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 .
DETAILED ACTION
This action is in response to the papers filed on November 12, 2024. Claims 1-5 are currently under examination.
Priority
The present application, filed on May 10, 2023, is a CON of PCT/CN2023/093246, filed May 10, 2023. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). Receipt is acknowledged of priority document electronically retrieved by USPTO from a participating IP Office on November 25, 2024. Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 3 7 CPR 41. l 54(b) and 41.202(e). Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Thus, the priority date is not yet perfected and the earliest possible priority for the instant
application is May 12, 2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on November 12, 2024 is acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim objections
Claim 1 is objected to and should be amended to recite: “A method of preparing natural killer (NK) cells”, in line 1.
Claim 1 is objected to and should be amended to recite: “preparation method comprises the following steps”, in line 3.
Claim 1 is objected to and should be amended to recite: “for knocking out the CD38 gene”, in line 4.
Claim 1 is objected to and should be amended to recite either: “a medium employed for continued culture” or “a medium employed for continuing the culture”, in line 17.
Claim 1 is objected to for the following informalities: in line 19, after and 1 vol% of double antibody to an MEM, there is no proper punctuation.
Claim 2 is objected to and should be amended to recite: “The method of preparing NK cells”, in line 1.
Claim 2 is objected to because of the following informalities: abbreviations such as
P3000 should be spelled out at the first encounter in the claims. Appropriate correction is
Required.
Claim 2 is objected to and should be amended to recite: “adding the mixture from the second EP tube to the first EP tube”, in line 7.
Claim 2 is objected to and should be amended to recite: “adding the mixture to the NK cells of step (2) dropwise after”, in line 8.
Claim 3 is objected to and should be amended to recite: “The method of preparing NK cells”, in line 1.
Claim 3 is objected to and should be amended to recite: “are prepared by the following steps”, in line 2.
Claim 3 is objected to and should be amended to recite: “culturing HEK 293T cells for lentivirus production”, in line 5.
Claim 3 is objected to because of the following informalities: To avoid any ambiguity, uncommon abbreviations should be fully defined on their first occurrence in each claim, unless they have been defined in a claim upon which the claim containing the abbreviation depends. Conversely, once an abbreviation has been identified, the abbreviation can be used after first occurrence. Claim 3 should be amended to recite “Multiplicity of Infection (MOI)”, in line 22.
Claim 3 is objected to because of the following informalities: “and continuing to culture, in which the third cell culture medium is employed to culture”, in line 25. This grammatically incorrect and should be amended to recite the relationship of the second cell culture medium to the third cell culture medium.
Claim 3 is objected to and should be amended to recite: “more than 10 times the starting number”, in lines 29-30.
Claim 4 is objected to and should be amended to recite: “A method of preparing NK cells”, in line 1.
Claim 4 is objected to and should be amended to recite: “(2-8); or wherein”, in lines 5-6.
Claim Interpretation
The claims are directed to “polynucleotide knocked out of CD38.” This recitation, in claim 1, is interpreted as targeting the CD38 gene in the NK92MI cell, rather than targeting the CD38 protein itself. This interpretation is consistent with the specification, which distinguishes the CD38 antigen or protein from the CD38 gene and describes utilizing the CRISPR-Cas editing system for knocking out the CD38 gene. Hence, the broadest reasonable interpretation is that the claimed knockout disrupts the cellular CD38 genetic locus to either reduce or eliminate CD38 expression.
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.
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 1-5 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 employing the specifically disclosed Cas9-based CRISPR editing system for knockout of CD38 in NK91MI cells, does not reasonably provide enablement commensurate with the full scope of the presently claimed methods without undue experimentation. Independent claim 1 broadly recites “constructing a CRISPR-Cas editing system for knocking out CD38 gene,” without limiting the CRISPR-Cas system to Cas9, the guide RNA disclosed in the specification, or the Cas9/gRNA configuration demonstrated by the disclosure, see instant specification [0089]. 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.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make or use the claimed invention, if not, whether an artisan would require undue experimentation to make and use the claimed invention and whether working examples have been provided. Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 USC § 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (CA FC 1988).
Wands states on page 1404,
"Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex parte Forman. They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skills of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims."
Claims 1-5 encompass a method in which essentially any “CRISPR-Cas editing system” directed at knocking out CD38 may be employed. However, the specification’s disclosure is limited to the Cas9 system ([0089]). The specification does not provide representative working examples employing other CRISPR-Cas nuclease systems, nor does it provide design parameters disclosing how the disclosed Cas9 guide, targeting configuration, transfection conditions, and selection procedure may be translated to other CRISPR-Cas effectors while obtaining the claimed CD38- knockout NK92MI cells. The breath of any CRISPR-Cas editing system substantially exceeds the disclosed Cas9 implementation.
The predictability of the art is limited, and the claimed “CRISPR-Cas editing system” encompasses CRISPR effectors having materially different targeting and cleavage requirements. For instance, Zetsche et al. (Cell. 2015 Oct 22;163(3):759-71. Epub 2015 Sep 25.) discloses that Cpf1 (Cas12a), unlike Cas9, lacks tracerRNA, employs a different guide-RNA architecture, recognizes a different PAM, and generates staggered DNA cleavage, demonstrating that CRISPR-Cas nucleases are not functionally interchangeable. Strecker et al. (Nat Commun. 2019 Jan 22;10(1):212.) further demonstrates that a Cas12b nuclease required engineering to achieve robust genome editing under mammalian-cell conditions, demonstrating that activity of one CRISPR-Cas system under specific cellular conditions does not establish operability of other CRISPR-Cas systems under those same conditions. Moreover, Kosicki et al. (Nat Commun. 2022 Jun 14;13(1):3422.) demonstrates that Cas9-induced editing itself can produce differing repair outcomes, including large deletions and smaller indels, depending upon cellular DNA repair pathways.
As summarized by Makarova et al. (Nat Rev Microbiol. 2020 Feb;18(2):67-83. Epub 2019 Dec 19.), CRISPR-Cas biological systems span multiple classes, types and subtypes, far beyond Cas 9 alone, and act through a variety of different effectors, targeting mechanisms, and complex cascades, not just a single endonuclease that creates a simple DNA DSB (Abstract; pg. 3, para. 4). The field recognizes that “CRISPR nuclease” is not a single, predictable toolset but a diverse family with distinct effectors and mechanisms. Makarova clarifies this biological system encompass class 1 systems and class 2 systems, with different types such as II, V and VI, each defined by a single-protein effector Cas9 (type II) uses two nuclease domains for DNA cleavage, Cas12 (type V) uses a single RUVC-like domain for DNA cleavage, and Cas13 (type VI) targets RNA rather than DNA (pg. 7, Section: CRISPR-Cas classification). These differences in nucleic acid substrate, domain architecture, and effector composition show that an artisan would not reasonably expect a single gene editing design or set of conditions to work across the full scope of “CRISPR nucleases” without undue experimentation, particularly where the claims read on both DNA-targeting (Cas9/Cas12) and RNA-targeting (Cas13) nucleases.
Hence, undue experimentation would be required because different CRISPR-Cas effectors are not merely interchangeable versions of Cas9. Different CRISPR systems can employ different guide architectures, targeting requirements, PAM requirements, cleavage mechanisms, and nucleic acid substrates. The disclosure of a Cas9 system directed to a particular CD38 target does not itself disclose or teach the substitution of another CRISPR-Cas effector and retain successful knockout of CD38 in the claimed NK9MI cells. Such a substitution would be unpredictable as it requires at least an identification of an appropriate CRISPR effector, compatible CD38 target site, guide design, delivery and editing conditions, and CD38 knockout verification. The specification does not provide working examples or design guidance for carrying out these steps with CRISPR-Cas systems other than the Cas9 system and specific guide disclsoed.
The breath of the CRISPR-Cas limitation therefore exceeds the scope of the working disclosure. The quantity of experimentation necessary to extend the disclosed Cas9 procedure to the full genus is significant, and the claimed genus encompasses genetic editing systems having materially different targeting and cleavage requirements. Therefore, the ordinary artisan would be required to engage in undue experimentation to determine which combinations of CRISPR-Cas systems, guides, target sites, and operating conditions would successfully provide the CD38 knockout NK91MI cells required by the claimed method. The enabled scope is therefore reasonably limited to the Cas9 based CD38 gene editing system, rather than the unrestricted genus of CRISPR-Cas editing systems presently encompassed by the claims.
Additionally, claims 2-5 do not reasonably provide enablement for the full scope of the claims across the genus and does not provide enabling disclosure commensurate with the full scope of the claimed transfection, culture, selection, and lentiviral-production conditions. Although the specification provides a working embodiment using particular Cas9 based editing, culture, and psPAX2/PMD2.G lentiviral conditions, the claims extend beyond that demonstrated embodiment by reciting a 15-100 µg amount for an undefined overall CRISPR-Cas editing system, culture decisions based on medium color and cell number without operative thresholds, downstream positive-cell selection and library establishment without sufficient procedural guidance, an alternative pLP1/pLP2/pLP/VSVG packaging system lacking comparable working examples, and polyethyleneimine-to-plasmid ratio that is not clearly demonstrated for that alternative packaging arrangement. In view of the limited working examples and lack of guidance for practicing these additional embodiments, the ordinary artisan would be required to develop operative component amounts, culture criteria, selection conditions, packaging parameters, and plasmid ratios through substantial experimentation.
Hence, undue experimentation would be required to practice the full scope of claims 1-5. The quantity of experimentation is significant because extending the disclosed Cas9 based CD38 knockout procedure of the full scope of “CRISPR-Cas editing systems” would require selection and testing of alternative Cas effectors, compatible target sites, guides, delivery conditions, and editing parameters not taught in the specification. Second, the amount of direction or guidance is limited because the operative disclosure is centered on the Cas9 system and does not provide general design rules for other CRISPR-Cas systems and claimed methodological steps and components such as the unidentified “double antibody” reagent, the culture decision criteria, or enablement commensurate with the full scope because the demonstrated procedures are directed to the disclosed Cas9 based editing and psPAX2/pMD2.G packaging system, while the claims encompass broader CRISPR-Cas systems and the alternative pLP1/pLP2/pLP/VSVG packaging system. The predictably of the art is limited because the claimed methods involve gene editing, NK cell culture, lentiviral production, transduction, cell selection, where changes in the editing components, reagents, culture conditions, and packaging constructs can materially affect the resulting cells. The breath of claims 1-5 exceeds the scope of the working disclosure. Accordingly, based on the lack of guidance in the art and specification, a person of ordinary skill would need to engage in undue experimentation to make and use the invention commensurate in scope with claims 1-5.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-5 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to set forth 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 1 is indefinite because the limitation “NK cells comprising a polynucleotide knocked out of CD38 and overexpressing CD16a” does not clearly establish a nexus between the asserted knock out of a polynucleotide and the CD38 protein nor the nexus to overexpression or what overexpresses CD16a. This recitation is indefinite because the phrase “a polynucleotide knocked out of CD38 and overexpressing CD16a” appears to employ slang language and associates “overexpressing CD16a” with the recited polynucleotide. Applicant may obviate the rejection by amending the claim to expressly identify the genetical material that is knocked out or disrupted or its relationships to CD38 and CD16a.
Claim 1 is additionally indefinite because step (5) recites “sampling and testing for a negative rate” without identifying what is being sampled or tested for. The specification discloses factors such as survival rate, growth rate, rate of NK cell transfection, CD38 rate, and flow cytometry rate ([0008-0011]; [0038]; [0045]; [0083]; [0093]). Applicant may obviate the rejection by amending the claim to clearly identify the characteristic being measured.
Claim 1 is additionally indefinite because each of the first, second and third culture media is required to contain a specified volume percentage of “double antibody,” but the claim does not define its composition with sufficient clarity. The ordinary artisan would not be reasonably apprised of the scope of what reagent must be present to meet the “double antibody” limitation as double antibody would be understood as a double antibody assay or method and the specification does not clearly identify a volumetric component as the “double antibody.” Applicant may obviate the rejection by amending the claim to identify what is the reagent or reagents are intended in the recitation of “double antibody.”
Claim 1 is further indefinite because each culture medium is recited as being “consisted by adding” specified components to the minimum essential media, or MEM. The ordinary artisan would not be able to reasonably determine whether the phrase defines a closed composition consisting only of MEM and the listed components or a medium prepared by adding the listed components to MEM that may contain additional constituents. A proper Markush-type claim recites alternatives in a format such as “selected from the group consisting of A, B and C.” See Ex parte Markush, 1925 C.D. 126 (Comm’r Pat. 1925). The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim, and the language “consisted by adding” is ambiguously drawn to structural media components and the methodological step of adding to a first cell culture media. Appropriate correction is required.
Claim 2 contains the trademark/trade name Lipofectamine. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a transfection reagent and, accordingly, the identification/description is indefinite.
Claim 2 is indefinite because it states that “step (3) is carried out as follows,” but the subsequently recited procedure concludes by adding the mixture to the NK cells. Claim 1 separately identifies addition of the mixture to the NK cells as step (4). This is unclear and the ordinary artisan would not be reasonably apprised of the scope of this limitation nor able to determine whether claim 2 further defines only step (3), further defines steps (3) and (4), replaces step (4), or is an additional step. Appropriate correction is required.
Claim 2 is indefinite in the recitation of “15 to 100 µg of the CRISPR-Cas editing system.” The CRISPR-CAS editing system is a gene editing tool. The ordinary artisan would not be reasonably apprised of the scope of this claim limitation because it does not properly identify which component or components are represented by the mass. This is ambiguous and the ordinary artisan would not be able to determine if this recited mass refers to the target plasmid, one component of the CRISPR-Cas editing system, gRNA, the combined components of the gene editing solution, etc. Applicant may obviate the rejection by amending the claim to identify the components what constitutes the 15-100 µg solution.
Claim 3 is indefinite because step (1) recites “a packaging plasmid,” and step (3) subsequently recites a “first packing plasmid” and a “second packaging plasmid,” and then generally refers to “the plasmid” in step (4), line 13, with no clear antecedent basis. It is unclear whether “a packaging plasmid” refers to a collective multi-packaging system, the first packaging system, the second or an additional plasmid. Applicant may obviate the rejection by amending step (1) to identify the intended packaging components such as a packaging-plasmid system comprising a first packaging plasmid and a second packaging plasmid.
Claim 3 is further indefinite in the recitation of “(9) centrifuging;” The ordinary artisan would not be reasonably able to determine what component is being centrifuged, the NK cells, lentivirus solution, the mixture, the second cell culture medium. Applicant may obviate the rejection by amending the claim to identify the material subjected to centrifugation.
Claim 3 is additionally indefinite because step (10) recites that “it is determined whether to semi-exchange liquid or subculture based on medium color and cell number during culture,” but the claim does not provide objective criteria for making the recited determination. The claim does not identify what medium color requires a semi-exchange of liquid, what medium color requires subculturing, what cell number requires either operation, or how the medium color and cell number are considered together in determining which operation is performed. Step (11) then recites that cells are evaluated when “subcultured to more than 10 times starting number,” but the cell population or process stage corresponding to the “starting number” is unclear. The ordinary artisan would not be reasonably apprised of the scope or able to determine when a semi-exchange is performed, when subculturing is performed, how cell number relates to the selection between those operations, or the reference cell quantity or number from which the subsequently recited 10-fold threshold is determined.
Claim 3 is additionally indefinite because step (11) recites “sampling and testing for a negative rate” without identifying what is being sampled or tested for. The specification discloses factors such as survival rate, growth rate, rate of NK cell transfection, CD38 rate, and flow cytometry rate ([0008-0011]; [0038]; [0045]; [0083]; [0093]). Applicant may obviate the rejection by amending the claim to clearly identify the characteristic being measured.
Claim 3 is also indefinite in the recitation of “establishing a library.” The ordinary artisan would not be able to determine what the library constitutes. The specification does not identify the type of library being established. The Applicant may obviate the rejection by indicating the collection being compiled, a cell library, clone library, cell bank, plasmid library, viral library, or another collection.
Claim 4 is indefinite in the recitation of “packaging plasmid,” “a first packaging plasmid,” “a second packaging system”, and “a third packaging system.” Parent claim 3 recites a lentiviral transfection system comprising a target plasmid having CD16a and “a packaging plasmid.” Claim 3 subsequently refers to “a first packaging plasmid” and “a second packaging plasmid.” Claim 4 further defines “the packaging plasmid” by reciting two alternative plasmid combinations. In the first alternative, the first and second packaging plasmid are identified as psPAX and pMD2. In the second alternative, the first, second, and third packaging plasmids are identified as pLP1, pLP2, and pLP/VSVG, respectively. It is unclear whether the singular “packaging plasmid” recited in parent claim 3 is intended to be one plasmid, or instead a packaging plasmid system comprising multiple distinct plasmids. If the “packaging plasmid” is intended to refer to a packaging plasmid system, it is unclear whether these are two mutually exclusive systems or whether the first, second and third packaging plasmids recited in different portions of claim 4 have some other relationship. The ordinary artisan would not be able to determine the number or identity of the packaging plasmids required by the claimed method or how those plasmids relate to the singular “packaging plasmid” introduced in parent claim 3.
Claim 5 is indefinite in the recitation of “a ratio between polyethyleneimine and a total of the plasmids containing packaging plasmids” without clearly identifying either the type of ratio or the plasmids included in the ratio. The ordinary artisan would not be able to determine whether the recited (1-10):1 ratio is a mass, molar, volume, or other ratio. Additionally, claim 5 recites the limitation "the plasmids containing packaging plasmids" in line 2. There is insufficient antecedent basis for this limitation in the claims. It is unclear what the total of the plasmids is in reference to since the parent claims recite a target plasmid, a packaging plasmid, second packaging plasmid, plasmid mixture,
Conclusion
Claims 1-5 are rejected. No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JOEL D LEVIN whose telephone number is (571)270-0616. The examiner be reached 8:00 am to 5:00 pm, Monday through Friday.
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/J.D.L./ Examiner, Art Unit 1633
/FEREYDOUN G SAJJADI/ Supervisory Patent Examiner, Art Unit 1699
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