DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. The present application claims benefit under 35 U.S.C. 119(e) to U.S. Provisional applications 63/332225, filed 4/18/2022, and 63/384764, filed 11/22/2022.
Election/Restrictions
Applicant’s election without traverse of Group I, encompassing claims 1-25 and 136-140, in the reply filed on 4/15/2026 is acknowledged. Claims 26-135 and 141-159 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Groups II-XI, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 6/21/2024.
Status of Claims
Claims 1-159 are pending; claims 26-135 and 141-159 are withdrawn; claims 1-25 and 136-140 are being examined on the merits.
Claim Objections
Claims 15 and 18 are objected to because of the following informalities: Applicants define a “chimeric antigen receptor” with the acronym “CAR” in claim 1, but then repeat the definition in claims 15 and 18. If the acronym is introduced in claim 1, it should then be used accordingly throughout the claims. Appropriate correction is required.
Claims 15 and 18 are objected to because of the following informalities: Applicants reference “hematopoietic progenitor cells” in claim 15, then define it with the acronym “HPC” in claim 18. If the acronym is going to be introduced, it should define the term after the first use of the term, and then be used accordingly throughout the claims. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph 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.
Claim 10 is 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The following quotation from section 2163 of the Manual of Patent Examination
Procedure is a brief discussion of what is required in a specification to satisfy the 35 U.S.C. 112 written description requirements for a generic claim covering several distinct inventions:
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... reduction to drawings...or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or 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 the applicant was in possession of the claimed genus... See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation
within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.
Thus, when a claim covers a genus of inventions, the specification must provide written description support for the entire scope of the genus. Support for a genus is generally found where the applicant has provided a number of examples sufficient so that one in the art would
recognize from the specification the scope of what is being claimed.
Claim 10 recites the genetically engineered macrophage of claim 1, wherein the anti-GD2 CAR has an amino acid sequence comprising SEQ ID NO: 6 or a sequence having at least 80% sequence identity to SEQ ID NO: 6. The CAR of SEQ ID NO: 6 is 696 amino acids in length; thus, at least 80% sequence identity allows for 139 amino acid residue substitutions within the full sequence of the CAR. The CAR of SEQ ID NO: 6 comprises, among other components, an anti-GD2 scFv, which comprises a VH and a VL domain linked by a G4S linker; specifically residues 22-284. Each of the VH and the VL domains comprise 3 complementary CDRs. Thus, the claim allows for up to 139 amino acid residue substitutions throughout the CAR of SEQ ID NO: 6, and including within the CDR regions of the anti-GD2 scFv binding domain of the CAR. There are 20 alternative amino acids that may be substituted at any one of the 139 residues of the 696 amino acid CAR construct of SEQ ID NO: 6 to generate variant CAR constructs having “at least 80%” sequence identity to SEQ ID NO: 6. Thus, the claim encompasses a vast number of alternative embodiments. The broadest reasonable interpretation of claim 10 is to a genus of anti-GD2 CAR constructs comprising unidentified VH CDRs 1-3 and VL CDRs 1-3 of the scFv of the anti-GD2 binding domain, with any number (up to 139) of amino acid point mutations, which may be with any alternative amino acid, and may occur in any combination, and may occur within the CDR binding domains of the CAR variants.
In support of the claimed genus of variants, the specifications disclose 3 anti-GD2 CAR constructs; CARa, CARb and CAR1 (specs., pg. 51, Table 3). CARb and CAR1 have identical scFv domain sequences, and differ in the transmembrane (TM) and intracellular (IC) domain components. CARa has a significantly different scFv sequence than either of CARb or CAR1. Thus, no guidance is provided as to which residues may be substituted, or with which alternative amino acids, or what the required CDRs of the binding domain are, or which of the CDR residues may be mutated with alternative amino acid residues. When determining the representative examples and the art, it is important to consider whether there is evidence of a singular shared structural feature which imparts the defining property of the claimed genus, and which would necessarily be present in every species of the claimed genus. In this case, none of the CARa, CARb or CAR1 have greater than 80% sequence identity to each other, thus the only 3 variants described are not encompassed by the claim limitations. No embodiments are described of CAR variants that have greater than 80% sequence identity to SEQ ID NO: 6. Further, there is no teaching of where the substitutions may be made in variant embodiments, or what the defining structural feature of all claimed species are required to have, in order to impart the required properties of functionally binding GD2.
Regarding the state of the art; it is known in the art that the antigen binding domain of an antibody-like CAR extracellular domains require the 6 complementarity determining regions (CDR) of the heavy and light chains, whereby the 3 CDRs of the heavy chain and the 3 CDRs of the light chain are structurally inter-dependent in forming the unique binding pocket of the antibody paratope region; and thus the CDRs constitute critical aspects of the binding domain paratope and ultimately impart the paratope-epitope binding functionality with regard to specificity and affinity (for review see MacCallum et al., 1996). However, the structure-to-function correlation continues to be highly unpredictable. For example, Chen et al., (1992) teaches that a single amino acid substitution in the VH CDR2 of PC-specific T15 antibody could increase, decrease or ablate binding the target antigen (abstract, Fig. 3), and this occurred in an unpredictable manner based on which residue was mutated. Similarly, a single point mutation in the heavy chain CDR3 region of the high affinity anti-VEGF antibody G6.31, could in some cases enhance, or otherwise completely ablate binding to the target antigen, and this also occurred in an unpredictable manner (Koenig et al., PNAS, 2017). That is, only screening each mutation individually provided insight as to the resulting changes in functionality. In some cases this extends even beyond the CDRs. Within the framework regions, Koenig et al. (PNAS, 2017) teaches that various amino acid point mutations can increase or decrease binding or neutralization capacity. Some amino acid residues are more tolerant to substitution, while other “conserved” residues are less tolerant, such that a single amino acid substitution may defunctionalize the antibody (pg. E487, Fig. 1). Thus, while CARs share certain characteristics such as TM, hinge regions or IC domains, these regions are not correlated with the binding function of the CAR. Conversely, the hyper-variable regions of an antibody or scFv, comprising the complementary set of 6 CDRs, are well established in the art as the portion of the binding regions which impart the specificity of the CAR; and yet, there is no way to look at an amino acid sequence and envision, a priori, whether the combination of six CDRs will bind a particular epitope, even when the CDRs are highly related, without teachings of the basic shared amino acid residues that are sufficient to impart functional binding across all variants. Further, even when provided with several related binding domains that bind the desired target, this does not represent the astronomical and potentially unknowable breadth of all possible amino acid sequences which will result in the desired binding properties. This is exemplified by the Court decision in Abbvie (Abbvie v Janssen 759 F.3d 1285 (Fed. Cir. 2014)), where Abbvie developed over 200 antibodies that shared 99.5% identity in the variable regions (pg. 7) and which bound the target, but in no way allowed one to envisage the unique structure of Centocor’s antibodies which bound the same target but shared only 50% sequence similarity. Thus, when claiming a genus of antibody-like binding domains based on their binding to a common target, the representative examples must cover the full scope of structural variabilities which encompass all species variants that would bind the target.
Section 2163(II)(A)(3)(a)(ii) of the MPEP states that the written description for a claimed genus may be satisfied through either a) a representative number of species, or b) disclosed correlation between function and structure. Here the applicants do not provide any variants of the claimed embodiments, in which alternative mutations were made, which were reduced to practice; nor do they identify the shared structural properties of the variants, such as the CDR residues, that would define the genus beyond the desired functionality. Currently the essential property of binding GD2 is imparted by the three specific CARs, with unidentified CDR sequences, that have been reduced to practice; and that accounts for only 3 examples out of millions of potential embodiments as claimed. Specifically, the physical features (or amino acid residues encoding said features) which impart the property of the binding domains of the CARs binding to GD2 should be disclosed. Further, a description of the type and number of amino acid residue substitutions that may be made at such identified positions within the sequence, that result in “at least 80%” sequence identity to a selected sequence, would be essential in determining the degree of variability that may be allotted in total sequence identity. This lack of definition complicates the determination of the boundaries of the claimed genus with regard to which, as of yet unidentified, species variants (CARs with 80% identical sequences) would be anticipated, a priori, by one skilled in the art, to fall within the scope of the claims. Without the identification of the necessary shared structural properties of all species variants that fall within the scope of the genus, it may be that an embodiment species with < 80% sequence identity, such as CARa or CAR1, would still bind GD2; or conversely, that CARs with > 80% sequence identity, but comprising a deleterious mutation in the scFv CDR regions would lose functional binding to GD2.
In view of this uncertainty and the lack of a representative number of examples of the claimed genus, claims 10 is rejected for lack of adequate written description support. Applicant is informed that one means of overcoming the rejection of the claims under 35 U.S.C. 112(a) is to amend claim 10 to remove the “80% sequence identity” language.
Claim Rejections - 35 USC § 103
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.
Claims 1-3, 5-9, 11-14 and 21-25 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., (from IDS of 5/7/2024, Cite No. 28; 2020) and Pule et al., (from IDS of 4/15/2026, US 20210403596; published 12/30/2021).
Zhang et al. teaches pluripotent stem cell-derived CAR-macrophage cells with antigen-dependent anti-cancer cell functions (title). Zhang developed induced pluripotent stem cell (iPSCs)-derived CAR-expressing macrophage cells; whereby the CAR expression confers antigen-dependent macrophage functions such as expression and secretin of cytokines, polarization toward the pro-inflammatory/anti-tumor state, enhanced phagocytosis of tumor cells and in vivo anticancer activity (abstract). Zhang teaches isolating single iPSC clones, genetically engineering them by lentiviral transduction of a CD19 CAR, and spurring differentiation into macrophage phenotypes (pg. 2, top). Zhang teaches the CAR-expressing macrophages can further be expanded to have a final yield of above 50-fold of the starting iPSCs, with high purity indicated by ~100% of CD11b and CD14 expression (pg. 2, col. 2; see also Fig. 1 a-b). Thus, Zhang teaches iPSCs transduced to express a CD19 CAR and induced to differentiate into CD11b+ and CD14+ macrophages expression a CD19 CAR. However, Zhang does not teach wherein the CAR is a GD2 CAR.
Pule et al. teaches CARs comprising a disialoganglioside (GD2)-binding domain and their expression in immune cells (abstract). Pule teaches cells that express the CAR may be cytolytic immune cells (pg. 3, para. 0063). Pule teaches that GD2 is expressed on tumors, and is comparatively has highly restricted expression on normal tissue; therefore the tumor specific expression of GD2 makes it a suitable target for immunotherapy (pg. 6, paras. 0113-0114). Pule teaches pharmaceutical compositions comprising CAR-expressing cells (pg. 11, para. 0136), as well as methods of treating cancer, specifically GD2 expressing cancers (pg. 11, para. 0139).
It would have been obvious to one of skill in the art to substitute the GD2 CAR in place of the CD19 CAR in the genetically engineered macrophages as taught by Zhang et al. One would have been motivated to do so in order to change the antigen-dependent specificity of the CAR-expressing macrophages, as taught by Zhang et al., to instead target alternative tumor associated antigens such as GD2, as taught by Pule et al. There would have been a reasonable expectation for success given that the GD2 CARs are also expressed in cytotoxic immune cells, whereby they are used in methods of treating GD2 expressing cancers, as taught by Pule et al. Thus, the invention was prima facie obvious to one of skill in the art at the time the invention was made.
Regarding claims 1-3; the anti-GD2 CAR-expressing macrophages of the combination of Zhang and Pule are described above. Zhang teaches that the macrophages are CD11b+ and CD14+, and thus makes obvious instant claim 1. Further, Zhang teaches that the macrophages are produced from iPSCs which are genetically engineered to express the CAR, and thus makes obvious instant claims 2-3.
Regarding claims 5-9; Zhang teaches the CAR-expressing macrophages have enhanced phagocytosis of tumor cells and in vivo anticancer activity, while Pule teaches the anti-GD2 CAR can direct the macrophages to tumor cells expressing GD2, such as neuroblastoma and melanoma (pg. 11, para. 0141). Thus, the combination of Zhang and Pule make obvious instant claims 5-6; and wherein the tumor is a solid tumor, not a blood cancer, and wherein the tumor is neuroblastoma, of instant claims 7-9.
Regarding claim 11; Pule teaches the anti-GD2 CAR comprises a scFv, an IgG1 spacer (hinge), a CD28 TM and a OX40-CD3zeta endodomain (pg. 1, para. 0014; Figure 2), and whereby a nucleic acid encodes the CAR (pg. 3, para. 0058). Thus, the combination of Zhang and Pule make obvious instant claim 11.
Regarding claim 12, Zhang teaches the macrophages has antigen-dependent incrase in M1 pro-inflammatory cytokine expression (pg. 3, col. 2, para. 1) and could be treated to further polarize CAR-iMAC cells toward M1 (pg. 3, col. 2, para. 2). Thus, the combination of Zhang and Pule make obvious instant claim 12.
Regarding claims 13-14; Zhang teaches the CAR-expressing macrophages can further be expanded to have a final yield of above 50-fold of the starting iPSCs, with high purity indicated by ~100% of CD11b and CD14 expression (pg. 2, col. 2). Thus, Zhang and Pule make obvious instant claims 13-14.
Regarding claims 21-25; Pule teaches the CAR-expressing cells in a pharmaceutical composition (pg. 11, para. 0136), and methods of treating a tumor (pg. 11, para. 0139), comprising administering the cells, whereby the therapeutic use is provided in order to lessen or reduce a symptom of the disease (pg. 11, para. 0142), including killing of cancer cells (i.e. reduced proliferation), and wherein the solid tumor is neuroblastoma or melanoma. Thus, the combination of Zhang and Pule make obvious the pharmaceutical composition of instant claim 21, as well as methods of treating or reducing proliferation of a tumor comprising administering the pharmaceutical composition or otherwise contacting the tumor with the CAR-expressing cells, wherein the tumor is neuroblastoma or melanoma, of instant claims 22-25.
Claims 1-9, 11-14, 21-25 and 136-140 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., (from IDS of 5/7/2024, Cite No. 28; 2020) and Pule et al., (from IDS of 4/15/2026, US 20210403596; published 12/30/2021) as applied to claims 1-3, 5-9, 11-14 and 21-25 above, and further in view of Gill et al., (from IDS of 4/15/2026; US 20220041688; published 2/10/2022).
The reasons why claims 1-3, 5-9, 11-14 and 21-25 are made obvious over a combination of Zhang and Pule is described above. Specifically, the combination makes obvious CD11b+ and CD14+ macrophages expressing an anti-GD2 CAR. However, Zhang and Pule are silent regarding the markers of the macrophages (re. claim 4) or where in the macrophage has inhibited SIRPa signaling (re. claims 136-140).
Gill teaches modified macrophages expressing CARs and methods for treating cancer (title, abstract), wherein the cell is a monocyte or macrophage which possess targeted effector activity (pg. 1, para. 0005). Gill teaches the tumor antigen target of the CAR may be GD2 (pg. 14, para. 0216). Regarding macrophages, Gill teaches macrophages are phenotypically plastic cells capable of adopting diverse function features, commonly separated into the M1 an M2 macrophage classifications- with M1 being inflammatory/activated, and M2 being immunosuppressive/tumor-promoting (pg. 30, para. 0388). Gill teaches activated macrophages have upregulated M1 marker CD80 and downregulated M2 markers CD163 and CD206 (pg. 30, para. 0388; pg. 22, para. 0282).
Regarding claim 4, the combination of Zhang and Pule make obvious a CD11b+ and CD14+ macrophage expressing a GD2 CAR of claim 1, and whereby the macrophage exhibits an M1 anti-cancer phenotype of claim 12, as described above. Gill provides evidence that the M1 phenotype of the GD2 CAR-expressing macrophages of Zhang and Pule would exhibit higher levels of CD80 and lower levels of CD163 and CD206 than a macrophage that does not express the CAR (e.g., a M2 macrophage). Thus, the combination of Zhang and Pule, as evidenced by Gill, makes obvious instant claim 4.
Further, Gill teaches macrophages expressing a CAR can be engineered for enhanced effector activity by inhibition of either CD47 or SIRPa activity (pg. 1, para. 0015; pg. 22, para. 0283). Gill teaches the CAR constructs may include the delivery of CRISPR/Cas9 gene editing material (pg. 2, para. 0022). Specifically, Gill teaches the specificity of CAR macrophages (CARMA) phagocytic enhancement in the presence of SIRPa blocking monoclonal antibody was tested by knocking out the SIRPa receptor on macrophages. CRISPR/Cas9 was used for SIRPa deletion; knocking out SIRPa enhanced CARMA function, and adding anti-SIRPa back to the knockout cells failed to further enhance phagocytosis (pg. 27, para. 0354, Fig. 5G).
It would have been obvious to one of skill in the art to augment the phagocytic activity of the GD2 CAR-expressing macrophages of Zhang and Pule, by genetically engineering the cells to have inhibited expression of SIRPa. One would have been motivated to do so given that knockout of SIRPa enhances macrophage phagocytic activity, as taught by Gill et al. There would have been a reasonable expectation for success given that Gill used CRISPR/Cas 9 gene editing to knockout SIRPa in CAR-expressing macrophages and demonstrated enhanced phagocytic activity. Thus the invention was prima facie obvious at the time the invention was made.
Regarding claims 136-140; the combination of Zhang, Pule and Gill make obvious GD2 CAR-expressing macrophages with inhibited SIRPa expression, and thus make obvious instant claim 136. Further, Gill teaches knocking out SIRPa by gene editing using a Cas enzyme, and thus makes obvious instant claims 137-138 and 140. Regarding claim 139, while Gill is silent as to which exon of the SIRPa gene is knocked out, it would be obvious to a skilled artisan to choose exon 3 of the SIRPa gene, given that the goal is to disrupt SIRPa expression and there are a limited number of exons of the SIRPa gene to choose from. MPEP 2143(I)(E) recites that it is obvious to try when choosing from a finite number of identified, predictable solutions with a reasonable expectation for success. Here, the goal is to disrupt expression of SIRPa by knocking out exons of the SIRPa gene. While knockout of alternatives to exon 3 may also work, a skilled artisan would experiment with different exons of the SIRPa gene and ultimately arrive at deletion of exon 3. Thus, the combination of Zhang, Pule and Gill make obvious instant claim 139.
Claims 1-3, 5-9 and 11-25 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., (from IDS of 5/7/2024, Cite No. 28; 2020) and Pule et al., (from IDS of 4/15/2026, US 20210403596; published 12/30/2021) as applied to claims 1-3, 5-9, 11-14 and 21-25 above, and further in view of Thomson et al., (from IDS of 5/7/2024; US 20200080059; published 3/12/2020), as evidenced by Gill et al., (from IDS of 4/15/2026; US 20220041688; published 2/10/2022).
The reasons why claims 1-3, 5-9, 11-14 and 21-25 are made obvious over a combination of Zhang and Pule is described above. Specifically, the combination makes obvious CD11b+ and CD14+ macrophages expressing an anti-GD2 CAR. However, Zhang and Pule do not teach the methods of producing the CAR expressing macrophages from progenitor cells of instant claims 15-20.
Thomson teaches methods for generation of hematopoietic progenitor cells from human pluripotent stem cells (title, abstract). Thomson teaches hematopoietic progenitor cells are obtained by a method of culturing mesoderm cells seeded at low density in a chemically-defined culture medium that comprises fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) among other agents, whereby a cell population comprising hemangioblast cells is obtained; and culturing the hemangioblast cells in culture medium whereby a cell population comprising CD34+ CD45+ hematopoietic progenitor cells is obtained (pg. 1, para. 0004). In some embodiments, the mesodermal cells are obtained by culturing human pluripotent stem cells; and in some embodiments the human pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells (iPSCs; pg. 1, para. 0005).
Thus, Thomson teaches a method of obtaining CD34+ CD45+ hematopoietic progenitor cells comprising culturing mesoderm cells seeded at low density in a first chemically-defined culture medium that comprises a FGF and a VEGF, whereby a cell population of hemangioblast cells is obtained; and culturing the hemangioblast cells in a second chemically-defined medium that comprises FGF and VEGF to obtain CD34+ CD45+ hematopoietic progenitor cells (pg. 15, claim 1). Thomson teaches the low cell culture density conditions were essential for generating hematopoietic progenitor cells (pg. 13, paras. 0093-0095). Further, Thomson teaches that these hematopoietic progenitor cells could be further differentiated to macrophages, NK cells and T cells in feeder-free and serum-free conditions (pg. 13, para. 0093). Thomson also teaches that the hematopoietic progenitor cells (HPCs) may be used in cancer immunotherapies or in the production of CAR T cells or CAR NK cells (pg. 10, para. 0066). Specifically, Thomson teaches the CAR is transfected in human pluripotent stem cells, mesoderm cells, hemangioblasts or HPCs for use in any of the methods described to produce CAR T cells or CAR NK cells (pg. 10, para. 0067).
It would have been obvious to one of skill in the art to modify the protocol of Zhang to produce CAR-expressing macrophages, wherein the CAR is the GD2 CAR of Pule, to follow the culturing steps as taught by Thomson et al. One would have been motivated to do so in order to generate macrophages expressing GD2 CAR for use as cancer therapeutics, as taught by Zhang and Pule. There would have been a reasonable expectation for success given that Zhang genetically engineered iPSCs to express the CAR and isolated differentiated macrophages from this population, and Thomson teaches that iPSCs can be transfected to produce a CAR, and the iPSCs can be cultured to produce CD11b+ CD14+ macrophages. Thus, the invention was prima facie obvious at the time the invention was made.
Note that as hemangioblasts primarily give rise to hemogenic endothelial cells which give rise to hematopoietic progenitor cells, and the methods of Thomson teach culturing mesodermal cells to produce hemangioblasts cells, and then culturing hemangioblasts to produce hematopoietic progenitor cells, it is interpreted that producing hematopoietic progenitor cells from hemangioblasts encompasses hemogenic endothelial cells. That is, for the purposes of the methods described by Thomson et al., a “hemanogioblast” cell is equivalent to “hemogenic endothelial cell”.
Regarding claims 15-17 and 19; Zhang and Pule teach genetically engineered GD2 CAR-expressing macrophages, as described above. Zhang teaches the method includes transfecting iPSCs with a lentivirus encoding the CAR, and then differentiating the iPSCs to macrophages. Thomson teaches methods of culturing iPSCs to obtain mesodermal cells, culturing mesodermal cells at low density to obtain hemogenic endothelial cells (i.e., hemangioblasts), culturing hemogenic endothelial cells to generate CD34+ CD45+ HPCs, and culturing HPCs to obtain CD11+ CD14+ macrophages. Thomson also teaches pluripotent stem cells can be transfected to express a CAR. Thus the combination of Zhang, Pule and Thomson make obvious the method of claim 15, and wherein the pluripotent stem cells are iPSCs, of claim 16. Gill provides evidence that M1 macrophages express higher levels of CD80 and lower levels of CD163 and CD206, as described above, and Zhang teaches inducing the CAR-expressing macrophages to the M1 phenotype, as described above. Thus, the combination of Zhang, Pule and Thomson, as evidenced by Gill, make obvious instant claim 17. As Zhang teaches the CAR-expressing macrophages can further be expanded to have a final yield of above 50-fold of the starting iPSCs, with high purity indicated by ~100% of CD11b and CD14 expression, as described for claim 13 above; the combination of Zhang, Pule and Thomson make obvious instant claim 19.
Regarding claims 18 and 20; Thomson teaches a method of generating HPCs from pluripotent stem cells which were previously differentiated into arterial hemogenic endothelium (pg. 13, Example 1, paras. 0092-0094). By culturing arterial endothelial cells in low density seeding culture, an “endothelial-to-hematopoietic” stem cell transition occurs, resulting in CD34+ CD45+ hematopoietic progenitor cells. Thomson teaches “these HPCs could be further differentiated to macrophages in feeder-free and serum-free conditions,” (pg. 13, para. 0093). Thus, the combination of Zhang, Pule and Thomson make obvious the methods of claim 18, and the isolated population of macrophages of claim 20.
Conclusion
No claims are allowed.
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/JAMES RYLAND MELCHIOR/Examiner, Art Unit 1644
/NELSON B MOSELEY II/Primary Examiner, Art Unit 1642
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