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 .
Response to Amendments
Applicant's amendments filed 6/10/2025 to claims 19 has been entered. Claims 19 and 29-37 remain pending and are being considered on their merits. References not included with this Office action can be found in a prior action. Any rejections of record not particularly addressed below are withdrawn in light of the claim amendments and applicant’s comments.
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.
Claims 19 and 29-37 remain rejected under 35 U.S.C. 103 as being unpatentable over Glicklis et al (2004, Biotechnol Bioeng, 20;86(6):672-80) in view of Maurel, T. (2010, Hepatocytes Methods and Protocols, MIMB, volume 640, Springer Protocols; 558 pages; 2/9/2024 IDS) and Prudencio et al (2008, Cellular Microbiology, 10(1), 218–224).
Glicklis teaches methods to make hepatocyte spheroid and a model to define the optimal spheroid size which enables maximal cell viability and hepatocellular functions (see abstract and col. 2 on page 678). Regarding claim 19, Glicklis teaches the method of making make hepatocyte spheroids comprises (a) introducing a cell suspension of hepatic cells (reads on single-cell suspension as Glisklis does not teach they are clustered) at a concentration of 5x105 cell/mL to rotating T-flasks or spinner flasks (both read on “an agitation- based culture system”) and (b) agitating the hepatic cells at a rate of 70 or 110 rpm to generate spheroids having diameters in the range of 50 to 200 micrometers (see abstract, col. 1 on page 763 and Figure 2). Regarding claim 32, Glicklis teaches the cells are agitated for periods between 1 and 2 weeks (see abstract, col. 1 on page 763 and Figure 2). Regarding claim 32, Glicklis teaches the cells are agitated for periods between 1 and 2 weeks (see abstract, col. 1 on page 763 and Figure 2). Regarding claim 35 and 36, Glicklis teaches the spheroids can be further assayed in 96-well plates (see col. 2 on page 763).
Glicklis does not teach the cells are a hepatic cell line that have been previously expanded in 2D culture, incubating the spheroids with a ratio of Plasmodium vivax or Plasmodium berghei with agitation for 2 hours, or in a Glicklis’ 96-well plate, or centrifugation, or that the pathogen is a reporter strain.
Regarding claim 19, Maurel teaches a method of making bioartificial liver model systems include forming aggregates of hepatocytes, and that these cell-containing spheres may then be utilized in bioreactors comprising rotary systems mimicking microgravity (see paragraph spanning pages 514-515). Regarding claim 19, Maurel teaches primary hepatocytes and hepatoma cell lines represent valuable models to investigate Plasmodium vivax infection and development and to evaluate drug candidates that may interfere with these processes, and that infection with P. vivax has demonstrated (see section 6.2 on page 29). Regarding claims 19 and 31, Maurel teaches the previously culture expanded human hepatoma cell line HepaRG cells, has advantages over primary hepatocytes (see pages 4-5). Regarding claims 19, 29-30 and 33-36, Maurel teaches there are limitations with infection efficiency, but that protocols can be optimized to increase infection efficiency (see pages 26-30). Regarding claims 19 and 37, Maurel teaches genetic manipulation of the pathogen has allowed marked progress in the understanding of hepatocyte infection, and that pathogens expressing the green fluorescent protein are used to determine infection efficiency and separate infected cells (reads on “reporter strain”) (see section 6.2 on page 29).
Regarding claims 19, 29-30, 33 and 35-37, Prudencio teaches methods for infection assays using a Plasmodium berghe reporter strain, and Prudencio teaches using 17.5x104 hepatic cells per well in a 24-well plate, and adding various numbers of sporozoites ranging from 4x103 to 24x103, and centrifuging at 1700xg for 7 min (see abstract, figures, and page 223).
It would have been obvious to combine Glicklis with Maurel and Prudencio to use Maurel’s HepaRG cells as the hepatocytes in Glicklis’s spheroid, and to infect the spheroid with either Maurel’s or Prudencio’s Plasmodium reporter strain, using either Glicklis’ agitation, Glicklis’ 96-well plate, or Prudencio’s centrifugation.
A person of ordinary skill in the art would have had a reasonable expectation of success in using Maurel’s HepaRG cells as the hepatocytes in Glicklis’s spheroid because Maurel’s HepaRG cells are taught to be a hepatoma cell lines, and Maurel teaches hepatoma cell lines represent valuable models to investigate Plasmodium vivax infection.
A person of ordinary skill in the art would have had a reasonable expectation of success in infecting the hepatic cell spheroid with either Maurel’s or Prudencio’s Plasmodium reporter strain, using either Glicklis’ agitation, Glicklis’ 96-well plate, or Prudencio’s centrifugation, because Glicklis’ agitation brings cells into contact, Glicklis’ 96-well plate is taught to be useful for assaying Glicklis’ spheroids, and Prudencio establishes that infection can be carried out using multi-well plates and with centrifugation.
The skilled artisan would have been motivated to use Maurel’s HepaRG cells as the hepatocytes in Glicklis’s spheroid, and to infect the spheroid with either Maurel’s or Prudencio’s Plasmodium reporter strain, using either Glicklis’ agitation, Glicklis’ 96-well plate, or Prudencio’s centrifugation because Maurel teaches infection of hepatoma cell lines with Plasmodium reporter strains represent valuable models to investigate infection and development and to evaluate drug candidates that may interfere with these processes, while Glicklis teaches agitation brings cells into contact and that a 96-well plate is useful for assaying Glicklis’ spheroids, and Prudencio establishes that infection can be carried out using multi-well plates and with centrifugation. Additionally, Maurel teaches there are limitations with infection efficiency, but that protocols can be optimized to increase infection efficiency, and that pathogens expressing the green fluorescent protein are used to determine infection efficiency and separate infected cells.
Regarding the ratio of sporozoites to hepatic cells, the number of cells per well, the length of time for infection, and the centrifugation speed, as stated above, Maurel teaches there are limitations with infection efficiency, but that protocols can be optimized to increase infection efficiency, and that pathogens expressing the green fluorescent protein are used to determine infection efficiency and separate infected cells. Additionally, Prudencio establishes that infection can be carried out using multi-well plates and with centrifugation, and that the ratio of cells to sporozoites can be varied. Therefore, the references establish that it is useful to optimize these infection parameters as they are result effective variables that effects the infection efficiency. It would be obvious in view of these teaching to adjust these result effective variables and use a centrifugation speed of 1800xg for 5 minutes (vs Prudencio’s 1700xg for 7 min), cell-to-sporozoite ratios of either 1:1, 1:2. 2:1, 3:2, or 5:2 (of which Prudencio’s amounts fall within), and 2.5x104 to 5x104 per well as each of these would affect the infection efficiency which is specifically taught to be a variable that can be both optimized and measured.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill at the time the invention was made.
Response to Arguments
Applicant's arguments filed 6/10/2025 have been fully considered but they are not persuasive.
Applicant highlights that amended claim 19 now emphasizes that the cells are human or primate hepatic cells. Applicant should note that since all of the originally recited hepatic cell lines in claim 19, namely HepG2 cells, HC-04 cells and HepaRG cells, are human hepatic cells, the amendment to claim 19 merely broaden the claim to include additionally options of human and primate hepatic cells.
Applicant highlights that Glicklis teaches their optimal spheroid diameter is 100 micrometers, as Glicklis found some negative effects on cell viability and function in spheroids with larger sizes. Applicant concludes that there are therefore limitations of applying the Glicklis’ model to other applications using larger spheroids. However, the claims are not drawn to using spheroids with sizes larger than those taught in Glicklis as Glicklis also exemplifies the use of spheroids with sizes of 100 and 200 micrometers, and found the most negative effects on spheroids with sizes of 600 micrometers. Importantly, as the claims are drawn to using spheroids having an average diameter of 50 to 200 micrometers, and Glicklis’ spheroids with sizes of 100 micrometers, which applicant recognizes Glicklis teaches are the optimal size, falls directly within the claimed range of spheroid diameter this argument is not persuasive.
Applicant highlights that Glicklis exemplifies using primary rat hepatocytes and not human hepatocytes as claimed. Applicant further highlights that Maurel emphasizes that for models to be clinically important, it is best to use human cells instead of cells from other species. Applicant concludes that because human hepatocytes spheroids are an ideal model, that there is no reason to rely on Glicklis’ model which uses rat hepatocytes. However, as stated in the above, the rejection is over the obviousness to use Maurel’s HepaRG cells (human hepatocytes) instead of the primary rat hepatocytes as the hepatocytes in Glicklis’s hepatocyte spheroids. As stated above, and as recognized by the applicant, Maurel teaches the human HepaRG hepatocyte cell line has advantages over primary hepatocytes which are used by Glicklis. Therefore, as Maurel provides both teaching and motivation to use HepaRG cells as the hepatocytes in Glicklis’s spheroid, this argument is not persuasive.
Applicant alleges that Prudencio does not cure the alleged deficiencies of Glicklis and Maurel. However, as applicant’s arguments alleging deficiencies in Glicklis and Maurel were not persuasive, this argument is not persuasive.
Conclusion
No claims are free of the art. No claims are 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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/S.A.M/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653