WO2021097289A1 - Pegylated recombinant immunotoxins - Google Patents

Abstract

Disclosed is a molecule comprising a first domain comprising a targeting moiety, wherein the targeting moiety comprises a first linker of 1 to 20 amino acid residues selected from the group consisting of glycine and serine, a second domain comprising a furin cleavage sequence, a third domain comprising an optionally substituted domain III from Pseudomonas exotoxin A, a fourth domain comprising 1 to 20 amino acid residues, and at least one ethylene glycol, wherein the molecule contains at least one cysteine, wherein the at least one cysteine is located within the first domain, the third domain, or the fourth domain, and wherein the at least one ethylene glycol is attached to the at least one cysteine. Related nucleic acids, recombinant expression vectors, host cells, populations of cells, pharmaceutical compositions, methods of producing the molecule, methods of using the molecule are also disclosed.

Description

PEGYLATED RECOMBINANT IMMUNOTOXINS CROSS-REFERENCE TO RELATED APPLICATION [0001] This patent application claims the benefit of copending U.S. Provisional Patent Application No.62/935,822, filed November 15, 2019, which is incorporated by reference in its entirety herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with Government support under project number Z01ZIA BC008753 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in this invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY [0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 34,563 Byte ASCII (Text) file named “750626_ST25.txt,” dated November 13, 2020. BACKGROUND OF THE INVENTION [0004] Recombinant Immunotoxins (“RITs”) are small chimeric proteins that include the variable domain (“Fv”) portion of an antibody fused to a bacterial toxin that kills the target cells. For example, the RITs can target mesothelin, a cell-surface glycoprotein that is robustly expressed in many common solid tumors. The bacterial toxin can be Pseudomonas exotoxin A (“PE”). PE is a bacterial toxin with cytotoxic activity that may be effective for destroying or inhibiting the growth of undesirable cells, e.g., cancer cells. Accordingly, PE may be useful for treating or preventing diseases such as, e.g., cancer. While PE can produce positive clinical responses in some cancer patients, there is a need for improved PE. BRIEF SUMMARY OF THE INVENTION [0005] An embodiment of the invention provides a molecule comprising a first domain comprising a targeting moiety, wherein the targeting moiety comprises a first linker of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine, a second domain comprising a furin cleavage sequence (FCS), wherein the FCS is cleavable by furin, a third domain comprising an optionally substituted domain III from PE, a fourth domain comprising 1 to 20 amino acid residues, and at least one ethylene glycol ((CH2OH)2), wherein the molecule contains at least one cysteine, wherein the first domain optionally comprises a second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and the at least one cysteine, wherein the at least one cysteine is located within the first domain, the third domain, or the fourth domain, and wherein the at least one ethylene glycol is attached to the at least one cysteine. [0006] Additional embodiments of the invention provide related nucleic acids, recombinant expression vectors, host cells, populations of cells, pharmaceutical compositions, and methods of producing the inventive molecule. [0007] Still another embodiment of the invention provides a method of treating or preventing cancer in a mammal comprising administering to the mammal the inventive molecule, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition, in an amount effective to treat or prevent cancer in the mammal. [0008] Another embodiment of the invention provides a method of inhibiting the growth of a target cell comprising contacting the cell with the inventive molecule, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition, in an amount effective to inhibit growth of the target cell. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [0009] Figure 1A is a schematic of molecules, or recombinant immunotoxin (“RIT”) constructs, SS1-dsFv-PE38 “SS1P,” SS1-LRggs “LMB-12,” SS1-scFV- LRggs “LMB-84,” E522C “LMB-179,” D406C “LMB-244,” Vl-C-Fur “LMB-203,” C-linker-Vh “LMB-249,” and S63C “LMB-163.” SS1P is an immunotoxin containing SS1 construct wherein the heavy chain variable region (Vh, SEQ ID NO: 4) and variable chain light fragment (Vl, SEQ ID NO: 5) (together, the variable domain or Fv) are fused to each other through disulfide bonds formed via cysteine residues (C). The Fv is attached to a 38 kDa portion of Pseudomonas exotoxin A (“PE38”, SEQ ID NO: 3) via a KASGG linker (SEQ ID NO: 14). LMB-12 is an immunotoxin containing SS1 construct wherein the Fv is fused to an 11 amino acid furin cleavage sequence (Fur, SEQ ID NO: 15), via a KASGG linker (SEQ ID NO: 14). The Vh and Vl of the Fv are fused to each other through disulfide bonds formed via cysteine residues (C). Fur is fused to a 24 kDa portion of PE (PE24, SEQ ID NO: 2) via linker GGS (SEQ ID NO: 11). LMB-84 contains a single-chain Fv instead of a dsFv and is the parental RIT from which the cysteine containing PEG-modified proteins were prepared. LMB-179, LMB-244, and LMB-203 have an additional 17-GS linker (SEQ ID NO: 9) that separates Vl from the furin cleavage site. LMB-249 has GS linker (SEQ ID NO: 12) placed at the N-terminus of the Vh. LMB-163 contains mutations that humanize the Fv portion (SEQ ID NOs: 6 (Vh) and 7(Vl)). For each construct, the cysteine for site-specific PEGylation is indicated by a “C” and an arrow. [0010] Figure 1B is another schematic of the RITs shown in Figure 1A. PEGs on cysteines are represented by wiggly lines and arrows. Domains are not drawn to scale. [0011] Figure 2A is a ribbon diagram that was generated with UCSF Chimera. The sites that were mutated to cysteine for each RIT are shown as spheres. [0012] Figure 2B is a ribbon diagram showing the RIT of Figure 2A bound to mesothelin (left arrow) and ADP-ribosylated elongation factor 2 (“EF2,” right arrow). [0013] Figure 2C is a ribbon diagram showing a hypothetical 20 kD linear PEG composed of 454 repeating units of ethylene glycol connected to the cysteine at D406 of PE24 (LMB-244). The structure of the illustrated PEG was random, and the overall size of the molecule was modeled to approximate the average radius of hydration measured for all five PEGylated RITs. [0014] Figure 3A depicts an image of a gel with bands following RIT elution from MONO Q^TM ion exchanger columns (GE Healthcare). Fractions A, B, and C correspond to separate peaks in the MONO Q^TM chromatogram and were pooled separately. [0015] Figure 3B is a schematic showing a site-specific PEGylation reaction which occurs by the formation of a thioester bond between a free thiol of a surface-exposed cysteine and a maleimide functional group of a PEG. [0016] Figure 3C depicts data following an exemplary high-performance liquid chromatography (“HPLC”) analysis of LMB-203/LMB-203-PEG. [0017] Figure 3D depicts an image of a non-reduced sodium dodecyl sulfate– polyacrylamide gel electrophoresis (“SDS-PAGE”) gel including bands indicating the molecular weights for parental, tris(2-carboxyethyl)phosphine (“TCEP”) TCEP-reduced, and PEGylated RITs. Gels were visualized by use of Coomassie Blue staining. Vertical white lines indicate the point of merge between separate gels. [0018] Figure 4A depicts experimental data illustrating the results of cytotoxicity assays after 3 days incubation of mesothelin-positive cancer cell lines with RITs LMB-249, LMB- 12, LMB-249-PEG, LMB-203, LMB-203-PEG, LMB-163, LMB-163-PEG, LMB-179, LMB-179-PEG, LMB-244, and LMB-244-PEG. KLM1 is a pancreatic cancer cell line. The Y-axis represents the % of viability of the target cells. The X-axis represents the concentration of immunotoxin in ng/ml. [0019] Figure 4B depicts experimental data illustrating the results of cytotoxicity assays after 3 days incubation of mesothelin-positive cancer cell lines with RITs LMB-163, LMB- 12, LMB-163-PEG, LMB-244, and LMB-244-PEG. A431/H9 is an epidermoid carcinoma cell line transfected with mesothelin cDNA and MKN28 is a gastric cancer cell line. The y- axis represents the % of viability of the target cells. The x-axis represents the concentration of immunotoxin in ng/ml. [0020] Figures 4C-4E depict graphs showing the IC₅₀ values of the PEGylated RITs normalized against the un-PEGylated controls which were set to 100%. The percent remaining activity is indicated in cells lines KLM1 (Figure 4C), A431/H9 (Figure 4D), and MKN28 (Figure 4E). [0021] Figures 5A-5E depict graphs showing properties of PEGylated RITs (i.e., a RIT with at least one ethylene glycol conjugated to to the RIT). Figure 5A is a graph depicting the pharmacokinetics (“PK”) of PEGylated RITs. LMB-203-PEG, LMB-244-PEG, LMB- 163-PEG, LMB-179-PEG, and LMB-249-PEG were injected into nu/nu mice and blood was collected at 5 min, 1 (LMB-163-PEG and LMB-179-PEG), 2, 4, 8 (LMB-203-PEG, LMB- 244-PEG, and LMB-249-PEG), and 24 hour time points. Enzyme-linked immunosorbent assay (“ELISA”) was performed to determine the level of RIT and plotted in two-phase decay using GraphPad. The amount of RIT at 5 min was set to 100% and used to normalize the remaining time points. LMB-12 was data from a previous study and plotted as one-phase decay. The AUC values are summarized. Figure 5B is a graph depicting radius of hydration (“Rh”) of PEGylated RITs as measured by Dynamic Light Scattering (“DLS”). 20μl aliquots of proteins (0.5-1mg/ml) were measured at least two times and Rh was plotted against half- life using GraphPad. The line of best fit was generated with R² value indicated. Figures 5C- 5E depict graphs showing the biodistribution of LMB-249-PEG and SS1P in mice. Serial dorsal and ventral 800 nm fluorescence images were obtained with focus placed on the tumor, kidney, and liver. The fluorescence intensity at each time point was quantified and plotted to show the accumulation in the kidney (Figure 5C), tumor (Figure 5D), and liver (Figure 5E). Plots were generated from the average results of 3 imaged mice. [0022] Figures 6A-6B depict graphs showing the anti-tumor activity of parental and PEGylated RITs. Figure 6A shows the antitumor activity of PEGylated RITs in nude mice (n = 10). Mice were implanted with mesothelin-expressing KLM1 tumor cells. When tumors reached ~100mm³, mice were intravenously (“IV”) injected with 10 μg/mouse PEGylated RITs x3 as indicated by arrows. Tumor burden was monitored over 2 weeks. Figure 6B shows the comparison of anti-tumor activities of LMB-12, LMB-163-PEG, and LMB-244- PEG. Groups of 10 mice were treated with 20 μg of each agent as shown by arrows and tumor burden was monitored over 2 weeks. [0023] Figures 7A-7B depict graphs showing the cell-killing activity of control RITs. Figure 7A depicts experimental data illustrating the results of cytotoxicity assays after 3 days incubation of mesothelin-positive cancer cell line KLM1 with LMB-84 or LMB-12. A water- soluble tetrazolium salt-8 (“WST8”) assay was performed to assess viability (using the tetrazolium salt WST-1, as described in International Patent Application Publication WO 2011/032022). The IC50 values are listed and also summarized in Table 7. Figure 7B depicts experimental data illustrating the results of cytotoxicity assays after 3 days incubation with A431 epidermoid carcinoma cell line devoid of cell-surface expressed mesothelin. [0024] Figure 8 depicts a set of graphs showing the mesothelin binding of PEGylated RITs. Serial dilution of PEGylated RITs was performed and mesothelin binding was determined by ELISA. Binding curves were generated using GraphPad and half-maximum binding was calculated. IC50 indicates the average of 2 independent measurements. [0025] Figure 9 depicts an image of a non-reduced SDS-PAGE gel including bands showing the ADP ribosylation activities of PEGylated RITs. The efficiencies of ADP- ribosylation were quantified using Image J, and LMB-12 was set to 100% in which the remaining samples were normalized against. [0026] Figures 10A-10B depict graphs showing the results of DLS analysis to determine Rh meaurements of the RITs. The Rhs of PEG-modified immunotoxins LMB-249, LMB- 203, and LMB-179 (Figure 10A), and LMB-163 and LMB-244 (Figure 10B) were measured along with the parental controls LMB-12 and LMB-203 (Figure 10B). Representative measurements are shown as %Mass versus Radius bar graph. For each sample, more than 90% of the total mass comes from the pegylated/unpegylated protein of interest. LMB-249, LMB-203, and LMB-179 have trace amounts of aggregate that also scattered lights. [0027] Figures 11A-11B depict images showing the biodistribution of LMB-249-PEG and SS1P. LMB-249-PEG (Figure 11A) and SS1P (Figure 11B) were labeled with a near- infrared fluorophore FNIR-Z-759. Serial dorsal and ventral 800 nm fluorescence images were obtained with a Pearl Imager at the times indicated to assess accumulation of RIT in the kidney (dorsal), tumor (dorsal), and liver (ventral). [0028] Figures 12A-12B depict graphs showing the weights of mice following treatment with PEGylated RITs. Mice weight was monitored every 2-3 days and mice that loss >10% weight were euthanized. The weights of mice (grams) treated with LMB-249-PEG, LMB- 203-PEG, LMB-163-PEG, LMB-179-PEG, and LMB-244-PEG is shown in Figure 12A. The weights of mice (grams) treated with LMB-163-PEG and LMB-244-PEG is shown in Figure 12B. [0029] Figure 13A is a schematic of a molecule, or recombinant immunotoxin (“RIT”) construct, “LMB-288.” LMB-288 contains a single-chain Fv in which a GS linker is placed at the N-terminus of the Vl. The scFv is connected to an albumin binding domain (ABD) and furin site by linkers. The toxin portion is PE24, a 24kDa portion of PE, containing a cysteine point mutation for PEG conjugation. Figure 13B depicts an image of a SDS-PAGE gel showing non-reduced, reduced and PEG-conjugated forms of LMB-288. Figure 13C is a ribbon diagram showing a structural view of LMB-288 with a cysteine mutation in PE24 shown as a sphere. [0030] Figure 14A depicts data from cytotoxicity assays of parental and pegylated immunotoxin, LMB-288, as well as LMB-12. The data presented in Figure 14A are the results of representative cytotoxicity assays after 72 hours of incubation of mesothelin- positive cancer cell lines with immunotoxins, in KLM1, (a pancreatic cancer cell line), A431/H9, (an epidermoid carcinoma cell line transfected with mesothelin cDNA), and MKN28, (a gastric cancer cell line). Figure 14B depicts a bar graph showing the IC₅₀ values of pegylated immunotoxin normalized against the parental which is set to 100%, with percent remaining activity as indicated. [0031] Figure 15 depicts data from assays measuring HSA binding of immunotoxins. ELISA was performed after overnight incubation of immunotoxins with albumin-coated affinity plates in order to determine the binding affinity to albumin. IC50 values were converted into molar affinity for comparison to controls. For LMB-288 reduced, TCEP was included in the dilution buffer to ensure the protein remained in monomer form during the binding step. LMB-164 is an ABD-containing immunotoxin that was shown to efficiently bind to albumin. LMB-12 and LMB-179 both lack ABD and were included as negative controls. [0032] Figure 16A depicts data from assays measuring half-life of immunotoxins.25ug of immunotoxins were I.V. injected into 4 nu/nu mice and blood was harvested at the indicated time points. ELISA was performed using the mesothelin-coated affinity plate to determine the amount of immunotoxin remaining in mouse blood serum at each time point. The half-life is represented as AUC. Figure 16B depicts data measuring the hydrodynamic volumes of immunotoxins using dynamic light scattering and plotted against the AUC. Albumin-bound LMB-164 and LMB-288-PEG were used for the measurement. The plotted shapes of points in Figure 16B correspond to the shapes of points in Figure 16A. [0033] Figure 17 depicts a graph showing the anti-tumor activity of a PEGylated RIT, LMB-288+20KDa PEG, in mice. Mice were implanted with mesothelin-positive KLM1 tumor cells. Mice were intravenously (“IV”) injected with varying dosages of LMB- 288+20KDa PEG (25 ug/mose, 10 ug/mouse and 75 ug/mouse) as indicated by arrows. Tumor burden was monitored over 2 weeks. [0034] Figure 18 depicts a graph showing the anti-tumor activity of a PEGylated RIT, LMB-288+20KDa PEG, and an unPEGylated RIT, LMB-12, in mice. Mice were implanted with mesothelin-positive A431/H9 tumor cells. Mice were intravenously (“IV”) injected with LMB-288+20KDa PEG (25 ug/mouse) or LMB-12 (20ug/mouse) as indicated by arrows. Tumor burden was monitored over 2 weeks. [0035] Figure 19 depicts a graph showing the anti-tumor activity of a PEGylated RIT, LMB-288+20KDa PEG, and an unPEGylated RIT, LMB-12, in mice. Mice were implanted with mesothelin-positive MKN28 tumor cells. Mice were intravenously (“IV”) injected with LMB-288+20KDa PEG (25 ug/mouse) or LMB-12 (20ug/mouse) as indicated by arrows. Tumor burden was monitored over 2 weeks. DETAILED DESCRIPTION OF THE INVENTION [0036] New molecules have been developed that have high cytotoxicity and high anti- tumor activity with improved PK chacteritics (e.g., half-life). The compounds include RITs with ethylene glycol molecules attached to them via cysteine residues (PEGylation). The cysteine residues are added to the RITs prior to PEGylation via site-specific mutagenesis. Although some cysteine residues may be present in the Fv regions of the molecules, the naturally occurring cysteine residues are buried and not available for reduction. [0037] RITs with modifications at specific positions were developed that produce PEGylated RITs with the improved characteritics. The PEGylation sites do not interfere with functional sites of the molecules, such as the site that binds to the target moiety (e.g., mesothelin), the EF2 site, or the furin cleavage site. It was also found that lysine residues could not be used to for PEGylation of the RITs because this led to poor control over the location of the PEGs. [0038] RITs may have a short half-life which may make it necessary to administer large amounts of the RITs to obtain high blood levels of RITs so that enough of the RITs can enter tumors and kill tumor cells. High blood levels of immunotoxin may, undesirably, cause non- specific toxicities. Accordingly, the increased half-life of the PEGylated RITs is desirable. [0039] The attachment of the PEGs to the RITs may, advantageously, not reduce or eliminate the cytotoxic activity of the RITs. Accordingly, the inventive RITs may kill target cells and/or achieve therapeutic efficacy with smaller dosages as compared to RITs without the PEGs attached to the RITs. [0040] An embodiment of the invention provides a molecule comprising: (a) a first domain comprising a targeting moiety, wherein the targeting moiety comprises a first linker of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine; (b) a second domain comprising a furin cleavage sequence (FCS), wherein the FCS is cleavable by furin; (c) a third domain comprising an optionally substituted domain III from Pseudomonas exotoxin A (PE); (d) a fourth domain comprising 1 to 20 amino acid residues; and (e) at least one ethylene glycol ((CH2OH)2), wherein the molecule contains at least one cysteine, wherein the first domain optionally comprises a second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and the at least one cysteine, wherein the at least one cysteine is located within the first domain, the third domain, or the fourth domain, and wherein the at least one ethylene glycol is attached to the at least one cysteine. Targeting Moiety [0041] An embodiment of the invention provides a molecule that includes a first domain comprising a targeting moiety. The term “targeting moiety” as used herein, refers to any molecule or agent that specifically recognizes and binds to a cell-surface marker, such that the targeting moiety directs the delivery of the inventive molecule to a population of cells on which surface the receptor is expressed. Targeting moieties include, but are not limited to, antibodies (e.g., monoclonal antibodies), or fragments thereof, peptides, hormones, growth factors, cytokines, and any other natural or non-natural ligands. The practice of conjugating compounds, e.g., inventive PEs, to targeting moieties is known in the art. [0042] The term “antibody,” as used herein, refers to whole (also known as “intact”) antibodies or antigen binding portions thereof that retain antigen recognition and binding capability. The antibody or antigen binding portions thereof can be a naturally-occurring antibody or antigen binding portion thereof, e.g., an antibody or antigen binding portion thereof isolated and/or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc. The antibody or antigen binding portion thereof can be in monomeric or polymeric form. Also, the antibody or antigen binding portion thereof can have any level of affinity or avidity for the cell surface marker. Desirably, the antibody or antigen binding portion thereof is specific for the cell surface marker, such that there is minimal cross- reaction with other peptides or proteins. [0043] The antibody may be monoclonal or polyclonal and of any isotype, e.g., IgM, IgG (e.g. IgG, IgG2, IgG3 or IgG4), IgD, IgA or IgE. Complementarity determining regions (“CDRs”) of an antibody or single chain variable fragments (“Fvs”) of an antibody against a target cell surface marker can be grafted or engineered into an antibody of choice to confer specificity for the target cell surface marker upon that antibody. For example, the CDRs of an antibody against a target cell surface marker can be grafted onto a human antibody framework of a known three dimensional structure to form an antibody that may raise little or no immunogenic response when administered to a human. In a preferred embodiment, the targeting moiety is a monoclonal antibody. [0044] The antigen binding portion can be any portion that has at least one antigen binding site, such as, e.g., the variable regions or CDRs of the intact antibody. Examples of antigen binding portions of antibodies include, but are not limited to, a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab, Fab’, Fv, or F(ab)2’ fragment; single domain antibodies (see, e.g., Wesolowski, Med Microbiol Immunol., 198(3): 157-74 (2009); Saerens et al., Curr. Opin. Pharmacol., 8(5):600-8 (2008); Harmsen and de Haard, Appl. Microbiol. Biotechnol., 77(1 ): 13-22 (2007), helix-stabilized antibodies; triabodies; diabodies; single-chain antibody molecules (“scFvs”); disulfide stabilized antibodies (“dsFvs”), and domain antibodies (“dAbs”). [0045] Methods of testing antibodies or antigen binding portions thereof for the ability to bind to any cell surface marker are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Kenneth Murphy (ed.), Janeway’s Immunobiology, 8th Ed., New York: Garland Science (2011)). [0046] Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods, EBV-hybridoma methods, bacteriophage vector expression systems, and methods of producing antibodies in non-human animals are described in, e.g., Murphy, supra. [0047] Phage display also can be used to generate the antibody that may be used in the molecules of the invention. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Green et al. (eds.), Molecular Cloning, A Laboratory Manual, 4^th Edition, Cold Spring Harbor Laboratory Press, New York (2012)). Phage encoding a variable region with the desired specificity are selected for specific binding to the desired antigen, and a complete or partial antibody is reconstituted comprising the selected variable domain. Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell used for hybridoma production, such that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Murphy, supra). [0048] Alternatively, antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example, Murphy, supra. [0049] Alternatively, the antibody can be a genetically-engineered antibody, e.g., a humanized antibody or a chimeric antibody. Humanized antibodies advantageously provide a lower risk of side effects and can remain in the circulation longer. Methods for generating humanized antibodies are known in the art and are described in detail in, for example, Murphy, supra). Humanized antibodies can also be generated using antibody resurfacing technology that is known in the art. [0050] The targeting moiety may specifically bind to any suitable cell surface marker. The choice of a particular targeting moiety and/or cell surface marker may be chosen depending on the particular cell population to be targeted. Cell surface markers are known in the art (see, e.g., Mufson et al., Front. Biosci., 11: 337-43 (2006) and Kreitman et al., AAPS Journal, 8(3): E532-E551 (2006)) and may be, for example, a protein or a carbohydrate. In an embodiment of the invention, the targeting moiety is a ligand that specifically binds to a receptor on a cell surface. Exemplary ligands include, but are not limited to, vascular endothelial growth factor (VEGF), Fas, TNF-related apoptosis-inducing ligand (TRAIL), a cytokine (e.g., IL-2, IL-15, IL-4, IL-13), a lymphokine, a hormone, and a growth factor (e.g., transforming growth factor (TGFa), neuronal growth factor, epidermal growth factor). [0051] The cell surface marker can be, for example, a cancer antigen. The term “cancer antigen” as used herein refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed or over-expressed by a tumor cell or cancer cell, such that the antigen is associated with the tumor or cancer. The cancer antigen can additionally be expressed by normal, non-tumor, or non-cancerous cells. However, in such cases, the expression of the cancer antigen by normal, non-tumor, or non-cancerous cells is not as robust as the expression by tumor or cancer cells. In this regard, the tumor or cancer cells can over-express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal, non-tumor, or non-cancerous cells. Also, the cancer antigen can additionally be expressed by cells of a different state of development or maturation. For instance, the cancer antigen can be additionally expressed by cells of the embryonic or fetal stage, which cells are not normally found in an adult host. Alternatively, the cancer antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host. [0052] The cancer antigen can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. The cancer antigen may be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, the cancer antigen may be a cancer antigen (e.g., may be characteristic) of more than one type of cancer or tumor. For example, the cancer antigen may be expressed by both breast and prostate cancer cells and not expressed at all by normal, non-tumor, or non-cancer cells. [0053] Exemplary cancer antigens to which the targeting moiety may specifically bind include, but are not limited to mucin 1 (MUC1), melanoma associated antigen (MAGE), preferentially expressed antigen of melanoma (PRAME), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), prostate specific membrane antigen (PSMA), granulocyte- macrophage colony-stimulating factor receptor (GM-CSFR), CD56, human epidermal growth factor receptor 2 (HER2/neu) (also known as erbB-2), CD5, CD7, tyrosinase tumor antigen, tyrosinase related protein (TRP)1, TRP2, NY-ESO-1, telomerase, mesothelin, and p53. In a preferred embodiment, the cell surface marker, to which the targeting moiety specifically binds, is selected from the group consisting of cluster of differentiation (CD) 19, CD21, CD22, CD25, CD30, CD33, CD79b, B-cell maturation antigen (BCMA), glypican 2 (GPC2), glypican 3 (GPC3), transferrin receptor, EGF receptor (EGFR), mutated EGFR, mesothelin, cadherin, and Lewis Y. CD22 is expressed in, e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), non-Hodgkin’s lymphoma, small lymphocytic lymphoma (SLL), and acute lymphatic leukemia (ALL). CD25 is expressed in, e.g., leukemias and lymphomas, including hairy cell leukemia and Hodgkin’s lymphoma. Lewis Y antigen is expressed in, e.g., bladder cancer, breast cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, and pancreatic cancer. CD33 is expressed in, e.g., acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CML), and myeloproliferative disorders. [0054] In an embodiment of the invention, the targeting moiety is an antibody that specifically binds to a cancer antigen. Exemplary antibodies that specifically bind to cancer antigens include, but are not limited to, antibodies against the transferrin receptor (e.g., HB21 and variants thereof), antibodies against CD22 (e.g., RFB4 and variants thereof), antibodies against CD25 (e.g., anti-Tac and variants thereof), antibodies against mesothelin (e.g., SS1, MORAb-009, SS, HN1, HN2, MN, MB, YP218, and variants thereof), antibodies against BCMA (BM24, BM306, and variants thereof), antibodies against GPC3 (YP7, HN3, and variants thereof), antibodies against GPC2 (LH1, LH4, LH7, and variants thereof), and antibodies against Lewis Y antigen (e.g., B3 and variants thereof). In this regard, the targeting moiety may be an antibody selected from the group consisting of B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21, MORAb-009, YP218, YP7, HN3, LH1, LH4, LH7, BM24, BM306, and antigen binding portions thereof. Further exemplary targeting moieties suitable for use in the inventive molecules are disclosed e.g., in U.S. Patents 5,242,824 (anti- transferrin receptor); 5,846,535 (anti-CD25); 5,889,157 (anti-Lewis Y); 5,981,726 (anti- Lewis Y); 5,990,296 (anti-Lewis Y); 7,081,518 (anti-mesothelin); 7,355,012 (anti-CD22 and anti-CD25); 7,368,110 (anti-mesothelin); 7,470,775 (anti-CD30); 7,521,054 (anti-CD25); and 7,541,034 (anti-CD22); U.S. Patent Application Publication 2007/0189962 (anti-CD22); Frankel et al., Clin. Cancer Res., 6: 326-334 (2000), and Kreitman et al., AAPS Journal, 8(3): E532-E551 (2006), each of which is incorporated herein by reference. In another embodiment, the targeting moiety may include the targeting moiety of immunotoxins known in the art. Exemplary immunotoxins include, but are not limited to, LMB-2 (Anti-Tac(Fv)- PE38), BL22 and HA22 (RFB4(dsFv)-PE38), SS1P (SS l (dsFv)-PE38), HB21-PE40, and variants thereof. In a preferred embodiment, the targeting moiety is the antigen binding portion of HA22. HA22 comprises a disulfide-linked Fv anti-CD22 antibody fragment conjugated to PE38. HA22 and variants thereof are disclosed in International Patent Application Publications U.S. Patent No.8,809,502 and U.S.Patent No.8,871,906, which are incorporated herein by reference. [0055] In an embodiment, the targeting moiety comprises any one or more of SEQ ID NOs: 4-7. In an embodiment, the targeting moiety comprises SEQ ID NOs: 4 and 5. In an embodiment, the targeting moiety comprises SEQ ID NOs: 6 and 7. [0056] In a further embodiment, the targeting moiety contains at least one amino acid substitution or insertion of at least one cysteine. An example of such a molecule is LMB-249 which includes a cysteine immediately after a methionine start codon (SEQ ID NO: 49, see Fig.1A and Table 5). Another example of such a molecule is LMB-163 which has a cysteine in the Vl region (SEQ ID NO: 5, see also Fig.1A and Table 6). The cysteine residue can be substituted or inserted by any known suitable means, including by site mutagenesis. [0057] The targeting moiety of the inventive molecule includes a first linker of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine. In some embodiments, the first linker is defined as (Xaa1)r, wherein each Xaa1 is selected independently from glycine and serine and r is an integer from 1 to 20. An example of such linker includes, but is not limited to GGGGS GGGGS GGGGS (SEQ ID NO: 12). [0058] The first domain optionally comprises a second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and cysteine. In some embodiments, the second linker is defined as (Xaa1)_r, wherein each Xaa1 is selected independently from methionine, glycine, serine, and cysteine and r is an integer from 1 to 20. In some embodiments, the second linker is defined as (Xaa1)r, wherein each Xaa1 is as defined in this paragraph and the second linker has no more than one methionine and no more than one cysteine. Examples of such linkers include, but are not limited to: MC (SEQ ID NO: 49), GSGSGSGSGSGSGSGSG (SEQ ID NO: 9), and MCGSGSGSGSGSGSGSGSG (SEQ ID NO: 50). Pseudomonas exotoxin A [0059] Pseudomonas exotoxin A (“PE”) is a bacterial toxin (molecular weight 66 kD) secreted by Pseudomonas aeruginosa. The native, wild-type PE sequence (SEQ ID NO: l) is set forth in U.S. Patent 5,602,095, which is incorporated herein by reference. Native, wild- type PE includes three structural domains that contribute to cytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding, domain II (amino acids 253-364) mediates translocation into the cytosol, and domain III (amino acids 400-613) mediates EF2. While the structural boundary of domain III of PE is considered to start at residue 400, it is contemplated that domain III may require a segment of domain Ib to retain ADP-ribosylating activity. Accordingly, functional domain III is defined as residues 395-613 of PE. The function of domain Ib (amino acids 365-399) remains undefined. Without being bound by a particular theory or mechanism, it is believed that the cytotoxic activity of PE occurs through the inhibition of protein synthesis in eukaryotic cells, e.g., by the inactivation of the EF2. [0060] Substitutions of PE are defined herein by reference to the amino acid sequence of PE. Thus, substitutions of PE are described herein by reference to the amino acid residue present at a particular position, followed by the position number, followed by the amino acid with which that residue has been replaced in the particular substitution under discussion. In this regard, the positions of the amino acid sequence of a particular embodiment of a PE are referred to herein as the positions as defined by SEQ ID NO: 1. When the positions are as defined by SEQ ID NO: 1, then the actual positions of the amino acid sequence of a particular embodiment of a PE are defined relative to the corresponding positions of SEQ ID NO: 1, and the positions as defined by SEQ ID NO: 1 may be different than the actual positions in the particular embodiment of PE under discussion. Thus, for example, substitutions refer to a replacement of an amino acid residue in the amino acid sequence of a particular embodiment of a PE corresponding to the indicated position of the 613-amino acid sequence of SEQ ID NO: 1 with the understanding that the actual positions in the respective amino acid sequences may be different. For example, when the positions are as defined by SEQ ID NO: 1, the term “R490” refers to the arginine normally present at position 490 of SEQ ID NO: 1, “R490A” indicates that the arginine normally present at position 490 of SEQ ID NO: 1 is replaced by an alanine, while “K590Q” indicates that the lysine normally present at position 590 of SEQ ID NO: 1 has been replaced with a glutamine. In the event of multiple substitutions at two or more positions, the two or more substitutions may be the same or different, i.e., each amino acid residue of the two or more amino acid residues being substituted can be substituted with the same or different amino acid residue unless explicitly indicated otherwise. [0061] The terms “Pseudomonas exotoxin” and “PE” as used herein include PE that has been modified from the native protein to reduce or to eliminate immunogenicity. Such modifications may include, but are not limited to, elimination of domain Ia, various amino acid deletions in domains Ib, II, and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus such as DEL (SEQ ID NO: 38) and REDL (SEQ ID NO: 39). See Siegall et al., J. Biol. Chem., 264: 14256-14261 (1989). In an embodiment, the PE may be a cytotoxic fragment of native, wild-type PE. Cytotoxic fragments of PE may include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell (e.g., as a protein or pre-protein). In a preferred embodiment, the cytotoxic fragment of PE retains at least about 20%, preferably at least about 40%, more preferably at least about 50%, even more preferably 75%, more preferably at least about 90%, and still more preferably at least about 95% of the cytotoxicity of native PE. In particularly preferred embodiments, the cytotoxic fragment has at least the cytotoxicity of native PE, and preferably has increased cytotoxicity as compared to native PE. [0062] Modified PE that reduces or eliminates immunogenicity includes, for example, PE4E, PE40, PE38, PE25, PE38QQR, PE38KDEL, PE-LR, PE35, and PE24. In an embodiment, the PE may be any of PE4E, PE40, PE38, PE25, PE38QQR (in which PE38 has the sequence QQR (SEQ ID NO: 40) added at the C-terminus), PE38KDEL (in which PE38 has the sequence KDEL (SEQ ID NO: 41) added at the C-terminus), PE-LR (resistance to lysosomal degradation), PE35, and PE24. [0063] In an embodiment, the PE is PE24 (SEQ ID NO: 2). PE24 contains only functional domain III (residues 395-613) of PE. In a further embodiment, PE is PE24 with cysteine at position 522 (SEQ ID NO: 47). In another embodiment, PE is PE24 with cysteine at position 406 (SEQ ID NO: 48). [0064] In an embodiment, the PE has been modified to reduce immunogenicity by deleting domain Ia as described in U.S. Patent 4,892,827, which is incorporated herein by reference. The PE may also be modified by substituting certain residues of domain Ia. In an embodiment, the PE may be PE4E, which is a substituted PE in which domain Ia is present but in which the basic residues of domain Ia at positions 57, 246, 247, and 249 are replaced with acidic residues (e.g., glutamic acid), as disclosed in U.S. Patent 5,512,658, which is incorporated herein by reference. [0065] PE40 is a truncated derivative of PE (Pai et al., Proc. Nat‘l Acad. Sci. USA, 88: 3358-62 (1991) and Kondo et al., Biol. Chem., 263: 9470-9475 (1988)). PE35 is a 35 kD carboxyl-terminal fragment of PE in which amino acid residues 1-279 have been deleted and the molecule commences with a Met at position 280 followed by amino acids 281-364 and 381-613 of native PE. PE35 and PE40 are disclosed, for example, in U.S. Patents 5,602,095 and 4,892,827, each of which is incorporated herein by reference. PE25 contains the 11- residue fragment from domain II and all of domain III. In some embodiments, the PE contains only domain III. [0066] In an embodiment, the PE is PE38 (SEQ ID NO: 3). PE38 contains the translocating and ADP ribosylating domains of PE but not the cell-binding portion (Hwang et al., Cell, 48: 129-136 (1987)). PE38 is a truncated PE pro-protein composed of amino acids 253-364 and 381-613 which is activated to its cytotoxic form upon processing within a cell (see e.g., U.S. Patent 5,608,039, which is incorporated herein by reference, and Pastan et al., Biochim. Biophys. Acta, 1333: C1-C6 (1997)). [0067] In another embodiment, the PE is PE-LR. PE-LR contains a deletion of domain II except for a furin cleavage sequence (FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 15)) and a deletion of amino acid residues 365-394 of domain Ib. Thus, PE-LR contains amino acid residues 274-284 and 395-613 of SEQ ID NO: 1. PE-LR is described in U.S. Patent No.8,871,906, which is incorporated herein by reference. The PE-LR may, optionally, additionally comprise a GGS (SEQ ID NO: 11) linking peptide between the FCS and amino acid residues 395-613 of SEQ ID NO: 1. [0068] As noted above, alternatively or additionally, some or all of domain Ib may be deleted with the remaining portions joined by a bridge or directly by a peptide bond. Alternatively or additionally, some of the amino portion of domain II may be deleted. Alternatively or additionally, the C-terminal end may contain the native sequence of residues 609-613 (REDLK) (SEQ ID NO: 42), or may contain a variation that may maintain the ability of the PE to translocate into the cytosol, such as KDEL (SEQ ID NO: 41) or REDL (SEQ ID NO: 39) and repeats of these sequences. See, e.g., U.S. Patents 5,854,044; 5,821,238; and 5,602,095 and International Patent Application Publication WO 1999/051643, which are incorporated herein by reference. [0069] The inventive molecule may further comprise a third domain that comprises an optionally substituted domain III from Pseudomonas exotoxin A (“PE”). Domain III from PE is well known in the art, and numerous substitutions are known as well. Suitable substitutions in PE include those which remove one or more B-cell epitopes and/or one or more T-cell epitopes. Suitable substitutions of PE are known to those of ordinary skill in the art and some are set forth, for example, in U.S. Patent No.9,206,240 and U.S. Patent No. 9,346,859, each of which is incorporated herein in its entirety by reference. In an embodiment of the invention, the third domain comprises a PE amino acid sequence, wherein the PE amino acid sequence has a substitution of one or more of amino acid residues R427, F443, R456, D463, R467, L477, R490, R494, R505, R538, and L552, as defined by reference to SEQ ID NO: 1. In an embodiment, the PE amino acid sequence has one or more of the following amino acid substitutions: R427A, F443A, R456A, D463A, R467A, L477H, R490A, R494A, R505A, R538A, and L552E, as defined by reference to SEQ ID NO: 1. See Mazor et al., Molecular Cancer Therapy, 14(12): 2789-96 (2015). [0070] The substitution of one or more amino acid residues within one or more T-cell epitopes may, advantageously, remove one or more T-cell epitopes and reduce T-cell immunogenicity as compared to a PE that lacks a substitution of one or more amino acid residues within one or more T-cell epitopes (e.g., wild-type PE). The substitution(s) may be located within any suitable PE T-cell epitope. Exemplary T-cell epitopes are disclosed in, for example, International Patent Application Publications WO 2012/170617, WO 2013/040141 and US Patent 9,206,240, each of which is incorporated herein by reference. In a preferred embodiment of the invention, the further substitution of one or more amino acid residues within one or more T-cell epitopes is a substitution of alanine, glycine, serine, or glutamine in place of one or more of amino acid residues L294, L297, Y298, L299, R302, R421, L422, L423, A425, L429, Y439, H440, F443, L444, A446, A447, I450, 464-466, 467, 468-480, 482-489, 490, 491-504, 505, 506-512, 513, 514-515, 517-519, L552, T554, I555, L556, and W558 of SEQ ID NO:1, wherein the amino acid residues L294, L297, Y298, L299, R302, R421, L422, L423, A425, L429, Y439, H440, F443, L444, A446, A447, I450, 464-466, 467, 468-480, 482-489, 490, 491-504, 505, 506-512, 513, 514-515, 517-519, L552, T554, I555, L556, and W558 are defined by reference to SEQ ID NO: 1. In an embodiment of the invention, the substitution of one or more amino acid residues at positions 464-466, 468-480, 482-489, 491-504, 506-512, 514-515, 517-519 may include, for example, a substitution of alanine, glycine, serine, or glutamine in place of one or more of amino acid residues at position 464, 465, 466, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 482, 483, 484, 485, 486, 487, 488, 489, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 506, 507, 508, 509, 510, 511, 512, 514, 515, 517, 518, and 519, as defined by reference to SEQ ID NO: 1. [0071] In an embodiment of the invention, any of the inventive molecules described herein has a further substitution of one or more amino acid residues within one or more B-cell epitopes. The substitution of one or more amino acid residues within one or more B-cell epitopes may, advantageously, remove one or more B-cell epitopes and reduce B-cell immunogenicity as compared to a PE that lacks a substitution of one or more amino acid residues within one or more B-cell epitopes (e.g., wild-type PE). The substitution(s) may be located within any suitable PE B-cell epitope. Exemplary B-cell epitopes are disclosed in, for example, International Patent Application Publications WO 2007/016150, WO 2009/032954, WO 2011/032022, WO 2012/170617, WO 2013/040141 and U.S. Patent 9,206,240, each of which is incorporated herein by reference. In an embodiment of the invention, the further substitution of one or more amino acid residues within one or more B-cell epitopes is a substitution of alanine, glycine, serine, or glutamine in place of one or more of amino acid residues E282, E285, P290, R313, N314, P319, D324, E327, E331, Q332, D403, D406, R412, R427, E431, R432, R456, R458, D461, D463, R467, Y481, R490, R505, R513, L516, E522, R538, E548, R551, R576, K590, Q592, and L597, wherein E282, E285, P290, R313, N314, P319, D324, E327, E331, Q332, D403, D406, R412, R427, E431, R432, R456, R458, D461, D463, R467, Y481, R490, R505, R513, L516, E522, R538, E548, R551, R576, K590, Q592, and L597 are defined by reference to SEQ ID NO: 1. In an embodiment of the invention, the amino acid residue at position 458 is arginine when amino acid residue R456 is substituted, preferably with alanine. Preferred mutations include those at positions R427, F443, R456, D463, R467, L477, R490, R494, R505, R538, and L552, including any combination of two or more (or all) of such. [0072] In an embodiment of the invention, any of the inventive molecules described herein has a deletion of one or more contiguous amino acid residues of residues 1-273 and 285-394 as defined by SEQ ID NO: 1. The deletion of one or more contiguous amino acid residues of residues 1-273 and 285-394 may, advantageously, further reduce B- and/or T-cell immunogenicity. [0073] In an embodiment of the invention, any of the inventive molecules described herein has a deletion of one or more contiguous amino acid residues of residues 253-273 and 285-394 as defined by SEQ ID NO: 1. The deletion of one or more contiguous amino acid residues of residues 253-273 and 285-394 may, advantageously, further reduce B- and/or T- cell immunogenicity. [0074] Any of the inventive molecules described herein may comprise PE with one or more further substitutions that may further increase cytotoxicity as disclosed, for example, in International Patent Application Publication WO 2007/016150, which is incorporated herein by reference. Increased cytotoxic activity and decreased immunogenicity can occur simultaneously, and are not mutually exclusive. Substitutions that both increase cytotoxic activity and decrease immunogenicity, such as substitutions of R490 to glycine or, more preferably, alanine, are especially preferred. Furin Cleavage Sequence [0075] The inventive molecule may further comprise a second domain that includes a furin cleavage sequence (“FCS”) that is cleavable by furin. The FCS may be a furin cleavage sequence of amino acid residues, which sequence is cleavable by furin and has an amino end and a carboxyl end. Without being bound by a particular theory or mechanism, it is believed that PEs containing the FCS undergo proteolytic processing inside target cells, thereby activating the cytotoxic activity of the toxin. The FCS of the inventive molecules may comprise any suitable furin cleavage sequence of amino acid residues, which sequence is cleavable by furin. Exemplary furin cleavage sequences are described in Duckert et al., Protein Engineering, Design & Selection, 17(1): 107-112 (2004) and U.S. Patent No. 8,871,906, each of which is incorporated herein by reference. In an embodiment of the invention, FCS comprises residues 274-284 of SEQ ID NO: 1 (i.e., RHRQPRGWEQL (SEQ ID NO: 15)). Other suitable FCS amino acid sequences include, but are not limited to: R-X1- X₂-R, wherein X₁ is any naturally occurring amino acid and X₂ is any naturally occurring amino acid (SEQ ID NO: 16), RKKR (SEQ ID NO: 17), RRRR (SEQ ID NO: 18), RKAR (SEQ ID NO: 19), SRVARS (SEQ ID NO: 20), TSSRKRRFW (SEQ ID NO: 21), ASRRKARSW (SEQ ID NO: 22), RRVKKRFW (SEQ ID NO: 23), RNVVRRDW (SEQ ID NO: 24), TRAVRRRSW (SEQ ID NO: 25), RQPR (SEQ ID NO: 26), RHRQPRGW (SEQ ID NO: 27), RHRQPRGWE (SEQ ID NO: 28), HRQPRGWEQ (SEQ ID NO: 29), RQPRGWE (SEQ ID NO: 30), RHRSKRGWEQL (SEQ ID NO: 31), RSKR (SEQ ID NO: 32), RHRSKRGW (SEQ ID NO: 33), HRSKRGWE (SEQ ID NO: 34), RSKRGWEQL (SEQ ID NO: 35), HRSKRGWEQL (SEQ ID NO: 36), and RHRSKR (SEQ ID NO: 37). [0076] In an embodiment of the invention, the FCS is represented by the formula P4-P3- P2-P1, wherein P4 is an amino acid residue at the amino end, P1 is an amino acid residue at the carboxyl end, P1 is an arginine or a lysine residue, and the sequence is cleavable at the carboxyl end of P1 by furin. [0077] In another embodiment of the invention, the FCS (i) further comprises amino acid residues represented by P6-P5 at the amino end, (ii) further comprises amino acid residues represented by P1’-P2’ at the carboxyl end, (iii) wherein if P1 is an arginine or a lysine residue, P2’ is tryptophan, and P4 is arginine, valine or lysine, provided that if P4 is not arginine, then P6 and P2 are basic residues, and (iv) the sequence is cleavable at the carboxyl end of P1 by furin. Fourth Domain [0078] The inventive molecule comprises a fourth domain comprising 1 to 20 amino acid residues. In some embodiments, the fourth domain is defined as (Xaa1)r, wherein each Xaa1 is selected independently from cysteine, lysine, glycine, alanine and serine and r is an integer from 1 to 20. In some embodiments, the fourth domain is defined as (Xaa1)r, wherein each Xaa1 is as defined in this paragraph and the fourth domain has no more than one cysteine, no more than one lysine, and no more than one alanine. Examples of sequences of the fourth domain include, but are not limited to: KASGG (SEQ ID NO: 14), GGGGS (SEQ ID NO: 10), GGGGS GGGGS GGGGS (SEQ ID NO: 12), SGG (SEQ ID NO: 43), KAS (SEQ ID NO: 13), GGGGS GGGGS GGGGS GG (SEQ ID NO: 44), GGS (SEQ ID NO: 11), GSGSGSGSGSGSGSGSG (SEQ ID NO: 9), GSGSGSGSCGSGSGSGS (SEQ ID NO: 8), and KASGSGSGSGSGSGSGSGSG (SEQ ID NO: 51). Fifth Domain [0079] The inventive molecule can optionally comprise a fifth domain comprising 1 to 20 amino acid residues. In some embodiments, the fourth domain is defined as (Xaa1)r, wherein each Xaa1 is selected independently from cysteine, lysine, glycine, alanine and serine and r is an integer from 1 to 20. In some embodiments, the fifth domain is defined as (Xaa1)r, wherein each Xaa1 is as defined in this paragraph and the fifth domain has no more than one cysteine, no more than one lysine, and no more than one alanine. Examples of sequences of the fifth domain include, but are not limited to: KASGG (SEQ ID NO: 14), GGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 12), SGG (SEQ ID NO: 43), KAS (SEQ ID NO: 13), GGGGSGGGGSGGGGS GG (SEQ ID NO: 44), GGS (SEQ ID NO: 11), GSGSGSGSGSGSGSGSG (SEQ ID NO: 9), GSGSGSGSCGSGSGSGS (SEQ ID NO: 8), and KASGSGSGSGSGSGSGSGSG (SEQ ID NO: 51). Ethylene Glycol [0080] The inventive molecule may further comprise at least one ethylene glycol ((CH₂OH)₂). In an embodiment, the at least one ethylene glycol is a polyethylene glycol of formula H−(O−CH2−CH2)n−OH, wherein n can be from about 100 to about 800 (e.g., from about 150 to about 750, from about 200 to about 700, from about 250 to about 650, from about 300 to about 600, from about 350 to about 550, from about 400 to about 500, from about 420 to about 480, from about 440 to about 460, or about 450). In this regard, the at least one ethylene glycol is a polyethylene glycol and comprises from about 100 to about 800 ethylene glycols, from about 150 to about 750 ethylene glycols, from about 200 to about 700 ethylene glycols, from about 250 to about 650 ethylene glycols, from about 300 to about 600 ethylene glycols, from about 350 to about 550 ethylene glycols, from about 400 to about 500 ethylene glycols, from about 420 to about 480 ethylene glycols, from about 440 to about 460 ethylene glycols, or about 450 ethylene glycols. [0081] In an embodiment, the at least one ethylene glycol is a polyethylene glycol and has a molecular weight from about 5 kDaltons to about 40 kDaltons. In this regard, the the at least one ethylene glycol has a molecular weight of from about 6 kDaltons to about 38 kDaltons, from about 8 kDaltons to about 35 kDaltons, from about 9 kDaltons to about 32 kDaltons, from about 11 kDaltons to about 30 kDaltons, from about 13 kDaltons to about 28 kDaltons, from about 15 kDaltons to about 25 kDaltons, from about 18 kDaltons to about 22 kDaltons, or about 20 kDaltons. [0082] The polyethylene glycol can be linear or branched. A brached polyethylene glycol is defined herein as two or more polyethylene glycol chains linked to a common center. In contrast, a linear polyethylene glycol defined herein as a polyethylene glycol that does not have any chains linked to a common center. Configuration of Molecule [0083] The inventive molecule may be configured in any of a variety of different ways. In an embodiment of the invention, the second domain is positioned between the first domain and the third domain. Alternatively or additionally, the second domain may be positioned between the first domain and the fifth domain. Alternatively or additionally, the second domain may be positioned between the fourth domain and the fifth domain. Alternatively or additionally, the second domain may be positioned between the fourth domain and the third domain. Alternatively or additionally, the fourth domain is positioned between the first domain and the second domain. Alternatively or additionally, the fourth domain is positioned between the first domain and the fifth domain. Alternatively or additionally, the fourth domain is positioned between the first domain and the third domain. Alternatively or additionally, the fifth domain is positioned between the first domain and the third domain. Alternatively or additionally, the fifth domain is positioned between the fourth domain and the third domains. Alternatively or additionally, the fifth domain is positioned between the second domain and the third domain. Alternatively or additionally, the fifth domain is positioned between the second domain and the third domain. Alternatively or additionally, the first domain is positioned at the amino terminus of the molecule. Alternatively or additionally, the third domain is positioned at the carboxyl terminus of the molecule. [0084] Another embodiment of the invention provides a molecule comprising, or consisting of, the following domains of the molecule in the following order, from the amino terminus to the carboxyl terminus: first, fourth, second, fifth, and third. [0085] In an embodiment, the inventive molecule does not contain a foreign protein domain which increases immunogenicity of the molecule. In an embodiment, the inventive molecule does not contain a moiety which specifically binds to serum albumin. However, in another embodiment, the molecule comprises an albumin binding domain (ABD). The albumin binding domain may, for example, be positioned between the targeting moiety and the FCS. The albumin binding domain may be flanked by linkers, as described herein with respect to other aspects of the invention. [0086] The ABD may be any moiety which specifically binds to serum albumin. The albumin, to which the ABD specifically binds, may be of any mammalian species. Preferably, the albumin is human serum albumin or mouse serum albumin. [0087] In an embodiment of the invention, the ABD is a peptide which specifically binds to serum albumin. Any peptide which specifically binds to serum albumin may be suitable to include in the inventive chimeric molecules. The peptide may be from any species, e.g., Streptococcus. In an embodiment of the invention, the peptide, which specifically binds to serum albumin, is a streptococcal protein G-derived serum albumin binding domain. Such peptides are described, for example, in Jonsson et al., Protein Engineering, Design & Selection, 21(8): 515-27 (2008). [0088] In an embodiment of the invention, the ABD is an antibody, or an antigen binding portion thereof, which specifically binds to serum albumin. In a preferred embodiment, the anti-albumin antibody is a single domain antibody. Antibodies which specifically bind to serum albumin are commercially available. The anti-albumin antibody may be of any species, e.g., llama. In an embodiment, the antibody, or an antigen binding portion thereof, which specifically binds to serum albumin, is a single domain antibody from llama as described, for example, in Coppieters et al., Arthritis & Rheumatism, 54(6): 1856-66 (2006). In an embodiment, the anti-albumin single domain antibody is ALB1 or MSA21, which are described in US 2007/0269422 and US 2006/0228355, respectively, each of which is incorporated herein by reference. [0089] Examples of molecules in accordance with embodiments of the invention are shown in Figures 1A-1B and in Tables 1-6. [0090] In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 4, 12, 5, 14, 15, 11, and 2. Another embodiment of the invention provides a molecule comprising, or consisting of, the following amino acid sequences presented in the order of the amino terminus to the carboxyl terminus: SEQ ID NOs: 4, 12, 5, 14, 15, 11, and 2 (see e.g., Table 1). Table 1

[0091] In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 4, 12, 5, 13, 9, 15, 11, and 47. In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 4, 12, 5, 51, 15, 11, and 47. Another embodiment of the invention provides a molecule comprising, or consisting of, t the following amino acid sequences presented in the order of the amino terminus to the carboxyl terminus: SEQ ID NOs: 4, 12, 5, 51, 15, 11, and 47 (see e.g., Table 2). Table 2

[0092] In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 4, 12, 5, 13, 9, 15, 11, and 48. In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 4, 12, 5, 51, 15, 11, and 48. Another embodiment of the invention provides a molecule comprising, or consisting of, the following amino acid sequences presented in the order of the amino terminus to the carboxyl terminus: SEQ ID NOs: 4, 12, 5, 51, 15, 11, and 48 (see e.g., Table 3). Table 3

[0093] In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 4, 12, 5, 8, 15, 11, and 2. Another embodiment of the invention provides a molecule comprising, or consisting of, the following amino acid sequences presented in the order of the amino terminus to the carboxyl terminus: SEQ ID NOs: 4, 12, 5, 8, 15, 11, and 2 (see e.g., Table 4). Another embodiment of the invention provides a molecule comprising, or consisting of, SEQ ID NO: 45. Table 4

[0094] In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 49, 9, 4, 12, 5, 14, 15, 11, and 2. In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 50, 4, 12, 5, 14, 15, 11, and 2. Another embodiment of the invention provides a molecule comprising, or consisting of, the following amino acid sequences presented in the order of the amino terminus to the carboxyl terminus: SEQ ID NOs: 50, 4, 12, 5, 14, 15, 11, and 2 (see e.g., Table 5). Table 5

[0095] In an embodiment, the inventive provides a molecule comprising, or consisting of, SEQ ID NOs: 6, 12, 7, 14, 15, 11, and 2. Another embodiment of the invention provides a molecule comprising, or consisting of, the following amino acid sequences presented in the order of the amino terminus to the carboxyl terminus: SEQ ID NOs: 6, 12, 7, 14, 15, 11, and 2 (see e.g., Table 6). Another embodiment of the invention provides a molecule comprising, or consisting of, SEQ ID NO: 46. Table 6

[0096] Included in the scope of the invention are functional portions of the inventive molecules described herein. The term “functional portion,” when used in reference to a molecule, refers to any part or fragment of the molecule of the invention, which part or fragment retains the biological activity of the molecule of which it is a part (the parent molecule). Functional portions encompass, for example, those parts of a molecule that retain the ability to specifically bind to and destroy or inhibit the growth of target cells or treat or prevent cancer, to a similar extent, the same extent, or to a higher extent, as the parent molecule. In reference to the parent molecule, the functional portion can comprise, for instance, about 10% or more, about 25% or more, about 30% or more, about 50% or more, about 68% or more, about 80% or more, about 90% or more, or about 95% or more, of the parent molecule. [0097] The functional portion can comprise additional amino acids at the amino or carboxyl terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent molecule. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., specifically binding to and destroying or inhibiting the growth of target cells, having the ability to treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent molecule. [0098] Included in the scope of the invention are functional variants of the inventive molecules described herein. The term “functional variant,” as used herein, refers to a molecule having substantial or significant sequence identity or similarity to a parent molecule, which functional variant retains the biological activity of the molecule of which it is a variant. Functional variants encompass, for example, those variants of the molecule described herein (the parent molecule) that retain the ability to specifically bind to and destroy or inhibit the growth of target cells to a similar extent, the same extent, or to a higher extent, as the parent molecule. In reference to the parent molecule, the functional variant can, for instance, be about 30% or more, about 50% or more, about 75% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more identical in amino acid sequence to the parent molecule. [0099] The functional variant can, for example, comprise the amino acid sequence of the parent molecule with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain chemical and/or physical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc. [00100] Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent molecule with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. Preferably, the non- conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent molecule. [00101] The molecule of the invention can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the functional variant, e.g., other amino acids, do not materially change the biological activity of the functional variant. [00102] The molecule of the invention (including functional portions and functional variants) of the invention can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N’-benzyl-N’-methyl-lysine, N’,N’-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α- aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ- diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert- butylglycine. [00103] The molecule of the invention (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated. Production Methods [00104] An embodiment of the invention provides methods of preparing the inventive molecules comprising incubating a molecule comprising the first domain, the second domain, the third domain, and the fourth domain with tris(2-carboxyethyl)phosphine (TCEP) to produce a recombinant immunotoxin with reduced cysteine, and adding methoxy- polyethylene glycol maleimide to the recombinant immunotoxin with reduced cysteine. [00105] As seen in Fig.3B, the at least one ethylene glycol (“PEG” in Fig.3B) can be conjugated to the molecule (“RIT” in Fig.3B) via a sulfhydryl on the molecule. Specifically, a sulfhydryl of the at least one cysteine of the molecule can be used to form a bond with a maleimide that is attached to the at least one ethylene glycol by virtue of its ability to form disulfide bonds. A simplied schematic of this reaction is provided below, wherein R is at least one ethylene glycol and R’ is the molecule with at least one cysteine. In this reaction, malemide is positioned between at least one cysteine of the molecule and the at least one ethylene glycol.

[00106] Prior to PEGylation, the molecule that includes the the first domain, the second domain, the third domain, and the fourth domain as described herein can be prepared by (a) recombinantly expressing the the first domain, the second domain, the third domain, and the fourth domain as described herein to produce a recombinant immunotoxin and (b) purifying the recombinant immunotoxin. The recombinant immunotoxins (including functional portions and functional variants) can be obtained by methods of producing proteins and polypeptides known in the art. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Dunn (ed.), Peptide Chemistry and Drug Design, 1st Ed., New York: Wiley (2015). Also, the recombinant immunotoxins can be recombinantly expressed using the nucleic acids described herein using standard recombinant methods. See, for instance, Green et al., supra. [00107] Once expressed, the recombinant immunotoxins may be purified in accordance with purification techniques known in the art. Exemplary purification techniques include, but are not limited to, ammonium sulfate precipitation, affinity columns, and column chromatography, or by procedures described in, e.g., Janson (ed.), Protein Purification: Principles, High Resolution Methods, and Applications, Springer-Verlag, NY (2011). [00108] In another embodiment of the invention, the recombinant immunotoxins may be produced using non-recombinant methods. For example, the recombinant immunotoxins described herein (including functional portions and functional variants) can be commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this respect, the recombinant immunotoxins can be synthetic, recombinant, isolated, and/or purified. Nucleic acids [00109] An embodiment of the invention provides a nucleic acid comprising a nucleotide sequence encoding the first domain, the second domain, the third domain, and/or the fourth domain of the molecule of any of the inventive molecules described herein. The term “nucleic acid,” as used herein, includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single- stranded or double-stranded, which can be synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural, or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions. [00110] Preferably, the nucleic acids of the invention are recombinant. As used herein, the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication. [00111] The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5’-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX). [00112] An embodiment of the invention also provides a nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein. [00113] The nucleotide sequence which hybridizes under stringent conditions preferably hybridizes under high stringency conditions. By “high stringency conditions” is meant that the nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions which would distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches, from a random sequence that happened to have only a few small regions (e.g., 3-10 bases) that matched the nucleotide sequence. Such small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions would include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70 °C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any of the inventive molecules. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide. Vectors [00114] The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention. For purposes herein, the term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The inventive recombinant expression vectors can comprise any type of nucleotide, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, which can be synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages does not hinder the transcription or replication of the vector. [00115] The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or for both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, e.g., a retroviral vector. [00116] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Green et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like. [00117] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based. [00118] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes. [00119] The recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the inventive molecule (including functional portions and functional variants), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the molecule. The selection of promoters, e.g., strong, weak, inducible, tissue-specific, and developmental- specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the ordinary skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus. [00120] The inventive recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression. Cells [00121] Another embodiment of the invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term “host cell” refers to a cell that can contain the inventive recombinant expression vector. For purposes of producing a recombinant inventive molecule, the host cell is preferably a prokaryotic cell (e.g., a bacteria cell), e.g., an E. coli cell. [00122] Also provided by the invention is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell which does not comprise any of the recombinant expression vectors. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly (e.g., consisting essentially of) host cells comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population of host cells comprising a recombinant expression vector as described herein. [00123] The inventive molecules (including functional portions and functional variants), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and populations of cells can be isolated and/or purified. The term “isolated,” as used herein, means having been removed from its natural environment. The term “purified,” as used herein, means having been increased in purity, wherein “purity” is a relative term, and not to be necessarily construed as absolute purity. For example, the purity can be about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100%. The purity preferably is about 90% or more (e.g., about 90% to about 95%) and more preferably about 98% or more (e.g., about 98% to about 99%). Compositions [00124] The inventive molecules (including functional portions and functional variants), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and populations of cells, all of which are collectively referred to as “inventive molecules” hereinafter, can be formulated into a composition, such as a pharmaceutical composition. In this regard, the invention provides a pharmaceutical composition comprising any of the molecules (including functional portions and functional variants), nucleic acids, recombinant expression vectors, host cells (including populations thereof), or populations of cells, and a pharmaceutically acceptable carrier. The inventive pharmaceutical composition containing any of the inventive molecules can comprise more than one inventive molecules, e.g., a polypeptide and a nucleic acid, or two or more different PEs. Alternatively, the pharmaceutical composition can comprise an inventive molecules in combination with one or more other pharmaceutically active agents or drugs, such as a chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. [00125] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. [00126] The choice of carrier will be determined in part by the particular inventive molecules, as well as by the particular method used to administer the inventive molecules. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral (e.g., subcutaneous, intravenous, intraarterial, intramuscular, intradermal, interperitoneal, and intrathecal) administration are exemplary and are in no way limiting. More than one route can be used to administer the inventive molecules, and in certain instances, a particular route can provide a more immediate and more effective response than another route. [00127] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The inventive molecules can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2- dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. [00128] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. [00129] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof. [00130] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the inventive molecules material in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The requirements for effective pharmaceutical carriers for parenteral compositions are well-known to those of ordinary skill in the art (see, e.g., Lloyd et al. (eds.), Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press (2012)). [00131] It will be appreciated by one of skill in the art that, in addition to the above- described pharmaceutical compositions, the inventive molecules of the invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. [00132] For purposes of the invention, the amount or dose of the inventive molecules administered should be sufficient to effect a desired response, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame. For example, the dose of the inventive molecules should be sufficient to inhibit growth of a target cell or treat or prevent cancer in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular inventive molecules and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated. [00133] Many assays for determining an administered dose are known in the art. An administered dose may be determined in vitro (e.g., cell cultures) or in vivo (e.g., animal studies). For example, an administered dose may be determined by determining the IC50 (the dose that achieves a half-maximal inhibition of symptoms), LD₅₀ (the dose lethal to 50% of the population), the ED50 (the dose therapeutically effective in 50% of the population), and the therapeutic index in cell culture and/or animal studies. The therapeutic index is the ratio of LD50 to ED50 (i.e., LD50/ED50). [00134] The dose of the inventive molecules also will be determined by the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular inventive molecules. Typically, the attending physician will decide the dosage of the inventive molecules with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive molecules to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the inventive molecules can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1 mg/kg body weight/day, from about 1 to about to about 1000 mg/kg body weight/day, from about 5 to about 500 mg/kg body weight/day, from about 10 to about 250 mg/kg body weight/day, about 25 to about 150 mg/kg body weight/day, or about 10 mg/kg body weight/day. [00135] The inventive molecules may be assayed for cytotoxicity by assays known in the art. Examples of cytotoxicity assays include a WST assay, which measures cell proliferation using the tetrazolium salt WST-1 (reagents and kits available from Roche Applied Sciences), as described in International Patent Application Publication WO 2011/032022. Treatment Methods [00136] It is contemplated that the inventive molecules, nucleic acids, recombinant expression vectors, host cell, population of cells, and pharmaceutical compositions can be used in methods of treating or preventing cancer. Without being bound by a particular theory or mechanism, it is believed that the inventive molecules destroy or inhibit the growth of cells. Without being bound to a particular theory or mechanism, it is believed that the inventive molecules recognize and specifically bind to cell surface markers, thereby delivering the cytotoxic PE to the population of cells expressing the cell surface marker with minimal or no cross-reactivity with cells that do not express the cell surface marker. In this way, the cytotoxicity of PE can be targeted to destroy or inhibit the growth of a particular population of cells, e.g., cancer cells. In this regard, an embodiment of the invention provides a method of treating or preventing cancer in a mammal comprising administering to the mammal any of the inventive molecules, nucleic acids, recombinant expression vectors, host cell, population of cells, or pharmaceutical compositions described herein, in an amount effective to treat or prevent cancer in the mammal. [00137] The terms “treat” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, for purposes herein, “prevention” can encompass delaying the onset of the disease, or a symptom or condition thereof. [00138] With respect to the inventive methods, the cancer can be any cancer, including any of adrenal gland cancer, sarcomas (e.g., synovial sarcoma, osteogenic sarcoma, leiomyosarcoma uteri, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma, and teratoma), lymphomas (e.g., small lymphocytic lymphoma, Hodgkin lymphoma, and non-Hodgkin lymphoma), hepatocellular carcinoma, glioma, head cancers (e.g., squamous cell carcinoma), neck cancers (e.g., squamous cell carcinoma), acute lymphocytic cancer, leukemias (e.g., hairy cell leukemia, myeloid leukemia (acute and chronic), lymphatic leukemia (acute and chronic), prolymphocytic leukemia (PLL), myelomonocytic leukemia (acute and chronic), and lymphocytic leukemia (acute and chronic)), bone cancer (osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing’s sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, and giant cell tumors), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, and retinoblastoma), fallopian tube cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva (e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma), myeloproliferative disorders (e.g., chronic myeloid cancer), colon cancers (e.g., colon carcinoma), esophageal cancer (e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma), cervical cancer (cervical carcinoma and pre-invasive cervical dysplasia), gastric cancer, gastrointestinal carcinoid tumor, hypopharynx cancer, larynx cancer, liver cancers (e.g., hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma), lung cancers (e.g., bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, and adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatous hamartoma, small cell lung cancer, non-small cell lung cancer, and lung adenocarcinoma), malignant mesothelioma, skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi’s sarcoma, nevi, dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids), multiple myeloma, nasopharynx cancer, ovarian cancer (e.g., ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid carcinoma, and clear cell adenocarcinoma), granulosa-theca cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, and malignant teratoma), pancreatic cancer (e.g., ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, and VIPoma), peritoneum, omentum, mesentery cancer, pharynx cancer, prostate cancer (e.g., adenocarcinoma and sarcoma), rectal cancer, kidney cancer (e.g., adenocarcinoma, Wilms tumor (nephroblastoma), and renal cell carcinoma), small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi’s sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma), soft tissue cancer, stomach cancer (e.g., carcinoma, lymphoma, and leiomyosarcoma), testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor, fibroma, fibroadenoma, adenomatoid tumors, and lipoma), cancer of the uterus (e.g., endometrial carcinoma), thyroid cancer, and urothelial cancers (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureter cancer, and urinary bladder cancer). In a preferred embodiment, the cancer is a cancer that is characterized by the expression or overexpression of CD22 (such as, for example, hairy cell leukemia, CLL, PLL, non-Hodgkin’s lymphoma, SLL, and ALL), BCMA (such as, for example, multiple myeloma and Hodgkin’s lymphoma), or mesothelin (such as, for example, mesothelioma and ovarian and pancreatic adenocarcinoma). [00139] As used herein, the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human. [00140] Also provided is a method of inhibiting the growth of a target cell comprising contacting the cell with any of the inventive molecules, nucleic acids, recombinant expression vectors, host cell, population of cells, or pharmaceutical compositions described herein, in an amount effective to inhibit growth of the target cell. The growth of the target cell may be inhibited by any amount, e.g., by about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100%. The target cell may be provided in a biological sample. A biological sample may be obtained from a mammal in any suitable manner and from any suitable source. The biological sample may, for example, be obtained by a blood draw, leukapheresis, and/or tumor biopsy or necropsy. The contacting step can take place in vitro or in vivo with respect to the mammal. Preferably, the contacting is in vitro. [00141] In an embodiment of the invention, the target cell is a cancer cell. The target cell may be a cancer cell of any of the cancers described herein. In an embodiment of the invention, the target may express a cell surface marker. The cell surface marker may be any cell surface marker described herein with respect to other aspects of the invention. The cell surface marker may be, for example, selected from the group consisting of CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor (EGFR), mutated EGFR, BCMA, glypican 2 (GPC2), glypican 3 (GPC3), mesothelin, cadherin, and Lewis Y. Examples of Non-Limiting Aspects of the Disclosure [00142] Aspects, including embodiments, of the present subject matter described herein may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-33 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below: [00143] (1) A molecule comprising: (a) a first domain comprising a targeting moiety, wherein the targeting moiety comprises a first linker of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine; (b) a second domain comprising a furin cleavage sequence (FCS), wherein the FCS is cleavable by furin; (c) a third domain comprising an optionally substituted domain III from Pseudomonas exotoxin A (PE); (d) a fourth domain comprising 1 to 20 amino acid residues; and (e) at least one ethylene glycol ((CH2OH)2), wherein the molecule contains at least one cysteine, wherein the first domain optionally comprises a second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and the at least one cysteine, wherein the at least one cysteine is located within the first domain or the third domain and wherein the at least one ethylene glycol is attached to the at least one cysteine. [00144] (2) A molecule comprising: (a) a first domain comprising a targeting moiety that specifically binds to mesothelin, wherein the targeting moiety comprises a first linker of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine; (b) a second domain comprising a furin cleavage sequence (FCS), wherein the FCS is cleavable by furin; (c) a third domain comprising an optionally substituted domain III from Pseudomonas exotoxin A (PE); (d) a fourth domain comprising 1 to 20 amino acid residues; and (e) at least one ethylene glycol ((CH₂OH)₂), wherein the molecule contains at least one cysteine, wherein the first domain optionally comprises a second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and the at least one cysteine, wherein the at least one cysteine is located within the first domain, the third domain or the fourth domain, and wherein the at least one ethylene glycol is attached to the at least one cysteine. [00145] (3) The molecule of aspect 1 or 2, wherein the molecule further comprises an albumin binding domain. [00146] (4) The molecule of any one of aspects 1 to 3, wherein the second domain is positioned between the first domain and the third domain. [00147] (5) The molecule of any one of aspects 1 to 4, wherein the targeting moiety specifically binds to a cell surface marker selected from the group consisting of CD19, CD21, CD22, CD25, CD30, CD33, CD79b, BCMA, glypican 2 (GPC2), glypican 3 (GPC3), transferrin receptor, EGF receptor (EGFR), mutated EGFR, mesothelin, cadherin, and Lewis Y. [00148] (6) The molecule of any one of aspects 1-5, wherein the targeting moiety comprises a single-chain Fv (scFv) with a Vh and Vl. [00149] (7) The molecule of aspect 6, wherein the first linker of the first domain is positioned between the Vh and Vl of the scFv. [00150] (8) The molecule of any one of aspects 1-7, wherein the first linker of the first domain comprises SEQ ID NO: 12. [00151] (9) The molecule of any one of aspects 1-8, wherein the targeting moiety comprises at least one of SEQ ID NOs: 4-7. [00152] (10) The molecule of any one of aspects 1-9, wherein the FCS comprises at least one of SEQ ID NOs: 15-37. [00153] (11) The molecule of any one of aspects 1-10, wherein the domain III from PE comprises a PE amino acid sequence, wherein the PE amino acid sequence has a substitution of one or more of amino acid residues R427, F443, R456, D463, R467, L477, R490, R494, R505, R538, and L552, as defined by reference to SEQ ID NO: 1. [00154] (12) The molecule of any one of aspects 1-11, wherein the domain III from PE comprises a PE amino acid sequence, wherein the PE amino acid sequence has one or more of the following amino acid substitutions: R427A, F443A, R456A, D463A, R467A, L477H, R490A, R494A, R505A, R538A, and L552E, as defined by reference to SEQ ID NO: 1. [00155] (13) The molecule of any one of aspects 1-12, wherein the domain III from PE comprises SEQ ID NO: 2. [00156] (14) The molecule of any one of aspects 1-13, wherein the fourth domain comprises 1 to 20 amino acid residues selected, independently, from the group consisting of glycine, serine, lysine, and alanine. [00157] (15) The molecule of any one of aspects 1-13, wherein the fourth domain comprises 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine, and optionally the at least one cysteine. [00158] (16) The molecule of any one of aspects 1-15, wherein the fourth domain comprises SEQ ID NO: 9. [00159] (17) The molecule of any one of aspects 1-15, wherein the molecule comprises a fifth domain. [00160] (18) The molecule of aspect 17, wherein the fifth domain comprises 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine, and optionally the at least one cysteine. [00161] (19) The molecule of any one of aspects 1-18, wherein the at least one ethylene glycol comprises from about 100 to about 800 ethylene glycols. [00162] (20) The molecule of any one of aspects 1-19, wherein the at least one ethylene glycol is from about 5 to about 40 kD. [00163] (21) The molecule of any one of aspects 1-20, wherein the at least one ethylene glycol is linear. [00164] (22) The molecule of any one of aspects 1-21, wherein the first domain comprises the at least one cysteine at position S63, as defined by reference to SEQ ID NO: 7. [00165] (23) The molecule of any one of aspects 1-22, wherein the third domain comprises the at least one cysteine at position E522 or D406, as defined by reference to SEQ ID NO: 1. [00166] (24) The molecule of any one of aspects 2-23, wherein the fourth domain comprises the at least one cysteine at position 9, as defined by reference to SEQ ID NO: 8. [00167] (25) The molecule of any one of aspects 1-24, wherein the first domain comprises the second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and the at least one cysteine. [00168] (26) The molecule of aspect 1, comprising SEQ ID NO: 45. [00169] (27) The molecule of aspect 1, comprising SEQ ID NO: 46. [00170] (28) A nucleic acid comprising a nucleotide sequence encoding the first domain, the second domain, the third domain, and the fourth domain of the molecule of any one of aspects 1-27. [00171] (29) A recombinant expression vector comprising the nucleic acid of aspect 28. [00172] (30) A host cell comprising the recombinant expression vector of aspect 29. [00173] (31) A population of cells comprising at least one host cell of aspect 30. [00174] (32) A pharmaceutical composition comprising (a) the molecule of any one of aspects 1-27, the nucleic acid of aspect 28, the recombinant expression vector of aspect 29, the host cell of aspect 30, or the population of cells of aspect 31, and (b) a pharmaceutically acceptable carrier. [00175] (33) The molecule of any one of aspects 1-27, the nucleic acid of aspect 28, the recombinant expression vector of aspect 29, the host cell of aspect 30, the population of cells of aspect 31, or the pharmaceutical composition of aspect 32 for use in treating or preventing cancer in a mammal. [00176] (34) An in vitro method of inhibiting the growth of a target cell, which method comprises contacting the cell with the PE of any one of aspects 1-27, the nucleic acid of aspect 28, the recombinant expression vector of aspect 29, the host cell of aspect 30, the population of cells of aspect 31, or the pharmaceutical composition of aspect 32, in an amount effective to inhibit growth of the target cell. [00177] (35) The method of aspect 34, wherein the target cell is a cancer cell. [00178] (36) The method of aspect 34 or 35, wherein the target cell expresses a cell surface marker selected from the group consisting of CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor (EGFR), mutated EGFR, BCMA, glypican 2 (GPC2), glypican 3 (GPC3), mesothelin, cadherin, and Lewis Y. [00179] (37) A method of preparing the molecule of aspect 1, comprising: (a) incubating a molecule comprising the first domain, the second domain, the third domain, and the fourth domain of aspect 1 with tris(2-carboxyethyl)phosphine (TCEP) to produce a recombinant immunotoxin with reduced cysteine; and (b) adding methoxy-polyethylene glycol maleimide to the recombinant immunotoxin with reduced cysteine to produce the molecule of aspect 1. [00180] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES [00181] Bacterial Strains and Plasmids: Escherichia coli DH5α (High Efficiency) was obtained from New England Biolabs for the propagation of plasmids. E. coli BL21(λDE3), which carries T7 RNA polymerase gene under the control of an inducible promoter on a λ prophage, was used as a host to express RIT. Plasmids that express RIT were under the control of T7 promoter and contain a single-chain Fv that was genetically fused to a 24 kD bacterial toxin PE by a flexible GS linker and a furin cleavage site. [00182] Construction, Expression, and Purification of RIT: A double-stranded gene fragment (gBlock, Integrated DNA Technologies) that contained the designated cysteine was ligated onto our standard laboratory RIT production vector (see, e.g., Pastan, et al., Methods Mol. Biol., 248: 503-18 (2004)) using Gibson Assembly Master Mix (New England Biolabs) according to the manufacturer’s protocol. The correct assembly of the plasmids was confirmed by cutting with the appropriate restriction enzymes and sequencing analysis. The plasmid was transformed into E. coli. BL21(λDE3) and the RIT were induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (“IPTG”) at OD 2.5 for 2 hours. All RITs were expressed and purified as inclusion bodies (see, e.g., Mazor, et al., Proc. Nat’l Acad. Sci. USA, 111(23): 8571-6 (2014). Briefly, inclusion bodies were dissolved in buffer (6M guanidine-HCl, 100mM Tris-HCl, 2mM ethylenediaminetetraacetic acid (“EDTA”)) for 19 hours, followed by refolding in 1000 ml 100 mM Tris-HCl, 1mM EDTA, 0.5M arginine and 0.5 M NDSB-201, pH 10.0 for 31 hours and dialysis against 50 liters of 30 mM Tris-HCl, 0.1 M urea for 19 hours. The refolded RITs were purified through Q SEPHAROSE^TM and MONO Q^TM ion exchanger columns (GE Healthcare). [00183] PEGylation of RITs: RITs were PEGylated in collaboration with Selecta Bioscience. Four mg of the RITs were buffer exchanged into 20 mM potassium phosphate, pH 8.0, 2mM EDTA using VIVASPIN^TM 20 centrifugal concentrator (5 kD MWCO, GE Healthcare) until the pH of the flow through matched that of the buffer. A 3-molar excess of tris(2-carboxyethyl)phosphine (“TCEP”) was added to the final 2mg/ml RIT and incubated at room temperature for 1 hour to reduce cysteine. Then a 10-molar excess of methoxy-PEG maleimide (molecular weight 20 kD, JenKem Technology) was added and left to react overnight. For purification, the PEGylated RIT was buffer exchanged into 10mM Tris-HCl, pH 8.0 using a VIVASPIN^TM column and applied to an anion exchange spin column (Pierce). Unreactive methoxy-PEG maleimide was washed away with four 10 ml washes of the same buffer and the RIT was eluted with 1x phosphate-buffered saline (PBS). Samples were collected before and after TCEP treatment and were analyzed on SDS-PAGE gels after the final elution. For high-performance liquid chromatography (“HPLC”) analysis, additional samples were collected during column washes to make sure methoxy-PEG maleimide was completely gone. [00184] HPLC Analysis: Reverse phase HPLC was performed to separate PEGylated RIT from the parental RIT and unreacted PEG. The column featured a non-polar C4 stationary phase with 3.5 μm particle size and 2.1 mm I.D. (Water Corporation). Mobile phases A and B contained 5% and 90% acetonitrile, respectively, and were supplemented with 0.1% trifluoroacetic acid. Five μl of about 1 mg/ml samples were injected into the gradient run, starting from 95% buffer A to 100% buffer B in 10 min. [00185] Mesothelin Binding: A 96-well microtiter plate was coated with 50 ul of 1 μg/ml MSLN-hFc, a fusion protein consisting of human IgG Fc and mesothelin protein and incubated at 4 °C overnight. The plate was blocked with blocking buffer (1x PBS supplemented with 25% Dulbecco’s Modified Eagle’s Medium, 25 mM (4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid), 0.5% bovine serum albumin, 0.1% Azide, 5% fetal bovine serum) and 50μl serial dilution of the PEGylated RITs was added in triplicate and 50 μl monoclonal anti-PE antibody (IP12) and horseradish peroxidase-labeled goat anti-mouse IgG were added as primary and secondary antibodies. After washing with blocking buffer, the plate was developed with 3,3’,5,5’-tetramethylbenzidine substrate kits (Thermos Scientific) and subsequently read with the plate reader at 640 and 450 nm wavelength. Binding curve was generated using GraphPad and 50% binding (IC50) was calculated. Table 7 summarizes average IC₅₀ values of two experiments. [00186] Adenosine diphosphate(ADP)-ribosylation: A 20 μl reaction was set up to contain 4 μl buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA), 1 ul 1M dithiothreitol (“DTT”), 1ul 20ng/ul RIT, 5 μg protein lysate (prepared from KLM1 cell) and 1 μl 250uM Biotin- nicotinamide-adenine-dinucleotide (“NAD,” Trevigen) and incubated at room temperature for 1 hour. Samples were analyzed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (“SDS-PAGE”) gels and western blot was performed. The polyvinylidene fluoride (“PVDF”) membrane was probed with horseradish peroxidase (“HRP”)-streptavidin followed by enhanced chemiluminescence (“ECL”) development to visualize the ADP- ribosylated elongation factor 2 (“EF2”). [00187] Cytotoxicity Assay: Human cell lines KLM1 (Hollevoet, et al., Mol. Cancer Ther., 13(8): 2040-9 (2014), L55 (Liu, et al., Oncotarget, 8(50): 87307-87316 (2017)) and A431/H9 (Ho, et al., Clin. Cancer Res., 11(10): 3814-20 (2005)) were described previously. Cells were grown in Roswell Park Memorial Institute (“RPMI”) media supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C until 70-78% confluency before they were trypsinized. Then 4,000 cells were seeded onto each well of a 96-well plate, and serial dilutions of the RIT were added in triplicate rows and incubated for 72 hours. Cytotoxicity activity was evaluated with a water-soluble tetrazolium salt-8 (“WST8”) assay according to the manufacturer’s protocol (Dojindo Molecular Technologies). Plates were read at 640 and 450 nm wavelength, and data were plotted using GraphPad to obtain IC50 values. [00188] Half-Life Assay: For each RIT, two mice (6-8 weeks old, 23-25 g) were intravenously (“IV”) injected with 25 μg of the RIT. Blood was harvested at 5 min, and 2, 4, 8, and 24 hours for LMB-203-PEG, LMB-244-PEG, and LMB-249-PEG and 5 min, and 1, 4, and 24 hours for LMB-163-PEG and LMB-179-PEG afterward, and serum was isolated. Enzyme-linked immunosorbent assay (“ELISA”) was used to determine the amount of RIT at each time point as described above. Half-life and area under the curve (“AUC”) were calculated using GraphPad, with two-phase decay fitting, except LMB-12 which was fitted with one-phase decay. The 5 min sample was used as the initial amount of RIT in the blood, and the later time points were normalized against this value. [00189] Radius of hydration (Rh): The Rh of five PEGylated proteins was measured using Dynamic Light Scattering (“DLS,” Wyatt Technology). Proteins in the range of 0.5-1 mg/ml were spun down at 13,000 rpm for 5 min to pellet aggregates and 20 μl was transferred to a quartz cuvette slowly to avoid bubbles. Each sample was measured at least two times, and each time constitutes an average of 30 measurements. Rh values were extracted and plotted against the half-life, cytotoxicity, and anti-tumor activity using GraphPad. Linear regression analysis was performed to obtain a line of best-fit and R² values. [00190] Biodistribution of RIT: 5.8 nmol of LMB-249-PEG and 4.0 nmol of SS1P were incubated with 23.1 nmol and 15.9 nmol of label FNIR-Z-759 in PBS buffer, pH 8.5, at room temperature for 1 hour, respectively. The labeled protein was purified with SEPHADEX^TM G25 column. Three mice bearing about 100 mm³ A431/H9 tumor were IV injected with 41 μg (580 pmol) of labeled LMB-249-PEG or 25 μg (400 pmol) of labeled SS1P, followed by serial dorsal and ventral 800 nm fluorescence imaging immediately and 15, 30 min, and 1, 2, 3, 4, 5, 6, 9, 12, 24, and 48 hours afterward. Fluorescence accumulation in the kidney, liver, and tumor was monitored. Background fluorescence was also calculated to obtain the target- to-background ratio. Plots were generated using GraphPad from the average results of 3 mice. [00191] Anti-tumor Experiment: Female (10 per group) nu/nu mice (6-week-old, 20-25 g) were injected subcutaneously (sc) in the flank with 5 x 10⁶ KLM1 cells. When the tumor reached 100 mm³, the mice were grouped based on weight and tumor size. RITs in PBS supplemented with 0.2% human serum albumin were IV injected at 10 ug/mouse three times per week for one week. Tumor volume and mice weight were monitored every two to three days. Mice were euthanized if they lost more than 10% of their body weight. The experiment was repeated for LMB-244-PEG and LMB-163-PEG (n = 10) and injected at 20 ug/mouse, two times per week for two weeks. Again, mouse weight was monitored every two to three days and the mice were euthanized if they lost more than 10% of their body weight. EXAMPLE 1 [00192] This example demonstrates the preparation of PEGylated RITs. [00193] Preparation of RITs. [00194] The RITs used in this study are shown in Figure 1A. SS1P was the first immunotoxin made to target mesothelin and contains an anti-mesothelin Fv attached to PE38 that contains domains II and III of PE (Chowdhury, et al., Nat. Biotechnol., 17(6): 568-72 (1999)). LMB-12 and LMB-84 both have domain II deleted and contain an 11-amino acids furin cleavage site that connects the Fv to domain III (PE24). In LMB-12, the Vl and Vh were connected by a disulfide bond and in LMB-84 they were connected by a flexible 15- residues (G4S)₃ linker (SEQ ID NO: 12). LMB-179, LMB-244, LMB-203, LMB-249, and LMB-163 were all derived from LMB-84, but have cysteines used for PEGylation. They also contain a 17-residue GS linker (SEQ ID NO: 9) placed either between the Vl and the furin cleavage site (LMB-179, LMB-244, and LMB-203) or at the N-terminus of the Vh (LMB- 249). LMB-163 is similar to LMB-84 but contains mutations that humanize the Fv portion. Figure 1B depicts cartoon representations of the RITs. The 20 kD PEG is shown as a wiggly line and an arrow. [00195] Site directed mutagenesis was used to insert cysteine residues at various locations in the RIT. To choose locations that were less likely to interfere with immunotoxin activity, the RIT was modeled using the existing structures of its various components. The model of immunotoxin is shown in Figure 2A. The structural complex of the immunotoxin bound to mesothelin and EF2 is shown in Figure 2B, and it was generated by superposing the templates of mesothelin-mAb (PDB 4F3F, Ma, et al., J. Biol. Chem., 287(40): 33123-31 (2012)), scFV (PDB 3GKZ, Celikel, et al., Protein Sci., 18(11): 2336-45 (2009)), PE-EF2 (PDB 1ZM4, Jorgensen, et al., Nature, 436(7053): 979-84 (2005)) and PE-NAD-AMP (PDB 1DMA, Li, et al., Proc. Nat’l Acad. Sci. USA, 92(20): 9308-12 (1995)) in UCSF Chimera (Pettersen, et al., J. Comput. Chem., 25(13): 1605-12 (2004)). Figure 2C shows a model of the 20 kD PEG, composed of 454 units of ethylene glycol, conjugated to the immunotoxin at D406 of domain III. Five different sites were mutated to cysteine for PEG conjugation as indicated by spheres. The five locations were selected in order to diminish interference with mesothelin binding, processing by furin protease, and ADP-ribosylation of EF2. [00196] LMB-179 and LMB-244 have cysteine residues located in domain III at E522 and D406, respectively. The cysteine residues were located on different sides of PE24 and were distant from the EF2 binding site (Figure 2A). LMB-203 had the cysteine placed in the middle of a 17 amino acid linker connecting the Fv to the furin cleavage peptide (Figure 1A). It was 8 residues away from the furin cleavage site in order to diminish effects on cleavage or mesothelin binding to the Fv or EF2 binding to domain III. In LMB-249 the cysteine residue was located on the amino terminus of a 17-residue GS linker attached to the first residue of the heavy chain of the Fv (Figures 1A-2B); this location was unlikely to interfere with the binding of mesothelin to the CDRs of the Fv. LMB-163 does not contain an extra 17-residue GS linker like the other four RITs. The cysteine was placed at S63 in the light chain (Figure 1 and 2). Position S63 was selected to mutate to cysteine because the residue was surface- exposed and distant from the CDRs and the furin cleavage peptide so the mutation was less likely to interfere with binding to mesothelin or cleavage of the RIT by furin protease. All of RITs were expressed in E. coli in the form of inclusion bodies, which were denatured, refolded, and purified through Q SEPHAROSE^TM and MONO Q^TM ion exchanger columns (GE Healthcare). [00197] PEGylation of RITs [00198] Because a free thiol can lead to dimer formation, the purification was stopped after the MONO Q^TM anion exchanger column, where a peak of monomer and another for dimer have been observed. Figure 3A shows MONO Q^TM anion exchanger column fractions of LMB-179, LMB-244, LMB-203, and LMB-249 that were mostly monomer (lanes 2, 5, 6, and 8) or dimer (lanes 3, 4, 7, and 9, respectively). LMB-163 only had monomer fractions (lanes 10 and 11). Monomer runs at about 50 kD, and dimer runs slightly above 100 kD, consistent with the predicted molecular weight of each species. A final purification on size exclusion chromatography was not used because both monomer and dimer can be reduced by TCEP and used for PEGylation. [00199] The PEGylation reaction was based on modifying a free thiol group of a surface- exposed cysteine to form a thioester bond with the maleimide functional group of a 20 kD linear PEG (Figure 3B). To determine the best conditions for conjugation, different conditions were examined and it was found that the reaction was most efficient in PBS at pH 8.0. TCEP may be useful to enhance PEGylation efficiency as compared to the standard reducing agents such as dithiothreitol and β-mercaptoethanol (Humphreys, et al., Protein Eng. Des. Sel., 20(5): 227-34(2007)). Since TCEP does not contain a free thiol, there was no need to remove it prior to PEGylation, and the yields of reduced protein were much higher than using traditional reducing reagents. A 10-fold molar excess of methoxy PEG-maleimide was added to the TCEP-treated protein for PEGylation overnight. HPLC analysis of the reaction mixture was performed to determine the PEGylation efficiency. An exemplary HPLC run was shown for LMB-203 and its PEGylated counterpart LMB-203-PEG in Figure 3C. The peaks were quantified and it was found that the PEGylation was about 90% complete. The unreacted methoxy-PEG-maleimide was removed on an anion exchange column and the PEGylated protein was eluted with PBS. [00200] Eluted proteins were analyzed on the SDS-PAGE gel along with pre and post- TCEP treated samples (Figure 3D). For LMB-179 and LMB-244, the starting materials were mostly dimer (lanes 2 and 5). For LMB-203 and LMB-249, there was a mixture of monomer and dimer (lanes 8 and 11). LMB-163 was all monomer (lane 14) perhaps because the SH group was not readily available for dimer formation. After reduction with TCEP, all of the RITs became monomers (lanes 3, 6, 9, 12 and 15). After addition of PEG, 90% or more of the TCEP-treated monomer was PEGylated (lanes 4, 7, 10, 13 and 16). The calculated molecular weight of a PEGylated RIT was about 72 kD; however, they all ran at about 100 kD indicating that the molecules were asymmetrical. The PEGylated proteins were quantified using Image J open source image processing program. The PEGylation efficiencies were about 90% overall, with LMB-203/LMB-203-PEG being 93-95%, which was consistent with the HPLC analysis. The PEG-modified proteins were frozen at -70 ºC in small aliquots and thawed as needed for assays. [00201] About 10% of each RIT could not be modified even when more methoxy-PEG- maleimide was added and the incubation time extended. Removing TCEP before the addition of methoxy-PEG-maleimide also did not improve the yield. [00202] Overall, the mutant RITs were PEGylated at a high efficiency (90% efficiency) and a high purity. The use of TCEP to reduce the protein followed by treating with a 20 kDa maleimide derivative of PEG at pH 8.0 assisted with the high efficiency rate. Such high efficiency rates were not achievable with DTT (see Tsutsumi, et al., Proc. Nat’l Acad. Sci. USA, 97(15): 8548-53 (2000)). EXAMPLE 2 [00203] This example demonstrates the cytotoxicity of the PEGylated RITs. To determine whether PEGylation hampers the activity of the RITs, cytotoxicity assays were performed on three mesothelin-expressing cancer cell lines. It was found that LMB-12 and LMB-84 have nearly identical cytotoxic activities (Figure 7A). LMB-84 had an IC50 of 0.1942 and LMB-12 had an IC₅₀ of 0.2214. LMB-12 was selected as the un-PEGylated control. PEGylated and control RITs were incubated with cancer cells for three days, and cell proliferation assays were performed. The results are shown in Figures 4A-4B. The cell- killing data was best fit to a sigmoidal curve. In general, the range of linearity in which the cell-killing activity was dependent on the concentration of RIT and occurs between 0.1-1 ng/ml. Using KLM1 cells, all of the cysteine containing proteins were as active as the LMB- 12 except for LMB-244 which was somewhat more active (Table 7). For KLM1 (pancreatic) cell line, parental RITs LMB-249, LMB-203, LMB-163 and LMB-179 have IC50 of 0.22 ± 0.06, 0.29 ± 0.02, 0.22 ± 0.03, and 0.21 ± 0.01ng/ml, respectively. LMB-244 had an IC50 of 0.16 ± 0.03 ng/ml. The PEG modified immunotoxins were 4-5-fold less active except for LMB-244-PEG, which lost only 35% activity. After PEGylation the IC50s for LMB-249- PEG, LMB-203-PEG, LMB-163-PEG, and LMB-179-PEG were 1.18 ± 0.03, 1.10 ± 0.19, 1.13 ± 0.19, and 1.09 ± 0.13 ng/ml, and LMB-244-PEG was 0.25 ± 0.05 ng/ml. The activities of LMB-244-PEG and LMB-163-PEG were also tested on A431/H9 epidermoid carcinoma cells and MKN28 stomach cancer cells. Consistent with the data from the KLM1 cell line, LMB-244 was the most active and lost less than 50% of its activity. The PEG- modified RITs were also tested on an A431 epidermoid carcinoma cell line that was not transfected with mesothelin cDNA. As seen in Figure 7B, no specific killing was observed. LMB-179-PEG, LMB-163-PEG, LMB-203-PEG, and LMB-244-PEG had IC50 values of about 318.7, 850.2, 365.1, and 667.0, respectively. Figures 4C-E show bar graphs of the activity of PEG-modified RITs relative to the parental control, which was set to 100%. It was evident that LMB-244-PEG was the most active. In three tumor cell lines, KLM1 (Figure 4C), A431/H9 (Figure 4D), and MKN28 (Figure 4E), LMB-244-PEG retains 65, 62 and 70% of the parental activity, respectively. Table 7. Summary of Properties of RITs

Table 7 notes: • “nt” indicates not tested • “n/a” indicates not applicable • bolded numbers refer to PEGylated RITs EXAMPLE 3 [00204] This example demonstrates that Mesothelin binding and ADP-ribosylation are not affected by PEGylation of the RITs. [00205] The first step in the action of RIT was binding of the mesothelin on the cell surface of the cancer cell by the Fv. To determine if binding was affected by the PEG, serial dilutions of each PEGylated RIT were tested for binding to mesothelin using ELISA. The results are shown in Figure 8 and 50% maximum binding was indicated. The binding of the PEGylated RITs was close to that of un-PEGylated LMB-12. The values were: LMB-12 (0.022 ± 0.001 ug/ml), LMB-249-PEG (0.027 ± 0.001 ug/ml), LMB-203-PEG (0.024 ± 0.004 ug/ml), LMB-163-PEG (0.029 ± 0.006 ug/ml), LMB-179-PEG (0.023 ± 0.002 ug/ml), and LMB-244-PEG (0.013 ± 0.001 ug/ml). Binding to mesothelin was similar for all PEG modified RITs, although there was a small loss of binding to mesothelin by the two RITs that had PEG added to the Fv (Figure 8). [00206] Another step in RIT action was the ADP-ribosylation and inactivation of EF2. This step can be measured by incubating EF2-containing cell lysate with the ADP precursor NAD-biotin and PEG-modified RIT and measuring the amount of biotin incorporated into EF2 using western blots and a Strep-HRP antibody. Figure 9 shows an ADP ribosylation assay in which the PEGylated RITs were incubated with the EF2-containing cell lysate. The resulting blots show a RIT-dependent modification of EF2, with only one major band that was consistent with the molecular weight of EF2 (indicated by arrow). The band was not detected in the negative control when the RIT was omitted (lane 16). The EF2-ADP bands were quantified and normalized each to LMB-12 positive control. For accuracy, 5, 10 and 15 μl of each reaction were loaded. All RITs retained about 100% of the original activity. This indicates that the loss of cytotoxic activity was not due to the inability of the immunotoxin to inactivate EF2. EXAMPLE 4 [00207] This example demonstrates that the PEGylated RITs have improved half-lives. [00208] The half-lives of the PEGylated RITs in the circulation were determined by IV injection of 25 μg of each protein into nu/nu mice and subsequent seum collection at 5 min and 2, 4, 8 and 24 hours for LMB-203-PEG, LMB-244-PEG, and LMB-249-PEG, and at 5 min and 1, 4 and 24 hours for LMB-163-PEG and LMB-179-PEG. ELISA was used to determine the amount of remaining RIT. To normalize the data, the data was plotted relative to the 5 min time point, which was set as 100% (Figure 5A). LMB-12, which had no cysteine added and was not PEGylated was used as an un-PEGylated control. The decay for LMB-12 was rapid and mono-exponential. The curves for the PEG-modified proteins best fit a two- phase decay function so a fast (⍺) and a slow (β) decay half-life was calculated for each protein (Table 7). AUC was also calculated to represent the amount in the blood present over time so that there was a single value to compare for each protein. Figure 5A shows that all PEGylated proteins have much longer half-lives than LMB-12. The AUC for LMB-12 was 0.2, while the PEGylated proteins have AUCs that were at least 10-fold higher and range from 2.2 for LMB-244-PEG to 5.9 for LMB-249-PEG. LMB-203-PEG had the second largest AUC of 5.1. LMB-163-PEG and LMB-179-PEG have AUCs of 4.3 and 3.9. Based on the AUC values, the order from the longest to the shortest half-life was LMB-249-PEG > LMB-203-PEG > LMB-163-PEG > LMB-179-PEG > LMB-244-PEG. The difference between LMB-244-PEG and LMB-249-PEG was about 3-fold even though the mass of each protein was identical. [00209] The PEGylated proteins have much longer β than ⍺ decay. LMB-249-PEG had the longest half-life with an ⍺ of 144 min and a β of 74,400 min. LMB-163-PEG had an ⍺ of 29 min and a β of 62,200 min. LMB-244-PEG had the shortest half-life, with an ⍺ of 25 min and a β of 256 min. In summary, PEGylation extends the half-life of RITs significantly, particularly the β phase. [00210] The half-life data of PEGylated RITs fits well to biexponential decay curves (Figure 5A). The ⍺ phase may be due to rapid equilibration with the extracellular compartment, and the β phase may be due to the metabolism by the kidney and liver. External imaging was used to determine which organs in mice take up SS1P, a small un- PEGylated RIT with a half-life of 19 min (AUC 0.2) and LMB-249-PEG with a long half-life and an AUC of 5.9. The data in Figure 5C and 5E show that SS1P was rapidly taken up by kidney and by liver, whereas the uptake of LMB-249-PEG was delayed. This delay leads to the greatly increased half-life and AUC. The initial uptake by tumors was the same for both RITs, but the uptake of SS1P was arrested as blood levels fell (Figure 5D), whereas the uptake of LMB-249-PEG increased for many hours. EXAMPLE 5 [00211] This example demonstrates that the hydrodynamic radius (Rh) of the PEGylated RITs impacts their half-lives. [00212] Dynamic Light Scattering was used to determine if the addition of PEG at different locations in the RIT changed the hydrodynamic radius. Each sample was measured at least two times and representative measurements are shown in Figures 10A-B and summarized in Table 7. The %Mass was plotted against the radius. Overall, the results show a monomodal size distribution for LMB-163-PEG, LMB-244-PEG, LMB-12 and LMB-203. For LMB-249-PEG, LMB-203-PEG, and LMB-179-PEG, polymodal size distributions were observed, which was due to the presence of trace amounts of aggregate that also diffracted laser light. LMB-12 and LMB-203 were included as un-PEGylated controls. LMB-12 had the smallest Rh of 3.4 nm. LMB-203 was slightly larger, with Rh of 3.7 nm (Figures 10A- 10B). With the addition of 20 kD PEG, the Rhs increased substantially to 7.1, 6.9, 5.9, 5.6, and 5.2 nm for LMB-249-PEG, LMB-203-PEG, LMB-179-PEG, LMB-163-PEG, and LMB- 244-PEG, respectively. Comparing LMB-203 and LMB-203-PEG, the increase in Rh was almost 2-fold. [00213] Because the same 20 kDa PEG was used to modify all the RITs, it was unexpected that the Rh values varied so widely from 5.2 to 7.1 indicating that the derivatized proteins adopted different conformations. Since the Rh values correlate with the half-life in the circulation (Figure 5B), and since kidney was the major organ responsible for removal of RITs, the data indicate that the glomerulus in the kidney may be sensitive to the size and shape of the proteins and therefore filters them at different rates. [00214] In Figure 5B, the Rh values were plotted against AUC. The result shows a good correlation between AUC and Rh with the R² value of 0.8. In general, larger Rh corresponds to longer half-life. The un-PEGylated LMB-12 was smallest protein and had a Rh of about 3.4. LMB-203 was slightly larger (Rh 3.7) having a 15 amino acid linker connecting the light and heavy chains and 17 amino acid linker connecting the Fv to the furin cleavage site (Figures 10A-10B). The PEGylated proteins were much larger, with average Rhs of 7.1, 6.9, 5.6, 5.9, 5.2 nm for LMB-249-PEG, LMB-203-PEG, LMB-163-PEG, LMB-179-PEG, and LMB-244-PEG, respectively. Therefore, our results indicate that the PEGylated RITs adopt distinct conformations governed by the position of the PEG, and such conformation influences the half-life. EXAMPLE 6 [00215] This example demonstrates the rate in which the PEGylated RITs were removed by the kidney and liver. [00216] Biodistribution studies were perfomed with LMB-249-PEG labelled with FNIR-Z- 759, a near-infrared fluorophore that allows external imaging of living mice (Sato, et al., Bioconjug. Chem., 27(2): 404-13 (2016)). Labeled LMB-249-PEG (41 ug, 580 pmol) was injected IV into tumor-bearing mice and the accumulation in the kidney, liver, and tumor was imaged over 12 hours. The images in the upper panel of Figure 11A were from a dorsal view, showing uptake by the kidneys and by tumor that were close to the dorsal side of the mouse. The images in the lower panel of Figure 11A show uptake by the liver which was close to the ventral side. To compare the biodistribution with an immunotoxin without PEG, the same experiment was performed with SS1P, which had a molecular weight of 63 kD, close to 72 kD for the PEG modified RITs. The upper panel of Figure 11B shows accumulation of SS1P in the kidney and tumor and the lower panel shows liver uptake. EXAMPLE 7 [00217] This example demonstrates the amounts in which the PEGylated RITs were taken up by kidney, liver, and tumor. [00218] To determine the amount of LMB-249-PEG and SS1P taken up by kidney, liver, and tumor, the images of Example 6 were scanned and quantified. Accumulation as a function of time for kidney was plotted in Figure 5C, for liver in Figure 5D, and for tumor in Figure 5E. The graphs show that uptake of LMB-249-PEG in kidney peaked at 6 hours whereas uptake of SS1P peaked much earlier at 2 hours. Liver uptake of LMB-249-PEG peaked late at 9 hours, while SS1P uptake peaked much earlier at 2 hours. The results for tumor follow a different pattern. Accumulation of LMB-249-PEG by tumor increased steadily over many hours and peaked at 9-12 hours, whereas uptake of SS1P was much smaller and peaked at 2-3 hours. EXAMPLE 8 [00219] This example demonstrates the anti-tumor activity of the PEGylated RITs. [00220] To do an initial assessment of the anti-tumor activity of the various PEGylated RITs, tumor-bearing mice IV were treated with 10 μg of PEG-modified RIT given every other day x3, when the tumors reached about about 100 mm³ in size. In the PBS treatment group, the tumors grew rapidly (Figure 6A). All treatment groups responded to PEGylated RITs. LMB-244-PEG and LMB-163-PEG were the most active and the tumors decreased in size to 69 mm³ and 79 mm³ on day 14 and the p-values were both 0.001, respectively. LMB- 179-PEG, LMB-203-PEG, and LMB-249-PEG slowed tumor growth; on day 14, the tumors reached 259 mm³ in the control group and only 152, 117, and 143 mm³ in LMB-179-PEG, LMB-203-PEG, and LMB-249-PEG, respectively. The weights of the treated mice decreased by less than 10% (Figure 12A), but two mice in the LMB-179-PEG group and one in the LMB-249-PEG died. [00221] LMB-244-PEG and LMB-163-PEG produced substantial tumor regressions (Figure 6A). The high anti-tumor activity of LMB-244-PEG reflects several factors. The starting protein, LMB-244, was almost 2-fold more active than the other proteins and PEG addition caused less than a 2-fold loss of activity. Also, LMB-164-PEG had the smallest Rh indicating it was more compact and probably enters tumors better than the other PEG modified proteins. However, the small Rh probably contributes to its low AUC of 2.2. LMB-163-PEG also had good tumor activity despite being 3-fold less cytotoxic than LMB- 264-PEG. [00222] Additional studies were performed with LMB-244-PEG and LMB-163-PEG using a dose and schedule that did not cause weight loss. The activity of LMB-244-PEG and LMB- 163-PEG was compared with the activity of LMB-12, which was similar to LMB-84 in structure and activity and was not PEG modified. Figure 6B shows that tumors in mice receiving five 20 μg doses given over 2 weeks of PEG modified RIT regressed to 46 mm³ and 48 mm³, whereas tumors in mice treated with LMB-12 increased in size but grew more slowly than tumors treated with PBS. The p-values for LMB-244-PEG and LMB-163-PEG compared to LMB-12 were 0.0049 and 0.0048, respectively. At this dosing schedule, the weight of the mice decreased by less than 10% and no mice died (Figure 12B). This data establishes that increasing half-life greatly increases anti-tumor activity. [00223] In summary, the most active of the PEG modified RITs was LMB-244-PEG (PEG attached to residue 406C in domain III of the toxin (Figure 1A)). LMB-244-PEG was tested on 3 cell lines and LMB-244-PEG retained more than 50% of its cytotoxic activity on all lines. When tested in mice, LMB-244-PEG had an 11-fold increase in residence time (AUC) in the circulation compared with the un-PEGylated protein LMB-12 and this change resulted in a very large increase in anti-tumor activity (Figure 6). LMB-244-PEG had the smallest Rh which may indicate that one or more steps in immunotoxin action were negatively affected by larger Rh of the protein and a smaller Rh is desirable. Further, LMB-244-PEG had a shorter half-life and AUC than the other PEGylated RITs indicating it was more efficiently filtered by the kidney glomerulus than RITs with higher Rh values. In addition, the PEG-modified proteins with the smallest Rh (e.g., LMB-244-PEG) had excellent anti-tumor activity, suggesting that entry into tumors was also affected by Rh. EXAMPLE 9 [00224] This example demonstrates the anti-tumor activity of a RIT, LMB-288, comprising an albumin binding domain. When LMB-288 is modified with PEG, it provides for an advantageously long serum half-life. [00225] The RIT LMB-288 is illustrated as a schematic at Figure 13A and as a ribbon diagram at Figure 13C. LMB-288 contains a single-chain Fv in which a GS linker is placed at the N-terminus of the Vl. The scFv is connected to an albumin binding domain (ABD) and furin site by linkers. The toxin portion is PE24, a 24kDa portion of PE, containing a cysteine point mutation for PEG conjugation. A SDS-PAGE gel showing non-reduced, reduced and PEG-conjugated forms of LMB-288 is depicted at Figure 13B. [00226] Figure 14A shows the results of cytotoxicity assays of parental and pegylated immunotoxin, LMB-288, as well as LMB-12. The data presented in Figure 14A represents the results of cytotoxicity assays after 72 hours of incubation of mesothelin-positive cancer cell lines with immunotoxins, in KLM1, (a pancreatic cancer cell line), A431/H9, (an epidermoid carcinoma cell line transfected with mesothelin cDNA), and MKN28, (a gastric cancer cell line). Figure 14B depicts a bar graph showing the IC50 values of pegylated immunotoxin normalized against the parental which is set to 100%, with percent remaining activity as indicated. [00227] Figure 15 depicts data from assays measuring HSA binding of several immunotoxins (LMB-288, LMB-288 Reduced, LMB-288-PEG, LMB-164, LMB-12, LMB- 179-PEG). ELISA was performed after overnight incubation of immunotoxins with albumin- coated affinity plates in order to determine the binding affinity to albumin. IC50 values were converted into molar affinity for comparison to controls. For LMB-288 reduced, TCEP was included in the dilution buffer to ensure the protein remained in monomer form during the binding step. LMB-164 is an ABD-containing immunotoxin that was shown to efficiently bind to albumin. LMB-12 and LMB-179 both lack ABD and were included as negative controls. [00228] Figure 16A depicts data from assays measuring half-life of immunotoxins (LMB- 12, LMB-244-PEG, LMB-164, LMB-288-PEG).25ug of immunotoxins were I.V. injected into 4 nu/nu mice and blood was harvested at the indicated time points. ELISA was performed using the mesothelin-coated affinity plate to determine the amount of immunotoxin remaining in mouse blood serum at each time point. The half-life is represented as AUC. Figure 16B depicts data measuring the hydrodynamic volumes of immunotoxins using dynamic light scattering and plotted against the AUC. Albumin-bound LMB-164 and LMB288-PEG were used for the measurement. The plotted shapes of points in Figure 16B correspond to the shapes of points in Figure 16A. Table 8. Amount of Immunotoxins Remaining at Select Time Points

[00229] The summary data of Table 8 shows the advantageously extended serum half-life of LMB-288-PEG by comparison to LMB-12, LMB-244-PEG and LMB-164. [00230] Figure 17-19 show the in vivo anti-tumor efficacy of LMB-288-PEG in mesothelin-positive KLM1 tumor cells, mesothelin-positive A431/H9 tumor cells, and mesothelin-positive MKN28 tumor cells. [00231] Figure 17 depicts a graph showing the anti-tumor activity of a PEGylated RIT, LMB-288+20KDa PEG, in mice. Mice were implanted with mesothelin-positive KLM1 tumor cells. Mice were intravenously (“IV”) injected with varying dosages of LMB- 288+20KDa PEG (25 ug/mose, 10 ug/mouse and 75 ug/mouse) as indicated by arrows. Tumor burden was monitored over 2 weeks. [00232] Figure 18 depicts a graph showing the anti-tumor activity of a PEGylated RIT, LMB-288+20KDa PEG, and an unPEGylated RIT, LMB-12, in mice. Mice were implanted with mesothelin-positive A431/H9 tumor cells. Mice were intravenously (“IV”) injected with LMB-288+20KDa PEG (25 ug/mouse) or LMB-12 (20ug/mouse) as indicated by arrows. Tumor burden was monitored over 2 weeks. [00233] Figure 19 depicts a graph showing the anti-tumor activity of a PEGylated RIT, LMB-288+20KDa PEG, and an unPEGylated RIT, LMB-12, in mice. Mice were implanted with mesothelin-positive MKN28 tumor cells. Mice were intravenously (“IV”) injected with LMB-288+20KDa PEG (25 ug/mouse) or LMB-12 (20ug/mouse) as indicated by arrows. Tumor burden was monitored over 2 weeks. Table 9. Summary Properties of RITs

[00234] As shown in Table 9, LMB-288-PEG demonstrates an advantageously extended serum half-life. [00235] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [00236] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [00237] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S): 1. A molecule comprising: (a) a first domain comprising a targeting moiety, wherein the targeting moiety comprises a first linker of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine; (b) a second domain comprising a furin cleavage sequence (FCS), wherein the FCS is cleavable by furin; (c) a third domain comprising an optionally substituted domain III from Pseudomonas exotoxin A (PE); (d) a fourth domain comprising 1 to 20 amino acid residues; and (e) at least one ethylene glycol ((CH₂OH)₂), wherein the molecule contains at least one cysteine, wherein the first domain optionally comprises a second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and the at least one cysteine, wherein the at least one cysteine is located within the first domain or the third domain, and wherein the at least one ethylene glycol is attached to the at least one cysteine.

2. A molecule comprising: (a) a first domain comprising a targeting moiety that specifically binds to mesothelin, wherein the targeting moiety comprises a first linker of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine; (b) a second domain comprising a furin cleavage sequence (FCS), wherein the FCS is cleavable by furin; (c) a third domain comprising an optionally substituted domain III from Pseudomonas exotoxin A (PE); (d) a fourth domain comprising 1 to 20 amino acid residues; and (e) at least one ethylene glycol ((CH2OH)2), wherein the molecule contains at least one cysteine, wherein the first domain optionally comprises a second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and the at least one cysteine, wherein the at least one cysteine is located within the first domain, the third domain, or the fourth domain and wherein the at least one ethylene glycol is attached to the at least one cysteine.

3. The molecule of claim 1 or 2, wherein the molecule further comprises an albumin binding domain.

4. The molecule of any one of claims 1 to 3, wherein the second domain is positioned between the first domain and the third domain.

5. The molecule of any one of claims 1 to 4, wherein the targeting moiety specifically binds to a cell surface marker selected from the group consisting of CD19, CD21, CD22, CD25, CD30, CD33, CD79b, BCMA, glypican 2 (GPC2), glypican 3 (GPC3), transferrin receptor, EGF receptor (EGFR), mutated EGFR, mesothelin, cadherin, and Lewis Y.

6. The molecule of any one of claims 1-5, wherein the targeting moiety comprises a single-chain Fv (scFv) with a Vh and Vl.

7. The molecule of claim 6, wherein the first linker of the first domain is positioned between the Vh and Vl of the scFv.

8. The molecule of any one of claims 1-7, wherein the first linker of the first domain comprises SEQ ID NO: 12.

9. The molecule of any one of claims 1-8, wherein the targeting moiety comprises at least one of SEQ ID NOs: 4-7.

10. The molecule of any one of claims 1-9, wherein the FCS comprises at least one of SEQ ID NOs: 15-37.

11. The molecule of any one of claims 1-10, wherein the domain III from PE comprises a PE amino acid sequence, wherein the PE amino acid sequence has a substitution of one or more of amino acid residues R427, F443, R456, D463, R467, L477, R490, R494, R505, R538, and L552, as defined by reference to SEQ ID NO: 1.

12. The molecule of any one of claims 1-11, wherein the domain III from PE comprises a PE amino acid sequence, wherein the PE amino acid sequence has one or more of the following amino acid substitutions: R427A, F443A, R456A, D463A, R467A, L477H, R490A, R494A, R505A, R538A, and L552E, as defined by reference to SEQ ID NO: 1.

13. The molecule of any one of claims 1-12, wherein the domain III from PE comprises SEQ ID NO: 2.

14. The molecule of any one of claims 1-13, wherein the fourth domain comprises 1 to 20 amino acid residues selected, independently, from the group consisting of glycine, serine, lysine, and alanine.

15. The molecule of any one of claims 1-13, wherein the fourth domain comprises 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine, and optionally the at least one cysteine.

16. The molecule of any one of claims 1-15, wherein the fourth domain comprises SEQ ID NO: 9.

17. The molecule of any one of claims 1-15, wherein the molecule comprises a fifth domain.

18. The molecule of claim 17, wherein the fifth domain comprises 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine, and optionally the at least one cysteine.

19. The molecule of any one of claims 1-18, wherein the at least one ethylene glycol comprises from about 100 to about 800 ethylene glycols.

20. The molecule of any one of claims 1-19, wherein the at least one ethylene glycol is from about 5 to about 40 kD.

21. The molecule of any one of claims 1-20, wherein the at least one ethylene glycol is linear.

22. The molecule of any one of claims 1-21, wherein the first domain comprises the at least one cysteine at position S63, as defined by reference to SEQ ID NO: 7.

23. The molecule of any one of claims 1-22, wherein the third domain comprises the at least one cysteine at position E522 or D406, as defined by reference to SEQ ID NO: 1.

24. The molecule of any one of claims 2-23, wherein the fourth domain comprises the at least one cysteine at position 9, as defined by reference to SEQ ID NO: 8.

25. The molecule of any one of claims 1-24, wherein the first domain comprises the second linker of 1 to 20 amino acid residues selected, independently, from the group consisting of methionine, glycine, serine, and the at least one cysteine.

26. The molecule of claim 1, comprising SEQ ID NO: 45.

27. The molecule of claim 1, comprising SEQ ID NO: 46.

28. A nucleic acid comprising a nucleotide sequence encoding the first domain, the second domain, the third domain, and the fourth domain of the molecule of any one of claims 1-27.

29. A recombinant expression vector comprising the nucleic acid of claim 28.

30. A host cell comprising the recombinant expression vector of claim 29.

31. A population of cells comprising at least one host cell of claim 30.

32. A pharmaceutical composition comprising (a) the molecule of any one of claims 1-27, the nucleic acid of claim 28, the recombinant expression vector of claim 29, the host cell of claim 30, or the population of cells of claim 31, and (b) a pharmaceutically acceptable carrier.

33. The molecule of any one of claims 1-27, the nucleic acid of claim 28, the recombinant expression vector of claim 29, the host cell of claim 30, the population of cells of claim 31, or the pharmaceutical composition of claim 32 for use in treating or preventing cancer in a mammal..

34. An in vitro method of inhibiting the growth of a target cell, which method comprises contacting the cell with the PE of any one of claims 1-27, the nucleic acid of claim 28, the recombinant expression vector of claim 29, the host cell of claim 30, the population of cells of claim 31, or the pharmaceutical composition of claim 32, in an amount effective to inhibit growth of the target cell.

35. The method of claim 34, wherein the target cell is a cancer cell.

36. The method of claim 34 or 35, wherein the target cell expresses a cell surface marker selected from the group consisting of CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor (EGFR), mutated EGFR, BCMA, glypican 2 (GPC2), glypican 3 (GPC3), mesothelin, cadherin, and Lewis Y.

37. A method of preparing the molecule of claim 1, comprising: (a) incubating a molecule comprising the first domain, the second domain, the third domain, and the fourth domain of claim 1 with tris(2-carboxyethyl)phosphine (TCEP) to produce a recombinant immunotoxin with reduced cysteine; and (b) adding methoxy-polyethylene glycol maleimide to the recombinant immunotoxin with reduced cysteine to produce the molecule of claim 1.