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 .
Priority
The current application claims priority to provisional application 63/236576, filed on 24 August 2021, and is 371 of PCT/US2022/075366, filed on 23 August 2022. The effective filing date is 24 August 2021.
Information Disclosure Statement
The information disclosure statement (IDS), filed on 06 June 2024, was considered by the examiner.
Status of Application, Amendments, and/or Claims
Claims 1-35 are the original claims. In the preliminary amendment of 04 September 2024, claims 9, 18, and 23-35 were canceled and claims 8, 10, 11, and 17 were amended. Claims 1-8, 10-17, and 19-22 are pending and the subject of this office action.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 3, 4, 6-8, and 10 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Dhar T, et al. (2011) Modification of transmembrane and GPI-anchored proteins on living cells by efficient protein trans-splicing using the Npu DnaE intein. Chem Commun (Camb). 2011 Mar 21;47(11):3063-5 (Dhar).
Dhar relates to the modification of transmembrane and GPI-anchored protein on living cells through the utilization of split Npu DnaE inteins (Abstract and Title). In this study, the authors disclose a strategy for efficient trans-splicing of cell surface proteins, using split inteins. In this model system, an exogenous GFP-Npu DnaE IntN fusion protein is added to cells expressing a fusion protein, comprising a Npu DnaE IntC, a transmembrane spanning helix (TM) from the platelet-derived growth factor (PDGF) receptor (a signaling receptor), and an intracellular mCherry reporter, as shown below (Relevant to instant claims 1, 3, 4, 6, 7, 8, and 10) (Figure 1a).
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It is demonstrated that two fusions proteins undergo protein trans-splicing, driven by the intein pairs, resulting in a GFP-TM-mCherry fusion, as confirmed by membrane localized GFP fluorescence and immune-blot analysis (Page 3064 paragraph 1 and Figure 2).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 21 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Dhar T, et al. (2011) Modification of transmembrane and GPI-anchored proteins on living cells by efficient protein trans-splicing using the Npu DnaE intein. Chem Commun (Camb). 2011 Mar 21;47(11):3063-5 (Dhar).
In regard to claim 21, Dhar teaches a method for synthesizing a fusion protein, comprising contacting a phospholipid bilayer-embedded transmembrane domain-split intein fusion protein with a second fusion protein, comprising the second half of the split intein, as described in the 35 U.S.C. 102(a)(1) rejections of claims 1, 3, 4, 6, 7, and 8.
Dhar does not explicitly teach a method, in which the transmembrane domain is contacted by a biologically active protein domain, as in instant claim 21.
Dhar teaches that membrane proteins play critical roles in various biological processes and are important drug targets (Page 3063 paragraph 1). It is also taught that site-specific labeling of these proteins is an important approach for studying their function, dynamics, and interactions with other molecules. Dhar goes onto briefly describe the shortcomings associated with the current crop of site-specific labelling techniques (i.e. inability to modify polypeptide backbone structure, the requirement of conjugation enzymes, and possible comprising of biological function) and goes onto detail some of the advantages associated with a split intein approach, such as the self-processing reaction and the traceless reaction products. Dhar et al is written with a bias towards labelling membrane proteins, but the authors do make note of the disclosed methods potential for other applications, as shown in the following excerpt: “Such backbone engineering would also be interesting for protein reconstitution or the creation of nanostructures in artificial systems, for example” (Page 3063 paragraph 1). Additionally, Char states that the disclosed method “is expected to be useful for the manipulation of the structure and primary sequence of membrane proteins in a post-translational fashion, for example to attach a modified extracellular domain back onto a truncated membrane protein”, which suggests that the system could be used for membrane proteins, not just the fluorescent proteins used in the disclosed model system, which were likely selected for ease of detection. Thus, contacting the transmembrane domain with a biologically active protein domain (i.e. extracellular domain of a membrane protein) would have been obvious to one skilled in the art.
In regard to claim 22, the Npu DnaE split intein system uses transthioesterification as a central step in protein trans-splicing, and as such would be inherent to any method utilizing the corresponding split inteins.
Claims 2 and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Dhar T, et al. (2011) Modification of transmembrane and GPI-anchored proteins on living cells by efficient protein trans-splicing using the Npu DnaE intein. Chem Commun (Camb). 2011 Mar 21;47(11):3063-5 (Dhar) in view of Zhao Y, et al. (2018) Antigen-Presenting Cell-Intrinsic PD-1 Neutralizes PD-L1 in cis to Attenuate PD-1 Signaling in T Cells. Cell Rep. 2018 Jul 10;24(2):379-390.e6 (herein Zhao).
Dhar teaches a method for synthesizing a fusion protein, comprising contacting a phospholipid bilayer-embedded transmembrane domain-split intein fusion protein with a second fusion protein, comprising a biologically active domain and second half of the split intein, as described in the 35 U.S.C. 103 rejections of claims 21 and 22. Dhar also suggests this method and the resulting fusion proteins may prove useful in the creation of nanostructures and artificial systems (Page 3063 paragraph 1).
Zhao et al relates to a study investigating the regulatory effect of PD-1-PD-L1 cis binding (Summary). In this study the extracellular domain of PD-1, comprising a His-tag, was non-covalently attached to a synthetic large uni-lamellar vesicles (LUV), comprising DGS-NTA-Ni (Relevant to instant claim 1) (Methods: LUVs Reconstitution and FRET assays and Figure 3). This system was designed to limit the complexity of the experimental system, because as stated by the authors “due to the complex environment of a cell membrane, it remains possible that the molecular proximity between PD-1 and its ligands is mediated by other proteins or is driven by lipid microdomains such as rafts” (Results: PD-1 and PD-L1 Bind to Each Other in cis).
Combining the teachings of Dhar (method for producing fusion proteins comprising a TM covalently linked to a biologically active domain) with those of Zhao (reconstituted LUV membranes containing PD-1) would have been obvious to one skilled in the art. Dhar suggests that the disclosed method, encompassing instant claim 1, could be used to produce an artificial system (Page 3063 paragraph 1). Both the method taught by Zhao and that taught by Dhar enable the attachment of a biologically active domain to a lipid bilayer, as demonstrated in Fig 3 (Zhao) and Figure 2 (Dhar). Zhao teaches a reconstituted artificial system, comprising a LUVs comprised of a biologically active domain (PD-1). Instant claims 2 and 5 are essentially the results of a simple substitution of methods, in which the method taught by Zhao, resulting in a LUV attached to the PD-1 extracellular domain, is substituted with the method of Dhar, yielding predictable results.
Claims 11-17, 19, and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Dhar T, et al. (2011) Modification of transmembrane and GPI-anchored proteins on living cells by efficient protein trans-splicing using the Npu DnaE intein. Chem Commun (Camb). 2011 Mar 21;47(11):3063-5 (Dhar) in view of Chen X, et al. (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69 (herein Chen) and Cronin M, et al. (2015) Dynamics differentiate between active and inactive inteins. Eur J Med Chem. 2015 Feb 16;91:51-62 (herein Cronin) with Zettler J, et al. (2007) The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 2009 Mar 4;583(5):909-14 (herein Zettler) providing additional evidentiary value.
In regard to claim 11, Dhar teaches Dhar teaches a method for synthesizing a fusion protein, comprising contacting a phospholipid bilayer-embedded transmembrane domain-split intein fusion protein with a second fusion protein, comprising the second half of the split intein and the extracellular domain of a biologically active protein, as described in the 35 U.S.C. 103 rejections of claims 20 and 21.
Dhar does not explicitly teach that the first intein is covalently attached to the transmembrane domain, described in claim, through a covalent linker. Chen and Cronin teach this deficiency.
Cronin relates to a study investigating the dynamics of active and inactive inteins (Abstract). This study employed experimental measurements and molecular dynamics simulations to probe the correlations between either protein dynamics or protein stability and intein function (Results: Sections 1-5). Structural models of the splicing domain of the Mtu RecA mini-intein, in which the loop the connecting the N-intein segment and the C-intein segment varied in length, and corresponding V67L mutant variants were used in molecular dynamics simulations, in order to assess both global protein dynamics (Results: Section 1 and Figure 3) and residue-specific dynamics (Results: Section 2 and Figure 4). It is taught that the V67L mutation restores activity to these mini-inteins, which lack the endonuclease domain that normally link the N- and C-terminal splicing domains (Introduction). The authors observed that structures containing the V67L mutation displayed higher global stability, as shown by lower global RMSD values for V67L structures compared to their V67 counterparts (Results: Section 1 and Figure 3). The authors also note that the length of the linker loop, connecting the splicing segments, appears to influence intein activity, as shown by the inactivity of the 96Δ402Leu (the mutant with the shortest linker loop) and the high activity of the 110Δ383Leu mutant (the mutant with the shortest linker loop) (Results: Section 2 and Figure 4). It is suggested that this difference in activity may be due to the differences in intein core region dynamics/flexibility, based on the higher calculated RMSF values of core region residues for the more active 110Δ383Leu structure (long linker) compared to the values of 96Δ402Leu (short linker). The authors go on to state “lengthening the linker loop allows the intein some flexibility, which is necessary for the splicing activity” and “shortening the loop too much will result in the intein structure becoming so rigid that it is unable to perform its splicing function” (Results: Section 2 and Discussion). Thus, Cronin establishes that flexibility between the intein splicing segments is requirements is required for intein activity and that this flexibility can be modulated through the moiety linking the two segments.
Chen relates to a review of fusion protein linker design (Abstract). Chen teaches that direct fusion of functional domains without a linker may lead to misfolding, low production yields, or impaired bioactivity, and that the use of domain linkers is often used to address these potential complications (Introduction paragraph 2). Chen also teaches that flexible linkers are often used when the joined domains require a certain degree of flexibility (Section 3.1). It is also taught that the most commonly used flexible linker consists of stretches of glycine and serine residues (GS linker), and provides (Gly-Gly-Gly-Gly-Ser)n as an example of such a linker, where n is the copy number, which can be optimized to achieve optimal domain spacing (Section 3.1). These teachings provide guidance for the design of flexible linkers in fusion proteins that require a certain degree of flexibility between domains.
It would have been obvious, to one skilled in the art, to combine the teachings of Dhar (a transmembrane domain fused to an intein) with those of Cronin (the importance of flexibility between splicing segments) and Chen (guidance regarding the design of linkers in fusion proteins). Cronin establishes that intein activity requires a degree of flexibility between the N- and C-terminal splicing domains (Results: Section 2 and Discussion). In the context of the fusion proteins taught by Dhar and the claimed invention of the current application, in which one of the split-inteins is fused to membrane-embedded TM domain, one of average skill in the art would recognize the crucial importance of Cronin’s teachings. In this scenario, the intein is fused to a transmembrane domain, which is relatively restrained in terms of flexibility owing to its placement in a lipid bi-layer, and as such the intein is relatively restrained/inflexible. Thus, to ensure that the fused intein is biologically active, one would have been motivated to increase the flexibility of the TM-fused intein, based on the teaching of Cronin. With this motivation, the incorporation of the teachings of Chen, which state that a flexible linker may afford a certain degree of flexibility between two functional domains, would have been obvious. Furthermore, the use of a GS linker also would have been obvious from these teaching, as Chen teaches that GS linkers, such as (Gly-Gly-Gly-Gly-Ser)n (Relevant to instant claims 12 and 13), are the most commonly used flexible linker, which implies a reasonable expectation of success (Section 3.1).
In regard to claims 14, Dhar teaches a method for synthesizing a fusion protein, comprising contacting a phospholipid bilayer-embedded transmembrane domain-split intein fusion protein with a second biologically active fusion protein, comprising the second half of the split intein, as discussed for the 35 U.S.C. 103 rejections of claims 21 and 22.
Dhar also provides as example of such a method, in which the split inteins are derived from NpuDnaE, with IntC being fused to the transmembrane domain and IntN being fused to the biologically active domain (Figure 1a), see 35 U.S.C. 102 rejection of claims 1, 3, 4, and 6-8. Additionally, Dhar, Cronin, and Chen teach that the transmembrane domain is attached to an intein through a (Gly-Gly-Gly-Gly-Ser)n linker, as discussed above. When combining these teachings, one would arrive at fusion proteins comprising the following structures:
NpuDnaE IntC -GGGSn-TM and;
BAD- NpuDnaE IntN
BAD – Biologically active domain
When combining these two fusion proteins, as taught in the method disclosed by Dhar, the product of the resulting reaction would be as follows:
BAD-CFN- GGGSn-TM
BAD – Biologically active domain
This product is inherent to the method taught by Dhar and the fusion proteins taught by Dhar, Cronin, and Chen, which renders instant claim 14 obvious. For additional clarification, the CFN sequence, referenced above, is the inherent intein scar amino sequence of NpuDnaE, as evidenced by the teachings of Zittler (Results: 3.1). Thus, a first linker of SEQ ID NO:10 is inherent to the method taught by Dhar (Relevant to instant claims 14 and 15). Additionally, the product, shown above, encompasses the limitations established in instant claims 16, 17, 19, and 20.
Conclusion
No claims allowed.
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/MATTHEW CURRAN METCALF/Examiner, Art Unit 1647 /JOANNE HAMA/Supervisory Patent Examiner, Art Unit 1647
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