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US-12616723-B1 - Modified bacteria having improved pharmacokinetics and tumor colonization enhancing antitumor activity

US12616723B1US 12616723 B1US12616723 B1US 12616723B1US-12616723-B1

Abstract

Bacterial strains are provided having at least one of a reduced size, a sialic acid coat, inducibly altered surface antigens, and expression of PD-L1 or CTLA-4 antagonists and/or tryptophanase. The bacteria may have improved serum half-life, increased penetration into tumors, increased tumor targeting and increased antitumor activity. The bacteria are useful for delivery of therapeutic agents that treat neoplastic diseases including solid tumors and lymphomas.

Inventors

  • David Gordon Bermudes

Assignees

  • David Gordon Bermudes

Dates

Publication Date
20260505
Application Date
20231113

Claims (14)

  1. 1 . A tumor targeting, gram negative, lipid A mutant bacterium with suppressed virulence and suppressed tumor necrosis factor α (TNF-alpha) induction, which is genetically engineered for non-lethal administration to a mammal or human, comprising at least one inducible gene adapted to produce an antineoplastic active amino acid-degrading enzyme, wherein the bacterium is further genetically engineered to secrete a peptide which inhibits at least one of programmed cell death protein 1 (PD1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).
  2. 2 . The bacterium according to claim 1 , wherein the antineoplastic amino acid-degrading enzyme is effective to reduce levels of the amino acid in a tumor, and produce a toxic metabolite of the amino acid in the tumor.
  3. 3 . The bacterium according to claim 1 , wherein the antineoplastic amino acid-degrading enzyme comprises tryptophanase.
  4. 4 . The bacterium according to claim 1 , wherein the antineoplastic amino acid-degrading enzyme comprises tyrosinase.
  5. 5 . The bacterium according to claim 1 , wherein the antineoplastic amino acid-degrading enzyme comprises asparaginase.
  6. 6 . The bacterium according to claim 1 , wherein the at least one inducible gene has at least one of an arabinose promoter and an acetylsalicylic acid inducible promoter.
  7. 7 . The bacterium according to claim 1 , wherein the at least one inducible gene is heterologous to the bacterium.
  8. 8 . The bacterium according to claim 1 , wherein the bacterium is of a genus selected from the group consisting of Salmonella, Staphylococcus, Streptococcus, Listeria , Proprionibacteria, Escherechia, Bacteriodies, Bifidobacterium, Bacillus, Enterococcus, Neisseria, Shigella, Yersinia, Xenorhabdus, Photorhabdus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus , and Vibrio.
  9. 9 . The bacterium according to claim 1 , wherein the bacterium has a maximum size of growth of about 650 nm.
  10. 10 . The bacterium according to claim 1 , wherein the bacterium further comprises a second inducible gene which produces a heterologous flagellar antigen.
  11. 11 . The bacterium according to claim 1 , in an intravenous pharmaceutical dosage form.
  12. 12 . The bacterium according to claim 1 , further comprising a genetically engineered construct for transforming the tumor targeting bacterium with at least one gene encoding an inducible promoter lined to an active amino acid-degrading enzyme.
  13. 13 . The bacterium according to claim 1 , further comprising at least one gene adapted to produce a protease inhibitor.
  14. 14 . A tumor targeting, gram negative, lipid A mutant bacterium, which is genetically engineered for non-lethal administration to a mammal or human in an intravenous pharmaceutical dosage form, comprising at least one inducible gene adapted to produce an antineoplastic active amino acid-degrading enzyme, wherein the bacterium is further genetically engineered to secrete a peptide which inhibits at least one of programmed cell death protein 1 (PD1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

Description

CROSS REFERENCE TO RELATED APPLICATIONS The present application is a: Continuation of U.S. patent application Ser. No. 17/093,133, filed Nov. 9, 2020, now U.S. Pat. No. 11,813,295, issued Nov. 14, 2023, which is aU.S. patent application Ser. No. 16/983,854, filed Aug. 3, 2020, now U.S. Pat. No. 11,633,435, issued Apr. 23, 2023, which is aU.S. patent application Ser. No. 16/659,168, filed Oct. 21, 2019, now U.S. Pat. No. 10,729,731, issued Aug. 4, 2020, which is aContinuation of U.S. patent application Ser. No. 16/410,627, filed May 13, 2019, now U.S. Pat. No. 10,828,356, issued Nov. 10, 2020, which is aContinuation of U.S. patent application Ser. No. 15/679,874, filed Aug. 17, 2017, now U.S. Pat. No. 10,449,237, issued Oct. 22, 2019, which is aContinuation of U.S. patent application Ser. No. 15/482,170, filed Apr. 7, 2017, now U.S. Pat. No. 10,286,051, issued May 14, 2019, which is aDivisional of U.S. patent application Ser. No. 14/858,810, filed Sep. 18, 2015, now U.S. Pat. No. 9,616,114, issued Apr. 11, 2017, which is aNon-provisional of, and claims benefit of priority under 35 U.S.C. § 119(e) from, U.S. Provisional Patent Application No. 62/052,252, filed Sep. 18, 2014,the entirety of which are expressly incorporated herein by reference. SEQUENCE LISTING This application contains a Sequence Listing that has been filed electronically in the form of a XML file, created Nov. 13, 2023, and named “Magna-211-8.xml” and is 20,122 bytes in size, the contents of which are incorporated herein by reference in their entirety. 1. FIELD OF THE INVENTION This invention is generally in the field of therapeutic delivery systems utilizing live bacteria for the diagnosis and treatment of neoplastic disease. 2. BACKGROUND OF THE INVENTION Citation or identification of any reference herein, or any section of this application shall not be construed as an admission that such reference is available as prior art to the present application. The disclosures of each of these publications and patents are hereby incorporated by reference in their entirety in this application, and shall be treated as if the entirety thereof forms a part of this application. Such references are provided for their disclosure of technologies to enable practice of the present invention, to provide basis for claim language, to make clear applicant's possession of the invention with respect to the various aggregates, combinations, and subcombinations of the respective disclosures or portions thereof (within a particular reference or across multiple references). The citation of references is intended to be part of the disclosure of the invention, and not merely supplementary background information. The incorporation by reference does not extend to teachings which are inconsistent with the invention as expressly described herein, and is evidence of a proper interpretation by persons of ordinary skill in the art of the terms, phrase and concepts discussed herein, without being limiting as the sole interpretation available. Cancer or neoplastic diseases including solid tumors, lymphomas, leukemias or leukemic bone marrow, is a devastating condition of uncontrolled cell growth, which often has the ability to spread throughout the body (metastases) resulting in death. Tumor-targeted bacteria offer tremendous potential advantages for the treatment of solid tumors, including the targeting from a distant inoculation site and the ability to express therapeutic agents directly within the tumor (Pawelek et al., 1997, Tumor-targeted Salmonella as a novel anticancer agent, Cancer Research 57: 4537-4544; Low et al., 1999, Lipid A mutant salmonella with suppressed virulence and TNF-alpha induction retain tumor-targeting in vivo, Nature Biotechnol. 17: 37-41), each of which is expressly incorporated herein by reference in its entirety. The primary shortcoming of tumor-targeted bacteria investigated in the human clinical trials (Salmonella strain VNP20009 and its derivative TAPET-CD; Toso et al., 2002, Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma, J. Clin, Oncol. 20: 142-152; Meir et al., 2001, Phase 1 trial of a live, attenuated Salmonella Typhimurium (VNP20009) administered by direct Intra-tumoral (IT) injection, Proc Am Soc Clin Oncol 20: abstr 1043); Nemunaitis et al., 2003, Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients, Cancer Gene Therapy 10: 737-744, each of which is expressly incorporated herein by reference in its entirety) was that no significant antitumor activity was observed, even in patients where the bacteria was documented to target the tumor. In addition, an important factor was also that bacterial colonization of tumors, both in the form of the percentage of tumors that were colonized and amount of the bacteria that accumulated within the tumors, was usually lower compared to the preclinical s