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 .
Claim Status
Claims 1-11 were cancelled (1/20/2023). Claims 12-31 are new (1/20/2023). No new matter was added. Thus, claims 12-31 are under examination.
Priority
Claims 12-31 receive a priority date of 7/29/2020, the filing date of PCT/CN2020/105621.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
The Information Disclosure Statements from 1/20/2023 and 5/22/2024 are considered.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the term “Tween-20” (p. 12), “NP-40” (p. 12), “Tritox-100” (p. 12), “MGI” (p. 15 and used throughout the Specification), “Thermo” (p. 15 and used throughout the Specification), “Trobot” (p. 15), “Eppendorf” (p. 15) is a trade name or mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 20 is objected to because of the following informality:
Claim 20 at steps (1) and (2): there are extra spaces between the numbers and wording.
Claim 31 is objected to because of the following informality:
Claim 31 at steps (5) and (7): step (6) was skipped.
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 15-27, 29 and 30-31 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 15 is rejected. Claim 15 recites the limitation “the size” in line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 15 is further rejected. Claim 15 recites the limitation “the maximum size” in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 15 is further rejected. Claim 15 recites the limitation “the spacing of the adjacent sites” in line 4. There is insufficient antecedent basis for this limitation in the claim.
Claim 16 is included in this rejection due to its dependency on claim 15.
Claim 16 is further rejected. Claim 16 recites the limitation “the time of amplification” in line 5. There is insufficient antecedent basis for this limitation in the claim.
Claim 17 is rejected. Claim 17 recites the limitation “the amount of dNTPs” in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 17 is further rejected. Claim 17 recites the limitation “the reaction mixture” in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 17 is further rejected. Claim 17 recites the limitation “the activity” in line 5. There is insufficient antecedent basis for this limitation in the claim.
Claim 17 is further rejected. Claim 17 recites the limitation “the nucleic acid polymerase” in line 6. There is insufficient antecedent basis for this limitation in the claim.
Claims 18-26 are included in this rejection due to their dependency on claim 17.
Claim 18 is further rejected. Claim 18 recites the limitation “the temperature” in line 6. There is insufficient antecedent basis for this limitation in the claim.
Claim 18 is further rejected. Claim 18 recites the limitation “the working temperature” in line 7. There is insufficient antecedent basis for this limitation in the claim.
Claim 18 is further rejected. Claim 18 recites the limitation “the pH” in line 8. There is insufficient antecedent basis for this limitation in the claim.
Claim 18 is further rejected. Claim 18 recites the limitation “the working pH” in line 9. There is insufficient antecedent basis for this limitation in the claim.
Claim 21 is further rejected. Claim 21 recites the limitation “the temperature” in line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 21 is further rejected. Claim 21 recites the limitation “the working temperature range” in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 22 is included in this rejection due to its dependency on claim 21.
Claim 22 is further rejected. Claim 22 recites the limitation “the upper limit of the working temperature” in lines 3-4. There is insufficient antecedent basis for this limitation in the claim.
Claim 22 is further rejected. Claim 22 recites the limitation “the lower limit of the working temperature” in lines 5-6. There is insufficient antecedent basis for this limitation in the claim.
Claim 23 is further rejected. Claim 23 recites the limitation “the pH of the reaction mixture” in line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 23 is further rejected. Claim 23 recites the limitation “the working pH range” in lines 2-3. There is insufficient antecedent basis for this limitation in the claim.
Claim 24 is included in this rejection due to its dependency on claim 23.
Claim 24 is further rejected. Claim 24 recites the limitation “the upper limit of the working pH range” in lines 3-4. There is insufficient antecedent basis for this limitation in the claim.
Claim 24 is further rejected. Claim 24 recites the limitation “the lower limit of the working pH range” in line 5. There is insufficient antecedent basis for this limitation in the claim.
Claim 26 is further rejected. Claim 26 contains the trademark/trade name “Tween-20”, “NP-40” and “Tritox-100.” Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a potential group of denaturing agents and, accordingly, the identification/description is indefinite.
Claim 27 is rejected. Claim 27 recites the limitation “the maximum size” in line 7. There is insufficient antecedent basis for this limitation in the claim.
Claim 27 is further rejected. Claim 27 recites the limitation “the nucleic acid nanoball” in line 7. There is insufficient antecedent basis for this limitation in the claim.
Claim 27 is further rejected. Claim 27 recites the limitation “the product” in line 20. Is this the same as the “amplification product” mentioned earlier in the claim? There is insufficient antecedent basis for this limitation in the claim.
Claim 29 is rejected. Claim 29 recites the limitation “the activity” in line 6. There is insufficient antecedent basis for this limitation in the claim.
Claim 29 is further rejected. Claim 29 recites the limitation “the temperature” in lines 19-20. There is insufficient antecedent basis for this limitation in the claim.
Claim 29 is further rejected. Claim 29 recites the limitation “the reaction mixture” in line 20. There is insufficient antecedent basis for this limitation in the claim.
Claim 29 is further rejected. Claim 29 recites the limitation “the working temperature range” in line 20. There is insufficient antecedent basis for this limitation in the claim.
Claim 29 is further rejected. Claim 29 recites the limitation “the pH” in line 24. There is insufficient antecedent basis for this limitation in the claim.
Claim 29 is further rejected. Claim 29 recites the limitation “the working pH range” in line 25. There is insufficient antecedent basis for this limitation in the claim.
Claim 30 is rejected. Claim 30 recites the limitation “the nucleic acid molecule” in lines 2-3. Is this the same as the nucleic acid previously amplified? There is insufficient antecedent basis for this limitation in the claim.
Claim 30 is further rejected. Claim 30 recites the limitation “the pH of solution” in line 13. There is insufficient antecedent basis for this limitation in the claim.
Claim 30 is further rejected. Claim 30 contains the trademark/trade name “Pluronic F68”, “NP-40” and “Tritox-100.” Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a reagent for loading the nucleic acid molecule onto the sold support and, accordingly, the identification/description is indefinite.
Claim 31 is included in this rejection due to its dependency on claim 30.
Claim 31 is further rejected. Claim 26 contains the trademark/trade name “Tween-20”, “NP-40” and “Tritox-100.” Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a potential group of denaturing agents and, accordingly, the identification/description is indefinite.
Claim 31 is further rejected. Claim 31 recites the limitation “the acidic or basic buffer” in line 22. Is this a different buffer from the acidic and alkaline buffer from parent claim 30? There is insufficient antecedent basis for this limitation in the claim.
Claim 31 is further rejected. Regarding claim 31, the phrase "for example" or “e.g.” renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 12-31 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (US PGPub 2005/0048578 A1; published 3/3/2005) and in view of Gunderson et al. (EP1923471B1; published 12/19/2012) and Liu et al. (“Whole Blood PCR Amplification with Pfu DNA Polymerase and Its Application in Single-Nucleotide Polymorphism Analysis”, Sage Journals, published; 2015), and in further view of Zhu et al. (“Guanine-rich sequences inhibit proofreading DNA polymerases”, Scientific Reports, published 6/28/2016).
Regarding claims 12-14, Zhang teaches methods for screening for a monoclonal antibody with a desirable activity, where the methods involve altering a nucleic acid encoding a selected parental humanized monoclonal antibody to make a library of nucleic acids, introducing the library into mammalian cells such that a library of monoclonal antibodies are produced on the surfaces of the mammalian cells, and sorting the cells to isolate a cell producing a humanized monoclonal antibody with a desirable activity, e.g., increased affinity for an binding partner as compared to the parental antibody, where this isolated cell may be cultured and used to reiterate the method (Abstract). Zhang further teaches ells producing a monoclonal antibody of interest may be isolated from the rest of the population of cells by their binding to a solid support. For example, cells producing a monoclonal antibody of interest may be separated from the population of cells using magnetic separation using paramagnetic particles that are coated with a binding partner (Kiesel et al. euk Res. 1987 11:1119-25). Zang also teaches that paramagnetic particles are available with a variety of surface derivatization chemistries to allow for the covalent attachment of a wide range of binding partners, e.g. a protein of interest or a cell producing the same on its surface, where mammalian cells producing surface-bound antibodies with high affinity for the binding partner bind to the magnetic particles and may be isolated in a strong magnetic field that attracts the magnetic beads (Paragraph 97, lines 1-5). Zang also teaches that alternatively, cells that bind to the particles may be separated in a continuous magnetic separator and in other embodiments, the variant antibody cell population is contacted with a solid support in which a binding partner is chemically immobilized, and cells that display antibodies that bind to the binding partner are retained on the solid support and following washing, cells which specifically bound to the support may be released using binding partner that is not immobilized to the support (Paragraph 97, lines 1-10). Zhang teaches that cells producing a monoclonal antibody of interest, once isolated from the population of cells may be cultured and subjected to further rounds of selection or used to isolate the nucleic acids encoding the monoclonal antibodies of interest, by e.g., plasmid rescue (if a plasmid vector is used), PCR (if a linear vector is used), or by isolation of virus particles produced by the cell (if a viral vector is used) and these nucleic acids may be used in a variety of ways (Paragraph 101, lines 1-5). Further, Zhang teaches that for example, the nucleic acids may be used to produce a monoclonal antibody of interest, as will be described below and in other embodiments, the nucleic acids may be used as starting materials for reiterating the above mutagenesis/selection process (in other words, the first monoclonal antibody of interest identified using the above methods may become a parental monoclonal antibody and the above methods may be repeated to produce a second monoclonal antibody of interest) (Paragraph 101, lines 5-10).
Zhang teaches that a first polynucleotide is "derived from" a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above. A first polynucleotide may be derived from a second polynucleotide if the first polynucleotide is used as a template for, e.g. amplification of the second polynucleotide (Paragraph 42, lines 1-5). Specifically, Zhang teaches that the previously discussed invention are kits for practicing the subject methods, as described above where the subject kits at least include one or more of: a library of mammalian cells displaying a plurality of different monoclonal antibodies, a library of mammalian cells displaying monoclonal antibodies, where each cell displays a different monoclonal antibody on its surface, a library of expression cassettes, for example linear or circular (e.g., in a retroviral vector) expression cassettes that provide for expression of a library of monoclonal antibodies in mammalian cells, or a parental monoclonal antibody (Paragraph 140, lines 1-5).
Zhang also teaches that the previously discussed invention also provides compositions, such as a library of mammalian cells displaying a plurality of different monoclonal antibodies (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more or 50 or more, etc.), a library of mammalian cells displaying monoclonal antibodies, where each cell displays a different monoclonal antibody on its surface, a library of expression cassettes, for example linear or circular (e.g., in a retroviral vector) expression cassettes that provide for expression of a library of monoclonal antibodies in mammalian cells (Paragraph 91, lines 1-5). Specifically, Zhang teaches that in these assays, each antibody of a plurality of antibodies is tested for its ability to bind specifically to a substrate where the term "specifically" in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific antigen i.e., a polypeptide, or epitopemany embodiments, the specific antigen is an antigen (or a fragment or subfraction of an antigen) used to immunize the animal host from which the antibody-producing cells were isolated (Paragraph 116, lines 1-5).
Regarding claims 15-20, Zhang teaches that the assay measures the specific inhibition of an antibody to an interaction between a first compound and a second compound (e.g. two biopolymeric compounds) or specifically inhibits a reaction (e.g. an enzymatic reaction) and in the interaction inhibition assay, one interaction substrate, usually a biopolymeric compound such as a protein e.g. a receptor, may be bound to a solid support in a reaction vessel (Paragraph 126, lines 1-5). Further, Zhang teaches that antibody is added to the reaction vessel followed by a detectable binding partner for the substrate, usually a biopolymeric compound such as a protein e.g. a radiolabeled ligand for the receptor where after washing the vessel, interaction inhibition may be measured by determining the amount of detectable binding partner present in the vessel and interaction inhibition occurs when binding of the binding partner is reduced greater than about 20%, greater than about 50%, greater than about 70%, greater than about 80%, or greater than about 90% or 95% or more, as compared to a control assay that does not contain antibody (Paragraph 126, lines 5-10).
Zhang also teaches that in the reaction inhibition assay, an enzyme may be bound to a solid support in a reaction vessel, where an antibody is usually added to the reaction vessel followed by a substrate for the enzyme. In many embodiments, the products of the reaction between the enzyme and the substrate are detectable, and, after a certain time, the reaction is usually stopped where after the reaction has been stopped, reaction inhibition may be measured by determining the level of detectable reaction product present in the vessel (Paragraph 127, lines 1-5). Also, Zhang teaches that reaction inhibition occurs when the rate of the reaction is reduced greater than about 20%, greater than about 50%, greater than about 70%, greater than about 80%, or greater than about 90% or 95% or more, as compared to a control assay that does not contain antibody (Paragraph 127, lines 5-10).
Zhang further teaches that kits for practicing the subject methods, as described above where the subject kits at least include one or more of: a library of mammalian cells displaying a plurality of different monoclonal antibodies, a library of mammalian cells displaying monoclonal antibodies, where each cell displays a different monoclonal antibody on its surface, a library of expression cassettes, for example linear or circular (e.g., in a retroviral vector) expression cassettes that provide for expression of a library of monoclonal antibodies in mammalian cells, or a parental monoclonal antibody and other optional components of the kit include: components for performing antibody binding assays, e.g. FACS assays, microtiter plates and ELISA reagents; buffers, nucleotides and reagents for performing amplifying heavy and light chain nucleic acids; and a humanized antibody framework-encoding nucleic acids that is operably linked to a cell surface targeting sequence (Paragraph 140, lines 1-10).
Regarding claims 21-22, Zhang teaches that with respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions (Paragraph 41, lines 1-5).
Regarding claims 23-24, Zhang teaches that immunoprecipitation protocols generally involve lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4.degree. C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4.degree. C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer where the loading ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis and one of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads) (Paragraph 119, lines 1-10).
Regarding claims 25-26, Zhang teaches that depending on the exact methodology used, labeled antigen may be added to the cell population at a non-saturating amount, or in combination (at a certain ratio) with unlabeled antigen, to facilitate the identification cells producing antibodies of interest and in other embodiments, a distinguishably labeled second binding partner which binds to all the monoclonal antibodies produced by the cells with equal affinity may be added to the cell population to identify the expression level of an antibody in a single cell and via the distinguishably labeled second binding partner, e.g., a labeled antibody that binds to an Fc region of the monoclonal antibody variants, the amount of binding of the first binding partner can be normalized to the amount of a monoclonal antibody produced by a cell (Paragraph 100, lines 1-10).
Buffers according to protocols for immunoprecipitation, Wester blots in gels include specific lysis buffers (i.e., Triton X-100, Tween-20) (Paragraphs 119-120).
Regarding claims 27-29, Zhang teaches that in some embodiments, cells producing a monoclonal antibody of interest may be isolated from the rest of the population of cells by their binding to a solid support and for example, cells producing a monoclonal antibody of interest may be separated from the population of cells using magnetic separation using paramagnetic particles that are coated with a binding partner where paramagnetic particles are available with a variety of surface derivatization chemistries to allow for the covalent attachment of a wide range of binding partners, e.g. a protein of interest or a cell producing the same on its surface (Paragraph 97, lines 1-5). Further Zhang teaches that mammalian cells producing surface-bound antibodies with high affinity for the binding partner bind to the magnetic particles and may be isolated in a strong magnetic field that attracts the magnetic beads and alternatively, cells that bind to the particles may be separated in a continuous magnetic separator. In other embodiments, the variant antibody cell population is contacted with a solid support in which a binding partner is chemically immobilized, and cells that display antibodies that bind to the binding partner are retained on the solid support. Following washing, cells which specifically bound to the support may be released using binding partner that is not immobilized to the support (Paragraph 97, lines 1-10).
Zhang also teaches methods for screening for a monoclonal antibody with a desirable activity, where the methods involve altering a nucleic acid encoding a selected parental humanized monoclonal antibody to make a library of nucleic acids, introducing the library into mammalian cells such that a library of monoclonal antibodies are produced on the surfaces of the mammalian cells, and sorting the cells to isolate a cell producing a humanized monoclonal antibody with a desirable activity, e.g., increased affinity for an binding partner as compared to the parental antibody, where this isolated cell may be cultured and used to reiterate the method (Abstract). Zhang further teaches ells producing a monoclonal antibody of interest may be isolated from the rest of the population of cells by their binding to a solid support. For example, cells producing a monoclonal antibody of interest may be separated from the population of cells using magnetic separation using paramagnetic particles that are coated with a binding partner (Kiesel et al. euk Res. 1987 11:1119-25). Zang also teaches that paramagnetic particles are available with a variety of surface derivatization chemistries to allow for the covalent attachment of a wide range of binding partners, e.g. a protein of interest or a cell producing the same on its surface, where mammalian cells producing surface-bound antibodies with high affinity for the binding partner bind to the magnetic particles and may be isolated in a strong magnetic field that attracts the magnetic beads (Paragraph 97, lines 1-5). Zang also teaches that alternatively, cells that bind to the particles may be separated in a continuous magnetic separator and in other embodiments, the variant antibody cell population is contacted with a solid support in which a binding partner is chemically immobilized, and cells that display antibodies that bind to the binding partner are retained on the solid support and following washing, cells which specifically bound to the support may be released using binding partner that is not immobilized to the support (Paragraph 97, lines 1-10). Zhang teaches that cells producing a monoclonal antibodies of interest, once isolated from the population of cells may be cultured and subjected to further rounds of selection or used to isolate the nucleic acids encoding the monoclonal antibodies of interest, by e.g., plasmid rescue (if a plasmid vector is used), PCR (if a linear vector is used), or by isolation of virus particles produced by the cell (if a viral vector is used) and these nucleic acids may be used in a variety of ways (Paragraph 101, lines 1-5). Further, Zhang teaches that for example, the nucleic acids may be used to produce a monoclonal antibody of interest, as will be described below and in other embodiments, the nucleic acids may be used as starting materials for reiterating the above mutagenesis/selection process (in other words, the first monoclonal antibody of interest identified using the above methods may become a parental monoclonal antibody and the above methods may be repeated to produce a second monoclonal antibody of interest) (Paragraph 101, lines 5-10).
Zhang teaches that a first polynucleotide is "derived from" a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above. A first polynucleotide may be derived from a second polynucleotide if the first polynucleotide is used as a template for, e.g. amplification of the second polynucleotide (Paragraph 42, lines 1-5). Specifically, Zhang teaches that the previously discussed invention are kits for practicing the subject methods, as described above where the subject kits at least include one or more of: a library of mammalian cells displaying a plurality of different monoclonal antibodies, a library of mammalian cells displaying monoclonal antibodies, where each cell displays a different monoclonal antibody on its surface, a library of expression cassettes, for example linear or circular (e.g., in a retroviral vector) expression cassettes that provide for expression of a library of monoclonal antibodies in mammalian cells, or a parental monoclonal antibody (Paragraph 140, lines 1-5).
Zhang also teaches that the previously discussed invention also provides compositions, such as a library of mammalian cells displaying a plurality of different monoclonal antibodies (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more or 50 or more, etc.), a library of mammalian cells displaying monoclonal antibodies, where each cell displays a different monoclonal antibody on its surface, a library of expression cassettes, for example linear or circular (e.g., in a retroviral vector) expression cassettes that provide for expression of a library of monoclonal antibodies in mammalian cells (Paragraph 91, lines 1-5). Specifically, Zhang teaches that in these assays, each antibody of a plurality of antibodies is tested for its ability to bind specifically to a substrate where the term "specifically" in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific antigen i.e., a polypeptide, or epitopemany embodiments, the specific antigen is an antigen (or a fragment or subfraction of an antigen) used to immunize the animal host from which the antibody-producing cells were isolated (Paragraph 116, lines 1-5).
Zhang teaches that with respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions (Paragraph 41, lines 1-5).
Regarding claims 30-31, Zhang teaches that the assay measures the specific inhibition of an antibody to an interaction between a first compound and a second compound (e.g. two biopolymeric compounds) or specifically inhibits a reaction (e.g. an enzymatic reaction) and in the interaction inhibition assay, one interaction substrate, usually a biopolymeric compound such as a protein e.g. a receptor, may be bound to a solid support in a reaction vessel (Paragraph 126, lines 1-5). Further, Zhang teaches that antibody is added to the reaction vessel followed by a detectable binding partner for the substrate, usually a biopolymeric compound such as a protein e.g. a radiolabeled ligand for the receptor where after washing the vessel, interaction inhibition may be measured by determining the amount of detectable binding partner present in the vessel and interaction inhibition occurs when binding of the binding partner is reduced greater than about 20%, greater than about 50%, greater than about 70%, greater than about 80%, or greater than about 90% or 95% or more, as compared to a control assay that does not contain antibody (Paragraph 126, lines 5-10).
Zhang also teaches that in the reaction inhibition assay, an enzyme may be bound to a solid support in a reaction vessel, where an antibody is usually added to the reaction vessel followed by a substrate for the enzyme. In many embodiments, the products of the reaction between the enzyme and the substrate are detectable, and, after a certain time, the reaction is usually stopped where after the reaction has been stopped, reaction inhibition may be measured by determining the level of detectable reaction product present in the vessel (Paragraph 127, lines 1-5). Also, Zhang teaches that reaction inhibition occurs when the rate of the reaction is reduced greater than about 20%, greater than about 50%, greater than about 70%, greater than about 80%, or greater than about 90% or 95% or more, as compared to a control assay that does not contain antibody (Paragraph 127, lines 5-10).
Zhang further teaches that kits for practicing the subject methods, as described above where the subject kits at least include one or more of: a library of mammalian cells displaying a plurality of different monoclonal antibodies, a library of mammalian cells displaying monoclonal antibodies, where each cell displays a different monoclonal antibody on its surface, a library of expression cassettes, for example linear or circular (e.g., in a retroviral vector) expression cassettes that provide for expression of a library of monoclonal antibodies in mammalian cells, or a parental monoclonal antibody and other optional components of the kit include: components for performing antibody binding assays, e.g. FACS assays, microtiter plates and ELISA reagents; buffers, nucleotides and reagents for performing amplifying heavy and light chain nucleic acids; and a humanized antibody framework-encoding nucleic acids that is operably linked to a cell surface targeting sequence (Paragraph 140, lines 1-10).
Zhang does not teach or suggest that nucleic acid amplification may be deliberately suspended and subsequently resumed through controlled manipulation of reaction conditions, such as pH adjustment or addition/removal of inhibitors, nor does Zhang contemplate a multi-stage amplification process involving an initial amplification, inhibition of polymerase activity and subsequent amplification of the resulting amplification product.
Gunderson teaches that the present invention is directed to methods for the use of microsphere arrays to detect and quantify a number of nucleic acid reactions where the invention finds use in genotyping, i.e. the determination of the sequence of nucleic acids, particularly alterations such as nucleotide substitutions (mismatches) and single nucleotide polymorphisms
(SNPs) and similarly, the invention finds use in the detection and quantification of a nucleic acid target using a variety of amplification techniques, including both signal amplification and target amplification (Paragraph 1). Gunderson further teaches that the methods of the invention can be used in nucleic acid sequencing reactions as well (Paragraph 1). Additionally, Gunderson teaches that in a preferred embodiment, as for most of the amplification techniques described herein, a second amplification reaction can be done using the complementary target sequence, resulting in a substantial increase in amplification during a set period of time where, a second primer nucleic add is hybridized to a second target sequence, that is substantially complementary to the find
target sequence, to form a second hybridization complex (Paragraph 102, lines 1-5). Gunderson also teaches that the addition of the enzyme, followed by disassociation of the second hybridization complex, results in the generation of a number of newly synthesized second strands (Paragraph 102, lines 5-10).
Further, Gunderson teaches an aspect, sensitivity to variations in stringency
parameters are used to determine either the identity of the nucleotide(s) at the detection position or the presence of a mismatch and as a preliminary matter, the use of different stringency conditions such as variations in temperature and buffer composition to determine the presence
or absence of mismatches in double stranded hybrids comprising a single stranded target sequence and a probe is well known (Paragraph 251, lines 1-5). Gunderson also teaches that stringent conditions are selected to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH where the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium) (Paragraph 253, lines 10-30).
Liu teaches point-of-care genetic analysis may require polymerase chain reaction (PCR) to be carried out on whole blood, however, human blood contains natural inhibitors of PCR such as hemoglobin, immunoglobulin G, lactoferrin, and proteases, as well as anticoagulant agents, including EDTA and heparin that can reduce whole blood PCR efficiency in order to develop a highly specific, direct whole blood single-nucleotide polymorphism (SNP) analysis method based on allele-specific (AS) PCR that is mediated by Pfu DNA polymerase and phosphorothioate-modified AS primers (Aims). Further, Liu teaches at high Mg2+ concentrations, Pfu DNA polymerase efficiently amplified genomic DNA in a reaction solution containing up to 14% whole blood where among the three anticoagulants tested, Pfu DNA polymerase showed the highest activity with sodium citrate and meanwhile, Triton X-100 and betaine inhibited Pfu DNA polymerase activity in whole blood PCR, whereas trehalose had virtually no effect (Results; Effects of [Mg2+] on the efficiency of direct whole blood PCR by Pfu DNA polymerase; Effects of PCR additives on whole blood PCR mediated by Pfu DNA polymerase). Specifically, Liu teaches that these findings provided for the development of a low-cost, simple, and fast direct whole blood genotyping method that uses Pfu DNA polymerase combined with phosphorothioate AS primers for CYP2C9*3 and VKORC1(−1639) loci (Results).
Zhu teaches DNA polymerases with proofreading activity are important for accurate amplification of target DNA, specifically showing that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequences such as GGGGG and GGGGHGG can cause PCR failure using proofreading DNA polymerases but not Taq DNA polymerase where the inhibitory effect of these G-rich sequences is caused by G-quadruplex and is dose dependent (Abstract). Further, Zhu teaches as secondary structure of the G-rich oligonucleotides are normally disrupted during PCR, and the inhibitory effect of G-rich sequences could be reduced by adding its complementary oligonucleotide (Fig. 4), one may conclude that the inhibition of proofreading DNA polymerases is caused by single-stranded G-rich sequences (Proofreading DNA polymerases specifically bind to G-quadruplex). Specifically, Zhu teaches that the inhibition effect to proofreading DNA polymerases is not significant when the G-rich primer is used below 0.1 μM (Fig. 4) and as the Sso7d fused proofreading DNA polymerases such as Phusion, Q5 and Cobuddy are recommended to use primers at a final concentration of 0.5 μM, PCR should be failed if a G-rich primer was used (Discussion).
In view of these teachings, one of ordinary skill in the art would have been motivated to combine the references because it was well known that nucleic acid amplification reactions are highly sensitive to reaction conditions and that controlling such conditions allow modulation of enzyme activity. Zhang teaches the use of buffers, detergents, and inhibitors in reaction environments, while Liu and Zhu teach that such components directly affect polymerase activity. Gunderson teaches the benefit of performing amplification in multiple stages. Therefore, it would have been obvious to a person of ordinary skill in the art to control polymerase activity between amplification steps by adjusting reaction conditions, including through the use of buffers or inhibitors, in order to regulate amplification, reduce background, or enabled staged amplification workflows.
Further, a person of ordinary skill in the art would have had a reasonable expectation of success in making this combination because the relationship between reaction conditions and enzyme activity was well understood and predictable. Liu and Zhu demonstrate that polymerase activity is affected in a predictable manner by reaction components and conditions, while Zhang demonstrates that such components are routinely used in biochemical systems. Gunderson demonstrates that amplification steps may be performed sequentially. Therefore, modifying reaction conditions to inhibit polymerase activity during one stage and allow amplification during another stage would have been expected to work as intended.
With respect to the claimed limitation of adjusting the temperature and pH of the reaction mixture to inhibit or inactivate the nucleic acid polymerase, it is well known in the art that enzymes, including DNA polymerases, function within defined pH and temperature ranges and that deviation from such ranges reduces or eliminates enzymatic activity. Zhang teaches the use of buffers such as Tris and phosphate buffers in reaction systems, and Liu teaches that reaction conditions affect polymerase activity. Therefore, adjusting the pH or temperature outside the working range of the polymerase to inhibit its activity constitutes no more than routine optimization of a known result-effective variable. Discovering the degree of deviation necessary to inhibit the enzyme would have required only routine experimentation. See MPEP 2144.05; In re Aller, 220 F. 2d 454 (CCPA 1955). Specifically, the extent that the claims recite specific pH or temperature ranges at least one, two or three degrees or units outside the working range of polymerase, such ranges would have been obvious as a matter of routine optimization. The art recognizes that enzyme activity varies as a function of pH or temperature, and recognizing at which inhibition occurs would have involved routine experimentation. See MPEP 2144.05; In Peterson, 315 F.3d 1325 (Fed. Cir. 2003).
Accordingly, the combination of Zhang, Gunderson, Liu and Zhu teaches or suggests all of the limitations of the claimed invention, and it would have been obvious to a person of ordinary skill in the art to combine these teachings to achieve controlled inhibition and subsequent amplification of nucleic acids by manipulating reaction conditions, including pH and the presence of inhibitors, with a reasonable expectation of success.
Conclusion
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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