DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communication filed 8-10-26.
Claims 8-15, 17, 21-23, 25-30 are pending in the instant application.
Election/Restrictions
Claims 13-15 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species or invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed 8-10-26.
Applicant’s election without traverse of Group II, an immune cell, claims 8-12, 17, 21-23, 25-30, in the reply filed on 8-10-26 is acknowledged.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Section 33(a) of the America Invents Act reads as follows:
Notwithstanding any other provision of law, no patent may issue on a claim directed to or encompassing a human organism.
Claims 8-12, 17, 21-23, 25-30 are rejected under 35 U.S.C. 101 and section 33(a) of the America Invents Act as being directed to or encompassing a human organism. See also Animals - Patentability, 1077 Off. Gaz. Pat. Office 24 (April 21, 1987) (indicating that human organisms are excluded from the scope of patentable subject matter under 35 U.S.C. 101). The claims recite genetically modified human cells, which includes modified human cells in a human organism.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 27 is 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.
In claim 27, there is insufficient antecedent basis for the term “truncated CAR” in line 3 of claim 27. Appropriate clarification is required.
The following is a quotation of the first paragraph of 35 U.S.C. 112(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 8-12, 17, 21-23, 25-30 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 pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The breadth of the claims:
The claims are drawn to genetically modified human cells optionally comprising immune cells comprising an exogenous nucleic acid sequence region encoding a chimeric antigen receptor (CAR) fragment which lacks a sequence encoding a functional intracellular domain of a CD3 zeta protein, which CAR fragment is integrated in-frame into an endogenous CD3 zeta gene, which cells express an in-frame fusion protein comprising the CAR fragment, an intracellular domain of the endogenous CD3 zeta gene, which intracellular domain optionally comprises one or more immunoreceptor tyrosine based activation motifs (ITAMs) which are optionally disrupted, which fusion gene is expressed under the control of an endogenous CD3 zeta promoter, which CAR expression is terminated by an endogenous CD3 zeta termination signal and an endogenous poly-A signal, which exogenous nucleic acid sequence region optionally further comprises an IRES or a protease cleavage site, which CAR fragment optionally comprises a nucleic acid sequence encoding an extracellular antigen binding domain, a transmembrane domain and/or one or more intracellular co-stimulatory domains, and which in-frame integration is into an exon or intron of the endogenous CD3 zeta gene, and is positioned upstream of the endogenous sequences encoding the truncated CAR, which exogenous nucleic acid optionally additionally comprises an immune regulating and/or therapeutic protein.
Teachings in the specification:
In Figure 2…Two days after activation, T cells were electroporated with sgRNAs targeting either the constant region of the T cell receptor alpha chain gene (TRAC, left panel) or the CD247 gene (CD3-zeta, right panel).
In Figure 3 … nuclease-assisted gene insertion of green fluorescent protein (GFP) reporter transgenes into the protein-coding reading frame of either the CD247 gene (encoding for CD3 zeta) or the TRAC gene (encoding for TCR alpha chain) was performed to investigate the effect on CD3-TCR surface expression.
Multiple Cas9 nucleases with different sgRNAs were used for CD247 exon 2 and 3 achieving similar results. Insertion of GFP into TRAC gene mirrored results seen in CD247, despite higher maximum GFP intensity
In Figure 5 In NK cells, transgenes can be expressed under control of CD247 but not TRAC …For this experiment, nuclease assisted gene insertion was repeated in Natural Killer (NK) cells (modeled by NK92 cell line) using preferred HDR templates and sgRNA-nuclease as before (Fig. 6, CD247: HDR template exon 2 + sgRNA 2.1; TRAC: exon 1 Charité + TRAC sg 1).
In Figure 6 Insertion of complete and CD3 zeta-deficient CAR transgenes into CD247 gene induce CAR protein surface expression: Nuclease assisted gene insertion was used to introduce complete or CD3 zeta-deficient (truncated) CAR transgenes into the CD247 gene. To perform this experiment, new HDR templates were designed. We rationally designed a 2nd generation CAR consisting of a murine CD19-specific single chain variable fragment (scFv), clone FMC63, an intermediate hinge CH3 spacer (derived from human Fc-IgG1), a CD28 transmembrane domain, a CD28 costimulatory intracellular domain and the CD3 cytoplasmic zeta chain. This CAR was cloned into HDR templates presented before (Fig. 3-5, Table 1 in Example 5).
For CD247 HDR templates, a full CAR expression module with CD3 zeta and poly-A terminator sequence as well as a short CAR sequence lacking a poly-A terminator sequence were generated…
In Figure 7 The effect of insert size (full CAR VS short zeta-less CARs) on absolute CAR+ T cell counts was compared after nuclease assisted gene insertion with two different non-viral dsDNA HDR template variants. HDR templates were either generated with regular homology arms (HA) (Fig. 3, previously described in PMID: 29995861) or with homology arms modified with truncated Cas9 targeting sequences (tCTS) (16 bp, previously described in PMID: 31819258).
In Figure 8 CAR expression level is regulated most strictly under control of the CD247 gene:
In this experiment, CAR expression levels were compared between CAR T cells generated by different strategies…
For overexpression controls, the CAR presented in Fig. 6 was cloned into an expression cassette commonly used for generation of CAR T cells via lentiviruses or via transposase-transposon technology. It includes an EF1-alpha promoter and a BGH poly-A terminator sequence. The expression cassette was cloned into an HDR template for insertion into the human safe harbor locus hAAVS1. Additionally, the CAR was cloned into the SIN epHIV7 lentiviral vector plasmid for virus preparation and lentiviral transduction was performed as previously described (PMID: 22707919).
In Figure 9
The only CAR T cell condition with reduced killing capacity at 2-1 and 1-1 T cell : target ratios was generated with the HDR template CD247 exon 3 inserting a full CAR expression cassette. To test the cytolytic activity of CAR T cells generated through transgene insertion into the CD247 locus…
In Figure 10
CAR T cells reprogrammed by insertion of truncated (zeta-deficient) CARs into the CD247 gene were able to eliminate tumor cells repeatedly… Both TRAC-integrated full-CAR equipped and CD247-integrated short CAR-reprogrammed T cells repeatedly eliminated tumor cells…
In Figure 11
Reduced cytokine production after antigen-engagement by truncated CARs integrated in the CD247 gene vs full CAR in the TRAC locus
In Figure 12
As presented before (e.g. Fig. 6), insertion of (truncated) CARs into the TRAC as well as the CD247 gene eliminates the CD3-TCR from the T cell surface.
In Figure 14…cells were stimulated on plate-bound anti-CD3/CD28 and nucleofected with either CRISPR-Cas9 ribonucleoproteins and HDR donor templates for AAVS1 or CD247… the NK92 cell line was transfected with CRISPR-Cas9 ribonucleoproteins and dsDNA donors. While TRAC-KI does not induce GFP/CAR expression, CD247 gene targeting allows expression of transgenes. (C) CD247- KI CAR enhances killing of Nalm-6 CD19+, but not CD19-negative control cells…
In Figure 15
NK cells were electroporated with CD247-specific RNP (incl. sgRNA 2.1).
In Figure 16
Primary NK cells expand with or without beads from human NK cell activation/expansion kit (Miltenyi) after nucleofection (A) without RNP (mock), (B) TRAC-specific RNP (TRAC KO, TRAC sg 1 (SEQ ID NO 55)), (C) CD247-specific RNP (zeta KO, CD247 sg2.1 (SEQ ID NO 52)) or (D) B2M-specific RNP (B2M KO, sgRNA target sequence: SEQ ID NO 65).
…
However B2M disruption inflicted severe fratricide due to loss of HLA-class 1 expression and consequently missing-self recognition.
In Figure 18
Comparison of the therapeutic efficacies between CD247-KI, TRAC-KI and lentivirally-transduced CD19-CAR T cells in a pre-B acute lymphoblastic leukemia xenograft mouse model…
… (ii) injected i.v. with 5x10⁵ CD19-CAR T cells generated via lentiviral transfer and consecutive TRAC KO ('LV-CD19CAR'), (iii) injected i.v. with 5x10⁵ CD19-specific TCR-deficient CAR T cells generated by TRAC-knockin with a complete CAR (TRAC-KICD19-CAR) or (iii) CD247-knockin of a short/truncated CD19-CAR (Zeta-KI CD19- CAR). … only TRAC-KI and CD247-KI show significantly reduced tumor burden in comparison to no treatment control, indicating a superior anti-tumor activity of CD247-KI over conventional lentiviral CAR T cells.
In Figure 19
Five HER2-specific CARs were cloned into the HDR templates for TRAC-KI or AAVS1-KI. The HER2-CARs differed in their respective scFv sequence with distinct affinity with Kd from 10e-11 to 10e-7 and shared the same generic 2nd CAR design (IgG1-hinge, CD28 transmembrane, CD28 intracellular signaling domain and CD3 zeta). For CD247-KI, the truncated CD3-deficient HER2 CAR was cloned into the CD247 HDR template for exon 2. HER2-CAR T cells were generated via co-electroporation of Cas9-sgRNA RNPs for the respective target gene and the dsDNA HDR templates.
… Spice analysis shows higher rate of exhaustion of CAR T cells when generated via knock-in into TRAC or hAAVS1 locus, however reduced exhaustion marker expressed after CD247-KI. The effect was seen with all four HER2 constructs…
In Figure 20
TRAC-KI and AAVS1-KI HER2 CAR T cells display higher proportion of cells with differentiated phenotypes (Tem, Temra). CD247-KI CAR T cells display highest proportion of cells with less-differentiated phenotype…
In Figure 21
Representative flow plots for HER2-CARs with highest affinity (Kd: 10e-11) and medium affinity (Kd: 10e-9) indicated severely reduced activation induced cell death of CD247- KI CAR T cells in comparison with CAR T cells generated via TRAC-KI or AAVS1-KI of expression cassettes driven by EF1alpha-LTR-promoter or CMV promoter.
In Figure 22
Mock electroporated, CD3 zeta KO and TRAC KO control NK cells all conferred to comparable dose- dependent lysis of K562 cancer cells indicating that the CD3 zeta protein is not essential for missing-self recognition and missing self tumor killing…
In Figure 23
Zeta-KI CAR-NK cells have superior killing capacity than CAR-NK that overexpress the CAR from an ectopic locus (hAAVS1)…
…As expected, unmodified NK92 or NK92-reprogrammed with a mock-CAR did not induce antigen-specific lysis of CD19-expressing targets over CD19-negative control Nalm-6 cells. However, NK92 cells reprogrammed via CD247-knockin of a short/truncated CD19-specific CAR (NK92 zeta-KI Y, SEQ ID NO 59 and 60) had superior antigen-specific lysis of targeted cells in comparison to CAR-NK92 generated via AAVS1 knock-in of an expression cassette of the complete CD19-specific CAR (NK92 AAVS1 CMV CD19CAR-Y, SEQ ID NO 61 and 62) or another CD19-CAR (NK92 AAVS1- CMV CD19CAR-K, SEQ ID NO 63 and 64).
In Example 2
In-frame insertion of GFP into different T cell receptor complex genes leads to loss of CD3-TCR complex surface expression
In these experiments as shown in Figure 2 and 3, nuclease-assisted gene insertion of green fluorescent protein (GFP) reporter transgenes into the protein-coding reading frame of either the CD247 gene (encoding for CD3 zeta) or the TRAC gene (encoding for TCR alpha chain) was performed to investigate the effect on CD3-TCR surface expression. As indicated by flow cytometric protein analysis, most GFP expression was observed in the CD3-TCR negative population (Y-axis). Multiple Cas9 nucleases with different sgRNAs were used for CD247 exon 2 and 3 achieving similar results. Insertion of GFP into TRAC gene mirrored results seen in CD247, despite higher maximum GFP intensity.
In Example 4
NK92 cell engineered with CD19 targeting CAR using the CD247 KI strategy (Fig. 14 C) and display superior antigen-specific cytotoxicity over CAR-NK92 generated via overexpression from the AAVS1 safe harbor locus (Fig. 23).
Further, primary NK cells expand after CD3 zeta KO (Fig. 16).
Example 5: Insertion of complete and zeta-deficient CAR transgenes into CD247 gene induce CAR protein surface expression
Nuclease assisted gene insertion was used to introduce complete or zeta-deficient (truncated) CAR transgenes into the CD247 gene (Fig. 6). To perform this experiment, new HDR templates were designed. We rationally designed a 2nd generation CAR consisting of a murine CD19-specific single chain variable fragment (scFv), clone FMC63, an intermediate hinge CH3 spacer (derived from human Fc-IgG1), a CD28 transmembrane domain, a CD28 costimulatory intracellular domain and the CD3 cytoplasmic zeta chain. This CAR was cloned into HDR templates presented before
(Fig. 3-5, Table 1). For CD247 HDR templates, a full CAR expression module with CD3 zeta and poly-A terminator sequence as well as a short CAR sequence lacking a poly-A terminator sequence were generated to test the hypothesis of this invention… (Note: for this experiment CD247 sg2.2 was used. For all subsequent experiment CD247 sg2.1 is used)…
Table 1: sgRNA sequences and respective locations/ donor templates. [sg = single guide RNA, AAVS1 = Adeno-associated virus integration site 1]…
In Example 6
Introducing shorter insert size tends to increase absolute CAR T cell generation after non-viral gene insertion The effect of insert size (full CAR vs short zeta-less CARs, Fig. 7) on absolute CAR+ T cell counts was compared after nuclease assisted gene insertion with two different non-viral dsDNA HDR template variants. HDR templates were either generated with regular homology arms (HA) (Fig. 3, previously described in PMID: 29995861) or with homology arms modified with truncated Cas9 targeting sequences (tCTS) (16 bp, previously described in PMID: 31819258).
In Example 7
In this experiment as presented by Figure 8, CAR expression levels were compared between CAR T cells generated by different strategies.
… CAR T cells generated through insertion of truncated CAR transgenes into the CD247 exon 2 (sg 2.1) display the lowest MFI compared to TRAC-integrated CARs or other controls. For overexpression controls, the CAR presented in Figure 6 was cloned into an expression cassette commonly used for generation of CAR T cells via lentiviruses or via transposase-transposon technology. It includes an EF1-alpha promoter and a BGH poly-A terminator sequence. The expression cassette was cloned into an HDR template for insertion into the human safe harbor locus hAAVS1. Additionally, the CAR was cloned into the SIN epHIV7 lentiviral vector plasmid for virus preparation and lentiviral transduction was performed as previously described (PMID: 22707919).
After repeated tumor re-challenge, CD247-KI CAR T cells express less exhaustion markers, such as Tim-3, PD-1, and LAG-3, versus overexpression with a Lenti-virus or an AAVS1 (Fig. 17). Results from the TRAC-KI and CD247-KI strategy are comparable. Cell fitness of CAR-T cell engineered with CD247 KI approach improved (Fig. 17).
[Emphases added] [Citations omitted].
The specification fails to provide the requisite guidance for making and using
the broad genus of genetically modified human cells. As listed above, the specification describes T cells treated with sgRNAs targeting the constant region of the T cell receptor alpha chain gene (TRAC) or the CD247 gene (CD3 zeta), the insertion of GFP into the CD247 gene or the TRAC gene, the insertion of complete and CD3 deficient CAR transgenes into CD237, a 2nd generation CAR consisting of a murine CD19-specific single chain variable fragment (scFv), clone FMC63, an intermediate hinge CH3 spacer (derived from human Fc-IgG1), a CD28 transmembrane domain, a CD28 costimulatory intracellular domain and the CD3 cytoplasmic zeta chain cloned into HDR templates, nuclease assisted gene insertion with two different non-viral dsDNA HDR template variants, HDR templates generated with regular homology arms (HA) (Fig. 3, previously described in PMID: 29995861) or with homology arms modified with truncated Cas9 targeting sequences (tCTS) (16 bp, previously described in PMID: 31819258), TRAC KO, TRAC sg 1 (SEQ ID NO 55)), CD247-specific RNP (zeta KO, CD247 sg2.1 (SEQ ID NO 52)), B2M-specific RNP (B2M KO, sgRNA target sequence: SEQ ID NO 65), TRAC-KI and AAVS1-KI HER2 CAR T cells, and five HER2-specific CARs cloned into the HDR templates for TRAC-KI or AAVS1-KI, where HER2-CARs differed in their respective scFv sequence with distinct affinity with Kd from 10e-11 to 10e-7 and shared the same generic 2nd CAR design (IgG1-hinge, CD28 transmembrane, CD28 intracellular signaling domain and CD3 zeta), and CD247-KI with the truncated CD3-deficient HER2 CAR cloned into the CD247 HDR template for exon 2. These examples fail to provide a representative number of species adequately describing the broad genus of genetically modified human cells claimed.
Since the disclosure fails to describe the common attributes and characteristics concisely identifying members of the proposed genus of genetically modified cells, and because the claimed genus is highly variant, the description provided is insufficient. One of skill in the art would reasonably conclude that the disclosure fails to provide a concise description of the broad genus of modified human cells instantly claimed.
Thus, Applicant was not in possession of the broadly claimed genus.
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.
Claim(s) 8-12, 17, 21-23, 26, 28 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Sadelain et l (WO 2017/180989).
Sadelain et l (WO 2017/180989) teach the modification of human T cells (para 0087 on page 23; para 00193 on pages 54-55) with a transgene that is integrated with the genome of the T cell, and the transgene is under control of an endogenous promoter of the T cell, and which transgene encodes a therapeutic protein or nucleic acid. The endogenous promoter is optionally constitutive or inducible, and optionally comprises a CD3zeta promoter (para 0012-0023, pages 5-8; para 00210 on page 63). The transgene optionally encodes a CAR (para 0037, page 11). Homologous recombination systems for integration target a desired site within the genome, optionally comprising the CD3 zeta chain (para 00218 on page 67).
Sadelain teaches the integration of truncated CARs into T cells (para 00241m on page 79). Transgenes expressing therapeutic molecules are integrated at sites within the genome of the T cell allowing expression of the transgene under control of an endogenous promoter of the T cell (para 00252-0025, pages 82-83). CARs comprise an extracellular antigen binding domain fused to a transmembrane domain which is fused to a cytoplasmic/intracellular domain from the CD3 zeta chain (para 00260 on pare 84). Antigens include cancer antigens (para 00267-00268 on pages 87-89, para 00270 on pages 91-92). The CAR optionally comprises a signaling domain derived from CD3 zeta (para 00273 on page 93).
Claim(s) 8-12, 17, 22 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Burleigh et al (WO 2020/223571).
Burleigh et al (WO 2020/223571) teach genetically modified T cells, including CD8+ or CD4+ T cells, expressing a chimeric antigen receptor (CAR) comprising an intracellular region comprising a CD3zeta signaling domain (para 0028 on page 7). A modified CD247 locus comprises a transgene sequence encoding a chimeric receptor or a portion thereof, which transgene is in frame with an open reading frame or a partial sequence thereof of the endogenous CD247 locus, the modified CD247 locus encodes a chimeric receptor that includes sequences encoded from the transgene sequence and sequences encoded from the endogenous CD247 locus. The intracellular region comprising the CD3 zeta signaling or a portion thereof is encoded by the genomic sequences including an open reading frame at the endogenous CD247 locus of the engineered cell (Abstract and text on page 2).
The intracellular region encoded by the transgene sequence comprises one or more costimulatory signaling domains. The modified CD247 locus encodes a chimeric receptor comprising, from the N to C terminus, an extracellular binding domain optionally comprising an antigen binding fragment capable of binding to a target antigen (para 0047-0048 on page 11), a spacer, a transmembrane domain and an intracellular signaling region, and which CD3zeta signaling domain is encoded by the genomic sequences at the endogenous CD247 locus (page 5). The chimeric antigen receptor is or comprises a non T cell or non TCR antigen receptor (para 0017, page 4) The extracellular region optionally comprises a binding domain capable of binding to a target antigen specific to a cell or tissue of a disease (para 0020 on page 4).
Burleigh teaches polynucleotides used for integration of a transgene sequence encoding a chimeric receptor into the CD247 locus. The polynucleotide comprises a nucleic acid sequence encoding a chimeric receptor or portion thereof, homology arms linked to the nucleic acid sequence, the chimeric receptor comprises an intracellular region comprising a CD3zeta signaling domain, the portion optionally does not include the full intracellular region of the chimeric receptor (para 0029 on page 7; para 0030 on page 8).
The engineered T cells optionally have a genetic disruption within a CD247 locus (para 0064-0065 on page 15). Genetic disrupting agents optionally include a DNA binding protein that specifically binds to a target site, a fusion protein comprising a DNA targeting protein and a nuclease, or an RNA guided nuclease (para 0077 on page 17). The polynucleotide sequence for integration into the CD247 locus optionally comprises the chimeric receptor that is in frame with one or more exons of the open reading frame of the CD247 locus (para 0072 on page 16).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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) 8-12, 17, 21-23, 25-30 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sadelain et l (WO 2017/180989) and Burleigh et al (WO 2020/223571), the combination in view of Attar et al (WO 2020/148677).
The claims are drawn to genetically modified human cells optionally comprising immune cells comprising an exogenous nucleic acid sequence region encoding a chimeric antigen receptor (CAR) fragment which lacks a sequence encoding a functional intracellular domain of a CD3 zeta protein, which CAR fragment is integrated in-frame into an endogenous CD3 zeta gene, which cells express an in-frame fusion protein comprising the CAR fragment, an intracellular domain of the endogenous CD3 zeta gene, which intracellular domain optionally comprises one or more immunoreceptor tyrosine based activation motifs (ITAMs) which are optionally disrupted, which fusion gene is expressed under the control of an endogenous CD3 zeta promoter, which CAR expression is terminated by an endogenous CD3 zeta termination signal and an endogenous poly-A signal, which exogenous nucleic acid sequence region optionally further comprises an IRES or a protease cleavage site, which CAR fragment optionally comprises a nucleic acid sequence encoding an extracellular antigen binding domain, a transmembrane domain and/or one or more intracellular co-stimulatory domains, and which in-frame integration is into an exon or intron of the endogenous CD3 zeta gene, and is positioned upstream of the endogenous sequences encoding the truncated CAR, which exogenous nucleic acid optionally additionally comprises an immune regulating and/or therapeutic protein.
Sadelain et l (WO 2017/180989) teach the modification of human T cells (para 0087 on page 23; para 00193 on pages 54-55) with a transgene that is integrated with the genome of the T cell, and the transgene is under control of an endogenous promoter of the T cell, and which transgene encodes a therapeutic protein or nucleic acid. The endogenous promoter is optionally constitutive or inducible, and optionally comprises a CD3zeta promoter (para 0012-0023, pages 5-8; para 00210 on page 63). The transgene optionally encodes a CAR (para 0037, page 11). Homologous recombination systems for integration target a desired site within the genome, optionally comprising the CD3 zeta chain (para 00218 on page 67).
Sadelain teaches the integration of truncated CARs into T cells (para 00241m on page 79). Transgenes expressing therapeutic molecules are integrated at sites within the genome of the T cell allowing expression of the transgene under control of an endogenous promoter of the T cell (para 00252-0025, pages 82-83). CARs comprise an extracellular antigen binding domain fused to a transmembrane domain which is fused to a cytoplasmic/intracellular domain from the CD3 zeta chain (para 00260 on pare 84). Antigens include cancer antigens (para 00267-00268 on pages 87-89, para 00270 on pages 91-92). The CAR optionally comprises a signaling domain derived from CD3 zeta (para 00273 on page 93).
Burleigh et al (WO 2020/223571) teach genetically modified T cells, including CD8+ or CD4+ T cells, expressing a chimeric antigen receptor (CAR) comprising an intracellular region comprising a CD3zeta signaling domain (para 0028 on page 7). A modified CD247 locus comprises a transgene sequence encoding a chimeric receptor or a portion thereof, which transgene is in frame with an open reading frame or a partial sequence thereof of the endogenous CD247 locus, the modified CD247 locus encodes a chimeric receptor that includes sequences encoded from the transgene sequence and sequences encoded from the endogenous CD247 locus. The intracellular region comprising the CD3 zeta signaling or a portion thereof is encoded by the genomic sequences including an open reading frame at the endogenous CD247 locus of the engineered cell (Abstract and text on page 2).
The intracellular region encoded by the transgene sequence comprises one or more costimulatory signaling domains. The modified CD247 locus encodes a chimeric receptor comprising, from the N to C terminus, an extracellular binding domain optionally comprising an antigen binding fragment capable of binding to a target antigen (para 0047-0048 on page 11), a spacer, a transmembrane domain and an intracellular signaling region, and which CD3zeta signaling domain is encoded by the genomic sequences at the endogenous CD247 locus (page 5). The chimeric antigen receptor is or comprises a non T cell or non TCR antigen receptor (para 0017, page 4) The extracellular region optionally comprises a binding domain capable of binding to a target antigen specific to a cell or tissue of a disease (para 0020 on page 4).
Burleigh teaches polynucleotides used for integration of a transgene sequence encoding a chimeric receptor into the CD247 locus. The polynucleotide comprises a nucleic acid sequence encoding a chimeric receptor or portion thereof, homology arms linked to the nucleic acid sequence, the chimeric receptor comprises an intracellular region comprising a CD3zeta signaling domain, the portion optionally does not include the full intracellular region of the chimeric receptor (para 0029 on page 7; para 0030 on page 8).
The engineered T cells optionally have a genetic disruption within a CD247 locus (para 0064-0065 on page 15). Genetic disrupting agents optionally include a DNA binding protein that specifically binds to a target site, a fusion protein comprising a DNA targeting protein and a nuclease, or an RNA guided nuclease (para 0077 on page 17). The polynucleotide sequence for integration into the CD247 locus optionally comprises the chimeric receptor that is in frame with one or more exons of the open reading frame of the CD247 locus (para 0072 on page 16).
Attar et al (WO 2020/148677) teach genetically modified T cells comprising a chimeric antigen receptor (CAR) or an exogenous T cell receptor to provide new antigen specificity on the modified T cell (para 0001, page 1). A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody linked to T cell signaling domains. The CARs comprise recombinant polypeptide constructs comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular domain. Intracellular signaling domains are functional portions of the protein acting by transmitting information within the cell to regulate cellular activity via defined signaling pathways resulting in modified cells expressing immune effector functions optionally comprising cytolytic activity and/or secretion of cytokines. Signaling motifs optionally comprise immunoreceptor tyrosine based activation motifs (ITAMs) (para 0054-0057, pages 13-14). Transmembrane domains optionally comprise the zeta chain of the T cell receptor (para 00175) on page 49).
Attar teaches the modification of any T cell, including Cd4+ helper T cells, CD8+ T cells, and natural killer cells (para 00219 on pages 65-66). Attar teaches the coadministration of other therapeutic agents along with the CAR-T cells (para 00256-000263, pages 75-78).
It would have been obvious to design and construct the instantly claimed nucleic acid constructs for integrating into T cells because the prior art routinely used all of the components claimed for generating chimeric antigen receptors One of ordinary skill would have reasonably expected that transformation of NK cells and a multitude of other T cells would provide genetically modified human cells with enhanced immunological properties for the treatment of cancers, relying on the combined teachings of Sadelain, Burleigh and Attar.
For these and the aforementioned reasons, the instant invention would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Conclusion
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
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Jane Zara
9-21-26
/JANE J ZARA/Primary Examiner, Art Unit 1637
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