DETAILED ACTION
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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 05/27/2025 has been entered.
Status of the claims
Claims 1-2, 4-7, 10-14, and 18-25 are pending; claims 3, 8-9, and 15-17 are cancelled; claims 1, 4, and 21 are amended; claim 25 is new. Claims 1-2, 4-7, 10-14, and 18-25 are examined below.
Priority
The present application was filed 10/19/2020. Acknowledgement is made of the present application as a proper National Stage (371) entry of PCT Application No. PCT/US2019/029066 filed 04/25/2019, which claims benefit to provisional application PRO 62/663,363 filed 04/27/2018.
Information Disclosure Statement
The information disclosure statement (IDS) filed 05/27/2025 has been considered, initialed and is attached hereto.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-2, 4-7, 10-14, and 18-25 are rejected under 35 U.S.C. 101 because the claimed invention is directed to abstract ideas without significantly more.
The U.S. Patent and Trademark Office recently revised the MPEP with regard to § 101 (see MPEP at 2106). Regarding the MPEP at 2106, in determining what concept the claim is “directed to” we first look to whether the claim recites:
Any judicial exceptions, including certain groupings of abstract ideas (i.e. mathematical concepts, certain methods of organizing human activity such as a fundamental economic practice, or mental processes); and
Additional elements that integrate the judicial exception into a practical application (see MPEP § 2106.05(a)-(c), (e)-(h)).
Only if a claim (1) recites a judicial exception and (2) does not integrate that exception into a practical application, do we then look to whether the claim contains an “’inventive concept’ sufficient to ‘transform’” the claimed judicial exception into a patent eligible application of the judicial exception. Alice, 573 U.S.at 2221 (quoting Mayo, 566 U.S. at 82). In so doing, we thus consider whether the claim:
Adds a specific limitation beyond the judicial exception that is not “well-understood, routine, conventional” in the field (see MPEP § 2106.05(d); or
Simply appends well-understood, routine, conventional activities previously known to the industry, specified at a high level of generality, to the judicial exception.
See MPEP 2106.
ELIGIBILITY STEP 2A: WHETHER A CLAIM IS DIRECTED TO A JUDICIAL EXCEPTION
Step 2A, Prong 1
Regarding claims 1, 22, and 25, the claims recite “wherein an increase in the cytokine production indicates expression of the disease-associated antigenic peptide in the infected APCs” (claims 1 and 25), “wherein an increase in IFNγ production indicates expression of the HPV 16 protein E7 tumor antigen” (claim 22). The language “wherein an increase” encompasses a comparison between a sample with a lower and a sample with an increase in the cytokine/IFNγ. The comparison step represents an abstract idea. Similar concepts involving comparing information regarding a sample or test subject to a control or target data have been held to be an "abstract mental process", as in University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 113 USPQ2d 1241 (Fed. Cir. 2014) which involved "comparing BRCA sequences and determining the existence of alterations", the collecting and comparing of known information in Classen, the comparing information regarding a sample or test subject to a control or target data in Ambry and Myriad CAFC, as well as Mayo (which also involved specific numerical cutoff levels). The claims, under broadest reasonable interpretation, cover performance of this step solely within the human mind.
Step 2A, Prong 1
The above discussed limitation wherein an increase in cytokine/IFNγ production indicates expression of a disease associated antigen, a step which encompasses a comparison, is insufficient to integrate into a practical application. Specifically, steps/limitations that are themselves the judicial exception(s) are not a practical application thereof.
Regarding the additional recited steps/limitations, claims 1, 22, and 25 further recite infecting APCs with the recombinant Listeria-based immunotherapeutic, culturing the infected APCs, and co-culturing the infected APCs with a population of T cells, claims 12 and 13 further recites “wherein determining a cytokine expression profile […] comprises measuring the level of IFNγ”, claim 22 further recites “washing the THP-1 cells”.
These are steps necessarily performed in order to determine the measured values, i.e. necessary data gathering steps (i.e., extra-solution activity) performed so that the comparison step can be performed. The purpose of these additional limitations is merely to obtain the data used for the mathematical concepts (for gathering the input). As such, these limitations fail to amount to an integration into a practical application. None of these recited steps/elements, apply, rely on or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception.
ELIGIBILITY STEP 2B: WHETER THE ADDITIONAL ALEMENTS CONTRIBUTE AN “INVENTIVE CONCEPT”
The additional elements of the claims (the active step method steps/limitations recited in addition to the judicial exceptions themselves) do not add significantly more to the judicial exception(s); the additional recited claim elements are recited at a high level of generality, and are not, for example limited to any particular machine or apparatus as claimed. See for example Sinnathamby et al. Priming and activation of human ovarian and breast cancer-specific CD8+ T cells by polyvalent Listeria monocytogenes-based vaccines. Journal of immunotherapy. 2009 Oct 1;32(8):856-69 (see PTO-892 05/25/2023), as cited in more detail below, teaches in vitro stimulation of peripheral blood mononuclear cells from a healthy donor using various ovarian cancer epitopes and comparing the number of IFNγ positive cells in cultures (Sinnathamby, page 861, Figure 3). Sinnathamby teaches an ELISpot assay to assess antigen stimulated IFNγ release (Sinnathamby, page 859, ‘ELISpot Assay’, lines 1-3). Geginat et al. Suppression of Acquired Immunity against Listeria monocytogenes by Amphotericin B-Mediated Inhibition of CD8 T Cell Function. The Journal of infectious diseases. 1999 Oct 1;180(4):1186-94 similarly teaches coculturing antigen presenting cells infected with Listeria monocytogenes with antigen specific CD8 T cells and measuring IFNγ concentration in the supernatants by ELISA (Geginat, page 1187, 2nd paragraph, lines 1-13). Letsch et al. Quantification and characterization of specific T-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining. Methods. 2003 Oct 1;31(2):143-9 (see PTO-892 05/25/2023) teaches quantification and characterization of specific T cells using an ELISPOT assay or intracellular cytokine staining (Letsch, see title). Letsch teaches intracellular cytokine staining and ELISPOT assay to detect T cells secreting IFNγ (Letsch, page 145, 2nd paragraph, lines 9-12).
In view of the above evidence, the claimed steps of measuring IFNγ by T cells do not add any feature that is more than well-understood, purely conventional, or routine in the field of diagnostics and biochemical assay methodologies.
There is no additional limitation, such as a particular or unconventional machine or a transformation of a particular article, in these steps that distinguishes them from well-understood, routine, and conventional activity previously engaged in by scientists prior to applicant’s invention, and at the time the application was filed, e.g., the recited steps/combination of steps are all routine and conventional techniques of detecting a protein using an automated immunoassay/mass spectrometry. See also MPEP 2106.05(g).
For all these reasons, the claims fail to include additional elements that are sufficient to amount to significantly more than the judicial exception(s).
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4-6, 12, and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Sinnathamby et al. Priming and activation of human ovarian and breast cancer-specific CD8+ T cells by polyvalent Listeria monocytogenes-based vaccines. Journal of immunotherapy. 2009 Oct 1;32(8):856-69 (see PTO-892 05/25/2023), in view of Petit et al., US PGPUB 2016/0220652 A1 (see PTO-892 05/25/2023), Drevets et al. Gentamicin kills intracellular Listeria monocytogenes. Infection and immunity. 1994 Jun;62(6):2222-8 (see PTO-892 02/20/2024), Badovinac et al. Intracellular staining for TNF and IFN-γ detects different frequencies of antigen-specific CD8+ T cells. Journal of immunological methods. 2000 Apr 21;238(1-2):107-17 (see PTO-892, 05/25/2023), and Miller et al. Attenuated Listeria monocytogenes vectors overcome suppressive plasma factors during HIV infection to stimulate myeloid dendritic cells to promote adaptive immunity and reactivation of latent virus. AIDS Research and Human Retroviruses. 2015 Jan 1;31(1):127-36 (see PTO-892, 05/25/2023).
Regarding Claim 1, Sinnathamby teaches a method of assessing potency of a Listeria-based immunotherapeutic (Sinnathamby, Summary, lines 7-10). Sinnathamby teaches infecting antigen presenting cells (APCs), specifically THP-1 cells, with a recombinant Listeria-based immunotherapeutic (Sinnathamby, Summary, lines 10-14). Sinnathamby further teaches comparing peptide pulsed APC with those APC infected with the Listeria-based immunotherapeutic, showing that the magnitude of the IFNγ+ T cell response is much lower when Listeria monocytogenes specific T cells are stimulated with peptide-pulsed APCs compared to being stimulated with APC infected with the recombinant Listeria construct, therefore determining a difference in potency (Sinnathamby, page 861, column 2, lines 7-14). Sinnathamby further teaches a Listeria-based immunotherapeutic comprising one or more ovarian cancer epitopes (disease associated antigenic peptide) derived from epitopes bound by HLA-A2 molecules and presented by ovarian cancer cells. Sinnathamby further teaches incubating (co-culturing) infected APCs with ovarian epitope-specific CD8 T cells that were generated in vitro from healthy donors and ovarian cancer patients (enriched for T cells having reactivity to the disease-associated antigenic peptide; Sinnathamby, page 857, 2nd column, lines 9-15). Sinnathamby further teaches characterizing a set of ovarian cancer-specific T cell epitopes delivered by live-attenuated recombinant Listeria monocytogenes (Lm; Sinnathamby, Summary, lines 7-9). Sinnathamby further teaches infecting the THP-1 cells with a multiplicity of infection of 10 (Sinnathamby, page 859, lines 8-9), a value that falls within the claimed range of 1-200, and therefore addresses the claim. Sinnathamby further teaches in vitro stimulation of peripheral blood mononuclear cells from a healthy donor using various ovarian cancer epitopes and comparing the number of IFNγ positive cells in cultures stimulated with antigen presenting cells infected with recombinant listeria monocytogenes (constructs expressing six different cancer antigens respectively) against cultures comprising uninfected antigen presenting cells (Sinnathamby, page 861, Figure 3).
Sinnathamby further teaches that to ascertain expression, appropriate processing and presentation of individual ovarian cancer epitopes from recombinant Listeria-based immunotherapeutics, one can use Listeria infected APC and use them as targets in ELISpot assays, measuring IFNγ production (determine cytokine production by T cells as indicating expression of disease-associated antigenic peptide; Sinnathamby, page 861, column one, lines 10-13, and Figure 3). Further, Sinnathamby teaches IFNγ production by T cells co-cultured with APC infected with a Listeria vector comprising a cancer antigen, but that T cells co-cultured with naïve APC (not infected) only secrete very low levels of IFNγ, demonstrating that there is an increase in cytokine production when the disease-associated antigenic peptide is presented by the infected APC (increase in cytokine production indicates expression of the disease-associated antigenic peptide in infected APC; Sinnathamby, Figure 2 and see ‘Results’, see entire 1st paragraph). Sinnathamby teaches using a fixed number of target cells, namely a concentration of 5000 cells/well (Sinnathamby, page 859, ‘ELISpot Assays’, lines 6-7). Sinnathamby teaches a using Listeria monocytogenes infected APC in ratios of APCs to T cells of 1:40, 1:20, and 1:10 and then measure cytokine production by T cells with an ELISpot assay (Sinnathamby, page 859, ‘ELISpot Assays’, lines 1-8).
Sinnathamby fails to teach that the Listeria monocytogenes strain comprises a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to the disease-associated antigenic peptide and fails to teach culturing the infected APCs in the absence of gentamicin. Sinnathamby further fails to teach a ratio of APCs to T cells that is 1:1 or 4:1.
Petit teaches a method of inducing an immune response against a disease using a recombinant Listeria monocytogenes strain (Petit, page 2, paragraph [0028], lin1-5). Petit teaches that the Listeria-based immunotherapeutic comprises a PEST-sequence, comprised in the LLO sequence (Petit, page 5, paragraph [0051], lines 23-24). Petit further teaches a Listeria strain that comprises a nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein (PEST sequence) fused to a heterologous antigen (disease-associated antigenic peptide), which causes an anti-tumor immune response (Petit, page 2, paragraph [0029], lines 10-20). Petit further teaches that Listeria monocytogenes constructs containing PEST regions induce a higher percentage of specific lymphocytes within a tumor (Petit, page 2, see entire paragraph [0021] and figure 6) and that fusing the antigen to the PEST containing sequence enhances cell mediated and anti-tumor immunity of the antigen (Petit et al., page 11, paragraph [0117], lines 19-21).
Drevets teaches infecting antigen presenting cells (mouse peritoneal macrophages) with Listeria monocytogenes, followed by washing the infected cells and resuspending them in a balanced salt solution (BSS) with 5% normal mouse serum (Drevets, page 223, ‘Bactericidal assay’, lines 1-10). Drevets further teaches incubating peritoneal macrophages for an additional 20-30 min and that after phagocytosis of bacteria, all cells were incubated in the absence or presence of various concentrations of gentamicin for a total of 60 min (Drevets, page2223, ‘Bactericidal assay’, 2nd paragraph, lines 1-12 and see Figure1, white squares). Drevets teaches that gentamicin causes normally nonbactericidal macrophages that had phagocytosed Listeria monocytogenes to kill Listeria monocytogenes (Drevets, page 2222, ‘Abstract’, lines 3-6). Drevets further teaches that the use of antibiotics in bactericidal assays is common, but controversial because antibiotics have numerous effects on both the phagocyte and bacteria and that in the case of Listeria monocytogenes the interactions between host, parasite, and antibiotic are even more complex (Drevets, page 2227, ‘Discussion’, lines 1-5).
Badovinac teaches a transfected APC that displays the Listeria antigen LLO on MHC class I molecules (Badovinac, page 108, see ‘2.2 Cell lines and cell culture’). Badovinac further teaches stimulating CD8 T cell lines specific for that antigen using 3 x 106 responder cells and 3 x 106 irradiated stimulator cells, a ratio of 1:1 T cell to APC (Badovinac, page 108, ‘2.3. Generation and maintenance of CD8 T cell lines’, lines 8-10) and further using the above-described method to stimulate T cells for intracellular cytokine staining (Badovinac, page 109, ‘2.5. Intracellular cytokine staining of CD8 T cell lines and splenocytes from infected mice’, see second paragraph).
Miller teaches infecting APC with a mutant Listeria monocytogenes vector (Miller, page 129, lines 1-2). Miller further teaches co-culturing T cells and APCs at a ratio of 5:1 and then assaying IFNγ in the supernatant (Miller, page 129, see ‘Naïve CD4 T cell coculture’) or by intracellular cytokine staining in response to peptide stimulation (Miller, page 129, ‘Expansion of gag-specific CD4 and CD8 memory T cell responses’, 2nd paragraph, lines 4-6 and see 2nd paragraph).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention, to have modified the vector in the method of Sinnathamby of assessing potency of a Listeria-based immunotherapeutic, to comprise a PEST sequence, because of the teaching of Petit that Listeria constructs containing PEST regions induce a higher percentage of specific lymphocytes and that fusing the antigen to the PEST containing sequence enhances cell mediated and anti-tumor immunity in response to the antigen. One having ordinary skill in the art would have been motivated to do so in order to increase the percentage of specific lymphocytes in the potency assay.
One of ordinary skill in the art would have had a reasonable expectation of success, because both Sinnathamby and Petit teach a method comprising a recombinant Listeria based immunotherapeutic that induces specific T cell responses.
It would have further been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Sinnathamby by culturing the Listeria infected cells without gentamicin because of the teaching of Drevets that gentamicin causes macrophages to kill Listeria monocytogenes and that antibiotics have numerous effects on both the phagocyte and bacteria. One of ordinary skill in the art would be motivated to culture the cells infected with a Listeria-based immunotherapeutic without Gentamicin in order to prevent the antigen presenting cells harboring the Listeria-based construct from destroying the immunotherapeutic.
One of ordinary skill in the art would have a reasonable expectation of success in modifying the method of Sinnathamby by culturing the cells without Gentamicin as taught by Drevets because both the methods of Sinnathamby and Drevets comprise infection of macrophages with Listeria monocytogenes or an immunotherapeutic Listeria monocytogenes construct.
It would have further been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the claimed ratio of 1:1 to 1:4 of APCs to T cells, because in the instant case, the variable (ratio) is a result effective variable, namely a variable selected to achieve release of IFNγ by the maximum number of antigen specific T cells in the assay as shown by Sinnathamby above. Put another way, the optimization of the ratio of APC and T cells allows for the detection of the largest number of antigen specific T cells possible and ensures that the outcome of the assay is not influenced by a mismatch in the number of antigen presenting cells. Since this is a result effective variable it would be obvious to optimize the ratio during the course of routine optimization, in order to achieve the desired release of IFNγ by the T cells, thereby arriving at the claimed ratio. See MPEP 2144.05. Considering the art teaching any of the ratios in the range of ratios as taught by the cited prior art above (see Sinnathamby teaches APC to T cell rations of 1:40, 1:20, and 1:10, Badovinac teaches a ratio of 1:1 and Miller teaches a ratio of 1:5 of APC to T cells), as usable for stimulating T cells for cytokine release, the same outcome as is investigated in the claimed invention, and considering the ratio is a result-effective variable, it would have been well within the skill level of one of ordinary skill in the art to have optimized the ratio of APCs to T cells to uncover a the optimum workable ratio (thereby arriving at the claimed range of a ratio of APCs to T cells of 1:1 to 4:1), given that an APC to T cell ratio on this same order of magnitude was recognized at the time as usable for this purpose (stimulating cytokine release by T cells), see Badovinac teaches a ratio of 1:1 Listeria antigen presenting APC to CD8 T cells and Miller teaches a ratio of 1:5 APC to T cell infected with a mutant Listeria monocytogenes vector.
Given that the art teaches a similar ratio on the same order of magnitude as that claimed and absent evidence that the claimed ratio achieves an unexpected result, one having ordinary skill would have a reasonable expectation of success optimizing the ratio and arriving at the claimed 1:1 to 1:4 to induce cytokine release.
Regarding claim 2, Sinnathamby teaches THP-1 cells as APC for co-culturing (Sinnathamby, page 859, lines 6-7).
Regarding claim 4¸ Sinnathamby teaches infecting the THP-1 cells with a multiplicity of infection of 10 (Sinnathamby, page 859, lines 8-9).
Regarding claim 5, Sinnathamby teaches infecting THP-1 cells for 1 hour (Sinnathamby, page 858, column 2, lines 30-32 and page 859, lines 7-9), 1 hour falls within the claimed range of 0.5-24 hours, and as such anticipates the claim.
Regarding claim 6, Sinnathamby teaches infecting THP-1 cells for 1 hour (Sinnathamby, page 858, column 2, lines 30-32 and page 859, lines 7-9).
Regarding claim 12, Sinnathamby teaches measuring IFNγ production by T cells (Sinnathamby, page 858, ‘ELISpot Assay’, lines 1-3). As such Sinnathamby teaches determining a cytokine expression profile.
Regarding claim 18, Sinnathamby and the cited art above teaches a method of assessing potency of a Listeria-based immunotherapeutic substantially as claimed.
Sinnathamby further teaches an increase in the number of IFNγ secreting cells at a 1:40 APC to T cell ratio as compared to a 1:20 ratio (Sinnathamby, page 863, see figure 5).
Sinnathamby and the cited art above fails to teach that the PEST containing peptide is listeriolysin O (LLO) or a fragment thereof and that the disease-associated antigenic peptide is HPV protein E7 or a fragment thereof.
Petit teaches a Listeria-based immunotherapeutic, comprising a PEST-sequence, comprised in the LLO sequence (Petit, page 5, paragraph [0051], lines 23-24). Petit further teaches a Listeria strain that comprises a nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein (PEST sequence) fused to a heterologous antigen (disease-associated antigenic peptide), which causes an anti-tumor immune response (Petit, page 2, paragraph [0029], lines 10-20). Petit further teaches that the heterologous antigen is Human Papilloma Virus-E7 (HPV-E7) antigen (Petit, page 8, paragraph [0093], lines 4-5) from HPV16 (Petit, page 8, which is associated with cervical cancers (Petit, page 9, paragraph [0095], lines 89). Petit teaches that immunization of mice with Lm-LLO-E7 confers long-term protection and induces specific T cells (Petit, page 1, paragraph [0006], lines 15-19).
It would have been prima facie obvious to one having ordinary skill before of the effective filing date of the claimed invention, to have modified the vector in the method of Sinnathamby to comprise a PEST sequence with the HPV-E7 antigen, because of the teaching Petit, that this combination induces specific T cells and confers long term protection.
The ordinary artisan would have had a reasonable expectation of success because both Sinnathamby and Petit teach a method comprising a recombinant Listeria-based immunotherapeutic that induces specific T cell responses because of the teaching of Petit that such a vector induces specific T cell responses because Sinnathamby teaches success in inducing and stimulating T cells with cancer antigen expressing listeria monocytogenes constructs even in peripheral blood mononuclear cells from healthy patients. As such one would expect success stimulating T cell cultures with the construct as modified by Petit to express the HPV-E7 antigen.
Regarding Claim 19, Sinnathamby teaches a method of assessing potency of a Listeria-based immunotherapeutic substantially as claimed.
Sinnathamby fails to teach an attenuated listeria monocytogenes strain comprising a deletion of or inactivating mutation in prfA, via an episomal plasmid comprising a second open reading frame encoding a D133V PrfA mutant protein.
Petit teaches an episomal expression vector (episomal plasmid; Petit, page 6, paragraph [0062], lines 1-2) and a mutant prfA gene with a D133VprfA mutation in a plasmid (Petit, see paragraph [0061]) encoded in a second open reading frame (Petit et al., page 2, paragraph [0029], lines 14-16). Petit further teaches that prfA is a positive regulatory factor that controls Listeria virulence factors and that the plasmid is being used to complement a prfA negative mutant so that in a live host, selection pressures would favor conservation of the plasmid, because without it the bacterium would be avirulent (Petit, page 24, paragraph [0229], lines 8-13).
It would have been prima facie obvious to one having ordinary skill before of the effective filing date of the claimed invention, to have modified the vector in the method of Sinnathamby to comprise a deletion or inactivating mutation in prfA in the plasmid in order to favor conservation of the plasmid and prevent the bacterium from becoming avirulent.
The ordinary artisan would have a reasonable expectation of success, because Petit teaches a Listeria based vector as in Sinnathamby and shows success inducing specific T cell responses with the construct (see above).
Claims 7 is rejected under 35 U.S.C. 103 as being unpatentable over Sinnathamby et al., in view of Petit et al., Drevets et al., Badovinac et al. and Miller et al., as applied to claim 1 above, and further in view of Booth et al. (2015) “Mucosal-Associated Invariant T Cells in the Human Gastric Mucosa and Blood: Role in Helicobacter pylori Infection”, Frontiers in Immunology, 6, article 466, pages 1-14, as evidenced by Caravella et al., US PGPUB 20150299325 A1, 10/22/2015.
Regarding claim 7, Sinnathamby and the cited art above teaches a method of assessing potency of a Listeria-based immunotherapeutic substantially as claimed.
Sinnathamby does not teach culturing the infected APCs for 18-24h after washing and prior to co-culture with the T cells.
Booth teaches infecting THP-1 cells with H. pylori for 4h, then washing them and culturing them overnight before co-culture with peripheral blood mononuclear cells. Booth further teaches that the resting step allows the cells to recover from the infection (Booth et al., page 3 of 14, ‘Culture, Differentiation, and Infection of THP-1’, lines 14-23). Incubating overnight (as taught by Booth) is the equivalent of 18h as evidenced by Caravella (Caravella, page 12, paragraph [0073], lines 5-6).
It would have been prima facie obvious to one having ordinary skill in the art before of the effective filing date of the claimed invention, to have modified the method of Sinnathamby et al. in order to culture the THP-1 cells overnight (for example 18 hours), after infecting the cells and washing them, but before co-culturing them with T cells, because of the teaching of Booth that this lets the antigen presenting cells recover from the infection. As such, the modification would be an obvious matter of applying a known technique to a known method (a known technique to suitably allow recovery).
The ordinary artisan would have a reasonable expectation of success, because of the teaching of Sinnathamby that THP-1 cells can successfully be infected by the Listeria-based immunotherapeutic and the teaching of Booth that such an added resting step after the infection allows the cells to recover from infection with bacteria.
Claims 10-11, 13 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Sinnathamby et al., in view of Petit et al., Drevets et al., Badovinac et al. and Miller et al., as applied to claim 1 above and further in view of Letsch et al. Quantification and characterization of specific T-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining. Methods. 2003 Oct 1;31(2):143-9 (see PTO-892 05/25/2023).
Regarding claims 10, 11, and 24 Sinnathamby and the cited art above teaches a method of assessing potency of a recombinant listeria based immunotherapeutic substantially as claimed.
Sinnathamby does not teach coculturing the antigen presenting cells with the T cells for 18-24 hours.
Letsch teaches quantification and characterization of specific T cells using an ELISPOT assay or intracellular cytokine staining (Letsch, see title). Letsch teaches successfully measuring cytokine release of peptide or tumor stimulated cells by both ELISpot (Letsch, page 145, see 4.1.2.2) and intracellular cytokine staining (Letsch et al., page 147, see 4.2.2.1. and page 146, Figure 3). Letsch teaches that intracellular cytokine staining seems to be more sensitive than the ELISPOT assay to detect T cells secreting low amounts of IFNγ, which may be seen with tumor-reactive T cells (Letsch, page 145, 2nd paragraph, lines 9-12). Letsch teaches the principle of intracellular cytokine staining assay for detecting cytokine secreting T cells. Letsch teaches that T cells and antigen presented by antigen presenting cells are co-cultured for 6-24h and that a protein secretion inhibitor (Brefeldin A) is added to avoid secretion of cytokines. Letsch further teaches that this results in cytokine accumulation within the cell and fluorescent antibodies can be used to detect cytokines, as well as other markers (Letsch, page 144, Figure 2, see entire legend).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention, to have modified the method of Sinnathamby to use intracellular cytokine staining for the detection of tumor specific IFNγ secreting T cells because of the teaching of Letsch that intracellular cytokine staining seems to be more sensitive than the ELISPOT assay to detect T cells secreting low amounts of IFNγ, which may be seen with tumor-reactive T cells. It would have further been obvious to have applied the timing of the co-culture and to have added the protein secretion inhibitor to the intracellular cytokine staining assay as taught by Letsch, because it allows for cytokine accumulation within the cell which in turn allows detection of cytokine producing T cells.
In particular, Letsch supports that incubation time for the coculturing step is a result effective variable, namely a variable which achieves a recognized result of accumulating cytokines by antigen specific T cells. Specifically, Letsch teaches a duration of 6-24 hour, the claimed range of 18-24 is a narrower range falling within the prior art disclosed duration of 6-24 hours. As such, it would have been prima facie obvious to one having ordinary skill to have arrived at the claimed duration as a matter of routine optimization of experimental procedures, namely optimizing to uncover the optimum time necessary to allow cytokine accumulation to allow detection of cytokine producing cells. It would have been well within the skill level of the ordinary artisan to optimize the duration, by selecting from times within the art disclosed 6-24 hours, thereby arriving 18-24h. Further, the claimed range (18-24h) lies inside the range disclosed by Letsch (6-24h) and therefore a prima facie case of obviousness exists. See MPEP 2144.05. "[A] prior art reference that discloses a range encompassing a somewhat narrower claimed range is sufficient to establish a prima facie case of obviousness." In re Peterson, 315 F.3d 1325, 1330, 65 USPQ2d 1379, 1382-83 (Fed. Cir. 2003). See also In re Harris, 409 F.3d 1339, 74 USPQ2d 1951 (Fed. Cir. 2005).
One having ordinary skill in the art would have a reasonable expectation of success because Sinnathamby teaches detection of IFNγ secreting T cells after co-culture with antigen presenting cells by ELISPOT and Letsch teaches that both ELISPOT and intracellular cytokine staining can be applied to detect IFNγ secreting T cells after co-culture with antigen presenting cells. One with ordinary skill in the art would therefore have a reasonable expectation of success using the protein excretion inhibitor and applying the timing as taught by Letsch, timing that encompasses a broader time range than which is claimed; as a result one would expect success using any incubation time within that range including the claimed range.
Regarding claim 13, Sinnathamby teaches detecting IFNγ secretion by activated T cells using an ELISpot assay.
Letsch teaches that that in an ELISpot assay, cells specifically release the cytokine of interest (secrete into a culture media) which is then bound by the antibody coated to the well (Letsch, page 144, figure 1). As such, Letsch provides evidence that the ELISpot performed by Sinnathamby comprises measuring the level of IFNγ produced by T cells in a culture media.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Sinnathamby et al., in view of Petit et al., Drevets et al., Badovinac et al. and Miller et al., as applied to claim 12 above, and further in view of Geginat et al. Suppression of Acquired Immunity against Listeria monocytogenes by Amphotericin B—Mediated Inhibition of CD8 T Cell Function. The Journal of infectious diseases. 1999 Oct 1;180(4):1186-94.
Regarding claim 14, Sinnathamby and the cited art above teaches a method of assessing potency of a Listeria-based immunotherapeutic substantially as claimed.
Sinnathamby teaches measuring cytokine secretion by ELISpot. While Sinnathamby does teach determining a cytokine profile (IFNγ), it does not teach using an ELISA assay to determine the cytokine, namely IFNγ, expression profile of T cells.
Geginat teaches a method of measuring IFNγ production by peptide-specific CD8 T cell lines and antigen presentation by Listeria monocytogenes infected antigen presenting cells (Geginat, page 1186, ‘Abstract’, lines 9-10). Geginat teaches coculturing antigen presenting cells infected with Listeria monocytogenes with antigen specific CD8 T cells and measuring IFNγ concentration in the supernatants by ELISA (Geginat, page 1187, 2nd paragraph, lines 1-13).
It would have been prima facie obvious to one having ordinary skill at the time of the effective filing date of the claimed invention, to have modified the method of Sinnathamby of detecting IFNγ expression by T cells by ELISpot with the method of Geginat of detecting IFNγ secretion by ELISA, as an obvious matter of a simple substitution of one art recognized method of detecting IFNγ secretion by T cells over another. The prior art contained the base invention (see as taught by Sinnathamby, the prior art already recognized a method of detecting IFNγ produced by T cells co-cultured with antigen presenting cells in vitro). Further, at the time the prior art recognized the ability to use either ELISPOT or ELISA to detect IFNγ secreted by T cells (as in either by Sinnathamby or Geginat). As such, it would have been obvious to have modified the method of Sinnathamby to use an ELISA instead of an ELISPOT assay as the ordinarily skilled artisan would appreciate one format is usable in place of the other. The results would have been predictable, namely in that both would be expected capable of detecting IFNγ secretion by T cells. One having ordinary skill in the art would have recognized that applying the known ELISA of Geginat would have predictably resulted in the detection of IFNγ in the supernatant and as a result, one would have had a reasonable expectation of success.
Claims 20 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Sinnathamby et al., in view of Petit et al., Drevets et al., Badovinac et al. and Miller et al., as applied to claim 1 above, and further in view of Wallecha et al. Lm‐LLO‐Based Immunotherapies and HPV‐Associated Disease. Journal of oncology. 2012;2012(1):542851 (see PTO-892, 05/25/2023).
Regarding claim 20, Sinnathamby teaches a method of assessing potency of a Listeria-based immunotherapeutic substantially as claimed.
Sinnathamby fails to teach an attenuated Listeria monocytogenes strain comprising a deletion or inactivation mutation in an actA, an alanine racemase enzyme (dal), or a D-amino acid transferase enzyme (dat) gene, comprising the dat or dal genes in a second open reading frame of the episomal plasmid also comprising LLO as a PEST containing peptide.
Petit teaches a Listeria-based immunotherapeutic, comprising a PEST-sequence, comprised in the LLO sequence (Petit, page 5, paragraph [0051], lines 23-24) as discussed previously in detail above. Petit further teaches the listeria-based immunotherapeutic is ADXS11-001 (Lm-LLO-E7; Petit, page 25, paragraph [0263], lines 1-2). Petit further teaches a mutation in the dal, dat, or actA genes and encoding of dat or dal in a second open reading frame of the plasmid (Petit, page 3, see paragraph [0039], lines 6-8).
Wallecha teaches the live attenuated Listeria monocytogenes-based immunotherapeutic (ADXS11-001) secreting an antigen-adjuvant fusion (Lm-LLO) protein fused to HPV16-E7 (Wallecha, page 2, see entire 4th paragraph). Wallecha teaches a Lm dal- dat- actA- (deletion of actA, dal, and dat) backbone which is cleared rapidly in vivo and highly attenuated. Wallecha further teaches that survival of a Listeria monocytogenes strain deficient in dal and dat genes depends upon the plasmid-based complementation of the dal gene, the vector/plasmid combination creating an antibiotic marker free plasmid delivery system which also attenuates the vector which can be used for immunotherapy (Wallecha et al., page 3, see entire first paragraph).
It would have been prima facie obvious to one having ordinary skill in the art at the time of the effective filing date of the claimed invention, to have modified the vector in the method of Sinnathamby to have a mutation in actA, dal, and dat, in order to create an attenuated vector with an antibiotic marker free plasmid delivery system that is rapidly cleared in vivo and highly attenuated in order to have a safe delivery system for immunotherapy.
The ordinary artisan would have a reasonable expectation of success modifying the vector of Sinnathamby with the vector of Petit and Wallecha because of the teaching of Petit that the vector induces specific T cells and because of the teaching of Sinnathamby that specific T cells induced by a Listeria-based vector can be stimulated to produce cytokines in vitro.
Regarding claim 21, Sinnathamby teaches a method of assessing potency of a Listeria-based immunotherapeutic substantially as claimed.
Sinnathamby fails to teach a specific Listeria monocytogenes strain genetically modified to express a fusion protein of listeriolysin O or a fragment thereof and a human papillomavirus 16 protein E7 rumor antigen and the T cell is an HPV-reactive T cell or an HPV-E7 reactive T cell.
Wallecha teaches a live attenuated listeria monocytogenes-based immunotherapeutic (ADX11-001), which secretes an antigen-adjuvant fusion protein consisting of a truncated fragment of the listeria monocytogenes protein listeriolysin O fused to HPV16-E7 (Wallecha, page 2, 4th paragraph, lines 1-6). Wallecha further teaches that this strain is currently evaluated in Phase 2 clinical trials and that the platform is stimulates profound pathogen-associated immune mechanisms, which are genetically conserved, highly efficacious, and resistant to tolerance (Wallecha, ‘Abstract’, lines 6-10). Wallecha further teaches E7 specific T cells (HPV-E7-reactive cell) as determined by tetramer staining (Wallecha, page 7, table 2).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention, to have used the ADXS11-001 strain of listeria monocytogenes in the method of Sinnathamby, because this modification stimulates a profound immune response, it is highly efficacious and resistant to tolerance. As such, it is known that APC infected with this recombinant vector stimulate IFNγ production in T cells if the antigen is presented on the APC surface. ADX11-001 can therefore be used to ascertain that the disease associated antigen is actually expressed. Further, it is currently evaluated in clinical trials, and as such, by using the method of Sinnathamby, one can test the potency of the immune response to this vector in individual patients by using patient-derived CD8 T cells, stimulating them with ADXS11-001-infected THP-cells, and measuring the cytokine release.
The ordinary artisan would have a reasonable expectation of success, because of the teaching of Sinnathamby that cytokine production of antigen specific T cells can be measured by stimulating the T cells using an antigen presenting cell infected with a recombinant listeria monocytogenes strain.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Sinnathamby et al., in view of Petit et al., Drevets et al., Wallecha et al., Booth et al., and Letsch et al.
Regarding claim 22, Sinnathamby teaches a method of assessing potency of a Listeria-based immunotherapeutic (Sinnathamby, Summary, lines 7-10). Sinnathamby teaches comparing peptide pulsed APC with those APC infected with the listeria based immunotherapeutic, showing that the magnitude of the IFNγ+ T cell response was much lower when listeria monocytogenes specific T cells were stimulated with peptide-pulsed APCs compared to being stimulated with APC infected with the recombinant listeria construct, therefore determining a difference in potency (Sinnathamby, page 861, column 2, lines 7-14). Further, Sinnathamby teaches infecting THP-1 cells (Sinnathamby, page 857, ‘Cell Lines and Primary Cells From Human Tissues’, line 1), with a recombinant Listeria-based immunotherapeutic (Sinnathamby et al., Summary, lines 12-14) with a multiplicity of infection of 10 (Sinnathamby et al., page 859, lines 8-9). A multiplicity of infection of 10 falls within the claimed range of 1-20 and therefore meets the limitation of the claim. Sinnathamby further teaches Listeria infected THP-1 cells as target cells (Sinnathamby, page 859, lines 6-8) and in vitro expanded T cells as effector cells in an assay to assess antigen stimulated IFNγ release (co-culturing THP-1 cells and T cells; Sinnathamby, page 859, ‘ELISpot Assays’, lines 1-9). Sinnathamby further teaches a Listeria-based immunotherapeutic comprising one or more ovarian cancer epitopes (disease associated antigenic peptide) derived from epitopes bound by HLA-A2 molecules and presented by ovarian cancer cells. Sinnathamby further teaches incubating (co-culturing) infected APCs with ovarian epitope-specific CD8 T cells that were generated in vitro from healthy donors and ovarian cancer patients (enriched for T cells having reactivity to the disease-associated antigenic peptide; Sinnathamby, page 857, 2nd column, lines 9-15). Sinnathamby further teaches measuring IFNγ production of T cells specific for the tumor antigen encoded by the Listeria-based immunotherapeutic, wherein T cells produce IFNγ if the cognate antigen is presented by the APC and fails to produce IFNγ if no antigen is presented by the APC (increase in IFNγ indicates expression of HPV antigen; Sinnathamby, page 861, Figure 3) as discussed previously in detail above.
Sinnathamby is silent on the duration of infection period of THP-1 cells and also silent on the duration of the co-culture of T cells with antigen presenting cells. Sinnathamby further fails to teach a listeria monocytogenes strain genetically modified to express a fusion protein of LLO or fragment thereof and the HPV 16 protein E7 or a fragment thereof. Sinnathamby further fails to teach washing infected THP-1 cells and culturing them for an additional 18-24h in the absence of gentamicin, then coculturing the infected THP-1 cells with T cells specific for HPV 16 E7.
Petit teaches infecting THP-1 cells with ADSX11-001 (Listeria monocytogenes strain genetically modified to express LLO or a fragment thereof and HPV 16 protein E7 tumor antigen; page 25, paragraph [0263], line 2) for 2h (Petit, page 25, Paragraph [0265] lines 5-6). Petit teaches that the method of infection above results in the activation of the STING pathway (Petit, page 25, paragraph [0267], lines 1-3). Petit further teaches that the STING pathway is a crucial part of the immune response that allows a host to detect threats such as infection or cancer cells (Petit, page 1, see paragraph [0004]). Still further, Petit teaches that Immunization of mice with ADSX11-001, a Listeria-based vector comprising LLO or a fragment thereof fused to the HPV16 protein E7, confers long-term protection and induces specific T cells (Petit, page 1, paragraph [0006], lines 15-19).
Drevets teaches infecting antigen presenting cells (mouse peritoneal macrophages) with Listeria monocytogenes, followed by washing the infected cells and resuspending them in a balanced salt solution (BSS) with 5% normal mouse serum (Drevets, page 2223, ‘Bactericidal assay’, lines 1-10). Drevets further teaches incubating peritoneal macrophages for an additional 20-30 min and that after phagocytosis of bacteria, all cells were incubated in the absence or presence of various concentrations of gentamicin for a total of 60 min (Drevets, page 2223, ‘Bactericidal assay’, 2nd paragraph, lines 1-12 and see Figure1, white squares). Drevets teaches that gentamicin causes normally nonbactericidal macrophages that had phagocytosed Listeria monocytogenes to kill Listeria monocytogenes (Drevets, page 2222, ‘Abstract’, lines 3-6). Drevets further teaches that the use of antibiotics in bactericidal assays is common, but controversial because antibiotics have numerous effects on both the phagocyte and bacteria and that in the case of Listeria monocytogenes the interactions between host, parasite, and antibiotic are even more complex (Drevets, page 2227, ‘Discussion’, lines 1-5).
Booth teaches infecting THP-1 cells with H. pylori for 4h, then washing them and culture them overnight (18-24h) in RPMI before co-culture. Booth further teaches that the resting step allows the cells to recover from the infection as discussed previously in detail above (Booth et al., page 3, ‘Culture, Differentiation, and Infection of THP-1’, lines 21-23).
Letsch teaches quantification and characterization of specific T cells using an ELISPOT assay or intracellular cytokine staining (Letsch, see title). Letsch et al. teaches successfully measuring cytokine release of peptide or tumor stimulated cells by both ELISpot (Letsch et al., page 145, see 4.1.2.2 and page 146, Figure 3) and intracellular cytokine staining (Letsch et al., page 147, see 4.2.2.1.). Letsch teaches that intracellular cytokine staining seems to be more sensitive than the ELISPOT assay to detect T cells secreting low amounts of IFNγ, which may be seen with tumor-reactive T cells (Letsch, page 145, 2nd paragraph, lines 9-12). Letsch teaches the principle of intracellular cytokine staining assay for detecting cytokine secreting T cells. Letsch teaches that T cells and antigen presented by antigen presenting cells are co-cultured for 6-24h and that a protein secretion inhibitor (Brefeldin A) is added to avoid secretion of cytokines. Letsch further teaches that this results in cytokine accumulation within the cell and fluorescent antibodies can be used to detect cytokines, as well as other markers (Letsch, page 144, Figure 2, see entire legend).
It would have been prima facie obvious to one having ordinary skill in the art at the time of the effective filing date of the claimed invention, to have infected the antigen presenting cells in the method of Sinnathamby for 2h as taught by Petit because of the teaching of Petit that this activates the STING pathway. The ordinary artisan would be motivated to do so because of the teaching of Petit that the STING pathway is a crucial part of the immune response that allows a host to detect threats such as infection or cancer cells. It would have further been obvious to modify the Listeria-based immunotherapeutic of Sinnathamby to express a fusion protein comprising a fragment LLO a fragment of HPV 16 protein E7 as taught by Petit because of the teaching of Petit that infection with ADSX11-001 confers long-term protection in mice and induces specific T cells.
It would have further been prima facie obvious for one having ordinary skill in the art at the time of the effective filing date of the invention to have modified the method of Sinnathamby to let the cells rest for an additional 18-24h because of the teaching of Booth that letting the cells rest for 18-24h helps them recover from an infection. It would have further been prima facie obvious to culture the infected cells without Gentamicin, for the same reasons as applied to claim 1 above, the same reasons apply here.
The ordinary artisan would have a reasonable expectation of success, because of the teaching of Sinnathamby that THP-1 cells can successfully be infected by the Listeria-based immunotherapeutic and the teaching of Booth that adding a rest step allows THP-1 cells to recover from infection with a bacteria. The ordinary artisan further would have a reasonable expectation of success culturing the infected macrophages without gentamycin as taught by Drevets because both the methods of Sinnathamby and Drevets comprise infection of macrophages with Listeria monocytogenes or a construct derived from it.
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Sinnathamby et al., in view of Petit et al., Drevets et al., Wallecha et al., Booth et al., and Letsch et al. as applied to claim 22 above and further in view of Ressing et al. Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A* 0201-binding peptides. Journal of immunology (Baltimore, Md.: 1950). 1995 Jun 1;154(11):5934-43. (see PTO-892, 05/25/2023).
Regarding claim 23¸ Sinnathamby teaches a method of assessing potency of a Listeria-based immunotherapeutic substantially as claimed.
Sinnathamby teaches that endogenously presented peptides from ovarian cancer cells can be effective in stimulating T cells ex vivo and that Lm strains engineered to express these epitopes can activate individual epitope-specific T cells in vitro, which provides the basis for a rationale to use the live-attenuated Lm encoding selected epitopes as a therapeutic vaccination strategy (Sinnathamby, page 868, see 3rd paragraph).
Sinnathamby et al. fails to teach an E7 tumor antigen comprising the sequence YMLDLQPETT (SEQ ID NO. 101).
Ressing et al. teach that the HPV E7 peptide with the sequence YMLDLQPETT represents a naturally processed human CD8 T cell epitope of HPV16, that is highly immunogenic and therefore could be used in vaccines for the prevention and treatment of cervical carcinoma (Ressing et al., Abstract, lines 8-16). Ressing further teaches that human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis (Ressing, page 5934, Abstract, line 1) and that HPV DNA (predominantly of the HPV16 and 18 genotypes) is detected in over 90% of squamous cell carcinomas of the human cervix and is believed to play a causative role in cervical carcinogenesis (Ressing, page 5934, 2nd paragraph, line 1- page 5935, line 1). Ressing further teaches that the identification of multiple human CTL epitopes encoded by HPV16 will enable the development of a peptide-based vaccine for the prevention and treatment of cervical carcinoma (Ressing, page 5941, last paragraph).
It would have been prima facie obvious to one having ordinary skill before the effective filing date of the claimed invention, to have modified the vector in the method of Sinnathamby, to comprise a peptide with the sequence YMLDLQPETT (SEQ ID NO. 101), because of the teaching of Ressing et al. that human papillomavirus 16 is strongly associated with cervical carcinogenesis and is believed to play a causative role in said process. One of ordinary skill in the art would be motivated to have modified the vector of Sinnathamby with the peptide sequence as taught by Ressing because of the teaching of Ressing that the identification of multiple human cytotoxic T lymphocyte epitopes encoded by HPV16 will enable the development of a peptide-based vaccine for the prevention and treatment of cervical carcinoma. It would have further been obvious to have modified the vector of Sinnathamby to comprise the peptide with the sequence YMLDLQPETT as an obvious matter of a simple substitution of one art recognized cancer related immunogenic peptide over another. The prior art contained the base invention (see as taught by Sinnathamby, the prior art already recognized a method of assessing the potency of a recombinant listeria-based immunotherapeutic). Further, at the time the prior art recognized a Listeria-based immunotherapeutic that is a listeria monocytogenes strain expressing a disease associated antigenic peptide, wherein the disease-associated antigenic peptide is a tumor-associated antigen (as in either Sinnathamby or Ressing). As such, it would have been obvious to modify the listeria-based immunotherapeutic of Sinnathamby to express a cervical cancer related antigenic peptide (as taught by Ressing)as the ordinarily skilled artisan would appreciate that one peptide is usable in place of the other to be used in a potency assay for a listeria-based immunotherapeutic that elicits a cytotoxic T lymphocyte response against a tumor antigen. The result would have been predictable seeing that both peptides elicit a specific response in cytotoxic T lymphocytes specific for the particular antigenic peptide. One having ordinary skill in the art would have recognized that modifying the listeria-based immunotherapeutic of Sinnathamby with the peptide of Ressing would have predictably resulted in cytokine release by YMLDLQPETT specific T cells because the method of Sinnathamby results in cytokine release in cytotoxic T cells specific for the antigenic peptide encoded by the listeria-based immunotherapeutic and because Ressing shows detecting T cells specific for the HPV16-antigenic peptide and as such one of ordinary skill in the art would have had a reasonable expectation of success.
One of ordinary skill further would have had a reasonable expectation of success, because of the teaching of Ressing that the peptide induces E7 specific T cells and because of the teaching of Sinnathamby, that specific T cells induced by a Listeria monocytogenes based vector can be stimulated to produce cytokines in vitro.
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Sinnathamby et al., in view of Drevets et al., Badovinac et al. and Miller et al. and further in view of Stritzker et al. Prodrug converting enzyme gene delivery by L. monocytogenes. BMC cancer. 2008 Apr 10;8(1):94, Meyer-Morse et al. Listeriolysin O is necessary and sufficient to induce autophagy during Listeria monocytogenes infection. PloS one. 2010 Jan 6;5(1):e8610, and Golovliov et al. An attenuated strain of the facultative intracellular bacterium Francisella tularensis can escape the phagosome of monocytic cells. Infection and immunity. 2003 Oct;71(10):5940-50..
Regarding claim 25, Sinnathamby teaches a method of assessing potency of a Listeria monocytogenes-based immunotherapeutic (Sinnathamby, Summary, lines 7-10). Sinnathamby teaches infecting antigen presenting cells (APCs), specifically THP-1 cells, with a recombinant listeria-based immunotherapeutic (Sinnathamby, Summary, lines 10-14). Sinnathamby further teaches a listeria based immunotherapeutic comprising one or more ovarian cancer epitopes (disease associated antigenic peptide) derived from epitopes bound by HLA-A2 molecules and presented by ovarian cancer cells. Sinnathamby further teaches incubating (co-culturing) infected APCs with ovarian epitope-specific CD8 T cells that were generated in vitro from healthy donors and ovarian cancer patients (enriched for T cells having reactivity to the disease-associated antigenic peptide; Sinnathamby, page 857, 2nd column, lines 9-15). Sinnathamby further teaches characterizing a set of ovarian cancer-specific T cell epitopes delivered by live-attenuated recombinant Listeria monocytogenes (Lm; Sinnathamby, Summary, lines 7-9). Sinnathamby further teaches infecting the THP-1 cells with a multiplicity of infection of 10 for 1h (Sinnathamby, page 859, lines 8-9).
Sinnathamby further teaches that to ascertain expression, appropriate processing and presentation of individual ovarian cancer epitopes from recombinant Listeria based immunotherapeutics, one can use Listeria infected APC and use them as targets in ELISpot assays, measuring IFNγ production (determine cytokine production by T cells; Sinnathamby, page 861, column one, lines 10-13, and Figure 3). Further, Sinnathamby teaches that IFNγ production by T cells co-cultured with APC infected with a Listeria vector comprising a cancer antigen, but that T cells co-cultured with naïve APC (not infected) only secrete very low levels of IFNγ, demonstrating that there is an increase in cytokine production when the disease-associated antigenic peptide is presented by the infected APC (increase in cytokine production indicates expression of the disease-associated antigenic peptide in infected APC; Sinnathamby, Figure 2 and see ‘Results’, see entire 1st paragraph). Sinnathamby teaches using a fixed number of target cells, namely a concentration of 5000 cells/well (Sinnathamby, page 859, ‘ELISpot Assays’, lines 6-7). Sinnathamby further teaches comparing peptide pulsed APC with those APC infected with the listeria based immunotherapeutic, showing that the magnitude of the IFNγ+ T cell response was much lower when Listeria specific T cells where stimulated with peptide-pulsed APCs compared to being stimulated with APC infected with the recombinant listeria construct, showing a difference in potency (assessing potency; Sinnathamby, page 861, column 2, lines 7-14). Sinnathamby teaches a using Listeria monocytogenes infected APC in ratios of APCs to T cells of 1:40, 1:20, and 1:10 and then measure cytokine production by T cells with an ELISpot assay (Sinnathamby, page 859, ‘ELISpot Assays’, lines 1-8).
Sinnathamby fails to teach that the Listeria monocytogenes comprises a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to the disease-associated antigenic peptide and fails to teach culturing the infected APCs in the absence of gentamicin. Sinnathamby further fails to teach an MOI of about 200 and an infection time of 5 hours. Sinnathamby further fails to teach a ratio of APCs to T cells that is 1:1 or 4:1 and fails to teach infecting the antigen presenting cells with a multiplicity of infection of about 200 for about 5 hours.
Petit teaches a method of inducing an immune response against a disease using a recombinant Listeria strain (Petit, page 2, paragraph [0028], lin1-5). Petit teaches that the Listeria-based immunotherapeutic comprises a PEST-sequence, comprised in the LLO sequence (Petit, page 5, paragraph [0051], lines 23-24). Petit further teaches a listeria strain that comprises a nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein (PEST sequence) fused to a heterologous antigen (disease-associated antigenic peptide), which causes an anti-tumor immune response (Petit, page 2, paragraph [0029], lines 10-20). Petit further teaches that Listeria constructs containing PEST regions induce a higher percentage of specific lymphocytes within a tumor (Petit, page 2, see entire paragraph [0021] and figure 6) and that fusing the antigen to the PEST containing sequence enhances cell mediated and anti-tumor immunity of the antigen (Petit et al., page 11, paragraph [0117], lines 19-21).
Drevets teaches infecting antigen presenting cells (mouse peritoneal macrophages) with listeria monocytogenes, followed by washing the infected cells and resuspending them in a balanced salt solution (BSS) with 5% normal mouse serum (Drevets, page 223, ‘Bactericidal assay’, lines 1-10). Drevets further teaches incubating peritoneal macrophages for an additional 20-30 min and that after phagocytosis of bacteria, all cells were incubated in the absence or presence of various concentrations of gentamicin for a total of 60 min (Drevets, page2223, ‘Bactericidal assay’, 2nd paragraph, lines 1-12 and see Figure1, white squares). Drevets teaches that gentamicin causes normally nonbactericidal macrophages that had phagocytosed listeria monocytogenes to kill listeria monocytogenes (Drevets, page 2222, ‘Abstract’, lines 3-6). Drevets further teaches that the use of antibiotics in bactericidal assays is common, but controversial because antibiotics have numerous effects on both the phagocyte and bacteria and that in the case of L. monocytogenes the interactions between host, parasite, and antibiotic are even more complex (Drevets, page 2227, ‘Discussion’, lines 1-5).
Badovinac teaches a transfected APC that displays the listeria antigen LLO on MHC class I molecules (Badovinac, page 108, see ‘2.2 Cell lines and cell culture’). Badovinac further teaches stimulating CD8 T cell lines specific for that antigen using 3 x 106 responder cells and 3 x 106 irradiated stimulator cells, a ratio of 1:1 T cell to APC (Badovinac, page 108, ‘2.3. Generation and maintenance of CD8 T cell lines’, lines 8-10) and further using the above-described method to stimulate T cells for intracellular cytokine staining (Badovinac, page 109, ‘2.5. Intracellular cytokine staining of CD8 T cell lines and splenocytes from infected mice’, see second paragraph).
Miller teaches infecting APC with a mutant listeria monocytogenes vector (Miller, page 129, lines 1-2). Miller further teaches co-culturing T cells and APCs at a ratio of 5:1 and then assaying IFNγ in the supernatant (Miller, page 129, see ‘Naïve CD4 T cell coculture’) or by intracellular cytokine staining in response to peptide stimulation (Miller, page 129, ‘Expansion of gag-specific CD4 and CD8 memory T cell responses’, 2nd paragraph, lines 4-6 and see 2nd paragraph).
Stritzker teaches a method of delivering a prodrug into cancer cells in culture by Listeria monocytogenes (Stritzker, page 1 of 10, ‘Methods’, lines 1-3). Stritzker further teaches infecting cells at a multiplicity of infection of 200 for 5 hours (Stritzker, page 2 of 10, 2nd column, 3rd paragraph, lines 1-2).
Meyer-Morse teaches intracellular growth curves of Listeria monocytogenes infected bone marrow derived macrophages. Meyer-Morse further teaches that the bacterial growth rapidly increases between 2 and 5 hours post-infection after which the number of colony forming units, i.e. number of bacteria, remains the same. (Meyer-Morse, page e8610, see Figure 5 d). Put another way, Meyer-Morse teaches that infection of bone marrow derived macrophages with Listeria monocytogenes results in increased bacterial growth in the period between 2 and 5 hours but does not result in more bacterial growth after 5 hours of infection.
Golovliov teaches determining an appropriate multiplicity of infection when infecting cells with intracellular bacteria (F. tularensis). Golovliov teaches using a multiplicity of infection (an MOI of 200) that results in a mean of 92% of cells containing bacteria.
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). (see MPEP 2144.05)
Further, it has been held that a particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before it the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation (also MPEP 2144.05).
In the present case, the prior art recognized regarding percentage of cells infected by extracellular bacteria, that the variables that are the multiple of infections and infection duration are result effective variables (see Golovliov teaches that MOI is a result effective variable, i.e. that an MOI of 200 results in a mean of 92% of cells containing bacteria and Meyer-Morse teaches that duration of infection is a result effective variable because the number of bacteria increases from 2-5h after infection and reaches a maximum of colony forming units 5h after infection). Specifically, such variables are variables to be optimized through routine experimentation in order to achieve infection of almost all cells in a culture (see Sinnathamby teaching an MOI of 10 for 1h and Stritzker teaching an MOI of 200 for 5h).
Absent evidence of criticality, it would have been prima facie obvious to one of ordinary skill in the art, when performing the method as taught by Sinnathamby, to have optimized MOI and time through routine experimentation in order to obtain the optimum workable conditions which achieve the highest percentage of infected cells because it was known in the art to infect cells with Listeria monocytogenes for 5h at an MOI of 200. One of ordinary skill in the art would further have a reasonable expectation of success since the prior art did recognize these variables to be variables routinely optimized in the assay art.
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention, to have modified the vector in the method of Sinnathamby of assessing potency of a Listeria-based immunotherapeutic, to comprise a PEST sequence, because of the teaching of Petit that Listeria constructs containing PEST regions induce a higher percentage of specific lymphocytes and that fusing the antigen to the PEST containing sequence enhances cell mediated and anti-tumor immunity of the antigen. One having ordinary skill in the art would have been motivated to do so in order to increase the percentage of specific lymphocytes in the potency assay.
One of ordinary skill in the art would have had a reasonable expectation of success, because both Sinnathamby and Petit teach a method comprising a recombinant Listeria based immunotherapeutic that induces specific T cell responses.
It would have further been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Sinnathamby by culturing the Listeria infected cells without Gentamicin because of the teaching of Drevets that gentamicin causes macrophages to kill Listeria monocytogenes and because of the teaching of Drevets that antibiotics have numerous effects on both the phagocyte and bacteria. One of ordinary skill in the art would be motivated to culture the cells infected with a Listeria based immunotherapeutic without Gentamicin in order to avoid the antigen presenting cells harboring the listeria based construct from destroying the immunotherapeutic.
One of ordinary skill in the art would have a reasonable expectation of success in modifying the method of Sinnathamby by culturing the cells without Gentamicin as taught by Drevets because both the methods of Sinnathamby and Drevets comprise infection of macrophages with listeria monocytogenes or an immunotherapeutic listeria monocytogenes construct.
It would have further been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the claimed ratio of 1:1 to 1:4 of APCs to T cells, because in the instant case, the variable (ratio) is a result effective variable, namely a variable selected to achieve release of IFNγ by the maximum number of antigen specific T cells in the assay as shown by Sinnathamby above. Put another way, the optimization of the ratio of APC and T cells allows for the detection of the largest number of antigen specific T cells possible and ensures that the outcome of the assay is not influenced by a mismatch in the number of antigen presenting cells. Since this is a result effective variable it would be obvious to optimize the ratio during the course of routine optimization, in order to achieve the desired release of IFNγ by the T cells, thereby arriving at the claimed ratio. See MPEP 2144.05. Considering the art teaching any of the ratios in the range of ratios as taught by the cited prior art above (see Sinnathamby teaches APC to T cell rations of 1:40, 1:20, and 1:10, Badovinac teaches a ratio of 1:1 and Miller teaches a ratio of 1:5 of APC to T cells), as usable for stimulating T cells for cytokine release, the same outcome as is investigated in the claimed invention, and considering the ratio is a result-effective variable, it would have been well within the skill level of one of ordinary skill in the art to have optimized the ratio of APCs to T cells to uncover a the optimum workable ratio (thereby arriving at the claimed range of a ratio of APCs to T cells of 1:1 to 4:1), given that an APC to T cell ratio on this same order of magnitude was recognized at the time as usable for this purpose (stimulating cytokine release by T cells), see Badovinac teaches a ratio of 1:1 Listeria antigen presenting APC to CD8 T cells and Miller teaches a ratio of 1:5 APC to T cell infected with a mutant Listeria monocytogenes vector.
Given that the art teaches a similar ratio on the same order of magnitude as that claimed and absent evidence that the claimed ratio achieves an unexpected result, one having ordinary skill would have a reasonable expectation of success optimizing the ratio and arriving at the claimed 1:1 to 1:4 to induce cytokine release.
Response to Arguments
Applicant's arguments filed 05/27/2025 have been fully considered but they are not persuasive.
Applicant argues starting at page 8 that claim 1 is directed to a method of assessing potency of a recombinant Listeria-based immunotherapeutic. Applicant further argues that the office action alleged that Sinnathamby teaches the elements of the claimed method with the exception of i (the construct comprising a PEST sequence) and ii (culturing infected antigen presenting cells in the absence of Gentamicin). Applicant argues that the office action further alleges that it would have been obvious to modify the method of Sinnathamby with PEST containing peptide as taught by Petit because of the teaching of Sinnathamby that there is a need for validated tumor antigens and methods to deliver these antigens in an optimal manner to stimulate T cell responses and that such motivation is irrelevant to the development of the claimed method of assessing potency.
The rejection of claim 1 is amended, see specifically the motivation to modify the method of Sinnathamby to comprise a PEST sequence is derived from the teaching of Petit that Listeria constructs containing PEST regions induce a higher percentage of specific lymphocytes and one having ordinary skill in the art would have been motivated to do so in order to increase the percentage of specific lymphocytes in the potency assay (see page 8 of the present office action). This is relevant in a potency assay because increasing the percentage of specific lymphocytes in an assay would be expected to potentially increase the difference between a specific response and non-specific background.
Applicant further argues that the present rejections can only be reached through impermissible hindsight in view of the present application because it fails to consider the invention as a whole.
This argument is not persuasive.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In the present case, the rejection relies only on the cited prior art (that which was known to those of ordinary skill in the art, see the rejections above), Sinnathamby teaches comparing IFNγ production by T cells in response to cells infected with a Listeria monocytogenes construct compared to control. As such Sinnathamby teaches a method of assessing potency of a recombinant Listeria immunotherapeutic because the teaching of Sinnathamby is directed to activation of CD8 T cells by a recombinant Listeria immunotherapeutic which is measured by T cell IFNγ expression in response to the specific antigen and the potency of an immunotherapeutic can be measured in the size of the T cell response to the specific antigen after treatment with the immunotherapeutic, wherein priming and activation of antigen specific CD8 T cells is measured by IFNγ expression in response to exposure to the antigen.
Applicant further argues starting on page 9 that the art cited is often different from the claimed subject matter beyond the single element for which it is cited, for example Letsch teaches ELISpot and intracellular cytokine cytometry methods, but utilizes fresh PBMCs, Booth teaches H.pylori, and Caravella is directed toward antibodies and antibody fragments.
This argument is not persuasive.
In response to Applicant’s argument, the Examiner notes Caravella is simply cited as an evidentiary reference relied upon to support that overnight incubation (as taught by Booth) is equivalent to 18 hours, and therefore Booth teaching overnight incubation for the purpose of resting the cells after infection does address the claims because as evidenced by Caravella, overnight is considered in the prior art to be 18 hours.
Further, regarding Letsch, Letsch teaches PBMCs which comprise CD8 T cells and measuring the number of IFNγ secreting cells in response to stimulation by peptide or tumor stimulated cells. Sinnathamby similarly teaches IFNγ secretion by tumor antigen specific T cells in response to their cognate antigen. As such both Sinnathamby and Booth teach measuring the number of IFNγ secreting cells in response to antigenic stimulation a method that is identical whether the T cells are primed and stimulated by an immunotherapeutic or another method (such as immunization and stimulation with a peptide) and therefore the teaching is relevant to the current application. Booth teaches the effect of bacterial infection and rest thereafter on infected cells, and as a result, this reference is therefore also relevant because the recombinant Listeria monocytogenes immunotherapeutic of Sinnathamby also infects cells like the bacterium it is derived from.
Applicant further argues on page 10 that there would be no motivation to look beyond the method of Sinnathamby because Sinnathamby has provided a fully functional method of assessing the potency and uses the assay to show that each epitope is individually capable of eliciting an immune response.
This argument is not persuasive.
The motivation to modify Sinnathamby comes from the secondary art such as to have modified the listeria monocytogenes based immunotherapeutic with adding the PEST sequence of Petit because of the teaching of Petit that this would increase the number of antigen specific lymphocytes.
Applicant further argues on page 10 that Sinnathamby shows that (i) CD8 T cells from healthy donors were stimulated by the constructs, that (ii) that CD8 T cells from healthy donor PBMCs generated by exposure to autologous monocyte-derived APCs infected with recombinant Lm expressing the epitopes were reactive to T2 cells pulsed with the respective epitopes, that (iii) CD8 T cells from ovarian cancer patients stimulated with a pool of 6 epitope peptides prior to exposure to THP-1 cells infected with recombinant Lm expressing each epitope, (iv) that CD8 T cells derived from healthy donors that were stimulated with each epitope peptide prior to exposure to antigen presenting cells infected with recombinant Lm expressing all 6 epitope peptides, and that (v) CD8 T cells derived from healthy donors that were generated by exposure to autologous monocyte-derived APCs infected with recombinant Lm expressing all 6 epitope peptides, which were reactive to T2 cells pulsed with each of the epitopes. Applicant argues that this would lead a skilled person to recognize that Sinnathamby’s vector effectively stimulates a broad CD8 T cell response from any individual, to naturally occurring peptides previously identified in ovarian cancer cells and that the particular potency of these immunotherapeutics has no bearing on the method of assaying their potency, nor can it provide motivation to modify such a method.
This argument is not persuasive.
Sinnathamby teaches a method of comparing the ability of a listeria monocytogenes based immunotherapeutic to stimulate IFNγ production by CD8 T cells that are specific for the antigen expressed by the Lm construct and can therefore be used to assess the potency of such a construct in inducing a specific T cell response and as such is a method of assaying the potency of the construct.
Applicant further argues on page 11 and 12 that there is no reason to modify the method of Sinnathamby because Sinnathamby notes that their work has met the need of selection of validated tumor antigens and selection of methods to deliver these antigens in an optimal manner nor would the ordinary artisan be motivated by the teaching of Petit that the modification would lead to a stronger therapeutic as well as a more robust assay.
This argument is not persuasive in view of the amended rejection of the claim set forth in detail above. The motivation to modify the method of Sinnathamby to comprise a PEST sequence is derived from the teaching of Petit that Listeria constructs containing PEST regions induce a higher percentage of specific lymphocytes and one having ordinary skill in the art would have been motivated to do so in order to increase the percentage of specific lymphocytes in the potency assay (see page 8 of the present office action).
Applicant further argues on page 12 that Benoun’s review is focused on CD4 mediated immune memory. However, Benoun is no longer relied on in the rejection under 35 U.S.C. 103 and as such the argument is moot.
For all the reasons stated above, the arguments are not persuasive.
Communication
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/STEFANIE J. KIRWIN/ Examiner, Art Unit 1677
/ELLEN J MARCSISIN/ Primary Examiner, Art Unit 1677