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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 March 13, 2026 has been entered.
Claims 1, 2, 4, 5, 10-16, 59, and 78 are now pending. Claims 16 and 59 remain withdrawn. Claims 1, 2, 4, 5, 10-15, and 78 are currently being examined. Claim 78 is new. Claim 1 is amended to limit CD73 low expression to immune cells.
New Rejection
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.
2. Claim 4 is 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 4 depends from canceled claim 3. There is insufficient antecedent basis for the limitations in the claim.
New Rejections with Additional References
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.
3. Claim(s) 1, 2, 5, and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Deumelandt et al (Cancer Immunol. Res., February 1, 2019, 7(2_Supplement), abstract B075); in view of Wainwright et al (Clinical Cancer Research, 2014, 20:5290-5301); Figueiro et al (Purinergic Signalling, 2016; 12:303–312); Jiang et al (BMC Cancer, 2018, 18:267, internet pages 1-10); Allard et al (Clinical Cancer Research, 2013, 19:5626-5635); Chen et al (Immunotherapy, 2019, 11:983-997, published online June 21, 2109); and Saha et al (Cancer Cell, 2017, 32:253-267).
Deumelandt teaches:
Immune checkpoint inhibitors are now implemented into the standard therapy of an increasing number of tumor entities and elicit remarkable durable therapy responses. However, gliomas seem resistant to checkpoint inhibition as recent evidence from a randomized clinical trial did not show a therapeutic benefit of PD-1 blockade in an unselected population of patients with recurrent glioblastoma.
This project investigates the mechanisms of response and resistance to checkpoint blockade targeting CTLA-4 and PD-1 in an experimental syngeneic Gl261 glioma model, where we found a clear and unanticipated dichotomy between responders and non-responders. We demonstrate that response to PD-1 and CTLA-4 blockade is driven by increased numbers and effector function of cytotoxic tumor-infiltrating T-cells as well as an enhanced TCRβ repertoire clonality of tumor infiltrating CD8 T-cells. Surprisingly, little overlap of the TCRβ repertoire between responder CD8 TILs was detected with only one shared TCRβ sequence motif, suggestive of a common tumor-antigen driving the expansion of reactive clones in responding mice.
Resistance to PD-1 and CTLA-4 blockade was associated with increased frequencies of intratumoral macrophages (TAMs) expressing high levels of immunosuppressive markers such as PD-L1, CD38 and CD73. TAMs of nonresponding mice induced enhanced suppression of CD4 T-cell proliferation which was partially restored by PD-L1 blockade. Strikingly, additional PD-L1 blockade enhanced response rates to PD-1 and CTLA-4 blockade in Gl261-bearing mice, potentially by inhibiting the ligation of PD-L1 on TAMs to its alternative interaction partner CD80 on TILs. Collectively, we suggest a syngeneic mouse model for assessing mechanisms of response and resistance to checkpoint blockade in gliomas demonstrating a surprising heterogeneity of the TCRβ repertoire of tumor-infiltrating CD8 T-cells despite strict syngeneicity. We also provide evidence for a suppressive TAM subset associated with resistance to immune checkpoint inhibition in glioma, providing a rationale for combinatorial therapy strategies to overcome resistance to checkpoint blockade.
Deumelandt recognizes glioblastoma demonstrates clinical resistance to PD-1 blockade and there is a need to identify the reason for resistance and to improve treatment. Deumelandt determined, using a mouse GI261 glioma model, that there was a clear dichotomy between responders and non-responders to CTLA-4 and PD-1 blockade. Deumelandt identified resistance to CTLA-4 and PD-1 blockade as correlated to increased frequencies of intratumoral macrophages (TAMs) expressing high levels of immunosuppressive markers such as PD-L1, CD38, and CD73 (therefore identifying comparative high versus low CD73 expression levels). Deumelandt (“strikingly”) was able to enhance response to PD-1 and CTLA-4 blockade and restore CD4 T cell proliferation by blocking PD-L1, teaching this blockade inhibited the ligation of PD-L1 on TAMs to its alternative interaction partner on CD80 on TILs. Deumelandt teach they provided evidence for a suppressive TAM subset associated with resistance to immune checkpoint inhibition in glioma, and suggests overcoming resistance with combination immune checkpoint blockade therapy.
Deumelandt recognizes CD73 as a marker of TAMs (immune cells) responsible for inducing resistance to PD-1 and CTLA-4 blockade in glioblastoma, and Deumelandt suggests and successfully demonstrates the solution to overcome resistance is to administer combination immune checkpoint blockade therapy.
However, Deumelandt does not demonstrate selecting patients having low CD73 immune cell expression and administering combination immune checkpoint blockade therapy comprising (i) PD-1 or PD-L1 antibody and (ii) CTLA-4 antibody. Deumelandt does not teach the CD73 expression was detected by immunoassay.
Wainwright also recognizes glioblastoma suffers from an immunosuppressive tumor environment with accumulation of tumor- and/or macrophage- expressed PD-L1 and increased dendritic cell expression of CD80 (abstract; p. 5290-5291). Wainwright suggests administering combination immune checkpoint therapy and demonstrates treating a GL261 glioma mouse model with anti-PD-L1 or anti-CTLA-4 antibody monotherapy, as well as combination antibody therapy. Wainwright demonstrates that combined anti-PD-L1 and anti-CTLA-4 antibody therapy resulted in significantly increased survival compared to either monotherapy. Wainwright concludes that combination blockade leads to prolonged T cell-mediated survival against brain tumors (Figure 3 below).
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Wainwright teach their data suggests combinatorially targeting immunosuppression in malignant glioma as a clinical strategy (abstract).
Figueiro teaches glioblastoma multiforme (GBM) is a deadly cancer characterized by a pro-tumoral immune response. T-regulatory (Treg) lymphocytes suppress effector immune cells through cytokine secretion and the adenosinergic system. Ecto-5′-nucleotidase/CD73 plays a crucial role in Treg-mediated immunosuppression in the GBM microenvironment (GME) (abstract). Figueiro demonstrates the immunosuppressive role of CD73 in glioblastoma (p. 307-311), and demonstrates known methods of immunoassay for detecting CD73 expression levels on immune cells (p. 306, “Surface protein staining”; Figure 4). Figueiro compares expression levels of CD73 on immune cells having lower CD73 expression levels and having higher CD73 expression (Figure 4), including determining a statistically significant difference between expression levels.
Jiang teaches (p. 1, col. 2 to p. 2, col. 1):
“CD73-derived adenosine mainly mediates immunosuppression via activation of A2A receptor on immune cells, especially natural killer (NK) cells and CD8+ T cells. Recent studies revealed that CD73 plays a pivotal role in tumor escape from immune surveillance. The mechanism can be summarized into three aspects: (i) inhibition of clonal expansion, activation and homing to tumor specific T cells; (ii) to increase a substantial component of the suppressive capabilities of regulatory T cells (Tregs) and Th17 cells; (iii) to accelerate the conversion of anti-tumor type 1 macrophages into pro-tumor type 2 macrophages [9]. Targeting CD73 results in favorable antitumor effects in preclinical studies and combination of CD73 blockade with other immune checkpoint inhibitors, such as anti-cytotoxic T-lymphocyte antigen (CTLA)-4 antibody or anti-programmed cell death protein (PD)-1/PD-1 ligand (PDL1) antibody, is particularly promising [9]. Increasing evidence suggested that CD73 highly expressed in a wide range of cancer types, including breast cancer, colorectal cancer, glioblastoma, melanoma, prostate cancer, ovarian cancer, and non-small-cell lung cancer (NSCLC). High CD73 expression was often associated with poor prognosis in different cancers.”
Jiang teaches that CD73 expression on tumor cells weakens the immune response to PD-1/PD-L1 inhibitors (p. 8, col. 2). Jiang teaches that the prior art (Allard) reported anti-CD73 antibody dramatically enhanced the effect of anti-CTLA-4 and PD-1 inhibitors against colon, prostate and breast cancer in mice models. The prior art (Iannone and colleagues) found that blockade of CD73 could enhance the efficacy of anti-CTLA-4 in a melanoma model. The prior art (Beavis) further showed that combination of CD73-A2A inhibition and anti-PD-1 mAb resulted in greater antitumor immune response through prolonged expression of IFN-gamma and granzyme B. These results suggested that CD73 was a potential biomarker for response to anti-PD-1/PD-L1 treatment (p. 8, col. 2). Jiang concludes that “CD73 is also a promising target in future cancer immunotherapy and has the potential significance as a biomarker for anti-PD-1/PD-L1 treatment” (p. 9, col. 1).
Allard, cited by Jiang above, teaches and demonstrates that inhibiting CD73 with anti-CD73 mAb significantly enhances the activity of both anti-CTLA-4 and anti-PD-1 antibodies against colon, prostate, and established metastatic breast cancer models, wherein anti-CD73 mAb synergized with anti-PD-1 mAb (abstract).
Chen reviews the known role of CD73 in checkpoint immunotherapy. Chen teaches CD73 is expressed by many cells, including tumor cells, endothelial cells, T cells (including Tregs which are TILs), macrophages, MDSCs, and other immune cells (p. 984-986). Chen teaches CD73, regardless of cell it is expressed by, performs the same function of generating extracellular adenosine that negatively regulates the activation and effector phases of antitumor T cell response, while also promoting T cell apoptosis. CD73:
Effects of CD73 activity by tumor cells
The extracellular adenosine generated by CD73-expressing tumor cells [24, 25] negatively regulates the activation and effector phases of the antitumor T cell response, while also promoting T cell apoptosis (see p. 984).
Effects of CD73 activity by nontumor cells
T cells
Regulatory T cells (Tregs; CD4+CD25+FoxP3+) mediate immunotolerance and help tumor cells evade immunosurveillance by suppressing the immune response. One of the main mechanisms for Treg-mediated tumor immunosuppression is dependent on the extracellular adenosine generated by CD73 [18] . CD73 is abundantly expressed by Tregs and is frequently coexpressed with CD39. CD73, in combination with CD39, renders an enzymatically driven accumulation of immunosuppressive adenosine by Tregs. Accordingly, Tregs derived from either CD73−/− or CD39−/− mice have impaired suppressive functions [64, 65]. Unlike WT murine Tregs, CD73−/− Tregs fail to promote tumor growth [18, 26] (see p. 985).
Macrophages
The expression levels of CD39 and CD73 regulates adenosine generation [90] . Macrophages are divided into two primary subsets, the pro-inflammatory M1 population and the anti-inflammatory M2 population. M1 macrophages express lower levels of CD39 and CD73 as compared with the M2 subset [90] . Changes in the ectoenzyme activities of CD39 and CD73 may fine-tune their functions in the inflammatory setting. In a murine model of myocardial infection, CD73 blockade by APCP augmented the predominance of the M1 subset [91] . Similarly, ablating CD73 activity in tumor-bearing mice decreases M2macrophages and increases the M1 subset [27] (see p. 986).
Chen also summarizes numerous examples of inhibiting CD73 in several different cancers, including glioblastoma, that resulted in reduced tumor cell proliferation, reduced metastasis, and enhanced anti-tumor immune response (Table 1). Chen summarizes clinical application of CD73 blockade in combination with anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody treatment of several different cancers (Table 2). Chen concludes the established function of CD73 is to generate adenosine that suppresses antitumor immunity and contributes to tumor outgrowth and/or metastasis (Conclusion p. 990). Chen suggests inhibiting CD73 will act synergistically with other immune checkpoint inhibitors (Executive summary box, p. 991).
Chen teaches the known higher expression of CD73 on immunosuppressive M2 macrophages as compared to pro-inflammatory M1 macrophages, teaching that inhibiting CD73 increases the proportion of M1 macrophages (see p. 986). Saha teaches treating a glioblastoma model by administering an agent that induced M1-like polarization in TAMs (reducing the immunosuppressive M2 macrophages), where the addition of anti-PD-1 and anti-CTLA-4 antibody therapy overcame the immunosuppressive tumor environment and eliminated tumors (p. 263, col. 2 to p. 264, col. 1; Fig. 7). Saha teaches the major hallmark of this combination therapy was an increase in M1-like polarized TAMS, suggesting this strategy is important to effectively treat glioblastoma and likely other checkpoint-inhibitor non-responding tumors outside the brain (p. 264, col. 1-2). Thus, Saha demonstrates that skewing the tumor microenvironment to contain CD73 low expressing M1-like TAMs significantly enhanced response to anti-PD-1 and anti-CTLA-4 antibody therapy.
Administer combined anti-PD-1/PD-L1 and anti-CTLA-4 antibody checkpoint inhibition therapy after the subject is determined to have low CD73 expression in an immune cell:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to administer combined anti-PD-1/PD-L1 and anti-CTLA-4 antibody checkpoint inhibition therapy after the subject is determined to have low CD73 expression in an immune cell in the method of Deumelandt. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Deumelandt specifically identified increased expression levels of CD73 in glioma as a marker of TAMs (immune cells) that contribute to immunosuppression and resistance to PD-1/PD-L1 and CTLA-4 blockade; (2) Deumelandt suggests improving treatment of glioma by administering combination immune checkpoint inhibiting therapy; (3) Deumelandt demonstrates successfully inhibiting the TAMs in a glioma model with anti-PD-L1 antibody and this inhibition enhanced glioma response to PD-1 and CTLA-4 blockade, (4) Deumelandt provided a clear correlation between increased CD73 levels/TAMs in glioma, increased immunosuppression in the tumor, and decreased response to PD-1 and CTLA-4 blockade; (5) Deumelandt provide a clear correlation between decreasing CD73 expressing TAMs with PD-L1 blockade in glioma and enhancing response to PD-1 and CTLA-4 blockade therapy; (6) Wainwright, like Deumelandt, also recognized the immunosuppressive tumor environment of gliomas and suggested and demonstrated successfully treating glioma with a combination of anti-PD-L1 and anti-CTLA-4 antibody, where the combination significantly prolonged T cell-mediated survival against brain tumors compared to monotherapy; (7) Figueiro also recognizes CD73 expression is a marker of immunosuppression in glioblastoma and several other cancers; (8) Jiang and Allard teach the prior art has established that CD73 is a biomarker of resistance to PD-1/PD-L1 blockade and demonstrate inhibiting CD73 with an anti-CD73 antibody dramatically enhanced the effect of anti-CTLA-4 and PD-1 inhibitors against several different mouse tumor models; (9) Chen teaches the function of CD73 is established and known as generating adenosine that suppresses antitumor immunity and contributes to tumor outgrowth and/or metastasis regardless of what cell type expresses it: tumor cell, Treg (TIL), or TAM (macrophage); (10) Chen, Jiang, and Allard demonstrate the decreasing CD73 expression/activity in combination with anti-CTLA-4 and PD-1 inhibitors predictably results in enhanced tumor treatment and enhanced anti-tumor immune response; (11) Chen and Saha teach it is known that M2 macrophages express high levels of CD73 compared to M1 subsets, M2 macrophages are immunosuppressive; and (12) Saha demonstrates that skewing the glioblastoma tumor to contain more M1-like macrophages and administering two checkpoint inhibitors against PD-1 and CTLA-4 synergistically cures glioblastoma, providing a clear correlation between increasing the presence of M1-like TAMS and enhanced response to PD-1 and CTLA-4 checkpoint inhibitors.
The cited prior art makes clear: (1) the association between increased CD73 expression on any cell type and an immunosuppressive tumor environment, (2) decreased CD73 expression in immune cells and increased response to anti-PD-1/PD-L1 and anti-CTLA-4 therapy, (3) combined anti-PD-1/PD-L1 and anti-CTLA-4 antibody therapy is superior to monotherapy for glioblastoma; and (4) skewing the glioblastoma tumor environment to contain more M1-like macrophages (known to have low CD73 expression compared to immunosuppressive M2 cells) and administering two checkpoint inhibitors against PD-1 and CTLA-4 synergistically cures glioblastoma.
Immunoassay for CD73 detection (claim 15):
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to utilize immunoassay to detect CD73 expression levels in the method of the cited references. One would have been motivated to, and have a reasonable expectation of success to, because Figueiro demonstrates successful immunoassay detection of CD73 expression levels in glioblastoma tissues and comparing their levels for statistically significant differences to identify higher and lower expression.
4. Claim(s) 13-14 are rejected under 35 U.S.C. 103 as being unpatentable over Deumelandt et al (Cancer Immunol. Res., February 1, 2019, 7(2_Supplement), abstract B075); Wainwright et al (Clinical Cancer Research, 2014, 20:5290-5301); Figueiro et al (Purinergic Signalling, 2016; 12:303–312); Jiang et al (BMC Cancer, 2018, 18:267, internet pages 1-10); Allard et al (Clinical Cancer Research, 2013, 19:5626-5635); Chen et al (Immunotherapy, 2019, 11:983-997, published online June 21, 2109); and Saha et al (Cancer Cell, 2017, 32:253-267); as applied to claims 1, 2, 5, and 15 above, and further in view of Wang et al (Journal of Clinical Medicine, 2019, 8:1526, internet pages 1-14) and Thibaudin et al (OncoImmunology, 2016, 5:e1055444).
Deumelandt; Wainwright; Figueiro; Jiang; Allard, Chen, and Saha (the combined references) teach a method of treating glioblastoma in a patient comprising administering to the patient combined anti-PD-1/PD-L1 and anti-CTLA-4 antibody immune checkpoint blockade therapy after the patient has been determined to have low expression of CD73 in an immune cell sample from the patient, as set forth above. As stated above, Figueiro teaches and demonstrates conducting immunoassays to measure and compare levels of CD73 in immune cells.
The combined references do not elaborate on methods for comparing CD73 expression levels to controls or cutoffs, or comparing to normalized low expression levels.
Wang, like the cited combined references, recognizes that CD73 plays an immunosuppressive role in cancer, especially glioblastoma (sections 3.5 and 4). Wang teaches and demonstrates measuring CD73 expression levels in glioblastoma biological samples, producing numerical cutoffs for high and low CD73 expression levels (sections 2.2 and 3.1), and exemplify identifying significantly higher expression levels in glioblastoma compared to normal brain tissue (section 3.1; Figure 1). Wang exemplifies identifying glioblastoma samples that had downregulated (low) CD73 expression levels that were correlated with longer overall survival than upregulated CD73 levels, and using cutoff to determine high and low expression (section 3.2; Figure 2).
Thibaudin also demonstrates measuring and comparing CD73/NT5E levels in patient biological samples, and normalizing levels to a standard human gene ACTB (see “Real-time quantitative PCR”). Thibaudin demonstrates measuring CD73 protein expression levels in patient immune cells with commercial anti-CD73 antibody (see “Western blotting” and “Immunofluorescence microscopy”). Thibaudin demonstrates known methods of statistical analysis to identify significant expression values (see “Statistical analyses”).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to identify high and low CD73 expression levels in immune cellss produced by normalized expression levels, cutoffs, and comparing to controls. One would have been motivated to, and have a reasonable expectation of success to, because the combined references demonstrate the need to identify high or low CD73 expression levels in patient tumor and immune cells to characterize tumor microenvironment, and Figueiro, Wang, and Thibaudin demonstrate such methods of analysis to identify high or low CD73 expression levels in patient cells are known and successfully practiced.
5. Claim(s) 4 and 78 are rejected under 35 U.S.C. 103 as being unpatentable over Deumelandt et al (Cancer Immunol. Res., February 1, 2019, 7(2_Supplement), abstract B075); Wainwright et al (Clinical Cancer Research, 2014, 20:5290-5301); Figueiro et al (Purinergic Signalling, 2016; 12:303–312); Jiang et al (BMC Cancer, 2018, 18:267, internet pages 1-10); Allard et al (Clinical Cancer Research, 2013, 19:5626-5635) Chen et al (Immunotherapy, 2019, 11:983-997, published online June 21, 2109); and Saha et al (Cancer Cell, 2017, 32:253-267); as applied to claims 1, 2, 5, and 15 above, and further in view of Thibaudin et al (OncoImmunology, 2016, 5:e1055444, internet pages 1-12) and Deng et al (International Journal of Cancer, 2018, 143:1494-1504).
Deumelandt; Wainwright; Figueiro; Jiang; Allard, Chen, and Saha (the combined references) teach a method of treating glioblastoma in a patient comprising administering to the patient combined anti-PD-1/PD-L1 and anti-CTLA-4 antibody immune checkpoint blockade therapy after the patient has been determined to have low expression of CD73 in an immune cell sample from the patient, as set forth above.
Biological sample is PBMC (claim 4)
The combined references do not teach the biological sample is PBMC.
As stated above, Deumelandt established CD73 is expressed on immune cells.
As stated above, Jiang recognizes the role of CD73 expression in immune cells and immunosuppression: “CD73 plays a pivotal role in tumor escape from immune surveillance. The mechanism can be summarized into three aspects: (i) inhibition of clonal expansion, activation and homing to tumor specific T cells; (ii) to increase a substantial component of the suppressive capabilities of regulatory T cells (Tregs) and Th17 cells; (iii) to accelerate the conversion of anti-tumor type 1 macrophages into pro-tumor type 2 macrophages.”
Thibaudin demonstrates successfully collecting cancer patient PBMCs, isolating immune cells, and testing the immune cells for CD73 expression (p. 8, col. 2, Materials and Methods; Figure 2). Thibaudin demonstrates that CD25hiTh17 immune cells exert adenosine dependent suppressive functions, and these cells express CD73 (Figure 2 and 3). CD25hiTh17 immune cells expressed CD73 at comparable levels to Treg cells, that are associated with immune suppression (p. 8, col. 1). Thibaudin teaches that CD25hiTh17 immune cells accumulated in breast cancer and suppress CD4+ and CD8+ T cell activation through adenosine release. Breast cancer patients with high IL-17+ cell infiltration had poor clinical prognosis, even in the case of high CD8+ T cell infiltration (p. 6, col. 2; Figure 1 and 6). Thibaudin teach that they have previously reported that Th17 cells can harbor immunosuppressive functions that relied primarily on CD73 ectonucleotidase expression (p. 6, col. 2). Thibaudin teaches intratumoral Th17 cells expressing CD73 compromise anticancer immune responses (abstract).
Deng teaches collecting PBMC samples and staining immune cells for CD73 expression (Materials and Methods). Deng determined that mice bearing tumors had significantly increased CD73 expression on CD4+ T cells and CD8+ T cells (p. 1496, col. 2; Figure 1; Results). Deng also determined that the CD4+ CD73hi subset and CD8+ CD73hi subset had a higher fraction of CTLA4 expression than the CD4+ CD73lo and CD8+ CD73lo subset (p. 1496, col. 2). Deng teaches that the CD73 expression is associated with “exhausted” phenotype of T cells in tumor-bearing mice (p. 1496, col. 2; Figure 2). Deng determined that CD73 is upregulated on the tumor infiltrating immune cells (TILs) in primary human HNSCC and associated with poor prognosis (p. 1500, col. 1; Figure 6). Inhibition of CD73 by anti-CD73 antibody in mice successfully and significantly reduced tumor growth (Figure 3) and reversed the “exhausted” phenotype of CD4+ and CD8+ T cells, reducing the CD73+ population of CD4+ and CD8+ T cells (Figure 4; p. 1497, col. 1-2). Exhausted T cells express multiple inhibitory receptors including PD-1 and CTLA-4, and lose effector functions (p. 1500, col. 2, Discussion).
Deng teaches (p. 1503, col. 1):
“Interestingly, the remaining CD73+ population express even higher levels of PD-1 and CTLA-4. This result, to some extent, supports that targeting CD73 enhances the therapeutic efficiency of PD-1 or CTLA-4 blockade. Thus, we hypothesize that anti-CD73 mAb treatment will reverse a large part of “exhausted” CD41 and CD81 T cells to immune-competent, however, the remaining CD73+CD4+ and CD73+CD8+ T cells, especially the CD73hi subset, gains more inhibitory markers and becomes even more dysfunctional. Thus, this subset of CD73hi T cells may represent a resistance to anti-CD73 mAb treatment. Ectopic overexpression of CD73 involved in tumor progression is frequently observed. In this study, using immunohistochemistry, we identify that CD73 is upregulated in the tumor infiltrating immune cells of HNSCC samples compared with normal mucosa. Increased expression of CD73 on tumor cells has been observed, and is associated with poor prognosis in human HNSCC. In our study, we found CD73 is also highly expressed on the tumor infiltrating immune cells in human HNSCC samples, and confers poor prognosis in our cohort. Tumor cells expressed CD73 could convert AMP to adenosine, which exerts potent immunosuppressive effect. However, CD73 highly expressed on T cell may be identified as an exhausted marker. Although there have been multiple reports relating CD73 to anti-tumor immunity in other tumors, only few of them are focused on HNSCC. Harnessing TCGA transcriptome sequencing data and R package TCGAbiolinks, we find out that NT5E mRNA level is also associated with negative immune regulation in HNSCC patients.”
Deng teaches (p. 1503, col. 2):
“Currently, The MEDI9447 (monoclonal antibody specific for CD73) alone and in combination with MEDI4736 (anti-PD-L1 monoclonal antibody) in select advanced solid tumor is recruiting participants (NCT02503774). In summary, we elucidate that the expression of CD73 on T cells represents an “exhausted” phenotype in competent HSNCC mouse model. Furthermore, in vivo assay showed the blockade of CD73 restrains tumor progression and reverses a large part of “exhausted” CD4+ and CD8+ T cells to immune-competent. Moreover, we showed a potential resistant population remaining CD73 positive during the anti-CD73 treatment. The human tumor samples reveal that CD73 is overexpressed in tumor-infiltrating immune cells, and the positive expression of CD73 is correlated with the poor prognosis in human HNSCC. Taken together, these data suggest that CD73 has a negative immunoregulatory function and it may prove to be a potential target for immunotherapy in HNSCC.”
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to utilize a PBMC biological sample comprising immune cells for determining CD73 expression levels in the method of the combined references. One would have been motivated to, and have a reasonable expectation of success to, because the combined references, Thibaudin, and Deng all recognize that CD73 expression in either tumor cells or immune cells correlates with the same immunosuppressive function and worse prognosis for cancer patients. Further, the combined references and Deng demonstrated that CD73 inhibition in tumor cells or immune cells successfully treats cancer, enhances T cell activation, and sensitizes tumor to anti-PD-1 and anti-CTLA-4 blockade, and Deng demonstrated that even with a reduction of CD73 immune cells, CTLA-4 and PD-1 expression on immune cells can remain or increase, suggesting these checkpoint proteins should be targeted to eliminate the “exhausted” cell phenotype. Deng teaches that cancer patients are clinically being treated with both anti-CD73 antibody and anti-PD-L1 antibody for this reason. Given the cited prior art recognize and established that: (1) increased CD73 expression on either tumor cells or immune cells indicates immunosuppression, exhausted immune cell phenotype, and worse prognosis for patients, (2) inhibiting or reducing CD73 expression in tumors or immune cells is beneficial, especially in conjunction with PD-L1/PD-1 or CTLA-4 blockade; it is well within the level of the ordinary skilled artisan to utilize a tumor biopsy sample or a PBMC immune cell sample for assessing low CD73 expression in order to identify patients that will benefit from combination immune checkpoint inhibition therapy, and with a reasonable expectation of success.
Immune cell is a TIL (claim 78):
The combined references established that high CD73 expression on any cell type, including TILs and TAMS, performs the same function if increased adenosine production and immunosuppressive function. As stated above, Figueiro demonstrates levels of CD73 expression on T lymphocyte immune cells in glioblastoma can vary from high to low, and it is known that high CD73 expression leads to increased adenosine production and immunosuppressive function. As stated above, Chen teaches T lymphocytes, including Tregs (TILs) are immunosuppressive due to high CD73 expression:
Effects of CD73 activity by nontumor cells
T cells
Regulatory T cells (Tregs; CD4+CD25+FoxP3+) mediate immunotolerance and help tumor cells evade immunosurveillance by suppressing the immune response. One of the main mechanisms for Treg-mediated tumor immunosuppression is dependent on the extracellular adenosine generated by CD73 [18] . CD73 is abundantly expressed by Tregs and is frequently coexpressed with CD39. CD73, in combination with CD39, renders an enzymatically driven accumulation of immunosuppressive adenosine by Tregs. Accordingly, Tregs derived from either CD73−/− or CD39−/− mice have impaired suppressive functions [64, 65]. Unlike WT murine Tregs, CD73−/− Tregs fail to promote tumor growth [18, 26] (see p. 985).
As stated above, Thibaudin teaches intratumoral T lymphocytes can express variable levels of CD73, where high expression compromises anticancer immune responses due to adenosine dependent suppressive functions.
As stated above, Deng demonstrates that TILs can have high CD73 expression that contribute to tumor immune suppression. Deng teaches blockade of CD73 reverses a large part of “exhausted” CD4+ and CD8+ T cells to become immune-competent, thereby demonstrating that decreasing CD73 on TILs through blockade enhances tumor immunity.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to administer combined ICB therapy to glioblastoma patients determined to have a TIL with low CD73 expression. One would have been motivated to, and have a reasonable expectation of success to, because the combined references, Thibaudin, and Deng all teach and recognize that immune cells, including TILs can have variable levels of CD73 expression, where high CD73 expression is immunosuppressive due to adenosine dependent suppressive functions, and that reducing CD73 by blockade enhances T cell tumor immune responses and enhances response to anti-PD-1 and anti-CTLA4 immune checkpoint blockade therapies. Therefore, it is well within the level of the ordinary skilled artisan to successfully treat a glioblastoma patient with combination immune checkpoint therapy after detecting a TIL immune cell having low CD73 expression indicative of enhanced immune cell competence and reduced adenosine-dependent suppressive functions.
6. Claim(s) 10-12 are rejected under 35 U.S.C. 103 as being unpatentable over Deumelandt et al (Cancer Immunol. Res., February 1, 2019, 7(2_Supplement), abstract B075); Wainwright et al (Clinical Cancer Research, 2014, 20:5290-5301); Figueiro et al (Purinergic Signalling, 2016; 12:303–312); Jiang et al (BMC Cancer, 2018, 18:267, internet pages 1-10); Allard et al (Clinical Cancer Research, 2013, 19:5626-5635); Chen et al (Immunotherapy, 2019, 11:983-997, published online June 21, 2109); and Saha et al (Cancer Cell, 2017, 32:253-267); as applied to claims 1, 3, 5, and 15 above, and further in view of Omuro et al (Neuro-Oncology, 2018, 20:674-686) and Romani et al (Frontiers in Oncology, 2018, 8:464).
Deumelandt; Wainwright; Figueiro; Jiang; Allard, Chen, and Saha (the combined references) teach a method of treating glioblastoma in a patient comprising administering to the patient combined anti-PD-1/PD-L1 and anti-CTLA-4 antibody immune checkpoint blockade therapy after the patient has been determined to have low expression of CD73 in a tumor immune cell sample from the patient, as set forth above.
The combined references do not teach the specific combination of anti-PD-1 antibody and anti-CTLA-4 antibody as nivolumab and ipilimumab, although Chen teaches known clinical administration of single and combined anti-PD-1/PD-L1 and anti-CTL-4 immune checkpoint inhibitors nivolumab, tremelimumab, durvalumab in combination with CD73 blockade to treat cancers (Table 2). The combined references do not teach additionally administering an anticancer therapy (chemotherapy/biological therapy) with the combined immune checkpoint inhibitors.
Omuro teaches clinically treating glioblastoma patients with nivolumab, and in combination with ipilimumab (Figure 1). Omuro recognizes that glioblastoma tumors generate immunosuppressive environment, and there is evidence that immune checkpoint inhibition has overcome the immunosuppressive CNS environment to provide antitumor responses in select patients (Box at top of page 675).
Romani demonstrate that glioblastoma is already being clinically treated with combined nivolumab and ipilimumab antibodies, as well as other anticancer agents such as Temozolomide or personalized vaccines (Table 1). Romani recognizes glioblastoma is characterized by immunosuppressive environment requiring treatment with combinations of immune checkpoint inhibitors (abstract).
Nivolumab and ipilimumab:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to administer nivolumab as the anti-PD-1 antibody or ipilimumab as the anti-CTLA-4 antibody in the method of the combined references. One would have been motivated to, and have a reasonable expectation of success to, because Omuro and Romani teach and demonstrate that these antibodies are clinically being used for treatment of glioblastoma, and are successful in treating glioblastoma patients by overcoming the immunosuppressive tumor environment.
Additional anticancer therapy:
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to additionally administer an anticancer agent. One would have been motivated to, and have a reasonable expectation of success to, because Romani teaches glioblastoma is already being clinically and successfully treated with ipilimumab, nivolumab, and additional anticancer agents such as Temozolomide or personalized vaccines.
Response to Relevant Arguments
7. Applicants argue that none of the cited references teach using “low CD73” expression on an immune cell to identify or select patients responsive to ICB. Applicants argue Deumelandt does not teach detecting CD73 expression on TILs. Applicants reiterate form their previous Remarks that they presented data demonstrating improved survival in GL261 and CT-2A mouse models with PD1/PD-L1 plus CTLA-4 antibody therapy if they have low expression of CD73. Applicants argue that they discovered GBM patients with low CD73 expression are responsive to ICB therapy. Applicants argue the claimed invention provides a significant improvement for the treatment of GBM by identifying a responsive population.
8. The arguments have been considered but are not persuasive. Many of the arguments presented by Applicants have been addressed in the new rejections above, adding Chen and Saha that further demonstrate the known, established, predictable correlation between high CD73 expression levels on tumor and immune cells and immunosuppressive tumor environment, as well as the known correlation between reducing CD73 expression through CD73 blockade and its synergistic therapeutic effects when combined with anti-CTLA4 and anti-PD-1 therapy. Saha demonstrates that skewing the glioblastoma tumor to contain more M1-like macrophages, known to have low CD73 expression compared to M2 subsets, and administering two checkpoint inhibitors against PD-1 and CTLA-4 synergistically cures glioblastoma. Chen suggests CD73 blockade (reducing CD73) is expected to act synergistically with other immune checkpoint inhibitors. Therefore low or inhibited levels of CD73 in the tumor environment (tumor cells or immune cells) provide a reasonable expectation that the tumor will have enhanced response to immune checkpoint inhibition. The cited prior art teaches and demonstrates that CD73 expression provides the same adenosine-induced immunosuppression in the tumor environment regardless of what cell expresses it, and the cited prior art provides a clear correlation between low CD73 expression and immune-competent TILs, and more favorable tumor immune environment for activating T cells.
Further, the claims as currently constituted recite treating a GBM subject determined to have “low expression of CD73” in an immune cell. The expression level identified as “low” is only relative to a level slightly higher than it, and the claims do not recite any specific levels of CD73 expression or control comparisons that would exclude the CD73 levels taught in the cited prior art. The cited prior art demonstrates that CD73 expression is variable in the immune cells of GBM patients, and given the significant success demonstrated by the prior art for treating GBM with anti-CTLA-4 and anti-PD-1 antibodies, one of ordinary skill in the art could predictably treat a GBM patient with anti-CTLA-4 and anti-PD-1 antibodies even when an immune cell is detected as having a relatively low CD73 expression.
The data argued by Applicants from the instant specification was based on CD73-/- mice rather than detecting a low CD73 expressing immune cell. Therefore, the data argued by Applicants is not commensurate in scope with the instantly claimed method (see MPEP 716.02(d)). Further, the cited prior art in the rejection provides a reasonable expectation of success to synergistically/significantly treat GBM with a combination of anti-PD-1 and anti-CTLA4 antibodies when immune cell CD73 expression is low, for the reasons stated in the rejection.
9. Conclusion: No claim is allowed. All other rejections stated in the office action mailed February 9, 2026 are withdrawn in view of the claim amendments.
10. 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
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