CTNF 18/566,782 CTNF 99432 DETAILED ACTION Notice of Pre-AIA or AIA Status 07-03-aia AIA 15-10-aia The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA. Priority 02-26 AIA Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Information Disclosure Statement 06-52 The information disclosure statement (IDS) was filed before the mailing date of the non-final first action on the merits. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner. Claim Objections Claims 4,6,9,12,14-16 are objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim cannot depend from any other multiple dependent claim. See MPEP § 608.01(n). 06-17 The numbering of claims is not in accordance with 37 CFR 1.126 which requires the original numbering of the claims to be preserved throughout the prosecution. When claims are canceled, the remaining claims must not be renumbered. When new claims are presented, they must be numbered consecutively beginning with the number next following the highest numbered claims previously presented (whether entered or not). Claim Rejections - 35 USC § 112 07-30-02 AIA 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. Claim 1 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. This is because in the first wherein clause, the language in the parentheticals, such as (nuclei), (oligodendrocytes), and (Astrocyte), is confusing as to whether it is claimed or exemplary. Improper Markush Grouping 08-40 Claim 2 is rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch , 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi , 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117. The Markush grouping of the extrinsic inhibitor being either “ a compound, a small molecule, a peptide, a nucleic acid, chondroitin sulfate proteoglycan, hyaluronan, fibronectin aggregate, myelin debris, inflammatory cytokine, bone morphogenetic protein, endothelin-1, semaphoring, environmental toxin and alcohol, tobacco or illicit or recreational drugs ” is improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons: The members of the Markush group defining the extrinsic factor represent a plurality of chemical classes with varying structures. To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use. Claim Rejections - 35 USC § 103 07-20-aia AIA 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. 07-23-aia AIA 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. 07-21-aia AIA Claim s 1-17 are rejected under 35 U.S.C. 103 as being unpatentable over Petersen et al ( Neuron,2017), in view of Lariosa ‑ Willingham et al ( BioMed Central, 2016), hereafter Willingham, Peppard et al ( Journal of Biomolecular Screening 2015), Su et al ( Journal of Neurotrauma), and Buchser et al (Assay Guidance Manual, 2014) . Regarding claims 1, 4, and 16 , Petersen et al demonstrate that fibrinogen is a potent extrinsic inhibitor of oligodendrocyte progenitor cells (OPC) differentiation and remyelination. To assess the effect of fibrinogen (i.e. extrinsic factor) on OPC differentiation, Petersen et al utilized an immunofluorescence assay to detect the presence of (MBP+), a marker of myelinating oligodendrocytes (OLs), and glial fibrillary acidic protein+ (GFAP+), a marker of astrocytes. (See Fig.2H). Petersen et al show that fibrinogen inhibits OPC differentiation into myelinating MBP+ oligodendrocytes (Ols) while promoting OPC differentiation to GFAP+ astrocyte-like cells rather than mature Ols. It should be noted that immunofluorescence for MBP and GFAP reads on obtaining two readouts in a single assay . Petersen et al also show that the inhibitory effect of fibrinogen on OPCs differentiation can be reversed by contacting the OPCs with DMH1, a dorsomorphin analog that inhibits the BMP type I receptor ACVR1. It should be emphasized that DMH1 reads on a test agent. Specifically, Petersen et al demonstrate that contacting a fibrinogen-treated OPCs (i.e. extrinsic factor) with DMH1 (i.e. test agent) increased the number of mature OLs, decreased the GFAP+ cells, and suppressed fibrinogen-induced Id1 gene expression, this reads on step (a) of instant claim. (Figures 3A and 3B). To assess the reversal effect of DHM1 on fibrinogen-treated OPCs, Petersen et al also obtained the immunofluorescence assay (i.e. two readouts in a single assay) to stain for MBP and GFAP, this reads on step (b) of instant claim. It should be emphasized that the immunofluorescence assay utilized by Petersen et al involves contacting OPCs after step (a) with DAPI dye to stain for nuclei, antibodies against MBP (oligodendrocytes), and antibodies against GFAP (astrocytes) to stain for MBP and GFAP, respectively. Petersen et al similarly used an increase in MBP+ OLs and a decrease in GFAP+ astrocytes relative to a control OPCs only contacted with the fibrinogen (extrinsic inhibitor) as a read out to show that DMH1 (test agent) overcome fibrinogen’s inhibition of remyelination. Taken together, Petersen et al teach an assay to screen for an agent that overcomes the inhibition exerted on OPCs by an intrinsic inhibitor, which involves contacting OPCs with fibrinogen and DHM1 and obtaining the immunofluorescence assay to s simultaneously stain for MBP and GFAP. It is noted that Petersen et al do not teach or suggest using a high-throughput, high content technique that involves the automated quantification of MBP and GFAP to screen for a test agent as required by instant claims. Willingham et al supplement Petersen et al by teaching a high throughput drug screening assay to identify compounds that promote oligodendrocyte differentiation using acutely dissociated and purified oligodendrocyte precursor cells. The method of Willingham et al involves plating OPCs in a differentiation media at 5000 cells/well into PDL/laminin coated 96-well plates, pre-incubating the OPCs for 1–2 h at 37 °C in 10 % CO2, followed by the addition of test compounds in quadruplicate. According to Willingham, on day 4, cells are fixed, immunostained for myelin basic protein (MBP) expression and DAPI, imaged and quantified using automated fluorescence microscopy. ( See Fig.1, and section “ Acute oligodendrocyte differentiation assay” on pages 11-12). Willingham et al suggest that high throughput screening could be used in large compound libraries for the identification of drugs for the treatment of MS and other demyelinating disease. Thus, providing an ordinary skill in the art with the teachings and motivation to modify the method of Petersen et al to screen for compounds that promote remyelination using high throughput, high-content screening. Peppard et al is another prior art that supplements Peterson et al by teaching an automated, cell-based, high-content screen for identifying small molecules eliciting the differentiation of OPCs after 3 days. Peppard et al disclose a secondary high-content assay for evaluating OPCs differentiation. In particular, CG-4 OPC like cells were contacted with test compounds and subsequently immunostained with an O4 antibody, an established marker of oligodendrocyte differentiation, together with Hoechst dye for nuclei stanning. The stained cells were then imaged and analyzed using an ArrayScan high-content imaging platform to quantify O4-positive cells and associated cellular morphology/processes complexity. Accordingly, Peppard et al teach automated high-content imaging and quantification analysis of immunofluorescence antibody to assess oligodendrocyte differentiation. ( See section Secondary Assays “ I.O4 staining of CG-4 cells” on pages 385-386). When taken together, instant claims would have been obvious to one of ordinary skill in the art, as there was some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill in the art to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. Petersen et al teach antibody-based immunofluorescence (i.e. two readouts in a single assay technique) to screen for an agent that overcomes remyelination inhibition exerted on OPCs by an extrinsic inhibitor, but use manual microscopy-based assessment instead of automated high-content screening. Willingham and Peppard et al teach the use of automated imaging and computation morphometric analysis to evaluate differentiation-associated phenotypes of OPCs exposed to test compounds. Thus an ordinary skill in the art at the time the invention was filed would be motivated to modify the teachings of Petersen et al and implement the high throughput, high content screening, taught by Willingham and Peppard, for screening test agents that would reverse the remyelination inhibition caused by the exposure of OPCs to extrinsic inhibitors. Moreover, the use of high-content, high throughput imaging system, was well known in the art for automated quantification of fluorescent cellular marker and morphology in cell-based screening assays. Thus an ordinary skill in the art would have been motivated to utilize such established approaches and with a reasonable expectation of success in order to improve the efficiency, and scalability of OPC differentiation screening assay. It is also noted that Willingham and Peppard do not explicitly state that 80% of the culture vessel is imaged and used in the quantification process . However, it is well recognized in the art that it would have been prima facie obvious for one with ordinary skill in the art to rely on routine experimentation when determining the appropriate coverage of well surface area during imaging. Because the appropriate coverage of well surface area would depend on the magnification lenses and the type of plate used during imaging. When the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimal or workable ranges through routine experimentation. See MPEP 2144.05. Regarding claims 2-3 , the combined teachings of Petersen, Willingham, and Peppard render obvious claim 1. However, none of the recited prior art teach a high throughput, high content screening for an agent that would overcome the remyelination inhibition exerted by solubleTNF-alpha. Su et al supplement the cited prior arts by teaching that TNFa, particularly when secreted by reactive astrocytes, functions as a potent inhibitor (i.e. extrinsic inhibitor) within the central nervous system microenvironment and negatively regulates OPCs survival and differentiation. Specifically, Su et al teach that exposure of OPCs to TNFa results in a reduction in OPC survival, characterized by increased apoptotic signaling and decreased cellular viability. In addition, Su et al demonstrate that TNFa exposure impairs differentiation, as evidenced by suppression of OPC differentiation into mature, MBP-positive oligodendrocytes. In other words, Su et al demonstrate that the exposure of OPC to TNFa impairs their differentiation, thereby decreasing the pool of myelinating oligodendrocytes. Accordingly, Su et al teach that elevated level of TNFa levels, such as those secreted from reactive astrocytes following demyelinating disease states, contribute to failed or incomplete remyelination by preventing the maturation of OPCs into functional oligodendrocytes. Given these teachings, a person of ordinary skill in the art at the time the invention was filed, would have recognized that TNFa act as an extrinsic inhibitor of remyelination and a key molecular barrier to effective oligodendrocyte lineage progression in demyelinating disease and injury context. ( See abstract, and Figs.5,7) Thus, instant claims 2-3 would have been obvious to one of ordinary skill in the art, as there was some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill in the art to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. Because Petersen et al in view of Willingham and Peppard render obvious the use of high throughput, high content screening for an agent that would overcome the fibrinogen-mediated inhibitory signaling in OPCs, but fail to teach screening for an agent that would reverse TNFa-mediated inhibitory effects on OPCs. Su et al teach that TNFa secreted by reactive astrocyte is a major extrinsic inhibitor of remyelination. Accordingly, it would have been obvious to an ordinary skill in the art at the time the invention was filed to modify the teachings of the cited prior arts to identify agents capable of reversing TNFa-mediated inhibitory signaling in OPCs, in order to restore OPC survival and differentiation and thereby promote remyelination. In view of these teachings, an ordinary skill in the art would have a reasonable expectation of success in employing routine-high content, high throughput screening methodology to identify agents that would reverse the effects of TNFa on OPCs signaling. Regarding claim 5, Petersen et al teach contacting OPCs with fibrinogen at a concentration of 2.5 mg/ml. ( See Method Details section “Fibrinogen and Pharmacologic Inhibitors”). Regarding claims 6-9, Petersen et al utilized primary OPCs isolated from the cerebral cortex of Sprague-Dawley rats, this reads on claim 6. ( See “Primary OPC Cultures” on page e2). Petersen et al also teach culturing primary OPCs in a proliferation medium comprising of PDGF-AA and NT3, this reads on claim 8. Petersen et al teach that the OPCs are cultured in proliferation medium for 2 days prior to the differentiation step. It should be noted that the method of Peterson et al involves contacting the OPC with the test agent and fibrinogen as the cells are switched to the differentiation medium. Thus, the teachings that OPCs are cultured in proliferation medium for 2 days prior to the differentiation step, reads on claim 7. (See section “ Primary OPC Cultures “ on pages e2-e3). Furthermore, Petersen et al teach releasing the OPCs from culture dish with trypsin, this reads on claim 9. Regarding claim 10, Petersen et al in view of Willingham et al and Peppard render obvious claim1. Willingham et al teach plating OPCs in a 96-well plate at a density of 5000/well. ( See section “ Acute oligodendrocyte differentiation assay” on pages 11-12). Regarding claim 11, Petersen et al teach culturing OPCs in a proliferating medium for 2 days prior the experiments. Petersen et al do not teach further replating the cells in a fresh culture plate and culturing it for 24 hours prior to step (a). However, it is well recognized that it is prima facie obvious for one of ordinary skill in the art to use routine experimentation to discover an optimum value of a result effective variable. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum of workable ranges by routine experimentation. The "discovery of an optimum value of a result effective variable in a known process is ordinarily within the skill of the art." Application of Boesch, 617 F.2d 272, 276, 205 USPQ 215, 218-219 (C.C.P.A. 1980). See MPEP 2144.05. In other words, and ordinary skill in the art would be able to arrive at the same time frame claimed in instant claim through routine experimentation, absent evidence to the contrary and the claim is considered obvious. Regarding claims 12-14, Petersen et al teach culturing OPCs in differentiating medium comprising CNTF , and triiodothyronine (T3), for up to 4 days . ( See section “Primary OPC Cultures” on page e2-e3) Regarding claim 15 , Petersen et al teach using labeling OPCs with primary antibodies against MBP and GFAP and fluorescent secondary antibodies, and obtaining immunofluorescence images of the cells with Axioplan II epifluorescence microscope. ( See the 2 nd paragraph on page e6, and Fig.2-3). Regarding claim 17, following the discussion above Petersen in view of Willingham and Peppard render obvious instant claim1. As discussed above, Peppard et al supplement Petersen et al by teaching automated imaging and quantitative morphometric analysis. Specifically, the method of Peppard et al involves staining cells with nuclear and cytoplasmic markers, and using automated high-content imaging system (e.g. , ArrayScan platform) to evaluate the impact of test agents on the OPCs differentiation. Peppard et al teach that the resulting images are analyzed using commercially available BioApplication software module that perform automated segmentation (i.e. ring extension) and feature extraction. It should be noted that automated segmentation includes nuclear identification and mask (i.e. ring) expansion-based cytoplasmic quantification. It is noted that Peppard et al do not explicitly teach the ring-expansion method recited in instant claim. However, it is clearly obvious that the automated ring/mask expansion in antibody-based high content screening is implemented as a standard feature in the analysis algorithm. ( See Supplementary Material -section “ 2. Screening data analysis”). This is further supported by the teachings of Buchser et al. Specifically Buchser et al teach that high-content screening quantify antibody-based immunofluorescence signals at the single-cell level using automated image segmentation and subcellular region identification. Buchser et al teach that cells are typically stained with fluorescently labeled antibody targeting intracellular or membrane associated proteins, and nuclei are counter-stained with DAPI or Hoechst to define primary cellular objects. ( See section “ Regional analysis”). Thus, an ordinary skill in the art would understand that image analysis software perform nuclear segmentation followed by computational expansion of the nuclear mask to generate a defined perinuclear or cytoplasmic ring region extending beyond the nucleus. An ordinary skill in the art who reviewed Petersen, Peppard, and Willingham, could have come across Buschser et al and immediately recognize the benefit of expanding the nuclear ring region, when employing the automated high content screening, to capture antibody signal localized outside the nucleus. An ordinary skill in the art would understand that this is relevant for antibody-based readouts where the target antigen is cytoplasmic, membrane-associated, or distributed in cell processes. Conclusion Any inquiry concerning this communication or earlier communications from the examiner should be directed to FATIMAH KHALAF MATALKAH whose telephone number is (703)756-5652. The examiner can normally be reached Monday-Friday,7:30 am-4:30 pm EST. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Tracy Vivlemore can be reached on 571-272-2914. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /FATIMAH KHALAF MATALKAH/Examiner, Art Unit 1638 /PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632 Application/Control Number: 18/566,782 Page 2 Art Unit: 1638 Application/Control Number: 18/566,782 Page 3 Art Unit: 1638 Application/Control Number: 18/566,782 Page 4 Art Unit: 1638 Application/Control Number: 18/566,782 Page 5 Art Unit: 1638 Application/Control Number: 18/566,782 Page 6 Art Unit: 1638 Application/Control Number: 18/566,782 Page 7 Art Unit: 1638 Application/Control Number: 18/566,782 Page 8 Art Unit: 1638 Application/Control Number: 18/566,782 Page 9 Art Unit: 1638 Application/Control Number: 18/566,782 Page 10 Art Unit: 1638 Application/Control Number: 18/566,782 Page 11 Art Unit: 1638 Application/Control Number: 18/566,782 Page 12 Art Unit: 1638
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