US 20080193373A1
`
`(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2008/0193373 A1
`Stritzker et al.
`(43) Pub. Date: Aug. 14, 2008
`
`
`(54) METHODS AND COMPOSITIONS FOR
`DETECTION OF MICROORGANISMS AND
`CELLS AND TREATMENT OF DISEASES AND
`DISORDERS
`
`(76)
`
`Inventors:
`
`Jochen Harald Stritzker, Kissing
`(DE); Phil Hill, Nottingham (GB);
`Aladara A. Szalay, Highland, CA
`(US); Yong A. Yu, San Diego, CA
`(US)
`
`Correspondence Address:
`Bell, Boyd & Lloyd LLP
`3580 Carmel Mountain Road, Suite 200
`San Diego, CA 92130
`
`(21) Appl. No.:
`
`11/827,518
`
`(22)
`
`Filed:
`
`Jul. 11, 2007
`
`Related US. Application Data
`
`(60) Provisional application No. 60/852,389, filed on Oct.
`16, 2006, provisional application No. 60/830,422,
`filed on Jul. 11, 2006.
`
`(30)
`
`Foreign Application Priority Data
`
`Jul. 11, 2007
`
`(US)
`
`................. PCT/US2007/000158
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`A61K 51/00
`A61K 49/00
`A61K 48/00
`C12N 7/00
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(52) us. Cl. ...................... 424/117; 424/92; 424/9321;
`435/2351
`
`(57)
`
`ABSTRACT
`
`Described herein are methods for detecting a microorganism
`or cell in a subject and methods for detecting, imaging or
`diagnosing a site, disease, disorder or condition in a subject
`using microorganisms or cells. Also described are methods
`which use microorganisms or cells for treating a disease,
`disorder or condition. Such sites, diseases and disorders
`include sites of cell proliferation, proliferative conditions,
`neoplasms, tumors, neoplastic disease, wounds and inflam-
`mation. Further described are microorganisms and cells for
`use in the methods and compositions, combinations and kits,
`including diagnostic and pharmaceutical compositions, con-
`taining a microorganism or cell. Microorganisms and cells
`described herein include those that bind, sequester or accu-
`mulate metal, such as those that provide for metal acquisition,
`transport, storage and/or metabolism. Additional imaging
`and therapy agents are also described.
`
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`US 2008/0193373 A1
`
`Aug. 14, 2008
`
`METHODS AND COMPOSITIONS FOR
`DETECTION OF MICROORGANISMS AND
`CELLS AND TREATMENT OF DISEASES AND
`DISORDERS
`
`RELATED APPLICATIONS
`
`[0001] Benefit ofpriority is claimed under 35 U.S.C. §119
`(e) to US. Provisional Application Ser. No. 60/852,389, to
`Jochen Strizker, Phil Hill, Aladar A. Szalay and Yong A. Yu,
`filed on Oct. 15, 2006, entitled “METHODS AND COMPO-
`SITIONS FOR DETECTION OF MICROORGANISMS
`AND CELLS AND TREATMENT OF DISEASES AND
`
`DISORDERS,” and to US. Provisional Application Ser. No.
`60/830,422, to Jochen Strizker, Phil Hill, Aladar A. Szalay
`and Yong A. Yu, filed on Jul. 11, 2006, entitled “METHODS
`AND COMPOSITIONS FOR DETECTION OF MICRO-
`ORGANISMS AND CELLS AND TREATMENT OF DIS-
`
`EASES AND DISORDERS.” The subject matter of each of
`these applications is incorporated by reference in its entirety.
`[0002] This application is related to International Applica-
`tion No. (Attorney Dkt. No. 17248-012WOl/4812PC) to
`Jochen Strizker, Phil Hill, Aladar A. Szalay and Yong A. Yu,
`filed on Jul. 11, 2007, entitled “METHODS AND COMPO-
`SITIONS FOR DETECTION OF MICROORGANISMS
`AND CELLS AND TREATMENT OF DISEASES AND
`
`DISORDERS,” which also claims priority to US. Provisional
`Application Ser. No. 60/852,389 and to US. Provisional
`Application Ser. No. 60/830,422.
`[0003] This application is related to US. application Ser.
`No. 11/238,025, to Aladar A. Szalay; Tatyana Timiryasova;
`Yong A. Yu; Qian Zhang, filed on Sep. 27, 2005, entitled
`“MICROORGANISMS FOR THERAPY,” which is a con-
`tinuation of US. application Ser. No. 10/872,156, to Aladar
`A. Szalay; Tatyana Timiryasova; Yong A. Yu; Qian Zhang,
`filed on Jun. 18, 2004, entitled “MICROORGANISMS FOR
`THERAPY,” which claims the benefit of priority under 35
`U.S.C. §119(a) to each ofEP 03 013 826.7, filed 18 Jun. 2003,
`entitled “RECOMBINANT VACCINIA VIRUSES USEFUL
`AS TUMOR-SPECIFIC DELIVERY VEHICLE FOR CAN-
`CER GENE THERAPY AND VACCINATIONg” EP 03 018
`478.2, filed 14 Aug. 2003, entitled “Method for the produc-
`tion of a polypeptide, RNA or other compound in tumor
`tissue,” and EP 03 024 283.8, filed 22 Oct. 2003, entitled
`“USE OF A MICROORGANISM OR CELL TO INDUCE
`AUTOIMMUNIZATION OF AN ORGANISMAGAINST A
`TUMOR.”
`
`[0004] This application also is related to International
`Application Serial No. PCT/USO4/ 19866, filed on Jun. 18,
`2004. This application also is related to US. Application filed
`Jun. 10, 2004 Ser. No. 10/866,606, entitled “LIGHT EMIT-
`TING MICROORGANISMS AND CELLS FOR DIAGNO-
`SIS AND THERAPY OF TUMORS,” which is a continuation
`of US. application Ser. No. 10/189,918, filed Jul. 3, 2002;
`US. Application filed May 19, 2004 Ser. No. 10/849,664,
`entitled, “LIGHT EMITTING MICROORGANISMS AND
`CELLS FOR DIAGNOSIS AND THERAPY OF DISEASES
`ASSOCIATED WITH WOUNDED OR INFLAMED TIS-
`
`SUE” which is a continuation of US. application Ser. No.
`10/163,763, filed Jun. 5, 2003; International PCT Application
`WO 03/014380, filed Jul. 31, 2002, entitled “Microorganisms
`and Cells for Diagnosis and Therapy ofTumors,” PCT Appli-
`cation W0 03/ 104485, filed Jun. 5, 2003, entitled, “LIGHT
`EMITTING MICROORGANISMS AND CELLS FOR
`DIAGNOSIS AND THERAPY OF DISEASES ASSOCI-
`
`ATED WITH WOUNDED OR INFLAMED TISSUE,” EP
`Application No. 01 118 417.3, filed Jul. 31, 2001, entitled
`“LIGHT-EMITTING MICROORGANISMS AND CELLS
`
`FOR TUMOUR DIAGNOSIS/THERAPY,” EP Application
`No. 01 125 911.6, filed Oct. 30, 2001, entitled “LIGHT
`EMITTING MICROORGANISMS AND CELLS FOR
`
`DIAGNOSIS AND THERAPY OF TUMORS,” EP Applica-
`tion No. 02 0794 632.6, filed Jan. 28,2004, entitled “MICRO-
`ORGANISMS AND CELLS FOR DIAGNOSIS AND
`
`THERAPY OF TUMORS,” and EP Application No. 02 012
`552.2,
`filed Jun. 5, 2002, entitled “LIGHT EMITTING
`MICROORGANISMS AND CELLS FOR DIAGNOSIS
`AND THERAPY OF DISEASES ASSOCIATED WITH
`
`WOUNDED OR INFLAMED TISSUE.” The subject matter
`of each of these applications is incorporated by reference in
`its entirety.
`[0005] This application also is related to US. Provisional
`Application Ser. No. 60/852,390, to Nanhai Chen; Aladar A.
`Szalay; Yong A. Yu; and Qian Zhang, entitled “MODIFIED
`VACCINIA VIRUS STRAINS FOR USE IN DIAGNOSTIC
`AND THERAPEUTIC METHODS,” filed Oct. 16, 2006.
`[0006] The subject matter of each the these applications,
`publications and international applications is incorporated by
`reference in its entirety.
`
`INCORPORATION BY REFERENCE OF A
`
`SEQUENCE LISTING PROVIDED ON COMPACT
`DISCS
`
`[0007] An electronic version on compact disc (CD-R) of
`the Sequence Listing is filed herewith in duplicate (labeled
`Copy # 1 and Copy # 2), the contents of which are incorpo-
`rated by reference in their entirety. The computer-readable
`file on each of the aforementioned compact discs, created on
`Jul. 9, 2007, is identical, 1,253 kilobytes in size, and entitled
`4812SEQ.001.txt.
`
`FIELD OF THE INVENTION
`
`[0008] Methods of detecting microorganisms and cells in a
`subject and treating diseases and disorders are provided
`herein. Methods of detecting or diagnosing sites of cellular
`proliferation associated with diseases, disorders and condi-
`tions, such as neoplasms, tumors, neoplastic diseases, can-
`cers, wounds and inflammation, are also provided. Further
`provided are microorganisms, cells and compositions, com-
`binations and kits containing the microorganisms or cells for
`use in the methods and use in the preparation of diagnostic
`and therapeutic or pharmaceutical compositions. Also pro-
`vided are methods for enriching a composition with respect to
`certain microorganisms and cells.
`
`BACKGROUND
`
`[0009] Various techniques have been reported for use of
`microorganisms, such as bacteria and viruses, in transferring
`substances, e.g., nucleic acids and proteins, to cells and
`organisms. Various techniques have also been reported for
`detection of microorganisms in samples and in organisms,
`including animals. Many such techniques involve incorpora-
`tion of nucleic acid encoding a foreign protein (e.g., a detect-
`able marker or protein of interest for transfer to a sample) into
`the microorganism. The amount of foreign protein or detect-
`able signal is related to, and thus can be dependent on, the
`amount of the microorganism present in the sample, the
`extent to which the microorganism is able to increase in
`
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`number, or replicate, in the sample and the degree of expres-
`sion of the foreign marker. Additionally, for some microor-
`ganisms, toxicity of the microorganism in the sample, par-
`ticularly an animal, can necessitate limiting the amount ofthe
`microorganism that is provided to the sample.
`[0010] Accordingly, there is a need for alternative methods
`and compositions to provide for enhanced detection ofmicro-
`organisms in a sample, such as an animal, potentially allow-
`ing for rapid growth in the sample, at lower initial concentra-
`tions, reduced toxicities and with decreased reliance on
`foreign protein expression.
`
`SUMMARY
`
`Provided herein are compositions containing a
`[0011]
`microorganism or cell and a diagnostic moiety. An exemplary
`microorganism or cell is, for example, the bacterial strain
`Escherichia coli Nissle 1917. In one embodiment, the bacte-
`ria contain a DNA sequence encoding a protein or polypep-
`tide produced by the bacteria (e.g., an enzyme involved in
`siderophore biosynthesis, including production of substrates
`for siderophore biosynthesis, an enzyme that modifies or
`processes a siderophore, a receptor, a transporter protein,
`such as a siderophore uptake protein (e.g., FepA protein, IroN
`protein, FyuA protein or IutA protein), iron acquisition mol-
`ecule, iron metabolism molecule, an iron transport protein, or
`an iron storage molecule, such as a ferritin or ferritin-like
`molecule) as a diagnostic moiety; the DNA sequence can be
`endogenous or exogenous to the microorganism. In another
`embodiment, the bacteria contain a DNA sequence encoding
`a protein or polypeptide (e.g., an enzyme) that cleaves a
`precursor siderophore to generate a mature siderophore. In
`one embodiment, the protein or polypeptide normally pro-
`duced by the microorganism or cell is also a therapeutic agent.
`Compositions provided herein can be further formulated with
`any ofthe therapeutic agents provided herein. In one embodi-
`ment, the microorganism or cell is recombinant. Provided
`herein is a composition containing a microorganism, such as
`Escherichia coli strain Nissle 1917, and a therapeutic agent.
`[0012]
`Provided herein is a composition containing a
`microorganism or cell (e.g., E. coli strain Nissle 1917), where
`the microorganism or cell contains a DNA sequence encoding
`a therapeutic agent (or encodes a protein or polypeptide
`responsible for the production of a therapeutic agent) and the
`therapeutic agent is, for example, one or more of the follow-
`ing molecules: a ferritin-like molecule, a siderophore (e.g.,
`enterobactin, salmochelin, yersiniabactin or aerobactin), iron
`acquisition molecule, a receptor, a transporter protein, such as
`a siderophore uptake protein (e.g., FepA protein, IroN pro-
`tein, FyuA protein or IutA protein), an iron metabolism mol-
`ecule, or an iron storage molecule, such as a ferritin or fer-
`ritin-like molecule, or
`a
`combination thereof. Such
`therapeutic agents deplete iron from the tumor environment.
`In one embodiment, the one or more molecule(s) is (are)
`endogenous to the microorganism, exogenous to the micro-
`organism or cell or a combination thereof. In one embodi-
`ment, the composition is formulated with an additional thera-
`peutic, such as any of those described herein or known in the
`art.
`
`Provided herein are compositions of microorgan-
`[0013]
`isms or cells that contain one or more heterologous nucleic
`acid molecules for the expression one or more gene products
`useful for therapy or for detection of the microorganisms or
`cells. Expression of such gene products can be controlled by
`operative linkage to a promoter. In some examples provided
`
`herein the promoter can be an inducible promoter. A non-
`limiting example of an inducible promoter provided herein
`for use in the compositions and methods provided is sugar-
`inducible promoter, such as an arabinose inducible promoter
`system, e.g. the araBAD system. According to the methods
`provided herein, induction of an arabinose inducible pro-
`moter can be carried out in vitro in cell culture or in vivo in a
`
`subject that has been administered the microorganisms or
`cells that contain the promoter. In vivo induction of an induc-
`ible promoter can be performed by administering an inducer,
`such as arabinose, to induce gene expression from the pro-
`moter. Provided herein are methods of inducing a promoter in
`vivo by administration of an inducer molecule by a method of
`administration, such as orally, for example, by feeding, or
`injected, such as by intravenous injection.
`[0014]
`Pharmaceutical compositions containing a microor-
`ganism or cell provided herein can be formulated with a
`pharmaceutically acceptable vehicle for use in the methods
`provided herein. The pharmaceutical compositions can be
`formulated for any mode of admini stration, including, but not
`limited to, systemic administration, such as for intravenous
`administration. The compositions can contain a delivery
`vehicle, such as a lipid-based carrier, including liposomes and
`micelles associated with the bacteria.
`
`Provided herein are methods of detecting and/or
`[0015]
`treating a site of proliferation or a proliferative condition,
`such as a tumor, tumor tissue, cancer or metastasis by admin-
`istering to a subject any of the compositions provided herein.
`Provided herein uses ofthe compositions provided herein for
`the preparation of a diagnostic or therapeutic composition for
`detecting and/or treating a site of proliferation or a prolifera-
`tive condition, such as a tumor,
`tumor tissue, cancer or
`metastasis. Provided herein are pharmaceutical compositions
`for detecting and/or treating a site of proliferation or a prolif-
`erative condition, such as a tumor, tumor tissue, cancer or
`metastasis.
`
`Provided herein are uses ofmicroorganisms or cells
`[0016]
`(e.g. Nissle) in the methods provided herein for detecting
`and/or treating a site of proliferation or a proliferative condi-
`tion, such as a tumor, tumor tissue, cancer or metastasis. Also
`provided are uses of microorganisms or cells (e.g. Nissle) for
`the formulation of compositions for use in the methods pro-
`vided herein for detecting and/or treating a site of prolifera-
`tion or a proliferative condition, such as a tumor, tumor tissue,
`cancer or metastasis.
`
`Provided herein are methods of detecting a site of
`[0017]
`proliferation or a proliferative condition, such as a tumor,
`tumor tissue, cancer or metastasis by administering to a sub-
`ject compositions, containing microorganism or cells, pro-
`vided herein, whereby detection of the microorganism or cell
`in the subject indicates the site of proliferation or a prolifera-
`tive condition. Provided herein are methods of detecting a site
`of proliferation or a proliferative condition, in which detec-
`tion is performed externally to the subject.
`[0018]
`Provided herein is a method of detecting a site of
`proliferation or a proliferative condition, such as a tumor,
`tumor tissue, cancer or metastasis, which includes adminis-
`tering to a subject a composition the contains a recombinant
`microorganism or cell, wherein the microorganism or cell
`contains one or more gene(s) encoding one or more molecule
`(s). Exemplary cancers to be treated include, but are not
`limited to, pancreatic cancer, lung cancer, ovarian cancer,
`breast cancer, cervical cancer, bladder cancer, prostate can-
`cer, glioma cancer, adenocarcinoma, liver cancer, skin cancer
`
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`or a combination thereof. In one embodiment, the tumor is
`metastatic. The microorganisms and cells used in the methods
`provided herein have been attenuated such that they are not
`lethal to the subject. Further, the microorganisms and cells
`preferentially accumulate in a tumor in the subject.
`[0019] Microorganisms or cells for the compositions,
`methods and uses provided herein can be, for example, bac-
`teria, Viruses and eukaryotic cells.
`[0020] Bacteria employed in the methods provided herein
`include, but are not limited to, mutual or commensal strains of
`Escherichia coli, Bacteroides, Eubacterium, Streptococcus,
`Actinomyces, Veillonella, Nesseria, Prevotella, Campylo—
`bacter, Fusobacterium, Eikenella, Porphyromonas and Prio—
`pionibacteria.
`the microorganism or cell
`[0021]
`In some examples,
`employed in the methods provided herein can be one that can
`form a mutual or commensal association with the subject,
`such as strains of Escherichia coli, Bacteroides, Eubacte-
`rium, Streptococcus, Actinomyces, Veillonella, Nesseria, Pre—
`votella, Campylobacter, Fusobacterium, Eikenella, Porphy—
`romonas and Propionibacteria, or one that is a probiotic
`strain ofEscherichia coli, Bacillus cereus, Bacillus licheni—
`form is, Bacillus pumilus, Bacillus clausii, Bacillus coagu—
`lans, Bacillus polyfermenticus, Brevibacillus laterosporus,
`Lactococcus, Lactobacillus reuteri, Lactobacillus amylo—
`vorus, Lactobacillus crispatus, Lactobacillus gallinarum,
`Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacil—
`lus bifidum, Lactobacillus helveticus, Bifidobacterium lactis,
`Bifidobacterium breve, Leuconostoc mesenteroides, Entero—
`coccus faecium, Pediococcus, Sporolactobacillus inulinus,
`Saccharomyces. In other examples, the bacteria employed in
`the methods provided herein are selected from nonpathogenic
`or attenuated E. coli strains of the 06 serotype, bacteria with
`five or more systems for acquiring iron.
`[0022]
`In a particular example, the bacterium employed in
`the methods provided herein is E. coli Nissle 1917. Deriva-
`tives of E. coli strain Nissle 1917 can be employed, such as,
`for example, Nissle strains that either do not produce
`lipopolysaccharide (LPS), or that produce LPS that lacks the
`myristic acid moiety of lipid A, strain that do not produce
`lipid acyl A transferase, strains that lack one or more cryptic
`plasmids, such as pMut1 and/or pMut2, strains that have
`reduced L-arabinose metabolism or strains in which the con-
`
`sensus nucleotide sequence for the Fur protein-binding
`region ofthe promoter of one or more genes that is negatively
`regulated by iron is inactivated.
`[0023] Viruses employed in the methods provided herein
`include, but are not limited to, vaccinia virus, baculovirus,
`sindbis virus, Sendai virus, adenovirus, AAV virus, parvovi-
`rus, retrovirus, Epstein-Barr virus, papillomavirus, SV40
`virus, cytomegalovirus, Newcastle Disease virus, bovine
`enterovirus,
`lymphocytic choriomeningitis virus,
`lentivi-
`ruses, a derivative ofthe Edmonton-B strain of measles virus,
`herpes simplex virus type 1, or yellow fever virus. Exemplary
`viruses include, for example, the Lister strain of vaccinia
`virus, MVM, H-1, MoMULV, HaMUSV, MuMTV, RSV or
`GaLV. In one embodiment, the virus is the LlVP strain of
`vaccinia virus.
`
`[0024] Eukaryotic cells employed in the methods provided
`herein include, but are not limited to, fibrosarcoma cells and
`stem cells.
`
`Provided herein is a method of diagnosing a site of
`[0025]
`proliferation or a proliferative condition, such as a tumor,
`tumor tissue, cancer or metastasis by administering to a sub-
`
`ject a composition containing E. coli Nissle 1917. E. coli
`Nissle 1917 can encode one or more or the following mol-
`ecule(s): an enzyme involved in siderophore biosynthesis,
`including production of substrates for siderophore biosynthe-
`sis, an enzyme that modifies or processes a siderophore, a
`receptor, a transporter protein, a ferritin-like molecule, and a
`combination thereof. Any ofthe diagnostic methods provided
`herein can be combined with any of the therapeutic methods
`provided herein. Such diagnostic methods provided herein
`can be used to monitor the efficacy ofthe therapeutic methods
`provided herein.
`[0026]
`Provided herein is a method of treating a site of
`proliferation or a proliferative condition, such as a tumor,
`tumor tissue, cancer or metastasis, by administering to a
`subject a composition containing a microorganism, wherein
`the microorganism is E. coli Nissle 1917. The microorganism
`or cell can contain one or more gene(s) encoding one or more
`molecule(s) chosen from among an enzyme involved in sid-
`erophore biosynthesis, including production of substrates for
`siderophore biosynthesis, an enzyme that modifies or pro-
`cesses a siderophore, a receptor, a transporter protein, a fer-
`ritin-like molecule or a combination thereof.
`
`[0027] A method of treating a site of proliferation or a
`proliferative condition, such as a tumor, tumor tissue, cancer
`or metastasis, provided herein can include administering to a
`subject a composition that contains a recombinant microor-
`ganism or cell, wherein the microorganism or cell contains (a)
`DNA sequence(s) encoding one or more molecule(s) selected
`from among an enzyme involved in siderophore biosynthesis,
`including production of substrates for siderophore biosynthe-
`sis, an enzyme that modifies or processes a siderophore, a
`receptor, a transporter protein, a ferritin-like molecule, and a
`combination thereof. In one embodiment, the microorganism
`or cell depletes iron from a tumor cell or a tumor environment,
`thereby dis-regulating iron hemostasis in the tumor cell or
`tumor environment. Ferritin-like molecules provided herein
`can be a ferritin produced by a prokaryotic microorganism, a
`eukaryotic cell or a virus-infected tumor cell. In a particular
`embodiment, the ferritin produced by a prokaryotic microor-
`ganism and is one or more of a bacterial ferritin, a bacteriof—
`erritin, a dodecameric ferritin, a rubrerythrin or a combina-
`tion thereof. Alternatively, ferritin-like molecules provided
`herein can be a ferritin produced by a eukaryotic cell and is
`one or more of an H chain, an L chain or a combination
`thereof. The one or more ferritin(s) can be endogenous to the
`microorganism, exogenous to the microorganism or cell or a
`combination thereof. In yet another embodiment, ferritin-like
`molecules provided herein can be a ferritin produced by a
`tumor cell that is infected with a recombinant virus. The one
`
`or more ferritin(s) can be one or more of a bacterial ferritin, a
`bacterioferritin, a dodecameric ferritin, a rubrerythrin, an H
`chain, an L chain or a combination thereof.
`[0028]
`Provided herein are microorganisms and cells con-
`taining DNA sequences for the expression of one or more
`siderophores (e.g., enterobactin, salmochelin, yersiniabactin
`or aerobactin). Such siderophores can be endogenous to the
`microorganism, exogenous to the microorganism or cell or a
`combination thereof. For example, a bacterium that expresses
`endogenous siderophores is provided herein. The bacterium
`can be further recombinantly engineered to express one or
`more exogenous siderophores and/or an enzyme involved in
`producing the exogenous siderophore. A microorganism or
`cell (e.g., bacterium, virus or eukaryotic cell) that does not
`normally express a siderophore can be recombinantly engi-
`
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`neered to express one or more exogenous siderophores and/or
`an enzyme involved in producing the exogenous siderophore.
`[0029] The one or more siderophore(s) can bind(s) to a
`receptor, and optionally, can be internalized by the microor-
`ganism. In such an embodiment, the microorganism or cell is
`a prokaryotic microorganism and the siderophore is intemal-
`ized. In another embodiment, the microorganism is a virus,
`and the siderophore binds to a receptor on the tumor cell
`surface in the form of a siderophore-metal complex. In yet
`another embodiment, the cell is a eukaryotic cell, and the
`siderophore binds to a receptor on the eukaryotic cell surface
`in the form of a siderophore-metal complex.
`[0030]
`Provided herein are methods for diagnosis and/or
`therapy of tumors wherein the microorganism or cell admin-
`istered to the subject expresses a recombinant receptor that
`binds a ligand. The recombinant receptor can be, for example,
`an outer membrane protein, such as OmpA or OmpC,
`expressing a recombinant peptide, such as a streptavidin
`binding peptide or an S-peptide. One embodiment further
`includes administering to the subject a composition that con-
`tains a ligand that binds to the recombinant peptide. Exem-
`plary ligands are conjugated to a molecule such as a detect-
`able moiety, a therapeutic agent or a combination thereof.
`[0031]
`In one embodiment, the method further provides for
`administering to a subject one or more molecule(s): a sidero-
`phore, a metal, a peptide ligand or a combination thereof.
`Siderophores can bind to a receptor on the injected microor-
`ganism or cell or on a tumor cell and, optionally, can be
`internalized. Molecules provided herein can be conjugated to
`a detectable moiety, a therapeutic agent or a combination
`thereof. In some embodiments, the detectable moiety and the
`therapeutic agent are the same. In one embodiment, where the
`detectable moiety and the therapeutic agent are different, the
`one or more molecule(s) can be conjugated to a detectable
`moiety and a therapeutic agent.
`[0032] The microorganisms and cells used in the methods
`herein allow for detection of a tumor, tumor tissue, cancer or
`metastasis based on a signal, such as a signal that is detectable
`magnetic resonance imaging (MRI), single-photon emission
`computed tomography (SPECT), positron emission tomog-
`raphy (PET), scintigraphy, gamma camera, a [3+ detector, a y
`detector, fluorescence imaging or bioluminescence imaging.
`[0033] The microorganisms and cells used in the methods
`herein allow for detection of a tumor, tumor tissue, cancer or
`metastasis through detection of electromagnetic radiation
`(e.g., light), such as, for example, by administering a micro-
`organism or cell containing a DNA sequence encoding a
`detectable protein or a protein capable of inducing a detect-
`able signal, such as a fluorescent protein or a luciferase. In
`some examples, the microorganism or cell contains a DNA
`sequence encoding one or more enzymes for the production
`of a substrate for the luciferase.
`
`In some embodiments, the microorganism or cell
`[0034]
`further acts as a therapeutic agent by depleting iron from the
`tumor environment or tumor cell, such as sequestration by
`siderophores, ferritin-like molecules, a receptor that scav-
`enges iron from host,
`transport proteins or combination
`thereof.
`
`[0035] Diagnostic methods provided herein can be com-
`bined with therapeutic methods provided herein. In one non-
`limiting example, after a tumor,
`tumor tissue, cancer or
`metastasis is detected using any of the imaging methods
`provided herein, the method further includes treating tumor,
`
`tumor tissue, cancer or metastasis, which includes adminis-
`tering to a subject a therapeutic agent.
`[0036]
`In the methods and uses provided herein, the ani-
`mals can be non-human animals or can include humans.
`
`[0037] Diagnostic moieties provided herein accumulate in
`a tumor cell or tumor environment. Diagnostic moieties
`include any of those described herein or known in the art. In
`some embodiments, the diagnostic moiety is also a therapeu-
`tic moiety. In other embodiments, a therapeutic agent can be
`administered to the subject. Compositions that can be used in
`the methods herein can be further formulated with a thera-
`
`peutic agent.
`[0038] Diagnostic moieties for the compositions, methods
`and uses provided herein can be any of those described or
`known in the art such as, for example, a bioluminescent
`moiety, a fluorescent moiety, a chemiluminescent moiety, a
`metal, a radionuclide or a combination thereof. Exemplary
`bioluminescent moieties include for example, a luciferin such
`as, but not limited to, click beetle luciferin, firefly luciferin,
`Cypridina
`luciferin, bacterial
`luciferin, derivatives of
`luciferins or a synthetic luciferin analog thereof. Exemplary
`fluorescent moieties include, for example, a green fluorescent
`protein (GFP), yellow fluorescent protein (YFP), orange fluo-
`rescent protein (GFP), cyan fluorescent protein (CFP), blue
`fluorescent protein (BFP), red fluorescent protein (RFP), far-
`red fluorescent protein, or near-infrared fluorescent protein.
`Exemplary chemiluminescent moieties include, for example,
`luminol, isoluminol, dioxetanes, acridinium esters, thioesters
`and sulfonamides, and phenanthradinium esters. Exemplary
`metals include, for example, iron, gadolinium, gold or gal-
`lium, which act as contrast agents in the methods and uses
`provided herein. Metals can, optionally, be labeled, such as
`with a radionuclide (e.g., llCarbon, llFluorine, 13Carbon,
`l3Nitrogen, 15Nitrogen,
`15 Oxygen, 18Flourine, 19Flour‘ine,
`24Sodium, 32Phosphate, 42Potassium, 51Chromium, 55Iron,
`59Iron, 57Cobalt, 6OCobalt, 64Copper, 67Gallium, 68Gallium,
`75Selenium, 81Krypton, 82Rubidium, 89Strontium, 92Stron-
`tium, 90Yttrium, 99Technetium, 103 Palladium, 106Ruthe-
`nium,
`111Indium,
`117Lutetium,
`123 Iodine,
`125Iodine,
`131Iodine, 133Xenon, 137Cesium, 153Samarium, 153Gado-
`linium, 165Dysprosium, 166Holmium, 169Ytterbium, 177Leu-
`tium 186Rhenium, 188Rhenium, 192Iridium, 198Gold, 201Thal-
`lium, 211Astatine, 212Bismuth or 213Bismuth).
`[0039] Diagnostic moieties also include those that are pro-
`duced by a microorganism, such as a bacterium or eukaryotic
`cell. Diagnostic moieties include, for example, a ferritin-like
`molecule, a siderophore, a receptor, a transporter protein, an
`enzyme involved in siderophore biosynthesis, including pro-
`duction of substrates for siderophore biosynthesis, an enzyme
`that modifies or processes a siderophore, and a combination
`thereof. Methods of recombinantly engineering a bacterium,
`a virus or a eukaryotic cell to carry a gene encoding such a
`type of diagnostic moiety are provided herein. Exemplary
`receptors include, for example, a recombinant outer mem-
`brane protein with an embedded peptide (e.g., a streptavidin
`binding peptide or an S-peptide) which, optionally, can be
`conjugated to a detectable label. Likewise, the ferritin-like
`molecules or siderophores can, optionally, be conjugated to a
`detectable moiety. In certain embodiments, the detectable
`moiety and the therapeutic agent are the same molecule. In
`some examples, the siderophore is conjugated to a metal, such
`as iron, gallium, gadolinium, manganese, cobalt, zinc, chro-
`mium, gold and indium or a radionuclide.
`
`Genome Ex. 1039
`
`Page 5 of 90
`
`Genome Ex. 1039
`Page 5 of 90
`
`

`

`US 2008/0193373 A1
`
`Aug. 14,2008
`
`[0040] Therapeutic agents for the compositions, methods
`and uses provided herein can be, for example, an anti-cancer
`agent. Anti-cancer agents provided herein include, but are not
`limited to, cytokines, growth factors, photosensitizing agents,
`radionuclides,
`toxins, .anti-metabolites, signaling modula-
`tors, anti-cancer antibiotics, anti-cancer antibodies, angio-
`genesis inhibitors, radiation therapy, chemotherapeutic com-
`~
`~
`~
`pounds, or a combination thereof. Exemplary therapeutic
`agents are provided herein. In some examples, therapeutic
`agents can be expressed by the microorganism or cell for use
`in the therapeutic methods provided herein. Such agents
`include, but are not limited to, a cytokine, a chemokine, an
`immunomodulatory molecule, a single chain antibody, anti-
`sense RNA, prodrug converting enzyme, s1RNA, angiogen-
`esis inhibitor, a toxin, an antitumor oligopeptides, an antimi-
`totic oligopeptide, an anti-cancer polypeptide antibiotic or
`.
`US$118 faCt0r~
`[0041]
`Provided herein are microorganisms and cells for
`use in Gene-Directed Enzyme Prodrug Therapy (GDEPT), in
`which microorganisms and cells are engineered to secrete an
`enzyme, or to express a cell-surface attached enzyme, that
`converts a non-toxrc prodrug into a cytotoxic drug. Followrng
`administration of the engineered bacteria, eukaryotic cells
`and/or viruses, the microorganisms and cells are allowed to
`proliferate and produce the prodrug converting enzyme at the
`site of the tumor. After a period of time the prodrug is deliv-
`ered intravenously, or by alternative delivery method, and the
`prodrug is converted to the cytotoxic form at the site of the
`tumor, causing toxicity in the surrounding tumor cells. Exem-
`plary prodrug converting enzymes with their prodrug partners
`are prov1ded herein.
`.
`.
`.
`.
`[0042]
`Prov1ded herein are combinations of a s1derophore,
`such as enterobactin, salmochelin, yersiniabactin or aerobac-
`tin, and a microorganism that does not produce a siderophore.
`Provided herein are combinations of a siderophore and a
`microorganism that expresses a siderophore uptake protein,
`such as Fe