Patent Grant
12090179
The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on Jan. 18, 2023, is named 84977-3-3-1_Sequence Listing.xml and is 2,981 bytes in size.
This disclosure relates to methods of treating cancer, and more particularly, to methods of treating cancer with attenuated Salmonella typhimurium.
Despite continuing efforts of fighting cancer, nearly 600,000 cancer-related deaths still occur in the United States each year, and over 8 million cancer-related deaths occur worldwide. Thus, according to the World Health Organization, cancer continues to be a leading cause of morbidity and mortality worldwide. In addition, it is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next two decades. The mainstay of cancer treatment is chemotherapy with the aim of curing or controlling this disease with the maximum tolerated dose (MTD) or the highest dose of a drug with tolerable side effects. Strategies employed to decrease the side effects include, for example, varying the combination of anti-neoplastic agents, metronomic dosing, and delivery of the chemotherapeutic agent directly to the affected organ. In the last several years, advancements have been made with immunotherapy for cancer treatment and many immunologic agents have demonstrated promise in this field. However, significant toxicities and tumor resistance limit this treatment strategy.
In general, this disclosure relates to methods of treating cancer with Salmonella typhimurium. A dose of a chemotherapy agent is administered in combination with a dose of attenuated Salmonella typhimurium. The dose of the chemotherapy agent is lower than a maximum effective dose of the chemotherapy agent. The attenuated Salmonella typhimurium can include a truncated interleukin-2 gene. The combination provides a synergistic effect that provides a greater reduction in tumor burden than the administration of an equivalent dose of the chemotherapy agent alone. As a result, a lower and less toxic dose of the chemotherapy agent can be used, which provides effective treatment while minimizing side effects caused by the toxicity of the chemotherapy agent.
In one embodiment, a method of treating cancer includes administering a combination of a dose of a chemotherapy agent and a dose of a composition consisting essentially of attenuated Salmonella typhimurium. The dose of the chemotherapy agent is lower than a maximum effective dose of the chemotherapy agent. The combination provides a synergistic reduction in tumor burden when compared to the reduction in tumor burden provided by administration of an equivalent dose of the chemotherapy agent without the composition consisting essentially of attenuated Salmonella typhimurium. The toxicity of the combination is lower than the toxicity of the maximum effective dose of the chemotherapy agent.
In another embodiment, a method of treating cancer includes administering a combination of a dose of a chemotherapy agent and a dose of attenuated Salmonella typhimurium containing a plasmid carrying a coding sequence encoding a truncated human interleukin-2, wherein the truncated human interleukin-2 consists of the amino acid sequence shown in SEQ ID NO: 2. The dose of the chemotherapy agent is lower than a maximum effective dose of the chemotherapy agent. The combination provides a synergistic reduction in tumor burden when compared to the reduction in tumor burden provided by administration of an equivalent dose of the chemotherapy agent without the attenuated Salmonella typhimurium containing the plasmid carrying the coding sequence encoding the truncated human interleukin-2. The toxicity of the combination is lower than the toxicity of the maximum effective dose of the chemotherapy agent.
In another embodiment, an anti-tumor agent for use in a method of treating cancer includes a combination of a dose of a chemotherapy agent and a dose of a composition consisting essentially of attenuated Salmonella typhimurium. The method includes administering the combination of the dose of the chemotherapy agent and the dose of the composition consisting essentially of attenuated Salmonella typhimurium. The dose of the chemotherapy agent is lower than a maximum effective dose of the chemotherapy agent. The combination provides a synergistic reduction in tumor burden when compared to the reduction in tumor burden provided by administration of an equivalent dose of the chemotherapy agent without the composition consisting essentially of attenuated Salmonella typhimurium. The toxicity of the combination is lower than the toxicity of the maximum effective dose of the chemotherapy agent.
In another embodiment, an anti-tumor agent for use in a method of treating cancer includes a combination of a dose of a chemotherapy agent and a dose of attenuated Salmonella typhimurium containing a plasmid carrying a coding sequence encoding a truncated human interleukin-2, wherein the truncated human interleukin-2 consists of the amino acid sequence shown in SEQ ID NO: 2. The method includes administering the dose of the chemotherapy agent in combination with the dose of attenuated Salmonella typhimurium containing the plasmid carrying the coding sequence encoding the truncated human interleukin-2. The dose of the chemotherapy agent is lower than a maximum effective dose of the chemotherapy agent. The combination provides a synergistic reduction in tumor burden when compared to the reduction in tumor burden provided by administration of an equivalent dose of the chemotherapy agent without the attenuated Salmonella typhimurium containing the plasmid carrying the coding sequence encoding the truncated human interleukin-2. The toxicity of the combination is lower than the toxicity of the maximum effective dose of the chemotherapy agent.
The following detailed description is exemplary in nature and is not intended to limit the scope, applicability, or configuration of the disclosure in any way. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the invention or testing, suitable methods and materials are described herein. The materials, methods and examples are illustrative only, and are not intended to be limiting. Those skilled in the art will recognize that many of the noted examples have a variety of suitable alternatives. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise.
“Attenuated,” as used herein, means bacteria selected or altered to greatly diminish its capacity to cause disease, but still able to retain its ability to colonize the gut associated lymphoid tissue.
“Coding sequence” and “coding region,” as used herein, are used interchangeably and refer to a polynucleotide that encodes a protein and, when placed under the control of appropriate regulatory sequences, expresses the encoded protein. The boundaries of a coding region are generally determined by a translation start codon at its 5′ end and a translation stop codon at its 3′ end.
“IL-2,” as used herein, means the protein human interleukin-2.
“NK” or “NK cell,” as used herein, means natural killer cell.
“Operably linked,” as used herein, refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulator sequence is operably linked to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.
“Regulatory Sequence,” as used herein, refers to a nucleotide sequence that regulates expression of a coding region to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, transcription initiation sites, translation start sites, translation stop sites and terminators.
Attenuated Salmonella typhimurium has been developed as a vector to deliver therapeutic agents to tumors. The potential of S. typhimurium is largely due to its reported propensity to accumulate at greater than 1,000-fold higher concentration in tumors relative to healthy tissues. In addition, the genetic manipulability of S. typhimurium allows for the expression of foreign recombinant proteins, making these bacteria an effective delivery system for proteins that may be toxic when administered systemically.
Interleukin-2 (IL-2) is a protein naturally produced by the human body which promotes lymphocyte proliferation and enhances the cytolytic function of T cells and natural killer (NK) cells. It is thus able to stimulate the immune system to produce cancer-destroying white blood cells. IL-2 based immunotherapy in certain types of cancer has been studied for years with limited success. The amino acid sequence (SEQ ID NO: 3) of the normal human IL-2 protein encoded by SEQ ID NO: 4 (the DNA sequence encoding normal human IL-2) is shown in FIG. 3 of U.S. patent application Ser. No. 13/524,503 now U.S. Pat. No. 8,647,618, which is a continuation of U.S. patent application Ser. No. 12/425,927, filed Apr. 17, 2009 now U.S. Pat. No. 8,221,739, which is a continuation in part of and claims priority of U.S. patent application Ser. No. 10/834,587, filed Apr. 29, 2004 now abandoned, the contents of which applications are hereby incorporated by reference in their entirety.
While IL-2 is naturally produced by the human body, its maximum effectiveness requires a higher concentration and more specific delivery vector to the disease site. However, high doses of IL-2 are found to result in severe toxicity in many patients. A solution to this problem is using a live bacterial strain of Salmonella typhimurium which was attenuated to greatly diminish its capacity to cause disease. S. typhimurium is used due to its natural ability to colonize the gut associated lymphoid tissue (GALT), liver and spleen. Colonization of the liver by the attenuated S. typhimurium further initiates a generalized cellular response against the bacteria or can persist as a carrier state. The χ4550 strain of S. typhimurium used in the present disclosure contains a gene deletion constructed by transposon mutagenesis with Tn10 followed by selection for furanic acid resistance. This method of genetic alteration leads to deletional loss of Tn10 and adjacent DNA sequences to produce a deletion of aspartate semialdehyde dehydrogenase (asd). This mutation imposes a requirement for diaminopimelic acid. The lack of the asd enzyme in these bacteria leads to the inability to construct a stable cell wall causing lethal lysis of the S. typhimurium. Thus, to insure stable expression of a desired protein, a plasmid (pYA292) was constructed which carries the asd gene.
A synthetic cDNA (SEQ ID NO: 5), coding for a truncated human IL-2 protein, optimized for expression in Escherichia coli was inserted into plasmid pYA292 using well known methods. The truncated cDNA (SEQ ID NO: 1) is a part of the synthetic IL-2 nucleotide sequence (SEQ ID NO: 5). This sequence is one nucleotide short of the sequence that was intended to code for a full-length mature human IL-2 protein. As used herein, “mature” means a protein lacking the beginning (N-terminal) 20 amino acid signal sequence that is cleaved off as the molecule is secreted from the a human cell. The mutation that occurred is a deletion of a “t” nucleotide between the “a” at position 272 and the “g” at position 273. This resulted in an in-frame taa stop codon at position 274 that truncated the resultant IL-2 protein. The resulting DNA nucleotide sequence is SEQ ID NO: 1 and the expressed protein is SEQ ID NO: 2.
Both the aspartate semialdehyde dehydrogenase (asd+) vector and the synthetic truncated human IL-2 cDNA were digested to completion with restriction enzymes EcoRI (Promega, Madison, Wis.) and HindIII (New England Biolabs, Beverly, Mass.). The about ˜3.4 kb linearized vector fragment of pYA292 and the EcoRI-HindIII fragment of the IL-2 gene were isolated following agarose gel electrophoresis using the PrepaGene Kit (BioRad, Hercules, Calif.). The IL-2 gene fragment was ligated into the pYA292 vector using T4 DNA ligase (Promega, Madison, Wis.) with a 3:1 molar excess of insert and incubating for 4 hours at 16° C. The ligation mix was then electroporated into the χ4550 strain of attenuated S. typhimurium. S. typhimurium, Δcya-1 Δcrp-1 ΔasdA1 strain χ4550 was grown in Luria Broth (Sigma, St. Louis, Mo.) containing 50 mg/ml diaminopimelic acid (DAP).
Cultures were grown to an absorbance of 0.200 at OD600 (approximately 108 colony forming units (cfu)/ml broth) and the cells were prepared for electroporation. Plasmid vector pYA292 and the ligation mix were electroporated into χ4550 utilizing an electroporation device (BioRad) with 0.2 cm disposable cuvettes. Cells were pulsed at 2.5 kV and 25 μF with a pulse controller at 200 ohms. Cells were then subsequently plated on Luria agar without DAP and recombinant clones were identified using the Magic Mini-Prep DNA Purification System (Promega), and restriction enzyme digestion with EcoRI and HindIII and gel electrophoresis with 1.2 agarose. The restriction enzyme mapping revealed a plasmid corresponding to that expected for an insert of the IL-2 fragment in pYA292 and the plasmid was renamed pIL2. The new transformant was renamed χ4550 (pIL2), also referred to herein as “SalpIL2.”
Transforming an asd deleted strain with the plasmid (pIL2) allows for the stable expression of IL-2. As discussed above, stability of this vector is maintained because the particular strain of S. typhimurium used here (χ4550) lacks the enzyme aspartate semialdehyde dehydrogenase (asd), which, conversely, the plasmid containing the IL-2 gene (pIL2) contains. Bacteria lacking asd cannot make diaminopimelic acid (DAP), an essential component of the bacterial cell wall and, thus, would not long survive. Thus, if the attenuated S. typhimurium were to attempt to revert to its wild-type strain and lose the plasmid, it would die a “DAP-less” death. Because the loss of the IL-2 containing plasmid would also result in the loss of the plasmid encoded asd, stable expression of the IL-2 gene is achieved.
In one embodiment, method 200 includes administering a first dose of attenuated S. typhimurium and a first dose of a chemotherapy agent (201) on a first day, administering a second dose of a chemotherapy agent (203) on a second day a week after the first day, and administering a third dose of a chemotherapy agent (205) on a third day a week after the second day. In this embodiment, a single dose of attenuated S. typhimurium is administered throughout the entire treatment period. In another embodiment, method 200 includes administering a first dose of attenuated S. typhimurium and a first dose of a chemotherapy agent (201) on a first day, administering a second dose of attenuated S. typhimurium (202) on a second day a week after the first day, administering a second dose of a chemotherapy agent (203) on the second day a week after the first day, and administering a third dose of a chemotherapy agent (205) on a third day a week after the second day. In this embodiment, two doses of attenuated S. typhimurium are administered throughout the entire treatment period.
In another embodiment, method 200 includes administering a first dose of attenuated S. typhimurium and a first dose of a chemotherapy agent (201) on a first day, administering a second dose of attenuated S. typhimurium (202) on a second day three weeks after the first day, administering a second dose of a chemotherapy agent (203) on the second day three weeks after the first day, administering a third dose of attenuated S. typhimurium (204) on a third day three weeks after the second day, and administering a third dose of a chemotherapy agent (205) on the third day three weeks after the second day. This embodiment further includes administering a fourth dose of attenuated S. typhimurium and a fourth dose of a chemotherapy agent on a fourth day three weeks after the third day, administering a fifth dose of attenuated S. typhimurium and a fifth dose of a chemotherapy agent on a fifth day three weeks after the fourth day, and administering a sixth dose of attenuated S. typhimurium and a sixth dose of a chemotherapy agent on a sixth day three weeks after the fifth day. In this embodiment, six doses of attenuated S. typhimurium are administered throughout the entire treatment period.
In some embodiments, administering the first dose of attenuated S. typhimurium (201) includes orally or intravenously administering the attenuated S. typhimurium. In one embodiment, administering a first dose of attenuated S. typhimurium (201) includes administering a oral dose of SalpIL2. In some embodiments, the dose is about 1×109 cfu. In another embodiment, administering a first dose of attenuated S. typhimurium (201) includes administering a intravenous (IV) dose of SalpNG.1. In some embodiments, the dose is about 2×106 cfu.
In some embodiments, administering a first dose of a chemotherapy agent (201) includes administering a IV dose of doxorubicin. In one embodiment, the dose of doxorubicin is 1.25 mg/kg. In another embodiment, the dose of doxorubicin is 2.5 mg/kg. In other embodiments, the chemotherapy agent can be carboplatin, cisplatin, cyclophosphamide, daunorubicin, oxaliplatin, 5-fluorouracil, gemcitabine, or any other appropriate chemotherapy agent.
Method 200 is advantageous, because the combination provides a synergistic effect that provides a greater reduction in tumor burden than the administration of the chemotherapy agent alone. As a result, a lower and less toxic dose of the chemotherapy agent can be used, which provides effective treatment while minimizing side effects caused by the toxicity of the chemotherapy agent.
BALB-neuT Tumor Treatment Model
The BALB-neuT model is a genetically engineered mouse model in which mammary tumor development is driven by expression of a constitutively activated rat homolog of human epidermal growth factor receptor 2. In this model, autochthonous tumors develop over several months and are palpable in the mammary pads of female mice around 16 weeks of age. The tumors closely resemble the aggressive Her2-driven cancer found in human patients.
BALB-neuT mice were maintained in specific pathogen free conditions and fed standard mouse chow (Harlan). Animals were cared for by the University of Minnesota's Research Animal Resources, and all animal use was approved by the University's Institutional Animal Care and Use Facility. Genotyping for the neu transgene was performed by Transnetyx on male and female pups. Breeding pairs consisted of heterozygous males and homozygous negative females. Female mice that were positive for the neu transgene were monitored for tumor development.
Methods
Female BALB-neu-T mice spontaneously developed palpable mammary fat pad tumors around 16 weeks of age (approximately 50-60 mm3). At this time (day 0), the mice typically had 1-3 palpable tumors. For each experiment, individual tumors were measured by caliper, and their volume was calculated. Individual tumor volumes were calculated as spheroid (L×W2×0.52) and combined to give a total tumor burden measurement for each mouse. Tumor burden data was gathered weekly from day 0 to day 35. Additionally, percent weight change data was gathered weekly for each mouse from day 0 to day 35. The percent weight change was calculated based on the baseline weight of each mouse.
Various embodiments of method 200, described above with respect to
One control group (301) received PBS alone. For this control group, the tumors in the mice enlarged over time, and new tumors appeared on the remaining fat pads, usually until each mammary pad developed a tumor. When left untreated, average total tumor burden per mouse reached 5.66 cm3 by day 35, at which point the mice were moribund and euthanized.
Three control groups received doxorubicin alone. The first control group (302) received the maximum tolerated dose (MTD) of intravenous (IV) 5 mg/kg doxorubicin on days 0, 7, and 14. The second control group (303) received IV 2.5 mg/kg (50% reduction in MTD) doxorubicin on days 0, 7 and 14. The third control group (304) received IV 1.25 mg/kg (75% reduction in MTD) doxorubicin on days 0, 7, and 14.
One control group (305) of mice received SalpIL2 alone. This control group received an oral dose of 1×109 cfu SalpIL2 on day 0 and did not receive any additional SalpIL2 or doxorubicin treatments. Another control group (306) received Salp NG.1 alone. This control group received an IV dose of 2×106 cfu SalpNG.1 on day 0 and did not receive any additional SalpNG.1 or doxorubicin treatments.
For combination therapy treatment with SalpIL2, one group of mice (307) received an oral dose of 1×109 cfu SalpIL2 as well as IV 1.25 mg/kg doxorubicin on day 0. Two additional doses of IV 1.25 mg/kg doxorubicin were administered on days 7 and 14. For combination therapy treatment with SalpNG.1, a first group of mice (308) received an IV dose of 2×106 cfu SalpNG.1 as well as IV 1.25 mg/kg doxorubicin on day 0. Two additional doses of IV 1.25 mg/kg doxorubicin were administered on days 7 and 14. A second group of mice (309) received an IV dose of 2×106 cfu SalpNG.1 as well as IV 2.5 mg/kg doxorubicin on day 0. Two additional doses of IV 2.5 mg/kg doxorubicin were administered on days 7 and 14.
Results
The group that received a combination of oral SalpIL2 and 1.25 mg/kg doxorubicin (307) and the group that received a combination of IV SalpNG.1 and 1.25 mg/kg doxorubicin (308) surprisingly showed a synergistic effect in tumor treatment. A statistically significant reduction in tumor burden is shown. The tumor burden by day 21 was less than double that of the group that received the MTD of doxorubicin (302), and was only slightly more than double by day 35. Unexpectedly, there was no difference in the combination treatment with the oral SalpIL2 and the combination treatment with the IV SalpNG.1. Thus, the combination treatments of 1.25 mg/kg of doxorubicin with either SalpIL2 or SalpNG.1 are nearly as effective as treatment with the MTD of doxorubicin alone and significantly more effective than treatment with 1.25 mg/kg of doxorubicin alone. The dose of doxorubicin in the combination treatments is only 25% of the MTD, which significantly reduces the toxic effects of the doxorubicin (explained in greater detail below with respect to
The group that received a combination of IV SalpNG.1 and 1.25 mg/kg doxorubicin (308) showed a synergistic effect in tumor treatment. A statistically significant reduction in tumor burden is shown. The tumor burden by day 21 was less than double that of the group that received the MTD of doxorubicin (302), and was only slightly more than double by day 35. The group that received a combination of IV SalpNG.1 and 2.5 mg/kg doxorubicin (309) also showed a synergistic effect in tumor treatment. A statistically significant reduction in tumor burden is also shown for this group. The tumor burden by day 21 was even less than the tumor burden in the group that received a combination of SalpNG.1 and 1.25 mg/kg, and by day 35 was barely double the tumor burden of the group that received the MTD of doxorubicin (302).
Thus, the combination treatments of SalpNG.1 with 1.25 mg/kg of doxorubicin or 2.5 mg/kg doxorubicin are nearly as effective as treatment with the MTD of doxorubicin alone and significantly more effective than treatment with 1.25 mg/kg of doxorubicin alone or 2.5 mg/kg of doxorubicin alone. This further demonstrates the synergistic effect of the combination treatment of IV SalpNG.1 and 1.25 mg/kg doxorubicin, as well as demonstrates the synergistic effect of the combination treatment of IV SalpNG.1 and 2.5 mg/kg of doxorubicin.
The group that received a combination treatment of oral SalpIL2 and 1.25 mg/kg of doxorubicin (307) did not show any weight loss by day 14 and showed about a 10% weight gain by day 35. The group that received a combination treatment of IV SalpNG.1 and 1.25 mg/kg of doxorubicin (308) showed a slight weight loss by day 14 and greater than a 5% weight gain by day 35. This data further supports the synergistic effect of combination treatments of doxorubicin with attenuated S. typhimurium with minimal or no toxicity.
As explained with respect to
This application is a divisional of U.S. application Ser. No. 16/467,338, filed Jun. 6, 2019, and entitled “Methods of Synergistic Treatment of Cancer,” which was filed under 35 U.S.C. § 371 and claims priority to International PCT Application No. PCT/US2017/064813, filed on Dec. 6, 2017 and entitled “Methods of Synergistic Treatment of Cancer,” which claims benefit to U.S. Provisional Application No. 62/430,962, filed Dec. 7, 2016, the entire contents of all which are incorporated herein by reference.
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| Number | Date | Country | |
|---|---|---|---|
| 20240091275 A1 | Mar 2024 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62430962 | Dec 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16467338 | US | |
| Child | 18159934 | US |
