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 .
Claim Status
1. The amendment filed 03/09/2026 has been entered. Claims 1 – 16, 21, 41, and 44 remain pending. Claims 1 – 16, 21, and 41 are under consideration.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 – 16, 21, and 41) in the reply filed on 07/18/2025 is acknowledged.
3. Claims 42 – 44 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/18/2025.
4. Claims 1 – 16, 21, and 41 are under consideration.
Priority
5. This application claims priority to United States Provisional Application No. 62/907,876, filed September 30, 2019.
Withdrawn Specification Objection
6. The objection to the specification for improper trade name or marker usage is withdrawn in view of Applicant’s amendment to the specification.
Claim Interpretation
7. For the purpose of applying prior art, claim 16 is interpreted as the tumor cells are from a primary tumor but have left the primary tumor such that the tumor cells are retrieved from a different site than the primary tumor.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Section 33(a) of the America Invents Act reads as follows:
Notwithstanding any other provision of law, no patent may issue on a claim directed to or encompassing a human organism.
8. Claims 1 – 16 and 41 remain rejected under 35 U.S.C. 101 because the claimed invention is directed to an indirect co-culture comprising bone marrow niche cells, tumor cell, and a culture medium without significantly more. The claim(s) recite(s) bone marrow niche cells and tumor cells which is not shown to differ from that in nature. Although maintained, the rejection is revised in view of the amendment to claim 1.
The claims are drawn to an indirect three-dimensional co-culture, comprising
isolated bone marrow niche cells, isolated tumor cells, a synthetic matrix, and a culture medium,
wherein the tumor cells are surrounded by the synthetic matrix, wherein the bone marrow niche
cells and the tumor cells are incubated in the culture medium without direct contact between the
bone marrow niche cells and the tumor cells, and wherein at least 65% of the tumor cells are
viable for at least 15 days.
The Office published Office's new guidance document entitled 2019 Revised Patent Subject Matter Eligibility Guidance, published January 7, 2019. Applicant is directed to the Federal Register, Volume 4, No. 4, pages 50-57 at page 74621.
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Step 1 of the USPTO' s eligibility analysis entails considering whether the claimed subject matter falls within the four statutory categories of patentable subject matter identified by 35 U.S.C. 101: Process, machine, manufacture, or composition of matter. The claims are directed to a composition of matter (Step 1, Yes).
Step 2A of the 2019 Revised Patent Subject Matter Eligibility Guidance is a two-prong inquiry. In Step 2A Prong One, examiners evaluate whether the claim recites a judicial exception. The composition of matter (bone marrow niche cells and tumor cells incubated in the same culture medium without physical contact with each other) is directed to a natural phenomenon because the claims read on naturally occurring paracrine signaling between bone marrow and tumor where the bone marrow cells and tumor cells are not in physical contact with each other (Step 2A, prong 1, Yes).
Because the claims recite a nature-based product limitation (bone marrow niche cells and tumor cells), the markedly different characteristics analysis is used to determine if the nature-based product limitation is a product of nature exception. The claim also recites “isolated”, “a synthetic matrix”, and “the bone marrow niche cells and the tumor cells are incubated in the culture medium without direct contact between the bone marrow niche cells and the tumor cells. The markedly different characteristics analysis is performed by comparing the nature-based product limitation in the claim to its naturally occurring counterpart to determine if it has markedly different characteristics from the counterpart. Here, the closest natural counterpart is naturally occurring bone marrow niche cells and tumor cells.
Zhang (Zhang, Xiaohui, et al. Leukemia & lymphoma 56.1 (2015): 211-218; previously cited) teaches isolation of bone marrow mesenchymal stem cells (BMSCs, bone marrow niche cells) and MM cells (tumor cells) from bone marrow from patients with multiple myeloma (MM) (page 212, left col. para. 2 – 3). Zhang teaches indirect co-culture of BMSCs and MM cells that mimics their interactions in the bone marrow microenvironment (page 212, right col. para. 2; page 215, right col. para. 2). Zhang teaches indirect co-culture of MM cells and BMSCs can increase expression of Cx43 in BMSCs while not changing Cx43 in MM cells which is similar to that observed in MM cells and BMSCs from MM patients (page 213, left col. para. 1 and 5; page 214, left col. para. 1). Zhang teaches after indirect co-culture, healthy BMSCs can display a phenotype similar to that of patient-derived MM-MSCs (page 215, right col. para. 2). Zhang teaches MM cells can interact with the BM microenvironment indirectly via stimulation of the autocrine/paracrine production of cytokines which can result in osteolytic lesions in the bone marrow (page 211, left col.; page 214, right col. para. 2; page 215, right col. para. 2). Zhang teaches BM mesenchymal stem cells (BMMSCs) and osteoblasts (bone marrow niche cells) can secrete IL-6, HGF, and SDF-1a and to increase MM cell proliferation and migration (page 215, right col. para. 2). Thus, Zhang teaches indirect co-culture of bone marrow niche cells and tumor cells show a phenotype of naturally occurring bone marrow niche cells and tumor cells not in direct contact in a patient with MM.
Kovacic (Kovacic, N., et. al. Calcif Tissue Int 94, 125–139 (2014); previously cited) teaches tumor cells with high skeletal homing affinity express numerous cell surface receptors that bind ligands produced in bone (Abstract). Kovacic teaches a bone matrix that releases proteins and growth factors that are produced by tumor cells residing in bone (Figure 2; page 131, left col. para. 3; page 132, right col. para. 2; page 134, left col. para. 2). Kovacic teaches upon arrival, these cells survive in the host environment, encompassed in close proximity to bone marrow cells (Abstract). Kovacic teaches interactions between tumor cells and cells of the host microenvironment are essential for tumor cell survival (Abstract). Kovacic teaches SDF-1 which binds to CXCR4 and CXCR7 receptors, is produced by bone marrow niche cells and attracts tumor cells into the bone marrow (page 126, left col. para. 2). Kovacic teaches clinical samples of breast cancer and prostate cancer cells express CXCR4 and are associated with metastatic disease (page 126, left col. para. 2). Kovacic teaches the SDF-1/CXCR4 axis is strongly associated with progressive myeloma bone disease in clinical samples (page 126, left col. para. 2 and right col. para. 1). Kovacic teaches PTHrp is produced by many tumors with affinity for bone, plays an important role in the development of bone metastasis, and has been identified in more than 90% of osteolytic breast cancer bone metastases (page 128, left col. para. 2). Kovacic teaches a number of cytokines and chemokines produced by tumor cells also act in both a paracrine and an autocrine fashion to alter osteoclastogenesis (bone marrow niche cells) and stimulate tumor growth (page 128, right col. para. 2 – 4; page 129, left col.; Figure 1). Kovacic teaches tumor cells interact with the bone marrow compartment to induce alterations to this compartment which are enhanced indirectly via locally residing host cells and through the release of tumor-promoting growth factors from the bone matrix (page 134, left col. para. 2).
Zhou (Zhou, Xuan, et al. "ACS applied materials & interfaces 8.44 (2016): 30017-30026; previously cited) teaches a synthetic bone matrix that facilitates the integration of human fetal osteoblasts, human bone marrow mesenchymal stem cells, and breast cancer cells in close proximity in an effort to create a bonelike microenvironment that provides cross-talk between these different cell types (page 30017, left col. para. 2). Zhou teaches the bone matrix can be a tool for investigating breast cancer bone invasion and metastasis (page 30025, right col. para. 2).
When the claimed bone marrow niche cells and tumor cells are compared to their naturally occurring counterpart, the comparison indicates that there are no differences in structure, function or other characteristics. Further, the claimed cells are not markedly different from the natural counterpart as a result not being in direct contact as Zhang teaches indirect co-culture of bone marrow niche cells and tumor cells show a similar phenotype to MM in a patient and Kovacic teaches both bone marrow niche cells and tumor cells release factors that affect each other through paracrine mechanisms. The claimed cells are not markedly different from the natural counterpart as a result of a synthetic matrix because Kovacic teaches tumor cells are present in the bone matrix and Zhou teaches isolated bone marrow niche cells and tumor cells in a synthetic bone matrix is a tool for investigating breast cancer bone invasion and metastasis and because “synthetic matrix” is not defined in Applicant’s specification such that it differs in structure from a naturally occurring matrix. Therefore, the claimed indirect co-culture of bone marrow niche cells and tumor cells is a product of nature exception and recites a judicial exception.
In Step 2A Prong Two, examiners evaluate whether the claim recites additional elements that integrate the exception into a practical application of that exception. This evaluation is performed by (a) identifying whether there are any additional elements recited in the claim beyond the judicial exception, and (b) evaluating those additional elements individually and in combination to determine whether the claim as a whole integrates the exception into a practical application. Besides the judicial exception, the claim recites that a culture medium. An evaluation of whether this limitation is insignificant extra-solution activity is then performed. Note that because Step 2A Prong Two analysis excludes consideration of whether a limitation is well-understood, routine, conventional activity, this evaluation does not take into account whether or not the limitation is well-known. When so evaluated, this additional element is insignificant extra-solution activity because “culture medium” and “synthetic matrix” is recited so generically. The claim does not recite the type of culture medium or the type of synthetic matrix or how the two types of cells are not in direct contact. Further, Applicant’s specification states that “a well-defined, synthetic 3D indirect co-culture model” mimics “both cell-matrix and cell-cell interactions in the bone marrow microenvironment” at page 6, lines 19 – 21 and the synthetic matrix is “bioinspired” at page 6, line 30 – 33. Therefore, the claim covers any possible indirect co-culture with any culture medium and any synthetic matrix which may not differ in structure from its naturally occurring counterpart and fails to meaningfully limit the claim because it is at best the equivalent of merely adding the words “apply it” to the judicial exception. Accordingly, the limitation of culture medium does not integrate the recited judicial exception into a practical application and the claim is therefore directed to the judicial exception (Step 2A, prong 2, No).
In Step 2B, the eligibility analysis evaluates whether the claim as a whole amounts to significantly more than the recited exception, i.e., whether any additional element, or combination of additional elements adds an inventive concept into the claim. As discussed with respect to Step 2A Prong Two, the claim recites a culture medium and synthetic matrix and that the two types of cells are not in direct contact, which is at best the equivalent of merely adding the words “apply it” to the judicial exception. Mere instructions to apply an exception cannot provide an inventive concept. At Step 2B, the evaluation of the insignificant extra-solution activity consideration takes into account whether or not the extra-solution activity is well-known. Here, recitation of culture medium and synthetic matrix and the cells not being in direct contact is recited at a high level of generality and does not amount to significantly more and does not provide an inventive concept (Step 2B: No).
In the instant case, the limitations of the claims do not impose limits on the claim scope such that they are not markedly different in structure from a naturally occurring product.
Markedly different characteristics can be expressed as the product' s structure, function, and/or other properties. In accordance with this analysis, a product that is purified or isolated, for example, will be eligible when there is a resultant change in characteristics sufficient to show a marked difference from the product' s naturally occurring counterpart. If the claim recites a nature-based product limitation that does not exhibit markedly different characteristics, the claim is directed to a ‘‘product of nature” exception (a law of nature or naturally occurring phenomenon), and the claim will require further analysis to determine eligibility based on whether additional elements add significantly more to the exception.
Limitations that were found not to be enough to qualify as ‘‘significantly more” when recited in a claim with a judicial exception include: Adding the words ‘‘apply it” (or an equivalent) with the judicial exception, or mere instructions to implement an abstract idea on a computer; simply appending well-understood, routine and conventional activities previously known to the industry, specified at a high level of generality, to the judicial exception, e.g., a claim to an abstract idea requiring no more than a generic computer to perform generic computer functions that are well-understood, routine and conventional activities previously known to the industry; adding insignificant extrasolution activity to the judicial exception, e.g., mere data gathering in conjunction with a law of nature or abstract idea; or generally linking the use of the judicial exception to a particular technological environment or field of use.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
9. Claim(s) 1, 14, 16, 21, and 41 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Dhawan (Dhawan, Abhishek, et al. Stem Cells 34.8 (2016): 2224-2235.), hereinafter Dhawan as evidenced by Koch (Koch MK, et. al. Adv Healthc Mater. 2023 Jun;12(14):e2202202), hereinafter Koch and Cailleau (Cailleau, Relda, In vitro 14.11 (1978): 911-915; previously cited), hereinafter Cailleau.
Claim 1 is drawn to an indirect three-dimensional co-culture, comprising isolated bone marrow niche cells, isolated tumor cells, a synthetic matrix, and a culture medium, wherein the tumor cells are surrounded by the synthetic matrix, wherein the bone marrow niche cells and the tumor cells are incubated in the culture medium without direct contact between the bone marrow niche cells and the tumor cells, and wherein at least 65% of the tumor cells are viable for at least 15 days.
Claim 21 is drawn to a method for preparing an indirect three-dimensional coculture, comprising surrounding tumor cells by a synthetic matrix, and incubating the bone marrow niche cells surrounded by the synthetic matrix and tumor cells in a culture medium without direct contact between the bone marrow niche cells and the tumor cells, wherein at least 65% of the tumor cells are viable for at least 15 days, whereby an indirect three-dimensional coculture is prepared.
Claim 41 is drawn to an indirect three-dimensional co-culture prepared according to the method of claim 21.
Regarding claims 1, 21, and 41, Dhawan teaches an indirect three-dimensional co-culture (claim 41) comprising isolated bone marrow mesenchymal stem cells (MSCs) (“isolated bone marrow niche cells” of claims 1 and 21), isolated breast cancer cells (“isolated tumor cells” of claims 1 and 21), a star-PEG-heparin hydrogel (“synthetic matrix” of claims 1 and 21), and osteogenic differentiation medium (“culture medium” of claims 1 and 21) prepared by encapsulating the MSCs and the breast cancer cells separately in the hydrogel (“wherein the tumor cells are surrounded by the synthetic matrix” of claim 1 and “surrounding tumor cells by a synthetic matrix” and “bone marrow niche cells surrounded by the synthetic matrix” of claim 21) and co-cultured in one well to mimic the individual microenvironment but allowing for paracrine signaling through soluble morphogens (“wherein the bone marrow niche cells and the tumor cells are incubated in the culture medium without direct contact between the bone marrow nice cells and the tumor cells” of claim 1 and “incubating” “without direct contact” of claim 21) (page 2230, right col.; page 2226, right col. para. 1 – 2; page 2231, right col.; page 2232, left col. para. 1; Figure 5C). At least 65% of breast cancer cells are viable for at least 15 days embedded in a star-PEG-heparin hydrogel as evidenced by Koch (Figure 9) (“wherein at least 65% of the tumor cells are viable for at least 15 days” of claims 1 and 21).
Regarding claim 14 and 16, Dhawan teaches the tumor cells are breast cancer cells that are MDA-MB231 (page 2226, right col. para. 1; Figure 5C). MDA-MB-231 tumor cells were obtained from a pleural effusion of a patient with breast cancer (claim 16) as evidenced by Cailleau (page 911, left col. para. 3; Table 1).
Therefore, Dhawan anticipates claims 1, 14, 16, 21, and 41.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
10. Claim(s) 1 –6, 14 – 16, 21, and 41 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dhawan (Dhawan, Abhishek, et al. Stem Cells 34.8 (2016): 2224-2235.), hereinafter Dhawan as evidenced by Koch (Koch MK, et. al. Adv Healthc Mater. 2023 Jun;12(14):e2202202), hereinafter Koch and Cailleau (Cailleau, Relda, In vitro 14.11 (1978): 911-915; previously cited), hereinafter Cailleau in view of Tivari (Tivari S, et. al. J Vis Exp. 2015 Jun 30;(100):e52672; previously cited), hereinafter Tivari in view of Zhou (Zhou, Xuan, et al. "ACS applied materials & interfaces 8.44 (2016): 30017-30026; previously cited), hereinafter Zhou.
Dhawan anticipates claim 1 as set forth above.
Regarding claim 15, Dhawan teaches indirect co-culture of MCF-7 breast cancer cells (“estrogen receptor-positive”) with MSCs (Figure 1; page 2228, right col. para. 1; page 2229, left col. para. 1; Figure 5A). Dhawan teaches co-culture of MCF-7 cells and MSCs increase MCF-7 proliferation (Figure 3C).
Dhawan does not teach “at least 80% of the tumor cells are dormant” of claim 2, “hFOBs” of claim 3, “a soluble dormancy factor” of claim 4 and 5, or “the soluble dormancy factor is secreted by the bone marrow niche cells” of claim 6. However, Dhawan teaches skeletal metastasis of breast cancer is associated with a poor prognosis and significant morbidity (Abstract). Dhawan teaches the interaction between disseminated breast cancer cells and the bone marrow microenvironment which harbors them has not been addressed comprehensively (Abstract). Dhawan teaches understanding the modulation of bone marrow microenvironment by metastatic breast cancer cells is important to identify clinical targets which can prevent tumor relapse and skeletal comorbidities (page 2232, left col. para. 2). Dhawan teaches the hydrogel co-culture demonstrated that breast cancer cells caused a significant reduction in ALP levels impairing osteogenic differentiation of MSCs and preloading of the MSC hydrogel compartment with heparin binding rhPDGF-BB could rescue the effect of breast cancer cells (page 2232, left col. para. 1 – 2; Figure 5D). Dhawan teaches co-culture of MSCs and breast cancer cells increased bFGF (FGF2) levels (Figure 2; page 2229, left col. para. 1 – 2). Dhawan teaches bFGF has been shown to regulate MSC proliferation as well as tumor survival (page 2229, left col. last para.).
Regarding “at least 80% of the tumor cells are dormant” of claim 2, Tivari teaches at least 80% of breast cancer cells grown in the presence of FGF-2 are dormant in an in vitro model of dormancy in estrogen-sensitive breast cancer cells in the bone marrow (Abstract; Figure 2; page 4, para. 1 of Representative Results; Figure 1).
Regarding “soluble dormancy factor” of claim 4 and “growth factor” of claim 5, Tivari teaches medium containing FGF-2 (“soluble dormancy factor” of claim 4; growth factor of claim 5) induces breast cancer cell dormancy (page 3, step 6, para. 1; Figure 2).
Regarding “the soluble dormancy factor is secreted by the bone marrow niche cells” of claim 6, Tivari teaches FGF-2 is a growth factor abundantly synthesized by and exported by bone marrow stroma, and heavily deposited in the extracellular matrix (Abstract; page 2, para. 2; page 8, para. 1 of Discussion).
Regarding ER+ breast cancer cells of claim 15, Tivari teaches treating ER+ breast cancer cell lines MCF-7 and T47D (page 2, para. 3; step 1 of Clonogenic Assay). Tivari teaches estrogen sensitive breast cancer cells are the type likely to remain dormant for extended periods (Abstract).
Tivari does not teach “hFOBs” of claim 3. However, Tivari teaches the study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment (Abstract). Tivari teaches ER+ breast cancer cells can remain dormant for up to 20 years with a steady, continuous rate of recurrence (page 1, para. 1). Tivari teaches while the differences in dormancy gene expression signatures between ER+ and ER- breast cancers reflect different dormancy potentials, interactions with bone marrow stroma likely represent a significant contribution to dormancy (page 1, para. 1). Tivari teaches the study of dormancy in vivo is exceptionally difficult and thus relevant models must be generated that provide in vitro data that can suggest mechanisms and generate testable hypotheses in vivo (page 1, para. 2). Tivari teaches their model is relatively sparse in its components but sufficiently robust to permit investigators to derive specific molecular mechanisms that affect significant functions of dormancy (page 1, last para.). One would have been motivated to combine the teachings of Dhawan and Tivari in a three-dimensional co-culture of breast cancer cells and bone marrow MSCs to study dormancy in breast cancer metastasis to the bone because both teach co-cultures of breast cancer cells and bone marrow niche cells and Dhawan teaches the co-culture increased FGF2 and Tivari teaches a model of dormancy by culturing cells in the presence of FGF2 and Dhawan teaches understanding the modulation of bone marrow microenvironment by metastatic breast cancer cells is important to identify clinical targets which can prevent tumor relapse and skeletal comorbidities and Tivari teaches the study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment.
Regarding hFOBs of claim 3, Zhou teaches an indirect co-culture of MDA-MB-231 cells and osteoblasts (ATCC CRL-11372; “hFOBs”) where osteoblasts were encapsulated in a hydrogel where osteoblast proliferation decreased suggesting osteoblast growth in co-culture could be restrained by breast cancer cell activity (Abstract; page 30018, right col.; Figure 1; page 30022, right col.). Zhou teaches breast cancer cell growth increased in co-culture with osteoblasts suggesting some bioactive molecules secreted by osteoblasts promote breast cancer cell growth (page 30023, left col. para. 2). Zhou teaches the composition of the hydrogel influenced the production of VEGF and alkaline phosphatase in a 2 week indirect co-culture of breast cancer cells and osteoblasts (page 30024, left col. para. 2 – 3 and right col. para. 1 and 3; page 30025, left col. para. 1 - 2).
It would have been obvious prior to the effective filing date of the invention as claimed for the person or ordinary skill in the art to combine the teachings of Dhawan regarding culturing an indirect three-dimensional co-culture of breast cancer cells and bone marrow MSCs each separately encapsulated in a PEG hydrogel and that co-culture of MCF-7 cells and MSCs increase FGF2 with the teachings of Tivari regarding at least 80% of breast cancer cells grown in the presence of FGF-2 are dormant in an in vitro model of dormancy in estrogen-sensitive breast cancer cells in the bone marrow with the teachings of Zhou regarding an indirect co-culture of breast cancer cells and osteoblasts to arrive at the claimed indirect co-culture wherein at least 80% of the tumor cells are dormant. One would have been motivated to combine the teachings of Dhawan, Tivari, and Zhou in an indirect co-culture modeling breast cancer dormancy as Dhawan teaches understanding the modulation of bone marrow microenvironment by metastatic breast cancer cells is important to identify clinical targets which can prevent tumor relapse and skeletal comorbidities and Tivari teaches the study of dormancy in vivo is exceptionally difficult and thus relevant models must be generated that provide in vitro data that can suggest mechanisms and generate testable hypotheses in vivo and Zhou teaches breast cancer cell growth increased in co-culture with osteoblasts suggesting some bioactive molecules secreted by osteoblasts promote breast cancer cell growth. One would have a reasonable expectation of success in combining the teachings as Dhawan teaches co-culture of MCF-7 and MSCs increased FGF2 and Tivari teaches breast cancer cells grown in the presence of FGF-2 are dormant in an in vitro model of dormancy.
11. Claim(s) 1, 7 – 11, 14, 16, 21, and 41 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dhawan (Dhawan, Abhishek, et al. Stem Cells 34.8 (2016): 2224-2235.), hereinafter Dhawan as evidenced by Koch (Koch MK, et. al. Adv Healthc Mater. 2023 Jun;12(14):e2202202), hereinafter Koch and Cailleau (Cailleau, Relda, In vitro 14.11 (1978): 911-915; previously cited), hereinafter Cailleau in view of Tivari-2018 (Tivari, Samir, et al. " Cell Communication and Signaling 16.1 (2018): 48; previously cited), hereinafter Tivari-2018.
Dhawan anticipates claim 1 as set forth above.
Regarding claim 8, Dhawan teaches the bone marrow niche cells are human MSCs (page 2225, left col. last para.).
Dhawan does not teach “at least 80% of the tumor cells are reactivated” of claim 7, “a soluble reactivation factor” of claim 9, “cytokines” of claim 10, or “the soluble reactivation factor is secreted by the bone marrow niche cells” of claim 11. However, Dhawan teaches co-culture of breast cancer cells and human bone marrow MSCs cause a perturbation in the cytokine profile of MSCs where TGFβ1 is upregulated (Figure 2). Dhawan teaches skeletal metastasis of breast cancer is associated with a poor prognosis and significant morbidity (Abstract). Dhawan teaches the interaction between disseminated breast cancer cells and the bone marrow microenvironment which harbors them has not been addressed comprehensively (Abstract). Dhawan teaches understanding the modulation of bone marrow microenvironment by metastatic breast cancer cells is important to identify clinical targets which can prevent tumor relapse and skeletal comorbidities (page 2232, left col. para. 2).
Regarding claim 7, Tivari-2018 teaches at least 80% of breast cancer cells are reactivated by IL-6, IL-18, or TGF-β1 at day 19 after being induced into dormancy by FGF-2 at day 0 in Figure 4B (page 5, right col. para. 2). Tivari-2018 teaches co-cultivation of breast cancer cells with bone marrow stroma and injury to bone marrow stroma results in secretion of IL-6 and IL-8 by the bone marrow stroma (page 3, right col. last para.; page 4, left col. para. 1; page 5, right col. last para.; Figure 7).
Regarding claim 8, Tivari-2018 teaches co-culture with bone marrow stroma including buffy coats from human bone marrow aspirates (page 3, right col. para. 3 – 4; page 4, left col. para. 1).
Regarding claim 9, Tivari-2018 teaches media contained IL-6, IL-8, or TGFβ1 (page 4, left col. para. 2; page 5, left col. para. 2 and right col. para. 1 – 2; Figure 4B).
Regarding “cytokines” of claim 10, Tivari-2018 teaches IL-6, IL-8, and TGFβ1 (page 4, left col. para. 2; page 5, left col. para. 2 and right col. para. 1 – 2; Figure 4B).
Regarding claim 11, Tivari-2018 teaches bone marrow stroma secretes IL-6 and IL-8 and activate (page 5, right col. last para.; page 6, left col.; Figure 5; Figure 9; page 8, left col.).
Tivari-2018 teaches breast cancer cells can settle in the bone marrow and become dormant and can reactivate for decades (page 2, left col. para. 1). Tivari-2018 teaches osteoblasts in the bone marrow support cancer cell dormancy (page 2, left col. para. 4). Tivari-2018 teaches relapse of breast cancer after years of dormancy remains a significant medical problem (page 2, right col. para. 3). Tivari-2018 teaches stromal inflammation reactivates the growth and motility of dormant breast cancer cells and reactivated cells have an impaired ability to re-enter dormancy (page 15, left col. last para.). Tivari-2018 teaches injury to the bone marrow stroma induces reactivation of dormant micrometastases, mediated by stromal inflammatory responses and this establishes a rationale for investigating methods for preventing stromal inflammation as a potential mechanism of suppressing recurrence of micrometastases of breast cancer (page 15, right col. para. 1).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Dhawan regarding culturing an indirect three-dimensional co-culture of breast cancer cells and human bone marrow MSCs each separately encapsulated in a PEG hydrogel and that co-culture of MCF-7 cells and MSCs increase TGFβ1 with the teachings of Tivari-2018 regarding a method of reactivation of dormant breast cancer cells by co-culturing with bone marrow stroma to arrive at the claimed invention where at least 80% of the breast cancer cells are reactivated. One would have been motivated to combine the teachings of Dhawan and Tivari-2018 in an indirect co-culture of reactivated breast cancer cells and hMSCs to develop therapies for breast cancer as Dhawan teaches understanding the modulation of bone marrow microenvironment by metastatic breast cancer cells is important to identify clinical targets which can prevent tumor relapse and skeletal comorbidities and Tivari-2018 teaches injury to bone marrow stroma induces reactivation of dormant breast cancer cells establishes a rationale for investigating methods for preventing stromal inflammation as a potential mechanism of suppressing recurrence of micrometastases of breast cancer. One would have a reasonable expectation of success in combining the teachings as Dhawan teaches co-culture of breast cancer cells and MSCs increases TGFβ1 and Tivari-2018 teaches at least 80% of breast cancer cells are reactivated by TGF-β1.
12. Claim(s) 1, 12 – 14, 16, 21, and 41 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dhawan (Dhawan, Abhishek, et al. Stem Cells 34.8 (2016): 2224-2235.), hereinafter Dhawan as evidenced by Koch (Koch MK, et. al. Adv Healthc Mater. 2023 Jun;12(14):e2202202), hereinafter Koch and Cailleau (Cailleau, Relda, In vitro 14.11 (1978): 911-915; previously cited), hereinafter Cailleau in view of Singh (Singh, Jagbir, et al. Cancer Research 76.14_Supplement (2016): 1334-1334.), hereinafter Singh.
Dhawan anticipates claim 1 as set forth above. Dhawan does not teach the culture media comprises an autophagy inhibitor (claim 12) that is hydroxychloroquine (claim 13). However, Dhawan teaches preloading of the MSC-hydrogel compartment with the heparin binding rhPDGF-BB could rescue the breast cancer cells’ mediated impairment in osteogenic differentiation of MSCs (page 2231, left col. para. 2; Figure 5D). Dhawan teaches skeletal metastasis of breast cancer is associated with a poor prognosis and significant morbidity (Abstract). Dhawan teaches the interaction between disseminated breast cancer cells and the bone marrow microenvironment which harbors them has not been addressed comprehensively (Abstract). Dhawan teaches understanding the modulation of bone marrow microenvironment by metastatic breast cancer cells is important to identify clinical targets which can prevent tumor relapse and skeletal comorbidities (page 2232, left col. para. 2).
Regarding claims 12 – 13, Singh teaches many anticancer drugs used to treat breast cancer trigger survival responses including the induction of autophagy (page 1, para. 1). Singh teaches hydroxychloroquine (HCQ) can improve the effects of anticancer drugs by inhibiting autophagy when used at high concentrations but these levels are difficult to attain in vivo (page 1, para. 1). Singh teaches a liposomal formulation of HCQ inhibited rapamycin-induced autophagy in non-tumor bearing mice (page 1, para. 2). Singh teaches the liposomal formulation of HCQ inhibited gefitinib-induced autophagy in mice with breast cancer xenografts and the combination of HCQ and gefitinib led to a significant inhibition of tumor growth compared to untreated controls (page 1, para. 2).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Dhawan regarding culturing an indirect three-dimensional co-culture of breast cancer cells and human bone marrow MSCs each separately encapsulated in a PEG hydrogel with the teachings of Singh regarding the autophagy inhibitor HCQ improves the effects of anticancer drugs to arrive at the claimed invention wherein the culture medium comprises HCQ. One would have been motivated to combine the teachings of Dhawan and Singh in an indirect co-culture of breast cancer cells and osteoblasts to evaluate the resistance breast cancer cells to anticancer drugs in the presence of HCQ as Dhawan teaches understanding the modulation of bone marrow microenvironment by metastatic breast cancer cells is important to identify clinical targets which can prevent tumor relapse and skeletal comorbidities and Singh teaches many anticancer drugs used to treat breast cancer trigger survival responses including the induction of autophagy. One would have a reasonable expectation of success in combining the teachings as Dhawan teaches the hydrogel can be modified with to reduce the breast cancer cells’ effect on the MSCs in the co-culture and Singh teaches HCQ inhibited autophagy in mice with breast cancer xenografts.
Applicant’s Arguments/ Response to Arguments
13. Applicant Argues: On page 7, Applicant asserts that as amended the claims are directed to an indirect co-culture with significantly more and the claims are directed to eligible subject matter.
Response to Arguments: The rejection is maintained because “synthetic matrix” is recited at a high level of generality and can include matrices that do not differ structurally from their naturally occurring counterpart. Should Applicant amend the claims to recite “crosslinked bioinert poly(ethylene glycol) (PEG) and bioactive peptides” (Applicant’s specification at page 13, line 12), the rejection may be overcome upon further consideration.
Applicant Argues: On page 8 – 12, Applicant traverses the rejection of the claims over the prior art because the prior art does not disclose “tumor cells surrounded by a synthetic matrix” of amended claims 1 and 21.
Response to Arguments: The previous rejection of the claims over prior art have been withdrawn in view of Applicant’s amendment to claims 1 and 21. In the new rejection of the claims set forth above, Dhawan teaches an indirect co-culture and a method of preparing the indirect co-culture comprising breast cancer cells encapsulated in a PEG hydrogel and human mesenchymal stem cells encapsulated in a PEG hydrogel separate from the encapsulated breast cancer cells and culturing these two separate cell encapsulated hydrogels in a culture medium where they are not in direct contact as shown in Figure 5C. Therefore, Dhawan anticipates claims 1, 14, 16, 21, and 41.
Conclusion
No claims allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Z.M.B./Examiner, Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632
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