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 .
Status of the Claims
Claims 1, 14-27, 29-30, 34-38, 45, 52-53, 55-64, 66, 87, and 95 are pending in the application.
Claims 1, 38, 45, 52-53, 55-64, 66, 87, and 95 are withdrawn.
Claims 14-27, 29-30, 34-37 are the subject of this office action.
Election/Restrictions
Applicant’s election without traverse of Group II, claims 14-27, 29-30, 34-37 in the reply filed on 21 January 2026 is acknowledged.
Claims 1, 38, 45, 52-53, 55-64, 66, 87, and 95 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 21 January 2026.
Priority
The instant application is a continuation of PCT/US2021/047537, filed 25 August 2021, which claims benefit of provision applications: 63/218,073, filed 2 July 2021; 63/085,113 filed 29 September 2020; and 63/070,211, filed 25 August 2020.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 20 July 2023, 5 September 2025, and 24 October 2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements have been considered by the examiner.
Drawings
The drawings are objected to because they contain sequences. As per MPEP 2412.06: “Pursuant to 37 CFR 1.83(a), sequences that are included in the Sequence Listing XML should not be duplicated in the drawing”. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Rejections - 35 USC § 112(a)
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 26-27, 29-30, and 34-37 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
A determination of enablement involves the consideration of the following factors: the breadth of the claims; the nature of the invention; the state of the prior art; level of one or ordinary skill; level of predictability in the art; amount of direction provided by the inventor; existence of working examples; and quantity of experimentation needed to make or use the invention based on the content of the disclosure.
The claims encompass a peptide exchange assay comprising an antibody complex comprising a FRET acceptor and a FRET emitter complex comprising a FRET emitter conjugated to a second label, wherein the claim states that detecting FRET emission of the second label thereby detects binding of a MHCI allele to a peptide. The claim does not limit or specifically define the second label.
This is not enabled because the second label is not a FRET acceptor or emitter, and would therefore not yield FRET emission, therefore it would not be possible to detect FRET emission of the second label in the reaction as an indication of binding of the MHCI allele to a peptide.
Assuming that step (b) is intended to instead recite a detection of FRET emission from the recited FRET acceptor (as appears to be shown in Fig. 8A, and as recited in claim 34), the claim is still not enabled in its full scope.
Claim 26 recites an antibody complex comprising an anti-MHCI allele antibody covalently attached to a FRET acceptor and a FRET emitter complex comprising a FRET emitter conjugated to a second label, and indicates the detection of FRET emission is indicative of binding of the MHCI allele to a peptide. Claim 25 from which claim 26 depends recites an MHCI complex comprising a first label, but neither the first label nor the second label are specifically defined in claims 25 and 26. If FRET emission is to be used to indicate binding of the MHCI allele to the test peptide, then there must be some mechanism by which the FRET emitter and the FRET acceptor are brought into proximity with one another in the assay. The FRET acceptor will bind to the MHCI/ligand peptide complex because it is covalently attached to an anti-MHCI allele antibody. However, the FRET emitter is only conjugated to a second label which is not defined and which may comprise any species of label.
The instant specification provides limited direction and no exact reduction to practice for this assay. One working example of a very similar FRET assay is discussed in the specification, as shown in Fig. 8A and discussed in associated example 3, starting at Par. 203. In this example the MHCI/peptide complex comprises a first label which is specifically biotin, and a second label which is specifically streptavidin conjugated to a FRET acceptor (APC). As such the streptavidin second label specifically binds to the biotin first label (attached to the MHCI). An antibody complex comprising an anti-B2M antibody and a FRET emitter also specifically binds to the MHCI, thereby producing FRET emission. However, this reduction to practice is noted to differ from the instant claim: the instant claim indicates that the anti-MHCI antibody is conjugated to a FRET acceptor, while the second label is conjugated to a FRET emitter; in contrast Fig 8A (and associated example 3) show an anti-MHCI antibody conjugated to a FRET emitter and a second label conjugated to a FRET acceptor.
Thus, FRET emission is enabled only if the first label and the second label can specifically bind to one another.
Regarding predictability of the art and the existence of working examples: predictability in the art is low because the number of possible species of labels encompassed by the claims is extremely broad, wherein most of these species of labels are not capable of specifically binding to one another, and wherein the use of a first and second label that do not specifically bind to one another in the claimed assay will not comprise any reliable mechanism for bringing the recited FRET acceptor and FRET emitter into proximity with one another in the presence of an MHCI allele bound to a peptide. The working example provided by known specific binding of biotin and avidin/avidin analog labels is insufficient to enable the entire genus of labels encompassed by the instant claims.
Claims 27, 29-30 and 34-37 provide further limitations on the FRET assay but do not resolve this issue. Notably, claim 29 recites “wherein the first label and the second label are independently streptavidin or biotin” but this is still not fully enabled as it encompasses embodiments wherein the first and second label are both biotin or wherein the first and second label are both streptavidin, wherein these embodiments would not facilitate specific binding of the first and second label to hold the FRET acceptor and FRET emitter in proximity to one another.
As such the quantity of experimentation needed to enable the full scope of the claims is undue because the claims encompass an extremely broad genera of labels, while the specification enables only one specific pairing of labels.
Claim Rejections - 35 USC § 112(b)
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 14-27, 29-30, 34-37 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 14 is rejected as indefinite over the recitation of “MHCI/-second peptide complex” in step (c) and “the second peptide” in step (d). It is unclear within the context of the claim whether the second peptide refers to the same “test peptide” introduced earlier in the claim, or whether it refers to a distinct different peptide. Additionally, if the second peptide does not refer to the same “test peptide” it is unclear how the assay is capable of “determining binding of a…MHCI allele to a test peptide” as recited in the preamble, since the active steps recited in the method indicate “determining whether the MHCI allele is bound to the second peptide” and not determining whether the MHCI allele is bound to the test peptide. For the purposes of applying prior art in this office action, “second peptide” is understood to refer to the same “test peptide” previously introduced in the claim, because this appears to be the only interpretation under which the assay as claimed is functional for its stated purpose of determining binding of a MHCI allele to a test peptide. Clarification is required.
Claims 15, 22, and 26 are rejected as indefinite over recitation of “the peptide” (claim 15), “each peptide” (claim 22), and “a peptide” (claim 26). There is introduction of multiple different peptides within the preceding claims, such that it is unclear exactly which peptide is being referenced by generic recitation of “peptide”. Clarification is required.
Claim 19 is vague regarding “the free second peptide”. There is no prior introduction of “a free second peptide” in claim 19 or in the claims from which it depends, therefore there is insufficient antecedent basis for this limitations in the claim.
Claim 20 is rejected as indefinite over a lack of clarity regarding “the second peptide”. As discussed above, claim 14 is indefinite regarding the second peptide because it is unclear whether the second peptide refers to the same “test peptide” or whether it refers to a distinct different peptide. This lack of clarity is compounded by claim 20 which recites that “presence of the second peptide as determined by HPLC and MS indicates that the MHCI is capable of binding to the second peptide”. This limitation may be true in an embodiment wherein the second peptide is present only in complex with MHCI, but it does not make sense if the second peptide can exist in a free state in the sample (wherein presence of a free second peptide as determined by HPLC and MS would therefore not necessarily indicate that MHCI is capable of binding to the second peptide). Clarification is required.
Claim 22 is rejected as indefinite because the chronology of the additional limitation within the method of the independent claim is unclear. That is, it is not clear whether the claim the test peptides are identified before or after the peptide exchange.
Claims 24 and 36 are rejected as indefinite because they depend from more than one claim, but not in the alternative.
Claim 24 is rejected as indefinite because it recites dependence on non-elected and canceled claims.
Claim 25 is vague regarding “the MHCI/peptide complex”. There is no prior introduction of an “MHCI/peptide complex”, therefore there is insufficient antecedent basis for this limitation in the claim. Additionally, because multiple peptides have been previously introduced in the claims, it is unclear which peptide this recitation refers to. Clarification is required.
Claim 26 is vague regarding “the labeled MHCI/ligand peptide complex”. There is no prior introduction of “a labeled MHCI/ligand peptide complex, therefore there is insufficient antecedent basis for this limitation in the claim. Additionally, it is unclear which complex is being referenced here because the name of the complex differs both from what is introduced in claim 25 and from what is introduced in claim 14. Clarification is required.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 27 recites the broad recitation “at least about 10 hours”, and the claim also recites “at least about 15 hours” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Dependent claims 15-23, 25-27, 29-30, 34-37 are rejected as indefinite because they depend from an indefinite claim and fail to remedy its deficiencies.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 14-15, 19, 21, and 23-25 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Hadrup et al ((2009) "High-throughput T-cell Epitope Discovery Through Mhc Peptide Exchange", Methods in Molecular Biology, 524:383-405.; IDS entered).
Regarding claims 14 and 24, Hadrup teaches a peptide exchange assay for determining binding of a MHCI allele to a test peptide (Abstract), comprising:
Providing a first composition comprising a test peptide and a MHCI/ligand complex comprising a MHCI molecule comprising an alpha chain, a beta chain, and a ligand, wherein the ligand is a peptide comprising a non-natural UV-cleavable amino acid (Fig. 2, MHC ligand discovery; Section 3.1.2; Section 3.2, Section 3.2.1 step 6: MHCI complex comprising MHC heavy chain (alpha chain) and B2M (beta chain) and a conditional ligand (a peptide comprising a non-natural UV-cleavable amino acid); Section 3.3.);
Exposing the first composition to UV light to cleave the ligand at the UV-cleavable amino acid (Section 3.3, Par. 1, and step 2: UV-mediated cleavage of the conditional ligand);
Incubating the first composition for a period of time to form a second composition comprising free test peptide, the alpha chain, the beta chain, and/or a MHCI/-second peptide complex (Section 3.3, steps 1-3);
Determining whether the MCHI allele is bound to the second peptide (Section 3.4: measuring peptide mediated MHC rescue by MHC ELISA; Section 3.4.3: analysis of peptide exchange reactions by gel filtration chromatography; Fig. 2).
Regarding claim 15, Hadrup further teaches the assay wherein MHCI allele binding to the peptide is determined by measuring a level of MHCI/peptide complex in the second composition (Section 3.4: measuring peptide mediated MHC rescue by MHC ELISA; Section 3.4.3: analysis of peptide exchange reactions by gel filtration chromatography; Fig. 2).
Regarding claim 19, Hadrup further teaches the assay wherein the free second peptide is removed from the second composition via size exclusion chromatography (Fig. 5; Section 3.4.3: gel filtration chromatography; Section 3.3: resulting complexes may be used to determine exchange efficiency by gel filtration chromatography).
Regarding claim 21, Hadrup further teaches the assay wherein a plurality of the MHCI/ligand complex is combined with at least two different test peptides (Pg. 403, step 17: parallel exchange reactions involving high numbers of peptides; Fig. 2: peptide library of potential MHC ligands; Abstract).
Regarding claim 23, Hadrup further teaches the assay wherein the test peptide is present in the first composition at a ratio of at least 10:1 (test peptide:MHCI) (Section 3.3: in routine experiment a 100 fold molar excess of peptide over MHC is used).
Regarding claim 25, Hadrup further teaches the assay wherein the MHCI/peptide complex further comprises a first label, thereby forming a labeled MHCI/peptide complex (Section 3.2.2: biotinylation of p*HLA A2.1 complexes; see also, e.g., Section 3.4.1 Protocol for 96-well MHC ELISA).
Claims 14-15, 18-21, and 23-25 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Toebes et al ((2006) "Design and Use of Conditional MHC Class I Ligands", Nature Medicine, 12(2):246-251.; IDS entered).
Regarding claims 14 and 24, Toebes teaches a peptide exchange assay for determining binding of a MHCI allele to a test peptide (Abstract), comprising:
Providing a first composition comprising a test peptide and a MHCI/ligand complex comprising a MHCI molecule comprising an alpha chain, a beta chain, and a ligand, wherein the ligand is a peptide comprising a non-natural UV-cleavable amino acid (Pg. 250, Col. 1, Par. 4: Peptide synthesis and preparation of recombinant MHC);
Exposing the first composition to UV light to cleave the ligand at the UV-cleavable amino acid (Pg. 250, Col. 1, last Par.-Col. 2, first Par.);
Incubating the first composition for a period of time to form a second composition comprising free test peptide, the alpha chain, the beta chain, and/or a MHCI/-second peptide complex (Pg. 250, Col. 1, last Par.-Col. 2, first Par.);
Determining whether the MCHI allele is bound to the second peptide (Fig. 2; Pg. 247, Col. 1, Par. 1-Col. 2, Par. 2).
Regarding claim 15, Toebes further teaches the assay wherein MHCI allele binding to the peptide is determined by measuring a level of MHCI/peptide complex in the second composition (Fig. 2; Pg. 247, Col. 1, Par. 1-Col. 2, Par. 2).
Regarding claims 18 and 20, Toebes further teaches the assay further comprising performing HPLC and MS to distinguish the MHCI and the second peptide; wherein presence of the second peptide as determined by HPLC and MS indicates that the MHCI is capable of binding to the second peptide (Fig. 2; Pg. 247, Col. 1, Par. 1-Col. 2, Par. 2).
Regarding claim 19, Toebes further teaches the assay wherein the free second peptide is removed from the second composition via size exclusion chromatography (Fig. 2; Pg. 247, Col. 1, Par. 1-Col. 2, Par. 2; gel filtration chromatography).
Regarding claim 21, Toebes further teaches the assay wherein a plurality of the MHCI/ligand complex is combined with at least two different test peptides (Pg. 249, Col. 1, last Par.-Col. 2, first Par.; Pg. 250, Col. 1, last Par.-Col. 2, Par. 2).
Regarding claim 23, Toebes further teaches the assay wherein the test peptide is present in the first composition at a ratio of at least 10:1 (test peptide:MHCI) (Pg. 450, Col. 1, last Par.-Col. 2, Par. 2: MHCI present at 0.5uM, test peptide present at 50uM).
Regarding claim 25, Toebes further teaches the assay wherein the MHCI/peptide complex further comprises a first label, thereby forming a labeled MHCI/peptide complex (Pg. 450, Col. 1, last Par.: biotinylated MHCI complex).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Hadrup et al ((2009) "High-throughput T-cell Epitope Discovery Through Mhc Peptide Exchange", Methods in Molecular Biology, 524:383-405.; IDS entered) as applied to claim 15 above, and further in view of Stoll et al (Two-dimension liquid chromatography: a state of the art tutorial. Analytical Chemistry 2017 89 (1), 519-531).
Hadrup teaches the assay of claim 15 as described above and discloses LC and MS analysis for analyzing and determining levels of MHCI/second peptide complex, but differs from instant claim 16 in that it does not teach 2D LC/MS.
Regarding 2D LC/MS, Stoll teaches that 1D LC is often unable to quickly separate mixtures of interest in two particular situations: (a) mixtures that are too complex in a general sense, and thus outstrip the ability of 1D LC to entirely separate the mixture into distinct components and (b) mixtures that are not necessarily complex per se, but contain several species of interest that are very difficult to resolve, either because there are just too many compounds to avoid overlap or due to the presence of closely related compounds (Pg. 519, Col. 2, Par. 1). Stoll teaches that 2D/LC is advantageous in separating and analyzing different analytes in a mixture based on two different dimensions of LC, and that 2D/LC is especially useful in peptide fingerprinting and identification in combination with MS (Pg. 519, Col. 2, Par. 1; Pg. 521, Col. 1, Par. 3; Pg. 527, Col. 1, Par. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the invention of Hadrup to further include wherein the level of MHCI/second peptide complex is measured by 2D LC/MS wherein 2D LC/MS comprises removing the free second peptide from the second composition. One would be motivated to make this modification because Stoll teaches that 2D LC is advantageous over 1D LC and is particularly useful and effective in separating and analyzing different components of mixtures comprising different species of interest, and teaches specifically that 2D LC can be used in combination with MS for peptide fingerprinting and identification. One of ordinary skill in the art would have a reasonable expectation of success in making this modification because Hadrup teaches that both LC and MS may be used for separation and analysis of components in the assay mixture.
Claims 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Toebes et al ((2006) "Design and Use of Conditional MHC Class I Ligands", Nature Medicine, 12(2):246-251.) as applied to claim 15 above, and further in view of Stoll et al (Two-dimension liquid chromatography: a state of the art tutorial. Analytical Chemistry 2017 89 (1), 519-531).
Toebes teaches the assay of claim 15 as described above and discloses LC and MS analysis for analyzing and determining levels of MHCI/second peptide complex, but differs from instant claim 16 in that it does not teach 2D LC/MS.
Regarding 2D LC/MS, Stoll teaches that 1D LC is often unable to quickly separate mixtures of interest in two particular situations: (a) mixtures that are too complex in a general sense, and thus outstrip the ability of 1D LC to entirely separate the mixture into distinct components and (b) mixtures that are not necessarily complex per se, but contain several species of interest that are very difficult to resolve, either because there are just too many compounds to avoid overlap or due to the presence of closely related compounds (Pg. 519, Col. 2, Par. 1). Stoll teaches that 2D/LC is advantageous in separating and analyzing different analytes in a mixture based on two different dimensions of LC, and that 2D/LC is especially useful in peptide fingerprinting and identification in combination with MS (Pg. 519, Col. 2, Par. 1; Pg. 521, Col. 1, Par. 3; Pg. 527, Col. 1, Par. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the invention of Toebes to further include wherein the level of MHCI/second peptide complex is measured by 2D LC/MS wherein 2D LC/MS comprises removing the free second peptide from the second composition. One would be motivated to make this modification because Stoll teaches that 2D LC is advantageous over 1D LC and is particularly useful and effective in separating and analyzing different components of mixtures comprising different species of interest, and teaches specifically that 2D LC can be used in combination with MS for peptide fingerprinting and identification. One of ordinary skill in the art would have a reasonable expectation of success in making this modification because Toebes teaches that both LC and MS may be used for separation and analysis of components in the assay mixture.
Claims 18 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Hadrup et al ((2009) "High-throughput T-cell Epitope Discovery Through Mhc Peptide Exchange", Methods in Molecular Biology, 524:383-405.; IDS entered).
Regarding claims 18 and 20, Hadrup further teaches performing HPLC or MS to distinguish the MHCI and the second peptide; wherein presence of the second peptide as determined by HPLC and MS indicates that the MHCI is capable of binding to the second peptide (Pg. 403, note 17: pMHC complexes can be analyzed by HPLC or MS; Section 3.4.3; Fig. 5).
Hadrup differs from instant claim 18 in that it does not explicitly teach performing HPLC and MS to distinguish the MHCI and the second peptide.
However, It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the invention of Hadrup to specifically comprise performing both HPLC and MS. One of ordinary skill in the art would be motivated to make this modification because redundancy of analysis by different methods confirms and improves accuracy of results, and Hadrup teaches that both HPLC and MS are valuable as independent methods for confirmation and analysis of peptide exchange (Pg. 403, note 17). One of ordinary skill in the art would have a reasonable expectation of success in making this modification because Hadrup teaches that HPLC and MS are appropriate for this application.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Hadrup et al ((2009) "High-throughput T-cell Epitope Discovery Through Mhc Peptide Exchange", Methods in Molecular Biology, 524:383-405.; IDS entered) as applied to claim 21 above, and further in view of Toebes et al ((2006) "Design and Use of Conditional MHC Class I Ligands", Nature Medicine, 12(2):246-251.; IDS entered).
Regarding claim 22, Hadrup teaches the method of claim 21, as described above. Hadrup further teaches that MS can be used for the analysis of parallel exchange reactions involving high numbers of peptides, and teaches that these techniques are valuable to follow the peptide exchange process by an independent method. Hadrup differs from the instant claim in that it does not explicitly teach that the test peptides are identified by MS based on the predicted mass of each peptide.
Toebes further teaches that MS can be used to verify the identity of the peptide bound to the MHCI complex after performance of the exchange assay, wherein verification is based on the expected mass of the test peptide (Pg. 247, Col. 2, last Par.-Pg. 248, Col. 1, first Par.: upon UV-light mediated cleavage, the sole detectable peptide mass associated with HLA-A2.1 corresponds to the mass of the CMV pp65(495-503) epitope).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the invention of Hadrup to further comprise identification of the multiple different test peptides by mass spectrometry based on the predicted mass of each test peptide, as taught by Toebes. One of ordinary skill in the art would be motivated to make this modification because the implementation of MS in this fashion allows the practitioner of the assay to verify the identity of the test peptide bound to the MHC complex, as taught by Toebes. One of ordinary skill in the art would have a reasonable expectation of success in making this modification because MS is a technique that is known and commonly used in the art to distinguish and identify proteins and peptides based on their mass, and because both Toebes and Hadrup are directed to peptide exchange assays used to screen pluralities of test peptides for their ability to bind to an MHCI complex.
Subject Matter Free of Prior Art
Claims 26-27, 29-30, and 34-37 are rejected as described above, but appear to be free of the prior art.
Regarding claims 26-27, 29-30, and 34-37, the closest prior art is Hadrup et al ((2009) "High-throughput T-cell Epitope Discovery Through Mhc Peptide Exchange", Methods in Molecular Biology, 524:383-405.; IDS entered) and Toebes et al ((2006) "Design and Use of Conditional MHC Class I Ligands", Nature Medicine, 12(2):246-251.), as described in the 102 rejections of claim 25 above, from which claim 26 depends. Additional prior art of interest is Axmann et al ("Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay," Journal of Visualized Experiments, 104:e53157 (2015) 17 pgs.; IDS entered), Weissbrich et al (US 2018/0042995 A1), and Rooney et al (WO 2020/132586 A1; IDS entered).
Both Hadrup and Toebes read on claim 25, as described above.
Hadrup further teaches an ELISA assay for detection of the binding of a MHCI allele to a peptide. The ELISA comprises detection of a biotinylated pMHC complex by capture of the biotinylated complex on a streptavidin coated plate and an antibody complex compriaing an anti-MHCI allele antibody labeled with HRP (Fig. 4). This is similar to instant claim 26 in that the assay comprises a labeled anti-MHCI allele antibody and a labeled (biotinylated) pMHC complex which binds to a second label (streptavidin).
Both Hadrup and Toebes differ from instant claim 26 in that they do not teach a FRET assay.
Regarding claim 26, Axmann and Weissbrich both teach assay comprising the use of FRET to detech binding of a T cell receptor to a pMHC complex (see Weissbrich, Fig. 12; see Axmann, Fig. 2; both references show a T cell or TCR labeled with either a FRET donor or acceptor, and an immobilized pMHC complex labeled with either a FRET donor or acceptor, such that the FRET labels are brought into proximity and produce detectable signal when the TCR binds to the immobilized pMHC).
Both Axmann and Weissbrich differ from the instant claim in that the FRET assay is used to detect binding of the TCR to the pMHC complex, rather than to detect binding of a peptide to an MHCI complex. Additionally, neither Axmann nor Weissbrich teaches an antibody complex comprising an anti-MHCI allele antibody covalently attached to a FRET acceptor or a FRET emitter conjugated to a second label.
Regarding claim 26, Rooney teaches an MHCII peptide exchange assay wherein peptide exchange is detected by TR-FRET (Par. 174, 345, 716, 721-722, Fig. 26A). However the FRET assay taught by Rooney differs from the instant claim in function and basic structure. The assay taught by Rooney comprises a first antibody conjugated to a first FRET label, wherein the antibody specifically binds to the placeholder peptide (i.e. the peptide that is replaced during the peptide exchange assay); a second FRET label is conjugated to a second antibody which is an anti-his antibody. As such, the Rooney’s assay comprises two antibodies which are brought into proximity by binding to both the placeholder peptide and the MHCII prior to peptide exchange, wherein proximity of the two FRET labels produces a detectable fluorescent signal. When peptide exchange successfully occurs, the placeholder peptide is displaced by the test peptide, such that the placeholder peptide and the first antibody and first FRET label dissociate from the MHC complex while the second anti-his antibody and the second FRET label remain bound to the MHC complex; this increases the distance between the two FRET labels, and as such, successful peptide exchange results in a decrease in fluorescent signal. This differs from the assay of claim 26 where increased FRET emission in the reaction composition indicates binding of the MHCI to the test peptide.
Rooney further differs from the instant claim in that it is directed to peptide exchange with MHCII rather than MHCI.
Conclusion
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/ELLIS FOLLETT LUSI/Examiner, Art Unit 1677
/CHRISTOPHER L CHIN/Primary Examiner, Art Unit 1677