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 .
Claims 1-12, 14-15 and 20-38 are pending.
Applicant’s election without traverse of Group I that read on (A) SEQ ID NO: 2 as the CAR, (B) CD4 as the transmembrane domain, and CD3 as the intracellular domain, (C) CHIR99021 as the GSK3β inhibitor, (D) SB431542 as the TGFβ inhibitor, (E) GM-CSF, IL-3, IL-6 as the stimulating factors, (F) AM580 as the retinoid acid receptor agonist, and G-CSF, in the reply filed on April 6, 2026 is acknowledged.
Upon reconsideration, the CAR amino acid sequence has been extended to include SEQ ID NO: 1 and 3 of claims 12, and 27.
Claims 28-38 are withdrawn from further consideration by the examiner, 37 C.F.R. 1.142(b) as being drawn to non-elected inventions.
Claims 1-12, 14-15 and 20-27, drawn to a stage-specific process for manufacturing a population of neutrophils from human pluripotent stem cells (hPSCs) and a process for manufacturing a population of chimeric antigen receptor (CAR) neutrophils form human pluripotent stem cells (hPSCs) that read on (A) SEQ ID NO: 1- 3 as the CAR, (B) CD4 as the transmembrane domain, and CD3 as the intracellular domain, (C) CHIR99021 as the GSK3β inhibitor, (D) SB431542 as the TGFβ inhibitor, (E) GM-CSF, IL-3, IL-6 as the stimulating factors, (F) AM580 as the retinoid acid receptor agonist, and G-CSF, are being acted upon in this Office Action.
Priority
Applicant’ claim priority to provisional application 63/124,125, filed December 11, 2020, is acknowledged.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on April 8, 2026, January 28, 2025 and June 9, 2023 have been considered by the examiner and an initialed copy of the IDS is included with this Office Action.
The listing of references in the specification at pages 31-31, 35 and 63-69 is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
The drawings were received on March 26, 2024. These drawings are acceptable.
Specification
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant's cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Claim Objection
Claim 5 is objected to because of the following informality: “said a retinoic acid” should have been “said retinoic acid”. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 3-5, 14-15, 21, 23 and 24 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
The recitation of “CD11b+/CD16+” in claims 1 and 21 is indefinite because it is not clear what is meant by “/”. One of ordinary skill in the art would not reasonably be apprised of the metes and bounds of the invention. For art rejection purpose, the claims have been interpreted to mean “CD11b+ or CD16+”.
The “similar chemicals” in claims 3, 4 and 5 is ambiguous and indefinite because one of ordinary skill in the art would not reasonably be apprised of the metes and bounds of the invention.
The recitation of “other human embryonic stem cells” in claims 14 and 23 as the sole means of identifying the hESCs renders the claim indefinite and ambiguous. Accordingly, one of ordinary skill in the art would not reasonably be apprised of the metes and bounds of the invention.
The recitation of “other induced pluripotent stem cells” in claims 15 and 24 as the sole means of identifying the iPSCs renders the claim indefinite because one of ordinary skill in the art would not reasonably be apprised of the metes and bounds of the invention.
Claim rejections under - 35 U.S.C. 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-10, 14-15 and 20-25 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Written Description Guidelines for examination of patent applications indicates, “the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical characteristics and/or other chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show applicant was in possession of the claimed genus.” (see MPEP 2163).
Claim 1 encompasses a stage-specific process for manufacturing a population of neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:
(a) preparing hPSCs;
(b) stimulating said hPSCs with a glycogen synthase kinase 3β (GSK3p) inhibitor to produce a population of CD34+ hemogenic endothelium cells;
(c) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor p (TGFP) inhibitor to produce a population of CD45+ hematopoietic cells; and
(d) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 6 (IL-6); and (e) inducing myeloid progenitor and neutrophil differentiation of the population of stimulated CD45+ hematopoietic cells with granulocyte-colony stimulating factor (G-CSF) and a retinoic acid receptor agonist to afford a population of CD11b+/CD16+ neutrophils.
Claim 2 encompasses the stage-specific process of claim 1, wherein said hPSCs comprise human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs).
Claim 3 encompasses the stage-specific process of claim 1, wherein said GSK3p inhibitor is CHIR99021, CHIR98014, or similar chemicals.
Claim 4 encompasses the stage-specific process of claim 1, wherein said TGFP inhibitor is SB431542, A83-01 or similar chemicals.
Claim 5 encompasses the stage-specific process of claim 1, wherein said a retinoic acid receptor agonist is AM80, AM50, or similar chemicals.
Claim 6 encompasses the stage-specific process of claim 1, wherein step b. is carried out in the presence of vascular endothelial growth factor (VEGF).
Claim 7 encompasses the stage-specific process of claim 1, wherein step c. is carried out in the presence of stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L).
Claim 8 encompasses the stage-specific process for manufacturing a population of neutrophils of claim 1, wherein preparing hPSCs comprises:
(i) knocking a CAR expression gene construct into the AAVS 1 safe harbor locus of an adeno-associated virus Si (AAVS1) plasmid in human pluripotent stem cells (hPSCs) via CRISPR/Cas9-mediated homologous recombination;
(ii) isolating successfully targeted single cell-derived hPSC colonies or hPSC cell mixture to afford a stable CAR-expressing hPSC cell line; and
(iii) preparing hPSCs from said stable CAR-expressing hPSC cell line; and wherein the population of CD11b+/CD16+ neutrophils comprises a population of CAR neutrophils.
Claim 9 encompasses the stage-specific process of claim 8, which further comprises the initial steps of: (a') preparing a CAR expression gene construct; and (a") constructing a AAVS1 plasmid.
Claim 10 encompasses the stage-specific process of claim 8, wherein the CAR comprises chlorotoxin or IL13, or other extracellular signaling domains, the transmembrane domain of CD4, CD28, CD32a or NKG2D, co-stimulatory domain 2B4, and the intracellular domain of CD3ζ or FcγR domains.
Claim 11 encompasses the stage-specific process of claim 10, wherein the CAR has the amino acid sequence of SEQ ID NO: 2.
Claim 12 encompasses the stage-specific process of claim 8, wherein the CAR has the amino acid sequence of SEQ ID NO: 1 or 3.
Claim 14 encompasses the stage-specific process of claim 2, where said hESCs comprises H9, H1 or other human embryonic stem cells.
Claim 15 encompasses the stage-specific process of claim 2, where said iPSCs comprises 6-9-9, 19-9-11 or other induced pluripotent stem cells.
Claim 20 encompasses the stage-specific process of claim 8, wherein step c. is carried out in the presence of stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L).
Claim 21 encompasses a process for manufacturing a population of chimeric antigen receptor (CAR) neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:
(a) constructing a PiggyBac transposon plasmid comprising a CAR expression gene;
(b) delivering the PiggyBac plasmid into a human pluripotent stem cell via nucleofection/electroporation;
(c) isolating successfully targeted single cell-derived human pluripotent stem cell (hPSC) colonies or hPSC cell mixture for stable CAR-expressing hPSC lines; and
(d) producing CAR-expressing neutrophils according to the process of:(i) preparing hPSCs;
(ii) stimulating said hPSCs with a glycogen synthase kinase 3 p (GSK3p) inhibitor to produce a population of CD34+ hemogenic endothelium cells;
(iii) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor p (TGFP) inhibitor to produce a population of CD45+ hematopoietic cells; and
(iv) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 6 (IL-6); and
(v) inducing myeloid progenitor and neutrophil differentiation of the population of stimulated CD45+ hematopoietic cells with granulocyte-colony stimulating factor (G-CSF) and a retinoic acid receptor agonist to afford a population of CD1lb+/CD16+ neutrophils.
Claim 22 encompasses the process of claim 21, wherein said hPSCs comprise human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs).
Claim 23 encompasses the process of claim 22, where said hESCs comprises H9, H1 or other human embryonic stem cells.
Claim 24 encompasses the process of claim 22, where said iPSCs comprises 6-9-9, 19-9-11 or other induced pluripotent stem cells.
Claim 25 encompasses the process of claim 21, wherein the CAR comprises chlorotoxin or IL13, or other extracellular signaling domains, the transmembrane domain of CD4, CD28, CD32a or NKG2D, co-stimulatory domain 2B4, and the intracellular domain of CD3ζ or FcγR domains.
The specification discloses a method of culturing and inducing differentiation of human pluripotent stem cells (hPSCs) to human neutrophils expressing CD11+ and CD16+ by growing the hPSCs in the presence of CHIR99021 on day 0, a GSK3β inhibitor to promote CD34+ expressing hemogenic endothelium (HE) cells, follows by SB431542 (SB) on day 4, which is a transforming growth factor beta (TGFβ) inhibitor to promote CD45+ expressing hematopoietic cells, then stimulating the CD45+ hematopoietic cells with various growth factors shown in Figure 1A below.
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However, the specification does not describe stimulating hPSCs with VEGF at step b as per claim 6 or Flt3L and SCF in step c as per claim 7.
The specification further discloses chlorotoxin (CLTX) specific chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 1 or 2 and a IL-3 specific chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 3. The CAR of SEQ ID NO: 1 comprises CLTX extracellular domain, an IgG4 Fc spacer, a CD4 transmembrane domain and a CD3zeta domain. The CAR of SEQ ID NO: 2 comprises CLTX extracellular domain, an IgG4 Fc spacer, a NKG2D transmembrane, a 2B4 and a CD3zeta domains. The CAR of SEQ ID NO: 3 comprises TQM-IL13, n IgG4 Fc spacer, a CD4 transmembrane domain and a CD3zeta domain.
The CAR constructs were knocked into the AAVS1 locus in hPSCs via Cas9-mediategd homologous recombination to produce CAR expressing hPSC derived neutrophils, see FIG. 2A to Fig. 2C.
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The CLTX-CAR in hPSC-derived neutrophils display enhanced activity against GBM in vivo using xenograft model.
However, the specification does not describe the structure, e.g., amino acid sequence of genus of chimeric antigen receptor expressed by hPSC cell line encompassed by the claimed processes. In the particular, other than the particular chlorotoxin and IL-13 specific CARs comprising SEQ ID NO: 1-3 above, the specification does not describe the structure of any and all possible CAR (claims 8, 21) comprising any and all extracellular signaling domains (claim 10) expressed by hPSCs derived neutrophils for treating any and all possible cancers to demonstrate possession of the genus at the time of filing. The specification does not teach the binding specificity of the CAR expressed by hPSCs derived neutrophils can treat any and all possible cancers.
Alrehaili et al (NPJ Precision Oncology 9: 382, 2025; PTO 892) that the success of chimeric antigen receptor (CAR) T-cell therapy is that the engineered T-cell recognize and eliminate tumor cells in an antigen-specific manner , see p. 9, right col. However, despite their efficacy in blood cancers, CAR-T therapies fail to demonstrate comparable success against solid tumors due to poor trafficking and infiltration into the tumor microenvironment (TME), see p. 1, left col, in particular. Other cells such as macrophages, monocytes cells have emerged as a promising alternative due to the success of CAR-based antigen recognition and the limitations of T-cells in solid tumors. As key components of the myeloid lineage, macrophages naturally infiltrate and persist within the TME, making them well-suited for solid tumor targeting.
Hou (Front Immunol 16: 1675807, 2005; PTO 892) teaches that CAR-T cell therapies have demonstrated significant clinical success in hematological malignancies, yet their application to solid tumors faces persistent challenges. Key limitations include the paucity of tumor-specific antigens, poor intratumoral infiltration, immunosuppressive tumor microenvironment (TME), and treatment-related toxicities such as cytokine release syndrome (CRS) and neurotoxicity. In contrast, CAR natural killer (CAR-NK) cells show promise in solid tumors such as ovarian, pancreatic, and glioblastoma, with encouraging preclinical and early clinical evidence, although limited persistence and antigen heterogeneity remain major challenges. Unlike CAR-T cells, CAR-NK therapies mediate tumor clearance through both cytotoxic (e.g., granzyme/perforin release) and cytokine-mediated mechanisms while mitigating toxicity risks. Their lack of human leukocyte antigen (HLA) dependency enables "off-the-shelf" manufacturing from allogeneic donors, circumventing patient-specific production bottlenecks. CAR-macrophage (CAR-M) therapies further address solid tumor barriers by leveraging innate phagocytic clearance, antigen-presenting functions, and TME penetration. Macrophages inherently infiltrate hypoxic tumor regions and remodel stromal barriers, enabling CAR-Ms to synergize with adaptive immunity by cross-priming T cells. Preclinical models highlight CAR-M efficacy in depleting immunosuppressive tumor-associated macrophages (TAMs) and reversing TME-driven immune evasion.
Give the lack of guidance as to the antigen-specificity encompassed by the claimed CAR, and insufficient in vivo working examples, it is unpredictable the process of producing any CAR expressing hPCS derived neutrophil has the ability to bind to all tumor antigens and, in turn, effective for treating any and all possible cancer or solid tumors. Further, cytokines storm e.g., IL-1, IL-6, TNF-α are potent drivers of chronic inflammation and can cause normal tissue injury. There are insufficient in vivo working examples.
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the written description inquiry, whatever is now claimed.” (See page 1117.) The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116.).
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016.
One cannot describe what one has not conceived. See Fiddles v. Baird, 30 USPQ2d 1481, 1483. In Fiddles v. Baird, claims directed to mammalian FGF’s were found unpatentable due to lack of written description for the broad class. The specification provided only the bovine sequence. Thus, the specification fails to describe these DNA sequences.
For genus claims, an adequate written description of a claimed genus requires more than a generic statement of an invention's boundaries. A patent must set forth either a representative number of species falling within the scope of the genus or structural features common to the members of the genus. Kubin, Exparte, 83 USPQ2d 1410 (Bd. Pat. App. & Int. 2007); Ariad Pharms., Inc. v. Eli Lilly& Co., 598 F.3d 1336, 1350 (Fed. Cir. 2010).
Therefore, only (1) a process for manufacturing a population of chlorotoxin or IL13 specific chimeric antigen receptor (CAR) neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:
(a) constructing a PiggyBac transposon plasmid comprising a CAR expression construct encoding the CAR comprising the amino acid sequence of SEQ ID NO: 1, 2 or 3;
(b) delivering the PiggyBac plasmid into a human pluripotent stem cell (hPSC) via nucleofection and electroporation;
(c) isolating the CAR-expressing hPSC cell; and
(d) producing CAR-expressing neutrophils according to the process of:
(i) preparing CAR-expressing hPSCs;
(ii) stimulating said hPSCs with a glycogen synthase kinase 3 β (GSK3β) inhibitor on day 0 selected from the group consisting of CHIR99021 and CHIR98014 to produce a population of CD34+ hemogenic endothelium (HE) cells;
(iii) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor β (TGFβ) inhibitor on day 4 wherein the TGFβ inhibitor is selected from the group consisting of SB431542 and A83-01 to produce a population of CD45+ hematopoietic cells; and
(iv) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 6 (IL-6); and
(v) inducing myeloid progenitor and neutrophil differentiation of the population of stimulated CD45+ hematopoietic cells with granulocyte-colony stimulating factor (G-CSF) and a retinoic acid receptor agonist selected from the group consisting of AM580 and AM80 on days 12 15 and 18 to afford a population of human PSC-derived CD1lb+/CD16+ neutrophils expressing said chlorotoxin or IL13 specific chimeric antigen receptor (CAR), (2) the process above wherein the CAR comprises chlorotoxin extracellular domain, an IgG4 Fc domain, a CD4 transmembrane domain, a 2B4 signaling domain and a CD3ζ domain or the CAR comprises an IL-13 extracellular domain, an IgG4 Fc domain, a CD4 transmembrane domain, a 2B4 and a CD3ζ signaling domains, (3) the process above wherein the human pluripotent stem cell (hPSC) is a human embryonic stem cell line (hESC) or induced pluripotent stem cell line (iPSC), (4) the process above wherein the hESC comprises H9 or H1 cell line, (5) the process above wherein the iPSC comprises 6-9-9 or 19-9-11 cell line, but not the full breadth of the claims meets the written description provision of 35 U.S.C. § 112, first paragraph.
Claims 1-10, 14-15 and 20-25 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for (1) a process for manufacturing a population of chlorotoxin or IL13 specific chimeric antigen receptor (CAR) neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:
(a) constructing a PiggyBac transposon plasmid comprising a CAR expression construct encoding the CAR comprising the amino acid sequence of SEQ ID NO: 1, 2 or 3;
(b) delivering the PiggyBac plasmid into a human pluripotent stem cell (hPSC) via nucleofection and electroporation;
(c) isolating the CAR-expressing hPSC cell; and
(d) producing CAR-expressing neutrophils according to the process of:
(i) preparing CAR-expressing hPSCs;
(ii) stimulating said hPSCs with a glycogen synthase kinase 3 β (GSK3β) inhibitor on day 0 selected from the group consisting of CHIR99021 and CHIR98014 to produce a population of CD34+ hemogenic endothelium (HE) cells;
(iii) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor β (TGFβ) inhibitor on day 4 wherein the TGFβ inhibitor is selected from the group consisting of SB431542 and A83-01 to produce a population of CD45+ hematopoietic cells; and
(iv) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 6 (IL-6); and
(v) inducing myeloid progenitor and neutrophil differentiation of the population of stimulated CD45+ hematopoietic cells with granulocyte-colony stimulating factor (G-CSF) and a retinoic acid receptor agonist selected from the group consisting of AM580 and AM80 on days 12 15 and 18 to afford a population of human PSC-derived CD1lb+/CD16+ neutrophils expressing said chlorotoxin or IL13 specific chimeric antigen receptor (CAR), (2) the process above wherein the CAR comprises chlorotoxin extracellular domain, an IgG4 Fc domain, a CD4 transmembrane domain, a 2B4 signaling domain and a CD3ζ domain or the CAR comprises an IL-13 extracellular domain, an IgG4 Fc domain, a CD4 transmembrane domain, a 2B4 and a CD3ζ signaling domains, (3) the process above wherein the human pluripotent stem cell (hPSC) is a human embryonic stem cell line (hESC) or induced pluripotent stem cell line (iPSC), (4) the process above wherein the hESC comprises H9 or H1 cell line, (5) the process above wherein the iPSC comprises 6-9-9 or 19-9-11 cell line, does not reasonably provide enablement for the processes as set forth in claims 1-10, 14-15 and 20-25. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). These factors include, but are not limited to: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. . In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988).
Claim 1 encompasses a stage-specific process for manufacturing a population of neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:
(a) preparing hPSCs;
(b) stimulating said hPSCs with a glycogen synthase kinase 3β (GSK3p) inhibitor to produce a population of CD34+ hemogenic endothelium cells;
(c) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor p (TGFP) inhibitor to produce a population of CD45+ hematopoietic cells; and
(d) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 6 (IL-6); and (e) inducing myeloid progenitor and neutrophil differentiation of the population of stimulated CD45+ hematopoietic cells with granulocyte-colony stimulating factor (G-CSF) and a retinoic acid receptor agonist to afford a population of CD11b+/CD16+ neutrophils.
Claim 2 encompasses the stage-specific process of claim 1, wherein said hPSCs comprise human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs).
Claim 3 encompasses the stage-specific process of claim 1, wherein said GSK3p inhibitor is CHIR99021, CHIR98014, or similar chemicals.
Claim 4 encompasses the stage-specific process of claim 1, wherein said TGFP inhibitor is SB431542, A83-01 or similar chemicals.
Claim 5 encompasses the stage-specific process of claim 1, wherein said a retinoic acid receptor agonist is AM80, AM50, or similar chemicals.
Claim 6 encompasses the stage-specific process of claim 1, wherein step b. is carried out in the presence of vascular endothelial growth factor (VEGF).
Claim 7 encompasses the stage-specific process of claim 1, wherein step c. is carried out in the presence of stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L).
Claim 8 encompasses the stage-specific process for manufacturing a population of neutrophils of claim 1, wherein preparing hPSCs comprises:
(i) knocking a CAR expression gene construct into the AAVS 1 safe harbor locus of an adeno-associated virus Si (AAVS1) plasmid in human pluripotent stem cells (hPSCs) via CRISPR/Cas9-mediated homologous recombination;
(ii) isolating successfully targeted single cell-derived hPSC colonies or hPSC cell mixture to afford a stable CAR-expressing hPSC cell line; and
(iii) preparing hPSCs from said stable CAR-expressing hPSC cell line; and wherein the population of CD11b+/CD16+ neutrophils comprises a population of CAR neutrophils.
Claim 9 encompasses the stage-specific process of claim 8, which further comprises the initial steps of: (a') preparing a CAR expression gene construct; and (a") constructing a AAVS1 plasmid.
Claim 10 encompasses the stage-specific process of claim 8, wherein the CAR comprises chlorotoxin or IL13, or other extracellular signaling domains, the transmembrane domain of CD4, CD28, CD32a or NKG2D, co-stimulatory domain 2B4, and the intracellular domain of CD3ζ or FcγR domains.
Claim 11 encompasses the stage-specific process of claim 10, wherein the CAR has the amino acid sequence of SEQ ID NO: 2.
Claim 12 encompasses the stage-specific process of claim 8, wherein the CAR has the amino acid sequence of SEQ ID NO: 1 or 3.
Claim 14 encompasses the stage-specific process of claim 2, where said hESCs comprises H9, H1 or other human embryonic stem cells.
Claim 15 encompasses the stage-specific process of claim 2, where said iPSCs comprises 6-9-9, 19-9-11 or other induced pluripotent stem cells.
Claim 20 encompasses the stage-specific process of claim 8, wherein step c. is carried out in the presence of stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L).
Claim 21 encompasses a process for manufacturing a population of chimeric antigen receptor (CAR) neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:
(a) constructing a PiggyBac transposon plasmid comprising a CAR expression gene;
(b) delivering the PiggyBac plasmid into a human pluripotent stem cell via nucleofection/electroporation;
(c) isolating successfully targeted single cell-derived human pluripotent stem cell (hPSC) colonies or hPSC cell mixture for stable CAR-expressing hPSC lines; and
(d) producing CAR-expressing neutrophils according to the process of:(i) preparing hPSCs;
(ii) stimulating said hPSCs with a glycogen synthase kinase 3 p (GSK3p) inhibitor to produce a population of CD34+ hemogenic endothelium cells;
(iii) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor p (TGFP) inhibitor to produce a population of CD45+ hematopoietic cells; and
(iv) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 6 (IL-6); and
(v) inducing myeloid progenitor and neutrophil differentiation of the population of stimulated CD45+ hematopoietic cells with granulocyte-colony stimulating factor (G-CSF) and a retinoic acid receptor agonist to afford a population of CD1lb+/CD16+ neutrophils.
Claim 22 encompasses the process of claim 21, wherein said hPSCs comprise human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs).
Claim 23 encompasses the process of claim 22, where said hESCs comprises H9, H1 or other human embryonic stem cells.
Claim 24 encompasses the process of claim 22, where said iPSCs comprises 6-9-9, 19-9-11 or other induced pluripotent stem cells.
Claim 25 encompasses the process of claim 21, wherein the CAR comprises chlorotoxin or IL13, or other extracellular signaling domains, the transmembrane domain of CD4, CD28, CD32a or NKG2D, co-stimulatory domain 2B4, and the intracellular domain of CD3ζ or FcγR domains.
The specification discloses a method of culturing and inducing differentiation of human pluripotent stem cells (hPSCs) to human neutrophils expressing CD11+ and CD16+ by growing the hPSCs in the presence of CHIR99021 on day 0, a GSK3β inhibitor to promote CD34+ expressing hemogenic endothelium (HE) cells, follows by SB431542 (SB) on day 4, which is a transforming growth factor beta (TGFβ) inhibitor to promote CD45+ expressing hematopoietic cells, then stimulating the CD45+ hematopoietic cells with various growth factors shown in Figure 1A below.
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However, the specification does not describe stimulating hPSCs with VEGF at step b as per claim 6 or Flt3L and SCF in step c as per claim 7.
The specification further discloses chlorotoxin (CLTX) specific chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 1 or 2 and a IL-3 specific chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 3. The CAR of SEQ ID NO: 1 comprises CLTX extracellular domain, an IgG4 Fc spacer, a CD4 transmembrane domain and a CD3zeta domain. The CAR of SEQ ID NO: 2 comprises CLTX extracellular domain, an IgG4 Fc spacer, a NKG2D transmembrane, a 2B4 and a CD3zeta domains. The CAR of SEQ ID NO: 3 comprises TQM-IL13, n IgG4 Fc spacer, a CD4 transmembrane domain and a CD3zeta domain.
The CAR constructs were knocked into the AAVS1 locus in hPSCs via Cas9-mediategd homologous recombination to produce CAR expressing hPSC derived neutrophils, see FIG. 2A to Fig. 2C.
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The CLTX-CAR in hPSC-derived neutrophils display enhanced activity against GBM in vivo using xenograft model.
However, the specification does not teach the structure, e.g., amino acid sequence of genus of chimeric antigen receptor expressed by hPSC cell line encompassed by the claimed processes. In the particular, other than the particular chlorotoxin and IL-13 specific CARs above, the specification does not describe the structure of any and all possible CAR (claims 8, 21) comprising any and all extracellular signaling domains (claim 10) expressed by any hPSCs to enable one of skill in the art to make and use the neutrophils for treating any and all cancers.
Hou (Front Immunol 16: 1675807, 2025; PTO 892) teaches that CAR-T cell therapies have demonstrated significant clinical success in hematological malignancies, yet their application to solid tumors faces persistent challenges. Key limitations include the paucity of tumor-specific antigens, poor intratumoral infiltration, immunosuppressive tumor microenvironment (TME), and treatment-related toxicities such as cytokine release syndrome (CRS) and neurotoxicity. In contrast, CAR natural killer (CAR-NK) cells show promise in solid tumors such as ovarian, pancreatic, and glioblastoma, with encouraging preclinical and early clinical evidence, although limited persistence and antigen heterogeneity remain major challenges. Unlike CAR-T cells, CAR-NK therapies mediate tumor clearance through both cytotoxic (e.g., granzyme/perforin release) and cytokine-mediated mechanisms while mitigating toxicity risks. Their lack of human leukocyte antigen (HLA) dependency enables "off-the-shelf" manufacturing from allogeneic donors, circumventing patient-specific production bottlenecks. CAR-macrophage (CAR-M) therapies further address solid tumor barriers by leveraging innate phagocytic clearance, antigen-presenting functions, and TME penetration. Macrophages inherently infiltrate hypoxic tumor regions and remodel stromal barriers, enabling CAR-Ms to synergize with adaptive immunity by cross-priming T cells. Preclinical models highlight CAR-M efficacy in depleting immunosuppressive tumor-associated macrophages (TAMs) and reversing TME-driven immune evasion.
Give the lack of antigen-specificity encompassed by the claimed CAR, and insufficient in vivo working examples, it is unpredictable the process of producing any CAR expressing hPCS derived neutrophil has the ability to home into cancer, in turn, effective for treating any and all possible cancer or solid tumors. Further, cytokines storm e.g., IL-1, IL-6, TNF-α are potent drivers of chronic inflammation and can cause normal tissue injury. There are insufficient in vivo working examples. It is unpredictable which undisclosed CAR expressed by hPSCs derived neutrophils is effective for treating any and all possible cancer in a subject.
In view of the quantity of experimentation necessary, the limited working examples, the unpredictability of the art, the lack of sufficient guidance in the specification, and the breadth of the claims, it would take undue trials and errors to practice the claimed invention.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103(a) 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 under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 1-7, 14 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Ng (WO2017/193177, published November 16, 2017; PTO 892) in view of Li et al (EMBO Molecular Medicine 8(11): 1340-1359, 2016; PTO 892), Valamehr (US20180320137, published November 8, 2018; PTO 892), and Saeki et al (Stem Cells 27: 59-67, 2009; PTO 892).
Claim 1 encompasses a stage-specific process for manufacturing a population of neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:
(a) preparing hPSCs;
(b) stimulating said hPSCs with a glycogen synthase kinase 3β (GSK3p) inhibitor to produce a population of CD34+ hemogenic endothelium cells;
(c) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor p (TGFP) inhibitor to produce a population of CD45+ hematopoietic cells; and
(d) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 6 (IL-6); and (e) inducing myeloid progenitor and neutrophil differentiation of the population of stimulated CD45+ hematopoietic cells with granulocyte-colony stimulating factor (G-CSF) and a retinoic acid receptor agonist to afford a population of CD11b+/CD16+ neutrophils.
Claim 2 encompasses the stage-specific process of claim 1, wherein said hPSCs comprise human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs).
Claim 3 encompasses the stage-specific process of claim 1, wherein said GSK3β inhibitor is CHIR99021, CHIR98014, or similar chemicals.
Claim 4 encompasses the stage-specific process of claim 1, wherein said TGFβ inhibitor is SB431542, A83-01 or similar chemicals.
Claim 5 encompasses the stage-specific process of claim 1, wherein said a retinoic acid receptor agonist is AM80, AM50, or similar chemicals.
Claim 6 encompasses the stage-specific process of claim 1, wherein step b. is carried out in the presence of vascular endothelial growth factor (VEGF).
Claim 7 encompasses the stage-specific process of claim 1, wherein step c. is carried out in the presence of stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L).
Regarding claims 1, 3, 4, 7, Ng teaches a method of isolating and in vitro differentiation of human pluripotent stem cells (hPSCs) to definitive hematopoiesis through exposure to (CHIR99021 (CHIR, p. 48, line 8), SB 431542, p. 48, line 9, see p. 20, lines 35-37, p. 23, line 10 to page 24, in particular. The media comprises GM-CSF (p. 28), IL-6 (see p. 28, line 36 to p. 29, line 4), IL-3 (p. 29, line 5-10), in particular. The cells appeared between differentiation day 8 and 11, initially expressing CD34, although by day 15, the cells expressed CD45, see p. 46, line 6-8, reference claims 9, 14-19, 25-26.
Regarding claim 2, Ng teaches that pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), see p. 24, line 8-12, reference claims 30-31. Suitable ESCs include H1 and H9 human ESCs, see p. 24, line
Regarding claim 6, Ng teaches that the method wherein the media comprises VEGF, see p. 28, lines 1-6, p. 36, line 14.
Regarding claim 7, Ng teaches that the method wherein the media comprises SCF (stem cell factor, see p. 28, lines 7-13, p. 36, line 15) and FLT3L (see p. 29, line 17-23, p. 36, line 27).
Regarding claim 14, Ng teaches that suitable human embryonic stem cell line includes H1 and H9 (see p. 24, line 19-20), and HES3, p. 36, line 4, in particular.
Regarding claim 15, Ng teaches that suitable human iPSCs include 19-9-7T, MIRJT6i-mND1-4, MIRJT7i-mND2-0, BM19-9, see p. 23, lines 27-32.
Regarding claim 1(e), Ng teaches the medium further comprises retinol retinoic acid analog, e.g., AM580 (aka instant retinoic acid receptor agonist) or EC23, see p. 23, lines 28-30, in particular.
Ng does not teach the method wherein the hPSCs are stimulated with a combination of retinoic acid receptor agonist and G-CSF as per claim 1.
However, Li teaches that the combination of AM80 and G-CSF synergies myeloid differentiation to generate functional neutrophils as per claim 1(e), see entire document, see p. 1343, right col. in particular. The AM80 and G-CSF promoted the highest neutrophil ROS production, bacterial killing, and morphogenic differentiation, see p. 1343, right col., Fig 3Dii-iv, in particular.
Valamehr teaches that the Wnt pathway activator CHIR99021 is responsible for the induction of the definitive hematopoietic program from hiPSCs. The effect of the modulation of CHIR during the induction phase of the hematopoietic differentiation protocol was assessed by treating the cultures with increasing concentrations of CHIR from about D3.75 to about D6. FIG. 5C demonstrates that while increasing the concentration of CHIR99021 increases the total percentage of CD34+ cells, para. [0088], in particular. Valamehr further teaches that TGFβ receptor inhibitor such as SB431542, see para. [0143].
Saeki et al (Stem Cells 27: 59-67, 2009; PTO 892) teaches feeder-free and efficient production of neutrophils from human Embryonic stem cells (hESCs) by culturing hESCs in the presence of VEGF, SCF, FLT3-L and TPO, see p. 60, right col. Saeki teaches that neutrophils differentiation at various stages is evaluate by the expression of neutrophil-specific makers of hematopoietic cells, e.g., CD34, CD45, CD11b and CD16b by flow cytometric analysis, see p. 63, Figure 4, in particular.
In view of the combined teachings of the references, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the teachings of Ng, Li and Valamehr by including human pluripotent cells, e.g., hiPSCs or hESCs into hemogenic endothelium (HE) expressing CD34+ with GSK3β inhibitor CHIR99021 as taught by Valamehr or Ng, follows by stimulating CD34+ HE cells with TGFβ receptor inhibitor such as SB431542 to give rise to CD45+ hematopoietic cells of Ng, then stimulating CD45+ hematopoietic cells of Ng with Li’s G-CSF and retinoic acid receptor agonist, e.g., AM580 and G-CSF and flow cytometry analysis of cell surface expression maker for differentiated mature neutrophils, e.g., CD34+, CD45+, CD11b+ followed by CD16e+ as taught by Saeki to arrive at the claimed invention with a reasonable expectation of success, e.g., induces differentiation of hematopoietic cells to functional neutrophils.
One of ordinary skill in the art would have been motivated to do so because Li teaches that the combination of AM80 and G-CSF synergies myeloid differentiation to generate functional neutrophils (see entire document, see p. 1343, right col. in particular), Valamehr teaches that pluripotent stem cell differentiation to hematopoietic lineage cells can be differentiate into CD34+ hemogenic endothelium (HE) with GSK3β inhibitor CHIR99021 and Ng teaches that human iPSC offer the advantage over embryonic stem cells as they eliminate a number of ethical issues associated with the use of embryonic issue, see p. 24, line 13-18.
One of ordinary skill in the art would be motivated to do so because hematopoietic stem progenitor cells (hPSC cell line) such as hECS and iPSCs have the capacity to generate large quantity of neutrophils in vitro, and hPSC derived neutrophils have the potential for cell-based therapies.
“The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007).
“The test of obviousness is not express suggestion of the cl aimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Claims 8-10 and 21-25 are rejected under 35 U.S.C. 103 as being unpatentable over Ng (WO2017/193177, published November 16, 2017; PTO 892) in view of Li (1) (EMBO Molecular Medicine 8(11): 1340-1359, 2016; PTO 892), Valamehr I (US20180320137, published November 8, 2018; PTO 892), and Saeki et al (Stem Cells 27: 59-67, 2009; PTO 892) as applied to claims1-7, 14 and 15 mentioned above and further in view of Valamehr II (US20180155717, published June 7, 2018; PTO 892), Li et al (Cell Stem cell 23: 181-192, 2018; PTO 892), Wang et al (Science Translational Medicine 12: 1-14, March 2020; PTO 1449) and/or Kim et al (Bioactive Materials 5: 624-635, available online May 7, 2020; PTO
The combine teachings of Ng, Li (1), Valaehr I, and Saeki have been discussed supra.
Regarding claim 22, Ng teaches that pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), see p. 24, line 8-12, reference claims 30-31. Suitable ESCs include H1 and H9 human ESCs, see p. 24, line
Regarding claim 23, Ng teaches that suitable human embryonic stem cell line includes H1 and H9 (see p. 24, line 19-20), and HES3, p. 36, line 4, in particular.
Regarding claim 24, Ng teaches that suitable human iPSCs include 19-9-7T, MIRJT6i-mND1-4, MIRJT7i-mND2-0, BM19-9, see p. 23, lines 27-32.
The references above do not teach the stage-specific process for manufacturing a population of neutrophils of claim 1, wherein preparing hPSCs comprises:
(i) knocking a CAR expression gene construct into the AAVS 1 safe harbor locus of an adeno-associated virus Si (AAVS1) plasmid in human pluripotent stem cells (hPSCs) via CRISPR/Cas9-mediated homologous recombination;
(ii) isolating successfully targeted single cell-derived hPSC colonies or hPSC cell mixture to afford a stable CAR-expressing hPSC cell line; and
(iii) preparing hPSCs from said stable CAR-expressing hPSC cell line; and wherein the population of CD11b+/CD16+ neutrophils comprises a population of CAR neutrophils as per claim 8, wherein the process further comprises the initial steps of: (a') preparing a CAR expression gene construct; and (a") constructing a AAVS1 plasmid as per claim 9, wherein the CAR comprises chlorotoxin or IL13, or other extracellular signaling domains, the transmembrane domain of CD4 (elected species), co-stimulatory domain 2B4, and the intracellular domain of CD3ζ domain.
However, Valaehr II teaches iPSC derived hematopoietic lineage cells, e.g., neutrophils, see para. [0187]. The reference method comprises genome editing such as the CRISPR/Cas system to facilitate the insertion (knock-in) of tolerogenic factors, e.g., PD-L1, CTLA-4-Ig shown in Table 2, into a safe harbor locus of an adeno-associated virus 1 (AAVS1) locus in iPSC, to actively inhibit immune rejection, see reference claims 1-2, 4, in particular.
Valaehr II does not teach knock-in a CAR expression gene construct into the AAVS1 safe harbor locus of AAVS1 plasmid and isolating CAR-expressing hPSC as per claim 8 and (a') preparing a CAR expression gene construct; and (a") constructing a AAVS1 plasmid as per claim 9.
However, regarding claim 10, Li II teaches human iPSC-derived natural killer cells engineered with various chimeric antigen receptor to enhance anti-tumor activity. Li teaches the various chimeric antigen receptors each comprises a scFv that binds to human mesothelin (aka other extracellular domains), a CD8 hinge, a NKG2D transmembrane domain, a costimulatory 2B4 domain, and a CD3zeta domain, see entire document, p. 184, Figure 2B, in particular.
Regarding claim 21, Li II teaches that the CARs were subcloned into a PiggyBac transposon vector with a cHS4 insulator element. These CAR-expressing iPSCs demonstrated a similar efficiency in NK cell production as non-CAR expressing iPSCs, see p. 183, e4, Molecular Constructs, in particular. The PiggyBac transposon plasmids were co-nucleofected with transposase such as SB100X or super piggyback transposes at transposon versus transposon ratio of 3:1 into iPSCs, see e4, cell culture, in particular. Li teaches that iPSC-derived NK cells have a normal karytotype and these cells can persist in vivo post-infusion, see p. 190, in particular. Li further teaches that the use of iPSCs or (hESCs) for NK cell production provides a more efficient means for gene modification compared with primary NK cells isolated from peripheral blood. Additionally, this can be done as a one-time gene modification, as done with the current CAR-T cell studies. iPSC-derived NK cells can also be produced o a large scale as a standardized cell product. This will allow multiple doses to be administered rather than the single cel dosing done with current CAR-T cell-based therapies. Additionally, hESC- and iPSC-derived NK cells can be engineered to express chimeric receptors to target chronic infectious disease, see p. 190.
Li II does not teach the CAR comprises chlorotoxin or IL13 as per claim 10.
However, Wang teaches a chimeric antigen receptor construct comprising a tumor-targeting CLTX, IgG4-Fc spacer/linker, a CD28 or CD4 transmembrane, a CD28 costimulatory domain and a CD3ζ signaling domain as per claim 10, see p. 4, Figure 2A, in particular. The construct is expressed in T cells to form CLTX-CAR T cells. Wang teaches that the chlorotoxin (CLTX), a peptide component of scorpion venom, can be successfully incorporated into a CAR construct to redirect cytotoxic T cells to target glioblastoma (GBMs), see p. 8, Discussion, in particular. Wang teaches that the chlorotoxin (CLTX) has been established to bind broadly and specifically to glioblastoma (GBM) and CLTX binding impairs GBM cell migration (see p. 1, right col).
Kim teaches an TQM-IL-13 specific chimeric antigen receptor (CAR) construct comprising TQM-IL13 binding domain, an IgG4 Fc domain, a CD4 transmembrane domain and a CD3zeta domain as per claim 10, see p. 627, vector design, p. 632, Fig. 6, in particular. The TQM-IL-13 specific CAR is expressed in T cells for treating brain cancer. Kim teaches that the advantage of TQM-IL-13 specific chimeric antigen receptor (CAR) T cell is that it reduces systemic toxicity and achieves higher drug bioavailability at the target site compared with traditional chemotherapy, see p. 633, in particular. Kim teaches that IL13-receptor-α2 (IL13Rα2), present in over 75% of glioblastoma (GBMs) and the IL13 (TQM-13) chimeric antigen receptor has a high binding affinity to IL13Rα2-expresssing glioblastoma with low off-target toxicity, see abstract, in particular.
In view of the combined teachings of the references, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to use Valaehr II’s method of (knock-in) the CAR constructs of Li II into the AAVS locus in hPSCs via caspase 9-mediated homologous recombination or follow Li’s non-viral method of expressing the functional CARs in iPSC cells by subcloned into a PiggBac transposon vector and delivering the PiggBac plasmid into human pluripotent stem cells via nucleofection and electroporation and then isolating the CAR expressing hPSC cells and producing the CAR-expressing hPSC derived neutrophil by chemical induced differentiation of Ng, Li, Valaehr I, Sweeney and Saeki with a reasonable expectation success, e.g., more efficient means for producing mesothelin-directed CAR in hPSC derived neutrophils.
In the alternative, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to substitute the extracellular domain in the chimeric antigen receptor of Li for Wang’s chlorotoxin or Kim’s high-affinity mutant IL-13, then use Valaehr II’s method of (knock-in) the CAR constructs of Li II into the AAVS locus in hPSCs via caspase 9-mediated homologous recombination or follow Li’s non-viral method of expressing the functional CARs in iPSC cells by subcloned into a PiggBac transposon vector and delivering the PiggBac plasmid into human pluripotent stem cells via nucleofection and electroporation and then isolating the CAR expressing hPSC cells and producing the CAR-expressing hPSC derived neutrophil by chemical induced differentiation of Ng, Li, Valaehr I and Saeki with a reasonable expectation success, e.g., more efficient means for producing chlorotoxin-directed CAR or IL-13-directged CAR in hPSC derived neutrophils.
The success of Li II for using iPSCs to provide a source of CAR-expressing NK cells, the success of Ng to differentiate hPSC to provide a source of neutrophils, the success of Valaehr II’s viral method or Li II’s non-viral method to genetically modified iPSCs would have led one of ordinary skill in the art to use human pluripotent stem cells (hPSCs) such as hESCs and iPSCs as the source for hPSC derived neutrophils production and genetically modified to express chimeric antigen receptor of interest. One of ordinary skill in the art would have been motivated to do so because Li II teaches that this can be done as a one-time gene modification event rather than requiring patient-specific gene modification and the iPSC-derived cells can be produced from a standardized cell population would provide a homogenous NK cells population that can be grown to clinical scale and reduced cost, see p. 182, left col, and p. 190.
One of ordinary skill in the art would have been motivated to substitute the mesothelin targeting domain the CAR of Li II for Wang’s chlorotoxin targeting domain because Wang teaches that the chlorotoxin (CLTX) has been established to bind broadly and specifically to glioblastoma (GBM) and CLTX binding impairs GBM cell migration (see p. 1, right col).
One of ordinary skill in the art would have been motivated to substitute the mesothelin targeting domain the CAR of Li II for Kim’s high affinity IL13 targeting domain because Kim teaches that IL13-receptor-α2 (IL13Rα2), present in over 75% of glioblastoma (GBMs) and the IL13 (TQM-13) chimeric antigen receptor has a high binding affinity to IL13Rα2-expresssing glioblastoma with low off-target toxicity, see abstract, in particular.
“The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007).
“The test of obviousness is not express suggestion of the cl aimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Conclusion
Claims 11, 12, 26 and 27 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PHUONG HUYNH whose telephone number is (571)272-0846. The examiner can normally be reached on 9:00 a.m. to 6:30 p.m. The examiner can also be reached on alternate alternative Friday from 9:00 a.m. to 5:30 p.m.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Misook Yu, can be reached at 571-272-0839. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/PHUONG HUYNH/ Primary Examiner, Art Unit 1641
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