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
1. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 October 21, 2025 has been entered.
Claims 40-55, 57-59 are pending. Claims 40, 42-48, 51-55 are amended. Claims 57-59 remain withdrawn. Claims 40-55 are currently being examined.
It is noted that claims 40 and 48 are amended to separate administration of the PD-1/PD-L1 immune checkpoint inhibitor from the dual kinase inhibitor targeting transforming growth factor β receptor type I and II (TGFBR1/TGFBR2 or TGFβI/TGFβII receptors) by listing them alternatively as second compounds administered in combination with first compound mithramycin.
Claim Objections
2. Claim 40 is objected to because of the following informalities: Claim 40 contains a typo where “Kiel-67” should be corrected to “Ki-67”.. Appropriate correction is required.
New Rejections
(necessitated by amendments)
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.
3. Claims 40-47 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 40 recites the limitation "the tumor growth" and “the tumor”. There is insufficient antecedent basis for this limitation in the claim. Claims 41-47 are rejected for encompassing the rejected limitation of claim 40.
4. Claims 40-55 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.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c).
In the present instance, claim 40 recites a range of “reducing weight of the tumor or reducing volume of the tumor by between about 45% to about 65%” and the claim also recites the different or narrower ranges of “preferably between about 65% to about 85%, more preferably between about 85% to about 95%”.
Additionally, claim 45 recites the broad recitation of “the expression of Ki-67 and Lgr5 are reduced by about 10% to about 95%”, and the claim also recites the narrower ranges of “preferably about 30% to about 50%, more preferably about 50% to about 70%, more preferably between about 70% to about 95%” which is the narrower statement of the range/limitation.
Additionally, claim 48 recites the broad recitation of “inflammatory factors inhibited by about 10% to about 95%”, and the claim also recites the narrower ranges of “preferably about 30% to about 50%, more preferably about 50% to about 70%, more preferably between about 70% to about 95%” which is the narrower statement of the range/limitation.
The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Claims 42-47 and 49-55 are rejected for encompassing the rejected limitation of claims 40 and 48.
5. Claims 45, 48-55 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 45 recites the expression of Ki-67 and Lgr5 are reduced by about a specific percentage as compared to a control. Claim 48 recites the inflammatory markers are inhibited by a specific percentage as compared to a control.
The claims do not recite what the control is, what it is derived from, or what its numerical value is. Therefore, one cannot determine a percent inhibition in the claimed ranges as compared to the claimed control because the value of the claimed control is unknown. The metes and bounds of the claims cannot be determined.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
6. Claims 40-41, 45, 47-49, 53, and 55 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Claims 40 and 48 limit the second compound to an immune checkpoint inhibitor targeting PD-1 or PD-L1 or a dual kinase inhibitor targeting transforming factor β receptor type I and II. Claims 40 and 48 subsequently claim the second compound is alemtuzumab, ipilimumab, ofatumumab, or rituximab. The antibodies alemtuzumab (anti-CD52 antibody), ipilimumab (anti-CTLA-4 antibody), ofatumumab (anti-CD20 antibody), and rituximab (anti-CD20 antibody) are not enabled to be an immune checkpoint inhibitor targeting PD-1 or PD-L1 or a dual kinase inhibitor targeting transforming factor β receptor type I and II because they cannot function as required of the second compound.
Claim Rejections - 35 USC § 103
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.
Mithramycin + PD-1/PDL-1 immune checkpoint inhibitor:
7. Claim(s) 40-42 and 45-47 are rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication 20180051085, Chang et al, published February 2018; in view of Goodman et al (Cancer Immunol. Research, October 1, 2019, 7:1570-1573); Ghiringelli et al (Frontiers in Immunology, August 6, 2019, 10:1816, internet pages 1-10); Halama et al (Journal of Clinical Oncology, May 26, 2019; 37:e14143); Suzuki et al (American Journal of Cancer Research, 2017, 7:2032-2040); Yu et al (Cellular & Molecular Immunology, 2019, 16:401-409; published online April 2018); Holmgaard et al (Journal for ImmunoTherapy of Cancer, 2018, 6:47, internet pages 1-15); Gao et al (OncoTargets and Therapy, August 28, 2019, 12:6961-6971); Zhao et al (Oncology Reports, 2013, 30:1782-1792); Kasagi et al (Cancer Research, 2016, 76:347-357); Jia 2010 (Cancer Research, 2010, 70:1111-1119); Jia 2007 et al (Cancer Research, 2007, 67:4878-4885); Baum (British Journal of Cancer, 22.2 (1968): 176-183); Luo et al (BMC Cancer, February 6, 2019, 19:123; internet pages 1-13); Jiang et al (BMC Cancer, 2015, 15:948, internet pages 1-10); Mao et al (Cancer Management and Research, 2018, 10; 3569-3577); and Murnane et al (International Journal of Cancer, 209, 2893-2902).
Chang suggests a method for treating cancer in a subject comprising administering a combination of anti-PD-1/PD-L1 antibody and anticancer drug mithramycin ([125-126]; claims 8-13). Chang teaches anti-PD-1 antibody pembrolizumab (also known as lambrolizumab) is FDA approved to treat a variety of cancers, and suggests combining the antibodies with their therapeutic regimen ([5]; [10]; [16]; [48]; [54]; [103]; claim 17). Chang teaches treating colorectal cancer in their method and teaches it is already known that anti-PD-1 antibodies are administered to treat colorectal cancer in the art ([47]; [50]; claim 28). Chang teaches inhibiting metastasis ([148]; [153-154]; Examples 5 and 6). Chang demonstrates that an anti-PD1 antibody alone was not effective at reducing tumor growth of CT26 colorectal cancer model, however, combining a second anti-cancer therapy with the anti-PD1 antibody resulted in synergistic treatment (Example 6, Figure 10).
Chang does not:
Exemplify combining mithramycin with pembrolizumab to treat MSS colorectal cancer and inhibiting tumor growth by reducing the weight or volume of the tumor by about 45% to about 65% (claim 40);
Teach treating MSS colon carcinoma results in a reduction in expression of Ki-67, Lgr5, CTLA-4 and pro-MMP9 and by about 10%-95% compared to a control (claims 40 and 45); or
Teach the dose of mithramycin is about 0.5 mg/kg to about 10 mg/kg or about 1 mg/kg (claims 40 and 47).
Goodman teaches anti-PD-1 antibody pembrolizumab is FDA approved for the treatment of microsatellite instability-high (MSI-H) tumors because of their inherently high mutational burden, however some microsatellite stable (MSS or MSI-L) tumors also have a high tumor mutational burden (TMB-H) and are expected to respond to checkpoint blockade, including colorectal cancer (CRC) (p. 1570, col. 2; abstract; Figure 1A). Goodman demonstrates that MSS/TMB-H tumors have significantly longer median progression-free survival (PFS) than MSS/TMB-Low tumors when treated with checkpoint inhibitors targeting PD-1/L1 or CTLA4 (p. 1572, col. 1-2). Goodman teaches that MSS cancers can comprise a TMB-H subset that should receive checkpoint blockade therapy (p. 1572, col. 2).
Ghiringelli reviews treatments of MSS colorectal cancer teaching that PD-1 or PD-L1 immune checkpoint inhibitors are often not effective as monotherapy in metastatic CRC with MSS tumors (abstract). Ghiringelli teaches combination therapy of immune checkpoint inhibitors with a variety of other chemotherapeutic agents has demonstrated improved or superior tumor growth suppression of MSS CRC (p. 5-6). Ghiringelli teaches pembrolizumab or anti-PD-1/PD-L1 antibodies are being clinically administered to patients with MMS tumors and in combination with various agents that induce tumor immunity or target MDSC and immunosuppressive macrophages (p. 6-7).
Halama demonstrates successfully clinically treating MMS metastatic colorectal cancer with a combination of pembrolizumab and a chemotherapeutic. Halama recognizes that PD-1 inhibition alone has not shown clinical benefit in MSS CRC patients that have impaired immune systems and high tumor load. Pembrolizumab combination therapy resulted in an induction of immune response and stable disease.
Suzuki demonstrates successfully treating MSS CRC tumors by combining administration of an anti-PD-1 antibody with a chemotherapeutic agent, resulting in synergistic tumor growth inhibition compared to either agent alone (98.4% inhibition with combined therapy versus 86.7% (single PD-1 antibody) and 52.7% (chemotherapeutic alone)) (abstract). Combination therapy induced a cytotoxic anti-tumor immune response and reduced immunosuppressive Treg cells (abstract; Results; Figures 1-3). Like Goodman, Ghiringelli, and Halama, Suzuki also recognizes that anti-PD-1 or checkpoint inhibition therapy is sometimes not effective in MSS ((MMR)-proficient) cancers, and teaches the solution is combining antitumor therapies with anti-PD-1 therapy. Suzuki teaches (p. 2033, col. 1):
“In a phase II clinical trial of pembrolizumab in 50 patients with colon cancer, RR was higher in the mismatch repair (MMR)-deficient group than in the MMR-proficient group, 40% versus 0% in the initial report [8]. Recently, pembrolizumab was approved for the treatment of adult and pediatric patients with unresectable or metastatic solid tumors that have been identified as microsatellite instability (MSI)-high or MMR-deficient. MMR deletion has become an important indicator of therapeutic efficacy of anti-PD-1 antibody. However, only 4% to 6% of metastatic colorectal cancers are associated with MSI-H [9, 10]. There is room for treatment development for MSS, which account for the majority.
To enhance the antitumor effect, many clinical trials have sought to determine the PD-1 inhibitors combination with other antitumor therapy, such as chemotherapy, targeted therapy, radiotherapy, or other immunotherapy [11].”
Yu recognizes that PD-1 blockade alone for MSS CRC is not very effective (abstract; Introduction). Yu suggests enhancing PD-1 therapy by combining it with a second chemotherapeutic that enhances immune response to tumors (abstract; Introduction). Yu demonstrates successfully treating CT26 CRC tumors by combining administration of an anti-PD-1 antibody with a chemotherapeutic agent, resulting in synergistic tumor growth inhibition compared to either agent alone, wherein tumor growth was reduced by at least 45% (Figure 7A and B), and combination treatment significantly enhanced survival (Figure 7C).
Holmgaard teaches a method of treating microsatellite stable (MSS) colon carcinoma (CT26 tumors) in mice, comprising administering either as a single agent or as a combination, an anti-PD-L1 antibody and a TGFβI kinase receptor inhibitor LY2157299, galunisertib, wherein single agents reduced tumor volume by at least about 45% compared to control IgG treatment, and combination treatment provided significantly increased benefit and reduced tumor volume compared to the control and either agent alone (p. 8-9; Figure 5). Figure 5a:
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To test immunologic memory, mice with complete responses were re-challenged with CT26 tumor cells 85 days after primary tumor challenge and all complete responders in monotherapy and combination treatment rejected the re-challenge with the CT26 tumors (p. 9-10; Figure 5b, left panel). Holmgaard teaches the ability of galunisertib to enhance the activity of anti-PD-L1 immunotherapy was confirmed in the MC38 tumor model (microsatellite instable, MSI), which is historically less responsive to checkpoint immunotherapy (PD-L1 insensitive) and considered to be more myeloid biology driven (p. 10, col. 1; Figure 5c). Holmgaard tested the gene expression profile of CT26 tumors during combination therapy and determined altered gene expression in the tumors progressed to an increase in genes indicative of T cell activation (Table 1; p. 11, col. 1; Figure 6). Holmgaard teaches that another study recently published demonstrated that combined treatment with galunisertib + anti-PD-L1 in murine colon cancer models resulted in pronounced immune responses which eradicated most metastases, prolonged recurrence-free survival, and was associated with disruption of a T-cell exclusion phenotype (p. 13, col. 1). Holmgaard teaches there are published reports demonstrating systemic treatment with monoclonal antibodies targeting the TGFβ ligands or the TGFβRII inhibit metastatic invasion of breast cancer cells in murine tumor models, and treatment blocking TGFβ signaling with small molecule inhibitors suppresses metastasis in murine pancreatic tumor models, and enhances radiation response and prolongs survival in glioblastoma xenograft models (p. 13, col. 2). Holmgaard concludes their data provide support for continued clinical development of galunisertib to target tumors dependent on TGFβ-driven biology for growth, metastasis, and immune evasion (p. 13, col. 2). Holmgaard teaches that galunisertib is already being clinically administered in combination with checkpoint inhibitors nivolumab and durvalumab in cancer patients with NSCLC, HCC, or pancreatic cancer (p. 2, col. 2; p. 14, col. 1).
Gao teaches and demonstrates successfully treating MSS colon carcinoma (CT26) and inhibiting metastasis by administering an anti-PD-1 antibody, wherein treatment resulted in enhance T-cell responses (abstract; p. 6966-6969). Gao demonstrates that treatment with anti-PD-1 antibody also significantly decreased TGF-β ligand expression (Figure 4E).
Zhao teaches mithramycin is a known SP1 inhibitor and demonstrates that mithramycin successfully inhibits SP1 expression in cancer stem cells (CSCs) derived from colorectal cancer cell lines, and led to marked suppression of CSC growth. SP1 is a biomarker of colon cancer cells and CSCs, and CSCs are known to persist in tumor masses as a distinct population that cause tumor relapse and metastasis. Mithramycin treatment of colorectal tumors in vivo at 25 mg/kg significantly reduced CD44 and CD166 expression, which are CSC markers (abstract; Introduction; p. 1782, col. 2 to p. 1783, col. 1; p. 1784, col. 2; Figure 6; p. 1789, col. 1-2; p. 1791, col. 1).
Kasagi teaches and successfully demonstrates that treatment of a MSS colon carcinoma (CT26) metastasis model with mithramycin and 5-FU successfully resulted in over 100 days long-term survival of 50% of animals treated (p. 355, col. 1; Figure 5 Group E). Mesenteric metastasis of CT26 cells was significantly inhibited by the pretreatment with mithramycin A (p. 354, col. 2; Figure 4F). Mesenteric tumor dissemination of human colorectal cancer cells was significantly abrogated by mithramycin A (p. 355, col. 1).
Baum teaches treating human patients having advanced cancers with a dose of 25 µg/kg (0.025 mg/kg) each day for 8 days, wherein the patients having rectal carcinoma had either major or minor tumor regression (p. 177-178). Baum suggests pursuing treatment with mithramycin for patients having rectal carcinomas (p. 183).
Combining mithramycin and pembrolizumab to treat MSS colorectal cancer and reducing tumor volume by about 45% to 65% and inhibit metastasis:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to combine mithramycin with anti-PD-1 antibody pembrolizumab for treatment of MSS colorectal cancer and inhibition of metastasis. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Chang suggests combining the two known anti-cancer agents for the treatment of colorectal cancer including metastatic cancer; (2) the cited references teach and demonstrate that each of the agents: mithramycin and anti-PD-1/PD-L1 antibody immune checkpoint inhibitors including pembrolizumab, successfully treat the same MSS colorectal cancer; (3) Chang, Goodman, Ghiringelli, Halama, Suzuki, Yu, and Holmgaard suggest and/or demonstrate combining second anti-cancer therapies with anti-PD-1 antibody/pembrolizumab in order to improve the effectiveness of anti-PD-1 immune checkpoint inhibition therapy of MSS tumors, demonstrating synergistic results with combination therapy, significant reduction in % tumor growth, and enhanced survival; and (4) Goodman recognizes that the MSS CRC patient population encompasses patients having high TMB that are responsive to anti-PD-1 treatment alone, resulting in significantly longer PFS.
Those of skill in the art recognize that the two anti-cancer agents, mithramycin, anti-PD-1 pembrolizumab immunotherapy: (1) each successfully treat the same MSS colorectal cancer and inhibit metastasis, wherein the combination of anti-PD-1 antibody/pembrolizumab antibody and second anti-cancer agents is demonstrated repeatedly as synergistic for inhibiting tumor growth and enhancing survival, and (2) could have been readily and predictably combined by known methods to perform the same function of treating MSS colorectal cancer, and that in combination, they would have merely would have performed the same cancer-treating function as they did separately, and expectedly and one of ordinary skill in the art would have recognized that the results of the combination would predictably treat MSS colon carcinoma and inhibit metastasis, as already demonstrated.
The instant situation is amenable to the type of analysis set forth in In re Kerkhoven, 205 USPQ 1069 (CCPA 1980) wherein the court held that: “It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition which is to be used for the very same purpose. In re Susi, 58 CCPA 1074, 1079-80, 440 F.2d 442, 445, 169 USPQ 423, 426 (1971); In re Crockett, 47 CCPA 1018, 1020-21, 279 F.2d 274, 276-77, 126 USPQ 186, 188 (1960). As this court explained in Crockett, the idea of combining them flows logically from their having been individually taught in the prior art.” In the instant case, it is prima facie obvious to combine mithramycin and anti-PD-1 antibody pembrolizumab for the treatment of MSS colorectal cancer, which is taught by the prior art to be useful for the same purpose of treating MSS colorectal cancer and inhibiting metastasis.
Dosing mithramycin at about 0.5 mg/kg to about 10 mg/kg or at about 1 mg/kg:
Jia 2010 teaches successfully administering mithramycin at a dose of 0.5 mg/kg to 1.5 mg/kg to treat cancer.
Jia 2007 teaches administering mithramycin in combination with antibody immunotherapy to treat cancer, and at a dose of 0.1, 0.2, or 0.4 mg/kg repeatedly, wherein combination therapy produced synergistic antitumor activity (p. 4880, col. 2 to p. 4881, col. 1; Figures 1-3; Materials and Methods p. 4879, col. 1). A dose of 0.4 mg/kg is reasonably encompassed by “about 0.5 mg/kg”.
Baum, as stated above, teaches treating human patients having advanced cancers with a dose of 25 µg/kg (0.025 mg/kg) each day for 8 days, wherein the patients having rectal carcinoma had either major or minor tumor regression (p. 177-178). Baum suggests pursuing treatment with mithramycin for patients having rectal carcinomas (p. 183).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to administer a dose of about 0.5 or about 1 mg/kg mithramycin to the MSS colorectal carcinoma patients in the method of the combined references. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Zhao, Kasagi, Jia 2010, Jia 2007, and Baum demonstrate mithramycin treatment is successful for colorectal cancer and other cancer treatment at a wide variety of doses; (2) Jia 2010 demonstrates doses ranging from 0.5 mg/kg to 1.5 mg/kg successfully treated cancer; and (3) Jia 2007 demonstrates that a dose of mithramycin of about 0.5 mg/kg is successful for cancer treatment and synergistic with immunotherapy.
Reducing expression of Ki-67, Lgr5, CTLA-4 pro-MMP9, and MMP2 with treatment and at about 10% to about 95% compared to a control:
Luo teaches the Ki-67 is a prognostic biomarker of colorectal cancer, wherein high Ki-67 expression serves as a valuable predictive method of poor prognosis of colorectal cancer patients, and reduced expression of Ki-67 indicates improved prognosis, improved survival, and improved disease-free survival (abstract; Figure 7 and 8; p. 12, col. 1).
Jiang teaches that Lgr5 is a prognostic biomarker of colorectal cancer, wherein Lgr5 overexpression was significantly associated with worse overall survival, deep invasion, distant metastasis, and lymph node metastasis (abstract; Table 3; Figure 4).
Mao teaches that increased CTLA-4 expression in colon cancer is associated with low tumor purity and worse survival/prognosis (abstract; p. 3571; p. 3574; Figure 4).
Murnane et al teaches that increase pro-MMP9 and MMP2 expression were associated with the presence of colorectal cancer, wherein lower levels were associated with normal tissue (abstract; Figure 1; Results on p. 2895, col. 1 to p. 2897, col. 1). Levels of pro-MMP9 were significantly higher in cancers versus normal tissue (abstract; Figure 4).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to achieve a result of reduced expression of biomarkers Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2, and to reduce their expression at about 10% to about 95% compared to a control in the method of treating MSS colorectal cancer taught by the cited combined references above. One would have been motivated to, and have a reasonable expectation of success to, because: (1) the cited combined references above teach the goal of their method is to treat colorectal cancer and enhance survival, (2) Luo, Jiang, Mao, and Murnane all teach reduction of expression in known biomarkers Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2 indicate improved prognosis for colorectal cancer and provide motivation to achieve such reduced expression; and (3) Luo, Jiang, Mao, and Murnane all teach known and routine methods for successfully detecting reduced expression of Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2 to assess prognosis. Given: (1) the known need to treat CRC and enhance survival; (2) the known biomarkers indicative of CRC treatment and enhanced survival; and (3) the known routine methods of measuring the biomarkers in CRC; one of ordinary skill in the art would be motivated to measure reduced levels of the known CRC biomarkers Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2, including reduced levels in the range of about 10%, as correlated to treatment response and improved prognosis with a reasonable expectation of success.
8. Claim(s) 48-50, 54 and 55 are rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication 20180051085, Chang et al, published February 2018; Goodman et al (Cancer Immunol. Research, October 1, 2019, 7:1570-1573); Ghiringelli et al (Frontiers in Immunology, August 6, 2019, 10:1816, internet pages 1-10); Halama et al (Journal of Clinical Oncology, May 26, 2019; 37:e14143); Suzuki et al (American Journal of Cancer Research, 2017, 7:2032-2040); Yu et al (Cellular & Molecular Immunology, 2019, 16:401-409; published online April 2018); Holmgaard et al (Journal for ImmunoTherapy of Cancer, 2018, 6:47, internet pages 1-15); Gao et al (OncoTargets and Therapy, August 28, 2019, 12:6961-6971); Zhao et al (Oncology Reports, 2013, 30:1782-1792); Kasagi et al (Cancer Research, 2016, 76:347-357); Jia 2010 (Cancer Research, 2010, 70:1111-1119); Jia 2007 et al (Cancer Research, 2007, 67:4878-4885); Baum (British Journal of Cancer, 22.2 (1968): 176-183); Luo et al (BMC Cancer, February 6, 2019, 19:123; internet pages 1-13); Jiang et al (BMC Cancer, 2015, 15:948, internet pages 1-10); Mao et al (Cancer Management and Research, 2018, 10; 3569-3577); and Murnane et al (International Journal of Cancer, 209, 2893-2902); as applied to claims 40-42 and 45-47 above, and further in view of Oladipo et al (British Journal of Cancer, 2011, 104:480-487); and Hsu et al (International Journal of Molecular Sciences, 2018, 19:2427; internet pages 1-17).
Chang, Goodman; Ghiringelli; Halama; Suzuki; Yu, Holmgaard; Gao; Zhao; Kasagi; Jia 2010; Jia 2007; Baum; Luo; Jiang; Mao; and Murnane (the combined references) teach a method of treating and inhibiting metastasis of MSS colorectal cancer in a subject comprising administering to the subject pembrolizumab + mithramycin, administering mithramycin at a dose of about 0.5 mg/kg to about 10 mg/kg, reducing tumor growth by about 45%-65%, and reducing expression of colorectal cancer markers Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2, as set forth above.
The combined references do not teach reducing the expression of colorectal cancer marker CXCL1, and from about 10% to about 95% compared to a control.
Oladipo teaches that CXCL1 is a marker increased in colorectal cancer compared to normal controls (Figure 2; abstract), and that decreasing CXCL1 expression levels correlated with increasing recurrence-free survival (Table 3; abstract). Oladipo demonstrates known methods for detecting and comparing levels of CXCL1 in CRC by immunohistochemistry (Materials and Methods).
Hsu demonstrates that inflammatory chemokine CXCL1 is present in large amounts in dendritic cells (DCs) isolated from colon cancer patients, and CXCL1 increases the metastatic ability of a cancer by enhancing cell migration, matrix metalloproteinase-7 expression and epithelial-to-mesenchymal transition (EMT) (abstract; Figure 7). Hsu demonstrates that CXCL1 expression increased the cancer stem cell properties of metastatic CRC cells (section 2.3; Figure 3). Hsu demonstrates CXCL1 enhanced cell migration and EMT transition of metastatic CRC cells (section 2.4, Figure 4). Hsu teaches (p. 2):
“A growing body of studies has shown inflammatory cytokines and chemokines can act as tumor growth and survival factors and promote tumor progression and metastasis by increasing angiogenesis and suppressing immune-mediated tumor elimination [14–16]. Elevated chemokine (C-X-C motif) ligand 1 (CXCL1) levels are found in CRC, and these increased levels are positively associated with cancer stage, metastasis and poor survival rates [17,18]. A recent study has revealed that CXCL1 contributes to the formation of a pre-metastatic niche in the liver by recruiting chemokine (C-X-C motif) receptor 2 (CXCR2)-positive myeloid-derived suppressor cells [19].”
Hsu teaches (p. 10-11):
“CXCL1 is a potent proinflammatory mediator of inflammatory diseases and infection, and is widely considered to both promote and exacerbate tumor growth and progression in several cancers [25,26]. CXCL1 is upregulated in various cancers and associated with cancer progression, such as cancer cell growth, proliferation, tumor angiogenesis and metastasis, after the activation of CXCR2 [27–29]. In addition, CXCL1 is not only involved in cancer progression, but also responsible for resistance to several chemotherapeutic drugs, such as oxaliplatin, doxorubicin, and cyclophosphamide [30–33]. CSCs are considered to display the clonogenic core of the cancer, since it is implied that these cells are involved in tumor propagation, progression, chemo-resistance and metastatic dissemination. In this study, we found that CXCL1 is not only released from malignant cells, but also secreted from cancer-conditioned DCs. TADCs express high levels of CXCL1, which in turn increases the tumorigenesis and chemo-resistance potential by increasing CSC-like properties. Furthermore, TADC-derived CXCL1 also enhances cancer migration and switches the epithelial phenotype to a mesenchymal characteristic, a key process of cancer metastasis. These findings suggest that TADC-derived CXCL1 may be a new candidate in conferring the ability for colon cancer to progress.
It is of interest to note that CXCL1-producing CD11c+ DCs were found to infiltrate the cancerous tissue of CT26-bearing mice. In addition, CD11c+ DCs isolated from patients with CRC produce high levels of CXCL1 when compared with CD11c+ DCs isolated from healthy donors. These results based on experimental cell studies, animal models, and clinical patients strongly suggest that TADCs are one of the critical effectors in CRC stroma enhancing the development of colon cancer by CXCL1 production.”
Hsu teaches (p. 11):
“In conclusion, our results further support the role of TADCs and their secreted CXCL1 in colon cancer progression. The findings reported here not only introduce a novel mechanism of immunosuppression, but also provide preliminary evidence of the potential utility of CXCL1 inhibition as a therapeutic strategy in fighting cancer.”
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to reduce expression of CXCL1 and reduce it about 10% to about 95% as compared to a control. One would have been motivated to, and have a reasonable expectation of success to, because: (1) the combined references teach the goal of their method is to treat colorectal cancer and enhance survival, (2) Oladipo teaches CXCL1 is an inflammatory marker of CRC where decreased levels correlated with increased recurrence-free survival; (3) Hsu teaches CXCL1 is a known marker upregulated in various cancers and associated with cancer progression, such as cancer cell growth, proliferation, tumor angiogenesis and metastasis; (4) Hsu demonstrates CXCL1 is a known inflammatory marker of CRC that increased the cancer stem cell properties of metastatic CRC cells, enhanced cell migration and EMT transition of metastatic CRC cells, and contributes to the development of colon cancer, and Hsu suggests reducing CXCL1 levels for treatment; and (5) Oladipo and Hsu demonstrate known methods for successfully detecting expression levels of CXCL1 in CRC.
Given: (1) the known function of CXCL1 as a marker of CRC and worse prognosis, (2) the known function of CXCL1 contributing to CRC tumorigenesis and metastatic phenotype, and (3) the known motivation to reduce levels of CXCL1 in CRC; it is well within the level of the ordinary skilled artisan to reduced levels of CXCL1 in the CRC patients of the combined references to achieve improved prognosis and treatment, and to reduce CXCL1 levels as low as possible from about 10% to about 95% as compared to a control.
Mithramycin + dual kinase inhibitor of TGFBR1 and TGFBR2:
9. Claim(s) 40, 41, 43-45 and 47 are rejected under 35 U.S.C. 103 as being unpatentable over Holmgaard et al (Journal for ImmunoTherapy of Cancer, 2018, 6:47, internet pages 1-15); in view of Tauriello et al (Nature, 2018, 554:538-543 and Extended Data); Schaer et al (Journal of ImmunoTherapy of Cancer, 2015, 3(Suppl):P402); Keedy et al Journal of Clinical Oncology, 2018, 36:3031); Zhao et al (Oncology Reports, 2013, 30:1782-1792); Kasagi et al (Cancer Research, 2016, 76:347-357); Baum (British Journal of Cancer, 22.2 (1968): 176-183); Zhang et al (Cancer Letters, 2009, 277:114-120); Jia 2010 (Cancer Research, 2010, 70:1111-1119); Jia 2007 et al (Cancer Research, 2007, 67:4878-4885); Luo et al (BMC Cancer, February 6, 2019, 19:123; internet pages 1-13); Jiang et al (BMC Cancer, 2015, 15:948, internet pages 1-10); Mao et al (Cancer Management and Research, 2018, 10; 3569-3577); and Murnane et al (International Journal of Cancer, 209, 2893-2902).
Holmgaard teaches a method of treating microsatellite stable (MSS) colon carcinoma (CT26 tumors) in mice, comprising administering single agent TGFβI kinase receptor inhibitor LY2157299, galunisertib, or a combination of an anti-PD-L1 antibody with galunisertib, wherein single agent galunisertib reduced tumor growth by at least 45%, and combination treatment provided significantly increased benefit compared to galunisertib treatment alone (p. 8-9; Figure 5). Figure 5a:
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To test immunologic memory, mice with complete responses were re-challenged with CT26 tumor cells 85 days after primary tumor challenge and all complete responders in monotherapy and combination treatment rejected the re-challenge with the CT26 tumors (p. 9-10; Figure 5b, left panel). Holmgaard teaches the ability of galunisertib to enhance the activity of anti-PD-L1 immunotherapy was confirmed in the MC38 tumor model (microsatellite instable, MSI), which is historically less responsive to checkpoint immunotherapy (PD-L1 insensitive) and considered to be more myeloid biology driven (p. 10, col. 1; Figure 5c). Holmgaard tested the gene expression profile of CT26 tumors during combination therapy and determined altered gene expression in the tumors progressed to an increase in genes indicative of T cell activation (Table 1; p. 11, col. 1; Figure 6). Holmgaard teaches that another study recently published demonstrated that combined treatment with galunisertib + anti-PD-L1 in murine colon cancer models resulted in pronounced immune responses which eradicated most metastases, prolonged recurrence-free survival, and was associated with disruption of a T-cell exclusion phenotype (p. 13, col. 1). Holmgaard teaches there are published reports demonstrating systemic treatment with monoclonal antibodies targeting the TGFβ ligands or the TGFβRII inhibit metastatic invasion of breast cancer cells in murine tumor models, and treatment blocking TGFβ signaling with small molecule inhibitors suppresses metastasis in murine pancreatic tumor models, and enhances radiation response and prolongs survival in glioblastoma xenograft models (p. 13, col. 2). Holmgaard concludes their data provide support for continued clinical development of galunisertib to target tumors dependent on TGFβ-driven biology for growth, metastasis, and immune evasion (p. 13, col. 2). Holmgaard teaches that galunisertib is already being clinically administered in combination with checkpoint inhibitors nivolumab and durvalumab in cancer patients with NSCLC, HCC, or pancreatic cancer (p. 2, col. 2; p. 14, col. 1).
Holmgaard does not teach the method treating MSS colon carcinoma (CT26) or metastases further comprises administration of mithramycin, or that the TGFβ receptor inhibitor inhibits both TGFβI/TGFβII receptors and is LY2109761 (claims 40, 43, 44).
Holmgaard does not teach combination therapy treating MSS colon carcinoma results in a reduction in expression of Ki-67, Lgr5, CTLA-4 and pro-MMP9 and reduced by about 10%-95% compared to a control (claims 40 and 45).
Tauriello teaches treatment of MSS CRC tumors with galunisertib, an inhibitor of TGFβRI, “unleashed a potent and enduring cytotoxic T-cell response against tumor cells that prevented metastasis” (abstract). Tauriello demonstrates galunisertib significantly inhibited metastasis of MSS CRC (Figure 2d, 2f, and 2h), and significantly enhanced survival (Figure 2e). Tauriello teaches the correlation between increased TGFB1 and TGFB2 expression and worse MSS CRC prognosis:
“In MSS CRCs, the ratio of TH1 to naive cells was inversely correlated to the mean expression of TGFB1, TGFB2 and TGFB3 genes or the CAF-specific gene expression program (Extended Data Fig. 9b–f) and predicted disease relapse (Extended Data Fig. 9d, g). Therefore, abrogated T-cell differentiation, and increased TGFβ and CAF gene expression characterize a substantial subset of patients with MSS CRC and a poor prognosis” (p. 541, col. 1).
Tauriello teaches the known mechanism of inhibiting TGFβ signaling in MSS CRC to enhance tumor immunity:
“It has been hypothesized that MSS CRCs are immunologically ‘cold’, that is, scarcely T-cell infiltrated and possibly non-immunogenic, and that they are therefore unlikely to benefit from immune therapies24 (Supplementary Discussion). By contrast, our data reveal that this class of CRCs can be killed effectively by the adaptive immune system through a CTL-dependent process, which CRC cells avert by increasing TGFβ levels. Consistent with the well-established role of TGFβ signalling in suppressing differentiation and activity of T cells25–28, we observed that a TGFβ-activated TME antagonizes the TH1-effector cell phenotype. We also show that such a TME excludes T cells from tumours, a phenomenon associated with poor outcomes across cancer types3,29,30. Enabling immune infiltration using TGFβ inhibitors is sufficient to confer susceptibility to anti-PD-1–PD-L1 checkpoint-based therapies, a strategy that may have broad applications for treatment of cancers that grow in a TGFβ-rich environment. These results strongly suggest that inhibition of TGFβ signalling could be promising as immunotherapy for patients with MSS, stroma-rich CRCs and a poor prognosis” (p. 542, col. 1-2).
Schaer demonstrates that treatment of CT26 tumors with galunisertib resulted in 75% tumor growth inhibition compared to control (abstract). Combination of galunisertib with anti-PD-L1 resulted in an enhanced tumor growth inhibition of 98%, suggesting synergy when targeting both PD-L1 and TGFβ pathways (abstract).
Keedy teaches successful clinical treatment of CMS4 subtype colorectal cancer (MSS CRC) by administering TGFBRI inhibitor vactosertib and suggests combining it with other therapeutics such as immune checkpoint inhibitors or conventional anti-tumor therapies.
Zhao teaches mithramycin is a known SP1 inhibitor and demonstrates that mithramycin successfully inhibits SP1 expression in cancer stem cells (CSCs) derived from colorectal cancer cell lines, and led to marked suppression of CSC growth. SP1 is a biomarker of colon cancer cells and CSCs, and CSCs are known to persist in tumor masses as a distinct population that cause tumor relapse and metastasis. Mithramycin treatment of colorectal tumors in vivo at 25 mg/kg significantly reduced CD44 and CD166 expression, which are CSC markers (abstract; Introduction; p. 1782, col. 2 to p. 1783, col. 1; p. 1784, col. 2; Figure 6; p. 1789, col. 1-2; p. 1791, col. 1).
Kasagi teaches and successfully demonstrates that treatment of a MSS colon carcinoma (CT26) metastasis model with mithramycin and 5-FU successfully resulted in over 100 days long-term survival of 50% of animals treated (p. 355, col. 1; Figure 5 Group E). Mesenteric metastasis of CT26 cells was significantly inhibited by the pretreatment with mithramycin A (p. 354, col. 2; Figure 4F). Mesenteric tumor dissemination of human colorectal cancer cells was significantly abrogated by mithramycin A (p. 355, col. 1).
Baum teaches treating human patients having advanced cancers with a dose of 25 µg/kg (0.025 mg/kg) each day for 8 days, wherein the patients having rectal carcinoma had either major or minor tumor regression (p. 177-178). Baum suggests pursuing treatment with mithramycin for patients having rectal carcinomas (p. 183).
Adding mithramycin treatment to anti-PD-1/PD-L1 immunotherapy + TGFβI kinase receptor inhibitor and reducing tumor growth at least 45%-65%:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to add mithramycin treatment to the method of treating MSS colon carcinoma and inhibiting metastasis taught by Holmgaard. One would have been motivated to, and have a reasonable expectation of success to, because: (1) the cited references teach and demonstrate that all of the agents: mithramycin and TGFβI kinase receptor inhibitor, successfully treat the same colon carcinoma and inhibit metastasis; (2) Holmgaard, Tauriello, Schaer, and Keedy demonstrate that TGFβI kinase receptor inhibitor therapy successfully treats MSS colon carcinoma, significantly reducing tumor growth greater than 45%; and (3) Holmgaard, Tauriello, Schaer, and Keedy suggest combining a second anti-cancer therapeutic with TGFβ inhibition and demonstrate that a variety of second anti-cancer agents combined with TGFβI kinase receptor inhibitor resulted in synergistic anti-tumor treatment.
Those of skill in the art recognize that the anti-cancer agents, mithramycin and TGFβI kinase receptor inhibitor: (1) each successfully treats the same MSS colon carcinoma and inhibit metastasis, wherein TGFβI kinase receptor inhibitor alone significantly reduces tumor growth, and (2) could have been readily and predictably combined by known methods to perform the same function of treating MSS colon carcinoma, and that in combination, they would have merely would have performed the same function as they did separately, and one of ordinary skill in the art would have recognized that the results of the combination would predictably treat MSS colon carcinoma and inhibit metastasis, as already demonstrated.
The instant situation is amenable to the type of analysis set forth in In re Kerkhoven, 205 USPQ 1069 (CCPA 1980) wherein the court held that: “It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition which is to be used for the very same purpose. In re Susi, 58 CCPA 1074, 1079-80, 440 F.2d 442, 445, 169 USPQ 423, 426 (1971); In re Crockett, 47 CCPA 1018, 1020-21, 279 F.2d 274, 276-77, 126 USPQ 186, 188 (1960). As this court explained in Crockett, the idea of combining them flows logically from their having been individually taught in the prior art.” In the instant case, it is prima facie obvious to combine mithramycin with the composition of Holmgaard, which is taught by the prior art to be useful for the same purpose, in order to be used for the very same purpose of treating MSS colon carcinoma and inhibiting metastasis.
Administering dual kinase TGFβI/ TGFβII receptor (TβRI,TβRII) inhibitor, LY2109761, in place of the TGFβI receptor inhibitor:
Like Holmgaard and Tauriello, Zhang teaches cancers can be driven by TGFβ, wherein TGFβ promotes tumor growth, invasion, and metastasis in advanced stages of colorectal cancer. Blocking the tumor-promoting effects of TGFβ provides an important therapeutic strategy for the treatment of colorectal cancer. The rational design of inhibitors that specifically counteract the tumor-promoting effects of TGFβ can be useful as a therapeutic strategy for the treatment of cancers. Small molecule inhibitors of TGFβ receptor kinases have demonstrated superior efficacy due to increased tissue penetration and cellular permeability (abstract; Discussion). The multifunctional effects of TGFβ are elicited through an oligomeric complex between TβRI and TβRII receptors. In response to TGFβ ligand, TβRII phosphorylates TβRI. TβRI then activates Smad2 and Smad3, which associates with Smad4 and translocates to the nucleus, where it modulates transcription of TGFβ target genes. Additionally, TGFβ can induce non-Smad pathways including p38MAPK, ERK, PI3K, JNK, and Rho, which are important for pro-oncogenic activities (Introduction). TGFβ small molecule inhibitors are reported to reduce metastasis of breast and pancreatic cancer cells. Reports of LY2109761, a TGFβ dual receptor kinase inhibitor, can reduce migration and invasion of hepatocellular carcinoma cells and suppress pancreatic cancer metastasis. Zhang successfully demonstrates that LY2109761 reduces TGFβ-mediated cell migration, invasion, tumorigenicity and metastasis of CT26 colon carcinoma cells, and significantly prolongs survival (Figures 2-5; Discussion). Zhang concludes that LY2109761 can reduce metastasis of colon cancer cells to the liver by attenuating TGFβ-induced oncogenic signaling pathways (Discussion, p. 120).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to administer small molecule kinase inhibitor LY2109761, in place of the small molecule inhibitor LY2157299, in the method of the cited combined references above. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Holmgaard, Tauriello, Schaer, and Keedy teach and demonstrate that inhibiting TGFβ signaling through inhibition of TGFβRI significantly or successfully treats MSS colon carcinoma; (2) Holmgaard, Tauriello, Schaer, and Keedy recognize that cancers are TGFβ-driven, and the prior art demonstrates TGFβI or TGFβII receptor inhibition inhibited metastatic invasion of breast cancer cells in murine tumor models, suppressed metastasis in murine pancreatic tumor models, and enhanced radiation response and prolongs survival in glioblastoma xenograft models; (3) Zhang also teaches treating cancer and inhibiting metastasis by inhibiting TGFβ signaling; and (4) Zhang explains the known mechanism of TGFβ-driven cellular pathways promoting oncogenesis, and successfully demonstrates that LY2109761, a known dual inhibitor of TGFβRI and TGFβRII kinase receptors, treats CT26 MSS colon carcinoma and inhibits metastasis. Given Holmgaard Tauriello, Zhao, Keedy and Zhang recognize inhibiting TGFβ-driven pathways through TβRI and TβRII kinase receptors treat cancer and inhibit metastasis, and they all successfully demonstrate small molecule inhibitors of TGFβRI and TGFβRII both treat the same MSS colon carcinoma, one of skill in the art could have substituted LY2109761 for LY2157299, in the method of the cited combined references, and predictably treated and inhibited metastasis of MSS colorectal cancer.
Dosing mithramycin at about 0.5 mg/kg to about 10 mg/kg or at about 1 mg/kg:
Jia 2010 teaches successfully administering mithramycin at a dose of 0.5 mg/kg to 1.5 mg/kg to treat cancer.
Jia 2007 teaches administering mithramycin to treat cancer, and at a dose of 0.1, 0.2, or 0.4 mg/kg repeatedly, wherein a combination therapy with mithramycin produced synergistic antitumor activity (p. 4880, col. 2 to p. 4881, col. 1; Figures 1-3; Materials and Methods p. 4879, col. 1). A dose of 0.4 mg/kg is reasonably encompassed by “about 0.5 mg/kg”.
Baum, as stated above, teaches treating human patients having advanced cancers with a dose of 25 µg/kg (0.025 mg/kg) each day for 8 days, wherein the patients having rectal carcinoma had either major or minor tumor regression (p. 177-178). Baum suggests pursuing treatment with mithramycin for patients having rectal carcinomas (p. 183).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to administer a dose of about 0.5 or about 1 mg/kg mithramycin to the MSS colorectal carcinoma patients in the method of the combined references. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Zhao, Kasagi, Jia 2010, Jia 2007, and Baum demonstrate mithramycin treatment is successful for colorectal cancer and other cancer treatment at a wide variety of doses; (2) Jia 2010 demonstrates doses ranging from 0.5 mg/kg to 1.5 mg/kg successfully treated cancer; and (3) Jia 2007 demonstrates that a dose of mithramycin of about 0.5 mg/kg is successful for cancer treatment and synergistic with a second anti-cancer agent.
Reducing expression of Ki-67, Lgr5, CTLA-4 pro-MMP9, and MMP2 with treatment and at about 10% to about 95% compared to a control:
Luo teaches the Ki-67 is a prognostic biomarker of colorectal cancer, wherein high Ki-67 expression serves as a valuable predictive method of poor prognosis of colorectal cancer patients, and reduced expression of Ki-67 indicates improved prognosis, improved survival, and improved disease-free survival (abstract; Figure 7 and 8; p. 12, col. 1).
Jiang teaches that Lgr5 is a prognostic biomarker of colorectal cancer, wherein Lgr5 overexpression was significantly associated with worse overall survival, deep invasion, distant metastasis, and lymph node metastasis (abstract; Table 3; Figure 4).
Mao teaches that increased CTLA-4 expression in colon cancer is associated with low tumor purity and worse survival/prognosis (abstract; p. 3571; p. 3574; Figure 4).
Murnane et al teaches that increase pro-MMP9 and MMP2 expression were associated with the presence of colorectal cancer, wherein lower levels were associated with normal tissue (abstract; Figure 1; Results on p. 2895, col. 1 to p. 2897, col. 1). Levels of pro-MMP9 were significantly higher in cancers versus normal tissue (abstract; Figure 4).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to achieve a result of reduced expression of biomarkers Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2, and to reduce their expression at about 10% to about 95% compared to a control in the method of treating MSS colorectal cancer taught by the cited combined references above. One would have been motivated to, and have a reasonable expectation of success to, because: (1) the cited combined references above teach the goal of their method is to treat colorectal cancer and enhance survival, (2) Luo, Jiang, Mao, and Murnane all teach reduction of expression in known biomarkers Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2 indicate improved prognosis for colorectal cancer and provide motivation to achieve such reduced expression; and (3) Luo, Jiang, Mao, and Murnane all teach known and routine methods for successfully detecting reduced expression of Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2 to assess prognosis. Given: (1) the known need to treat CRC and enhance survival; (2) the known biomarkers indicative of CRC treatment and enhanced survival; and (3) the known routine methods of measuring the biomarkers in CRC; one of ordinary skill in the art would be motivated to measure reduced levels of the known CRC biomarkers Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2, including reduced levels in the range of about 10%, as correlated to treatment response and improved prognosis with a reasonable expectation of success.
10. Claim(s) 48, 49, 51-53 and 55 are rejected under 35 U.S.C. 103 as being unpatentable over Holmgaard et al (Journal for ImmunoTherapy of Cancer, 2018, 6:47, internet pages 1-15); Tauriello et al (Nature, 2018, 554:538-543 and Extended Data); Schaer et al (Journal of ImmunoTherapy of Cancer, 2015, 3(Suppl):P402); Keedy et al Journal of Clinical Oncology, 2018, 36:3031); Zhao et al (Oncology Reports, 2013, 30:1782-1792); Kasagi et al (Cancer Research, 2016, 76:347-357); Baum (British Journal of Cancer, 22.2 (1968): 176-183); Zhang et al (Cancer Letters, 2009, 277:114-120); Jia 2010 (Cancer Research, 2010, 70:1111-1119); Jia 2007 et al (Cancer Research, 2007, 67:4878-4885); Luo et al (BMC Cancer, February 6, 2019, 19:123; internet pages 1-13); Jiang et al (BMC Cancer, 2015, 15:948, internet pages 1-10); Mao et al (Cancer Management and Research, 2018, 10; 3569-3577); and Murnane et al (International Journal of Cancer, 209, 2893-2902) as applied to claims 40, 41, 43-45 and 47 above, and further in view of Oladipo et al (British Journal of Cancer, 2011, 104:480-487); and Hsu et al (International Journal of Molecular Sciences, 2018, 19:2427; internet pages 1-17).
Holmgaard; Tauriello; Schaer; Keedy; Zhao; Kasagi; Baum; Zhang; Jia 2010; Jia 2007; Luo; Jiang; Mao; and Murnane (the combined references) teach a method of
treating and inhibiting metastasis of MSS colorectal cancer in a subject comprising administering to the subject a dual inhibitor of TGFβI/TGFβII receptors + mithramycin, administering mithramycin at a dose of about 0.5 mg/kg to about 10 mg/kg, reducing tumor growth by about 45%-65%, and reducing expression of colorectal cancer markers Ki-67, Lgr5, CTLA-4, pro-MMP9, and MMP2, as set forth above.
The combined references do not teach:
reducing the expression of colorectal cancer marker CXCL1, and from about 10% to about 95% compared to a control (claim 48); or
synergistic inhibition of metastases with dual inhibitors of TGFβI/TGFβII receptors + mithramycin (claim 53).
Reducing the expression of colorectal cancer marker CXCL1, and from about 10% to about 95% compared to a control:
Oladipo teaches that CXCL1 is a marker increased in colorectal cancer compared to normal controls (Figure 2; abstract), and that decreasing CXCL1 expression levels correlated with increasing recurrence-free survival (Table 3; abstract). Oladipo demonstrates known methods for detecting and comparing levels of CXCL1 in CRC by immunohistochemistry (Materials and Methods).
Hsu demonstrates that inflammatory chemokine CXCL1 is present in large amounts in dendritic cells (DCs) isolated from colon cancer patients, and CXCL1 increases the metastatic ability of a cancer by enhancing cell migration, matrix metalloproteinase-7 expression and epithelial-to-mesenchymal transition (EMT) (abstract; Figure 7). Hsu demonstrates that CXCL1 expression increased the cancer stem cell properties of metastatic CRC cells (section 2.3; Figure 3). Hsu demonstrates CXCL1 enhanced cell migration and EMT transition of metastatic CRC cells (section 2.4, Figure 4). Hsu teaches (p. 2):
“A growing body of studies has shown inflammatory cytokines and chemokines can act as tumor growth and survival factors and promote tumor progression and metastasis by increasing angiogenesis and suppressing immune-mediated tumor elimination [14–16]. Elevated chemokine (C-X-C motif) ligand 1 (CXCL1) levels are found in CRC, and these increased levels are positively associated with cancer stage, metastasis and poor survival rates [17,18]. A recent study has revealed that CXCL1 contributes to the formation of a pre-metastatic niche in the liver by recruiting chemokine (C-X-C motif) receptor 2 (CXCR2)-positive myeloid-derived suppressor cells [19].”
Hsu teaches (p. 10-11):
“CXCL1 is a potent proinflammatory mediator of inflammatory diseases and infection, and is widely considered to both promote and exacerbate tumor growth and progression in several cancers [25,26]. CXCL1 is upregulated in various cancers and associated with cancer progression, such as cancer cell growth, proliferation, tumor angiogenesis and metastasis, after the activation of CXCR2 [27–29]. In addition, CXCL1 is not only involved in cancer progression, but also responsible for resistance to several chemotherapeutic drugs, such as oxaliplatin, doxorubicin, and cyclophosphamide [30–33]. CSCs are considered to display the clonogenic core of the cancer, since it is implied that these cells are involved in tumor propagation, progression, chemo-resistance and metastatic dissemination. In this study, we found that CXCL1 is not only released from malignant cells, but also secreted from cancer-conditioned DCs. TADCs express high levels of CXCL1, which in turn increases the tumorigenesis and chemo-resistance potential by increasing CSC-like properties. Furthermore, TADC-derived CXCL1 also enhances cancer migration and switches the epithelial phenotype to a mesenchymal characteristic, a key process of cancer metastasis. These findings suggest that TADC-derived CXCL1 may be a new candidate in conferring the ability for colon cancer to progress.
It is of interest to note that CXCL1-producing CD11c+ DCs were found to infiltrate the cancerous tissue of CT26-bearing mice. In addition, CD11c+ DCs isolated from patients with CRC produce high levels of CXCL1 when compared with CD11c+ DCs isolated from healthy donors. These results based on experimental cell studies, animal models, and clinical patients strongly suggest that TADCs are one of the critical effectors in CRC stroma enhancing the development of colon cancer by CXCL1 production.”
Hsu teaches (p. 11):
“In conclusion, our results further support the role of TADCs and their secreted CXCL1 in colon cancer progression. The findings reported here not only introduce a novel mechanism of immunosuppression, but also provide preliminary evidence of the potential utility of CXCL1 inhibition as a therapeutic strategy in fighting cancer.”
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to reduce expression of CXCL1 and to reduce it about 10% to about 95% as compared to a control. One would have been motivated to, and have a reasonable expectation of success to, because: (1) the combined references teach the goal of their method is to treat colorectal cancer and enhance survival, (2) Oladipo teaches CXCL1 is an inflammatory marker of CRC where decreased levels correlated with increased recurrence-free survival; (3) Hsu teaches CXCL1 is a known marker upregulated in various cancers and associated with cancer progression, such as cancer cell growth, proliferation, tumor angiogenesis and metastasis; (3) Hsu demonstrates CXCL1 is a known inflammatory marker of CRC that increased the cancer stem cell properties of metastatic CRC cells, enhanced cell migration and EMT transition of metastatic CRC cells, and contributes to the development of colon cancer, and Hsu suggests reducing CXCL1 levels for treatment; and (4) Oladipo and Hsu demonstrate known methods for successfully detecting expression levels of CXCL1 in CRC. Given: (1) the known function of CXCL1 as a marker of CRC and worse prognosis, (2) the known function of CXCL1 contributing to CRC tumorigenesis and metastatic phenotype, and (3) the known motivation to reduce levels of CXCL1 in CRC; it is well within the level of the ordinary skilled artisan to reduced levels of CXCL1 in the CRC patients of the combined references to achieve improved prognosis and treatment, and to reduce CXCL1 levels as low as possible from about 10% to about 95% as compared to a control.
Synergistic inhibition of metastasis:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to produce synergistic inhibition of metastases in the method of the combined references. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Holmgaard, Tauriello, and Schaer demonstrate that combination therapy with TGFβI receptor inhibitor and second anti-cancer agent synergistically treats CRC, wherein TGFβI receptor inhibitor alone is demonstrated to significantly treat CRC and inhibit metastasis; (2) Holmgaard teaches there are published reports demonstrating systemic treatment with inhibitors of TGFβRII inhibit metastatic invasion of breast cancer cells in murine tumor models, (3) Zhao demonstrates that mithramycin successfully suppresses of CRC cancer stem cell growth that normally contributes to metastasis; and (4) Kasagi successfully demonstrates that treatment of a MSS colon carcinoma (CT26) metastasis model with mithramycin and 5-FU successfully resulted in over 100 days long-term survival of 50% of animals treated, mesenteric metastasis of CT26 cells was significantly inhibited by the pretreatment with mithramycin A, and mesenteric tumor dissemination of human colorectal cancer cells was significantly abrogated by mithramycin A. Given the significant reduction of CRC metastasis by either kinase inhibitor or mithramycin alone, and given they serve the same function of inhibiting metastases, one of ordinary skill in the art could have combined a dual inhibitor of TGFβRI and TGFβRII with mithramycin to synergistically inhibit metastases with a reasonable expectation of success.
11. All other rejections recited in the Office Action mailed August 28, 2025 are hereby withdrawn in view of amendments. The amendments to remove reference to the dual kinase inhibitor as an immunotherapy, and to remove the requirement to combine all three agents (dial kinase inhibitor, anti-PD-1/PD-L1 immunotherapy, and mithramycin) obviate the new matter rejection previously of record, as well as the prior rejections made under 35 USC 103.
12. Conclusion: No claim is allowed.
13. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
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/Laura B Goddard/Primary Examiner, Art Unit 1642