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 .
DETALED ACTION
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12/3/2025 has been entered.
Applicant’s response filed 12/3/25 is acknowledged and has been entered.
2. Applicant is reminded of Applicant's election of Group I in Applicant’s response filed 1/8/19.
Claims 31, 33, 34, 37, 39, 41, 42 and 45-49 are presently being examined.
3. In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
4. 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.
5. Claims 31, 33, 34, 37, 39, 41, 42 and 45-49 are rejected under 35 U.S.C. 103 as being obvious over US 20100168390 A1 (of record) in view of Andersen et al (Nature Protocols, 2012, 7: 891-902, of record), US 2021/0239698 A1 (priority to 2011), Brakmann, S. (Angew. Chem. Int. Ed. 2004, 43: 5730-5734), and WO 2013/137737 A1 (of record).
Claim Interpretation: instant base claim 31 recites that eight or more MHC molecules are coupled to the backbone and a nucleic acid molecule comprising the barcode with primer regions of part “iii” is coupled to the backbone; these limitations are being interpreted to mean that the MHC molecules and the nucleic acid molecule comprising the barcode with primer regions are directly or indirectly attached or coupled to the backbone. The specification does not disclose a limiting definition for “conjugating” as in “conjugating the nucleic acid label to the backbone” as is recited in instant dependent claim 46. The said limitation is therefore being interpreted as is it is known in the art to mean ‘to join together’ or ‘chemically join together’. See for example, evidentiary reference Biology Online (2024,10 pages, of record). The definition of “barcode” in the instant specification is: “In the present context, a nucleic acid barcode is a unique oligo-nucleotide sequence ranging for [from] 10 to more than 50 nucleic acids. The barcode has shared amplification sequences in the 3’ and 5’ ends, and a unique sequence in the middle. This sequence can be revealed by sequencing and can serve as a specific barcode for a given molecule.” (see page 5 at lines 21-26).
US 2010/0168390 A1 discloses peptide/MHC class I molecules or tetramers or other multimers thereof bound to fluorophore-labeled dextran carrier molecules (or other polysaccharides such as derivatized dextrans, scleroglucan (i.e., a glucan), streptavidin, streptavidin tetramers, or avidin); and when the complexes are bound to streptavidin, attachment is via biotin/streptavidin attachment chemistries. The streptavidin can also be attached to a derivatized dextran or other polysaccharide. US 2010/0168390 A1 also discloses other carriers such as magnetic or other beads, including those comprising dextran-coated beads comprising the MHC dextramers. US 2010/0168390 A1 discloses that the MHC molecule can be a recombinant molecule wherein the MHC class I heavy chain comprises a C-terminal target peptide sequence for biotinylation, and the chemically biotinylated MHC can then bind to streptavidin coupled to the carrier molecule. US 2010/0168390 A1 discloses that in making the MHC peptide molecule recombinantly, the heavy chain of MHC class I and the b2m light chain may be expressed separately and added together during in vitro refolding. US 2010/0168390 A1 discloses that peptide epitopes presented by MHC molecules can be presented to T cells and activates the T cells, wherein each T cell expresses one unique specificity of TCR which recognizes one specific MHC/peptide epitope complex. US 2010/0168390 A1 discloses that the dextran backbone can further comprise one or more than one detectable label and tags such as for example, a His tag, metal-ion tag, or other selectable tags and labels such as detectable labels. US 2010/0168390 A1 discloses that the labeling molecule many be any labeling molecule such as a nucleic acid molecule, including DNA, or nucleic acid analogs, (e.g.,[0488]) and it may be attached to the MHC multimer directly or indirectly, covalently or noncovalently; it can be attached to the MHC multimer, to the multimerization domain, or to the dextran backbone. US 2010/0168390 A1 discloses that the labelling compound can be attached via a suitable linker and that such linkers are readily known by the person skilled in the art.
US 2010/0168390 A1 discloses that the number of MHC molecules can be at least two, at least four, or at least eight, up to a plurality depending on the capacity and nature of the multimerization domain(s), and the MHC can harbor the same or a different peptide; in the latter case, the composition can be used to detect several types of MHC recognizing T cells simultaneously. US 2010/0168390 A1 discloses that one of ordinary skill in the art can determine the number of binding entities (pMHC multimers) that can be attached to the multimerization domains. US 2010/0168390 A1 discloses that the MHC multimers may be comprised of single chain MHC/peptide complexes, that the peptides that bind to MHC class | molecules are typically 8-11 amino acid residues in length.
US 2010/0168390 A1 discloses that different MHC multimers can be differently labeled enabling visualization of different target MHC-recognizing T cells; if several different MHC multimers with different labels are present, it is possible simultaneously to identify more than one specific T cell receptor, if each of the MHC multimers present a different peptide. US 2010/0168390 A1 discloses using groups of MHC multimers that are labeled with different labels together in the same preparation.
US 2010/0168390 A1 discloses that MHC multimers, including those comprising single chain MHC/peptide monomers attached to one or more multimerization domains, provide increased affinity and half-life on interaction with a cognate TCR as compared with that to the monomer MHC/peptide complex; the MHC multimers bind with high avidity to cognate T cell receptors (TCRs). US 2010/0168390 A1 discloses that the increased valences of the compounds of the invention produce surprisingly higher avidity in comparison to oligo-valent complexes such as tetramers known from the prior art, allowing for quantitative analysis of even small T cell populations, with the increased binding avidity of the MHC multimers of the invention allowing detection of MHC-recognizing T cells expressing low affinity T cell receptors. US 2010/0168390 A1 discloses that this augmented interaction also allows detection of very small MHC recognizing cell populations in blood samples without the need for in vitro expansion, and the MHC multimers of the invention are therefore useful for direct monitoring of all types of MCH recognizing cells in blood samples. US 2010/0168390 A1 discloses that these carriers are useful for binding and identifying cognate T cells comprising cognate T cell receptors on their surfaces, including for identifying low affinity binding T cells.
US 2010/0168390 A1 discloses that the MHC multimers can be labelled, for example, with one or more fluorophores and used in flow cytometry to label T cells carrying specific TCRs that bind the MHC multimers, including individual T cells or populations of T cells. US 2010/0168390 A1 discloses that the flow cytometer can also separate and collect particular types of cells, i.e., by “cell sorting’, and the MHC multimers in combination with sorting on a flow cytometer can be used to isolate antigen specific T cell populations. That is, US 2010/0168390 A1 discloses that the MHC multimers in addition to being useful for binding and identifying cognate T cells comprising cognate T cell receptors on their surfaces, are also useful for isolating the T cells for identification, further study, monitoring the antigen specific T cell response to a vaccine, or for adoptive transfer. US 2010/0168390 A1 discloses that p/MHC-specific T cells can be isolated using fluorescence activated cell sorting (FACs) when fluorescent label(s) is/are also attached to the multimer backbones. US 2010/0168390 A1 discloses that p/MHC-specific T cells can also be counted/quantified by FACs analysis (e.g., [0663], [0667], [0668], [0759], [0859], [0855], [0866], [0894]). US 2010/0168390 A1 discloses that the p/MHC I multimers of the invention also allow for better separation of specific and unspecific MHC recognizing cells ([0772]). US 2010/0168390 A1 discloses that an advantage of sorting the p/MHC specific T cells is that the relevant population of cells are selected for expansion, avoiding polyclonal expansion of T cell populations that include a multitude of irrelevant T cell specificities ([0900], [0901]). (See entire reference, especially abstract, [0003], [0013], [ [0042]-[0047], [0054], [0062],[0066]-[0086], [0094], [0095], [0134], [0145],[0161], [0195], [0196],[0200], [0207]-[0211], [(0213],[0214], [0220], [0236], [0242], [0252], [0308]-[0323],[0326], [0358],[0401 ]-[0405], [0411], [0415]-[0417], [0487], [0488], [0659], [0770], [0771], [0845], [0874]).
US 2010/0168390 A1 does not disclose that the different DNA labels are DNA barcodes comprising at least 10 nucleotides and comprised within 5’ and 3’ universal primer regions, nor wherein the composition comprises 1,000 to 10,000 different subsets of multimeric MHC, nor wherein the complexes are produced by UV peptide exchange. US 2010/0168390 A1 does not disclose wherein the coupling of the nucleic acid label to the backbone is through a streptavidin-biotin binding as is recited in instant dependent claim 49.
Andersen et al teach that producing pMHC tetramers by UV peptide exchange. Andersen et al teach that UV exchange technology enables the parallel production of large panels of peptide/MHC complexes, allowing the generation of sets of thousands of different peptide/MHC complexes within hours. Andersen et al teach that a method of using one to four dimensional fluorescent color codes (i.e., color barcodes) at the higher number of color combinations results in lower sensitivity, as the use of all possible color combinations precludes the elimination of background events that are caused by signal in only one channel or in many channels. In addition, for each additional dimension added, a consequent reduction in fluorescence intensity of each individual color takes place, and the use of four color codes and to some extent three color codes can make it difficult to distinguish antigen-specific T cells. Andersen et al teach a protocol that allows the detection of 27 different (combinatorially encoded) antigen-specific T cell populations in a single sample, wherein the protocol uses p/MHC tetramers labeled with different combinations of three QDOT fluorescent labels (color barcodes) by FACs analysis. Although Andersen et al teach that the method is more sensitive than conventional MHC multimer staining, they also teach that limitations of their method include: lower sensitivity with an often limited sample size, variation in lot to lot fluorescence, sometimes observed optical overlap in labels despite narrow emission spectra, the configuration of the flow cytometer limits the number of labels used depending upon the particular flow cytometer, differences in fluorochrome intensities exist between different labels and degradation of labels occurs over time, the QDOT fluorescent labeled-streptavidin conjugates are expensive, and structurally related peptides cannot be placed in the same panel. (See entire reference, especially page 891 at the second paragraph, sentence spanning pages 892-893, first two full paragraphs at column 1 on page 893, spanning paragraph at columns 1-2, paragraph spanning pages 893-894, first three paragraphs at the column 1 on page 894, paragraph spanning pages 894-895).
Thus, Andersen et al teach that their method of using one to four dimensional fluorescent color codes (i.e., use of color barcodes) to label MH multimers comprising MHC molecules bound to different peptides is more sensitive than conventional pMHC multimer staining (such as that disclosed by US 2010/0168390 A1), there are still limitations such as lower sensitivity with an often limited sample size, variation in lot to lot fluorescence, optical overlap in labels, dependence of limitations of the particular flow cytometer used, differences in fluorochrome intensities between different labels, label degradation over time, expense, and that structurally related peptides cannot be placed in the same panel. Andersen et al teach a protocol that allows for the parallel detection of 27 different (combinatorially encoded) antigen-specific T cell populations in a single sample. Conversely, Andersen et al teach that the UV exchange technology enables the parallel production of large panels of pMHC complexes, allowing for the generation of sets of hundreds or thousands of different pMHC complexes within hours. Thus, Andersen et al teach that they can produce hundreds to thousands of different pMHC complexes within hours, but that they can only analyze 27 different antigen-specific T cell populations in parallel, and including with the limitations they teach in such a detection assay. Andersen et al teach that there are serious limitations as to the number of different complexes that can be tested, the number of different particular p/MHC-specific TCRs that can be detected, and the sensitivity of the assay, making it difficult to identify peptide antigen/MHC-specific T cells, including wherein often the sample size is not optimal for such detection.
US2021/0239698 A1 discloses linking a peptide (that is bound to its necessary components such as b2m and MHC class I heavy chain such as in a single chain format that optionally includes flexible linker peptides between the components, e.g., [0046], [0075]-[0077]) to the said peptide’s encoding DNA through attachment of both to any suitable support carrier. US2021/0239698 A1 discloses that this is advantageous because methods that are available to sequence DNA are far more sophisticated than those available to sequence protein, it is less expensive and much more rapid, and can be successful on very small samples, both in terms of length and molar amounts. In addition DNA samples can be easily amplified to provide more DNA if needed. DNA sequencing can be undertaken by traditional Sanger based methodology or by various high throughput sequencing approaches ([0006]. US2021/0239698 A1 discloses that the aim of the invention is to provide a system to screen for ligands, in particular, to screen for ligands for cell surface receptors such as for TCRs ([0007]) that recognize and bind to a MHC molecule presenting a peptide epitope ([0008]). US2021/0239698 A1 discloses that a carrier may be advantageously be multivalent, i.e., it may carry multiple copies of each peptide/MHC complex (including single chain b2m/MHC heavy chain/peptide complex) and the peptides encoding DNA (with primer region for amplifying the templated DNA encoding the peptide), increasing the chance of its interaction with a TCR and improving the rate of recovery ([0030]). The peptide may be randomly generated or derived from a source library, and the nucleic acid may then be analyzed to determine the peptide it encodes ([0034]-[0036]). US2021/0239698 A1 discloses HLA class I molecules and that a complex thereof comprises the HLA class I heavy chain, b2m and peptide, and the complexes thereof may comprise the individual components or a single chain construct ([0039]-[0047]) and the complex and the DNA encoding the peptide are attached to a carrier (e.g., claims 1-5, 8-10). US2021/0239698 A1 discloses that a biotin moiety may be attached to the DNA when using a carrier having streptavidin disposed thereon (e.g., [0058]). Thus, US2021/0239698 A1 inherently discloses that biotin is a binding partner for streptavidin. US2021/0239698 A1 discloses that the MHC molecule may be covalently or non-covalently attached to the carrier (e.g., [0055]) including attachment to a streptavidin treated or coated carrier (e.g., [0056]). US2021/0239698 A1 discloses that preferably multiple copies of the peptide and its encoding DNA are attached to the carrier, preferably at least 10, 100, 1000 or more copies are attached ([0078]). US2021/0239698 A1 discloses that preferably each carrier comprises multiple copies of the same MHCI/peptide complex and encoding DNA, while multiple carriers may be used together and comprise different populations of carriers, each of the said carriers having a different MHC I/peptide complex/encoding DNA from one another (e.g., [0052]). See entire reference, including claims.
Thus, US2021/0239698 A1 discloses linking a peptide-b2m-MHC I single chain molecule to the peptide’s encoding DNA (i.e., a type of barcode DNA) through attachment to a same carrier, the DNA comprising a primer region, and the advantages of doing so in terms of rapid, sensitive, high throughput, parallel detection of cognate TCRs and identification of the cognate peptide ligands in the MHC molecules by isolating and sequencing the encoding DNA.
Brakmann teaches that interaction of an antibody protein disposed on a carrier that recognizes an antigen advantageously comprises one or more copies of a marker DNA [comprising PCR primer reactive sequences flanking barcode DNA, wherein the barcode DNA codes for a same particular antigen of interest), and whereby the DNA can be amplified using PCR, and wherein using multiple copies of DNA increases the sensitivity of detection of the protein of interest (for example, the ratio of recognition of the protein to marker DNA is about 1:100). Brackmann teaches that the carriers were functionalized with DNA barcodes. Brackmann teaches that if every antigen is coded by a distinct marker DNA sequence (“bio-barcodes”), parallel analysis of multiple analytes may be accomplished (see entire reference, especially barcodes for the identification of proteins section, Figure 1C).
Thus, Brakmann links a protein binding specificity to a DNA barcode sequence on a same carrier.
Likewise, WO 2013/137737 A1 cited below links a binding specificity to a DNA or other unique nucleic acid barcode. The DNA barcode is flanked by universal (i.e., same) primer regions which allow for parallel, high-throughput screening of binding region pools, including those from libraries of from 10 molecules up to one million molecules, as is enunciated below.
WO 2013/137737 A1 teaches compositions comprising library binding regions connected or covalently attached with a specific PCR-amplifiable DNA, PNA, LNA or other artificial nucleotide molecule, wherein the nucleic acid molecule can be flanked at both ends by a universal primer binding site to which primers can hybridize, serving as the starting point for amplification. WO 2013/137737 A1 teaches that the size of the unique identifier barcode is typically from 2-100 nucleotides in length, preferably 12-25 nucleotides usually being sufficient. WO 2013/137737 A1 teaches that the library may vary in size, with a lower limit of 10 molecules up to 1,000,000 molecules. WO 2013/137737 A1 teaches that the target molecule corresponding to the target of the binding region may be a receptor, including a cell surface receptor. WO 2013/137737 A1 teaches that each binding region is attached to a specific nucleotide sequence identifier and that the constructs can be placed in pools, wherein each nucleotide sequence identifier is a pool-specific sequence identifier termed a DNA barcode. WO 2013/137737 A1 teaches that the library binding region constructs are used in a method of screening of the binding regions for potential interaction with target molecules(s) including one on a cell surface, and wherein the identifying binding comprises amplifying the DNA barcodes in parallel through the universal primal primer regions an sequencing the barcodes in parallel, preferably through high throughput sequencing. WO 2013/137737 A1 teaches that the constructs may also be labeled with a tag such as a fluorescent tag (see entire reference, especially Figure 1, abstract, [22], [24], [30], [31], [40], [41], [45], [59], [60], [63], [67], claims).
Thus, the art reference US2021/0239698 A1 teaches a different type of barcode, a DNA barcode that uniquely identifies the peptide comprised in a pMHC complex and it comprises the particular peptides encoding DNA with both the pMHC complex and the encoding DNA attached to any suitable support carrier, and that the carrier may be advantageously and preferably configured to be multivalent, carrying multiple copies of each pMHC complex, including at least 10 or more copies of the pMHC and its encoding DNA (barcode) are attached. The DNA comprises primer regions for amplifying the DNA encoding the peptide. The art reference Brakmann also links a protein binding specificity to a DNA barcode, and art reference WO 2013/137737 A1 teaches compositions comprising library binding regions attached with a specific PCR-amplifiable DNA or other nucleic acid molecule that is a unique identifier barcode that is flanked at both ends by a universal primer binding site for parallel, high throughput sequencing along with their use in screening for potential interaction with target molecules, including those on a cell surface. WO 2013/137737 A1 that the library may vary in size with a lower limit of 10 molecules up to 1,000,000 molecules, indicating the combinatorial encoding power of DNA barcodes attached to a binding specificity.
It would have been prima facie obvious to one of ordinary skill in the art before the filing date of the claimed invention to have used unique DNA barcode sequences as is disclosed by US2021/0239698 A1 and taught by Brakmann, either random or corresponding to the peptide sequence, and including with 5’ and 3’ primer sequences flanking the DNA label as is taught by Brakmann, particularly those taught by WO 2013/137737 A1 having universal PCR- amplifiable 3’ and 5’ flanking primers, as the DNA label on the multimeric carriers of the primary art reference that are disclosed to have MHC class I complexes attached thereto along with a DNA label, and also a fluorescent (selectable) label.
One of ordinary skill in the art would have been motivated to do this in order to reap the aforementioned advantages of using unique barcode molecules to make a carrier that is useful and improved in identifying cognate TCRs for MHC class I/peptide complexes (e.g., sequencing DNA is far more sophisticated than protein sequencing, it is less expensive and much more rapid, it can be successful on very small samples, both in terms of length and molar amounts, and DNA samples can be easily amplified to provide more DNA if needed), and in addition, when using DNA barcodes that encode the peptide, for identifying the peptide through DNA sequences, and with a reasonable expectation of success in doing so, as the primary art reference is silent as to the identity of the different DNA labels, Brakmann and US2021/0239698 A1 teach or disclose, respectively, the advantageous use of barcode DNA labels that connect a binding specificity to its encoding DNA, while Andersen et al teach that the use of fluorescent labels to distinguish binding specificities of MHC/peptide complexes to TCRs is associated with disadvantages, (e.g., limited in the number of labels that can be used, lower sensitivity with a limited sample size, variation in lot to lot fluorescence, sometimes observed optical overlap in labels despite narrow emission spectra, differences in fluorochrome intensities exist between different labels and degradation of labels occurs over time, the QDOT fluorescent labeled-streptavidin conjugates are expensive, and structurally related peptides cannot be placed in the same panel).
It would have been prima facie obvious to one of ordinary skill in the art before the filing date of the claimed invention to have used UV peptide exchange as is taught by Anderson et al in constructing the composition of the combined references.
One of ordinary skill in the art would have been motivated to do this, and with a reasonable expectation of success in doing so, in order to produce large numbers of peptide/MHC complexes quickly, as Andersen et al teach that UV exchange technology enables the parallel production of large panels of peptide/MHC complexes, allowing the generation of sets of thousands of different peptide/MHC complexes within hours.
Instant claim 42 is included in this rejection because the primary art reference discloses that peptide may be randomly generated or derived from a source library, and one of ordinary skill in the art was aware of the size of random or source libraries, the tens of thousands of human MHC class I molecules alone (see for example, evidentiary reference HLA Nomenclature 2015, of record) as well as the repertoire of peptides that can be bound from the universe of proteins, while US2021/0239698 A1 also discloses library screening and that the method of using such a carrier with attached MHC class I/peptide complexes and the barcode DNA is high-throughput. In addition, WO 2013/137737 A1 teaches that the library may vary in size, with a lower limit of 10 molecules up to 1,000,000 molecules.
With regard to the limitation recited in instant base claim 31 “wherein the barcode comprises at least 10 nucleotides”, the instant claims are included in this rejection because the primary art reference discloses that peptides that typically bind to MHC class I molecules are 8-11 amino acid residues in length, and wherein the barcodes encode the peptide, the length of the barcode nucleotides would therefore range from 24 to 33. Wherein the barcodes are unique identifiers that don’t correspond to the actual sequence of the peptide, it would have been prima facie obvious to one of ordinary skill in the art to have used a number of nucleotides around the same size as those encoding a MHC class I binding peptide. One of ordinary skill in the art would have been motivated to do this, and with a reasonable expectation of success in doing so, as the art teaches that encoding DNA of such lengths may be used as DNA barcodes.
Applicant’s arguments have been fully considered but are not persuasive. Applicant’s said arguments are of record in the amendment and response filed 12/3/25 on pages 5-6.
Applicant alleges hindsight reconstruction. However, this said argument has been rebutted in the prosecution history. Applicant further argues that even if each of the rejections is sufficient for a prima facie showing of obviousness, they are outweighed by the advantages of the present[ly] claimed compositions, which would have been unexpected at the time of the invention over the teachings of the cited prior art. Applicant argues that the requirement of eight or more MHC molecules per multimer heightens the sensitivity of detection by introducing a high degree of binding cooperativity, for example enabling the detection of T cell populations having a lower binding affinity for a given peptide than is possible using fewer MHC molecules per multimer.
However, as is enunciated in the instant rejection, the art reference US 2010/0168390 A1 discloses that the increased valencies of the compounds of the invention produce surprisingly higher avidity in comparison to oligo-valent complexes such as tetramers known from the prior art, allowing for quantitative analysis of even small T cell populations, with the increased binding avidity of the MHC multimers of the invention allowing detection of MHC-recognizing T cells expressing low affinity receptors, and allowing for quantitative analysis of even small cell populations by for example, flow cytometry (e.g., [0659]). In addition, said art reference discloses that the number of MHC molecules can be at least eight, up to a plurality depending on the capacity and nature of the multimerization domain(s), i.e., a teaching that an optimal number of p/MHC molecules depends upon the capacity and nature of the multimerization domains (e.g., the actual backbone and its size and/or average molecular weight, the orientation of pMHC on the backbone) (e.g., [0062], [0075]). The instant specification does not appear to disclose optimal numbers of pMHC for particular backbones from those recited in the claims, nor in the examples in the specification. Although the Examiner agrees that avidity can be increased when disposing pMHC at optimized number thereof for a particular backbone (and sensitivity can additionally be increased by choice of flurophore(s)), the art indicates that this is not unexpected. The art reference also discloses using the fluorescent label as a basis for fluorescence activated cell sorting, quantifying and/or isolating/separating cognate T cells bound to the constructs (e.g., [0663], [0667], [0668], [0759], [0859], [0855], [0866], [0894]). The art reference also discloses that the pMHC multimers can comprise more than one label or more than one type of label.
Applicant’s further argument as to an exemplary use of the multimers by attaching a fluorescent label to the backbone of only one or more specific groups of MHC multimers is not persuasive, as all of the multimers in the claimed composition must necessarily comprise one or more fluorescent labels.
Applicant argues that the combination of fluorescent labeling of the backbone with nucleic acid barcoding offers two independent levels of labeling, analysis, and isolation of cells, combined with the detection of low affinity peptide-binding cell populations resulting from the use of eight or more MHC molecules per multimer, which was not possible or even suggested by the cited references. Applicant argues that the recited combination of features provides synergistic benefits that are uniquely suited to allow the tremendous diversity of immune cell specificity to be analyzed and exploited in ways that are not disclosed or suggested by any combination of the cited references.
However, the said art reference US 2010/0168390 A1 discloses that if several different MHC multimers with different labels are present, it is possible simultaneously to identify more than one specific TCR if each of the MHC multimers presents a different peptide. The art reference also discloses that the pMHC multimers can comprise more than one label or more than one type of label, including a nucleic acid label such as a DNA label.
As is also enunciated in the instant rejection, Andersen et. al. teach that their method of using one to four dimensional fluorescent color codes (i.e., use of color barcodes) to label MH multimers comprising MHC molecules bound to different peptides is more sensitive than conventional pMHC multimer staining (such as that disclosed by US 2010/0168390 A1), there are still limitations such as lower sensitivity with an often limited sample size, variation in lot to lot fluorescence, optical overlap in labels, dependence of limitations of the particular flow cytometer used, differences in fluorochrome intensities between different labels, label degradation over time, expense, and that structurally related peptides cannot be placed in the same panel. Andersen et. al. teach a protocol that allows for the parallel detection of 27 different (combinatorially encoded) antigen-specific T cell populations in a single sample. Conversely, Andersen et. al. teach that the UV exchange technology enables the parallel production of large panels of pMHC complexes, allowing for the generation of sets of hundreds or thousands of different pMHC complexes within hours. Thus, Andersen et. al. teach that they can produce hundreds to thousands of different pMHC complexes within hours, but that they can only analyze 27 different antigen-specific T cell populations in parallel, and including with the limitations they teach in such a detection assay.
The art reference US2021/0239698 A1 teaches a different type of barcode, a DNA barcode that uniquely identifies the peptide comprised in a pMHC complex and it comprises the particular peptides encoding DNA with both the pMHC complex and the encoding DNA attached to any suitable support carrier, and that the carrier may be advantageously and preferably configured to be multivalent, carrying multiple copies of each pMHC complex, including at least 10 or more copies of the pMHC and its encoding DNA (barcode) are attached. The DNA comprises primer regions for amplifying the DNA encoding the peptide. The art reference Brakmann also links a protein binding specificity to a DNA barcode, and art reference WO 2013/137737 A1 teaches compositions comprising library binding regions attached with a specific PCR-amplifiable DNA or other nucleic acid molecule that is a unique identifier barcode that is flanked at both ends by a universal primer binding site for parallel, high throughput sequencing along with their use in screening for potential interaction with target molecules, including those on a cell surface. WO 2013/137737 A1 that the library may vary in size with a lower limit of 10 molecules up to 1,000,000 molecules, indicating the combinatorial encoding power of DNA barcodes attached to a binding specificity.
Thus, the art indicates that an increase in avidity using an optimal number of pMHC, including at least eight, is not surprising for identifying low affinity TCRs, that pMHC constructs labeled with fluorochrome(s) (including for cell sorting of populations of T cells), including using several in tandem as color barcodes, can enable the use of cell sorting but is limited in its ability to identify a large number of different pMHC constructs, but that a different type of barcode (DNA or nucleic acid barcode) has the power to identify the sequence of the different peptides comprised in different pMHC complexes and matching the ability to easily and quickly produce hundreds to thousands of different pMHC complexes in parallel, rebutting Applicant’s argument of unexpected results.
6. Claims 31, 33, 34, 37, 39, 41, 42 and 46-49 are rejected under 35 U.S.C. 103 as being obvious over US2021/0239698 A1 (of record) in view of US 2010/0168390 A1 (of record), Brakmann (of record), and WO 2013/137737 A1 (of record).
Claim Interpretation: instant base claim 31 recites that eight or more MHC molecules are coupled to the backbone and a nucleic acid molecule comprising the barcode with primer regions of part “iii” is coupled to the backbone; these limitations are being interpreted to mean that the MHC molecules and the nucleic acid molecule comprising the barcode with primer regions are directly or indirectly attached or coupled to the backbone. The specification does not disclose a limiting definition for “conjugating” as in “conjugating the nucleic acid label to the backbone” as is recited in instant dependent claim 46. The said limitation is therefore being interpreted as is it is known in the art to mean ‘to join together’ or ‘chemically join together’. See for example, evidentiary reference Biology Online (2024,10 pages, of record). The definition of “barcode” in the instant specification is: “In the present context, a nucleic acid barcode is a unique oligo-nucleotide sequence ranging for [from] 10 to more than 50 nucleic acids. The barcode has shared amplification sequences in the 3’ and 5’ ends, and a unique sequence in the middle. This sequence can be revealed by sequencing and can serve as a specific barcode for a given molecule.” (see page 5 at lines 21-26).
US2021/0239698 A1 discloses linking a peptide (that is bound to its necessary components such as b2m and MHC class I heavy chain such as in a single chain format that optionally includes flexible linker peptides between the components, e.g., [0046], [0075]-[0077]) to the peptide-encoding DNA through attachment of both to any suitable solid support carrier is advantageous because methods that are available to sequence DNA are far more sophisticated than those available to sequence protein, it is less expensive and much more rapid and can be successful on very small samples, both in terms of length and molar amounts. In addition DNA samples can be easily amplified to provide more DNA if needed. DNA sequencing can be undertaken by traditional Sanger based methodology or by various high throughput sequencing approaches ([0006]. US2021/0239698 A1 discloses that the aim of the invention is to provide a system to screen for ligands, in particular, to screen for ligands for cell surface receptors such as for TCRs ([0007]) that recognize and bind to a MHC molecule presenting a peptide epitope ([0008]). US2021/0239698 A1 discloses that a carrier may be advantageously be multivalent, i.e., it may carry multiple copies of each peptide/MHC complex (including single chain b2m/MHC heavy chain/peptide complex) and the peptides encoding DNA (with primer region for amplifying the templated DNA encoding the peptide), increasing the chance of its interaction with a TCR and improving the rate of recovery ([0030]). The peptide may be randomly generated or derived from a source library, and the nucleic acid may then be analyzed to determine the peptide it encodes ([0034]-[0036]). US2021/0239698 A1 discloses HLA class I molecules and that a complex thereof comprises the HLA class I heavy chain, b2m and peptide, and the complexes thereof may comprise the individual components or a single chain construct ([0039]-[0047]) and the complex and the DNA encoding the peptide are attached to a carrier (e.g., claims 1-5, 8-10). US2021/0239698 A1 discloses that a biotin moiety may be attached to the DNA when using a carrier having streptavidin disposed thereon (e.g., [0058]). Thus, US2021/0239698 A1 inherently discloses that biotin is a binding partner for streptavidin. US2021/0239698 A1 discloses that the MHC molecule may be covalently or non-covalently attached to the carrier (e.g., [0055]) including attachment to a streptavidin treated or coated carrier (e.g., [0056]). US2021/0239698 A1 discloses that preferably multiple copies of the peptide and its encoding DNA are attached to the carrier, preferably at least 10, 100, 1000 or more copies are attached ([0078]). US2021/0239698 A1 discloses that preferably each carrier comprises multiple copies of the same MHCI/peptide complex and encoding DNA, while multiple carriers may be used together and comprise different populations of carriers, each of the said carriers having a different MHC I/peptide complex/encoding DNA from one another (e.g., [0052]). See entire reference, including claims.
Thus, US2021/0239698 A1 teaches a DNA barcode that uniquely identifies the peptide comprised in a pMHC complex and it comprises the particular peptide’s encoding DNA with both the pMHC complex and the encoding DNA attached to any suitable support carrier, and that the carrier may be advantageously and preferably configured to be multivalent, carrying multiple copies of each pMHC complex, including at least 10 or more copies of the pMHC and its encoding DNA (barcode) are attached. The DNA comprises primer regions for amplifying the DNA encoding the peptide.
US2021/0239698 A1 does not disclose that the carrier is one of a polysaccharide, a glucan, a dextran or a streptavidin, nor does US2021/0239698 A1 disclose that the DNA label that encodes the peptide also comprises 3’ and 5’ universal primer regions, nor that the barcode DNA comprises at least 10 nucleotides. US2021/0239698 A1 does not disclose that the carrier further comprises one or more fluorescent labels. US2021/0239698 A1 does not disclose wherein the composition comprises 1,000 to 10,000 different subsets of multimeric MHC. Although US2021/0239698 A1 discloses that the carrier has at least 10 copies of the peptide and its encoding DNA, it does not disclose that the lower limit includes at least 8 pMHC molecules coupled to the carrier.
US 2010/0168390 A1 discloses similar constructs, but wherein the carrier is a dextran carrier molecule or other polysaccharides such as derivatized dextrans, scleroglucan, streptavidin, streptavidin tetramers or avidin, and wherein the construct may comprise a DNA label and a fluorescent label as is enunciated below.
US 2010/0168390 A1 discloses peptide/MHC class I molecules or tetramers or other multimers thereof bound to fluorophore-labeled dextran carrier molecules (or other polysaccharides such as derivatized dextrans, scleroglucan (i.e., a glucan), streptavidin, streptavidin tetramers, or avidin); and when the complexes are bound to streptavidin, attachment is via biotin/streptavidin attachment chemistries. The streptavidin can also be attached to a derivatized dextran or other polysaccharide. US 2010/0168390 A1 also discloses other carriers such as magnetic or other beads, including those comprising dextran-coated beads comprising the MHC dextramers. US 2010/0168390 A1 discloses that the MHC molecule can be a recombinant molecule wherein the MHC class I heavy chain comprises a C-terminal target peptide sequence for biotinylation, and the chemically biotinylated MHC can then bind to streptavidin coupled to the carrier molecule. US 2010/0168390 A1 discloses that in making the MHC peptide molecule recombinantly, the heavy chain of MHC class I and the b2m light chain may be expressed separately and added together during in vitro refolding. US 2010/0168390 A1 discloses that peptide epitopes presented by MHC molecules can be presented to T cells and activates the T cells, wherein each T cell expresses one unique specificity of TCR which recognizes one specific MHC/peptide epitope complex. US 2010/0168390 A1 discloses that the dextran backbone can further comprise one or more than one detectable labels and tags such as for example, a His tag, metal-ion tag, or other selectable tags and labels such as detectable labels. US 2010/0168390 A1 discloses that the labeling molecule many be any labeling molecule such as a nucleic acid molecule, including DNA, or nucleic acid analogs, (e.g.,[0488]) and it may be attached to the MHC multimer directly or indirectly, covalently or noncovalently; it can be attached to the MHC multimer, to the multimerization domain, or to the dextran backbone. US 2010/0168390 A1 discloses that the labelling compound can be attached via a suitable linker and that such linkers are readily known by the person skilled in the art.
US 2010/0168390 A1 discloses that the number of MHC molecules can be at least two, at least four, or at least eight, up to a plurality depending on the capacity and nature of the multimerization domain(s), and the MHC can harbor the same or a different peptide; in the latter case, the composition can be used to detect several types of MHC recognizing T cells simultaneously. US 2010/0168390 A1 discloses that one of ordinary skill in the art can determine the number of binding entities (pMHC multimers) that can be attached to the multimerization domains. US 2010/0168390 A1 discloses that the MHC multimers may be comprised of single chain MHC/peptide complexes, that the peptides that bind to MHC class | molecules are typically 8-11 amino acid residues in length.
US 2010/0168390 A1 discloses that different MHC multimers can be differently labeled enabling visualization of different target MHC-recognizing T cells; if several different MHC multimers with different labels are present, it is possible simultaneously to identify more than one specific T cell receptor, if each of the MHC multimers present a different peptide. US 2010/0168390 A1 discloses using groups of MHC multimers that are labeled with different labels together in the same preparation.
US 2010/0168390 A1 discloses that MHC multimers, including those comprising single chain MHC/peptide monomers attached to one or more multimerization domains, provide increased affinity and half-life on interaction with a cognate TCR as compared with that to the monomer MHC/peptide complex; the MHC multimers bind with high avidity to cognate T cell receptors (TCRs). US 2010/0168390 A1 discloses that the increased valences of the compounds of the invention produce surprisingly higher avidity in comparison to oligo-valent complexes such as tetramers known from the prior art, allowing for quantitative analysis of even small T cell populations, with the increased binding avidity of the MHC multimers of the invention allowing detection of MHC-recognizing T cells expressing low affinity T cell receptors. US 2010/0168390 A1 discloses that this augmented interaction also allows detection of very small MHC recognizing cell populations in blood samples without the need for in vitro expansion, and the MHC multimers of the invention are therefore useful for direct monitoring of all types of MCH recognizing cells in blood samples. US 2010/0168390 A1 discloses that these carriers are useful for binding and identifying cognate T cells comprising cognate T cell receptors on their surfaces, including for identifying low affinity binding T cells.
US 2010/0168390 A1 discloses that the MHC multimers can be labelled, for example, with one or more fluorophores and used in flow cytometry to label T cells carrying specific TCRs that bind the MHC multimers, including individual T cells or populations of T cells. US 2010/0168390 A1 discloses that the flow cytometer can also separate and collect particular types of cells, i.e., by “cell sorting’, and the MHC multimers in combination with sorting on a flow cytometer can be used to isolate antigen specific T cell populations. That is, US 2010/0168390 A1 discloses that the MHC multimers in addition to being useful for binding and identifying cognate T cells comprising cognate T cell receptors on their surfaces, are also useful for isolating the T cells for identification, further study, monitoring the antigen specific T cell response to a vaccine, or for adoptive transfer. US 2010/0168390 A1 discloses that p/MHC-specific T cells can be isolated using fluorescence activated cell sorting (FACs) when fluorescent label(s) is/are also attached to the multimer backbones. US 2010/0168390 A1 discloses that p/MHC-specific T cells can also be counted/quantified by FACs analysis (e.g., [0663], [0667], [0668], [0759], [0859], [0855], [0866], [0894]). US 2010/0168390 A1 discloses that the p/MHC I multimers of the invention also allow for better separation of specific and unspecific MHC recognizing cells ([0772]). US 2010/0168390 A1 discloses that an advantage of sorting the p/MHC specific T cells is that the relevant population of cells are selected for expansion, avoiding polyclonal expansion of T cell populations that include a multitude of irrelevant T cell specificities ([0900], [0901]). (See entire reference, especially abstract, [0003], [0013], [ [0042]-[0047], [0054], [0062],[0066]-[0086], [0094], [0095], [0134], [0145],[0161], [0195], [0196],[0200], [0207]-[0211], [(0213],[0214], [0220], [0236], [0242], [0252], [0308]-[0323],[0326], [0358],[0401 ]-[0405], [0411], [0415]-[0417], [0487], [0488], [0659], [0770], [0771], [0845], [0874]).
Brakmann teaches that interaction of an antibody protein disposed on a carrier that recognizes an antigen advantageously comprises one or more copies of a marker DNA [comprising PCR primer reactive sequences flanking barcode DNA, wherein the barcode DNA codes for a same particular antigen of interest), and whereby the DNA can be amplified using PCR, and wherein using multiple copies of DNA increases the sensitivity of detection of the protein of interest (for example, the ratio of recognition of the protein to marker DNA is about 1:100). Brackmann teaches that the carriers were functionalized with DNA barcodes. Brackmann teaches that if every antigen is coded by a distinct marker DNA sequence (“bio-barcodes”), parallel analysis of multiple analytes may be accomplished (see entire reference, especially barcodes for the identification of proteins section, Figure 1C). Thus, Brakmann link a binding specificity to a DNA barcode sequence on a same carrier.
Likewise, WO 2013/137737 A1 links a binding specificity to a DNA or other nucleic acid barcode that is flanked by universal primer regions which allows for parallel, high-throughput screening of binding region pools, including those from libraries of from 10 molecules up to one million molecules, as is enunciated below. WO 2013/137737 A1 that the library may vary in size with a lower limit of 10 molecules up to 1,000,000 molecules, indicating th