USOO96770.55B2

(12) United States Patent (10) Patent No.: US 9,677,055 B2 Jones et al. (45) Date of Patent: Jun. 13, 2017

(54) MODIFIED FORMS OF PSEUDOMONAS 2008.0125363 A1 5/2008 Filpula et al. EXOTOXINA 2008/O193976 A1 8, 2008 Harding 2009, O142341 A1 6/2009 Pastan et al. 2009/0305411 A1 12/2009 FitzGerald et al. (71) Applicant: Intrexon Corporation, Blacksburg, VA 2010, 0215656 A1 8, 2010 Pastan et al. (US) 2012/0263674 A1 10, 2012 Pastan et al. (72) Inventors: Timothy David Jones, Cambs (GB); FOREIGN PATENT DOCUMENTS Francis Joseph Carr, Aberdeen (GB) WO WO 88,02401 A1 4f1988 Assignee: Interxon Corporation, Blacksburg, VA WO WO 89.10971 A1 11, 1989 (73) WO WO 90,12592 A1 11, 1990 (US) WO WO 91,09949 A1 7, 1991 WO WO 91,09965 A1 7, 1991 (*) Notice: Subject to any disclaimer, the term of this WO WO 91 18018 A1 11F1991 patent is extended or adjusted under 35 WO WO91f18099 A1 11, 1991 U.S.C. 154(b) by 0 days. WO WO 91.18100 A1 11F1991 WO WO92fO9613 A1 6, 1992 WO WO 93.07.286 A1 4f1993 (21) Appl. No.: 15/234,297 WO WO 93,25690 A1 12, 1993 WO WO 94,04689 A1 3, 1994 (22) Filed: Aug. 11, 2016 WO WO 97,13529 A1 4f1997 WO WO 98/20135 A2 5, 1998 (65) Prior Publication Data WO WO 98/41641 A1 9, 1998 WO WO 99,28471 A2 6, 1999 US 2017/OO37386 A1 Feb. 9, 2017 WO WO O2/40545 A2 5, 2002 WO WO 2004/050849 A2 6, 2004 Related U.S. Application Data (Continued) (63) Continuation of application No. 14/984,006, filed on Dec. 30, 2015, now Pat. No. 9,447,387, which is a OTHER PUBLICATIONS continuation of application No. 14/561,707, filed on Siegall et al., “Cell-Specific Toxicity of a Chimeric Protein Com Dec. 5, 2014, now Pat. No. 9,371,517, which is a posed of -6 and Pseudomonas Exotoxin (IL6-PE40) on continuation of application No. 13/604,173, filed on Tumor Cells.” Mol. Cell. Biol., vol. 10, No. 6, pp. 2443-2447, Sep. 5, 2012, now Pat. No. 8,932,586. American Society for Microbiology, Jun. 1990. Pastan et al., “Recombinant Toxins for Cancer Treatment,” Science (60) Provisional application No. 61/531,576, filed on Sep. Mag., vol. 254, pp. 1173-1177, Nov. 22, 1991. Retrieved on Aug. 6, 2011. 29, 2011 from www.sciencemag.org. Siegallet al., “Analysis of Sequences in Domain II of Pseudomonas Exotoxin A Which Mediate Translocation,” Biochem., vol. 30, No. (51) Int. C. 29, pp. 7154-7159. American Chemical Society, 1991. CI2N 9/10 (2006.01) Theuer et al., “Immunotoxins Made with a Recombinant Form of (52) U.S. C. Pseudomonas Exotoxin A That Do Not Require Proteolysis for CPC. CI2N 9/1077 (2013.01); CI2Y 204/02036 Activity.” Cancer Res., vol. 53, pp. 340-347, American Assoc. for Cancer Research, Jan. 15, 1993. Retrieved on Jan. 9, 2014 from (2013.01) www.cancerres.aacjournals.org. (58) Field of Classification Search None (Continued) See application file for complete search history. Primary Examiner — Amber D Steele (56) References Cited Assistant Examiner — Schuyler Milton (74) Attorney, Agent, or Firm — Intrexon Corporation U.S. PATENT DOCUMENTS (57) ABSTRACT 4,545,985 A 10, 1985 Pastan et al. 4,892,827 A 1/1990 Pastan et al. Pseudomonas exotoxin A or “PE is a 66 kD. highly potent, 5,458,878 A 10, 1995 Pastan et al. cytotoxic protein secreted by the bacterium Pseudomonas 5,512,658 A 4, 1996 Pastan et al. 5,602,095 A 2f1997 Pastan et al. aeruginosa. Various forms of PE have been coupled to other 5,696,237 A 12/1997 FitzGerald et al. proteins, such as , to generate therapeutically 5,705,156 A 1/1998 Pastan et al. useful cytotoxin conjugates that selectively target cells of a 5,705,163 A 1/1998 Pastan et al. desired phenotype (Such as tumor cells). In the present 5,821,238 A 10, 1998 Pastan et al. invention, peptides spanning the sequence of an approxi 5,854,044 A 12/1998 Pastan et al. 5,863,745 A 1/1999 Fitzgerald et al. mately 38 kD form of Pseudomonas exotoxin A protein were 5,980,895 A 11/1999 Pastan et al. analyzed for the presence of immunogenic CD4+ 5,981,726 A 11/1999 Pastan et al. epitopes. Six immunogenic T cell epitopes were identified. 6,051.405 A 4/2000 FitzGerald et al. Residues were identified within each epitope for introduc 6,074,644 A 6, 2000 Pastan et al. 6.426,075 B1 7/2002 Fitzgerald et al. tion of targeted amino acid Substitutions to reduce or prevent 7,314.632 B1 1/2008 Fitzgerald immunogenic T-cell responses in PE molecules which may 7,750,136 B2 7, 2010 Baker et al. be administered to a heterologous host. 8,092,809 B2 1/2012 FitzGerald 2006/0051359 A1 3, 2006 Pastan et al. 23 Claims, 12 Drawing Sheets US 9,677,055 B2 Page 2

(56) References Cited mor Activity in Mice,” J Immunother; vol. 33, No. 3, pp. 297-304. Lippincott Williams & Wilkins, Apr. 2010. Hwang et al., “Functional Domains of Pseudomonas Exotoxin FOREIGN PATENT DOCUMENTS Identified by Deletion Analysis of the Gene Expressed in E. coli,” Cell, vol. 48, pp. 129-136, Cell Press, Jan. 16, 1987. WO WO 2006/065867 A2 6, 2006 Kreitman et al., “Recombinant immunotoxins and other therapies WO WO 2007/O16150 A2 2, 2007 for relapsed/refractory hairy cell leukemia,” Leukemia & Lym WO WO 2009/032954 A1 3, 2009 WO WO 2011/032022 A1 3, 2011 phoma, vol. 52, No. S2, pp. 82-86, Informa UK. Ltd., Jun. 2011. WO WO 2012/154530 A1 11, 2012 Shapira et al., “An Immunoconjugate of Anti-CD24 and WO WO 2012/170617 A1 12/2012 Pseudomonas Exotoxin Selectively Kills Human Colorectal Tumors WO WO 2013/040141 A1 3, 2013 in Mice,” Gastroenterology, vol. 140, No. 3, pp. 935-946, Mar. 2011. Chaudhary et al., “Pseudomonas exotoxin contains a specific OTHER PUBLICATIONS sequence at the carboxyl terminus that is required for cytotoxicity.” Benhar et al., “Pseudomonas Exotoxin A Mutants.” The Journal of Proc. Natl. Acad. Sci. USA, vol. 87, pp. 308-312, Jan. 1990. Biol. Chem., vol. 269, No. 18, pp. 13398-13404, May 6, 1994. Hu et al., “Investigation of a plasmid containing a novel Thomas et al., “Abrogation of Head and Neck Squamous Cell immunotoxin VEGF165-PE38 gene for antiangiogenic therapy in a Carcinoma Growth by Epidermal Growth Factor Receptor Ligand malignant glioma model.” Int. J. Cancer, vol. 127, pp. 2222-2229, Fused to Pseudomonas Exotoxin Transforming Growth Factor 2010. O-PE38.” Clin. Cancer Res., vol. 10, pp. 7079-7087, American Itoi et al., “Targeting of Locus Ceruleus Noradrenergic Neurons Assoc. for Cancer Research, Oct. 15, 2004. Retrieved on Aug. 29. Expressing Human Interleukin-2 Receptor O-Subunit in Transgenic 2012 from www.clincancerres.aacjournals.org. Mice by a Recombinant Immunotoxin anti-Tac(Fv)-PE38: A Study Bang et al., “HA22 (R490A) Is a Recombinant Immunotoxin with for Exploring Noradrenergic Influence upon Anxiety-Like and Increased Antitumor Activity without an Increase in Animal Tox Depression-Like Behaviors.” The Journal of Neuroscience, vol. 31. icity.” Clin. Cancer Res., vol. 11, pp. 1545-1550, American Assoc. No. 16, pp. 6132-6139, Society for Neuroscience, Apr. 20, 2011. for Cancer Research, Feb. 15, 2005. Retrieved on Jan. 9, 2014 from Kuan et al., “Affinity-matured anti-glycoprotein NMB recombinant www.clincancerres.aacjournals.org. immunotoxins targeting malignant gliomas and melanomas,” Ini J Onda et al., “An immunotoxin with greatly reduced immunogenic Cancer; vol. 129, No. 1, pp. 111-121. International Union Against ity by identification and removal of B cell epitopes.” PNAS, vol. Cancer, Jul. 1, 2011. 105, No. 32, pp. 11311-11316. Aug. 12, 2008. Mareeva et al., “A Novel Composite Immunotoxin That Suppresses Weldon et al., “A protease-resistant immunotoxin against CD22 Rabies Production by the Infected Cells,”J Immunol Methods, with greatly increased activity against CLL and diminished animal vol. 353, Nos. 1-2, pp. 1-18, Elsevier B.V., Feb. 28, 2010. toxicity,” Blood, vol. 113, No. 16, pp. 3792-3800, Apr. 16, 2009. Nagata, S. and Pastan, I., “Removal of B cell epitopes as a practical Retrieved on Jan. 9, 2014 from www.bloodjournal. approach for reducing the immunogenicity of foreign protein-based hematologylibrary.org. therapeutics.” Adv. Drug Deliv Rey, vol. 61, No. 11, pp. 977-985, Baker et al., “Pre-Clinical Considerations in the Assessment of Elsevier Science Publishers B.V., Sep. 30, 2009. Immunogenicity for Protein Therapeutics.” Current Drug Safety, Seetharam et al., “Increased Cytotoxic Activity of Pseudomonas vol. 5, No. 4, pp. 1-6, Bentham Science Publishers Ltd., Feb. 19. Exotoxin and Two Chimeric Toxins Ending in KDEL.” The Journal 2010. of Biological Chemistry, vol. 266, No. 26, pp. 17376-17381, Ameri Kreitman et al., “ Fusion Proteins: Anti-CD22 Recombi can Society for Biochemistry and Molecular Biology, Sep. 15, 1991. nant Immunotoxin Moxetumomab Pasudotox,” Clin. Cancer Res., Stish et al., “Design and modification of EGF4KDEL 7Mut, a novel vol. 17, No. 20, pp. 6398-6405, American Assoc. for Cancer bispecific ligand-directed toxin, with decreased immunogenicity Research, Oct. 15, 2011. and potent anti-mesothelioma activity.” British Journal of Cancer, Onda et al., “Recombinant immunotoxin against B-cell malignan vol. 101, Cancer Research UK, Sep. 15, 2009. cies with no immunogenicity in mice by removal of B-cell Theuer et al., “A Recombinant Form of Pseudomonas Exotoxin epitopes.” PNAS, vol. 108, No. 14, pp. 5742-5747, Apr. 5, 2011. Directed at the Epidermal Growth Factor Receptor That Is Pastan et al., “Immunotoxins with decreased immunogenicity and Cytotoxic without Requiring Proteolytic Processing.” The Journal improved activity.” Leukemia & Lymphoma, vol. 52, No. S2, pp. of Biological Chemistry, vol. 267, No. 24, pp. 16872-16877, Ameri 87-90, Informa UK, Ltd., Jun. 2011. Retrieved on Jul. 11, 2011 from can Society for Biochemistry and Molecular Biology, Aug. 25. www.informahealthcare.com. 1992. Weldon et al., “A guide to taming a toxin recombinant Zielinski et al..." Affitoxin-A Novel Recombinant, HER2-Specific, immunotoxins constructed from Pseudomonas exotoxin A for the Anti-Cancer Agent for Targeted Therapy of HER2-Positive treatment of cancer.” The FEBS Journal, vol. 278, pp. 4683-4700, Tumors,” J Immunother; vol. 32, No. 8, pp. 817-825, Lippincott FEBS, 2011. Williams & Wilkins, Oct. 2009. Liu et al., “Recombinant immunotoxin engineered for low Fontana et al., “Secreted Bacterial Effectors That Inhibit Host immunogenicity and antigenicity by identifying and silencing Protein Synthesis Are Critical for Induction of the Innate Immune human B-cell epitopes.” PNAS, vol. 109, No. 29, pp. 11782-11787, Response to Virulent Legionella pneumophila, ' PLOS Pathog, vol. Jul. 17, 2012. 7, No. 2, Public Library of Science, Feb. 17, 2011. Hansen et al., “A Recombinant Immunotoxin Targeting CD22 With International Search Report, mailed Jan. 28, 2013, for International Low Immunogenicity, Low Nonspecific Toxicity, and High Antitu Application No. PCT/US2012/53868, filed Sep. 6, 2012.

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it US 9,677,055 B2 1. 2 MODIFIED FORMS OF PSEUDOMONAS nism by which immunogenicity is triggered remains unclear EXOTOXINA although the tolerance to self proteins may be broken by a number of factors linked to both the product and the patient CROSS-REFERENCE TO RELATED (reviewed in Chester et al., 2006; Baker and Jones, 2007). APPLICATIONS For the product, these include dose, frequency of adminis tration, route, immunomodulatory capacity of the protein This application is a continuation of U.S. application Ser. therapeutic, and the formulation (Jaber and Baker, 2007). No. 14/984,006 filed Dec. 30, 2015 (now U.S. Pat. No. For the patient, factors such as immune competence (i.e. 9.447.387), which is a continuation of U.S. application Ser. whether the patient is receiving immunosuppressive treat No. 14/561,707 filed Dec. 5, 2014 (now U.S. Pat. No. 10 ment), patient's MHC haplotype and intrinsic tolerance to 9,371.517), which is a continuation of U.S. application Ser. the protein therapeutic will influence immunogenicity. No. 13/604,173 filed Sep. 5, 2012 (now U.S. Pat. No. Regardless of how immunogenicity is triggered, one of the 8,932.586), which claims priority benefit of U.S. Applica single most important factors in the development of an tion No. 61/531,576 filed Sep. 6, 2011. ensuing immune response is the presence of epitopes that are 15 able to effectively stimulate a potent CD4+ T cell response REFERENCE TO RELATED APPLICATION (reviewed Baker and Jones, 2007). T cells or T lymphocytes are a subset of white blood cells This application claims benefit of and priority based on known as lymphocytes. (The abbreviation “T” in T cell is for U.S. Provisional Patent Application Ser. No. 61/531,576, “thymus' since this is the primary organ responsible for T filed Sep. 6, 2011, the contents of which are herein incor cell maturation.) T cells play a central role in cell-mediated porated by reference in their entirety. immunity. They can be distinguished from other types of lymphocytes (such as B cells and natural killer cells (NK NAMES OF THE PARTIES IN A JOINT cells)), by the presence of cell-surface proteins called T cell RESEARCH AGREEMENT receptors (TCRs). Different types of T cells have also been 25 identified; these can be distinguished based on the differing The claimed invention was made pursuant to a joint functions they serve (e.g., CD4+ T cells (a.k.a., T or T research agreement, as defined in 35 U.S.C.S 103 (c)(3), that helper cells), CD8+ cytotoxic T cells (CTLs), memory T was in effect on or before the date the claimed invention was cells, regulatory T cells (T cells), natural killer cells (NK made, and as a result of activities undertaken within the cells), and gamma delta T cells (Y& T cells)). scope of the joint research agreement, by or on behalf of the 30 T helper (T) cells are so named because they aid other Intrexon Corp. (Foster City, Calif., U.S.A.) and Antitope white blood cells in immunologic processes including, inter Ltd. (Cambridge, UK). alia, assisting the maturation of B cells into plasma and B memory cells, and activation of cytotoxic T cells and mac REFERENCE TO SEQUENCE LISTING rophages. T cells are also known as CD4+ T cells because SUBMITTED ELECTRONICALLY 35 they express CD4 protein on the cell-surface. CD4+ T cells are activated when peptide are presented by MHC A Sequence Listing is submitted electronically via EFS class II molecules expressed on the Surface of Web as an ASCII formatted sequence listing in a file named Presenting Cells (APCs). Once activated, CD4+ T cells “OT050-PCT SEQLIST.txt”, created on Sep. 4, 2012, and divide rapidly and secrete chemokines that further assist in having a file size of 295,678 bytes which is filed concur 40 activating or regulating immune responses. rently with the present specification, claims, abstract and T cell epitope analysis is becoming increasingly important figures provided herewith. The sequence listing contained in particularly in the pre-clinical analysis of biologies and may, this ASCII formatted document is part of the specification in time, become a requirement for regulatory approval for and is herein incorporated by reference in its entirety. clinical trials. To this end, a pre-clinical ex vivo T cell assay 45 (EPISCREENTM) has been used to provide an effective BACKGROUND OF THE INVENTION technology for predicting T cell immunogenicity by identi fying linear T cell epitopes present in protein sequences. and T Cell Epitopes Synthetic overlapping peptides typically of about 15 amino acids in length are tested against a cohort of community Immune responses to biological therapeutic agents are 50 blood donors carefully selected based on MHC class II wide ranging, and can be directed against agents that are haplotypes to provide a quantitative analysis of T cell both non-human and human in origin. These responses epitopes present in protein sequences. This technology has include those that elicit a weak clinical effect and those that been used successfully to compare protein variants for the limit efficacy which can occasionally result in morbidity or potential to induce an immune response in vivo. By provid even mortality in patients. In particular, serious complica 55 ing a high degree of sensitivity along with high reproduc tions can arise with the production of neutralizing antibod ibility, the EPISCREENTM assay allows an accurate pre ies, especially when they target recombinant self proteins clinical assessment of the potential for immunogenicity of and therefore have the potential to cross react with the biologies. See, Baker & Carr, “Preclinical Considerations in patients own endogenous protein (Lim, 2005). Problems the Assessment of Immunogenicity for Protein Therapeu associated with immunogenicity to biologies (i.e., therapeu 60 tics. Current Drug Safety 5(4): 1-6 (2010), Bryson et al., tic medical products; Such as, antibodies and recombinant “Prediction of Immunogenicity of Therapeutic Proteins: proteins/polypeptides) have been reduced largely due to Validity of Computational Tools. Biodrugs 24(1)1-8 advances in molecular biology. There are, however, many (2010), Holgate & Baker, “Circumventing Immunogenicity recombinant protein biologies that are identical to endog in the Development of Therapeutic Antibodies.” IDrugs enously expressed human sequences that still elicit potent 65 12(4):233-237 (2009); Perry et al., “New Approaches to neutralizing immune responses in patients (Hochuli, 1997: Prediction of Immune Responses to Therapeutic Proteins Schellekens et al., 1997: Namaka et al., 2006). The mecha during Preclinical Development,” Drugs R D 9(6):385-396 US 9,677,055 B2 3 4 (2008); and, Baker & Jones, “Identification and removal of Kuan et al., “Affinity-matured anti-glycoprotein NMB immunogenicity in therapeutic proteins. Current Opinion recombinant immunotoxins targeting malignant in Drug Discovery & Development 10(2):219-227 (2007). gliomas and melanomas.” Int. J. Cancer, 129(1): 111-21 Pseudomonas Exotoxin A (July 2011): Pseudomonas exotoxin A (PE-A) is a highly potent, 66 Hu et al., “Investigation of a plasmid containing a novel kD, cytotoxic protein secreted by the bacterium Pseudomo immunotoxin VEGF165-PE38 gene for anti angiogenic nas aeruginosa. PE-A causes cell death by inhibiting protein therapy in a malignant glioma model.” Int. J. Cancer, synthesis in eukaryotic cells via inactivation of translation 127(9):2222-2229 (November 2010): elongation factor 2 (EF-2), which is mediated by PE-A Mareeva et al., “A novel composite immunotoxin that catalyzing ADP-ribosylation of EF-2 (i.e., transfer of an 10 ADP ribosyl moiety onto EF-2). PE-A typically produces Suppresses rabies virus production by the infected death by causing liver failure. cells,” J. Immunol. Methods, 353(1-2): 78-86 (February PE-A has at least three different structural domains 2010); responsible for various biological activities (FIG. 1). See Zielinski et al., “Aflitoxin a novel recombinant, HER2 e.g., Siegall et al., Biochemistry, vol. 30, pp. 7154-7159 15 specific, anticancer agent for targeted therapy of (1991); Theuer et al., Jour: Biol. Chem. Vol. 267, no. 24, pp. HER2-positive tumors,” J. Immunother. 32(8):817-825 16872-16877 (1992); and, U.S. Pat. No. 5,821,238. PE-A (October 2009); domain IA (amino acids 1-252 (see e.g., SEQ ID NO:133)) Theuer et al., J. Biol. Chem., 267(24): 16872-16877 is responsible for cell binding. Domain II (amino acids (1992); 253-364 (see e.g., SEQ ID NO:133)) is responsible for Pastan et al., “Recombinant toxins for cancer treatment,” translocation of PE-A into the cell cytosol. Domain III, the Science, 254:1173-1177(1991); cytotoxic domain (amino acids 400-613 (see e.g., SEQ ID U.S. Pat. No. 5,821.238 (“Recombinant Pseudomonas NO:133)), is responsible for ADP ribosylation of Elongation Exotoxins with Increased Activity”); and Factor 2 (EF2); which thereby inactivates EF2, subsequently U.S. Pat. No. 4,892,827 (“Recombinant Pseudomonas causing cell death. Additionally, a function for domain IB 25 Exotoxins: Construction of an Active Immunotoxin (amino acids 365-399 (SEQ ID NO:139)) has not been with Low Side Effects”). established. Indeed, it has been reported that amino acids A significant disadvantage in using PE-A for treatment of 365-380 (SEQID NO: 138) within domain IB can be deleted disease, however, is that it is a foreign (non-self) protein without producing an identifiable a loss of function. See, being introduced into a heterologous host (e.g., a human). Siegall et al., Biochemistry, vol. 30, pp. 7154-7159 (1991). 30 It has also been reported that PE-A may comprise any one Introduction of non-self proteins into heterologous hosts of at least three different carboxy-terminal tails (FIG. 1); commonly elicits host immune reactions, such as the gen these appear to be essential for maintaining or recycling eration of antibodies (“neutralizing antibodies’) or immune proteins into the endoplasmic reticulum. See. Theuer et al., cell reactions (e.g., cytotoxic T cell responses) which are J. Biol. Chem., vol. 267, no. 24, pp. 16872-16877 (1992); 35 directed at eliminating the non-self protein (i.e., PE-A). Chaudhary et al., Proc. Natl. Acad. Sci. USA, Vol. 87, pp. Accordingly, it would be advantageous if elements of PE-A 308-312 (1990); and, Seetharam et al., Jour: Biol. Chem. (PE-A epitopes) which are recognized and targeted as “non vol. 266, 17376-17381 (1991). In particular, in correspon self could be removed prior to use of this molecule as a dence with the exemplary sequence shown in FIG. 1 (SEQ therapeutic agent. ID NO:133) these alternative carboxy-terminal tails com 40 Deimmunization of PE prise amino acid sequences: Some investigators have previously attempted to identify 609-REDLK-613 (SEQ ID NO:135): and remove immunogenic determinants from PE-A (i.e., to 609-REDL-612 (SEQ ID NO:136); and “deimmunize” PE-A). See, for example: 609-KDEL-612 (SEQ ID NO:137). Pastan et al., “Immunotoxins with decreased immunoge Variants of PE-A, modified to lack the cell binding 45 nicity and improved activity, Leukemia and Lym domain but coupled to heterologous cell-specific targeting phoma, 52(S2):87-90 (June 2011): molecules (e.g., antibodies), have been shown to have Onda et al., “Recombinant immunotoxin against B-cell reduced levels of non-specific toxicity. See e.g., U.S. Pat. malignancies with no immunogenicity in mice by No. 4,892,827. removal of B-cell epitopes.” Proc. Natl. Acad. Sci. Various forms of PE-A (e.g., truncated/deletion forms 50 USA, 108(14):5742-5747 (April 2011): with molecular weights of -37 kD, 38 kD, 40 kD, etcetera) Hansen et al., “A recombinant immunotoxin targeting have been combined with a number of growth factors, CD22 with low immunogenicity, low nonspecific tox antibodies, and other proteins to generate cytotoxins which icity, and high antitumor activity in mice.” J. Immu selectively target cells of a desired phenotype. See, for nother: 33(3):297-304 (April 2011): example: 55 Stish et al., “Design and modification of EGF4KDEL Kreitman et al., “Recombinant immunotoxins and other 7Mut, a novel bispecific ligand-directed toxin, with therapies for relapsed/refractory hairy cell leukemia,” decreased immunogenicity and potent anti-mesothe Leuk. Lymphoma. Suppl. 2:82-86 (June 2011): lioma activity.” Br. J. Cancer, 101(7): 1114-1123 (Octo Itoi et al., “Targeting of locus ceruleus noradrenergic ber 2009); neurons expressing human interleukin-2 receptor 60 Nagata et al., “Removal of B cell epitopes as a practical C-Subunit in transgenic mice by a recombinant immu approach for reducing the immunogenicity of foreign notoxin anti-Tac(Fv)-PE38.” J. Neurosci., 31(16): protein-based therapeutics.” Adv. Drug Deliv Rev., 6132-6139 (April 2011): 61 (11):977-985 (September 2009); Shapira et al., “An immunoconjugate of anti-CD24 and Onda et al., “An immunotoxin with greatly reduced Pseudomonas exotoxin selectively kills human col 65 immunogenicity by identification and removal of B cell orectal tumors in mice.” Gastroenterology, 140(3):935 epitopes.” Proc. Natl. Acad. Sci. USA, 105(32): 11311 946 (March 2011): 11316 (August 2008); and US 9,677,055 B2 5 6 Pastan et al., “Mutated Pseudomonas Exotoxins with FIG. 6. Alignment of peptides 81 and 82 showing the Reduced Antigenicity, U.S. Patent Application No. potential HLA-DR core 9 mer binding register. Predicted 2009/O142341. core 9 mer sequences are bracketed by p1 and p9 anchor Despite progress in the area of deimmunization of PE-A, residues. Peptides that stimulated positive T cell responses there remains a need for the development of optimized, less 5 in the adjusted data set are shown. Amino acid numbering immunogenic or non-immunogenic, biologically active (residues 244 and 252) correspond to SEQ ID NO:2 (PE38 forms of this useful cytotoxin. The invention described of SEQ ID NO:1 plus amino-terminal linker herein addresses this need. GGGGGSGGGGGS (SEQ ID NO:3)). FIG. 7. Alignment of peptides 110 and 111 showing a BRIEF SUMMARY OF THE INVENTION 10 predicted HLA-DR core 9 mer binding register. Predicted core 9 mer sequences are bracketed by p1 and p9 anchor Peptides spanning the sequence of an approximately 38 residues. Peptides that stimulated positive T cell responses kD (predicted molecular weight) form of Pseudomonas in the adjusted data set are shown. Amino acid numbering exotoxin A protein (SEQ ID NO: 1) were analyzed for the (residues 333 and 341) correspond to SEQ ID NO:2 (PE38 presence of immunogenic CD4+ T cell epitopes. A total of 15 of SEQ ID NO:1 plus amino-terminal linker 120 overlapping 15 mer peptides spanning this sequence GGGGGSGGGGGS (SEQ ID NO:3)). (SEQ ID NO:1), but also including an amino terminal FIG. 8. Position of CD4+ T cell epitopes within the PE38 (Glys-Ser), linker sequence (SEQ ID NO:3) to produce a sequence. T cell epitopes identified by EPISCREENTMT cell 359 amino acid Gly-Ser-PE38 polypeptide sequence (SEQ epitope mapping are shown as shaded bars above the ID NO:2), were tested against a cohort of healthy human sequence. The frequency of donors responding (SIa2.00, donors. CD4+ T cell responses against individual peptides p-0.05) to each epitope are indicated by the shading of the were measured via proliferation assays. Assay data was used bars; lightgrey <10%, mid grey 10-14%; dark grey >=14%. to compile a T cell epitope map of the PE38 sequence. Six Numbers assigned to each individual donor (that responded immunogenic T cell epitopes were identified. Residues were to a corresponding epitope) are included within each shaded then identified within each of these epitopes for use in 25 bar. targeted amino acid substitutions to reduce or prevent PE38 FIG. 9. In vivo Transcription/Translation (IVTT) shows induced immunogenicity. Reduction or prevention of PE that circular plasmid expression vector encoding PE38-IL2 immunogenicity should allow for multiple therapeutic was slightly better at inhibiting Luciferase administrations of cytotoxic PE for use, for example, in the protein synthesis compared to linearized plasmid encoding targeted destruction of cancer cells in vivo (Such as when 30 the same PE38-IL2 fusion protein. administered as an immunoconjugate or cell-Surface tar FIG. 10. Luciferase activity measure in counts per second geted fusion protein). (CPS) in In vitro Transcription/Translation (IVTT) assays of genes encoding either Wild-Type (WT) PE or encoding BRIEF DESCRIPTION OF THE DRAWINGS amino acid substituted PE. 35 FIG. 11. Analysis of production of IL-2 and FIG. 1. Example of a Pseudomonas exotoxin A protein IL-6 stimulated in response to expression of genes encoding and domains which may be contained therein. either Wild-Type (WT) PE or encoding amino acid substi FIG. 2. Comparison of the frequency of donor allotypes tuted PE. expressed in the IEX01 study cohort (n=52) and the world population. 40 DETAILED DESCRIPTION OF THE FIGS. 3A & 3B. CD4+ T cell epitope map of IEX01 PE38 INVENTION sequence using overlapping 15 mer peptides tested against 52 healthy donors. The non-adjusted (FIG. 3A) and adjusted I. Definitions and Descriptions (FIG. 3B) proliferation assay data for the PE38 sequence is shown. Peptides inducing positive (SI-2.00, p<0.05, includ 45 Unless specifically indicated otherwise, as used herein the ing borderline responses) T cell proliferation responses at a term “PE” or “PE-A' is intended to indicate a polypeptide frequency above the background response rate (mean posi comprising a cytotoxic polypeptide sequence derived from a tive T cell responses plus SD) contain T cell epitopes (dotted wild-type or naturally occurring form of Pseudomonas line indicates the background response threshold). KLH aeruginosa exotoxin A protein. In addition to cytotoxic induced positive responses in (SI-2.00, p<0.05) 75% of 50 polypeptide sequences, PE polypeptides may comprise addi (non-adjusted) donors. tional naturally occurring or heterologous polypeptide FIG. 4. Alignment of peptides 50, 52 and 53 showing the sequences. Additional naturally occurring polypeptide predicted HLA-DR core 9 mer binding register. Predicted sequences may include sequences such as are found in core 9 mer sequences are bracketed by p1 and p9 anchor full-length Pseudomonas exotoxin A protein, for example, residues. Peptides that stimulated positive T cell responses 55 amino acid sequences responsible for cytosolic translocation in the adjusted data set are shown. Amino acid numbering and cell-specific targeting (as discussed further herein)). (residues 153 and 161) correspond to SEQ ID NO:2 (PE38 Additional heterologous polypeptide sequences may include of SEQ ID NO: 1 plus amino-terminal linker sequences with which at least a PE cytotoxic polypeptide is GGGGGSGGGGGS (SEQ ID NO:3)). fused to impart additional functions or properties. (For FIG. 5. Alignment of peptides 65, 67 and 68 showing one 60 example, a PE cytotoxic polypeptide may be fused to predicted HLA-DR core 9 mer binding register. Predicted antigen binding polypeptide sequences such as an Schv core 9 mer sequences are bracketed by p1 and p9 anchor antibody.) Examples of sequences comprising a cytotoxic residues. Peptides that stimulated positive T cell responses portion of PE can be found in SEQ ID NO:1 and SEQ ID in the adjusted data set are shown. Amino acid numbering NO:4 spanning amino acid residues Phe-134 to Lys-347. (residues 196 and 204) correspond to SEQ ID NO:2 (PE38 65 Examples of sequences comprising a cytotoxic portion of PE of SEQ ID NO: 1 plus amino-terminal linker can also be found in SEQID NO:133 and SEQID NO:134 GGGGGSGGGGGS (SEQ ID NO:3)). spanning amino acid residues Phe-400 to Lys-613. US 9,677,055 B2 7 8 As used herein in reference to PE, unless indicated 45% or more, 45% or more, about 50% or more, 50% or otherwise, a “cytotoxic polypeptide' or “cytotoxic polypep more, about 60% or more, 60% or more, about 70% or more, tide sequence' is intended to indicate a polypeptide (or 70% or more, about 75% or more, 75% or more, about 80% portion thereof) which is capable of inactivating translation or more, 80% or more, about 85% or more, 85% or more, elongation factor 2 (EF-2), mediating ADP-ribosylation of 5 about 90% or more, 90% or more, about 95% or more, 95% EF-2, inhibiting protein synthesis, or inducing cell death. or more, about 100%, or 100% of at least one biological For example, it has been demonstrated that PE domain III, activity compared to a corresponding wild-type, naturally comprised of amino acid residues 400-613 of SEQ ID occurring, or non-amino acid Substituted forms of PE or NO:133, is sufficient to mediate ADP-ribosylation of EF-2 PE-A. and thereby cause cell death. See, Theuer et al., J. Biol. 10 As used herein, the term “wild-type' Pseudomonas exo Chem., vol. 267, no. 24, pp. 16872-16877 (1992) and Hwang toxin A (PE-A) (or “wild-type' PE) biological activity is et al., Cell, vol. 48, pp. 129-136 (1987). intended to indicate at least one or more biological activities Cytotoxic polypeptide sequences in the present invention exhibited by naturally occurring forms of the Pseudomonas may also comprise alternative carboxy-terminal sequences. exotoxin A (PE-A) or PE polypeptides. These include, for See, Theuer et al., Chaudhary et al. and, Seetharam et al. In 15 example, without limitation, activities such as cell killing or particular embodiments, examples of carboxy-terminal tails cell cytotoxic activity (a.k.a., cell cytotoxicity), inactivation of PE38 in the present invention may comprise sequences as of translation elongation factor EF-2, ADP-ribosylation of shown in FIG. 1 (SEQ ID NO:133). Hence, exemplary EF-2, and inhibition of protein synthesis. Two examples, alternative carboxy-terminal tails may comprise amino acid without limitation, of polypeptide sequences representing Sequences: “wild-type' or non-amino acid substituted forms of PE-A 609-REDLK-613 (SEQID NO: 135; numbers 609-613 cor are shown in SEQ ID NO:133 and SEQ ID NO:134. Two respond to SEQ ID NO:133) examples, without limitation, of polypeptide sequences rep 609-REDL-612 (SEQ ID NO:136; numbers 609-612 corre resenting “wild-type' or non-amino acid substituted forms spond to SEQ ID NO:133); and of PE are shown in SEQID NO: 1 (PE38) and SEQID NO:4 609-KDEL-612 (SEQ ID NO:137; numbers 609-612 corre 25 (variant of PE38). spond to SEQ ID NO:133). As used or claimed herein the term “a” or “an in Unless specifically indicated otherwise, as used herein the reference to the subsequent recited entity refers to one or term “PE38 is intended to indicate a Pseudomonas aerugi more of that entity, for example, “a PE38 antibody' or “a nosa exotoxin A (PE (or PE-A)) molecule comprising an polynucleotide encoding PE38 is understood to indicate amino acid sequence as shown in SEQID NO:1. The amino 30 one or more PE38 antibody molecules and one or more acid sequence used to generate peptide sequences referenced polynucleotides encoding PE38, not a single PE38 antibody in the Examples is shown in SEQID NO:2. SEQID NO:2 molecule nor a single polynucleotide molecule encoding comprises an amino terminal GGGGGSGGGGGS linker PE38, respectively. As such, the terms “a” (or “an'), “one or sequence (SEQ ID NO:3) fused to the PE38 amino acid more,” and “at least one' can be used interchangeably sequence of SEQ) ID NO:1. A variant form of PE38 is 35 herein. shown in SEQID NO:4. SEQID NO:4 differs from SEQID Likewise, as used herein, the term “polypeptide' is NO:1 by comprising a Ser-to-ASn change at position 114, a intended to encompass a singular “polypeptide' as well as Ile-to-Val change at position 141, and a Gly-to-Ser change plural “polypeptides,” and refers to a molecule composed of at position 249. monomers (amino acids) linked by amide bonds (also As used herein, unless specifically stated otherwise, “bio 40 known as peptide bonds). The term “polypeptide' refers to logical activity in reference to Pseudomonas exotoxin A any chain or chains of two or more amino acids, and does not (PE-A), PE or PE38 is intended to indicate at least one of the refer to a specific length of the product. Thus, peptides, biological activities exhibited by naturally occurring forms dipeptides, tripeptides, oligopeptides, “protein,” “amino of the Pseudomonas aeruginosa exotoxin A molecule. These acid chain,” or any other term used to refer to a chain or activities include, for example, cell killing or cell cytotoxic 45 chains of two or more amino acids, are included within the activity (a.k.a., cell cytotoxicity), inactivation of translation definition of “polypeptide,” and the term “polypeptide' may elongation factor EF-2, ADP-ribosylation of EF-2, and inhi be used instead of, or interchangeably with any of these bition of protein synthesis. The biological activity of PE and terms. The term “polypeptide' is also intended to refer to the PE38 polypeptides (and modified forms thereof; e.g., PE and products of post-expression modifications of the polypep PE38 amino acid substituted variants and fusion proteins) 50 tide. Such as, but without limitation glycosylation, acety can be measured using assays and experiments which are lation, phosphorylation, amidation, et cetera. A "polypep well-known and routinely used by those skilled in the art. tide' unless specifically described otherwise herein, may be Examples of some of these assays and experiments are derived from a natural biological source or produced by further described and referenced herein, without limitation, recombinant technology, but is not necessarily translated in the Examples sections included herein. 55 from a designated nucleic acid sequence. It may be gener As used herein, the term “having Pseudomonas exotoxin ated in any manner, including by chemical synthesis. A (PE-A) biological activity” (or “PE biological activity') is Polypeptides may have a defined three-dimensional struc intended to indicate molecules exhibiting about 5% or more ture, although they do not necessarily have such structure. of at least one biological activity compared to a correspond Polypeptides with a defined three-dimensional structure may ing wild-type, naturally occurring, or non-amino acid Sub 60 be referred to as “folded” or having a “tertiary’ structure. stituted form of PE or PE-A. In some embodiments, mol Polypeptides not configured into a three-dimensional struc ecules “having Pseudomonas exotoxin Abiological activity” ture, are referred to as unfolded. As used herein, the term (or “PE biological activity”) exhibit 5% or more, about 10% glycoprotein refers to a protein coupled to at least one or more, 10% or more, about 15% or more, 15% or more, carbohydrate moiety attached to the protein via a covalent about 20% or more, 20% or more, about 25% or more, 25% 65 bond. or more, about 30% or more, 30% or more, about 35% or The term "isolated” is intended to indicate a biological more, 35% or more, about 40% or more, 40% or more, about component no longer in its naturally occurring milieu. For US 9,677,055 B2 10 example, an "isolated polypeptide' or "isolated polynucle tially) polynucleotides in solution. Isolated RNA molecules otide' is intended to indicate a polypeptide or polynucle include in vivo or in vitro synthesized RNA molecules: otide, respectively, which has been removed from its natu including synthetically produced molecules. rally occurring milieu and which may have been inserted As used herein, a "coding region' is a portion of nucleic within a non-naturally occurring milieu. By way of example, acid containing codons which may be translated into amino this would include, without limitation, a polynucleotide acids, although “stop codons’ (TAG, TGA, or TAA) are not which has been removed from a naturally occurring location translated into an amino acids, but may also be considered within a host genome, and Subsequently inserted, for to be part of a coding region. Unless Stated otherwise herein, example, into an expression vector or inserted into a new promoters, ribosome binding sites, transcriptional termina host genome location or into the genome of a heterologous 10 tors, introns, and the like, are not considered part of a coding host organism. The "isolation of a polypeptide or poly region. Two or more coding regions of the present invention nucleotide, as used herein, requires no particular level of can be present in a single polynucleotide construct, e.g., on purification. For example, recombinantly produced polypep a single vector, or in separate polynucleotide constructs, e.g., tides expressed in host cells are considered isolated for on separate (different) vectors. Furthermore, any vector may purposes of the invention, as are native or recombinant 15 contain a single coding region, or may comprise two or more polypeptides which have been separated, fractionated, or coding regions, e.g., a single vector may separately encode partially or Substantially purified by any Suitable technique. an immunoglobulin heavy-chain variable region and an Polypeptide embodiments also include fragments, deriva immunoglobulin chain variable region. In addition, a tives, analogs, variants and fusion proteins; preferably but vector, polynucleotide, or nucleic acid embodiments may not necessarily wherein such embodiments retain one or encode heterologous coding regions, either fused or unfused more biological activities associated with a corresponding to a nucleic acid encoding a different heterologous polypep full-length or naturally occurring polypeptide. Fragments tide. Heterologous coding regions include without limitation include proteolytic fragments, deletion fragments, and frag specialized elements or motifs, such as a secretory signal ments encoded by synthetically or recombinantly produced peptide or a heterologous functional domains. polynucleotides. Variants may occur naturally or be non 25 In certain embodiments, the polynucleotide or nucleic naturally occurring. Non-naturally occurring variants may acid is DNA. In the case of DNA, a polynucleotide com be produced using art-known mutagenesis techniques. Vari prising a nucleic acid which encodes a polypeptide normally ant polypeptides may comprise conservative or non-conser may include a promoter and/or other transcription or trans Vative amino acid Substitutions, deletions, or additions. lation control elements operably associated with one or more Derivatives include, but are not limited to, polypeptides 30 coding regions. An operable association is when a coding which contain one or more non-naturally occurring amino region for a gene product, e.g., a polypeptide, is associated acids, non-standard amino acids, and amino acid analogs. with one or more regulatory sequences in such a way as to Polypeptide embodiments may comprise amino acid place expression of the gene product under the influence or sequences which are at least 60% identical, at least 70% control of the regulatory sequence(s). Two DNA fragments identical, at least 80% identical, at least 85% identical, at 35 (such as a polypeptide coding region and a promoter asso least 90% identical, at least 95% identical, at least 97% ciated therewith) are “operably associated if induction of identical, at least 98% identical, or at least 99% identical to promoter function results in the transcription of mRNA SEQ ID NO:1. encoding the desired gene product. Thus, a promoter region Unless specifically defined otherwise, the term “poly would be operably associated with a nucleic acid encoding nucleotide' is intended to indicate nucleic acid molecules or 40 a polypeptide if the promoter was capable of effecting constructs as routinely used and understood by those of skill transcription of that nucleic acid. Other transcription control in the art. For example, nucleic acids include, but are not elements, besides a promoter, include for example, but limited to, molecules such as messenger RNA (mRNA), without limitation, enhancers, operators, repressors, and plasmid DNA (pDNA), complementary DNA (cDNA), and transcription termination signals, can be operably associated genomic DNA (gDNA). A polynucleotide may comprise a 45 with the polynucleotide to direct cell-specific transcription. conventional phosphodiester bond or a non-conventional Suitable promoters and other transcription control regions bond (e.g., an amide bond, Such as found in peptide nucleic are disclosed herein. acids (PNA)). The terms “polynucleotide' and “nucleic The terms “antibody' and “immunoglobulin' may be acid are intended to include embodiments wherein any one used interchangeably herein. An antibody or immunoglobu or more sequences of polynucleotide or nucleic acid seg 50 lin comprises at least the antigen-binding elements (e.g., ments are contained, or comprised within, a larger poly complementarity determining regions or CDRs) of the vari nucleotide or nucleic acid sequence. For example, but with able domain of a heavy chain and/or of the variable domain out limitation, and unless stated otherwise to the contrary of a light chain. Basic immunoglobulin structures in verte herein, reference to a nucleic acid Such as “a polynucleotide brate systems are well understood by those of skill in the art. encoding PE38 is intended to include nucleic acids com 55 See, e.g., Harlow & Lane. Using Antibodies: A Laboratory prising “a polynucleotide encoding PE38 wherein such Manual, (Cold Spring Harbor Laboratory Press, 1999 (ISBN polynucleotide may also be part of a larger nucleic acid or 0879695447)); see also, Harlow & Lane, Antibodies: A polynucleotide, Such as an expression vector or a polynucle Laboratory Manual, (Cold Spring Harbor Laboratory Press, otide/nucleic acid encoding an PE fusion protein. 2nd ed. 1988). The term “immunoglobulin' or “antibody' An "isolated nucleic acid or polynucleotide is intended a 60 comprises various broad classes of antibody molecules, such nucleic acid molecule, DNA or RNA, which has been as, but without limitation, IgG, IgM, IgA IgG, and IgE removed from its native environment. For example, a classes of antibodies; as well as antibody Subclasses (iso recombinant polynucleotide encoding an antibody contained types). Such as, IgG1, IgG2, IgG3, IgG4, IgA1, etcetera. in a vector is considered isolated for the purposes of the Antibodies or antigen-binding fragments, variants, or present invention. Further examples of an isolated poly 65 derivatives thereof of the invention include, but are not nucleotide include recombinant polynucleotides maintained limited to, polyclonal, monoclonal, multispecific, human, in heterologous host cells or purified (partially or Substan humanized, primatized, or chimeric antibodies, single chain US 9,677,055 B2 11 12 antibodies, epitope-binding fragments, e.g., Fab., Fab' and it binds to an epitope other than the one that induced its F(ab'), Fd, FVs, single-chain Fvs (scEv), single-chain anti formation. The cross reactive epitope generally contains bodies, disulfide-linked FVS (SdPV), fragments comprising many of the same complementary structural features as the either a VL or VH domain, fragments produced by a Fab inducing epitope, and in some cases, may actually fit better expression library, and anti-idiotypic (anti-Id) antibodies. than the original. As used herein, an “epitope' or “antigenic determinant' is As used herein, the terms “linked,” “fused’ or “fusion' the part of a polypeptide, antigen, or molecule that is may be used interchangeably. These terms refer to the recognized by the immune system, specifically by antibod joining together of two more elements or components, by ies, B cells, or T cells. Epitopes of polypeptide antigens may whatever means including chemical conjugation or recom function as conformational epitopes or linear epitopes. A 10 binant means. An "in-frame fusion” refers to the joining of conformational epitope is comprised of non-linear sections two or more polynucleotide open reading frames (ORFs) to of a target molecule (such as that formed via the tertiary form a continuous longer ORF, in a manner that maintains structure of a folded polypeptide). In contrast, amino acids the correct translational reading frame of the original ORFs. that make up a linear epitope may be comprised of a Thus, a recombinant fusion protein is a single protein continuous sequence of amino acids or may be comprised 15 containing two ore more segments that correspond to poly only of particular amino acid residues critical to antibody/B peptides encoded by the original ORFs (which segments are cell/T cell binding. not normally so joined in nature.) Although the reading By “specifically binds, it is generally meant that an frame is thus made continuous throughout the fused seg antibody binds to an epitope via its antigen binding domain, ments, the segments may be physically or spatially separated and that the binding entails some complementarity between by, for example, in-frame linker sequence. For example, the antigen binding domain and the epitope. According to polynucleotides encoding the CDRS of an immunoglobulin this definition, an antibody is said to “specifically bind' to variable region may be fused, in-frame, but be separated by an epitope when it binds to that epitope, via its antigen a polynucleotide encoding at least one immunoglobulin binding domain more readily than it would bind to a random, framework region or additional CDR regions, as long as the unrelated epitope. The term “specificity' may be used herein 25 “fused CDRs are co-translated as part of a continuous to qualify the relative affinity by which a certain antibody polypeptide. binds to a certain epitope. For example, antibody “A” may In the context of polypeptides, a "linear sequence' or a be deemed to have a higher specificity for a given epitope "sequence' is an order of amino acids in a polypeptide in an than antibody “B,” or antibody “A” may be said to bind to amino to carboxyl terminal direction in which residues that epitope “C” with a higher specificity than it has for related 30 neighbor each other in the sequence are contiguous in the epitope “D.” primary structure of the polypeptide. By "preferentially binds,” it is meant that the antibody A “variant of a polypeptide or protein refers to any specifically binds to an epitope more readily than it would analogue, fragment, derivative, or mutant which is derived bind to a related, similar, homologous, or analogous epitope. from a polypeptide or protein and which retains at least one Thus, an antibody which “preferentially binds to a given 35 biological property of the polypeptide or protein. Different epitope would more likely bind to that epitope than to a variants of the polypeptide or protein may exist in nature or related epitope, even though Such an antibody may cross may be generated artificially (e.g., via synthetic or genetic react with the related epitope. engineering). Variants may be allelic variations character An antibody is said to competitively inhibit binding of a ized by differences in the nucleotide sequences of the reference antibody to a given epitope if it preferentially 40 structural gene coding for the protein, or may involve binds to that epitope to the extent that it blocks, to some differential splicing or post-translational modification. The degree, binding of the reference antibody to the epitope. skilled artisan can produce variants having single or multiple Competitive inhibition may be determined by any method amino acid Substitutions, deletions, additions, or replace known in the art, for example, competition ELISA assays. ments. Variants may include, interalia: (a) Variants in which An antibody may be said to competitively inhibit binding of 45 one or more amino acid residues are substituted with, for the reference antibody to a given epitope by at least 90%, at example, conservative amino acids, non-conservative amino least 80%, at least 70%, at least 60%, or at least 50%. acids, or amino acid analogs (b) variants in which one or As used herein, the term “affinity” refers to a measure of more amino acids are added to the polypeptide or protein, (c) the strength of the binding of an individual epitope with the variants in which one or more of the amino acids includes a CDR of an immunoglobulin molecule. See, e.g., Harlow et 50 Substituent group, and (d) variants in which the polypeptide al., Antibodies: A Laboratory Manual, (Cold Spring Harbor or protein is fused with another polypeptide Such as serum Laboratory Press, 2nd ed., 1988) at pages 27-28. As used albumin. The techniques for obtaining these variants, includ herein, the term “avidity” refers to the overall stability of the ing genetic (Suppressions, deletions, mutations, etc.), chemi complex between a population of immunoglobulins and an cal, and enzymatic techniques, are known to those of skill in antigen, that is, the functional combining strength of an 55 the art. immunoglobulin mixture with the antigen. See, e.g., Harlow The term “expression” as used herein refers to a process at pages 29-34. Avidity is related to both the affinity of by which a gene produces a biochemical, for example, an individual immunoglobulin molecules in the population RNA or polypeptide. It includes without limitation transcrip with specific epitopes, and also the Valencies of the immu tion of the gene into RNA molecules such as, for example, noglobulins and the antigen. For example, the interaction 60 messenger RNA (mRNA), transfer RNA (tRNA) or any between a bivalent and an antigen with other RNA product, and the translation of mRNA into a highly repeating epitope structure, such as a polymer, polypeptide(s). would be one of high avidity. Expression of a gene produces a “gene product.” As used The term “cross-reactivity” refers to the ability of an herein, a gene product can be either a nucleic acid, e.g., a antibody, specific for one antigen, to react with a second 65 messenger RNA produced by transcription of a gene, or a antigen; a measure of relatedness between two different polypeptide which is translated from a transcript. Gene antigenic Substances. Thus, an antibody is cross reactive if products described herein further include nucleic acids with US 9,677,055 B2 13 14 post transcriptional modifications, e.g., polyadenylation, or plasmid derivatives, or the Bluescript vector. Another polypeptides with post translational modifications, e.g., example of vectors that are useful in the present invention is methylation, glycosylation, the addition of lipids, associa the Ultra VectorTM Production System (Intrexon Corp., tion with other protein subunits, proteolytic cleavage, et Blacksburg, Va.) as described in WO 2007/038276, incor Cetera. porated by reference herein. For example, the insertion of As used herein, the term “gene' refers to a polynucleotide the DNA fragments corresponding to response elements and comprising nucleotides that encode a functional molecule, promoters into a suitable vector for in vitro and/or in vivo including functional molecules produced by transcription expression of modified forms of PE (and fragments thereof) only (e.g., a bioactive RNA species) or by transcription and as described herein (including fusion proteins, conjugates, translation (e.g. a polypeptide). The term 'gene' encom 10 and otherwise linked forms) can be accomplished by ligating passes cDNA and genomic DNA nucleic acids. “Gene' also the appropriate DNA fragments into a chosen vector that has refers to a nucleic acid fragment that expresses a specific complementary cohesive termini. Alternatively, the ends of RNA, protein or polypeptide, including regulatory the DNA molecules may be enzymatically modified or any sequences preceding (5' non-coding sequences) and follow site may be produced by ligating nucleotide sequences ing (3' non-coding sequences) the coding sequence. “Native 15 (linkers) into the DNA termini. Such vectors may be engi gene' refers to a gene as found in nature with its own neered to contain selectable marker genes that provide for regulatory sequences. "Chimeric gene' refers to any gene the selection of cells that have incorporated the marker into that is not a native gene, comprising regulatory and/or the cellular genome. Such markers allow identification and/ coding sequences that are not found together in nature. or selection of host cells that incorporate and express the Accordingly, a chimeric gene may comprise regulatory proteins encoded by the marker. sequences and coding sequences that are derived from Viral vectors, and particularly retroviral vectors, have different Sources, or regulatory sequences and coding been used in a wide variety of gene delivery applications in sequences derived from the same source, but arranged in a cells, as well as living animal Subjects. Viral vectors that can manner different than that found in nature. A chimeric gene be used to express embodiments of the invention described may comprise coding sequences derived from different 25 herein include, but are not limited to, retrovirus, adeno Sources and/or regulatory sequences derived from different associated virus, pox, baculovirus, vaccinia, herpes simplex, Sources. "Endogenous gene' refers to a native gene in its Epstein-Barr, adenovirus, geminivirus, and caulimovirus natural location in the genome of an organism. A “foreign' vectors. Non-viral vectors include plasmids, liposomes, gene or "heterologous gene refers to a gene not normally electrically charged lipids (cytofectins), DNA-protein com found in the host organism, but that is introduced into the 30 plexes, and biopolymers. In addition to a nucleic acid, a host organism by gene transfer. Foreign genes can comprise vector may also comprise one or more regulatory regions, native genes inserted into a non-native organism, or chime and/or selectable markers useful in selecting, measuring, and ric genes. A “transgene' is a gene that has been introduced monitoring nucleic acid transfer results (e.g., monitoring into the genome by a transformation procedure. transfer to target or non-target tissues, duration of expres “RNA transcript refers to the product resulting from 35 Sion, etcetera). RNA polymerase-catalyzed transcription of a DNA The term “plasmid' refers to an extra-chromosomal ele sequence. When the RNA transcript is a perfect comple ment often carrying a gene that is not part of the central mentary copy of the DNA sequence, it is referred to as the metabolism of the cell, and usually in the form of circular primary transcript or it may be a RNA sequence derived double-stranded DNA molecules. Such elements may be from post-transcriptional processing of the primary tran 40 autonomously replicating sequences, genome integrating script and is referred to as the mature RNA, "Messenger sequences, phage or nucleotide sequences, linear, circular, or RNA (mRNA) refers to the RNA that is without introns and supercoiled, of a single- or double-stranded DNA or RNA, that can be translated into protein by the cell. “cDNA refers derived from any source, in which a number of nucleotide to a double-stranded DNA that is complementary to and sequences have been joined or recombined into a unique derived from mRNA. “Sense” RNA refers to RNA transcript 45 construction which is capable of introducing a promoter that includes the mRNA and so can be translated into protein fragment and DNA sequence for a selected gene product by the cell. “Antisense RNA refers to a RNA transcript that along with appropriate 3' untranslated sequence into a cell. is complementary to all or part of a target primary transcript A “cloning vector” refers to a “replicon, which is a unit or mRNA and that blocks the expression of a target gene. length of a nucleic acid, preferably DNA, that replicates The complementarity of an antisense RNA may be with any 50 sequentially and which comprises an origin of replication, part of the specific gene transcript, i.e., at the 5' non-coding Such as a plasmid, phage or cosmid, to which another nucleic sequence, 3' non-coding sequence, or the coding sequence. acid segment may be attached so as to bring about the A “vector” refers to any vehicle for the cloning of and/or replication of the attached segment. Cloning vectors may be transfer of a nucleic acid into a host cell. A vector may be capable of replication in one cell type and expression in a replicon to which another DNA segment may be attached 55 another (“shuttle vector). Cloning vectors may comprise So as to bring about the replication of the attached segment. one or more sequences that can be used for selection of cells A “replicon refers to any genetic element (e.g., plasmid, comprising the vector and/or one or more multiple cloning phage, cosmid, chromosome, virus) that functions as an sites for insertion of sequences of interest. The term “expres autonomous unit of DNA replication in vivo, i.e., capable of sion vector” refers to a vector, plasmid or vehicle designed replication under its own control. The term “vector includes 60 to enable the expression of an inserted nucleic acid sequence both viral and nonviral vehicles for introducing the nucleic following transformation into the host. The cloned gene, i.e., acid into a cell in vitro, ex vivo or in vivo. A large number the inserted nucleic acid sequence, is usually placed under of vectors known in the art may be used to manipulate the control of control elements such as a promoter, a nucleic acids, incorporate coding sequences into genes, et minimal promoter, an enhancer, or the like. Initiation control cetera. Possible vectors include, for example, plasmids or 65 regions or promoters, which are useful to drive expression of modified including, for example bacteriophages Such a nucleic acid in the desired host cell are numerous and as lambda derivatives, or plasmids such as pBR322 or puC familiar to those skilled in the art. Virtually any promoter US 9,677,055 B2 15 16 capable of driving expression of these genes can be used in Vectors and polynucleotides of the invention may be an expression vector, including but not limited to, viral introduced in vivo by lipofection. For example, via use of promoters, bacterial promoters, animal promoters, mamma liposomes for encapsulation and transfection of nucleic lian promoters, synthetic promoters, constitutive promoters, acids in vitro. Synthetic cationic lipids designed to limit the tissue specific promoters, pathogenesis or disease related 5 difficulties encountered with liposome-mediated transfec promoters, developmental specific promoters, inducible pro tion can be used to prepare liposomes for in vivo transfection moters, light regulated promoters; CYC1, HIS3, GAL1, of a gene encoding a marker (Feigner et al. Proc. Natl. Acad. GAL4, GAL1O, ADH1, PGK, PH05, GAPDH, ADC1, Sci USA. 84:7413 (1987); Mackey et al. Proc. Natl. Acad. TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase pro Sci USA 85:8027 (1988); and Ulmer et al, Science 259:1745 10 (1993)). Use of cationic lipids may promote encapsulation moters (useful for expression in Saccharomyces); AOX1 of negatively charged nucleic acids, and also promote fusion promoter (useful for expression in Pichia); 3-lactamase, lac, with negatively charged cell membranes (Feigner et al. ara, tet, trp, IP, IP, T7, tac, and trc promoters (useful for Science 337:387 (1989)). Particularly useful lipid com expression in Escherichia coli), light regulated-, seed spe pounds and compositions for transfer of nucleic acids are cific-, pollen specific-, ovary specific-, cauliflower mosaic 15 described in WO95/18863, WO96/17823 and U.S. Pat. No. virus 35S, CMV 35S minimal, cassava vein mosaic virus 5,459,127. (CsVMV), chlorophyll a?b binding protein, ribulose 1.5- Other molecules are also useful for facilitating transfec bisphosphate carboxylase, shoot-specific, root specific, chi tion of a nucleic acid in Vivo, Such as a cationic oligopeptide tinase, stress inducible, rice tungro bacilliform virus, plant (e.g., WO95/21931), peptides derived from DNA binding Super-promoter, potato leucine aminopeptidase, nitrate proteins (e.g., WO96/25508), or a cationic polymer (e.g., reductase, mannopine synthase, nopaline synthase, ubiq WO95/21931). uitin, Zein protein, and anthocyanin promoters (useful for It is also possible to introduce a vector in vivo as a naked expression in plant cells); animal and mammalian promoters DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and known in the art including, but are not limited to, the SV40 5,580,859). Receptor-mediated DNA delivery approaches early (SV40e) promoter region, the promoter contained in 25 can also be used (Curiel et al., Hum. Gene Ther. 3:147 the 3' long terminal repeat (LTR) of Rous sarcoma virus (1992); and Wu et al., J. Biol. Chem. 262:4429 (1987)). (RSV), the promoters of the E1A or major late promoter The term “transfection” refers to the uptake of exogenous (MLP) genes of adenoviruses (Ad), the cytomegalovirus or heterologous RNA or DNA by a cell. A cell has been (CMV) early promoter, the herpes simplex virus (HSV) “transfected by exogenous or heterologous RNA or DNA thymidine kinase (TK) promoter, a baculovirus IE1 pro 30 when such RNA or DNA has been introduced inside the cell. moter, an elongation factor 1 alpha (EF1) promoter, a “Transformation” refers to the transfer of a nucleic acid phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) fragment into the genome of a host organism, resulting in promoter, an albumin promoter, the regulatory sequences of genetically stable inheritance. Host organisms containing the mouse metallothionein-L promoter and transcriptional the transformed nucleic acid fragments are referred to as control regions, the ubiquitous promoters (HPRT, vimentin, 35 “transgenic' or “recombinant’ or “transformed' organisms. C.-actin, tubulin and the like), the promoters of the interme In addition, recombinant vector comprising polynucle diate filaments (desmin, neurofilaments, keratin, GFAP, and otides of the invention may include one or more origins for the like), the promoters of therapeutic genes (of the MDR, replication in the cellular hosts in which their amplification CFTR or factor VIII type, and the like), pathogenesis or or their expression is sought, markers or selectable markers. disease related-promoters, and promoters that exhibit tissue 40 The term “selectable marker” refers to an identifying specificity and have been utilized in transgenic animals, factor, usually an antibiotic or chemical resistance gene, that Such as the elastase I gene control region which is active in is able to be selected for based upon the marker gene's pancreatic acinar cells; insulin gene control region active in effect, i.e., resistance to an antibiotic, resistance to a herbi pancreatic beta cells, immunoglobulin gene control region cide, colorimetric markers, enzymes, fluorescent markers, active in lymphoid cells, mouse mammary tumor virus 45 and the like, wherein the effect is used to track the inheri control region active in testicular, breast, lymphoid and mast tance of a nucleic acid of interest and/or to identify a cell or cells; albumin gene, Apo AI and Apo AII control regions organism that has inherited the nucleic acid of interest. active in liver, alpha-fetoprotein gene control region active Examples of selectable marker genes known and used in the in liver, alpha 1-antitrypsin gene control region active in the art include: genes providing resistance to amplicillin, Strep liver, beta-globin gene control region active in myeloid cells, 50 tomycin, gentamycin, kanamycin, hygromycin, bialaphos myelin basic protein gene control region active in oligoden herbicide, Sulfonamide, and the like; and genes that are used drocyte cells in the brain, myosin light chain-2 gene control as phenotypic markers, i.e., anthocyanin regulatory genes, region active in skeletal muscle, and gonadotropic releasing isopentanyl transferase gene, and the like. hormone gene control region active in the hypothalamus, The term “reporter gene' refers to a nucleic acid encoding pyruvate kinase promoter, Villin promoter, promoter of the 55 an identifying factor that is able to be identified based upon fatty acid binding intestinal protein, promoter of the Smooth the reporter gene's effect, wherein the effect is used to track muscle cell C-actin, and the like. In addition, these expres the inheritance of a nucleic acid of interest, to identify a cell sion sequences may be modified by addition of enhancer or or organism that has inherited the nucleic acid of interest, regulatory sequences and the like. and/or to measure gene expression induction or transcrip Vectors comprising polynucleotides of the invention may 60 tion. Examples of reporter genes known and used in the art be introduced into the desired host cells by methods known include: Luciferase (Luc), green fluorescent protein (GFP), in the art, e.g., transfection, electroporation, microinjection, chloramphenicol acetyltransferase (CAT), B-galactosidase transduction, cell fusion, DEAE dextran, calcium phosphate (Lacz), B-glucuronidase (Gus), and the like. Selectable precipitation, lipofection (lysosome fusion), use of a gene marker genes may also be considered reporter genes. gun, or a DNA vector transporter (see, e.g., Wu et al., J. Biol. 65 “Promoter and “promoter sequence' are used inter Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 changeably and refer to a DNA sequence capable of con (1988); and Hartmut et al., Canadian Patent No. 2,012,311). trolling the expression of a coding sequence or functional US 9,677,055 B2 17 18 RNA. In general, a coding sequence is located 3' to a NO:141), where N(n) can be one or more spacer nucleotides promoter sequence. Promoters may be derived in their (see D'Avino et al., Mol. Cell. Endocrinol. 113:1 (1995)); entirety from a native gene, or be composed of different and GGGTTGAATGAATTT (SEQ ID NO: 142) (see Anton elements derived from different promoters found in nature, iewski et al., Mol. Cell Biol. 14:4465 (1994)). or even comprise synthetic DNA segments. It is understood The terms “operably linked.” “operably associated.” by those skilled in the art that different promoters may direct “through operable association,” and the like refer to the the expression of a gene in different tissues or cell types, or association of nucleic acid sequences on a single nucleic at different stages of development, or in response to different acid fragment so that the function of one is affected by the environmental or physiological conditions. Promoters that other. For example, a promoter is operably linked with a cause a gene to be expressed in most cell types at most times 10 are commonly referred to as “constitutive promoters.” Pro coding sequence when it is capable of affecting the expres moters that cause a gene to be expressed in a specific cell sion of that coding sequence (i.e., that the coding sequence type are commonly referred to as “cell-specific promoters' is under the transcriptional control of the promoter). Coding or “tissue-specific promoters.” Promoters that cause a gene sequences can be operably linked to regulatory sequences in sense or antisense orientation. to be expressed at a specific stage of development or cell 15 differentiation are commonly referred to as “developmen The terms “cassette,” “expression cassette' and “gene tally-specific promoters' or “cell differentiation-specific expression cassette' refer to a segment of DNA that can be promoters.” Promoters that are induced and cause a gene to inserted into a nucleic acid or polynucleotide at specific be expressed following exposure or treatment of the cell restriction sites or by homologous recombination. The seg with an agent, biological molecule, chemical, ligand, light, ment of DNA comprises a polynucleotide that encodes a or the like that induces the promoter are commonly referred polypeptide of interest, and the cassette and restriction sites to as “inducible promoters” or “regulatable promoters.” It is are designed to ensure insertion of the cassette in the proper further recognized that since in most cases the exact bound reading frame for transcription and translation. “Transfor aries of regulatory sequences have not been completely mation cassette' refers to a specific vector comprising a defined, DNA fragments of different lengths may have 25 polynucleotide that encodes a polypeptide of interest and identical promoter activity. having elements in addition to the polynucleotide that facili The promoter sequence is typically bounded at its 3' tate transformation of a particular host cell. Cassettes, terminus by the transcription initiation site and extends expression cassettes, gene expression cassettes and transfor upstream (5' direction) to include the minimum number of mation cassettes of the invention may also comprise ele bases or elements necessary to initiate transcription at levels 30 detectable above background. Within the promoter sequence ments that allow for enhanced expression of a polynucle will be found a transcription initiation site (conveniently otide encoding a polypeptide of interest in a host cell. These defined for example, by mapping with nuclease S1), as well elements may include, but are not limited to: a promoter, a as protein binding domains (consensus sequences) respon minimal promoter, an enhancer, a response element, a ter sible for the binding of RNA polymerase. 35 minator sequence, a polyadenylation sequence, and the like. A coding sequence is “under the control of transcrip For purposes of expressing polynucleotides and polypep tional and translational control sequences in a cell when tides under control of a gene Switch mechanism, the term RNA polymerase transcribes the coding sequence into “gene switch” refers to the combination of a response mRNA, which is then trans-RNA spliced (if the coding element associated with a promoter, and a ligand-dependent sequence contains nitrons) and translated into the protein 40 transcription factor-based system which, in the presence of encoded by the coding sequence. one or more ligands, modulates the expression of a gene into “Transcriptional and translational control sequences which the response element and promoter are incorporated. refer to DNA regulatory sequences, such as promoters, Stated otherwise, a “gene switch” refers to a peptide, protein enhancers, terminators, and the like, that provide for the or polypeptide complex that functions to (a) bind an acti expression of a coding sequence in a host cell. In eukaryotic 45 Vating ligand, and (b) regulate the transcription of a gene of cells, polyadenylation signals are control sequences. interest in a ligand-dependent fashion. The term “response element” (“RE) refers to one or more As used herein with respect to gene Switch regulation cis-acting DNA elements which confer responsiveness on a systems, the term “dimerizes with the ligand binding domain promoter mediated through interaction with the DNA-bind that binds an activating ligand’ refers to a selective protein ing domains of a transcription factor. This DNA element 50 protein interaction that is induced by the presence of acti may be either palindromic (perfect or imperfect) in its Vating ligand. sequence or composed of sequence motifs or half sites As used herein, the term “ligand binding domain that separated by a variable number of nucleotides. The half sites binds an activating ligand’ refers to an amino acid sequence can be similar or identical and arranged as either direct or that selectively binds an activating ligand. In the methods inverted repeats or as a single half site or multimers of 55 disclosed herein, an activating ligand binds to a ligand adjacent half sites in tandem. The response element may binding domain, e.g., an ecdysone receptor ligand binding comprise a minimal promoter isolated from different organ domain, that is part of a ligand-dependent transcriptional isms depending upon the nature of the cell or organism into activation complex that regulates the expression of a poly which the response element will be incorporated. The DNA nucleotide sequence that encodes a gene of interest. Hence, binding domain of the transcription factor binds, in the 60 the expression of the gene of interest is regulated in a presence or absence of a ligand, to the DNA sequence of a ligand-dependent fashion. response element to initiate or Suppress transcription of The term “ecdysone receptor-based, with respect to a downstream gene(s) under the regulation of this response gene Switch, refers to a gene Switch comprising at least a element. Examples of DNA sequences for response elements functional part of a naturally occurring or synthetic of the natural ecdysone receptor include: RRGG/TTCANT 65 ecdysone receptor ligand binding domain and which regu GAC/ACYY (SEQ ID NO:140) (see Cherbaset. al., Genes lates gene expression in response to a ligand that binds to the Dev. 5:120-131 (1991)): AGGTCAN(n)AGGTCA (SEQ ID ecdysone receptor ligand binding domain. US 9,677,055 B2 19 20 The terms “modulate' and “modulates' mean to induce, (“USP) proteins (see Yao et al., Nature 366:476 (1993)); reduce or inhibit nucleic acid or gene expression, resulting Yao et al., Cell 71:63 (1992)). A functional LDTFC such as in the respective induction, reduction or inhibition of protein an EcR complex may also include additional protein(s) Such or polypeptide production. as immunophilins. Additional members of the nuclear recep Polynucleotides or vectors comprising sequences encod tor family of proteins, known as transcriptional factors (such ing polypeptides of the present invention may further com as DHR38, betaFTZ-1 or other insect homologs), may also prise at least one promoter Suitable for driving expression of be ligand dependent or independent partners for EcR and/or a gene in a modified cell. USP. A LDTFC such as an EcR complex can also be a Enhancers that may be used in embodiments of the heterodimer of EcR protein and the vertebrate homolog of invention include but are not limited to: an SV40 enhancer, 10 ultraspiracle protein, retinoic acid-X-receptor (“RXR) pro a cytomegalovirus (CMV) enhancer, an elongation factor 1 tein or a chimera of USP and RXR. The terms “LDTFC and (EF1) enhancer, yeast enhancers, viral gene enhancers, et “EcR complex' also encompass homodimer complexes of Cetera. the EcR protein or USP, as well as single polypeptides or "Regulatory region” refers to a nucleic acid sequence that tamers, tetramer, and other multimers serving the same regulates the expression of a second nucleic acid sequence. 15 function. A regulatory region may include sequences which are natu ALDTFC such as an EcR complex can be activated by an rally responsible for expressing a particular nucleic acid (a active ecdysteroid or non-steroidal ligand bound to one of homologous region) or may include sequences of a different the proteins of the complex, inclusive of EcR, but not origin that are responsible for expressing different proteins excluding other proteins of the complex. As used herein, the or even synthetic proteins (a heterologous region). In par term “ligand, as applied to LDTFC-based gene switches ticular, the sequences can be sequences of prokaryotic, e.g., EcD complex based gene Switches, describes Small and eukaryotic, or viral genes or derived sequences that stimu soluble molecules having the capability of activating a gene late or repress transcription of a gene in a specific or Switch to stimulate expression of a polypeptide encoded non-specific manner and in an inducible or non-inducible therein. Examples of ligands include, without limitation, an manner. Regulatory regions include origins of replication, 25 ecdysteroid, such as ecdysone, 20-hydroxyecdysone, pon RNA splice sites, promoters, enhancers, transcriptional ter asterone A, muristerone A, and the like, 9-cis-retinoic acid, mination sequences, and signal sequences which direct the synthetic analogs of retinoic acid, N,N'-diacylhydrazines polypeptide into the secretory pathways of the target cell. such as those disclosed in U.S. Pat. Nos. 6,013,836, 5,117, The term "exogenous gene' or "heterologous gene’ 057: 5,530,028; 5,378,726, and 7,304,161 and U.S. Pat. No. means a gene foreign to the Subject or organism, that is, a 30 7,456,315; oxadiazolines as described in U.S. Pat. No. gene which is introduced into the Subject through a trans 7.304,162; dibenzoylalkyl cyanohydrazines such as those formation process, an unmutated version of an endogenous disclosed in European Patent No. 461,809B1; N-alkyl-N,N'- mutated gene or a mutated version of an endogenous unmu diaroylhydrazines such as those disclosed in U.S. Pat. No. tated gene. The method of transformation is not critical to 5.225,443; N-acyl-N-alkylcarbonylhydrazines such as those this invention and may be any method suitable for the 35 disclosed in European Patent No. 234,99481; N-aroyl-N- Subject known to those in the art. Exogenous genes can be alkyl-N'-aroylhydrazines such as those described in U.S. either natural or synthetic genes which are introduced into Pat. No. 4,985,461; amidoketones such as those described in the subject in the form of DNA or RNA which may function U.S. Pat. No. 7,375,093; each of which is incorporated through a DNA intermediate such as by reverse tran herein by reference and other similar materials including Scriptase. Such genes can be introduced into target cells, 40 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O- directly introduced into the subject, or indirectly introduced acetylharpagide, oxysterols, 22(R) hydroxy cholesterol, by the transfer of transformed cells into the subject. 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, Polynucleotides and polypeptides of the invention may be 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ke expressed in vivo under control of a “gene switch” control tocholesterol-3-sulfate, famesol, bile acids, 1,1-biphospho mechanism, such as those described in, for example, but not 45 nate esters, juvenile hormone III, and the like. Examples of limited to: diacylhydrazine ligands useful in the present invention WO 2009/045370 (PCT/US2008/011270); include RG-115819 (3,5-Dimethyl-benzoic acid N-(1-ethyl WO 2009/025866 (PCT/US2008/010040); 2,2-dimethyl-propyl)-N'-(2-methyl-3-methoxy-benzoyl)- WO 2008/073154 (PCT/US2007/016747); hydrazide), RG-115932 ((R)-3,5-Dimethyl-benzoic acid WO 2005/108617 (PCT/US2005/015089); 50 N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hy WO 2003/0/27289 (PCT/US2002/005026); drazide), and RG-115830 (3,5-Dimethyl-benzoic acid N-(1- WO 2002/066615 (PCT/US2002/005708); tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydraz WO 2003/027266 (PCT/US/2002/05234); ide). See, e.g., U.S. Pat. No. 8,076,517 (Publication No. WO 2002/066612 (PCT/US2002/005090); 2009/0163592), and PCT Appl. No. PCT/US2008/006757 WO 2002/066614 (PCT/US/2002/005706); 55 (WO 2008/153801), both of which are incorporated herein WO 2002/066613 (PCT/US2002/005090); by reference in their entireties. WO 2002/029075 (PCT/US2001/030608); and A LDTFC such as an EcR complex includes proteins WO 2001/070816 (PCT/US2001/090500), which are members of the nuclear receptor superfamily each of which are incorporated by reference herein. wherein all members are characterized by the presence of The term “ligand-dependent transcription factor com 60 one or more polypeptide subunits comprising an amino plex” or “LDTFC refers to a transcription factor compris terminal transactivation domain (“AD.” “TD, or “TA. ing one or more protein Subunits, which complex can used interchangeably herein), a DNA binding domain regulate gene expression driven by a “factor-regulated pro (“DBD”), and a ligand binding domain (“LBD). The AD moter as defined herein. A model LDTFC is an “ecdysone may be present as a fusion with a "heterodimerization receptor complex generally refers to a heterodimeric pro 65 partner or “HP. A fusion protein comprising an AD and HP tein complex having at least two members of the nuclear of the invention is referred to herein as a “coactivation receptor family, ecdysone receptor (“EcR) and ultraspiracle protein' or “CAP.” The DBD and LBD may be expressed as US 9,677,055 B2 21 22 a fusion protein, referred to herein as a “ligand-inducible ments, elixirs, and injectable compositions. Pharmaceutical transcription factor (“LTF). The fusion partners may be compositions may contain from 0.01% to 99% by weight of separated by a linker, e.g., a hinge region. Some members of the ligand. Compositions may be either in single or multiple the LTF family may also have another transactivation dose forms. The amount of ligand in any particular phar domain on the carboxy-terminal side of the LBD. The DBD maceutical composition will depend upon the effective dose, is characterized by the presence of two cysteine Zinc fingers that is, the dose required to elicit the desired gene expression between which are two amino acid motifs, the P-box and the or Suppression. D-box, which confer specificity for ecdysone response ele Suitable routes of administering the pharmaceutical ments. These domains may be either native, modified, or preparations include oral, rectal, topical (including dermal, chimeras of different domains of heterologous receptor 10 buccal and Sublingual), vaginal, parenteral (including Sub proteins. cutaneous, intramuscular, intravenous, intradermal, intrath EcR ligands, when used with a LDTFC, e.g., an EcR ecal and epidural) and by nasogastric tube. It will be complex, which in turn is bound to the response element understood by those skilled in the art that the preferred route linked to an exogenous gene (e.g., a reporter gene), provide of administration will depend upon the condition being the means for external temporal regulation of expression of 15 treated and may vary with factors such as the condition of the exogenous gene. The order in which the various com the recipient. ponents bind to each other, that is, ligand to receptor As used herein, the terms “treat' or “treatment” refer to complex and receptor complex to response element, is not both therapeutic treatment and prophylactic or preventative critical. Typically, modulation of expression of the exog measures, wherein the object is to prevent or slow down enous gene is in response to the binding of a LDTFC, e.g., (lessen) an undesired physiological change or disorder, Such an EcR complex, to a specific control, or regulatory, DNA as the development, progression or spread (i.e., metastasis) element. The EcR protein, like other members of the nuclear of cancer. Beneficial or desired clinical results include, but receptor family, possesses at least three domains, an AD, a are not limited to, alleviation of symptoms, diminishment of DBD, and a LBD. This receptor, like a subset of the nuclear extent of disease, stabilized (i.e., not worsening) state of receptor family, also possesses less well-defined regions 25 disease, delay or slowing of disease progression, ameliora responsible for heterodimerization properties (referred to tion or palliation of the disease state, and remission (whether herein as a "heterodimerization partner' or “HP). Binding partial or total). “Treatment can also mean prolonging of the ligand to the ligand binding domain of a LTF, e.g., an Survival as compared to expected Survival if not receiving EcR protein, after heterodimerization with a CAP including, treatment. Those in need of treatment include those already e.g., an AD and/or an HP, e.g., a USP or RXR protein, 30 with the condition or disorder as well as those prone to have enables the DNA binding domains of the heterodimeric the condition or disorder or those in which the condition or proteins to bind to the response element in an activated form, disorder is to be prevented. thus resulting in expression or Suppression of the exogenous The terms “subject,” “individual,” “animal,” “patient,” or gene. This mechanism does not exclude the potential for “mammal,” is meant any Subject, particularly a mammalian ligand binding to individual Subunits, e.g., LTF or CAP, e.g., 35 Subject, for whom diagnosis, prognosis, or therapy is an EcR or USP, and the resulting formation of active desired. Mammalian Subjects include, without limitation, homodimer complexes (e.g. EcR+EcR or USP+USP). In one humans, domestic animals, farm animals, and Zoo, sports, or embodiment, one or more of the receptor domains can be pet animals such as dogs, cats, guinea pigs, rabbits, rats, varied producing a chimeric gene Switch. Typically, one or mice, horses, cattle, cows, etcetera. more of the three domains may be chosen from a source 40 The terms “hyperproliferative disease or disorder is different than the source of the other domains so that the intended to encompass all neoplastic cell growth and pro chimeric receptor is optimized in the chosen host cell or liferation, whether malignant or benign, including all trans organism for transactivating activity, complementary bind formed cells and tissues and all cancerous cells and tissues. ing of the ligand, and recognition of a specific response Hyperproliferative diseases or disorders include, but are not element. In addition, the response element itself can be 45 limited to, precancerous lesions, abnormal cell growths, modified or substituted with response elements for other tumors (whether benign or malignant), "cancer and other DNA binding protein domains such as the GAL-4 protein hyperplasias. from yeast (see Sadowski et al., Nature 335:563 (1988) or The term “cancer includes, but is not limited to, primary LexA protein from E. coli (see Brent et al., Cell 43:729-736 malignant cells or tumors (e.g., those whose cells have not (1985)) to accommodate chimeric LDTFCs, e.g., EcR com 50 migrated to sites in the subjects body other than the site of plexes. Another advantage of chimeric systems is that they the original malignancy or tumor) and secondary malignant allow choice of a promoter used to drive the exogenous gene cells or tumors (e.g., those arising from metastasis, the according to a desired end result. Such double control can be migration of malignant cells or tumor cells to secondary particularly important in areas of gene therapy, especially sites that are different from the site of the original tumor). when cytotoxic proteins are produced, because both the 55 A tumor or tumor tissue may also comprise "tumor timing of expression as well as the cells wherein expression associated non-tumor cells', e.g., vascular cells which form occurs can be controlled. When exogenous genes, opera blood vessels to supply the tumor or tumor tissue. Non tively linked to a suitable promoter, are introduced into the tumor cells may be induced to replicate and develop by cells of the Subject, expression of the exogenous genes is tumor cells, for example, the induction of angiogenesis in a controlled by the presence of the ligand of this invention. 60 tumor or tumor tissue. Promoters may be constitutively or inducibly regulated or Some examples of cancer include, but are not limited to, may be tissue-specific (that is, expressed only in a particular carcinoma, lymphoma, blastoma, sarcoma, and leukemia or type of cell) or specific to certain developmental stages of lymphoid malignancies. More particular examples of Such the organism. cancers are noted below and include: squamous cell cancer For in vivo use, the ligands described herein may be taken 65 (e.g. epithelial squamous cell cancer), lung cancer including up in pharmaceutically acceptable carriers, such as, for Small-cell lung cancer, non-Small cell lung cancer, adeno example, Solutions, Suspensions, tablets, capsules, oint carcinoma of the lung and squamous carcinoma of the lung, US 9,677,055 B2 23 24 cancer of the peritoneum, hepatocellular cancer, gastric or Primary Metastatic Squamous Neck Cancer, Oropharyngeal stomach cancer including gastrointestinal cancer, pancreatic Cancer, Osteo-fMalignant Fibrous Sarcoma, Osteosarcoma/ cancer, glioblastoma, cervical cancer, ovarian cancer, liver Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant cancer, bladder cancer, hepatoma, breast cancer, colon can Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, cer, rectal cancer, colorectal cancer, endometrial cancer or 5 Ovarian Germ Cell Tumor, Ovarian Low Malignant Poten uterine carcinoma, Salivary gland carcinoma, kidney or renal tial Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, cancer, prostate cancer, Vulval cancer, thyroid cancer, Parathyroid Cancer, Pheochromocytoma, Pituitary Tumor, hepatic carcinoma, as well as head (e.g., brain) and neck Plasma Cell Neoplasm/Multiple Myeloma, Primary Central CaCC. Nervous System Lymphoma, Primary Liver Cancer, Prostate Other examples of cancers or malignancies include, but 10 are not limited to: Acute Childhood Lymphoblastic Leuke Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and mia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Sali Leukemia, Acute Myeloid Leukemia, Adrenocortical Carci vary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syn noma, Adult (Primary) Hepatocellular Cancer, Adult (Pri drome, Skin Cancer, Small Cell Lung Cancer, Small Intes mary) Liver Cancer, Adult Acute Lymphocytic Leukemia, 15 tine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Adult Acute Myeloid Leukemia, Adult Hodgkin’s Disease, Stomach Cancer, Supratentorial Primitive Neuroectodermal Adult Hodgkin’s Lymphoma, Adult Lymphocytic Leuke and Pineal Tumors, T cell Lymphoma, Testicular Cancer, mia, Adult Non-Hodgkin’s Lymphoma, Adult Primary Liver Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lym Renal Pelvis and Ureter, Transitional Renal Pelvis and phoma, AIDS-Related Malignancies, Anal Cancer, Astrocy Ureter Cancer, Trophoblastic Tumors, Ureter and Renal toma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of Sarcoma, Vaginal Cancer, Visual Pathway and Hypotha the Renal Pelvis and Ureter, Central Nervous System (Pri lamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobu mary) Lymphoma, Central Nervous System Lymphoma, linemia, Wilms Tumor, and any other hyperproliferative Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical 25 disease, besides neoplasia, located in an organ system listed Cancer, Childhood (Primary) Hepatocellular Cancer, Child above. hood (Primary) Liver Cancer, Childhood Acute Lympho Naturally Occurring Amino Acid Substitutions blastic Leukemia, Childhood Acute Myeloid Leukemia, List of naturally occurring amino acids and some of their Childhood Brain Stem Glioma, Childhood Cerebellar Astro biochemical properties. cytoma, Childhood Cerebral Astrocytoma, Childhood 30 Extracranial Germ Cell Tumors, Childhood Hodgkin’s Dis ease, Childhood Hodgkin’s Lymphoma, Childhood Hypo 3- 1- Side-chain thalamic and Visual Pathway Glioma, Childhood Lympho Letter Letter Side-chain charge (pH Hydropathy blastic Leukemia, Childhood Medulloblastoma, Childhood Amino Acid Code Code polarity* 7.4)* index* Non-Hodgkin’s Lymphoma, Childhood Pineal and 35 Alanine Ala A nonpolar neutra 1.8 Supratentorial Primitive Neuroectodermal Tumors, Child Arginine Arg R polar positive -4.5 hood Primary Liver Cancer, Childhood Rhabdomyosar Asparagine ASn N polar lel 8 -3.5 Aspartic acid Asp D polar negative -3.5 coma, Childhood Soft Tissue Sarcoma, Childhood Visual Cysteine Cys C polar lel 8 2.5 Pathway and Hypothalamic Glioma, Chronic Lymphocytic Glutamic Glu E polar negative -3.5 Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, 40 acid Cutaneous T cell Lymphoma, Endocrine Pancreas Islet Cell Glutamine Gln Q polar lel 8 -3.5 Glycine Gly G nonpolar neutra -0.4 Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Histidine His H polar positive (10%) -3.2 Cancer, Esophageal Cancer, Ewing's Sarcoma and Related neutral (90%) Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Isoleucine Ile I nonpolar neutra 4.5 Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic 45 Leucine Leu L nonpolar neutra 3.8 Lysine Lys K polar positive -3.9 Bile Duct Cancer, Eye Cancer. Female Breast Cancer, Gau Methionine Met M nonpolar neutra 1.9 cher's Disease, Gallbladder Cancer, Gastric Cancer, Gastro Phenylalanine Phe F nonpolar neutra 2.8 intestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Proline Pro P nonpolar neutra -1.6 Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Serine Ser S polar lel 8 -0.8 50 Threonine Thr T polar lel 8 -O.7 Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Tryptophan Trp W nonpolar neutra -0.9 Hodgkin’s Disease, Hodgkin's Lymphoma, Hypergamma Tyrosine Tyr Y polar lel 8 -1.3 globulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Valine Wal V nonpolar neutra 4.2 Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pan *Hausman & Cooper, (2004), The Ceii: A Molecular Approach, Washington, D.C: ASM creatic Cancer, Kaposi’s Sarcoma, Kidney Cancer, Laryn Press. p. 51 (2004) (ISBN 0-87893-214-3). geal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, 55 **Kyte & Doolittle, “A simple method for displaying the hydropathic character of a Lung Cancer, Lymphoproliferative Disorders, Macroglobu protein,” Journal of Molecular Biology, 157 (1): 105-132 (May 1982). linemia, Male Breast Cancer, Malignant Mesothelioma, Conservative Amino Acid Substitutions Malignant Thymoma, Medulloblastoma, Melanoma, Meso Polypeptides may be made to differ by introduction of thelioma, Metastatic Occult Primary Squamous Neck Can conservative or non-conservative amino acid changes. Con cer, Metastatic Primary Squamous Neck Cancer, Metastatic 60 servative amino acid Substitutions refer to the interchange Squamous Neck Cancer, Multiple Myeloma, Multiple ability of residues having similar amino acid side chains. Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syn “Conservative amino acid substitutions' refer to substitu drome, Myelogenous Leukemia, Myeloid Leukemia, tions of one or more amino acids in a native amino acid Myeloproliferative Disorders, Nasal Cavity and Paranasal sequence (e.g., wild-type or naturally occurring form of PE) Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, 65 with other amino acid(s) having similar side chains (e.g., Non-Hodgkin’s Lymphoma During Pregnancy, Nonmela side chains similar in terms of size, charge, element com noma Skin Cancer. Non-Small Cell Lung Cancer. Occult position, and/or hydrophobicity/hydrophilicity). US 9,677,055 B2 25 26 Conserved substitutes for an amino acid within a native of Pseudomonas exotoxin A, or a fragment thereof, wherein amino acid sequence can be selected from other members of said modified form, or fragment thereof, comprises an the group to which the naturally occurring amino acid epitope selected from the group consisting of: belongs. For example, conservative amino acid residue Substitution groups include: (1) Alanine (A)-Glycine (G)-Serine (S)-Threonine: (epitope 1; SEQ ID NO: 5) (2) Aspartic acid (D)-Glutamic acid (E); ISFSTRGTO; (3) Asparagine (N)-Glutamine (Q); (4) Arginine (R)-Lysine (K)-Histidine (H), (epitope 2; SEQ ID NO : 6) GTONWTVER; (5) Isoleucine (I)-Leucine (L)-Methionine (M)-Valine 10 (V); and (epitope 3; SEQ ID NO: 7) (6) Phenylalanine (F)-Tyrosine (Y)-Tryptophan (W). IVFGGVRAR; Other Substitution groups of amino acids can be envi Sioned. For example, amino acids can be grouped by similar (epitope 4; SEQ ID NO: 8) function or chemical structure or composition (e.g., acidic, ARSODLDAI; 15 basic, aliphatic, aromatic, Sulfur-containing). For example, (epitope 5; SEQ ID NO: 9) an Aliphatic grouping may comprise: Glycine (G), Alanine LRVYVPRSS; (A), Valine (V), Leucine (L), Isoleucine (I). Other groups and containing amino acids that are considered conservative substitutions for one another include: (epitope 6; SEQ ID NO: 10 Aromatic: Phenylalanine (F)-Tyrosine (Y)-Tryptophan IPDKEQAIS (W); wherein one or more amino acid residues in any one or Sulfur-containing: Methionine (M)-Cysteine (C); more of these epitopes are substituted with a different Basic: Arginine (R)-Lysine (K)-Histidine (H): amino acid residue. Acidic: Aspartic acid (D)-Glutamic acid (E); Embodiments of the invention include isolated polypep Non-polar uncharged residues: Cysteine (C)-Methionine 25 tides (proteins) comprising or consisting of a modified form (M)-Proline (P); and of Pseudomonas exotoxin A, or a fragment thereof, wherein Hydrophilic Uncharged Residues: Serine (S)-Threonine said modified form, or fragment thereof, comprises an (T)-Asparagine (N)-Glutamine (Q). epitope selected from the group consisting of: Exemplary embodiments of conservative amino acid Sub stitutions include the interchangeability of valine-leucine, 30 valine-isoleucine-leucine, phenylalanine-tyrosine, lysine-ar (peptide 50 (epitope 1); SEQ ID NO: 60 ginine, alanine-Valine, aspartic acid-glutamic acid, and GDGGDISFSTRGTON, asparagine-glutamine. (peptide 52 (epitope 2); SEQ ID NO: 62.) Examples of Amino Acid Analogs and Non-Standard SFSTRGTONWTVERL; Amino Acid Residues 35 Examples of a few of the many possible amino acid (peptide 53 (epitope 2); SEQ ID NO: 63) analogs routinely known to those of skill in the art include, TRGTONWTVERLLOA; for example, but without limitation, analogs such as: 4-hy (peptide 65 (epitope 3) ; SEQ ID NO: 75) droxyproline which may be substituted for proline, 5-hy AQSIWFGGWRARSOD; droxylysine which may be substituted for lysine: 3-methyl 40 histidine which may be substituted for histidine; homoserine (peptide 67 (epitope 4); SEQ ID NO: 77) which may be substituted for serine; and ornithine which GGWRARSODLDAIWR; may be substituted for lysine. (peptide 68 (epitope 4); SEQ ID NO: 78) Examples of a few of the many possible non-standard RARSODLDAIWRGFY; amino acids routinely known to those of skill in the art 45 include, for example, but without limitation, molecules Such (peptide 81 (epitope 5); SEQ ID NO: 91) as: Ornithine, citrulline, lanthionine, 2-aminoisobutyric acid, NGALLRVYVPRSSLP; dehydroalanine, Y-aminobutyric acid, B-alanine (3-amino (peptide 82 (epitope 5); SEQ ID NO: 92) propanoic acid), selenocysteine and pyrrolysine. LLRVYVPRSSLPGFY; Substitution mutations may be made by any technique for 50 (peptide 110 (epitope 6); SEQ ID NO: 12 O mutagenesis known in the art including, for example, but not LDPSSIPDKEQAISA; limited to, in vitro site-directed mutagenesis (Hutchinson et al, J. Biol. Chem. 255:6551 (1978); Zoller et al, DNA 3:479 (peptide 111 (epitope 6); SEQ ID NO: 121 (1984); Oliphant et al. Gene 44:177 (1986); Hutchinson et SSIPDKEQAISALPD; al, Proc. Natl. Acad. Sci. USA 83:710 (1986)), use of TAB(R) 55 (overlapping epitopes 1 and 2; SEQ ID NO: 131) linkers (Pharmacia), restriction endonuclease digestion/frag ISFSTRGTONWTVER; ment deletion and substitution, PCR-mediated/oligonucle otide-directed mutagenesis, etcetera. PCR-based techniques (overlapping epitopes 3 and 4; SEQ ID NO: 132) are preferred for site-directed mutagenesis (see Higuchi, IWFGGWRARSODLDAI 1989, “Using PCR to Engineer DNA”, in PCR Technology: 60 wherein one or more amino acid residues in any one or Principles and Applications for DNA Amplification, H. more of these epitopes are substituted with a different Erlich, ed., Stockton Press, Chapter 6, pp. 61-70). amino acid residue. Embodiments of the invention include isolated polypep Embodiments of the Invention tides (proteins) comprising or consisting of a modified form 65 of Pseudomonas exotoxin A, or a fragment thereof, wherein Embodiments of the invention include isolated polypep said modified form, or fragment thereof, comprises an tides (proteins) comprising or consisting of a modified form epitope selected from the group consisting of: US 9,677,055 B2 27 28 a) ISFSTRGTQ (SEQ ID NO:5), wherein amino acid b) GTQNWTVER (SEQ ID NO:6), wherein amino acid residues at one or more of positions 1, 6 and 9 are residues at one or more of positions 3, 4 and 6 are Substituted with a different amino acid residue; Substituted with a conservative amino acid substitution; b) GTQNWTVER (SEQ ID NO:6), wherein amino acid c) IVFGGVRAR (SEQ ID NO:7), wherein amino acid residues at one or more of positions 3, 4 and 6 are residues at one or more of positions 1 and 6 are Substituted with a different amino acid residue; Substituted with a conservative amino acid substitution; c) IVFGGVRAR (SEQ ID NO:7), wherein amino acid d) ARSQDLDAI (SEQ ID NO:8), wherein amino acid residues at one or more of positions 1 and 6 are residues at one or more of positions 4 and 7 are Substituted with a different amino acid residue; Substituted with a conservative amino acid substitution; 10 e) LRVYVPRSS (SEQ ID NO:9), wherein amino acid d) ARSQDLDAI (SEQ ID NO:8), wherein amino acid residues at one or more of positions 1, 2 and 9 are residues at one or more of positions 4 and 7 are Substituted with a conservative amino acid substitution; Substituted with a different amino acid residue; f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid e) LRVYVPRSS (SEQ ID NO:9), wherein amino acid residues at one or more of positions 1, 4, 6 and 7 are residues at one or more of positions 1, 2 and 9 are 15 Substituted with a conservative amino acid substitution; Substituted with a different amino acid residue; g) ISFSTRGTQNWTVER (SEQ ID NO:131), wherein f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid amino acid residues at one or more of positions 1, 6, 9, residues at one or more of positions 1, 4, 6 and 7 are 10 and 12 are substituted with a conservative amino Substituted with a different amino acid residue; acid Substitution; and g) ISFSTRGTQNWTVER (SEQ ID NO:131), wherein h) IVFGGVRARSQDLDAI (SEQ ID NO:132), wherein amino acid residues at one or more of positions 1, 6, 9, amino acid residues at one or more of positions 1, 6, 11, 10 and 12 are substituted with a different amino acid and 14 are Substituted with a conservative amino acid residue; and Substitution, h) IVFGGVRARSQDLDAI (SEQ ID NO:132), wherein wherein the conservative amino acid Substitution at one or amino acid residues at one or more of positions 1, 6, 11, 25 more of said positions in a) through f) is selected from and 14 are substituted with a different amino acid the group consisting of: residue. 1) A is substituted with any one of G. I. L. S. T or V: Embodiments of the invention include isolated polypep 2) D is substituted with E: tides (proteins) comprising or consisting of a modified form 3) I is substituted with any one of L, M or V: 30 4) K is substituted with any one of H or R: of Pseudomonas exotoxin A, or a fragment thereof, wherein 5) L is substituted with any one of A, G, I, M or V: said modified form, or fragment thereof, comprises an 6) N is substituted with any one of S, T or Q: epitope selected from the group consisting of: 7) Q is substituted with any one of S, T or N: a) ISFSTRGTQ (SEQ ID NO:5), wherein amino acid 8) R is substituted with any one of K or H: residues at one or more of positions 1, 6 and 9 are 35 9) S is substituted with any one of A, G., N, T or Q: Substituted with a conservative amino acid substitution; 10) T is substituted with any one of A. G. N. Q or S; and b) GTQNWTVER (SEQ ID NO:6), wherein amino acid 11) V is substituted with any one of A, G, I, L or M. residues at one or more of positions 3, 4 and 6 are Embodiments of the invention also comprise or consist of Substituted with a conservative amino acid substitution; isolated polypeptides and peptides comprising or consisting c) IVFGGVRAR (SEQ ID NO:7), wherein amino acid 40 of the above-referenced amino acids sequences, except residues at one or more of positions 1 and 6 are wherein one or more amino acids have been substituted with Substituted with a conservative amino acid substitution; conservative amino acids substitutions. Embodiments of the d) ARSQDLDAI (SEQ ID NO:8), wherein amino acid invention further comprise or consist of isolated polypep residues at one or more of positions 4 and 7 are tides (proteins) and peptides comprising or consisting of the Substituted with a conservative amino acid substitution; 45 above-referenced amino acids sequences, except wherein e) LRVYVPRSS (SEQ ID NO:9), wherein amino acid one or more amino acids have been Substituted with amino residues at one or more of positions 1, 2 and 9 are acids which are naturally occurring, non-naturally occur Substituted with a conservative amino acid substitution; ring, non-standard amino acids, or amino acid analogs. f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid Embodiments of the invention include isolated polypep residues at one or more of positions 1, 4, 6 and 7 are 50 tides (proteins) comprising or consisting of a modified form Substituted with a conservative amino acid substitution; of Pseudomonas exotoxin A, or a fragment thereof, wherein g) ISFSTRGTQNWTVER (SEQ ID NO:131), wherein said modified form, or fragment thereof, comprises an amino acid residues at one or more of positions 1, 6, 9, epitope selected from the group consisting of: 10 and 12 are substituted with a conservative amino a) ISFSTRGTQ (SEQ ID NO:5), wherein amino the acid acid Substitution; and 55 residue at position 1 (I) is substituted with A. N. T. Q h) IVFGGVRARSQDLDAI (SEQ ID NO:132), wherein or H, or wherein the amino acid residue at position 6 amino acid residues at one or more of positions 1, 6, 11, (R) is substituted with Q, or wherein the amino acid and 14 are substituted with a conservative amino acid residue at position 9 (Q) is substituted with N or T, or Substitution. wherein the amino acid sequence ISFSTRGTQ (SEQ Embodiments of the invention include isolated polypep 60 ID NO:5) comprises two or more of said substitutions tides (proteins) comprising or consisting of a modified form in any combination; of Pseudomonas exotoxin A, or a fragment thereof, wherein b) GTQNWTVER (SEQ ID NO:6), wherein the amino said modified form, or fragment thereof, comprises an acid residue at position 3 (Q) is substituted with N or epitope selected from the group consisting of: T, wherein amino the acid residue at position 4 (N) is a) ISFSTRGTQ (SEQ ID NO:5), wherein amino acid 65 Substituted with K or R, or wherein the amino acid residues at one or more of positions 1, 6 and 9 are residue at position 6 (T) is substituted with Kor R, or Substituted with a conservative amino acid substitution; wherein the amino acid sequence GTONWTVER US 9,677,055 B2 29 30 (SEQ ID NO:6) comprises two or more of said substi toxin A, or a fragment thereof, wherein said modified form, tutions in any combination; or fragment thereof, comprises one or more amino acid c) IVFGGVRAR (SEQID NO:7), wherein amino the acid Substitutions selected from the group consisting of: residue at position 1 (I) is substituted with A or N, or a) I at position 141 changed to any amino acid residue; (epitope 1) wherein the amino acid residue at position 6 (V) is b) R at position 146 changed to any amino acid residue. Substituted with D, M, or N, or wherein the amino acid (epitope 1) sequence IVFGGVRAR (SEQ ID NO:7) comprises c) O at position 149 changed to any amino acid residue; Substitutions at both positions in any combination of (epitope 1) amino acid residues A or N at position 1 (I) and D. M. 10 d) Nat position 150 changed to any amino acid residue; or N at position 6 (V); (epitope 2) d) ARSQDLDAI (SEQID NO:8), wherein amino the acid e) T at position 152 changed to any amino acid residue; residue at position 4 (Q) is substituted with Kor R, or (epitope 2) wherein the amino acid residue at position 7 (D) is f) I at position 184 changed to any amino acid residue; Substituted with K or R, or wherein the amino acid 15 (epitope 3) g) V at position 189 changed to any amino acid residue; sequence ARSQDLDAI (SEQ ID NO:8) comprises (epitope 3) substitutions with K or R in any combination at both h) Q at position 194 changed to any amino acid residue. positions 4 (Q) and 7 (D): (epitope 4) e) LRVYVPRSS (SEQID NO:9), wherein amino the acid i) Dat position 197 changed to any amino acid residue; residue at position 1 (L) is substituted with A, or (epitope 4) wherein the amino acid residue at position 2 (R) is j) L at position 233 changed to any amino acid residue, Substituted with D, S or A, or wherein the amino acid (epitope 5) residue at position 9 (S) is substituted with D, E, N, K, k) R at position 234 changed to any amino acid residue. P or T, or wherein the amino acid sequence 25 (epitope 5) 1) S at position 241 changed to any amino acid residue; LRVYVPRSS (SEQ ID NO:9) comprises two or more (epitope 5) of said Substitutions in any combination; m) I at position 321 changed to any amino acid residue. f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid (epitope 6) residues at one or more of positions 1, 4, 6 and 7 are 30 n) K at position 324 changed to any amino acid residue. Substituted with a different amino acid residue, wherein (epitope 6) amino the acid residue at position 1 (I) is substituted o) Qat position 326 changed to any amino acid residue: with A. N. T. Q or H, or wherein the amino acid residue (epitope 6) at position 4 (K) is substituted with T, or wherein the p) A at position 327 changed to any amino acid residue. amino acid residue at position 6 (Q) is substituted with 35 (epitope 6) D, or wherein the amino acid residue at position 7 (A) q) any combination of one or more of a) through ao), is substituted with D, or wherein the amino acid wherein the amino acid numbering corresponds to SEQ sequence IPDKEQAIS (SEQ ID NO:10) comprises ID NO:1. Embodiments of the invention include an isolated poly two or more of said Substitutions in any combination; 40 peptide comprising a modified form of Pseudomonas exo g) ISFSTRGTQNWTVER (SEQ ID NO:131), wherein toxin A, or a fragment thereof, wherein said modified form, amino acid residues at one or more of positions 1, 6, 9, or fragment thereof, comprises one or more amino acid 10 and 12 are substituted with a different amino acid Substitutions selected from the group consisting of: residues wherein amino the acid residue at position 1 a) I at position 141 changed to A, (epitope 1) (I) is substituted with A. N. T. Q or H, or wherein the 45 b) I at position 141 changed to N: (epitope 1) amino acid residue at position 6 (R) is substituted with c) I at position 141 changed to T, (epitope 1) Q, or wherein the amino acid residue at position 9 (Q) d) I at position 141 changed to Q, (epitope 1) is substituted with N or T, or wherein amino the acid e) I at position 141 changed to H. (epitope 1) residue at position 10 (N) is substituted with Kor R, or f) R at position 146 changed to Q, (epitope 1) 50 g) O at position 149 changed to N, (epitope 1) wherein the amino acid residue at position 12 (T) is h) Q at position 149 changed to T; (epitope 1) Substituted with K or R, or wherein the amino acid i) Nat position 150 changed to R; (epitope 2) sequence ISFSTRGTQNWTVER (SEQ ID NO:131) j) Nat position 150 changed to K; (epitope 2) comprises two or more of said Substitutions in any k) T at position 152 changed to R; (epitope 2) combination; and 55 1) T at position 152 changed to K; (epitope 2) h) IVFGGVRARSQDLDAI (SEQ ID NO:132), wherein m) I at position 184 changed to A, (epitope 3) amino the acid residue at position 1 (I) is substituted n) I at position 184 changed to N: (epitope 3) with A or N, or wherein the amino acid residue at o) V at position 189 changed to D; (epitope 3) position 6 (V) is substituted with D. M. or N, wherein p) V at position 189 changed to M, (epitope 3) amino the acid residue at position 11 (Q) is substituted 60 q) V at position 189 changed to N: (epitope 3) with K or R, or wherein the amino acid residue at r) Q at position 194 changed to R; (epitope 4) position 14 (D) is substituted with Kor R, or wherein S) Q at position 194 changed to K; (epitope 4) the amino acid sequence IVFGGVRARSQDLDAI t) Dat position 197 changed to R; (epitope 4) (SEQ ID NO:132) comprises two or more of said u) D at position 197 changed to K; (epitope 4) Substitutions in any combination. 65 v) L at position 233 changed to A; (epitope 5) Embodiments of the invention include an isolated poly w) R at position 234 changed to D; (epitope 5) peptide comprising a modified form of Pseudomonas exo x) Rat position 234 changed to S.; (epitope 5) US 9,677,055 B2 31 y) R at position 234 changed to A, (epitope 5) - Continued Z) S at position 241 changed to D. (epitope 5) (peptide 16; SEQ ID NO: 26) ab) S at position 241 changed to E: (epitope 5) LEOCGYPWORLVALY; ac) S at position 241 changed to N: (epitope 5) (peptide 17; SEQ ID NO: 27) ad) S at position 241 changed to K; (epitope 5) 5 CGYPWORLVALYLAA; ae) S at position 241 changed to P. (epitope 5) (peptide 18; SEQ ID NO: 28) af) S at position 241 changed to T; (epitope 5) PWORLVALYLAARLS; ag) I at position 321 changed to A, (epitope 6) ah) I at position 321 changed to N: (epitope 6) (peptide 19; SEQ ID NO: 29) ai) I at position 321 changed to T; (epitope 6) 10 RLVALYLAARLSWNQ; ak) I at position 321 changed to Q, (epitope 6) (peptide 20; SEQ ID NO: 30) al) I at position 321 changed to H; (epitope 6) ALYLAARLSWNOWDO; am) K at position 324 changed to T; (epitope 6) an) Qat position 326 changed to D. (epitope 6) (peptide 21; SEQ ID NO: 31) ao) A at position 327 changed to D; (epitope 6) 15 LAARLSWNOWDOWIR; ap) any combination of one or more of a) through ao), (peptide 22; SEQ ID NO: 32) wherein the amino acid numbering corresponds to SEQ RLSKNOWDOWIRNAL; ID NO:1. (peptide 23; SEQ ID NO: 33) Embodiments of the invention comprise isolated polypep WNOWDOWIRNALASP; tides as described above, including polypeptides comprising (peptide 24; SEQ ID NO. 34) amino acid Substitutions introduced at each of amino acid WDQVIRNALASPGSG; positions 141, 146, 149, 150, 152, 184, 189, 194, 197,233, (peptide 25; SEQ ID NO: 35) 234, 241, 321, 324, 326 and 327 (in comparison to the amino WIRNALASPGSGGDL; acid sequence of SEQ ID NO:1). 25 Embodiments of the invention include isolated polypep (peptide 26; SEQ ID NO: 36) tides (proteins) and peptides comprising, or consisting of NALASPGSGGDLGEA; the following amino acid sequences: (peptide 27; SEQ ID NO: 37) ASPGSGGDLGEAIRE; 30 (peptide 1; SEQ ID NO: 11) (peptide 28; SEQ ID NO: 38) GGGGGSGGGGGSPEG; GSGGDLGEAIREQPE; (peptide 2; SEQ ID NO: 12) (peptide 29; SEQ ID NO: 39) GGSGGGGGSPEGGSL; GDLGEAIREOPEQAR; 35 (peptide 3; SEQ ID NO: 13) (peptide 3O; SEQ ID NO: 40) GGGGGSPEGGSLAAL; GEAIREOPEQARLAL; (peptide 4; SEQ ID NO: 14) (peptide 31; SEQ ID NO: 41) GGSPEGGSLAALTAH; IREOPEQARLALTLA; (peptide 5; SEQ ID NO: 15) 40 (peptide 32; SEQ ID NO: 42) PEGGSLAALTAHOAC; QPEQARLALTLAAAE; (peptide 6; SEQ ID NO: 16) (peptide 33; SEQ ID NO: 43) GSLAALTAHOACHLP; QARLALTLAAAESER; (peptide 7; SEQ ID NO: 17) 45 (peptide 34; SEQ ID NO: 44) AALTAHOACHLPLET; LALTLAAAESERFVR; (peptide 8; SEQ ID NO: 18) (peptide 35; SEQ ID NO: 45) TAHOACHLPLETFTR; TLAAAESERFVROGT; (peptide 9; SEQ ID NO: 19) 50 (peptide 36; SEQ ID NO: 46) QACHLPLETFTRORQ; AAESERFVROGTGND; (peptide 10; SEQ ID NO: 2O) (peptide 37; SEQ ID NO: 47) HLPLETFTRHROPRG; SERFVROGTGNDEAG;

(peptide 11; SEQ ID NO: 21) 55 (peptide 38; SEQ ID NO: 48) LETFTRHROPRGWEO; FVROGTGNDEAGAAS; (peptide 12; SEQ ID NO: 22) (peptide 39; SEQ ID NO: 49) FTRHROPRGWEOLEO; QGTGNDEAGAASGPA; (peptide 40; SEQ ID NO: 50) (peptide 13; SEQ ID NO. 23) 60 HRQPRGWEOLEOCGY; GNDEAGAASGPADSG; (peptide 14; SEQ ID NO: 24) (peptide 41; SEQ ID NO: 51) PRGKEOLEOCGYPWQ; EAGAASGPADSGDAL; (peptide 15; SEQ ID NO: 25) (peptide 42; SEQ ID NO: 52) WEQLEQCGYPWORLV; 65 AASGPADSGDALLER;

US 9,677,055 B2 36 - Continued NO:1 or SEQID NO:4; and amino acids corresponding (peptide 105; SEQ ID NO: 115 to Gly-381 to Glu-399 in SEQ ID NO:133 or SEQ ID IPSAIPTDPRNVGGD; NO:134; see e.g., FIG. 1); (peptide 106; SEQ ID NO: 116) (c) Amino-terminal portion of Domain IB (e.g., amino AIPTDPRNVGGDLDP; acids corresponding to Ala-365 to Ala-380 in SEQ ID (peptide 107; SEQ ID NO: 117 NO:133 or SEQ ID NO:134; see e.g., FIG. 1); TDPRNVGGDLDPSSI; (d) Domain IB (i.e., amino acid sequences intervening (peptide 108; SEQ ID NO: 118 between Domains II and III; e.g., amino acids corre RNVGGDLDPSSIPDK; 10 sponding to Ala-365 to Glu-399 in SEQ ID NO:133 or (peptide 100; SEQ ID NO: 119 SEQ ID NO:134; see e.g., FIG. 1); GGDLDPSSIPDKEQA; (e) a carboxy-terminal tail selected from the group con (peptide 2; SEQ ID NO: 122) sisting of: PDKEOAISALPDYAS; 15 (peptide 3; SEQ ID NO: 123) (i) EQAISALPDYASQPG; (SEQ ID NO: 135) Arg-Glu-Asp-Leu-Lys; (peptide 4; SEQ ID NO: 124) ISALPDYASQPGKPP; (ii) (SEQ ID NO: 136) (peptide 5; SEQ ID NO: 125 Arg-Glu-Asp - Lleu; LPDYASOPGKPPRED; and

(peptide 6; SEQ ID NO: 126 (iii) YASOPGKPPREDLN; (SEO ID NO : 137) 25 Lys-Asp-Glu-Leu, (peptide 7, SEO ID NO: 127 ITGPEEEGGRLDTIL; wherein one or more of said domains has been modified (peptide 8; SEQ ID NO: 128 with amino acid Substitutions, as described herein, to PEEEGGRLDTILGKP; reduce or eliminate immunogenicity. 30 (peptide 9; SEQ ID NO: 129 Embodiments of the invention further comprise PE vari EGGRLDTILGWPLAE; ants. For example, such variants include, without limitation, and PE polypeptide examples as shown in SEQ ID Nos: 143 to (peptide 120; SEQ ID NO: 130 163 and SEQ ID No: 175. RLDTILGWPLAERTW 35 Embodiments of the invention comprise any one or more Embodiments of the invention also comprise or consist of of the PE-A domains indicated in the preceding paragraphs, isolated polypeptides (proteins) and peptides comprising or wherein said one or more domains are chemically linked, consisting of the above-referenced amino acids sequences, covalently coupled, or fused (i.e., as in-frame fusion pro except wherein one or more amino acids have been Substi 40 teins) with a heterologous polypeptide (for example, Such as tuted with conservative amino acids substitutions. Embodi ligand or antigen-binding polypeptide). ments of the invention also comprise or consist of isolated Embodiments of the invention include polypeptides com polypeptides (proteins) and peptides comprising or consist prising PE wherein one or more amino acids are substituted ing of the above-referenced amino acids sequences, except with any combination of one or more conservative amino wherein one or more amino acids have been substituted with 45 acid substitutions, non-conservative amino acid substitu amino acids which are naturally occurring, non-naturally tions, non-naturally occurring amino acid Substitutions, non occurring, non-standard amino acids, or amino acid analogs. standard amino acids, and/or substitutions with amino acid Embodiments of the invention include polypeptides com analogs and further wherein said polypeptides are non prising a PE-ADomain III (i.e., a cytotoxic domain; see e.g., immunogenic or exhibit reduced immunogenicity as deter FIG. 1). Examples of sequences comprising a cytotoxic 50 mined and assayed by comparison to immunogenicity of portion of PE can be found in SEQ ID NO:1 and SEQ ID corresponding non-amino acid Substituted forms of PE; as NO:4 spanning amino acid residues Phe-134 to Lys-347. measured using in vitro or in vivo assays. In particular Examples of sequences comprising a cytotoxic portion of PE embodiments, amino acid substituted forms of PE are at can also be found in SEQID NO:133 and SEQID NO:134 55 least 25%, at least about 25%, at least 50%, at least about spanning amino acid residues Phe-400 to Lys-613. 50%, at least 75%, or at least about 75% less immunogenic Embodiments of the invention include polypeptides com compared to corresponding non-amino acid Substituted prising a PE-ADomain III (i.e., a cytotoxic domain) and one forms of PE. In particular embodiments, amino acid substi or more PE-A domains selected from the group consisting tuted forms of PE are at least 2-fold, at least about 2-fold, at of: 60 least 3-fold, at least about 3-fold, at least 4-fold, at least (a) Domain II (i.e., a cytosolic translocation domain, e.g., about 4-fold, at least 5-fold, at least about 5-fold, at least amino acids corresponding to Gly-3 to Ser-114 in SEQ 10-fold, at least about 10-fold, at least 50-fold, at least about ID NO:1 or amino acids corresponding to Gly-3 to 50-fold, at least 100-fold, at least about 100-fold, at least Asn-114 in SEQ ID NO:4, see e.g., FIG. 1); 65 500-fold, at least about 500-fold, at least 1000-fold, or at (b) Carboxy-terminal portion of Domain IB (e.g., amino least about 1000-fold less immunogenic compared to cor acids corresponding to Gly-115 to Glu-133 in SEQ ID responding non-amino acid Substituted forms of PE. In one US 9,677,055 B2 37 38 embodiment, amino acid substituted forms of PE are non bition of protein synthesis as measured using in vitro or in immunogenic or exhibit undetectable immunogenicity com Vivo assays. In particular embodiments, amino acid Substi pared to corresponding non-amino acid Substituted forms of tuted forms of PE exhibit 100% or about 100% of biological PE. activity compared to corresponding non-amino acid Substi The immunogenicity of Substituted peptides may be mea tuted forms of PE. In particular embodiments, amino acid Sured via assays routinely known and used by those of skill substituted forms of PE exhibit at least 95%, or at least about in the art. For example, immunogenicity may be assayed by 95% of biological activity compared to corresponding non methods including, but not limited to, the proliferation amino acid substituted forms of PE. In particular embodi assays described in Example 1 herein. 10 ments, amino acid substituted forms of PE exhibit at least Additionally, methods for predicting, and assays for 90%, at least about 90%, at least 85%, at least about 85%, assessing, immunogenicity include those methods and at least 80%, at least about 80%, at least 75%, at least about assays such as described or referenced in: 75%, at least 70%, at least about 70%, at least 60%, at least Baker M P and Jones T. D. Identification and removal of about 60%, at least 50%, or at least about 50% of biological immunogenicity in therapeutic proteins. Curr: Opin. 15 activity compared to corresponding non-amino acid Substi Drug. Disc. Dev. 2007 10(2): 219-227. tuted forms of PE. Bryson C J, Jones T D. Baker M. P. Prediction of immuno Embodiments of the invention further comprise fusion genicity of therapeutic proteins: validity of computational proteins, conjugates, covalently-linked, and non-covalently tools. BioDrugs. 2010; 24(1): 1-8. linked amino acid substituted forms of PE, or fragments Chester, K. Baker, M P and Mayer A. Overcoming the thereof, as described herein. Amino acid substituted forms of immunologic response to foreign enzymes in cancer PE may be fused, conjugated or otherwise linked with any therapy. Expert Rev. Clin. Immunol 2006 1(4): 549-559. artificial, recombinant, or naturally occurring molecule or Hochuli E. immunogenicity: technical evaluation polypeptide to modify PE activity and/or PE localization/ of interferon-alpha2a.J Interferon Res. 1997 17 25 targeting, Such as by conferring to PE, via said fusion or Suppl 1:S15-21. conjugation, the tissue targeting, cell targeting, or Sub Jaber A and Baker MP Assessment of the immunogenicity cellular localization properties of the molecule to which PE of different interferon beta-1a formulations using ex vivo is fused, conjugated or otherwise linked. For example, but T cell assays. J Pharm Biomed Anal 2007 43(4): 1256-61. 30 without limitation, amino acid Substituted forms, or frag Perry L. C. Jones T D and Baker M. P. New approaches to ments thereof of PE may be fused, conjugated, or otherwise prediction of immune responses to therapeutic proteins linked with any type of antibody or antigen-binding frag during preclinical development. Drugs R D. 2008 9(6): ments thereof, cell-surface receptor, secreted or cell-surface 385-96. ligand, or fragments thereof. Schellekens, H., Ryff, J. C., and Van Der Meide, P. H. Assays 35 In one embodiment, amino acid substituted forms of PE, for antibodies to human interferon-alpha: the need for including amino acid substituted forms of PE fused, conju standardization. J. Interferon Cytokine Res. 1997 gated or otherwise linked to another molecule or polypeptide 17(Suppl. 1), S5-S8. are useful in the treatment of cancer, including, but not Embodiments of the invention include polypeptides com 40 limited to, types of cancer described herein. In one embodi prising PE wherein one or more amino acids are substituted ment, amino acid substituted forms of PE as described with any combination of one or more conservative amino herein are useful for the preparation of a medicament for the acid substitutions, non-conservative amino acid substitu treatment of cancer, including, but not limited to, types of tions, non-naturally occurring amino acid Substitutions, non cancer described herein. standard amino acids, and/or substitutions with amino acid 45 In one embodiment, amino acid substituted forms of PE, analogs and further wherein said polypeptides retain bio or fragments thereof, may be fused, conjugated, or otherwise logical activity as determined and assayed by comparison to linked, without limitation, antigen-binding moieties such as biological activities of corresponding non-amino acid Sub antibodies, or fragments thereof, which specifically or pref stituted forms of PE; such as, but not limited to, cell killing erentially bind to disease associated antigens. Such mol activity, cell cytotoxicity, inactivation of the translation ecules include, for example, but without limitation, antibod elongation factor EF-2, ADP-ribosylation of EF-2, and inhi ies indicated in Table 1. TABLE 1. Examples of Antibodies and Therapeutic Uses TRADE Putative Antigen Example(s) of Therapeutic NAME NAME Targets Use

3F8 GD2 neuroblastoma ABAGOVOMAB CA-125 ovarian cancer (imitation) ABCIXIMAB REOPRO CD41 ( platelet aggregation alpha-IIb) inhibitor HUMIRA TNF-C. etc. ADECATUMUMAB EpCAM prostate and breast cancer TNF-C. Sepsis US 9,677,055 B2 39 40 TABLE 1-continued Examples of Antibodies and Therapeutic Uses TRADE Putative Antigen Example(s) of Therapeutic NAME NAME Targets Use AFUTUZUMAB CD2O lymphoma ALACIZUMAB VEGFR2 C8Ce PEGOL ALD518 IL-6 rheumatoid arthritis CAMPATH, CD52 CLL, CTCL MABCAMPATH ALTUMOMAB HYBRI CEA colorectal cancer PENTETATE CEAKER (diagnosis) ANATUMOMAB TAG-72 non-Small cell lung MAFENATOX carcinoma L-13 antigen-induced pulmonary inflammation, asthma ATOLIZUMAB HLA-DR hematological cancers ARCITUMOMAB CEA-SCAN CEA gastrointestinal cancers (diagnosis) L-selectin Severely injured patients (CD62L) ATLIZUMAB ACTEMRA, L-6 receptor rheumatoid arthritis ROACTEMRA Rhesus factor hemolytic disease of the newborn BAPINEUZUMAB beta amyloid Alzheimer's disease SIMULECT CD25 (C. chain of prevention of organ L-2 receptor) transplant rejections BAVITUXIMAB phosphatidylserine cancer, viral BECTUMOMAB LYMPHOSCAN CD22 non-Hodgkin's lymphoma (detection) BENLYSTA, BAFF non-Hodgkin lymphoma LYMPHOSTATB etc. CD12S asthma ERTILIMUMAB CCL11 (eotaxin-1) Severe allergic disorders SCINTLMUN CEA-related inflammatory lesions and antigen metastases (detection) EVACIZUMAB AVASTIN VEGF-A metastatic cancer CIROMAB FIBRISCENT fibrin II, beta chain thromboembolism (diagnosis) VATUZUMAB CD44 w8 Squamous cell carcinoma ERTANSINE LINATUMOMAB CD19 C8Ce RENTUXIMAB CD30 (TNFRSF8) hematologic cancers EDOTIN RIAKINUMAB IL-I2, IL-23 psoriasis, rheumatoid arthritis, inflammatory bowel diseases, multiple Sclerosis ILARIS IL-1 rheumatoid arthritis CANTUZUMAB mucin CanAg colorectal cancer etc. MERTANSINE CAPROMAB PROSTASCINT prostatic prostate cancer (detection) PENDETIDE carcinoma cells CATUMAXOMAB REMOVAB EpCAM, CD3 ovarian cancer, malignant ascites, gastric cancer CC49 TAG-72 tumor detection CD4 prevention of organ transplant rejections, treatment of autoimmune diseases CERTOLIZUMAB CIMZIA TNF-ct, Crohn's disease PEGOL ETUXIMAB ERBITUX EGFR metastatic colorectal cancer and head and neck cancer TATUZUMAB EpCAM ovarian cancer and other OGATOX Solid tumors CXUTUMUMAB IGF-1 receptor Solid tumors LENOLIXIMAB CD4 rheumatoid arthritis LIVATUZUMAB MUC1 pancreatic cancer ETRAXETAN ONATUMUMAB TRAIL-R2 R6261 Influenza. A infectious diseasefinfluenza. A hemagglutinin DACETUZUMAB CD40 hematologic cancers ZENAPAX CD25 (C. chain of prevention of organ IL-2 receptor) transplant rejections US 9,677,055 B2 41 TABLE 1-continued Examples of Antibodies and Therapeutic Uses TRADE Putative Antigen Example(s) of Therapeutic NAME NAME Targets Use DARATUMUMAB CD38 (cyclic ADP myleoma, CD38-positive ribose hydrolase) multiple myeloma PROLLA RANKL osteoporosis, bone metastases etc. DETUMOMAB B-lymphoma cell ymphoma DORLIMOMAB auto immune auto immune disorders ARITOX associated antigen DORLIXIZUMAB CD3 type 1 diabetes, autoimmune diseases ECROMEXIMAB GD3 ganglioside malignant melanoma SOLIRIS C5 paroxysmal nocturnal hemoglobinuria Endotoxin Sepsis caused by Gram negative bacteria EDRECOLOMAB PANOREX EpCAM colorectal carcinoma RAPTIVA LFA-1 (CD11a) psoriasis (blocks T-cell migration) MYCOGRAB Hsp90 invasive Candida ELOTUZUMAB SLAMF7 multiple myeloma L-6 Lymphoma, myeloma ENLIMOMAB, CAM-1 (CD54) stroke ENLIMOMAB PEGOL EPITUMOMAB Episialin C8Ce CITUXETAN CD22 cancer, SLE TGB2 (CD18) heart attack, stroke, traumatic shock ERTUMAXOMAB REXOMUN HER2/neu, CD3 breast cancer etc. ETARACIZUMAB ABEGRIN integrin CVB3 melanoma, prostate cancer, ovarian cancer etc. hepatitis B surface hepatitis B antigen EANOLESOMAB NEUTROSPEC CD15 appendicitis (diagnosis) interferon receptor autoimmune disorders EARLETUZUMAB olate receptor 1 ovarian cancer respiratory respiratory syncytial virus syncytial virus infection L-22 rheumatoid arthritis, psoriasis FIGITUMUMAB GF-1 receptor adrenocortical carcinoma, non-Small cell lung carcinoma etc. HUZAF FN-y Crohn's disease etc. rabies virus rabies (prophylaxis) glycoprotein TGF-B idiopathic pulmonary fibrosis, focal segmental glomerulosclerosis, cancer CD8O B-cell lymphoma GANTENERUMAB beta amyloid Alzheimer's disease CD147 () graft versus host disease GEMTUZUMAB MYLOTARG CD33 acute myelogenous OZOGAMICIN leukemia GIRENTUXIMAB RENCAREX carbonic clear cell renal cell anhydrase 9 (CA- carcinoma IX) GLEMBATUMUMAB GPNMB melanoma, breast cancer VEDOTIN SIMPONI TNF-ct, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis GOMILIXIMAB CD23 (IgE allergic asthma receptor) CD4 HIV infection IBRITUMOMAB ZEVALIN CD2O non-Hodgkin's lymphoma TIUXETAN IGOVOMAB INDIMACIS- CA-125 ovarian cancer (diagnosis) 125 IMCIROMAB MYOSCINT cardiac myosin cardiac imaging REMICADE TNF-ct, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, Crohn's disease, ulcerative colitis US 9,677,055 B2 43 44 TABLE 1-continued Examples of Antibodies and Therapeutic Uses TRADE Putative Antigen Example(s) of Therapeutic NAME NAME Targets Use NTETUMUMAB CD51 Solid tumors (prostate cancer, melanoma) NOLIMOMAB CD25 (C. chain of graft versus host disease IL-2 receptor) NOTUZUMAB CD22 C8Ce OZOGAMICIN PILIMUMAB YERVOY CD152 melanoma RATUMUMAB CD30 (TNTRSF8) Hodgkin's lymphoma KELIXLMAB CD4 chronic asthma LABETUZUMAB CEA-CIDE CEA colorectal cancer IL-13 asthma NCA-90 diagnostic agent (granulocyte antigen) TGFbeta 2 reduction of Scarring after glaucoma Surgery LEXATUMUMAB TRAIL-R2 C8Ce hepatitis B surface hepatitis B antigen LINTUZUMAB CD33 C8Ce LORVOTUZUMAB CDS6 C8Ce MERTANSINE LUCATUMUMAB CD40 multiple myeloma, non Hodgkin's lymphoma, Hodgkin's lymphoma CD23 (IgE chronic lymphocytic receptor) leukemia MAPATUMUMAB TRAIL-R1 C8Ce T-cell receptor autoimmune disorders MATUZUMAB EGFR colorectal, lung and stomach cancer BOSATRIA IL-5 asthma and white blood cell diseases TGFbeta 1 systemic scleroderma MILATUZUMAB CD74 multiple myeloma and other hematological malignancies MINRETUMOMAB TAG-72 C8Ce MITUMOMAB GD3 ganglioside Small cell lung carcinoma Rhesus factor disease antigen NUMAX respiratory respiratory syncytial virus syncytial virus (prevention) MUROMONAB- ORTHOCLONE CD3 prevention of organ CD3 OKT3 transplant rejections NACOLOMAB C242 antigen colorectal cancer TAFENATOX NAPTUMOMAB ST4 non-Small cell lung ESTAFENATOX carcinoma, renal cell carcinoma TYSABRI integrin C4 multiple sclerosis, Crohn's disease Endotoxin Sepsis NECITUMUMAB EGFR non-Small cell lung carcinoma TNF-ct, auto immune disorders NIMOTUZUMAB THERACIM, EGFR Squamous cell carcinoma, THERALOC head and neck cancer, nasopharyngeal cancer, glioma NOFETUMOMAB VERLUMA cancer-associated cancer (diagnosis) MERPENTAN antigen CD2O rheumatoid arthritis, lupus erythematosus etc. LFA-1 (CD11a) prevention of organ transplant rejections, immunological diseases OFATUMUMAB ARZERRA CD2O chronic lymphocytic leukemia OLARATUMAB PDGF-RC C8Ce XOLAIR IgE Fc region allergic asthma OPORTUZUMAB EpCAM C8Ce MONATOX OREGOWOMAB OVAREX CA-125 ovarian cancer CD3 diabetes mellitus type 1 US 9,677,055 B2 45 46 TABLE 1-continued Examples of Antibodies and Therapeutic Uses TRADE Putative Antigen Example(s) of Therapeutic NAME NAME Targets Use lipoteichoic acid Sepsis (Staphylococcus) SYNAGIS, respiratory respiratory syncytial virus ABBOSYNAGIS syncytial virus (prevention) PANITUMUMAB VECTIBIX EGFR colorectal cancer Pseudomonas Pseudomonas aeruginosa aeruginosa infection IL-4 asthma REMTUMOMAB THERAGYN MUC1 C8Ce PERTUZUMAB OMNITARG HER2/neu. C8Ce C5 reduction of side effects of cardiac Surgery PINTUMOMAB adenocarcinoma adenocarcinoa antigen CD4 Crohn's disease, multiple Sclerosis PRITUMUMAB vimentin brain cancer PRO 140 CCR5 HIV infection rabies virus rabies (prophylaxis) glycoprotein RAMUCIRUMAB VEGFR2 Solid tumors RANIBIZUMAB LUCENTIS VEGF-A macular degeneration (wet form) anthrax toxin, anthrax (prophylaxis and protective antigen treatment) cytomegalovirus cytomegalovirus infection glycoprotein B IL-5 inflammations of the airways, skin and gastrointestinal tract RILOTUMUMAB HGF Solid tumors MABTHERA, CD2O lymphomas, leukemias, RITUXAN Some autoimmune disorders ROBATUMUMAB IGF-1 receptor C8Ce IFN-O. systemic lupus erythematosus LEUKARREST CD11, CD18 haemorrhagic shock ANTOVA CD154 (CD4OL) rheumatic diseases SATUMOMAB TAG-72 C8Ce PENDETIDE cytomegalovirus cytomegalovirus infection SIBROTUZUMAB FAP C8Ce IFN-O. SLE, dermatomyositis, polymyositis SILTUXIMAB IL-6 C8Ce CD2 psoriasis, graft-versus-host disease (prevention) SOLANEZUMAB beta amyloid Alzheimer's disease SONEPCIZUMAB sphingosine-1- choroidal and retinal phosphate neovascularization SONTUZUMAB episialin disease antigen STAMULUMAB myostatin muscular dystrophy SULESOMA LEUKOSCAN NCA-90 osteomyelitis (imaging) (granulocyte antigen) TACATUZUMAB AFP-CIDE alpha-fetoprotein C8Ce TETRAXETAN TADOCIZUMAB integrin C.IIbf83 percutaneous coronary intervention IgE allergic reaction TANEZUMAB NGF pain TAPLITUMOMAB CD19 C8Ce PAPTOX AUREXIS clumping factor A Staphylococcusatiretts infection TELIMOMAB autoimmune autoimmune disorders ARITOX antigen TENATUMOMAB tenascin C C8Ce CD40 autoimmune disorders CD3 diabetes mellitus type 1 TGN1412 CD2 chronic lymphocytic leukemia, rheumatoid arthritis US 9,677,055 B2 47 48 TABLE 1-continued Examples of Antibodies and Therapeutic Uses TRADE Putative Antigen Example(s) of Therapeutic NAME NAME Targets Use

TICILIMUMAB CTLA-4 C8Ce TIGATUZUMAB TRAIL-R2 C8Ce TNX-6SO IL-13 Hodgkin's lymphoma ACTEMRA, IL-6 receptor rheumatoid arthritis ROACTEMRA CD154 (CD4OL) rheumatoid arthritis, lupus nephritis TOSITUMOMAB BEXXAR CD2O follicular lymphoma TRASTUZUMAB HERCEPTIN HER2/neu. breast cancer CTLA-4 C8Ce TUCOTUZUMAB EpCAM C8Ce CELMOLEUKIN hepatitis B virus chronic hepatitis B Escherichia coi diarrhoea caused by E. coli STELARA IL-12, IL-23 multiple Sclerosis, psoriasis, psoriatic arthritis WAPALIXIMAB AOC3 (VAP-1) autoimmune disorders integtin C437 Crohn's disease, ulcerative colitis VELTUZUMAB CD2O non-Hodgkin's lymphoma AOC3 (VAP-1) inflammation NUVION CD3 Crohn's disease, ulcerative colitis VOLOCIXIMAB integrin C.531 Solid tumors WOTUMUMAB HUMASPECT tumor antigen colorectal tumors CTA A16.88 ZALUTUMUMAB HUMAX- EGFR Squamous cell carcinoma of EGFR the head and neck HUMAX-CD4 CD4 rheumatoid arthritis, psoriasis, T-cell lymphoma ZIRALIMUMAB CD147 (basigin) autoimmune disorders ZOLIMOMAB CD5 systemic lupus ARITOX erythematosus, graft-versus host disease

In certain embodiments, amino acid Substituted forms of 40 In certain embodiments, amino acid Substituted forms of PE, or fragments thereof, may be fused, conjugated, or PE, or fragments thereof, may be generated as in-frame otherwise linked, without limitation, to naturally occurring polypeptide fusion proteins with molecules (such as, but not normal or disease related molecules such as secreted, extra limited to, those referenced above) wherein the PE moiety is cellular, intracellular, transmembrane, or cell-surface-bound either an amino-terminal portion or a carboxyl-terminal molecules or fragments thereof (or non-naturally occurring 45 portion of the fusion protein. Determination of which of variants and fragments thereof). Such as without limitation: these two configurations provides the desired results and/or ligands, receptors, receptor extracellular domains, cytok biological activities may be determined by routine experi ines, growth factors, cell signaling proteins, extracellular mentation practiced by those skilled in the art. and intracellular enzymes, structural proteins, cell adhesion 50 In certain embodiments, amino acid Substituted forms of proteins and molecules, cluster of differentiation (CD) mol PE, or fragments thereof, may be generated as fusion ecules, mitogens, cell division regulating molecules, cancer/ proteins wherein heterologous amino acid sequences (such tumor markers and antigens, et cetera. In certain embodi as cell targeting sequences) are inserted within the amino ments, molecules which are normally transmembrane and 55 acid substituted form of PE (i.e., heterologous amino acids cell-surface bound polypeptides may be fused or conjugated are flanked at the amino terminus and at the carboxy to amino acid substituted forms of PE as polypeptide frag terminus by PE amino acid sequences). An example of a ments lacking at least their transmembrane domains or non-amino acid substituted form of PE in Such a configu polypeptide regions responsible for cell-surface binding. ration is demonstrated in U.S. Pat. No. 8,854,044 wherein a In certain embodiments, molecules may be fused or 60 TGF-C. polypeptide is incorporated at amino acid residues conjugated to amino acid substituted forms of PE wherein 607 to 604 within a “PE37 polypeptide sequence. See e.g., Such molecules possess or retain the ability (even as fusion U.S. Pat. No. 8,854,044, FIG. 1. proteins or protein conjugates) to form multimeric com Some examples of molecules which may be fused, con plexes (such as hetero- and homopolymers including, but 65 jugated, or otherwise linked to amino acid substituted forms not limited to, dimers, trimers, tetramers, pentamers, hex of PE, include for example, but without limitation, those amers, et cetera.) such as indicated in Table 2. US 9,677,055 B2 49 50 TABLE 2 Examples of Potential Compounds and Indications to which Amino Acid Substituted Forms of PE May Be Fused or Conjugated for Therapeutic Use Note: The potential indications and nucleic acid and amino acid sequences shown in Table 2 (as well as accession numbers listed) are presented for purposes of providing a few illustrative examples only. Thus, embodiments of the invention may or may not comprise these indications and sequences. Accordingly, it is envisioned that embodiments of the invention comprise other indication uses as well as other molecules and sequence variants (e.g., naturally occurring variants (such as allelic or polymorphic variants) and non-naturally occurring variants (such as genetically engineered or mutated variants) of these sequences wherein one or multiple amino acids are changed and/or wherein only a fragment or fragments of such sequences are fused or conjugated to amino acid substituted forms of PE. Hence, the examples shown in Table 2 should in no manner be considered limiting with respect to potential therapeutic indications or protein fusions and conjugates HSY-AES-ASEA-AAEEEof amino acid modified SS-S-S-E- forms of PE Example Molecule (Nucleotide Accession) Protein Potential Accession* * Indications Example Amino Acid Sequence Mesothelin Pancreatic cancer MALPTARPLLGSCGTPALGSLLFLLFSLGWWOPS (NM 013404) Ovarian cancer RTLAGETGOEAAPLDGVLANPPNISSLSPROLLG |NP 037536 FPCAEWSGLSTERVRELAVALAOKNVKLSTEOLR CLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPOA CTRFFSRITKANVDLLPRGAPERQRLLPAALACW GWRGSLLSEADWRALGGLACDLPGRFWAESAEWL LPRLVSCPGPLDODOOEAARAALOGGGPPYGPPS TWSVSTMDALRGLLPVLGOPIIRSIPOGIVAAWR ORSSRDPSWROPERTILRPRFRREVEKTACPSGK KAREIDESLIFYKKWELEACVDAALLATOMDRVN ATPFTYEOLDVLKHKLDELYPOGYPESVIOHLGY (FLKMSPEDIRKWNWTSLETLKALLEWNKGHEMS POAPRRPLPOVATLIDRFWKGRGOLDKDTLDTLT AFYPGYLCSLSPEELSSVPPSSIWAVRPODLDTC DPROLDWLYPKARLAFONMNGSEYFWKIOSFLGG APTEDLKALSOONVSMDLATFMKLRTDAVLPLTV AEWOKLLGPHVEGLKAEERHRPVRDWILRORODD LDTLGLGLOGGIPNGYLVLDLSMOEALSGTPCLL GPGPWLTVLALLLASTLA (SEQ ID NO: 167)

CD24 Liver cancer MGRAMVARLGLGLLLLALLLPTOTYSSETTTGTS (NM 013230) Colorectal cancer SNSSQSTSNSGLAPNPTNATTKAAGGALOSTASL AAH64619 Pancreatic cancer FWSLSLLHLYS (SEQ ID NO: 168) CD22 Hairy Cell Leukemia WRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEM (ABO13 007) Chronic Lymphocytic NIPRTG (SEO ID NO: 169 BAA36576 Leukemia Non-Hodgkin's Lymphoma

CD25 a.k.a., Hodgkin's MDSYLLMWGLLTFIMVPGCOAELCDDDPPEIPHA Lymphoma TFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCT receptor, Hairy Cell Leukemia GNSSHSSWDNOCOCTSSATRNTTKOWTPOPEEOK alpha chain Chronic ERKTTEMOSPMOPVDOASLPGHCREPPPWENEAT (NM 000417) Lymphocytic ERIYHFVVGOMVYYQCVOGYRALHRGPAESWCKM (NP 000408 Leukemia THGKTRWTOPOLICTGEMETSOFPGEEKPOASPE Cutaneious T-cell GRPESETSCLVTTTDFOIOTEMAATMETSIFTTE Lymphoma YOVAVAGCVFLLISWLLLSGLTWORRORKSRRTI Adult T-cell (SEO ID NO : 17 O) leukemia

CD174 Bladder cancer MDPLGAAKPOWPWRRCLAALLFOLLWAWCFFSYL a.k.a. , Breast cancer RVSRDDATGSPRAPSGSSRODTTPTRPTLLILLW Lewis Y, Colorectal cancer TWPFHIPVALSRCSEMVPGTADCHITADRKWYPO galactoside Esophageal cancer ADTVIVHHWDIMSNPKSRLPPSPRPOGORWIWFN 3 (4)-L- Gastric cancer LEPPPNCOHLEALDRYFNLTMSYRSDSDIFTPYG fucosyl- Pancreatic cancer WLEPWSGOPAHPPLNLSAKTELVAWAVSNWKPDS transterase Lung cancer ARVRYYOSLOAHLKVDWYGRSHKPLPKGTMMETL (NM_000149) SRYKFYLAFENSLHPDYITEKLWRNALEAWAWPW (NP_000140 WLGPSRSNYERFLPPDAFIHVDDFOSPKDLARYL OELDKDHARYLSYFRWRETLRPRSFSWALDFCKA CWKLOOESRYOTVRSIAAWFT (SEO ID NO: 171) US 9,677,055 B2 51 52 TABLE 2 - continued

Examples of Potential Compounds and Indications to which Amino Acid Substituted Forms of PE May Be Fused or Conjugated for Therapeutic Use Note: The potential indications and nucleic acid and amino acid sequences shown in Table 2 (as well as accession numbers listed) are presented for purposes of providing a few illustrative examples only. Thus, embodiments of the invention may or may not comprise these indications and sequences. Accordingly, it is envisioned that embodiments of the invention comprise other indication uses as well as other molecules and sequence variants (e.g., naturally occurring variants (such as allelic or polymorphic variants) and non-naturally occurring variants (such as genetically engineered or mutated variants) of these sequences wherein one or multiple amino acids are changed and/or wherein only a fragment or fragments of such sequences are fused or conjugated to amino acid substituted forms of PE. Hence, the examples shown in Table 2 should in no manner be considered limiting with respect to potential therapeutic indications or protein fusions and conjugates of amino acid modified forms of PE, Example Molecule (Nucleotide Accession) Protein Potential Accession* * Indications Example Amino Acid Sequence TPBG Non Small cell lung MPGGCSRGPAAGDGRLRLARLALWLLGWWSSSSF a.k.a., Calice TSSASSFSSSAPFLASAVSAOPPLPDQCPAL,CEC onco fetal Renal carcinoma SEAARTVKCVNRNLTEVPTDLPAYVRNLFLTGNO antigen 5T4, Pancreatic cancer LAWLPAGAFARRPPLAELAALNLSGSRLDEWRAG 5T4 onco fetal AFEHLPSLROLDLSHNPLADLSPFAFSGSNASVS trophoblast APSPLVELILNHIVPPEDERONRSFEGMVVAALL glycoprotein AGRALOGLRRLELASNHFLYLPRDVLAOLPSLRH (NM 0.06670) LDLSNNSLWSLTYWSFRNLTHLESLHLEDNALKW CAA09930 LHNGTLAELOGLPHIRVFLDNNPWVCDCHMADMV TWLKETEVVOGKDRLTCAYPEKMRNRVLLELNSA DLDCDPILPPSLOTSYWFLGIWLALIGAIFLLVL YLNRKGIKKWMHNIRDACRDHMEGYHYRYEINAD PRLTNLSSNSDV (SEO ID NO : 172)

CD56 Small cell lung MLOTKDLIWTLFFLGTAVSLOVDIVPSOGEISVG a.k.a. NCAM1 cancer ESKFFLCOVAGDAKDKDISWFSPNGEKLTPNOOR neural cell Merkel cell ISWWWNDDSSSTLTIYNANIDDAGIYKCWWTGED adhesion carcinoma GSESEATVNVKIFOKLMFKNAPTPOEFREGEDAV molecule 1 Ovarian cancer IWCDWWSSLPPTIIWKHKGRDWILKKDWRFIWLS isoform 1 Neuroendocrine NNYLOIRGIKKTDEGTYRCEGRILARGEINFKDI precursor tumors OWIVNVPPTIOARONIVNATANLGOSVTLVCDAE (NM 000615) Multiple Myeloma GFPEPTMSWTKDGEOIEQEEDDEKYIFSDDSSOL (NP 000606 TIKKVDKNDEAEYICIAENKAGEODATIHLKVFA KPKITY VENOTAMELEEOVTLTCEASGDPIPSIT WRTSTRNISSEEKTLDGHMWWRSHARWSSLTLKS IOYTDAGEYICTASNTIGODSOSMYLEVOYAPKL OGPWAVYTWEGNOVNITCEVFAYPSATISWFRDG OLLPSSNYSNTKIYNTPSASYLEVTPDSENDFGN YNCTAVNRIGOESLEFILVOADTPSSPSIDOVEP YSSTAOVOFDEPEATGGVPILKYKAEWRAVGEEV WHSKWYDAKEASMEGIWTIWGLKPETTYAWRLAA LNGKGLGEISAASEFKTOPVOGEPSAPKLEGOMG EDGNSIKVNLIKODDGGSPIRHYLVRYRALSSEW KPEIRLPSGSDHVMLKSLDWNAEYEVYWVAENOO GKSKAAHFVFRTSAOPTAIPANGSPTSGLSTGAI WGILIWIFWLLLWWWDITCYFLNKCGLFMCIAWN LCGKAGPGAKGKDMEEGKAAFSKDESKEPIWEWR TEEERTPNHDGGKHTEPNETTPLTEPEKGPWEAK PECOETETKPAPAEWKTVPNDATOTKENESKA (SEO ID NO : 173)

C-type lectin- Acute myeloid MWIDFFTY5SMSEEWTYADLOFON5SEMEKIPEI like leukemia GKFGEKAPPAPSHWWRPAALFLTLLCLILLIGIG molecule-1 VLASMFHVTLKIEMKKMNKLONISEELORNISLO a.k.a. CLL-1 LMSNMNISNKIRNLSTTLOTIATKLCRELYSKEO US 9,677,055 B2 53 54 TABLE 2- Continued Examples of Potential Compounds and Indications to which Amino Acid Substituted Forms of PE May Be Fused or Conjugated for Therapeutic Use Note: The potential indications and nucleic acid and amino acid sequences shown in Table 2 (as well as accession numbers listed) are presented for purposes of providing a few illustrative examples only. Thus, embodiments of the invention may or may not comprise these indications and sequences. Accordingly, it is envisioned that embodiments of the invention comprise other indication uses as well as other molecules and sequence variants (e.g., naturally occurring variants (such as allelic or polymorphic variants) and non-naturally occurring variants (such as genetically engineered or mutated variants) of these sequences wherein one or multiple amino acids are changed and/or wherein only a fragment or fragments of such sequences are fused or conjugated to amino acid substituted forms of PE. Hence, the examples shown in Table 2 should in no manner be considered limiting with respect to potential therapeutic indications or protein fusions and conjugates of amino acid modified forms of PE Example Molecule (Nucleotide Accession) Protein Potential Accession* * Indications Example Amino Acid Sequence (AY547296) EHKCKPCPRRWIWHKDSCYFLSDDVOTWOESKMA AAT11783) CAAONASLLKINNKNALEFIKSQSRSYDYWLGLS PEEDSTRGMRWDNIINSSAWWIRNAPDLNNMYCG YINRLY VOYYHCTYKORMICEKMANPVOLGSTYF REA (SEQ ID NO: 174) *"Nucleotide Accession" refers to the NCBI Reference Sequence accession number associated with the Corresponding nucleic acid sequence as found in the "Nucleotide" database provided for public access and searching (via the Internet) through the National Center for Biotechnology information, U.S. National Library of Medicine (8600 Rockville Pike, Bethesda MD 20894 USA (www.ncbi.nlm.nih.gov)). **"Protein. Accession" refers to the NCBI Reference Sequence accession number associated with the Corresponding amino acid sequence as found in the "Protein" database provided for public access and searching (via the Internet through the National Center for Biotechnology Information, U.S. National Library of Medicine (86OO Rockville Pike, Bethesda MD 20894 USA (www.ncbi.nlm.nih.gov)).

In one embodiment, the present invention includes iso 35 which nucleic acid(s) encoding modified forms of PE may lated nucleic acids and methods of expressing nucleic acids be introduced into a cell. These examples are in no way encoding any of the herein-referenced modified forms of PE, intended to limit the scope of that may be used for gene including fusions, conjugates, and otherwise linked mol delivery and expression of modified forms of PE in cells, ecules; whether Such forms are expressed from a single or tissues, or organisms; these examples are presented to illus one more separate polynucleotide sequences; whether Such 40 trate the many available methods. polynucleotide sequences are expressed from a single or one —Viral-Based Delivery of Target Nucleic Acids— or more separate expression vectors. Gene therapy based methods can be used to deliver (into Expression Vectors a host cell, tissue or organism) target nucleic acids encoding In one embodiment, the present invention includes meth 45 modified forms of PE (and other polynucleotides, as needed, ods of making and using recombinant expression vectors to to allow expression of the same). As one example, poly express nucleic acids encoding polypeptides comprising any nucleotides operably encoding the target nucleic acid can be of the herein-referenced modified forms of PE, including delivered to a tissue or organism either as “naked nucleic fusions, conjugates, and otherwise linked molecules. Use of acid' or as part of an expression vector. The term vector a wide variety of expression vectors are well-known and 50 includes for example, but is not limited to, Vectors such as routinely used by those skilled in the art. A few examples of plasmid vectors, cosmid vectors, artificial chromosome vec the types of expression vectors which may be used include, tors, and viral vectors. Some examples of viral vectors but are not limited to: derivatives of human or animal viruses include adenovirus, herpes simplex virus (HSV), alphavirus, (such as retrovirus, adeno-associated virus, pox, baculovi simian virus 40, picomavirus, vaccinia virus, retrovirus, rus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, 55 lentivirus, and adeno-associated virus. Vectors encoding geminivirus, and caulimovirus vectors) and insect viruses modified forms of PE may be capable of replication in a cell (such as baculovirus); yeast vectors; bacteriophage vectors in which it is introduced, or it may be preferred that the (e.g., bacteriophage lambda); plasmids; cosmids; artificial vector is not capable of replication. Vectors encoding modi chromosomes; liposomes; electrically charged lipids (cyto fied forms of PE may be capable of integration into the fectins); DNA-protein complexes, and biopolymers. 60 genomic DNA of a cell (and Subsequent expression there Gene Delivery and Expression Systems from), or it may be preferred that the vector is not capable A wide variety of methods (i.e., gene delivery systems) of integrating into the host genome. An example of a vector are available and well-known to those of skill in the art; any that can integrate into the genomic DNA of a cell is a of Such methods may be used for introducing nucleic acids retroviral vector, in which an integrase enzyme mediates encoding modified forms of PE into a cell, tissue, or organ 65 integration of the retroviral vector sequences. A vector may ism for in vitro, in vivo, in situ, or ex vivo expression. The also contain transposon sequences that facilitate integration methods referenced below represent examples of ways in of the coding region into the genomic DNA of a host cell. US 9,677,055 B2 55 56 Liposomes represent another manner in which target DNA Many other expression vectors can also be used to deliver may be delivered to a subject. (into a host cell, tissue or organism) target nucleic acids Selection of a vector depends upon a variety of desired encoding modified forms of PE (and other polynucleotides, characteristics in the resulting construct, such as a selection as needed, to allow expression of the same). For example, marker, vector replication rate, type of target host cell, 5 vectors derived from viruses such as vaccinia viruses, her species of host organism, desired duration of protein expres pesviruses, equine encephalitis viruses, hepatitis viruses and Sion. An expression vector optionally includes expression lentiviruses can be used. Methods of constructing and using control sequences operably linked to the coding sequence viral expression vectors as gene delivery systems are well Such that the coding region is expressed in the cell. The known to those of skill in the art. The examples of such invention is not limited by the use of any particular pro 10 vectors referenced herein are not intended to be limiting moter, and a wide variety is known. Promoters act as with respect to the means by which modified forms of PE regulatory signals that bind RNA polymerase in a cell to may be delivered and expressed in various host cells, tissues, initiate transcription of a downstream (3' direction) operably or organisms. linked coding sequence. The promoter used in the invention - Non-Viral Delivery of Modified Target Nucleic may be a constitutive or an inducible promoter. It can be, but 15 Acids— need not be, heterologous with respect to the cell to which In addition to viral delivery of modified target nucleic it is introduced. acid, the following are additional methods of recombinant In certain embodiments, adenovirus expression vectors gene delivery can be used to deliver (into a host cell, tissue can be used to deliver (into a host cell, tissue or organism) or organism) target nucleic acids encoding modified forms target nucleic acids encoding modified forms of PE (and of PE (and other polynucleotides, as needed, to allow other polynucleotides, as needed, to allow expression of the expression of the same). Methods of constructing and using same). The terms “adenovirus expression vector” is meant to non-viral gene delivery systems are well-known to those of include those constructs containing nucleic acid sequences skill in the art. See, for example, Al-Dosari et al., “Nonviral Sufficient to (a) Support packaging of the construct and (b) to gene delivery: principle, limitations, and recent progress.” ultimately express a recombinant gene construct that has 25 AAPS Journal, 11(4):671-681 (2009), and references cited been inserted therein. In contrast to retroviruses, use of therein. adenovirus vectors does not result in chromosomal integra In certain embodiments, electroporation can be used to tion because adenovirus DNA replicates in an episomal deliver (into a host cell, tissue or organism) target nucleic manner. Moreover, adenoviruses are considered to be struc acids encoding modified forms of PE (and other polynucle turally stable with no genome rearrangement occurring even 30 otides, as needed, to allow expression of the same). Methods after extensive virus reproduction and amplification. Meth of using electroporation are well-known to those of skill in ods of constructing and using adenovirus vectors as gene the art. See, for example, Bodles-Brakhop et al., “Electropo delivery systems are well-known to those of skill in the art. ration for the delivery of DNA-based vaccines and immu In certain embodiments, adeno-associated virus (AAV) notherapeutics: current clinical developments.” Mol. Ther., expression vectors can be used to deliver (into a host cell, 35 17(4):585-592 (2009); and references cited therein. See also, tissue or organism) target nucleic acids encoding modified Golzio et al., “Observations of the mechanisms of electro forms of PE (and other polynucleotides, as needed, to allow mediated DNA uptake from vesicles to tissues. Curr expression of the same). AAV may be desirable for a number Gene Ther, 10(4):256-266 (2010); and references cited or reasons; for example, because AAV vectors exhibit a high therein. See also, Andre et al., "Nucleic acids electrotransfer frequency of integration, can infect nondividing cells, and 40 in Vivo: mechanisms and practical aspects. Curr Gene have a broad host range. AAV is a dependent parvovirus in Then, 10(4):267-280 (2010), and references cited therein. that it requires coinfection with another virus (either adeno See also, Wells, “Electroporation and ultrasound enhanced virus or a member of the herpes virus family) to undergo a non-viral gene delivery in vitro and in vivo. Cell Biol productive infection in cultured cells. In the absence of Toxicol., 26(1):21-28 (2010); and references cited therein. coinfection with helper virus, the wild-type AAV genome 45 In certain embodiments, particle bombardment can be integrates through its ends into a human chromosome where used to deliver (into a host cell, tissue or organism) target it resides as a latent provirus. When a cell containing latent nucleic acids encoding modified forms of PE (and other AAV provirus is superinfected with a helper virus, the AAV polynucleotides, as needed, to allow expression of the genome is “rescued from the chromosome and a normal same). This method depends on the ability to accelerate productive infection is established. Methods of constructing 50 nucleic acid-coated microprojectiles to a sufficient Velocity and using AAV vectors as gene delivery systems are well to allow them to pierce cell membranes, thereby delivering known to those of skill in the art. nucleic acid “payloads, without killing them. Some typical In certain embodiments, retrovirus expression vectors can microprojectiles consist of biologically inert Substances be used to deliver (into a host cell, tissue or organism) target Such as tungsten, platinum, and gold beads. Methods of nucleic acids encoding modified forms of PE (and other 55 using particle bombardment are well-known to those of skill polynucleotides, as needed, to allow expression of the in the art. See, for example, Klein et al., “Particle bombard same). Retroviruses are a group of single-stranded RNA ment: a universal approach for gene transfer to cells and viruses characterized by the ability to convert their genomic tissues.” Curr. Opin. Biotechnol., 4(5):583-590 (1993); and RNA to double-stranded DNA in infected cells through a references cited therein. reverse-transcription process. The resulting DNA stably 60 In certain embodiments, a variety of methods incorporat integrates into cellular chromosomes as a provirus and ing calcium phosphate co-precipitation can be used to directs synthesis of viral proteins. Retroviral integration deliver (into a host cell, tissue or organism) target nucleic results in the retention of viral gene sequences in the acids encoding modified forms of PE (and other polynucle recipient cell and in its descendants. Retroviral vectors are otides, as needed, to allow expression of the same). Methods able to infect a broad variety of cell types. Methods of 65 of using calcium phosphate co-precipitation are well-known constructing and using retroviruses as gene delivery systems to those of skill in the art. See, for example, Uskokovi? et al., are well-known to those of skill in the art. “Nanosized hydroxyapatite and other calcium phosphates: US 9,677,055 B2 57 58 chemistry of formation and application as drug and gene WO 2003/027266 (PCT/US/2002/05234); WO 201Of delivery agents. J. Biomed. Mater. Res. B Appl. Biomater, 042189 (PCT/US2009/005510); and 96(1): 152-191 (2011), and references cited therein. See also, WO 2002/066612 (PCT/US2002/005090); WO 2011/ Colosimo et al., “Transfer and expression of foreign genes 119773 (PCT/US2011/029682). in mammalian cells. Biotechniques, 29(2):314-8, 320-322 For purposes of expressing polynucleotides and polypep (2000); and references cited therein. tides under control of a gene Switch mechanism, the term In certain embodiments, microinjection and Sonication “gene switch” refers to the combination of a response methods can be used to deliver (into a host cell, tissue or element associated with a promoter, and a ligand-dependent organism) target nucleic acids encoding modified forms of transcription factor-based system which, in the presence of PE (and other polynucleotides, as needed, to allow expres 10 one or more ligands, modulates the expression of a gene into sion of the same). Methods of using microinjection and which the response element and promoter are incorporated. sonication are well-known to those of skill in the art. See, for Stated otherwise, a “gene switch” refers to a peptide, protein example, Rochlitz et al., “Gene therapy of cancer. Swiss or polypeptide complex that functions to (a) bind an acti Med. Wkly, 131(1-2):4-9 (2001); and references cited 15 Vating ligand, and (b) regulate the transcription of a gene of therein. See also, Donnelly et al., “Microneedle-based drug interest in a ligand-dependent fashion. delivery systems: microfabrication, drug delivery, and In one embodiment, the polynucleotide encoding a gene safety.” Drug Deliv., 17(4): 187-207 (2010); and references Switch is a recombinant polynucleotide, i.e., a polynucle cited therein. See also. Miller et al., “Sonoporation: otide, that has been engineered, by molecular biological mechanical DNA delivery by ultrasonic cavitation'. Somat. manipulation, to encode the gene Switch. In another embodi Cell Mol. Genet., 27(1-6): 115-34 (2002); and references ment, the recombinant polynucleotide is a synthetic poly cited therein. nucleotide. In certain embodiments, liposomes and lipid formulations As used herein with respect to gene Switch regulation can be used to deliver (into a host cell, tissue or organism) systems, the term “dimerizes with the ligand binding domain target nucleic acids encoding modified forms of PE (and 25 that binds an activating ligand’ refers to a selective protein other polynucleotides, as needed, to allow expression of the protein interaction that is induced by the presence of acti same). Liposomes are vesicular structures characterized by Vating ligand. a phospholipid bilayer membrane and an inner aqueous As used herein, the term “ligand binding domain that medium. Multilamellar liposomes have multiple lipid layers binds an activating ligand’ refers to an amino acid sequence separated by aqueous medium. They form spontaneously 30 when phospholipids are suspended in an excess of aqueous that selectively binds an activating ligand. In the methods solution. An example of a commonly used, commercially disclosed herein, an activating ligand binds to a ligand available lipid formulation is Lipofectamine (Gibco BRL). binding domain, e.g., an ecdysone receptor ligand binding Methods of using liposomes and lipid formulations to domain, that is part of a ligand-dependent transcriptional deliver nucleic acids to cells, tissues and organisms are 35 activation complex that regulates the expression of a poly well-known to those of skill in the art. See, for example, nucleotide sequence that encodes a gene of interest. Hence, Xiong et al., “Cationic liposomes as gene delivery system: the expression of the gene of interest is regulated in a transfection efficiency and new application. Pharmazie, ligand-dependent fashion. 66(3):158-64 (2011); and references cited therein. See also, The term “ecdysone receptor-based, with respect to a Pichon et al., “Chemical vectors for gene delivery: uptake 40 gene Switch, refers to a gene Switch comprising at least a and intracellular trafficking. Curr Opin Biotechnol., 21(5): functional part of a naturally occurring or synthetic 640-645 (2010); and references cited therein. See also, ecdysone receptor ligand binding domain and which regu Pathak et al., “Recent trends in non-viral vector-mediated lates gene expression in response to a ligand that binds to the gene delivery,” Biotechnol J., 4(11):1559-1572 (2009). ecdysone receptor ligand binding domain. Expression of Modified Forms of PE Via Gene Switch 45 As used herein, 'selective binding of an activating ligand Modulation Systems to a ligand binding domain in a gene Switch means that the Expression of modified forms of PE, including fusions, ligand has an EC50 of about 700 nanomolar (nM), 650 nM, conjugates, and otherwise linked molecules, may be 600 nM, 550 nM, 500 nM, 450 nM, 400 nM, 350 nM, 300 expressed in host cells, tissues, and organisms using gene nM, 250 nM, 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, Switch expression systems. Some examples, without limita 50 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM 70 nM, 65 tion, of Such gene expression systems, and genetically nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, engineered cells comprising gene Switch expression sys 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, tems, which can be used to express polynucleotides and 5 nM, 4 nM, 3 nM, 2 nM or 1 nM, or less, in a gene switch polypeptides of the present invention, are described in the assay. following publications; each of which are hereby incorpo 55 As used herein, “EC50' is the “half maximal effective rated by reference herein: concentration,” which refers to the concentration of an WO 2009/045370 (PCT/US2008/011270); activating ligand that induces a gene Switch-regulated WO 2009/025866 (PCT/US2008/010040), WO 2002/ change in expression of a polynucleotide encoding an gene 066614 (PCT/US/2002/005706); of interest (e.g., modified forms of PE, including fusions, WO 2008/073154 (PCT/US2007/016747); WO 2002/ 60 conjugates, etcetera), that is halfway between the baseline 066613 (PCT/US2002/005090); level of expression and the maximum level of expression WO 2005/108617 (PCT/US2005/015089); WO 2002/ after a specified exposure time. Examples of cellular assays 029075 (PCT/US2001/030608); for measuring gene Switch-regulated gene expression are WO 2003/0/27289 (PCT/US2002/005026), WO 2001/ well known to those of skill in the art. See, for example, 070816 (PCT/US2001/090500); 65 Karzenowski et al., BioTechniques 39:191-200 (2005). WO 2002/066615 (PCT/US2002/005708), WO 2009/ In one embodiment, the ligand binding domain that binds 048560 (PCT/US2008/011563); an activating ligand, e.g., an ecdysone receptor ligand bind US 9,677,055 B2 59 60 ing domain, dimerizes with another ligand binding domain, fruit fly Drosophila melanogaster ecdysone receptor ligand e.g., a retinoid X receptor ligand binding domain, to form a binding domain, a mosquito Aedes aegypti ecdysone recep protein-protein complex. tor ligand binding domain, a blowfly Lucilia capitata In one embodiment, the expression of the gene of interest ecdysone receptor ligand binding domain, a blowfly Lucilia is regulated by an activating ligand in an on/off fashion that cuprina ecdysone receptor ligand binding domain, a blowfly is independent of the concentration or dosage of an activat Caliphora vicinia ecdysone receptor ligand binding ing ligand. In another embodiment, the expression of the domain, a Mediterranean fruit fly Ceratitis capitata gene of interest is regulated by an activating ligand in a ecdysone receptor ligand binding domain, a locust Locusta concentration (or dosage)-dependent fashion, i.e., there is a migratoria ecdysone receptor ligand binding domain, an dose-response relationship between the concentration (or 10 aphid Myzus persicae ecdysone receptor ligand binding dosage) of an activating ligand and the level of gene domain, a fiddler crab Celuca pugilator ecdysone receptor expression of the gene of interest. See, e.g., US Patent ligand binding domain, an ixodid tick Amblyomma ameri Publication No. 2009/0123441 (see also, WO 2009/0485.60 canum ecdysone receptor ligand binding domain, a whitefly (PCT/USUS2008/011563)). Bamecia argentifoli ecdysone receptor ligand binding The term “operably linked’ refers to the association of 15 domain, a leafhopper Nephotetix cincticeps ecdysone recep polynucleotide sequences on a single polynucleotide so that tor ligand binding domain, or a mutant thereof that binds An the function of one is affected by the other. For example, a activating ligand. promoter is operably linked with a coding sequence when it In another embodiment, the ecdysone receptor ligand is capable of affecting the expression of that coding binding domain is a spruce budworm Choristoneura sequence (i.e., that the coding sequence is under the tran filmiferana ecdysone receptor ligand binding domain, for Scriptional control of the promoter). Coding sequences can which the amino acid sequence is: Leu Thr Ala Asn Gln Gln be operably linked to regulatory sequences in sense or Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu antisense orientation. Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp In one embodiment, an activating ligand, or a composition Gln Gln Ala Asp Asp Glu ASn Glu Glu Ser Asp Thr Pro Phe thereof, is administered to a subject orally. In another 25 Arg Glin Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val embodiment, an activating ligand, or a composition thereof, Glu Phe Ala Lys Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln is administered to a subject parenterally. In another embodi Pro Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Val ment, an activating ligand, or a composition thereof, is Met Met Leu Arg Val Ala Arg Arg Tyr Asp Ala Ala Ser Asp administered Subcutaneously, intramuscularly, intrave Ser Val (position 107) Leu Phe Ala ASn Asn Gln Ala Tyr Thr nously, intraperitoneally, transdermally, or intratumorally. 30 Arg Asp Asn Tyr Arg Lys Ala Gly Metala Tyr (position 127) In one embodiment the ligand binding domain in the gene Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser switch is a Group H nuclear receptor ligand binding domain, Metala Leu Asp ASn Ile His Tyr Ala Leu Leu Thr Ala Val or a mutant thereof that binds an activating ligand. In Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu another embodiment, the Group H nuclear receptor ligand Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg Ile binding domain is selected from the group consisting of an 35 Tyr Ile Leu ASn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile ecdysone receptor ligand binding domain, a ubiquitous Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu receptor ligand binding domain, an orphan receptor-1 ligand Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys binding domain, an NER-1 ligand binding domain, a recep Asn Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val tor-interacting protein-15 ligand binding domain, a liver X (SEQID NO:1), which is also set forth as SEQ NO:1 in U.S. receptor-3 ligand binding domain, a steroid hormone recep 40 Patent Publication No. 2006/0100416 A1 (see also, WO tor-like protein ligand binding domain, a liver X receptor 2002/066612 (PCT/US2002/005090)). ligand binding domain, a liver X receptor ligand binding Exemplary ecdysone receptor ligand binding domains domain, a farnesoid X receptor ligand binding domain, a include those disclosed, for example, in U.S. Pat. No. receptor-interacting protein-14 ligand binding domain, and a 7,935,510 (see also, WO 2003/0/27289 (PCT/US2002/ farnesol receptor ligand binding domain ligand binding 45 005026)); U.S. Pat. No. 7,919,269 (see also, WO 2003/ domain, or a mutant thereof that binds an activating ligand. 027266 (PCT/US/2002/05234)); U.S. Pat. No. 7,563.879 In another embodiment, the Group H nuclear receptor (see also, WO 2003/0/27289 (PCT/US2002/005026)); and ligand binding domain is an ecdysone receptor ligand bind in U.S. Patent Publication No. 2006/0100416A1 (see also, ing domain, or a mutant thereof that binds an activating WO 2002/066612 (PCT/US2002/005090)), each of which is ligand. In another embodiment, the ecdysone receptor ligand 50 hereby incorporated by reference in its entirety. binding domain is selected from the group consisting of an In one embodiment, the ecdysone receptor ligand binding Arthropod ecdysone receptor ligand binding domain a Lepi domain is a mutant of an ecdysone receptor ligand binding dopteran ecdysone receptor ligand binding domain, a Dip domain that binds the activating compound. In another teran ecdysone receptor ligand binding domain, an embodiment, the ecdysone receptor ligand binding domain Orthopteran ecdysone receptor ligand binding domain, a 55 is a mutant of the spruce budworm Choristoneura Homopteran ecdysone receptor ligand binding domain and filmiferana ecdysone receptor ligand binding domain that a Hemipteran ecdysone receptor ligand binding domain, a binds the activating compound. spruce bud worm Choristoneura filmiferana ecdysone In one embodiment, the gene Switch comprises a Chori receptor ligand binding domain, a beetle Tenebrio molitor stoneura filmiferana ecdysone receptor ligand binding ecdysone receptor ligand binding domain, a Manduca sexta 60 domain that is engineered to contain the mutations V107I ecdysone receptor ligand binding domain, a Heliothies vire and Y127E of the Choristoneura filmiferana ecdysone scens ecdysone receptor ligand binding domain, a midge receptor sequence as set forth in SEQ ID NO:1 of U.S. Chironomus tentans ecdysone receptor ligand binding Patent Publication No. 2006/0100416 (see also, WO 2002/ domain, a silk moth Bombyx mori ecdysone receptor ligand 066612 (PCT/US2002/005090)). The term “V107I means binding domain, a squinting bush brown Bicyclus any nana 65 that the valine amino acid residue at position 107 (a as set ecdysone receptor ligand binding domain, a buckeye forth in SEQ ID NO:1 of U.S. Patent Publication No. Junonia Coenia ecdysone receptor ligand binding domain, a 2006/0100416) is changed to isoleucine. The term “Y127E” US 9,677,055 B2 61 62 means that the tyrosine amino acid residue at position 127 comprises helices 1-6, helices 1-7, helices 1-8, helices 1-9, (as set forth in SEQID NO:1 of U.S. Patent Publication No. helices 1-10, helices 1-11, or helices 1-12 of a first species 2006/0100416) is changed to glutamate. of retinoid X receptor, and the second polypeptide fragment Exemplary mutant ecdysone receptor ligand binding of the chimeric retinoid X receptor ligand binding domain domains are disclosed, for example, in US 2006/0100416A1 comprises helices 7-12, helices 8-12, helices 9-12, helices (see also, WO 2002/066612 (PCT/US2002/005090)) and 10-12, helices 11-12, helix 12, or F domain of a second U.S. Pat. No. 7,935,510 (Pub. No. 2005/0266457) (see also, species of retinoid X receptor, respectively. WO 2005/108617 (PCT/US2005/015089)) each of which is In another embodiment, the first polypeptide fragment of incorporated by reference in its entirety. the chimeric retinoid X receptor ligand binding domain In one embodiment, the gene Switch comprises a ligand 10 binding domain that dimerizes with the ligand binding comprises helices 1-6 of a first species RXR according to the domain that binds an activating ligand. In one embodiment, disclosure, and the second polypeptide fragment of the the ligand binding domain that dimerizes with the ligand chimeric retinoid X receptor ligand binding domain com binding domain that binds an activating ligand is a Group B prises helices 7-12 of a second species of retinoid X recep tOr. nuclear receptor ligand binding domain. In another embodi 15 ment, the Group B nuclear receptor ligand binding domain In another embodiment, the first polypeptide fragment of is selected from the group consisting of a retinoid X receptor the chimeric retinoid X receptor ligand binding domain ligand binding domain, an H-2 region II binding protein comprises helices 1-7 of a first species retinoid X receptor ligand binding domain, a nuclear receptor co-regulator-1 according to the disclosure, and the second polypeptide ligand binding domain, an ultraspiracle protein ligand bind fragment of the chimeric retinoid X receptor ligand binding ing domain, a 2C1 nuclear receptor ligand binding domain, domain comprises helices 8-12 of a second species retinoid and a chorion factor 1 ligand binding domain. In another X receptor. embodiment, a ligand binding domain that dimerizes with In another embodiment, the first polypeptide fragment of the ligand binding domain that binds an activating ligand is the chimeric retinoid X receptor ligand binding domain not an ecdysone receptor ligand binding domain. 25 comprises helices 1-8 of a first species of retinoid X recep In one embodiment, the ligand binding domain that tor, and the second polypeptide fragment of the chimeric dimerizes with the ligand binding domain that binds an retinoid X receptor ligand binding domain comprises helices activating ligand is a retinoic X receptor ligand binding 9-12 of a second species of retinoid X receptor. domain. In another embodiment, the retinoic X receptor In another embodiment, the first polypeptide fragment of ligand binding domain is a vertebrate retinoic X receptor 30 the chimeric retinoid X receptor ligand binding domain ligand binding domain. In another embodiment, the retinoic comprises helices 1-9 of a first species of retinoid X recep X receptor ligand binding domain is a Homo sapiens retinoic tor, and the second polypeptide fragment of the chimeric X receptor ligand binding domain. In another embodiment, retinoid X receptor ligand binding domain comprises helices the retinoic X receptor ligand binding domain is a retinoic X 10-12 of a second species of retinoid X receptor. receptor C. isoform. In another embodiment, the retinoic X 35 In another embodiment, the first polypeptide fragment of receptor ligand binding domain is a retinoic X receptor B the chimeric retinoid X receptor ligand binding domain isoform. In another embodiment, the retinoic X receptor comprises helices 1-10 of a first species of retinoid X ligand binding domain is a retinoic X receptor Y isoform. receptor, and the second polypeptide fragment of the chi In another embodiment, the retinoic X receptor ligand meric retinoid X receptor ligand binding domain comprises binding domain is an invertebrate retinoic X receptor ligand 40 helices 11-12 of a second species of retinoid X receptor. binding domain. In another embodiment, the invertebrate In another embodiment, the first polypeptide fragment of retinoic X receptor ligand binding domain is a Locusta the chimeric retinoid X receptor ligand binding domain migratoria retinoic X receptor ligand binding domain. comprises helices 1-11 of a first species of retinoid X In another embodiment, the invertebrate retinoic X recep receptor, and the second polypeptide fragment of the chi tor ligand binding domain is a non-Lepidopteran, non 45 meric retinoid X receptor ligand binding domain comprises Dipteran retinoic X receptor ligand binding domain. helix 12 of a second species of retinoid X receptor. In one embodiment, the retinoid receptor ligand binding In another preferred embodiment, the first polypeptide domain is a vertebrate retinoid X receptor ligand binding fragment of the chimeric retinoid X receptor ligand binding domain, an invertebrate retinoid X receptor ligand binding domain comprises helices 1-12 of a first species of retinoid domain, an ultraspiracle protein ligand binding domain, or a 50 X receptor, and the second polypeptide fragment of the chimeric retinoid X receptor ligand binding domain. chimeric retinoid X receptor ligand binding domain com In one embodiment, the chimeric retinoid X receptor prises an F domain of a second species of retinoid X ligand binding domain comprises two polypeptide frag receptor. ments, wherein the first polypeptide fragment is from a In one embodiment, the first polypeptide fragment in the vertebrate retinoid X receptor ligand binding domain, an 55 chimeric retinoid X receptor ligand binding domain is invertebrate retinoid X receptor ligand binding domain, or human retinoid X receptor sequence, and the second poly an ultraspiracle protein ligand binding domain, and the peptide fragment in the chimeric retinoid X receptor ligand second polypeptide fragment is from a different vertebrate binding domain is invertebrate retinoid X receptor sequence. retinoid X receptor ligand binding domain, a different inver In another embodiment, the invertebrate retinoid X receptor tebrate retinoid X receptor ligand binding domain, or a 60 sequence is Locusta migratoria retinoid X receptor different ultraspiracle protein ligand binding domain. Sequence. In another embodiment, the chimeric retinoid X receptor In another embodiment, the first polypeptide fragment of ligand binding domain is one that is disclosed in U.S. Pat. the chimeric retinoid X receptor ligand binding domain No. 7,531,326, which is hereby incorporated by reference in comprises helices 1-8 of a human retinoid X receptor, and its entirety. 65 the second polypeptide fragment of the chimeric retinoid X In another embodiment, the first polypeptide fragment of receptor ligand binding domain comprises helices 9-12 of the chimeric retinoid X receptor ligand binding domain Locusta migratoria retinoid X receptor. US 9,677,055 B2 63 64 In one embodiment, the gene Switch further comprises a In another embodiment, the gene Switch comprises a DNA binding domain (“DBD). In another embodiment, the DNA binding domain, an ecdysone receptor ligand binding DBD is selected from the group consisting of a GAL4 DBD, domain, a retinoid X receptor ligand binding domain, and a a Lex ADBD, a transcription factor DEM), a steroid/thyroid transactivation domain. hormone nuclear receptor superfamily member DBD, a 5 In another embodiment, the gene Switch comprises a bacterial Lacz DBD, and a yeast DBD. DNA binding domain, an ecdysone receptor ligand binding In one embodiment, the gene Switch further comprises a domain, a chimeric vertebrate/invertebrate retinoid X recep transactivation domain (“TD). In another embodiment, the tor ligand binding domain, and a transactivation domain. transactivation domain is selected from the group consisting In another embodiment, the gene Switch comprises a of a VP16 TD, a GAL4 TD, an NF-kB TD, a BP64 TD, and 10 GAL4 DNA binding domain, a Choristoneura filmiferana a B42 acidic TD. ecdysone receptor ligand binding domain that is engineered to contain the mutations V107 I and Y127E of the Choris In one embodiment, a DNA binding domain, the ligand toneura filmiferana ecdysone receptor sequence set forth in binding domain that binds an activating ligand, a ligand SEQID NO:1, a chimeric Homo sapiens/Locusta migratoria binding domain that dimerizes with the ligand binding 15 retinoid X receptor ligand binding, and a VP16 transactiva domain that binds an activating ligand, and a transactivation tion domain. domain are encoded by polynucleotide sequences that are In another embodiment, the host cell further comprises a contained in the same polynucleotide. polynucleotide encoding a peptide, protein or polypeptide In another embodiment, a DNA binding domain, a ligand whose expression is regulated by the gene Switch. A pro binding domain that binds an activating ligand, a ligand moter that binds the gene switch complex is operably linked binding domain that dimerizes with the ligand binding to the polynucleotide encoding a peptide, protein or poly domain that binds an activating ligand, and a transactivation peptide whose expression is regulated by the gene Switch. domain are encoded by polynucleotide sequences that are In another embodiment, the polynucleotide encoding a contained in two or more separate polynucleotide sequences. peptide, protein or polypeptide whose expression is regu In another embodiment, a DNA binding domain, a ligand 25 lated by the gene Switch is contained in the same polynucle binding domain that binds an activating ligand, a ligand otide as a polynucleotide that encodes one or more of a DNA binding domain that dimerizes with the ligand binding binding domain, the ligand binding domain that binds an domain that binds an activating ligand, and a transactivation activating ligand, a ligand binding domain that dimerizes domain are encoded by polynucleotide sequences that are with the ligand binding domain that binds an activating contained in two separate polynucleotide sequences. 30 ligand, and a transactivation domain. Such constructs are disclosed, for example, in U.S. Patent Publication No. In another embodiment, a DNA binding domain and a 2009/0123441 (see also, WO 2009-048560 (PCT/ ligand binding domain that binds an activating ligand are USUS2008/011563)). encoded by polynucleotide sequences that are contained in In another embodiment, the polynucleotide encoding a a first polynucleotide sequence, and a ligand binding domain 35 peptide, protein or polypeptide whose expression is regu that dimerizes with the ligand binding domain that binds an lated by the gene switch is contained in a different nucleic activating ligand and a transactivation domain are encoded acid molecule than a nucleic acid molecule that encodes one by polynucleotide sequences that are contained in a second or more of a DNA binding domain, the ligand binding polynucleotide sequence. domain that binds an activating ligand, a ligand binding In another embodiment, a DNA binding domain and a 40 domain that dimerizes with the ligand binding domain that ligand binding domain that dimerizes with the ligand bind binds an activating ligand, and a transactivation domain. ing domain that binds an activating ligand are encoded by In one embodiment, the gene Switch is more sensitive to polynucleotide sequences that are contained in a first poly an activating ligand than to a steroid hormone. In another nucleotide sequence, and a ligand binding domain that binds embodiment, the gene Switch is more sensitive to an acti an activating ligand and a transactivation domain are 45 Vating ligand than to another diacylhydrazine compound. encoded by polynucleotide sequences that are contained in The sensitivity of a gene Switch to an activating ligand, a second polynucleotide sequence. relative to another ligand, can readily be determined in an in In embodiments in which one or more of the DNA binding vitro assay, for example, an in vitro assay that employs a domain, a ligand binding domain that binds an activating reporter gene. Such as firefly Luciferase. Examples of Such ligand, a ligand binding domain that dimerizes with the 50 in vitro assays are well known to those of ordinary skill in ligand binding domain that binds an activating ligand, and a the art. See, for example, Karzenowski et al., BioTechniques transactivation domain are encoded by polynucleotide 39:191-200 (2005). sequences that are contained in one or more separate poly In one embodiment, the polynucleotide encoding the gene nucleotide sequences, then the one or more separate poly Switch is contained in a vector. In one embodiment, the nucleotide sequences are operably linked to one or more 55 vector selected from the group consisting of a plasmid, an separate promoters. In another embodiment, the one or more expression vector, a replicon, a phage vector, a cosmid, a separate polynucleotide sequences are operably linked to viral vector, a liposome, an electrically charged lipid (e.g., a one or more separate enhancer elements. In another embodi cytofectin), a DNA-protein complex, and a biopolymer. ment, the promoter(s) and/or the enhancer(s) are constitu In another embodiment, the vector is a retroviral vector. In tively active. In another embodiment, the promoter(s) and/or 60 another embodiment, the vector is selected from the group the enhancer(s) are tissue specific promoters and/or enhanc consisting of an adeno-associated viral vector, a pox viral CS. vector, a baculoviral vector, a vaccinia viral vector, a herpes In one embodiment, the gene Switch comprises a DNA simplex viral vector, an Epstein-Barr viral vector, an adeno binding domain, an ecdysone receptor ligand binding viral vector, a gemini viral vector, and a caulimo viral vector. domain, a ligand binding domain that dimerizes with the 65 In one embodiment, a composition of the invention com ecdysone receptor ligand binding domain, and a transacti prises one or more polynucleotides that encode two or more Vation domain. orthogonal gene Switches. Two or more individually oper US 9,677,055 B2 65 66 able gene regulation systems are said to be “orthogonal to the ligand binding domain of the gene Switch encoded by when (a) modulation of each of the given gene Switches by the polynucleotide in the composition or unit dosage. its respective ligand results in a measurable change in the The present invention also provides a drug label for the magnitude of expression of the gene that is regulated by that composition or unit dosage of the present invention. In one 5 embodiment, the drug label displays an expiration date for gene Switch, and (b) the change is statistically significantly the composition or unit dosage. In another embodiment, the different than the change in expression of all other gene drug label displays instructions for using the composition or switches that are in the host cell. In one embodiment, unit dosage. In another embodiment, the drug label displays regulation of each individually operable gene Switch system the approved indication(s) for the composition or unit dos effects a change in gene expression at least 2-fold, 3-fold, age. In another embodiment, the said label is in paper form. 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 70-fold, 100-fold, 10 In another embodiment, the drug label is in digital or 200-fold, 300 fold, 400-fold or 500-fold greater than all of computer-readable form. the other operable gene switches in the host cell. Non The term “activating ligand’ as used herein refers to a limiting examples of orthogonal gene Switch systems are set compound that shows activity as an ecdysone receptor agonist, i.e., a compound that is able to mimic 20-hy forth in U.S. Pat. No. 8,105,825 (Publication No. US 2002/ 15 0110861 A1). droxyecdysone biological activity, and binds to a gene As used herein, an “activating ligand is a compound that Switch ligand binding domain. Activating ligands for use in binds selectively to the ligand binding domain of a gene the present invention include both ecdysteroids and non switch. steroidal compounds, e.g., tebufenozide and methoxyfenoZ ide. In one embodiment, the activating ligand is administered In one embodiment, the activating ligand is an ecdysone to the subject within an hour of the time at which the priming receptor agonist disclosed in U.S. Pat. No. 8,076,517 (Pub dosage is administered to the Subject. In another embodi lication No. 2009/0163592), No. 2009/0298.175, No. 2005/ ment, the activating ligand is administered to the Subject 0228.016 and in U.S. Pat. No. 6,258,603, U.S. Pat. No. within about 24, 48, 96, 120, 144 or 168 hours of the time 7,375,093, No. 7,456,315, No. 7,304,161, and No. 7,304, at which the priming dosage is administered to the Subject. 25 162; each of which are hereby incorporated by reference In another embodiment, the activating ligand is administered herein. to the subject within about 1, 2, 3, 4 or 5 weeks of the time In certain embodiments, the activating ligand is a com at which the priming dosage is administered to the Subject. pound having Formula I: In one embodiment, the activating ligand is administered to the subject within an hour of the time at which the first of 30 the at least one boosting dosage is administered to the subject. In another embodiment, the activating ligand is administered to the subject within about 24, 48, 96, 120, 144 or 168 hours of the time at which the first of the at least one --- boosting dosage is administered to the Subject. In another 35 H embodiment, the activating ligand is administered to the r O subject within about 1, 2, 3, 4 or 5 weeks of the time at which the first of the at least one boosting dosage is administered wherein: to the subject. A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally In another embodiment, a composition of the invention is 40 substituted aryl or optionally substituted heteroaryl; contained within a container. In one embodiment, the con B is optionally substituted aryl or optionally substituted tainer is a vial. In another embodiment the container is a heteroaryl; multiple-use vial. In another embodiment, the container E is CRRR: displays an expiration date for the composition. In another R" is optionally substituted alkyl, arylalkyl, hydroxyalkyl, embodiment, the container contains instructions for using 45 haloalkyl, optionally substituted cycloalkyl, optionally Sub the composition. stituted alkenyl, optionally substituted alkynyl, optionally In one embodiment, a composition of the invention is a substituted heterocycle, optionally substituted aryl or unit dosage composition. In one embodiment, a unit dosage optionally substituted heteroaryl; and composition is a composition that is manufactured to Supply R° and Rare independently hydrogen, optionally substi a single dosage of the composition of the invention. In 50 tuted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally another embodiment, the unit dosage composition is manu substituted cycloalkyl, optionally substituted alkenyl, factured to provide more than one measured dosages of the optionally substituted alkynyl, optionally substituted hetero composition of the invention. cycle, optionally substituted aryl or optionally substituted The present application also provides an article of manu heteroaryl; or facture comprising more than one of the unit dosage com 55 R" and R taken together form an optionally substituted positions of the invention. In one embodiment, the article of alkenyl group. manufacture is a container. In another embodiment, the In one embodiment, the activating ligand is a compound article of manufacture is a box. In another embodiment, the article of manufacture displays an expiration date for the having Formula I: unit dosage composition. 60 The present invention also provides a comprising more than one of the composition or unit dosage of the present invention. In one embodiment, the kit displays an expiration date for the composition or unit dosage. In another embodi ment, the kit displays and/or or contains instructions for 65 using the composition or unit dosage. In another embodi ment, the kit also comprises an activating ligand that binds

US 9,677,055 B2 79 80 (R)-3,5-Dimethyl-benzoic acid N'-(4-ethyl-benzoyl)-N-(1- (C-C)alkenylthio, halo(C-C)alkenylthio, (C-C) phenethyl-but-3-enyl)-hydrazide. alkynylthio, halo(C-C)alkynylthio, (C-C)alkyl In another embodiment, the activating ligand is a com- Sulfinyl, halo(C-C)alkylsulfinyl, (C-C)alkylsulfo pound having Formula IV: nyl, halo(C-C)alkylsulfonyl, (C-C)alkylamino, di (C-C)alkylamino, (C-C)alkoxy(C-C)alkyl, (C- IV C.)alkylthio(C-C)alkyl, (C-C)alkylsulfinyl (C-C) R5 alkyl, (C-C)alkylsulfonyl(C-C)alkyl, (C-C)alky lamino(C-C)alkyl, di (C-C)alkylamino(C-C) 10 alkyl, (C-C)alkylcarbonyl, (C-C) alkylaminocarbonyl, di(C-C)alkylaminocarbonyl, or R3 (C-C)alkoxycarbonyl, optionally substituted phenyl, optionally Substituted 1-naphthyl, optionally Substi R2 tuted 2-maphthyl, optionally Substituted phenyl (C-C) R10 1. 15 alkyl, optionally Substituted phenyl(C-C)alkenyl, or optionally substituted heterocycle. In another embodiment, the activating ligand is a com wherein: pound having Formula IV wherein: Q is O or S: R" is selected from the group consisting of hydrogen, 20 Q is O: (C-C)alkyl, (C-C)cycloalkyl, (C-C)cycloalkyl R" is selected from the group consisting of 4-fluorophe (C-C)alkyl, (C-C)haloalkyl, (C-C)alkenyl, (C- nyl, 3-fluorophenyl, 4-fluoro-3-methylphenyl, C)cycloalkenyl, (C-C)haloalkenyl, (C-C)alky 4-fluoro-3-(trifluoromethyl)phenyl, 4-fluoro-3-iodo nyl, (C-C)alkoxy(C-C)alkyl, (C-C)alkylthio(C- 25 phenyl, 3-fluoro-4-iodophenyl, 3,4-di-fluorophenyl, C.)alkyl, (C-C)alkoxycarbonyl. Succinimidylmethyl, 4-ethylphenyl, 3-fluoro-4-methylphenyl, 3-fluoro-4- ethylphenyl, 3-chloro-4-fluorophenyl, 3-fluoro-4-chlo benZoSuccinimidylmethyl, optionally substituted phe rophenyl, 2-methyl-3-methoxyphenyl, 2-ethyl-3- nyl, optionally Substituted 1-naphthyl, optionally Sub methoxyphenyl, 2-ethyl-3,4-ethylenedioxyphenyl, stituted 2-maphthyl, optionally Substituted phenyl (C- 30 3-nitrophenyl, 4-iodophenyl, 3-fluoro-4-trifluorometh C.)alkyl, optionally Substituted phenyl(C-C)alkenyl, ylphenyl, 3-methylphenyl, 4-methylphenyl, 4-chloro optionally substituted naphthyl(C-C)alkyl, optionally phenyl, 3-trifluoromethylphenyl, 3-methoxyphenyl, Substituted phenoxy(C-C)alkyl, optionally Substi 3-chloro-6-pyridyl, 2-chloro-4-pyridyl, phenylamino, tuted phenylamino, and optionally Substituted hetero 35 3-chlorophenylamino, 3-methylphenylamino, 4-chlo cycle; rophenylamino, and 4-methylphenylamino; RandR are each independently selected from hydrogen, (C-C)alkyl, and (C-C)haloalkyl: R’ is hydrogen, methyl or CF; R" is hydrogen, (C-C)alkyl, or (C-C)haloalkyl; R is hydrogen, methyl or CF; Rand Rare each independently selected from the group 40 R is hydrogen; consisting of hydrogen, (C-C)alkyl, (C-C)cy R is optionally substituted phenyl, wherein the substitu cloalkyl, (C-C)haloalkyl, (C-C)alkenyl, (C-C) ents are selected from the group consisting of cyano, cycloalkenyl, (C-C)haloalkenyl, (C-C)alkynyl, nitro, halogen, (C-C)alkyl, halo(C-C)alkyl, (C-C) (C-C)alkoxy(C-C)alkyl, (C-C)alkylthio(C-C) alkoxy, halo(C-C)alkoxy, (C)alkenyloxy, (C)alky alkyl, aminocarbonyl, aminothiocarbonyl, formyl, (C- 45 nyloxy, (C-C)alkylthio, halo(C-C)alkylthio, (C- C.)alkylsulfinyl, (C-C)alkylsulfonyl, (C-C)alkyl C.)alkylsulfinyl, halo(C-C)alkylsulfinyl, (C-C) carbonyl, cyclo(C-C)alkylcarbonyl, halo(C-C) alkylsulfonyl, halo(C-C)alkylsulfonyl, (C-C) alkylcarbonyl, (C-C)alkylaminocarbonyl, di(C-C) alkoxy(C-C)alkyl, (C-C)alkylthio(C-C)alkyl, and alkylaminocarbonyl, (C-C)alkoxycarbonyl, (C-C) (C-C)alkoxycarbonyl: alkoxycarbonylcarbonyl, O phenyl (C-C) 50 R is selected from the group consisting of hydrogen, alkenylcarbonyl, optionally Substituted phenyl, formyl, (C-C)alkylcarbonyl, and cyclo(C-C)alkyl optionally Substituted 1-naphthyl, optionally Substi carbonyl; and tuted 2-maphthyl, optionally Substituted phenyl(C-C) R. R. R. R'' are independently selected from the group alkyl, optionally substituted phenyl(C-C)alkenyl, 55 consisting of hydrogen, cyano, nitro, chlorine, fluorine, optionally Substituted phenylcarbonyl, or optionally methyl, trifluoromethyl, difluoromethyl, methoxy, trif luoromethoxy, difluoromethoxy, methylthio, trifluo substituted heterocycle: romethylthio, difluoromethylthio, methylsulfinyl, trif R. R. R. and R' are each independently selected from luoromethylsulfinyl, difluoromethylsulfinyl, the group consisting of hydrogen, cyano, nitro, halo methylsulfonyl, trifluoromethylsulfonyl, difluorometh gen, (C-C)alkyl, (C-C)cycloalkyl, (C-C)ha 60 ylsulfonyl, methoxymethyl, and methoxycarbonyl, or loalkyl, (C-C)alkenyl, (C-C)cycloalkenyl, (C- R7/R, R/R, or R/R' form a 5- or 6-membered C.)haloalkenyl, (C-C)alkynyl, halo(C-C)alkynyl, heterocyclic ring. hydroxy, (C-C)alkoxy, halo(C-C)alkoxy, (C-C) In another embodiment, the activating ligand is a com alkenyloxy, halo(C-C)alkenyloxy, (C-C)alkyny 65 pound having Formula IV wherein Q is O, R is methyl, and loxy, halo(C-C)alkynyloxy, aryloxy, (C-C)alkoxy R. R. R. R. R. R. R. and R'' are defined according to (C-C)alkyl, (C-C)alkylthio, halo(C-C)alkylthio. Table 4.

US 9,677,055 B2 89 90 optionally substituted phenyl, optionally substituted C.)halocycloalkylamino, di (C-C)alkylamino, phenyl(C-C)alkyl, optionally substituted benzoyl, di(C-C)cycloalkylamino, di(C-C)haloalky optionally substituted naphthyl, optionally Substi lamino, di(C-C)halocycloalkylamino, (C-C) tuted heterocycle, and optionally substituted hetero alkoxy(C-C)alkyl, (C-C)althylthio(C-C)alkyl, cyclylcarbonyl, or (C-C)alkylsulfinyl (C-C)alkyl, (C-C)alkylsul R° and Rare joined together with the carbon to which fonyl(C-C)alkyl, (C-C)alkylamino(C-C)alkyl, they are attached to form an unsubstituted or substi di (C-C)alkylamino(C-C)alkyl, (C-C)alkylcar tuted, partially unsaturated or saturated optionally bonyl(C-C)alkyl, cyano(C-C)alkyl, optionally substituted 3-, 4-, 5-, 6-, 7- or 8-membered carbo substituted phenyl, optionally substituted phenyl (C- cyclic or heterocyclic ring, wherein the heterocyclic 10 C.)alkyl, optionally substituted heterocycle, option ring contains from one to three heteroatoms selected ally substituted phenoxy, optionally substituted het from O, N, or S; erocycloxy, optionally Substituted phenylthio. R" is selected from the group consisting of (C-C) optionally substituted heterocyclylthio, optionally alkyl, (C-C)cycloalkyl, (C-C)haloalkyl, (C-C) substituted naphthyl, optionally substituted phe halocycloalkyl, (C-C)alkenyl, (C-C)haloalkenyl, 15 nylamino, optionally Substituted heterocyclylamino, (C-C)alkynyl, (C-C)haloalkynyl, (C-C)alkoxy, optionally substituted N-phenyl-N-(C-C)alky (C-C)cycloalkoxy, (C-C)haloalkoxy, (C-C)ha lamino, and optionally substituted N-heterocyclyl locycloalkoxy, (C-C)alkenyloxy, (C-C)alkyny N—(C-C)alkylamino. loxy, (C-C)alkylthio, (C-C)cycloalkylthio, (C- In another embodiment, the activating ligand is a com C.)haloalkylthio, (C-C)halocycloalkylthio, (C- pound having Formula V, wherein: C.)alkylamino, (C-C)cycloalkylamino, (C-C) Q is O: haloalkylamino, (C-C)halocycloalkylamino, R" is substituted phenyl wherein the substituents are di (C-C)alkylamino, di(C-C)cycloalkylamino, independently selected from the group consisting of di (C-C)haloalkylamino, di(C-C)halocycloalky 25 (C-C)alkyl and (C-C)alkoxy; or two adjacent posi lamino, (C-C)alkoxy(C-C)alkyl, (C-C)althyl tions are joined together with the atoms to which they thio(C-C)alkyl, (C-C)alkylsulfinyl (C-C)alkyl, are attached to form an unsubstituted or substituted, unsaturated, partially unsaturated, or saturated 5-, 6- or (C-C)alkylsulfonyl(C-C)alkyl, (C-C)alky 7-membered carbocyclic or heterocyclic ring, wherein lamino(C-C)alkyl, di(C-C)alkylamino(C-C) 30 the heterocyclic ring contains from one to two oxygen alkyl, (C-C)alkylcarbonyl(C-C)alkyl, cyano (C- atoms and one to four substituents are independently C.)alkyl, optionally substituted phenyl, optionally selected from the group consisting of cyano, (C-C) substituted 1-naphthyl, optionally substituted alkyl, (C-C)alkylamino, di(C-C)alkylamino, (C- 2-naphthyl, optionally substituted phenyl (C-C) C.)alkoxycarbonyl, (C-C)alkylaminocarbonyl, alkyl, optionally Substituted phenyl(C-C)alkenyl, 35 di(C-C)alkylaminocarbonyl, oxo, and methoxy optionally Substituted naphthyl(C-C)alkyl, option imino; ally Substituted phenoxy(C-C)alkyl, optionally R° and R are independently selected from the group Substituted phenyl amino, and optionally Substituted consisting of (C-C)alkyl, (C-C)cycloalkyl, halo heterocycle: 40 (C-C)alkyl, (C-C)alkoxy(C-C)alkyl, (C-C)alth R is selected from the group consisting of (C-C) ylthio(C-C)alkyl, (C-C)alkylsulfinyl (C-C)alkyl, alkyl, (C-C)cycloalkyl, (C-C)haloalkyl, (C-C) (C-C)alkylsulfonyl(C-C)alkyl, (C-C)alkylamino halocycloalkyl, (C-C)alkenyl, (C-C)haloalkenyl, (C-C)alkyl, di(C-C)alkylamino(C-C)alkyl, (C- (C-C)alkynyl, (C-C)haloalkynyl, (C-C)alkoxy C.)alkylcarbonyl, (C-C)alkylcarbonyl(C-C)alkyl, (C-C)alkyl, (C-C)althylthio(C-C)alkyl, (C- 45 (C-C)alkylaminocarbonyl, di(C-C)alkylaminocar C.)alkylsulfinyl (C-C)alkyl, (C-C)alkylsulfonyl bonyl, (C-C)alkylaminocarbonyl(C-C)alkyl, di(C- (C-C)alkyl, (C-C)alkylamino(C-C)alkyl, C.)alkylaminocarbonyl(C-C)alkyl, (C-C)alkylcar di (C-C)alkylamino(C-C)alkyl, (C-C)alkylcar bonylamino(C-C)alkyl, (C-C)alkoxycarbonyl, and bonyl(C-C)alkyl, cyano(C-C)alkyl, optionally C-C)alkoxycarbonyl(C-C)alkyl, or Substituted phenyl, optionally substituted 1-naph 50 thyl, optionally Substituted 2-maphthyl, optionally R° and R may be joined together with the carbon to Substituted phenyl (C-C)alkyl, optionally Substi which they are attached to form an unsubstituted or tuted phenyl (C-C)alkenyl, optionally Substituted Substituted, partially unsaturated or Saturated 5-, 6- or naphthyl(C-C)alkyl, optionally substituted phe 7-membered carbocyclic or heterocyclic ring, wherein noxy(C-C)alkyl, optionally Substituted phe 55 the heterocyclic ring contains one heteroatom selected nylamino, and optionally Substituted heterocycle; from O or S; and one to four substituents are indepen and dently selected from the group consisting of (C-C) R and R are independently selected from the group alkyl, (C-C)alkylamino, di(C-C)alkylamino, (C- consisting of (C-C)alkyl, (C-C)cycloalkyl, (C- C.)alkoxy carbonyl, (C-C)alkylaminocarbonyl, and C.)haloalkyl, (C-C)halocycloalkyl, (C-C)alk 60 di(C-C)alkylaminocarbonyl; and enyl, (C-C)haloalkenyl, (C-C)alkynyl, (C-C) R" is selected from optionally substituted phenyl or haloalkynyl, (C-C)alkoxy, (C-C)cycloalkoxy, pyridyl wherein the substituents are independently (C-C)haloalkoxy, (C-C)halocycloalkoxy, (C- selected from the group consisting of (C-C)alkyl and C.)alkenyloxy, (C-C)alkynyloxy, (C-C)alkyl (C-C)alkoxy; thio, (C-C)cycloalkylthio, (C-C)haloalkylthio. 65 In another embodiment, the activating ligand is a com (C-C)halocycloalkylthio, (C-C)alkylamino, (C- pound having Formula V, wherein Q is oxygen, and R. R. C.)cycloalkylamino, (C-C)haloalkylamino, (C- R, and R are defined according to Table 5.

US 9,677,055 B2 103 104 allyl ether, 20-hydroxyecdysone-22-benzyl ether; 20-hy turkesterone-11C.-acetate; turkesterone-2.11 C-diacetate; droxyecdysone-22-(28RS)-2'-ethyloxiranyl ether; turkesterone-11C.-propionate; turkesterone-11C.-butanoate; ponasterone A-2-methyl ether, ponasterone A-14-methyl turkesterone-11C.-hexanoate; turkesterone-11C.-decanoate; ether, ponasterone A-22-methyl ether, ponasterone A-2.22 turkesterone-11C.-laurate; turkesterone-11C-myristate; dimethyl ether, ponasterone A-3,22-dimethyl ether, ponas turkesterone-11C.-arachidate, 22-dehydro-123-hydroxynors terone A-14.22-dimethyl ether, dacryhainansterone-22 engosterone; 22-dehydro-123-hydroxycyasterone; 22-de methyl ether; 25,26-didehydroponasterone A (iso hydro-12B-hydroxysengosterone; 14-deoxy(14C-H)-20-hy stachysterone C (A25(26))); shidasterone (stachysterone D), droxyecdysone; 20-hydroxyecdysone-14-methyl ether; 14C.- stachysterone C, 22-deoxy-20-hydroxyecdysone (taxister perhydroxy-20-hydroxyecdysone; 20-hydroxyecdysone-2, one); ponasterone A; polyporusterone B: 22-dehydro-20 10 hydroxyecdysone; 20-hydroxyecdysone; pterosterone; 3.14.22-tetramethyl ether; (20S)-22-deoxy-20.21 (25R)-inokosterone, (25S)-inokosterone; pinnatasterone: dihydroxyecdysone; 22.25-dideoxyecdysone; (22S)-20-(2, 25-fluoroponasterone A; 24(28)-dehydromakisterone A: 2'-dimethylfuranyl)ecdysone, (22R)-20-(2,2'- 24-epi-makisterone A, makisterone A; 20-hy dimethylfuranyl)ecdysone; 22-deoxyecdysone; droxyecdysone-22-methyl ether; 20-hydroxyecdysone-25 15 25-deoxyecdysone; 22-dehydroecdysone; ecdysone; 22-epi methyl ether, abutasterone; 22.23-di-epi-geradiasterone; ecdysone; 24-methylecaysone (20-deoxymakisterone A); 20,26-dihydroxyecdysone (podecdysone C), 24-epi-abutas ecdysone-22-hemisuccinate; 25-deoxyecdysone-22-B-D- terone; geradiasterone; 29-norcyasterone; ajugasterone B; glucopyranoside; ecdysone-22-myristate; 22-dehydro-20 24(28)Z-dehydroamarasterone B; amarasterone A, makis iso-ecdysone; 20-iso-ecdysone; 20-iso-22-epi-ecdysone; terone C: rapisterone C; 20-hydroxyecdysone-22,25-dim 2-deoxyecdysone; sileneoside E (2-deoxyecdysone 33-glu ethyl ether, 20-hydroxyecdysone-22-ethyl ether; carthamos coside, blechnoside A); 2-deoxyecdysone-22-acetate; 2-de terone; 24(25)-dehydroprecyasterone; leuZeasterone; oxyecdysone-3,22-diacetate; 2-deoxyecdysone-22-3-D-glu cyasterone; 20-hydroxyecdysone-22-allyl ether, 24(28)Z- copyranoside; 2-deoxyecdysone 25-3-D-glucopyranoside; dehydro-29-hydroxymakisterone C; 20-hydroxyecdysone 2-deoxy-21-hydroxyecdysone; 3-epi-22-iso-ecdysone; 3-de 22-acetate; Viticosterone E (20-hydroxyecdysone 25-ac 25 hydro-2-deoxyecdysone (silenosterone); 3-dehy etate); 20-hydroxyecdysone-22-n-propyl ether; droecdysone; 3-dehydro-2-deoxyecdysone-22-acetate; 24-hydroxycyasterone; ponasterone A 22-hemisuccinate; ecdysone-6-carboxymethyloxime; ecdysone-2,3-acetonide; 22-acetoacetyl-20-hydroxyecdysone; canescensterone; 14-epi-20-hydroxyecdysone-2,3-acetonide; 20-hy 20-hydroxyecdysone-22-hemisuccinate; inokosterone-26 droxyecdysone-2,3-acetonide; 20-hydroxyecdysone-20.22 hemisuccinate; 20-hydroxyecdysone-22-benzoate; 20-hy 30 acetonide; 14-epi-20-hydroxyecdysone-2.3.20.22-diac droxyecdysone-22-3-D-glucopyranoside; 20-hy etonide; paxillosterone-20.22-p-hydroxybenzylidene acetal; droxyecdysone-25-f-D-glucopyranoside; sileneoside A (20 poststerone; (20R)-dihydropoststerone; (20S)dihydroposts hydroxyecdysone-22C-galactoside); 3-deoxy-1B.20 terone; poststerone-20-dansylhydrazine; (20S)-dihydropost dihydroxyecdysone (3-deoxyintegristerone A); sterone-2,3,20-tribenzoate; (20R)-dihydropoststerone-2,3, 2-deoxyintegristerone A, 1-epi-integristerone A: integrister 35 20-tribenzoate: (20R)dihydropoststerone-2,3-acetonide; one A, sileneoside C (integristerone A 22O-galactoside); (2020S)dihydropoststerone-2,3-acetonide; (5C.-H)-dihydro 2.22-dideoxy-20-hydroxyecdysone: 2-deoxy-20-hy rubrosterone; 2,14.22.25-tetradeoxy-5C.-ecdysone; 5C.-keto droxyecdysone; 2-deoxy-20-hydroxyecdysone-3-acetate; diol, bombycosterol. 2C.3C.22S,25-tetrahydroxy-5C.- 2-deoxy-20.26-dihydroxyecdysone: 2-deoxy-20-hy cholestan-6-one; (5C.-H)-2-deoxy-21-hydroxyecdysone, droxyecdysone-22-acetate; 2-deoxy-20-hydroxyecdysone 40 castasterone; 24-epi-castasterone; (5C. -H)-2-deoxyinteg 3.22-diacetate, 2-deoxy-20-hydroxyecdysone-22-benzoate: risterone A; (5C.-H)-22-deoxyintegristerone A; (5C.-H)-20 ponasterone A 2-hemisuccinate; 20-hydroxyecdysone-2-ac hydroxyecdysone; 24.25-didehydrodacryhaninansterone; etate; 20-hydroxyecdysone-2-hemisuccinate; 20-hy 25,26-didehydrodacryhainansterone; 5-deoxykaladasterone droxyecdysone-2-3-D-glucopyranoside; 2-dansyl-20-hy (dacryhainansterone); (14C-H)-14-deoxy-25-hydroxydacry droxyecdysone; 20-hydroxyecdysone-2.22-dimethyl ether; 45 hainansterone; 25-hydroxydacryhainansterone; rubroster ponasterone A 3.3-D-xylopyranoside (limnantheoside B); one; (5B-H)-dihydrorubrosterone; dihydrorubrosterone 20-hydroxyecdysone-3-methyl ether; 20-hydroxyecdysone 173-acetate, sidisterone; 20-hydroxyecdysone-2.3.22 3-acetate; 20-hydroxyecdysone-3?-D-xylopyranoside (lim triacetate, 14-deoxy(14B-H)-20-hydroxyecdysone; 14-epi nantheoside A); 20-hydroxyecdysone-3-3-D-glucopyrano 20-hydroxyecdysone: 9C.20-dihydroxyecdysone; side; sileneoside D (20-hydroxyecdysone-3C.-galactoside); 50 malacosterone, 2-deoxypolypodine B-3-3-D-glucopyrano 20-hydroxyecdysone 3f-D-glucopyranosyl-(1-3-3-D-xy side; ajugalactone; cheilanthone B; 23.33.6C-trihydroxy lopyranoside (limnantheoside C); cyasterone-3-acetate; 5 B-cholestane; 2B.3?.6?-trihydroxy-5 B-cholestane: 14-de 2-dehydro-3-epi-20-hydroxyecdysone; 3-epi-20-hy hydroshidasterone; stachysterone B; 2B.3?.9C.20R.22R,25 droxyecdysone (coronatasterone); rapisterone D, 3-de hexahydroxy-5B-cholest-7, 14-dien-6-one: kaladasterone: hydro-20-hydroxyecdysone; 53-hydroxy-25,26-didehydro 55 (14 B-H)-14-deoxy-25-hydroxydacryhainansterone: 4-de ponasterone A; 5 B-hydroxystachysterone C; hydro-20-hydroxyecdysone; 14-methyl-12-en-shidasterone: 25-deoxypolypodine B; polypodine B: 25-fluoropolypodine 14-methyl-12-en-15.20-dihydroxyecdysone; podecdysone B: 5B-hydroxyabutasterone; 26-hydroxypolypodine B: B; 23.3f2OR,22R-tetrahydroxy-25-fluoro-5B-cholest-8, 14 29-norsengosterone, Sengosterone; 6B-hydroxy-20-hy dien-6-one (25-fluoropodecdysone B); calonysterone: droxyecdysone; 6O-hydroxy-20-hydroxyecdysone; 20-hy 60 14-deoxy-14, 18-cyclo-20-hydroxyecdysone; 9C, 14C-ep droxyecdysone-6-oxime; ponasterone A 6-carboxymethyl oxy-20-hydroxyecdysone; 9BC.143-epoxy-20-hy Oxime; 20-hydroxyecdysone-6-carboxymethyloxime; droxyecdysone; 9C, 14C-epoxy-20-hydroxyecdysone 2.3.20, ajugasterone C, rapisterone B: muristerone A. atrotosterone 22-diacetonide; 28-homobrassinolide; and iso B; atrotosterone A, turkesterone-2-acetate; punisterone (rha homobrassinolide. pontisterone); turkesterone; atrotosterone C; 25-hydroxya 65 The disclosure of all patents, patent applications, and trotosterone B: 25-hydroxyatrotosterone A. paxillosterone: publications cited herein are incorporated by reference in turkesterone-2.22-diacetate; turkesterone-22-acetate; their entireties. US 9,677,055 B2 105 106 The following examples are illustrative, but not limiting, glycerol or Sorbitol. The push-fit capsules can contain the of the methods of the present invention. Other suitable active compounds in the form of granules or nanoparticles modifications and adaptations of the variety of conditions which may optionally be mixed with fillers such as lactose, and parameters normally encountered in medical treatment binders such as starches, and/or lubricants such as talc or and gene expression systems and which are obvious to those magnesium Stearate and, optionally, stabilizers. In one skilled in the art are within the spirit and scope of the embodiment, the is dissolved or Suspended in Suitable liq invention. uids, Such as fatty oils, or liquid paraffin, optionally with Pharmaceutical Compositions stabilizers. In certain embodiments, polynucleotides and polypep Fatty oils may comprise mono-, di- or triglycerides. tides of the invention can be administered as part of a 10 medicament or pharmaceutical composition. Medicaments Mono-, di- and triglycerides include those that are derived and pharmaceutical compositions of the invention comprise from C6, C8, C10, C12, C14, C16, C18, C20 and C22 acids. one or more pharmaceutically acceptable carriers, diluents, Exemplary diglycerides include, in particular, diolein, excipients or additives. dipalmitolein, and mixed caprylin-caprin diglycerides. Pre The term “excipient’ as used herein is typically an inert 15 ferred triglycerides include vegetable oils, fish oils, animal Substance added to a composition to facilitate processing, fats, hydrogenated vegetable oils, partially hydrogenated handling, administration, et cetera of a pharmaceutically vegetable oils, synthetic triglycerides, modified triglycer acceptable composition. Useful excipients include, but are ides, fractionated triglycerides, medium and long-chain tri not limited to, adjuvants, anti-adherents, binders, carriers, glycerides, structured triglycerides, and mixtures thereof. disintegrants, fillers, flavors, colors, diluents, lubricants, glidants, preservatives, Sorbents, solvents, Surfactants, and Exemplary triglycerides include: almond oil; babassu oil; SWeetenerS. borage oil, blackcurrant seed oil; canola oil; castor oil; A few examples of pharmaceutically acceptable carriers, coconut oil; corn oil; cottonseed oil; evening primrose oil; diluents, excipients and additives include, without limita grapeseed oil; groundnut oil; mustard seed oil, olive oil; tion, water, saline, Ringer's solution, dextrose solution, 25 palm oil; palm kernel oil; peanut oil, rapeseed oil; safflower buffers (such as phosphates (e.g., calcium phosphates Such oil; sesame oil; shark liver oil; soybean oil; Sunflower oil; as tricalcium phosphate or calcium hydrogen phosphate)), hydrogenated castor oil; hydrogenated coconut oil; hydro citrate. Succinate, acetic acid, and other organic acids or their genated palm oil; hydrogenated Soybean oil; hydrogenated salts), antioxidants, proteins and other high molecular vegetable oil, hydrogenated cottonseed and castor oil; par weight molecules (such as serum albumin, gelatin, or immu 30 tially hydrogenated soybean oil; partially soy and cottonseed noglobulins), hydrophilic polymers (such as polyvinylpyr oil; glyceryl tricaproate; glyceryl tricaprylate; glyceryl tri rolidone), amino acids (such as glycine, glutamic acid, aspartic acid, and arginine), Saccharides (for example mono caprate; glyceryl triundecanoate; glyceryl trilaurate; glyc saccharides, disaccharides, lactose, Sucrose, mannitol, Sor eryl trioleate; glyceryl trilinoleate; glyceryl trilinolenate, bitol, other carbohydrates and Sugar-alcohols, cellulose or its 35 glyceryl tricaprylate/caprate; glyceryl tricaprylate/caprate/ derivatives, glucose, mannose, and dextrins), chelating laurate; glyceryl tricaprylate/caprate/linoleate; and glyceryl agents (such as EDTA); Sugar alcohols (such as mannitol or tricaprylate/caprate/stearate. Sorbitol), counterions (such as sodium), Surfactants (such as In one embodiment, the triglyceride is the medium chain polysorbates, poloxamers, or polyethylene glycol (PEG)), triglyceride available under the trade name LABRAFAC and binders (such as starch paste (e.g., maize starch, wheat 40 CC. Other triglycerides include neutral oils, e.g., neutral starch, rice starch, potato starch)), gelatin, tragacanth, plant oils, in particular fractionated coconut oils such as methyl cellulose, hydroxypropylmethylcellulose, sodium known and commercially available under the trade name carboxymethylcellulose, and/or polyvinyl pyrrolidone). MIGLYOL, including the products: MIGLYOL 810; MIG Pharmaceutically acceptable carriers, diluents, excipients LYOL 812; MIGLYOL 818; and CAPTEX 355. Other and additives may include: disintegrating agents such as the 45 triglycerides are caprylic-capric acid triglycerides such as above-mentioned Starches as well as compounds such as known and commercially available under the trade name carboxymethyl-Starch, cross-linked polyvinyl pyrrolidone, MYRITOL, including the product MYRITOL 813. Further agar, or alginic acid or a salt thereof. Such as sodium triglycerides of this class are CAPMUL MCT, CAPTEX alginate; and, flow-regulating agents and lubricants, for 200, CAPTEX 300, CAPTEX 800, NEOBEE M5 and example, silica, talc, Stearic acid or salts thereof Such as 50 MAZOL. 1400. magnesium Stearate or calcium Stearate, and/or polyethylene Pharmaceutical compositions comprising triglycerides glycol. In one embodiment, dragee cores are provided with may further comprise lipophilic and/or hydrophilic Surfac Suitable coatings which, if desired, are resistant to gastric tants which may form clear Solutions upon dissolution with juices. For this purpose, concentrated saccharide solutions an aqueous solvent. One Such surfactant is tocopheryl poly may be used, which may optionally contain gum arabic, talc, 55 ethylene glycol 1000 succinate (vitamin ETPGS). Examples polyvinyl pyrrolidone, polyethylene glycol and/or titanium of such compositions are described in U.S. Pat. No. 6,267, dioxide, lacquer Solutions and Suitable organic solvents or 985. Solvent mixtures. In order to produce coatings resistant to In another embodiment, the pharmaceutically acceptable gastric juices, solutions of Suitable cellulose preparations carrier comprises LABRASOL (Gattefosse SA), which is Such as acetyl cellulose phthalate or hydroxypropylmethyl 60 PEG-8 caprylic/capric glycerides. In another embodiment, cellulose phthalate, are used. Dye stuffs or pigments may be the pharmaceutically acceptable carrier comprises PL90G, added to the tablets or dragee coatings, for example, for vitamin E TPGS, and Miglyol 812N. identification or in order to characterize combinations of Pharmaceutical compositions can be administered in any active compound doses. suitable manner as determined by those skilled in the art, Pharmaceutical preparations which can be used orally 65 Such as, but without limitation, by oral, rectal, vaginal, include push-fit capsules made of gelatin, as well as Soft, topical (including dermal, buccal and Sublingual), parent sealed capsules made of gelatin and a plasticizer Such as eral, intravenous, intraperitoneal, intramuscular, intratu US 9,677,055 B2 107 moral, intraarticular, Subcutaneous, intranasal, inhalation, intradermal, intrathecal, epidural, and by nasogastric routes. a) Methods and compositions for preparation, formulation, (SEO ID NO; 5) and delivery of pharmaceutically acceptable compositions ISFSTRGTO; and medicaments are well-known and routinely practiced by b) those skilled in the art. A few examples of textbooks and (SEQ ID NO : 6) manuals providing information and instruction on Such GIONWTVER; methods and compositions include: Rowe et al. (Editor), c) “Handbook of Pharmaceutical Excipients.” Pharmaceutical (SEO ID NO: 7) Press, 6' Ed. (August 2009); University of the Sciences in 10 IWFGGVRAR; Philadelphia (Editor), “Remington. The Science and Prac d) tice of Pharmacy” Lippincott Williams & Wilkins, 21 Ed. (SEQ ID NO: 8) (2005): “Physicians' Desk Reference 2011.” PDR Network ARSODLDAI; (2010): “Physicians’ Desk Reference 2012, PDR Network 15 e) (2011); O'Neil, “The Merck Index: An Encyclopedia of (SEO ID NO: 9) Chemicals, Drugs, and Biologicals, "14" Ed. (2006); Allen LPNYWPRSS; et al. (Editor) “Ansel's Pharmaceutical Dosage Forms and f) Drug Delivery Systems.” Lippincott Williams & Wilkins; 9" (SEQ ID NO: 10) Ed. (2011); and, Ash et al. (Editor), “Handbook of Pharma IPDKEQAIS; ceutical Additives, Third Edition.” Synapse Information g) Resources, Inc.; 3" Ed. (2007). (SEQ ID NO: 131) Protocols for general molecular biology methods can be ISFSTRGTONWTVER; found in: Methods in Molecular Biology, series editor J M and Walker, Humana Press, New York. 25 h) Embodiments of the invention comprise any amino acid (SEQ ID NO: 132) substituted form of PE as indicated by, or represented in, IWFGGWRARSODLDAI Table 13. Embodiments of the invention further comprise any amino acid substituted form of PE which may comprise wherein one or more amino acid residues in any one or 30 more of said epitopes in a) through h) are substituted any combination of amino acid Substitutions indicated by, or with a different amino acid residue. represented in, Table 13. E2. An isolated polypeptide having Pseudomonas exo Embodiments of the invention also comprise variants, toxin A biological activity, wherein said polypeptide derivatives, or biologically active fragments of any amino comprises an epitope selected from the group consist acid substituted form of PE as indicated by, or represented 35 ing of in, Table 13, wherein said variant, derivative, or biologically a) ISFSTRGTQ (SEQ ID NO:5), wherein amino acid active fragment of PE is at least 80% identical, at least 85% residues at one or more of positions 1, 6 and 9 are identical, at least 90% identical, at least 95% identical, at substituted with a different amino acid residue; least 97% identical, at least 98% identical, at least 99% b) GTQNWTVER (SEQ ID NO:6), wherein amino identical, or is at least 100% identical to an amino acid 40 acid residues at one or more of positions 3, 4 and 6 are substituted with a different amino acid residue; substituted form of PE, or a fragment thereof, as indicated c) IVFGGVRAR (SEQ ID NO:7), wherein amino acid by, or represented in, Table 13. For example, embodiments residues at one or more of positions 1 and 6 are of the invention comprise variants, derivatives, or biologi substituted with a different amino acid residue; cally active fragments of any amino acid Substituted form of d) ARSQDLDAI (SEQ ID NO:8), wherein amino acid PE as indicated by, or represented in, Table 13, wherein said 45 residues at one or more of positions 4 and 7 are variant, derivative, or biologically active fragment of PE is substituted with a different amino acid residue; at least 80% identical, at least 85% identical, at least 90% e) LRVYVPRSS (SEQ ID NO:9), wherein amino acid identical, at least 95% identical, at least 97% identical, at residues at one or more of positions 1, 2 and 9 are least 98% identical, at least 99% identical, or is at least substituted with a different amino acid residue; 100% identical to PE constructs, or fragments thereof, as 50 f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid represented by plEX02-003 through plEX02-248 in Table residues at one or more of positions 1, 4, 6 and 7 are 13 (such as, for example, as shown in SEQ ID NO:177 substituted with a different amino acid residue; (pIEX02-228), SEQID NO:178 (plEX02-244), and SEQID g) ISFSTRGTQNWTVER (SEQID NO:131), wherein NO: 179 (p1EX02-246)). amino acid residues at one or more of positions 1, 6. Embodiments of the invention include methods of mak 55 9, 10 and 12 are substituted with a different amino ing, methods of using, methods of treatment using, medi acid residue; and caments comprising, pharmaceutically acceptable composi h) IVFGGVRARSQDLDAI (SEQ ID NO:132), tions comprising, therapeutically useful compositions wherein amino acid residues at one or more of comprising, and kits comprising any of the amino acid positions 1, 6, 11, and 14 are substituted with a substituted forms of PE referenced, or otherwise described 60 different amino acid residue. or provided for, herein. E3. The isolated polypeptide of embodiment E1 or E2. Embodiments of the invention also include (where “E” wherein said different amino acid residue is a conser indicates "embodiment'): Vative amino acid Substitution. E1. An isolated polypeptide having Pseudomonas exo E4. The isolated polypeptide of embodiment E3, wherein toxin A biological activity, wherein said polypeptide 65 said conservative amino acid Substitution is one or comprises an epitope selected from the group consist more Substitutions selected from the group consisting ing of of: US 9,677,055 B2 109 110 a) A is substituted with any one of G. I. L. S. T or V: g) ISFSTRGTQNWTVER (SEQID NO:131), wherein b) D is substituted with E: amino acid residues at one or more of positions 1, 6. c) I is substituted with any one of L, M or V: 9, 10 and 12 are substituted with a different amino d) K is substituted with any one of H or R: acid residues wherein amino the acid residue at e) L is substituted with any one of A, G, I, M or V: 5 position 1 (I) is substituted with A. N. T. Q or H, or f) N is substituted with any one of S, T or Q; wherein the amino acid residue at position 6 (R) is g) O is substituted with any one of S, T or N: substituted with Q, or wherein the amino acid resi h) R is substituted with any one of K or H: due at position 9 (Q) is substituted with N or T, or i) S is substituted with any one of A, G., N, T or Q: wherein amino the acid residue at position 10 (N) is 10 substituted with Kor R, or wherein the amino acid j) T is substituted with any one of A, G. N. Q or S, and residue at position 12 (T) is substituted with Kor R. k) V is substituted with any one of A, G, I, L or M. or wherein the amino acid sequence ISFSTRGTQN E5. An isolated polypeptide having Pseudomonas exo WTVER (SEQ ID NO:131) comprises two or more toxin A biological activity, wherein said polypeptide of said Substitutions in any combination, and comprises an epitope selected from the group consist 15 h) IVFGGVRARSQDLDAI (SEQ ID NO:132), ing of wherein amino the acid residue at position 1 (I) is a) ISFSTRGTQ (SEQ ID NO:5), wherein amino the substituted with A or N, or wherein the amino acid acid residue at position 1 (I) is substituted with A. N. residue at position 6 (V) is substituted with D. M, or T. Q or H, or wherein the amino acid residue at N, wherein amino the acid residue at position 11 (Q) position 6 (R) is substituted with Q, or wherein the is substituted with Kor R, or wherein the amino acid amino acid residue at position 9 (Q) is substituted residue at position 14 (D) is substituted with Kor R. with N or T, or wherein the amino acid sequence or wherein the amino acid sequence IVFGGVRAR ISFSTRGTQ (SEQID NO:5) comprises two or more SQDLDAI (SEQ ID NO:132) comprises two or of said Substitutions in any combination; more of said Substitutions in any combination. b) GTQNWTVER (SEQID NO:6), wherein the amino 25 E6. An isolated polypeptide having Pseudomonas exo acid residue at position 3 (Q) is substituted with N or toxin A biological activity, wherein said polypeptide T, wherein amino the acid residue at position 4 (N) comprises an epitope selected from the group consist is substituted with Kor R, or wherein the amino acid ing of residue at position 6 (T) is substituted with Kor R. a) I at position 141 is a different amino acid; or wherein the amino acid sequence GTONWTVER 30 b) R at position 146 is a different amino acid; (SEQ ID NO:6) comprises two or more of said c) Q at position 149 is a different amino acid; substitutions in any combination; d) Nat position 150 is a different amino acid: c) IVFGGVRAR (SEQ ID NO:7), wherein amino the e) T at position 152 is a different amino acid, acid residue at position 1 (I) is substituted with A or f) I at position 184 is a different amino acid; N, or wherein the amino acid residue at position 6 35 g) V at position 189 is a different amino acid; (V) is substituted with D. M. or N, or wherein the h) Q at position 194 is a different amino acid; amino acid sequence IVFGGVRAR (SEQID NO:7) i) D at position 197 is a different amino acid; comprises Substitutions at both positions in any j) L at position 233 is a different amino acid, combination of amino acid residues A or N at posi k) R at position 234 is a different amino acid; tion 1 (I) and D. M. or N at position 6 (V); 40 1) S at position 241 is a different amino acid; d) ARSQDLDAI (SEQ ID NO:8), wherein amino the m) I at position 321 is a different amino acid; acid residue at position 4 (Q) is substituted with Kor n) K at position 324 is a different amino acid; R, or wherein the amino acid residue at position 7 o) Q at position 326 is a different amino acid; (D) is substituted with Kor R, or wherein the amino p) A at position 327 is a different amino acid; acid sequence ARSQDLDAI (SEQ ID NO:8) com 45 q) any combination of one or more of a) through p), prises substitutions with Kor R in any combination wherein the amino acid numbering corresponds to SEQ at both positions 4 (Q) and 7 (D): ID NO:1. e) LRVYVPRSS (SEQ ID NO:9), wherein amino the E7. An isolated polypeptide comprising an amino acid acid residue at position 1 (L) is substituted with A, or sequence identical to SEQ ID NO:1, except for one or wherein the amino acid residue at position 2 (R) is 50 more amino acid Substitutions selected from the group substituted with D, S or A, or wherein the amino acid consisting of: residue at position 9 (S) is substituted with D, E, N. a) I at position 141 is substituted with a conservative K., P or T, or wherein the amino acid sequence amino acid Substitution; LRVYVPRSS (SEQ ID NO:9) comprises two or b) Rat position 146 is substituted with a conservative more of said Substitutions in any combination; 55 amino acid Substitution; f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid c) O at position 149 is substituted with a conservative residues at one or more of positions 1, 4, 6 and 7 are amino acid Substitution; substituted with a different amino acid residue, d) Nat position 150 is substituted with a conservative wherein amino the acid residue at position 1 (I) is amino acid Substitution; substituted with A. N. T. Q or H, or wherein the 60 e) T at position 152 is substituted with a conservative amino acid residue at position 4 (K) is Substituted amino acid Substitution, with T. or wherein the amino acid residue at position f) I at position 184 is substituted with a conservative 6 (Q) is substituted with D, or wherein the amino amino acid Substitution; acid residue at position 7 (A) is substituted with D. g) V at position 189 is substituted with a conservative or wherein the amino acid sequence IPDKEQAIS 65 amino acid Substitution; (SEQ ID NO:10) comprises two or more of said h) Q at position 194 is substituted with a conservative Substitutions in any combination; amino acid Substitution d: US 9,677,055 B2 111 112 i) D at position 197 is substituted with a conservative -continued amino acid Substitution; ah) I at position 321 is N: j) L at position 233 is substituted with a conservative ai) I at position 321 is T: amino acid Substitution; alk) I at position 321 is Q; k) Rat position 234 is substituted with a conservative al) I at position 321 is H: amino acid Substitution; am) Kat position 324 is T. an) Q at position 326 is D; 1) Sat position 241 is substituted with a conservative ao) A at position 327 is D; amino acid Substitution; ap) any combination of one or m) I at position 321 is substituted with a conservative more of a) through ao), amino acid Substitution; 10 n) K at position 324 is substituted with a conservative wherein the amino acid numbering corresponds to SEQ amino acid Substitution; ID NO:1. o) Qat position 326 is substituted with a conservative E10. The polypeptide in any one of embodiments E1 to amino acid Substitution; 15 E9, comprising a number of amino acid Substitutions p) A at position 327 is substituted with a conservative Selected from the group consisting of: amino acid Substitution; q) any combination of one or more of a) through p), wherein the amino acid numbering corresponds to SEQ a) 1 amino acid Substitution; ID NO:1. b) 2 amino acid Substitutions; E8. The isolated polypeptide of embodiment E7, wherein c) 3 amino acid Substitutions; said conservative amino acid Substitution is one or d) 4 amino acid substitutions; e) 5 amino acid Substitutions; more substitutions selected from the group consisting f) 6 amino acid Substitutions; of: g) 7 amino acid Substitutions; a) A is substituted with any one of G. I. L. S. T or V: h) 8 amino acid Substitutions; 25 i) 9 amino acid Substitutions; b) D is substituted with E: j) 10 amino acid Substitutions; c) I is substituted with any one of L, M or V. k) 11 amino acid substitutions; d) K is substituted with any one of H or R: l) 12 amino acid Substitutions; e) L is substituted with any one of A, G, I, M or V: m) 13 amino acid Substitutions; n) 14 amino acid Substitutions; f) N is substituted with any one of S, T or Q; 30 o) 15 amino acid Substitutions; and g) O is substituted with any one of S, T or N: p) 16 amino acid Substitutions. h) R is substituted with any one of K or H: i) S is substituted with any one of A, G., N, T or Q: j) T is substituted with any one of A, G. N. Q or S; and E11. The polypeptide of embodiment E9, comprising k) V is substituted with any one of A, G, I, L or M. amino acid Substitutions present at each of amino acid E9. An isolated polypeptide comprising an amino acid 35 positions 141, 146, 149, 150, 152, 184, 189, 194, 197, sequence identical to SEQ ID NO:1, except for one or 233, 234, 241, 321, 324, 326 and 327. more amino acid Substitutions selected from the group E12. The polypeptide of any one of embodiments E1 to consisting of: E11, wherein said polypeptide comprises the amino acid sequence in SEQID NO:1, except for amino acid 40 Substitutions indicated in embodiments E1 to E11. a) I at position 141 is A: E13. The polypeptide in any one of embodiments E1 to b) I at position 141 is N: E11, wherein said polypeptide is a variant or fragment c) I at position 141 is T. of a Pseudomonas exotoxin A polypeptide. d) I at position 141 is Q; e) I at position 141 is H: E14. The polypeptide of embodiment E13, wherein said R at position 146 is Q; 45 variant or fragment comprises a number of epitopes g) Q at position 149 is N: Selected from the group consisting of: h) Q at position 149 is T: a) at least one epitope; i) Nat position 150 is R: ) Nat position 150 is K: b) at least two epitopes; k) Tat position 152 is R; c) at least three epitopes; ) Tat position 152 is K: 50 d) at least four epitopes; m) I at position 184 is A: n) I at position 184 is N: e) at least five epitopes; and o) V at position 189 is D; f) at least six epitopes. p) V at position 189 is M: E15. The polypeptide of embodiment E14, wherein said q) V at position 189 is N: polypeptide comprises a contiguous number of amino r) Q at position 194 is R: 55 acids selected from the group consisting of s) Q at position 194 is K: ) D at position 197 is R; a) at least 20 contiguous amino acids, u) D at position 197 is K: b) at least 30 contiguous amino acids; v) L at position 233 is A: c) at least 40 contiguous amino acids; w) Rat position 234 is D: d) at least 50 contiguous amino acids; X) Rat position 234 is S: y) Rat position 234 is A: 60 e) at least 60 contiguous amino acids, Z) Sat position 241 is D; f) at least 70 contiguous amino acids. ab) Sat position 241 is E: g) at least 80 contiguous amino acids; ac) Sat position 241 is N: h) at least 90 contiguous amino acids; ad) Sat position 241 is K: ae) Sat position 241 is P: i) at least 100 contiguous amino acids; af) Sat position 241 is T: 65 j) at least 125 contiguous amino acids, ag) I at position 321 is A: k) at least 150 contiguous amino acids; 1) at least 175 contiguous amino acids, US 9,677,055 B2 113 114 m) at least 200 contiguous amino acids; E23. The polypeptide in any one of embodiments E 16 to n) at least 225 contiguous amino acids, E22, wherein said polypeptide is a variant or fragment o) at least 250 contiguous amino acids; of a Pseudomonas exotoxin A polypeptide. p) at least 275 contiguous amino acids; E24. The polypeptide of any one of embodiments E1 to q) at least 300 contiguous amino acids; E23, wherein said polypeptide has one or more bio r) at least 325 contiguous amino acids; and logical activities selected from the group consisting of: S) at least 350 contiguous amino acids. a) eukaryotic cell killing activity (cell cytotoxicity); E16. An isolated polypeptide comprising a Pseudomonas b) inhibits translation elongation factor EF-2 biological exotoxin A (PE-A) cytotoxic domain (Domain III), activity; 10 c) induces or catalyzes ADP-ribosylation of EF-2; and wherein the cytotoxic domain comprises one or more d) inhibits protein synthesis. amino acid substitutions which prevent or reduce host E25. The polypeptide of any one of embodiments E1 to immunogenic responses compared to the same poly E24, wherein said one or more amino acid substitutions peptide without said one or more amino acid Substitu prevent or reduce host immunogenic responses com tions. 15 pared to the same polypeptide without the correspond E17. The polypeptide of embodiment E16, wherein said ing said one or more amino acid substitutions. one or more amino acid Substitutions are introduced E26. The polypeptide of any one of embodiments E1 to into a cytotoxic domain sequence selected from the E24, wherein said one or more amino acid substitutions group consisting of prevent or reduce host immunogenic responses com (a) amino acid residues Phe-134 to Lys-347 of SEQID pared to a polypeptide comprising an amino acid NO:1; sequence selected from the group consisting of: (b) amino acid residues Phe-134 to Lys-347 of SEQID (a) SEQ ID NO:1; NO:4: (b) SEQ ID NO:4: (c) amino acid residues Phe-400 to Lys-613 of SEQID (c) SEQ ID NO:133; and NO:133; and 25 (d) SEQ ID NO:134. (d) amino acid residues Phe-400 to Lys-613 of SEQID E27. The polypeptide of any one of embodiments E1 to NO:134. E26, wherein said polypeptide is a fusion protein. E18. The polypeptide of embodiment E17, wherein the E28. The fusion protein of embodiment E27, wherein the last five amino acids in said cytotoxic domain are amino-terminal end of said polypeptide in any one of replaced with an amino acid sequence selected from the 30 embodiments E1 to E26 is fused to the carboxyl group consisting of terminal end of a different polypeptide. E29. The fusion protein of embodiment E27, wherein the carboxyl-terminal end of said polypeptide in any one of (i) embodiments E1 to E26 is fused to the amino-terminal (SEQ ID NO: 136) 35 end of a different polypeptide. Arg-Glu-Asp-Lieu; E30. The fusion protein in embodiment E28 or E29, and wherein said different polypeptide comprises an anti (ii) gen binding moiety. (SEO ID NO : 137) E31. The fusion protein of embodiment E30, wherein said Lys-Asp-Glu-Lieu. 40 antigen binding moiety is an antibody or fragment thereof. E19. The polypeptide in any one of embodiments E 16 to E32. The fusion protein of embodiment E31, wherein said E18, wherein said polypeptide further comprises one or antibody, or fragment thereof is an antibody selected more PE-A domains selected from the group consisting of: from the list in Table 1, or is a fragment thereof. 45 E33. The fusion protein of embodiment E31, wherein said (a) a cytosolic translocation domain (Domain II); antibody, or fragment thereof, specifically binds to a (b) a carboxy-terminal portion of Domain IB; cancer-specific or tumor-specific antigen. (c) an amino-terminal portion of Domain IB; and E34. The fusion protein of embodiment E33, wherein said (d) a complete Domain IB. cancer-specific or tumor-specific antigen is a breast wherein one or more of said domains has been modified 50 cancer antigen. with amino acid Substitutions, as described herein, to E35. The fusion protein of embodiment E34, wherein said reduce or eliminate immunogenicity. breast cancer antigen is HER2. E20. The polypeptide of embodiment E19, wherein said E36. The fusion protein of embodiment E31, wherein said cytosolic translocation domain (Domain II) comprises antibody, or fragment thereof is selected from the group an amino acid sequence selected from the group con 55 consisting of: sisting of: a) ERTUMAXOMAB (Rexomun): (a) amino acids Gly-3 to Ser-114 of SEQID NO:1; and b) PERTUZUMAB (Omnitarg); and (b) amino acids Gly-3 to Asn-114 of SEQ ID NO:4. c) TRASTUZUMAB (Herceptin). E21. The polypeptide of embodiment E19, wherein said E37. The fusion protein of any one of embodiments E27 carboxy-terminal portion of Domain IB comprises the 60 to E29, wherein said different polypeptide comprises a amino acid sequence of Gly-115 to Glu-133 of SEQID polypeptide selected from the group consisting of: NO:1 or wherein said amino-terminal portion of a) Mesothelin; Domain IB comprises the amino acid sequence of b) CD24; Ala-365 to Ala-380 of SEQ ID NO: 133. c) CD22: E22. The polypeptide of embodiment E19, wherein said 65 d) CD25, complete Domain IB comprises the amino acid e) CD174; sequence of Ala-365 to Glu-399 of SEQ ID NO:133. f) TPBG: US 9,677,055 B2 115 116 g) CD56; and E48. The method of embodiment E47, wherein the poly h) C-type lectin-like molecule-1. peptide or fusion protein is delivered to the subject by E38. An isolated polynucleotide encoding the polypeptide first administering the first, second, and third poly or fusion protein in any one of embodiments E1 to E37. nucleotides. E39. An expression vector comprising the polynucleotide 5 E49. The method of embodiment E47, wherein the poly of embodiment E38. peptide or fusion protein is delivered to the subject by E40. A host cell comprising the expression vector of first administering the expression vector or expression embodiment E39. vectors of embodiments E43 or E44. E41. A method of producing the polypeptide or fusion E50. The method of embodiment E47, wherein said 10 polypeptide or fusion protein is delivered to the subject protein in any one of embodiments E1 to E37, wherein by first administering the host cell of embodiment E45. said method comprises: E51. A pharmaceutical composition comprising the poly a) obtaining a host cell comprising a polynucleotide peptide or fusion protein in any one of embodiments E1 encoding said polypeptide or fusion protein; to E37, comprising the polynucleotide of embodiment b) exposing said host cell to conditions wherein said 15 E38, comprising the expression vector or expression polypeptide or fusion protein is produced. vectors in any one of embodiments E39, E43 or E44, or E42. A method of producing the polypeptide or fusion comprising the host cell of embodiments E40 or E45, protein in any one of embodiments E1 to E37, wherein and a pharmaceutically acceptable carrier, diluent or said method comprises use of an expression system excipient. comprising: 2O E52. A medicament comprising the polypeptide or fusion (A) a first polynucleotide encoding a first hybrid poly protein in any one of embodiments E1 to E37, com peptide comprising: prising the polynucleotide of embodiment E38, com (i) a first ligand binding domain; and prising the expression vector or expression vectors in (ii) a DNA-binding domain; any one of embodiments E39, E43 or E44, or compris (B) a second polynucleotide encoding a second hybrid 25 ing the host cell of embodiments E40 or E45. polypeptide comprising: E53. Use of the medicament of embodiment E52, wherein (i) a second ligand binding domain, and said use is for the treatment of a disease or disorder. (ii) a transactivation domain; E54. Use of the medicament according to embodiment (C) a third polynucleotide encoding the polypeptide or E53, wherein the disease or disorder is cancer. 30 E55. A polypeptide having at least one Pseudomonas fusion protein in any one of embodiments E1 to E37, exotoxin A (PE-A) biological activity, wherein said wherein said third polynucleotide is operably asso polypeptide comprises one or more amino acid substi ciated with a response element capable of being tutions compared to a wild-type PE-A polypeptide, bound by the DNA-binding domain of said first wherein said one or more amino acid Substitutions is a hybrid polypeptide; 35 Substitution of a different amino acid at one or more wherein the first ligand binding domain and the second positions corresponding to amino acid residues in the ligand binding domain are capable of ligand-induced polypeptide of SEQID NO:1, wherein said substitution dimerization, positions are selected from the group consisting of wherein expression of the polypeptide or fusion protein in a) isoleucine (I) at position 141; any one of embodiments E1 to E37 is modulated by a 40 b) arginine (R) at position 146; ligand which induces dimerization of said first and said c) glycine (G) at position 147; second ligand binding domains, d) glutamine (Q) at position 149: wherein the polypeptide or fusion protein in any one of e) asparagine (N) at position 150; embodiments E1 to E37 is produced by allowing said f) threonine (T) at position 152; ligand to contact said first and said second ligand 45 g) valine (V) at position 189: binding domains. h) arginine (R) at position 192: E43. A single expression vector or two or more expression i) glutamine (Q) at position 194; vectors comprising the first, second, and third poly j) aspartic acid (D) at position 197; nucleotides of embodiment E42. k) serine (S) at position 241: 1) isoleucine (I) at position 321; and E44. The expression vector or expression vectors of m) glutamine (Q) at position 326. embodiment E43, wherein one or more of the vectors E56. The polypeptide of embodiment E55, wherein said is a viral expression vector. one or more amino acid Substitutions is a conservative E45. A host cell comprising the expression vector or amino acid Substitution. expression vectors of embodiments E43 or E44. 55 E57. The polypeptide of embodiment E55, wherein said E46. A method of treating a disease or disorder compris one or more amino acid substitutions is selected from ing administering to a Subject in need thereof the the group consisting of: polypeptide or fusion protein in any one of embodi a) isoleucine (I) at position 141 is substituted with ments E1 to E37, the polynucleotide of embodiment alanine (A), threonine (T), or histidine (H): E38, the vector of embodiment E39, the host cell of 60 b) arginine (R) at position 146 is substituted with embodiment E40, or a polypeptide or fusion protein glutamine (Q) or alanine (A): produced by the method of embodiment E41. c) glycine (G) at position 147 is substituted with serine E47. A method of treating a disease or disorder compris (S); ing delivering to a Subject in need thereof a polypeptide d) glutamine (Q) at position 149 is substituted with or fusion protein produced by the method of embodi- 65 threonine (T); ment E42, wherein said method comprises administra e) asparagine (N) at position 150 is substituted with tion of the ligand to said Subject. alanine (A): US 9,677,055 B2 117 118 f) threonine (T) at position 152 is substituted with tion 141, a Substitution of arginine (R) or alanine (A) alanine (A) or arginine (R): for threonine (T) at position 152, a substitution of g) valine (V) at 189 is substituted with alanine (A): lysine (K) for aspartic acid (D) at position 197, a h) arginine (R) at position 192 is substituted with substitution of threonine (T) for serine (S) at position alanine (A) or glutamine (Q); 5 241, and a substitution of glutamic acid (E) for gluta i) glutamine (Q) at position 194 is substituted with mine (Q) at position 326. arginine (R): E65. The isolated polypeptide of embodiment E55, j) aspartic acid (D) at position 197 is substituted with wherein said polypeptide comprises a substitution of lysine (K); alanine (A) for isoleucine (I) at position 141, a Substi k) serine (S) at position 241 is substituted with threo 10 nine (T), asparagine (N), lysine (K), or proline (P): tution of arginine (R) for threonine (T) at position 152, 1) isoleucine (I) at position 321 is substituted with a substitution of lysine (K) for aspartic acid (D) at alanine (A), asparagine (N), histidine (H), threonine position 197, a substitution of threonine (T) for serine (T), or glutamine (Q); and (S) at position 241, and a substitution of glutamic acid m) glutamine (Q) at position 326 is substituted with 15 (E) for glutamine (Q) at position 326. glutamic acid (E). E66. The polypeptide of embodiment E55, wherein said E58. The polypeptide of embodiment E55, wherein said polypeptide comprises a Substitution of alanine (A) for polypeptide comprises a Substitution for isoleucine (I) isoleucine (I) at position 141, a Substitution of alanine at position 141, a substitution for threonine (T) at (A) for threonine (T) at position 152, a substitution of position 152, a Substitution for arginine (R) at position lysine (K) for aspartic acid (D) at position 197, a 192, a substitution for aspartic acid (D) at position 197, substitution of threonine (T) for serine (S) at position a Substitution for serine (S) at position 241, and a 241, and a substitution of glutamic acid (E) for gluta substitution for glutamine (Q) at position 326. mine (Q) at position 326. E59. The polypeptide of embodiment E55, wherein said E67. The polypeptide of embodiment E55, wherein said polypeptide comprises a Substitution of threonine (T) or 25 polypeptide comprises a Substitution of threonine (T) alanine (A) for isoleucine (I) at position 141, a Substi for isoleucine (I) at position 141, a substitution of tution alanine (A) or arginine (R) for threonine (T) at alanine (A) for threonine (T) at position 152, a substi position 152, a Substitution of alanine (A) for arginine tution of lysine (K) for aspartic acid (D) at position 197, (R) at position 192, a substitution of lysine (K) for a substitution of threonine (T) for serine (S) at position aspartic acid (D) at position 197, a substitution of 30 241, and a substitution of glutamic acid (E) for gluta threonine (T) for serine (S) at position 241, and a substitution of glutamic acid (E) for glutamine (Q) at mine (Q) at position 326. position 326. E68. The polypeptide in any one of embodiments E55 to E60. The polypeptide of embodiment E55, wherein said E67, wherein the at least one Pseudomonas exotoxin A polypeptide comprises a Substitution of threonine (T) 35 (PE-A) biological activity comprises the ability to for isoleucine (I) at position 141, a Substitution alanine inhibit in vitro transcription/translation compared to a (A) for threonine (T) at position 152, a substitution of corresponding wild-type or non-substituted PE-A poly alanine (A) for arginine (R) at position 192, a Substi peptide, wherein said ability to inhibit in vitro tran tution of lysine (K) for aspartic acid (D) at position 197, Scription/translation is in an amount selected from the a substitution of threonine (T) for serine (S) at position 40 group consisting of 241, and a Substitution of glutamic acid (E) for gluta (a) at least 5% inhibition; mine (Q) at position 326. (b) at least 10% inhibition; E61. The polypeptide of embodiment E55, wherein said (c) at least 15% inhibition, polypeptide comprises a Substitution of alanine (A) for (d) at least 20% inhibition; isoleucine (I) at position 141, a Substitution alanine (A) 45 (e) at least 25% inhibition; for threonine (T) at position 152, a substitution of (f) at least 30% inhibition: alanine (A) for arginine (R) at position 192, a Substi (g) at least 40% inhibition, tution of lysine (K) for aspartic acid (D) at position 197, (h) at least 50% inhibition; a substitution of threonine (T) for serine (S) at position (i) at least 60% inhibition; 241, and a Substitution of glutamic acid (E) for gluta 50 () at least 70% inhibition; mine (Q) at position 326. (k) at least 80% inhibition; E62. The polypeptide of embodiment E55, wherein said (1) at least 90% inhibition: polypeptide comprises a Substitution for isoleucine (I) (m) at least 100% inhibition; at position 141, a substitution for threonine (T) at (n) about 100% inhibition; and position 152, a substitution for aspartic acid (D) at 55 (o) 100% inhibition. position 197, a substitution for serine (S) at position E69. The polypeptide in any one of embodiments E55 to 241, and a Substitution for glutamine (Q) at position E68, comprising a number of amino acid Substitutions 326. Selected from the group consisting of: E63. The polypeptide of embodiment E55, wherein said a) 1 amino acid substitution; polypeptide comprises a Substitution for isoleucine (I) 60 b) 2 amino acid substitutions; at position 141, a substitution for threonine (T) at c) 3 amino acid substitutions; position 152, a Substitution for arginine (R) at position d) 4 amino acid substitutions; 192, a substitution for aspartic acid (D) at position 197, e) 5 amino acid substitutions; and and a substitution for serine (S) at position 241. f) 6 amino acid Substitutions. E64. The isolated polypeptide of embodiment E55, 65 E70. The polypeptide in any one of embodiments E55 to wherein said polypeptide comprises a substitution of E69, wherein said polypeptide comprises one or more alanine (A) or threonine (T) for isoleucine (I) at posi amino acid Substitutions which prevent or reduce host US 9,677,055 B2 119 120 immunogenic responses compared to the same poly E81. The fusion protein in embodiment E79 or E80, peptide without said one or more amino acid Substitu wherein said different polypeptide comprises an anti tions. gen binding moiety. E71. The polypeptide of embodiment E70, wherein host E82. The fusion protein of embodiment E81, wherein said immunogenic responses are prevented or reduced in a 5 antigen binding moiety is an antibody or fragment human host. thereof. E72. The polypeptide in any one of embodiments E55 to E83. The fusion protein of any one of embodiments E78 E71, wherein the last five or six amino acids in said to E82, wherein said antibody, or fragment thereof, is polypeptide comprise one or more amino acid an antibody selected from the list in Table 1, or is a sequences selected from the group consisting of: 10 fragment thereof. (i) Arg-Glu-Asp-Leu-Lys; E84. The fusion protein of embodiment E82, wherein said (ii) Arg-Glu-Asp-Leu, antibody, or fragment thereof, specifically binds to a (iii) Lys-Asp-Glu-Leu, cancer-specific or tumor-specific antigen. (iv) Glu-Asp-Leu-Lys; and 15 E85. The fusion protein of embodiment E84, wherein said (v) a dimer, trimer, pentamer, hexamer, septamer, or cancer-specific or tumor-specific antigen is a breast octamer of (i), (ii), or (iii), or any combination cancer antigen. thereof. E86. The fusion protein of embodiment E85, wherein said E73. The polypeptide of any one of embodiments E55 to breast cancer antigen is HER2. E72, wherein said polypeptide has one or more bio E87. The fusion protein of embodiment E82, wherein said logical activities selected from the group consisting of: antibody, or fragment thereof is selected from the group a) eukaryotic cell killing activity (cell cytotoxicity); consisting of: b) inhibits translation elongation factor EF-2 biological a) ERTUMAXOMAB (Rexomun): activity; b) PERTUZUMAB (Omnitarg); and c) induces or catalyzes ADP-ribosylation of EF-2; and 25 c) TRASTUZUMAB (Herceptin). d) inhibits protein synthesis. E88. The fusion protein of any one of embodiments E78 E74. The polypeptide of any one of embodiments E55 to to E80, wherein said different polypeptide comprises a E72, wherein said one or more amino acid substitutions polypeptide selected from the group consisting of: prevent or reduce host immunogenic responses com a) Mesothelin; pared to the same polypeptide without the correspond 30 b) CD24; ing said one or more amino acid substitutions, c) CD22: E75. A polypeptide comprising a biologically active frag d) CD25; ment of the polypeptide in any one of embodiments e) CD174; E55 to ET4. f) TPBG: E76. A polypeptide comprising a variant or derivative of 35 g) CD56; and the polypeptide in any one of embodiments E55 to E75, h) C-type lectin-like molecule-1. wherein said variant or derivative shares amino acid E89. A polynucleotide encoding the polypeptide or fusion sequence identity with the polypeptide in any one of protein in any one of embodiments E55 to E88. embodiments E55 to E75, wherein said shared amino E90. An expression vector comprising the polynucleotide acid sequence identity is selected from the group con 40 of embodiment E89. sisting of: E91. A host cell comprising the expression vector of a) at least 80% identity; embodiment E90. b) at least 85% identity: E92. A method of producing the polypeptide or fusion c) at least 90% identity; protein in any one of embodiments E55 to E88, wherein d) at least 95% identity; 45 said method comprises: e) at least 97% identity: a) obtaining a host cell comprising a polynucleotide f) at least 98% identity; and encoding said polypeptide or fusion protein; g) at least 99% identity. b) exposing said host cell to conditions wherein said E77. The polypeptide of any one of embodiments E55 to polypeptide or fusion protein is produced. E76, wherein said one or more amino acid substitutions 50 E93. A method of producing the polypeptide or fusion prevent or reduce host immunogenic responses com protein in any one of embodiments E55 to E88, wherein pared to a polypeptide comprising an amino acid said method comprises use of an expression system sequence selected from the group consisting of: comprising: (a) SEQ ID NO:1; (A) a first polynucleotide encoding a first hybrid poly (b) SEQ ID NO:4: 55 peptide comprising: (c) SEQ ID NO:133; and (i) a first ligand binding domain; and (d) SEQ ID NO:134. (ii) a DNA-binding domain; E78. The polypeptide of any one of embodiments E55 to (B) a second polynucleotide encoding a second hybrid E77, wherein said polypeptide is a fusion protein. polypeptide comprising: E79. The fusion protein of embodiment E78, wherein the 60 (i) a second ligand binding domain; and amino-terminal end of said polypeptide in any one of (ii) a transactivation domain; embodiments E55 to E78 is fused to the carboxyl (C) a third polynucleotide encoding the polypeptide or terminal end of a different polypeptide. fusion protein in any one of embodiments E55 to E80. The fusion protein of embodiment E78, wherein the E88, wherein said third polynucleotide is operably carboxyl-terminal end of said polypeptide in any one of 65 associated with a response element capable of being embodiments E55 to E78 is fused to the amino-terminal bound by the DNA-binding domain of said first end of a different polypeptide. hybrid polypeptide; US 9,677,055 B2 121 122 wherein the first ligand binding domain and the second position corresponding to amino acid position E184 in ligand binding domain are capable of ligand-induced SEQ ID NO:1 (or position E196 in SEQ ID NO:2) dimerization, wherein an isoleucine at position E184 (or position 196 wherein expression of the polypeptide or fusion protein in in SEQID NO:2) is substituted with a different amino any one of embodiments E55 to E88 is modulated by a acid. ligand which induces dimerization of said first and said E107. The polypeptide of embodiment E 106, wherein second ligand binding domains, said polypeptide does not have PE-A biological activ wherein the polypeptide or fusion protein in any one of ity. embodiments E55 to E88 is produced by allowing said E108. A method for assaying the immunogenicity of a ligand to contact said first and said second ligand 10 binding domains. mutated form of Pseudomonas exotoxin A (PE-A), E94. A single expression vector or two or more expression wherein said method comprises: vectors comprising the first, second, and third poly (a) contacting immune cells with a mutated form of nucleotides of embodiment E93. PE-A; and E95. The expression vector or expression vectors of 15 (b) assaying immune cell stimulation, embodiment E94, wherein one or more of the vectors wherein said mutated form of PE-A comprises a mutation is a viral expression vector. at a position corresponding to amino acid position E184 E96. A host cell comprising the expression vector or in SEQ ID NO:1 (or position 196 in SEQ ID NO:2) expression vectors of embodiments E94 or E95. wherein an isoleucine at position E184 (or position 196 E97. A method of treating a disease or disorder compris in SEQID NO:2) is substituted with a different amino ing administering to a Subject in need thereof the acid, and wherein said mutated form of PE-A also polypeptide or fusion protein in any one of embodi comprises one or more additional amino acid Substitu ments E55 to E88, the polynucleotide of embodiment tions compared to a wild-type form of PE-A. E89, the expression vector or expression vectors in any E109. The method of embodiment E 108, wherein said one of embodiments E90, E94 or E95, the host cell of 25 immune cells are human immune cells. embodiments E91 or E96, or a polypeptide or fusion E 110. The method of embodiment E109, wherein said protein produced by the method of embodiment E92. immune cells are human T-cells, cells of a human T-cell E98. A method of treating a disease or disorder compris lineage, human B-cells, or cells of a human B-cell ing delivering to a Subject in need thereof a polypeptide lineage. or fusion protein produced by the method of embodi 30 E111. The method of embodiment E47, wherein the ment E93, wherein said method comprises administra ligand is a compound having Formula I, or a pharma tion of the ligand to said subject. ceutically acceptable salt thereof. E99. The method of embodiment E44, wherein the poly E112. The method of embodiment E47, wherein the peptide or fusion protein is delivered to the subject by ligand is a compound having Formula II, or a pharma first administering the first, second, and third poly 35 ceutically acceptable salt thereof. nucleotides. E 113. The method of embodiment E47, wherein the E100. The method of embodiment E98, wherein the ligand is a compound having Formula III, or a phar polypeptide or fusion protein is delivered to the subject maceutically acceptable salt thereof. by first administering the expression vector or expres E114. The method of embodiment E47, wherein the sion vectors of embodiments E94 or E95. 40 ligand is a compound of Table 3, or a pharmaceutically E101. The method of embodiment E98, wherein said acceptable salt thereof. polypeptide or fusion protein is delivered to the subject E115. The method of embodiment E47, wherein the by first administering the host cell of embodiments E91 ligand is a compound having Formula III, wherein: or E96. A is: E102. A pharmaceutical composition comprising the 45 polypeptide or fusion protein in any one of embodi ments E55 to E88, comprising the polynucleotide of embodiment E89, comprising the expression vector or expression vectors in any one of embodiments E90. E94 or E95, or comprising the host cell of embodiments 50 E91 or E96, and a pharmaceutically acceptable carrier, diluent or excipient. E103. A medicament comprising the polypeptide or fusion protein in any one of embodiments E55 to E88, comprising the polynucleotide of embodiment E89, 55 comprising the expression vector or expression vectors in any one of embodiments E90. E94 or E95, or comprising the host cell of embodiments E91 or E96. E104. Use of the pharmaceutical composition of embodi ment E 102 or the medicament of embodiment E 103, 60 wherein said use is for the treatment of a disease or disorder. E105. Use of the pharmaceutical composition or the medicament according to embodiment E104, wherein the disease or disorder is cancer. 65 E106. An Pseudomonas exotoxin A (PE-A) polypeptide, wherein said polypeptide comprises a mutation at a US 9,677,055 B2 123 124 R3a, R3, R3, R3s, R3e, R3?, R3s, R3h, R3i and R3, a (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(6- independently selected from hydrogen, halo, (C-C) chloro-pyridine-3-carbonyl)-hydrazide; alkyl, or (C-C)alkoxy; (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3- R" is (C-C)alkyl, hydroxy(C-C)alkyl, or (C-C) methoxy-2-methyl-benzoyl)-hydrazide; alkenyl; and (R)-3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl R’ is optionally substituted (C-C)alkyl, or a pharma butyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; and ceutically acceptable salt thereof. (R)-3,5-Dimethyl-benzoic acid N'-(4-ethyl-benzoyl)-N-(1- E116. The method of embodiment E47, wherein the phenethyl-but-3-enyl)-hydrazide, ligand is a compound selected from the group consist or a pharmaceutically acceptable salt thereof. ing of 10 (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2- EXAMPLES ethyl-3-methoxy-benzoyl)-hydrazide; (R)-3,5-Dimethyl-benzoic acid N-benzoyl-N-(1-tert-butyl Example 1: T Cell Epitope Mapping of PE butyl)-hydrazide: (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2- 15 Peptides spanning the sequence of an approximately 38 methyl-benzoyl)-hydrazide: kD form of Pseudomonas exotoxin A protein (“PE38”) were (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2- analyzed for the presence of immunogenic CD4+ T cell methoxy-benzoyl)-hydrazide; epitopes using EPISCREENTM T cell epitope mapping (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2- analysis (Antitope Ltd, Cambridge, UK). fluoro-benzoyl)-hydrazide; EPISCREENTM is a proprietary technology commercially (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2- available through Antitope Ltd, Cambridge, UK, to map T chloro-benzoyl)-hydrazide; cell epitopes within a protein sequence to determine poten (R)-3,5-Dimethyl-benzoic acid N'-(2-bromo-benzoyl)-N-(1- tial for immunogenicity (based on the number and potency tert-butyl-butyl)-hydrazide; 25 of T cell epitopes within a sequence). EPISCREENTM T cell (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3- epitope mapping typically uses CD8+ T cell depleted methyl-benzoyl)-hydrazide: PBMCs from a minimum of 50 HLA-typed donors (selected (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3- to represent the human population of interest). Typically, 15 methoxy-benzoyl)-hydrazide; mer peptides with 12 amino acid overlaps spanning a protein (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3- 30 sequence are analyzed in a large number of replicate cultures chloro-benzoyl)-hydrazide; for in vitro CD4+ T cell stimulation by 3H TaR incorpora (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(4- tion. CD4+ T cell stimulation is often detected in two or methyl-benzoyl)-hydrazide: three adjacent and overlapping peptides since the core 9 mer (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(4- that binds the MHC class II binding groove will be present ethyl-benzoyl)-hydrazide; 35 (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(4- in more than one peptide sequence. Following identification methoxy-benzoyl)-hydrazide; of peptides that stimulate CD4+ T cells in vitro, in silico (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(4- technology can be used to design epitope-depleted (deim chloro-benzoyl)-hydrazide; munized) variants by determining the precise location of (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2, 40 core 9 mer sequences and the location of key MHC class II 6-difluoro-benzoyl)-hydrazide: anchor residues. (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2, A total of 120 overlapping 15 mer peptides spanning the 6-dichloro-benzoyl)-hydrazide; entire PE38 sequence (SEQ ID NO:2), including 4 peptides (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3. covering a null mutation and 4 peptides spanning an N-ter 4-dimethoxy-benzoyl)-hydrazide; 45 minal linker sequence (SEQ ID NO:3) were tested against a (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3. cohort of 52 healthy donors. CD4+ T cell responses against 5-difluoro-benzoyl)-hydrazide: individual peptides were measured using proliferation (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3. assays (3H-thymidine incorporation). The proliferation 5-dimethoxy-4-methyl-benzoyl)-hydrazide: assay data was used to compile a T cell epitope map of the (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(4- 50 PE38 sequence and six T cell epitopes were identified. methyl-benzo. 1.3dioxole-5-carbonyl)-hydrazide; EPISCREENTM Donor Selection (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(5- Peripheral blood mononuclear cells (PBMC) were iso methyl-2,3-dihydro-benzo 1.4 dioxine-6-carbonyl)-hy lated from healthy community donor buffy coats (from drazide; blood drawn within 24 hours) obtained from the UK (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(5- 55 National Blood Transfusion Service (Addenbrooke's Hos ethyl-2,3-dihydro-benzo 1.4 dioxine-6-carbonyl)-hy pital, Cambridge, UK) and according to approval granted by drazide; Addenbrooke's Hospital Local Research Ethics Committee. (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'- PBMC were isolated from buffy coats by LYMPHOPREPTM (naphthalene-1-carbonyl)-hydrazide; (Axis-Shield UK, Dundee, Scotland) density centrifugation. (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'- 60 (LYMPHOPREPTM is a ready-made, sterile and endotoxin (naphthalene-2-carbonyl)-hydrazide; tested Solution for the isolation of human mononuclear cells (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'- from blood. See, Axis-Shield, package insert for LYM (thiophene-2-carbonyl)-hydrazide: PHOPREPTM density gradient media No. 619. March 2003. (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2, Div.-1114740.) CD8+ T cells were depleted using CD8+ 5-dimethyl-furan-3-carbonyl)-hydrazide; 65 ROSETTESEPTM (STEMCELLTM Technologies inc, Man (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2- chester, UK) to remove CD8+ cells from the isolated mono chloro-pyridine-3-carbonyl)-hydrazide; nuclear cells. See e.g., StemCell Technologies Inc., ROSET US 9,677,055 B2 125 126 TESEPTM procedure for Human CD8+ T Cell Enrichment TABLE 7-continued Cocktail (Catalog #15023/15063, Procedure version 1.3.0, “#28572 (May 2011)). Donor Haplotypes and Responses HLA allotypes of donors were characterized using the KLH Biotest HLA SSP-PCR tissue-typing kit (Biotest, Solihull, UK, catalogue number 826215). T cell responses to a Donor Haplotype Test 1 IEXO1 reproducibility control neo-antigen were also determined 35 DRB1*12: DRB3*: DRB48 3.51 2.55 using Imject maricutlure keyhole limpet haemocyanin 36 DRB1*07: DRB3*: DRB48 6.63 8.90 (KLH) (Pierce (Perbio Science UK, Ltd)), Cramlington, UK, 37 DRB1*04, DRB1*04: DRB4* 44.94 6.28 10 38 DRB1*01, DRB1*15; DRB3*: DRB4* 1.36 1.30 catalogue number 77600) with the KLH diluted to a final 39 DRB1*07, DRB1*13; DRB3*: DRB4 12.62 2.29 concentration of 100 ug/ml. PBMC were then frozen and 40 DRB1*03, DRB1*04: DRB3*: DRB4* 1.39 1.37 stored in liquid nitrogen until required. 41 DRB1*07, DRB1*08: DRB4* 340 3.47 A cohort of 52 donors was selected to best represent the 42 DRB1*07, DRB1*13; DRB3*: DRB4* 40.32 7.36 number and frequency of HLA-DR allotypes expressed in 43 DRB1*13, DRB1*15; DRB3*: DRB5* 3.56 3.21 15 44 DRB1*11, DRB1*14: DRB3* 1.15 2.86 the world population. Analysis of the allotypes expressed in 45 DRB1*03, DRB1*13; DRB3* 8.78 8.72 the cohort against those expressed in the world population 46 DRB1*03, DRB1*13; DRB3* 1147 3.11 revealed that coverage of >80% was achieved and that all 47 DRB1*03, DRB1*04: DRB3*: DRB4* 6.27 2.03 major HLA-DR alleles (individual allotypes with a fre 48 DRB1*04, DRB1*15; DRB5* 10.29 3.77 quency >5% expressed in the world population) were well 49 DRB1*07, DRB1*15; DRB4*: DRB5* 2.59 232 represented. Details of individual donor haplotypes and a 50 DRB1*04, DRB1*15; DRB4*: DRB5* 2.49 2.42 comparison of the frequency of MHC class II haplotypes 51 DRB1*11; DRB3*: DRB48 8.30 2.09 expressed in the world population and the sample population 52 DRB1*03, DRB1*13; DRB3* 3.99 6.22 are shown in Table 7 and FIG. 2, respectively. Table 7. EPISCREENTM Analysis: Proliferation Assay Donor details and haplotypes. Donor responses (SI) to PBMC from each donor were thawed, counted and viabil KLH are shown for two independent proliferation assays. 25 ity was assessed. Cells were revived in room temperature Test 1 was performed using KLH on freshly isolated PBMC AIM V(R) culture medium (Invitrogen, Paisley, UK) before and IEXO1 is the KLH re-test performed in the current study adjusting the cell density to 2-3x10 PBMC/ml (prolifera on PBMC recovered from liquid nitrogen storage as indi tion cell Stock). Peptides were synthesized on a 1-3 mg scale cated above. Responses that did not produce the same result with free N-terminal amine and C-terminal carboxylic acid. (i.e. positive including borderline SI >1.90 p-0.05 or nega 30 Peptides were dissolved in DMSO to a concentration of 10 tive) in both tests are donors 3, 7, 9, 33 and 44. mM and peptide culture stocks prepared by diluting into AIMV(R) culture medium to a final concentration of 5 uM in TABLE 7 the well. For each peptide and each donor, sextuplicate Donor Haplotypes and Responses 35 cultures were established in a flat bottomed 96 well plate. Both positive and negative control cultures were also tested KLH in sextuplicate. For each donor, three control antigen/pep tides (KLH protein and peptides derived from Influenza A Donor Haplotype Test 1 IEXO1 and Epstein Barr viruses) were also included. 1 DRB1*15, DRB1*16; DRB5* 18.10 1.97 Cultures were incubated for a total of 6 days before 2 DRB1*03, DRB1*07: DRB3*: DRB4 2.49 5.74 40 adding 0.75 uCi 3H]-thymidine (PERKIN ELMER(R), Bea 3 DRB1*11, DRB1*13; DRB3*: DRB4* O.81 4.00 consfield, UK) to each well. Cultures were incubated for a 4 DRB1*03: DRB3* 1.73 1.78 5 DRB1*01, DRB1*13; DRB3*: DRB4 O.99 1.OS further 18 hours before harvesting onto filter mats using a 6 DRB1*03, DRB1*14: DRB3* 2.91 2.01 TOMTEC MACHR III cell harvester (TOMTECR), Ham 7 DRB1*13, DRB1*14: DRB3*: DRB4 3.13 120 den, Conn., USA). Counts per minute (cpm) for each well 8 DRB1*01, DRB1*07: DRB4* 2.49 5.74 45 were determined by MeltilexTM (PERKIN ELMER(R) scin 9 DRB1*03, DRB1*07: DRB3*: DRB4 O.81 4.00 tillation counting on a Microplate Beta Counter (PERKIN 10 DRB1*03, DRB1*15; DRB5 6.16 6.16 ELMER(R) in paralux, low background counting mode. 11 DRB1*01, DRB1*13; DRB3* 7.15 17.34 12 DRB1*13; DRB4*: DRB5* 7.98 2.76 EPISCREENTM Data Analysis 13 DRB1*13, DRB1*14: DRB4* 1.OO 1.81 For proliferation assays, an empirical threshold of a 14 DRB1*03, DRB1*13; DRB3* 228 2.70 50 stimulation index (SI) equal to or greater than 2 (SI-2.00) 15 DRB1*04, DRB1*11; DRB3*: DRB4* 8.96 1.91 has been previously established whereby samples inducing 16 DRB1*04, DRB1*14: DRB3*: DRB4* 5.85 4.01 proliferative responses above this threshold are deemed 17 DRB1*13, DRB1*15; DRB3*: DRB5* 1916 2.69 18 DRB1*11, DRB1*13; DRB3* 10.32 6.48 positive (where included, borderline SI-1.90 are high 19 DRB1*04, DRB1*15; DRB4* 2.70 3.39 lighted). Extensive assay development and previous studies 20 DRB1*04, DRB1*07: DRB4* O.S1 1.27 55 have shown that this is the minimum signal to noise thresh 21 DRB1*01, DRB1*04: DRB4* 1.71 1.OS old allowing maximum sensitivity without detecting large 22 DRB1*03: DRB3* 1.OS 1.59 numbers of false positive responses. Positive responses are 23 DRB1*04, DRB1*15; DRB4*: DRB5* 2.83 2.34 24 DRB1*O1 1.63 1.09 defined by the following statistical and empirical thresholds: 25 DRB1*04, DRB1*15; DRB4*: DRB5* 1.12 1.44 1. Significance (p<0.05) of the response by comparing 26 DRB1*03, DRB1*07: DRB3*: DRB4 1.18 O.84 cpm of test wells against medium control wells using 27 DRB1*11, DRB1*13; DRB3* 8.80 14.30 60 unpaired two sample Student's t-test. 28 DRB1*01, DRB1*07: DRB4* 3.68 453 2. Stimulation index greater than 2.00 (SI 2.00), where 29 DRB1*12, DRB1*13; DRB3* 3.68 240 30 DRB1*11; DRB3* 7.68 2.71 SI mean cpm of test wells/mean cpm medium control 31 DRB1*03, DRB1*11; DRB3* 3.04 4.16 wells. Data presented in this way is indicated as 32 DRB1*13; DRB3* 1.96 2.22 SI-2.00, p<0.05. 33 DRB1*15; DRB48 1.31 3.13 65 In addition, intra-assay variation was assessed by calcu 34 DRB1*03, DRB1*04: DRB3*: DRB4* 0.97 1.35 lating the coefficient of variance and standard deviation (SD) of the raw data from replicate cultures. US 9,677,055 B2 127 128 Proliferation assays were set up in Sextuplicate cultures adjusted data and 10.7% in the adjusted data (where maxi “non-adjusted data'). To ensure that intra-assay variability mum and minimum values were removed and the mean cpm was low, the data was also analyzed after removing the calculated on the remaining four wells). Thus, peptides containing a T cell epitope induced positive T cell prolif maximum and minimum cpm values (“adjusted data') and eration responses (SI 2.00, p<0.05) in 6 donors in the the SI of donor responses was compared using both data non-adjusted and adjusted data sets. Inter-assay variability SetS. was assessed using KLH as a reproducibility control where T cell epitopes were identified by calculating the average the frequency of positive T cell responses against KLH were frequency of positive responses (defined above) to all pep compared in two separate EPISCREENTM assays (Table 7). tides in the study plus standard deviation (SD) to give a The results show that inter-assay variability is within the background response threshold. Any peptide that induced a 10 acceptable range and consistent with previous studies frequency of positive proliferation responses above this (s.10%). The frequency of T cell responses against the two threshold in both the adjusted and non-adjusted data was control peptides C3 (EBNA derived epitope) and C32 (Influ considered to contain an immunogenic T cell epitope (and, enza derived epitope) ranged between 23-31% (non-ad thus, potentially represents an immunogenicity inducing justed) and between 21-29% (adjusted) for the two peptides, epitope which could give rise to immunogenic responses in 15 respectively (FIG. 3). This is within the typical range vivo). observed for these two peptides in T cell epitope mapping In Silico Analysis of Peptides studies. The sequences of peptides that were positive in the The output from non-adjusted and adjusted data analysis proliferation assay were analyzed using Antitope's predic was examined to ensure that intra-assay variability was low tive iTOPETM software (Perry et al. 2008). This software and that positive responses were not the result of spurious predicts favorable interactions between amino acid side proliferation in individual wells. The results from each chains of the peptide and specific binding pockets within the analysis showed, in most cases, only small differences MHC class II binding groove. Analysis of the peptide between the methods and donor responses for both non sequences using iTOPETM was performed with overlapping adjusted and adjusted analysis. Table 10 provides a Summary 9 mers spanning the peptides which were tested against each of individual donor responses to each of the peptides. The of the 34 MHC class II alleles. Each 9 mer was scored based 25 proliferation assay data showing the frequency of positive on the potential fit and interactions with the MHC class II donor responses to each peptide is shown in FIG. 3. For all molecules. 9 mers that produced a high mean binding score peptides that induced a high frequency of positive (SI 2.00, were identified and, from the T cell proliferation data, 9 mers p-0.05, including borderline responses) T cell proliferation which were considered as critical to T cell responses (“core responses above the background response threshold, addi 9 mers') were highlighted. iTOPETM analysis was then 30 tional in silico analysis was performed to aid in the identi repeated with a range of amino acid changes in the core 9 fication of the precise location of MHC class II core 9 mer mers in order to determine preferred amino acid substitu binding registers (using iTOPETM), and to identify peptides tions for use in deimmunization. that are homologous to sequences containing T cell epitopes Results and Discussion that have been tested in previous EPISCREENTM T cell A total of 120 peptides were synthesized spanning the 35 epitope mapping assays (using TCEDTM). entire PE38 sequence. The peptides were designed as 15 Table 8. mers to span the sequence in overlapping increments of 12 Summary of individual donor responses to PE38 peptides. amino acids. These peptides were then tested for the pres Positive responses (SI-2.00, p<0.05, including borderline ence of CD4+ T cell epitopes by EPISCREENTM T cell responses) are indicated by the donor number and individual epitope mapping analysis. Positive T cell responses were SI are shown in parentheses next to the corresponding donor. defined by donors that produced a significant (p<0.05) 40 The background response rate was 10.8% in the non response with a SI-2.00 to any given peptide (SI 2.00, adjusted data and 10.7% in the adjusted data peptides p-0.05). T cell epitopes were identified by calculating the inducing positive T cell proliferation above this frequency average frequency of the positive responses to all peptides in (positive response in 26 donors) contained T cell epitopes the study plus SD (termed background response thresh (i.e., peptides 50, 52, 53, 65, 67, 68, 81, 82 and 110 (also as old). This was calculated to be 10.8% in the raw non indicated in FIGS. 3-6)). TABLE 8 Donor Responses to PE38 Peptides Peptide # Proliferation Non-Adjusted Proliferation Adjusted Peptide Sequence 1 11 (2.25), 19(2.42), 25 (3.24), 11 (2.34), 19(2.72), GGGGGSGGGGGSPEG 35(2.77), 36(2.21) 25(3.33), 35 (2.76), (SEQ ID NO: 11) 36(2.10) 2 19(2.44), 36(1.94) 19(2.67), 48(2.06) GGSGGGGGSPEGGSL (SEQ ID NO: 12) 3 11 (1.98), 16(1.92), 19(2.54) 11 (1.99), 19(2.78), GGGGGSPEGGSLAAL 38(5.37) (SEQ ID NO: 13) 4 16(2.33), 17 (2.02), 19(2.29), 16(2.13), 17 (2.02), GGSPEGGSLAALTAH 35(2.33) 19(2.46), 35 (2.35), (SEQ ID NO: 14) 38(3.71) 5 7(1.97), 10(2.24), 17 (2.24), 7(2.05), 10(2.46), PEGGSLAALTAHQAC 24(1.90) 17(2.20) (SEQ ID NO: 15) 6 3(2.00) GSLAALTAHQACHLP (SEQ ID NO: 16 7 3(2.11), 17(2.39), 24(2.20), 3(2.30), 17(2.22), AALTAHQACHLPLET 45 (2.11) 24(2.11), 45 (1.90) (SEQ ID NO: 17) 8 17(2.23), 45 (2.12) 17(2.04) TAHQACHLPLETFTR (SEQ ID NO: 18)

US 9,677,055 B2 135 136 TABLE 8-continued Donor Responses to PE38 Peptides Peptide # Proliferation Non-Adjusted Proliferation Adjusted Peptide Sequence O 8(2.07), 9(2.35), 11(2.27), 9(2.46), 10(2.04), LDPSSIPDKEQAISA 13(2.13), 16(1.91), 19(3.00), 13(2.11), 19(1.99), (SEQ ID NO: 120) 35 (1.90) 35(1.94), 38(1.95), 50(2.01) 1 3(2.29), 8(2.20), 9(1.93), 3 (2.33), 8(2.74), 9(2.02), SSIPDKEQAISALPD 11 (2.08), 16(2.19), 19(2.60) 13(1.98) (SEQ ID NO: 121) 2 11 (2.47), 16(3.07), 19(2.61) 9(1.90), 16(2.11), PDKEQAISALPDYAS 50(1.97) (SEQ ID NO: 122) 3 3(2.07), 11(2.61), 16(2.44), 3(2.04), 11(1.93), EQAISALPDYASQPG 19(2.62) 45(1.90) (SEQ ID NO: 123) 4 19(2.04) ISALPDYASQPGKPP (SEQ ID NO: 124) 5 16(1.99) LPDYASQPGKPPRED (SEQ ID NO: 125) 6 YASQPGKPPREDLK (SEQ ID NO: 126) 7 ITGPEEEGGRLDTIL (SEQ ID NO: 127) 8 9(2.04), 11(2.26), 16(2.12), 9(2.27) PEEEGGRLDTILGWP 39(1.93), 5 (SEQ ID NO: 128) 9 16(2.11), 39(2.13) 14(1.90), 38(1.96), EGGRLDTILGWPLAE 39(2.10) (SEQ ID NO: 129) 2O 11 (2.13), 39(2.05) 39(2.07) RLDTILGWPLAERTW (SEQ ID NO: 130)

T Cell Epitope Map cell response. Peptide 50 induced lower magnitude T cell Epitopes 1 and 2– proliferation responses where the mean SI were 2.23 and Peptides 50, 52 and 53 induced a high number of positive 30 2.28 in the non-adjusted and adjusted datasets respectively T cell proliferation responses in the study cohort (Table 8 Suggesting that this peptide may induce a naive T cell and FIG. 3). Peptide 50 showed the highest number of response (Table 9). positive responses with 15.38% donors responding in the Epitopes 3 and 4 non-adjusted dataset, and 15.38% in the adjusted data set, 35 A cluster of T cell responses were observed around (SI-2.00, p<0.05). From in silico analysis, the proposed core peptides 65-68 and the subsequent analysis revealed the 9 mer in this region is ISFSTRGTQ (SEQ ID NO:5). Peptides 52 and 53 induced lower frequencies of response presence of two T cell epitopes in this region. Peptide 65 with 11.54% and 13.46% positive donor responses in the stimulated positive T cell proliferation responses in 15.38% non-adjusted dataset, and 13.46% and 11.54% in the 40 of the study cohort for both non-adjusted and adjusted adjusted datasets, respectively. A core 9 mer was identified datasets (Table 8 and FIG. 3) (SI 2.00, p<0.05). The posi in peptide 50 but was only partially present in peptides 52 tive responses were high magnitude (mean SI of positive responses ranged from 3.04-2.89 in the non-adjusted and and 53 Suggesting that these peptides must contain a differ adjusted data sets) suggesting that the T cell precursor ent T cell epitope. In silico analysis of peptides 52 and 53 did 45 not identify any core HLA-DR restricted 9 mers so it is frequency in healthy donors against this epitope is high likely that the positive T cell responses seen are due to a (Table 9). In silico analysis revealed a potential core 9 mer HLA-DQ restricted T cell epitope. comprising IVFGGVRAR (FIG. 5: SEQID NO:7). Peptides The magnitude of T cell proliferation responses can 67 and 68 induced frequencies of response with 15.38% and 13.46% positive donor responses in the non-adjusted data provide an indication as to the T cell precursor frequency. In 50 set, and 13.46% and 15.38% in the adjusted datasets, respec general, peptides that induce high frequency (of positive tively. In silica analysis of these peptides did not identify any responses in the study cohort) and high magnitude T cell core HLA-DR 9 mers so it is likely that the positive T cell proliferation responses are a characteristic of recall-like T responses seen are due to a HLA-DQ restricted T cell cell responses in which the T cell pre-cursor frequency is 55 epitope. high. In contrast, naive T cell responses are generally Epitope 5 characterized by low magnitude T cell proliferation Peptides 81 and 82 stimulated a number of T cell responses (with low T cell precursor frequencies). Peptides responses in the study cohort (Table 8 and FIG. 3). Peptide 52 and 53 induced moderately high magnitude T cell pro- 60 81 had the highest frequency of response of all the peptides liferation responses where the mean SI for positive (SI 2.00, tested with a frequency of positive responses of 19.23% in p-0.05) T cell responses in the non-adjusted and adjusted the non-adjusted and 17.31% in the adjusted data set. For data sets were 3.09-2.89 (peptide 52) and 2.51-2.55 (peptide peptide 82, the frequency of positive response was 17.31% 53) (Table 9). Thus these peptides may induce T cell 65 and 15.38% in the non-adjusted and adjusted data sets responses in clones that are present in high frequencies in respectively. The positive responses were relatively low in healthy individuals and may be indicative of a memory T magnitude (mean SI of positive responses ranged from 2.12 US 9,677,055 B2 137 138 to 2.22 in the non-adjusted and adjusted data sets) suggest the study cohort. This analysis was only carried out for ing that the T cell precursor frequency in healthy donors peptides that induced positive responses above the back against this epitope is relatively low (Table 9). Adjacent ground response rate in the adjusted data in the study cohort and was also restricted to allotypes expressed at higher peptide 80 induced a sub-threshold response. In silico analy 5 sis of peptides 81 and 82 suggested a core 9 mer of frequencies (>5%) in the study population. LRVYVPRSS (FIG. 6: SEQ ID NO:9). Analysis of responding donor allotypes (Table 10 and Epitope 6 FIG. 8) revealed that there was a possible association Peptide 110 induced positive T cell responses in 13.46% between T cell responses to peptides 81, and 82 and MHC of the study cohort in non-adjusted and 13.46% in adjusted 10 class II allotype HLA DRB1*07 which was expressed at datasets (Table 8 and FIG. 3). The magnitude of positive twice the percentage of positively responding donors com proliferation responses was low with a mean SI of 2.23 for pared to the study population. Peptide 53 also had a possible the non-adjusted dataset and 2.07 for the adjusted dataset association with DRB1*11, and peptides 82 and 110 showed (Table 9). There was also a sub-threshold response to 15 possible associations with DRB1*15. It should be noted that peptide 111. In silico analysis of the peptides sequence further studies (such as MHC class II binding analysis) revealed a core 9 mer. IPDKEQAIS (FIG. 7, SEQ ID would be required to show conclusively that responses to the NO:10) which, in addition to peptide 110, was also present T cell epitope are associated with these allotypes as the in peptide 111. present analysis was performed on a small group of respond Table 9. Summary of magnitude (mean SI and standard ing donors. deviation) and frequency (% donor response) of positive T Table 10. Frequency (expressed as a percentage) of cell proliferation responses against peptides containing T responding donor allotypes compared to the frequency of cell epitopes for PE38. The position of p1 in potential core 25 allotypes expressed in the IEX01 study cohort. An associa 9 mers are shown as underlined/bolded text (as predicted by tion between MHC class II allotype and a response to a iTOPETM) in peptides 50, 65, 81, 82 and 110. particular epitope was considered if the frequency of the TABLE 9 Macinitude and Fred uency of Donor Responses

Mean Response Fredulency Mean (SD) (SD) Non- Non-Adjusted Adjusted Peptide Peptide Sequence Adjusted Adjusted Data Data

SO GDGGDISFSTRGTON 5.38% 5.38% 2.23 - 0.28 2.28 O. 26 (SEQ ID NO: 6O)

52 SFSTRGTONWTVERL 1.54& 3.46& 3. O9 1.99 2.89 - 150 (SEQ ID NO: 62)

53 TRGTONWTVERLLOA 3.46& 1.54& 2.51 - 1.18 2.55 O. 98 (SEQ ID NO: 63)

65 AQSIVFGGVRARSQD 5.38% 5.38% 3. O4 - 2. O4 2.89 - 1. 64 (SEO ID NO: 75)

67 GGWRARSODLDAIWR 5.38% 3.46& 2.79 1.65 2. 69 - 1.15 (SEO ID NO: 77)

68 RARSODLDAIWRGFY 3.46& 5.38% 2. 4 O - O. 62 2.28 O. 54 (SEO ID NO: 78)

81 NGALLRVYVPRSSLP 9.23% 7.31% 2.22 - O.36 2.12 it O. 12 (SEQ ID NO: 91)

82 LLRVYVPRSSLPGFY 7.31% 5.38% 2.14 - O. 19 2.12 - O - 17 (SEQ ID NO: 92)

11O LDPSSIPDKEQAISA 3.46& 3.46& 2.23 - 0.38 2. Of O. 18 (SEQ ID NO: 12O)

60 HLA Analysis allotype within the responding population was double the Analysis of the responding donor haplotypes was per frequency observed in the study population in the adjusted formed whereby an association between MHC class II data set. Possible associations are indicated in heavily bor allotype and a response to a particular peptide was consid 65 dered boxes. The analysis has been restricted to allotypes ered possible if the frequency of the allotype within the expressed at higher frequencies (>5%) in the study popula responding population was double the frequency observed in tion. US 9,677,055 B2 139 140 TABLE 10 Frequency of responding donor allotypes versus frequency of allotypes in the IEXO1 study cohort. Frequency (%) of HLA alleles expressed DRB1 DRB1* DRB1* DRB1* DRB1*1 DRB DRB DRB within: *03 04 O7 11 5 3 4 5 Study 8 8 7 5 7 2O 18 6 population Peptide 50 8 8 O 8 23 15 4 Peptide 52 4 8 8 4 24 2O O Peptide 53 5 10 5 24 19 O Peptide 65 12 O 8 O 32 16 O Peptide 67 8 13 8 8 25 21 O Peptide 68 7 7 4 7 25 21 O Peptide 81 7 O 7 7 21 21 7 Peptide 82 8 8 O 15 23 8 Peptide 110 4 13 O 13 25 8

Results TABLE 11-continued The results show that six T cell epitopes were present in the PE38 sequence. Table 6 Table 11 and FIG. 8 summarize Location of Core 9-mers and Projected Mutations the location of the putative core 9 mers in each sequence Amino along with the frequency and magnitude of T cell responses 25 Epi- Acids in Anchor Projected against each epitope. The T cell epitopes identified in PE38 tope Sequence Residues Mutations Notes: were prioritized according to their potency based on the V 7 preferred. frequency and magnitude (mean SI) of positive donor responses to each peptide. However since the responding R 9 30 3 1 A, N P1 is buried, therefore A donor magnitudes were similar (Table 4 Table 9) for most V is preferred. epitopes, the ranking was mainly based on frequency of P6V is partially exposed. positive donor responses (from highest to lowest): G 4 All mutations should be G olerated. Preference is Epitope 5>Epitope 4-Epitope 3>Epitope 1 >Epitope V 6 D, M, N D > M > N. 2>Epitope 6 35 R 7 Deimmunization Strategy A. The six epitope core 9 mer sequences were analyzed by R 9 4 A. 1 HLA-DQ epitopes have a proprietary software (iTOPETM) in order to identify muta R strong negative preference tions that remove the T cell epitopes by eliminating or S or positively charged significantly reducing binding to MHC class II (Table 11). Q 4 K, R residues in key anchor As part of the strategy as to which residues to mutate, 40 D positions. All four 6 mutations are equally location within the structure was considered, especially D 7 K, R preferred. whether the residue is buried, on the surface, or near active A. sites. I 9 Table 11. Projected mutations to remove MHC class II 5 1 A. P1 is buried and close in 45 R D, S, A the structure to epitope 3 binding (based upon iTOPETM and crystal structure data). V P1, therefore changes are Y 4 imited. For this epitope, TABLE 11 V changes at P2 affect C 6 binding (D > S > A). Location of Core 9-mers and Projected Mutations R 7 P9 is mostly surface 50 S exposed. Preferred Amino S 9 D, E, N, changes are D, E, N, then Epi- Acids in Anchor Projected P T K > P > T. tope Sequence Residues Mutations Notes: 6 I 1 A. N. T., P1 I is partially surface s exposed, therefore all 1 1 A. N. T., P1 (Ile) is partially P alternatives should be Q, H surface exposed, therefore 55 D possible. S all alternatives should K 4 T P4, P6 and P9 changes are be possible. E ess preferred than P1 S 4 P6 and P9 changes Q 6 D changes. P6 D > P7 D > perform equally well, A. 7 D P4 T. R 6 Q but are less preferred I S 9 G 7 than P1 changes. 60 Q 9 N, T 2 G 1 HLA-DQ epitopes have a Conclusions strong negative preference Q for positively charged EPISCREENTM T cell epitope mapping of 120 overlap N 4 K, R residues in key anchor ping 15mer peptides including 112 spanning the entire PE38 W positions. All four 65 sequence Suggested six novel T cell epitopes. In silico 6 K, R mutations are equally analysis was used to identify potential core 9 mers for MHC binding and, together with structural analysis, was used as a US 9,677,055 B2 141 142 basis for design of changes for re-engineering and deimmu dure for Human CD8+ T Cell Enrichment Cocktail (Catalog nizing PE38 in particular, and PE molecules in general. #15023/15063; Procedure version 1.3.0, “H28572 (May 2011)). Example 2: T Cell Epitope Mapping of Donors HLA-DR haplotypes are determined using meth Deimmunized/Amino Acid Substituted Forms of PE 5 ods or kits well-known and routinely used by those skilled in the art. For example, Donors HLA-DR haplotypes are The immunogenicity of amino acid substituted forms of determined using a Biotest HLASSP-PCR tissue-typing kit PE can be assessed using the same procedures as described (Biotest, Solihull, UK, catalogue number 826215). T cell in Example 1. Accordingly, EPISCREENTM T cell epitope responses to a reproducibility control antigen are measured mapping analysis (Antitope Ltd, Cambridge, UK) analysis 10 using, for example neo-antigen, using Imject mariculture permits identification of amino acid Substituted epitopes in keyhole limpet haemocyanin (KLH) (Pierce (Perbio Science PE polypeptides, wherein the introduced amino acid UK, Ltd), Cramlington, UK, catalogue number 77600), or changes result in reduced or undetectable immunogenicity other similar control antigen (such antigens and methods are (i.e., for generating deimmunized forms of PE) as compared well known and routinely used by those skilled in the art). to epitopes in corresponding forms of non-amino acid Sub 15 PBMC are frozen and stored in liquid nitrogen until ready stituted PE polypeptides. for use in to measuring immunogenicity of amino acid EPISCREENTM is a proprietary technology commercially substituted forms of PE. available through Antitope Ltd, Cambridge, UK, to map T A cohort of donors are selected to best represent the cell epitopes within a protein sequence to determine poten number and frequency of HLA-DR allotypes expressed in tial for immunogenicity (based on the number and potency the world population. It is desirable that allotypes expressed of T cell epitopes within a sequence). EPISCREENTM T cell in the cohort represent a coverage of >80% of all major epitope mapping typically uses CD8+ T cell depleted HLA-DR alleles in the world population (i.e., individual PBMCs from a minimum of 50 HLA-typed donors (selected allotypes with a frequency >5% expressed in the world to represent the human population of interest). Typically, 15 population are well represented). Records of individual mer peptides with 12 amino acid overlaps spanning a protein 25 donor haplotypes and comparison of the frequency of MHC sequence are analyzed in a large number of replicate cultures class II haplotypes expressed in the world population and the for in vitro CD4+ T cell stimulation by 3H TaR incorpora sample population are recorded and assessed. tion. CD4+ T cell stimulation is often detected in two or Donor responses (SI) to a control antigen (such as KLH) three adjacent and overlapping peptides since the core 9 mer are assessed by comparing two independent proliferation that binds the MHC class II binding groove will be present 30 assays. Test-1 is performed using the control antigen (Such in more than one peptide sequence. Following identification as KLH) on freshly isolated PBMC and Test-2 is the control of peptides that stimulate CD4+ T cells in vitro, in silico antigen re-test performed on PBMC recovered from liquid technology can be used to design epitope-depleted (deim nitrogen storage, the latter of which are used in assessing munized) variants by determining the precise location of immunogenicity of amino acid Substituted epitopes in PE. core 9 mer sequences and the location of key MHC class II 35 Responses that do not produce the same result in these two anchor residues. tests (i.e. positive including borderline SD 1.90 p<0.05 or In this case, amino acid substituted PE peptides are negative) in both tests are disregarded. analyzed for the presence of immunogenic CD4+ T cell EPISCREENTM Analysis: Proliferation Assay epitopes using EPISCREENTM T cell epitope mapping PBMC from each donor are thawed, counted and viability analysis. For example, amino acid Substituted 15 mer pep 40 is assessed. Cells are revived in room temperature AIMV(R) tides (compared to non-substituted 15 mer peptides corre Culture Medium (INVITROGENTM, Paisley, UK) before sponding to a non-amino acid Substituted form of PE) are adjusting cell density to 2-3x10 PBMC/ml (proliferation tested against a cohort of healthy donors. CD4+ T cell cell stock). Peptides are synthesized on a 1-3 mg scale with responses against individual peptides are measured using free N-terminal amine and C-terminal carboxylic acid. Pep proliferation assays (3H-thymidine incorporation). Prolif 45 tides are dissolved in DMSO to a concentration of 10 mM eration assay data is used to compile a T cell epitope map of and peptide culture stocks are prepared by diluting into AIM varying responses to amino acid Substituted forms of PE to V(R) Culture Medium to a final concentration of 5 uM per determine those amino acid changes producing reduced or well. For each peptide and each donor, Sextuplicate cultures abrogated immunogenic responses. are established in a flat bottomed 96 well plate. Both positive EPISCREENTM Donor ASSessments 50 and negative control cultures are tested in Sextuplicate. For Peripheral blood mononuclear cells (PBMC) are isolated each donor, three control antigen/peptides (KLH protein and from healthy donor buffy coats (e.g., from blood drawn peptides derived from Influenza A and Epstein Barr viruses) within 24 hours). For example, PBMC are isolated from are also included. buffy coats using density gradient centrifugation using Cultures are incubated for 6 days before adding 0.75 uCi LYMPHOPREPTM (Axis-Shield UK, Dundee, Scotland) or a 55 3H]-thymidine (PERKIN ELMER(R), Beaconsfield, UK) to similar density gradient centrifugation media for the isola each well. Cultures are incubated a further 18 hours before tion of human mononuclear cells from blood (such methods, harvesting onto filter mats using a TOMTEC MACHR III media and products are well known and routinely used by cell harvester (TOMTECR), Hamden, Conn., USA). Counts those skilled in the art). See e.g., Axis-Shield, package insert per minute (cpm) for each well are determined by MELT for LYMPHOPREPTM density gradient media No. 619. 60 ILEXTM (PERKIN ELMER(R) scintillation counting on a March 2003. Div.-1114740.) To remove CD8+ cells from the Microplate Beta Counter (PERKIN ELMER(R) in paralux, isolated mononuclear cells, CD8+ T cells are depleted using low background counting mode. CD8+ ROSETTESEPTM kit (STEMCELLTM Technologies EPISCREENTM Data Analysis Inc, Manchester, UK) or similar CD8+ selection methods In proliferation assays, an empirical threshold of stimu and techniques (such methods, media and products are well 65 lation index (SI) equal to or greater than 2 (SI-2.00) is known and routinely used by those skilled in the art). See considered to represent an induced proliferative response; e.g., StemCell Technologies Inc., ROSETTESEPTM proce samples registering values above this threshold are deemed US 9,677,055 B2 143 144 positive (values of SI<2.00 but a 1.90 are considered bor ing to methods routinely used and well-known to those of derline). Extensive assay development and previous studies skill in the art. Measured biological activities of deimmu have shown that this is the minimum signal to noise thresh nized (“DI) forms of PE (“DI-PE'), in particular, or PE old allowing maximum sensitivity without detecting large molecules, in general, may include, for example, assays to numbers of false positive responses. Positive responses are CaSU defined by the following statistical and empirical thresholds: a) general or specific inhibition of protein synthesis (i.e., 1. Significance (p<0.05) of the response by comparing measuring inhibition of synthesis of a specific protein cpm of test wells against medium control wells using (or specific proteins) or inhibition of overall (mass) unpaired two sample Student's t-test. protein synthesis; 2. Stimulation index greater than 2.00 (SI 2.00), where SI mean cpm of test wells/mean cpm medium control 10 b) inhibition of translation elongation factor EF-2 bio wells. Thus, data presented is indicated as SI-2.00, logical activity; p<0.05. c) induction or catalysis of ADP-ribosylation of EF-2; and In addition, intra-assay variation is assessed by calculat d) eukaryotic cell killing activity (cell cytotoxicity). ing the coefficient of variance and standard deviation (SD) Assays for Biological Activity: Inhibition of Protein Syn of raw data from replicate cultures. 15 thesis Proliferation assays are set up in Sextuplicate cultures In one example, measurement of inhibition of protein from which “non-adjusted data' is gathered. To ensure synthesis may be done via use of in vitro transcription/ intra-assay variability is low, data is also analyzed after translation assays (which are routinely used and well-known removing maximum and minimum cpm values (to produce to those of skill in the art). For example, a cell-free assay “adjusted data”) and the SI of donor responses is compared may be used to measure DI-PE induced inhibition of in vitro using both data sets. transcription/translation of a target plasmid (such as, but not Reactive T cell epitopes are identified by calculating the limited to, T7-luc). In the case of using a T7-luc transcrip average frequency of positive responses (defined above) to tion/translation assay, the biological activity readout would all peptides in the study plus standard deviation (SD) to give be chemiluminescent measurement of luciferase activity a background response threshold. Any peptide inducing a 25 wherein amino acid substituted forms of PE are compared to frequency of positive proliferation responses above the corresponding non-amino acid substituted forms of PE for threshold in both adjusted and non-adjusted data is consid ered to contain an immunogenic T cell epitope (and, thus, ability/inability to inhibit translation of the luciferase potentially represents an immunogenicity inducing epitope enzyme in vitro. In Such assays, the PE polypeptides being which could give rise to immunogenic responses in vivo). assayed can be introduced via expression from template Output from non-adjusted and adjusted data is examined to 30 DNA (e.g., a PCR product) encoding the toxin-conjugate ensure that intra-assay variability is low and that positive gene, or by directly introducing quantified amounts of PE responses are not the result of spurious proliferation in proteins. Such assays may be used to assess IC50 values of individual wells. An example of this type of analysis is the various forms of PE tested (IC50=concentration at provided in Example 1. which 50% of protein synthesis is inhibited versus standard A comparison of corresponding forms of non-amino acid 35 ized control samples lacking PE). Substituted PE immunogenic epitope responses versus Some examples of kits and reagents available for in vitro responses obtained with amino acid substituted PE peptides transcription/translation assays include, but are not limited is used to assess and predict the effects of various amino acid tO: Substitutions in reducing or eliminating the immunogenicity TNT(R) SP6 Coupled Reticulocyte Lysate System (e.g., of PE polypeptides (i.e., for making deimmunized forms of 40 PROMEGAR) catalog #L4610 (PROMEGAR) Corp., PE). Madison, Wis., USA)) allows for eukaryotic cell-free Assays for measuring and testing the immunogenicity of protein expression in a single-tube, as a coupled tran amino acid substituted forms of PE may also be done as scription/translation process. More traditional rabbit described and exemplified in Example 1 (i.e., via prolifera reticulocyte lysate translations commonly use RNA tion assays quantitating CD4+ T cell responses) wherein 45 synthesized in vitro from SP6, T3 or T7 RNA poly amino acid substituted forms of PE (i.e., “deimmunized PE merase promoters and require three separate reactions or “DI-PE), and/or DI-PE conjugates and fusion proteins with several steps between each reaction. The TNT(R) (e.g., fusions of DI-PE to antibodies or antigen-binding System bypasses many of these steps by incorporating fragments thereof) are tested and measured for the presence transcription directly into the translation mix. See e.g., and potency of immunogenic responses compared to 50 PROMEGAR Technical Bulletin HTB 126 (Revised responses induced by corresponding forms of non-amino December 2010) which is incorporated by reference acid substituted PE peptides, polypeptides, and fusion or herein. See also, Pelham et al., Eur: J. Biochem. 67, conjugation constructs. 247-56 (1976), Krieg et al., (1984) Nucl. Acids Res. 12, Assays for measuring immunogenicity of amino acid 7057-7070 (1984). See also, U.S. Pat. Nos. 5,324,637; substituted forms of PE specifically (as indicated above), or 55 5,492.817: 5,641,641, and, 5,650,289. PE molecules, generally, may also be done according to TNTR. T7 Quick Coupled Transcription/Translation Sys methods routinely used and well-known to those of skill in tem (e.g., PROMEGAR) catalog #L1170 (PRO the art. For example, immunogenicity of amino acid Substi MEGAR) Corp., Madison, Wis., USA)) further simpli tuted forms of PE, in particular, or PE molecules, in general, fies in vitro transcription/translation reactions by (as indicated above) may be measured in vivo in non-human 60 combining RNA polymerase, nucleotides, salts and primates and/or in transgenic mouse model systems. Recombinant RNasin(R) Ribonuclease Inhibitor with the reticulocyte lysate to form a single TnT.R. Quick Master Example 3: Measuring Biological Activity of Mix. The TnT.R. Quick Coupled Transcription/Transla Amino Acid Substituted Forms of PE tion System may be used with plasmids for transcrip 65 tion and translation of genes cloned downstream from Assays for measuring the biological activity of amino acid either the T7 or SP6 RNA polymerase promoters. The substituted/deimmunized forms of PE, may be done accord TnT.R. Quick System includes a luciferase-encoding US 9,677,055 B2 145 146 control plasmid and Luciferase Assay Reagent, which or at least about 50% of biological activity compared to can be used in a non-radioactive assay for rapid (<30 corresponding forms of non-amino acid substituted PE. seconds) detection of functionally active luciferase Assays for Biological Activity: Cell Cytotoxic Activity protein. Starting with either circular plasmid DNA or In one example, measurement of cell cytotoxic activity PCR-generated DNA, in vitro transcription/translation 5 may be done via use of in vitro cell based assays wherein results may be obtained in 5-6 hours. See e.g., PRO deimmunized PE (DI-PE)-antibody conjugates are assayed MEGAR Technical Bulletin HTM045 (Revised May in comparison to non-amino acid Substituted PE-antibody 2011) which is incorporated by reference herein. conjugates. The antibody portion of Such conjugates would STEADY-GLOR. Luciferase Assay System (e.g., PRO be antibodies, or antigen-binding fragments thereof which MEGAR) catalog #E2510) (PROMEGAR) Corp., Madi 10 specifically bind antigens expressed on the cell-Surface of son, Wis., USA)) allows for high-throughput quantita cell types used in Such in vitro assays. Cell cytotoxicity may tion of firefly (Photinus pyralis) luciferase expression be quantitated, for example, by measuring cell lysis wherein in mammalian cells via batch processing of 96- and the biological readout is represented by measurement of for 384-well plates. The STEADY-GLOR. Luciferase example, based on chemiluminescent (LUMI), fluorometric Assay System provides signal half-lives of over 5 hours 15 (FL), and colorimetric (COL) outputs; such as can be in commonly used cell culture media without prior practiced using commercially available kits routinely used sample processing. Throughput rates of several thou and well-known to those of skill in the art. sand samples per hour may be achieved with high Some examples of kits available for measurement and reproducibility under standard laboratory conditions. comparison of DI-PE versus non-amino acid substituted PE See e.g., PROMEGAR Technical Bulletin HTM051 cell cytotoxicity include, without limitation: (Revised March 2009 & Revised September 2011) TOXILIGHTR BioAssay Kit (e.g., Catalog #LT07-117 which is incorporated by reference herein. See also, (Lonza Rockland, Inc., Rockland, Me., USA)) is a U.S. Pat. Nos. 5,641,641; 5,650,289; 5,583,024; 5,674, non-destructive bioluminescent cytotoxicity assay that 713; and 5,700,673. quantitatively measures release of Adenylate Kinase Full protocols for use of such kits are provided by the 25 (AK) from damaged mammalian cells and cell lines in manufacturer with each kit. A brief example of a typical vitro. The assay is based on the bioluminescent mea experimental procedure may include: surement of AK which is present in all cells. A loss of Assembling kit reagents (except target T7-luc plasmid), cell integrity, through damage to the plasma membrane, plus PE test samples (using an experimentally deter results in the leakage of a number of factors from cells mined titration of PE test samples; e.g., in a range of 30 cultured in vitro into the surrounding medium. The 0-500 ng DNA per reaction for PCR templates or using measurement of the release of AK from the cells allows a PE protein titre in a range to be determined experi the accurate and sensitive determination of cytotoxicity mentally), in a total volume of 12.5 ul RNAse-free and cytolysis. The reaction involves two steps. The first water in PCR tubes or cell wells on plates. involves the addition of ADP as a substrate for AK. In For plasmid DNA: Pre-incubate for required time (e.g. 35 the presence of the enzyme, AK, the ADP is converted 30-60 min, time to be determined experimentally) at to ATP for assay by bioluminescence. The biolumines 30° C. to allow pre-reaction transcription/translation to cent part of the assay utilizes the enzyme Luciferase, OCCU. which catalyses the formation of light from ATP and For purified protein: No pre-incubation step required. luciferin. By combining these two reactions, the emit Add target plasmid T7-luc (e.g. 250 ng/reaction, deter 40 ted light intensity is linearly related to the AK concen mined experimentally) and incubate further (e.g. 30-60 tration and can be measured using a luminometer or min, time to be determined experimentally) at 30° C. beta counter. See, “TOXILIGHTR BioAssay Kit: Stop reaction by placing on ice. Increase sample Volume Instructions for Use.' (C2007 Lonza Rockland, Inc., to 50 u1 with RNAse-free water. which is incorporated by reference herein. See also, Add luciferase reagent (e.g. SteadyGlo, 50 ul per well) to 45 Crouch, et al., J. Immunol. Methods, 160(1): 81-88 each well, incubate according to manufacturers (1993); Olsson, T. et al., J. Appl. Biochem 5,347-445 instructions, transfer to 96 well black/white plate and (1983); and, Squirrell et al., A Practical Guide to read chemiluminescent signal via chemiluminescence Industrial Uses of ATP Luminescence in Rapid Micro platereader. biology, p. 107-113 (1997). Compare to zero toxin control samples (i.e., no PE 50 CYTOTOX-GLOR) (e.g., PROMEGAR catalog #G9290 present) to determine the '% inhibition of transcription/ (PROMEGAR) Corp., Madison, Wis., USA)) is a lumi translation (i.e., as a function of inhibition of luciferase nescent cytotoxicity assay that measures the relative activity). number of dead cells in cell populations. The assay Compare inhibition of transcription/translation values of measures extracellular activity of a distinct intracellular amino acid substituted/deimmunized forms of PE com 55 protease activity (dead-cell protease) when the protease pared to corresponding forms of non-amino acid Sub is released from membrane-compromised cells. Alumi stituted PE. nogenic cell-imperimeant peptide Substrate (AAF-ami Comparative protein synthesis inhibition values may noluciferin) is used to measure dead-cell protease activ show that various forms of DI-PE exhibit 100% or about ity. The liberated aminoluciferin product is measured as 100% of biological activity (inhibition of protein synthesis) 60 “glow type” luminescence generated by ULTRA compared to corresponding forms of non-amino acid Sub GLOTM Recombinant Luciferase provided in the assay stituted PE. Comparative protein synthesis inhibition values reagent. The AAF-aminoluciferin Substrate cannot may also show that various forms of DI-PE exhibit at least cross the intact membrane of viable cells and does not 95%, or at least about 95%, at least 90%, at least about 90%, generate appreciable signal from the live-cell popula at least 85%, at least about 85%, at least 80%, at least about 65 tion. The amount of luminescence directly correlates 80%, at least 75%, at least about 75%, at least 70%, at least with the percentage of cells undergoing cytotoxic about 70%, at least 60%, at least about 60%, at least 50%, stress. With the addition of a lysis reagent (provided US 9,677,055 B2 147 148 with the kit), the CYTOTOX-GLOTM Assay provides a Plus Kit may be used to measure a wide range of luminescent signal associated with the total number of biological activities effecting cell viability. The kit is cells in each assay well. Viability can be calculated by formulated for use with a microtitre plate reading Subtracting the luminescent dead-cell signal from the luminometer for assay automation. The assay is based total luminescent value, thus allowing normalization of 5 on bioluminescent measurement of ATP is present in all assay data to cell number and mitigation of assay metabolically active cells. The bioluminescent method interferences. The cytotoxicity protease biomarker is utilizes an enzyme, Luciferase, which catalyses the constitutive and conserved across cell lines. See e.g., formation of light from ATP and luciferin according to PROMEGAR Technical Bulletin Nos. TB359 (Revised the following reaction: May 2009 & Revised October 2011) which is incor 10 ATP+Luciferin+O2-Luciferase/Mg’->Oxyluciferin+ porated by reference herein. See also, Niles, A. et al. AMP+PPi--CO+LIGHT (2007) Anal. Biochem., 366, 197-206 (2007) and U.S. The emitted light intensity is linearly related to the ATP Pat. Nos. 6,602,677 and 7,241,584. concentration and can be measured using a luminom CYTOTOX-ONETM kit (e.g., PROMEGAR) catalog eter or beta counter. The assay is conducted at ambient #G7891 (PROMEGAR) Corp., Madison, Wis., USA)) 15 temperature (18° C.-22 C.), the optimal temperature allows performance of homogeneous membrane integ for luciferase enzymes. See, “VIALIGHTR Plus Kit: rity assays wherein a fluorometric method may be used Instructions for Use.' (C2007 Lonza Rockland, Inc, to estimate the number of nonviable cells present in which is incorporated by reference herein. multiwell plates. This assay measures the release of Full protocols for use of such kits are provided by the lactate dehydrogenase (LDH) from cells with damaged manufacturer with each kit. A brief example of a typical membranes. LDH released into the culture medium is experimental procedure may include: measured with a coupled enzymatic assay that results in Plate cells to test plate (e.g., 96 well plates) in growth the conversion of resaZurin into a fluorescent resorufin medium. product. The amount of fluorescence produced is pro Incubate cells with titrations of amino acid substituted portional to the number of lysed cells (which may be 25 forms of PE-toxin conjugates (including Zero toxin and monitored using a 96- or 384-well plate formats). The non-amino acid Substituted PE controls (up to a maxi CYTOTOX-ONETM Reagent does not damage normal mum toxicity point, e.g. 100% cell lysis) for required healthy cells. Therefore, reactions to measure released time (determined experimentally, e.g. 48-72 hr). quantities of LDH can be performed directly in a Add kit reagents for cytotoxicity measurements as per homogeneous format in assay wells containing a mixed 30 manufacturers instructions. population of viable and damaged cells. See e.g., Transfer test samples to 96 well black/white walled plate PROMEGAR Technical Bulletin #TB306 (Revised (as appropriate) and read reaction signal output. May 2009) which is incorporated by reference herein. Compare cell cytotoxicity values obtained for substituted/ See also, U.S. Pat. Nos. 6,982,152 and 7,282,348. deimmunized forms of PE versus corresponding non CELLTITER GLOR. Luminescent Cell Viability Assay 35 amino acid substituted forms of PE. (e.g., PROMEGAR catalog #G7571 (PROMEGAR Comparative cell cytotoxicity values may show that vari Corp., Madison, Wis., USA)) provides a homogeneous ous forms of DI-PE exhibit 100% or about 100% of bio method for determining the number of viable cells in a logical activity (induction of cell cytotoxicity) compared to culture based on quantitation of the amount of ATP corresponding forms of non-amino acid Substituted PE. present (an indicator of metabolically active cells). The 40 Comparative cell cytotoxicity values may also show that CELLTITER GLOR Assay is particularly useful for various forms of DI-PE exhibit at least 95%, or at least about automated high-throughput Screening (HTS), cell pro 95%, at least 90%, at least about 90%, at least 85%, at least liferation and cytotoxicity assays. The homogeneous about 85%, at least 80%, at least about 80%, at least 75%, assay procedure involves adding the single reagent at least about 75%, at least 70%, at least about 70%, at least (CELLTITER GLOR) Reagent) directly to cells cul 45 60%, at least about 60%, at least 50%, or at least about 50% tured in serum-Supplemented medium. The assay of biological activity compared to corresponding forms of allows for detection of as few as 15 cells/well in a non-amino acid substituted PE. 384-well format in 10 minutes after adding reagent and mixing. The homogeneous "add-mix-measure' format Example 4: Measuring Ability of Deimmunized PE results in cell lysis and generation of a luminescent 50 Variants to Inhibit Protein Synthesis signal proportional to the amount of ATP present (which is directly proportional to the number of cells Quantitative in vitro transcription/translation (IVTT) present in culture). The CellTiter-Glo R Assay gener assays to assess the biological activity of deimmunized ates a 'glow-type” luminescent signal, which has a variants of PE in inhibiting protein synthesis (i.e., possess half-life generally greater than five hours, depending on 55 wild-type PE biological activity) may be performed using cell type and medium used. See e.g., PROMEGAR the TNTR Quick Coupled Transcription/Translation Sys Technical Bulletin Nos. TB288 (Revised June 2009 & tems assay from PROMEGAR) Corp. (Madison, Wis., Revised August 2011) which are incorporated by ref USA). See, PROMEGAR Technical Bulletin #TB126 (Re erence herein. See also: U.S. Pat. Nos. 6,602,677: vised December 2010) which is incorporated by reference 7,241,584; 7,700,310; 7,083,911; 7,452,663; 7,732, 60 herein. 128; 7,741,067: 5,583,024, 5,674,713; and 5,700,673. VIALIGHTR Plus Kit (e.g., Catalog #LT07-221 (Lonza Example 5: Measuring Ability of a PE-IL2 Fusion Rockland, Inc., Rockland, Me., USA)) may be used for Protein to Inhibit Protein Synthesis in an In Vitro rapid detection of cytotoxicity in mammalian cells and Transcription/Translation (IVTT) Assay cell lines in culture via determination of ATP levels. 65 Any form of cell injury results in a rapid decrease in A preliminary experiment was performed to compare the cytoplasmic ATP levels. Therefore, the VIALIGHTR ability of a PE-IL2 fusion protein to inhibit protein synthesis US 9,677,055 B2 149 150 in an in vitro transcription/translation assay when a com Restriction Digest: mercially available PE-IL fusion protein is translated in vitro In PCR tubes, the following were combined: following transcription from either a circular plasmid VVN-52431 was linearized by combining the following: expression vector or a linearized plasmid expression vector. The PE-IL2 expression vector in this experiment is referred NF HO VVN52431 Fsp I Reaction to as “VVN-52431.” A few examples of IL2-PE fusion RXn dig. final Product construct are shown in SEQ ID NO: 164, 165 and 166. The (LL) (LL) aim of this experiment was to determine if circular or 1 5 5 (5 g) O 0.5 gful Circular linearized plasmids produced significantly different quanti Vector - No 10 Restriction ties of PE-IL protein in the PROMEGAR) Corp. TNTR) enzyme Quick Coupled Transcription/Translation Systems assay. A added 5 (5 g) 1 0.5 gful Linearized commercially available T7 Promoter/Luciferase expression Vector vector (PROMEGAR) Corp.; hereinafter “T7-Luc DNA”) was used to measure the ability of PE-IL2 to inhibit protein synthesis in vitro. 15 Reactions were incubated at 37° C. for 60 min. Reactions were heat inactivated at 65° C. for 20 min. Based on a pilot IVTT experiment, it was determined that IVTT Reactions: 0.2 ug T7-Luc DNA provided optimal RLU (Relative Light 1. In nuclease-free 1.5 ml eppendorf tubes, the following Units) in a 90 minute IVTT reaction. In this experiment, were combined according to the chart below: VVN-52431 was linearized using the restriction enzyme Diluted T7-Luc DNA in NF (nuclease free) water at Fsp-I. Linearized and circular VVN-52431 DNA were used 1:5. Final DNA concentration=0.1 g/ul. as templates in the IVTT reactions. Reactions were done in Serial diluted unlinearized and linearized VVN-52431 triplicate, using 0.5, 1 and 2 ug of DNA. The T7 control DNA (0.5 ug/ul) in NF water at 1:5. Final concen reaction was performed using 1 g DNA. Reactions were tration 0.5 Lig/ul, 0.1 ug/ul, 0.02 ugful. analyzed via SDS-PAGE and by Luciferase assay. 25 Materials: NF T7 Luc Meth- T7 TNT HO DNA VVN52431 Fsp online ex RXn (IL) (LL) (IL) I dig. (IL) (IL) Item Vendor Lot # 30 1 7 2 (0.2 g) 1 40 Nuclease-free water (1000 ml) Ambion 1105062 2 7 2 (1 Lig) 1 40 TNT T7 Quick Coupled TT system PROMEGA (R) 328577 3 7 2 (1 Jug) -- 1 40 4 5 2 (0.2 g) 2 (1 Lig) 1 40 T7 luciferase plasmid DNA (From PROMEGA (R) 5 5 2 (0.2 g) 2 (0.2 g) 1 40 same kit) 6 5 2 (0.2 g) 2 (0.04 g) 1 40 Fsp I NEB 0571 101 35 7 5 2 (0.2 g) 2 (1 Lig) -- 1 40 Dual Glo (R) Luciferase Assay PROMEGA (R) 322310 8 5 2 (0.2 g) 2 (0.2 g) -- 1 40 System 9 5 2 (0.2 g) 2 (0.04 g) -- 1 40 Ultrapure Water GIBCO 896656 10 9 1 40 Tris-Glycine SDS Sample Buffer, nvitrogen 743995 2x 10x Reducing Agent nvitrogen 897034 40 2. Reactions were incubated at 30° C. for 90 minutes in a Criterion Tris HCl 4-15%. 1 mm, Bio-Rad 4OOOS9499 water bath. 12 + 2 well 3. Reactions were analyzed for the synthesis of functional Precision Plus Protein Standards, Bio-Rad 31 OOO9928 Luciferase using a standard Luciferase assay. Kaleidoscope Luciferase Assay: 10x Tris/Glycine/SDS Buffer Bio-Rad 21 OOO7884 45 1. Luciferase assay Substrate was prepared according to Gelcode Blue Safe Protein Stain ThermoFisher LL152043 manufacturers instructions: Reagent Kit was thawed at room temperature. Dual-Glo R. Luciferase Buffer was transferred into the Equipment: Dual-Glo R. Luciferase Substrate bottle and shaken 50 slightly to ensure the substrate dissolved. Dual-Glo R Stop & Glo(R) substrate was transferred into Item Vendor ID # the Dual-Glo(R) Stop & Glo R buffer and mixed well. P20, P200, P1,000 Rainin NA Rehydrated reagent was aliquoted into 15 ml centrifuge Water bath tube (10 ml/tube) and wrapped with Aluminum foil. Luminometer 55 Power Pac HC Bio-Rad NA Rehydrated reagent was stored at -80° C. until ready Heat block VWR NA for use (the reagent is good for 6 months). Microcentrifuge, refrigerated Eppendorf NA 2.5ul of reaction end products/well were transferred into Platform Adjustable Tilt Rocker Labnet NA a 96-well white plate Thermal cycler MJ Research NA 3. 100 uL of the Luciferase Assay Reagent was dispensed 60 per well and mixed by pipetting 2-3x. Procedure: 4. RLU's for each well on plate were read within 10 minutes. Per manufacturers instructions: Except for the actual Results: transcription/translation incubation, all handling of the Results are shown in FIG. 9. Circular plasmid expression TNT.R. Quick Master Mix was performed at 4°C. Unused 65 vector encoding PE-IL2 fusion protein was slightly better at Master Mix was refrozen as soon as possible after thawing inhibiting Luciferase protein synthesis compared to linear to minimize loss of translational activity. ized plasmid encoding the same (at all Luciferase vector: US 9,677,055 B2 151 152 VVN-52431 vector ratios). These results also demonstrate Reagent Preparation: the ability of to test and compare the biological activity of Luciferase Preparation: PE-fusion proteins in inhibiting protein synthesis. 1. Thaw reagent Kit on ice or at 4 degrees C. In addition to measuring inhibition of protein synthesis as 2. Transfer entire Dual-Glo R. Luciferase Buffer into Dual a measure of light production catalyzed by Luciferase, Glo(R. Luciferase Substrate bottle and shake bottle quantitative analysis of inhibition of protein synthesis was also performed by separating polypeptide reaction products slightly to ensure Substrate completely dissolved. on SDS-PAGE gels, staining, and assessing amounts of 3. Transfer entire Dual-Glo R Stop & Glo(R) substrate into polypeptide produced (data not shown). Dual-Glo R Stop & Glo(R) buffer and mix well. Assays such as these may be used to compare the ability 10 4. Aliquot rehydrated reagent into 15 ml centrifuge tube of amino acid substituted (e.g., deimmunized) forms of PE (10 ml/tube) and wrap tube with Aluminum foil. (alone or as fusion proteins) to retain biological activity 5. Store rehydrated reagent in -80° C. Freezer (reagent is (such as inhibition of protein synthesis) compared to corre good for 6 months) sponding non-amino acid Substituted forms of PE (alone or Procedure: as fusion proteins). 15 Per manufacturer's recommendations: Except for the actual transcription/translation incubation, all handling of Example 6: In Vitro Transcription/Translation TNTR Quick Master Mix should be done at 4° C. Any (IVTT) Assay to Measure and Compare unused Master Mix should be refrozen as soon as possible Ribosylation Activity of Amino Acid Substituted after thawing to minimize loss of translational activity. Do Variants of PE not freeze-thaw the Master Mix more than two times. Plasmid DNA Dilution: Purpose: This protocol provides an example of they type Dilute plasmid DNA and Luc plasmid DNA in nuclease of methods which may be used to measure and compare the free water to final concentration of 0.1 g/ul. ribosylation activity (i.e., inhibition of protein synthesis) of 25 IVTT Reactions amino acid substituted forms of PE compared to correspond 1. In 1.5 ml NF (nuclease free) eppendorf tubes, the ing non-amino acid substituted forms of PE. following are combined for each test sample: Background: a. 5 uL of NF HO: The IVTT assay measures PE mediated inhibition of in 30 b. 2 ul, 0.1 g/ul test plasmid (for increased accuracy of vitro transcription/translation of a target plasmid, T7-Luc. results test a dilution series of samples); The level of inhibition (or lack thereof) is determined by c. 1 ul, 1 mM Methionine: chemiluminescent measurement of Luciferase activity (i.e., d. 40 uL TNT quick master mix: the transcribed and translated protein). In this assay, a e. Negative control (reaction mix only, NF water 9 ul, lowered level of transcription and translation (and thereby, 35 lowered levels of chemiluminescent light output) corre methionine 1 ul, reaction mixture 40 ul; sponds to increased inhibition of protein synthesis, IVTT 2. Incubate reaction mixes at 30° C. for 15 minutes in can be performed using template DNA encoding PE, or by water bath; directly using quantified protein. This assay may be used to 3. Add 2 ul, 0.1 ug/ul Luc plasmid to each reaction mix: different PE variants against each other and to compare 40 4. Incubate reactions at 30° C. for 90 minutes in water their biological activities to corresponding non-amino acid bath; and substituted forms. 5. Transfer all samples onto ice to stop reaction. Materials: Luciferase Assay Test sample: Vectors comprising either circular plasmids 6. Transfer 5 ul of end product/well into 96-well white with an SP6 promoter or linearized plasmids with a T7 45 plates in triplicate; promoter. 7. Dispense 100 uL of Luciferase Assay Reagent per well. Mix by pipetting 2-3x; Reagents and Materials Vendor 8. Read entire plate within 10 minutes. 50 Calculations Nuclease-free water (1000 ml) Ambion TNT SP6 Quick Coupled TT system PROMEGA (R) 1. Calculate percent inhibition based on relative lumines SP6 luciferase plasmid DNA (From PROMEGA (R) cence units (RFU) of the test sample divided by the same kit) RFU of the LUC plasmid with no test sample, then RNase-free 1.5 ml microfuge tubes Ambion subtract the result from 100. Dual Glo (R) Luciferase Assay System PROMEGA (R) 55 2. Calculate the percent of activity of non-amino acid substituted PE by percent inhibition of the test sample Equipment: divided by the percent inhibition of non-amino acid substituted, then multiply the result by 100. 60 3. If a dilution series of samples is tested, calculate the Item Vendor IC50 (half maximal inhibition concentration) for each P20, P200, P1,000 Rainin sample using the RFU of the test sample divided by the 96 well white plate Costar RFU of the LUC plasmid alone, then subtract the result Water bath (circulation) from 100. Determine the concentration which results in Luminometer Microcentrifuge, refrigerated Eppendorf 65 50% inhibition. 4. Calculate the percent of non-amino acid substituted PE inhibition by dividing the IC50 of the non-amino acid US 9,677,055 B2 153 154 substituted PE by the IC50 of the test sample and mice expressing human immunoglobulin genes), rats, rab multiplying the result by 100. bits, dogs, goats, sheep, horses, cows (and other bovine species), non-human primates, and humans. Example 7: Ex Vivo Assays to Assess Immunogenicity of Amino Acid Substituted Forms Examples 9-13: Oligonucleotides Referenced in the of PE (i.e. Deimmunized PE) Versus Corresponding Following Examples are Listed in Table 12 Non-Amino Acid Substituted Forms Example 9: Generation of cDNAs Encoding Amino 10 Acid Substituted Forms of PE The immunogenicity of amino acid substituted forms of PE (alone or as PE-fusion proteins) are assessed using The Kozak sequence in vector plT14b (EMD Millipore methods well-known and routinely used by those skilled in catalog #69660, Darmstadt, Germany) was modified by the art. For example, ELISA assays are used wherein serum 15 introducing a linker made up of annealed oligonucleotides is assayed ex vivo (following extraction from organisms in 5'-CATGGT which amino acid substituted forms of PE, or non-amino GGCTCTCCTTCTTAAAGTTAAACAAAATTATTT-3' acid substituted PE, (alone or as fusion proteins) are admin (SEQ ID NO:239)(OL2216 in Table 12) and istered) to determine whether or not antibodies that specifi 5'-CTAGAAATAATTTTGTTTAACTT cally bind the administered protein are produced. It is noted TAAGAAGGAGAGCCAC-3' (SEQ ID NO:240)(OL2217 that in the case of PE-fusion proteins it is necessary to use, in Table 12) (underlined letters indicate nucleotides changed as the ELISA assay target antigen, not only intact PE-fusion in the Kozak sequence) via NcoI and Xbal restriction sites proteins (i.e., amino acid Substituted or non-amino acid into vector pET14b resulting in a modified Kozak sequence substituted PE), but to also test the PE component and the (SEQ ID NO:176) by mutation of three nucleotides at polypeptide fusion component separately to determine 25 positions 587 to 589. The resulting vector was named whether or not antibodies produced specifically bind the PE pET14b-K. portion or the fusion polypeptide portion (e.g., IL2 as used Kozak sequence=(gcc)gccRccAUGG (SEQ ID NO:286), in a previous example of a PE-IL2 fusion protein). Accord where R is a purine (i.e., adenine or guanine) three bases ingly, it is most desirable to identify amino acid Substituted 30 upstream of the start codon (AUG), which is followed by forms of PE which do not result in host production of another G'. See, Kozak, Nucleic Acids Res. 15 (20): 8125 antibodies that specifically bind modified forms of PE (i.e., 8148 (1987). deimmunized forms of PE). Oligonucleotides for generation of genes encoding amino Organisms in which amino acid substituted forms of PE acid Substituted forms of PE are listed as OL2164 to OL2194 may be administered (alone or as fusion proteins) include, 35 and OL2281 to OL2366 in Table 12. A wild-type (WT) PE for example, without limitation: mice (including transgenic gene (SEQ ID NO: 1) was made by gene synthesis and mice expressing human immunoglobulin genes), rats, rab amplified using oligonucleotides: 5'-ATTGTCCATATGC bits, dogs, goats, sheep, horses, cows (and other bovine CAGAAGGCGGTAGCCTGGC-3' (SEQ ID NO:215) species), non-human primates, and humans. (OL2154 in Table 12) to introduce a Ndel site, and 40 5'-ATCCTCGAGTTACTTCAGGTCCTCACGCGGCG 3' (SEQ ID NO:222)(OL2167 in Table 12) to introduce Example 8: In Vitro and In Vivo Assays to Assess a XhoI site. The resulting DNA fragment was sub Cytotoxicity of Amino Acid Substituted Forms of cloned into pGEMTR-T (PROMEGAR) catalogue PE #A1360, PROMEGAR, Southampton, UK). Colonies 45 were screened by PCR using M13 primers OL0001 and OL0002 (Table 12). Subsequently the wild-type (WT) The cytotoxicity of amino acid substituted forms of PE PE gene was subcloned into pET14b-K using Ndel and (alone or as PE-fusion proteins) are assessed using methods XhoI restriction enzymes (Fermentas catalog #FD0583 and FD0695, respectively) and resulting in a gene well-known and routinely used by those skilled in the art. 50 For example, the cytotoxic effects of amino acid substituted encoding an N-terminal His6 tag fused to the WT PE forms of PE (alone or as PE-fusion proteins) administered to sequence. The resulting vector was named pET14b-K- cells in vitro or organisms in Vivo, may be assessed with WT PE. reference to cytotoxic (cell killing) effects on target cells, Oligonucleotides for generation of genes encoding amino acid Substituted forms Genes encoding amino acid Substi organs, tissues, or tumors against which PE or PE fusion 55 tuted forms of PE were generated using overlapping PCR proteins are expected to produce a cytotoxic effect. For with the WT PE gene in pET14b-K as template. Pairs of example, the therapeutically beneficial cytotoxic effects of primers from the oligonucleotides of Table 12 (as noted in amino acid substituted PE-Mesothelin fusions may be the “application' column of Table 12) were annealed to WT assessed by monitoring and measuring reduction or elimi PE DNA and the amino acid substituted genes were PCR nation of tumor or cancer cells or tissues (in vitro or in vivo) 60 amplified using terminal oligonucleotides: 5'-ATCTC in response to administration of amino acid Substituted CCTCTAGAAATAATTTTGTTTAACTTTAAGAAG-3' forms of PE-Mesothelin versus wild-type PE-Mesothelin (SEQ ID NO:241)(OL2268 in Table 12) and fusion. 5'-ATCCTCGAGTTACTTCAGGTCCTCACGCGGCG Organisms in which amino acid substituted forms of PE 65 3' (SEQ ID NO:216)(OL2161 in Table 12). PCR frag may be administered (alone or as fusion proteins) include, ments were and cloned into pET14b-K using Xbaland for example, without limitation: mice (including transgenic XhoI restriction sites.

US 9,677,055 B2 163 164 TABLE 12- Continued Oligonueleotides Name sequence length application

OL 2323 CAAGCTGGAGATCAAAGCTC 44 IEXO2 FOR to create ATGGGGGCAGCCATCATCATC RFB4 - 6xHis PE38 fusions ATC (SEO ID NO: 284) (pLEXO2-302 and pIEXO2 3O4) to pair with OL2161

OL 2324 GATGATGATGATGGCTGCCCC 44 IEXO2 REW to create CATGAGCTTTGATCTCCAGCT RFB4 - 6xHis PE38 fusions TG (SEO ID NO: 285) (pLEXO2-302 and pIEXO2 3O4) to pair with OL2318

15 Example 10: Analysis of Genes Encoding Amino from 2.5 nanograms of DNA encoding amino acid Substi Acid Substituted Forms of PE by an In Vitro tuted PE compared to expression from 2.5 nanograms WT Transcription/Translation (IVTT) Assay PE DNA as shown in Table 13 (data expressed as % inhibition of IVTT for the DNA encoding amino acid The cell-free in vitro transcription/translation (IVTT) substituted PE compared to wild-type PE). In order to assay was performed with a TnT.R. T7 Coupled Reticulocyte identify “inhibitory” amino acid substituted PE polypeptides Lysate System (PROMEGAR) catalog #L4610) following (i.e., genes encoding amino acid Substituted forms of PE the procedure described in the User's Manual provided in which inhibit IVTT), selected mutations in each T cell the kit. See, PROMEGAR), Technical Bulletin HTB 126, 25 epitope as identified in Table 11 were initially tested using Revised December 2010, pp. 1-28 (2010) which is incorpo various combinations of epitope 5 mutations (e.g., corre rated by reference herein. sponding to S241N, S241 K, S241P and S241T in SEQ ID WT PE in plasmid plT14b-K was used as standard on NO: 1) along with mutations in either: epitope 4: epitopes 4 every plate and tested at concentrations ranging from 0.08 and 3; epitopes 4 and 1; epitopes 4 and 2; or, epitopes 4 and ng to 10 ng in a 12.5 microliter final volume reaction. All test 30 6 (see, Table 13). For all combinations except those includ samples were run in triplicate. DNAs encoding WT or amino ing amino acid substitutions in epitope 3 at I 184 (SEQ ID acid substituted PE in plasmid pET14b-K were added to the NO:1; or, I196 in SEQ ID NO:2) (which produced 0% IVTT reaction mix supplemented with NAD+ (final concen inhibition), one or more inhibitory PE polypeptides were tration 0.15 mM; Fisher Scientific catalog #BPE9746-212) identified (Table 13). (Note: “Inhibitory PE polypeptides’ and incubated at 30° C. for 15 min. Following a subsequent 35 indicates amino acid substituted forms of PE which exhibit cooling step at 4°C. for 5 min, 250 ng of T7 Luciferase PE biological activity in the inhibition of IVTT). From plasmid (Luciferase T7 control DNA supplied in the TnT(R) structural analysis of PE, it was noted that residue I184 T7 Coupled Reticulocyte Lysate System) was added to each (SEQ ID NO:1; or, I196 in SEQ ID NO:2) (anchor residue reaction and incubated at 30° C. for 90 min. The reaction 1, Table 11) was located within the active enzymatic site of was stopped by placing the samples on ice. Samples were 40 PE. In view of this result, alternative mutations distal to the analyzed using the STEADY-GLOR. Luciferase Assay active site were sought at anchor residues 6 and 9 (V189 and (PROMEGAR) catalog #E2510) according to the protocol R192 in SEQ ID NO:1; or, V201 and R204 in SEQ ID provided by the manufacturer. See, PROMEGAR),Technical NO:2). Alternative epitope 3 mutations at V189 and R192 in Bulletin HTM051, revised September 2011, pp. 1-23 (2011) SEQID NO:1 were tested in combination with other epitope which is incorporated by reference herein. Luminescence 45 mutations. These mutations confirmed that inhibitory PE was measured in a FLUOstar OPTIMA plate reader (BMG polypeptides with epitope 3 mutations could also be gener Labtech Ltd., Aylesbury, UK) ated (Table 13, pIEX02-173 to -248). A range of combina A representative result is shown in FIG. 10 which shows tions of DNAs encoding multiple amino acid substituted the results expressed in CPS (counts per second as read from forms of PE were tested progressively leading to a final the FLUOstar OPTIMA fluorescence plate reader) for 50 analysis of mutations in four of six, five of six, and six of six luciferase activity from IVTT assays of genes encoding identified immunogenic epitopes. See, Table 13, "Quadru either WT PE (pIEX02-001 (SEQ ID NO:1)) or encoding plicates”, “Quintuplicates' and “Sextuplicates”. In this amino acid substituted PE (pIEX02-228 (SEQID NO:177), regard, quadruplicate epitope mutations were identified pIEX02-244 (SEQ ID NO:178), pIEX02-246 (SEQ ID which exhibited IVTT inhibitory activity ranging from 0% NO:179)); which were expressed as fusion proteins com 55 to about 70%. Quintuplicate mutations were identified that prising a histidine polymer and a linker sequence preceding exhibited IVTT inhibitory activity ranging from about 5% to a sequence of WT PE or amino acid substituted PE; see, about 35%. Sextuplicate mutations were identified that pIEX02-001 PEWT (SEQID NO: 189 (DNA) and SEQ ID exhibited IVTT inhibitory activity ranging from about 5% to NO:190 (AA)), pIEX02-228 amino acid substituted PE about 20%. It is also noted that multiple single, double, and (SEQ ID NO:193 (DNA) and SEQ ID NO: 194 (AA)), 60 triple epitope mutations also resulted in amino acid Substi pIEX02-244 amino acid substituted PE (SEQ ID NO:197 tuted forms of PE exhibiting PE biological activity in the (DNA) and SEQID NO:198 (AA)), pIEX02-246 amino acid inhibition of IVTT such that the percent (%) inhibitory substituted PE (SEQ ID NO:201 (DNA) and SEQ ID activity ranged from 0% to 100% (or about 100%); see Table NO:202 (AA)). See also, Table 13. 13. For the analysis of various amino acid substituted PEs, the 65 Three different “candidates' (i.e., amino acid substituted potency of each mutated PE in inhibiting IVTT was forms of PE or DNA constructs encoding the same) were expressed as relative inhibition exhibited via expression selected for use as examples in performing Subsequent US 9,677,055 B2 165 166 experiments described further herein. In particular, addi 20%, and 36%) in amino acid substituted forms of PE may tional experiments were performed using the Sextuplicate provide a therapeutically effective polypeptide. See, for AA substituted candidate plEX02-244 (SEQID NO:178; see example, Thomas et al., “Abrogation of Head and Neck also. Table 13); which retained approximately 20% of the Squamous Cell Carcinoma Growth by Epidermal Growth Factor Receptor Ligand Fused to Pseudomonas Exotoxin WTPE inhibitory activity. Likewise, additional experiments Transforming Growth Factor C.-PE38.” Clin. Cancer Res. were also performed using the Sextuplicate AA Substituted 10:7079-7087 (2004); Siegall et al., “Cell-specific toxicity candidate plEX02-246 (SEQID NO:179; see also, Table 13) of a chimeric protein composed of interleukin-6 and which retained approximately 8% of the WT PE inhibitory activity; and using the quintuplicate AA Substituted candi Pseudomonas exotoxin (IL6-PE40) on tumor cells’, Mol. 10 Cell. Biol. 10(6): 2443-2447 (1990); and, Weldon & Pastan, date plEX02-228 (SEQ ID NO:177; see also, Table 13) 'A Guide to Taming a Toxin-Recombinant Immunotoxins which retained approximately 36% of the WT PE inhibitory Constructed From Pseudomonas Exotoxin A for the Treat activity. These were expressed as fusion proteins comprising ment of Cancer, FEBS Journal 278(23):4683-4700(2011). a histidine polymer and a linker sequence preceding a Moreover, it is noted that in view of the highly cytotoxic sequence of WT PE or amino acid substituted PE; see, 15 nature of wild-type PE, IVTT inhibition activity (i.e., cyto pIEX02-001 PEWT (SEQID NO: 189 (DNA) and SEQ ID toxicity) as low as about 5% (or higher) of WT (e.g., 8%, NO:190 (AA)), pIEX02-228 amino acid substituted PE 20%, and 36%) in amino acid substituted forms of PE may (SEQ ID NO:193 (DNA) and SEQ ID NO: 194 (AA)), provide a therapeutically effective polypeptide. See, for pIEX02-244 amino acid substituted PE (SEQ ID NO:197 example, Thomas et al., “Abrogation of Head and Neck (DNA) and SEQID NO:198 (AA)), pIEX02-246 amino acid Squamous Cell Carcinoma Growth by Epidermal Growth substituted PE (SEQ ID NO:201 (DNA) and SEQ ID Factor Receptor Ligand Fused to Pseudomonas Exotoxin NO:202 (AA)). These AA substituted PE polypeptides, and Transforming Growth Factor C.-PE38.” Clin. Cancer Res. DNA constructs encoding, them may be referenced herein as 10:7079-7087 (2004); Siegall et al., “Cell-specific toxicity “228”, “244 or “246” using simply these three numbers, or of a chimeric protein composed of interleukin-6 and using these numbers and a prefix or Suffix included there 25 Pseudomonas exotoxin (IL6-PE40) on tumor cells’, Mol. with. Cell. Biol. 10(6): 2443-2447 (1990); and, Weldon & Pastan, Moreover, it is noted that in view of the highly cytotoxic 'A Guide to Taming a Toxin-Recombinant Immunotoxins nature of wild-type PE, IVTT inhibition activity (i.e., cyto Constructed From Pseudomonas Exotoxin A for the Treat toxicity) as low as about 5% (or higher) of WT (e.g., 8%, ment of Cancer, FEBD Journal 278(23):4683-4700 (2011). TABLE 13 Examples of Amino Acid Substituted Forms of PE and ASSociated Cell Cytotoxic Activity. Epitopes pIEXO2 - Epitope Epitope Epitope Epitope Epitope Epitope %. Inhibition changed #H# 5 4 3 1 2 6 of IVTT Wild-Type (WT)

OO1 100.00 (WT) Single Substitutions

5 OO3 S241N 100.00 S253N 100.00

5 OOS S241P 99.71 S253P 99.99

Double Substitutions

4, 5 OO7 S241N Q194R 41.63 S253N Q206R 4, 5 O08 S241K Q194R 69.51 S2S3K Q206R 4, 5 O09 S241P Q194R 20.58 S253P Q206R 4, 5 O10 S241T Q194R 21.44 S253T Q206R 4, 5 O11 S241N D197K 99.88 S253N D209K 4, 5 O12 S241K D197K 42.49 S2S3K D209K 4, 5 O13 S241P D197K 74.87 S253P D209K 4, 5 O14 S241T D197K 98.84 S253T D209K Triple Substitutions

3, 4, 5 O15 S241N Q194R I184A O.OO S253N Q206R LI196A) 3, 4, 5 O16 S241K Q194R I184A O.OO S2S3K Q206R LI196A)

US 9,677,055 B2 171 172 TABLE 13-continued Examples of Anino Acid Substituted Forms of PE and ASSociated Cell Cytotoxic Activity. Epitopes pIEXO2 - Epitope Epitope Epitope Epitope Epitope Epitope %. Inhibition changed #H# 5 4 3 2 6 of IVTT

226 S241T D197K 41A N1SOA Q326E 11.23 S253T D209K S3A N162A Q338E. 1, 2, 4, 5, 6 228 S241T D197K 41A T152R Q326E 36.27 S253T D209K S3A T164R) Q338E. 1, 2, 4, 5, 6 229 S241T D197K 41A T152A Q326E 18.11 S253T D209K S3A T164A) Q338E. 1, 2, 3, 4, 5 221 S241T Q194R R192A 41A T152R 4.79 S253T Q206R R204A S3A T164R) 1, 2, 4, 5, 6 242 S241T D197K 41T T152A Q326E 21.64 S253T D209K 53T T164A) Q338E. Sextuplicate Substitutions

1-6 244 S241T D197K R192A 41T T152A Q326E 20.53 S253T D209K R204A 53T T164A) Q338E. 1-6 246 S241T D197K R192A 41A T152A Q326E 7.95 S253T D209K R204A S3A T164A) Q338E. 1-6 248 S241T D197K R192A 41A T152R Q326E 4.45 S253T D209K R204A S3A T164R) Q338E.

Note: Non-bracketed amino acid substitution positions correspond to the PE polypeptide sequence in SEQID NO: 1. Bracketed amino acid substitution positions correspond to the PE polypeptide sequence in SEQD NO:2 (comprising an N-terminal 12-amino acid linker).

25 Example 11: Ex Vivo Human T Cell Assay to ducing Eag site) and OL2098, introducing HindIII site Assess Immunogenicity of Wild-Type (WT) and (Table 12). The resulting PCR fragment was subcloned into Amino Acid Substituted PE pACYC184 (NEB catalog #E4152S) which was then trans formed into SHuffleTM T7 with selection for chlorampheni Ex vivo human T cell assays (EPISCREENTM: see e.g., 30 col resistance. The PE E287D (SEQ ID NOs: 180 to 183) preceding Examples) were performed to assess the immu genes in pET14b-K were transformed into the SHuffleTM nogenicity of whole proteins corresponding to pIEX02-244 T7/GroEL/ES strain with selection for amplicillin resistance. (SEQ ID NO:178) plEX02-246 (SEQ ID NO: 179) and Single colonies were grown in 2XYT medium (Sigma pIEX02-228 (SEQ ID NO:177) (Example 10). In order to Aldrich catalog #Yr2627-1KG) and protein expression was avoid direct cytotoxicity to cells used in the assay, “null 35 induced at ODoo 1.0 by adding isopropyl-f-D-thio mutants” were generated for the three candidates and WT PE galactoside (IPTG) to a final concentration of 0.4 mM. by overlapping PCR as in Example 9 using primers OL2162 Cultures were then grown at 16 degrees C. for 17 h before and 2163 (Table 12) to introduce an amino acid substitution harvesting by centrifugation. Cell pellets were resuspended of E287D in the three candidates and WT PE (to give SEQ 40 in 35 ml of binding buffer (50 mM Tris pH 8.0, 500 mM ID NOs: 180 to 183). (Note: “Null mutants” is intended to NaCl and 10 mM imidazole) supplemented with protease indicate mutated forms of PE which lack cell cytotoxic inhibitors (cComplete protease inhibitor tablets, Roche cata biological activity; the amino acid Substitution used to log #11873580001, Roche Diagnostics Ltd., Burgess Hill, generate null mutants corresponds to a change of E287D in UK (mixture of several protease inhibitors for inhibition of SEQID NO:1 or E299 in SEQID NO:2.) The E287D (SEQ 45 serine and cysteine proteases)). Cells were lysed by Sonica ID NOs: 180 to 183) encoding genes were cloned into tion (SONICATORR), Misonix catalog #XL2020, Misonix pET14b-K as in Example 9. WT PE sequence is shown in Inc., Farmingdale, N.Y.), and cell debris and insoluble SEQID NO:180; plEX02-228 sequence is shown in SEQID material removed by centrifugation. Proteins were purified NO:181; plEX02-244 sequence is shown in SEQ ID from the soluble fraction by nickel chelate affinity chroma NO:182; and, pIEX02-246 sequence is shown in SEQ ID 50 tography using HISTRAPRFF Crude columns (GE Health NO:183. These were expressed as fusion proteins compris care catalog #11-004-58, GE Healthcare Life Sciences, ing a histidine polymer and a linker sequence preceding a Little Chalfont, UK). After loading, the columns were “null mutant sequence of WTPE or amino acid substituted washed with 50 mM Tris (pH 8.0) containing 500 mM. NaCl PE; see, pIEX02-001 PEWT null mutant (SEQ ID NO:191 and 20 mM imidazole and bound protein was eluted with 50 (DNA) and SEQ ID NO.192 (AA)), pIEX02-228 null 55 mM Tris (pH 8.0) containing 500 mM. NaCl and 500 mM mutant (SEQ ID NO:195 (DNA) and SEQ ID NO:196 imidazole. Following buffer exchange to 20 mM Tris (pH (AA)), pIEX02-244 null mutant (SEQ ID NO:199 (DNA) 8.0) using Zeba Spin desalting columns (7K MWCQ, Pierce and SEQID NO:200 (AA)), pIEX02-246 null mutant (SEQ catalog #89893), a negative purification step was employed ID NO:203 (DNA) and SEQ ID NO:204 (AA)). using anion exchange chromatography on Q-Sepharose (1 The host cell for expression of the PE E287D genes was 60 ml. HISTRAPR) Q FF column (GE Healthcare catalog an Escherichia coli BL21 derivative Strain called SHuffleTM #17-5053-01) with 20 mM Tris pH 8.0 and an NaCl gradient T7 Express (NEB catalog #C3029H, New England Biolabs form O M to 1.5 M. For each protein, the column flow UK Ltd., Knowl Piece, UK) which was altered to overex through was concentrated using an AMICONR) Ultra cen press the chaperonins GroEL/ES by amplification of the trifugal filter (EMD Millipore catalog #UFC 800 396, EMD GroEL/ES operon, including its promoter/regulatory sites, 65 Millipore, Feltham, UK) and further purified by size-exclu from E. coli DH5alphaTM (Invitrogen catalog #18265-017, sion chromatography (120 ml, HiLoad 16/60 SUPERDEX(R) Life Technologies Ltd., Paisley, UK) using OL2097 (intro 75 pg (GE Healthcare catalog #28-9893-33)) using 1xPBS