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 .
Office Action: Notice
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 1/26/2026 has been entered.
Claim Status
Claims 88, 90, 94, 98-101, 103-207 have been amended (1/26/2026). Claims 1-87, 95-97, and 102 are cancelled. No new matter was added. Thus, claims 88-94, 98-101, and 103-107 are under examination (1/26/2026).
Priority
Claims 88-94, 98-101, and 103-107 are given a priority date of 3/1/2019.
Objections Withdrawn
Claims:
The minor grammatical objections to claims 88, 94 and 107 are withdrawn in view of Applicant’s amendments.
Rejections Withdrawn
Claim Rejections - 35 USC § 112(b)
The rejection of claims 101 and 102-104 under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 2nd paragraph, is withdrawn in view of Applicant’s amendments of claim 101 and cancellation of claim 102.
New Rejections
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.
Claims 88-94, 98-101, and 103-107 are 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.
Independent claims 88 and 98 require, inter alia, a phage library expressing deep mutation scanning (DMS) proteins or peptides comprising a plurality of peptide fragments generated by systematically shifting starting positions along substantially the length of a viral protein, bacterial protein, fungal protein, or cancer antigen while maintaining fragment length constant and introducing at least a single amino acid mutation, and determining residues responsible for binding or non-binding based on sequencing-derived enrichment factors and reproducibility thresholds.
The claims further require that the DMS proteins or peptides collectively span proteins derived from multiple distinct biological classes (viral, bacterial, fungal, and cancer antigens) and that residue-level functional mapping be achieved through comparative sequencing analysis following selection.
The MPEP states that the purpose of the written description requirement is to ensure that the inventor had possession, as of the filing date of the application, of the specific subject matter later claimed by him. The courts have stated:
To fulfill the written description requirement, a patent specification must describe an invention and do so in sufficient detail that one skilled in the art can clearly conclude that "the inventor invented the claimed invention." Lockwood v. American Airlines, Inc., 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (Fed. Cir. 1997); In re Gostelli, 872 F.2d 1008, 1012, 10 USPQ2d 1614, 1618
(Fed. Cir. 1989) ("[T]he description must clearly allow persons of ordinary skill in the art to recognize that [the inventor] invented what is claimed."). Thus an applicant complies with the written description requirement "by describing the invention, with all its claimed limitations, not that which makes it obvious" and by using "such descriptive means as words, structures, figures, diagrams, formulas, etc., that set forth the claimed invention." Lockwood, 107 F.3d at 1572, 41 USPQ2d at 1966; Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
Further, the presently claimed requirement of determining binding or non-binding residues based on an enrichment factor together with a reproducibility threshold constitutes a broad functional analytical genus. For such generic functional limitations, the specification must provide adequate written description demonstrating possession of the claimed analytical framework, for example through disclosure of representative calculations, defined statistical parameters, or structural correlations between sequencing data outputs and residue determining criteria. See Reagents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398 (Fed. Cir. 1997); MPEP 2163.
A written description of an invention involving a chemical genus, like a description of a chemical species, "requires a precise definition, such as by structure, formula, [or] chemical name," of the claimed subject matter sufficient to distinguish it from other materials. Fiers v. Revel, 984 F.2d at 1171,25 USPQA2d, 1601; In re Smyth, 480 F.2d 1376,1383, 178 USPQ 279,284 (CCPA 1973) ("In
other cases, particularly but not necessarily, chemical cases, where there is an unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
The MPEP further explains that where a biomolecule or analytical framework is described primarily by a functional characteristic, without disclosure of a correlation between the claimed function and defining structural, mathematical, or procedural features, such functional description alone is not sufficient for written description purposes. See MPEP 2163. For a broad generic limitation, the disclosure must provide a sufficient number of representative species or identifying characteristics demonstrating possession of the claimed genus, particularly where the genus exhibits substantial variation.
The MPEP lists factors that can be used to determine if sufficient evidence of possession has been furnished in the disclosure of the application. These include: (1) Actual reduction to practice, (2) Disclosure of drawings or structural chemical formulas, (3) Sufficient relevant identifying characteristics (such as: i. Complete structure, ii. Partial Structure, iii. Physical and/or chemical properties, iv. Functional characteristics when coupled with a known or disclosed structure, and v. Correlation between function and structure), (4) Method of making the claimed invention, (5) Level of skill and knowledge in the art, and (6) Predictability in the art.
A "representative number of species" means that the species, which are adequately described, are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. This disclosure of only one or a few species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure indicates that the patentee has invented species sufficient to constitute the gen[us]." See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 115; Noelle v. Lederman, 355 F.3d, 1343, 1350, 69 USPO2d 1508, 1514 (Fed. Cir. 2004) ("[A] patentee of a biotechnological invention cannot necessarily claim a genus after only describing a limited number of species because there may be unpredictability in the results obtained from species other than those specifically enumerated.").
In the present case, the claimed requirement of determining binding or non-binding residues based on an enrichment factor together with a reproducibility threshold encompasses a wide range of possible sequencing normalization strategies, statistical enrichment models, replicate analysis methodologies, and threshold selection criteria. Minor differences in these analytical parameters can result in substantially different residue determinations and biological interpretations.
Adequate written description requires that the specification convey with reasonable clarity to those skilled in the art that, as of the filing date, the inventor was in possession of the invention now claimed. See Vas-Cath Inc. v Mahurkar, 19 USPQ2d 1111 (Fed. Cir. 1991). The inquiry focuses on whether the disclosure clearly allows persons of ordinary skill in the art to recognize that the inventor invented what is now claimed.
The description of an enrichment factor in combination with a reproducibility threshold, are thereby defined by a genus of sequencing-based analytical methodologies with a functional outcome. However, the specification does not disclose sufficient analytical detail that would allow one of ordinary skill in the art to envision the scope of enrichment-based residue determination frameworks and/or algorithms encompassed by the claims. The disclosure does not set forth specific statistical models, quantitative formulas, replicate analysis strategies, or defined threshold criteria that would distinguish the claimed analytical approach from other possible sequencing data interpretation models.
The rejected claims thus comprise a mere statement that such enrichment-based determination is part of the invention or reference to a potential method of obtaining sequencing data via an algorithm. Rather, the analytical framework must be sufficiently described. Absent disclosure of defining quantitative or procedural characteristics enabling recognition of the claimed methodology, the specifical does not reasonably demonstrate possession of the full scope of the enrichment factor and reproducibility threshold limitation. To provide adequate written description and evidence of possession of a claimed genus, the
specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof.
The specification states the following paragraphs [144-145]: “In particular embodiments, a bioinformatics analysis method can include determining a zero-inflated generalized Poisson significant-enrichment assignment algorithm that can be used to generate a -log10(p-value) for enrichment of each clone across all samples. A reproducibility threshold can be established to call 'hits' in technical replicate pairs by first calculating the log10(-log10(p-value)) for each clone in Replicate 1. These values can then be surveyed in Replicate 2 by using a sliding window of width 0.01 from -2 to the maximum log10(-log10(p-value)) value in Replicate 1. For all clones that fall within each window, the median and median absolute deviation of log10(-log10(p-values)) in Replicate 2 can be calculated and plotted against the window location. The reproducibility threshold can be set as the window location where the median was greater than the median absolute deviation. The distribution of the threshold -log10(p-values) is centered around a median of 2.2. In sum, a phage clone is called a 'hit' if the -log10(p value) is at least 2.2 in both replicates. Beads-only samples, which serve as a negative control for non- specific binding of phage, can be used to identify and eliminate background hits. Peptides called as hits are then aligned using Clustal Omega. The shortest amino acid sequence present in all of the hits is defined as the "minimal binding epitope" of a candidate binding molecule (Larman, et al., Nat Biotechnol, 29(6):535-541, 2011). [0145] In particular embodiments, a bioinformatics analysis method can include determining the position weight matrix (PWM) spanning the epitope region to determine the motifs that are enriched in the presence of the protein of interest. A matrix of the frequency of each amino acid at every position is determined by observing the number of clones with a specific amino acid enriched by the protein of interest, as compared to the background. The log2 of the relative frequency of an amino acid can be plotted on a logo plot, and the motif displayed corresponds to the epitope of the protein of interest (Stormo, et al., Nucleic Acids Res., 10(9): 2997-3011, 1982 and Xia, Scientifica, Volume 2012, 2012).”
Even if one accepts these embodiments as meeting certain aspects of the claimed analytical limitation, the disclosure is limited to a specific statistical implementation of enrichment analysis and threshold determination. The claims, in contrast, broadly require determining residues responsible for binding or non-binding based on an enrichment factor and a reproducibility threshold without restriction to the particular statistical model, replicate analysis methodology, threshold calculation procedure, or decision boundary described in the specification. Thus, the examples described in paragraphs 144-145 are only representative of a narrow subset or possible enrichment-based analytical approaches, and are not necessarily predictive of how alternative enrichment metrics, normalization strategies, replicate consistency frameworks, or statistical thresholding methods would perform in identifying binding residues. Minor variations in enrichment calculation methodology or reproducibility criteria can lead to substantially different residue identification outcomes.
Further, the prior art does not appear to offset the deficiencies of the instant specification in that it does not describe the specifications or individualization involved in the application of enrichment factors and/or reproducibility threshold algorithms to determine binding or non-binding residues. Specifically, Fowler et al. (“Measuring the activity of protein variants on a large scale using deep mutational scanning”, Nat Protoc., published 8/2014; from IDS 9/1/2021) teaches that deep mutational scanning marries selection for protein function to high-throughput DNA sequencing in order to quantify the activity of variants of a protein on a massive scale via appropriate selection system for the protein function of interest (Abstract). Further, Fowler teaches that mutagenesis paradigms include targeted, systematic and random mutagenesis through examining a limited number of protein variants with an unbiased library of variants (a short protein in a phage-display format) (Introduction: Paragraph 1; Protocol Overview and Experimental Design). Specifically, Fowler teaches that many mutagenesis methods to create virally-transformed or modified cells (Transforming and Selecting the Library: Paragraphs 1-2) can be used to construct diversity libraries, including those based on error-prone PCR, oligonucleotide-directed mutagenesis or degenerate oligonucleotide assembly (Designing and creating a diversity library: Paragraph 1). Therefore, while Fowler teaches generation and screening of variant libraries using sequencing-based approaches, Fowler does not disclose calculating enrichment factors together with applying a reproducibility threshold to identify binding residues. And, thus, Fowler does not remedy the lack of written description support for the specific analytical limitation recited in the claims.
Additionally, Larman et al. (“Autoantigen discovery with a synthetic human peptidome”, Nature Biotechnology, published 5/22/2011, from IDS 9/1/2021) teaches that they used a two-parameter, generalized Poisson model to approximate the distribution of the abundance of immunoprecipitated clones, as recently demonstrated for RNA-Seq data12 and because the sample preparation and sequencing steps introduce similar biases, this distribution family fits the data quite well (Fig. 2b). Further Larman teaches that they calculated the generalized Poisson parameter values for each input abundance level13 and regressed these parameters to form our null model for the calculation of enrichment significance (P-values) of each clone (Fig. 2c) and through comparing the two PhIP-Seq replicates for patient A revealed that the most significantly enriched clones were the same in both replicas (Fig. 2d), highlighting the reproducibility of the assay, where this contrasts dramatically with a comparison of two different patients, as the enrichment of peptides is highly patient-specific (Supplementary Fig. 3). Larman also teaches that a performing a negative-control PhIP-Seq experiment identified phage capable of binding to the slurry of Protein A and Protein G magnetic beads in the absence of patient antibodies and thus defined patient A–positive clones as those clones with a reproducible –log10 P-value greater than a cutoff (Fig. 2d, dashed red line) but not significantly enriched on beads alone (P < 10−3) (Table 2; Analysis of a PND patient with NOVA autoantibodies). Therefore, Larman’s teachings are directed to a particular PhIP-Seq statistical workflow for identifying enriched clones and do not provide sufficient disclosure of a generalized enrichment factor and reproducibility threshold analytical framework for determining binding residues. Thus, Larman does not overcome the written description deficiency of the claimed analytical limitation.
Further, Stormo et al. (“Use of the ‘Perception’ algorithm to distinguish translational initiation sites in E. coli”, Nucleic Acids Res., 1982, from IDS 9/1/2021) teaches the use of a "Perceptron" algorithm to find a weighting function which distinguishes E. coli translational initiation sites from all other sites in a library of over 78,000 nucleotides of mRNA sequence, where the "Perceptron" examined sequences as linear representations (Abstract). Also, Stormo teaches that they tried to define a probability function based solely on linear sequences where the goal was to fund a mathematical function W, for weighting function, such that applying W to any sequence will give a value, and the magnitude of that value will determine whether that particular sequence is a ribosome binding site (Introduction: Paragraphs 2-5). Therefore, although Stormo describes applying a mathematical weighting function to sequence analysis, the teaching is directed to identifying ribosome binding sites and does not describe or support a sequencing-based enrichment factor and reproducibility threshold methodology for residue determination. And, thus, Stormo does not remedy the written description deficiency of the claimed analytical limitation.
Neither the prior art nor the specification describes with sufficient clarity the specific analytical parameters or methodological structures capable of determining binding or non-binding residues based on enrichment factor in combination with a reproducibility threshold across the full scope of the claims. If one of ordinary skill in the art cannot reasonably envision the quantitative framework, statistical decision criteria or analytical boundaries required to implement the claimed residue determination methodology, then possession of the claimed invention is not demonstrated. Accordingly, the skilled artisan would have reasonably concluded that Applicants were not in possession of the full scope of the enrichment-based and reproducibility-threshold residue determination limitation recited in the claims 88-94, 98-101, and 103-107. Therefore, these claims are rejected under 35 USC 112(a) for lack of adequate written description.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 88-94, 98-101, and 103-107 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
The claims recite determining residues responsible for binding or non-binding based on calculating an enrichment factor and applying a reproducibility threshold using sequencing-derived data. This limitation involves mathematical calculations, statistical analysis, comparison of datasets, and evaluation of probabilities or threshold criteria, which fall within the category of mathematical concepts and mental processes, including observation, evaluation and judgement.
Such data analysis steps constitute an abstract idea because they involve processing and interpreting information to reach a determination, rather than reciting a specific technological improvement or particularized machine implementation.
The integration of the judicial exception into the claims does not render them patent eligible because the claims are written at a high level of generality and merely use well-known, routine, and conventional techniques in the field.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES.
The claims provide for a method comprising:
Generating and sequencing variant libraries and obtaining molecular interaction data;
Calculating enrichment factors for variants based on sequencing-derived abundance information;
Comparing enrichment values across replicates and applying a reproducibility threshold to identify binding residues; and
Analyzing the resulting data to determine residues associated with binding activity.
Thus, the claims are directed to statutory categories (i.e., processes).
Step 2A, Prong One — Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES.
Abstract ideas have been identified by the courts by way of example, including fundamental economic practices, certain methods of organizing human activities, an idea ‘of itself,’ and mathematical relationships/formulas. The claims recite a judicial exception. The “mental process” directed to calculating enrichment factors and applying a reproducibility threshold constitutes the use of mathematical relationships. Specifically, the claimed steps involve evaluating sequencing-derived numerical data, performing comparative calculations, and determining whether values satisfy a threshold criterion to identify correlations between sequence variants and binding activity which corresponds to “an abstraction” (an idea having no particular concrete or tangible form). Thus, the claimed invention describes a judicial exception, which correspond to abstractions (ideas, having no particular concrete or tangible form).
Step 2A, Prong Two — Does the Claim Recite an Additional Elements that Integrate the Judicial Exception into a Practical Application? NO.
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. However, absent are any additional elements recited in the claim beyond the judicial exceptions which integrate the exception into a practical application of the exception. The “integration into a practical application” requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception.
The claim limitations are considered to be; (a) generating variant libraries and performing sequencing to obtain abundance data, and (b) calculating enrichment factors and comparing such values across replicates to determine whether a reproducibility threshold is satisfied for identifying binding residues, are recited at a high level of generality and represent well-understood, routine, and conventional techniques used to collect data for subsequent data correlation analysis.
While the claims recite steps of “preparing libraries”, “sequencing variants”, and “obtaining molecular interaction measurements”, these steps are recited at a high level of generality and amount to mere data gathering steps. There are no additional steps which meaningfully limit the application of the identified judicial exception into a practical application or integrate the abstract mathematical analysis into a practical application. Thus, the claims do not provide for any element/step that integrates the abstraction into a practical application.
Step 2B - Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO.
The Supreme Court has identified a number of considerations for determining whether a claim with additional elements amounts to “significantly more” than the judicial exception(s) itself. The claims as a whole are analyzed to determine whether any additional element/step, or combination of additional elements/steps, in addition to the identified judicial exception(s) is sufficient to ensure that the claim amounts to “significantly more” than the exception(s).
However, the additional elements of the instant application, individually and in combination, do not amount to “significantly more.” Under the Step 2B analysis, the “physical” elements/steps of, generating variant libraries, introducing variants into cells, performing selection assays, and sequencing variant populations to obtain abundance measurements are “physical” steps telling a practitioner to simply implement the abstract idea and are considered to be within the purview of one in the art as being routine and conventional in the art when applying a reproducibility threshold to identify binding residues for obtaining molecular interaction or functional activity data.
For example, Fowler et al. (“Measuring the activity of protein variants on a large scale using deep mutational scanning”, Nat Protoc., published 8/2014; from IDS 9/1/2021) discloses that deep mutational scanning marries selection for protein function to high-throughput DNA sequencing in order to quantify the activity of variants of a protein on a massive scale via appropriate selection system for the protein function of interest (Abstract). Further, Fowler teaches that mutagenesis paradigms include targeted, systematic and random mutagenesis through examining a limited number of protein variants with an unbiased library of variants (a short protein in a phage-display format) (Introduction: Paragraph 1; Protocol Overview and Experimental Design). Specifically, Fowler teaches that many mutagenesis methods to create virally-transformed or modified cells (Transforming and Selecting the Library: Paragraphs 1-2) can be used to construct diversity libraries, including those based on error-prone PCR, oligonucleotide-directed mutagenesis or degenerate oligonucleotide assembly (Designing and creating a diversity library: Paragraph 1), establishing these as conventional techniques.
Further, Tripathi et al. (“Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing”, Current Opinion in Structural Biology, published 2/2014) discloses that multiple methods currently exist for rapid construction and screening of single-site saturation mutagenesis (SSM) libraries in which every codon or nucleotide in a DNA fragment is individually randomized, where nucleotide sequences of each library member before and after screening or selection can be obtained through deep sequencing and the relative enrichment of each mutant at each position provides information on its contribution to protein activity or ligand-binding under the conditions of the screen (Abstract). Tripathi also discloses that recent developments in highly parallelized sequencing methodologies permit rapid analysis of the relative populations of individual DNA sequence variants in a large pool, where various such deep sequencing platforms are available (Use of deep sequencing to characterize mutant enrichment during screening: Paragraph 1). Specifically, Tripathi discloses that reads are filtered for quality as well as the absence of insertion and deletions, and the frequency of every amino acid at each position in the mutagenized region is calculated, where the enrichment of mutants at each condition, relative to the starting library as well as relative to the WT codon can be obtained from this information (Use of deep sequencing to characterize mutant enrichment during screening: Paragraph 1). These detailed sequencing applications demonstrate that practitioners were well-versed in applying complex enrichment factor to sequencing data.
Further, Larman et al. (“Autoantigen discovery with a synthetic human peptidome”, Nature Biotechnology, published 5/22/2011, from IDS 9/1/2021) teaches that they used a two-parameter, generalized Poisson model to approximate the distribution of the abundance of immunoprecipitated clones, as recently demonstrated for RNA-Seq data12 and because the sample preparation and sequencing steps introduce similar biases, this distribution family fits the data quite well (Fig. 2b). Further Larman teaches that they calculated the generalized Poisson parameter values for each input abundance level13 and regressed these parameters to form our null model for the calculation of enrichment significance (P-values) of each clone (Fig. 2c) and through comparing the two PhIP-Seq replicates for patient A revealed that the most significantly enriched clones were the same in both replicas (Fig. 2d), highlighting the reproducibility of the assay, where this contrasts dramatically with a comparison of two different patients, as the enrichment of peptides is highly patient-specific (Supplementary Fig. 3). Larman also teaches that a performing a negative-control PhIP-Seq experiment identified phage capable of binding to the slurry of Protein A and Protein G magnetic beads in the absence of patient antibodies and thus defined patient A–positive clones as those clones with a reproducible –log10 P-value greater than a cutoff (Fig. 2d, dashed red line) but not significantly enriched on beads alone (P < 10−3) (Table 2; Analysis of a PND patient with NOVA autoantibodies). Therefore, Larman’s teachings are establishing a high level of routine and convention in regards to the application of statistical-based algorithms (i.e., reproducibility thresholds) to sequencing data.
Therefore, the prior art establishes a high level of routine and conventional activity with respect to applying statistical-based algorithms, including enrichment methods and reproducibility thresholding, to sequencing-derived variant abundance data. Accordingly, generating variant libraries, performing functional selection assays, sequencing variant populations, and determining enrichment metrics or probabilities associated with variant performance constituted well-understood and conventional activities prior to the effective filing date of the claimed invention.
Simply appending routine and conventional activities previously known to the industry specified at a high level of generality to the judicial exception and/or generally linking the use of the judicial exception(s) to a particular technological environment or field of use, are not found to be enough to qualify as “significantly more.” Nothing is added by identifying the techniques to be used (i.e., ““preparing libraries”, “sequencing variants”, and “obtaining molecular interaction measurements”) because those techniques were well-understood, routine, and conventional techniques that a practitioner would have thought of when instructed to apply a reproducibility threshold to identify binding residues for obtaining molecular interaction or functional activity data.
In context with the other recited claim limitations, the language directed to determining enrichment factors for protein variants and applying reproducibility thresholds across replicate sequencing datasets indicates whether a statistical relationship or correlation exists between variant abundance measurements and functional activity. This information simply tells a practitioner about the relevant mathematical correlation, at most adding a suggestion that the researcher should take those relationships into account when analyzing sequencing-derived data.
Thus, when viewed both individually and as an ordered combination, the claimed elements/steps in addition to the identified judicial exception are found insufficient to supply an inventive concept because the elements/steps (i.e., generating variant libraries, introducing variants into cells, performing selection assays, sequencing variant populations, and comparing enrichment values across replicates) are considered conventional and specified at a high level of generality. The claim limitations do not transform the abstract idea that they recite into patent-eligible subject matter because “the claims simply instruct the practitioner to implement the abstract idea with routine, conventional activity.”
Accordingly, the claims do not qualify as patent-eligible subject matter.
Rejections Maintained
Claim Rejections - 35 USC § 103
Claims 88-94, 98 and 100-101, 103-107 are rejected under 35 U.S.C. 103 as being unpatentable over Henley et al. (WO 2018/081476 A2, published 5/3/2018), in view of Fowler et al. (“Measuring the activity of protein variants on a large scale using deep mutational scanning”, Nat Protoc., published 8/2014; from IDS 9/1/2021) and in further view of Tripathi et al. (“Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing”, Current Opinion in Structural Biology, published 2/2014). This rejection is modified, as necessitated by Applicants' amendments.
Regarding claims 88-98 and 100-107, Henley teaches a kit or collection of parts for methods of producing a population of genetically modified cells using viral or non-viral vectors for the treatment of cancer (Abstract). Further, Henley teaches that the previously mentioned targeted viral vectors include; an anti-DNA sensing protein may also be a DNA sensing protein (i.e., TREX1), cellular FLICE-inhibitory protein (c-FLiP), Human cytomegalovirus tegument protein (HCMV pUL83), dengue virus specific NS2B-NS3 (DENV NS2B-NS3), Protein E7-Human papillomavirus type 18 (HPV18 E7), hAd5 El A, Herpes simplex virus immediate-early protein ICP0 (HSV1 ICP0), Vaccinia virus B 13 (VACV B 13), Vaccinia virus C16 (VACV C16), three prime repair exonuclease 1 (TREX1), human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV) (Paragraph 436, lines 1-10). Further, Henley teaches that the previously described kit includes a population or phage library consisting of genetically modified cells or sequenced proteins of interest comprising: providing a population of cells from a human subject; introducing a clustered regularly interspaced short palindromic repeats (CRISPR) system comprising a guide polynucleic acid to said population of cells, wherein said guide polynucleic acid specifically binds to a candidate binding molecule (i.e., Cytokine Inducible SH2 Containing Protein (CISH)) (Paragraph 6, lines 1-10) following gene transfer or a gene delivery system derived of a viral vector via recombinant techniques (i.e., adeno-associated virus (AA V), helper-dependent adenovirus, hybrid adenovirus, Epstein-Bar virus, retrovirus, lentivirus, herpes simplex virus, hemmaglutinating virus of Japan (HVJ), Moloney murine leukemia virus, poxvirus, and HIV-based virus) (Figure 3; Paragraph 230, lines 1-5). Further, Henley teaches that these peptides may be derived from phage display or synthetic peptide library (Paragraph 313, lines 15-20). Henley also teaches that the sequenced proteins of interest or population of modified cells is separated from the candidate binding molecule via activation of the plasma membrane of a eukaryotic cell, in the presence of its binding target, followed by expression where a target can also be soluble (i.e., not bound to a cell) (Paragraph 318, lines 1-5), followed by isolation from the PEG-precipitated or immunoprecipitated supernatant by low-speed centrifugation followed by CsCl gradient (Paragraph 268, lines 5-10). Henley also teaches that the bound and non-bound or isolated phage or population of modified cells then undergoes lysing and sequencing (Paragraph 562, lines 5-10) to identify a cancer-related target sequence, for example, a Neoantigen, from a sample obtained from a cancer patient using an in vitro assay (i.e., whole- exomic sequencing) (Figure 1; Paragraph 34, lines 1-5).
Henley teaches that previously described modified cells or proteins of interest undergo genomic transplant to a binding domain that can be activated or expanded or linked by co-culturing with tissue or cells, while improving their potency and function and engineered to express any gene for T cell activation (i.e., a bead, a cell, a protein, an antibody, a cytokine, or any combination) (Paragraph 247, lines 1-5).
Henley also teaches that the previously described modified cells or proteins of interest linked to a binding domain can catalyze insertion of foreign DNA into a host genome; including site-specific recombinases clusters or complexes (in which DNA is covalently attached to a tyrosine residue) and serine recombinases (where covalent attachment occurs at a serine residue) to the candidate binding molecule (Paragraph 299, lines 1-10).
Further, Henley teaches that the derived modified cells or proteins of interest originate from a cancer antigen; including, elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferase fusion protein, HLA-A2d, HLA-A1 Id, hsp70-2, KIAAO205, MART2, ME1, MUM- If, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1- or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-1, GAGE-1, 2, 8, Gage 3, 4, 5, 6, 7, GnTVf, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, MAGE- Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-C2, mucink, NA-88, NY-ESO-l/LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-1, TAG-2, TRAG-3, TRP2-INT2g, XAGE-lb, CEA, gpl00/Pmell7, Kallikrein 4, mammaglobin-A, Melan-A/MART- 1, NY-BR-1, OA1, PSA, RAB38/NY-MEL-1, TRP-l/gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BCMA, BING-4, CPSF, cyclin Dl, DKK1, ENAH (hMena), EP-CAM, EphA3, EZH2, FGF5, G250/MN/CALX, HER- 2/neu, IL13Ralpha2, intestinal carboxyl esterase, alpha fetoprotein, M-CSFT, MCSP, mdm-2, MMP-2, MUC1, p53, PBF, PRAME, PSMA, RAGE-1, RGS5, RNF43, RU2AS, secernin 1, SOX10, STEAP1, survivin, Telomerase, VEGF, and/or WT1 (Paragraph 336, lines 1-15). Further, Henley teaches that tumor-associated antigens may be antigens not normally expressed by the host; they can be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they can be identical to molecules normally expressed but expressed at abnormally high levels; or they can be expressed in a context or environment that is abnormal, as well as proteins or protein-fragments (Paragraph 336, lines 15-20).
Henley also teaches that the derived modified cells or proteins of interest includes a DNA sensing pathway involved in the detection of intracellular nucleic acids, limited via the candidate binding molecule (i.e., Z-DNA-binding protein 1 (ZBP1)) (Paragraph 432, lines 1-5).
Henley teaches that the previously described candidate binding molecule includes specialized delivery to targeted tissues; EnGeneIC delivery vehicles (EDVs) using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV through acting to bring the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis (Paragraph 474, lines 5-10). Further, Henley teaches those anti-angiogenic agents can also be used (i.e., anti-VEGF antibodies, including humanized and chimeric antibodies, anti- VEGF aptamers and antisense oligonucleotides; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor) (Paragraph 501, lines 1-5). Henley also teaches that following the previously described targeted sequencing of the phage library expressed via proteins of interest or modified cells, start sites of GUIDE-Seq reads mapped back to the genome enable localization of the double-strand break to within a few base pairs, divided into groups and then divided by the number of libraries to be pooled together for sequencing followed by mapping reads for the on- and off-target sites (Paragraph 563, lines 1-10). Specifically, Henley teaches that mapping state- specific enhancers in exhausted T cells can enable improved genomic editing for adoptive T cell therapy to make T cells resistant to exhaustion may improve adoptive T cell therapy (Figure 154-155; Paragraph 467, lines 15-25).
Henley teaches that up to 100% of a population of genetically modified cells (i.e., amino acids) comprises integration or substitution of at least one exogenous transgene at a break in a targeted gene or protein of interest (Paragraph 28, lines 1-5; Paragraphs 365-367). Additionally, Henley teaches that a protein can have one or more mutations (i.e., deletion, insertion, amino acid replacement, or rearrangement) compared to a wild-type polypeptide comprising about 20 or more transgenes or amino acid substitutions (Paragraph 315, lines 30-40).
Henley further teaches that another mutagenesis technique that can be used in methods of the present disclosure is DNA shuffling or staggering via the creation of random fragments of members of a gene family and their recombination to yield many new combinations (Paragraph 272, lines 1-5). Specifically, Henley teaches that a guide RNA can target a nucleic acid sequence or targeted peptide with a length of about 20 nucleotides via spacer sequences or staggered fragments located 1, 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to about 20 base pairs or amino acids away from an individualized target (Paragraph 383, lines 1-5). Further, Henley teaches that the spacer sequence or staggered fragment can be a length ranging 25 to 30 or more nucleotides (Paragraph 386, lines 10-15). See MPEP § 2144.05.
Henley teaches that the previously described modified cells or proteins of interest do not have to comprise a barcode sequence, unless unambiguous identification of the polynucleotide is required (Paragraph 286, lines 1-5).
Henley teaches that the proteins or peptides of interest or modified cells include a functional sequence or targeted DNA sequence that can form a complex, comprising a guide sequence, or spacer sequence, that specifies a target site and guides an RNA/Cas complex to a specified target DNA or protein/peptide of interest (Paragraph 374, lines 1-5). Further, Henley teaches that the DNA sequence encoding a guide RNA can also be part of a vector (i.e., plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors), including additional expression control sequences (i.e., enhancer sequences, Kozak sequences,
polyadenylation sequences, transcriptional termination sequences) and/or selectable marker sequences (i.e., antibiotic resistance genes) (Paragraph 391, lines 1-10). Further, Henley teaches that the functional sequence or targeted DNA sequence are potential sites for phosphorylation and subsequent poly-ubiquitination which serves as a cue for proteasomal degradation of capsid proteins (Paragraph 273, lines 5-10) via a transport sequence (i.e., indoleamine 2,3-dioxygenase 1 (IDOl), TCR, killer cell immunoglobulin-like receptor sequence) (Paragraph 355, lines 1-5).
Henley does not teach or suggest the use of deep mutational scanning proteins (DMS) or peptides of interest to create a phage library or the use of enrichment factors and/or reproducibility thresholds determined via sequencing results.
Fowler teaches that deep mutational scanning marries selection for protein function to high-throughput DNA sequencing in order to quantify the activity of variants of a protein on a massive scale via appropriate selection system for the protein function of interest (Abstract). Further, Fowler teaches that mutagenesis paradigms include targeted, systematic and random mutagenesis through examining a limited number of protein variants with an unbiased library of variants (a short protein in a phage-display format) (Introduction: Paragraph 1; Protocol Overview and Experimental Design). Specifically, Fowler teaches that many mutagenesis methods to create virally-transformed or modified cells (Transforming and Selecting the Library: Paragraphs 1-2) can be used to construct diversity libraries, including those based on error-prone PCR, oligonucleotide-directed mutagenesis or degenerate oligonucleotide assembly (Designing and creating a diversity library: Paragraph 1).
Tripathi teaches multiple methods currently exist for rapid construction and screening of single-site saturation mutagenesis (SSM) libraries in which every codon or nucleotide in a DNA fragment is individually randomized, where nucleotide sequences of each library member before and after screening or selection can be obtained through deep sequencing and the relative enrichment of each mutant at each position provides information on its contribution to protein activity or ligand-binding under the conditions of the screen (Abstract). Further, Tripathi teaches that such saturation scans have been applied to diverse proteins to delineate hot-spot residues, stability determinants, and for comprehensive fitness estimates (Abstract). Tripathi also teaches that recent developments in highly parallelized sequencing methodologies permit rapid analysis of the relative populations of individual DNA sequence variants in a large pool, where various such deep sequencing platforms are available (Use of deep sequencing to characterize mutant enrichment during screening: Paragraph 1). Specifically, Tripathi teaches that reads are filtered for quality as well as the absence of insertion and deletions, and the frequency of every amino acid at each position in the mutagenized region is calculated, where the enrichment of mutants at each condition, relative to the starting library as well as relative to the WT codon can be obtained from this information (Use of deep sequencing to characterize mutant enrichment during screening: Paragraph 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the phage library kit of Henley to incorporate the deep mutational scanning (DMS) approach taught by Fowler to create a comprehensive system for protein residue mapping. The motivation to combine these references stems from Fowler’s teaching that DMS enables systematic and high-throughput analysis of protein variants, which would significantly enhance the protein analysis capabilities of Henley’s system for identifying binding residues. A person of ordinary skill would have had a reasonable expectation of success in combining these prior art elements because; both references deal with related technologies of protein analysis using phage systems and Henley already teaches the major components needed (i.e., phage libraries, separation techniques, binding molecules, and sequencing). Further, Fowler provides explicit protocols for implementing DMS with phage display and the combination would predictably yield a more comprehensive and efficient system for mapping protein binding residues. This combination simply applies known techniques (DMS from Fowler) to a known system (Henley’s library/kit) to yield predictable results, since DMS library construction (i.e., amino acid substitutions, staggered fragments) would have been obvious extensions of the DMS methodology taught by Fowler when applied to Henley’s protein analysis system.
Additionally, it would have been obvious to one of ordinary skill in the art to apply the residue-level enrichment analysis and systematic substitution strategies taught by Tripathi to the phage-based protein analysis and sequencing workflow of Henley, as further refined by Fowler’s deep mutational scanning framework, in order to improve resolution in identifying binding or non-binding residues and to generate comprehensive mutational landscapes for proteins or peptide fragments. The motivation to combine arises from the shared objective of all references to enable high-throughput functional mapping of protein variants, and Tripathi expressly teaches that deep sequencing-derived enrichment metrics provide a predictable and scalable means of determining residue contributions to activity or binding.
Such modification would merely involve the routine implementation of known saturation mutagenesis library design parameters (i.e., systematic amino-acid substitutions, positional fragment shifting and quantitative enrichment analysis) within Henley’s existing phage-display selection and sequencing system, as guided by Fowler’s DMS protocols, yielding no more than predictable results. Therefore, the combined teachings render obvious the amended limitations directed to determining residues responsible for binding based on sequencing-derived enrichment factors, generating libraries comprising multiple peptide fragments or variants spanning a protein sequence, and performing comprehensive residue mapping.
Applicant’s Response: The Applicant argues that the combination of references, Henley in view of Fowler, do not teach or suggest all the claimed elements, particularly the use of deep mutational scanning (DMS) proteins in phage libraries and the required separation and sequencing steps, specifically in view of enrichment factors. The Applicant further asserts that Henley is non-analogous art, since its field of endeavor is cancer therapeutics, not identifying amino acid residues important for protein binding. The Applicant also argues that Henley does not disclose incubating DMS proteins with binding molecules, separating bound from unbound phage, or sequencing to determine binding residues. Finally, the Applicant asserts there is no motivation to combine Henley with Fowler, as Fowler’s teachings do not remedy Henley’s deficiencies and would not reasonably lead to the claimed invention.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive, as discussed below.
Specifically, the Applicant argues that Henley does not teach or suggest the use of DMS proteins or peptides in phage libraries and that Fowler does not cure this deficiency, as previously discussed. The Applicant further argues that Henley is non-analogous art, since its field of endeavor is cancer therapeutics, not identifying amino acid residues responsible for protein binding.
Firstly, the Applicant’s argument that Henley is outside the field of endeavor is not persuasive. The test for analogous art under MPEP 2141.01(a) is whether a reference is (1) from the same field of endeavor or (2) reasonably pertinent to the problem faced by the inventor. Henley teaches systematic mutagenesis, creation of mutant protein libraries, and analysis of variants to identify important functional residues (Paragraphs 28, 315, 336, 365), which is reasonably pertinent to the problem of identifying amino acid residues responsible for binding interactions. One of ordinary skill in the art would have naturally consulted Henley for its teachings on variant generation and mapping functional protein interactions.
Further, while Henley teaches creating and using mutagenized protein libraries for identifying functional residues, Fowler teaches high-throughput DMS-based approaches for mapping protein function and binding (Transforming and Selecting the Library: Paragraphs 1-2; Designing and creating a diversity library: Paragraph 1). Additionally, new reference, Tripathi teaches multiple methods currently exist for rapid construction and screening of single-site saturation mutagenesis (SSM) libraries in which every codon or nucleotide in a DNA fragment is individually randomized, where nucleotide sequences of each library member before and after screening or selection can be obtained through deep sequencing and the relative enrichment of each mutant at each position provides information on its contribution to protein activity or ligand-binding under the conditions of the screen (Abstract). Specifically, Tripathi teaches that reads are filtered for quality as well as the absence of insertion and deletions, and the frequency of every amino acid at each position in the mutagenized region is calculated, where the enrichment of mutants at each condition, relative to the starting library as well as relative to the WT codon can be obtained from this information (Use of deep sequencing to characterize mutant enrichment during screening: Paragraph 1). Under MPEP 2143, the combination of references may be relied upon to show obviousness when each teaches part of the claimed subject matter and there is a reason to combine them. A person of ordinary skill in the art would have recognized that applying the systematic DMS methods of Fowler to the protein analysis systems of Henley in view of the enrichment factors of Tripathi would yield predictable results, namely more comprehensive and effective mapping of binding residues.
Additionally, the Applicant’s focus on individual claim steps (i.e., incubating mutant proteins with binding molecules, separating unbound/bound phage, sequencing variants) is also found unpersuasive. Henley already discloses contacting variants with binding partners and analyzing retained or lost binding activity (Paragraphs 313, 318 and 562). Fowler further reinforces these techniques with explicit DMS protocols (Introduction: Paragraph 1; Protocol Overview and Experimental Design), which can be further enhanced by Tripathi’s enrichment methodology. As per MPEP 2144, it is not necessary for a reference to disclose the invention per ipissimis verbis, only to render the claimed subject matter obvious to one of ordinary skill.
The Applicant also argues that there is no reason to combine Henley with Fowler. However, Fowler expressly teaches that DMS enables systematic, high-throughput analysis of protein variants to identify functional residues, which would directly enhance Henley’s system for mapping binding residues. Per MPEP 2143(A), explains that if the combination of familiar elements according to known methods yields predictable results, obviousness is established. The predictable benefit of combining Fowler’s DMS methodology with Henley’s mutagenesis libraries is an expanded and efficient protein mapping system.
Therefore, the Applicant’s arguments are not found persuasive. Henley, in view of Fowler and Tripathi, teaches or suggests each of the limitations of claims 88-94, 98 and 100-101, 103-107, and one of ordinary skill in the art would have had a reasonable expectation of success in combining these teachings. Accordingly, the 35 USC 103 rejection of claims 88-94, 98 and 100-101, 103-107 is upheld.
Claim 99 is rejected under 35 U.S.C. 103 as being unpatentable over Henley et al. (WO 2018/081476 A2, published 5/3/2018), in view of Fowler et al. (“Measuring the activity of protein variants on a large scale using deep mutational scanning”, Nat Protoc., published 8/2014; from IDS 9/1/2021) and Tripathi et al. (“Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing”, Current Opinion in Structural Biology, published 2/2014), as applied to claims 88-98 and 100-107, and in further view of Acharya et al. (“Recognition of HIV-inactivating peptide triazoles by the recombinant soluble Env trimer, BG505 SOSIP.664”, Proteins, published 1/5/2017).
As discussed above, Henley and Fowler teach the use of DMS to create an unbiased library of protein variants (i.e., a short protein in a phage-display format) on a massive scale via appropriate selection system for the protein function of interest (Fowler: Abstract; Protocol Overview and Experimental Design). Further, Henley and Fowler teach that mutagenesis paradigms include targeted, systematic and random mutagenesis via virally-transformed or modified cells (Henley: Paragraph 563, lines 1-10; Fowler: Transforming and Selecting the Library: Paragraphs 1-2). Additionally, Tripathi teaches multiple methods currently exist for rapid construction and screening of single-site saturation mutagenesis (SSM) libraries in which every codon or nucleotide in a DNA fragment is individually randomized, where nucleotide sequences of each library member before and after screening or selection can be obtained through deep sequencing and the relative enrichment of each mutant at each position provides information on its contribution to protein activity or ligand-binding under the conditions of the screen (Abstract). Specifically, Tripathi teaches that reads are filtered for quality as well as the absence of insertion and deletions, and the frequency of every amino acid at each position in the mutagenized region is calculated, where the enrichment of mutants at each condition, relative to the starting library as well as relative to the WT codon can be obtained from this information (Use of deep sequencing to characterize mutant enrichment during screening: Paragraph 1).
Regarding claim 99, Henley teaches a kit or collection of parts for methods of producing a population of genetically modified cells using viral or non-viral vectors for the treatment of cancer (Abstract). Further, Henley teaches that the sequenced proteins of interest or population of modified cells is separated from the candidate binding molecule via activation of the plasma membrane of a eukaryotic cell, in the presence of its binding target, followed by expression where a target can also be soluble (i.e., not bound to a cell) (Paragraph 318, lines 1-5), followed by isolation from the PEG-precipitated or immunoprecipitated supernatant by low-speed centrifugation followed by CsCl gradient (Paragraph 268, lines 5-10). Henley also teaches that the bound and non-bound or isolated phage or population of modified cells then undergoes lysing and sequencing (Paragraph 562, lines 5-10) to identify a cancer-related target sequence, for example, a Neoantigen, from a sample obtained from a cancer patient using an in vitro assay (i.e., whole- exomic sequencing) (Figure 1; Paragraph 34, lines 1-5). Henley further teaches that following the previously described targeted sequencing of the phage library expressed via proteins of interest or modified cells, start sites of GUIDE-Seq reads mapped back to the genome enable localization of the double-strand break to within a few base pairs, divided into groups and then divided by the number of libraries to be pooled together for sequencing followed by mapping reads for the on- and off-target sites (Paragraph 563, lines 1-10).
Henley does not teach or suggest specified DMS proteins or proteins that are derived from BF520.W14.C2; BG505.W6M.C2.T332N; BG505 SOSIP Env trimer; BL035.W6M.ENV.C1;SF162; ZM109F.PB4; C2-94UG114; SIV/mac239; resurfaced Env core protein (RSC3); CD4- binding site defective mutant (RSC3 ∆3711); 2J9C-ZM53_V1V2; a 1FD6-Fc-ZM109_V1V2 scaffold peptide; a V3 consensus peptide of ConAl and ConB; MN gp41 monomer; ectodomain ZA.1197/MB; Q23; QA013.701.Env.H1; QA013.385M.Env.R3 677; QB850.73P.C14;QB850.632P.B10; Q461.D1; or QC406.F3.
Acharya teaches that human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS, and via host cell infection, the HIV-1 envelope (Env) trimeric complex of glycoproteins gp120 andgp41 mediates virus entry by interaction with host cell receptors (Introduction: Paragraph 1). Further, Acharya teaches that potent inhibitors are used to derive improved insights of how Env antagonists of the PT class are able to inactivate the virus upon Env protein binding (i.e., binding of PTs to a trimeric Env constructBG505 SOSIP.664.gp140 (SOSIP), a stabilized HIV-1 Envtrimeric derivative) (Introduction: Paragraphs 2-3). Acharya also teaches that through specific and high affinity-binding (Figures 2 and 3), structural models of the Envbinding mode were developed, thus allowing flexible movement of the W112 side-chain on the gp140 protomer extracted from the crystal structure of BG505 SOSIP.664 to further investigate an additional potential target for gp-120 binding ligand-associated behaviors (Flexible Docking: Paragraph 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the DMS phage library system of Henley, Fowler and Tripathi to incorporate the specific HIV envelope constructs taught by Acharya (i.e., BG505. SOSIP Env Trimer). The motivation to do so would have been to apply the powerful DMS methodology to clinically relevant HIV envelope proteins that are known targets for therapeutic development, as explicitly taught by Acharya. A person of ordinary skill would recognize that applying DMS techniques to these specific HIV envelope variants would provide valuable insights into binding restudies that could inform vaccine or therapeutic design. Further, a person of ordinary skill would have had a reasonable expectation of success in combining these references because Henley, Tripathi and Fowler establish the methodology for protein residue mapping using phage display and DMS and enrichment factors, while Acharya demonstrates that the specific HIV envelope constructs were already well-characterized and available for experimental use. Thus, this combination (DMS library/kit for protein analysis from Fowler, Henley and Tripathi) to known proteins (HIV envelopes from Acharya) using established methods, in the same field of protein analysis, would yield predictable results in identifying critical binding residues in these HIV envelope variants for clear applications in developing therapeutics and vaccines.
Applicant’s Response: The Applicant argues that the combination of references, Henley in view of Fowler, do not teach or suggest all the claimed elements, particularly the use of deep mutational scanning (DMS) proteins in phage libraries and the required separation and sequencing steps. The Applicant also argues that Henley does not disclose incubating DMS proteins with binding molecules, separating bound from unbound phage, or sequencing to determine binding residues. Finally, the Applicant asserts that Acharya does not remedy the deficiencies listed above of Henley and Fowler, and therefore would not reasonably lead to the claimed invention.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are not found persuasive, as discussed below.
As previously discussed, Henley teaches the generation and use of mutant protein libraries for mapping functional residues (Paragraphs 28, 315, 336, 365), while Fowler teaches systematic DMS methods and high-throughput sequencing approaches for identifying residue-level contributions to protein function (Transforming and Selecting the Library: Paragraphs 1-2; Designing and creating a diversity library: Paragraph 1). Additionally, new reference, Tripathi teaches multiple methods currently exist for rapid construction and screening of single-site saturation mutagenesis (SSM) libraries in which every codon or nucleotide in a DNA fragment is individually randomized, where nucleotide sequences of each library member before and after screening or selection can be obtained through deep sequencing and the relative enrichment of each mutant at each position provides information on its contribution to protein activity or ligand-binding under the conditions of the screen (Abstract). Specifically, Tripathi teaches that reads are filtered for quality as well as the absence of insertion and deletions, and the frequency of every amino acid at each position in the mutagenized region is calculated, where the enrichment of mutants at each condition, relative to the starting library as well as relative to the WT codon can be obtained from this information (Use of deep sequencing to characterize mutant enrichment during screening: Paragraph 1). Acharya further teaches methods of analyzing variant proteins in the context of residue mapping, including high-throughput analysis of protein stability and function (Figures 2 and 3; Introduction: Paragraphs 2-3). Acharya’s teachings of experimentally measuring functional consequences of amino acid substitution (Flexible Docking: Paragraph 2) reinforces the combination of Henley and Fowler by providing an established protocol for applying mutational scanning to protein mapping.
Under MPEP 2143, a rejection under 35 USC 103 may be based on a combination of references where each reference teaches a portion of the claimed subject matter and a reason exists to combine them. One of ordinary skill in the art would have been motivated to apply Acharya’s detailed protein variant analysis techniques to the combined teachings of Henley, Fowler and Tripathi to further enhance residue mapping accuracy. The combination of reference represents the predictable use of prior art elements according to their established functions, yielding no more than predictable results, consistent with KSR Int’l Co. v. Teleflex Inc., 550 US 398, 416 (2007).
Therefore, the Applicant’s arguments are not found persuasive. Henley, Fowler, Tripathi and Acharya collectively teach or suggest the subject matter of instant claim 99, and one of ordinary skill in the art would have had a reasonable expectation of success in combining these teachings. Accordingly, the rejection of claim 99 under 35 USC 103 is upheld.
Conclusion
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684