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 without comment of Group I (claims 1-9 and 11) and the below-listed species in the reply filed on 04/21/2026 is acknowledged.
Elected Species:
Intracellular activating domain FcRγ;
Intracellular costimulatory domain 4-1BB; and
Immunostimulatory cytokine IL-15.
Because Applicant did not distinctly and specifically point out any errors in the restriction requirement, the election has been treated as an election without traverse (MPEP 818.03(a)).
Claim Status
Claims 3 and 5-15 have been amended as requested in the preliminary amendment filed on 12/26/2023. Following the amendment, claims 1-15 are pending in the instant application.
Claims 10 and 12-15 stand as withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, and claims 5-6 stand as withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species of invention (i.e., comprise a CD3ζ intracellular activation domain) in the Response filed 04/21/2026, there being no allowable generic or linking claim.
Claims 1-4, 7-9, and 11 are under examination in the instant office action.
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.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Furthermore, it is noted that the certified copy of the foreign priority document is in English. As such, the claim to foreign priority has been perfected.
Claims 1-3, 6-9, and 11 have an effective filing date of December 15, 2020 corresponding to EP20214352.5.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 06/14/2023 and 11/18/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code at Pages 8-9. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The disclosure is further objected to for the use of the terms, for example, FACS, ELISPOT, CytoFLEX, Prism (GraphPad software), which are trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Interpretation
With regard to claim 2, under broadest reasonable interpretation (BRI) the claim is being interpreted such that an NK cell as defined in claim 1 that (i) further comprises the intramembrane domain of CD27 or (ii) lacks all or a portion of the intracellular domain of CD27 is sufficient to meet the claim.
With regard to claim 3, under BRI the claim is being interpreted such that an NK cell as defined in claim 1 that further comprises at least one intracellular activation domain is sufficient to meet the claim; an intracellular costimulatory domain is optionally present, and is therefore not required.
With regard to claim 4, under BRI the claim is being interpreted such that an NK cell as defined in claim 3 that comprises at least one intracellular activation domain further comprises (i) CD3ζ as the specific intracellular activation domain or (ii) a 4-1BB intracellular costimulatory domain meets the claim.
With regard to claim 7, under BRI the claim is being interpreted such that an NK cell that has been (i) isolated from a subject or (ii) has been in vitro differentiated from a hematopoietic stem cell or from an induced pluripotent stem cell meets the claim; in vitro culture of cells isolated from a subject is optional, and is therefore not required.
With regard to claim 9, under BRI the claim is being interpreted such that the CAR of the NK cell as defined in claim 1 is constitutively or inducibly expressed. It is noted that “the optional one or more immunostimulatory cytokines” lacks antecedent basis, as detailed below, and is optional and therefore not required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
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, 8-9, and 11 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 isolated NK cells engineered to express a chimeric antigen receptor (CAR), does not reasonably provide enablement for non-isolated NK cells engineered to express a CAR. 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 the invention commensurate in scope with these claims.
The Breadth of the Claims
Claim 1 is drawn to an NK cell that is engineered to express, generally, a CAR specific to CD70. Page 26 of the specification indicates that "a cell engineered to express" a protein of interest (i.e., a CAR) may in particular denote a cell altered or manipulated by man to comprise a nucleic acid encoding the protein of interest, such as a cell into which an exogenous nucleic acid encoding the protein of interest has been introduced or inserted by available technical means. Thus, the claim is broadly drawn to a host cell (i.e., NK cell) comprising a nucleic acid encoding a CAR specific to CD70. As such, the claim broadly reads on a cell within a transgenic animal given that the term "isolated" is not denoted in describing the host cell (i.e., NK cell).
The State of the Prior Art/Level of Predictability in the Art
With respect to the unisolated host cells of the instant claims discussed above, the state of the art at the time of filing was such that one of skill could not predict the phenotype of transgenics. The art of transgenic animals has for many years stated that the unpredictability lies, in part, with the site or sites of transgene integration into the target genome and that "the position effect" as well as unidentified control elements are recognized to cause aberrant expression of a transgene (Wall et. al., Theriogenology, Vol. 45, Pg. 57-68, 1996).
The elements of the particular construct used to make transgenic animals are also held to be critical, and they must be designed case by case without general rules to obtain good expression of a transgene; e.g., specific promoters, presence or absence of introns, etc. (Houdebine et. al., Journal of Biotechnology, Vol. 34, Pg. 269- 287, 1994). Furthermore, transgenic animals are regarded to have within their cells, cellular mechanisms that prevent expression of the transgene, such as methylation or deletion from the genome (Kappell et. al.., Current Opinions in Biotechnology, Vol. 3, Pg. 548-553, 1992). Houdebine (Comparative Immunology, Microbiology, and Infectious Diseases, Vol. 32, Pg. 107-121, 2009) teaches progress has been made in the field of transgenic animals for production of foreign proteins (Abstract); however, constructing an efficient expression vector to produce a therapeutic protein is not a standard operation (Pg. 116, Paragraph, second). Therefore, undue experimentation is required to make and use a transgene and transgenic animal to produce the antibody and antibody fragments of the instant claims.
At the time of filing, the phenotype of a transgene and transgenic cell contained within any animal was unpredictable. The claims as written, encompassing a transgene and cell in a transgenic animal, is not adequately described in the specification as to prevent excessive experimentation by the public to generate and use the invention. Applicants can obviate the instant rejection by amending the claim to recite the term "isolated" before the recitation, "host cell" and by amending the vector and polynucleotide claims to specify they are not in a transgenic animal. Applicant may consider using purified in such claims if description is appropriate for such a term and it is not redefined away from standard meaning. Method claims using these products should also carry the appropriate adjectives above.
The Amount of Direction Provided by the Inventor/Existence of Working Examples
Pages 58-59 of the instant specification details the generation of CD70-directed CAR mRNA, the development/validation of CD70-directed CAR-NK-92 cells, and the evaluation of in vitro cytotoxic potential. Specifically, NK-92 cells (an isolated cell line) were stimulated with IL-2 and electroporated in OptiMEM™ medium with CAR mRNA. The generated CAR-NK-92 cells were then validated using flow cytometry. The cytotoxic potential of the CAR-NK-92 cells was evaluated by co-culturing different effector conditions with different CD70+ cell lines. Thus, the only working example of CAR-NK cells in the instant specification are generated from isolated NK cells (i.e., an NK cell line). There are no working examples reading on non-isolated host cells and/or transgenic animals.
In view of the lack of the predictability of the art to which the invention pertains as evidenced by the art above, the lack of guidance and direction provided by Applicant, and the absence of working examples, undue experimentation would be required to make transgenes and transgenic animals encompassed by the instant claims, with a reasonable expectation of success, absent a specific and detailed description in Applicant’s specification of how to effectively make the claimed host cells and/or transgenic animals encompassed by the instant claims. Thus, claim 1 and subsequently dependent claims 2-4, 8-9, and 11 are rejected here.
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 2-3 and 6-9 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.
Regarding claims 3 and 7-8, the phrase “such as” renders the claims indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). For example, claim 3 recites “at least one intracellular activation domain, such as a CD3ζ or FcRγ intracellular activation domain”; it is unclear as to if the recitation of the specific CD3ζ or FcRγ intracellular activation domains are intended to limit the intracellular activation domain, or if they are merely exemplary embodiments and are not intended to be limiting. For the purpose of examination, the limitations following the phrase “such as” are being interpreted as exemplary embodiments which are not limiting.
Regarding claims 2-3 and 6-8, the phrase(s) “preferably”, “more preferably”, and/or “particularly preferably” render the claims indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). For example, claim 2 recites “comprises the intramembrane domain of CD27 and/or lacks all or a portion of the intracellular domain of CD27, preferably lacks all of the intracellular domain of CD27”; it is unclear as to if the recitation of “preferably lacks all of the intracellular domain of CD27” is intended to limit the intramembrane domain of CD27, or it is merely an exemplary embodiment not intended to be limiting. For the purpose of examination, the limitations following the phrase(s) “preferably”, “more preferably”, and/or “particularly preferably” are being interpreted as exemplary embodiments which are not limiting.
Regarding claim 9, the claim recites the limitation "the optional one or more immunostimulatory cytokine" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim because there is no prior reference to an optional one or more immunostimulatory cytokine as pertains to the NK cell and CAR recited in claim 1 from which claim 9 depends. As such, it is unclear as to what “the optional one or more immunostimulatory cytokine” is referring to.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-4, 7, 9, and 11 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by WO 2021/055437 A1 (herein after referred to as "Leick") as evidenced by UniProt Entry P26842 (sequence last updated in 2009; herein after referred to as “P26842”).
Leick teaches CD70 targeting chimeric antigen receptors and engineered immune cells comprising such CAR (Abstract). In some embodiments, a CAR comprises at least an extracellular ligand domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and in some embodiments, the CAR comprises an optional leader sequence (also referred to as "signal peptide"), an extracellular antigen binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain; the domains in the CAR are (i) in the same polypeptide chain, e.g., comprise a chimeric fusion protein or (ii) not contiguous with each other (Page 9, Lines 19-27). In some embodiments, the CAR of the invention comprises an extracellular target binding domain comprising a polypeptide that binds Cluster of Differentiation 70 (CD70); expression of CD70 is highly restricted in normal human (noncancer) tissues, but CD70 is expressed in numerous cancers (Line 28 of Page 9 through Line 6 of Page 10). The extracellular domain of CD70 is a ligand for CD27; in some embodiments, the polypeptide that binds CD70 comprises a CD70-binding domain of Cluster of Differentiation 27 (CD27) also called the CD27 antigen (Page 10, Lines 7-9). The CD27 protein has extracellular, transmembrane, and cytoplasmic domains, and in some embodiments, the CD70 binding domain is located within the extracellular signaling domain of CD27; in some embodiments, (i) the CD70-binding domain in CD27 comprises a peptide comprising the amino acid sequence of TRPHCESCRHCN (SEQ ID NO: 9) that is located in the extracellular domain of CD27, (ii) the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 80%, at least 90%, or at least 95% identical) to the amino acid sequence of SEQ ID NO: 9, or (iii) the extracellular targeting binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 9 (Page 10, Lines 17-29). In some embodiments, the extracellular targeting binding domain of the CAR comprises a polypeptide comprising the extracellular domain of CD27; in some embodiments, the extracellular targeting binding domain of the CAR comprises (i) a polypeptide comprising an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 80%, at least 90%, or at least 95% identical) to the amino acid sequence of SEQ ID NO: 1, or (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (Line 30 of Page 10 through Line 3 of Page 11). The cytoplasmic domain or region of the CAR includes one or more intracellular signaling domains; an intracellular signaling domain is capable of activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced (Page 14, Lines 24-27); the intracellular signaling domain can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell or CAR-expressing NK cell (Page 15, Lines 4-6). The one or more intracellular signaling domains comprise a primary intracellular signaling domain, wherein exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation; in some embodiments, a primary intracellular signaling domain comprises a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM, wherein examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 theta, CD3 eta, CDS, CD22, CD79a, CD79b, CD278 ("ICOS "), FceRI, CD66d, DAP10, and DAP12 (Page 15, Lines 15-23; emphasis added). In some embodiments, the one or more intracellular signaling domain comprise a costimulatory intracellular domain (Page 15, Lines 32-33); costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response, wherein various examples of such molecules are provided on Page 16, and in some embodiments, the co-stimulatory domain of the CARs of the invention comprise one or more signaling domains from one or more co-stimulatory protein or cytokine receptor selected from CD28, 4-lBB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor, and SynNotch (Page 15, Lines 8-31; emphasis added). In some embodiments, the intracellular signaling domain of the CAR comprises the primary signaling domain, e.g., an ITAM containing domain, by itself or combined with a costimulatory signaling domain (e.g., a costimulating domain from one or more co-stimulatory protein or cytokine receptor selected from CD28, 4-lBB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor, and SynNotch) (Page 17, Lines 4-8). In some embodiments, the CAR of the invention comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 80%, at least 90%, or at least 95% identical) to the amino acid sequence of any one of SEQ ID NOs: 2-7 or the CAR of the invention comprises the amino acid sequence of any one of SEQ ID NOs: 2-7 (Page 17, Lines 22-25). It is specifically noted that Leick SEQ ID NOs: 2 and 3 each comprise the extracellular domain of CD27 and the costimulatory domain of 4-1BB; Leick SEQ ID NOs: 2 and 3 each only comprise residues 1-192 of Leick SEQ ID NO: 8 corresponding to CD27 and, as evidenced by P26842, residues 1-192 do not include any portion of the cytoplasmic/intracellular domain of CD27 (see Features under Subcellular Location in P26842). Thus, Leick teaches CARs that target CD70 wherein the CAR comprises the extracellular domain of CD27 and lacks all of the intracellular portion of CD27, as evidenced by P26842, and further comprises a costimulatory domain that is 4-1BB. Leick further discloses that the invention provides nucleic acid molecules (e.g., vectors) for expressing CARs in cells, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a CAR of the invention (Page 18, Lines 7-9). In some embodiments, expression of natural or synthetic nucleic acids CARs is typically achieved by operably linking a nucleic acid encoding the CAR to a promoter, and incorporating the construct into an expression vector; the vectors can be suitable for replication and integration into eukaryotes and typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence (Page 18, Lines 23-28). One example of suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequences, which is a strong constitutive promoter sequence (Page 19, Lines 7-9); inducible promoters are also contemplated wherein the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired (Page 19, Lines 18-21). Aspects of the invention provide engineered immune cell comprising the CAR or the nucleic acid encoding the CAR; in some embodiments, the immune cell is (i) a mammalian immune cell or (ii) a human immune cell wherein an "immune cell" can be a T-cell, an NK cell, a dendritic cell, a macrophage, a B-cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloid-derived suppressor cell, a mesenchymal stem cell, or combinations thereof, or any precursor, derivative, or progenitor cells thereof (Line 29 of Page 20 through Line 1 of Page 21). Immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors; the immune cells may also be generated from induced pluripotent stem cells or hematopoietic stem cells or progenitor cells (Page 21, Lines 3-7). Other aspects of the invention provide compositions comprising any one of the engineered immune cells; in some embodiments, the composition is a pharmaceutical composition, wherein the composition further comprises a pharmaceutically acceptable carrier, excipients or stabilizers typically employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives (Line 33 of Page 22 through Line 3 of Page 23). Thus, Leick anticipates an NK cell engineered to express a CAR, wherein the CAR comprises the extracellular domain of CD27 (see claim 1) wherein said CAR (i) lacks all of the intracellular domain of CD27 (see claim 2); (ii) further comprises at least one intracellular activation domain (which may include FcRγ) (see claim 3); and/or (iii) comprises an intracellular activation domain and further comprises a 4-1BB costimulatory domain (see claim 4) and wherein the NK cell has been isolated from a patient’s peripheral blood mononuclear cells or cord blood (see claim 7) and expression of the CAR may be constitutive or inducible (see claim 9). Leick further anticipates pharmaceutical compositions comprising such an engineered NK cell and a pharmaceutically acceptable carrier (see claim 11).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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, 7-9, and 11 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2021/055437 A1 (herein after referred to as "Leick") in view of UniProt Entry P26842 (sequence last updated in 2009; herein after referred to as “P26842”) and non-patent literature by Sahm et. al. (Cancer Immunol. Immunother., 2012, 61, 1451-1461; herein after referred to as “Sahm”).
Leick teaches CD70 targeting chimeric antigen receptors and engineered immune cells comprising such CAR (Abstract). In some embodiments, a CAR comprises at least an extracellular ligand domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and in some embodiments, the CAR comprises an optional leader sequence (also referred to as "signal peptide"), an extracellular antigen binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain; the domains in the CAR are (i) in the same polypeptide chain, e.g., comprise a chimeric fusion protein or (ii) not contiguous with each other (Page 9, Lines 19-27). In some embodiments, the CAR of the invention comprises an extracellular target binding domain comprising a polypeptide that binds Cluster of Differentiation 70 (CD70); expression of CD70 is highly restricted in normal human (noncancer) tissues, but CD70 is expressed in numerous cancers (Line 28 of Page 9 through Line 6 of Page 10). The extracellular domain of CD70 is a ligand for CD27; in some embodiments, the polypeptide that binds CD70 comprises a CD70-binding domain of Cluster of Differentiation 27 (CD27) also called the CD27 antigen (Page 10, Lines 7-9). The CD27 protein has extracellular, transmembrane, and cytoplasmic domains, and in some embodiments, the CD70 binding domain is located within the extracellular signaling domain of CD27; in some embodiments, (i) the CD70-binding domain in CD27 comprises a peptide comprising the amino acid sequence of TRPHCESCRHCN (SEQ ID NO: 9) that is located in the extracellular domain of CD27, (ii) the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 80%, at least 90%, or at least 95% identical) to the amino acid sequence of SEQ ID NO: 9, or (iii) the extracellular targeting binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 9 (Page 10, Lines 17-29). In some embodiments, the extracellular targeting binding domain of the CAR comprises a polypeptide comprising the extracellular domain of CD27; in some embodiments, the extracellular targeting binding domain of the CAR comprises (i) a polypeptide comprising an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 80%, at least 90%, or at least 95% identical) to the amino acid sequence of SEQ ID NO: 1, or (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (Line 30 of Page 10 through Line 3 of Page 11). The cytoplasmic domain or region of the CAR includes one or more intracellular signaling domains; an intracellular signaling domain is capable of activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced (Page 14, Lines 24-27); the intracellular signaling domain can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell or CAR-expressing NK cell (Page 15, Lines 4-6). The one or more intracellular signaling domains comprise a primary intracellular signaling domain, wherein exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation; in some embodiments, a primary intracellular signaling domain comprises a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM, wherein examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 theta, CD3 eta, CDS, CD22, CD79a, CD79b, CD278 ("ICOS "), FceRI, CD66d, DAP10, and DAP12 (Page 15, Lines 15-23; emphasis added). In some embodiments, the one or more intracellular signaling domain comprise a costimulatory intracellular domain (Page 15, Lines 32-33); costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response, wherein various examples of such molecules are provided on Page 16, and in some embodiments, the co-stimulatory domain of the CARs of the invention comprise one or more signaling domains from one or more co-stimulatory protein or cytokine receptor selected from CD28, 4-lBB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor, and SynNotch (Page 15, Lines 8-31; emphasis added). In some embodiments, the intracellular signaling domain of the CAR comprises the primary signaling domain, e.g., an ITAM containing domain, by itself or combined with a costimulatory signaling domain (e.g., a costimulating domain from one or more co-stimulatory protein or cytokine receptor selected from CD28, 4-lBB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor, and SynNotch) (Page 17, Lines 4-8). In some embodiments, the CAR of the invention comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 80%, at least 90%, or at least 95% identical) to the amino acid sequence of any one of SEQ ID NOs: 2-7 or the CAR of the invention comprises the amino acid sequence of any one of SEQ ID NOs: 2-7 (Page 17, Lines 22-25). It is specifically noted that Leick SEQ ID NOs: 2 and 3 each comprise the extracellular domain of CD27 and the costimulatory domain of 4-1BB; Leick SEQ ID NOs: 2 and 3 each only comprise residues 1-192 of Leick SEQ ID NO: 8 corresponding to CD27. It is specifically noted that P26842 teaches that residues 1-192 of CD27 do not include any portion of the cytoplasmic/intracellular domain of CD27 (see Features under Subcellular Location in P26842). Thus, Leick teaches CARs that target CD70 wherein the CAR comprises the extracellular domain of CD27 and lacks all of the intracellular portion of CD27, as taught by P26842, and further comprises a costimulatory domain that is 4-1BB. Leick further discloses that the invention provides nucleic acid molecules (e.g., vectors) for expressing CARs in cells, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a CAR of the invention (Page 18, Lines 7-9). In some embodiments, expression of natural or synthetic nucleic acids CARs is typically achieved by operably linking a nucleic acid encoding the CAR to a promoter, and incorporating the construct into an expression vector; the vectors can be suitable for replication and integration into eukaryotes and typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence (Page 18, Lines 23-28). One example of suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequences, which is a strong constitutive promoter sequence (Page 19, Lines 7-9); inducible promoters are also contemplated wherein the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired (Page 19, Lines 18-21). Aspects of the invention provide engineered immune cell comprising the CAR or the nucleic acid encoding the CAR; in some embodiments, the immune cell is (i) a mammalian immune cell or (ii) a human immune cell wherein an "immune cell" can be a T-cell, an NK cell, a dendritic cell, a macrophage, a B-cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloid-derived suppressor cell, a mesenchymal stem cell, or combinations thereof, or any precursor, derivative, or progenitor cells thereof (Line 29 of Page 20 through Line 1 of Page 21). Immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors; the immune cells may also be generated from induced pluripotent stem cells or hematopoietic stem cells or progenitor cells (Page 21, Lines 3-7). Other aspects of the invention provide compositions comprising any one of the engineered immune cells; in some embodiments, the composition is a pharmaceutical composition, wherein the composition further comprises a pharmaceutically acceptable carrier, excipients or stabilizers typically employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives (Line 33 of Page 22 through Line 3 of Page 23). Thus, Thus, Leick teaches NK cells engineered to express a CAR, wherein the CAR comprises the extracellular domain of CD27 wherein said CAR (i) lacks all of the intracellular domain of CD27; (ii) further comprises at least one intracellular activation domain (which may include FcRγ); and/or (iii) comprises an intracellular activation domain and further comprises a 4-1BB costimulatory domain and wherein the NK cell has been isolated from a patient’s peripheral blood mononuclear cells or cord blood and expression of the CAR may be constitutive or inducible. Leick further teaches pharmaceutical compositions comprising such an engineered NK cell and a pharmaceutically acceptable carrier.
However, Leick does not teach or suggest an NK cell engineered to express a CAR comprising the extracellular domain of CD27 (or a CD70-binding portion thereof) further engineered to express one or more immunostimulatory cytokines. This deficiency is remedied by Sahm.
Sahm teaches that Natural killer (NK) cells represent another valuable effector cell population suitable for the generation of tumor-specific variants by means of genetic modification and donor-derived primary NK cells as well as continuously growing NK
cell lines such as NK-92 are being developed for adoptive cancer immunotherapy; utilizing retroviral transfer of CAR sequences, NK-92 variants with specificity for different tumor-associated surface antigens have been generated and it was demonstrated that such cells were potent and exhibited highly selective antitumoral activity against otherwise NK-resistant targets derived from solid tumors as well as enhanced cytotoxicity against moderately NK-sensitive malignant cells of hematologic or neuroectodermal origins (Last Paragraph of Page 1451 through First Paragraph of 1452). The authors investigated the consequences of ectopic expression
of IL-15 on growth and activity of established NK cells, and NK cells co-expressing a tumor-specific chimeric antigen receptor; transduction of established human NK cells with a lentiviral vector encoding human IL-15 resulted in predominantly intracellular expression of IL-15 and maintained proliferation and activity of the producer cells in the absence of IL-2, wherein the growth of non-transduced bystander cells was not supported, allowing rapid enrichment of gene-modified cells solely by IL-2 withdrawal (Page 1452, Column 1, Second Paragraph). This was also the case upon transduction of NK cells with a bicistronic lentiviral vector encoding IL-15 and a second-generation CAR targeting the pancarcinoma antigen epithelial cell adhesion molecule (EpCAM); CAR-expressing effector cells isolated from the resulting IL-2-independent
cell pool continued to proliferate in the absence of exogenous cytokines and displayed high and selective cell-killing activity against EpCAM-expressing breast carcinoma cells that were resistant to the natural cytotoxicity of unmodified NK cells (Id.). Isolation, genetic modification and expansion of primary NK and T cells are labor intensive and may yield variable
results. Clinically applicable cytotoxic cell lines such as NK-92 could bypass the need for separate genetic modification of effector cells for each individual patient, especially
in cases where autologous effector cells cannot be employed and suitable donors are not available; co-expression of an EpCAM-specific CAR together with IL-15 allowed rapid isolation and long-term maintenance of NK cells that demonstrated durable antigen-specific cytotoxicity and this strategy may therefore complement existing approaches for GMP-compliant large-scale production of established lines such as NK-92 and extend the potential clinical utility of these effector cells for a wider range of disease indications (Page 1460, Column 1, Second Full Paragraph). Thus, Sham suggests that engineering NK cells, including NK cells engineered to express CARs targeting tumor/cancer specific antigens, to express IL-15 allows for rapid isolation and long-term maintenance of NK cells while maintaining durable antigen-specific cytotoxicity.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to modify the NK cells engineered to express a CAR comprising the extracellular domain of CD27 (or a CD70-binding portion thereof) of Leick such that the NK cell is further engineered to express an immunostimulatory cytokine, such as IL-15 as suggested by Sahm. One would have been motivated to further modify the CAR-expressing NK cell of Leick to express IL-15 with the motivation of allowing for more rapid isolation and long-term maintenance of the NK cells while preserving cytotoxicity. One of ordinary skill in the art would have a reasonable expectation of success because Sahm demonstrates that it is possible to engineer NK cells to express (i) a tumor/cancer targeting CAR and (ii) IL-15 and that such engineered NK cells are functional and clinically applicable.
In the test of whether it is “obvious to try” there must be:
(1) a finding in the art at the time of filing of the invention that there had been a recognized problem or need in the art;
(2) a finding that there had been a finite number of identified, predictable potential solutions to the recognized need or problem;
(3) a finding that one of ordinary skill in the art could have pursued the known potential solutions with a reasonable expectation of success.
Sahm recognizes the need to improve clinical applicability/functionality of engineered NK cells in applications such as cancer therapy. Given the recognized need to improve NK cell-based therapeutics and given the known benefit of engineering NK cells to express IL-15 (alone or in combination with a cancer/tumor specific CAR), one of skill in the art could have pursued modifying the NK cells engineered to express a CAR comprising the extracellular domain of CD27 (or a CD70-binding portion thereof) of Leick such that they further express IL-15, as suggested by Sahm, with a reasonable expectation of success.
Conclusion
Claims 1-15 are pending. Claims 5-6, 10, and 12-15 are withdrawn. Claims 1-4, 7-9, and 11 are rejected. No claims are allowed.
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/ALYSSA RAE STONEBRAKER/Examiner, Art Unit 1642
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