Patent Grant
11013784
A sequence listing in electronic (ASCII text file) format is filed with this application and incorporated herein by reference. The name of the ASCII text file is “2016_0823A_ST25.txt”; the file was created on Aug. 18, 2016; the size of the file is 74 KB.
Proteolytic enzymes and their regulatory networks, including cofactors, activators, and endogenous inhibitors, are frequently dysregulated in tumors resulting in increased protease activities that contribute to progression of disease [1]. Manipulation of tumor-promoting proteases is a promising approach for the development of anti-tumor therapies [2,3]. While the targeting of proteases has been approached in several ways [4], prodrug-like protease substrates that are activated by overexpressed proteases are an extremely efficient approach to increasing selectivity and efficacy while reducing off-target effects [5].
Anthrax toxins requiring proteolytic activation have been engineered to target proteases overexpressed by tumor cells. Anthrax toxin is a cytotoxic pore-forming exotoxin secreted by Bacillus anthracis. Consisting of protective antigen (PrAg), lethal factor (LF), and edema factor (EF), the toxin (the combination of PrAg and LF and/or EF) causes cellular cytotoxicity through a well-characterized mechanism [6], whereas individually these proteins are non-toxic. PrAg binds to either of two cell-surface receptors, tumor endothelial marker-8 (TEM8, ANTXR1) and capillary morphogenesis gene-2 (CMG2, ANTXR2), of which CMG2 is expressed on nearly all cell types. PrAg (83 kDa) bound to its cell-surface receptor(s) is proteolytically cleaved and activated by the protease furin (FURIN) or furin-like proprotein convertases in an exposed flexible loop to generate an active C-terminal 63-kDa PrAg fragment. The newly-generated 63-kDa PrAg fragment remains receptor bound and catalyzes the formation of a PrAg/receptor oligomer that presents docking sites to enable up to four molecules of LF or EF to bind and translocate into the cytosol of a cell, through an endosomal PrAg-formed pore, wherein LF/EF then have potent cytotoxic effects [7].
As a highly efficient protease-activated delivery system, PrAg can be engineered to deliver different payloads or co-factors into the cytosol [8-14]. Additionally, PrAg can be engineered to be activated specifically by proteases other than furin. Since furin is ubiquitously expressed, it is advantageous to narrow the cellular protease targets for drug delivery applications. Alteration of the furin protease cleavage site within PrAg to amino acid sequences recognized by either urokinase-type plasminogen activator (uPA, PLAU) [15], matrix metalloproteinase 2 (MMP2), or matrix metalloproteinase 9 (MMP9) [16] renders PrAg a potent uPA- or MMP2/9-activated prodrug that has been shown to target tumors that overexpress any of these proteases [17-26]. An engineered anthrax inter-complementing toxin has also been created that requires combined activation by these protease systems for function and killing of tumor cells [20,27].
While such uPA- or MMP2/9-activated prodrugs may be useful in some applications, in addition to their roles in tumor biology the uPA and MMP protease systems play leading roles in immune regulation and physiological tissue remodeling [4,28]. Therefore, while these engineered anthrax protein prodrugs are effective when used to target tumors in vivo, it is possible that paracrine association of the tumor-secreted proteases with other non-tumor cells in or near the tumor microenvironment could contribute to off-target effects of these toxin systems. Therefore, the use of existing protease-activated PrAg proteins is limited, and the development of new, targeted proteins is needed. The present application is directed to this and to other important goals.
The present application provides engineered, protease-activated, anthrax toxin protective antigen (PrAg) protein prodrugs and means for their use in therapeutic applications. These engineered PrAg protein prodrugs can be targeted to cells overexpressing membrane-anchored serine proteases, such as many tumor cells. As demonstrated herein, the targeting of such cells allows for a highly-specific, more efficient approach to directed cell targeting and tumor cell killing by engineered anthrax toxins than previous in systems.
In a first aspect, the invention generally relates to engineered PrAg proteins comprising the native anthrax PrAg amino acid sequence where the furin activation site is replaced by a membrane-anchored serine protease activation site. These activation sites are domains within the proteins that are recognized and cleaved by membrane-anchored serine proteases. The engineered PrAg proteins of the invention thus comprise the amino acid sequence set forth in SEQ ID NO:1, wherein the furin activation site is replaced by a membrane-anchored serine protease activation site. The PrAg protein includes an N-terminal, 29 amino acid signal peptide. Therefore, the engineered PrAg proteins of the invention also comprise the amino acid sequence set forth in SEQ ID NO:1, wherein the furin activation site is replaced by a membrane-anchored serine protease activation site and wherein the N-terminal, 29 amino acid signal peptide has been removed. The engineered PrAg proteins of the invention further include sequence variants having 90% or more sequence identity over their entire length to one of the engineered PrAg proteins defined herein. In aspects of the invention, the furin activation site consists of amino acids 193-200 of SEQ ID NO:1. In other aspects, the membrane-anchored serine protease activation site is one or more sequences selected from the group consisting of FTFRSARL (PCIS; SEQ ID NO:28), IPSRIVGG (TAS; SEQ ID NO:4), PQARITGG (PAS; SEQ ID NO:5), and PRFRITGG (UAS; SEQ ID NO:6).
In a second aspect, the invention generally relates to methods of inducing pore formation in a cell using the engineered PrAg proteins described herein. The invention thus includes methods of inducing pore formation in a cell comprising contacting a cell with an engineered PrAg protein, as defined herein, under conditions promoting pore formation in the cell, where the cell expresses an anthrax toxin PrAg protein receptor and a membrane-anchored serine protease. The receptor may be, but is not limited to, one or more of tumor endothelial marker-8 (TEM8) and capillary morphogenesis gene-2 (CMG2). The membrane-anchored serine protease may be, but is not limited to, one or more of testisin, hepsin, and matriptase.
In a third aspect, the invention generally relates to methods of inducing translocation of a selected co-factor into a cell using the engineered PrAg proteins described herein. The invention thus includes methods of inducing translocation of a selected co-factor into a cell, comprising (a) contacting a cell with an engineered PrAg protein, as defined herein, under conditions promoting pore formation in the cell, wherein the cell expresses an anthrax toxin PrAg protein receptor and a membrane-anchored serine protease, and (b) contacting the cell of (a) with a selected co-factor under conditions promoting translocation of the selected co-factor into the cell. The receptor may be, but is not limited to, one or more of tumor endothelial marker-8 (TEM8) and capillary morphogenesis gene-2 (CMG2). The membrane-anchored serine protease may be, but is not limited to, one or more of testisin, hepsin, and matriptase. The selected co-factor may be, but is not limited to, a diagnostic co-factor or a therapeutic co-factor. Exemplary diagnostic co-factors include, but are not limited to, imaging agents and markers. The therapeutic co-factor may be a cytotoxic co-factor or a non-cytotoxic co-factor. Exemplary cytotoxic co-factors include, but are not limited to, one or more of LF, EF, FP59, and LFn-CdtB. Exemplary non-cytotoxic co-factors include, but are not limited to, one or more of peptide fragments, antigens and epitopes, growth factors, enzymes, and antibodies and functional fragments or mimetics thereof.
In a fourth aspect, the invention generally relates to methods of treating cancer in a subject using the engineered PrAg proteins described herein and a co-factor that has a cytotoxic effect on a cancer cell. The invention thus includes methods of treating cancer in a subject comprising administering a pharmaceutical formulation to a subject in need thereof wherein the pharmaceutical formulation comprises a therapeutically effective amount of an engineered PrAg protein, as defined herein, and a therapeutically effective amount of a therapeutic co-factor, thereby treating cancer in the subject. The method may also be practiced by administering the engineered PrAg protein and the therapeutic co-factor in separate formulations. The invention thus includes methods of treating cancer in a subject comprising (a) administering a first pharmaceutical formulation to a subject in need thereof wherein the first pharmaceutical formulation comprises a therapeutically effective amount of an engineered PrAg protein, as defined herein, and (b) administering a second pharmaceutical formulation to the subject of (a) wherein the second pharmaceutical formulation comprises a therapeutically effective amount of a therapeutic co-factor, thereby treating cancer in the subject. In particular aspects, the cancer is a cancer characterized by cells expressing an anthrax toxin PrAg protein receptor and a membrane-anchored serine protease. The receptor may be, but is not limited to, one or more of tumor endothelial marker-8 (TEM8) and capillary morphogenesis gene-2 (CMG2). The membrane-anchored serine protease may be, but is not limited to, one or more of testisin, hepsin, and matriptase. The cancer may be, but is not limited to ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, and lung cancer. The cancer may be a benign cancer or a metastatic cancer. The cancer may be one that is resistant to other treatments, such as a cancer resistant to radiotherapy or chemotherapy. The therapeutic co-factor may be, but is not limited to, one or more cytotoxic co-factors selected from the group consisting of EF, LF, FP59 and LFn-CdtB.
The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technical references.
As used herein, “a” or “an” may mean one or more. As used herein, when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.
As used herein, “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., +/−5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.
Membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the surface of a cell via transmembrane domains or GPI-anchors [29,30]. Overexpressed in ovarian and other tumors, with pro-tumorigenic properties, they are attractive targets for anti-tumor therapies [31-48]. However, developed drugs targeted against the catalytic mechanism of serine proteases can lead to unacceptable non-target effects due to involvement of the proteases in many essential physiological processes [92]. Presented herein is an alternative approach for exploiting these enzymes in the therapeutic targeting of tumors and the treatment of cancer.
Rather that blocking the activity of membrane-anchored serine proteases expressed by tumor cells, this alternative approach relies on the protease activity of the enzymes. By taking advantage of the fact that protease overexpression is associated with many types of tumor cells, the prodrugs disclosed herein can have a targeted effect on tumor cells. In particular, the prodrugs disclosed herein are activated by these overexpressed membrane-anchored serine proteases. Because activation is largely centered on tumor cells, cytotoxic co-factors that function in concert with the prodrugs can be functionally restricted to the tumor cell microenvironment.
These prodrugs are engineered, anthrax toxin protective antigen (PrAg) proteins. Anthrax toxin is a cytotoxic pore-forming exotoxin secreted by Bacillus anthracis. Consisting of protective antigen (PrAg), lethal factor (LF), and edema factor (EF), the toxin (the combination of PrAg and LF and/or EF) causes cellular cytotoxicity. PrAg binds to either of two cell-surface receptors, tumor endothelial marker-8 (TEM8, ANTXR1) and capillary morphogenesis gene-2 (CMG2, ANTXR2), of which CMG2 is expressed on nearly all cell types. Upon receptor binding, PrAg (83 kDa) is proteolytically cleaved and activated by the protease furin (FURIN) or furin-like proprotein convertases at an activation site to generate an active C-terminal 63-kDa PrAg fragment. The newly-generated 63-kDa fragment remains receptor bound and catalyzes the formation of a PrAg/receptor oligomer that presents docking sites to enable up to four molecules of LF or EF to bind and translocate into the cytosol of a cell, through an endosomal PrAg-formed pore, wherein LF/EF then have potent cytotoxic effects [7].
The engineered PrAg proteins disclosed herein are based on the native anthrax PrAg polypeptide, but possess an activation site recognized and cleaved by a selected membrane-anchored serine protease in place of the furin activation site. Upon application to tumor cells in vitro or administration to a subject having cancer, activation of the engineered PrAg proteins via the membrane-anchored serine protease activation site is concentrated on tumor cells overexpressing the corresponding serine protease. The engineered PrAg proteins disclosed herein are bound by the same cell surface receptors as native anthrax PrAg (e.g., TEM8 and CMG2). Furthermore, once activated the engineered PrAg proteins exhibit the same activity as the native protein which includes catalyzing the formation of a PrAg/receptor oligomer pores in the cell that allow translocation of co-factors, such as LF and EF, into the cell. Thus, when a cytotoxic co-factor such as LF or EF is administered with the engineered PrAg proteins, the co-factors can induce a tumoricidal effect.
Engineered PrAg Proteins
The present invention is thus directed, in part, to engineered PrAg proteins. The engineered PrAg proteins of the invention include polypeptides comprising the amino acid sequence of the native anthrax PrAg protein, wherein the furin activation site has been replaced by a membrane-anchored serine protease activation site. The engineered PrAg proteins of the invention also include sequence variants of these polypeptides.
The full-length Bacillus anthracis anthrax toxin PrAg protein is shown in SEQ ID NO:1 and it comprises 764 amino acids. The polypeptide undergoes processing to release a 29 amino acid, N-terminal signal peptide. The resulting mature PrAg protein comprises 735 amino acids and it is set forth in SEQ ID NO:3. The polynucleotide sequence encoding the full-length anthrax toxin PrAg protein is shown in SEQ ID NO:2.
The engineered PrAg proteins of the invention include both full-length and mature anthrax PrAg proteins in which the furin activation site has been replaced by a membrane-anchored serine protease activation site. For sake of convenience, the engineered PrAg proteins of the invention are generally defined herein based on the sequence of the full-length anthrax PrAg protein set forth in SEQ ID NO:1. It should be understood that the engineered PrAg proteins of the invention also include mature forms where the 29 amino acid signal sequence has been removed.
The furin activation site (i.e., the domain within native PrAg recognized and cleaved by the protease furin) may be generally defined as encompassing amino acids 189-204 of SEQ ID NO:1 (i.e., the full-length PrAg protein). The furin activation site may also be defined as encompassing amino acids 189-203, amino acids 189-202, amino acids 189-201, amino acids 189-200, amino acids 190-204, amino acids 190-203, amino acids 190-202, amino acids 190-201, amino acids 190-200, amino acids 191-204, amino acids 191-203, amino acids 191-202, amino acids 191-201, amino acids 191-200, amino acids 192-204, amino acids 192-203, amino acids 192-202, amino acids 192-201, amino acids 192-200, amino acids 193-204, amino acids 193-203, amino acids 193-202, amino acids 193-201, or amino acids 193-200 of SEQ ID NO:1. In a particular aspect of the invention, the furin activation site is RKKRSTSA (SEQ ID NO:56), which consists of amino acids 193-200 of SEQ ID NO:1 (amino acids 164-171 of SEQ ID NO:3).
The identity of the membrane-anchored serine protease activation site that is used in place of the furin activation site in the engineered PrAg proteins is limited only in that it confers on the engineered PrAg protein the ability to be cleaved and activated by a selected membrane-anchored serine protease. Suitable membrane-anchored serine protease activation sites include activation sites recognized by one or more of the membrane-anchored serine proteases shown in Table 1. In particular aspects, the engineered PrAg proteins of the invention contain protease activation sites recognized by one or more of testisin, hepsin, and matriptase.
Non-limiting examples of membrane-anchored serine protease activation sites that may be used in the engineered PrAg proteins of the invention include those shown in Table 2. This table provides two groups of activation sites, i.e., domains that are recognized and cleaved by one or more of the membrane-anchored serine protease of Table 1. The first group encompasses the activation sites defined as SEQ ID NOs:4-27. These are zymogen activation sites of various proteases. The second group encompasses the activation sites defined as SEQ ID NOs:28-47. These are reactive center loop sites of various serpins. In particular aspects, the membrane-anchored serine protease activation sites are one or more of FTFRSARL (PCIS; SEQ ID NO:28), IPSRIVGG (TAS; SEQ ID NO:4), PQARITGG (PAS; SEQ ID NO:5), and PRFRITGG (UAS; SEQ ID NO:6).
In particular aspects, the engineered PrAg proteins of the invention comprise the amino acid sequence set forth in SEQ ID NO:1 where the furin activation site consisting of amino acids 193-200 is replaced by a membrane-anchored serine protease activation site selected from the group consisting of FTFRSARL (PCIS; SEQ ID NO:28), IPSRIVGG (TAS; SEQ ID NO:4), PQARITGG (PAS; SEQ ID NO:5), and PRFRITGG (UAS; SEQ ID NO:6), and sequence variants thereof having about 90% or more sequence identity over their entire length. The engineered PrAg proteins of the invention also can be defined a comprising the amino acid sequences set forth in: SEQ ID NO:54 (PrAg-PCIS), SEQ ID NO:48 (PrAg-TAS), SEQ ID NO:50 (PrAg-PAS), and SEQ ID NO:52 (PrAg-UAS).
As indicated above, the invention includes engineered PrAg proteins may defined based on the mature form of the PrAg protein lacking the signal peptide and thus the engineered PrAg proteins of the invention also comprise the amino acid sequence set forth in SEQ ID NO:3 where the furin activation site consisting of amino acids 164-171 is replaced by a membrane-anchored serine protease activation site selected from the group consisting of FTFRSARL (PCIS; SEQ ID NO:28), IPSRIVGG (TAS; SEQ ID NO:4), PQARITGG (PAS; SEQ ID NO:5), and PRFRITGG (UAS; SEQ ID NO:6), and sequence variants thereof having about 90% or more sequence identity over their entire length.
Sequence Variants
Because amino acid alterations to the native anthrax PrAg protein and the protease activation sites can often be made without adversely affecting the activity of the engineered PrAg proteins, sequence variants of the engineered PrAg proteins disclosed herein are encompassed within the scope of the invention. The sequence variants have amino acid alterations that include individual amino acid insertions, substitutions (e.g., conservative and/or non-conservative), and/or additions, and combinations thereof.
Examples of conservative substitutions within different groups of amino acids include basic amino acid substitutions (i.e. between arginine, lysine and histidine), acidic amino acid substitutions (i.e. between glutamic acid and aspartic acid), polar amino acid substitutions (i.e. between glutamine and asparagine), hydrophobic amino acid substitutions (i.e. between leucine, isoleucine and valine), aromatic amino acid substitutions (i.e. between phenylalanine, tryptophan and tyrosine), and small amino acid substitutions (i.e. between glycine, alanine, serine, threonine and methionine). Amino acid substitutions known to have minimal effect on specific activity are described [93]. Specific exchanges included within the scope of the invention include Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
In addition to conservative and non-conservative substitutions, amino acids used in the preparation of the sequence variants include non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) and unnatural amino acids, e.g., those that have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids include, but are not limited to, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.
Amino acids essential for the structure and activity of the engineered PrAg proteins can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis [94,95]. Sites of protein interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids [96-98]. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides which are related to an engineered PrAg protein disclosed herein.
Alternations to the amino acid sequence of the engineered PrAg proteins may be accomplished via a number of techniques known to those of ordinary skill in the art, including mutagenesis, recombination, and/or shuffling, which can be confirm be sequencing or other relevant screening procedures [99-102].
The invention thus includes sequence variants of the engineered PrAg proteins disclosed herein, wherein the sequence variants have about 90% or more sequence identity over the entire length of the amino acid sequence to the amino acid sequence of an engineered PrAg protein defined herein. The sequence variants also include those having about 91% or more sequence identity, about 92% or more sequence identity, about 93% or more sequence identity, about 94% or more sequence identity, about 95% or more sequence identity, about 96% or more sequence identity, about 97% or more sequence identity, about 98% or more sequence identity, or about 99% or more sequence identity over the entire length of the amino acid sequence to the amino acid sequence of an engineered PrAg protein defined herein.
The amino acid alterations in the sequence variants can be limited to particular regions or domains of the proteins. For example, amino acid alterations may be excluded from the protease activation sites. Alternatively, or in addition, the amino acid alterations may be excluded from PrAg receptor binding sites.
The sequence variants of the invention retain the ability to be activated by a membrane-anchored serine protease, to be bound by an anthrax PrAg receptor, and to form PrAg/receptor oligomer pores in a cell for translocation of co-factors.
Co-Factors
The engineered PrAg proteins of the invention, by themselves, are generally benign and non-toxic. But because these proteins form pores in a cell, they can be used to introduce one or more selected co-factors (as they work in conjunction with the engineered PrAg proteins) into a cell. Co-factors can be selected based on the activity they have once inside of a cell. Relevant activities include, but are not limited to, signaling, therapeutic, and cytotoxic activities on or in the cell. It will be apparent that the co-factors can be used in diagnostic and therapeutic applications, thus the co-factors can be diagnostics and therapeutics.
As shown in the Examples provided herein, engineered PrAg proteins can bind to tumor cells where they are activated by the enzymatic activities of cell surface serine proteases. When introduced to the cells along with a cytotoxic co-factor, the combination induces death of the tumor cells. Moreover, the Examples demonstrate the several different engineered PrAg proteins have been established that are cytotoxic in combination with the co-factors to multiple human tumors, including pancreatic, prostate, lung and ovarian tumors, that each express variable levels of membrane-anchored serine proteases.
The co-factors that may be used in combination with the engineered PrAg proteins of the invention are only limited in that (i) they can enter a cell through a PrAg-induced cellular pore, and (ii) they have a desired effect once in the cytosol.
The co-factors may be, but are not limited to, diagnostic co-factors and therapeutic co-factor.
Exemplary diagnostic co-factors include, but are not limited to, imaging agents such as green fluorescent protein and AlexaFluor545, as well as markers, such as a radioactive moiety. The diagnostic co-factors are commonly administered in the context of a chaperone. For example, a non-cytotoxic variant of LF or EF can be conjugated to or labeled with an imaging agent or a marker.
The therapeutic co-factor may be a cytotoxic co-factor or a non-cytotoxic co-factor. Cytotoxic co-factors induce cell death, while non-cytotoxic co-factors alter a selected characteristic or activity of a cell, but do not kill the cell. The cytotoxic co-factors include those that are cytotoxic only after entry into the cytosol, as well as cytotoxic co-factors that are active both outside and inside of a cell.
Cytotoxic co-factors include, but are not limited to, anthrax toxin lethal factor (LF) and anthrax toxin edema factor (EF). Cytotoxic co-factors also include fusions between LF or EF, or functional portions thereof, and agents that have a lethal effect in or on a cell. As an example, FP59 is a fusion protein consisting of LF and the catalytic domain of Pseudomonas aeruginosa exotoxin A that has a cytotoxic effect when translocated into a cell via a PrAg/receptor oligomeric pore [67]. Additional fusions between LF or EF, or functional portions thereof, include fusions with one or more of diphtheria toxin A chain (DTA), Shiga toxin A chain (STA), listeriolysin O epitope (LLO), ricin toxin A chain, cytolethal distending toxin B (CdtB), doxorubicin, monomethyl auristatin F, docetaxel, and antibodies and functional fragments or mimetics thereof. See also the listing provided in Table 1 of [103]. Functional portions of LF include the N-terminal domain of LF (LFn, comprising amino acids 1-254 of the protein). In one example, a fusion between LFn and CdtB (LFn-CdtB) may be used.
Non-cytotoxic co-factors include, but are not limited to, beta-lactamase, dihydrofolate reductase (DHFR), gp120, peptide fragments, antigens and epitopes, growth factors, enzymes, and antibodies and functional fragments or mimetics thereof. Such non-cytotoxic co-factors will commonly be fused to a functional fragment of EF or LF, such as LFn.
Methods of Inducing Pore Formation
It will be apparent to the skilled artisan that the engineered PrAg proteins of the invention can be used in a number of different applications. In one aspect, the engineered PrAg proteins are used to induce formation of pores in a cell, both in vitro as well as in vivo. Alteration of the furin activation site of the native anthrax PrAg protein does not affect the ability of the engineered PrAg proteins disclosed herein to oligomerize with PrAg receptors on the cell surface and to form membrane pores.
The invention thus includes methods for inducing pore formation in a cell. The methods comprise contacting a cell with an engineered PrAg protein, as defined herein, under conditions promoting pore formation in the cell. It will be apparent that cells on which the method may be practiced are cells that express an anthrax toxin PrAg protein receptor as well as a membrane-anchored serine protease that acts on the activation site engineered into the PrAg protein. When the method is practiced in vitro, conditions promoting pore formation in a cell include culture conditions typical associated with the cell culture in vitro, such as 5% CO2, 95% relative humidity, and 37° C.
Suitable PrAg protein receptors include, but are not limited to, tumor endothelial marker-8 (TEM8) and capillary morphogenesis gene-2 (CMG2).
Suitable membrane-anchored serine proteases include, but are not limited to, one or more of testisin, hepsin, and matriptase.
Methods of Inducing Translocation of Selected Factors
In another aspect, the engineered PrAg proteins are used to induce translocation of co-factors into a cell. The engineered PrAg proteins disclosed herein retain the ability to oligomerize with PrAg receptors on the cell surface and to form membrane pores. They also retain the ability to induce translocation of selected co-factors into the cytosol of the cell.
The invention thus includes methods for inducing translocation of a selected co-factor into a cell, comprising contacting a cell with an engineered PrAg protein, as defined herein, under conditions promoting pore formation in the cell, and then contacting the cell with a selected co-factor under conditions promoting translocation of the selected co-factor into the cell. These methods can be practiced on cells in vitro and in vivo. It will be apparent that cells on which the method may be practiced are cells that express an anthrax toxin PrAg protein receptor as well as a membrane-anchored serine protease that acts on the activation site engineered into the PrAg protein. When the method is practiced in vitro, conditions promoting pore formation in a cell include culture conditions typical associated with the cell culture in vitro, such as 5% CO2, 95% relative humidity, and 37° C.
Suitable PrAg protein receptors include, but are not limited to, tumor endothelial marker-8 (TEM8) and capillary morphogenesis gene-2 (CMG2).
Suitable membrane-anchored serine proteases include, but are not limited to, one or more of testisin, hepsin, and matriptase.
The selected co-factor is only limited in that it can enter a cell through PrAg-induced cellular pores and have a desired effect therein. The selected co-factor may be, but is not limited to, a diagnostic co-factor or a therapeutic co-factor. Exemplary diagnostic co-factors include, but are not limited to, imaging agents and markers. The therapeutic co-factor may be a cytotoxic co-factor or a non-cytotoxic co-factor. Exemplary cytotoxic co-factors include, but are not limited to, one or more of LF, EF, FP59, and LFn-CdtB. Exemplary non-cytotoxic co-factors include, but are not limited to, one or more of peptide fragments, antigens and epitopes, growth factors, enzymes, and antibodies and functional fragments or mimetics thereof.
Methods of Treatment
In a further aspect, the engineered PrAg proteins are used in therapeutic applications, e.g., methods of medical treatment of a subject. Because tumor cells have been shown to overexpress certain membrane-anchored serine proteases, the engineered PrAg proteins of the invention are especially suitable for methods of treating diseases such as cancer in a subject. However, it should be apparent that the engineered PrAg proteins may also be used in methods of treating other disease and conditions.
The methods of treatment encompassed by the invention include methods where the engineered PrAg proteins alone are administered to a subject. The methods of treatment encompassed by the invention also include methods where the engineered PrAg proteins and a one or more selected co-factors are administered to a subject. When also administered to the subject, the identity of the co-factors will depend on the particular disease or condition to be treated.
Methods of treatment encompassed by the invention include those that comprise administering a therapeutically effective amount of an engineered PrAg protein, as defined herein, to a subject in need thereof, such as a subject having a disease or condition, including, but not limited to, cancer. Methods of treatment encompassed by the invention also include those that comprise administering a therapeutically effective amount of an engineered PrAg protein, as defined herein, and a therapeutically effective amount of therapeutic co-factor, as defined herein, to a subject in need thereof.
In a specific aspect, the invention includes methods of treating cancer in a subject comprising administering a therapeutically effective amount of an engineered PrAg protein, as defined herein, to a subject in need thereof. The invention also includes methods of treating cancer in a subject comprising administering a therapeutically effective amount of an engineered PrAg protein, as defined herein, and a therapeutically effective amount of therapeutic co-factor, as defined herein, to a subject in need thereof.
In the methods of treatment disclosed herein, the engineered PrAg protein may be in a pharmaceutical formulation. The therapeutic co-factor may also be in a pharmaceutical formulation. In some aspects, the engineered PrAg protein and the therapeutic co-factor are in the same pharmaceutical formulation.
In another selected aspect, the invention includes methods of treating cancer in a subject comprising (a) administering a first pharmaceutical formulation to a subject in need thereof wherein the first pharmaceutical formulation comprises a therapeutically effective amount of an engineered PrAg protein, as defined herein, and (b) administering a second pharmaceutical formulation to the subject wherein the second pharmaceutical formulation comprises a therapeutically effective amount of a therapeutic co-factor, as defined herein, thereby treating cancer in the subject.
In a further selected aspect, the invention includes methods of treating cancer in a subject comprising administering a pharmaceutical formulation to a subject in need thereof comprising a therapeutically effective amount of an engineered PrAg protein, as defined herein, and a therapeutically effective amount of a therapeutic co-factor, as defined herein, thereby treating cancer in the subject.
The order in which the engineered PrAg proteins and therapeutic co-factors are administered to a subject when the methods of the invention are practiced may vary. Thus, a portion or all of the engineered PrAg protein may be administered to the subject before administration of the therapeutic co-factor begins. Similarly, a portion or all of the therapeutic co-factor may be administered to the subject before administration of the engineered PrAg protein begins. Alternatively, the engineered PrAg proteins and therapeutic co-factors may be co-administered, such as when administered in the same pharmaceutical formulation.
It will be apparent that the cancer on which the methods may be practiced will comprise cells that express an anthrax toxin PrAg protein receptor as well as a membrane-anchored serine protease that acts on the activation site engineered into the PrAg protein.
PrAg protein receptors expressed by the cancer cells include, but are not limited to, tumor endothelial marker-8 (TEM8) and capillary morphogenesis gene-2 (CMG2).
Suitable membrane-anchored serine proteases expressed by the cancer cells include, but are not limited to, one or more of testisin, hepsin, and matriptase.
The methods of treatment provided herein can be used to treat a variety of diseases and conditions, limited only in that cells associated with the disease or condition, such as tumor cells of a cancer, express an anthrax toxin PrAg protein receptor as well as a membrane-anchored serine protease that acts on the activation site engineered into the PrAg protein. Exemplary diseases and conditions include, but are not limited to, cancer and tumors. Cancers that may be treated using the methods of the invention potential include all cancers, including all solid tumors, as well as hematological tumors, such as leukemia. In one aspect, the cancers that may be treated using the methods of the invention include, but are not limited to, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, and lung cancer. The cancer may be a benign cancer or a metastatic cancer. The cancer may be one that is resistant to other treatments, such as a cancer resistant to radiotherapy or chemotherapy.
The therapeutic co-factor that may be used in these methods is limited only in that it can enter a cell through PrAg-induced cellular pores and have a therapeutic effect on the cell. The therapeutic co-factor may be a cytotoxic co-factor or a non-cytotoxic co-factor. Exemplary cytotoxic co-factors include, but are not limited to, one or more of LF, EF, FP59, and LFn-CdtB. Exemplary non-cytotoxic co-factors include, but are not limited to, one or more of peptide fragments, antigens and epitopes, growth factors, enzymes, and antibodies and functional fragments or mimetics thereof.
Polynucleotide, Expression Vectors, Host Cells and Method of Making
The present invention also includes polynucleotide sequences encoding each of the engineered PrAg proteins defined herein, as well as complementary strands thereof. These polynucleotide sequences include those encoding engineered PrAg proteins having the amino acid sequence set forth in SEQ ID NO:1 where the furin activation site consisting of amino acids 193-200 is replaced by a membrane-anchored serine protease activation site selected from the group consisting of FTFRSARL (PCIS; SEQ ID NO:28), IPSRIVGG (TAS; SEQ ID NO:4), PQARITGG (PAS; SEQ ID NO:5), and PRFRITGG (UAS; SEQ ID NO:6), and sequence variants thereof having about 90% or more sequence identity over their entire length.
These polynucleotide sequences also include those encoding engineered PrAg proteins having the amino acid sequence set forth in SEQ ID NO:3 where the furin activation site consisting of amino acids 164-171 is replaced by a membrane-anchored serine protease activation site selected from the group consisting of FTFRSARL (PCIS; SEQ ID NO:28), IPSRIVGG (TAS; SEQ ID NO:4), PQARITGG (PAS; SEQ ID NO:5), and PRFRITGG (UAS; SEQ ID NO:6), and sequence variants thereof having about 90% or more sequence identity over their entire length.
Specific polynucleotide sequences encompassed within the scope of the invention include the polynucleotide sequences set forth in SEQ ID NO:55 (PrAg-PCIS), SEQ ID NO:49 (PrAg-TAS), SEQ ID NO:51 (PrAg-PAS), and SEQ ID NO:53 (PrAg-UAS).
The skilled artisan will understand that due to the redundancy of the genetic code, there are a large number of different polynucleotide sequences that may encode the engineered PrAg proteins of the invention. The invention therefore also encompasses sequence variants of the polynucleotides defined herein. These sequence variants include those having about 90% or more sequence identity over their entire length, as well as those having about 91% or more sequence identity, about 92% or more sequence identity, about 93% or more sequence identity, about 94% or more sequence identity, about 95% or more sequence identity, about 96% or more sequence identity, about 97% or more sequence identity, about 98% or more sequence identity, or about 99% or more sequence identity over their entire length.
The invention also includes cloning and expression vectors comprising the polynucleotide sequences defined herein, as well as host cells comprising the cloning and expression vectors. Suitable expression vectors include, e.g., E. coli Bacillus expression plasmids pYS5 or pYS5-PA33. Suitable host cells include, e.g., B. anthracis strains, attenuated B. anthracis strains, B. anthracis strain BH460.
The invention further includes methods of producing the engineered PrAg proteins defined herein, comprising culturing the host cells under conditions promoting expression of the engineered PrAg proteins encoded by the expression vectors, and recovering the engineered PrAg proteins from the cell cultures.
Pharmaceutical Formulations
While the engineered PrAg proteins may be administered directly to a subject, the methods of the present invention are preferably based on the administration of a pharmaceutical formulation comprising one or more engineered PrAg proteins and a pharmaceutically acceptable carrier or diluent. Thus, the invention includes pharmaceutical formulations comprising one or more of the engineered PrAg proteins defined herein and a pharmaceutically acceptable carrier or diluent.
Pharmaceutically acceptable carriers and diluents are commonly known and will vary depending on the particular engineered PrAg protein being administered and the mode of administration. Examples of suitable carriers and diluents include saline, buffered saline, dextrose, water-for-injection, glycerol, ethanol, and combinations thereof, propylene glycol, polysorbate 80 (Tween-80™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g. Cremophor EL), poloxamer 407 and 188, a cyclodextrin or a cyclodextrin derivative (including HPCD ((2-hydroxypropyl)-cyclodextrin) and (2-hydroxyethyl)-cyclodextrin), hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes, other stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents. The formulations comprising engineered PrAg proteins will typically have been prepared using engineered PrAg proteins from cultures prepared in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).
The pharmaceutical formulations of the present invention may be formulated, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of drug formulations can be used to effect such administration.
Parenteral formulations using hydrophobic carriers include, for example, fat emulsions and formulations containing lipids, lipospheres, vesicles, particles and liposomes. Fat emulsions include in addition to the above-mentioned excipients, a lipid and an aqueous phase, and additives such as emulsifiers (e.g. phospholipids, poloxamers, polysorbates, and polyoxyethylene castor oil), and osmotic agents (e.g. sodium chloride, glycerol, sorbitol, xylitol and glucose). Liposomes include natural or derived phospholipids and optionally stabilizing agents such as cholesterol.
Depending on the means of administration, the dosage may be administered all at once, such as with an oral formulation in a capsule or liquid, or slowly over a period of time, such as with an intramuscular or intravenous administration.
The amount of engineered PrAg proteins, alone or in a pharmaceutical formulation, administered to a subject is an amount effective for the treatment of a disease or condition, such as cancer. Thus, therapeutically effective amounts of the engineered PrAg proteins are administered to subjects when the methods of the present invention are practiced. In general, between about 0.1 ug/kg and about 1000 mg/kg of the engineered PrAg protein per body weight of the subject is administered. Suitable ranges also include between about 50 ug/kg and about 500 mg/kg, and between about 10 ug/kg and about 100 mg/kg. However, the amount of engineered PrAg protein administered to a subject will vary between wide limits, depending upon the location, source, extent and severity of the disease, the age and condition of the subject to be treated, etc. A physician will ultimately determine appropriate dosages to be used.
The amount of the selected compound or co-factor administered in conjunction with the engineered PrAg proteins, alone or in a pharmaceutical formulation, is also an amount effective for the treatment of a disease or condition, such as cancer, in the subject. Thus, therapeutically effective amounts of the selected compound or co-factor are administered to subjects when the methods of the present invention are practiced. While the amount of the selected compound or co-factor administered to a subject will vary widely depending on the identity of the selected compound or co-factor, as well as the disease or condition being treated, in general, between about 0.001 ug/kg and about 1000 mg/kg of the selected compound or co-factor per body weight of the subject is administered. Suitable ranges also include between about 50 ug/kg and about 500 mg/kg, and between about 10 ug/kg and about 100 mg/kg.
Administration frequencies of the engineered PrAg proteins and pharmaceutical formulations comprising the engineered PrAg proteins will vary depending on factors that include the location of the disease, the identity of the disease, the severity of the disease, and the mode of administration, among other factors. As non-limiting examples, each formulation may be independently administered 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly and bi-monthly. The concentration of the protein in the formulation may vary or be the same in each formulation.
The duration of treatment will depend on relevant factors concerning the disease and will be best determined by the attending physician. However, continuation of treatment is contemplated to last for a number of days, weeks, or months.
In each embodiment and aspect of the invention, the subject is a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
The invention also provides a kit comprising one or more containers filled with one or more engineered PrAg proteins or pharmaceutical formulations comprising one or more engineered PrAg proteins. The kit may also comprise one or more containers filled with one or more co-factors or pharmaceutical formulations comprising one or more co-factors. The kit may further include instructions for use. Associated with the kit may further be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
The membrane-anchored serine protease testisin (PRSS21) is synthesized with a 17-amino acid carboxy-terminal hydrophobic extension that is post-transcriptionally modified with a glycosyl-phosphatidylinositol (GPI) linkage that serves to anchor the protease to the extracellular side of the plasma membrane [49-52]. Testisin has remarkably specific tissue distribution, being constitutively expressed in abundance only in spermatocytes, where it has a specific role in male fertility [53-55]. Yet, testisin possesses the characteristics of a Cancer/Testis Antigen (CTA), a group of proteins whose expression is normally restricted to testis, but which are frequently aberrantly activated in tumors [56,57].
Testisin is strongly overexpressed in human invasive epithelial ovarian cancers, as well as cervical cancers, while being undetectable in normal ovarian or cervical tissues. In an RT-PCR study of ovarian tumors, Shigemasa et al. [58] reported that testisin was present in 80-90% of stage 2 or 3 disease. Bignotti et al. [59] also found testisin expressed in primary and metastatic ovarian tumors. Overexpression of testisin in ovarian tumor cells resulted in increased colony formation in soft agar and increased xenograft tumor growth in severe combined immunodeficient (SCID) mice [60]. Its increased expression has also been found to enhance matrigel invasion of cervical cancer cells [61]. Conversely, reduction of endogenous testisin expression via siRNA-mediated knockdown in ovarian and cervical tumor cell lines led to reduced colony formation, reduced invasion in cell culture, and reduced cellular resistance to the chemotherapy drug adriamycin [60,61]. The selective expression of testisin by human tumors relative to its normally restricted expression in testis, combined with the relationship of testisin expression to tumorigenic processes, suggested that testisin is an attractive target for anti-tumor therapeutic approaches.
In light of these studies, an engineered PrAg protein comprising a testisin cleavage site in place of the furin cleavage site was produced. This engineered PrAg protein is termed PrAg-PCIS. In particular, eight amino acids flanking the native furin cleavage site within PrAg (RKKRSTSA; SEQ ID NO:56) were replaced with a sequence that can be cleaved by testisin. The amino acid of PrAg-PCIS is provided in SEQ ID NO:54 and the nucleic acid sequence encoding the protein is provided in SEQ ID NO:55.
As shown in below, alteration of the cleavage site abrogated furin activation and resulted in a potent anti-tumor prodrug. The engineered PrAg-PCIS protein is a testisin substrate that is cleaved and activated by testisin in vitro and in cell culture, and it has potent anti-tumor cell activity when combined with a recombinant LF-Pseudomonas exotoxin based payload (FP59). Moreover, in vivo administration of the toxin inhibited growth of established xenograft tumors in mice by inducing tumor necrosis and reducing tumor cell proliferation.
Materials and Methods
Reagents
Enzymes for recombinant DNA preparation were purchased from New England BioLabs. Recombinant mouse testisin (6820-SE-10), human hepsin (4776-SE-10), human prostasin (4599-SE), and HAT (2695-SE) were purchased from R&D Systems. Each protease was activated according to the manufacturer's instructions. Recombinant human thrombin (470HT) and recombinant human uPA (ADG125N) were purchased from American Diagnostica. Recombinant human PCI and mouse anti-PCI antibody were prepared as previously described [80,81]. Briefly, recombinant PCI was prepared in Escherichia coli and purified using Ni2+-chelate and heparin-sepharose affinity chromatography, as in [81]. Recombinant human furin was provided by Dr. Iris Lindberg (University of Maryland School of Medicine, Baltimore, Md.) [82]. Recombinant human matriptase was provided by Dr. Richard Leduc (Universite de Sherbrooke, Quebec, Canada) [83]. Human aPC was provided by Dr. Li Zhang (University of Maryland Baltimore School of Medicine, Baltimore, Md.) [84]. Aprotinin (A1153) was purchased from Sigma-Aldrich. Rabbit anti-PrAg antibody (no. 5308) was prepared as previously described [16]. Additional antibodies included goat anti-HAI-1 (AF1048) and goat anti-HAI-2 (AF1106) (R&D Systems), rabbit anti-matriptase (IM1014) (Calbiochem), mouse anti-prostasin (612172) (BD Transduction Laboratories), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (14C10) (Cell Signaling Technologies), rabbit anti-hepsin (100022) (Cayman Chemical); anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch Laboratories), and anti-goat HRP-conjugated antibody (KPL). Mouse Pro1.4.C25.1 anti-testisin antibody was produced by standard procedures from a hybridoma cell line (PTA-6076) (ATCC).
Real-Time Quantitative PCR (qPCR)
RNA was isolated from cell lines using the RNeasy Kit (Qiagen). Reverse transcription was performed using Taqman Reverse Transcription Reagents (Applied Biosystems). qPCR was performed using testisin (Hs00199035_m1), hepsin (Hs01056332_m1), matriptase (Hs00222707_m1), GAPDH (Hs02758991_g1) and beta-actin (β-actin) (Hs99999903) primers and Taqman RT-PCR reagents (Applied Biosystems). mRNA expression levels were normalized to GAPDH or β-actin.
Cell Lysis and Immunoblotting
Cells were lysed in cell lysis buffer (150 mM NaCl, 10 mM CaCl2), 50 mM HEPES (pH 7.3), 0.5% Triton X-100, 0.5% NP-40, Complete Mini-EDTA Protease Inhibitor Cocktail (Roche)), and protein concentrations determined by Bradford assay. Samples containing equal protein were heated at 95° C. for 5 minutes in Laemmli sample buffer containing 10% beta-mercaptoethanol and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), using 4-12% or 10% NuPage Bis-Tris pre-cast gels (Life Technologies), followed by immunoblotting using PVDF membranes (Life Technologies). Membranes were blocked for 30 minutes in 5% (w/v) non-fat milk and then sequentially incubated with primary and HRP-conjugated secondary antibodies. HRP activity was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
Plasmids and Mutagenesis
A two-step overlap PCR strategy was employed to mutate the cDNA bases encoding the furin cleavage site in the PrAg expression plasmid pYS5-PA33 [85]. pYS5-PA33 (SEQ ID NO:67) served as the template for the first round of PCR using the primers denoted ‘A’ (below). The resulting PCR reaction was digested with DpnI and the mutant plasmid cloned by standard techniques and used as the template for the second round of PCR using primers denoted ‘B’ (below). The resulting PCR reaction was digested with DpnI and the final mutant plasmid cloned and verified by DNA sequencing.
Expression plasmids encoding human matriptase [86], human HAI-1 [86], and human HAI-2 [87] were provided by Dr. Chen-Yong Lin (Georgetown University, Washington D.C.). cDNA encoding human testisin (GPI-testisin) [51], cloned into pcDNA3.1 expression plasmid (Life Technologies), was mutated by site-directed mutagenesis using the primers denoted below using the QuikChange Mutagenesis kit (Stratagene) to create ‘zymogen-locked’ activation site (R41A-testisin) and catalytic triad (S238A-testisin) mutants of testisin. Similarly, cDNA encoding human hepsin (WT-hepsin) [88], cloned into pcDNA 3.1, was mutated to create a catalytic triad S353A-hepsin mutant (S353A-hepsin). Cloning and mutagenesis accuracy was verified by DNA sequencing.
Expression and Purification of PrAg Proteins
Recombinant anthrax toxin protective antigens (PrAg-WT, PrAg-PCIS), recombinant LF, and FP59 were generated and purified as previously described [16,66]. Briefly, expression plasmids containing PrAg sequences contained in the E. coli Bacillus expression plasmids pYS5 or pYS5-PA33, were transformed into the non-virulent B. anthracis strain BH460. The proteins were secreted into the culture supernatants and purified by ammonium sulfate precipitation and chromatography on a Mono-Q column to high yield and purity, as described [66]. The LF and FP59 used herein have the native N-terminal sequence of AGG [89].
PCI Cleavage Assay
Recombinant hepsin or matriptase (50 nM) were incubated with 50 nM recombinant PCI. Recombinant testisin (50 nM) was incubated with 500 nM recombinant PCI. After 30 minutes of incubation at room temperature, in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 10 mM CaCl2), or at indicated intervals, Laemmli sample buffer containing 10% beta-mercaptoethanol was added to the reactions. Samples were immunoblotted for PCI cleavage or protease-PCI complex formation using anti-PCI, anti-hepsin, or anti-matriptase antibodies.
Peptide Assays
Peptide cleavage assays were performed using 10 nM recombinant testisin, 10 nM recombinant hepsin, or 100 nM recombinant matriptase, and 100 nM chromogenic succinyl-AAPR-p-nitroaniline peptide (Bachem). Reaction absorbance (abs) values were measured at 420 nm using a spectrophotometer (TECAN) at times indicated in the figure legend. The change in absorbance units is relative to the absorbance measured in the absence of peptide substrate. The absorbance of peptide substrate alone did not increase in the absence of protease over time.
In Vitro PrAg Cleavage Assays
Recombinant PrAg proteins (1 μM) were incubated with recombinant proteases (50 nM) for 2.5 hours, or indicated intervals, at 30° C., in 50 mM HEPES (pH 7.3), 10 mM CaCl2), 150 mM NaCl, and 0.05% (v/v) Brij-35. Reactions were stopped by addition of Laemmli sample buffer containing 10% beta-mercaptoethanol to the samples. PrAg cleavage was analyzed by SDS-PAGE followed by immunoblotting using anti-PrAg antibody.
For densitometry of PrAg processing, all values were measured using Image J software and normalized to GAPDH expression. Individual PrAg 83-kDa and 63-kDa values for each timepoint are calculated relative to the sum value of PrAg 83-kDa and 63-kDa at that timepoint, which was set equal to 1.
Cell Culture and Transfections
Human cell lines were purchased from American Type Culture Collection (ATCC), with the exception of NCI/ADR-Res cells, which were purchased from the NCI-DCTD repository (Frederick, Md.). Cell lines were cultured and maintained at 37° C. in a 5% CO2/95% air environment in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. All cells were routinely tested and confirmed to be free of mycoplasma contamination. HEK293T cells were transfected with expression plasmids encoding full-length human GPI-anchored testisin (HEK/GPI-testisin), S238A-testisin catalytic triad mutant (HEK/S238A-testisin), R41A-testisin ‘zymogen-locked’ activation site mutant (HEK/R41A-testisin), or vector alone (HEK/vector) using Lipofectamine 2000 (Life Technologies). Two stably-transfected pools of each transfection were obtained by selection in hygromycin and testisin/mutant expression determined by immunoblot (data not shown). HeLa cells were transiently transfected or co-transfected with expression plasmids encoding matriptase (WT-matriptase), prostasin [90], hepsin (WT-hepsin), S353A-hepsin catalytic triad mutant (S353A-hepsin), HAI-1, HAI-2, or vector alone (vector) using Lipofectamine 2000.
Knockdown by RNA Interference
HeLa cells were transfected with 20 nM testisin-specific STEALTH siRNAs (HSS116894; HSS173992) (Life Technologies) or 20 nM luciferase-specific negative control (Luc-siRNA) (Life Technologies) using Dharmafect 1 (Dharmacon). After 48 hours, cells were harvested for analysis of testisin mRNA and protein expression, or used in MTT cytotoxicity assays. The efficiency of testisin knockdown was analyzed by qPCR and immunoblotting.
MTT Cytotoxicity Assays
Cells were incubated with various concentrations of PrAg-PCIS or PrAg-WT (as indicated in figure legends) and FP59 (50 ng/mL) in growth media for indicated times. Media was replaced with fresh media and cell viability was assayed from 24-48 hours later (as indicated in the figure legends) by adding MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Millipore) to a final concentration of 1.25 mg/mL, and incubating for 45 minutes to one hour at 37° C. MTT was dissolved in growth media and filtered through a 0.22 μm syringe filter. The formed pigment was solubilized with 0.5% (w/v) SDS, 25 mM HCl, in 90% (v/v) isopropanol. Absorbance was measured using a spectrophotometer (TECAN) at 550 nm and 620 nm (reference wavelength). Values obtained for incubation of cells with PrAg toxins were normalized to those obtained for the cells incubated with FP59 alone (100%). EC50 is defined as the concentration (derived from the viability plots) of PrAg toxin required to kill 50% of the cells.
In Vivo Tumor Xenograft Models
Female athymic nude mice (NU/NU) (6-8 wks old) (Charles River) were housed and monitored according to Institutional Animal Care and Use Committee guidelines, given free access to food and water, and maintained in a 12 hour dark/light environment. 2.5×106 HeLa tumor cells were injected subcutaneously into the right hind flanks of the mice. Upon measurable tumor growth (˜50-200 mm3), mice were distributed into cohorts containing mice bearing approximately equal individual tumor volumes and approximately equal average tumor volumes. Each mouse received a 100 μL intratumoral injection, injected into multiple spots in the tumor, every three days for a total of three injections. Tumor dimensions were measured with calipers at indicated timepoints in a blinded manner with respect to tumor treatment. Tumor volume was calculated using the formula 0.5×length×width2. Experiments were concluded when one or more mice reached predetermined endpoints (weight gain>10%, tumor diameter>1 cm, tumor ulceration). Mice were then euthanized and tumors were removed, weighed (in a treatment-blinded manner), fixed in 10% zinc buffer, and stored in 70% ethanol for histology and immunohistochemical analysis.
Histopathological Analysis
Zinc-fixed tumor specimens were embedded in paraffin and 5 μm-thick sections were cut, deparafinized, and stained with hematoxylin and eosin (H&E) using standard procedures, or subjected to immunohistochemical analysis. For immunohistochemistry, samples were rehydrated, endogenous peroxidase activity blocked with 3% hydrogen peroxide in methanol, subjected to antigen retrieval in boiling sodium citrate, and then non-specific binding sites blocked with 5% goat serum. Sections were incubated overnight at 4° C. with 1:100 dilutions of rabbit anti-Ki67 (ab16667) (Abeam), rabbit anti-human activated caspase-3 (9661S) (Cell Signaling Technology), or rat anti-mouse CD31 (553370) (BD Pharmingen), followed by incubation for 30 minutes with 1:200 anti-rat or anti-rabbit biotinylated secondary antibodies. Antibody binding was detected using a Vectastain ABC Kit (Vector Laboratories). Sections were counterstained with hematoxylin, dehydrated, and mounted. Control slides were incubated with primary or secondary antibodies only. Images were obtained using an EVOS FL Auto Cell Imaging System (Life Technologies). Composite images of the whole tumor sections were obtained with a 10× objective and stitched together using the EVOS software, while individual fields were taken using 20× (H&E, Ki67, activated caspase-3) or 40× (CD31) objectives, respectively. Staining was quantified using Image J software (H&E, Ki67, activated caspase-3) and Photoshop (Adobe) (CD31). Quantification, performed in a treatment-blinded manner, was performed by outlining the tumors in the composite images and analyzing the tumor sections for % viable area (H&E) or % positive staining for the immunostained sections. Percentages were calculated using the ratio of viable area or stained area of the tumor to the total tumor area (areas determined by pixel count), as described in [91].
Statistical Analysis
Quantitative data are represented as mean values with their respective standard errors (SEM). Significance (relative to vector or vehicle control groups) was tested using unpaired two-tailed Student's t test, which was calculated using GraphPad software. p values<0.05 were considered statistically significant.
Results
Engineering the Mutant PrAg-PCIS Protein
The eight amino acid sequence 164RKKRSTSA (SEQ ID NO:56), containing the furin cleavage site (furin cleaves the peptide bond between R-S) in the mature wild-type PrAg protein (PrAg-WT; SEQ ID NO:3), was replaced with the sequence 164FTFRSARL (SEQ ID NO:28) to create PrAg-PCIS using an overlap PCR strategy. This new substrate sequence was derived from a region of protein C inhibitor (PCI, SERPINA5), within the reactive center loop and close to the C-terminus, and is known to be cleaved by testisin [62], as confirmed (see
PrAg-PCIS Toxin is Cytotoxic to a Broad Range of Human Tumor Cells
The combination of PrAg and FP59, a fusion protein consisting of the PrAg binding domain of LF and the catalytic domain of Pseudomonas aeruginosa exotoxin A, has been shown to efficiently kill tumor cells following PrAg activation [67]. When translocated into the cytosol by activated PrAg, FP59 induces cytotoxicity by ADP-ribosylation and inhibition of translation elongation factor-2, resulting in inhibition of protein synthesis and the induction of cell death [67-69]. FP59 does not induce cytotoxicity alone, but must be delivered into cells via an activated PrAg protein to induce cell death. To compare the abilities of PrAg-PCIS and PrAg-WT to be activated by tumor cells and to deliver FP59, cytotoxicity assays were performed on a range of human tumor cell lines after treatment with FP59 in combination with PrAg-PCIS (PrAg-PCIS toxin) or PrAg-WT (PrAg-WT toxin). All tumor cell lines showed a dose-dependent sensitivity to the PrAg-PCIS toxin. In 7 of the 9 tumor lines (NCI/ADR-Res, SKOV3, ES-2, OVCAR3, LnCAP, DU-145, and PC3), the PrAg-PCIS toxin showed potent killing effects at doses similar to the PrAg-WT toxin (
Protease Selectivity of PrAg-PCIS
Many pericellular proteases, including the membrane-anchored serine proteases, have preferred recognition sequences for substrate cleavage. Yet, there exists promiscuity in sequence recognition and cleavage, particularly with regard to the amino acids adjacent to the cleavage site. Incubation of PrAg-PCIS with the recombinant catalytic domains of several membrane-anchored serine proteases and other potentially reactive pericellular serine proteases resulted in activation cleavage of PrAg-PCIS from the 83-kDa to the 63-kDa form by the membrane-anchored serine proteases testisin, hepsin (HPN), matriptase (ST14), and to a lesser extent human airway trypsin-like protease (HAT, TMPRSS11D) (
The observation that PrAg-PCIS was susceptible to cleavage by hepsin and matriptase suggested that in addition to native PCI being a substrate of testisin, PCI might be a substrate of these proteases. PCI is a member of the serpin family, whose structure and inhibitory mechanism has been well-characterized [70,71]. Cleavage of the serpin reactive center loop (RCL) can result in the formation of a protease-inhibitory complex, consisting of PCI covalently bound to the serine protease or production of lower molecular weight cleaved forms of PCI [70,71]. Incubation of hepsin and matriptase recombinant catalytic domains with PCI resulted in the appearance of cleaved forms of PCI, as well as higher molecular weight complexes representing SDS-resistant serpin-serine protease inhibitory complexes (
Processing of PrAg-PCIS by Cell-Expressed GPI-Anchored Testisin
Following activation cleavage on the cell surface, the cleaved PrAg forms an oligomer which is internalized by the cell. To confirm that testisin anchored on a tumor cell surface can process PrAg-PCIS to an activated form, HEK293T cells stably expressing full-length human testisin (HEK/GPI-testisin) or vector alone (HEK/vector) were exposed to PrAg-PCIS or PrAg-WT for various times up to 6 hours and assayed for the appearance of the 63-kDa activation product. The processing of PrAg-PCIS to the 63-kDa form was detectable in HEK/GPI-testisin cells within 30 minutes and these levels increased with time (
PrAg-PCIS Toxin is Cytotoxic to Cells Expressing Active GPI-Anchored Testisin
To investigate potential tumor cell killing resulting from testisin activation of PrAg-PCIS, cytotoxicity assays were performed using HEK/GPI-testisin and HEK/vector cells. HEK/GPI-testisin cells showed a dose-dependent sensitivity to killing by PrAg-PCIS toxin (i.e., PrAg-PCIS and FP59) (
Tumor Cells Expressing Endogenous Testisin are Killed by the PrAg-PCIS Toxin
To investigate the activation of PrAg-PCIS toxin (i.e., PrAg-PCIS and FP59) by endogenous testisin in a natural tumor cell system, HeLa cervical cancer cells, which constitutively express testisin [60,74], were treated with the PrAg-PCIS and FP59. Increasing concentrations of PrAg-PCIS resulted in substantial HeLa cell death that was dose-dependent, although HeLa cells were less sensitive to the PrAg-PCIS toxin than to the PrAg-WT toxin (
PrAg-PCIS Toxin is Cytotoxic to Tumor Cells Expressing Active Hepsin, but not Matriptase
The activation cleavage of PrAg-PCIS by both recombinant matriptase and hepsin in vitro suggested that the full-length forms of these membrane-tethered enzymes could be additional activators of PrAg-PCIS. To test the role of cell-expressed hepsin in activating PrAg-PCIS, HeLa cells were transfected with expression plasmids encoding full-length hepsin or an inactive S353A-hepsin catalytic mutant (
To test the role of cell-expressed matriptase in activating PrAg-PCIS, full-length matriptase was expressed in HeLa cells. Efficient matriptase expression required co-expression with hepatocyte growth factor activator inhibitor-1 (HAI-1, SPINT1) and prostasin (PRSS8), to enhance matriptase trafficking to the cell surface [75,76] and increase matriptase zymogen activation [77,78] (
PrAg-PCIS Toxin Inhibits Tumor Growth in a Preclinical Xenograft Mouse Model
The ability of the PrAg-PCIS toxin to inhibit tumor growth in vivo was examined using a xenograft mouse model. Athymic female nude mice bearing subcutaneous HeLa tumors received three intratumoral injections (one every three days) of PrAg-PCIS toxin (10 μg PrAg-PCIS and 5 μg LF) or vehicle alone (PBS), and tumor growth was assessed by caliper measurements. LF was used in vivo in place of FP59 to avoid any off-target effects that may be associated with non-specific uptake of the very effective protein translation inhibitor FP59 [27]. After the first injection of PrAg-PCIS toxin, tumor growth arrested and did not increase compared with vehicle treated tumors, over the course of the experiment (
The dose-dependence of tumor growth inhibition by PrAg-PCIS toxin was also investigated using this xenograft model. Cohorts of mice bearing subcutaneous HeLa tumors received three injections (one every three days) composed of 10 μg, 5 μg, 1 μg PrAg-PCIS toxin, or vehicle (5 μg LF in PBS). Tumor growth as assessed by caliper measurements again showed tumor growth arrest in all 3 cohorts treated with PrAg-PCIS toxin compared with vehicle alone treated animals over the course of the experiment (
Quantitative histomorphometric analyses were performed on serial sections of the harvested tumors to investigate the mechanistic basis for the potent anti-tumor activity of the PrAg-PCIS toxin. Microscopic analysis of sections stained with hematoxylin/eosin (H&E) showed that tumors exposed to either 10 μg PrAg-PCIS toxin or 5 μg PrAg-PCIS toxin presented with substantial areas of necrosis, as indicated by reduced staining of the tissue and the presence of patches of destroyed tumor with loss of nuclei (
The methods, reagents and techniques described in detail in Example 1 above where use to generate three additional engineered PrAg proteins. The engineered PrAg-TAS protein had the native furin activation site replaced by the testisin zymogen activation site (IPSRIVGG; SEQ ID NO:4). The amino acid of PrAg-TAS is provided in SEQ ID NO:48 and the nucleic acid sequence encoding the protein is provided in SEQ ID NO:49. The engineered PrAg-PAS protein had the native furin activation site replaced by the prostration zymogen activation site (PQARITGG; SEQ ID NO:5). The amino acid of PrAg-PAS is provided in SEQ ID NO:50 and the nucleic acid sequence encoding the protein is provided in SEQ ID NO:51. The engineered PrAg-UAS protein had the native furin activation site replaced by a modified uPA zymogen activation site (PRFRITGG; SEQ ID NO:6). The amino acid of PrAg-UAS is provided in SEQ ID NO:52 and the nucleic acid sequence encoding the protein is provided in SEQ ID NO:53. Details regarding these three proteins, along with the PrAG-PCIS protein and the wild-type anthrax PrAg protein are provided in Table 3. The peptide bond that is cleaved within each of the sequences follows the arginine residue in the P1 position and is designated by a dash and the vertical arrow.
A. Establishing Orthotopic Xenograft Models of Metastatic Ovarian Cancer.
Using ovarian tumor cell lines transduced with luciferase for in vivo imaging (ES-2-luc), an i.p. orthotopic ovarian xenograft tumor model was established. Published literature indicated that 1×107 ES-2 cells injected i.p. form overwhelming tumor burden, with ascites, within two to three weeks of injection [104]. Therefore, in order to assess the in vivo i.p. growth of the ES-2-luc cells, establish an optimal cell density for cell injection, and determine whether the luciferase activity levels in the ES-2-luc cells were indeed sufficient to enable in vivo imaging, cohorts of female athymic nude mice (n=2) were injected i.p. with either 1×106, 5×106, or 1×107 ES-2-luc cells, respectively. Ovarian tumor burden was imaged using the IVIS imaging system. Mice injected with 1×107 ES-2-luc cells developed significant tumor burden in approximately 2 weeks (data not shown), as determined by IVIS imaging, and required euthanasia shortly thereafter due to tumor-induced weight gain, as well as mild cachexia and jaundice, and ascites accumulation. Mice injected with 5×106 ES-2-luc cells also developed significant tumor burden (data not shown), and similar symptoms, with slower onset, requiring euthanasia approximately a week later. One mouse injected with 1×106 ES-2-luc cells developed significant tumor burden (data not shown) after approximately 4 weeks and was euthanized due to similar symptoms, while the other mouse did not develop substantial tumor burden.
At the time of euthanizing the mice, necropsies were performed to visualize the characteristics and extent of ES-2-luc ovarian tumor growth in the peritoneal cavity. In all cases, when significant tumor burden was observed by IVIS imaging, substantial tumor burden was also observed by gross visualization. The ES-2-luc tumor cells were distributed throughout the abdominal cavity, both floating in the ascites as spheroids and attached to various organs and the body wall (data not shown). Due to the aggressive growth kinetics and tumor characteristics of the 5×106 ES-2-luc cell dose in female athymic nude mice, this cellular density was chosen as optimal for further experiments.
B. Establishment of a Well-Tolerated Dose(s) for Mutant PrAg Toxin Treatment.
To determine a well-tolerated dose to treat i.p. xenograft ovarian tumor-bearing mice, cohorts of female athymic nude mice were injected i.p. with increasing doses of PrAg-PAS toxin (PrAg proteins combined with LF). LF was used in place of FP59 to avoid any off target effects that may be associated with non-specific uptake of the very effective protein translation inhibitor FP59 [27]. PrAg-PAS toxin was chosen for these experiments because PrAg-PAS was an engineered PrAg protein that was cleaved to an activated form by testisin, hepsin, and matriptase in vitro. Moreover, PrAg-PAS toxin was able to be activated by testisin, hepsin, and matriptase to increase ovarian tumor cell cytotoxicity. Cohorts of female athymic nude mice (n=3) received six i.p. injections of PrAg-PAS toxin over the course of two weeks. Treatment with PrAg-PAS toxin was very well-tolerated. None of the mice treated with the highest dose of PrAg-PAS toxin exhibited any apparent toxicity (data not shown). Based on these results, PrAg-PAS 45/15 (45 μg PrAg-PAS combined with 15 μg LF) was identified as the maximum PrAg-PAS toxin dose for further experiments.
C. Treatment with PrAg-PAS Toxin Reduces Tumor Growth and Metastasis in an Orthotopic Xenograft Model of Metastatic Ovarian Cancer.
To determine whether treatment with PrAg-PAS toxin could inhibit ovarian xenograft tumor growth and metastasis, female athymic nude mice were injected i.p. with 5×106 ES-2-luc ovarian tumor cells. After four days, when established tumors were visible by IVIS imaging, mice were divided into four cohorts of five mice, with all mice bearing approximately equal tumor burden. Each cohort received four i.p. injections of PrAg-PAS toxin (15 μg PrAg-PAS combined with 5 μg LF), 15 μg PrAg-PAS alone, 5 μg LF alone, or vehicle alone (PBS). During the course of the experiment, tumor growth was assessed by imaging with the IVIS system (
The results showed that in the mice treated with vehicle alone, ES-2-luc tumor growth proceeded rapidly (
While tumor burden was significant and widespread in vehicle treated mice, mice treated with PrAg-PAS toxin showed significant reductions in average tumor burden over the course of the experiment (
Mice treated with PrAg-PAS toxin also showed drastically less tumor burden at the time of euthanasia and performance of necropsies (data not shown). PrAg-PAS toxin-treated mice did not develop ascites, did not present with ovarian tumor cells covering the diaphragm or the tissue surrounding the mesentery arteries, and did not present with any symptoms of cachexia or jaundice (data not shown). Moreover, PrAg-PAS toxin-treated mice did not have tumor nodules abundant on the body cavity wall, tumor nodules spread throughout the abdominal cavity, or swollen gallbladders. While mice receiving PrAg-PAS alone had reductions in tumor burden over the course of the experiment (
As observed when establishing a tolerated dose, all treatments of PrAg-PAS toxin, or the components alone, were well tolerated. Mice experienced no treatment specific weight loss, symptoms, or gross organ damage as visualized upon performing necropsies. The substantial decrease in tumor burden (by IVIS and necropsy) in mice treated with PrAg-PAS toxin indicated that PrAg-PAS toxin was very effective in reducing ovarian tumor burden and metastasis in vivo in this model. Additionally, the data indicated that the mechanism of effective ovarian tumor killing by PrAg-PAS toxin requires the co-administration of both PrAg-PAS and LF, and is not due to the action of either component in the absence of the other.
D. Treatment with PrAg-PAS Toxin Reduces Established Ovarian Tumor Burden.
To determine whether treatment with PrAg-PAS toxin could reduce established ovarian tumor burden, rather than early ovarian tumor growth (treatment beginning on day 4 after ES-2-luc tumor cell injection), female athymic nude mice were injected i.p. with 5×106 ES-2-luc ovarian tumor cells. After ten days, when significant tumor burden was present, mice were divided into three cohorts of five mice, with all mice bearing approximately equal tumor burden. Each cohort received two i.p. injections of either of two different doses of PrAg-PAS toxin (45 μg PrAg-PAS, 15 μg PrAg-PAS, and 15 μg LF, or 5 μg LF, respectively), or vehicle (PBS). During the course of the experiment, tumor growth was assessed by imaging with the IVIS system (
The results showed that in the mice treated with vehicle alone, ES-2-luc tumor growth proceeded rapidly (
Mice treated with the two different doses of PrAg-PAS toxin also showed less tumor burden at the time of euthanasia and performance of necropsies (data not shown). PrAg-PAS toxin-treated mice presented with reduced tumor burden specifically covering the tissue surrounding the mesenteric arteries. Tumor burden was also reduced on the body wall and the diaphragm. As observed when establishing a tolerated dose, all treatments of PrAg-PAS toxin, were well tolerated. The decrease in tumor burden (by IVIS and necropsy) in mice treated with the two different doses of PrAg-PAS toxin indicated that PrAg-PAS toxin was able to reduce established tumor burden, in addition to reducing early stage tumor burden.
E. Anti-Ovarian Tumor Effect of Mutant PrAg-PAS Toxin is Dependent Upon Proteolytic Activation.
To determine whether the anti-tumor mechanism of PrAg-PAS toxin requires its proteolytic activation, cohorts of female athymic nude mice bearing approximately equal ES-2-luc xenograft ovarian tumor burden received six i.p. treatments of PrAg-PAS toxin, vehicle (PBS), or an un-activatable PrAg toxin, termed PrAg-U7, in which the amino acid sequence that functions as the cleavage site mediating activation of PrAg-PAS was replaced with the amino acid sequence PGG [15]. The replacement with the PGG amino acid sequence renders PrAg-U7 unable to be proteolytically cleaved and activated and therefore unable to oligomerize and ultimately deliver proteins (LF, EF, FP59) into the cytosol to cause cell death. A cohort of mice was also treated with a mutant PrAg toxin that requires activation by both uPA and MMP2/9, termed PrAg-IC (intercomplementing toxin), which contains the same activation sequences as the PrAg-L1 and PrAg-U2 engineered toxins. PrAg-IC had not previously been tested for anti-ovarian tumor efficacy, but had been shown to be efficacious in reducing tumor burden in multiple other tumor models [20, 27]. PrAg-IC was used to assess the relative effectiveness of PrAg-PAS toxin in reducing ovarian tumor burden, and to investigate whether PrAg-PAS toxin was more efficacious in reducing ovarian tumor burden than PrAg-IC toxin.
Treatment with PrAg-PAS toxin significantly reduced the average tumor burden of the ES-2-luc tumor-bearing mice, relative to cohorts treated with vehicle (
When the mice were euthanized and necropsies were performed, mice treated with PrAg-PAS toxin or PrAg-IC toxin had substantially less tumor burden within the peritoneal cavity than did mice treated with vehicle or PrAg-U7 toxin (data not shown). PrAg-PAS toxin- and PrAg-IC toxin-treated animals possessed few, if any, tumors attached to the diaphragm, wall of the peritoneal cavity, tissue surrounding the mesentery arteries, intestinal tract, or surrounding the major organs. Vehicle- and PrAg-U7 toxin-treated mice had significant tumor accumulation and tumor attachment to these areas. Mice in the vehicle- and PrAg-U7 toxin-treated cohorts also presented with enlarged gall bladders, whereas this was not seen in mice treated with PrAg-PAS toxin or PrAg-IC toxin. As before, all toxin treatments were well tolerated. Mice displayed no treatment-specific weight loss, outward signs of toxicity, or gross organ damage due to treatment with the toxins.
These data demonstrate that proteolytic activation of the PrAg-PAS toxin is required for its anti-ovarian tumor effect, and suggest that in the absence of proteolytic activation, the mutant PrAg toxins are relatively inactive. These data also suggest that PrAg-IC toxin, not previously demonstrated to be effective at treating preclinical models of ovarian cancer, also requires proteolytic activation, and is effective at reducing i.p. ovarian tumor burden and metastasis in this mouse model.
Human Ovarian Tumor Cell Lines Possess Cell-Surface Trypsin-Like Serine Protease Activity
To investigate the expression of cell-surface serine protease activities that might be capable of activating the mutant PrAg toxins, ovarian tumor cell lines were incubated with a fluorogenic peptide that functions as a substrate for membrane-anchored serine proteases. In the presence or absence of the serine protease inhibitor AEBSF, cleavage of the peptide by each of the tumor cell lines resulted in an AEB SF-sensitive increase in fluorescent signal intensity, indicating that each of the tumor cells possessed serine proteases capable of cleaving the peptide (
PrAg-PAS Toxin Treatment Extends Survival in a Murine Xenograft Tumor Model.
Based on the results indicating that PrAg-PAS toxin could significantly reduce i.p. xenograft ovarian tumor burden, it was determined whether the PrAg-PAS toxin-mediated reductions in tumor burden could translate into an extension of mouse survival, and if so whether this activity could exhibit dose-dependence. Therefore, female athymic nude mice injected i.p. with ES-2-luc tumor cells, upon tumor development, were divided into cohorts of equal average tumor burden. The cohorts of mice then received nine i.p. injections composed of different doses of PrAg-PAS toxin (45 μg, 15 μg, or 6 μg PrAg-PAS, in combination with 15 μg, 5 μg, or 2 μg LF, respectively) or vehicle (PBS). Mice were euthanized when they exhibited substantial weight gain (>10%), were moribund, or exhibited other signs of significant malaise and/or distress due to tumor burden. An increase in body weight of >10% was chosen as the primary endpoint in the absence of health conditions caused by tumor burden because it is typical weight gain suggestive of excess tumor burden in the relevant literature.
Tumor-bearing mice treated with either of the two highest doses of PrAg-PAS toxin (15 μg or 45 μg of PrAg-PAS, combined with 5 μg or 15 μg of LF, respectively) exhibited significant 2.04-fold and 2.06-fold increases in survival over the course of the experiment, relative to mice treated with vehicle (
Human Ovarian Tumor Cell Lines are Susceptible to Killing by the Mutant PrAg Toxins.
To test the efficacy of the toxins to kill a range of human ovarian cell lines including ADR-Res, OvCAR3, and SKOV3 which are resistant to clinically relevant concentrations of cisplatinin (typical of recurrent ovarian cancers), cell lines were treated with the mutant PrAg toxins and MTT cytotoxicity assays were performed (
Cisplatin Resistant Ovarian Tumor Cells are Killed by Engineered Anthrax Toxins.
To confirm the resistance of SKOV-3 cells to cisplatin, SKOV3-Luc ovarian tumor cells (SKOV3 cells expressing luciferase) were treated with varying doses of cisplatin (1-1000 μM) for 24 hrs and the IC50 calculated using a non-linear regression best fit model (
Treatment with PrAg-PAS Toxin Reduces NCI/ADR-Res-Luc Ovarian Tumor Burden.
To test the in vivo efficacy of the PrAg-PAS toxin to kill platinin-resistant cell line ADR-Res, cohorts of female athymic nude mice were injected with 1.5×107 NCI/ADR-Res-Luc ovarian tumor cells and the tumor allowed to grow for 29 days. This tumor is slower growing that the ES-2-luc cells. Cohorts of mice bearing tumors received 6 injections, 3 per week×2 weeks (day 1, 4, 6, 8, 11, and 13) of i.p. treatments with PrAg-PAS toxin (15 ug), PrAg-PAS toxin (45 ug), or vehicle alone (FP59 control), starting at day 1. Tumors were imaged 6 days later at day 22. Treatment with PrAg-PAS toxin significantly reduced the average tumor burden of the NCI/ADR-Res-Luc tumor-bearing mice, relative to control cohorts treated with vehicle, at both doses tested (
Human Pancreatic Cancer Cell Lines are Susceptible to Engineered Mutant Anthrax Toxins.
To test the efficacy of the toxins to kill human pancreatic cancer lines were treated with the toxins and viability measured by MTT cytotoxicity assays. Pancreatic cancer cell lines were incubated with engineered anthrax toxins (0-500 ng/mL) and FP59 (50 ng/mL) for 48 hours after which cell viability was evaluated by MTT assay. Each of the pancreatic tumor cell lines showed a dose-dependent susceptibility to killing by the mutant PrAg toxins (PrAg-PAS, PrAg-UAS, PrAg-PCIS or the PrAg-WT toxin (data not shown). These results show that pancreatic tumor cell lines are susceptible to killing by the mutant PrAg toxins.
Human Lung Tumors are Susceptible to Killing by the Mutant PrAg Toxins.
To test the efficacy of the mutant toxins to kill lung tumors, the A549 human lung cancer line was treated with each of the toxins and viability measured by MTT cytotoxicity assay. The lung cancer cell line A549 was incubated with engineered anthrax toxins (0-500 ng/mL) and FP59 (50 ng/mL) for 48 hours after which cell viability was evaluated by MTT assay. The lung tumor line showed a dose-dependent susceptibility to killing by all of the mutant PrAg toxins (data not shown). These data show that lung tumors, which exhibit varying expression levels of proteases and the anthrax toxin receptors are likely to be susceptible to killing by the mutant PrAg toxins.
While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.
All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. Each cited patent and publication is incorporated herein by reference in its entirety. All of the following references have been cited in this application:
This invention was made with government support under Grant Numbers HL118390 and HL084387 awarded by the National Institutes of Health. The government has certain rights in the invention.
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| 20200155643 A1 | May 2020 | US |
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| 62207100 | Aug 2015 | US |
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| Parent | 15747255 | US | |
| Child | 16751864 | US |
