Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election with traverse of Group I (Claims 1-16; drawn to a bacterial cell and pharmaceutical composition comprising the cell) in the reply filed on February 9, 2026, is acknowledged.
Claims 17-26 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention (Groups II), there being no allowable generic or linking claim.
Applicant further elected the following species with traverse:
a. An exogenous inducible promoter lined to an endogenous gene.
In light of the Applicant’s elected species, claim 11 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Response to Arguments
Applicant's arguments filed February 9, 2026, are acknowledged.
Applicant argues that Groups I and II should be rejoined as there would be no search burden for examination of Groups I and II together (page 6, paragraphs 6-7). Applicant argues that there wouldn’t be a search burden for a search of all the alleged species as the results of any such search would likely bear on all the alleged species (page 7, paragraph 2).
Applicant's arguments have been fully considered but they are not persuasive.
The restriction was not predicated on US practice (i. e. search burden). The application was filed under 371 practice so the criteria for restriction is lack of unity. The previous restriction, mailed December 9, 2025, recited the reasons for lack of unity.
DETAILED ACTION
The amended claims filed on February 9, 2026, have been acknowledged. In light of the Applicant’s elected invention and species, claims 11 and 17-26 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 1-10 and 12-16 are pending and examined on the merits.
Priority
The applicant claims domestic priority from U.S. provisional application No. 63/147,506, filed on February 9, 2021. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Claims 1-10 and 12-16 receive domestic benefit from U.S. provisional application No. 63/147,506, filed on February 9, 2021.
Information Disclosure Statement
The information disclosure statements (IDS) filed on August 10, 2023, December 13, 2023, December 8, 2025, and February 9, 2026, have been considered.
Claim Objections
Claim 9 is objected to because of the following informalities:
In line 1, “The cell claim 1”, should read “The cell of claim 1”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-10 and 12-16 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the term “intracellularly induced Salmonella promoter” and claim 6 further specifies that "the intracellularly induced Salmonella promoter is a promoter for one of the genes in Salmonella pathogenicity island 2 type III secretion system (SPI2-T3 SS) …”. However, the specification does not provide a definition of the term "intracellularly induced Salmonella promoter". According to the instant specification, the Pssej promoter intracellular activity was four times greater than extracellular activity and PsifA also exhibited intracellular and extracellular activity (page 7, paragraph 2 and Figure 2A). Claim 6 lists both SseJ and SifA as intracellularly induced Salmonella promoters. Therefore, it is difficult to determine the what the metes and bounds of a Salmonella promoter that falls within the scope of the "intracellularly induced Salmonella promoter''. Based on the results with SifA and SseJ promoter in the instant specification, it is unclear whether the term ''intracellularly induced Salmonella promoter" can be interpreted to mean a Salmonella promoter that is specifically activated intracellularly or if it only encompasses promoters that exhibit intracellular and extracellular activity and increased intracellular activity compared to extracellular activity as seen with SifA and SseJ. Accordingly, the term intracellularly induced Salmonella promoter lacks clarity.
Claims 2-10 and 12-16 are also rejected because of their dependency on claim 1.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-6 and 13-16 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No: 20170333490 (Forbes) further in view of Ohlson et al. (Infection and Immunity 73: 6249-6259. 2005).
Forbes teaches a bacterial cell comprising a lysis gene or lysis cassette operably linked to an intracellularly induced Salmonella promoter. Forbes teaches that their bacterial cell is used as a platform for controlled gene and protein deliver into cancer cells to treat cancer (claim 1 and paragraphs 0006-0020, 0088-0091, and 0123-0124). Forbes teaches that the bacterial cell can further comprise a sifA deletion (claim 2).
Forbes does not teach wherein the bacterial cell further comprises deletion of SseJ.
However, Ohlson teaches that full virulence requires SseJ, as ΔsseJ mutants are mildly defective for intracellular replication in cultured cells and have a virulence defect in the mouse infection model. SseJ is one of only a few SPI2 effector proteins, including SifA, that lead to a virulence phenotype when deleted, which underscores the importance of this protein to Salmonella pathogenesis (abstract and page 6249, column 2, paragraph 2-page 6250, column 1, paragraph 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the bacterial cell of Forbes by deleting SseJ, as identified by Ohlson, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Ohlson teaches that SseJ deletion reduces the virulence of the bacterial cell. Furthermore, Forbes teaches that their bacterial cell can further comprise deletion of SifA, which is also known to reduce virulence when deleted. Therefore, it would have been obvious that the bacterial cell of Forbes could be modified to include an SseJ deletion as Forbes already contemplates a SifA deletion which is known to lead to a similar phenotype, reduced virulence, as the SseJ mutation, and reduced virulence is an important consideration for treating cancer. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 2, Forbes teaches that the bacterial cell is an intratumoral bacterial cell (paragraphs 0006-0040).
Regarding claims 3-4, Forbes teaches that the bacterial cell can be a Salmonella cell (paragraph 0088 and claim 1).
Regarding claim 5, Forbes teaches that the lysis cassette is Lysin E from phage phiXl 74, the lysis cassette of phage iEPS5, or the lysis cassette from lambda phage (claim 18).
Regarding claim 6, Forbes teaches that the intracellularly induced promoter is a promoter for one of the genes in Salmonella pathogenicity island 2 type III secretion system (SPI2-T3SS) selected from the group SpiC/SsaB, SseF, SseG, SseI, SseJ,
SseKl, SseK2, SifA, SifB, PipB, Pip 82, SopD2, GogB, SseL, SteC, SspHl, SspH2, or SirP (claim 3).
Regarding claims 13-15, Forbes teaches that the cell can comprise a plasmid that encodes a therapeutic peptide, such as amino acids 143-224 of NIPP1 (paragraph 0013 and claims 7-10).
Regarding claim 16, Forbes teaches that the bacterial cell can be part of a pharmaceutical composition (paragraph 0020).
Claims 1 and 7-8 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No: 20170333490 (Forbes) further in view of Ohlson et al. (Infection and Immunity 73: 6249-6259. 2005) as applied to claim 1 above, and further in view of Gauger et al. (Infection and Immunity 75: 3315-3324. 2007) and United States Patent Application No. 20200270613 (Thanos).
The teachings of Forbes and Ohlson are as discussed above.
Forbes and Ohlson do not teach wherein the bacterial cell does not comprise endogenous flhDC.
However, Gauger teaches that bacteria with flhDC deletions grew 10 to 30% faster. The deletions appear to be selected for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by flhDC but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth (whole document).
Thanos teaches that it has been shown that selective inhibition of QseC by LED209 inhibits bacterial virulence without suppressing S. typhimurium growth, by inhibiting the QseC-mediated activation of virulence-related gene expression (e.g.,flhDC, sifA and sopB), and partially protects mice from death following infection with S. typhimurium or Francisella tularensis. QseC blockade was found to inhibit caspase-1 activation, IL-1β release, and S. typhimurium-induced pyroptosis of macrophages, by inhibiting excess inflammasome activation in the infected macrophages. Inhibition of QseC also suppresses flagellar gene expression and motility, and suppresses the invasion and replication capacities of S. typhimurium in epithelial cells. Thus, modification of the bacteria, to mutate or knockout the gene encoding QseC, can enhance the anti-tumor immune response by focusing S. typhimurium infection to non-epithelial cells, and by reducing cell death in immune
cells, such as by preventing pyroptosis in macrophages (paragraph 0487).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the bacterial cell of the combined teachings of Forbes and Ohlson by further deleting the flhDC gene, as identified by Gauger, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Gauger teaches that flhDC deletions grew 10 to 30% faster and Thanos teaches that reducing flhDC expression through QseC inhibition inhibits bacterial virulence without suppressing Salmonella typhimurium growthInhibition of QseC also suppresses flagellar gene expression and motility and can enhance the anti-tumor immune response. Therefore, it would have been obvious to further modify the bacterial cells of Forbes and Ohlson to improve cell growth and anti-tumor response. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1, 9-10, and 12 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No: 20170333490 (Forbes) further in view of Ohlson et al. (Infection and Immunity 73: 6249-6259. 2005) as applied to claim 1 above, and further in view of Raman et al. (Journal of ImmunoTherapy of Cancer 7: 1-16. 2019) and Stritzker et al. (International Journal of Medical Microbiology 300: 449–456. 2010).
The teachings of Forbes and Ohlson are as discussed above.
Forbes and Ohlson do not teach wherein the bacterial cell comprises an exogenous inducible promoter operably linked to an endogenous flhDC.
However, Raman teaches that overexpression of flhDC using an inducible arabinose promoter (PBAD) increased intracellular accumulation and tumor colonization 2.5 and 5 times more than control Salmonella, respectively. When flhDC was selectively induced after penetration into tumor masses, Salmonella both accumulated intracellularly and colonized tumor masses 2 times more than controls. Mathematical modeling of tumor colonization dynamics demonstrated that intracellular accumulation increased retention of Salmonella in tumors by effectively causing the bacteria to bind to cancer cells and preventing leakage out of the tumors. These results demonstrated that increasing intracellular bacterial density increased overall tumor colonization and that flhDC could be used to control both (abstract and Table 1).
Raman teaches that Induction of flhDC increased the accumulation inside cancer cells. Controlling intracellular accumulation by inducing flhDC would increase tumor colonization. It would be beneficial to suppress flagellar expression outside of tumors. Flagella biosynthesis is an energetically costly process and can consume as much as 2% of bacterial energy. In addition , Salmonella flagellin is an immunogenic agonist that facilitates accelerated bacterial clearance. Inducing flhDC selectively after initial penetration into tumors would improve fitness prior to administration, while promoting invasion and colonization within tumors (Fig. 7, and page 13, column 1, paragraph 2-page 14, column 2, paragraph 1)
Stritzker teaches that motility does not play a significant role in tumor colonization
and bacterial distribution within the tumor. Rather, the whole colonization and intratumoral migration process seems to be a passive mechanism that is influenced by the reticuloendothelial system of the host, by the tumor microenvironment and by the bacterial metabolism. These conclusions were supported by experimental data demonstrating that disruption of the basic branch of the aromatic amino acid biosynthetic pathway and depletion of macrophages, in contrast to flagellar mutations, led to significant changes in bacterial accumulation in tumors of live mice (whole document). As can be seen in Figure 2, motility deficient mutants of Salmonella enterica serovar Typhimurium showed the same tumor colonization compared to wild type bacteria.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the bacterial cell of the combined teachings of Forbes and Ohlson by expressing the endogenous flhDC gene under an exogenous PBAD arabinose inducible promoter, as identified by Raman, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Raman specifically identifies that it would be beneficial to suppress flagellar expression outside of tumors as flagella biosynthesis is an energetically costly process and can consume as much as 2% of bacterial energy. In addition , Salmonella flagellin is an immunogenic agonist that facilitates accelerated bacterial clearance. Inducing flhDC selectively after initial penetration into tumors would improve fitness prior to administration, while promoting invasion and colonization within tumors. Furthermore, Stritzker identifies that motility does not play a significant role in tumor colonization and bacterial distribution within the tumor as motility deficient mutants of Salmonella enterica serovar Typhimurium showed the same tumor colonization compared to wild type bacteria. Therefore, it would have been obvious to express the endogenous flhDC gene under the control of an exogenous PBAD arabinose inducible promoter to improve fitness prior to administration, while promoting invasion and colonization within tumors and Stritzker has already shown that lack of motility (which would occur outside the tumor without flhDC expression) did not impact tumor colonization. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-10, 13-14, and 16 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6, 9-14, 18-21, 28-29, and 32-33 of copending Application No. 18851628 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding claim 1, ‘628 claims a non-pathogenic bacterial cell expressing an exogenous immunogenic protein intracellularly, wherein the cell comprises a lysis gene or lysis cassette operably linked to an intracellularly induced Salmonella promoter, wherein expression of SseJ has been reduced (claims 1 and 6).
Regarding claim 2, ‘628 claims the bacterial cell is an intratumoral bacteria cell (claim 9).
Regarding claims 3-4, ‘628 claims the bacterial cell is a Clostridium, Bifidus, Escherichia coli or Salmonella cell (claim 10).
Regarding claim 5, ‘628 claims the lysis cassette is Lysin E from phage phiX174, the lysis cassette of phage iEPS5, or the lysis cassette from lambda phage (claim 11).
Regarding claim 6, ‘628 claims the intracellularly induced Salmonella promoter is for one of the genes in Salmonella pathogenicity island 2 type III secretion system (SPI2-T3SS) selected from the group SpiC/SsaB, SseF, SseG, SseI, SseJ, SseKl, SseK2, SifA, SifB, PipB, PipB2, SopD2, GogB, SseL, SteC, SspHl, SspH2, or SirP (claim 12).
Regarding claims 7-8, ‘628 claims wherein the cell does not comprise endogenous flhDC (claim 13).
Regarding claims 9-10, ‘628 claims wherein the cell comprises an exogenous inducible promoter operably linked to an endogenous flhDC (claim 14).
Regarding claims 13-14, ‘628 claims wherein the expressed protein is coded for by an expression plasmid and claims multiple therapeutic protein (claims 2-4).
Regarding claim 16, ‘628 claims a composition comprising a population of cells of claim 1 and a pharmaceutically acceptable carrier (claim 18).
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1 and 12 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6, 9-14, 18-21, 28-29, and 32-33 of copending Application No. 18851628 (reference application) in view of Raman et al. (Journal of ImmunoTherapy of Cancer 7: 1-16. 2019).
The claims of ‘628 are as discussed above.
‘628 is silent regarding the exogenous inducible promoter.
However, Raman teaches that overexpression of flhDC using an inducible arabinose promoter (PBAD) increased intracellular accumulation and tumor colonization 2.5 and 5 times more than control Salmonella, respectively. When flhDC was selectively induced after penetration into tumor masses, Salmonella both accumulated intracellularly and colonized tumor masses 2 times more than controls. Mathematical modeling of tumor colonization dynamics demonstrated that intracellular accumulation increased retention of Salmonella in tumors by effectively causing the bacteria to bind to cancer cells and preventing leakage out of the tumors. These results demonstrated that increasing intracellular bacterial density increased overall tumor colonization and that flhDC could be used to control both (abstract and Table 1).
Raman teaches that Induction of flhDC increased the accumulation inside cancer cells. Controlling intracellular accumulation by inducing flhDC would increase tumor colonization. It would be beneficial to suppress flagellar expression outside of tumors. Flagella biosynthesis is an energetically costly process and can consume as much as 2% of bacterial energy. In addition , Salmonella flagellin is an immunogenic agonist that facilitates accelerated bacterial clearance. Inducing flhDC selectively after initial penetration into tumors would improve fitness prior to administration, while promoting invasion and colonization within tumors (Fig. 7, and page 13, column 1, paragraph 2-page 14, column 2, paragraph 1).
Therefore, it would have been obvious to one of ordinary skill in the art that the PBAD arabinose inducible promoter could be used because it has previously been used to induce flhDC expression with improved tumor colonization, a similar goal of ‘628 (see claim 9) and inducing flhDC selectively after initial penetration into tumors would improve fitness prior to administration, while promoting invasion and colonization within tumors. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
This is a provisional nonstatutory double patenting rejection.
Claims 1 and 15 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6, 9-14, 18-21, 28-29, and 32-33 of copending Application No. 18851628 (reference application) in view of United States Patent Application No: 20170333490 (Forbes).
The claims of ‘628 are as discussed above.
‘628 does not teach wherein the therapeutic peptide is NIPP1.
However, Forbes teaches a bacterial cell comprising a lysis gene or lysis cassette operably linked to an intracellularly induced Salmonella promoter. Forbes teaches that their bacterial cell is used as a platform for controlled gene and protein deliver into cancer cells to treat cancer (claim 1 and paragraphs 0006-0020, 0088-0091, and 0123-0124). Forbes teaches that the cell can comprise a plasmid that encodes a therapeutic peptide, such as amino acids 143-224 of NIPP1 (paragraph 0013 and claims 7-10). Forbes teaches that bacterial delivery of a NIPP1-based polypeptide induces cancer cell death (paragraph 0030).
Therefore, it would have been obvious to one of ordinary skill in the art that the therapeutic protein could be NIPP1 because Forbes teaches a similar bacterial cell that is used to induce cancer cell death and has identified that expressing NIPP1 induces cancer cell death and ‘628 directly claims that their cell can be intratumoral and wants to use their cell to treat cancer (claim 21). Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
This is a provisional nonstatutory double patenting rejection.
Claims 1-6 and 13-16 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-14 of U.S. Patent No. 11103538 in view of Ohlson et al. (Infection and Immunity 73: 6249-6259. 2005).
Regarding claims 1-4, and 6, ‘538 claims an attenuated Salmonella strain comprising a lysis gene or cassette operably linked to an intracellularly induced Salmonella promoter, wherein the intracellularly induced Salmonella promoter induces lysis of the Salmonella strain when in mammalian cytosol without an extracellular inducer agent, wherein the promoter is selected from the group consisting of SseJ, SseK2, SifB, SopD2, SseL or SteC. ‘538 claims that the bacterial cell can further comprise a sifA deletion (claim 2). ‘538 claims that their bacterial cell can be used to treat cancer (claims 12-15).
‘538 does not teach wherein the bacterial cell further comprises deletion of SseJ.
However, Ohlson teaches that full virulence requires SseJ, as ΔsseJ mutants are mildly defective for intracellular replication in cultured cells and have a virulence defect in the mouse infection model. SseJ is one of only a few SPI2 effector proteins, including SifA, that lead to a virulence phenotype when deleted, which underscores the importance of this protein to Salmonella pathogenesis (abstract and page 6249, column 2, paragraph 2-page 6250, column 1, paragraph 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the bacterial cell of ‘538 by deleting SseJ, as identified by Ohlson, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Ohlson teaches that SseJ deletion reduces the virulence of the bacterial cell. Furthermore, ‘538 claims that their bacterial cell can further comprise deletion of SifA, which is also known to reduce virulence when deleted. Therefore, it would have been obvious that the bacterial cell of ‘538 could be modified to include an SseJ deletion as ‘538 already contemplates a SifA deletion which is known to lead to a similar phenotype, reduced virulence, as the SseJ mutation, and reduced virulence is an important consideration for treating cancer. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 5, ‘538 claims that the lysis cassette is Lysin E from phage phiXl 74, the lysis cassette of phage iEPS5, or the lysis cassette from lambda phage (claim 16).
Regarding claims 13-15, ‘538 claims that the cell can comprise a plasmid that encodes a therapeutic peptide, such as amino acids 143-224 of NIPP1 (claims 6-9).
Regarding claim 16, ‘538 claims that the bacterial cell can be part of a pharmaceutical composition (claim 11).
Claims 1 and 7-8 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-14 of U.S. Patent No. 11103538 in view of Ohlson et al. (Infection and Immunity 73: 6249-6259. 2005) as applied to claim 1 above, and further in view of Gauger et al. (Infection and Immunity 75: 3315-3324. 2007) and United States Patent Application No. 20200270613 (Thanos).
The teachings of ‘538 and Ohlson are as discussed above.
‘538 and Ohlson do not teach wherein the bacterial cell does not comprise endogenous flhDC.
However, Gauger teaches that bacteria with flhDC deletions grew 10 to 30% faster. The deletions appear to be selected for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by flhDC but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth (whole document).
Thanos teaches that it has been shown that selective inhibition of QseC by LED209 inhibits bacterial virulence without suppressing S. typhimurium growth, by inhibiting the QseC-mediated activation of virulence-related gene expression (e.g.,flhDC, sifA and sopB), and partially protects mice from death following infection with S. typhimurium or Francisella tularensis. QseC blockade was found to inhibit caspase-1 activation, IL-1β release, and S. typhimurium-induced pyroptosis of macrophages, by inhibiting excess inflammasome activation in the infected macrophages. Inhibition of QseC also suppresses flagellar gene expression and motility, and suppresses the invasion and replication capacities of S. typhimurium in epithelial cells. Thus, modification of the bacteria, to mutate or knockout the gene encoding QseC, can enhance the anti-tumor immune response by focusing S. typhimurium infection to non-epithelial cells, and by reducing cell death in immune
cells, such as by preventing pyroptosis in macrophages (paragraph 0487).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the bacterial cell of the combined teachings of ‘538 and Ohlson by further deleting the flhDC gene, as identified by Gauger, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Gauger teaches that flhDC deletions grew 10 to 30% faster and Thanos teaches that reducing flhDC expression through QseC inhibition inhibits bacterial virulence without suppressing Salmonella typhimurium growthInhibition of QseC also suppresses flagellar gene expression and motility and can enhance the anti-tumor immune response. Therefore, it would have been obvious to further modify the bacterial cells of ‘538 and Ohlson to improve cell growth and anti-tumor response. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1, 9-10, and 12 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-14 of U.S. Patent No. 11103538 in view of Ohlson et al. (Infection and Immunity 73: 6249-6259. 2005) as applied to claim 1 above, and further in view of Raman et al. (Journal of ImmunoTherapy of Cancer 7: 1-16. 2019) and Stritzker et al. (International Journal of Medical Microbiology 300: 449–456. 2010).
The teachings of ‘538 and Ohlson are as discussed above.
‘538 and Ohlson do not teach wherein the bacterial cell comprises an exogenous inducible promoter operably linked to an endogenous flhDC.
However, Raman teaches that overexpression of flhDC using an inducible arabinose promoter (PBAD) increased intracellular accumulation and tumor colonization 2.5 and 5 times more than control Salmonella, respectively. When flhDC was selectively induced after penetration into tumor masses, Salmonella both accumulated intracellularly and colonized tumor masses 2 times more than controls. Mathematical modeling of tumor colonization dynamics demonstrated that intracellular accumulation increased retention of Salmonella in tumors by effectively causing the bacteria to bind to cancer cells and preventing leakage out of the tumors. These results demonstrated that increasing intracellular bacterial density increased overall tumor colonization and that flhDC could be used to control both (abstract and Table 1).
Raman teaches that Induction of flhDC increased the accumulation inside cancer cells. Controlling intracellular accumulation by inducing flhDC would increase tumor colonization. It would be beneficial to suppress flagellar expression outside of tumors. Flagella biosynthesis is an energetically costly process and can consume as much as 2% of bacterial energy. In addition , Salmonella flagellin is an immunogenic agonist that facilitates accelerated bacterial clearance. Inducing flhDC selectively after initial penetration into tumors would improve fitness prior to administration, while promoting invasion and colonization within tumors (Fig. 7, and page 13, column 1, paragraph 2-page 14, column 2, paragraph 1)
Stritzker teaches that motility does not play a significant role in tumor colonization
and bacterial distribution within the tumor. Rather, the whole colonization and intratumoral migration process seems to be a passive mechanism that is influenced by the reticuloendothelial system of the host, by the tumor microenvironment and by the bacterial metabolism. These conclusions were supported by experimental data demonstrating that disruption of the basic branch of the aromatic amino acid biosynthetic pathway and depletion of macrophages, in contrast to flagellar mutations, led to significant changes in bacterial accumulation in tumors of live mice (whole document). As can be seen in Figure 2, motility deficient mutants of Salmonella enterica serovar Typhimurium showed the same tumor colonization compared to wild type bacteria.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the bacterial cell of the combined teachings of ‘538 and Ohlson by expressing the endogenous flhDC gene under an exogenous PBAD arabinose inducible promoter, as identified by Raman, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Raman specifically identifies that it would be beneficial to suppress flagellar expression outside of tumors as flagella biosynthesis is an energetically costly process and can consume as much as 2% of bacterial energy. In addition , Salmonella flagellin is an immunogenic agonist that facilitates accelerated bacterial clearance. Inducing flhDC selectively after initial penetration into tumors would improve fitness prior to administration, while promoting invasion and colonization within tumors. Furthermore, Stritzker identifies that motility does not play a significant role in tumor colonization and bacterial distribution within the tumor as motility deficient mutants of Salmonella enterica serovar Typhimurium showed the same tumor colonization compared to wild type bacteria. Therefore, it would have been obvious to express the endogenous flhDC gene under the control of an exogenous PBAD arabinose inducible promoter to improve fitness prior to administration, while promoting invasion and colonization within tumors and Stritzker has already shown that lack of motility (which would occur outside the tumor without flhDC expression) did not impact tumor colonization. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Conclusion
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/KEENAN A BATES/Examiner, Art Unit 1631
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