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 .
Nucleotide and/or Amino Acid Sequence Disclosures
Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures
37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted:
1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying:
a. the name of the XML file
b. the date of creation; and
c. the size of the XML file in bytes; or
2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying:
a. the name of the XML file;
b. the date of creation; and
c. the size of the XML file in bytes.
SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS:
Specific deficiency - Sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.831(c). Sequence identifiers for sequences (i.e., “SEQ ID NO:X” or the like) must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Amended drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required sequence identifiers (i.e., “SEQ ID NO:X” or the like) into the Brief Description of the Drawings, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1 – 3, 7 – 9, 14 – 15, and 20 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by US Patent Application Publication US 20170145058 A1 to Griebenow et. al. (herein after Griebenow’058; cited on the IDS dated December 29th, 2023).
Regarding claims 1 – 3, 7 – 9, 14 – 15, and 20, Griebenow’058 teach a novel process for the targeted conjugation of peptides and proteins which are characterized by the pairwise C2-bridging of cysteine amino acids via their via their thiol groups (page 1 paragraph 0001). Additionally, Griebenow’058 teach a process for preparing homogeneous peptide and protein conjugates, which process is characterized in that a peptide or protein or the formula (II) in which S1 and S2 represent cysteine sulfur atoms of this peptide or protein which are bonded in a disulfide bridge is converted under reducing conditions into a peptide or protein of the formula (III) and this is then reacted under free-radical reaction conditions with an alkyne derivative of the formula (IV) (page 3 paragraph 0018) to give a peptide or protein conjugate of the formula (I) (page 4 paragraph 0033) summarized here:
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(page 4 paragraph 0034). Specifically, Griebenow’058 teach example 8 the C2-bridging thiol-yne reaction with an antibody Fab fragment (claim 14) (page 19 paragraph 0227) of structure
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(claims 1, 9, and 20) (Sheet 7 of 7 Figure 13) formed by 1st reacting 108.4 µL of a solution of Fab fragment M14-G07 with the reducing agent 4 µL of a solution of TCEP-HCl prepared from dissolving 4.60 mg of TCEP-HCI (claim 8) in 400 µL of buffer before being further reacted with hept-6-ynoic acid of the structure
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(claims 7, and 15), LAP and irradiated with 365 nm UV light (claims 2 – 3) (page 19 paragraph 0228).
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.
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 4 – 6 are rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication US 20170145058 A1 to Griebenow et. al. (herein after Griebenow’058; cited on the IDS dated December 29th, 2023) in view of Geel et.al. ((2012), Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide−Alkyne Cycloaddition, Bioconjugate Chemistry, 23, 392 – 398).
The teachings of Griebenow’058 as they relate to claim 1, from which claims 4 – 6 depend, are given previously in this office action and are fully incorporated here.
However, Griebenow’058 is silent about the use of a strained ring dibenzocyclooctyne (claims 5 – 6) moiety of the modifying reagent (claim 4).
Nevertheless, Geel et. al. teach that click chemistry refers to a set of chemical reactions that proceed rapidly in a highly specific and efficient manner, even within a complex chemical environment (page 392 column 1 paragraph 1). Moreover, Geel et. al. teach the use of a highly reactive strained cyclooctyne allows efficient and specific labeling under physiological conditions without the toxic copper(I) catalyst, enabling labeling of biomolecules in vivo (page 392 column 1 paragraph 2). Furthermore, Geel et. al. teach that is has been noted that strained alkynes can bind to a varying extent to biological functionalities in an azide-independent manner, thereby limiting the sensitivity of assays based on this reaction (page 392 column 2 paragraph 2). Additionally, Geel et. al. teach a panel of three strained cyclooctynes which include dibenzocyclooctyne (DIBO) (claims 4 – 6) in train-promoted azide−alkyne cycloaddition (SPAAC) (page 392 column 2 paragraph 2). Now while Geel et. al. taught the use of cyclooctynes in the context of optimizing SPAAC reactions Geel et. al. does teach that without the iodoacetamide (IAM) catalyst non-specific labeling of cysteine can occur along the alkyne bond of DIBO (page 392 abstract figure). In fact, Geel et. al. teach that all of the cyclooctynes in the study showed higher reactivities than observed for the terminal alkyne, suggesting that additional instability introduced by the ring-strain and/or the increased hydrophobicity enhances the efficiency by which they, that is the cyclooctynes, react with proteins (page 394 column 1 paragraph 4 and column 2 paragraph 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the instant application to modify the method of Griebenow’058 for a method of disulfide bridging cysteines of an antibody in view of Geel et. al. that is to use dibenzocyclooctyne (DIBO) as the strained ring moiety of the modifying reagent. One of ordinary skill in the art would have been motivated to make this modification to efficiently and specifically label an antibody under physiological conditions without the use of toxic copper(I) catalyst. One of ordinary skill in the art would have had a reasonable expectation of success because strained cyclooctynes, such as (DIBO) showed higher reactivities than those observed for the terminal alkynes.
Claims 10 – 13 are rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication US 20170145058 A1 to Griebenow et. al. (herein after Griebenow’058; cited on the IDS dated December 29th, 2023) in view of Hall, E. A. ((2014), Antibody Conjugates via Disulfide Bridging: Towards therapeutic and diagnostic applications [Doctoral dissertation, University College London]. UCL Discovery, https://discovery.ucl.ac.uk/id/eprint/1461131/).
The teachings of Griebenow’058 as they relate to claim 1, from which claims 4 – 6 depend, are given previously in this office action and are fully incorporated here.
However, Griebenow’058 fails to teach a method of modifying an antibody wherein an alkyne moiety reacts with a sulfur moiety on a second heavy chain of the antibody and a sulfur moiety on a second light chain of the antibody, thereby synthetically crosslinking the second heavy chain to the second light chain (claim 10); a sulfur moiety on a first heavy chain of the antibody and a sulfur moiety on a second heavy chain of the antibody, thereby synthetically crosslinking the first heavy chain to the second heavy chain (claim 11); a second sulfur moiety on the first heavy chain of the antibody and a second sulfur moiety on the second heavy chain of the antibody, thereby forming a second synthetic bridge between the first heavy chain and the second heavy chain (claim 12); and a first sulfur moiety on a heavy chain of the antibody and a second sulfur moiety on the heavy chain of the antibody, thereby forming an intrachain synthetic crosslink in the heavy chain (claim 13).
Nevertheless, Hall teach that currently insertion of unnatural amino acids and enzymatic labelling for the site-specific introduction of bio-orthogonal click chemistry reagents has proved the most successful strategy for producing homogenous antibody conjugates in high yield (page 46 paragraph 2). Furthermore, Hall teach that in the case of antibodies and antibody fragments with no free cysteine residues, site-directed conjugation has been attempted via interchain disulfide bond reduction and subsequent conjugation of the free cysteines (page 46 paragraph 2). Additionally, Hall teach that for many diseases, antibodies act as a therapeutic agent by blocking the function of their target (page 19 paragraph 3). Moreover, Hall teach that while there has been some success using unmodified mAbs many biological barriers must be overcome for significant accumulation in the target tissue (page 19 paragraph 3). Thus, Hall teach antibodies and antibodies fragments as targeting agents for administering cytotoxic substances or delivering potent effectors (page 19 paragraph 3). Specifically, Hall teach a sequential and in situ conjugation strategies for the modification of monoclonal IgG1 antibody Trastuzumab with a doxorubicin payload shown here
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(page 55 Scheme 1.15) wherein the there is a disulfide bridge crosslinking a 1st heavy chain with a 1st light chain; a 2nd heavy chain with a 2nd light chain, and two disulfide bridges crosslinking the 1st heavy chain with the 2nd heavy chain. In terms of the reaction conditions, Hall suggest the formation of a homogenous antibody with intrachain disulfide bridges formed through the complete reduction by TCP in molar excess in situ. Moreover, Hall suggest the obtainability of the reaction through changes to the reaction set up or the amount of reduction agents used.
Therefore it would have been obvious before the effective filing date of the instant application to modify the method of Griebenow’058 for a method of disulfide bridging cysteines of an antibody in view of Hall, that is to use the in situ protocol with excess of the alkyne moiety and excess of the reducing agent. One of ordinary skill in the art would have been motivated to make this modification to overcome significant accumulation issue by having the maximum number of disulfide bridges with maximum payload. One of ordinary skill in the art would have had a reasonable expectation of success because the method was proven effective in trastuzumab, an example the monoclonal IgG1 antibody with a doxorubicin payload.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication US 20170145058 A1 to Griebenow et. al. (herein after Griebenow’058; cited on the IDS dated December 29th, 2023) in view of Wu et. al. ((2012), Reactive Polymer Coatings: A General Route to Thiol-ene and Thiol-yne Click Reactions, Macromol. Rapid Commun., 33, 922 – 927).
The teachings of Griebenow’058 as they relate to claim 1, from which claim 16 depend, are given previously in this office action and are fully incorporated here.
However, Griebenow’058 fails to teach a method further comprising attaching the antibody to a solid support via the attachment moiety (claim 16).
Nevertheless, Wu et. al. teach that radical thiol-ene and thiol-yne reactions combine the benefits of click reactions, which offer regiospecificity, stereospecificity, mild reaction conditions, high yield, and can be activated through thermal or photochemical processes to proceed with selected times and locations, and being a process free of heavy metal catalysts (page 922 column 2 paragraph 2). Wu et. al. teach that thiol-ene/-yne chemistry have been found in dendrimers synthesis, polymers functionalization, particles modification, protein conjugation, microarray fabrication, and functional silanes, and difunctionalization of materials (page 922 column 2 paragraph 2). Specifically, Wu et. al. teach the following reaction scheme
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wherein Poly(4-vinyl- p -xylylene- co - p -xylylene) (polymer 1 ) and poly(4- ethynyl- p -xylylene- co - p -xylylene) (polymer 2 ) are coated on various substrates via CVD polymerization process followed by surface conjugation with designed thiol-terminated poly(ethylene glycol) (thiol-PEG) model molecules through thiol-ene and thiol-yne click reactions that are activated by UV irradiation (page 924 Figure 1). Thus Wu et. al. teach a method wherein the alkyne moiety is a modifying regent with an attachment moiety, that is the alkyne group which is attached to a solid support as recited (page 924 Figure 1). Furthermore, Wu et. al. teach that a homogeneous cell distribution with high cell selectivity was demonstrated on the selectively modified GRGDYC/thiol-PEG surfaces created by sequential thiolene click reactions on the designed surfaces (page 926 column 1 paragraph 1 and Figure 4a-c).that surface design via thiol-ene coupling reaction on reactive polymers can achieve controlled biological responses with effectiveness and accuracy, and the reported modification route is simple and versatile (page 926 column 1 paragraph 2).
Now while Wu et. al. uses a thiol-terminated poly(ethylene glycol) (thiol-PEG) as model molecules it would be within the purview of one of ordinary skill in the pharmaceutical or medicinal chemistry arts to use an antibody with reduced cysteines as the “thiol” portion of the click reaction. Therefore, it would have been obvious before the effective filing date of the instant application to modify the method of Griebenow’058 for a method of disulfide bridging cysteines of an antibody in view of Wu et. al., that is to attached the antibody to a solid support via the attachment moiety. One of ordinary skill in the art would have been motivated to achieve controlled biological responses. One of ordinary skill in the art would have had a reasonable expectation of success because a homogeneous cell distribution with high cell selectivity.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication US 20170145058 A1 to Griebenow et. al. (herein after Griebenow’058; cited on the IDS dated December 29th, 2023) in view of Macias-Contreras et. al. ((2020), SNAP/CLIP-Tags and Strain-Promoted Azide−Alkyne Cycloaddition (SPAAC)/Inverse Electron Demand Diels−Alder (IEDDA) for Intracellular Orthogonal/Bioorthogonal Labeling, Bioconjugate Chem., 31, 1370 – 1381).
The teachings of Griebenow’058 as it relates to claim 1, from which claim 18 depend, is given previously in this office action and are fully incorporated here.
However, Griebenow’058 fail to teach a method further comprising attaching the antibody to a structured nucleic acid particle via the attachment moiety (claim 18).
Nevertheless, Macias-Contreras et. al. teach that an important undertaking of chemical biology is the development of methods to selectively introduce chemical modifications to biomolecules of interest in their native biological environments for example, enable the illuminations of intracellular substructures or the dynamics of proteins (page 1370 column 1 paragraph 1). Furthermore, Macias-Contreras et. al. teach that a commonly used approach to accomplish this consists of labeling a protein-of-interest (POI) with a fluorescent protein which allows for the visualization of the localization and distribution of the POI using fluorescence microscopy (page 1370 column 1 paragraph 1). However, Macias-Contreras et. al. teach that FPs are constrained by the photophysical properties of their intrinsically amino acid-derived fluorophores, such as broad absorption and emission profiles and low photon outputs (page 1370 column 1 paragraph 1).
Thus Macias-Contreras et. al. teach a technique where the POI is connected via genetic means with a polypeptide, which then reacts with a cargo-carrying synthetic substrate that can be made to possess desirable photophysical properties but with substantially expanded labeling potential (page 1370column 1 paragraph 2 and column 2 paragraph 1). Macias-Contreras et. al. teach that the mutant of the human O6-alkylguanine-DNA alkyl transferase (hAGT), a single-turnover enzyme that repairs alkylated DNA (SNAP), takes up O6-benzylguanine (BG) derivatives as substrates; while CLIP-tag, which is mutated from SNAP-tag that reacts exclusively with O2-benzylcytosine (BC) derivatives (page 1370 column 1 paragraph 1). Moreover, Macias-Contreras et. al. teach that the use of SNAP- and CLIP-tags that have orthogonal substrate specificities, thus enabling the simultaneous labeling reactions with little cross-reactivity (page 1370 column 2 paragraph 2). Additionally, Macias-Contreras et. al. teach the synthesis of the BG derivatives wherein BG-NH2 reacts with compound 1 to get BG-Azide shown here
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(page 1372 Scheme 2). Moreover, Macias-Contreras et. al. teach example Fluorescent Reporters such as DBCO-PEG4-5/6-TAMRA of structure
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,DBCO-PEG4-5/6-FAM of structure
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, and DBCO-PEG4-5/6-Sulfo-Cy5 of structure
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which contain label portions useful for fluorescent microscopy (page 1373 Scheme 3). Furthermore, Macias-Contreras et. al. teach the generic synthetic scheme for the Dual orthogonal – bioorthogonal labeling shown here
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(page 1377 Scheme 4). Moreover, Macias-Contreras et. al. teach that the SNAP/CLIP-tag technology can be used in conjunction with the bioorthogonal reactions SPAAC and IEDDA to specifically and simultaneously label two proteins of interest inside live HeLa cells (page 1378 column 1 Figure 8) and fixed Hela cells (page 1378 Figure 9).
Regarding claim 18, recitation for a method further comprising attaching the antibody to a structured nucleic acid particle via the attachment moiety (claim 18), as taught above Macias-Contreras et. al. taught the generic synthetic scheme for the synthesis of the BG derivatives wherein BG-NH2 reacts with compound 1 to get BG-Azide shown here
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(page 1372 Scheme 2) and the Dual orthogonal – bioorthogonal labeling shown here
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(page 1377 Scheme 4). Moreover, given that one or ordinary skill in the art would know be knowledgeable about organic reactions and synthetic scheme, it would have been within the purview of one of ordinary skill in the art to substitute reagents thereby modifying the reaction to get a desired product.
Therefore, it would have been obvious to one of ordinary skill in the arts before the effective filing date of the instant application to modify the method of Griebenow’058 for a method of forming disulfide bridged cysteines in an antibody in view of Macias-Contreras et. al., that is to react BG-NH2 with hept-6-ynoic acid of the structure
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to form a BG-alkyne moiety that can react with either SNAP-LifeAct or CLIP-LaminA to subsequently react with the reduced Fab fragment M14-G07 of Griebenow’058. One of ordinary skill in the art would have been motivated to make this modification to selectively introduce chemical modifications to biomolecules of interest in their native biological environments. One of ordinary skill in the art would have had a reasonable expectation of success because SNAP- and CLIP-tag technology was shown to specifically and simultaneously label two proteins of interest inside live and fixed cells.
Claims 17 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over US Patent Application Publication US 20170145058 A1 to Griebenow et. al. (herein after Griebenow’058; cited on the IDS dated December 29th, 2023) and Geel et.al. ((2012), Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide−Alkyne Cycloaddition, Bioconjugate Chemistry, 23, 392 – 398) and in further view of Macias-Contreras et. al. ((2020), SNAP/CLIP-Tags and Strain-Promoted Azide−Alkyne Cycloaddition (SPAAC)/Inverse Electron Demand Diels−Alder (IEDDA) for Intracellular Orthogonal/Bioorthogonal Labeling, Bioconjugate Chem., 31, 1370 – 1381).
The teachings of Griebenow’058 as they relate to claim 1, from which claims 17 and 19 depend, and Geel et. al. are given previously in this office action and are fully incorporated here.
However, Griebenow’058 and Geel et. al. fail to teach a method further comprising attaching the antibody to a label via the attachment moiety (claim 17) wherein the modifying reagent comprises a label moiety (claim 19).
Nevertheless, Macias-Contreras et. al. teach that an important undertaking of chemical biology is the development of methods to selectively introduce chemical modifications to biomolecules of interest in their native biological environments for example, enable the illuminations of intracellular substructures or the dynamics of proteins (page 1370 column 1 paragraph 1). Furthermore, Macias-Contreras et. al. teach that a commonly used approach to accomplish this consists of labeling a protein-of-interest (POI) with a fluorescent protein which allows for the visualization of the localization and distribution of the POI using fluorescence microscopy (page 1370 column 1 paragraph 1). However, Macias-Contreras et. al. teach that FPs are constrained by the photophysical properties of their intrinsically amino acid-derived fluorophores, such as broad absorption and emission profiles and low photon outputs (page 1370 column 1 paragraph 1).
Thus Macias-Contreras et. al. teach a technique where the POI is connected via genetic means with a polypeptide, which then reacts with a cargo-carrying synthetic substrate that can be made to possess desirable photophysical properties but with substantially expanded labeling potential (page 1370column 1 paragraph 2 and column 2 paragraph 1). Macias-Contreras et. al. teach that the mutant of the human O6-alkylguanine-DNA alkyl transferase (hAGT), a single-turnover enzyme that repairs alkylated DNA (SNAP), takes up O6-benzylguanine (BG) derivatives as substrates; while CLIP-tag, which is mutated from SNAP-tag that reacts exclusively with O2-benzylcytosine (BC) derivatives (page 1370 column 1 paragraph 1). Moreover, Macias-Contreras et. al. teach that the use of SNAP- and CLIP-tags that have orthogonal substrate specificities, thus enabling the simultaneous labeling reactions with little cross-reactivity (page 1370 column 2 paragraph 2). Additionally, Macias-Contreras et. al. teach the synthesis of the BG derivatives wherein BG-NH2 reacts with compound 1 to get BG-Azide shown here
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(page 1372 Scheme 2). Moreover, Macias-Contreras et. al. teach example Fluorescent Reporters such as DBCO-PEG4-5/6-TAMRA of structure
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,DBCO-PEG4-5/6-FAM of structure
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, and DBCO-PEG4-5/6-Sulfo-Cy5 of structure
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which contain label portions useful for fluorescent microscopy (claims 17 – 19) (page 1373 Scheme 3). Furthermore, Macias-Contreras et. al. teach the generic synthetic scheme for the Dual orthogonal – bioorthogonal labeling shown here
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(page 1377 Scheme 4). Moreover, Macias-Contreras et. al. teach that the SNAP/CLIP-tag technology can be used in conjunction with the bioorthogonal reactions SPAAC and IEDDA to specifically and simultaneously label two proteins of interest inside live HeLa cells (page 1378 column 1 Figure 8) and fixed Hela cells (page 1378 Figure 9).
Although claims 17 and 19, recite a method further comprising attaching the antibody to a label via the attachment moiety (claim 17) wherein the modifying reagent comprises a label moiety (claim 19); the prior art of Macias-Contreras et. al. teach example fluorescent reporters such as DBCO-PEG4-5/6-TAMRA of structure
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,DBCO-PEG4-5/6-FAM of structure
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, and DBCO-PEG4-5/6-Sulfo-Cy5 of structure
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which contain label portions useful for fluorescent microscopy and an attachment moiety which contains an alkyne moiety. Now while the prior art of Macias-Contreras et. al. used these derivatives to react with the BG or BC derivatives, as taught by the prior art of Geel et. al. non-specific labeling of cysteine can occur along the alkyne bond of DIBO without the use of IAM catalysis (page 392 abstract figure). Moreover, given that one or ordinary skill in the art would know be knowledgeable about organic reactions and synthetic scheme, it would have been within the purview of one of ordinary skill in the art to substitute reagents thereby modifying the reaction to get a desired product.
Therefore, it would have been obvious to one of ordinary skill in the arts before the effective filing date of the instant application to modify the method of Griebenow’058 for a method of forming disulfide bridged cysteines in an antibody in view of Geel et. al. that is to use the alkyne portion of dibenzocyclooctyne (DIBO) in further view of Macias-Contreras et. al., that is to use the DIBO containing fluorescent reporters of DBCO-PEG4-5/6-TAMRA, DBCO-PEG4-5/6-FAM, or DBCO-PEG4-5/6-Sulfo-Cy5 as the strained ring moiety modifying reagent to get an antibody with disulfide bridges further containing DBCO-PEG4-5/6-TAMRA, DBCO-PEG4-5/6-FAM, or DBCO-PEG4-5/6-Sulfo-Cy5. One of ordinary skill in the art would have been motivated to make this modification to selectively introduce chemical modifications to biomolecules of interest in their native biological environments. One of ordinary skill in the art would have had a reasonable expectation of success because SNAP- and CLIP-tag technology was shown to specifically and simultaneously label two proteins of interest inside live and fixed cells.
Conclusion
Claims 1 – 20 are rejected.
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/DAWANNA SHAR-DAY WHITE/Examiner, Art Unit 1627