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 .
Response to Amendment
The Amendment filed 10/21/2025 has been entered. Claims 1-62 have been cancelled and new claims 63-66, 68-87 have been added and are examined herein. Claim 67 is missing from the newly added claim set and therefore Applicant should cancel this claim in the next response.
Status of Objections and Rejections
The objection to the drawings has been withdrawn in view of Applicant's amendment.
The objection to the claims is obviated by Applicant's cancellation.
All rejections from the previous office action are obviated by Applicant's cancellation.
New grounds of claim objection are necessitated by the amendments.
New grounds of rejection under 35 U.S.C. 112 (b) are necessitated by the amendments.
New grounds of rejection under 35 U.S.C. 101 are necessitated by the amendments.
New grounds of rejection under 35 U.S.C. 103 are necessitated by the amendments.
Response to Arguments
Applicant's arguments, see pages 7-12, filed 10/21/2025, with respect to the rejections of claims 1-3, 6-10, 13, 14, 17, 18, 21, 25, 26, 31-36, 42, 49-51, 53, and 54, under 35 U.S.C. 101 and 35 U.S.C. 103, have been fully considered but they are not persuasive.
Applicant’s arguments (p. 10) with respect to claim(s) 63-66, 68-87 toward the amendment of “target antigen” and not just “target” have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
In regard to the rejection under 35 U.S.C. 101, Applicant argues (pp. 8-9) that amended claims 63-87 are not directed to an abstract idea or mental process because the claims recite a specific, laboratory-based method involving concrete biochemical steps that cannot practically be performed in the human mind. In particular, the claims require applying stress to a biological sample, forming antigen-binding protein-antigen complexes, separating bound and unbound fractions by size exclusion chromatography (SEC), digesting proteins, performing LC-MS peptide mapping, quantifying structural abundance, and calculating a ratio to determine whether a structure negatively affects binding. These steps require physical manipulation of samples and the use of specialized analytical instrumentation, and therefore all fall outside the scope of a mental process. Applicant points out that “comparing” is not a term used in either the rejected or amended claims although the Office Action alleges that a comparison is made between the abundances of unbound and bound fractions to determine if the structure negatively affects the interaction between the therapeutic protein and the target and, therefore, constitutes an abstract idea. Even if the ratio calculation were viewed as involving a comparison, the claims integrate that calculation into a specific biochemical workflow requiring SEC separation and LC-MS peptide mapping of physical samples. The ratio is calculated from measured abundances obtained through defined laboratory techniques and is used to determine structural attributes of therapeutic antigen-binding proteins that negatively affect binding to target antigens. Accordingly, the alleged abstract idea is meaningfully applied within a technological process and is integrated into a practical application under Step 2A, Prong Two. The Applicant argues, overall, that this integrated analytical workflow enhances the field of biochemical analysis of antigen-binding proteins by improving the accuracy and physiological relevance of detecting such structural changes compared to conventional methods.
The Examiner respectfully disagrees.
As stated in the rejection below, using a mathematical calculation (a ratio) and a correlative determination or comparing (if >1) to determine if a structure negatively affects binding is a judicial exception through the abstract ideas of mental processing, mathematical calculations, and natural correlations. Although the SEC and LC-MS instrumentation in the claims execute the separating, identifying and quantifying steps of independent claims 63 and 76, the ratio of structural abundances can still be calculated through the human mind using data gathered from routine laboratory techniques in sample manipulation using that instrumentation. The active data gathering steps (e.g. (a)-(e) in claim 63) are not the abstract idea, but rather steps used to provide information to ultimately apply the abstract idea. The Applicant argues that the claims do not recite “comparing” and therefore do not recite a mental process. However, the rejection of claims under 35 U.S.C. 101 in the prior Office Action was not based on the presence of specific terminology, but rather on the substance of the claimed limitations. As previously explained, the claims require evaluating quantified abundances in bound and unbound fractions and determining whether one value exceeds another to infer a negative effect on interaction. The absence of the word “compare” does not alter the fact that the claimed conditional relationship embodies an evaluative determination based on relative measured values. The requirement that the ratio be “greater than 1” establishes a threshold-based evaluative criterion that determines whether a structural feature negatively affects binding. The amended claim’s calculation of a ratio <1 embodies a comparative analysis that assesses the relationship between two data sets and correlates that relationship to a binding outcome (negatively affecting binding). The absence of the word “comparing” does not alter the underlying analytical operation, as the ratio calculation compares the abundances to reach the stated conclusion regarding binding impact.
In order to transform this abstract idea into a practical application, MPEP 2106.05(a) explains that an improvement must be made to the functioning of a computer or other technology or a technical improvement in a technological field, not merely a better result from applying an abstract idea. If the claim only improves the accuracy of a mathematical correlation (a structure negatively affects binding if a ratio is >1) but does not improve the underlying technology (e.g., LC-MS, SEC instrumentation) then it does not qualify as a technological improvement under 2106.05(a). The claims recite other process steps of applying a stress to the sample, creating a mixture with another sample, separating the mixture, and identifying and quantifying a structure within fractions of the separation step. These steps alone are insufficient to transform the claim into a practical application and are merely data gathering. None of these steps alter the therapeutic protein for therapeutic use, manufactures a new composition, or improves the separation technology itself. Instead, each step is performed to obtain data about structural attributes and their relative abundances. The claimed workflow culminates in calculating a ratio (or evaluating whether one abundance excess another) to determine whether a structure negatively affects binding, an abstract idea. Thus, the physical steps serve as preparatory and information collecting steps that feed into an evaluative determination based on mathematical comparison.
Under Step 2B of Alice/Mayo framework, the additional elements beyond calculating a ratio represent well-understood, routine, and conventional techniques in the art.
The additional claimed steps of independent claims 63 and 76 state: applying a stress to a sample, separating antigen-binding protein-antigen complexes into bound and unbound fractions via SEC, subjecting the fractions to digestion, and analyzing them via LC-MS constitute well-understood, routine, and conventional techniques in the art.
Reference Nowak (“Forced degradation of recombinant monoclonal antibodies: A practical guide”; 2017) explains that applying a stress to sample of antigen-binding proteins (monoclonal antibodies) through forced oxidation is commonly used in the art to assess a change in the residue of an amino acid sequence that may affect binding and potency (p. 1217, col. 1, para, 1)(p. 1220, col. 2, Oxidation)(Paragraph [0042] of the instant specification (US 20220260584 A1) also admits that stresses such as “Enzymatic modifications are known in the art”).
Paragraphs [0061] and [0068] of the instant specification establish that the use of SEC and LC-MS/MS peptide mapping protocols (including reduction, alkylation, and trypsin digestion) is conventional, admitting that “Suitable methods and techniques for separating mixtures into fractions are known in the art. See, e.g….Size Exclusion Chromatography,” and “Protocols for LC-MS peptide mapping are known in the art”. Applicant cites reference Mouchahoir et al. “Development of an LC-MS/MS peptide mapping protocol for the NISTmAb”; 2017). Likewise, reference Pollastrini states that “nearly five decades ago an equilibrium SEC method was introduced for measuring weak protein–protein interactions…this method has been successfully applied for measuring the stoichiometries of IgG and FcRn molecules from several species” (p. 90, col. 2, para. 2, ll. 6-14).
Finally, structural immunology literature confirms that quantification of structural changes in amino acid residues of antibodies are conventionally studied in both free and antigen-bound states, reinforcing that distinguishing and analyzing these states is standard in the field (Sela-Culang et al., “A Systematic Comparison of Free and Bound Antibodies Reveals Binding-Related Conformational Changes”; 2012)(p. 4893, col. 2, para. 5). Taken together, the cited art and the specification’s own admissions establish that independent claims 63 and 76 do not include additional elements that are sufficient to amount to significantly more than the judicial exception.
Applicant argues (pp. 11-12) that the claimed methods provide unpredictable technical advantages not suggested by the prior art. Specifically, Applicant contends that the application demonstrates for the first time that structural attributes at particular amino acid positions affecting antigen binding can be identified by fractionating bound and unbound molecules. Applicant further asserts that the method enables high-throughput detection of multiple modification types (e.g., oxidation, isomerization, deamidation), that the identified positions correlate with crystal structure data, that the approach is broadly applicable across antibody formats, and that it addresses a long-felt need in therapeutic antibody development in a cost and time-efficient manner.
The Examiner respectfully disagrees.
The asserted advantages relate to the results obtained from applying known biochemical techniques: stress induction, fractionation, peptide mapping, and quantitative analysis to identify structures that impact binding. However, the claims are evaluated based on the recited steps, not on alleged novelty of results or experimental confirmation. The claimed data gathering steps listed above were well-established in the art, as evidenced by the cited references. The fact that the application demonstrates correlation with crystal structures or applicability across formats reflects expected outcomes of applying conventional analytical workflows to protein characterization. Such empirical confirmation does not convert routine laboratory techniques into an inventive concept under 35 U.S.C. 101. Moreover, assertions of high throughput, efficiency, or long-felt need are not recited in the claims and therefore cannot supply significantly more. The claims do not recite any technological improvement to separation, detection, or structural analysis methods; rather, they employ known techniques to gather and analyze data. Accordingly, the alleged technical advantages do not alter the 35 U.S.C. 101 analysis, as the claimed subject matter remains directed to an abstract evaluative concept implemented with routine and conventional laboratory steps.
In regard to the rejection under 35 U.S.C. 103, Applicant argues (p. 11) that Pollastrini does not teach or suggest using SEC to analyze antigen-binding protein complexes because SEC failed to detect binding of full-length IgG to FcRn and Pollastrini criticized equilibrium SEC (Hummel-Dreyer) as having practical limitations. Applicant contends that Pollastrini instead favors AF4 as the appropriate method for studying weak binding interactions and therefore teaches away from SEC. Accordingly, Applicant asserts that Pollastrini, alone or in combination with Pan, does not suggest the claimed method of identifying structures that affect antigen-binding protein interactions.
The Examiner respectfully disagrees.
While certain practical limitations are noted (e.g., diffusion, ligand excess, experimental complexity; See p. 90, col. 2, para. 2), identifying disadvantages of a known technique does not amount to teaching away. A reference teaches away only when it discourages or discredits the claimed approach, not when it merely discusses trade-offs. Pollastrini identified transient dissociation during conventional SEC (Emphasis added) as the reason full-length IgG/FcRn complexes were not detected and acknowledges equilibrium SEC (Hummel-Dreyer chromatography)(Emphasis added) as a known method as a way “to avoid this problem” (See p. 90, col. 2, para. 2). Pollastrini does not state that equilibrium SEC is unsuitable (Emphasis added); rather, it recognizes it as an established method (Hummel-Dreyer chromatography)(See p. 90, col. 2, para. 2 It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have applied equilibrium SEC conditions to detect full-length IgG/FcRn complexes. This represents the predictable substitution of a known SEC variant (equilibrium) for conventional SEC to address a recognized problem (See MPEP 2143(I)(B)), the use of a known technique to improve a similar analytical method for weak protein-protein interactions (See MPEP 2143(I)(C)), and an obvious-to-try approach where the art identifies a specific problem and a finite number of predictable solutions with a reasonable expectation of success (See MPEP 2143(I)(E). Selection of AF4 as an orthogonal or “softer” method does not exclude the obviousness of using equilibrium SEC (p. 90, col. 2, para. 3). Both AF4 and SEC are known size- based separation techniques for resolving protein complexes under native conditions. Accordingly, modifying Pollastrini’s SEC analysis to employ equilibrium conditions for detecting full-length IgG/FcRn binding would have been an obvious analytical refinement rather than a departure from the teachings of the reference.
Drawings
The drawings were received on 10/21/2025. These drawings are Figs. 1B, 5A-5B, 8A-8C, 10A, 11A-11D, and 14. These drawings are acceptable.
Claim Objections
Claims 63-66, 68-57 are objected to because of the following informalities:
Claim 63, ll. 8-9 states “separating the mixture into (i) an unbound fraction comprising unbound antigen-binding proteins and target antigens,” (Emphasis added); however, the abstract of the instant publication (US 20220260584 A1) gives the option of the unbound fraction comprising either the antigen-binding proteins (therapeutic proteins) or the target antigens (unbound targets) stating “an unbound fraction comprises unbound therapeutic proteins or unbound targets” (Emphasis added). The Applicant may correct the claim by changing “and” to “or” to read “separating the mixture into (i) an unbound fraction comprising unbound antigen-binding proteins or target antigens”. The Examiner interprets the claim to read as the latter as examined below.
Claims 64-66, 68-75 are objected to upon dependency of objected claim 63.
Claim 76, ll. 8-9 states “separating the mixture into (i) an unbound fraction comprising unbound antigen-binding proteins and target antigens,” however, the abstract of the instant publication (US 20220260584 A1) gives the option of the unbound fraction comprising either the antigen-binding proteins (therapeutic proteins) or the target antigens (unbound targets) stating “an unbound fraction comprises unbound therapeutic proteins or unbound targets” (Emphasis added). The Applicant may correct the claim by changing “and” to “or” to read “separating the mixture into (i) an unbound fraction comprising unbound antigen-binding proteins or target antigens”. The Examiner interprets the claim to read as the latter as examined below.
Claims 77-87 are objected to upon dependency of objected claim 76.
Claim 66 claims dependency upon claim 5. However, claim 5 has been cancelled. Claim 66 will be examined with dependency upon independent claim 63.
Claim 64, ll. 1-2 states “the structure is comprised by a paratope”. The phrase “comprised by” is objected to as informal and non-standard claim terminology. Applicant may correct the claim to read “the structure is part of a paratope” in order to clarify the intended scope of the claim.
Claim 77, ll. 1-2 states “the structure is comprised by a paratope”. The phrase “comprised by” is objected to as informal and non-standard claim terminology. Applicant may correct the claim to read “the structure is part of a paratope” in order to clarify the intended scope of the claim.
Appropriate correction is required.
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 68 and 80 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.
Regarding claim 68, independent claim 63, line 17 recites “f. calculating a ratio”. However, claim 68 further recites “repeating (a)- (f), calculating a plurality of ratios, and determining the statistical significance of the plurality of ratios,” thereby reintroducing the step of calculating a ratio without clearly specifying whether the plurality of ratios refers to: (i) multiple repetitions of the same ratio calculation of step (f) created from the “repeating” step, (ii) different ratios than that recited in step (f), or (iii) an additional, distinct ratio calculated beyond step (f). Applicant may correct claim 68 to read “wherein the method comprises repeating (a)- (e), to obtain a plurality of ratios calculated in step (f), and determining the statistical significance of the plurality of ratios based on calculation of a p-value”. The Examiner interprets the plurality of ratios to refer to: (i) multiple repetitions of the same ratio calculation of step (f) created from the “repeating” step).
Regarding claim 80, independent claim 76 line 16 recites “e. calculating a ratio”. However, claim 68 further recites “repeating (a)- (f), calculating a plurality of ratios, and determining the statistical significance of the plurality of ratios,” thereby reintroducing the step of calculating a ratio without clearly specifying whether the plurality of ratios refers to: (i) multiple repetitions of the same ratio calculation of step (f) created from the “repeating” step, (ii) different ratios than that recited in step (f), or (iii) an additional, distinct ratio calculated beyond step (f). Applicant may correct claim 68 to read “wherein the method comprises repeating (a)- (e), to obtain a plurality of ratios calculated in step (f), and determining the statistical significance of the plurality of ratios based on calculation of a p-value”. The Examiner interprets the plurality of ratios to refer to: (i) multiple repetitions of the same ratio calculation of step (f) created from the “repeating” step).
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 63-66, 68-87 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (Step 2A/1)(i.e., a law of nature, a natural phenomenon, or an abstract idea) without practical application (Step 2A/2) or significantly more (Step 2B) (See MPEP 2106). Although the instant claims encompass a process (Step 1), they are directed to the following abstract ideas through mental processes and mathematical calculations (Step 2, Prong 1):
Claims 63 and 76 recite “calculating a ratio of the abundance of the structure in the unbound fraction to the abundance of the structure in the bound fraction that is greater than 1, indicating that the structure negatively affects the interaction between the antigen-binding protein and the target antigen”.
Using a mathematical calculation (a ratio) and a correlative determination or comparing (if >1) to determine if a structure negatively affects binding is a judicial exception through the abstract ideas of mental processing, mathematical calculations, and natural correlations. Although the SEC and LC-MS instrumentation in the claims execute the separating, identifying and quantifying steps of independent claims 63 and 76, the ratio of structural abundances can still be calculated through the human mind using data gathered from routine laboratory techniques in sample manipulation using that instrumentation. Calculating a ratio of structure abundance between the unbound and bound fractions and determining if the structure negatively affects the interaction between the therapeutic protein and the target encompasses the user mentally analyzing one value against another to draw a conclusion. This can be performed in the mind using a mathematical equation of a variable less than or greater than a threshold of 1 which categorizes this recitation as an abstract idea through mathematical calculations and mental processes. There is no active step in the “indicating” clause, such as determining or comparing (although “determining” is present within the preamble of claim 76); however, the statement is reflective of an abstract idea through passive conclusion.
Claims 68 and 80 recite: “calculating a plurality of ratios, and determining the statistical significance of the plurality of ratios based on calculation of a p-value”.
The recited steps introduce further mathematical calculations through computing multiple ratios and applying statistical analysis (e.g., p-value determination) which fall within the categories of mathematical concepts and mental processes. The claims ultimately determine significance which constitutes an abstract idea.
Step 2A/2: This judicial exceptions are not integrated into a practical application.
After calculating ratios and statistical significance of the results and concluding if a structure negatively affects the interaction between the therapeutic protein and the target, there is no further action. The claims recite other process steps of applying a stress to the sample, creating a mixture with another sample, separating the mixture, and identifying and quantifying a structure within fractions of the separation step. These steps alone are insufficient to transform the claim into a practical application and are merely data gathering. Although the identifying and quantifying step cannot be performed in the mind, since these are microscopic structures that will require the use of instrumentation, this step only enables the data to be gathered in order to draw the conclusion of the effect of a structure through use of a mathematical calculation that can be performed by hand or in the mind. None of these steps alter the therapeutic protein for therapeutic use, manufactures a new composition, or improves the separation technology itself. Instead, each step is performed to obtain data about structural attributes and their relative abundances. The claimed workflow culminates in calculating a ratio (or evaluating whether one abundance excess another) to determine whether a structure negatively affects binding, an abstract idea. Thus, the physical steps serve as preparatory and information collecting steps that feed into an evaluative determination based on mathematical comparison. There is no further practical application after this conclusion is drawn and therefore fails to satisfy Step 2A/2.
Step 2B: The claims do not include additional elements that are sufficient to
amount to significantly more than the judicial exception.
The additional claimed steps of independent claims 63 and 76 state: applying a stress to a sample, separating antigen-binding protein-antigen complexes into bound and unbound fractions via SEC, subjecting the fractions to digestion, and analyzing them via LC-MS constitute well-understood, routine, and conventional techniques in the art.
Reference Nowak (“Forced degradation of recombinant monoclonal antibodies: A practical guide”; 2017) explains that applying a stress to sample of antigen-binding proteins (monoclonal antibodies) through forced oxidation is commonly used in the art to assess a change in the residue of an amino acid sequence that may affect binding and potency (p. 1217, col. 1, para, 1)(p. 1220, col. 2, Oxidation)(Paragraph [0042] of the instant specification (US 20220260584 A1) also admits that stresses such as “Enzymatic modifications are known in the art”).
Paragraphs [0061] and [0068] of the instant specification establish that the use of SEC and LC-MS/MS peptide mapping protocols (including reduction, alkylation, and trypsin digestion) is conventional, admitting that “Suitable methods and techniques for separating mixtures into fractions are known in the art. See, e.g….Size Exclusion Chromatography,” and “Protocols for LC-MS peptide mapping are known in the art”. Applicant cites reference Mouchahoir et al. “Development of an LC-MS/MS peptide mapping protocol for the NISTmAb”; 2017). Likewise, reference Pollastrini states that “nearly five decades ago an equilibrium SEC method was introduced for measuring weak protein–protein interactions…this method has been successfully applied for measuring the stoichiometries of IgG and FcRn molecules from several species” (p. 90, col. 2, para. 2, ll. 6-14).
Finally, structural immunology literature confirms that quantification of structural changes in amino acid residues of antibodies are conventionally studied in both free and antigen-bound states, reinforcing that distinguishing and analyzing these states is standard in the field (Sela-Culang et al., “A Systematic Comparison of Free and Bound Antibodies Reveals Binding-Related Conformational Changes”; 2012)(p. 4893, col. 2, para. 5). Taken together, the cited art and the specification’s own admissions establish that independent claims 63 and 76 do not include additional elements that are sufficient to amount to significantly more than the judicial exception.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
Claims 63, 65-66, 68-76, 78--87 U.S.C. 103 are rejected under 35 U.S.C. 103 as being unpatentable over Pollastrini et al. (“Field flow fractionation for assessing neonatal Fc receptor and Fcϒ receptor binding to monoclonal antibodies in solution”; 2011), in view of Houde et al. (“Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding” and Supplementary Data; 2010) and in further view of Pan et al. (“Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn1”; 2008).
Regarding claim 63, Pollastrini teaches a method of identifying structures (modifications; p. 94, col. 2, para. 2, l. 1) of an antigen-binding protein (IgG; p. 94, col. 2, para. 2, l. 1) that affect an interaction between the antigen-binding protein and a target (FcRn; p. 94, col. 2, para. 2, l. 2)(To understand the effect of specific modifications of IgG on the binding affinity with FcRn,," wherein IgG is comprised of “two variable antigen-binding (Fab) regions and the constant (Fc) region,” and is therefore an antigen-binding protein; p. 94, col. 2, para. 2, ll. 1-2; p. 88, col. 1, para. 1, ll. 7-10; Table 2)(The examiner interprets an attribute to be a type of structure/modification with examples given in Table A of the instant publication US 20220260584 A1 and will therefore be used interchangeably through the office action; See also paragraphs [0039] and [0078]), said method comprising:
a. applying a stress (the degradation of an IgG2 was accelerated by treatment with UV light (see Materials and Methods) to increase oxidation of amino acids; p. 94, col. 2, para. 2, ll. 2-4) to a first sample (The oxidized sample; p. 95, col. 1, l. 3) comprising antigen-binding proteins (IgG molecules; p. 95, col. 1, l. 2)
b. contacting the first sample with a second sample comprising the target to form a mixture (“The oxidized sample and the control material were incubated with FcRn,” wherein the FcRn is the second sample; p. 95, col. 1, ll. 3-4) comprising
(i) antigen-binding protein-target complexes (IgG/FcRn complex; p. 95, col. 1, l. 8; Green peak around 22 min. in Fig. 10)
(ii) unbound antigen-binding proteins (Pollastrini states there to be a decrease in binding ability between the oxidized IgG and FcRn, therefore, there would naturally exist unbound IgG at equilibrium; See ; p. 95, col. 1, ll. 11-13), and
(iii) unbound target (unbound FcRn; p. 95, col. 1, l. 10; Green peak around 14 min. in Fig. 10);
c. separating the mixture into (i) an unbound fraction comprising unbound antigen-binding proteins and [sic] targets and (ii) a bound fraction comprising antigen-binding protein-target complexes (Pollastrini explains that “the retention time of the IgG/FcRn complex, for the oxidized sample, was shifted by approximately 1 min earlier relative to the control sample. In addition, the amount of unbound FcRn was increased (_8%) in the oxidized sample,” thereby demonstrating separation of all components into fractions via asymmetrical flow field flow fractionation (AF4) as shown in the spectrum in Fig. 10; p. 95, col. 1, ll. 7-11)(The Examiner interprets the unbound fraction to have the option of being comprised of either the antigen-binding proteins or targets. See objection above).
Pollastrini fails to teach steps (d)-(f) and SEC analysis in step (c) as well as an interaction between the antigen-binding protein and a target antigen (Emphasis added), and instead teaches the interaction is between the (Fc) region of the antigen-binding protein IgG and an FcRn receptor. However, Pollastrini does teach that IgG comprises two variable antigen-binding (Fab) regions responsible for specificity toward a target antigen (p. 88, col. 1, ll. 7-10) and separation via AF4 (See p. 94, col. 2, para. 2).
Houde teaches an interaction between the antigen-binding protein and a target antigen (The IgG1 analyzed here binds to a membrane receptor (antigen) on the surface of lymphoma cells; p. 1717, col. 2, Cell-based Apoptosis Assay, ll. 1-2).
Houde is considered to be analogous to the claimed invention because it is in the same field of endeavor for identifying attributes of antigen-binding proteins. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have incorporated the antigen-binding assessment described by Houde into the IgG characterization framework of Pollastrini in order to ensure that structural modifications, including methionine oxidation, do not detrimentally affect antigen-binding functionality. Pollastrini is directed to identifying structural modifications, such as methionine oxidation, that affect the binding affinity between FcRn and the Fc region of IgG (p. 96, col. 2, last para. ll. 1-5; Table 2)(p. 88, col.1, para. 1; p. 90, col. 1, last para., ll. 1-3) as a way to assess mAb product quality attributes (Abstract, last 2 ll.). However, Pollastrini does not disclose evaluating whether these structural modifications affect antigen-binding activity of IgG to a target antigen (Emphasis added). Even so, Pollastrini does acknowledge that IgG comprises variable antigen-binding (Fab) regions responsible for specificity toward a target antigen interactions (p. 88, col.1, para. 1, ll. 7-10).
Reference Houde likewise concerns assessment of mAb IgG product quality attributes (p. 1717, para. 2, ll. 4-7) and specifically evaluates the impact of post-translational modifications (PTMs), including methionine oxidation within the Fc region, on antibody functionality (p. 1717, para. 2, ll. 1-8). Houde explains that “to ensure such Fc-region modifications did not cripple the antigen binding activity and render the antibody completely non-functional, a cell-based apoptosis induction assay was utilized” to confirm antigen binding to a cell-surface antigen (p. 1718, last para. – p. 1719, first para.). Because both references address the same class of antibodies (IgG), the same type of structural modifications (methionine oxidation), and the same objective of assessing mAb product quality attributes, incorporating antigen-binding testing into Pollastrini’s quality attribute evaluation would have yielded the predictable result of confirming whether such modifications impact antigen recognition (See MPEP 2143(I)(A)).
Modified Pollastrini fails to teach steps (d)-(f) and SEC analysis in step (c) but does teach separation via AF4 (See p. 94, col. 2, para. 2).
In regard to step (c), Pollastrini teaches analysis of Fc/FcRn and IgG/FcRn interactions using both SEC and AF4. Specifically, Pollastrini performed SEC analyses of Fc/FcRn and full-length IgG/FcRn mixtures, demonstrating that SEC successfully differentiated Fc from FcRn and resolved the Fc/FcRn complex, with integrated peak areas indicating a 1:1 stoichiometry (p. 90, cols, 1-2). However, when full-length IgG1 and IgG2 were analyzed by SEC, no apparent IgG/FcRn binding was detected, which Pollastrini attributes to transient dissociation of weakly bound complexes during SEC due to on-column dilution and matrix interactions. Pollastrini further acknowledges that equilibrium SEC (Hummel-Dreyer chromatography) was previously developed to remedy the problem of weak protein-protein interactions, including IgG/FcRn stoichiometry (p. 90, col. 2, para. 2), thereby recognizing SEC as a suitable size-based technique when performed under equilibrium conditions. Pollastrini ultimately deployed AF4 as an orthogonal “softer” method to preserve weak complexes (p. 90, col. 2, para. 3). Given that SEC was already demonstrated to separate Fc/FcRn complexes and was recognized as adaptable to weak binding through equilibrium methods, It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted equilibrium SEC for conventional SEC to separate bound and unbound antigen binding protein-target complexes (IgG/FcRn) in oxidized (stressed) samples, as this represents the predictable substitution of a known SEC variant (equilibrium) for conventional SEC to address a recognized problem (See MPEP 2143(I)(B)), the use of a known technique to improve a similar analytical method for weak protein-protein interactions (See MPEP 2143(I)(C)), and an obvious-to-try approach where the art identifies a specific problem and a finite number of predictable solutions with a reasonable expectation of success (See MPEP 2143(I)(E).
Modified Pollastrini fails to teach steps (d)-(f).
In regard to steps (d-e), Pollastrini teaches performing tryptic digestion followed by RP-HPLC-MS to characterize antibody modifications of a stressed IgG sample for peptide mapping as shown in Table 2 (p. 90, col. 1, para. 2)(p. 94, col. 2, para. 2, ll. 4-6). This sample is later mixed with FcRn and then separated by asymmetrical flow field flow fractionation (AF4) into bound IgG/FcRn complexes and unbound FcRn and IgG (p. 95, col. 1, para. 1; Fig. 10). However, digestion and RP-HPLC-MS is not mentioned as being performed for these bound and unbound fractions. After AF4 analysis of the bound and unbound fractions, Pollastrini concludes that “These results, along with the data presented in Table 2, provide a strong correlation between specific amino acid modifications and the decrease in overall binding of FcRn to the degraded IgG sample” (p. 95, col. 1, para. 1, last 4 ll). Because Pollastrini already employs proteolytic digestion and RP-HPLC-MS as a tool for structural characterization of the stressed IgG sample and separately teaches fraction collection and analysis after AF4 separation, It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the separation method taught by Pollastrini with the addition of protein digestion in the bound and unbound fractions separated by AF4/SEC followed by RP-HPLC–MS so as to compare structural differences between interacting and non-interacting species and to assess its impact on binding under the same analysis conditions which represents a predictably analytical extension of the quantitative methods already disclosed (See MPEP 2143(I)(A)).
Modified Pollastrini fails to teach:
f. calculating a ratio of the abundance of the structure in the unbound fraction to the abundance of the structure in the bound fraction that is greater than 1, indicating that the structure negatively affects the interaction between the antigen-binding protein and the target antigen (the italicized portion does not further limit step d. and is merely an intended result of step d. and is therefore not given patentable weight (Minton v. Nat' l Ass' n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003) See MPEP 2111.04).
Modified Pollastrini does mention that the binding affinity Kd between IgG and FcRn reinforces the conclusion that there is “a strong correlation between specific amino acid modifications and the decrease in overall binding of FcRn to the degraded IgG sample,” based on the bound and unbound results of Fig. 10 and data from Table 2; page 95, column 1, paragraph 1, last 3 lines; p. 95, col. 2, ll. 13-18)
A goal of reference, Pan, it to “quantitatively assess the impact of Fc methionine oxidation on Protein A binding” (page 428, column 1, last 3 lines; See Table I with each methionine and %). Pan teaches that oxidation of specific Fc methionine residues (Met252 and Met428) decreases binding affinity to FcRn and Protein A, and quantifies this reduction through measurable differences in binding constants (Kd and EC50) derived from relative amounts of bound versus unbound species (p. 429, last 3 ll.-p. 430, para.1). Pan specifically states that “Full oxidation of Met 252 and Met 428 resulted in about two-fold increase in the association rate and 10-fold increase in the dissociation rate of the antibody and Protein A, which led to a 4.2-fold increase in the dissociation equilibrium constant (KD),” and therefore “oxidation of Met 252 and Met 428 reduces the affinity of the antibody with Protein A" (page 428, paragraph 1, last 6 lines; page 428, column 2, paragraph 1, lines 32-33; See binding constants in Table 2 wherein the unbound and bound fractions are, mathematically, a rearrangement of the dissociation equilibrium constant). Pollastrini likewise states that “These data suggest that the degraded sample had decreased binding ability to FcRn, likely due to the increase in oxidized methionine residues, consistent with previous results [42],” wherein reference [42] is Pan (Pollastrini, page 95, column 1, paragraph 1, lines 10-13). It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the mab product assessment method taught by Pollastrini in view of Houde with the addition of calculating a ratio of the abundance of a modified structure in the bound fraction relative to its abundance in the bound fraction, where a ratio greater than 1 indicates reduced binding because Pollastrini already teaches quantifying relative bound versus unbound species to assess binding strength and Pan teaches that specific structural modifications decrease binding affinity in a quantifiable manner. Expressing the effect as a ratio greater than 1 is merely a routine mathematical expression of the already measure quantities and represents a predictable way to numerically characterize reduced binding based on relative abundance.(See MPEP 2143(I)(A)).
Regarding claim 65, Modified Pollastrini teaches the method of claim 63, wherein the first sample comprises one or more species of the antigen-binding protein (IgG2; Pollastrini, p. 94, col. 2, para. 2, l. 2)(Paragraph [0009] of the instant publication US 20220260584 A1 explains “Each therapeutic protein having a different profile of attributes is considered a species”).
Regarding claim 66, Modified Pollastrini teaches the method of claim 5 [sic] (Claim 66 will be examined within dependence upon claim 63. See objection above), wherein the first sample comprises serum or a serum fraction (“mixtures at different ratios of FcRn, HSA, and/or IgG were prepared and analyzed by AF4,” wherein HSA is human serum albumin; Pollastrini, p. 95, col.1-2).
Regarding claim 68, Modified Pollastrini teaches the method of claim 63, wherein the method comprises repeating (a)- (e), calculating a plurality of ratios, (Changes in these regions were reproducible in replicate experiments, and overlapping peptides displayed similar behavior, giving us high confidence that the changes observed were real and significant; Houde, p. 1723, col. 2, ll. 17-20) and determining the statistical significance of the plurality of ratios based on calculation of a p-value (See association rate, dissociate rates and equilibrium constants of Table II’s binding experiment with 95% confidence intervals shown in the brackets of Pan which corresponds to a p-value of 0.05)(Calculation of a confidence interval naturally requires data from repetitive steps of the experiment)(The Examiner interprets the plurality of ratios to refer to: (i) multiple repetitions of the same ratio calculation of step (e))(See 112(b) rejection above).
Regarding claim 69, Modified Pollastrini teaches the method of claim 63, wherein the structure comprises a chemical modification (quantifying deamidation and oxidation of the stressed antibody used in this study; Pollastrini, page 97, paragraph 2, lines 3-4).
Regarding claim 70, Modified Pollastrini teaches the method of claim 69, wherein the chemical modification alters the mass-to-charge ratio (m/z) of charged ions of an amino acid of the antigen-binding protein (Supplemental Figure S2 of Houde’s Supplementary Data shows “XIC chromatograms (left) and MS/MS spectra (right) showing oxidized (top panels) and nonoxidized (bottom panels) heavy chain peptide DTLM253ISRTPEVTCVVVDVSHEDPEVK,” which reveal two distinct m/z ratio profiles; See also pp. 2-3).
Regarding claim 71, Modified Pollastrini teaches the method of claim 69, wherein the chemical modification is a
Regarding claim 72, Modified Pollastrini teaches the method of claim 63, wherein the stress is an exposure to ultra- violet light (For the forced oxidation study, the samples were exposed to visible and ultraviolet (UV) light; p. 89, Materials, ll. 9-11; Table 2 of Pollastrini),
Regarding claim 73, Modified Pollastrini teaches the method of claim 72, wherein the change in pH is greater than 1.0 or greater than 2.0 (This claim is directed to a non-elected species and will not be examined).
Regarding claim 74, Modified Pollastrini teaches the method of claim 72, wherein the change in temperature is greater than 2 degrees Celsius or greater than 5 degrees Celsius (This claim is directed to a non-elected species and will not be examined).
Regarding claim 75, Modified Pollastrini teaches the method of claim 63, wherein the antigen-binding protein comprises an antibody (IgG naturally comprises IgG1 or IgG2 which are antibodies; Pollastrini, p. 90, col. 1, last para., ll. 1-3), an antigen-binding antibody fragment, a Bi-specific T cell engager molecule, a bispecific antibody, a trispecific antibody, or an Fc fusion protein.
Regarding claim 76, Pollastrini teaches a method of determining an effect of a known structure (modifications; p. 94, col. 2, para. 2, l. 1; See Table 2 showing Percentages of major amino acid modifications occurring in Fc portion of IgG from forced oxidation) present on an antigen-binding protein (IgG; p. 94, col. 2, para. 2, l. 1) on an interaction between the antigen-binding protein and a target (FcRn; p. 94, col. 2, para. 2, l. 2)(To understand the effect of specific modifications of IgG on the binding affinity with FcRn,," wherein IgG is comprised of “two variable antigen-binding (Fab) regions and the constant (Fc) region,” and is therefore an antigen-binding protein; p. 94, col. 2, para. 2, ll. 1-2; p. 88, col. 1, para. 1, ll. 7-10; Table 2)(The examiner interprets an attribute to be a type of structure/modification with examples given in Table A of the instant publication US 20220260584 A1 and will therefore be used interchangeably through the office action; See also paragraphs [0039] and [0078]), said method comprising:
a. contacting a first sample comprising an antigen-binding protein comprising a known structure with a second sample comprising target antigens to form a mixture (“The oxidized sample and the control material were incubated with FcRn,” wherein the the oxidized sample is the first sample od IgG with known structures of Table 2, and FcRn is the second sample; p. 95, col. 1, ll. 3-4) comprising
(i) antigen-binding protein-target antigen complexes (IgG/FcRn complex; p. 95, col. 1, l. 8; Green peak around 22 min. in Fig. 10),
(ii) unbound antigen-binding proteins (Pollastrini states there to be a decrease in binding ability between the oxidized IgG and FcRn, therefore, there would naturally exist unbound IgG at equilibrium; See ; p. 95, col. 1, ll. 11-13), and
(iii) unbound target antigens (unbound FcRn; p. 95, col. 1, l. 10; Green peak around 14 min. in Fig. 10);
b. separating the mixture into (i) an unbound fraction comprising unbound antigen-binding proteins and target antigens and (ii) a bound fraction comprising antigen-binding protein-target antigen complexes (Pollastrini explains that “the retention time of the IgG/FcRn complex, for the oxidized sample, was shifted by approximately 1 min earlier relative to the control sample. In addition, the amount of unbound FcRn was increased (_8%) in the oxidized sample,” thereby demonstrating separation of all components into fractions via asymmetrical flow field flow fractionation (AF4) as shown in the spectrum in Fig. 10; p. 95, col. 1, ll. 7-11)(The Examiner interprets the unbound fraction to have the option of being comprised of either the antigen-binding proteins or targets. See objection above).
Pollastrini fails to teach steps (c)-(e) and SEC analysis in step (b) as well as an interaction between the antigen-binding protein and a target antigen (Emphasis added), and instead teaches the interaction is between the (Fc) region of the antigen-binding protein IgG and an FcRn receptor. However, Pollastrini does teach that IgG comprises two variable antigen-binding (Fab) regions responsible for specificity toward a target antigen (p. 88, col. 1, ll. 7-10) and separation via AF4 (See p. 94, col. 2, para. 2).
Houde teaches an interaction between the antigen-binding protein and a target antigen (The IgG1 analyzed here binds to a membrane receptor (antigen) on the surface of lymphoma cells; p. 1717, col. 2, Cell-based Apoptosis Assay, ll. 1-2).
Houde is considered to be analogous to the claimed invention because it is in the same field of endeavor for identifying attributes of antigen-binding proteins. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have incorporated the antigen-binding assessment described by Houde into the IgG characterization framework of Pollastrini in order to ensure that structural modifications, including methionine oxidation, do not detrimentally affect antigen-binding functionality. Pollastrini is directed to identifying structural modifications, such as methionine oxidation, that affect the binding affinity between FcRn and the Fc region of IgG (p. 96, col. 2, last para. ll. 1-5; Table 2)(p. 88, col.1, para. 1; p. 90, col. 1, last para., ll. 1-3) as a way to assess mAb product quality attributes (Abstract, last 2 ll.). However, Pollastrini does not disclose evaluating whether these structural modifications affect antigen-binding activity of IgG to a target antigen (Emphasis added). Even so, Pollastrini does acknowledge that IgG comprises variable antigen-binding (Fab) regions responsible for specificity toward a target antigen interactions (p. 88, col.1, para. 1, ll. 7-10).
Reference Houde likewise concerns assessment of mAb IgG product quality attributes (p. 1717, para. 2, ll. 4-7) and specifically evaluates the impact of post-translational modifications (PTMs), including methionine oxidation within the Fc region, on antibody functionality (p. 1717, para. 2, ll. 1-8). Houde explains that “to ensure such Fc-region modifications did not cripple the antigen binding activity and render the antibody completely non-functional, a cell-based apoptosis induction assay was utilized” to confirm antigen binding to a cell-surface antigen (p. 1718, last para. – p. 1719, first para.). Because both references address the same class of antibodies (IgG), the same type of structural modifications (methionine oxidation), and the same objective of assessing mAb product quality attributes, incorporating antigen-binding testing into Pollastrini’s quality attribute evaluation would have yielded the predictable result of confirming whether such modifications impact antigen recognition (See MPEP 2143(I)(A)).
Modified Pollastrini fails to teach steps (c)-(e) and SEC analysis in step (b) but does teach separation via AF4 (See p. 94, col. 2, para. 2).
In regard to step (b), Pollastrini teaches analysis of Fc/FcRn and IgG/FcRn interactions using both SEC and AF4. Specifically, Pollastrini performed SEC analyses of Fc/FcRn and full-length IgG/FcRn mixtures, demonstrating that SEC successfully differentiated Fc from FcRn and resolved the Fc/FcRn complex, with integrated peak areas indicating a 1:1 stoichiometry (p. 90, cols, 1-2). However, when full-length IgG1 and IgG2 were analyzed by SEC, no apparent IgG/FcRn binding was detected, which Pollastrini attributes to transient dissociation of weakly bound complexes during SEC due to on-column dilution and matrix interactions. Pollastrini further acknowledges that equilibrium SEC (Hummel-Dreyer chromatography) was previously developed to remedy the problem of weak protein-protein interactions, including IgG/FcRn stoichiometry (p. 90, col. 2, para. 2), thereby recognizing SEC as a suitable size-based technique when performed under equilibrium conditions. Pollastrini ultimately deployed AF4 as an orthogonal “softer” method to preserve weak complexes (p. 90, col. 2, para. 3). Given that SEC was already demonstrated to separate Fc/FcRn complexes and was recognized as adaptable to weak binding through equilibrium methods, It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted equilibrium SEC for conventional SEC to separate bound and unbound antigen binding protein-target complexes (IgG/FcRn) in oxidized (stressed) samples, as this represents the predictable substitution of a known SEC variant (equilibrium) for conventional SEC to address a recognized problem (See MPEP 2143(I)(B)), the use of a known technique to improve a similar analytical method for weak protein-protein interactions (See MPEP 2143(I)(C)), and an obvious-to-try approach where the art identifies a specific problem and a finite number of predictable solutions with a reasonable expectation of success (See MPEP 2143(I)(E).
Modified Pollastrini fails to teach steps (c)-(e).
In regard to steps (c-d), Pollastrini teaches performing tryptic digestion followed by RP-HPLC-MS to characterize antibody modifications of a stressed IgG sample for peptide mapping as shown in Table 2 (p. 90, col. 1, para. 2)(p. 94, col. 2, para. 2, ll. 4-6). This sample is later mixed with FcRn and then separated by asymmetrical flow field flow fractionation (AF4) into bound IgG/FcRn complexes and unbound FcRn and IgG (p. 95, col. 1, para. 1; Fig. 10). However, digestion and RP-HPLC-MS is not mentioned as being performed for these bound and unbound fractions. After AF4 analysis of the bound and unbound fractions, Pollastrini concludes that “These results, along with the data presented in Table 2, provide a strong correlation between specific amino acid modifications and the decrease in overall binding of FcRn to the degraded IgG sample” (p. 95, col. 1, para. 1, last 4 ll). Because Pollastrini already employs proteolytic digestion and RP-HPLC-MS as a tool for structural characterization of the stressed IgG sample and separately teaches fraction collection and analysis after AF4 separation, It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the separation method taught by Pollastrini with the addition of protein digestion in the bound and unbound fractions separated by AF4/SEC followed by RP-HPLC–MS so as to compare structural differences between interacting and non-interacting species and to assess its impact on binding under the same analysis conditions which represents a predictably analytical extension of the quantitative methods already disclosed (See MPEP 2143(I)(A)).
Modified Pollastrini fails to teach:
e. calculating a ratio of the abundance of the structure in the unbound fraction to the abundance of the structure in the bound fraction that is greater than 1, indicating that the structure negatively affects the interaction between the antigen-binding protein and the target antigen (the italicized portion does not further limit step d. and is merely an intended result of step d. and is therefore not given patentable weight (Minton v. Nat' l Ass' n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003) See MPEP 2111.04).
Modified Pollastrini does mention that the binding affinity Kd between IgG and FcRn reinforces the conclusion that there is “a strong correlation between specific amino acid modifications and the decrease in overall binding of FcRn to the degraded IgG sample,” based on the bound and unbound results of Fig. 10 and data from Table 2; page 95, column 1, paragraph 1, last 3 lines; p. 95, col. 2, ll. 13-18)
A goal of reference, Pan, it to “quantitatively assess the impact of Fc methionine oxidation on Protein A binding” (page 428, column 1, last 3 lines; See Table I with each methionine and %). Pan teaches that oxidation of specific Fc methionine residues (Met252 and Met428) decreases binding affinity to FcRn and Protein A, and quantifies this reduction through measurable differences in binding constants (Kd and EC50) derived from relative amounts of bound versus unbound species (p. 429, last 3 ll.-p. 430, para.1). Pan specifically states that “Full oxidation of Met 252 and Met 428 resulted in about two-fold increase in the association rate and 10-fold increase in the dissociation rate of the antibody and Protein A, which led to a 4.2-fold increase in the dissociation equilibrium constant (KD),” and therefore “oxidation of Met 252 and Met 428 reduces the affinity of the antibody with Protein A" (page 428, paragraph 1, last 6 lines; page 428, column 2, paragraph 1, lines 32-33; See binding constants in Table 2 wherein the unbound and bound fractions are, mathematically, a rearrangement of the dissociation equilibrium constant). Pollastrini likewise states that “These data suggest that the degraded sample had decreased binding ability to FcRn, likely due to the increase in oxidized methionine residues, consistent with previous results [42],” wherein reference [42] is Pan (Pollastrini, page 95, column 1, paragraph 1, lines 10-13). It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the mab product assessment method taught by Pollastrini in view of Houde with the addition of calculating a ratio of the abundance of a modified structure in the bound fraction relative to its abundance in the bound fraction, where a ratio greater than 1 indicates reduced binding because Pollastrini already teaches quantifying relative bound versus unbound species to assess binding strength and Pan teaches that specific structural modifications decrease binding affinity in a quantifiable manner. Expressing the effect as a ratio greater than 1 is merely a routine mathematical expression of the already measure quantities and represents a predictable way to numerically characterize reduced binding based on relative abundance.(See MPEP 2143(I)(A)).
Regarding claim 78, Modified Pollastrini teaches the method of claim 76, wherein the first sample comprises one or more species of the antigen-binding protein (IgG2; Pollastrini, p. 94, col. 2, para. 2, l. 2)(Paragraph [0009] of the instant publication US 20220260584 A1 explains “Each therapeutic protein having a different profile of attributes is considered a species”).
Regarding claim 79, Modified Pollastrini teaches the method of claim 76, wherein the first sample comprises serum (“mixtures at different ratios of FcRn, HSA, and/or IgG were prepared and analyzed by AF4,” wherein HSA is human serum albumin; Pollastrini, p. 95, col.1-2) or a serum fraction.
Regarding claim 80, Modified Pollastrini teaches the method of claim 76, wherein the method comprises repeating (a)- (e), calculating a plurality of ratios, (Changes in these regions were reproducible in replicate experiments, and overlapping peptides displayed similar behavior, giving us high confidence that the changes observed were real and significant; Houde, p. 1723, col. 2, ll. 17-20) and determining the statistical significance of the plurality of ratios based on calculation of a p-value (See association rate, dissociate rates and equilibrium constants of Table II’s binding experiment with 95% confidence intervals shown in the brackets of Pan which corresponds to a p-value of 0.05)(Calculation of a confidence interval naturally requires data from repetitive steps of the experiment)(The Examiner interprets the plurality of ratios to refer to: (i) multiple repetitions of the same ratio calculation of step (e))(See 112(b) rejection above).
Regarding claim 81, Modified Pollastrini teaches the method of claim 76, wherein the structure comprises a chemical modification (quantifying deamidation and oxidation of the stressed antibody used in this study; Pollastrini, page 97, paragraph 2, lines 3-4).
Regarding claim 82, Modified Pollastrini teaches the method of claim 81, wherein the chemical modification alters the mass-to-charge ratio (m/z) of charged ions of an amino acid of the antigen-binding protein (Supplemental Figure S2 of Houde’s Supplementary Data shows “XIC chromatograms (left) and MS/MS spectra (right) showing oxidized (top panels) and nonoxidized (bottom panels) heavy chain peptide DTLM253ISRTPEVTCVVVDVSHEDPEVK,” which reveal two distinct m/z ratio profiles; See also pp. 2-3).
Regarding claim 83, Modified Pollastrini teaches the method of claim 81, wherein the chemical modification is a
Regarding claim 84, Modified Pollastrini teaches the method of claim 76, wherein the stress is an exposure to ultra- violet light (For the forced oxidation study, the samples were exposed to visible and ultraviolet (UV) light; p. 89, Materials, ll. 9-11; Table 2 of Pollastrini)
Regarding claim 85, Modified Pollastrini teaches the method of claim 84, wherein the change in pH is greater than 1.0 or greater than 2.0 (This claim is directed to a non-elected species and will not be examined).
Regarding claim 86, Modified Pollastrini teaches the method of claim 84, wherein the change in temperature is greater than 2 degrees Celsius or greater than 5 degrees Celsius (This claim is directed to a non-elected species and will not be examined).
Regarding claim 87, Modified Pollastrini teaches the method of claim 76, wherein the antigen-binding protein comprises an antibody (IgG naturally comprises IgG1 or IgG2 which are antibodies; Pollastrini, p. 90, col. 1, last para., ll. 1-3) , an antigen-binding antibody fragment, a Bi-specific T cell engager molecule, a bispecific antibody, a trispecific antibody, or an Fc fusion protein.
Claims 64, 77 are rejected under 35 U.S.C. 103 as being unpatentable over Pollastrini et al. (“Field flow fractionation for assessing neonatal Fc receptor and Fcϒ receptor binding to monoclonal antibodies in solution”; 2011), in view of Houde et al. (“Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding” and Supplementary Data; 2010) and Pan et al. (“Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn1”; 2008), as applied to claims 63 and 76 above, and in further view of Majumder et al. (“Utility of High Resolution NMR Methods to Probe the Impact of Chemical Modifications on Higher Order Structure of Monoclonal Antibodies in Relation to Antigen Binding”; 07-01-2019)
The effective filing date for claim is 01/31/2020 via provisional application no. 62/968,682 since provisional application 62/857,637 does not disclose the limitation of “the structure is comprised by a paratope of the antigen-binding protein”.
Regarding claim 64, Modified Pollastrini teaches the method of claim 63.
Modified Pollastrini is silent to teaching the structure is comprised by a paratope of the antigen-binding protein (Paragraph [0057] of the instant publication US 20220260584 A1 states “the paratope or binding site is the site at which the target interacts with or binds to the therapeutic protein,” and therefore the Examiner interprets the paratope to be the Fab region of the antigen-binding protein IgG since the target is an antigen as claimed in claim 63).
Instead, modified Pollastrini teaches the structure is comprised by an epitope of the antigen-binding protein (Fc region of IgG)(Pollastrini; Abstract; Table 2)(Houde, p. 1716, col. 1, ll. 15-20) of the antigen-binding protein yet still checks the Fab region (paratope) for structural modifications after forced oxidation (See Supplementary data from Houde p. 3, para. 2) to which were nominal.
Majumder teaches that structural modifications (e.g. deamidation) occur within the Fab region (paratope) of monoclonal antibodies and evaluates the impact of such modifications on antigen binding using structural and functional assays (mAbs were subjected to forced deamidation stress…The extent of deamidation in the mAb domains were quantified by LC-MS/MS… The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged.; Abstract).
Majumder is considered to be analogous to the claimed invention because it is in the same field of endeavor for identifying attributes of antigen-binding proteins. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted forced deamination targeting residues in the Fab region of IgG as taught by Majumder for the forced oxidation of Fc-region methionine residues taught by Pollastrini in view of Houde and Pan in order to evaluate effects on binding because both deamination and oxidation are well-known stress-induced chemical modifications of antibodies, and both are recognized in the art as attributes capable of altering protein structure and binding interactions. Pollastrini acknowledges that IgG comprises variable antigen-binding (Fab) regions responsible for specificity toward a target antigen (p. 88, col.1, para. 1, ll. 7-10) while Houde verifies that forced oxidation caused nominal structural modifications in the Fab regions of IgG. Houde goes on to teach that structural modifications such as oxidation and deamidation, may impact antigen-binding functionality and therefore evaluates whether antigen binding is preserved following modification (pp. 1716-1717). Although Houde’s forced oxidation is confined to Fc methionine residues, Houde’s peptide mapping workflow analyzes modifications from both the Fc and the Fab portion of IgG (Houde, Supplementary Data; p. 3, para. 2, ll. 1-4) and concludes that the oxidation only significantly affects the Fc portion (epitope) of IgG and not the Fab portion. Both Pollastrini and Houde are concerned with assessing structural attributes that affect antibody interactions as part of mAb product quality evaluation, and a person of ordinary skill in the art would have recognized the benefit in applying the same structural characterization and stress-testing methods to structural attributes located within the antigen-binding Fab region (paratope). As taught in Houde, oxidative stress was used to induce Fc methionine oxidation and assess its impact on Fc receptor binding; however, deamidation of asparagine and glutamine residues is likewise a known and common degradation pathway in antibodies, particularly within variable regions. Substituting one known chemical stress modification (oxidation) with another known stress modification (deamidation) represents the predictable substitution of one known degradation mechanism for another to assess its impact on binding, consistent with MPEP 2143(I)(B) because both modifications alter amino acid side chains and can influence protein conformation and interaction affinity. A person of ordinary skill in the art would have reasonably expected that inducing deamination in the Fab region and evaluating its effect on binding would yield predictable information regarding structure-function relationships just as oxidation was used to evaluate Fc-mediated binding.
Regarding claim 77, Modified Pollastrini teaches the method of claim 76,
Modified Pollastrini is silent to teaching the structure is comprised by a paratope of the antigen-binding protein (Paragraph [0057] of the instant publication US 20220260584 A1 states “the paratope or binding site is the site at which the target interacts with or binds to the therapeutic protein,” and therefore the Examiner interprets the paratope to be the Fab region of the antigen-binding protein IgG since the target is an antigen as claimed in claim 63).
Instead, modified Pollastrini teaches the structure is comprised by an epitope of the antigen-binding protein (Fc region of IgG)(Pollastrini; Abstract; Table 2)(Houde, p. 1716, col. 1, ll. 15-20) of the antigen-binding protein yet still checks the Fab region (paratope) for structural modifications after forced oxidation (See Supplementary data from Houde p. 3, para. 2) to which were nominal.
Majumder is considered to be analogous to the claimed invention because it is in the same field of endeavor for identifying attributes of antigen-binding proteins. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted forced deamination targeting residues in the Fab region of IgG as taught by Majumder for the forced oxidation of Fc-region methionine residues taught by Pollastrini in view of Houde and Pan in order to evaluate effects on binding because both deamination and oxidation are well-known stress-induced chemical modifications of antibodies, and both are recognized in the art as attributes capable of altering protein structure and binding interactions. Pollastrini acknowledges that IgG comprises variable antigen-binding (Fab) regions responsible for specificity toward a target antigen (p. 88, col.1, para. 1, ll. 7-10) while Houde verifies that forced oxidation caused nominal structural modifications in the Fab regions of IgG. Houde goes on to teach that structural modifications such as oxidation and deamidation, may impact antigen-binding functionality and therefore evaluates whether antigen binding is preserved following modification (pp. 1716-1717). Although Houde’s forced oxidation is confined to Fc methionine residues, Houde’s peptide mapping workflow analyzes modifications from both the Fc and the Fab portion of IgG (Houde, Supplementary Data; p. 3, para. 2, ll. 1-4) and concludes that the oxidation only significantly affects the Fc portion (epitope) of IgG and not the Fab portion. Both Pollastrini and Houde are concerned with assessing structural attributes that affect antibody interactions as part of mAb product quality evaluation, and a person of ordinary skill in the art would have recognized the benefit in applying the same structural characterization and stress-testing methods to structural attributes located within the antigen-binding Fab region (paratope). As taught in Houde, oxidative stress was used to induce Fc methionine oxidation and assess its impact on Fc receptor binding; however, deamidation of asparagine and glutamine residues is likewise a known and common degradation pathway in antibodies, particularly within variable regions. Substituting one known chemical stress modification (oxidation) with another known stress modification (deamidation) represents the predictable substitution of one known degradation mechanism for another to assess its impact on binding, consistent with MPEP 2143(I)(B) because both modifications alter amino acid side chains and can influence protein conformation and interaction affinity. A person of ordinary skill in the art would have reasonably expected that inducing deamination in the Fab region and evaluating its effect on binding would yield predictable information regarding structure-function relationships just as oxidation was used to evaluate Fc-mediated binding.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Oda et al., 2008 (instant PTO-892) teaches digesting each fraction of sample separated via chromatography.
Van Eyk et al., 2011 (instant PTO-892) uses native SEC to separate serum and protein complexes originating from IgG.
Nowak et al., 2017 (instant PTO-892) teaches forced oxidation is commonly used in the art to assess a change in the residue of an amino acid sequence that may affect binding and potency.
Sela-Culang et al., 2017 (instant PTO-892) teaches comparing structural changes and binding efficiency in bound and unbound species.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/V.S./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758