DETAILED ACTION
Claims 60-81 are currently pending and under examination. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . An action on the merits follows.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 9/1/23 and 12/22/25 are in compliance with the provisions of 37 CFR 1.97 and 1.98. Accordingly, the information disclosure statements have been considered by the examiner and initialed and signed copies of the 1449s are attached to this action.
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 60-81 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 the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Independent claim 60 recites, “[a]t least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of: ETS1, TBET, NFIL3 and EOMES”. As phrased, it is unclear whether the product encompasses one or more polynucleotides where each of the polynucleotides encodes at least three of the recited transcription factors, or whether the product encompasses, for example, three separate polynucleotides where each polynucleotide encodes a single transcription factor and the three separate polynucleotides together encode the combination of the three transcription factors. As such, the metes and bounds of claim 60 cannot be determined. Claims 61-81 depend on claim 60 and thus are included in this rejection.
In the interests of compact prosecution, the product of claim 60 has been given its broadest reasonable interpretation of encompassing either a single polynucleotide encoding all three transcription factors, more than one polynucleotide where each polynucleotide encodes all three transcription factors, or three, or more, polynucleotides where each polynucleotide encodes a different transcription factor and the three polynucleotides together encode the combination of three or more transcription factors.
Claim 68 is further indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 68 recites, “[a] construct or vector according to claim 65, wherein the construct or vector further comprises a CRISPR/CAS activation system”. Claim 65 recites a construct or vector comprising at least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of: ETS1, TBET, NFIL3 and EOMES. Claim 68 is confusing based on description of a CRISPR/CAS activation system in the instant specification. The instant specification makes only a single reference to “CRISPR/CAS activation system” on page 14, where it is stated, “[i]n one embodiment, the construct or vector is a CRISPR/CAS activation system. A person skilled in the art will understand that this system is able to activate endogenous transcription factors, enabling the use of a plasmid without transcription factors”. This disclosure states that where the construct or vector is a CRISPR/CAS activation system, the plasmid/vector/construct does not include the actual transcription factor polynucleotide sequences as the CRISPR/Cas activation system apparently activates endogenous transcription factors. Thus, it does not appear that the specification actually discloses a construct or vector comprising the polynucleotide(s) encoding the at least three transcription factors AND a CRISPR/CAS activation system. Further, it is unclear what elements are encompassed by the “CRISPR/CAS activation system”. Is it a system that activates a separate “CRISPR/CAS” system not present in the construct or vector, or does the CRISPR/CAS system itself function by “activating” something else, such as perhaps endogenous transcription factors. The specification provides no guidance as to what CRISPR/CAS elements such a system may encompass which allow it to function as an “activation” system, and/or does not define what elements may be present in a construct or vector to “activate” a separate CRISPR/CAS system. As such the metes and bounds of the claim cannot be determined.
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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.
Claim 68 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.
Claim 68 depends on claim 65, which in turn depends on independent claim 60. Claim 68 recites, “[a] construct or vector according to claim 65, wherein the construct or vector further comprises a CRISPR/CAS activation system”. Claim 65 recites a construct or vector comprising at least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of: ETS1, TBET, NFIL3 and EOMES. Claim 68 was added by preliminary amendment on 12/13/23. Original claim 28, which is the closest claim in language to claim 68, recited, “[t]he construct or vector according to any one of claims 16 to 27, wherein the construct or vector is a CRISPR/CAS activation system”. Thus, the original claim 28 recited that the construct or vector “is” a CRISPR/CAS activation system, whereas new claim 68 recites that the construct or vector “further comprises a CRISPR/CAS activation system”. These are not equivalent disclosures. Furthermore, the instant specification makes only a single reference to “CRISPR/CAS activation system” on page 14, where it is stated, “[i]n one embodiment, the construct or vector is a CRISPR/CAS activation system. A person skilled in the art will understand that this system is able to activate endogenous transcription factors, enabling the use of a plasmid without transcription factors”. Thus, according to the specification, where the construct or vector is a CRISPR/CAS activation system, the plasmid/vector/construct does not include the actual transcription factor polynucleotide sequences as the CRISPR/Cas activation system activates endogenous transcription factors. Based on the single sentence present in the specification on page 14, the specification does not actually disclose a construct or vector comprising the polynucleotide(s) encoding the at least three transcription factors AND a CRISPR/CAS activation system. As such, the new limitation, “wherein the construct or vector further comprises a CRISPR/CAS activation system” recited in claim 68 is considered new matter not supported by the as filed specification.
In addition, it is further noted that the specification does not provide adequate guidance for any “CRISPR/CAS activation system”. The specification is silent as to the elements/components of any such system. While the single sentence on page 14 states that the “CRISPR/CAS activation system” can activate endogenous transcription factors, the specification provides no description of how any endogenous transcription factor can be targeted for activation using the CRISPR/CAS activation system, or what is encompassed by any such “activation”. As discussed in the rejection of claim 68 for indefiniteness under 35 U.S.C. 112(b) above, it is unclear what elements are encompassed by the “CRISPR/CAS activation system”. Is it a system that activates a separate “CRISPR/CAS” system not present in the construct or vector, or does the CRISPR/CAS system itself function by “activating” something else, such as perhaps endogenous transcription factors. The specification provides no guidance as to what CRISPR/CAS elements such a system may encompass which allow it to function as an “activation” system, and/or does not define what elements may be present in a construct or vector to “activate” a separate CRISPR/CAS system. As such, the specification fails to provide adequate guidance for any “CRISPR/CAS activation system”, and the single sentence description is not sufficient to adequately describe and demonstrate possession of the invention as set forth in claim 68.
The following guidance provided in MPEP 2163 is informative. To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. 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 (see i)(A) above), reduction to drawings (see i)(B) above), 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 i)(C) above). See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. An invention described solely in terms of a method of making and/or its function may lack written descriptive support where there is no described or art-recognized correlation between the disclosed function and the structure(s) responsible for the function. In other words, describing a composition by its function alone typically will not suffice to sufficiently describe the composition. See for example Eli Lilly, 119 F.3 at 1568, 43 USPQ2d at 1406 (Holding that description of a gene’s function will not enable claims to the gene "because it is only an indication of what the gene does, rather than what it is."); see also Fiers, 984 F.2d at 1169-71, 25 USPQ2d at 1605-06 (discussing Amgen Inc. v. Chugai Pharm. Co., 927 F.2d 1200, 18 USPQ2d 1016 (Fed. Cir. 1991)). An adequate written description of a chemical invention also requires a precise definition, such as by structure, formula, chemical name, or physical properties, and not merely a wish or plan for obtaining the chemical invention claimed. See, e.g., Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 927, 69 USPQ2d 1886, 1894-95 (Fed. Cir. 2004) (The patent at issue claimed a method of selectively inhibiting PGHS-2 activity by administering a non-steroidal compound that selectively inhibits activity of the PGHS-2 gene product, however the patent did not disclose any compounds that can be used in the claimed methods. As such, based on the single sentence disclosure related to a “CRISPR/CAS activation system” present on page 14 of the instant specification which presents a desired function without any structural information or physical properties, the skilled artisan would not have concluded that the applicant was in possession of the claimed invention of claim 68.
Claims 77-81 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 method of inducing human embryonic fibroblasts to differentiate into CD34+ cells comprising transduction of the human embryonic fibroblasts in vitro with four lentiviral vector encoding one of the four different human transcription factors ETS1, TBET, NFIL3 and EOMES followed by culture of the cells for at least 6 days up to at least 12 days and 2) a method of inducing human embryonic fibroblasts to differentiate into CD56+ NK cells comprising transduction of the human embryonic fibroblasts in vitro with four lentiviral vector encoding one of the four different human transcription factors ETS1, TBET, NFIL3 and EOMES followed by culture in NK cell differentiation and activation media comprising at least IL-7, SCF, IL-3, IL-15, and FLT3L for at least 6 days up to at least 12 days, does not reasonably provide enablement for inducing or reprogramming any type of germ cell or somatic cell to generate a CD34+ cell or a CD56+ NK cell or any type of progenitor cell by expressing three or four of the transcription factors ETS1, TBET, NFIL3 and EOMES, or for treating any cancer by administering any construct or vector encoding three or four of the transcription factors ETS1, TBET, NFIL3 and EOMES in vivo in a subject. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The claims are broadly drawn to methods of reprogramming of inducing a cell into a natural killer cell or progenitor cell comprising transducing the cell with a construct or vector comprising at least one polynucleotide(s) encoding at least three of the transcription factors ETS1, TBET, NFIL3 and EOMES, and culturing the cells in media to obtain reprogrammed cells, or a method of treating cancer in a subject by administering a construct or vector comprising at least one polynucleotide(s) encoding at least three of the transcription factors ETS1, TBET, NFIL3 and EOMES. Claims 77-80 read broadly on the differentiation or transdifferentiation of any cell, such as a germ cell, any kind of stem or progenitor cell, or any kind of differentiated cell into either a progenitor cell or a natural killer cell. Note that claim 77 does not identify the progenitor cell as a progenitor cell or an NK cell, but rather reads broadly on any kind of progenitor cell such as a liver progenitor cell, a muscle progenitor cell, a kidney progenitor cell etc. The specification provides a general disclosure of the method in broad terms which focus on the use of three or four of the a construct or vector comprising at least one polynucleotide(s) encoding at least three of the transcription factors ETS1, TBET, NFIL3 and EOMES to induce or reprogram a cell which may be any of a large number of discloses cell types including stem cell, progenitor cell, mature differentiated cells, and terminally differentiated cells. The specification also generally discloses the administration of a vector or construct encoding the three or four transcription factors to a subject to treat a variety of cancers.
Contrary to the bread of the claims and the generic nature of the disclosure in the specification, the working examples provide only two specific examples of reprogramming/induction. The working examples teach the induction of human embryonic fibroblasts to differentiate into CD34+ cells comprising transduction of the human embryonic fibroblasts in vitro with four lentiviral vector encoding one of the four different human transcription factors ETS1, TBET, NFIL3 and EOMES followed by culture of the cells for at least 6 days up to at least 12 days, and further a method of inducing human embryonic fibroblasts to differentiate into CD56+ NK cells comprising transduction of the human embryonic fibroblasts in vitro with four lentiviral vector encoding one of the four different human transcription factors ETS1, TBET, NFIL3 and EOMES followed by culture in NK cell differentiation and activation media comprising at least IL-7, SCF, IL-3, IL-15, and FLT3L for at least 6 days up to at least 12 days. The working examples demonstrate that they were able to produce a small number of CD34+ cells from human embryonic fibroblasts transduced with four separate vector encoding individually all four of the transcription factors ETS1, TBET, NFIL3 and EOMES after 6 days of culture post-transduction. The CD34+ cells are not further characterized such that it is unclear what type of progenitor cell was produced using this method. In a separate example, human embryonic fibroblasts transduced with four separate vector encoding individually all four of the transcription factors ETS1, TBET, NFIL3 and EOMES were cultured for 6 days post-transduction in the presence of NK cell differentiation and activation media comprising at least IL-7, SCF, IL-3, IL-15, and FLT3L resulting in the generation of a small number of CD56+ NK cell like cells. It is noted that these cells were not further characterized or tested for any NK cell functionality. The working examples also include a prophetic example which simply states that a variety of cancer can be treated by cells produced by the disclosed methods. Note in this case that this prophetic example does not teach to treat cancer by administering the transcription factors in vivo. Thus, aside from two specific examples for inducing/reprogramming human embryonic fibroblasts into either CD34+ cells or CD56+ cells, the working examples do not demonstrate or provide any specific guidance for inducing/reprogramming any cell other than an embryonic fibroblast cell into any kind of progenitor cell or NK cell.
At the time of filing, the four transcription factors ETS1, TBET, NFIL3 and EOMES were known to be important mediators of NK cell differentiation and maturation. Male et al., for examples, teaches the transduction of E4pb4 -/- common lymphoid precursor/stem (CLP) cells with a retroviral expression vector pMSCV encoding E4bp4 (NIFL3) under transcriptional control of the MSCV promoter which resulted in the development of NK cells (Male et al. (2014) J. Exp. Med., Vol. 211(4), 635-642, page 638). Male et al. teaches that the effects of E4bp4 on NK development were only effective in CLP cells and not in cells at a later stage of lymphoid or NK cell development (Male et al., page 638). Male et al. further generated a number of additional retroviral expression vectors encoding transcription factors, including EOMES, T-BET, Id2, Gata3, and ETS1, and tested them in E4pb4 -/- common lymphoid precursor (CLP) cells and in lineage depleted bone marrow cells, demonstrating that EOMES, T-BET, Id2, and ETS1 were each individually effective in rescuing NK cell development measured by expression of the NK1.1 marker protein, which Male et al. concludes show that they act downstream of E4bp4 in the hierarchy of NK cell development and contribute to NK cell development (Male et al., page 639 and Figure 4). It is also noted that Male et al. teaches in vitro development involved transducing the CLIP or lin- bone marrow cells with the retroviral vector encoding the transcriptional factor followed by culture in media comprising cytokines such as IL-7 and SCF (Male et al., page 641). Thus, while Male et al. teaches the importance of the expression of at least 4 transcription factors, E4pb4/NFL3, EOMES, T-BET, and ETS1 for NK cell development, Male et al. shows that NK cell development required the expression of the transcription factors in specific progenitor cells, either CLIP cells, or lineage-negative bone marrow cells (lin-CD34+), and further required culture in media with the cytokines IL-7 and SCF. Neither Male et al. nor the prior art at the time of filing as a whole taught that any three of all of these four transcription factors was capable of inducing an stem or progenitor cell to differentiate into a CD34+ cell or an NK cell, or of reprogramming any differentiated cell into a CD34+ cell or an NK cell. Further at the time of filing, the differentiation or transdifferentiation of one cell type into another was considered unpredictable. Grath et al., in a review of cellular reprogramming, teaches that the main challenge when inducing transdifferentiation is choosing what transcription factors (TFs) to overexpress, and that many studies use a guess-and-check method, where TFs are chosen based on logical conclusions, such as the use of TFs that are active during the development of a cell type or drive the differentiation of stem cells into a specific cell type are often investigated first (Grath et al. (2019) J. Biol. Engineer., Vol. 13, 1-15, see page 3). It is further noted that the prior art is silent as to the use of any of these transcription factors for direct in vivo delivery to a subject for the treatment of any cancer. As such, the prior art at the time of filing taught the unpredictability of using transcription factors for reprogramming cells, requiring a guess-and-check approach in order to establish the effects of any one or more transcription factors on a cells ability to differentiate or be reprogrammed into another type of cell.
Therefore, in view of the high level of unpredictability in the prior art at the time of filing with regards to reprogramming/transdifferentiation using transcription factors, the general nature of the disclosure which broadly discloses the invention, the limitation of the working examples to two specific methods for reprogramming human embryonic fibroblasts into two specific cell types, and the breadth of the claims, it would have required undue experimentation to practice the scope of the methods as claimed.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 60-64 are rejected under 35 U.S.C. 101 because the claimed invention is directed to is directed to a judicial exception which is a product of nature without significantly more. The claims are product claims which recites, “[a]t least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of: ETS1, TBET, NFIL3 and EOMES”. The phrase “at least one polynucleotide” reads on a single polynucleotide sequence or multiple nucleotide sequences such as one or more genomic sequences present, for example, within the chromosomes of a cell. Naturally occurring TS1, TBET, NFIL3, and EOMES genes are all present in the genome of cells including human cells, and the human amino acid sequences encoded by these nucleotide sequences for each of human ETS1, TBET, NFIL3, and EOMES were known at the time of filing, including amino acid sequences with 100% sequence identity to instant SEQ ID NOS 1-4 respectively. For examples, the amino acid sequence of human ETS1 with 100% sequence identity to SEQ ID NO:1 was reported by Uniprot Accession Number Q6FG54_HUMAN (19-JUL-2004), the amino acid sequence of human TBET (TBX21) with 100% sequence identity to SEQ ID NO:2 was reported by Uniprot Accession Number Q9UL17 TBX21_Human (5-1-2000), the amino acid sequence of human NIFL3 with 100% sequence identity to SEQ ID NO:3 was reported by Uniprot Accession Number D91VD5_HUMAN (5-OCT-2010), and the amino acid sequence of the long isoform1 of human EOMES with 100% sequence identity to SEQ ID NO:4 was reported by NCBI Accession Number NP_001265111.1. and NM_001278182.2 (2025), with references for the sequence beginning in 1999, see also EP3258268-A1 (20-DEC-2017), Luking et al., SEQ ID NO:111. It is also noted that the human genome comprises a number of additional transcription factors including those listed in instant claim 64. Thus, the at least one polynucleotide encoding the combination of three or four or more transcription factors as claimed encompass a human genome which is a naturally occurring phenomenon, i.e. product of nature.
In addition, there is no evidence of record that the at least one polynucleotide has any markedly different characteristics than the polynucleotides encoding these transcription factors found in nature, i.e. there is no evidence that isolation of the polynucleotide changes the essential characteristics of the polynucleotides in terms of structure or function such that it is materially different than the naturally occurring polynucleotide sequences present in the human genome.
Further, this judicial exception is not integrated into a practical application because the claims are directed to a product and not a method of using the product. Also, the claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception. The claims do not recite any additional elements other than the polynucleotide(s).
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.
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.
Claims 60-67, and 71-80 are rejected under 35 U.S.C. 103 as being unpatentable over Male et al. (2014) J. Exp. Med., Vol. 211(4), 635-642, in view of Zhang et al. (2018) Eur. J. Immunol., Vol. 48, 738-750, Renoux et al. (2015) Immunity, Vol. 43, 394-407, Uniprot Accession Number Q6FG54_HUMAN (19-JUL-2004), Uniprot Accession Number Q9UL17 TBX21_Human (5-1-2000), Uniprot Accession Number D91VD5_HUMAN (5-OCT-2010), EP3258268-A1 (20-DEC-2017), hereafter referred to as Luking et al., KR2012061005-A (2012), hereafter referred to as Im et al., US Patent No. 6,812,339 (2004), hereafter referred to as Venter et al., WO 2002/28999 (2002), hereafter referred to as Beazer-Barclay, Hlavety et al. (2005) J. Virol., Vol. 341, 1-11, Zhang et al. (2014) Curr. Gene Ther., Vol. 14,10-23, hereafter referred to as Zhang 2014, and Fux et al. (2004) Biotech. & Bioengineer., Vol. 86(2), 174-187.
Male et al. teaches that ETS1, T-BET, and E4bp4 (NIFL3) are all important transcription factors which are required for NK cell committed NK progenitor development, and the generation of NK1.1 positive NK cells (Male et al., pages 635, and pages 638-639). In particular, Male et al. teaches that E4bp4 is particularly important early in NK cell development, and that EOMES, Id2, IRF2, and MF are important in acting later in NK development and maturation (Male et al., page 635). Male et al. further teaches the transduction of E4pb4 -/- common lymphoid precursor/stem (CLP) cells with a retroviral expression vector pMSCV encoding E4bp4 (NIFL3) under transcriptional control of the MSCV promoter which resulted in the development of NK cells (Male et al., page 638). Male et al. teaches that the effects of E4bp4 on NK development were only effective in CLP cells and not in cells at a later stage of lymphoid or NK cell development (Male et al., page 638). Male et al. further generated a number of additional retroviral expression vectors encoding transcription factors, including EOMES, T-BET, Id2, Gata3, and ETS1, and tested them in E4pb4 -/- common lymphoid precursor (CLP) cells and in lineage depleted bone marrow cells, demonstrating that EOMES, T-BET, Id2, and ETS1 were each individually effective in rescuing NK cell development measured by expression of the NK1.1 marker protein, which Male et al. concludes show that they act downstream of E4bp4 in the hierarchy of NK cell development and contribute to NK cell development (Male et al., page 639 and Figure 4). It is also noted that Male et al. teaches in vitro development involved transducing the CLIP or lin- bone marrow cells with the retroviral vector encoding the transcriptional factor followed by culture in media comprises cytokines such as IL-7 and SCF (Male et al., page 641). Thus, Male et al. teaches the importance of the expression of at least 4 transcription factors, E4pb4/NFL3, EOMES, T-BET, and ETS1, and two additional transcription factors ID2, and Gata3, in NK cell development from lymphoid stem cells such as CLP cells or lineage depleted bone marrow cells, thus providing motivation to express all four of E4pb4/NFL3, EOMES, T-BET, and ETS1 in CLP cells or lineage depleted bone marrow cells in order to induce development of NK cells. Male et al. tested the effects of the expression of these transcription factors on mouse cells; however, Zhang et al. teaches that mouse and human cells follow a similar path of NK cell development from CLIP cells (referred to as CILP cells in Zhang), including the importance of expression of T-BET and EOMES for later stage mature NK cell development (Zhang et al., page 743, Figure 3). In particular, Zhang et al. teaches that T-BET and EOMES cooperate to promote NK cell maturation and homeostasis (Zhang et al., page 745). Renoux et al. further supplements Male et al. by teaching that CLP cells and lin- bone marrow cells express CD34 (Renoux et al., graphical abstract). Renoux et al. further demonstrates that human lin-CD34+ bone marrow develop in vivo in NOD/SCID mice into mature CD56+ NK cells and that such mature CD56+ NK cells express both T-BET and EOMES (Renoux et al., pages 397, 402-403, and Figure 4). Thus, in view of the of teachings of Male as to the importance of all four of E4pb4/NFL3, EOMES, T-BET, and ETS1 in NK cell development from CLP cells, the further teachings of Male et al. for the importance of additional transcriptional factors in NK cell development such as ID-2 and Gata3, the teachings of Zhang et al. that mouse and human NK development are similar and that T-BET and EOMES cooperate in NK cell development, particularly the development of mature NK cells in both mouse and humans, the teachings of Male et al. that retroviral vectors encoding transcription factors including E4pb4/NFL3, EOMES, T-BET, and ETS1 can be used to transduce CLP cells or lineage depleted bone marrow cells in order to induce development of NK cells in vitro in the presence of cytokines, and the teachings of Renoux et al. that human lin- bone marrow cells and CLP cells are CD34+ and develop into NK cells that express CD56, it would have been prima facie obvious to the skilled artisan at the time of filing to transduce mouse or human CD34+ CLP cells or lin-CD34+ bone marrow stem cells in vitro with retroviral vectors encoding E4pb4/NFL3, EOMES, T-BET, and ETS1, or E4pb4/NFL3, EOMES, T-BET, ETS1, ID2, and Gata3 in order to induce the differentiation of these cells into CD56+ NK cells with a reasonable expectation of success.
While Male et al. in view of Zhang et al. and Renoux et al. transcription factors ETS1, T-BET, E4pb4/NFL3, and EOMES which are important in both mouse and human NK cell development, none of these references including Male et al. specifically teaches the amino acid sequences of these transcription factors, or specific polynucleotide sequences which encode each of ETS1, T-BET, E4pb4/NFL3, and EOMES. However, the amino acid sequences of both mouse and human ETS1, T-BET, E4pb4/NFL3, and EOMES were well known at the time of filing, including the sequences of human ETS1, T-BET, E4pb4/NFL3, and EOMES with the amino acid sequences set forth in SEQ ID NOS 1-4 respectively. For example, the amino acid sequence of human ETS1 with 100% sequence identity to SEQ ID NO:1 was reported by Uniprot Accession Number Q6FG54_HUMAN (19-JUL-2004), the amino acid sequence of human TBET (TBX21) with 100% sequence identity to SEQ ID NO:2 was reported by Uniprot Accession Number Q9UL17 TBX21_Human (5-1-2000), the amino acid sequence of human NIFL3 with 100% sequence identity to SEQ ID NO:3 was reported by Uniprot Accession Number D91VD5_HUMAN (5-OCT-2010), and the amino acid sequence of the long isoform1 of human EOMES with 100% sequence identity to SEQ ID NO:4 was reported by EP3258268-A1 (20-DEC-2017), Luking et al., as SEQ ID NO:111. Further, the prior art teaches polynucleotide sequence encoding these transcription factors where the polynucleotides are more than 95% identical to SEQ ID NOS 22-26 respectively. KR2012061005-A (2012), hereafter referred to as Im et al. teaches a polynucleotide sequence for Human protein C-ets-1 (Ets1) identified as SEQ ID 6 which is 99.2% identical to SEQ ID NO:22. US Patent No. 6,812,339 (2004), hereafter referred to as Venter et al. teaches the polynucleotide sequence of SEQ ID NO: 3434 which is 99.7% identical to SEQ ID NO:23. WO 2002/28999 (2002), hereafter referred to as Beazer-Barclay teaches the polynucleotide sequence of SEQ ID NO: 1190 which is 99.9% identical to SEQ ID NO:24. EP3258268-A1 (2017), hereafter referred to as Luking et al., teaches a polynucleotide sequence for human EOMES with 100% sequence identity to SEQ ID NO:25. Therefore, in view of the well known amino acid sequences for ETS1, T-BET, E4bp4/NFL3, and EOMES which are 100% identical to SEQ ID NOS: 1-4 respectively, and polynucleotide sequences encoding these amino acid sequences which are greater than 95% identical to SEQ ID NOS 22-25 respectively, it would have been prima facie obvious to the skilled artisan at the time of filing to transduce mouse or human CD34+ CLP cells or lin-CD34+ bone marrow stem cells in vitro with retroviral vectors encoding ETS1, T-BET, E4bp4/NFL3, and EOMES as taught by Male et al. in view of Zhang et al. and Renoux et al. with the amino acid sequences of SEQ ID NO:1-4 respectively, and/or where the polynucleotide sequences for each transcription factor are selected from the polynucleotide sequences taught by Im et al., Venter et al., Beazer-Barclay et al., and Luking et al. respectively, in order to induce the differentiation of these cells into CD56+ NK cells with a reasonable expectation of success.
While Male et al. teaches to express the transcription factors E4pb4/NFL3, EOMES, T-BET, and ETS1 in a retroviral vector, Male et al. does not teach the use of a PRE in the vector, or the use of a naked alphavirus replicon(s) to express the transcription factors, or to express the transcription factors using a single vector encoding three or four of the transcription factors. Hvalaty et al. supplements Male et al. by teaching that the inclusion of a PRE such as the woodchuck post transcriptional regulatory element (WPRE) in a retroviral vector substantially enhanced transgene expression in transduced cells (Hvalaty et al., pages 1 and 8). Thus, based on the motivation provided by Hvalaty et al. to include a PRE such as WPRE in a retroviral vector to enhance transgene expression in transduced cells, it would have been prima facie obvious to the skilled artisan at the time of filing to include an WPRE in the retroviral vectors encoding the transcription factors E4pb4/NFL3, EOMES, T-BET, and ETS1 according to Male in view of Zhang et al. and Renoux et al. with a reasonable expectation of success.
In regards to the use of a vector other than a retroviral vector, while Male et al. teaches to generate and use a retroviral vector encoding a transcriptional factor, other vector were known in the prior art which have benefits over retroviral vectors. For example, Zhang 2014 teaches that in contrast to integrating viral vectors such as retroviral vectors which can risk insertional mutagenesis in cells, autonomously replicating genetic elements such as alphavirus RNA replicons, in particular Semliki virus replicons, are non-integrating autonomous replicating vectors which are maintained episomally and can express therapeutic sequences in target cell and tissues (Zhang 2014, pages 10 and 12). Zhang 2014 also teaches to deliver naked SFV RNA replicons (Zhang 2014, page 12). Thus, based on the advantages to using a Semliki virus replicon, particularly a naked Semliki virus replicon over a retroviral vector to express a desired sequence in a target cell as taught by Zhang et al., it would have been prima facie obvious to the skilled artisan at the time of filing to utilize the naked Semliki virus replicon to encode and express the E4pb4/NFL3, EOMES, T-BET, and ETS1 transcription factors according to Male in view of Zhang et al. and Renoux et al. with a reasonable expectation of success.
Finally, while Male et al. teaches individual retroviral vectors encoding each of E4pb4/NFL3, EOMES, T-BET, and ETS1, other vectors for expressing more than one gene of interest in cells were available at the time of filing. Fux et al. teaches the a multi-cistronic expression vector which can express three or four different genes from a single promoter (Fux et al., page 179). Fux et al. teaches that the pTRIDENT plasmid can be used to transfect cells or that the expression cassette of the pTRIDENT vector can be inserted into a lentiviral vector for cell transduction (Fux et al., page 180-181 and Figure 4, and pages 185-186 and Figure 7). In experiments using a tri-cistronic version of the plasmid or lentivirus, Fux et al. shows the comparable expression of all three encoded genes in cells (Fux et al., Figures 4 and 7). Fux et al. teaches that the advantages of the multicistronic vector are simultaneous high-level expression of multiple genes in a single cell (Fux et al., page 174). Thus, based on the advantages to using a single multicistronic cells to express multiple genes in a single cell as taught by Fux et al., it would have been prima facie obvious to the skilled artisan at the time of filing to utilize the a multicistronic plasmid or lentiviral vector as taught by Fux et al. to encode and express the E4pb4/NFL3, EOMES, T-BET, and ETS1 transcription factors according to Male in view of Zhang et al. and Renoux et al. with a reasonable expectation of success. Furthermore, as Fux et al. shows comparable expression of the genes from each cistron present in the multicistronic vector, it would have been prima facie obvious to the skilled artisan at the time of filing to include the four polynucleotide sequences for E4pb4/NFL3, EOMES, T-BET, and ETS1 in any order in the multicistronic vector taught by Fux et al. with a reasonable expectation that all four transgenes would be capable of being expressed from the vector in cells.
No claims are allowed.
Any inquiry concerning this communication from the examiner should be directed to Anne Marie S. Wehbé, Ph.D., whose telephone number is (571) 272-0737. If the examiner is not available, the examiner’s supervisor, Maria Leavitt, can be reached at (571) 272-1085. For all official communications, the technology center fax number is (571) 273-8300. Please note that all official communications and responses sent by fax must be directed to the technology center fax number. For informal, non-official communications only, the examiner’s direct fax number is (571) 273-0737. For any inquiry of a general nature, please call (571) 272-0547.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634