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 01/22/2026 has been entered. Claims 1 – 6 and 16 – 20 remain pending. Claims 15 and 21 have been cancelled.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 – 8 and 10 – 15) in the reply filed on 10/17/2024 is acknowledged.
3. Claims 16 – 20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 10/17/2024.
4. Applicant has amended claim 21 to depend from claim 15 which is in the elected Group I. Therefore, claim 21 is examined in this office action along with claims 1 – 8 and 10 – 15.
5. Claims 1 – 6 are under consideration.
Priority
6. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in parent Application No. CN201910467062.7, filed on 05/16/2019.
7. Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Withdrawn Claim Rejections
8. The rejection of claim 15 under 35 U.S.C. 101 is rendered moot in view of Applicant’s cancellation of the claim.
9. The rejection of claims 15 and 21 under 35 U.S.C. 102(a)(1) is rendered moot in view of Applicant’s cancellation of these claims.
Claim Interpretation
10. For the purpose of applying prior art, claim 1 is interpreted as a process comprising the following steps:
(a) obtaining an ex vivo bronchoscopic brushed-off biopsy tissue;
(b) digesting the bronchoscopic brushed-off biopsy tissue and collecting the bronchial basal cells after terminating the digestion;
(c) primary culture of the digested cells at 37 °C in 4 – 8% CO2 on a culture plate pre-coated with feeder layer cells that cover 50 – 70% of the culture plate comprising (i) resuspending the digested bronchial basal cells in a culture medium; (ii) plating the resuspended cells on the culture plate pre-coated with feeder layer cells and adding antibiotics; (iv) replacing the culture medium every other day; (v) collecting the cells when clones; (d) amplification culture of primary cultured cells on a culture plate pre-coated with feeder layer cells that cover 50 – 70% of the culture plate in culture medium at 37 °C;
(e) passage culture of amplified primary cultured cells when the cells grow to cover 50% - 90% of the surface area of the culture plate;
(f) collecting passage cultured cells by digesting, collecting adherent cells and washing when the passage cultured cells grow to cover 85%-95% of the surface area of the culture plate;
wherein the passage culture comprises 1 – 7 passages where passage 1 and 2 are performed with a culture dish pre-coated with matrix gel and supernatant is removed from passage 1 cells when they cover 50-90% of the culture dish surface, the cells are washed once with 1 x DPBS, trypsin is added, a single cell suspension is prepared by pipetting up and down when most of the cells are detached from the bottom of the culture container (see Applicant’s remarks at page 11, paragraph 7), terminating the digestion by adding a stop buffer, centrifuging the cell suspension and removing the supernatant, re-suspending the cells in a culture medium and plating the suspension into a culture dish pre-coated with feeder layer cells and replacing the culture medium every other day; wherein after passage 0 (when n=2), culture supernatant and cells are collected to carry out tests
wherein the culture medium has DMEM, F12, FBS, L-glutamine, insulin, epidermal growth factor, adenine , and hydrocortisone with a formulation consisting of 225 mL of DMEM, 225 mL of F12, 20 – 70 mL of FBS, 0.2 – 2 mM of L-glutamine, 1 – 14 ng/mL of insulin, 0.1 – 1 ng/mL of epidermal growth factor, 5 – 30 µg/mL adenine, and 2 – 20 µg/mL hydrocortisone.
Maintained Claim Rejections
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.
11. Claim(s) 1 – 6 remain rejected under 35 U.S.C. 103 as being unpatentable over Zhang (CN107267437A; Filed 04/08/2016; Published 10/20/2017; previously cited), hereinafter Zhang which is cited on the IDS filed 01/31/2022 in view of Fulcher (Fulcher, M. Leslie, et al. Human cell culture protocols. Totowa, NJ: Humana Press, 2005. 183-206; previously cited), hereinafter Fulcher in view of Gowers (Gowers, Kate HC, et al. Journal of Tissue Engineering and Regenerative Medicine 12.1 (2018): e313-e317; previously cited), hereinafter Gowers which is cited on the IDS filed 09/21/2022 in view of in view of He (CN-107974429-A; Published 05/01/2018; previously cited) which is cited on the IDS filed 01/31/2022 in view of Ma (Ma Q, et. al. Protein Cell. 2018 Mar;9(3):267-282. Epub 2018 Jan 1; previously cited), hereinafter Ma which is cited on the IDS filed 01/31/2022 inpreviously cited), hereinafter Reynolds which is cited on the IDS filed 09/21/2022 as evidenced by GibcoGlutamine (Gibco, “L-Glutamine (200 mM)”, Retrieved from: https://www.thermofisher.com/order/catalog/product/25030081; previously cited), hereinafter GibcoGlutamine and as evidenced by Sigma (Sigma, “Insulin solution human”, Retrieved from: https://www.sigmaaldrich.com/US/en/product/sigma/i9278; previously cited), hereinafter Sigma. A machine translation of CN107267437 is provided. The translation was performed on 10/20/2025. A machine translation of CN-107974429-A was previously provided. The translation was performed on 05/28/2025 of pages 1 – 15 of the original document. A copy of Gowers is provided that includes the Supplementary Methods, which was not included in the copy submitted with the IDS.
Regarding “tissue preparation” of claim 1, Zhang teaches obtaining a tissue sample containing lung stem cells using a cell brush with bronchoscopy (page 21, para. 3; page 25 – 26, para. 0115).
Regarding “enzymolysis” of claim 1, Zhang teaches digesting the tissue sample and collecting the digested cells after adding the stop solution (page 29, para. 0136).
Regarding “plating and amplification culture” of claim 1, Zhang teaches following digestion, the cells were resuspended in culture medium and plated onto irradiated human embryonic fibroblasts and cultured at 36 – 38 °C in 4% - 6.5% CO2 (page 30, para. 0136). Zhang teaches after 2 – 5 days, colonies composed of lung stem cells were observed (page 30, 0136). Zhang teaches the lung stem cells obtained were continuously expanded (page 34, para. 0161). Zhang does not teach adding antibiotics or replacing the culture medium every other day or the feeder layer percentage or stem cell percentage for passaging or cell collecting or passage culture.
Regarding “carry out tests… KRT5 cells percentage is ≥90%” of claim 1, Zhang teaches lung stem cells had good cell activity, high purity, and no bacterial and mycoplasma contamination (page 34, para. 0159). Zhang teaches a method for determining bacterial contamination and the culture medium was clear indicating no bacterial contamination (page 29, para. 0139). Zhang teaches a method for detecting mycoplasma contamination and the final value of the test is less than 0.8 indicating that there is no mycoplasma contamination (page 31, 0140). Zhang teaches detecting the expression markers Krt5 and p63 by immunofluorescence showing that a large number of clones expressed Krt5 and p63 (page 30, 0137; Figure 2A and 2B). Zhang does not teach measuring HOPX or endotoxin.
Regarding “the culture medium has a formulation consisting of” of claim 1, Zhang teaches 90% RPMI1640, 10% FBS, 2 mM L-glutamine, β-mercaptoethanol, leukemia inhibitor factor, and 3.5 ng/mL EGF (page 9 – 10, para. 0041 – 0047). Zhang does not teach DMEM, F12, insulin, adenine or hydrocortisone.
Regarding claim 2, Zhang teaches the digestion is carried out at 37 °C for 1 – 5 hours with a digestion solution comprising 1 U/mL of DNase, 0.3 mg/mL proteinase XIV (page 14 – 15, para. 0069 – 0077) but does not teach DMEM/F12, or trypsin.
Regarding claim 3, Zhang teaches the stop solution contains 90% RPMI with 10% fetal bovine serum but does not teach DMEM (page 29, para. 0136). Zhang teaches some lung diseases such as pulmonary fibrosis and COPD are increasingly affecting human health and survival (page 2, 0004).
Regarding claim 4, Zhang teaches obtaining biopsies of tracheal wall (page 21, para. 0102) but not lung lobe.
Regarding claim 15, Zhang teaches obtaining lung stem cells that had good cell activity, high purity, and no bacterial and mycoplasma contamination (page 34, para. 0159).
Regarding claim 21, Zhang teaches infusing the cells into the lungs of immunodeficient mice with lung damage induced by bleomycin (page 34, para. 0161). Zhang teaches the cells differentiated into human bronchi and alveoli and other tissue structures indicating that the stem cells obtained using the method can be used for clinical stem cell transplantation therapy (page 35, para. 0162 – 0163).
Zhang does not teach amplification or passaging culture or measuring HOPX or endotoxin test or culture medium formulation of claim 1 or DMEM/F12 or trypsin of claim 2 or DMEM of claim 3 or freezing cells of claim 5 or thawing cells of claim 6. Zhang teaches apart from lung transplantation, there is currently no effective treatment for some lung diseases such as pulmonary fibrosis and COPD (page 2, 0004). Zhang teaches lung stem cells when transplanted can integrate into mouse lungs and form alveolar-like structures at the site of damage (page 2, 0005). Zhang teaches it has long been difficult to isolate and obtain lung stem cells that can be expanded in large quantities from adult tissues and organs (page 2, 0005). Zhang teaches the method is simple to operate, has low implementation cost, and is easy to promote (page 13, 0068; page 194, last para.).
Regarding “adding antibiotics” and “replacing the culture medium every other day” of “plating and amplification culture” of claim 1, Fulcher teaches a method of obtaining primary and amplified airway cell cultures (P1, P2) in Figure 1 where ex vivo tissue samples are digested with protease XIV and cultured on collagen-coated supports. Fulcher teaches it is assumed that many primary human tissues contain yeasts, fungi, or bacteria and media for primary cultures can be supplemented with gentamicin and amphotericin where fewer episodes of contamination will result from using amphotericin (page 191, last para.). Fulcher teaches to generate P1 or P2, primary cells can be plated on collagen-coated plastic dishes and primary cell media should be supplemented with additional antibiotics for the first 3 days after plating and should be changed every 2 – 3 days or as needed to prevent acidification (page 197, para. 1).
Regarding “cell collecting” of claim 1, Fulcher teaches when primary cultures reach 70 – 90% confluence they are ready for passage and cells are harvested by digestion with trypsin and incubation at 37 °C followed by rinsing with PBS (page 15, para. 3).
Regarding “the culture medium has a formulation consisting of” of claim 1, Fulcher teaches BEGM is used when the initial cell harvests are plated on collagen-coated plastic dishes or to expand passaged cells on plastic (page 186, para. 3). Fulcher teaches the composition of BEGM in Table 1. Fulcher teaches BEGM contains insulin (0.87 µM), hydrocortisone (0.1 µM), EGF (25 ng/mL), and additional components in Table 1. Fulcher teaches ALI media to support growth and differentiation on porous supports where ALI differs from BEGM in that ALI contains DMEM and 0.5 ng/mL EGF as shown in Table 2. Fulcher teaches BEGM is prepared using 100% LHC basal medium (page 190) and ALI medium uses a 50:50 mix of DMEM: LHC (page 191). Fulcher teaches LHC basal media contains adenine at 16 g/550 L batch in Table 4.
Regarding claim 2, Fulcher teaches 0.1% trypsin for cell digests (page 192, para. 5; page 197, para. 3).
Regarding claim 5, Fulcher teaches cryopreservation of trypsinized cells (page 198, para. 2).
Regarding claim 6, Fulcher teaches thawing cells (page 198, para. 3).
Fulcher teaches airway cells will facilitate studies of both basic pathophysiology of asthma and chronic bronchitis and the development of novel therapies (page 183, last para.). Fulcher teaches airway cells can be extracted from bronchi (page 186, para. 2). Fulcher does not teach the feeder layer percentage of claim 1. One would have been motivated to combine Zhang and Fulcher because both teach methods of isolating and culturing ex vivo airway cells.
Regarding the feeder layer percentage of claim 1, Gowers teaches isolation and expansion of human airway basal cells from endobronchial biopsy samples where endobronchial brushing samples were cultured with 3T3-J2 feeder cells plated at a density of 2 x 104 cells/cm2 (Abstract; Supplementary Methods page 2 and page 3 para. 1; Figure 1a). Gowers teaches continuous production of secreted factors by feeder cells was important for epithelial support and could not be reproduced by feeding cells with 3T3-J2 conditioned medium (page 316, left col. para. 2). Gowers teaches using trypsin for 60 seconds at 37 °C to separate cells from feeder cells (Supplementary Methods page 3, para. 1).
Regarding “the culture medium has a formulation consisting of” of claim 1, Gowers teaches culture media consisted of DMEM and F12 in a 3:1 ratio with pen/strep, 5% FBS, Y-27632, 25 ng/mL hydrocortisone, 0.125 ng/mL EGF, 5 ug/mL insulin, cholera toxin, amphotericin B and gentamycin (Supplementary Methods page 3, para. 1).
Regarding claim 2 and 3, Gowers teaches digesting endobronchial samples with 0.1% trypsin at 37 C for 30 minutes with agitation by pipetting every 10 minutes and neutralization with FBS to a final concentration of 10% (Supplementary Methods page 2, para. 2).
Regarding claim 5 and 6, Gowers teaches cryopreservation of samples prior to culture (Supplementary Methods page 4, last para.). Gowers teaches primary epithelial cell cultures were initiated from endobronchial biopsy samples that had been cryopreserved before the initiation of cell cultures, suggesting that cryopreservation could eliminate the requirement for close proximity between the clinical facility in which biopsy samples are taken and the specialist laboratory in which cells are cultured (Abstract; page 316, left col. last para.; page 317, left col. para. 1). Gowers teaches generating cell cultures from cryopreserved samples with 100% efficiency for brushings (page 316, left col. para. 3; Figure 3a – b).
Gowers teaches autologous airway epithelial cells have been used in clinical tissue-engineered airway transplantation procedures with a view to assisting mucosal regeneration and restoring mucociliary escalator function but limited time is available for epithelial cell expansion due to the urgent nature of these interventions (Abstract). Gowers teaches clinical protocols have recognized the importance of epithelial cell replacement but a standardized method to expand these cells is lacking, largely due to the varied clinical scenarios in which transplantation is indicated (page 313, right col.). Gowers teaches such technology would be invaluable to inform the development of “standalone” respiratory mucosal epithelial replacement treatments for a range of diseases (page 313, right col.). Gowers teaches samples obtained by brushing and cultured on a feeder layer showed cells positive for Krt5 (Figure 1f; page 315, right col. para. 1). Gowers teaches the cell yield with initiating cultures from a cell suspension by collecting cells by brush biopsy was 94% culture success with 1.8 x 106 cells after 12 days (page 316, left col. para. 4). Gowers teaches an estimate of 3 – 4 weeks for isolation of cells from a cell suspension in 3T3+Y where only basal cells were to be transplanted (page 316, left col. para. 4). One would have been motivated to combine the teachings of Zhang and Gowers because both teach methods of isolating and culturing ex vivo airway cells.
Regarding “passage culture” of claim 1, He teaches obtaining primary cultured airway epithelial cells (P0) and subculture when the cells reach 80 – 90% where the culture medium is aspirated, the cells are rinsed with PBS buffer and digested where after the cells become rounded, the side of the culture dish is gently tapped and when the cells are fully suspended, the digestion is terminated (page 6, 0018 – 0019). He teaches the cell suspension after terminating the digestion is collected, centrifuged and the pellet is suspended in optimized culture medium (page 7, 0019). He teaches the suspended cells are inoculated onto rat tail collagen-coated cell culture dishes in optimized culture medium and incubated for 2 – 3 days and then the medium is replaced to obtain passage 1 (P1 (page 7, 0020). He teaches after the P1 cells reach 80 – 90%, subculture is performed as described and after 80 days of continuous subculture, cells at or above P20 are obtained (page 7, 0020). He teaches the survival rate of human airway cells isolated and cultured reached 80 – 95% and the optimized culture medium can enable the isolated human airway cells to be subcultured for more than 20 generations, which will provide sufficient cells for in vitro cell biology and tissue regeneration engineering research (page 22, para. 0079). He teaches an optimized culture medium contains DMEM (70 – 75%), F-12 (15 – 30%), 0.5 mg/mL hydrocortisone, FBS (5 – 8%), 25 ug/mL EGF, 5 mg/mL insulin, cholera toxin, ROCK1 inhibitor, and BMP4 antagonist (page 8 – 9, para. 0023 – 0032).
Regarding “the culture medium has a formulation consisting of” of claim 1, He teaches an optimized culture medium for culturing human airway cells includes 70 – 75% DMEM, 15 – 30% F12, 0.5 mg/mL hydrocortisone (0.001 – 0.005%), FBS 5 – 8%, 25 µg/mL EGF (0.001 – 0.005%), 5 mg/mL insulin (0.05 – 0.2%), cholera toxin, ROCK1 inhibitor, BMP antagonist (page 8, para. 0023 - 0032).
Regarding claim 2, He teaches performing digestion with a digestion solution containing 99% DMEM (page 15, 0055).
Regarding claim 3, He teaches resuspending cells in DMEM with 10% FBS after digestion to terminate digestion (page 16, 0059).
Regarding claim 5, He teaches cryopreservation of cells by digesting the cells and resuspending in freezing solution and transferring to liquid nitrogen for long-term storage (page 17 – 18, para. 0065).
Regarding claim 6, He teaches resuscitation of frozen cells by thawing and seeding onto collagen coated dishes where the cells were ready for passage after 2 – 3 days (page 18 – 19, para. 0067).
He does not teach testing for HOPX or endotoxin or the culture medium of claim 1. However, He teaches current airway cell isolation methods and culture conditions cannot meet research needs (page 2, para. 0004). He teaches it is particularly important to seek simple and effective methods and culture media for culturing human airway cells in order to obtain sufficient amounts of epithelial cells (page 3, para. 0005). One would have been motivated to combine the teachings of Zhang and He because both teach methods of isolating and culturing ex vivo airway cells where Zhang teaches primary culture and He teaches amplification and expansion culture.
Regarding “HOPX” and “endotoxin” of claim 1, Ma teaches cells were cultured according to GMP by qualified operators with GCP certificate and the quality of clinically used cells (bacteria, endotoxin and pyrogen-free) was confirmed by ILAC-MRA recognized third-party organization (Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement). Ma teaches staining cells for HOPX (page 278, left col.; Figure 2F; Figure 3F). Ma teaches a method of obtaining basal cells (BCs) that are progenitor cells comprising obtaining an ex vivo bronchoscopic brushed-off biopsy tissue (page 277, right col. paragraph 2; “Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement; Figure 1A; page 268, right col. paragraph 1). Ma teaches cell pellets of the digested cells were collected by centrifugation and plated onto feeder cell coated plates and cultured in media comprising antibiotics under 7.5% CO2 at 37 °C where colonies emerged 3 – 5 days after plating and were collected for passaging (page 277, right col. paragraph 2; “Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement; Figure 1A; page 268, right col. paragraph 2). Ma teaches to obtain single cell-derived clone, cells are digested into single cells and seeded with extremely low density (page 277, right col. paragraph 2; page 268, right col. paragraph 2). Ma teaches culturing onto dishes pre-coated with Matrigel for further expansion and Ma teaches two passages of clones and Ma teaches passage 0, passage 1, and passage 2 of cells (page 270, right col. paragraph 1; page 277, right col. paragraph 2; Figure 1A; Supplementary Figure 3C). Ma teaches 100% of the colonies contain 40 – 100 cells grown on feeder cells in Figure 1C and Supplementary Figure 4B. Ma teaches digesting with trypsin (page 277, right col. paragraph 2).
Regarding “the culture medium has a formulation consisting of” of claim 1, Ma teaches a culture medium of DMEM/F12, 10% FBS and growth (page 277, right col. paragraph 2).
Regarding claim 2, Ma teaches digesting was performed at 37 ◦C for an hour with a solution comprising DMEM/F12, 2 mg/mL protease XIV, 0.01% trypsin and 10 ng/mL DNase I (page 277, right col. paragraph 2). 0.01% trypsin is 0.01 g/100 mL and claim 2 recites a concentration range of trypsin inclusive of 100 ng/mL which is equivalent to 10,000 ng/100 mL or 0.01 g/100 mL which is 0.01%.
Regarding claim 4, Ma teaches the biopsy was taken from bronchi in the right or left lobe (page 268, right col. paragraph 1; page 277, left col. last paragraph).
Regarding claim 15, Ma teaches clinical grade autologous bronchial basal cells were obtained by the method (page 279, right col. last paragraph; Figure 1). Ma teaches cells were cultured according to GMP by qualified operators with GCP certificate and the quality of clinically used cells (bacteria, endotoxin and pyrogen-free) was confirmed by ILAC-MRA recognized third-party organization (Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement). Ma teaches the cells are HOPX and KRT5 positive (Figure 1A, F, G, H).
Regarding claim 21, Ma teaches treating bronchiectasis comprising administering clinical grade autologous bronchial basal cells in 0.9% sterile saline to patients with bronchiectasis (page 272, right col. last paragraph; page 279, right col. last paragraph; “Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement; Figure 7).
Ma teaches cells of 75% confluence were karyotyped and all cells used for clinical purpose were subjected to karyotyping prior to transplantation (page 278, right col. paragraph 1). Ma teaches 0.67 – 3.2 x 107 cells were transplanted into patients (Figure 7B). Ma teaches bronchiectasis is a lethal lung disease where lung transplant surgery is limited due to the extreme lack of donor lung as well as severe side effects due to immune-rejection and patients are in urgent need of a new strategy to cure bronchiectasis and stem/progenitor cell-based regenerative therapy is likely to be the biggest hope for them (page 267, right col. last paragraph; page 268, left col. first paragraph). Ma teaches transplantation of autologous BCs could repair the damaged lung in bronchiectasis patients in both pulmonary structure and function (page 277, left col. paragraph 1). One would have been motivated to combine the teachings of Zhang and Ma because both teach methods of isolating and culturing ex vivo airway cells.
Regarding “the culture medium has a formulation consisting of” of claim 1, Reynolds teaches F media for the culturing airway basal cells collected from the human nasal airway by a cytology brush from the posterior surface of the inferior turbinate that comprises DMEM, FBS, F-12, L-glutamine, hydrocortisone/EGF, adenine, insulin, cholera toxin, and Y-27632 (page 324, right col. last paragraph; page 334, right col. paragraph 2; page E8 of Supplementary Material). Reynolds teaches culture media for airway cells contains 1.9 mM L-glutamine (“0.2 – 2 mM L-glutamine”) (page E7 of Supplementary Material; Sub-media 1) as evidenced by GibcoGlutamine (page 1). Reynolds teaches adding 500 µL of insulin (product number I9278 from Sigma-Aldrich) (9.5-11.5 mg/mL as evidenced by Sigma, page 2) to make F media which results in a final concentration of 9.5 µg/mL of insulin (page E8 of Supplementary Material, Sub-media 5). Reynolds teaches a sub-media 2 at 1000X comprising hydrocortisone (25 µg/mL) and EGF (0.125 µg/mL) (page E8 of Supplementary Material; Sub-media 2). When diluted to a working concentration of 1X the final concentration of hydrocortisone would be 25 ng/mL and the final concentration of EGF would be 0.125 ng/mL (page E8 of Supplementary Material, F Media components). Reynolds teaches 8 mL of adenine at 1.5 mg/mL is added to make F media which results in a final concentration of 24 µg/mL ([1.5 mg/mL x 8 mL]/499.0043 mL) (page E8 of Supplementary Material, F Media components). Reynolds teaches the culture method allowed the production of 7.1 x 1010 cells after 4 passages, approximately 379 times more cells than were generated by the standard BEGM media and the cells had normal basal cell markers (Abstract). One of ordinary skill in the art would be motivated to combine the teachings of Zhang, Fulcher, Gowers, He, Ma, and Reynolds to arrive at the claimed culture medium because each teaches methods of isolating and culturing ex vivo airway cells. A rationale for arriving at the claimed culture media comes from the teachings of Zhang, Fulcher, Ma and Reynolds where Ma teaches culturing the basal cells on feeder cells and then shifting to feeder-free culture conditions for preparation of cells for delivery to patients in the clinical trial where cells were cultured according to GMP and the clinical quality was verified and Reynolds teaches the culture media and Reynolds teaches culturing cells in DMEM/F12 with FBS, L-glutamine, insulin, EGF, adenine, and hydrocortisone. Additionally, Fulcher teaches BEGM contains insulin, hydrocortisone, EGF, and adenine and is used when the initial cell harvests are plated on collagen-coated plastic dishes or to expand passaged cells on plastic while Reynolds teaches the culture method allowed the production of 7.1 x 1010 cells after 4 passages, approximately 379 times more cells than were generated by the standard BEGM media and the cells had normal basal cell markers. A rationale for omitting cholera toxin and Y-27632 from the media of Reynolds comes from the teachings of Zhang where the media does not contain either cholera toxin or Y-27632 and Zhang teaches the cells differentiated into human bronchi and alveoli and other tissue structures indicating that the stem cells obtained using the method can be used for clinical stem cell transplantation therapy. Taken together, Zhang, Fulcher, Ma and Reynolds provide a rationale for one of ordinary skill in the art to arrive at the claimed culture medium with a reasonable expectation of success as Zhang teaches the cells differentiated into human bronchi and alveoli in vivo and Ma teaches the cells were used for clinical trials.
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 Zhang regarding a method of preparing lung stem cells from airway biopsies with the teachings of Fulcher regarding a method of preparing airway cells from airway biopsies with the teachings of Gowers regarding isolation and expansion of human airway basal cells from endobronchial biopsy samples where endobronchial brushing samples were cultured with 3T3-J2 feeder cells with the teachings of He regarding primary and passaging culture of airway epithelial cells with the teachings of Ma regarding a method of producing bronchial basal cells with the teachings of Reynolds regarding a method of produce clinical-grade autologous bronchial basal cells to arrive at the claimed preparation process of clinical-grade autologous bronchial basal cells. One would have been motivated to combine the teachings of Zhang, Fulcher, Gowers, He, Ma and Reynolds in a method to produce clinical-grade autologous bronchial basal cells as Zhang teaches apart from lung transplantation, there is currently no effective treatment for some lung diseases such as pulmonary fibrosis and COPD and Fulcher teaches airway cells will facilitate studies of both basic pathophysiology of asthma and chronic bronchitis and the development of novel therapies and Gowers teaches autologous airway epithelial cells have been used in clinical tissue-engineered airway transplantation procedures but limited time is available for epithelial cell expansion due to the urgent nature of these interventions and Gowers teaches clinical protocols have recognized the importance of epithelial cell replacement but a standardized method to expand these cells is lacking, and such technology would be invaluable to inform the development of “standalone” respiratory mucosal epithelial replacement treatments for a range of diseases and He teaches it is particularly important to seek simple and effective methods and culture media for culturing human airway cells in order to obtain sufficient amounts of epithelial cells and Ma teaches bronchiectasis is a lethal lung disease where lung transplant surgery is limited due to the extreme lack of donor lung as well as severe side effects due to immune-rejection and patients are in urgent need of a new strategy to cure bronchiectasis and stem/progenitor cell-based regenerative therapy is likely to be the biggest hope for them. One would have a reasonable expectation of success in combining the teachings as Zhang teaches the method is simple to operate, has low implementation cost, and is easy to promote and the cells produced by the method when transplanted into mice developed into human bronchi and alveolar structures and Gowers teaches the cell yield with initiating cultures from a cell suspension by collecting cells by brush biopsy was 94% culture success with 1.8 x 106 cells and He teaches the survival rate of human airway cells isolated and cultured reached 80 – 95% and the optimized culture medium can enable the isolated human airway cells to be subcultured for more than 20 generations, which will provide sufficient cells for in vitro cell biology and tissue regeneration engineering research and Ma teaches transplantation of autologous basal cells could repair the damaged lung in bronchiectasis patients in both pulmonary structure and function and Reynolds teaches the culture method allowed the production of 7.1 x 1010 cells after 4 passages, approximately 379 times more cells than were generated by the standard BEGM media and the cells had normal basal cell markers.
Applicant Argues/ Response to Arguments
12. Applicant Argues: On page 7, Applicant asserts that claim 1 differs from Zhang in that (1) Zhang does not teach adding antibiotics or replacing the culture medium every other day or the feeder layer percentage or stem cell percentage for passaging or cell collecting or passage culture; (2) Zhang does not teach measuring HOPX or endotoxin; (3) Zhang does not teach the recited components of the culture medium.
Response to Arguments: In the maintained rejection set forth above regarding adding antibiotics and passage culture in (1), Fulcher teaches a method of obtaining primary and amplified airway cell cultures (P1, P2) in Figure 1 where ex vivo tissue samples are digested with protease XIV and cultured on collagen-coated supports. Fulcher teaches it is assumed that many primary human tissues contain yeasts, fungi, or bacteria and media for primary cultures can be supplemented with gentamicin and amphotericin where fewer episodes of contamination will result from using amphotericin (page 191, last para.). Fulcher teaches to generate P1 or P2, primary cells can be plated on collagen-coated plastic dishes and primary cell media should be supplemented with additional antibiotics for the first 3 days after plating and should be changed every 2 – 3 days or as needed to prevent acidification (page 197, para. 1). Fulcher teaches when primary cultures reach 70 – 90% confluence they are ready for passage and cells are harvested by digestion with trypsin and incubation at 37 °C followed by rinsing with PBS (page 15, para. 3). Gowers teaches isolation and expansion of human airway basal cells from endobronchial biopsy samples where endobronchial brushing samples were cultured with 3T3-J2 feeder cells plated at a density of 2 x 104 cells/cm2 (Abstract; Supplementary Methods page 2 and page 3 para. 1; Figure 1a). Gowers teaches continuous production of secreted factors by feeder cells was important for epithelial support and could not be reproduced by feeding cells with 3T3-J2 conditioned medium (page 316, left col. para. 2). Gowers teaches using trypsin for 60 seconds at 37 °C to separate cells from feeder cells (Supplementary Methods page 3, para. 1). He teaches obtaining primary cultured airway epithelial cells (P0) and subculture when the cells reach 80 – 90% where the culture medium is aspirated, the cells are rinsed with PBS buffer and digested where after the cells become rounded, the side of the culture dish is gently tapped and when the cells are fully suspended, the digestion is terminated (page 6, 0018 – 0019).
Regarding HOPX or endotoxin of item (2), Ma teaches staining cells for HOPX (page 278, left col.; Figure 2F; Figure 3F). Ma teaches cells were cultured according to GMP by qualified operators with GCP certificate and the quality of clinically used cells (bacteria, endotoxin and pyrogen-free) was confirmed by ILAC-MRA recognized third-party organization (Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement).
Regarding the culture medium of item (3), one of ordinary skill in the art would be motivated to combine the teachings of Zhang, Fulcher, Gowers, He, Ma, and Reynolds to arrive at the claimed culture medium because each teaches methods of isolating and culturing ex vivo airway cells. A rationale for arriving at the claimed culture media comes from the teachings of Zhang, Fulcher, Ma and Reynolds where Ma teaches culturing the basal cells on feeder cells and then shifting to feeder-free culture conditions for preparation of cells for delivery to patients in the clinical trial where cells were cultured according to GMP and the clinical quality was verified and Reynolds teaches the culture media and Reynolds teaches culturing cells in DMEM/F12 with FBS, L-glutamine, insulin, EGF, adenine, and hydrocortisone. Additionally, Fulcher teaches BEGM contains insulin, hydrocortisone, EGF, and adenine and is used when the initial cell harvests are plated on collagen-coated plastic dishes or to expand passaged cells on plastic while Reynolds teaches the culture method allowed the production of 7.1 x 1010 cells after 4 passages, approximately 379 times more cells than were generated by the standard BEGM media and the cells had normal basal cell markers. A rationale for omitting cholera toxin and Y-27632 from the media of Reynolds comes from the teachings of Zhang where the media does not contain either cholera toxin or Y-27632 and Zhang teaches the cells differentiated into human bronchi and alveoli and other tissue structures indicating that the stem cells obtained using the method can be used for clinical stem cell transplantation therapy. Taken together, Zhang, Fulcher, Ma and Reynolds provide a rationale for one of ordinary skill in the art to arrive at the claimed culture medium with a reasonable expectation of success as Zhang teaches the cells differentiated into human bronchi and alveoli in vivo and Ma teaches the cells were used for clinical trials.
Claim 1 is constructed to contain multiple “wherein” clauses, beginning at line 7, instead of active method steps. These “wherein” clauses are not active steps and do not limit the method because they do no give meaning and purpose to the active steps to prepare clinical-grade autologous bronchial basal cells (see MPEP 2111.04 (I)).
Should Applicant amend the claims to recite active method steps instead of “wherein” clauses, the rejections of the claims over the prior art may be overcome upon further search and consideration.
Applicant Argues: On page 8 paragraph 1, Applicant asserts that Fulcher disclosed primary cell culture to obtain differentiated cells for in vitro research, fundamentally opposing the present invention’s objective of producing undifferentiated basal cells for in vivo transplantation. Applicant asserts that incorporating components from Fulcher lacks inspiration in technical logic. Applicant asserts that He did not disclose the phased testing plan tied to clinical-grade quality control in the present application and He did not disclose the timing of P0 cell passage and the ratio of feeder layer cells.
Response to Arguments: This is not found persuasive because the preamble of claim 1 recites “bronchial basal cells” and does not recite “undifferentiated basal cells” and Fulcher teaches a method of obtaining primary and amplified airway cell cultures (P1, P2) in Figure 1. Therefore, one of ordinary skill in the art would logically look to the method of Fulcher to obtain and amplify primary airway cells. Regarding “phased testing plan”, it is noted that claim 1 does not recite an active method step for testing and instead recites within a “wherein” clause “to carry out” tests and further recites within a “wherein” clause a preferred outcome of the tests. Zhang teaches lung stem cells had good cell activity, high purity, and no bacterial and mycoplasma contamination (page 34, para. 0159). Zhang teaches a method for determining bacterial contamination and the culture medium was clear indicating no bacterial contamination (page 29, para. 0139). Zhang teaches a method for detecting mycoplasma contamination and the final value of the test is less than 0.8 indicating that there is no mycoplasma contamination (page 31, 0140). Fulcher teaches it is assumed that many primary human tissues contain yeasts, fungi, or bacteria and media for primary cultures can be supplemented with gentamicin and amphotericin where fewer episodes of contamination will result from using amphotericin (page 191, last para.). Ma teaches cells were cultured according to GMP by qualified operators with GCP certificate and the quality of clinically used cells (bacteria, endotoxin and pyrogen-free) was confirmed by ILAC-MRA recognized third-party organization (Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement). Ma teaches staining cells for HOPX (page 278, left col.; Figure 2F; Figure 3F). Therefore, again, one of ordinary skill in the art would logically look to the method of Fulcher to obtain and amplify primary airway cells and minimize bacterial contamination.
Applicant Argues: On page 8, paragraph 3, Applicant asserts that Ma does not disclose specific operatable testing items, time point, or pass/fail criteria and the testing plan in the instant application constitutes a whole technical solution that cannot be summarized as a simple “cultivation according to GMP”.
Response to Arguments: This is not found persuasive because claim 1 does not recite an active method step for testing and instead recites within a “wherein” clause “to carry out” tests and further recites within a “wherein” clause with preferred outcome of tests. Ma teaches cells positive for KRT5 and HOPX were cultured according to GMP by qualified operators with GCP certificate and the quality of clinically used cells (bacteria, endotoxin and pyrogen-free) was confirmed by ILAC-MRA recognized third-party organization (Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement; page 268, left col. last para.; page 278, left col.; Figure 2F; Figure 3F) which were used in a clinical trial. Therefore, the culture method according to GMP by Ma yields clinical-grade cells that were used in a clinical trial. Should Applicant amend the claims to recite active method steps regarding testing instead of “wherein” clauses, an intent to carry out tests and preferred outcomes, the rejections of the claims over the prior art may be overcome upon further search and consideration.
Applicant Argues: On page 9 – 15, Applicant asserts that the culture medium components in claim 1 is limited in a closed manner. Applicant asserts that applying the medium formulation or its components intended for differentiation endpoints to a culture process aimed at maintaining an undifferentiated state is scientifically illogical. Applicant asserts that the cells cultured in Reynolds are distinct cell types from the basal layer cells derived from the bronchi. Applicant asserts that the effect of Zhang is incomparable to that demonstrated in the present application. Applicant asserts that a person skilled in the art would not obtain the culture medium formulation, nor could they foresee that the culture medium of the present application could effectively control cell morphology, thereby enabling the prepared cells to be used for clinical treatment with improved therapeutic effects.
Response to Arguments: This is not found persuasive because the components of the culture medium are recited in a “wherein” clause and do not give patentable weight to the method because the culture medium is used in passaging that are not active steps and the medium is not required for the clinical-grade autologous bronchial basal cells. Again, the preamble of claim 1 recites “bronchial basal cells” and does not recite “undifferentiated basal cells” and the first active step of claim 1 broadly recites “getting an ex vivo bronchoscopic brushed-off biopsy tissue” and Fulcher teaches a method of obtaining primary and amplified airway cell cultures (P1, P2) in Figure 1. Therefore, one of ordinary skill in the art would logically look to the culture method of Fulcher to obtain and amplify primary airway cells. Further, Ma teaches a method of obtaining basal cells (BCs) that are progenitor cells comprising obtaining an ex vivo bronchoscopic brushed-off biopsy tissue (page 277, right col. paragraph 2; “Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement; Figure 1A; page 268, right col. paragraph 1) and Ma teaches the biopsy was taken from bronchi in the right or left lobe (page 268, right col. paragraph 1; page 277, left col. last paragraph). Ma teaches cells were cultured according to GMP by qualified operators with GCP certificate and the quality of clinically used cells (bacteria, endotoxin and pyrogen-free) was confirmed by ILAC-MRA recognized third-party organization (Procedure of SOX9+ BC transplantation” in Clinical Trial Information supplement). Ma teaches a culture medium of DMEM/F12, 10% FBS and growth (page 277, right col. paragraph 2). Ma teaches 0.67 – 3.2 x 107 cells were transplanted into patients (Figure 7B). Ma teaches bronchiectasis is a lethal lung disease where lung transplant surgery is limited due to the extreme lack of donor lung as well as severe side effects due to immune-rejection and patients are in urgent need of a new strategy to cure bronchiectasis and stem/progenitor cell-based regenerative therapy is likely to be the biggest hope for them (page 267, right col. last paragraph; page 268, left col. first paragraph). Ma teaches transplantation of autologous BCs could repair the damaged lung in bronchiectasis patients in both pulmonary structure and function (page 277, left col. paragraph 1). Therefore, the combined teachings give inspiration for the culture medium of claim 1 to prepare clinical-grade BCs for treating bronchiectasis. Should Applicant amend the claims to recite active method steps regarding preparing the claimed formulation of culture medium and suspending/formulating the clinical-grade autologous bronchial basal cells in the claimed formulation of the culture medium instead of “wherein” clauses, the rejections of the claims over the prior art may be overcome upon further search and consideration.
Conclusion
No claims allowed.
THIS ACTION IS MADE FINAL. 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