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 .
Claims 1-17 are pending and have been considered on the merits herein.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1-17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Deprey et al., (Methods in Enzymology, vol. 641, p. 277-309, 2020) in view of Kritzer et al. (US2018/0188260, IDS), Richter et al. (Nature, vol. 545, p. 299-304, 2017, IDS) and Ke et al (J. of Bacteriology, vol. 198, p. 1035-1043, 2016).
Deprey teaches the chloroalkane penetration assay (CAPA) to determine cytosolic localization of molecules. CAPA uses a HaloTag protein, which is a mutant bacterial haloalkane dehalogenase modified to react irreversibly with a chloroalkane ligand. CAPA uses a cell line, i.e. mammalian cell, to express the HaloTag, which is then pulsed with a chloroalkane tagged molecule of interest (ct-molecule). If the ct-molecule reaches the cytosol, it reacts with the HaloTag and blocks HaloTag reactive sites. A chase agent, i.e. a chloroalkane tagged dye (ct-dye) reacts with the open HaloTag active sites (Fig. 1, section 2.1 p. 281-282, section 4.2 steps 4, 7, Fig. 4). The more cell-penetrant the molecule, the less fluorescence that is detected (p. 281 section 2.1-p. 282 whole page, p. 283, section 3.1-p. 2841st parag.). Thus, CAPA reads cytosolic localization of the molecules and allows for large-scale analysis of thousands of molecules with an automated high-throughput screening platform (section 7.1) Fluorescence is measured directly as background fluorescence on the cells with dye and without dye.
Thus, the reference teaches contacting a small molecule linked to a chloroalkane moiety with a cell expressing a mutant form of bacterial haloalkane dehalogenase and contacting that cell with a fluorophore linked to a chloroalkane moiety, detecting the fluorescence signal of the cells and determining that a low fluorescent signal relative to the background fluorescence correlates to high accumulation of the molecule within the cell and high fluorescence correlates to low accumulation of the molecule within the cell (see section 2.1, p. 281-283, Fig. 1-3).
Regarding claims 2 and 3, the fluorophore is a rhodamine dye, i.e. chloroalkane-tetramethylrhodamine (ct-TMR) (section 3.3.3, 4.2 (steps 4, 7), Fig. 1, 3, 4).
Regarding claim 4, the chloroalkane moiety linked to the fluorophore is of the formula claimed, see Fig 3C for example and section 3.3.3.
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Regarding claims 5-8, 12-15, the chloroalkane moiety linked to the molecule is of the formulas according to the claims. See Fig. 2 and 3 teaching the synthesis of the chloroalkane tag (see compounds 2, 3), comprising a strained alkyne, DBCO moiety (according to claims 5-7, 12, 13) linked to a chloroalkane wherein alk-Cl is a C1-C20 alkyl optionally interrupted to 1-3 or 1-6 oxygen atoms according to claims 5-7, 14,15 (see Compound 3 and ct-DBCO), and wherein the linker is an amide bond (see section 3.2, 3.3, Fig. 3). Regarding the limitation of claim 8, drawn to a small molecule linked to an azide moiety, the reference teaches the cycloaddition of chloroalkane-DBCO with an 18-mer oligonucleotide with a 5’ azide, i.e. an azide-alkyne click reaction (p. 286, section 3.3, 3.3.4).
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The reference differs from the claimed invention in that mammalian cells are used in CAPA, not bacterial cells according the claims 1-17.
Kritzer teaches a method for localizing and quantifying molecules/agents (0018) which penetrate a cell expressing chloroalkane dehydrogenase, i.e. a mutant bacterial haloalkane dehalogenase (0008-0010, 0061, 0062), wherein the cell is contacted with an agent conjugated to a chloroalkane, and contacting the cell with chloroalkane conjugated to a detectable moiety. The method is the chloroalkane penetration assay i.e. CAPA (0042, 0043, 0060), which uses the HaloTag system comprising a HaloEnzyme (cell engineered to express a mutant bacterial haloalkane dehalogenase) and a chloroalkane linker)(0061, 0062). Cells which express the HaloTag/Enzyme are treated with a small molecule conjugated to chloroalkane moiety to determine if the molecule can penetrate the cell membrane, and if it reacts with the HaloTag/enzyme, it will block its active sites, the small molecule is then “chased” with a chloroalkane moiety linked to a fluorophore, i.e. HTag-TAMRA (0073, 0079). A fluorescence signal is detected after the “chase” indicating cell penetration of the molecule/agent of interest (0008) which is inversely proportional to the degree of penetration (0060, 0073). The reference teaches that the cell expressing a HaloTag is treated with a cell-penetrant molecule/agent (HTag-cTMP or HTag-DD5o) and chased with a HaloTag or HTag linked to a fluorophore, i.e. HTag-TAMRA (0043, 0045, 0080). The reference teaches that the CAPA method can be applied to any chemically tractable molecule including small molecules, and since the cell is engineered to express the HaloEnzyme, it can be directed to any cellular compartment for specific quantitation of access to any cellular compartment or organelle, thus measuring penetration of molecules (0074).
Regarding claims 1, 8, 16, 17, the reference teaches that the cell can be a eukaryotic or prokaryotic bacteria cell (0011, 0016), and that the HaloTag/enzyme system can be successfully applied in many systems including bacteria (0062) and E. coli (0064).
Regarding claims 2 and 3, the detectable moiety/fluorophore is a fluorescent tag including HaloTag coumarin, tetrazine-tetramethyl rhodamine (TAMRA) or tetramethyl rhodamine (TMR) (0016, 0071).
Regarding claims 4 and 5, the fluorophore, and small molecule or agent is covalently linked to a chloroalkane moiety of the formulas of claims 4, 5 and has an alk according to claims 5-7. See Fig. 1D. HTag itself is the chloroalkane moiety having the claimed alk according to claims 4-7.
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It is known that small molecules have difficulty crossing the outer and inner membranes of Gram-negative bacteria, specifically E. coli. Further support is provided by Richter who teach that because of this difficulty, the discovery of drugs, specifically antibiotics, against these pathogens is challenging (abstract). Gram-negative bacteria possess two cellular membranes, and the lipopolysaccharide coated outer membrane is very challenging for small molecules to cross, thus there is limited understanding of small-molecule accumulation in these bacteria and effective antibiotics. The reference studies accumulating compounds in E. coli cells.
Ke teaches the use of the HaloTag in E. coli to visualize proteins within the periplasmic and cytoplasmic compartments of the cells. Ke teaches the difficulty of targeting molecules to the periplasmic compartment of E. coli, particularly the difficulties of crossing the inner and outer membranes (abstract, p. 1035, 2nd full parag.) Ke teaches that HaloTag covalently bind chloroalkane ligands, including the fluorophore tetramethylrhodamine (TMR) (p. 1036, 1st col, last parag.-2nd col. 1st parag.). The use of the HaloTag effectively allowed the visualization of periplasmic and cytoplasmic proteins inside the bacteria (p. 1037, Results parag., p. 1041, Discussion section, 1st parag.).
Therefore, before the effective filing date of the claimed invention, the difficulty of small molecules traversing the membrane and accumulating within Gram-negative bacteria was known. Thus, there is an art recognized need to discover drugs against the pathogens.
Kritzer teaches that the CAPA method can be applied to bacteria including E. coli, and Ke demonstrates the use of the HaloTag to visualize molecules within the difficult to cross pathogen E. coli. Therefore, it would have been obvious to one of ordinary skill in the art to apply the CAPA method to bacterial cells to discover and determine small molecules including antibiotics which can cross and accumulate within bacterial cells and a phosita would have had a reasonable expectation in view of the teachings of Deprey, Kritzer and Ke, that the HaloTag and CAPA method could be used not just in mammalian cells but also in bacterial cells including E. coli cells to determine the accumulation of small molecules within the bacteria. Thus, in light of the teachings of Deprey, Kritzer and Ke, it would have been obvious to apply the CAPA method to bacterial cells to discover and determine small molecules including antibiotics which can cross and accumulate within the cells.
Conclusion
No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIFFANY MAUREEN GOUGH whose telephone number is (571)272-0697. The examiner can normally be reached M-Thu 8-5.
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/TIFFANY M GOUGH/ Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651
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