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 .
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.
Status of the Claims
Claims 3-5, 7, 8, 11, 12, 14, and 15 have been previously amended. Claims 1-15 are pending and examined herein.
Priority
This application, 18/704,515, filed 04/25/2024, is a 371 of PCT/EP2022/080265 filed on 10/28/2022, and claims benefit of EP22167875.8 filed on 04/12/2022, and EP21205185.8 filed on 10/28/2021. This priority is acknowledged and the claims examined herein are treated as having an effective filing date of 10/28/2021.
Information Disclosure Statement
The Information Disclosure Statements filed on 04/25/2024 are acknowledged and have been considered.
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, 5, 9, and 15 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 recites “…particularly an enzyme selected from…”. The recitation of “particularly” represents exemplary language that renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention, or represent a description of examples or preferences which can lead to confusion over the intended scope of the claim. See MPEP § 2173.05(d).
Similarly, claim 5 recites “…more particularly a fluorescent label”, rendering the claim indefinite.
Similarly, claim 15 recites “…particularly wherein the…”, rendering the claim indefinite.
Claim 9 recites “…contacting the sample with…”. The term “the sample” lacks antecedent basis because it is not recited or defined previously in claim 9, nor does the recited language make evident that “the sample” is specifically in reference to “cell lysate” recited previously in claim 9.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 15 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a written description rejection.
Claim 15 is are directed towards a method for determining a phosphorylation status of a 7TMR polypeptide, using a first antibody that is capable of specifically binding to a target sequence that is selected from a group of phosphorylated sequences comprising SEQ ID NO 1-51 and SEQ ID NO 139-141 and SEQ ID NO 145- 178, or a target sequence comprising SEQ ID NO 52-54 and SEQ ID NO 179-182, and is not capable of binding to the corresponding unphosphorylated sequence.
The MPEP lists factors that can be used to determine if sufficient evidence of possession has been furnished in the disclosure of an application. These include “level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention. Disclosure of any combination of such identifying characteristics that distinguish the claimed invention from other materials and would lead one of skill in the art to the conclusion that the applicant was in possession of the claimed species is sufficient.” MPEP § 2163. While all of the factors have been considered, a sufficient amount for a prima facie case are discussed below.
Further, to provide evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include: a) the scope of the invention; b) actual reduction to practice; c) disclosure of drawings or structural chemical formulas; d) relevant identifying characteristics including complete structure, partial structure, physical and/or chemical properties, and structure/function correlation; e) method of making the claimed compounds; f) level of skill and knowledge in the art; and g) predictability in the art.
The U.S. Court of Appeals for the Federal Circuit (Federal Circuit) decided Amgen v. Sanofi, 872 F.3d 1367 (Fed. Cir. 2017), which concerned adequate written description for claims drawn to antibodies. Amgen v. Sanofi describes how when an antibody is claimed, 35 U.S.C. § 112(a) requires adequate written description of the antibody itself; not just a description of the sequence to which the antibody binds. Amgen, 872 F.3d at 1378-79.
Office guidance requires that adequate written description of a newly characterized antigen alone is not considered adequate written description of a claimed antibody to that newly characterized antigen, even when preparation of such an antibody is routine and conventional. See the Memorandum dated February 22, 2018 entitled “Clarification of Written Description Guidance For Claims Drawn to Antibodies and Status of 2008 Training Materials” available at https://www.uspto.gov/sites/default/files/documents/amgen_22feb2018.pdf.
As claimed, these methods lack written description. The specification fails to provide enough evidence to show that applicant has possession of the entire scope of the antibodies that are capable of specifically binding to the target sequences recited in the claimed invention.
Disclosure of drawings or structural chemical formulas/ Actual Reduction to Practice
The specification and drawings only provide disclosure of an actual reduction to practice for the claimed methods using antibodies targeting a subset of phosphorylated GPCR sequences, not of the whole scope of the claimed invention. The scope of claim 15’s sequences represent over 180 phosphorylated sequences/antibodies corresponding to over 50 distinct receptors, which fall into 3 different classes. However, the specification provides working examples for 25 phosphosite specific antibodies, and all of them except for one (PAC1 – B1 class) represent a Class A GPCR. Only 5 of those 25 antibodies target one of the specific sequences recited in claim 15 of the instant application, and those sequences represent only 3 different receptors. No working examples are provided for an antibody binding to a Class B2 GPCR sequence which are recited in claim 15. Therefore, the claim recites a very broad genus of antibodies but the specification only describes and provides a reduction to practice for a subset (3 receptors representing only 2 classes) with no evidence that the genus is contemplated. However, a small number of species would not put one skilled in the art in possession of any other antibodies having the claimed functional properties.
The specification provides merely generic information regarding the structure of the antibodies. The specification recites “the term antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region of IgG is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, the term encompasses a so-called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain (page 6, lines 16-28).
In this case, the disclosure of some species is insufficient to represent the claimed genus of antibodies having the recited functional properties. There is no disclosed partial structure or other common structural features, common to the members of the genus, which are responsible for conferring the desired function.
Similarly, a description of a method for the generation of the claimed antibodies is limited. The specification merely recites that “In certain embodiments, each member specifically binding to a different phosphorylated sequence is a polyclonal antibody. A polyclonal antibody comprises a plurality of antibody molecules which were obtained by immunizing an animal with the corresponding target sequence” (page 8, lines 3-6). The specification further recites that “For immunization, cysteines within the sequence marked in bold were replaced by Abu (a-Aminobutyric acid). For all sequences, a cysteine was added to the N-terminus of the peptide (not shown) for immunization”.
This description fails to provide enough evidence to show that the applicant has possession of the entire scope of the antibodies that are capable of specifically binding to the target sequences recited in the claimed invention. The recitation describes merely a very broad method of antibody generation and a single modification made to the peptide, which would not allow one of ordinary skill in the art to generate the claimed antibodies without undue experimentation.
A claimed invention may not be adequately described where an invention is described solely in terms of a method of its making coupled with its function and there is no described or art recognized correlation or relationship between the structure of the invention and its function. A biomolecule defined solely by its ability to perform a function, such as to serve as an antigen recognizing construct, without a known or disclosed correlation between that function and the structure of the sequence, normally is not a sufficient identifying characteristic for written description purposes, even when accompanied by a method of obtaining the biomolecule of interest. See MPEP 2163, which states that “A biomolecule sequence described only by a functional characteristic, without any known or disclosed correlation between that function and the structure of the sequence, normally is not a sufficient identifying characteristic for written description purposes, even when accompanied by a method of obtaining the claimed sequence”.
Recent court cases have indicated that recitation of an antibody which has specific functional properties in the absence of knowledge of the antibody sequences that give rise to said functional properties do not satisfy the requirements for written description. See AbbVie Deutschland GmbH v. Janssen Biotech. Inc. as well as Amgen v. Sanofi, as discussed above. Indeed, in Amgen the court indicates that that it is improper to allow patentees to claim antibodies by describing something that is not the invention, i.e. the antigen, as knowledge of the chemical structure of an antigen does not give the required kind of structure-identifying information about the corresponding antibodies, with the antibody-antigen relationship be analogized as a search for a key on a ring with a million keys on it.
The description requirement of the patent statue requires a description of an invention, not an indication of a result that one might achieve if one made that invention. See In re Wilder, 736, F.2d 1516, 1521, 222 USPQ 369, 372-73 (Fed. Cir. 1984) (affirming rejection because the specification does “little more than outlin[e] goals appellants hope the claimed invention achieves and the problems the invention will hopefully ameliorate.”).
Level of skill and knowledge in the art/predictability in the art
The level of ordinary skill in the art of measuring phosphorylated 7TMR polypeptides using immunoassay methods is high, as an ordinary artisan in this art needs specialized knowledge for proper phosphosite-specific primary antibody and detection moiety determination for various 7TMR polypeptide types. In particular, methods for making and/or screening monoclonal antibodies with desired binding properties were well known in the art at the time of the invention. At the same time, however, it was not within the skill of the art to predict whether a given antibody would bind specifically to a particular epitope.
The level of unpredictability in the art is high. In particular, the art is unpredictable with regard to whether a phosphosite specific antibody for a sequence will be able to successfully differentiate between the phosphorylated and unphosphorylated versions, have high binding affinity, and work properly within the chosen immunoassay method. As an illustration of the unpredictability in the art, Brown et al. (1996). “Tolerance of single, but not multiple, amino acid replacements in antibody VH CDR 2: a means of minimizing B cell wastage from somatic hypermutation?”, J Immunol., 156(9):3285-91, describes how a one amino acid change in the VHCDR2 of a particular antibody was tolerated whereas, the antibody lost binding upon introduction of two amino changes in the same region (at 3290 and Tables 1 and 2). Furthermore, with regards to antibodies recognizing phosphorylated sites specifically, most bind to only one or two phosphorylation sites on a given protein, and many key proteins have a significant number of sites, each involved in different aspects of cellular activity, sometimes even having opposite effects, as taught by Stewart et al. (2017). “Utilizing the Luminex magnetic bead-based suspension array for rapid multiplexed phosphoprotein quantification”. In Kinase Signaling Networks (pp. 119-131). New York, NY: Springer New York. Stewart teaches that this makes a priori knowledge of the targeted pathway vital before designing the experiment and limits the scope for unexpected discovery (page 120, 2nd paragraph).
In summary, the specification fails to provide adequate written description for the genus of the antibodies claimed. The specification does not reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the full scope of the claimed invention.
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.
Claims 1-6 and 11-14 are rejected under 35 U.S.C. 103 as being unpatentable over Hüttenrauch et al. (2005). “G protein-coupled receptor kinases promote phosphorylation and β-arrestin-mediated internalization of CCR5 homo-and hetero-oligomers”. Journal of Biological Chemistry, 280(45), 37503-37515, (herein referred to as Hüttenrauch), in view of Kamikubo et al. (2018). “Co-immunoprecipitation Analysis of GPCR Complexes in the Central Nervous System”. In Co-Immunoprecipitation Methods for Brain Tissue (pp. 49-64). New York, NY: Springer New York, (herein referred to as Kamikubo), and Stewart et al. (2017). “Utilizing the Luminex magnetic bead-based suspension array for rapid multiplexed phosphoprotein quantification”. In Kinase Signaling Networks (pp. 119-131). New York, NY: Springer New York, (herein referred to as Stewart).
Regarding claims 1 and 6, Hüttenrauch teaches the stimulation with CCL5 of RBL cells, which co-expressed CCR5 together with an N-terminally truncated CCR5-ΔNT mutant, resulted in both protein kinase C (PKC)- and G protein-coupled receptor (GPCR) kinase (GRK)-mediated cross-phosphorylation of the mutant unligated receptor, as determined by phosphosite-specific monoclonal antibody (abstract). Hüttenrauch teaches immunoprecipitation and immunoblotting where RBL cell lines stably expressing HA-tagged N-terminally truncated (CCR5-ΔNT), phosphorylation-deficient CCR5 (CCR5-P–), C5aR alone or in various combinations were lysed by treatment with detergent buffer (0.5% Nonidet P-40, 150 mm NaCl, 50 mm Tris-HCl, pH 7.9, 5 mm EDTA containing phosphatase and protease inhibitors as described, Ref. 17) for 30 min on ice (page 37504, column 2, 2nd full paragraph). Hüttenrauch teaches that following centrifugation receptors were immunoprecipitated with 12 μg of anti-CCR5 R22/7, anti-C5aR S5/1, or anti-HA 12CA5 antibodies and protein G-Sepharose (page 37504, column 2, 2nd full paragraph). Hüttenrauch teaches that immunoblots were performed using phospho-CCR5-specific monoclonal anti-pSer337 V14/2 or anti-pSer349 E11/19 (5 μg/ml) antibodies or anti-C5aR P12/1 mAb in Tris-buffered saline containing 0.1% Tween 20/5% nonfat dry milk, and that enhanced chemiluminescence detection of antigens was achieved with horseradish peroxidase-conjugated secondary antibodies, and that afterward membranes were stripped and reprobed for total cellular receptors with anti-HA 12CA5 peroxidase conjugate (page 37504, column 2, 2nd full paragraph).
Hüttenrauch also teaches that ligand-induced phosphorylation of CCR5 C-terminal serine residues at positions 337 and 349 were determined by ELISA, where 1.5 106 RBL cells expressing CCR5 and C5aR variants were incubated in 40-mm wells for various times at 37 °C with stimulus and scraped in 0.7 ml of lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% SDS with phosphatase and protease inhibitors). Hüttenrauch teaches that following centrifugation, supernatants were applied into wells of microtiter plates coated with the anti-CCR5 mAb T21/8 (5 g/ml). Hüttenrauch teaches that after washing twice, wells were incubated with biotinylated mAb E11/19 and V14/2 with specificities for phosphorylated Ser349 and Ser337, respectively. Hüttenrauch teaches that immune complexes were detected with streptavidin-horseradish peroxidase and 2.2-azino-di-(3-ethylbenzthiazoline sulfonate) as substrate (page 37504, column 2, 3rd full paragraph). Hüttenrauch also teaches that receptor expression levels on transfected cell lines and on monocytes were determined by quantitative FACS analysis, performed by converting the mean channel of fluorescence (MCF) into antibody binding sites (ABS) per cell by using a standardized microbeads kit (page 37505, column 1, 2nd paragraph). Hüttenrauch teaches that this kit consists of a mixture of four separate microbead populations with incremental capacities to bind mouse Ig, and that beads were incubated with the same saturating concentrations of antibodies as the samples and treated like the samples being quantitated, and FITC-conjugated anti-mouse Ig was used as secondary detecting reagents (page 37505, column 1, 2nd paragraph).
However, Hüttenrauch does not teach the use of magnetic beads coated with the first binder as the solid-phase, and then contacting the magnetic beads with the second binder.
Kamikubo teaches co-immunoprecipitation analysis of GPCR complexes in the central nervous system (title). Kamikubo teaches that magnetic beads are significantly smaller than agarose beads to increase binding surface, and that by using the magnetic beads, they can use smaller volumes of sample and increase washing efficiency because of much smaller packed bead volumes (page 52, 1st paragraph). Kamikubo teaches that physical stress on samples may be reduced through magnetic separation, comparing to repeated centrifugation for agarose beads, and that for these reasons, they use magnetic beads such as Dynabeads Protein A for Co-IP of GPCR oligomer (page 52, 1st paragraph). Kamikubo teaches that they perform IP using a direct or indirect method, and that the direct method allows us to cross-link the antibody onto the beads to prevent antibody leakage upon elution, which is beneficial for protein detection and identification by reducing background (page 56, 5th paragraph). Kamikubo also teaches a protocol for the preparation of antibody-bound magnetic beads, where in the direct method, the antibody is bound to the agarose or magnetic beads through protein A or G in advance and then incubated with the lysate (page 57, 1st paragraph). Kamikubo teaches that first the antibody specific to the target and an appropriate control antibody are added to each tube containing washed beads and incubated for 1 h at room temperature on a rotator, and then the supernatant containing unbound antibody is removed, and the beads are washed twice with washing buffer and then twice with lysis buffer (page 57, 1st paragraph).
Stewart teaches a method/protocol for using the 96-well plate-based xMAP® platform from Luminex for rapid multiplexed measurement of phosphorylated proteins (abstract). Stewart teaches that the protocol uses antibody-bound free-floating magnetic spheres which can easily be removed from suspension via magnetization, and that there are 100 unique bead sets (moving up to 500 bead sets for the most recent system) identified by the ratio of two dyes coating the microsphere (abstract). Stewart teaches that each bead set is conjugated to a specific antibody which allows targeted protein extraction from low-concentration lysate solution, and that biotinylated secondary antibodies/streptavidin-R-phycoerythrin (SAPE) complexes provide the quantification mechanism for the phosphoprotein of interest (abstract). Stewart teaches that the study of protein phosphorylation is critical for the advancement of our understanding of cellular responses to external and internal stimuli (abstract). Stewart also teaches that the cellular phosphoprotein pathway dysregulation by mutation or chromosomal instability can often give the cell a selective advantage and lead to cancer, so the understanding of these systems is of huge importance to the field of oncology (abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the a method of measuring a phosphorylated 7TMR polypeptide as recited by Hüttenrauch, to use magnetic beads coated with the first binder and then contacting the magnetic beads with the second binder, as taught by Stewart, as a matter of using a known technique to improve similar methods in the same way. Hüttenrauch contains the “base method”, which the claimed invention can be seen as an improvement, because although the method of Hüttenrauch similarly uses phosphorylated protein specific antibodies, secondary binding, and chemiluminescent detection, the solid-phase where the primary antibody is bound is wells of microtiter plates, rather than magnetic beads. However, Stewart teaches a comparable method in that it uses specific primary antibodies, secondary antibodies, and chemiluminescent detection to measure phosphorylated proteins, but it has been improved in the same way as the claimed invention because the primary antibodies are bound to magnetic beads.
A person of ordinary skill would have been motivated to make this modification to allow for separation of target molecules and reduce background, and Kamikubo teaches and that by using the magnetic beads specifically, smaller volumes of sample can be use and there is an increase in washing efficiency because of much smaller packed bead volumes, and also that physical stress on samples may be reduced through magnetic separation, comparing to repeated centrifugation for agarose beads. A person of ordinary skill would have had a reasonable expectation of success in making this modification because Hüttenrauch teaches the use of commercially available antibodies for phosphorylated 7TMR polypeptides, and Stewart supplies a detailed protocol for constructing and using magnetic bead-antibody complexes to measure phosphorylated proteins.
Regarding claim 2, Hüttenrauch teaches wherein the lysis buffer comprises an inhibitor of phosphatases and/or an inhibitor of proteases (page 37505, column 1, 2nd paragraph).
Regarding claim 3, Hüttenrauch teaches that RBL cells expressing CCR5 and C5aR variants were incubated in 40-mm wells for various times at 37 °C with stimulus and scraped in 0.7 ml of lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% SDS with phosphatase and protease inhibitors). Hüttenrauch teaches that following centrifugation, supernatants were applied into wells of microtiter plates coated with the anti-CCR5 mAb T21/8 (5 g/ml) (page 37505, column 1, 2nd paragraph).
Regarding claims 4 and 5, Hüttenrauch teaches the use of FITC (fluorescein isothiocyanate)-conjugated anti-mouse Ig as secondary detecting reagents in their method of quantitative FACS analysis for measurement of receptor expression levels using beads (page 37505, column 1, 2nd paragraph). Hüttenrauch teaches that in their method of phosphorylation assay, wells were incubated with biotinylated mAb E11/19 and V14/2 (1 g/ml PBS-0.5% Tween) with specificities for phosphorylated Ser349 and Ser337, respectively, and immune complexes were detected with streptavidin-horseradish peroxidase and 2.2-azino-di-(3-ethylbenzthiazoline sulfonate) as substrate (page 37504, column 2, 3rd full paragraph).
Regarding claim 11, Hüttenrauch teaches that to determine whether these receptor kinases also phosphorylate CCR5/C5aR heterodimers, we assessed CCR5 phosphorylation in whole RBL-CCR5/C5aR cells in response to either C5a or CCL5 ligands (page 37507, column 2, 2nd paragraph). Hüttenrauch also teaches that different RBL cell transfectants were therefore exposed to PMA or CCL5 and CCR5 (ligands) phosphorylation at either the PKC site (Ser337) or the GRK site (Ser349) was documented using phosphosite-specific antibodies. (page 37507, column 1, 1st paragraph).
Regarding claim 12, Hüttenrauch teaches that they monitored C5a-induced CCR5 phosphorylation in RBL-CCR5/C5aR cells using phosphosite-specific antibodies. Hüttenrauch teaches that treatment with increasing concentrations of C5a for 3 min resulted in CCR5 phosphorylation by both PKC-(EC50 ∼0.7 nm) as well as GRK-mediated (EC50 ∼30 nm) mechanisms. Hüttenrauch teaches that when cells were exposed to C5a for longer periods of time, the dose response curve of PKC-mediated CCR5 phosphorylation significantly shifted to higher concentrations of C5a, and a kinetic analysis showed that C5a-mediated CCR5 phosphorylation at these two sites proceeds with distinct kinetics (Fig. 7C). Hüttenrauch teaches that whereas PKC phosphorylates CCR5 rapidly (t½ <0.5 min) but transiently, especially at low agonist concentrations, C5a-stimulated CCR5 phosphorylation at the GRK site is slower (t½ ∼3 min) but sustained. Hüttenrauch teaches that the dose dependence and the kinetics of PKC- and GRK-mediated heterologous phosphorylation of CCR5 closely resembles our earlier findings in homologous CCL5-induced CCR5 phosphorylation, and thus support our conclusion that GRKs phosphorylate unligated receptors within activated hetero-oligomers (page 37509, column 1, 3rd paragraph).
Regarding claim 13, Hüttenrauch teaches performing phosphorylation assays using RBL cells expressing CCR5 and cells expressing C5aR variants (page 37504, column 2, 4th paragraph).
Regarding claim 14, Hüttenrauch teaches that 32P-labeled RBL cells that co-express C5aR together with CCR5 were pretreated with or without 2 μm bisindolylmaleimide (BIM) and exposed to medium (M), 200 nm PMA, 20 nm C5a, or 100 nm CCL5 for 10 min. CCR5 were immunoprecipitated with T21/8 anti-CCR5 mAb and resolved by SDS-PAGE (Fig. 7A). Hüttenrauch also teaches that pretreatment with 2 M bisindolylmaleimide completely blocked PMA-induced receptor phosphorylation, but only partially inhibited CCR5 phosphorylation following CCL5 or C5a stimulation (page 37509, column 1, 1st paragraph).
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Hüttenrauch in view of Kamikubo and Stewart as applied to claims 1-6 and 11-14 above, and further in view of Burd et al. (US9696309B2), (herein referred to as Burd).
The teachings of Hüttenrauch in view of Kamikubo and Stewart are incorporated herein.
Regarding claim 7, Hüttenrauch in view of Kamikubo and Stewart teaches all the limitations of claim 1 of the instant application, but does not teach splitting the cell lysate in half and contacting the first half with the first antibody and the second half with a binder for the 7TMR that does not depend on the phosphorylation status of the 7TMR.
Burd teaches a method for diagnosing perinatal brain injury in a patient comprising determining the ratio of phosphorylated nNOS to unphosphorylated nNOS in a sample collected from the patient using an ELISA (abstract). Burd teaches a method for diagnosing brain injury in a patient comprises the steps of (a) obtaining a sample from the patient; (b) determining the ratio of phosphorylated neuronal nitric oxide synthase (nNOS) to unphosphorylated nNOS in the sample; and (c) correlating the ratio to a patient having brain injury or to a patient not having brain injury, thereby providing the diagnosis (column 3, lines 49-55). Burd teaches that a sample obtained from a patient can be divided and only a portion may be used for diagnosis (column 8, lines 28-30). Burd teaches that 100 μl of samples were added in duplicates into wells coated with pan-NOS and p-NOS respectively, and incubated for 2 hours at room temperature, with primary antibodies for pan-NOS and p-NOS biotinylated according to manufacturer's protocol (column 27, lines 4-10). Burd teaches that streptavidin-HRP was diluted to the working concentration, added in the amount of 100 μl to each well, and incubated for 30 min. at room temperature (column 27, lines 15-19). Burd teaches that phosphorylated/dephosphorylated at Ser 852 ratio was calculated for each sample and presented using GraphPadPrizm5 software (column 27 lines 27-29). Burd teaches that the coating of microplates with phospho-specific antibodies and probing with pan-NOS antibodies provides better results in terms of higher sensitivity and lower background (column 26, lines 60-63).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the a method of measuring a phosphorylated 7TMR polypeptide as taught by Hüttenrauch in view of Kamikubo and Stewart, to split the cell lysate in half and contacting the first half with the first antibody and the second half with a binder for the target protein that does not depend on the phosphorylation status of the target protein, as taught by Burd, as it would be “obvious to try”. Hüttenrauch teaches several methods of normalizing the measured phosphorylated 7TMR levels to total 7TMR levels, namely reprobing the membrane with an anti-HA 12CA5 antibody for total receptor amounts (immunoblotting method), calibration with a synthetic bovine serum albumin 7TMR (CCR5-(phospho)peptide) standard protein with the results expressed in relative units (ELISA method), and comparison with a regression curve (QFACS/bead method). Burd teaches that an alternative method of calculating actual values for the phosphorylated and total target protein amounts and normalizing the results in their method is through the generation of a standard curve (column 27, lines 34-67). Therefore, it would be expected that the split lysate method of claim 7 could be applied to the method of Hüttenrauch in view of Kamikubo and Stewart, as the method already teaches the required antibodies to measure phosphorylated and total 7TMR proteins in the sample.
A person of ordinary skill would have been motivated to make this modification because Burd teaches that using this method to determine a normalized phosphorylated to total 7TMR protein ratio provides better results in terms of higher sensitivity and lower background. A person of ordinary skill would have had a reasonable expectation of success in making this modification because Hüttenrauch teaches the use of commercially available antibodies for phosphorylated 7TMR polypeptides and antibodies that bind both phosphorylated and non-phosphorylated 7TMR polypeptides, and the modification would merely require partitioning the lysate which there are no barriers to om the method of Hüttenrauch.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Hüttenrauch in view of Kamikubo, Stewart, and Burd as applied to claim 7 above, and further in view Yeliseev et al. (2007). “Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor”. Protein expression and purification, 53(1), 153-163, (herein referred to as Yeliseev).
The teachings of Hüttenrauch in view of Kamikubo, Stewart, and Burd are incorporated herein.
Regarding claim 8, Hüttenrauch in view of Kamikubo, Stewart, and Burd teaches all the limitations of claim 1 of the instant application, as well as using an affinity tag and using a ratio between a signal of the first and second binder to indicate an amount of phosphorylation of the 7TMR.
However, Hüttenrauch in view of Kamikubo, Stewart, and Burd does not teach the use of a 7TMR construct with two affinity tags.
Yeliseev teaches the use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor (title). Yeliseev teaches that the human peripheral cannabinoid receptor (CB2) (a GPCR) was expressed as a fusion with the maltose-binding protein (at the N-terminus), thioredoxin A (at the C-terminus) and two small affinity tags (a Strep-tag and a polyhistidine tag) (abstract). Yeliseev teaches that the use of dual affinity tags for expression and purification of recombinant proteins has become an increasingly popular method that simplifies purification and yields homogenous preparations of the proteins of interest (page 155, column 1, 1st full paragraph). Yeliseev teaches that in addition to the polyhistidine tag at the C-terminus of the fusion, they attached a small StrepTag directly to the N-terminus of CB2 (page 154, column 2, 2nd full paragraph). Yeliseev teaches that the HisProbe-HRP kit for detection of polyhistidine fusion proteins was obtained from Pierce, the Ni–NTA resin from Qiagen, and the StrepTactin agarose resin from Novagen (page 155, column 1, 2nd full paragraph). Yeliseev teaches that the expression levels of the various CB2-fusions were assessed by Western blot analysis, and the presence of the His-tag, CB2, and MBP in the fusion proteins was visualized by probing protein extracts from bacterial plasma membranes with HisProbe reagent, anti-CB2 or anti-MBP antibody, respectively (page 157, column 2, 3rd paragraph). Yeliseev teaches that introduction of the StrepTag II (construct 122) or StrepTag III (construct 125) into the fusion did not change expression levels of the CB2-fusion significantly (page 157, column 2, 3rd paragraph). Yeliseev teaches that aliquots of the reaction mixture were withdrawn at several time points, and the protein composition of the mixture analyzed by SDS–PAGE and Western blot, probed either with antibodies against CB2 or with Streptactin con jugated to HRP (page 159, column 1, 2nd paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of measuring a phosphorylated 7TMR polypeptide as taught by Hüttenrauch in view of Kamikubo, Stewart, and Burd, to use a 7TMR construct with two affinity tags, as taught by Yeliseev, as a matter of using a known technique to improve similar methods in the same way. Hüttenrauch contains the “base method”, which the claimed invention can be seen as an improvement, because although the method of Hüttenrauch similarly uses phosphorylated protein specific antibodies, secondary binding, Western blots, and chemiluminescent detection, the method uses only one affinity tag. However, Yeliseev teaches a comparable method in that they use specific primary antibodies, secondary antibodies, Western blots, and chemiluminescent detection to measure phosphorylated proteins, but it has been improved in the same way as the claimed invention because the their 7TMR construct expresses two affinity tags allowing a primary antibody and a detection moiety to bind independently to the phosphorylated protein.
A person of ordinary skill would have been motivated to make this modification to allow for the measurement of total target protein levels when the proper antibody is not available, by using detection moieties conjugated to the second tag targeting antibody. Stewart teaches that the antibody availability, affinity, and specificity for 7TMR/GPCR is limited (page 120, 1st paragraph). This would allow for quantitative measurement of the phosphorylated protein of interest, allowing for normalization and the use of a large number of unique reporter molecules, as taught by Stewart (page 123, 1st full paragraph). A person of ordinary skill would have had a reasonable expectation of success in making this modification because Hüttenrauch teaches the use of commercially available antibodies for tagged phosphorylated 7TMR polypeptides, and the use of an affinity-tag targeting antibody conjugated to a detection moiety (anti-HA 12CA5 peroxidase conjugate). Furthermore, Yeliseev supplies a method for constructing a 7TMR (CB2) fusion protein with two affinity tags that was successfully used to measure the phosphorylated protein with immunoblotting methods similar to Hüttenrauch.
Claims 9 and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Hüttenrauch in view of Harbert et al. (2008). “Development of a HTRF® kinase assay for determination of Syk Activity”. Current Chemical Genomics, 1, 20, (herein referred to as Harbert).
The teachings of Hüttenrauch are incorporated herein.
Regarding claim 9, Hüttenrauch teaches a method for determining a phosphorylation status of a 7TMR polypeptide, said method comprising the steps:- providing a cell comprising a 7TMR construct, wherein the 7TMRconstruct comprises a 7TMR polypeptide, and an affinity tag; - in a lysis step, lysing the cell with a lysis buffer yielding a cell lysate; - in a binding step, contacting the sample with i. an affinity binder; and ii. a first antibody specifically binding to said 7TMR polypeptide in phosphorylated status, but not in unphosphorylated status. Hüttenrauch also teaches the us of an emission moiety and an excitation moiety, wherein the emission moiety is capable of emitting light at a wavelength which corresponds to the excitation wavelength of the excitation moiety. Specifically, Hüttenrauch teaches that for the BRET studies, the CCR5, C5aR, or AT1a R (17) coding sequences without their stop codons were amplified using sense and antisense primers containing unique HindIII or BamHI sites, respectively, and then the fragments were then cloned in-frame into the HindIII/ BamHI sites of pRluc-N2 and pGFP2-N2 (page 37504, column 1, 4th paragraph). Hüttenrauch teaches that the BRET signal is expressed as the ratio of light emitted by the GFP2 construct (500–530 nm) over the light emitted by the hRluc construct (370–450 nm), corrected for Rf, which corresponds to the signal in cells that express only the hRluc construct in the same exper iment. Total GFP fluorescence and luminescence signals were determined, in parallel, for all samples using coelenterazine (page 37505, column 1, 4th paragraph – column 2, 1st paragraph).
However, Hüttenrauch does not teach that one of the affinity binder and the first antibody is coupled to the emission moiety, and the other one of the binder and the first antibody is coupled to the excitation moiety.
Harbert teaches the use of HTRF®, a TR-FRET technology, which includes a phosphospecific antibody conjugated to Eu3+ cryptate (donor fluorophore), and an anti-tag or affinity molecule conjugated to XL665 or d2 (acceptor fluorophore) which recognizes the substrate, irrespective of its phosphorylation state (page 20, column 2, 1st paragraph). Harbert teaches that the time-resolved characteristic of HTRF® technology allows for the removal of nearly all environmental and compound effects, and the use of a FRET-based readout has an additional advantage of specificity for the intended biological moiety under investigation (page 20, column 2, 1st paragraph). Harbert teaches that detection of phosphorylation events have been validated using HTRF® on small peptides as well as large protein substrates (page 20, column 2, 2nd paragraph). Harbert teaches that the enzyme reaction was stopped by EDTA-containing buffer, which also contained europium-conjugated anti-phosphoresidue antibody and streptavidin-XL665(SA-XL665) to allow for detection of the phosphorylated peptide product (Page 22, column 1, 3rd paragraph). Harbert teaches that following 1 hour incubation at room temperature fluorescence was measured with excitation of 337 nm and dual emission of 665 and 620 nm on the RubyStar microplate reader, and signal was expressed in terms of HTRF ratio (fluorescence intensity @ 665 nm/fluorescence intensity @ 620nm x 10,000) (Page 22, column 1, 3rd paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of measuring a phosphorylated 7TMR polypeptide as taught by Hüttenrauch, to have the emission and excitation moieties coupled to an affinity binder and antibody, as taught by Harbert, as a matter of using a known technique to improve similar methods in the same way. Hüttenrauch contains the “base method”, which the claimed invention can be seen as an improvement, because although Hüttenrauch teaches a method of protein measurement using an emission and excitation moiety (BRET), the method however uses moieties cloned into the 7TMR constructs themselves rather than coupled to binders. However, Harbert teaches a comparable method in that they use specific anti-phosphoresidue antibodies, generic affinity tags on the substrate molecule of interest, and specific anti-tag reagents for substrate catching to measure phosphorylated proteins, but it has been improved in the same way as the claimed invention because their method uses emission and excitation moieties.
A person of ordinary skill would have been motivated to make this modification Harbert teaches that their FRET technology allows for the removal of nearly all environmental and compound effects, and the use of a FRET-based readout has an additional advantage of specificity for the intended biological moiety under investigation. A person of ordinary skill would have had a reasonable expectation of success in making this modification because Hüttenrauch teaches the use of commercially available antibodies for tagged phosphorylated 7TMR polypeptides, and the use of an affinity-tag targeting antibody conjugated to a detection moiety, and Harbert provides the necessary assay protocol using all commercially available materials.
Regarding claim 10, Hüttenrauch teaches the emission moiety and the excitation moiety are a BRET pair (page 37505, column 1, 4th paragraph – column 2, 1st paragraph).
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Hüttenrauch in view of Kamikubo and Stewart as applied to claims 1 above, and further in view of Doll et al. (2011). “Agonist‐selective patterns of µ‐opioid receptor phosphorylation revealed by phosphosite‐specific antibodies”. British journal of pharmacology, 164(2), 298-307, (herein referred to as Doll), and Prihandoko et al. (2015). “Determination of GPCR phosphorylation status: establishing a phosphorylation barcode”. Current Protocols in Pharmacology, 69(1), 2-13, (herein referred to as Prihandoko).
The teachings of Hüttenrauch in view of Kamikubo and Stewart are incorporated herein.
Regarding claim 15, Hüttenrauch in view of Kamikubo and Stewart teaches all the limitations of claim 1 of the instant application, and teaches a first antibody that is capable of specifically binding a phosphorylated target sequence, and not capable of binding to the corresponding unphosphorylated sequence.
However, Hüttenrauch in view of Kamikubo and Stewart does not teach an antibody binding to one of the specific SEQ ID’s recited in claim 15.
Doll teaches the generation of phosphosite-specific antibodies for the carboxyl-terminal residues serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), which enabled them to selectively detect either the Ser363-, Thr370- or Ser375-phosphorylated form of the receptor (abstract). Doll teaches that phosphosite-specific antibodies for the Ser363-phosphorylated form of the µ receptor were generated against the following sequence that contained a phosphorylated serine residue: EQQN(pS)ARIRQ, which corresponds to amino acids 359–368 of the mouse, and 361–370 of the human µ-opioid receptor, respectively. Doll teaches that phosphosite-specific antibodies for the Thr370-phosphorylated form of the µ receptor were generated against the following sequence that contained a phosphorylated threonine residue: IRQN(pT)REHP, which corresponds to amino acids 366–374 of the mouse, and 368–376 of the human µ-opioid receptor, respectively. Doll teaches that phosphosite-specific antibodies for the Ser375-phosphorylated form of the µ receptor were generated against the following sequence that contained a phosphorylated serine residue: REHP(pS)TANTV, which corresponds to amino acids 371–380 of the mouse, and 373–382 of the human µ-opioid receptor respectively. Doll teaches that the peptides were purified by HPLC and coupled to keyhole limpet haemocyanin, and the conjugates were mixed 1:1 with Freund's adjuvant and injected into groups of four rabbits {3198–3201} for anti-pSer363 antibody production, {3194–3197} for anti-pT370 antibody production and {2493–2497} for anti-pS375 antibody production. Doll teaches that the animals were injected at 4-week intervals, and serum was obtained 2 weeks after immunizations beginning with the second injection. Doll teaches that the specificity of the antisera was initially tested using dot-blot analysis, and for subsequent analysis, antibodies were affinity purified against their immunizing peptide, as well as against the non-phosphorylated peptide using the SulfoLink kit (page 299, column 2, 1st paragraph).
Doll also teaches that cells were transfected with a plasmid encoding for a HA-tagged mouse m-opioid receptor using LipofectAMINE 2000 according to the instructions of the manufacturer (page 299, column 2, 2nd paragraph). Doll teaches that the cells were lysed in detergent buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10mM disodium pyrophosphate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS] in the presence of protease and phosphatase inhibitors Complete mini and PhosSTOP (page 300, column 1, 1st paragraph). Doll teaches that samples were split, resolved on 7.5% SDS–polyacrylamide gels, and after electroblotting, membranes were incubated with either 0.1 mg·mL-1 anti-pSer363 {3199}, 0.1 mg·mL-1 anti-pThr370 {3196} or 0.1 mg·mL-1 anti-pSer375 {2493} followed by detection using an enhanced chemiluminescence detection system (page 31409, column 2, 1st paragraph).
Prihandoko teaches detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated (abstract). Prihandoko teaches that these techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype (abstract). Prihandoko teaches a description of MS-based approaches that can precisely identify the phosphorylation sites on GPCRs (see Basic Protocol 2), and an approach that can inform the design of phospho-specific antibodies (see Basic Protocol 3) (page 2, 2nd paragraph). Prihandoko teaches that these antibodies, once fully characterized (see Basic Protocols 3 and 4), can be employed to probe the phosphorylation status of GPCRs in primary cultures and native tissues (page 2, 2nd paragraph). Prihandoko teaches that the receptor used as an example in this unit is the M3 muscarinic acetylcholine receptor stably expressed in Chinese hamster ovary (CHO) cells at 1.2 pmol/mg protein (page 2, 2nd paragraph). Prihandoko teaches that G protein−coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization (abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of measuring a phosphorylated 7TMR polypeptides as taught by Hüttenrauch in view of Kamikubo and Stewart, to use an antibody that is capable of specifically binding one of the phosphorylated target sequences recited in claim 15 of the instant application, as a matter of applying a known technique to a known method ready for improvement to yield predictable results. Hüttenrauch in view of Kamikubo and Stewart contains the “base method”, which the claimed invention can be seen as an improvement, because although the method of Hüttenrauch in view of Kamikubo and Stewart similarly uses phosphorylated protein specific antibodies, secondary binding, magnetic beads, and chemiluminescent detection, and antibody generation, the method does not teach using an antibody specific to one of the phosphorylated target sequences recited in claim 15 of the instant application. While Doll and Prihandoko do not teach antibodies specifically targeting one of these phosphosite sequences, they both teach methods of generating such antibodies specifically for GPCRs, and Prihandoko teaches a method of MS-based approaches that can precisely identify the phosphorylation sites on GPCRs, specifically in the context of informing the design of phospho-specific antibodies. Therefore, armed with this knowledge available in the art of how to specifically identify phosphorylation sites of 7TMR polypeptides and generate antibodies against those sequences, one could apply these techniques to the method of Hüttenrauch in view of Kamikubo and Stewart in order to use an antibody that is capable of specifically binding one of the phosphorylated target sequences recited in claim 15 of the instant application.
A person of ordinary skill would have been motivated to make this modification because the use of such phosphosite-specific antibodies allows for determination of site-specific patterns of phosphorylation of a GPCR, which are different depending if it is in basal state or stimulated, as taught by Doll which showed distinct agonist-selective patterns of GPCR phosphorylation following activation by internalizing or non-internalizing agonists. This has important implications for pharmacology, as desensitization occurs when GPCRs are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling, as taught by Prihandoko. A person of ordinary skill would have had a reasonable expectation of success in making this modification because Hüttenrauch already teaches the use of phosphosite-specific antibodies for 7TMR polypeptides, and Prihandoko provides detailed methods of determining the phosphosite specific sequences and how to generate an antibody against these sequences, and teaches that they are well-understood, routine, and conventional activities in the field.
Conclusion
For all the reasons discussed above, claims 1-15 are rejected and therefore no claims are allowed.
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/ALEXANDER J. HOFFMAN/Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 September 30, 2026
