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 .
1. Claims 1, 2, 4, 7 – 10, 13 – 15, 17, 18, and 44 remain pending. Claims 1, 2, 4, and 7 – 10 are under consideration.
Election/Restrictions
2. Newly submitted claims 13 – 15 (drawn to a method of making a humanized animal) and 17 – 18 (drawn to a method of producing human immune cells in a humanized animal) are directed to an invention that lacks unity with the invention originally claimed for the following reasons:
The amended claims contains the following inventions or groups of inventions which are not so linked as to form a single general inventive concept:
Group I, claims 1, 2, 4, 7 – 10, and 44 drawn to a method of making a bioengineered thymus organoid and producing human immune cells.
Group II, claims 13 – 15, drawn to a method of making a humanized animal.
Group III, claims 17 and 18, drawn to a method of producing human immune cells in a humanized animal.
Groups I – III lack unity of invention because even though the inventions of these groups require the technical feature of a bioengineered thymus organoid made by steps (a) – (g), this technical feature is not a special technical feature as it does not make a contribution over the prior art in view of Gu (Gu, Qi, et al. Advanced healthcare materials 6.17 (2017): 1700175.), hereinafter Gu in view of Gai (Gai, Hui, et. al. Blood 130 (2017): 2445.), hereinafter Gai in view of Fan (Fan Y, et. al. Mol Ther. 2015 Jul;23(7):1262-1277; previously cited), hereinafter Fan which is cited on the IDS filed 05/24/2022 and 03/19/2026. Gu teaches encapsulating hiPSCs in a medium that separates the hiPSCs into single cells (step (a)) and expansion culturing of the hiPSCs in a growth medium without differentiation (step (b)) (Figure 1 – 3; page 2, left col. para. 1 – 2 and right col. para. 2 – 5; page 9, left col. para. 2 and right col. para. 2). Gu teaches the method overcomes reputed difficulties with maintaining and differentiating iPSCs (page 8, left col. para. 2). Gu teaches the encapsulated hiPSCs could be differentiated in situ (step (c)) into all germ lineages indicating the potential to form multiple cell and tissue types within and from the bioprinted constructs (page 3 – 4; Figure 4; page 5, left col. para. 1; page 6, left col. para. 2 – 3; page 8, left col. para. 1), but does not teach differentiation into hTEPCs or hTECs. Gu teaches extracting encapsulated cells with EDTA and fluxing with a pipette (step (d)) (page 10, left col. last para. and right col. para. 2) but does not teach the cells comprise hTEPCs or hTECs or both. Gu teaches encapsulation of the iPSCs provides for direct and complete contact with extracellular elements that more closely mimic the native cell microenvironment (page 2, left col. para. 1).
Gai teaches differentiating hPSCs to TEPCs by first induction of definitive endoderm followed by induction of anterior foregut, and pharyngeal pouch endoderm (step (c)) (page 2, para. 2). Gai teaches the goal of differentiating hPSCs to TEPCs is for use as a stem cell-based therapy for thymic regeneration and restoration of immune competence in hematopoietic stem cell transplantation (HSCT) recipients (page 2, para. 2; Figure 1). Gai teaches thymic function naturally declines with age with little to no endogenous potential to regenerate (page 1).
Fan teaches a method of combining TECs harvested from the thymus with Lin- progenitors at a 1:1 ratio (step (e)) and injecting the combination into decellularized thymus scaffolds to form reconstructed thymus organoids (step (f)) that were cultured (step (g)) (page 1263, left col. last para. and right col. para. 1; page 1274, right col. last para.; page 1275, left col. para. 1 – 3; page 1265). Fan teaches one of the major clinical challenges to manipulate postnatal TECs for clinical application is their dependency on a properly configured 3D ECM microenvironment for survival and proliferation (page 1271, right col.). Fan teaches the complement of Lin- progenitors was used to ensure the continuity of cross talk between TECs and the developing thymocytes that is essential for the survival of TECs (page 1266, left col. para. 1). Fan teaches the decellularized thymus scaffold environment provided a suitable and essential niche for long-term survival of adult TECs in vitro as the TECs in the scaffold could retain their specific molecular properties for up to 8 weeks (page 1270, left col. para. 1; page 1273, left 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 Gu, Gai, and Fan to expand and in situ differentiate encapsulated hiPSCs to hTEPCs to make a bioengineered thymus organoid in order to provide the hiPSCs and hiPSC-derived hTEPCs an environment that mimics the native cell microenvironment for improved differentiation and survival of these cells.
Since applicant has received an action on the merits for the originally presented invention, this invention has been constructively elected by original presentation for prosecution on the merits. Accordingly, claims 13 – 15 and 17 – 18 are withdrawn from consideration as being directed to a non-elected invention. See 37 CFR 1.142(b) and MPEP § 821.03.
To preserve a right to petition, the reply to this action must distinctly and specifically point out supposed errors in the restriction requirement. Otherwise, the election shall be treated as a final election without traverse. Traversal must be timely. Failure to timely traverse the requirement will result in the loss of right to petition under 37 CFR 1.144. If claims are subsequently added, applicant must indicate which of the subsequently added claims are readable upon the elected invention.
Should applicant traverse on the ground that the inventions are not patentably distinct, applicant should submit evidence or identify such evidence now of record showing the inventions to be obvious variants or clearly admit on the record that this is the case. In either instance, if the examiner finds one of the inventions unpatentable over the prior art, the evidence or admission may be used in a rejection under 35 U.S.C. 103 or pre-AIA 35 U.S.C. 103(a) of the other invention.
Information Disclosure Statement
3. The information disclosure statement (IDS) submitted on 03/19/2026 AND 06/18/2026 are acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Withdrawn Claim Rejections
4. The rejection of claims 13 – 15 and 17 – 18 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to claims 13 and 17 to make these independent claims. Claims 13 – 15 and 17 – 18 are withdrawn and not further considered.
5. The rejection of claims 13 – 15, 17, and 18 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 13 and 17 to make these independent claims. Claims 13 – 15 and 17 – 18 are withdrawn and not further considered.
6. The rejection of claims 7 – 10 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to claim 7 to make it an independent claim.
7. The rejection of claim 5 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of the claim.
8. The rejection of claims 1, 2, 4, and 44 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1 to require steps (a) – (d).
9. The rejection of claims 7 – 10 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 7 to make it an independent claim.
Rejections Necessitated by Amendment
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, 2, 4, 5, and 44 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gu (Gu, Qi, et al. Advanced healthcare materials 6.17 (2017): 1700175.), hereinafter Gu in view of Gai (Gai, Hui, et. al. Blood 130 (2017): 2445.), hereinafter Gai in view of Fan (Fan Y, et. al. Mol Ther. 2015 Jul;23(7):1262-1277; previously cited), hereinafter Fan which is cited on the IDS filed 05/24/2022 and 03/19/2026.
Regarding steps (a) – (b) of claim 1, Gu teaches encapsulating hiPSCs in a medium that separates the hiPSCs into single cells (step (a)) and expansion culturing of the hiPSCs in a growth medium without differentiation (step (b)) (Figure 1 – 3; page 2, left col. para. 1 – 2 and right col. para. 2 – 5; page 9, left col. para. 2 and right col. para. 2). Gu teaches the method overcomes reputed difficulties with maintaining and differentiating iPSCs (page 8, left col. para. 2).
Regarding step (c) of claim 1, Gu teaches the encapsulated hiPSCs could be differentiated in situ into all germ lineages indicating the potential to form multiple cell and tissue types within and from the bioprinted constructs, and could be differentiated into neural tissue (page 3 – 4; Figure 4; page 5, left col. para. 1; page 6, left col. para. 2 – 3; page 8, left col. para. 1), but does not teach differentiation into hTEPCs or hTECs. However, Gu teaches without specific medium supplements, pluripotent stem cells have a tendency to differentiate to derivatives of the three germ lineages, alternative media compositions can promote differentiation toward one or another lineage (page 6, left col. para. 2).
Regarding step (d) of claim 1, Gu teaches extracting encapsulated cells with EDTA and fluxing with a pipette (page 10, left col. last para. and right col. para. 2) but does not teach the cells comprise hTEPCs or hTECs or both.
Gu does not teach “differentiating the hiPSCs to generate” hTEPCs or hTECs of step (c) or “obtaining a cell population comprising hTEPCs or hTECs or both of step (d) or steps (e) – (g) of claim 1 or “wherein the hiPSCs or the hHSCs are derived from one or more donor individuals” of claim 2 or “the de-cellularized thymus scaffold is from a donor animal” of claim 4 or “wherein the hTEPCs or the hTECs are at least partially HLA-matched with the hHSCs” of claim 44. However, Gu teaches the encapsulated hiPSCs proliferation increased from the time of encapsulation and peaked at day 9 and maintained pluripotency markers (page 2, right col. para. 4 – 5). Gu teaches their method of encapsulating hiPSCs, expanding the encapsulated hiPSCs, and differentiating the encapsulated hiPSCs will be useful in iPSC translation for pharmaceuticals development and regenerative medicine (Abstract). Gu teaches 3D co-printing is a single-step approach to rapidly fabricate a 3D cellularized construct whereby iPSCs are immediately integrated with biomaterials by encapsulation for direct and complete contact with extracellular elements that more closely mimic the native cell microenvironment (page 2, left col. para. 1). Gu teaches the ability to 3D print hiPSCs to then expand and generate cells of different lineages provides an unprecedented opportunity to form different, authentic, and renewable body tissues (page 8, right col. para. 2). Gu teaches further refinement of the method is expected to enhance tissue identity, architecture, and function to better model development and diseases, for pharmaceuticals screening and assessing in vivo function and safety in animal models toward transplantation therapies (page 8, right col. para. 2).
Regarding “differentiating the hiPSCs to generate” hTEPCs or hTECs of step (c) and “obtaining a cell population comprising hTEPCs or hTECs or both of step (d) of claim 1, Gai teaches differentiating hPSCs to TEPCs by first induction of definitive endoderm followed by induction of anterior foregut, and pharyngeal pouch endoderm (page 2, para. 2). Gai teaches the TEPCs derived in vitro from hPSCs express the key thymic transcription factor FOXN1 along with Keratin 5 and Keratin 8, the primary cytoskeletal building blocks of medullary and cortical TECs (page 2, para. 2; Figure 2).
Regarding claim 2, Gai teaches the potential applications of in vitro derived TEPCs are not confined to restoring thymic function in the HSCT setting, because TEPCs derived from solid organ donors hold the promise of inducing tolerance to donor tissues, thereby preventing rejection of the transplanted donor organ (page 2, last para.). As Gai teaches deriving TEPCs from hPSCs and TEPCs derived from donors, Gai makes obvious hiPSCs are derived from one or more donor individuals.
Gai does not teach steps (e) – (g) of claim 1 or “the de-cellularized thymus scaffold is from a donor animal” of claim 4 or the hTECs are at least partially HLA-matched with the hHSCs of claim 44. However, Gai teaches the goal of differentiating hPSCs to TEPCs is for use as a stem cell-based therapy for thymic regeneration and restoration of immune competence in hematopoietic stem cell transplantation (HSCT) recipients (page 2, para. 2; Figure 1). Gai teaches the outcome of a HSCT and the recipient thymic function are intimately intertwined where the production of mature T cells occurs in the thymus and depends on the interaction of hematopoietic progenitor cells with TECs (page 1). Gai teaches the thymus gives rise to regulatory T-cells which confine immune responses and further promote central tolerance (page 1). Gai teaches thymic function naturally declines with age with little to no endogenous potential to regenerate (page 1). Gai teaches current options for thymic injury during HSCT are limited to symptomatic therapy and therefore novel approaches to cure irreversible thymic injury are critically needed (page 2, para. 1). Gai teaches the potential applications of in vitro derived TEPCs are not confined to restoring thymic function in the HSCT setting, because TEPCs derived from solid organ donors hold the promise of inducing tolerance to donor tissues, thereby preventing rejection of the transplanted donor organ (page 2, last para.). Gai teaches leveraging the body’s own ability to educate T-cells through regenerating thymic tissues could represent a novel approach to addressing key challenges in transplantation immunology (page 3, para. 1). One would have been motivated to combine the teachings of Gu and Gai to restore thymic function in subjects with thymic injury as Gu teaches the ability to 3D print hiPSCs to then expand and generate cells of different lineages provides an unprecedented opportunity to form different, authentic, and renewable body tissues and assess in vivo function and safety in animal models toward transplantation therapies and Gai teaches novel approaches to cure irreversible thymic injury are critically needed.
Regarding steps (e) – (g) of claim 1, Fan teaches a method of combining the TECs with Lin- progenitors at a 1:1 ratio (step (e)) and injecting the combination into decellularized thymus scaffolds to form reconstructed thymus organoids (step (f)) that were cultured (step (g)) (page 1263, left col. last para. and right col. para. 1; page 1274, right col. last para.; page 1275, left col. para. 1 – 3; page 1265).
Regarding claim 4, Fan teaches the decellularized thymus scaffold is from a mouse (page 1274, right col. para. 4).
Regarding claim 44, Fan teaches the TECs and HSCs are from B6 (H-2b) mice and were combined with TECs from CBA/J mice to make thymus constructs that were then transplanted into B6 mice (page 1265, right col. last para.; page 1266, left col. para. 1; page 1268, right col. para. 2). Fan teaches including donor TECs in the reconstruction of thymus organoids might be a clinical applicable way to induce donor-specific immune tolerance (page 1268, right col. para. 2).
Fan teaches one of the major obstacles in organ transplantation is to establish immune tolerance of allografts (Abstract). Fan teaches long-term allograft survival has not improved (page 1270, left col. para. 2). Fan teaches conventional pharmacological drugs can cause severe side effects including increased risk of cardiovascular death, diabetes, and kidney disease (page 1270, left col. para. 2). Fan teaches induction of thymic central tolerance to allografts remains challenging largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering and when transplanted into mice supported thymopoiesis (Abstract). Fan teaches one of the major challenges to manipulate postnatal TECs for clinical application is their dependency on a properly configured 3D ECM microenvironment for survival and proliferation (page 1271, right col.). Fan teaches the complement of Lin- progenitors was used to ensure the continuity of cross talk between TECs and the developing thymocytes that is essential for the survival of TECs (page 1266, left col. para. 1). Fan teaches the decellularized thymus scaffold environment provided a suitable and essential niche for long-term survival of adult TECs in vitro as the TECs in the scaffold could retain their specific molecular properties for up to 8 weeks (page 1270, left col. para. 1; page 1273, left col. para. 1). Fan teaches the TECs remained viable for > 3 weeks in the thymic scaffolds (page 1262, right col. para. 1). Fan teaches transplantation of bioengineered thymus organoids with donor and recipient TECs confer allo-skin tolerance (page 1269, right col. para. 2). Fan teaches donor TECs can be generated from induced pluripotent stem cells (page 1274, left col. para. 3).
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 Gu regarding a method of encapsulating hiPSCs, expanding the encapsulated hiPSCs, and differentiating the encapsulated hiPSCs with the teachings of Gai regarding a method of differentiating hPSCs to TEPCs with the teachings of Fan regarding a method of combining TECs and HSCs in a decellularized thymus scaffold to arrive at the claimed method for making a bioengineered thymus organoid, comprising (a) encapsulating human induced pluripotent stem cells (hiPSCs) in a suspension medium that separates the hiPSCs into single cells; (b) culturing the hiPSCs in a growth medium to increase the number thereof without differentiation; (c) differentiating the hiPSCs to generate human thymic epithelial progenitor cells (hTEPCs) or human thymic epithelial cells (hTECs) or both in an encapsulation medium; (d) freeing the hTEPCs or hTECs or both from the encapsulation medium, thereby obtaining a cell population comprising hTEPCs or hTECs or both, (e) combining the cell population with human hematopoietic stem cells (hHSCs) in a defined ratio to form a combination; (f) seeding the combination into an extracellular matrix of a de-cellularized thymus scaffold to generate a thymus construct; and (g) culturing the thymus construct under conditions permitting cellular attachment onto the extracellular matrix, thereby making the bioengineered thymus organoid. One would have been motivated to combine the teachings of Gu, Gai, and Fan in a method of making a bioengineered thymus organoid to restore thymic function in subjects with thymic injury as Gu teaches the ability to 3D print hiPSCs to then expand and generate cells of different lineages provides an unprecedented opportunity to form different, authentic, and renewable body tissues and Gu teaches further refinement of the method is expected to allow for assessing in vivo function and safety in animal models toward transplantation therapies and Gai teaches in vitro derived TEPCs could prevent rejection of a transplanted donor organ and HSCT and Fan teaches induction of thymic central tolerance to allografts remains challenging largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering and Fan teaches one of the major challenges to manipulate postnatal TECs for clinical application is their dependency on a properly configured 3D ECM microenvironment for survival and proliferation and because the method of Gai precludes the isolation of TEPCs/TECs from donor thymuses. One would have a reasonable expectation of success in combining the teachings as Gu teaches the encapsulated hiPSCs proliferation increased from the time of encapsulation and peaked at day 9 and maintained pluripotency markers and the encapsulated hiPSCs could be differentiated in situ and Gai teaches the TEPCs derived in vitro from hPSCs express the key thymic transcription factor FOXN1 along with Keratin 5 and Keratin 8, the primary cytoskeletal building blocks of medullary and cortical TECs and Fan teaches the 3D scaffold environment of the decellularized thymus can support the long-term survival of TECs in vitro and enable them to retain the thymic specific patterns of molecule expression that is essential for T-cell development and Fan teaches transplantation of bioengineered thymus organoids with donor and recipient TECs confer allo-skin tolerance and Fan teaches donor TECs can be generated from induced pluripotent stem cells.
11. Claim(s) 7 – 10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gu (Gu, Qi, et al. Advanced healthcare materials 6.17 (2017): 1700175.), hereinafter Gu in view of Gai (Gai, Hui, et. al. Blood 130 (2017): 2445.), hereinafter Gai in view of Fan (Fan Y, et. al. Mol Ther. 2015 Jul;23(7):1262-1277; previously cited), hereinafter Fan which is cited on the IDS filed 05/24/2022 and 03/19/2026.
in view of Hamilton (US11371000B2; Filed 03/30/2017; Published 06/28/2022; previously cited), hereinafter Hamilton in view of Mamonkin (Mamonkin, Maksim, et al. Blood, The Journal of the American Society of Hematology 126.8 (2015): 983-992; previously cited), hereinafter Mamonkin.
Regarding steps (a) – (b) of claim 7, Gu teaches encapsulating hiPSCs in a medium that separates the hiPSCs into single cells (step (a)) and expansion culturing of the hiPSCs in a growth medium without differentiation (step (b)) (Figure 1 – 3; page 2, left col. para. 1 – 2 and right col. para. 2 – 5; page 9, left col. para. 2 and right col. para. 2). Gu teaches the method overcomes reputed difficulties with maintaining and differentiating iPSCs (page 8, left col. para. 2).
Regarding step (c) of claim 7, Gu teaches the encapsulated hiPSCs could be differentiated in situ into all germ lineages indicating the potential to form multiple cell and tissue types within and from the bioprinted constructs, and could be differentiated into neural tissue (page 3 – 4; Figure 4; page 5, left col. para. 1; page 6, left col. para. 2 – 3; page 8, left col. para. 1), but does not teach differentiation into hTEPCs or hTECs. However, Gu teaches while without specific medium supplements pluripotent stem cells have a tendency to differentiate to derivatives of the three germ lineages, alternative media compositions can promote differentiation toward one or another lineage (page 6, left col. para. 2).
Regarding step (d) of claim 7, Gu teaches extracting encapsulated cells with EDTA and fluxing with a pipette (page 10, left col. last para. and right col. para. 2) but does not teach the cells comprise hTEPCs or hTECs or both.
Gu does not teach “differentiating the hiPSCs to generate” hTEPCs or hTECs of step (c) or “obtaining a cell population comprising hTEPCs or hTECs or both of step (d) or steps (e) – (h) of claim 7 or “wherein the bioengineered thymus organoid comprises immune cells” of claim 8 or “wherein the immune cells comprise B-cells and T-cells” of claim 9 or “wherein the immune cells are T-cells, and wherein the T-cells are transduced with a viral vector encoding a chimeric antigen receptor (CAR)” of claim 10. However, Gu teaches the encapsulated hiPSCs proliferation increased from the time of encapsulation and peaked at day 9 and maintained pluripotency markers (page 2, right col. para. 4 – 5). Gu teaches their method of encapsulating hiPSCs, expanding the encapsulated hiPSCs, and differentiating the encapsulated hiPSCs will be useful in iPSC translation for pharmaceuticals development and regenerative medicine (Abstract). Gu teaches 3D co-printing is a single-step approach to rapidly fabricate a 3D cellularized construct whereby iPSCs are immediately integrated with biomaterials by encapsulation for direct and complete contact with extracellular elements that more closely mimic the native cell microenvironment (page 2, left col. para. 1). Gu teaches the ability to 3D print hiPSCs to then expand and generate cells of different lineages provides an unprecedented opportunity to form different, authentic, and renewable body tissues (page 8, right col. para. 2). Gu teaches further refinement of the method is expected to enhance tissue identity, architecture, and function to better model development and diseases, for pharmaceuticals screening and assessing in vivo function and safety in animal models toward transplantation therapies (page 8, right col. para. 2).
Regarding “differentiating the hiPSCs to generate” hTEPCs or hTECs of step (c) and “obtaining a cell population comprising hTEPCs or hTECs or both of step (d) of claim 7, Gai teaches differentiating hPSCs to TEPCs by first induction of definitive endoderm followed by induction of anterior foregut, and pharyngeal pouch endoderm (page 2, para. 2). Gai teaches the TEPCs derived in vitro from hPSCs express the key thymic transcription factor FOXN1 along with Keratin 5 and Keratin 8, the primary cytoskeletal building blocks of medullary and cortical TECs (page 2, para. 2; Figure 2).
Gai does not teach steps (e) – (h) of claim 7 or “wherein the bioengineered thymus organoid comprises immune cells” of claim 8 or “wherein the immune cells comprise B-cells and T-cells” of claim 9 or “wherein the immune cells are T-cells, and wherein the T-cells are transduced with a viral vector encoding a chimeric antigen receptor (CAR)” of claim 10. However, Gai teaches the thymus gives rise to regulatory T-cells which confine immune responses and further promote central tolerance (page 1). Gai teaches the goal of differentiating hPSCs to TEPCs is for use as a stem cell-based therapy for thymic regeneration and restoration of immune competence in hematopoietic stem cell transplantation (HSCT) recipients (page 2, para. 2; Figure 1). Gai teaches thymic function naturally declines with age with little to no endogenous potential to regenerate (page 1). Gai teaches current options for thymic injury during HSCT are limited to symptomatic therapy and therefore novel approaches to cure irreversible thymic injury are critically needed (page 2, para. 1). Gai teaches the potential applications of in vitro derived TEPCs are not confined to restoring thymic function in the HSCT setting, because TEPCs derived from solid organ donors hold the promise of inducing tolerance to donor tissues, thereby preventing rejection of the transplanted donor organ (page 2, last para.). Gai teaches leveraging the body’s own ability to educate T-cells through regenerating thymic tissues could represent a novel approach to addressing key challenges in transplantation immunology (page 3, para. 1). One would have been motivated to combine the teachings of Gu and Gai to expand encapsulated hiPSCs and differentiate the encapsulated hiPSCs in situ to TEPCs to restore thymic function in subjects with thymic injury as Gu teaches the ability to 3D print hiPSCs to then expand and generate cells of different lineages provides an unprecedented opportunity to form different, authentic, and renewable body tissues and assess in vivo function and safety in animal models toward transplantation therapies and Gai teaches novel approaches to cure irreversible thymic injury are critically needed.
Regarding steps (e) – (g) of claim 7, Fan teaches a method of combining the TECs with Lin- progenitors at a 1:1 ratio (step (e)) and injecting the combination into decellularized thymus scaffolds to form reconstructed thymus organoids (step (f)) that were cultured (step (g)) (page 1263, left col. last para. and right col. para. 1; page 1274, right col. last para.; page 1275, left col. para. 1 – 3; page 1265).
Regarding claim 8 and “T-cells” of claim 10, Fan teaches culturing the thymus organoid in vitro produces T cells (page 1265, left col. and right col. para. 1). Fan does not teach “wherein the T-cells are transduced with a viral vector encoding a chimeric antigen receptor (CAR)” of claim 10.
Regarding claim 9, Fan teaches culturing the thymus organoid in vitro produces T cells (page 1265, left col. and right col. para. 1). Regarding “B-cells”, Fan teaches one of the essential roles of T-cells in adaptive immunity is their helper function for humoral immunity, namely to mediate Ig class switch in antibody producing B-cells (page 1266, right col. last para.). Fan teaches to examine whether T-cells from the bioengineered thymus can provide helper function to humoral responses, the authors immunized thymus recipients with chicken ovalbumin (OVA) and as expected, high titers of anti-OVA antibodies were detectable by ELISA in sera of B6 mice postimmunization (Figure 4f; page 1266, right col. last para.). Fan teaches comparable levels of anti-OVA IgG isotypes were found in serum samples harvested from mice transplanted with the thymus organoid, whereas seroactivities against OVA remained at the background levels in immunized mice transplanted with empty thymus scaffolds (page 1266, right col. last para.). Fan teaches these results suggest that T-cells generated from the thymus organoids can support B-lymphocyte Ig class switch to mount efficient humoral responses (page 1266, right col. last para.). Therefore, thymus organoid transplanted into mice comprise B-cells and T-cells.
Fan does not teach step (h) of claim 7 or “wherein the T-cells are transduced with a viral vector encoding a chimeric antigen receptor (CAR)” of claim 10. However, Fan teaches one of the major obstacles in organ transplantation is to establish immune tolerance of allografts (Abstract). Fan teaches conventional pharmacological drugs can cause severe side effects including increased risk of cardiovascular death, diabetes, and kidney disease (page 1270, left col. para. 2). Fan teaches induction of thymic central tolerance to allografts remains challenging largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering and when transplanted into mice supported thymopoiesis (Abstract). Fan teaches one of the major challenges to manipulate postnatal TECs for clinical application is their dependency on a properly configured 3D ECM microenvironment for survival and proliferation (page 1271, right col.). Fan teaches three-dimensional scaffolds generated from mouse thymus can support TEC survival in culture and maintain their unique molecular properties (Abstract). Fan teaches the TECs remained viable for > 3 weeks in the thymic scaffolds (page 1262, right col. para. 1). Fan teaches the mouse thymus scaffolds can be stored at 4 °C for up to 1 month before use and it is likely that human thymus scaffolds can be preserved in a similar (page 1274, right col. para. 1). Fan teaches the 3D scaffold environment of the decellularized thymus can support the long-term survival of TECs in vitro and enable them to retain the thymic specific patterns of molecule expression that is essential for T-cell development (page 1264, right col.; page 1265, right col. para. 1). Fan teaches transplantation of bioengineered thymus organoids with donor and recipient TECs confer allo-skin tolerance (page 1269, right col. para. 2). Fan teaches the thymus organoids can support the development of T cells in vitro and can support lymphopoiesis in vivo (page 1265, right col.; page 1266, left col.). Fan teaches the thymus-reconstructed mice did not display any pathological sign of autoimmunity (page 1266, left col. para. 3). Fan teaches donor TECs can be generated from induced pluripotent stem cells (page 1274, left col. para. 3). Fan teaches the thymus is a rather vulnerable organ and anticancer drug treatments can irreversibly compromise its function (page 1262, right col. para. 1). One would have been motivated to combine the teachings of Gu, Gai, and Fan in a method of making a bioengineered thymus organoid to restore thymic function in subjects with thymic injury where TEPCs are derived from iPSCs instead of harvested thymus tissue as Fan teaches the thymus is a rather vulnerable organ and anticancer drug treatments can irreversibly compromise its function and Fan teaches donor TECs can be generated from iPSCs.
Regarding step (h) of claim 7, Hamilton teaches Thymus-on-Chip that is a microfluidic platform that is in fluid communication with Cancer-on-Chips (col. 25, lines 35; col. 35, lines34 – 41; col. 38, lines 37 – 54; Figure 17). Hamilton teaches the microfluidic device can comprise an inlet channel connecting an inlet fluid port to a first chamber where the inlet channels and inlet ports can be used to introduce cells and culture media into the first chamber (col. 44, lines 32 – 37). Hamilton teaches the tumor cells in the microfluidic device are in close proximity with at least one type of immune cells including T cells (col. 2, lines 23 – 48). Hamilton teaches in Example 3 studying immune cell migration in relation to cancer cell/tumor growth (col. 52, lines 30 – 50). Hamilton teaches microfluidic platforms or chips for testing and understanding cancer where the interaction between cancer cells and immune cells can be tested by linking a cancer chip to another chip (col. 1, lines 59 – 66). Hamilton teaches the interaction with circulating immune cells recruited to the tumor site will be enabled to allow testing of immunomodulatory agents, confirm immune surveillance (or lack thereof), and provide a platform for testing of immunotherapeutics (col. 2, lines 1 – 6). Hamilton teaches microfluidic platforms can increase our understanding of tumor growth and all other aspects of cancer including the role of ECM on this process, resistance to immune surveillance, and development of metastatic disease (col. 2, lines 6 – 12). Hamilton teaches resident immune cells (B cells, T cells, dendritic cells, macrophages, and innate lymphoid cells) may be isolated from cancer patients and incorporated in the chip (col. 29, lines 45 – 63).
Hamilton does not teach “wherein the T-cells are transduced with a viral vector encoding a chimeric antigen receptor (CAR)” of claim 10. However, Hamilton teaches CAR T cell therapy has dramatically improved the outcomes of blood cancer patients with advanced forms of leukemia and lymphoma but the full potential of CARs for treating solid tumors has not been reached and many challenges remain (col. 42, lines 40 – 45). Hamilton teaches having more predictive, human relevant systems to study human tumor biology and the interactions of the human immune system with the tumor would advance our knowledge and help to provide the most robust and precise preclinical platforms for drug discovery and enable the advancement of immunotherapies (col. 42, lines 45 – 52). One would have been motivated to combine the teachings of Fan and Hamilton in a method of making a thymus organoid in a flow cell to study the interaction of immune cells produced by the organoid with cancer cells and anticancer drugs as Fan teaches anticancer drug treatments can irreversibly compromise thymus function and Hamilton teaches microfluidic platforms can increase our understanding of tumor growth and all other aspects of cancer including the role of ECM on this process, resistance to immune surveillance, and development of metastatic disease.
Regarding “wherein the T-cells are transduced with a viral vector encoding a chimeric antigen receptor (CAR)” of claim 10, Mamonkin teaches transduction of T cells with a CD5 CAR (page 983, right col. para. 3). Mamonkin teaches CD5 CAR T cells recognize and eliminate malignant T cells in vitro (page 984, right col. para. 3 – 5). Mamonkin teaches CD5 CAR T cells kill primary T-ALL cells from patients (page 985, right col. para. 4 – 5; page 986, left col. para. 1). Mamonkin teaches prognosis of patients with primary chemotherapy-refractory or relapsed lymphoid malignancies remains poor (page 983, left col. para. 1). Mamonkin teaches CAR T cell therapy that targeted a tumor antigen shared between normal and malignant T cells might lead to fratricide of the CAR T cells thus jeopardizing their therapeutic efficacy (page 983, left col. para. 1). Mamonkin teaches CD5 CAR can effectively redirect human T cells to target malignant CD5+ cells and this may provide novel treatment options for patients with refractory or relapsed CD5+ T-cell neoplasms (page 991, right col. 3).
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 Gu regarding a method of encapsulating hiPSCs, expanding the encapsulated hiPSCs, and differentiating the encapsulated hiPSCs with the teachings of Gai regarding a method of differentiating hPSCs to TEPCs with the teachings of Fan regarding a method of combining TECs and HSCs in a decellularized thymus scaffold with the teachings of Hamilton regarding Thymus-on-Chip with the teachings of Mamonkin regarding transducing T cells with a CAR to treat cancer to arrive at the claimed method producing human immune cells, the method comprising: (a) encapsulating hiPSCs in a suspension medium that separates the hiPSCs into single cells; (b) culturing the hiPSCs in a growth medium to increase the number thereof without differentiation; (c) differentiating the hiPSCs to generate hTEPCs or hTECs or both in an encapsulation medium; (d) freeing the hTEPCs or hTECs or both from the encapsulation medium, thereby obtaining a cell population comprising hTEPCs or hTECs or both, (e) combining the cell population with hHSCs in a defined ratio to form a combination; (f) seeding the combination into an extracellular matrix of a de-cellularized thymus scaffold to generate a thymus construct; (g) culturing the thymus construct under conditions permitting cellular attachment onto the extracellular matrix, thereby making a bioengineered thymus organoid; and (h) placing the bioengineered thymus organoid into a flow cell with a continuous feed of nutrients and human cells, thereby producing the human immune cells. One would have been motivated to combine the teachings of Gu, Gai, Fan, Hamilton, and Mamonkin in a method for making a thymus organoid to restore thymic function in subjects with thymic injury and test immunotherapeutics on immune cell function and thymus function as Gu teaches further refinement of the method is expected to enhance tissue identity, architecture, and function to better model development and diseases, for pharmaceuticals screening and assessing in vivo function and safety in animal models toward transplantation therapies and Gai teaches novel approaches to cure irreversible thymic injury are critically needed and Fan teaches the thymus is a vulnerable organ where anticancer drug treatments can irreversibly compromise its function and Hamilton teaches CAR T cell therapy has dramatically improved the outcomes of blood cancer patients with advanced forms of leukemia and lymphoma but the full potential of CARs for treating solid tumors has not been reached and many challenges remain, and Hamilton teaches having more predictive, human relevant systems to study human tumor biology and the interactions of the human immune system with the tumor would advance our knowledge and help to provide the most robust and precise preclinical platforms for drug discovery and enable the advancement of immunotherapies. One would have a reasonable expectation of success in combining the teachings as Fan teaches in vitro culture of TECs with HPCs produces immune cells and Hamilton teaches studying immune cell migration in relation to cancer cell/tumor growth with the chips and Mamonkin teaches the T cells express the CAR and can target and kill malignant cells.
Applicant’s Arguments/ Response to Arguments
12. Applicant Asserts: Applicant asserts that Examiner stated that incorporating the limitations of claim 5 into independent claim 1 and reciting said limitations as active steps would address the obviousness rejection.
Response to Argument: In response, the Examiner stated that incorporating the limitations of claim 5 into claim 1 and changing “wherein” to active steps, these limitations would not be interpreted as product-by-process limitations as stated on the Interview Summary mailed 06/01/2026.
Applicant Asserts: Applicant asserts that independent claims 1, 7, 13, and 17 as amended are non-obvious over the prior art.
Response to Argument: This is not found persuasive because as set forth above, claim 1, 2, 4 and 44 are obvious over the combined teachings of Gu, Gai, and Fan; claims 7 – 10 are obvious over the combined teachings of Gu, Gai, Fan, Hamilton, and Mamonkin.
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
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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