inhibits protein synthesis and elicits a reduced T cell response as compared to wild type mitogillin or restrictocin (SEQ ID NO:26 or SEQ ID NO:37).

[00305] 26. The modified mitogillin or restrictocin polypeptide of embodiment 24, further comprising at least a second mutation, wherein the at least second mutation is within a second T cell epitope consisting of the sequence of SEQ ID NO:35, and wherein the second T cell epitope is replaced with a deimmunized T cell epitope from the different Aspergillus ribotoxin having the at least second mutation.

[00306] 27. The modified clavin polypeptide of any one of embodiments 24-26, wherein the different Aspergillus ribotoxin is selected from the group consisting of sarcin, clavin, and gigantin.

[00307] 28. A method of deimmunizing a first Aspergillus ribotoxin polypeptide, the method comprising replacing a first wild type T cell epitope and/or a second wild type T cell epitope within the first Aspergillus ribotoxin with a first deimmunized T cell epitope and/or a second deimmunized T cell epitope from a second Aspergillus ribotoxin, wherein the second Aspergillus ribotoxin is different from the first Aspergillus ribotoxin, wherein the first wild type T cell epitope consists of an amino acid selected from the goup consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:31 , and SEQ ID NO:36 and the second wild type T cell epitope consists of an amino acid selected from the goup consisting of SEQ ID NO:4, SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:35.

[00308] 29. A method of deimmunizing a wild type sarcin ribotoxin polypeptide having the amino acid sequence of SEQ ID NO: 1 , the method comprising replacing a first wild type T cell epitope and/or a second wild type T cell epitope within the sarcin ribotoxin polypeptide with a first deimmunized T cell epitope and/or a second deimmunized T cell epitope from an Aspergillus ribotoxin other than sarcin, wherein the first wild type T cell epitope consists of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6 and wherein the second wild type T cell epitope consists of the amino acid sequence of SEQ ID NO:4. [00309] 30. The method of embodiment 29, wherein the Aspergillus ribotoxin other than sarcin is selected from the group consisting of clavin, gigantin, mitogillin, and restrictocin.

[00310] 31 . The method of embodiment 30, wherein the first deimmunized T cell epitope is a deimmunized version of SEQ ID NO:31 or SEQ ID NO:36.

[00311] 32. The method of any one of embodiments 29-31 , wherein second deimmunized T cell epitope is a deimmunized version of SEQ ID NO:27, SEQ ID NO:32, or SEQ ID NO:35.

[00312] 33. The method of any one of embodiments 29-32, wherein both the first and second T cell epitopes are replaced with the first and second deimmunized T cell epitopes, respectively.

[00313] 34. A method of deimmunizing a wild type clavin ribotoxin polypeptide having the amino acid sequence of SEQ ID NO:24, the method comprising replacing a first wild type T cell epitope and/or a second wild type T cell epitope within the clavin ribotoxin polypeptide with a first deimmunized T cell epitope and/or a second deimmunized T cell epitope from an Aspergillus ribotoxin other than clavin, wherein the first wild type T cell epitope consists of the amino acid sequence of SEQ ID NO:5 and wherein the second wild type T cell epitope consists of the amino acid sequence of SEQ ID NO:27.

[00314] 35. The method of embodiment 34, wherein the Aspergillus ribotoxin other than clavin is selected from the group consisting of sarcin, gigantin, mitogillin, and restrictocin.

[00315] 36. The method of embodiment 35, wherein the first deimmunized T cell epitope is a deimmunized version of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:31 , or SEQ ID NO:36.

[00316] 37. The method of any one of embodiments 34-36, wherein second deimmunized T cell epitope is a deimmunized version of SEQ ID NO:4, SEQ ID NO:32, or SEQ ID NO:35.

[00317] 38. The method of any one of embodiments 34-37, wherein both the first and second T cell epitopes are replaced with the first and second deimmunized T cell epitopes, respectively.

[00318] 39. A method of deimmunizing a wild type gigantin ribotoxin polypeptide having the amino acid sequence of SEQ ID NO:25, the method comprising replacing a first wild type T cell epitope and/or a second wild type T cell epitope within the gigantin ribotoxin polypeptide with a first deimmunized T cell epitope and/or a second deimmunized T cell epitope from an Aspergillus ribotoxin other than gigantin, wherein the first wild type T cell epitope consists of the amino acid sequence of SEQ ID NO:31 and wherein the second wild type T cell epitope consists of the amino acid sequence of SEQ ID NO:32.

[00319] 40. The method of embodiment 39, wherein the Aspergillus ribotoxin other than gigantin is selected from the group consisting of sarcin, clavin, mitogillin, and restrictocin.

[00320] 41 . The method of embodiment 40, wherein the first deimmunized T cell epitope is a deimmunized version of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36.

[00321] 42. The method of any one of embodiments 39-41 , wherein second deimmunized T cell epitope is a deimmunized version of SEQ ID NO:4, SEQ ID NO:27, or SEQ ID

NO:35.

[00322] 43. The method of any one of embodiments 39-42, wherein both the first and second T cell epitopes are replaced with the first and second deimmunized T cell epitopes, respectively.

[00323] 44. A method of deimmunizing a wild type mitogillin or restrictocin ribotoxin polypeptide having the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:37, the method comprising replacing a first wild type T cell epitope and/or a second wild type T cell epitope within the mitogillin or restrictocin ribotoxin polypeptide with a first deimmunized T cell epitope and/or a second deimmunized T cell epitope from an Aspergillus ribotoxin other than mitogillin or restrictocin, wherein the first wild type T cell epitope consists of the amino acid sequence of SEQ ID NO:36 and wherein the second wild type T cell epitope consists of the amino acid sequence of SEQ ID NO:35. [00324] 45. The method of embodiment 44, wherein the Aspergillus ribotoxin other than mitogillin or restrictocin is selected from the group consisting of sarcin, clavin, and gigantin.

[00325] 46. The method of embodiment 45, wherein the first deimmunized T cell epitope is a deimmunized version of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:31 .

[00326] 47. The method of any one of embodiments 44-46, wherein second deimmunized T cell epitope is a deimmunized version of SEQ ID NO:4, SEQ ID NO:27, or SEQ ID NO:32.

[00327] 48. The method of any one of embodiments 44-47, wherein both the first and second T cell epitopes are replaced with the first and second deimmunized T cell epitopes, respectively.

[00328] ADDITIONAL EMBODIMENTS - NUCLEIC ACID ENCODING MODIFIED

SARCIN

[00329] 1 . A nucleic acid encoding a modified sarcin protein, wherein the modified sarcin protein has a reduced propensity to elicit an immune response, as compared to the wild type sarcin protein (SEQ ID NO: 1 ), wherein the amino acid sequence of the modified sarcin protein comprises:

AVTWTCLNTXi KNX2KX3X4KX5ET KRLLYNQNKA ESNSHHAPLS

DGKTGSSYPH WFTNGYDGDG KLPKGRTPIK FGKSDCDRPP KHSKDGNGKT DHYLLEFPTF PDGHDYKFDS KKPKENPGPA RVIYTYPNKV FCGXelAHTXyXs NTGELKLCSH,

wherein Xi through Xs can be any amino acid (SEQ ID NO: 12), provided that the amino acid sequence of the modified sarcin protein is not identical to the wild

type sarcin protein (SEQ ID NO:1 ).

[00330] 1 .1 . A nucleic acid encoding a modified sarcin protein, wherein the modified sarcin protein has a reduced propensity to elicit an immune response, as compared to the wild type sarcin protein (SEQ ID NO: 1 ), wherein the amino acid sequence of the modified sarcin protein comprises:

AVTWTCLNX1X2 KNIKX3X4KX5ET KRLLYNQNKA ESNSHHAPLS

DGKTGSSYPH WFTNGYDGDG KLPKGRTPIK FGKSDCDRPP KHSKDGNGKT DHYLLEFPTF PDGHDYKFDS KKPKENPGPA RVIYTYPNKV FCGXelAHTXyXs NTGELKLCSH,

wherein Xi through Xs can be any amino acid (SEQ ID NO: 13), provided that the amino acid sequence of the modified sarcin protein is not identical to the wild

type sarcin protein (SEQ ID NO:1 ).

[00331] 1 .2. The nucleic acid encoding a modified sarcin protein according to embodiment 1 or 1 .1 , wherein the modified sarcin protein inhibits protein synthesis on ribosomes.

[00332] 2. The nucleic acid encoding a modified sarcin protein according to embodiment 1 , wherein Xi is Q, K, R, or A; X2 is P or I; Xs is T, G, Q, or H; X₄ is N, R, K or A; Xs is Y, H, K, or R; Xe is I or A; X₇ is K, D, E, G, Q, H, or N; and X₈ is E or D (SEQ ID NO:41 ).

[00333] 2.1 . The nucleic acid encoding a modified sarcin protein according to embodiment 1 .1 , wherein Xi is D, A, or T; X2 is Q, K, R, or A; X3 is T, G, Q, or H; X₄ is N, R, K or A; Xs is Y, H, K, or R; Xe is I or A; Xy is K, D, E, G, Q, H, or N; and X₈ is E or D (SEQ ID NO:14).

[00334] 3. The nucleic acid encoding a modified sarcin protein according to embodiment 1 or 1 .1 , wherein said immune response is T cell activity.

[00335] 4. A nucleic acid encoding a cytotoxin comprising: (a) a nucleic acid encoding a targeting moiety attached to; (b) a nucleic acid encoding the modified sarcin protein of embodiment 1 or 1 .1 .

[00336] 5. A nucleic acid encoding a cytotoxin comprising: (a) a nucleic acid encoding a ligand that binds to a target attached to; (b) a nucleic acid encoding the modified sarcin protein of embodiment 1 or 1 .1 .

[00337] 6. The nucleic acid encoding a cytotoxin of embodiment 5, wherein the ligand is an antibody or antibody fragment that binds to the target.

[00338] 7. The nucleic acid encoding a cytotoxin of embodiment 6, wherein the antibody or antibody fragment binds to Ep-CAM on the surface of the cancer cell.

[00339] 8. The nucleic acid encoding a cytotoxin of embodiment 7, wherein the antibody or antibody fragment that binds to Ep-CAM is a humanized antibody or antibody fragment that binds to the extracellular domain of human Ep-CAM and comprises complementarity determining region sequences derived from a MOC-31 antibody.

[00340] 9. The nucleic acid encoding a cytotoxin of embodiment 7, wherein the variable region of the cancer-binding ligand attached to the modified sarcin protein is 4D5MOCB.

[00341] 10. The nucleic acid encoding a cytotoxin of embodiment 6, wherein the antibody or antibody fragment binds to a tumor-associated antigen on the surface of the cancer cell. [00342] ADDITIONAL EMBODIMENTS - FUSION PROTEINS

[00343] 1 A ribotoxin fusion protein compris

(a) a modified sarcin molecule having reduced immunogenicity in humans as compared to wild type a-sarcin, wherein the modified sarcin molecule comprises a first amino acid modification and a second amino acid modification,

wherein the first amino acid modification is D9T or P 131 and the second amino acid modification is Q142T; and

(b) a targeting molecule linked to the modified sarcin molecule, the targeting molecule is effective for binding a target.

[00344] 2. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule is linked to the N-terminus of the modified sarcin molecule.

[00345] 3. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule is linked to the C-terminus of the modified sarcin molecule.

[00346] 4. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule is incorporated within the modified sarcin molecule.

[00347] 5. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule and the modified sarcin molecule are linked via a linker.

[00348] 5.1 . The ribotoxin fusion protein of embodiment 5, wherein the linker comprises an amino acid or a peptide.

[00349] 5.2. The ribotoxin fusion protein of embodiment 5.1 , wherein the linker comprises glycine and serine amino acids.

[00350] 6. The ribotoxin fusion protein of embodiment 5 or 5.1 , wherein the linker is between 1 and 20 amino acids in length. [00351] 7. The ribotoxin fusion protein of embodiment 5 or 5.1 , wherein the linker is between 3 and 20 amino acids in length.

[00352] 8. The ribotoxin fusion protein of embodiment 5 or 5.1 , wherein the linker is between 4 and 30 amino acids in length.

[00353] 9. The ribotoxin fusion protein of embodiment 5, wherein the linker comprises a discrete polyethylene glycol (dPEG).

[00354] 9.1 . The ribotoxin fusion protein of embodiment 9, wherein the dPEG is linked to the modified sarcin molecule at either one of a serine, tyrosine, cysteine, or lysine of the modified sarcin molecule or a glycosylation site of the modified sarcin molecule.

[00355] 9.2. The ribotoxin fusion protein of embodiment 9, wherein the dPEG is linked to the targeting molecule at either one of a serine, tyrosine, cysteine, or lysine of the targeting molecule or a glycosylation site of the targeting molecule.

[00356] 9.3. The ribotoxin fusion protein of embodiment 9, wherein the dPEG is between about 200 to 10,000 daltons.

[00357] 9.4. The ribotoxin fusion protein of embodiment 1 , wherein a branched dPEG molecule is linked to the targeting molecule.

[00358] 9.5. The ribotoxin fusion protein of embodiment 9.3, wherein between 1 and 12 sarcin molecules are attached to one or more branches of the branched dPEG molecule.

[00359] 10. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises a peptide.

[00360] 1 1 . The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises an antibody, an antibody fragment, a single chain variable fragment (scFv), a nanobody, an abdurin, a CH2 domain molecule, a CH2 domain fragment, a CH3 domain molecule, a CH3 domain fragment, a protein scaffold, a hormone, a receptor-binding peptide, or a combination thereof. [00361] 1 1 .1 . The nbotoxin fusion protein of embodiment 1 1 , wherein the targeting molecule targets Her2 receptor, PMSA, nucleolin, or a death receptor.

[00362] 1 1 .2. The ribotoxin fusion protein of embodiment 1 1 .1 , wherein the death receptor is a Fas receptor or tumor necrosis factor receptor

[00363] 1 1 .3. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises a binding moiety, the binding moiety comprises a VH domain, a VL domain, a camelid VHH domain, a tenth type three domain of fibronectin, a designed ankyrin repeat protein, a centyrin scaffold, a peptide ligand, a protein ligand, a receptor, hormone, an enzyme, a cytokine, a small molecule, a fragment thereof, or a combination thereof.

[00364] 12. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises an antigen binding region.

[00365] 13. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule is a CH2 domain molecule having a molecular weight less than about 20 kDa.

[00366] 14. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises at least one functional FcRn binding site.

[00367] 15. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is a monospecific molecule.

[00368] 16. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is a bispecific molecule.

[00369] 17. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is a trispecific molecule.

[00370] 18. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises at least a first paratope specific for a first epitope. [00371] 19. The nbotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises at least two first paratopes each specific for a first epitope.

[00372] 20. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises a first paratope specific for a first epitope and a second paratope specific for a second epitope.

[00373] 21 . The ribotoxin fusion protein of embodiment 1 , further comprising a second targeting molecule linked to either the targeting molecule or the modified sarcin molecule.

[00374] 21 .1 . The ribotoxin fusion protein of embodiment 1 , further comprising at least one additional targeting molecule.

[00375] 22. The ribotoxin fusion protein of embodiment 21 , wherein the second targeting molecule is linked to the N-terminus of the modified sarcin molecule and the targeting molecule is linked to the C-terminus of the modified sarcin molecule.

[00376] 23. The ribotoxin fusion protein of embodiment 21 , wherein the second targeting molecule is linked to the C-terminus of the modified sarcin molecule and the targeting molecule is linked to the N-terminus of the modified sarcin molecule.

[00377] 24. The ribotoxin fusion protein of embodiment 21 , wherein the second targeting molecule comprises a first paratope specific for the first epitope.

[00378] 25. The ribotoxin fusion protein of embodiment 21 , wherein the second targeting molecule comprises a second paratope specific for a second epitope.

[00379] 26. The ribotoxin fusion protein of embodiment 1 , wherein the targeting molecule comprises a third paratope specific for the first epitope or a fourth paratope specific for a third epitope.

[00380] 27. The ribotoxin fusion protein of embodiment 1 further comprising a second modified sarcin molecule. [00381] 27.1 . The nbotoxin fusion protein of embodiment 1 further comprising at least one additional modified sarcin molecule.

[00382] 28. The ribotoxin fusion protein of embodiment 27, wherein the second modified sarcin molecule is linked to the modified sarcin molecule.

[00383] 29. The ribotoxin fusion protein of embodiment 27, wherein the second modified sarcin molecule is linked to the targeting molecule.

[00384] 29.1 The ribotoxin fusion protein of embodiment 1 , wherein the ribotoxin fusion protein comprises a cleavable linker linking the modified sarcin molecule to the targeting molecule.

[00385] 29.2. The ribotoxin fusion protein of embodiment 29.1 , wherein the cleavable linker can be cleaved in the cytosol.

[00386] 29.3. The ribotoxin fusion protein of embodiment 29.1 , wherein the cleavable linker can be cleaved in the endosome.

[00387] 29.4. The ribotoxin fusion protein of embodiment 29.1 , wherein the cleavable linker is not cleaved in serum.

[00388] 30. The ribotoxin fusion protein of embodiment 27, wherein the second modified sarcin molecule is linked to the modified sarcin molecule or the targeting molecule via a linker.

[00389] 31 . The ribotoxin fusion protein of embodiment 30, wherein the linker comprises a discrete polyethylene glycol (dPEG).

[00390] 32. The ribotoxin fusion protein of embodiment 31 , wherein the dPEG is linked to the modified sarcin molecule at either one of a serine, tyrosine, cysteine, or lysine of the modified sarcin molecule or a glycosylation site of the modified sarcin molecule.

[00391] 33. The ribotoxin fusion protein of embodiment 31 , wherein the dPEG is linked to the targeting molecule at either one of a serine, tyrosine, cysteine, or lysine of the targeting molecule or a glycosylation site of the targeting molecule.

[00392] 34. The ribotoxin fusion protein of embodiment 31 , wherein the dPEG is between about 200 to 10,000 daltons.

[00393] 35. The ribotoxin fusion protein of embodiment 1 , further comprising a

pharmaceutical carrier.

[00394] 36. The ribotoxin fusion protein of embodiment 1 , further comprising an imaging reagent, an isotope, a drug, an immunoconjugate, or a combination thereof.

[00395] 37. The ribotoxin fusion protein of embodiment 36, wherein the imaging reagent, isotope, drug, or immunoconjugate is linked to the modified sarcin molecule.

[00396] 38. The ribotoxin fusion protein of embodiment 36, wherein the imaging reagent, isotope, drug, or an immunoconjugate is linked to the targeting molecule.

[00397] 39. The ribotoxin fusion protein of embodiment 1 , wherein the target comprises a receptor.

[00398] 40. The ribotoxin fusion protein of embodiment 1 , wherein the target comprises a cell, a tumor cell, an immune cell, a protein, a peptide, a molecule, a bacterium, a virus, a protist, a fungus, or a combination thereof.

[00399] 41 . The ribotoxin fusion protein of embodiment 1 , further comprising a second targeting molecule.

[00400] 42. The ribotoxin fusion protein of embodiment 41 , further comprising a third targeting molecule.

[00401] 43. The ribotoxin fusion protein of embodiment 1 , further comprising a fourth targeting molecule. [00402] 44A. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein has increased cell permeability as compared to wild type a-sarcin.

[00403] 44B. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein has increased cell permeability as compared to the targeting molecule alone.

[00404] 44C. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein has increased cell permeability as compared to the modified sarcin molecule alone.

[00405] 44D. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is modified to increase cell permeability as compared to wild type a-sarcin.

[00406] 44E. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is modified to increase cell permeability as compared to the targeting molecule alone.

[00407] 44F. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is modified to increase cell permeability as compared to the modified sarcin molecule alone.

[00408] 45A. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein has increased cell retention as compared to wild type a-sarcin.

[00409] 45B. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein has increased cell retention as compared to the targeting molecule alone.

[00410] 45C. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein has increased cell retention as compared to the modified sarcin molecule alone.

[00411] 45D. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is modified to increase cell retention as compared to wild type a-sarcin.

[00412] 45E. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is modified to increase cell retention as compared to the targeting molecule alone.

[00413] 45F. The ribotoxin fusion protein of embodiment 1 , wherein the fusion protein is modified to increase cell retention as compared to the modified sarcin molecule alone.

[00414] 46. The modified sarcin molecule of embodiment 1 expressed in an expression system.

[00415] 47. The modified sarcin molecule of embodiment 46, wherein the expression system is an E. coli expression system or a Pichia pastoris expression system.

Claims

What is claimed:

1 . A modified sarcin polypeptide, wherein the modified sarcin polypeptide comprises at least two mutations as compared to a wild type a-sarcin polypeptide (SEQ ID NO: 1 ), wherein the at least two mutations comprise a first mutation within a first T cell epitope and a second mutation within a second T cell epitope of the wild type a-sarcin polypeptide, wherein the first T cell epitope consists of the amino acid sequence DQKNPKTNKY (SEQ ID NO:6), and the second T cell epitope consists of the amino acid sequence IIAHTKENQ (SEQ ID NO:4) and wherein the first mutation is D9T or P13I and the second mutation is Q142T.

2. The modified sarcin polypeptide of claim 1 , wherein the first mutation is D9T and the second mutation is Q142T.

3. The modified sarcin polypeptide of claim 1 , wherein the first mutation is P13I and the second mutation is Q 142T.

4. A composition comprising the modified sarcin polypeptide of any of the preceeding claims and a pharmaceutically acceptable excipient or carrier.

5. A fusion protein comprising the modified sarcin polypeptide of claim 1 conjugated or fused to a targeting molecule.

6. The fusion protein of claim 5, wherein the targeting molecule is an antibody or antigen-binding fragment thereof.

7. The fusion protein of claim 6, wherein the modified sarcin polypeptide is fused or linked to the antibody or antigen-binding fragment thereof via a linker.

8. The fusion protein of claim 7, wherein the linker comprises an amino acid or a peptide.

The fusion protein of claim 8, wherein the linker comprises glycine and serine ) acids.

10. The fusion protein of claim 8 or 9, wherein the linker is between 1 and 20 amino acids in length.

1 1 . The fusion protein of claim 10, wherein the linker is between 3 and 20 amino acids in length.

12. An isolated nucleic acid encoding the modified sarcin polypeptide of claim 1 or the fusion protein of claim 5.

13. An expression vector comprising the nucleic acid of claim 12.

14. A host cell transformed with an expression vector of claim 13.

15. A method of producing a modified sarcin polypeptide of claim 1 or the fusion protein of claim 5, comprising culturing a host cell according to claim 14 and

purifying the modified sarcin polypeptide or fusion protein expressed from the host cell.