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 .
Response to Amendment
The amendment filed 16 January 2026 is acknowledged. Claim 114, 118, 119, and 127 are amended. Claims 137-139 are new. Claims 115-117 are cancelled.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 114-131 in the reply filed on 2 September 2025 is acknowledged.
Claims 132-136 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 2 September 2025.
Applicant further elects the species of (i) a CAR, reading on claims 114-122 and 125-131; (ii) an ER localization tag, reading on claims 114-131; and (iii) a hepatitis C virus protease cleavage site, reading on claims 114-131.
Claims 123 and 124 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 2 September 2025.
Claim Status
Claims 114 and 118-139 are pending. Claims 123-124 and 132-136 are withdrawn from consideration as described in the Non-Final Rejection mailed 17 October 2025 and above. Claims 114, 118-122, 125-131, and 137-139 are under examination in the instant office action.
Withdrawal of Rejections
The rejection of claim 119 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite is withdrawn in view of the amendment to the claims.
The rejection of claims 114, 115, 116, 128, 129, 130, and 131 under 35 U.S.C. 101 because the claimed invention is directed to a natural product without significantly more is withdrawn in view of the amendment to the claims.
The rejection of claim(s) 114-117 and 120-131 under 35 U.S.C. 102(a)(2) as being anticipated by WO2020154635 to Frimannsson et. al. effectively filed 25 January 2019 (of record, cited in IDS dated 10/13/2023) is withdrawn in view of the amendment to the claims.
The rejection of claim 118 under 35 U.S.C. 103 as being unpatentable over WO2020154635 to Frimannsson et. al. effectively filed 25 January 2019 (of record, cited in IDS dated 10/13/2023) as applied to claims 114, 115, and 117 above, and further in view of WO2016012623 to Agaugue et. al. published 28 January 2016 is withdrawn in view of the amendment to the claims.
The rejection of claim 119 under 35 U.S.C. 103 as being unpatentable over WO2020154635 to Frimannsson et. al. effectively filed 25 January 2019 (of record, cited in IDS dated 10/13/2023) in view of WO2016012623 to Agaugue et. al. published 28 January 2016 as applied to claim 118 above, and further in view of US 20190038733 to Campana et. al. published 7 February 2019 is withdrawn in view of the amendment to the claims.
Claim Rejections - 35 USC § 112(a)- Written Description- Maintained/Modified, necessitated by amendment
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 114, 118-122, 125-131, and 137-139 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.
Regarding claim 114, the claims are broadly directed towards a recombinant polypeptide comprising from N-terminus to C-terminus: a protein of interest, wherein the protein of interest is any chimeric antigen receptor (CAR) or any T cell receptor (TCR); and any protease cleavage site; and any protein localization tag, wherein the protein localization tag is an ER localization tag or a Golgi localization tag, wherein the TCR is heterologous to the protease cleavage site and the protein localization tag. Regarding the dependent claims, no single dependent claim narrows the breadth of the claimed elements such that the particular structure of the invention described in the specification is claimed. For example, claim 126 narrows the recombinant polypeptide such that the viral protease cleavage site is for a viral protease derived from hepatitis C virus (HCV) nonstructural protein 3 (NS3). While this narrows the structure to proteins of interest with the particular cleavage site used in the invention, it does not narrow the claim from any protein localization tag in that order such that it defines the inventive concept as described in the specification; therefore, the claims encompass an unknown number of undescribed CAR and TCR proteins with “ER localization tags” of unknown structure and function, such as those yet to be discovered, or those that are not known to work on the C-terminus.
Scope of the claimed genus
Claim 114 is broadly directed towards a recombinant polypeptide comprising from N-terminus to C-terminus a protein of interest, wherein the protein of interest is any chimeric antigen receptor (CAR) or any T cell receptor; and any protease cleavage site; any protein localization tag wherein the protein localization tag is an ER localization tag or a Golgi localization tag. Regarding any protein localization tags, the instant specification defines a protein localization tag as “As used herein, the term "protein localization tag" refers to an amino acid sequence that directs the cellular localization of the recombinant polypeptide (and in turn, the protein of interest) to a particular cellular compartment” (Specification p. 7 lines 13-15). Regarding any protease cleavage site, the instant specification describes: “The term "cleavage site" refers to the bond (e.g., a scissile bond) cleaved by an agent, e.g., a protease” (Specification p. 17 lines 3-4).
Claim 118 narrows the type of protein localization to ER localization tags. Claim 119 claims that the ER localization tag comprises 85% or greater identity to SEQ ID NO: 1; Claim 139 requires that the ER localization tag comprises SEQ ID NO: 1; and Claim 137 requires the ER localization tag comprise SEQ ID NO: 2. Claims 120-122 narrow the protein of interest to describe the CAR and the elements required of the CAR, but do not narrow the protein localization tag or protease sequence. Claims 125 and 126 recite wherein the protease site is a viral protease cleavage site and wherein the viral protease cleavage site is for a viral protease derived from hepatitis C virus (HCV) nonstructural protein 3 (NS3). Regarding the claim interpretation of the phrase “derived from” in claim 126, the instant specification defines “derived from” as: “By "derived from HCV NS3" is meant the protease is the serine protease domain of HCV NS3 or a proteolytically active variant thereof capable of cleaving a cleavage site for the serine protease domain of HCV NS3” (Specification p. 18 lines 7-9). Therefore, regarding the “derivatives” of claim 126, the claim requires that the HCV NS3 derivative be a proteolytically active variant of the HCV NS3 wherein the variant cleaves the cleavage site for the serine protease domain of HCV NS3; the HCV NS3 derivative may not be infinitely derivatized such that it loses this essential function of the original protein; Claim 139 requires that the viral protease cleavage site is for an HCV NS3. Claim 126 recites wherein the recombinant polypeptide further comprises a protease and wherein the protease cleavage site is a cleavage site for said protease. Claims 128-130 recite a nucleic acid encoding the recombinant polypeptide of claim 114, a cell comprising the nucleic acid, and wherein the cell is a T cell or NK cell. Claim 131 recites a pharmaceutical composition comprising a population of the cells of claim 130 and a pharmaceutically acceptable carrier.
State of the Relevant Art
Regarding T-cell receptors, these are protein complexes in the art. As described in Suzuki 1992, T cell receptors comprise a clonotypic ɑβ heterodimer which is non-covalently associated with the CD3 complex: CD3ε, γ, and δ, with a disulfide-linked CD3ζ2 homodimer (p. 31-32).
Regarding chimeric antigen receptors, although many chimeric antigen receptors are known in the art, there is no one single art-accepted definition that encompasses the entire class of chimeric antigen receptors. For example, Zhang C, Liu J, Zhong JF, Zhang X. Engineering CAR-T cells. Biomark Res. 2017 Jun 24;5:22. doi: 10.1186/s40364-017-0102-y. PMID: 28652918; PMCID: PMC5482931, published 24 June 2017 (PTO-892 dated 10/17/2025) teaches that CAR-T cells generally comprise an extracellular antigen-binding domain comprising an scFv, a spacer, a transmembrane domain, and an intracytoplasmic domain (See Figure 1). Zhang et. al. teaches that the engineering of CARs has evolved to include costimulatory modules such as CD137 (Fig. 2). Accordingly, second generation CARs added intracellular signaling domains from various co-stimulatory protein receptors to the cytoplasmic tail of the CARs to provide additional signals to the T cell, such as CD28 or CD137(4-1BB and CD134(OX40)), which can improve the proliferation, cytotoxicity, and sustained response, and prolong the life of CAR-T cells in vivo [16,17,18]” (See “Second generation” section). However, their structures and functions are so diverse that it cannot be said that every plausible CAR, have been invented such that the applicant is in possession of a genus. First, regarding the function of binding, many diverse types of domains have been used to perform the binding function in a CAR. For example, Siegler E, et. al. Designed Ankyrin Repeat Proteins as Her2 Targeting Domains in Chimeric Antigen Receptor-Engineered T Cells. Hum Gene Ther. 2017 Sep;28(9):726-736. doi: 10.1089/hum.2017.021. Epub 2017 Jun 22. PMID: 28796529; PMCID: PMC5582591 (PTO-892 dated 10/17/2025) teaches a chimeric antigen receptor which binds to HER2 using two different designed ankyrin repeat proteins (DARPins) rather than an scFv. Pham-Danis C, et. al. Restoration of LAT activity improves CAR T cell sensitivity and persistence in response to antigen-low acute lymphoblastic leukemia. Cancer Cell. 2025 Mar 10;43(3):482-502.e9. doi: 10.1016/j.ccell.2025.02.008. PMID: 40068599 (PTO-892 dated 10/17/2025), although not prior art, teaches an entirely different type of CAR incorporating the intracellular domain of LAT. Even the transmembrane domain, which might at once been regarded to have played only a structural role, has been found to impart particular functional properties upon the CAR. Bridgeman JS, et. al. The optimal antigen response of chimeric antigen receptors harboring the CD3zeta transmembrane domain is dependent upon incorporation of the receptor into the endogenous TCR/CD3 complex. J Immunol. 2010 Jun 15;184(12):6938-49. doi: 10.4049/jimmunol.0901766. Epub 2010 May 17. PMID: 20483753 (PTO-892 dated 10/17/2025) found, for example, that CARs containing the CD3z transmembrane domain can form a complex with the endogenous TCR that may be beneficial for optimal T cell activation, a property that could be abolished by altering the structure of the transmembrane domain by amino acid substitution (see entire document; e.g., the abstract).
Regarding protein localization tags, many types of these are known in the art. For example, Moot, Robert, et al. "Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors." Molecular Therapy-Oncolytics 3 (2016) teaches a CAR that comprises a CD8 leader sequence using lamprey derived variable lymphocyte receptors rather than an scFv, wherein the backbone comprised the “IL-2 signal peptide” (PTO-892 dated 10/17/2025) as evidenced by Uniprot “Keywords- Signal” (PTO-892 dated 10/17/2025), “The signal sequence (usually 20-30 amino acids long) interacts with the signal recognition particle and directs the ribosome to the endoplasmic reticulum where co-translational insertion takes place. Signal peptides are highly hydrophobic but have some positively charged amino acids. Normally, the signal sequence is removed from the growing peptide chain by specific peptidases (signal peptidases) located on the cisternal face of the endoplasmic reticulum”. Therefore, a traditional signal peptide used in chimeric antigen receptors reads on both the instant “protein localization tag” and the “protease cleavage site”, except it is not predictable which of the signal peptides, absent direct testing, would have a claimed ER targeting effect when the sequence is localized to a non-native portion of the protein (such as an N-terminal ER localization tag on the C-terminus). US 20180179509 to Lin et. al. published 28 June 2018 (Of record, IDS dated 4/8/2022) teaches fusion constructs comprising a degron connected to a protein of interest through a cleavable linker comprising a hepatitis C virus protease site (Abstract). A degron sequence “promotes degradation of an attached polypeptide through either the proteasome or autophagy-lysosome pathways” and thus reads on the instant protein localization sequence. Protein localization tags are a large genus, wherein some act during translation to direct a ribosome to the correct subcellular compartment for that particular protein, some are part of the endosomal sorting pathway directing transmembrane proteins through to various secretory pathway compartments such as the plasma membrane and Golgi, and some act as “stop” signals to prevent the default targeting if a protein is improper folded. Suzuki, Carolyn Kiyoko. Structural and functional determinants for the retention of T-cell antigen receptor alpha chain variants within the endoplasmic reticulum. The Johns Hopkins University, 1992 (PTO-892 dated 10/17/2025) teaches that the TCR alpha chain comprises a protein localization tag that results in the ER retention if it is improperly folded or truncated versions of the TCR-alpha protein. US20190038733 to Campana et. al. teaches intracellular retention sequences for ER or Golgi retention [0051] (PTO-892 dated 10/17/2025). Rivera, Victor M., et al. "Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum." Science 287.5454 (2000): 826-830 teaches that proteins with induced aggregation domains can cause retention of a fusion protein, such as a secretory pathway protein, in the endoplasmic reticulum.
Regarding protease cleavage sequences, some of these are known in the art. Song, Jiangning, et al. "iProt-Sub: a comprehensive package for accurately mapping and predicting protease-specific substrates and cleavage sites." Briefings in bioinformatics 20.2 (2019): 638-658 (PTO-892 dated 10/17/2025) teaches the identification of protease substrates using a bioinformatic cleavage site prediction model. Song et. al. teaches “The specificity of proteases can vary significantly, depending on the protease and the active sites, with the cleavage site selectivity ranging from preferences for limited and specific amino acids at specific positions, to more general preferences with little discrimination. Current experimental methods for proteolytic cleavage characterization include one-dimensional and two-dimensional gel-based methods (used for identifying the substrates) [12], N-terminal peptide identification methods (for identifying both substrates and cleavage sites), methods using mass spectrometry, as well as quantitation methods of proteolysis to better understand the dynamics and extent of proteolytic events such as the TAILS method [13]. Despite the advances of these experimental methods, they are labor intensive, expensive and time-consuming, and are often limited to the investigation of one protease each time” (Introduction ¶2). Song et. al. further teaches “Third, the accuracy of protease-specific cleavage site prediction varies substantially between different proteases and different protease families. The difficult cases include cleavage site prediction of the MMP family and other proteases (e.g. thrombin) whose activities are also regulated by confounding factors such as the presence of exosites (sites that are located outside the active sites)” (Results and discussion, “Performance evaluation based on a different sequence encoding schemes”, ¶2). Thus, it is difficult to predict and describe all protease sites for all proteases, since each protease requires different, sometimes unpredictable, structural features to both the cleavage site and sometimes to other aspects of the target protein.
Overall, the instant claims are so broad in terms of the features (the protein of interest, protease cleavage site, and protein localization tag) and the structure of the recombinant polypeptide claimed such that an artisan would not be able to identify all of the proteins that would function as a “Stash” tag of the instant invention as described in the specification. It would not be predictable, solely from the claimed genus, which of the species of recombinant proteins would demonstrate the instantly described properties of regulatable cellular localization of cell surface receptors.
Summary of Species disclosed in the original specification
The original species teaches examples wherein the protein of interest is a CAR, wherein the target site is an HCV NS3 protease target site linked on the C-terminal end of the CAR to particular organellar retention signals (2 ER variants, one Golgi variant, one lysosome variant, and a control proteasome targeted variant called “Smash” tag) (See example 1 and Table 1 p. 21-24). In the working embodiments described, the required drug-regulatable HCV NS3 protease is expressed as an additional polypeptide that is “intracellularly tethered” and therefore cleaves the retention tags off of the target CARs in the absence of a protease inhibitor, grazoprevir. Example 3 demonstrates that this localization-tag based modulation is sufficient to regulate the cell-surface activity of the CAR. There are no working examples with T cell receptors, which is a multi-protein complex; therefore, it is unclear which of the TCR polypeptides may be used to regulate T-cell activity by ER retention in this manner. There are no working examples with protease cleavage sites other than the HCV NS3 protease target site.
Do the disclosed species represent a genus?
To provide adequate written description and evidence of possession of the claimed recombinant protein genus, the instant specification can structurally describe representative polypeptides that have the disclosed regulatable cellular localization, that function as claimed, or describe structural features common to the members of the genus, which features constitute a substantial portion of the genus. Alternatively, the specification can show that the claimed invention is complete by disclosure of sufficiently detailed, relevant identifying characteristics, functional characteristics when coupled with a known or disclosed correlation between function and structure, or some combination of such characteristics (see University of California v. Eli Lilly and Co., 119 F.3d 1559, 43 USPQ2d 1398 (Fed. Cir. 1997) and Enzo Biochem, Inc. V. Gen-Probe Inc.).
Although Applicants may argue that it is possible to screen for recombinant proteins that have the desired protease-inhibitor regulated subcellular localization function, the court found in (Rochester v. Searle, 358 F.3d 916, Fed Cir., 2004) that screening assays are not sufficient to provide adequate written description for an invention because they are merely a wish or plan for obtaining the claimed chemical invention. “As we held in Lilly, “[a]n adequate written description of a DNA … ‘requires a precise definition, such as by structure, formula, chemical name, or physical properties,’ not a mere wish or plan for obtaining the claimed chemical invention.” 119 F.3d at 1566 (quoting Fiers, 984 F.2d at 1171). For reasons stated above, that requirement applies just as well to non-DNA (or RNA) chemical inventions.” Knowledge of experimental methods of determining the appropriate localization tag and proteasomal cleavage site provides no information about the structure of any future recombinant yet to be discovered that may function as claimed. The functional descriptions of the proteasomal cleavage site and the localization tag add no additional information about how the claimed recombinant proteins will function with currently unknown variants and variants with unpredictable protease cleavage sites or with protein localization tags that function differently than the endosomal retention signal domains tested in the instant application. For example, it is unclear how and whether the instantly claimed recombinant protein would function in the context of an N-terminal signal peptide transferred to the C-terminus of a protein as described in the “State of the Art” section above.
The instant specification fails to describe fails to describe structural features common to the members of the genus, which features constitute a substantial portion of the genus because the instant specification discloses only the above that function as claimed (e.g. “target to a cellular compartment”). A definition by function does not suffice to define the genus because it is only an indication of what the agents do, rather than what they are. The specification fails to provide sufficient structural features coupled to the claimed functional characteristics. Accordingly, in the absence of sufficient recitation of distinguishing identifying characteristics, the specification does not provide an adequate written description of the claimed genus of agents that function as claimed.
Summary
A genus of species is not present in the instant specification or prior art that would demonstrate a structure/activity relationship would be known for the entire genus of recombinant polypeptides comprising any chimeric antigen receptor or TCR, any protein localization tag, and any protease cleavage site disposed between the CAR or TCR and the protein localization tag. There is a lack of an appropriate number of species with i) alternate protein localization tags; ii) alternate protease cleavage sites, and iii) alternate structure and configurations of the claimed elements. One of skill in the art would reasonably conclude that the applicant was not in possession of the genus of recombinant proteins of the polypeptide of claim 114 at the time of filing. Regarding claims 118-131 and 137-139, the claims are ultimately dependent on the rejected claim 114 without sufficiently narrowing the claimed subject matter and thus are also rejected.
Response to Arguments
Applicant’s arguments have been fully considered but are not persuasive. Applicant states “CARs and TCRs are well known. Although not required to fulfill the enablement requirement, the specification provides numerous examples of CARs and TCRs (e.g. see, page 150). Similarly, protease cleavage sites are well known. Additionally, the specification provides written description of protease cleavage sites (e.g. see, page 20) and the corresponding proteases (e.g. see pages 17-20). Similarly, ER localization tags or Golgi localization tags have been previously described and are also described in the specification at, e.g. pages 7-8” (Remarks 1/16/2026 p. 7). This is not persuasive. As described above, the genus of species of the claimed elements is so large and encompasses individual pieces that are only claimed by function such that it is unclear whether they function in the whole polypeptide system of described in the specification. For example, the majority of the claims are directed at a polypeptide comprising a CAR and no disclosed polypeptide; or claim 125, for example is directed towards viral protease cleavage sites. These claims, therefore, extend to a significant number of CARs that would not have the essential function of being regulatable that the inventors lay out in the specification because the polypeptide is claimed individually and not in a system of polypeptides that has the regulatable protease; in this case, the CAR would not be expressed on the surface except in the potential circumstance that the virus infection the CAR-expressing cell and the viral protease was expressed. Additionally, the claims are still directed towards any protease cleavage site, where some proteases are highly promiscuous and will cleave at many sites across a protein.
MPEP 2163 states that a “representative number of species” means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. The specification p. 20 as pointed to by applicant teaches particular HCV NS3 or NS4 variant protease cleavage sites SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. Given the sheer breadth of protease and therefore protease cleavage sites, the disclosure by applicant of 5 particular examples for one specific genus of proteases is not sufficient to convey to a person of ordinary skill in the art possession of all protease target sequences for any protease. Additionally, as described in the written description rejection above, for some proteases it is not predictable a priori what sites are substrates for that particular protease and which may require additional structure such as an exosite. Further, regarding the “ER localization tag” and “Golgi localization tag” on p. 7-8, these again cannot be considered adequate to describe the entire genus of any amino acid sequence that “directs” a protein to the ER compartment or the Golgi compartment. The examiner notes that p.7 attempts to incorporates by reference a database websites comprising protein localization information (LocSigDB; DBSubLoc; LOCATE; LocDD; eSLDB; “and/or any other convenient database of protein localization tags”); however, these are a moving record that are constantly updated with new experimental information. Additionally, although some of these sequences are known in the art, it would not be discernable a priori whether any sequence that directs protein localization in the ER would work in the context of the instant invention, which particularly focuses on sequences known to cause ER retention. Finally, some of these databases are annotations of proteins known to localize to particular locations rather than identifying a portion of a sequence that is responsible for ER targeting (For example, see Rastogi S, Rost B. LocDB: experimental annotations of localization for Homo sapiens and Arabidopsis thaliana. Nucleic Acids Res. 2011 Jan;39(Database issue):D230-4. doi: 10.1093/nar/gkq927. Epub 2010 Nov 11. PMID: 21071420; PMCID: PMC3013784), suggesting that the claims encompass fusion proteins wherein an entire full-length ER-targeted protein is used as the ER localization tag. Page 8 recites 21 ER localization tags which comprise the known ER localization sequences KDEL, KKMP, and KKXX, except for SEQ ID NOs: 21-23. One Golgi localization tag is disclosed as SEQ ID NO: 24. Again, given the breadth of amino acid sequences that can be considered to “direct” localization to the ER or Golgi, some of which have yet to be discovered, these examples would not be considered representative of the entire genus by a person of ordinary skill in the art. Lastly, as described above, there is insufficient structure/function correlation between the described species that a person of ordinary skill would be able to discern the genus by an understanding of a known or disclosed correlation between function and structure. Therefore, the 112(a) is maintained/modified as necessitated by amendment, as described above. The examiner suggests that this rejection may be overcome by amendments narrowing the structure and function of the polypeptide such that it would be predictable that the individual domains would function as described in the specification in the scope of the entire polypeptide; that is to say, sufficiently specifying the structure and/or function of the polypeptide domains such that, for example, it would not apply to non-specific protease cleavage sites that are found ubiquitously in polypeptides (e.g., trypsin, proteinase K) and non-specific ER/Golgi localization tags that do not function to retain a protein in the ER or Golgi.
Claim Rejections - 35 USC § 103- New, necessitated by amendment
Claims 114, 118, 120-122, 125, and 128-131 are rejected under 35 U.S.C. 103 as being unpatentable over WO2017181119 to Golosov et. al. published 19 October 2017 as evidenced by Rivera, Victor M., et al. "Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum." Science 287.5454 (2000): 826-830.
Claim interpretation: Regarding “protein localization tag”, the instant specification defines protein localization tag as “As used herein, the term "protein localization tag" refers to an amino acid sequence that directs the cellular localization of the recombinant polypeptide (and in turn, the protein of interest) to a particular cellular compartment” (Specification p. 7 lines 13-15). Therefore, the broadest reasonable interpretation of “ER localization tag” and “Golgi localization tag” are an amino acid sequence that directs that “directs” (e.g. causes a protein to be localized to) either the ER or the Golgi.
Regarding claims 114, 118, and 120, Golosov et. al. teaches fusion proteins including two protein domains separated by a heterologous protease cleavage site; in particular a fusion protein comprising a conditional expression domain, a domain containing a protein of interest, and a protease cleavage domain separating the two; in one embodiment the fusion protein is a temporally regulated CAR (Abstract). Golosov et. al. teaches that the conditional expression domain may be N- or C-terminal to the protein of interest (p. 7 lines 30-31; p. 28 lines 27-p. 29 line 2; p. 52 lines 4-11). Regarding conditional expression domains, Golosov et. al. teaches that a conditional expression domain is “a domain of a fusion protein that has a first state and a second state, e.g., states of aggregation or conformational states, e.g., states of stabilization/destabilization, or states of folding/misfolding. The first state is associated with, causes, or mediates cell surface expression or extracellular expression of one or more (e.g., all) portions of the fusion protein at a first rate or level and the second state is associated with, causes, or mediates cell surface expression or extracellular expression of one or more (e.g., all) portions of the fusion protein at a second rate or level” (p. 29 lines 1-10; p. 201 lines 13-p. 203 line 31). Golosov et. al. teaches a particular embodiment wherein the aggregation domain FKBP12F36M causes CAR molecules to spontaneously aggregate in the endoplasmic reticulum, preventing the CAR molecules from presenting on the surface of the cell until the solubilizing compound is present (Fig. 26; p. 24 lines 18-30). Golosov et. al. teaches that when the aggregation domain is derived from FKBP, the deaggregation compound can be AP21998 or AP22542 and that FKBP F36M dimers can be dissociated by FK506, rapamycin, or shield-1 and cites Rivera et. al. One particular embodiment of Golosov et. al. uses furin protease cleavage site to separate the CAR and the aggregation domain and therefore requires an N-terminal deaggregation domain so the ER and Golgi resident protease can cleave the domain off in the lumen. Golosov et. al. teaches that in the case of a C-terminal fusion, the protease cleavage site has to be oriented towards the compartment where the protease resides (p. 28 line 27-p. 29 line 2). Golosov et. al. teaches that after washout of the drug, the percent of cells expressing CAR and the MFI of the CAR in the T cells was reduced (Fig. 5). Golosov et. al. teaches that the baseline % killing of anti-CD19 CAR FKBPFD was completely abrogated (less than UTD cells) in the absence of shield-1, but was higher than control CD19 CAR-T when 1µM shield-1 was added (Fig. 9).
Regarding claim 121, Golosov et. al. teaches that the CAR comprises a primary and secondary intracellular signaling domain, wherein the primary signaling domain is CD3-zeta and the secondary signaling domain is a costimulatory molecule chosen from a group including 4-1BB and CD28 (p. 31 lines 24-33).
Regarding claim 122, Golosov et. al. teaches the CAR binding to CD19 expressed on cancer cells (p. 32 lines 13-16; p. 46 lines 1-15).
Regarding claims 128 and 129, Golosov et. al. teaches nucleic acids and vectors encoding the CARs of the invention and host cells comprising the nucleic acids (p. 54 lines 13-22; p. 55 lines 5-7; p. 197 line 32; p. 227 lines 6-p. 230 line 33; p. 241 line 30-p. 242 line 12).
Regarding claim 130, Golosov et. al. teaches that the cells are immune effector cells such as T cells (p. 241 line 30-p. 242 line 12; Example 10 p. 304).
Regarding claim 131, Golosov et. al. teaches pharmaceutical compositions comprising the T cells and pharmaceutically acceptable carriers (Example 10 p. 304; p. 434 claim 18-19; p. 282 lines 25-p. 284 lines 22).
As evidenced by Rivera et. al., pharmacological control can be established through small-molecule induced disaggregation of a protein that is otherwise engineered to accumulate as aggregates in the endoplasmic reticulum (Abstract; Fig. 1A). Rivera et. al. teaches that the FKBP fusion proteins are retained in the ER (Fig. 2). Therefore, the FKBP aggregation domain of Golosov et. al. reads on “ER localization tag”.
Golosov et. al. as evidenced by Rivera et. al. does not explicitly teach a CAR wherein the order from N-to-C terminus is a protein of interest wherein the protein of interest is a CAR, a protease cleavage site, and a protein localization tag wherein the protein localization tag is an ER localization tag.
It would have been obvious for a person of ordinary skill in the art, before the effective filing date, to make a variant of the CAR of Golosov et. al. and Rivera et. al. by moving the FKBP-protease cleavage domain to the C-terminus and substituting the protease cleavage site for a cytosol-expressed protease as taught by Golosov et. al. because Golosov et. al. teaches that they are obvious variants and explicitly discloses that the conditional expression domain and the protease cleavage site may be C-terminal. There is a reasonable expectation of success because Golosov et. al. teaches that an artisan may select a suitable protease based on the compartment the aggregation domain is facing and the orientation of the protein of interest.
Regarding claim 125, Golosov et. al. teaches that the protease cleavage site may be a TEV protease cleavage site (reads on viral protease) (p. 200 line 9; p. 6 lines 21-32; p. 5 lines 21-24). Golosov et. al. teaches that the cleavage site can be designed to be cleaved by any site-specific protease that is expressed in a cell of interest either through recombinant expression or endogenous expression to cleave the conditional expression domain and that in embodiments the protease cleavage site is chosen to correspond to a protease natively or by virtue of cell engineering to be present in a cellular compartment of a protein of interest.
It would have been obvious for a person of ordinary skill in the art, before the effective filing date, to engineer the fusion protein of modified Golosov et. al. as evidenced by Rivera et. al. with a viral cleavage site to benefit from viral proteases that can be engineered to reside in a particular cellular compartment (e.g. cytosol or ER) as taught by Golosov et. al. in order to use a highly specific protease site with compartment-controlled protease expression as taught by Golosov et. al. This would have a reasonable expectation of success because Golosov et. al. teaches embodiments including viral protease cleavage sites as equivalent protease cleavage sites and teaches that a person of skill in the art would be able to engineer the relevant recombinant expression and protease combination.
Claims 126, 127, and 139 are rejected under 35 U.S.C. 103 as being unpatentable over WO2017181119 to Golosov et. al. published 19 October 2017 as evidenced by Rivera, Victor M., et al. "Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum." Science 287.5454 (2000): 826-830 as applied to claims 114 and 125 above, and further in view of Juillerat, Alexandre, et al. "Modulation of chimeric antigen receptor surface expression by a small molecule switch." BMC biotechnology 19.1 (2019): 44 published 3 July 2019 (PTO-892 dated 3 July 2025).
The teachings of Golosov et. al. as evidenced by Rivera et. al. in regards to claims 114 and 125 are in the 103 rejection above.
Golosov et. al. as evidenced by Rivera et. al. does not teach the polypeptide comprising a protease cleavage site for a viral protease wherein the viral protease cleavage site is for a viral protease derived from hepatitis C virus (HCV) nonstructural protein 3 (NS3), or wherein the viral protease cleavage site is for a HCV NS3 protease; or wherein the recombinant polypeptide further comprises a protease disposed between the protease cleavage site and the protein localization tag, and wherein the protease cleavage site is a cleavage site for said protease.
This deficiency is resolved by Juillerat et. al.
Juillerat et. al. teaches a CAR-control system comprising, from N-to-C terminus, a CAR, a protease target site, a protease, and a degron (Fig. 1) wherein the CAR is controlled using HCV NS3 protease and its inhibitor asunaprevir which created a switch off CAR or “SWIFF-CAR” (p. 2 left column ¶1). Juillerat et. al. teaches that the ASN is functionally inert to CAR-T cells (p. 2 right column, Fig. 2). Juillerat et. al. teaches that CAR surface intensity could be modulated by the compound ASN and that the modulation of levels correlated with cytotoxicity to target cells (Fig. 3). Juillerat states that “switch-off systems with (slower) non-lethal off-kinetics may provide alternative benefits through reversible and progressive control […] Although additional work is required, we anticipate that manufacturing CAR T-cells with the CAR in an off-state (not present at the cell surface) could also decrease or completely eliminate the non-specific activation with the benefit to prevent T-cell differentiation, exhaustion or fratricide, overall improving their future in vivo functions” (p. 6 left column- right column ¶1).
It would have been obvious for a person of ordinary skill in the art, before the effective filing date, to substitute the viral protease sequence of Golosov et. al. that can be recombinantly expressed with the HCV NS3 protease target sequence and to incorporate the HCV NS3 protease as taught by Juillerat et. al. and , resulting in a CAR from N-to-C-terminus: CAR-protease cleavage site-protease-aggregation domain. An artisan would be motivated to combine these in order to benefit from the regulatable HCV NS3 protease as taught by Juillerat because Golosov et. al. teaches that an artisan can choose a suitable protease and protease site and to benefit from a Switch-OFF version of the switched on system of Golosov et. al. An artisan would understand that designing the system with an on-board protease as taught by Juillerat would result in cleavage prior to aggregation and ER retention and therefore turn the default state of the CAR to “ON” with the removal of the aggregation domain and ER retention signal. This would have a reasonable expectation of success because both Golosov et. al. Juillerat et. al., and are teaching protease-regulatable target proteins, Golosov et. al. and Juillerat et. al. both teach that the target protein is a CAR and successful regulation of the surface concentration of a CAR. Golosov et. al. teaches that a reasonable expectation that an artisan can design the regulatable systems with different proteases and recombinant expression of the protease to control the compartment and the system. Lastly, Golosov et. al. teaches a reasonable expectation that an artisan would be able to use a degradation domain such as a degron or an aggregation domain as alternate embodiments that both allow control of the cell-surface expression of a protein.
Claims 119, 137, and 138 are rejected under 35 U.S.C. 103 as being unpatentable over WO2017181119 to Golosov et. al. published 19 October 2017 as evidenced by Rivera, Victor M., et al. "Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum." Science 287.5454 (2000): 826-830 as applied to claims 114 and 118 above, and further in view of WO2016012623 to Agaugue et. al. published 28 January 2016 and US 20190038733 to Campana et. al. published 7 February 2019 (IDS dated 10/13/2023).
The teachings of Golosov et. al. as evidenced by Rivera et. al. in regards to claims 114 and 125 are in the 103 rejection above.
Golosov et. al. does not teach the CAR wherein the ER localization tag comprises 85% or greater identity to SEQ ID NO: 1, wherein the ER localization tag is SEQ ID NO: 1, or wherein the ER localization sequence comprises SEQ ID NO: 2.
These deficiencies are resolved by Agaugue et. al., and Campana et. al.
Agaugue et. al. teaches an invention CAR constructs with regulatable expression which prevents proper processing and release of the CAR to the cell membrane (Abstract). Agaugue teaches that this regulation is achieved by the CAR binding of a hook peptide which comprises an endoplasmic retention domain [143]. Agaugue et. al. teaches: “CARMIN 2.0: development of a switchable on/off system which is based on a W protein anchored to the membrane of the endoplasmic reticulum (ER) through a hook, and its binding partner Y introduced into the CAR structure. The interaction between the X-hook (e.g., Streptavidin) and the Y (e.g., Streptavidin-Binding Protein)- CAR allows the CAR retention inside the ER. The addition of a Z protein (e.g., Biotin) displaces the equilibrium of binding of X towards Z instead of Y, thus leading to the release of the CAR from the ER and its expression to the cytoplasmic membrane. The release of the CAR will stop with Z exhaustion (or antagonist) and remaining cells can be easily reactivated through reintroduction of the Z inducer” [255]. Agaugue et. al. teaches that the ER retention domain on the hook can control the surface expression of the CAR in a biotin-inducible manner (Fig 6 and 7, [48-49]). Agaugue et. al. teaches that “It would be better to be able quickly turn off the expression of the CAR at the surface of the cells to prevent adverse events and be able reactivate the system after it has been turned off. Thus, a need exists in the art for improved vectors and methods for treatment of humans” [29].
Campana et. al. teaches the regulation of T-cell receptor expression in therapeutic T-cells using an anti-CD3 antibody linked to an ER retention domain: “The present invention provides compositions comprising a protein expression blocker or PEBL comprising a target-binding molecule and localizing domain, and methods of using such compositions in cancer therapy. PEBLs are useful as a blockade of expression of target surface receptors (peptides or antigens) in immune cells” (Abstract). According to Campbell, “The best PEBLs in our study contained either the KDEL (SEQ ID NO:32) or KKXX [SEQ ID NO:35; such as, but not limited to, KKMP (SEQ ID NO:33) or KKTN (SEQ ID NO:43)] retention domains, which anchor associated luminal ER proteins, preventing their secretion or membrane expression.32,33 Thus, our anti-CD3ε PEBLs blocked CD3ε assembly with the other components of the CD3/TCRαβ complex and its surface expression” [0274]. Campana et. al. teaches SEQ ID NO: 21 which is 100% identical to instant SEQ ID NO: 1.
It would have been obvious for a person of ordinary skill in the art, before the effective filing date, to substitute the ER aggregation domain of Golosov et. al. for SEQ ID NO: 21 of Campana et. al. in order to benefit from a protease-regulatable CAR that blockades the expression of a target surface receptor such as a CAR as taught by Agaugue et. al. and Campana et. al. in a single protease-regulatable CAR peptide as taught by Golosov et. al. This would have a reasonable expectation of success because Golosov et. al. teaches that the proteases and conditional expression domains may be designed with different proteases and different conditional expression domains in mind and Agaugue et. al. teaches that an ER-resident protein retention domain can be used to conditionally express a CAR; therefore, a person of ordinary skill in the art would be able to determine what proteases (for example, a recombinant non-native protease such as TEV as taught by Golosov) would work to make a regulatable CAR with a non-aggregation ER retention sequence. Additionally, Campbell et. al. teaches that the KKMP (SEQ ID NO: 33) expression domains are the best for anchoring a protein in the ER and therefore an artisan would use the strongest ER anchor domains to design a better secretion-pathway regulatable CAR as taught by Golosov et. al.
Response to Arguments
Applicant’s arguments with respect to claim(s) 118 and 119 (Remarks 1/16/2026 p. 8-10) have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/KATHLEEN CUNNINGCHEN/ Examiner, Art Unit 1646
/GREGORY S EMCH/ Supervisory Patent Examiner, Art Unit 1678