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 traverse of Group I (i.e., claim 1-14, 20, 28-29, 40, and 44 drawn to a population of cells comprising a scaffold protein) in the reply filed on June 29, 2026, is acknowledged. Additionally, Applicant’s election without traverse of Species A (i.e., a single and specific fusion protein as dNGFR as the scaffold protein, a series of linear epitopes separated by an amino acid spacer sequence of AU5 (SEQ ID NO: 26), AU1 (SEQ ID NO: 25), and V5 (SEQ ID NO: 24); and E (SEQ ID NO: 28), HA (SEQ ID NO: 21), and V5 (SEQ ID NO: 24) as the epitopes, and were each cell in the population does not comprise a protein of interest) in the reply filed on June 29, 2026, is acknowledged.
Please note that the election of the scaffold protein is expanded to include GFP in light of the Examiner’s search. Thus, claims 6-7 are rejoined and examined below. Also please note that Applicants elected species of a series of linear epitopes separated by an amino acid spacer sequence of AU5 (SEQ ID NO: 26), AU1 (SEQ ID NO: 25), and V5 (SEQ ID NO: 24); and E (SEQ ID NO: 28), HA (SEQ ID NO: 21), and V5 (SEQ ID NO: 24) as the epitopes is free of the prior art. Although each of these epitopes is known in the art (See discussion of Kimple et al., Curr. Protoc. Protein Sci. 73:26 pages (2015) at Table 9.9.1), the specific combination of epitopes is not known and there is no teaching and/or suggestion in the art to arrive the specific combination of epitopes. Thus, the species of epitopes is expanded as discussed below.
Status of Claims
Claims 1-43 were originally filed on November 2, 2023.
The amendment received on March 6, 2024, canceled claims 15-19, 21-27, 31-39, and 41-43; amended claims 1-14, 20, 28-30, and 40; and added new claim 44.
Claims 1-14, 20, 28-30, 40, and 44 are currently pending and claims 1-7 are under consideration as claims 8-14, 20, 28-29, 40, and 44 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, and claim 30 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 June 29, 2026.
Priority
The present application is a continuation of US Application No. 16/641,959, filed on February 25, 2020, which claims status as a 371 (National Stage) of PCT/US2018/047996 filed August 24, 2018, and claims priority under 119(e) to U.S. Provisional Application No. 62/550,086 filed on August 25, 2017.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on November 2, 2023; March 6, 2024; and August 25, 2026, are being considered by the examiner.
Sequence Interpretation
For claim 2, please note that the Examiner is interpreting the scope as closed-ended requiring 100% identity and the same length to at least two of the recited sequences. However, it is noted that the scope of claim 1 is interpreted as open-ended. Thus, the series of two or more distinct linear epitopes encompasses additional residues and/or components, e.g., amino acid linker sequences, but the epitope sequences themselves in claim 2 are limited to these sequences.
Claim Interpretation
For purposes of applying prior art, the claim scope has been interpreted as set forth below per the guidance set forth at MPEP § 2111. If Applicant disputes any interpretation set forth below, Applicant is invited to unambiguously identify any alleged misinterpretations or specialized definitions in the subsequent response to the instant action. Applicant is advised that a specialized definition should be properly supported and specifically identified (see, e.g., MPEP § 2111.01(IV), describing how Applicant may act as their own lexicographer).
For claim 1, Pursuant to MPEP 2111, the pending claims must be "given their broadest reasonable interpretation consistent with the specification." The Federal Circuit’s en banc decision in Phillips v. AWH Corp., 415 F.3d 1303, 1316, 75 USPQ2d 1321, 1329 (Fed. Cir. 2005) expressly recognized that the USPTO employs the "broadest reasonable interpretation" standard:
The Patent and Trademark Office ("PTO") determines the scope of claims in patent applications not solely on the basis of the claim language, but upon giving claims their broadest reasonable construction "in light of the specification as it would be interpreted by one of ordinary skill in the art." In re Am. Acad. of Sci. Tech. Ctr., 367 F.3d 1359, 1364[, 70 USPQ2d 1827, 1830] (Fed. Cir. 2004). Indeed, the rules of the PTO require that application claims must "conform to the invention as set forth in the remainder of the specification and the terms and phrases used in the claims must find clear support or antecedent basis in the description so that the meaning of the terms in the claims may be ascertainable by reference to the description." 37 CFR 1.75(d)(1).
With respect to the broadest reasonable interpretation of the structural requirements for the claimed population of cells, it is noted that each cell is structurally required to contain and/or express a fusion protein that requires two components; namely, a reporter protein as a scaffold protein, and two or more distinct linear epitopes where the two or mor epitopes are fused to the scaffold protein. The instant specification defines a reporter protein as referring to a protein that is heterologous to a target cell and whose presence indicates successful gene transfer from a vector to the target cell (See instant, [0028]). Structurally speaking, the definition only requires the reporter protein to be heterologous to a host cell. The presence indicating successful gene transfer from a vector to the cell constitutes an inherent property of the reporter protein that would necessarily be present since the reporter protein is heterologous to the cell. As such, it must follow that any heterologous protein’s presence would indicate successful transfection into a cell. Thus, the claimed reporter protein is not structurally limited other than it must not occur naturally in the target cell. Moreover, the instant specification defines an epitope as referring to the portion of an antigenic molecule that is specifically bound by the antigen binding domain of an antibody or antibody fragment (See instant, [0031]). Linear epitopes are formed from contiguous residues and are typically retained upon exposure to a denaturing solvent (See instant, [0031]). As such, the claimed two or more distinct linear epitopes structurally requires two amino acid sequences with differing amino acid sequences where the sequences are made of contiguous residues and where each of the epitopes is fused to the reporter protein. Each epitope must also exhibit the function of being antigenic and able to bind to an antigen binding domain of an antibody or antibody fragment. However, although claim 1 recites the at least distinct linear epitopes are part of a “series”, there is no structural requirement that the at least distinct linear epitopes are directly bound together. In fact, the scope of dependent claim 3 encompasses amino acid spacer sequences that separate the epitopes. Thus, there is no structural requirement limiting the location of the epitopes fused to the scaffold protein, e.g., FLAG epitope bound to the N-terminus of reporter protein and HA epitope bound to the C-terminus of the reporter protein.
With respect to where the distinct linear epitopes each being recognized by distinct antibodies and wherein the series of linear epitopes forms a detectable protein barcode tag unique to each fusion protein, it is noted that these claim limitations constitute functional properties of the at least two distinct linear epitopes. As long as a prior art reference teaches and/or suggests the structural requirement of the at least two distinct linear epitopes, the epitopes must be recognized by distinct antibodies and form a detectable protein barcode tag unique to each fusion protein. Thus, the claiming of a new use, new function or unknown property which is inherently present in the prior art does not necessarily make the claim patentable. In re Best, 562 F.2d 1252, 1254, 195 USPQ 430, 433 (CCPA 1977). There is no requirement that a person of ordinary skill in the art would have recognized the inherent disclosure at the time of invention, but only that the subject matter is in fact inherent in the prior art reference. Schering Corp. v. Geneva Pharm. Inc., 339 F.3d 1373, 1377, 67 USPQ2d 1664, 1668 (Fed. Cir. 2003).
With respect to where each of the cells in the population are distinguishable from each other, it is noted that there is no limit as to the number of cells in the population. In order words, a population can contain two cells, where one cell contains/expresses one fusion protein, and a second cell that contains/expresses a second fusion protein that contains a different combination of at least two epitopes, e.g., cell 1 = FLAG tag – dNFGR – V5 tag, and cell 2 = FLAG tag – dNFGR – HA tag; or cell 1 = dNFGR – FLAG tag – V5 tag, and cell 2 = FLAG tag – V5 tag – dNFGR. Note: even though the first example contains FLAG in both cells, the cells remain distinguishable because given the second epitope is different; and in the second example, the orientation of the sequences is distinguishable.
For claim 3, please note that the Examiner is interpreting the scope such that the spacer sequence is any sequence ranging from a single residue, e.g., glycine, up to any number of residues. Notably, the spacer sequence is not limited and encompasses any sequence including an antibody fragment.
For claim 5, please note that the “dNGFR” corresponds to a known mutant NGFR. Wroblewska et al. teaches that dNGFR refers to a truncated receptor without an intracellular domain (See Wroblewska et al., Cell 175:1141-1155 (2018) at pg. 5, 3rd paragraph). As evidenced by UniProt P08138, the human dNGFR amino acid sequence would correspond to the deletion of residues 273-427 since these residues correspond to the cytoplasmic domain (See UniProt P08138, 11 pages (accessed on 9/9/26) at pg. 4). Thus, an ordinary skilled artisan would be well-aware that the mutant NGFR referred to in claim 5 is a NGFR where the intracellular domain is deleted.
Drawings
The drawings; in particular, Figure 2B, are objected to because of the following reason:
The drawings have a line quality that is too light to be reproduced (weight of all lines and letters must be heavy enough to permit adequate reproduction) or text that is illegible (reference characters, sheet numbers, and view numbers must be plain and legible) see 37 CFR 1.84(l) and (p)(1)); See Figure(s) 2B.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because of the following informalities: paragraph [0121[, discusses amino acid sequences without an accompanying SEQ ID NO. Pursuant to MPEP 2422 and 37 CFR 1.821(a), any amino acid sequence at least 4 amino acids in length requires a sequence identifier. Additionally, please updated the Sequence Listing if necessary.
Appropriate correction is required.
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 4-7 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. Claim 4 is directed to where the scaffold protein is a cell surface protein, and claim 6 is directed to where the scaffold protein is an intracellular protein. However, claims 4 and 6 are each dependent upon claim 1, which recites that a scaffold protein is a reporter protein. It is unclear whether the cell surface protein of claim 4 or the intracellular protein of claim 6 also constitute a reporter protein. Although the instant specification teaches that dNGFR and GFP are well known reporter proteins, these examples are not part of the definition of a scaffold or reporter protein. As such, it is unclear if the scaffold protein is a reporter protein AND a cell surface protein/intracellular protein or whether the cell surface protein/intracellular protein of claims 4 and 6 are also a reporter protein. Thus, an ordinary skilled artisan would be unable to ascertain the metes and bounds of the presently claimed invention with respect to the relationship between the reporter protein and cell surface protein/intracellular protein of claims 1, 4, and 6.
Please note that the Examiner suggests one way to over the rejection is to amend claims 4 and 6 such that each claim recites, “wherein the reporter protein is….” Also please note that the Examiner is interpreting the scope of claims 4 and 6 in this manner. Claims 5 and 7 are rejected by virtue of their dependency.
Claim Rejections - 35 USC § 102
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.
Claims 1 and 6-7 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Zhang et al., Molec. Biotechnol. 19:313-321 (2001).
For claims 1 and 6-7, Zhang et al. discloses two fusion proteins expressed transiently in a mammalian cell line; namely, one being p1xFLAG-GFP-c-Myc and the second being p3xFLAG-GFP-c-Myc (See Zhang, pg. 314, col. 1, 2nd paragraph). Zhang et al. teaches that each fusion protein was transfected into COS-7 cells, which extracts were obtained from and analyzed via Western blot analysis (See Zhang, pg. 314, col. 1, last paragraph; col. 2, 2nd paragraph). Moreover, immunohistochemical analysis was performed by seeding equal number of COS-7 cells transfected with each fusion protein (See Zhang, pg. 314, col. 2, last paragraph). Of a total of 8 chambers, two were seeded with cells expressing one fusion protein, another two were seeded with cells expressing the other fusion protein, and the other four chambers were used for Western blot analysis to assess expression level of both fusion proteins (See Zhang, pg. 314, col. 2, last paragraph). As such, GFP constitutes a scaffold protein that is a reporter protein such as an intracellular protein as recited in instant claims 1 and 6-7; and the FLAG and c-Myc constitute a series of two distinct linear epitope tags that are fused to the scaffold protein and where each are recognized by distinct antibodies as recited in instant claim 1. Plus, since there is no limit as to the number of cells in the claimed population of cells, a cell expressing one fusion protein and a second cell expressing the second fusion protein where one chamber contains the transfected p1xFLAG-GFP-c-Myc fusion protein and a second chamber contains the transfected p3xFLAG-GFP-c-Myc fusion protein constitute a population of cells where each cell is distinguishable from each other as recited in instant claim 1. In other words, two of the eight chambers constitute a population of cells where each cell comprises a fusion protein as described supra, where each of the cells in the population is distinguishable from each other. It is further noted that even though each fusion protein contains FLAG and c-Myc, there is no structural requirement that each cell contain/express distinct linear epitopes. Rather, the structural requirement, as stated in the “Claim Interpretation” section supra, is that each cell is distinguishable and where each fusion protein contains distinct linear epitopes. Since one fusion protein contains three copies of FLAG and the other contains one copy, it must then follow that a cell that contains/expresses each fusion protein is distinguishable from each other, and each fusion protein contains at least two distinct linear epitopes, i.e., FLAG and c-Myc.
Furthermore, with respect to where the distinct linear epitopes are each recognized by distinct antibodies and where the series of linear epitopes forms a detectable protein barcode tag unique to each fusion protein, since Zhang et al. expressly discloses a fusion protein comprising a series of two distinct linear epitopes fused to the scaffold protein thereby satisfying the structural limitations of the claim, functional properties not expressly disclosed by Zhang et al. would inherently be present. Thus, the claiming of a new use, new function or unknown property which is inherently present in the prior art does not necessarily make the claim patentable. In re Best, 562 F.2d 1252, 1254, 195 USPQ 430, 433 (CCPA 1977). There is no requirement that a person of ordinary skill in the art would have recognized the inherent disclosure at the time of invention, but only that the subject matter is in fact inherent in the prior art reference. Schering Corp. v. Geneva Pharm. Inc., 339 F.3d 1373, 1377, 67 USPQ2d 1664, 1668 (Fed. Cir. 2003). Therefore, Zhang’s disclosure satisfies the functional limitations recited in instant claim 1.
Accordingly, Zhang’s disclosure anticipates instant claims 1 and 6-7.
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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under pre-AIA 35 U.S.C. 103(a) 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 under 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of 35 U.S.C. 103(c) and potential 35 U.S.C. 102(e), (f) or (g) prior art under 35 U.S.C. 103(a).
103 - KSR Examples of 'Rationales' Supporting a Conclusion of Obviousness(Consistent with the "Functional Approach" of Graham)
Further regarding 35 USC 103(a) rejections, the Supreme Court in KSR International Co. v. Teleflex Inc., 550 U.S. 398, 127 S. Ct. 1727, 82 USPQ2d 1385, 1395-97 (2007) (KSR) identified a number of rationales to support a conclusion of obviousness which are consistent with the proper "functional approach" to the determination of obviousness as laid down in Graham. The key to supporting any rejection under 35 U.S.C. 103 is the clear articulation of the reason(s) why the claimed invention would have been obvious. The Supreme Court in KSR noted that the analysis supporting a rejection under 35 U.S.C. 103 should be made explicit.
Exemplary rationales that may support a conclusion of obviousness include:
(A) Combining prior art elements according to known methods to yield predictable results;
(B) Simple substitution of one known element for another to obtain predictable results;
(C) Use of known technique to improve similar devices (methods, or products) in the same way;
(D) Applying a known technique to a known device (method, or product) ready for improvement to yield predictable results;
(E) "Obvious to try" - choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success;
(F) Known work in one field of endeavor may prompt variations of it for use in either the same field or a different one based on design incentives or other market forces if the variations are predictable to one of ordinary skill in the art;
(G) Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention.
Note that the list of rationales provided is not intended to be an all-inclusive list. Other rationales to support a conclusion of obviousness may be relied upon by Office personnel.
Also, a reference is good not only for what it teaches by direct anticipation but also for what one of ordinary skill in the art might reasonably infer from the teachings. (In re Opprecht 12 USPQ 2d 1235, 1236 (Fed Cir. 1989); In re Bode 193 USPQ 12 (CCPA) 1976).
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., Molec. Biotechnol. 19:313-321 (2001) in view of Kimple et al., Curr. Protoc. Protein Sci. 73:26 pages (2015).
For claim 2, with respect to the incorporated limitations of claim 1, please see discussion of Zhang et al. supra. Briefly, it is noted that Zhang et al. teaches two fusion proteins expressed transiently in a mammalian cell line; namely, one being p1xFLAG-GFP-c-Myc and the second being p3xFLAG-GFP-c-Myc. The amino acid sequence of the FLAG epitope sequence is DYKDDDDK as evidenced by Kimple et al. (See Kimple, Table 9.9.1, at pg. 19). When comparing instant SEQ ID NO: 22 with the FLAG amino acid sequence, there is 100% identity. As such, one of the two distinct linear epitopes taught by Zhang et al. constitute one of instantly claimed epitopes recited in instant claim 2. However, Zhang et al. does not teach or suggest where the epitope tag is one of the other epitope tag sequences such as VSVg (SEQ ID NO: 23), V5 (SEQ ID NO: 24), AU1 (SEQ ID NO: 25), AU5 (SEQ ID NO: 26), HSV (SEQ ID NO: 40), or S-tag (SEQ ID NO: 46) recited in instant claim 2.
Kimple et al. teaches that relatively short epitope tags such as FLAG, HA, c-Myc, T7, and Glu-Glu, among other depicted in Table 9.9.1, which are used for the detection of fusion proteins in vitro and in cell culture (See Kimple, pg. 7, 4th paragraph). Their short, linear recognition motifs rarely affect the properties of the heterologous protein of interest and are usually very specific for their respective primary antibodies (See Kimple, pg. 7, 4th paragraph). Table 9.9.1 depicts a number of affinity tags including other short epitope tags including VSVg, V5, AU1, AU5, HSV, FLAG, c-Myc, and S-tag (See Kimple, Table 9.9.1). When comparing the amino acid sequences depicted in Table 9.9.1 for VSVg, V5, AU1, AU5, HSV, and S-tag with instant SEQ ID NOs: 23-26, 40, and 46, respectively, there is 100% identity. As such, Kimple et al. teaches that short, linear epitope tags including FLAG, c-Myc, HA, VSVg, V5, AU1, AU5, HSV, and S-tag function similarly for detection purposes.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant application to modify the teachings of Zhang et al. and substitute the short, linear epitope tag of c-Myc with VSVg, V5, AU1, AU5, HSV, or S-tag thereby forming the fusion proteins of p1xFLAG-GFP-(VSVg/V5/AU1/AU5/HSV/S-tag) and p3xFLAG-GFP-(VSVg/V5/AU1/AU5/HSV/S-tag) in order to examine the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope. One of ordinary skill in the art at the time the invention was made would have been motivated to do so because VSVg, V5, AU1, AU5, HSV, FLAG, c-Myc, and S-tag were known to be short, linear epitope tags useful for detection purposes as taught by Kimple et al. One of ordinary skill in the art at the time the invention was made would have had a reasonable expectation of success given that the fusion proteins of Zhang et al. comprised two distinct linear epitopes of p1xFLAG/p3xFLAG and c-Myc used to examine the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope, and therefore, substituting c-Myc with any one of VSVg, V5, AU1, AU5, HSV, or S-tag thereby forming the fusion proteins of p1xFLAG-GFP-(VSVg/V5/AU1/AU5/HSV/S-tag) and p3xFLAG-GFP-(VSVg/V5/AU1/AU5/HSV/S-tag) would support the examination of the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope by constituting the simple substitution of one known element for another to obtain predictable results and/or some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention pursuant to KSR.
Thus, the invention as a whole is prima facie obvious over the references, especially in the absence of evidence to the contrary.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., Molec. Biotechnol. 19:313-321 (2001) in view of Rondon et al. US 2010/0184612 A1 published on July 22, 2010 (cited in the IDS received on 11/2/23).
For claim 3, with respect to the incorporated limitations of claim 1, please see discussion of Zhang et al. supra. Briefly, it is noted that Zhang et al. teaches two fusion proteins expressed transiently in a mammalian cell line; namely, one being p1xFLAG-GFP-c-Myc and the second being p3xFLAG-GFP-c-Myc. Although the GFP is separating the two distinct linear epitopes in Zhang et al., since the scope of claim 3 requires that the fusion protein further comprises amino acid spacer sequences separating the linear epitopes, the claimed fusion protein must contain amino acid spacer sequences in addition to the scaffold protein. However, there is no structural requirement that as to the residues that make up the amino acid spacer sequences and no structural requirement that the spacer sequences are directly fused to the linear epitopes. Thus, Zhang et al. does not expressly teach or suggest where the fusion protein further comprises an amino acid spacer between the two linear epitopes.
Rondon et al. teaches a triple tag sequence, either directly linked together (SEQ ID NO: 4) or via amino acid spacer sequences (SEQ ID NO: 5), to a protein of interest where the triple tag sequence comprises a 6x histidine sequence, a c-myc sequence, and a V5 sequence (See Rondon specification, paragraph [0006]-[0008], [0013]-[0015]) where the tagged protein is detected in a sample, e.g., cell culture, by utilizing antibodies directed to the distinct tag sequences (See Rondon specification, paragraph [0018]-[0019], [0034], [0049]). A specific example of a protein of interest is a single chain antibody fused to this triple tag sequence, either directly or via an amino acid spacer sequences (See Rondon specification, paragraph [0013]-[0015], [0030]). Examples of the amino acid spacer sequences are GAA, KAA, or SEQ ID NO: 5 (See Rondon, [0006], [0014], [0049], [0051], [0131]). The protein of interest can also be linked to the triple tag sequence via a spacer (See Rondon, [0031], [0054]). Rondon et al. teaches that the protein of interest to be fused to the triple tag sequences can be any protein known in the art such as an antibody including a single chain antibody or a scaffold protein (See Rondon specification, paragraphs [0091]-[0094]). Thus, Rondon et al. suggests a fusion protein comprising a scaffold protein fused to a triple tag sequence where the scaffold protein and/or each of the epitopes of the triple tag sequence can be separated via an amino acid spacer sequence.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant application to modify the teachings of Zhang et al. and insert an amino acid spacer sequence such as GAA or KAA between the epitope tags and the GFP thereby forming the fusion proteins of p1xFLAG-GAA/KAA-GFP-GAA/KAA-c-Myc and p3xFLAG-GAA/KAA-GFP-GAA/KAA-c-Myc in order to examine the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope. One of ordinary skill in the art at the time the invention was made would have been motivated to do so because fusion proteins comprising a scaffold protein fused to a triple epitope tag sequence was known to contain amino acid spacer sequences such as GAA and KAA where the spacers can be inserted between the epitope tag and the scaffold protein or between the epitopes and where the fusion proteins were known to be detected in a sample, e.g., cell culture, by utilizing antibodies directed to the distinct tag sequences as taught by Rondon et al.
One of ordinary skill in the art at the time the invention was made would have had a reasonable expectation of success given that the fusion proteins of Zhang et al. are used to examine the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope in vitro, and therefore, inserting an amino acid spacer sequence such as GAA or KAA between the epitope tags and the GFP thereby forming the fusion proteins of p1xFLAG-GAA/KAA-GFP-GAA/KAA-c-Myc and p3xFLAG-GAA/KAA-GFP-GAA/KAA-c-Myc would support the examination of the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope in vitro by constituting the simple substitution of one known element for another to obtain predictable results and/or some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention pursuant to KSR.
Thus, the invention as a whole is prima facie obvious over the references, especially in the absence of evidence to the contrary.
Claims 4-5 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., Molec. Biotechnol. 19:313-321 (2001) in view of Fehse et al., Human Gene Ther. 8:1815-1824 (1997).
For claim 5, with respect to the incorporated limitations of claims 1 and 4, please see discussion of Zhang et al., Rondon et al., and Huang et al. supra. Briefly, it is noted that Zhang et al. teaches two fusion proteins expressed transiently in a mammalian cell line; namely, one being p1xFLAG-GFP-c-Myc and the second being p3xFLAG-GFP-c-Myc. As such, Zhang et al. teaches utilizing GFP as a scaffold protein. The combination of Rondon et al. and Huang et al. suggest substituting the GFP of Zhang et al. with a cell surface protein such as a T cell receptor. However, Zhang et al., Rondon et al., and Huang et al. do not teach or suggest where the cell surface protein is dNFGR.
Fehse et al. that human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat disease (See Fehse, abstract). To rapidly characterize transduced cells and to isolate these from residual non-transduced, but biologically equivalent, cells, Fehse used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (delta-LNGFR) cDNA as a marker gene (See Fehse, abstract; pg. 1815, col. 1, last paragraph). Fast and convenient quantitation of the transduction efficiency by florescence-activated cell sorting (FACS) analysis as well as enrichment of transduced cells by immunoadhesion were possible through the ectopically expressed transgene (See Fehse, pg. 1815, col. 1, last paragraph). Expression of deltaLNGFR in hematopoietic progenitor cells will be a useful tool for the selection of transgene-expressing cells and for the design of novel and further development of existing retroviral vectors because gene transfer efficiency can be measured almost immediately after transduction (See Fehse, pg. 1815, col. 1, last paragraph to col. 2, 1st paragraph). Moreover, Fehse et al. teaches that other methods to determine the number of transduced cells include the PCR or in vitro, ex vivo, or in situ staining with substrates for the bacterial beta-galactosidase, the human placental alkaline phosphatase or GFP (See Fehse, pg. 1816, col. 1, 2nd paragraph). An alternative strategy has relied on the expression of transgene-encoded cell-surface antigens such as CD24, its murine homolog heat-stable antigen (HSA), murine CD2, human CD4ζ, the multidrug resistance gene mdr-1, and the full-length or intracytoplasmically truncated human low-affinity nerve growth factor (LNGFR or deltaLNGFR) (See Fehse, pg. 1816, col. 1, 2nd paragraph). As indicated in the “Claim Interpretation” section supra, the dNGFR encompassed by claim 5 encompasses any mutant NGFR that is truncated such that the intracellular domain has been deleted. As such, Fehse’s deltaLNGFR constitutes a species of dNGFR as recited in instant claim 5. Thus, Fehse et al. teaches that reporter proteins include GFP and cell surface proteins such as a mutant NGFR such as deltaLNGFR. Therefore, Fehse et al. suggests that a mutant NGFR that is truncated to remove the intracellular domain functions is a cell surface antigen that functions as a reporter protein (i.e., a protein that is heterologous to a target cell and where its presence is indicative of successful gene transfer from a vector to the target cell) similar to GFP by allowing for efficient gene transfer that can be measured almost immediately.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant application to modify the teachings of Zhang et al. and substitute GFP with a cell surface protein such as deltaLNGFR as a reporter protein thereby forming the fusion proteins of p1xFLAG-deltaLNGFR-c-Myc and p3xFLAG-deltaLNGFR-c-Myc in order to examine the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope. One of ordinary skill in the art at the time the invention was made would have been motivated to do so because reporter proteins such as GFP and deltaLNGFR were known to be used in retroviral vectors and transduced into a target cell in order to measure gen transfer efficiency as taught by Fehse et al. One of ordinary skill in the art at the time the invention was made would have had a reasonable expectation of success given that the fusion proteins of Zhang et al. utilized GFP as a reporter scaffold protein to examine the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope, and therefore, substituting a cell surface protein such as deltaLNGFR as a reporter protein instead of GFP thereby forming the fusion proteins of p1xFLAG-deltaLNGFR-c-Myc and p3xFLAG-deltaLNGFR-c-Myc would support the examination of the detection sensitivity of using a triple FLAG epitope compared to a single FLAG epitope by constituting the simple substitution of one known element for another to obtain predictable results and/or some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention pursuant to KSR.
Thus, the invention as a whole is prima facie obvious over the references, especially in the absence of evidence to the contrary.
Conclusion
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/THEA D' AMBROSIO/ Primary Examiner, Art Unit 1654
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