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 .
Election/Restrictions
Applicant's election with traverse of the species election of A) adaptor protein which is DAP10 in the reply filed on 09/22/2025 is acknowledged. Applicant states on the record that “Applicant respectfully disagrees with the Examiner’s assertion above and submits that DAP10 and DAP12 are obvious variants of each other based on the current record.” This is a clear admission on the record that these are patentably indistinct species. Therefore, if the examiner finds one of the species unpatentably over the prior art, the admission may be used in a rejection under 35 USC 103 of the other species.
Applicant’s election of species of B) effector domain which is 4-1BB, C) artificial receptor which is a CAR and D) costimulatory domain of the CAR which is 4-1BB in the reply filed on 09/22/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
The requirement is still deemed proper and is therefore made FINAL.
Alternative Names
CD3zeta is also referred to as CD3z or CD3ζ here.
Specification
Abstract
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
Applicant is reminded of the proper content of an abstract of the disclosure.
A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art.
If the patent is of a basic nature, the entire technical disclosure may be new in the art, and the abstract should be directed to the entire disclosure. If the patent is in the nature of an improvement in an old apparatus, process, product, or composition, the abstract should include the technical disclosure of the improvement. The abstract should also mention by way of example any preferred modifications or alternatives.
Extensive mechanical and design details of an apparatus should not be included in the abstract. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length.
See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
The instant Abstract begins with “The present invention concerns” and refers to purported merits and comparison with the prior art by saying the CNKs “have improved sensitivity” and display “enhanced sensitivity and superior cytotoxicity against tumor cells.”
Title
The title of the invention is not descriptive. A new title is required that is clearly indicative of the invention to which the claims are directed.
The following title is suggested: Immune Cell Comprising a Polynucleotide Encoding a Chimeric Natural Killer Cell Receptors
SPECIFICATION
The disclosure is objected to because of the following informalities: in [0011] the extracellular domain is identified as SEQ ID NO:12, however, this is the intracellular domain and new SEQ ID NO:14 is human NKG2D extracellular domain (see below).
Appropriate correction is required.
Nucleotide and/or Amino Acid Sequence Disclosures
It is noted that instant SEQ ID NO:8 and 9, designated as DAP10 transmembrane domain and DAP12 transmembrane domain, respectively, have been exchanged relative to the original parent specification in US Application 16/827,697. That is, SEQ ID NO:8, DAP10 transmembrane sequence, now has the sequence of original SEQ ID NO:9, and vice versa. Basis for this exchange is an obvious error and finds support in, for example, the human DAP10 Uni-ProtKB/Swiss-Prot Dabase entry Q9UBK5 and the human DAP12 Uni-ProtKB/Swiss-Prot Dabase entry O43914 (attached and cited on the accompanying PTO-892).
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 120 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
This application repeats a substantial portion of prior Application No. 16/827,697, filed 03/23//2020, and adds disclosure not presented in the prior application. Because this application names the inventor or at least one joint inventor named in the prior application, it may constitute a continuation-in-part of the prior application. Should applicant desire to claim the benefit of the filing date of the prior application, attention is directed to 35 U.S.C. 120, 37 CFR 1.78, and MPEP § 211 et seq. The presentation of a benefit claim may result in an additional fee under 37 CFR 1.17(w)(1) or (2) being required, if the earliest filing date for which benefit is claimed under 35 U.S.C. 120, 121, 365(c), or 386(c) and 1.78(d) in the application is more than six years before the actual filing date of the application. There is no support in the parent application as filed for a human NKG2D intracellular domain that is amino acids 1-52 of SEQ ID NO:1, which is the same as current SEQ ID NO:12. The disclosure added after the filing date of application 16/827,697 is SEQ ID NO:12, designated “Human NKG2D intracellular domain”.
The prior-filed applications did not originally disclose SEQ ID NO:12 per se, “Human NKG2D intracellular domain" as defined at the end of the instant specification and in the sequence listing. It did not disclose the amino acid ranges for each NKG2D domain, even though it disclosed the full-length “Human NKG2D” as SEQ ID NO:1. Nevertheless, there is no basis in parent application 16/827,697 as originally filed for the intracellular domain of SEQ ID NO:1 being amino acids 1-52 thereof, i.e., being SEQ ID NO:12, nor is there support in the prior art for this as an inherent feature. What is now SEQ ID NO:12 was added after the filing date of that application. The prior art only supports the intracellular domain as amino acids 1-51. For example, Uni-ProtKB/Swiss-Prot Database Accession No. P26718, version 174 (see “TOPO_DOM”; Feb. 2019; attached and cited on the accompanying PTO-892), sets forth the intracellular domain as amino acids 1-51 not 1-52. Similarly, NCBI GenBank Database Accession No. NP_031386.2 (Feb.2019) also sets forth the intracellular domain as 1-51 (see “FEATURES”, attached and cited on the accompanying PTO-892). As a result, it appears the instant application may constitute a continuation-in-part of the prior application.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Objections
Claims 1, 2, 11 and 18 are objected to because of the following informalities: The first occurrence of an abbreviation should be accompanied by the full meaning. In claim 1, the first occurrence of “NK” should be accompanied by the full name of natural killer. In claim 2, “ED” apparently be instead “ECD” and should be accompanied by the term extracellular domain, “TMD” by the term transmembrane domain and “ICD” by the term intracellular domain (see end of [0031]). In claim 11, “ED” should likewise apparently be “ECD”. In claim 18, “TAA” should be accompanied by the term tumor-associated antigen.
Appropriate correction is required.
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, 4 and 19 and dependent claims 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.
Claims 1, 4 and 19 recite “the NKG2D protein”, however, this term is not clearly defined in claim 1. Independent claims 1 and 19 first recite “a natural killer group 2 membrane D (NKG2D) protein domain”, and then refer to “the NKG2D protein” generated after cleavage at the self-cleavage peptide. Dependent claim 4 also recites “the NKG2D protein”. It is unclear if the “NKG2D protein” is the same as the “NKG2D protein domain” or if it refers to the full-length NKG2D protein comprising the domain. Therefore, the claims are indefinite.
Similarly, claim 1 uses both the terms “adaptor protein domain” and “adaptor protein”. It is unclear what the difference is between the adaptor protein and a domain thereof, thus rendering the claim indefinite.
Claim Rejections - 35 USC § 112(a)
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 1-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are drawn to a human immune cell comprising a polynucleotide encoding a chimeric natural killer cell (NK) receptor polypeptide (CNK) comprising 1) a NKG2D protein domain linked by a self-cleavage peptide to 2) an adaptor protein domain comprising DAP10 or DAP12 fused director of via a linker to a cytoplasmic effector domain. Dependent claim 2 further limits the NKG2D protein domain to one comprising a human NKG2D extracellular domain (ED or ECD), ii) human or mouse NKG2D transmembrane domain (TMD) and iii) human intracellular domain (ICD). No other claims other than claims 11-13, defining the ECD, ICD and full amino acid sequence of NKG2D, respectively, limit the NKG2D protein domain. Claim 4, which depends from claim 1, recites the NKG2D protein homodimerizes to form a homodimer of NKG2D protein, and the adaptor protein homodimerizes to form a homodimer of the adaptor protein, wherein the CNK complex is a hexamer comprising a NKG2D homodimer and two adaptor protein homodimers, wherein each NKG2D protein is associated with a homodimer of the adaptor protein. Claim 5 depends from claim 4 and recites the CNK complex “is capable of enhancing the cytotoxicity of the human immune cell against a target cell"; however, it is unclear under what conditions the CNK complex can enhance said cytotoxicity. Again, there is no limitation in claims 1 or 4 related to the structure of the NKG2D protein (domain) such that it is able to homodimerize nor to what the effector domain is. Further, the CNK complex is not limited to the recited components because of the use of the open language term “comprising” in connect with the components of the CNK complex. For example, there could be an exogenous dimerizing domain that promotes homodimerization of the NKG2D protein. Additionally, the written description of the effector domain which is part of the adaptor protein domain is limited.
The specification focuses only on a NKG2D protein having an extracellular (ECD), transmembrane (TMD) and intracellular domain (ICD). There is no disclosure of the CNK comprising a NKG2D that does not have all three domains, even though the domains may be from different mammalian species (mouse and human). However, there is no limitation in claim 1 concerning the structure of the NKG2D protein domain, e.g., whether it is the full protein or only, for example, the ECD and TMD of NKG2D, while the specification discloses a CNK having only a full-length human NKG2D or full-length chimeric NKG2D, i.e., wherein the ECD, TMD and ICD are a combination of mouse and human domains (e.g., [0011]-[0013]). In order to be able to use the encoded CNK polypeptide as disclosed by the specification, i.e., binding cells overexpressing NKG2D ligands and initiating cytotoxicity against those cells (e.g.¸ [0003]), it must be able to bind NKG2D ligands and transmit a signal upon that binding to initiate the cytotoxicity against the ligand-expressing target cells and further to physically associated with DAP10 or DAP12 as disclosed in [0008].
The only two adaptor molecules discussed are DAP 10 and DAP 12 (e.g., [0008]). The specification states in [0009], “Whether the potential advantage can be realized is not known and cannot be predicted by conjecturing, and simple experimentations cannot provide answers due to the myriad amounts of experimental designs. Thus it remains to be a highly desirable but unpredictable goal to transform native T cells to CNK-T for recognizing and killing the tumor cells via NKG2D signaling using a chimeric NK cell receptor that contains NKG2D ligand binding domain, an adaptor domain (chimeric DAP 10/12), and an effector domain (such as CD3 zeta).” There is no support for a NKG2D protein that associates with an adaptor protein domain comprising DAP10 or DAP12 wherein the association is by other than a NKG2D ICD (see, e.g., Figs. 1A-1F). The statement in [0009] supports the inability of the skilled artisan to readily envision chimeric NK receptor polypeptides having other than a full-length NKG2D, either human or chimeric, physically associated with DAP10 and/or DAP 12, and with the addition to the adaptor protein domain of a cytoplasmic effector domain comprising the intracellular domain of CD3zeta (e.g., [0037]). The order of the domains and protein regions within the CNK is critical (see Fig. 1). The intracellular and extracellular domains of NKG2D cannot be connected without a transmembrane domain between. For the adaptor protein, the cytoplasmic effector domain must result in being expressed so that it has an intracellular position.
The only actual reduction to practice of the adaptor protein domain is wherein it comprises DAP10 or DAP12 fused to a cytoplasmic effector domain which is CD3Z (e.g., [0055]). There is recitation of the adaptor protein domain alternatively or additionally being fused to another stimulatory signaling domain which is CD28, 4-1BB or OX40 ([0010], [0024], [0037]). The prior art supports a reasonable expectation of success for the addition of an ICD of one of these three co-stimulatory proteins. However, paragraph [0017] provides a laundry list of effector domains, including of CD35, Light, NOTCH1, and IL15, for which there is no description to support their functional presence as an effector domain in an adaptive protein as claimed. There is nothing in the specification or prior art to support the full scope of the genus of effector domains of [0017].
As mentioned above, the order of the domains and protein regions within the CNK complex is critical (see Fig. 1). With the exception of the CNK receptor polypeptides as illustrated by the expression construct of Figs. 2A-2D, the skilled artisan cannot envision the detailed chemical structure of the encompassed polypeptides, and therefore conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method of isolation. Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method of isolating it. The polynucleotide able to encode the CNK receptor polypeptide itself is required. See Fiers v. Revel, 25 USPQ2d 1601 at 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Lid., 18 USPQ2d 1016 (Fed. Cir. 1991). Only those particular encoded proteins in the necessary order would support the function of the enhancing cytotoxicity of the engineered human immune cell against target cell as set forth in claim 5.
Vas-Cath Inc. v. Mahurkar, 1{9USPQ2d 1111 (Fed. Cir. 1991), clearly states that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the ‘written description’ inquiry, whatever is now claimed.” (See page 1117.) The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116).
Therefore, only a polynucleotide encoding the CNK complex wherein the NKG2D protein domain is a full or full-length chimeric NKG2D protein linked by a self-cleaving peptide to an adaptor protein is fused to a cytoplasmic effector domain, i.e., wherein the adaptor protein comprises DAP10 or DAP12 that is fused directly or via a linker to a cytoplasmic effector domain which is CD3z and optionally also 4-1BB, CD28 or OX40 (see Fig. 1C-1D and the Brief Description thereof in [0010], as well as [0037], for basis), but not the full breadth of the claim meets the written description provision of 35 U.S.C. § 112(a). Applicant is reminded that Vas-Cath makes clear that the written description provision of 35 U.S.C. § 112 is severable from its enablement provision (see page 1115).
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows: The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later- filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AJA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc.,38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994) The disclosure of the prior-filed application, provisional Application No. US 62/919,750, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application.
There is disclosure in provisional application 62/919,750 of the chimeric NK receptor polypeptide that recognizes an NKG2D ligand and triggering subsequent down signaling. The provisional application describes a NK chimeric antigen receptor (NK CAR2) comprising a full-length NKG2D and adaptor protein DAP10 and/or DAP12, optionally fused to a T cell activation signal (e.g., CD3Z as illustrated in Fig. 1). “[P]olynucleotides that encode the receptor, vectors and host cells encoding a chimeric receptor” is set forth. It reasonably appears “the receptor” refers to the chimeric NK receptor. It does not describe of a self-cleave peptide as part of the CNK polypeptide. Also, no sequences are disclosed.
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.
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.
Claim(s) 1-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over US Patent 7,994,298 B2 (‘298) in view of VanSeggelen et al. (Mol. Ther. 23(10):1600-1610, Oct. 2015), Karimi et al. (J. Immunol. 175(12):7819-7828, 2005), US Patent 9,511,092 B2 (‘092) and US 11,845,804 B2 (‘804).
US 7,994,298 teaches two proteins that can make a complex: wildtype NKG2D and chDAP10, which comprises wildtype DAP10 fused to the cytoplasmic domain of CD3ζ (Fig. 1, referred to hereafter as either chDAP10 or chDAP10-CD3ζ). In col. 11, lines 16-18, and col. 17, lines 10-14, a particular embodiment of a chimeric construct which is a fusion between full-length DAP10 and the cytoplasmic domain of CD3ζ, e.g., chDAP10 is described. ‘298 teaches a human chimeric NKG2D receptor comprising human NKG2D and human CD3zeta, as well as human DAP10 and human CD3zeta (e.g., col. 8, lines 20-28, and col. 17, lines 24-25). Additionally, it was taught that only when both NKG2D and DAP10 were transfected in the same cell (Bosc23) did NKG2D cell surface expression result (col. 8, lines 28-34). When human chimeric NKG2D (having a human NKG2D ECD)-bearing CD8+T cells were contacted with a variety of human cancer cell lines, the T cells secreted IFNγ (Table 2 and col. 5, lines 40-50). The amount of IFNγ produced when chimeric NKG2D-expressing human CD8+ T cells are brought into contact with human tumor cells is about 5-times higher than for wildtype NKG2D-expressing T cells (Table 2 and col. 5, lines 40-51). Figure 2 shows that the highest amount of target cell lysis occurs when the target cells are co-cultured with effector T cells that have been transfected with murine chDAP10>> chNKG2D> wtDAP12> wtNKG2D≈wtDAP10 (col. 3, lines 47-57, and col. 6, lines 9-44). Both chNKG2D- and chDAP10-transduced T cells recognized NKG2D ligands as shown by production of higher amounts of IFNγ after co-culture with cells expressing NKG2D ligands but not with cells that did not express ligand (col. 5, lines 10-20). Human chimeric NKG2D also produced a high amount of IFNᵞ in response to NKG2D ligands (col. 9, lines 21-32). Blocking human chNKG2D with an anti-NKG2D antibody prevented killing of tumor cells by chNKG2D-transduced human T cells (Table 7, sentence bridging cols. 8-9). It is disclosed (col. 2, lines 8-10) that the prior art has established that “NKG2D is associated with the transmembrane adaptor protein DAP10”. The invention is designed for the production of tumor-specific T cells by increasing the cytotoxic activity of activated NKG2D receptors (e.g., col. 3, lines 61-64). It is taught that the CD3ζ may be that of GENBANK accession number human NM_198053; SEQ ID NO:6 (col. 11, lines 10-12). The DAP protein may be human DAP10 with the sequence of GENBANK accession number AJ001383; SEQ ID NO:4 (encoding a protein identical to instant SEQ ID NO:3), or human DAP12 with the sequence of GENBANK accession number AF019562; SEQ ID NO:5 (which encodes a protein identical to instant SEQ ID NO:4; col. 10, lines 55-60). GENBANK accession number BC039836 encodes human NKG2D protein (SEQ ID NO:2, differing by one amino acid from instant SEQ ID NO:1; col. 10, lines 36-43). Also, ‘298 teaches introduction of exogenous chDAP10 (DAP10-CD3zeta) and chNKG2D (NKG2D-CD3zeta) into primary murine immune T cells led to cytotoxicity when NKG2D ligand-expressing tumor cells were present, with in most cases the chDAP10-transfected cells having the highest specific lysis activity (Fig. 2B-D, and col. 2, lines 47-57). Further, murine spleen T cells genetically engineered to express wildtype or chimeric NKG2D or DAP10 showed that none of the wildtype constructs induced significant surface expression of NKG2D, while chNKG2D showed 42% increased expression and chDAP10 induced 64% increased expression, noting that the NKG2D surface expression by chDAP10 was of endogenous NKG2D and by chNKG2D was a combination of endogenous and exogenous NKG2D (col. 4, line 59, through col. 5, line 9). Also, looking at Table 1 of ‘298, which shows the amount of the cytokine IFNγ produced by chNKG2D-expressing murine T cells co-transfected with a variety of constructs, use of chDAP10 induced the largest amount of IFNγ production, nearly twice as much as chNKG2D. A similar study was done (col. 9 lines 21-45) using chNKG2D-transduced human T cells, showing that high amounts of IFNγ were produced when the T cells were co-cultured with NKG2D ligand-expressing tumor cells (chDAP10 co-transfection was not tested), supporting the reasonable expectation that human and murine immune cells expressing NKG2D function similarly in response to a target cell expressing NKG2D ligand. ‘298 does not teach wherein all or part of the NKG2D was human in a complex with a chimeric DAP10- CD3ζ adaptor protein, or wherein the DAP10 and/or CD3ζ in the NKG2D complex were human. Nor is a chimeric DAP12-effector explicitly taught. ‘298 does not teach a protein comprising a NKG2D domain linked by a self-cleavage peptide to a DAP10 adaptor protein comprising a CD3ζ effector domain and optionally a 4-1BB costimulatory domain.
VanSeggelen et al. teach a chimeric NKG2D comprising NKG2D fused to CD3Z (NKz), a NKG2D CAR comprising the extracellular domain of NKG2D fused to a CD8 hinge, and CD28 and CD38 intracellular signaling domains, as well as a construct comprising DAP10 linked to the NKz by a self-cleaving p2A peptide (NKz10) (Fig. 1a). This NKz10 polypeptide showed protein expression of 7-10 fold higher than a construct without the DAP10 domain (Fig. 1a, and p. 1602, col. 1, first paragraph). Its expression in T cells led to significant killing of tumor target cells in vitro (e.g., p. 602, col. 2, third paragraph).
Karimi et al. used interfering RNA (RNAi) to silence human NKG2D, DAP10 or DAP12 in activated CD8+ T cells and NK cells in vitro to disrupt the NKG2D/DAP complex to investigate the role of each component in cytokine release and cytotoxicity (e.g., p. 7821, col. 2, end of second paragraph). Further, in vivo it was shown that NKG2D silencing in expanded activated human CD8+ T cells administered to a mouse tumor model significantly increased tumor progress (p. 7825, col. 2 middle). Human NKG2D forms a hexameric complex with DAP10, with a NKG2D homodimer interacting with four DAP10 proteins in the transmembrane domains (p. 7824, col. 2, middle). Activated human CD8+ T cells transduced with DAP10 RNAi lost 75% of cytolytic activity compared to ineffective DAP10 shRNA or empty vector (p. 7823, col. 1, first paragraph), but was less effective than silencing NKG2D (p. 7823, col. 1, start of second paragraph). Silencing DAP12 in activated human CD 8+ T cells showed a significant though reduced reduction in cytotoxicity compared to silenced DAP10 or NKG2D (p. 7823, col. 2, first paragraph). When both DAP10 and DAP12 were knocked down in activated human CD8+ T cells, suppression of in vitro cytolysis was greater than 80% compared to negative controls, “reflecting more potent suppression than silencing DAP10 or DAP12 alone.” (p. 7824, paragraph bridging cols. 1-2). The ability to coprecipitate DAP12 and NKG2D from activated CD8+ T cells whether or not DAP10 is silenced indicates that human NKG2D can pair with DAP12 when DAP10 is absent (p. 7825, col. 1, first paragraph).
US 9,511,092 B2 teaches a chimeric NKG2D receptor made of NKG2D linked to CD3zeta and expressed with DAP10, all encoded by a single polynucleotide (Fig. 1A). It is discussed that the prior art has shown that NKG2D associates with DAP10, which promotes and stabilizes its surface expression, and that NKG2D signal transduction occurs only through its association with DAP10 (col. 2, lines 18-34). In the same section it references US ‘298 (supra) as teaching use of chimeric receptors comprising NKG2D or DAP10 fused to CD3zeta for expression in T cells. Transduction of the encoded chimeric receptor of ‘092 led to increased NKG2D surface expression in NK cells, which were more cytotoxic than mock-transduced cells against leukemia and sold tumor cell lines (col. 56, line 36, through line 63). It also led to increased cytokine secretion (Table 1, e.g., of IFNγ). These chimeric receptor-expressing NK cells had antitumor activity in a mouse osteosarcoma xenograft model (col. 56, line 64, through col. 57, line 17). A vector comprising the polynucleotide can be used for expression in the host cell (e.g., col. 3, lines 15-16). As shown in Fig. 1A, the NKG2D and DAP10 encoding portions of the polynucleotide are separated by a ribosome entry site (IRES) that functions to allow translation initiation in the middle of a mRNA sequence so that the NKG2D and DAP10 proteins are expressed as independent proteins (col. 11, lines 40-44). The NKG2D may be human, having the sequence of SEQ ID NO:22 (col. 7, lines 39-40; which is identical to instant SEQ ID NO:1).
US 11,845,804 teaches a chimeric antigen receptor comprising an antigen-binding domain (AD) which comprises a tumor-associated antigen (TAA) and is an scFv in the form of anti-CD30 scFv AC10, a hinge domain (HD) comprising an IgG4 hinge having the sequence of SEQ ID NO:10 (identical to instant SEQ ID NO:7), a transmembrane domain (TMD) and an intracellular domain (ICD) comprising costimulatory domains which are CD28 and 4-1BB and a cytoplasmic domain of CD3zeta of SEQ ID NO:23 (identical to instant SEQ ID NO:5; col. 7, line 32, through col. 8, line 57, and claim 1). Claim 1 is drawn to the same CAR except wherein the costimulatory domain comprises CD3zeta, CD28 and OX40 ICDs. Claim 10 is drawn to a nucleic acid encoding the CD30 CAR of claim 1. Claim 12 is drawn to a vector comprising the nucleic acid and claim 13 is drawn to a cell comprising the vector. Treatment of a CD30+ expressing cancer, such as CD30-expressing tumors, including lymphoid malignancies, leukemia and solid tumors, by administration of the encoding nucleic acid to a subject is taught (claim 17 and col. 1, lines 19-23). CAR-T cells comprising the CAR are taught (col. 27, line 44, through col. 28, 65).
It would have been obvious to the artisan of ordinary skill before the effective filing date of the instant application to combine for expression in a single polynucleotide an encoded NKG2D protein and the chDAP10-CD3ζ of ’298 for transduction into a T cell for expression of the NKG2D/chDAP10-CD3ζ complex taught by ‘298, wherein NKG2D is separated from the chDAP10-effector molecule by a self-cleavage peptide as taught for a similar construct by VanSeggelen et al. Based on the success of VanSeggelen, there would have been a reasonable expectation of successful expression and NKG2D receptor DAP10-CD3ζ complex formation in human T cells that could function cytotoxically. Both ‘298 and VanSeggelen et al. showed it was advantageous to express DAP10 when NKG2D was expressed because it led to increased surface expression of NKG2D. This is also supported by ‘092, which showed that expression of a similar construct in NK cells also lead to cytotoxicity of cancer cells. It would have been obvious wherein all the components of the single polypeptide were human in order to reduce the chance of immunogenicity for anti-cancer human T cell therapy. Therefore, it would have been obvious to have an immune cell expressing a chimeric receptor NKG2D/chDAP10-CD3ζ in order to produce a cytotoxic effect on tumor cells expressing of NKG2D ligand(s).
It further would have been obvious wherein DAP10 was substituted with DAP12 because Karimi et al. discussed that both DAP10 and DAP12 complex with NKG2D to induce cell lysis, but through different signaling pathways. The findings of Karimi et al. showed that even in the absence of DAP10, DAP12 still exercised some tumor cell cytotoxicity. Because the transmembrane domain of NKG2D regulates complex formation with DAP10 or DAP12, and both DAP proteins signal through different pathways, it would have been desirable wherein either or both signaling pathways were enabled by NKD2D ligand activation in order to induce T or NK cell cytotoxicity toward tumor cells. One of ordinary skill in the art would have expected an increase in activity of either pathway, e.g., by increased cell surface expression of NKG2D on T or NK cells, would have led to increased target cell lysis and IFNγ release (e.g., Karimi et al., ‘092 and ‘298). Karimi et al. provides the reasonable expectation that the NKG2D/DAP10-CD3Z complex would have been in a hexameric configuration. As shown by the prior art, genetic engineering of immune cells to comprise a polynucleotide encoding protein capable of forming a NKG2D/DAP10-CD3z complex would have been obvious and desirable wherein components of the chimeric receptor protein, NKG2D and either DAP10-CD3zeta or DAP12--CD3ζ, were of human origin for the same purpose, i.e., to cytotoxically target tumor cells by the NKG2D/chDAP12-CD3ζ complex expressed in T or NK cells. One would have desired this to analyze the therapeutic potential of the immune cells with the complex and with a therapeutic goal. There would have been a reasonable expectation of success because ‘298 showed that chDAP-CD3ζ with wildtype NKG2D in T cells caused an increased amount of IFNγ release after co-culture with cells expressing NKG2D ligand, and the chDAP10-CD3ζ led to significantly higher NKG2D surface expression on the transduced T cells. Further, when murine T cells were co-cultured with murine NKG2D ligand-expressing target cells, chDAP10-CD3ζ induced the greatest amount of target cell lysis, closely followed by chNKG2D-CD3ζ (‘293 Fig. 2). Similarly, when human cancer target cells were contacted with human chimeric NKG2D-expressing T cells, IFNγ was released, which did not occur in the absence of the tumor cells (Table 2 of ‘298). As referenced in ‘904, the ICD human CD3ζ sequence (SEQ ID NO:23) is identical to instant SEQ ID NO:5. The amino acid translations of the ‘298 GENBANK human DAP10 and DAP12 are identical to instant SEQ ID NO:3 and 4, respectively. The human NKG2D of ‘092 is identical to instant SEQ ID NO:1. The sequence of human NKG2D of ‘092 is identical to instant SEQ ID NO:1 and it would have been obvious to use this NKG2D protein in the DAP10-associated chimeric receptor. However, it would have been obvious to use any wildtype human CD3ζ intracellular domain NKG2D, DAP10 or DAP12 protein (unless expressing the CNK in a mouse, in which case use of wildtype murine sequences necessary for function would have been obvious to use). It further would have been obvious to have the genetically engineered immune cell also express a CAR, such as taught by ‘804, for the treatment of CD30+ tumors, such as lymphoid malignancies, leukemia and sold tumors, or substituting any other TAA-binding site for CD30 for the same expected TAA-directed cytotoxic function.
Prior Art
The prior art made of record and not relied upon is considered pertinent to Applicant's disclosure.
Wang et al. (Canc. Immunol. Res. 3(7):815-826, 2015) teaches a polynucleotide encoding a single chain scFv that binds mesothelin fused to a KIR2DS2 TMD and ICE separated by a T2A self-cleavage peptide from DAP12 (Figs. 1A-B). It is cited to show the state of the art for polynucleotides encoding a polypeptide with a self-cleavage peptide for use of DAP12 for enhanced antitumor activity.
Carter (Undergraduate Research Thesis: The Ohio State University, Retrieved online: <URL: https://kb.osu.edu/bitstreams/3d397ddc-15a0-5ef1-8c50-ca847496e522/download>[retrieved on 07/10/2023], May 2018) teaches a chimeric NKG2D protein comprising the extracellular domain of human NKG2D, transmembrane domain (TMD) of murine NKG2D-S and intracellular domain of murine NKG2D-S (p.22, middle, and Fig. 7). The chimeric NKG2D construct is expected to bind to both NKG2D ligands and anti-human NKG2D antibodies, (p. 22). A chimeric NKG2D comprising only the transmembrane and extracellular NKG2D domains were shown to have the expected binding activity (p. 37, second paragraph). Carter mentions that if they are unable to show NKG2D activation in a cytotoxicity assay or IFNγ assay, then they will consider co-transducing human DAP10 into murine NK cells with the full length human NKG2D receptor. However, it does not teach or suggest transduction of a full length NKG2D receptor (human or chimeric) with an adaptor protein comprising DAP10 and an effector domain.
Bolsee et al. (Cancer Res. (2018) 78 (13_Supplement): 3583) teaches NKG2D CAR constructs comprising full-length NKG2D and the cytoplasmic domain of CD3zeta (third paragraph). Second generation CARs were also made by including a CD28 or 4-1BB costimulatory domain (third paragraph). While co-expression with DAP10 in the CAR-expressing cells is taught (fourth paragraph), an adaptor protein domain comprising DAP10 and a cytoplasmic effector domain is not taught.
Chang et al (Canc. Res. 73(6):1777-1796, 2013) includes authors who were the inventors of US Patent ‘092 above. It teaches a chimeric NKG2D receptor made of NKG2D linked to CD3zeta and expressed with DAP10, all encoded by a single polynucleotide (Fig. 1). Transduction of the encoded chimeric receptor led to increased NKG2D surface expression in NK cells, which were more cytotoxic than mock-transduced cells against leukemia and sold tumor cell lines (p. 1779, col. 1, paragraph 4, through col. 2, paragraph 2). These chimeric receptor-expressing NK cells had antitumor activity in a mouse osteosarcoma xenograft model (p.1782, col. 1). Chang et al. is cumulative with US Patent 9,511,092 B2 relied upon above for supporting simultaneous transduction of an immune cell with both NKG2D and DAP10 for antitumor activity.
Conclusion
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Claire Kaufman
/CLAIRE KAUFMAN/Primary Examiner, Art Unit 1674 November 17, 2025