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 .
Application Status
This action is written in response to applicant’s correspondence received March 17, 2026. Claims 1-4, 6, 10-15, 17-22, 24-26, and 29-35 are amended. Claims 5, 7-9, 16, 23, 27, 28, and 36-48 are cancelled. Claims 49-65 are new. Accordingly, claims 1-4, 6, 10-15, 17-22, 24-26, 29-35, and 49-65 are currently pending and under consideration.
Withdrawn Claim Rejections 35 USC 102
Applicant’s arguments “Roth teaches gene editing of T cells after stimulation and culture in expansion conditions which include stimulatory cytokines such as IL-2, see page 14 paragraph 4, filed March 17, 2026, with respect to the rejection(s) of claim(s) 1-18, 26-28, 30-36 under 102(a)(1) have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of Seki et al, Oh et al, Nussing et al and Roth et al (see full citations in section 17).
Information Disclosure Statement
The information disclosure statement filed 23 Feb, 2023 fails to comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. It has been placed in the application file, but the information referred to therein has not been considered.
The information disclosure statement (IDS) submitted on March 17, 2026 was filed after the mailing date of the Non-final Office Action on December 17, 2025. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Priority
Acknowledgment is made of applicant's claim for domestic priority based on a US provisional application No. 63/071,236 filed on 27 Aug, 2020.
Claim Objections
Claim 22 is objected to because of the following informalities: Claim 22 recites “wherein the antigen-binding polypeptide is a CAR”; however, this is redundant because this recitation has already been recited in claim 19. Appropriate correction is required.
Claim Interpretation
Independent claims 1 and 3 recite the limitation “a viral vector free method”, and the specification discloses a “vector” comprises an isolated nucleic acid, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compound, plasmids, and viruses, as well as non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells ([0276]). Therefore, the limitation of “a viral vector free method’ is interpreted as a method comprising an isolated nucleic acid that is not a viral vector, which include, but are not limited to, linear polynucleotides, non-viral plasmids, and non-viral compounds.
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 2, 19-22, 24-25, 32, and 59-61 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.
Claim 2 recites the limitation "the unstimulated immune cells within the population”. It is unclear whether it is referring to the population of unstimulated immune cells in (a) or (b) of claim 1. It is unclear whether about 10% or higher of the population of (a) are modified to express the antigen-binding polypeptide because that is the percentage of unstimulated immune cells subjected to transfection. It is unclear whether about 10% or higher of the population of (b) are modified to express the antigen-binding polypeptide due to transfection efficiency and cell survival rate post-transfection. Further, the method of claim 1 does not require (a) and (b) to be performed in chronological order; thus, under the broadest reasonable interpretation, (b) can be performed prior to (a), which further supports the lack of antecedent basis for the recited limitation in claim 2. In addition, claim 1 recites wherein the HDR template facilitates HDR at the target site to generate at least one modified unstimulated immune cell. It is unclear whether about 10% or higher of unstimulated immune cells resulted as modified unstimulated immune cells, which is supported by the instant disclosure (FIG. 7). For compact prosecution, claim 2 is interpreted as wherein about 10% of higher are modified unstimulated immune cells expressing the antigen-binding polypeptide, which is measured as transfection efficiency.
Claim 19 recites the antigen-binding polypeptide is a chimeric antigen receptor (CAR) that comprises an antigen-binding domain, or an exogenous T cell receptor (TCR). It is unclear whether the CAR comprises either an antigen-binding domain or an exogenous TCR, meaning that these two are alternatives of each other. It is unclear whether the antigen-binding polypeptide is a CAR or an exogenous TCR. The specification teaches a CAR comprises an antigen-binding domain ([0138]), a transmembrane domain may be derived from the TCR ([0150]), and an intracellular domain that includes a domain from TCR ([0174). However, there is no mention of TCR in the teachings of antigen-binding domain. Therefore, the specification does not support a CAR that comprises an antigen-binding domain or an exogenous TCR. Further, the specification teaches “in some instances, the antigen-binding polypeptide is a CAR. In some instances, the antigen-binding polypeptide is a TCR”. Based on this teaching, claim 19 is interpreted as wherein the antigen-binding polypeptide is an exogenous TCR or a CAR that comprises an antigen-binding domain. However, an appropriate amendment is required.
Under this interpretation of claim 19 and in the interest of compact prosecution, dependent claim 21 is interpreted as wherein the CAR comprises a transmembrane domain, dependent claim 22 is interpreted as wherein the CAR comprises a 4-1BB costimulatory domain and a human CD3 zeta chain cytoplasmic signaling domain, dependent claim 25 is interpreted as wherein the exogenous TCR comprises a TCR alpha chain and a TCR beta chain, and dependent claim 61 is interpreted as wherein the exogenous TCR is a wild-type TCR, a high affinity TCR, or a chimeric TCR. However, appropriate amendments are required.
Claim 32 recites “comprising delivering a second guide RNA into the unstimulated immune cells” which lacks antecedent basis in the claim. It is unclear which population of unstimulated immune cells, (a) or (b) the second guide RNA is delivered into. Is it unclear whether the second guide RNA is delivered into the population of (a) along with components (i), (ii), and (iii) or claim 32 is reciting an additional step in the method of claim 1 by delivering a second guide RNA to the population of (b) after culturing the cells under non-expansion condition. For compact prosecution, claim 32 is interpreted as delivering a second guide RNA into the population of (a) along with components (i), (ii), and (iii).
Those claims included in the statement of rejection but not otherwise discussed are rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
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.
Claims 1-4, 10-13, 17-21, 24, 26, 29-35, 49-54, and 57-58 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 a method of preparing a population of modified unstimulated CD4+ and CD8+ T cells, does not reasonably provide enablement for a method of preparing a population of any modified unstimulated immune cells not limited to CD4+ and CD8+ T cells. 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.
The test of enablement is whether one skilled in the art could make and use the claimed invention from the disclosures in the specification coupled with information known in the art without undue experimentation (United States v. Telectronics., 8 USPQ2d 1217 (Fed. Cir. 1988)). Whether undue experimentation is needed is not based upon a single factor but rather is a conclusion reached by weighing many factors. These factors were outlined in Ex parte Forman, 230 USPQ 546 (Bd. Pat. App. & Inter. 1986) and again in In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988), and the most relevant factors are indicated below:
Nature of the Invention and Breadth of the Claims
Claims are drawn to a method of preparing a population of modified unstimulated immune cells, which encompasses a broad genus of immune cell types. Dependent claims 14-15, 6, and 55-56 drawn to modified unstimulated T cells only represent a subgenus within the broad genus of immune cells. Thus, the breadth of the claims extends beyond unstimulated T cells to all immune cell populations from different hosts and possessing distinct biological properties, functions, activation requirements, and susceptibilities to genome editing.
Guidance of the Specification
The specification discloses “in some embodiments…the population of unstimulated immune cells: (a) comprises unstimulated T cells, unstimulated Natural Killer (NK) cells, unstimulated natural killer T (NKT) cells, or a combination hereof” ([0016]). However, the specification teaches only one working example involving unstimulated CD4+ and CD8+ T cells, wherein a fluorescent green protein donor HDR template was introduced using CRISPR Cas9 RNP complex (FIG. 7). The specification does not demonstrate successful modification of other unstimulated immune cell species within the claimed genus, nor does it provide guidance to make and use the claimed method spanning the breadth of all immune cells.
State of the Art
At the time of the filling, the state of the art did not establish that diverse unstimulated immune cells can be modified using a common workflow. Seki et al (Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells; Journal of Experimental Medicine, 2018, 215(3):985-997; IDS received on 11/22/2024, Cite No. D2) teach that naïve (i.e., unstimulated) mouse T cells required culture with cytokine factor IL-7 to maintain cell viability without inducing activation, and that IL-7 conditions were identified through empirical screening among additional cytokine factors. Seki et al further teach “in contrast, IL-2 and IL-15 induced proliferation in CD8+ T cells but did not preserve cell viability in CD4+ T cells (pg. 990, col. 1, para. 2). If unstimulated T cells themselves require cytokine screening and tailored culture conditions merely to survive and maintain unstimulated cell state prior to transfection, one ordinary skilled in the art would not reasonably extrapolate guidance from the specification to the full genus of unstimulated immune cells.
In addition, Rautela et al (Drug target validation in primary human natural killer cells using CRISPR RNP; J Leukoc Biol, 2020, 108:1397-1408) teach “[natural killer] cells are notoriously difficult to infect with retro/lentiviral particles meaning that tried and tested genetic modifications approaches for human T cells are not appropriate for human NK cells” (pg. 1398, col. 1, para. 1). Rautela et al further teach a viral-free CRISPR-RNP gene-editing strategy for primary human NK cells that required NK cells to be stimulated with cytokine IL-15 prior to transfection (pg. 1398, col. 1, para. 2), which is a cytokine found not effective to preserve cell viability in CD4+ T cells taught by Seki et al (pg. 990, col. 1, para. 2). Thus, the state of the art does not support a reasonable expectation that claimed method demonstrated only in unstimulated CD4+ and CD8+ T cells would be broadly applicable to all unstimulated immune cells.
Experimentation Required
In order to practice the claimed invention, an immense amount of experimentation would be required. For example, it would be necessary for one of ordinary skill in the art to conduct substantial screening and culturing conditions analogous to the IL-7 optimization taught by Seki et al across distinct immune cell populations and establish which specific immune cell types can maintain unstimulated state while still allow viral vector-free HDR-mediated genome modifications.
Conclusion
Taking into consideration the factors outlined above, including the nature of the invention, the breadth of the claims, the state of the art, the guidance provided by the applicant and the specific examples, it is the conclusion that an unreasonable amount experimentation would be required to make and use the invention as claimed. Therefore, claims 1-4, 10-13, 17-21, 24, 26, 29-35, 49-54, and 57-58 are not considered to be fully enabled by the instant disclosure.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 6, 10-15, 17-18, 26, 30-35, 49, 52-58, and 63-65 are rejected under 35 U.S.C. 103 as being unpatentable over Seki et al (Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells; Journal of Experimental Medicine, 2018, 215(3):985-997; IDS received on 11/22/2024, Cite No. D2) in view of Oh et al (Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells; Current Protocols in Immunology, 2018, 124(e69):1-18), Nussing et al (Efficient CRISPR/Cas9 gene editing in uncultured naïve mouse T cells for in vivo studies; The Journal of Immunology, 2020, 204(8):2308-2315; IDS received on 02/23/2023, Cite No. A3) and Roth et al (WO 2018/232356 A1; Published Date: 20 Dec, 2018).
Regarding claims 1, 12, 13, 18, 30, 31, 32, 53-56, 58, and 63, Seki et al teach a method of preparing a population of modified unstimulated immune cells (e.g., naïve T cells), comprising:
(a) delivering into naïve T cells (i.e. a population of unstimulated immune cells) isolated from spleens and lymph nodes (i.e. biological samples) of mice (pg. 995, col. 1, para. 3) (i) ribonucleoprotein (RNP) complex containing CRISPR Cas9 nuclease (i.e. a gene editing nuclease) and (ii) one or more single guide RNAs (sgRNA) by electroporation (i.e. a transfection method), wherein the RNP complex generate a double-stranded break at a target site within at least one unstimulated T cell (pg. 986, col. 2, para. 2; pg. 988, col. 2, para. 2; Fig. 4A); and
(b) culturing the population of unstimulated immune cells under non-expansion conditions for 24 hours (i.e. 18 hours to about 72 hours), which under the broadest reasonable interpretation, this step can be performed before or after step (a). Seki et al teach culturing resting murine CD4+ and CD8+ T cells in complete T cell media containing IL-7 before transfection, which drastically improved cell viability without inducing proliferation or strong up-regulation of activation markers (pg. 990, col. 1, para. 2). Seki et al also teach culturing the naïve T cells in complete T cell media for several days post-transfection without the need for TCR or cytokine stimulation, specifically 48-72 h (pg. 988, col. 2, para. 1).
Seki et al does not teach delivering (iii) a homology-directed repair (HDR) template comprising a polynucleotide encoding an antigen-binding polypeptide, and wherein the HDR template facilitates HDR at the target site to generate at least one modified unstimulated immune cell expressing the antigen-binding polypeptide within the population.
However, Seki et al explicitly teach “the ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function, but also holds great promise for T cell-based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T-cells”, and previous attempts of generating CAR-T cels “required T cell receptor stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation” (abstract). Seki et al further teach a strategy to develop CAR-T cells involve disruption of inhibitory receptors such as CTLA-4 or PD-1 (pg. 985, paragraph bridging col 1 and 2).
Oh et al teach each step of the method of Seki et al in details, from purifying naïve T cells from mice spleen and lymph nodes, preparing RNP complexes, electroporation, culturing electroporated naïve T cells under non-expansion conditions to stimulating the modified naïve T cells and expanding under stimulation conditions (whole document). Oh et al further teach the method of Seki et al can successfully disrupt inhibitory receptors such as CTLA-4 and PD-1 in naïve human CD4+ and CD8+ T cells (Fig. 3, pg. 7), positively demonstrating that the method of Seki et al can be used for development of CAR-T cells.
In addition, Nussing et al teach a method of preparing a population of modified unstimulated immune cells (e.g., naïve T cells) comprising delivering into naïve T cells isolated from spleens and lymph nodes of mice (supplemental material, pg. 2-3), “without in vitro T cell activation” (pg. 2308, col. 2, para. 2) a RNP complex containing CRISPR Cas9 nuclease, one or more sgRNAs, and a viral vector-free HDR template by electroporation (pg. 2309, col. 1, para. 3 and col. 2, para. 2). Nussing et al further teach the viral vector-free HDR template is a double-stranded DNA generated via PCR amplification (pg. 2309, col. 1, para. 3) and encodes for CD90.2 marker with a single nucleotide difference to convert endogenous CD90.2 into the CD90.1 congenic marker (pg. 2312, col. 2, para. 1). Nussing et al further teach that HDR editing efficiencies achieved in naïve T cells are “similar to that observed with the template in in vitro cultured and activated T cells”, thereby demonstrating that HDR editing can be successfully performed in naïve T cells using CRISPR Cas9 RNP complexes and results are comparable to those employed in stimulated or activated T cells (pg. 2313, col. 2, para. 1).
Further, Roth et al teach a method of preparing a population of modified T cells comprising: (b) culturing a population of unstimulated or naïve T cells obtained from a biological sample under non-expansion conditions (e.g., “cultured in media without stimulation”) for one day ([0100], [0023]), stimulating the naïve T cells, followed by delivering into a population of stimulated T cells RNP complexes comprising a Cas9 nuclease, a guide RNA, and a HDR template ([0010], [0068]), wherein the Cas9 nuclease and guide RNA form a complex to generate a double-stranded break at a target site and wherein the HDR template facilitates HDR at the target site to generate modified immune cells ([0058]). Roth further teaches the HDR template can encode a polypeptide or fragment thereof ([0058]), and this method can be used for generation of recombinant T cells (0089] wherein “the T cell receptor can be replaced with a heterologous T cell receptor, with a polypeptide having a different receptor domain, such as an antibody or antibody fragment” ([0065]), and with a chimeric antigen receptor ([0065]). Thus, Roth’s method comprises a HDR template encoding for an antigen-binding polypeptide to develop CAR-T cells.
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to modified Seki’s method to develop CAR-T cells comprising delivering a HDR template in naïve T cells taught by Nussing et al because would have merely amounted to a simple combination of prior art elements to yield predictable results. Each element in the combination (CRISPR Cas9 RNP and HDR template) performs the same function as it does separately (CRISPR Cas9 nuclease generates a double-stranded break at a target site in the naïve T cell and the HDR template provides the polynucleotide to be inserted at the target site to express the encoded antigen-binding polypeptide). Following this modification, it would have also been obvious to substitute the HDR template of Nussing et al with a HDR template encoding for an antigen-binding polypeptide, particularly a chimeric antigen receptor taught by Roth et al because it would have merely amounted to a simple substitution of prior art elements to yield predictable results. The substituted components (HDR template encoding a CD90.1 congenic marker to a HDR template encoding a chimeric antigen receptor) and their functions were known in the art and the results of the substitution would have been predictable (generation of modified CAR-T cells). One would have been motivated to have done so for the advantage of developing next-generation CAR-T cells suggested by Seki et al through modifying naïve T cells without T cell receptor stimulation and preclusion of key genes involved in T cell activation or differentiation. One would have had a reasonable expectation of success in doing so because Seki et al, Oh et al, and Nussing et al teach methods of preparing modified unstimulated T cells by CRISPR Cas9 RNP and electroporation, and Nussing et al expressly teach that naïve T cells are amenable not only to knockout editing, but also to HDR-mediated genome modifications. Further, Sekit et al, Oh et al, and Roth et al teach methods wherein the edited T cells after electroporation are subsequently cultured under activation and expansion conditions, which positively demonstrating that stimulation and expansion following CRISPR Cas9 genome editing are conventional aspects of T cell engineering workflows and are not limited to methods involving naïve T cells. Moreover, the confirmation by Nussing et al that HDR editing efficiencies in naïve T cells “is similar to that observed with the template in in vitro cultured and activated T cells” (pg. 2313, col. 2, para. 1) provides a reasonable expectation of success for substituting the HDR template to one encoding for an antigen-binding polypeptide taught by Roth et al.
Regarding claim 2, Nussing et al teach wherein 4.5% of naïve T cells are modified (Fig. 6), and “homology arms of at least 250 bp either side of the Cas9 cut side were optimal, however, this may need to be optimized independently for each experiment” (supplemental material, pg. 6, step 1), demonstrating that transfection efficiency can be optimized. Roth et al also teach transfection of T cells via CRISPR Cas9 RNP and HDR template achieve transfection efficiency exceeding 10% ([0073] and Fig. 12b). Thus, in view of the obviousness discussed above as applied to claim 1, a person of ordinary skilled in the art would have reasonably expected that optimization of the methods of Seki et al and Nussing et al would yield transfection efficiency about 10 % or higher because this efficiency is a result-effective variable that is dependent on parameters including HDR template homology arm length demonstrated by Nussing et al, RNP complex amount, Cas9 and sgRNA ratios, HDR template concentrations, electroporation conditions demonstrated by Seki et al (Fig. 2A and Fig. 3C).
Regarding claims 3, 4, 6, and 65, the obviousness of modifying the method of Seki et al to introduce HDR at a target site in unstimulated immune cells is discussed above as applied to claims 1 and 2.
Regarding claims 10, Nussing et al teach the following concentrations are used for preparation of RNP complex for delivery into naïve T cells:
(a) 3x1012 copies of 984-bp sized DNA HDR template in a final volume of 5 µL, which is equivalent to 1.0 µM (i.e., about 1 pM to about 10 mM) (pg. 2313, col. 2, para. 1; supplemental material pg. 4, step 2), and
(b) 0.6 µL of Cas9 protein (10 mg/mL) in a final volume of 5 µL, which is equivalent to 7.5 µM (i.e., about 1 pM to about 10 mM) given the molecular weight of SpCas9 protein is about 16 kDa (supplemental material pg. 4, step 2), and
(c) 1 µL of sgRNA (0.3 nmol/µL) in a final volume of 5 µL, which is equivalent to 60 µM (i.e., about 1 pM to about 10 mM) (supplemental material pg. 4, step 2).
Regarding claims 11, 49, based on the calculations in claim 10, Nussing et al teach wherein the ratio of Cas9 nuclease to sgRNA is 1:8. However, Seki et al teach a 1:3 ratio of Cas9 nuclease to sgRNA dramatically increased efficiency compared to a 1:1 ratio (pg. 986, col. 2, para. 2), which suggests that the ratio of Cas9 and sgRNA is routinely optimized to achieve desired editing efficiency. Thus, in view of the obviousness discussed above as applied to claim 2, one would have reasonably expected that optimization of the methods of Seki et al and Nussing et al would comprise of ratios of Cas9 to sgRNA to about 2:1 and about 1:2 as Seki et al already teach a ratio of about 1:2 indeed yielded increased efficiency.
Regarding claims 14 and 15, all three methods of modifying naïve T cells teach naïve CD4+ or CD8+ T cells are enriched using anti-CD4 or anti-CD8 labeled microbeads (Seki et al: pg. 995, col. 1, para. 3; Oh et al: pg. 3-7; Nussing et al: pg. 2309, col. 1, para. 3 and supplemental material, pg. 3, steps 8-10).
Regarding claim 17, Seki et al teach wherein the modified unstimulated immune cells are cultured under non-expansion conditions for 24 h before transfection (pg. 995, col. 1, para. 5) and 48-72 h after transfection considering that “human T cells can be cultured in complete T cell media for several days without the need for TCR or cytokine stimulation” (pg. 988, col. 2, para. 2).
Regarding claim 26, the obviousness to modify the HDR template of Nussing et al to encode an antigen-binding polypeptide taught by Roth et al is discussed above as applied to claim 1. Nussing et al further teach wherein the HDR template comprises at least 250 bp on either side of the Cas9 cut site (supplemental material, pg. 6, step 1) which indicates that the HDR template comprises a 5’ and a 3’ homology arm upstream and downstream of the polynucleotide encoding the antigen-binding polypeptide that is homologous to a genomic region of the target site.
Regarding claim 33, Seki et al teach wherein the biological sample is an apheresis sample (pg. 993, col. 1, para. 5).
Regarding claims 34, 35, and 64, Oh et al teach after RNP transfection, the modified unstimulated T cells are stimulated in solution containing anti-CD28 or anti-CD28 plus IL-2 and cultured under expansion conditions for up to 72 hours (pg. 12, subsection “polyclonal T cell receptor-stimulation of RNP-transfected T cells”).
Regarding claim 52, Nussing et al teach wherein the HDR template is a double-stranded DNA template (pg. 2309, paragraph bridging col. 1 and 2).
Regarding claim 57, Seki et al modified unstimulated human T cells are cultured in non-expansion conditions, followed by transfection efficiency analysis and stimulation (pg. 988, col. 2, para. 2 and Fig. 2A).
Seki et al do not teach wherein the modified unstimulated immune cells are further resuspended in a cryopreservant solution and cryo-frozen.
Roth et al teach unstimulated T cells can be frozen in Bambaker freezing medium (i.e., cryopreservant solution and cryo-frozen) for storage and usage at desired time by following manufacturer’s instructions to thaw and culture in media without stimulation for one day ([0100]).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to modified the method of Seki et al to resuspend the modified unstimulated immune cells in a cryopreservant solution because it merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. One would have been motivated to have done so for the advantage of freezing modified unstimulated T cells for long-term storage and downstream analysis at a desired time. One would have had a reasonable expectation of success in doing so because Roth et al teach unstimulated T cells can be frozen and recovered by following specific instructions ([0100]) and Seki et al teach “loss of target protein expression may take 48-72 h or longer. Human T cells can be cultured in complete T cell media for several days without the need for TCR or cytokine stimulation” (pg. 988, col. 2, para. 2).
Claims 19-22, 24-25, 29, 59-60, and 61-62 are rejected under 35 U.S.C. 103 as being unpatentable over Seki et al (Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells; Journal of Experimental Medicine, 2018, 215(3):985-997; IDS received on 11/22/2024, Cite No. D2) in view of Oh et al (Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells; Current Protocols in Immunology, 2018, 124(e69):1-18), Nussing et al (Efficient CRISPR/Cas9 gene editing in uncultured naïve mouse T cells for in vivo studies; The Journal of Immunology, 2020, 204(8):2308-2315; IDS received on 02/23/2023, Cite No. A3) and Roth et al (WO 2018/232356 A1; Published Date: 20 Dec, 2018) as applied to claim 1, and in view of Brandt (WO 2019/070541 A1; Published Date: 11 Apr, 2019).
Regarding claims 19, 24, and 59, the obviousness of modifying the method of Seki et al to introduce a HDR template encoding for an antigen-binding polypeptide as taught by Roth et al is discussed above as applied to claim 1.
Roth et al teach the antigen-binding polypeptide comprises a chimeric antigen receptor (CAR) ([0065]).
Roth does not teach wherein the CAR comprises an antigen-binding domain that binds to a tumor antigen.
Brandt teaches a method of modifying T cells using CRISPR Cas9 gene-editing and HDR templates to express antigen-binding polypeptides such as antigen receptor (i.e., antigen-binding domain), cell surface receptor, recombinant CARs, TCR-like CARs, and CD19 marker ([0428] and [0364]), which is a tumor antigen associated with hematologic malignancy.
It would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to modified the HDR template of Roth et al with the antigen-binding polypeptides taught by Brandt because it would have merely amounted to a simple substitution of Roth’s recombinant CAR with Brandt’s antigen-binding polypeptides according to known methods to yield predictable results. The substituted components and their functions are known in the art. Further, the substituted components, chimeric antigen receptor and additional antigen-binding polypeptides taught by Brandt serve the same purpose. One would have been motivated to have done so for the advantage of obtaining modified T cells expressing various types of antigen-binding polypeptides for uses in adoptive cell therapy, such as treatment of diseases and disorders associated with HPV 16 E6 or E7 expression as taught by Brandt. One would have had a reasonable expectation of success in doing so because both Roth and Brandt teach methods of modifying T cells using CRISPR Cas9 nuclease and HDR insertions, especially Brandt teach T cells can be modified via CRISPR RNP and HDR templates to express these antigen-binding polypeptides.
Regarding claims 20 and 60, the obviousness to modify the antigen-binding polypeptide of Roth et al that comprises a CAR to comprise an antigen-binding domain binding to a tumor antigen or an exogenous T cell receptor taught by Brandt is discussed above as applied to claim 19. Brandt further teach the antigen-binding domain comprises a single-chain variable fragment (scFv) ([0375]).
Regarding claims 21 and 22, Brandt teaches the antigen-binding polypeptide is a CAR that comprises transmembrane domain derived from alpha, beta or zeta chain of the T-cell receptor ([0411]), and an intracellular domain with a 4-1BB costimulatory ([0423]) and a CD3 zeta chain cytoplasmic signaling domain ([0418]) that mediates T-cell activation and cytotoxicity. The obviousness to modify the antigen-binding polypeptide of Roth et al that comprises a CAR to comprise an antigen-binding domain binding taught by Brandt is discussed above as applied to claim 19. One would have also been motivated to modify the CAR to comprise a transmembrane domain and an intracellular domain for the advantage of improving T-cell activation and target efficiency.
Regarding claims 25 and 61, the obviousness to modify the antigen-binding polypeptide of Roth et al to antigen-binding polypeptides taught by Brandt is discussed above as applied to claim 19. Further, Brandt teaches an antigen-binding polypeptide that is an exogenous TCR comprising an alpha chain and a beta chain (]0992]) and recombinant TCRs as chimeric TCRs containing a mouse constant region ([1042]).
Regarding claims 29 and 62, Roth teaches wherein the target site is in the RAB11A locus.
Roth does not explicitly teach wherein the target site is in the TRAC locus.
Brandt teaches the target site is at or near TRAC locus ([0581]), and specifically between the most 5’ nucleotide of exon 1 and upstream of the most 3’ nucleotide of exon 1 ([0629]).
Thus, following the obviousness to modify the HDR template of Roth et al to express CAR that comprises exogenous TCR, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to target the TRAC locus because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substituted locus, their locations, and functions are known in the prior art and serve the same purpose of substituting endogenous receptor expression with recombinant TCR. One would have been motivated to have done so for the advantage of eliminating the endogenous TCR expressed from the TRAC locus to prevent cross pairing between the exogenous and endogenous TCRs. One would have had a reasonable expectation of success in doing so because Roth et al and Brandt teach method of generating modified CAR-T cells using CRISPR Cas9 RNP and HDR template.
Claims 50 and 51 are rejected under 35 U.S.C. 103 as being unpatentable over Seki et al (Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells; Journal of Experimental Medicine, 2018, 215(3):985-997; IDS received on 11/22/2024, Cite No. D2) in view of Oh et al (Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells; Current Protocols in Immunology, 2018, 124(e69):1-18), Nussing et al (Efficient CRISPR/Cas9 gene editing in uncultured naïve mouse T cells for in vivo studies; The Journal of Immunology, 2020, 204(8):2308-2315; IDS received on 02/23/2023, Cite No. A3) and Roth et al (WO 2018/232356 A1; Published Date: 20 Dec, 2018) as applied to claim 1, and in view of Schumann et al (Generation of knock-in primary human T cells using Cas9 ribonucleoproteins; PNAS, 2015, 112(33):10437-10442).
Regarding claims 50 and 51, the obviousness of modifying the method of Seki et al to introduce a HDR template encoding for an antigen-binding polypeptide as taught by Roth et al is discussed above as applied to claim 1. Nussing et al teach wherein the ratio the HDR template to RNP complex and HDR template to nuclease is about 7:1.
Nussing et al do not teach wherein the ratios are about 5:1 to about 1:1.
Schumann et al teach a method of generating a population of modified T cells using CRISPR Cas9 RNP of 50 pmol and a HDR template of 50, 100, and 200 pmol (pg. 10438, col. 1, para. 1 and pg. 10439, col. 1, para. 2), which is equivalent to a ratio of the HDR template to RNP complex of about 1:1 and a ratio of the HDR template to nuclease of about 1:1.
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to modified method of Nussing et al with the ratios taught by Schumann et al because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substituted ratios is a parameter for optimization of HDR editing efficiency and they serve the same purpose. One would have been motivated to have done so for the advantage of obtaining the optimal conditions to achieve highest HDR-mediated editing in T cells as taught by Schumann et al. One would have had a reasonable expectation of success in doing so because Schumann et al teach this optimization works to CRISPR Cas9 RNP and HDR-mediated genome modifications in T cells.
Response to the Arguments
Applicant’s arguments, received on March 17, 2026, with respect to claim(s) 19-25, and 29 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument. In this case, Applicant’s arguments “Roth does not teach the editing of unstimulated immune cells” (pg. 15, para. 3) and “Brandt is silent regarding the editing of unstimulated immune cells and provides no motivation to edit immune cells prior to stimulation (pg. 16, para. 1) are not persuasive to overcome the current prior art rejection in view of Seki et al, Oh et al, and Nussing et al who teach methods of modifying unstimulated T cells via delivery of Cas9 ribonucleoprotein complexes, and Nussing et al explicitly teach delivering a HDR template to facilitate HDR at a target site in the genome of unstimulated T cells (see sections 17 and 18).
Conclusion
No claims are allowable.
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QIWEN SU-TOBON
Examiner
Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
Prosecution Insights