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 .
Elections/Restrictions
Applicant’s election without traverse of Group I in the reply filed on May 20, 2026 is acknowledged. Claims 33,36-37,39,41,49-51,54 and 57 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected Group 2, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 5/20/2026. Thus, claims 1-2, 5-6, 8-11, 13-14, 17-18, 28-30, 81, 84, and 141-148 are under examination (5/20/2026).
Claim Status
Claims 3-4, 7, 12, 15-16, 19-27, 31-32, 34-35, 38, 40, 42-48, 52-53, 55-56, 58-80, 82-83 and 85-140 are canceled (5/20/2026). Claims 1-2,5-6,8-11,13-14,17-18,28-30,33,36-37,39,41,49-51,54,57,81,84 and 141-148 are pending (5/20/2026). Claim 13 has been amended (5/20/2026). Applicant previously selected Group I without traverse in response to the Restriction Requirement (3/26/2026), thus, claims 1-2, 5-6, 8-11, 13-14, 17-18, 28-30, 81, 84, and 141-148 are under examination (5/20/2026).
Priority
Claims 1-2, 5-6, 8-11, 13-14, 17-18, 28-30, 81, 84, and 141-148 receive a priority date of 5/26/2020, the effective filing date of US Provisional Patent 63/030,056.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure
statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information
submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be
incorporated into the specification but must be submitted in a separate paper." Therefore, unless
the references have been cited by the examiner on form PTO-892, they have not been
considered.
Information disclosure statements (IDS) were submitted on 5/23/2023 and 5/20/2026 and are being considered by the examiner.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the terms “Illumina” (p. 16, 64, 68), “Life Technologies” (p. 36), “New England BioLabs” (p. 65-68), “Bio-Rad” (p. 65), “Twist Bioscience” (p. 65), “Agilent” (p. 66-67), “Thermo Fisher” (p. 66-68), “IBA LifeSciences” (p. 68), and “Promega” (p. 69) which are trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 at part (a), step (iii) at line 6 :wherein the 3’ end of RNA transcripts are joined” should be “is joined” because “the 3’ end” is singular.
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 1-2, 5-6, 8-11, 13-14, 17-18, 28-29 and 30 and 141-148 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 1 is rejected. Claim 1 recites the limitation “the RNA transcripts" in claim 1, step (c), line 1. There is insufficient antecedent basis for this limitation in the claim. Specifically, earlier in the claim, the RNA transcripts are modified by joining to puromycin-tagged RNA transcripts, and step (b) is performed on the puromycin-tagged RNA transcripts. However, step (c) recites separating the polypeptide-linked RNA complexes from “the RNA transcripts” without specifying whether this refers to the original population of RNA transcripts, the puromycin-tagged RNA transcripts, or another set of RNA transcripts. Accordingly, it is unclear which RNA transcripts are intended.
Claims 5-6, 8-11, 28-29 and 30 are dependent on this rejection due to their dependency on claim 1.
Claim 2 is rejected. Claim 2 recites the limitation “the RNA transcripts" in claim 2, step (c), line 1. There is insufficient antecedent basis for this limitation in the claim. Specifically, earlier in the claim, the RNA transcripts are modified by joining to puromycin-tagged RNA transcripts, and step (b) is performed on the puromycin-tagged RNA transcripts. However, step (c) recites separating the polypeptide-linked RNA complexes from “the RNA transcripts” without specifying whether this refers to the original population of RNA transcripts, the puromycin-tagged RNA transcripts, or another set of RNA transcripts. Accordingly, it is unclear which RNA transcripts are intended.
Claims 141-148 are dependent on this rejection due to their dependency on claim 2.
Claim 13 is rejected. Claim 13 recites the limitation “the RNA transcripts" in claim 13, step (6), line 1. There is insufficient antecedent basis for this limitation in the claim. Specifically, earlier in the claim, the RNA transcripts are modified by joining to puromycin-tagged RNA transcripts, and step (b) is performed on the puromycin-tagged RNA transcripts. However, step (c) recites separating the polypeptide-linked RNA complexes from “the RNA transcripts” without specifying whether this refers to the original population of RNA transcripts, the puromycin-tagged RNA transcripts, or another set of RNA transcripts. Accordingly, it is unclear which RNA transcripts are intended.
Claim 17 is included in this rejection due to its dependency on claim 13.
Claim 14 is rejected. Claim 14 recites the limitation “the RNA transcripts" in claim 14, step (g), line 1. There is insufficient antecedent basis for this limitation in the claim. Specifically, earlier in the claim, the RNA transcripts are modified by joining to puromycin-tagged RNA transcripts, and step (b) is performed on the puromycin-tagged RNA transcripts. However, step (c) recites separating the polypeptide-linked RNA complexes from “the RNA transcripts” without specifying whether this refers to the original population of RNA transcripts, the puromycin-tagged RNA transcripts, or another set of RNA transcripts. Accordingly, it is unclear which RNA transcripts are intended.
Claim 18 is included in this rejection due to its dependency on claim 14.
Claim 28 is further rejected. Claim 28 recites the limitation “the amplification products" in claim 28, lines 1-2. There is insufficient antecedent basis for this limitation in the claim. Specifically, claim 6, from which claim 28 depends, recites “to generate an amplification product”, therefore making it unclear whether the plural form of this in the dependent claim includes other products.
Claim 143 is further rejected. Claim 143 recites the limitation “the library of RNA transcripts" in claim 143, line 1. There is insufficient antecedent basis for this limitation in the claim. Specifically, claim 2, from which claim 143 depends recites a method of generating a “library of in-frame cording region fragments from a population of RNA transcripts”, therefore making it unclear whether the library in the dependent claim is composed of the same RNA transcript population.
Claims 144-146 are included in this rejection due to their dependency on claim 143.
Claim 147 is further rejected. Claim 147 recites the limitation “the amplification products" in claim 147, lines 1-2. There is insufficient antecedent basis for this limitation in the claim. Specifically, claim 142, from which claim 147 depends, recites “to generate an amplification product”, therefore making it unclear whether the plural form of this in the dependent claim includes other products.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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 1-2, 5-6, 8-11, 13-14, 17-18, 28-30, 81, 84, and 141-148 are rejected under 35 U.S.C. 103 as being unpatentable over Deering et al. (WO 2018/213803 A1, published: 11/22/2018) and Govarts et al. (“Frameshifting in the P6 cDNA Phage Display System”, molecules, published 2010), and further in view of Kipnis et al., (USPGPub 2021/0311076 A1, filed 7/15/2019).
Regarding claims 1-2, 5-6, Deering teaches methods and compositions for identifying immunogenic neoantigens and methods of using such neoantigens for the treatment of disease and for producing therapeutics for the treatment of disease (Abstract). Further, Deering teaches in some aspects, provided herein is a method for identifying a subject-specific and tumor-specific polypeptide sequence or polynucleotide sequence encoding the subject-specific and tumor-specific polypeptide sequence for preparing an immunogenic composition, the method comprising: contacting an antigen presenting cell (APC) of a plurality of APCs with a T cell of a plurality of T cells, forming one or more APC:T cell conjugates, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; isolating an APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both; and sequencing the target polynucleotide that encodes the target polypeptide from the APC of the APC:T cell conjugate; wherein the target polypeptide comprises a neoantigen sequence encoded by a nucleic acid of a tumor cell sample of a subject and that is not encoded by a nucleic acid of a non-tumor cell sample of the subject (Paragraphs 5-9). Deering also teaches that in some embodiments, the cell is an antigen presenting cell (APC), where in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; and isolating the APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both (Paragraph 10, lines 1-5).
Deering also teaches that in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; and selectively enriching for the target polynucleotide from the APC of the APC:T cell conjugate, wherein the enriching comprises transcribing or amplifying the target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide (Paragraph 13, lines 1-5). Deering teaches that in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide comprising at most about 100 amino acids encoded by a target polynucleotide, wherein the target polynucleotide is an in vitro transcribed target RNA, and wherein the APC and/or the T cell is from a subject with a disease or condition; and identifying target RNA enriched from cellular RNA isolated from the APC of the APC:T cell conjugate to encode for an immunogenic polypeptide (Paragraph 14, lines 1-5). And, further, Deering teaches that in some embodiments, the APC comprises an amplifier polynucleotide comprising a sequence encoding an amplifier polypeptide that transcribes the target polynucleotide and in some embodiments, the amplifier polynucleotide is linear or, the amplifier polynucleotide is an in vitro transcribed RNA, or the target polynucleotide is an in vitro transcribed RNA, or the target polynucleotide is DNA, or the target polynucleotide is linear or, the target polypeptide comprises at most about 100 amino acids, or the target polypeptide comprises at least about 20 amino acids or, the target polypeptide comprises from about 20 to about 90 amino acids or, the target polypeptide or a fragment thereof is presented on the APC (Paragraph 16, lines 1-10). Deering also teaches that in some embodiments, the method further comprises contacting the antigen presenting cell (APC) with a T cell, or in some embodiments, the method further comprises forming an APC:T cell conjugate or, the method further comprises isolating an APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both or, the method further comprises sequencing a target polynucleotide that encodes the target polypeptide from an APC of an APC:T cell conjugate or, the method further comprises identifying a target polynucleotide to encode for an immunogenic target polypeptide or selecting an immunogenic target polypeptide (Paragraph 16, lines 10-15). Deering also teaches that in some embodiments, the immunogenic target polypeptide elicits in an immune response when administered to a subject with a disease or condition or, the immunogenic target polypeptide elicits an immune response when administered to a subject with a disease or condition or, the immunogenic target polypeptide increases an immune response when administered to a subject with a disease or condition or, the method further comprises contacting the immunogenic target polypeptide or a nucleic acid encoding the immunogenic target polypeptide to a plurality of cells or, the plurality of cells comprises a plurality of APCs or, the method further comprises administering one or more cells of the plurality of APCs to a subject or, the method further comprises contacting a plurality of T cells to the plurality of APCs (Paragraph 16, lines 15-30). Further, Deering teaches that in some embodiments, the plurality of APCs is from a subject or, the plurality of APCs is from a cell line or, the plurality of APCs is engineered cells or, the plurality of T cells is from a subject or, the plurality of T cells is from a cell line or, the plurality of T cells is engineered cells. In some embodiments, the method further comprises expanding one or more T cells of the plurality of T cells (Paragraph 16, lines 30-40).
Further, Deering teaches that in some embodiments, the method further comprises administering one or more cells of the plurality of T cells to a subject or, the plurality of T cells and the plurality of APCs are from the same subject or, the method is performed ex vivo or, the method further comprises enriching the target polynucleotide from cellular R A from the APC of the APC:T cell conjugate or, the method further comprises selectively enriching for the target polynucleotide from an APC of an APC:T cell conjugate, wherein the enriching comprises transcribing or amplifying the target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide where, the APC is from a subject with a disease or conditions and, the target polypeptide comprises a neoantigen or a neoepitope. In some embodiments, the target polypeptide comprises an immunogenic peptide or an immunogenic epitope or, the target polypeptide comprises a mutant peptide, or the target polypeptide comprises a cancer peptide, a viral peptide, an infectious disease peptide, or a non-cancer disease-associated peptide (Paragraph 16, lines 40-50). Also, Deering teaches that in some embodiments, the non-cancer disease-associated peptide is a peptide associated with a disease selected from the group consisting of diabetes, Crohn's, ulcerative colitis and IBD, and arthritis where in some embodiments, the target polypeptide activates a T cell or stimulates a T cell to proliferate. In some embodiments, the target polypeptide activates a CD4.sup.+ T cell or a CD8.sup.+ T cell or, the target polypeptide stimulates a CD4.sup.+ T cell or a CD8.sup.+ T cell to proliferate or, the target polypeptide stimulates a CD4.sup.+ T cell or a CD8.sup.+ T cell to proliferate when an APC comprising an MHC bound to the target peptide is contacted to the CD4.sup.+ T cell or the CD8.sup.+ T cell, or the T cell is from a subject with a disease or condition or, the target polynucleotide is incorporated into the genome of the APC where the genome of the APC comprises the target polynucleotide or, the APC is a CRISPR-Cas engineered APC comprising the target polynucleotide (Paragraph 16, lines 50-60). Deering also teaches that in some embodiments, the target polynucleotide further comprises a sequence encoding a reporter tag or wherein the target polypeptide is linked to a reporter tag or, the reporter tag comprises a reporter tag that can be detected in a cell optically or, the reporter tag comprises a fluorescent reporter tag or, the reporter tag comprises a fluorescent protein, e.g., GFP. In some embodiments, isolating an APC:T cell conjugate comprises isolating APC:T cell conjugates comprising the reporter tag where, APC:T cell conjugates without the reporter tag are not isolated (Paragraph 16, lines 60-65). Further, Deering teaches that in some embodiments, the method comprises selecting a target polynucleotide of an APC:T cell conjugate where in some embodiments, the selecting comprises selecting a target polynucleotide of an APC:T cell conjugate, or the APC:T cell conjugate comprises a plurality of APC:T cell conjugates and the selecting comprises selecting a target polynucleotide based on a frequency of the target polynucleotide or a barcode of a target polynucleotide in the plurality of APC:T cell conjugates where, the T cell comprises an engineered T cell or, the engineered T cell is engineered to express a TCR of interest (Paragraph 16, lines 65-70). Deering further teaches that in some embodiments, the method comprises identifying a target polypeptide that interacts with a TCR of interest expressed by the engineered T cell. In some embodiments, the target polynucleotide comprises one or more target polynucleotides encoding a library of neoantigens where in some embodiments, the method comprises identifying a neoantigen of the library of neoantigens that binds to a TCR of interest expressed by the engineered T cell and in some embodiments, the APC is a first APC, and wherein the method further comprises expressing a polypeptide from a selectively transcribed target RNA in a second antigen presenting cell (APC); contacting the second APC to a second T cell, thereby forming a second APC:T cell conjugate; isolating cellular RNA from the second APC of the second APC:T cell conjugate; and enriching for the target RNA from the cellular RNA where, the method further comprises identifying a target RNA enriched from cellular RNA isolated from an APC of an APC:T cell conjugate to encode for a polypeptide that elicits an immune response when administered to a subject with a disease or condition or, the isolated APC:T cell conjugate is isolated by flow cytometry or magnetic beads and, the method further comprises isolating polynucleotides from the APC and/or T cell of the APC:T cell conjugate where, the isolated polynucleotides comprise cellular RNA. In some embodiments, the isolated polynucleotides comprise mRNA, or , the isolated polynucleotides comprise the target polynucleotide or the method further comprises reverse transcribing a target polynucleotide from the APC of an APC:T cell conjugate (Paragraph 16, lines 70-80).
Deering also teaches that in some embodiments, the method comprises transcribing or amplifying a target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide and in some embodiments, the APC comprises a plurality of APCs and in some embodiments, the APC is a dendritic cell (DC) where, the dendritic cell is an immature dendritic cell (iDC), where in some embodiments, the T cell comprises a plurality of T cells. In some embodiments, the T cell and the APC are from the same subject (Paragraph 17, lines 1-5). Further, Deering teaches that in some embodiments, the target polynucleotide comprises a plurality of target polynucleotides where, the target polynucleotide is RNA or, the target polynucleotide is mRNA or, the target polynucleotide encodes a candidate immunogenic neoantigen or, the target polynucleotide and the amplifier polynucleotide are mixed at a molar concentration ratio of from about 1:2 to 1 : 1000 or from about 1 :20 to 1 : 1000 or from about 1 :2 to 1 : 100 before contacting with the APC (Paragraph 17, lines 5-10). Deering also teaches that in some embodiments, the method is performed in vitro or ex vivo where, the target polynucleotide and the amplifier polynucleotide are delivered into the APC by nucleofection or transfection or electroporation where, the amplifier polynucleotide is transcribed from an amplifier expression polynucleotide comprising a sequence encoding the amplifier polynucleotide and in some embodiments, the target polynucleotide is transcribed from a target expression polynucleotide comprising a sequence encoding the target polynucleotide or, the amplifier expression polynucleotide and/or the target expression polynucleotide is a vector and, the target expression polynucleotide comprises a target polynucleotide ID barcode sequence where in some embodiments, the target polynucleotide ID barcode sequence is barcoded to the sequence encoding the target polynucleotide or the target polypeptide encoded by the target polynucleotide and, the target polynucleotide ID barcode sequence of each target polynucleotide encoding a different target polypeptide is unique (Paragraph 17, lines 10-20).
Deering further teaches that the previously described method can in some embodiments be used in junction with a neoantigen therapeutic and administered as a combination therapy with two or more therapeutic agents uses agents that work by different mechanisms of action, although this is not required and can result in additive or synergetic effects and can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s) and can decrease the likelihood that resistant cancer cells will develop where, combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells (Paragraph 575, lines 1-10). Further, Deering teaches that therapeutic agents that can be administered in combination with the neoantigen therapeutic described herein include chemotherapeutic agents and thus, in some embodiments, the method or treatment involves the administration of an agent described herein in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents (Paragraph 580, lines 1-5). Specifically, Deering teaches that useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor (Paragraph 581, lines 1-5).
Regarding claims 8-11, Deering teaches that the present disclosure also provides non-limiting examples of applications where the methods and compositions described herein can be used and these examples include 1) functional ranking of in silico- predicted antigens to inform immunogenic antigen selection; 2) query both CD4.sup.+ T cell responses and CD8.sup.+ T cell responses to antigen specific responses to epitopes simultaneously; 3) link epitopes with cognate TCR sequences for development of T cell therapies; 4) rapid screening of low-frequency memory responses from patient peripheral blood to monitor responses to immunotherapy; 5) functional ranking predicted antigens to inform immunogenic antigen selection for any immunotherapy, including infectious disease immunotherapies (Paragraph 143, lines 1-5). Further, Deering teaches that generally, one or more candidate antigen sequences can be first determined, e.g., in silico (FIG. 1) and nucleotide sequences, such as DNA or RNA sequences encoding identified antigen sequences can then be synthesized and neoantigen encoding sequences can then be assembled into a vector sequence, e.g., a self- amplifying mRNA vector, under control of a promoter, e.g., a replicon subgenomic promoter, where RNA encoding neoantigen sequences can then be produced, e.g., by in vitro transcription (Paragraph 146, lines 1-10). Further Deering teaches that the RNA encoding neoantigen sequences can then be delivered, e.g., in a pooled fashion, to antigen presenting cells (APCs), e.g., primary dendritic cells (DCs) and after the neoantigen sequences are processed by the APCs, the APCs presenting the processed neoantigen sequences can be contacted to T cells, e.g., autologous T cells and after exposure of the APCs to the T cells, APC:T cell conjugates can be isolated, e.g., by using T cell activation as a functional bait (FIG. 2), where RNA, e.g., total polyadenylated mRNA, can then be isolated from one or more of a pool of the APC:T cell conjugates (Paragraph 146, lines 10-15). Deering also teaches that neoantigen-encoding replicon RNA can then be specifically synthesized, e.g., by in vitro transcription using constant sequence features on replicon molecules and the method can enrich for RNA sequences from APCs to which T cells specifically respond, where enriched RNA sequences can be sequenced to identify these RNA sequences from APCs to which the T cells specifically respond and to continue to narrow the immunogenic neoantigens by selective RNA enrichment, RNA sequences isolated from APC:T cell conjugates can be tested in a subsequent round of the RaFT method by re -introducing the RNA sequences to a new culture of matched APCs and in some embodiments, the RaFT method can be performed for one round, or the RaFT method can be performed for more than one round or, mRNA encoding a neoantigen sequence can be a linear mRNA or, the linear mRNA encoding a neoantigen sequence is in vitro transcribed or, the linear mRNA encoding a neoantigen sequence can be delivered into a APC (Paragraph 146, lines 15-20).
Further, Deering teaches that in both animals and humans, mutated epitopes can be potentially effective in inducing an immune response or activating T cells and in one embodiment, the potentially immunogenic epitopes of an infectious agent in a subject, such as a virus, can be determined where, the potentially immunogenic mutated epitopes of a subject with a disease, such as cancer, can be determined an in some embodiments, a potentially immunogenic neoantigen for use in the methods described herein can be a differentiation antigen expressed in a tumor and cells of the type of tissue from which they are generated or, a potentially immunogenic neoantigen for use in the methods described herein can be a cancer/germ line antigens not expressed in another differentiated tissue or, a potentially immunogenic neoantigen for use in the methods described herein can be a mutated antigen (Paragraph 148, lines 1-10). Deering further teaches that for example, a candidate immunogenic neoantigen for use in the methods described herein can comprise a missense-point mutation or a neoantigen of a fusion protein generated through tumor specific translocation of a gene segment and in some embodiments, a potentially immunogenic neoantigen for use in the methods described herein can be an over- expressed antigen, or, a potentially immunogenic neoantigen can be found in tumors where for example, a potentially immunogenic neoantigen for use in the methods described herein can include a protein whose expression is strictly regulated in cells of differentiated normal tissue. (Paragraph 148, lines 10-15).
Regarding claims 13-14, 17-18 and 28-30, Deering teaches methods and compositions for identifying immunogenic neoantigens and methods of using such neoantigens for the treatment of disease and for producing therapeutics for the treatment of disease (Abstract). Further, Deering teaches in some aspects, provided herein is a method for identifying a subject-specific and tumor-specific polypeptide sequence or polynucleotide sequence encoding the subject-specific and tumor-specific polypeptide sequence for preparing an immunogenic composition, the method comprising: contacting an antigen presenting cell (APC) of a plurality of APCs with a T cell of a plurality of T cells, forming one or more APC:T cell conjugates, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; isolating an APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both; and sequencing the target polynucleotide that encodes the target polypeptide from the APC of the APC:T cell conjugate; wherein the target polypeptide comprises a neoantigen sequence encoded by a nucleic acid of a tumor cell sample of a subject and that is not encoded by a nucleic acid of a non-tumor cell sample of the subject (Paragraphs 5-9). Deering also teaches that in some embodiments, the cell is an antigen presenting cell (APC), where in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; and isolating the APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both (Paragraph 10, lines 1-5).
Deering also teaches that in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; and selectively enriching for the target polynucleotide from the APC of the APC:T cell conjugate, wherein the enriching comprises transcribing or amplifying the target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide (Paragraph 13, lines 1-5). Deering teaches that in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide comprising at most about 100 amino acids encoded by a target polynucleotide, wherein the target polynucleotide is an in vitro transcribed target RNA, and wherein the APC and/or the T cell is from a subject with a disease or condition; and identifying target RNA enriched from cellular RNA isolated from the APC of the APC:T cell conjugate to encode for an immunogenic polypeptide (Paragraph 14, lines 1-5). And, further, Deering teaches that in some embodiments, the APC comprises an amplifier polynucleotide comprising a sequence encoding an amplifier polypeptide that transcribes the target polynucleotide and in some embodiments, the amplifier polynucleotide is linear or, the amplifier polynucleotide is an in vitro transcribed RNA, or the target polynucleotide is an in vitro transcribed RNA, or the target polynucleotide is DNA, or the target polynucleotide is linear or, the target polypeptide comprises at most about 100 amino acids, or the target polypeptide comprises at least about 20 amino acids or, the target polypeptide comprises from about 20 to about 90 amino acids or, the target polypeptide or a fragment thereof is presented on the APC (Paragraph 16, lines 1-10). Deering also teaches that in some embodiments, the method further comprises contacting the antigen presenting cell (APC) with a T cell, or in some embodiments, the method further comprises forming an APC:T cell conjugate or, the method further comprises isolating an APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both or, the method further comprises sequencing a target polynucleotide that encodes the target polypeptide from an APC of an APC:T cell conjugate or, the method further comprises identifying a target polynucleotide to encode for an immunogenic target polypeptide or selecting an immunogenic target polypeptide (Paragraph 16, lines 10-15). Deering also teaches that in some embodiments, the immunogenic target polypeptide elicits in an immune response when administered to a subject with a disease or condition or, the immunogenic target polypeptide elicits an immune response when administered to a subject with a disease or condition or, the immunogenic target polypeptide increases an immune response when administered to a subject with a disease or condition or, the method further comprises contacting the immunogenic target polypeptide or a nucleic acid encoding the immunogenic target polypeptide to a plurality of cells or, the plurality of cells comprises a plurality of APCs or, the method further comprises administering one or more cells of the plurality of APCs to a subject or, the method further comprises contacting a plurality of T cells to the plurality of APCs (Paragraph 16, lines 15-30). Further, Deering teaches that in some embodiments, the plurality of APCs is from a subject or, the plurality of APCs is from a cell line or, the plurality of APCs is engineered cells or, the plurality of T cells is from a subject or, the plurality of T cells is from a cell line or, the plurality of T cells is engineered cells. In some embodiments, the method further comprises expanding one or more T cells of the plurality of T cells (Paragraph 16, lines 30-40).
Further, Deering teaches that in some embodiments, the method further comprises administering one or more cells of the plurality of T cells to a subject or, the plurality of T cells and the plurality of APCs are from the same subject or, the method is performed ex vivo or, the method further comprises enriching the target polynucleotide from cellular R A from the APC of the APC:T cell conjugate or, the method further comprises selectively enriching for the target polynucleotide from an APC of an APC:T cell conjugate, wherein the enriching comprises transcribing or amplifying the target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide where, the APC is from a subject with a disease or conditions and, the target polypeptide comprises a neoantigen or a neoepitope. In some embodiments, the target polypeptide comprises an immunogenic peptide or an immunogenic epitope or, the target polypeptide comprises a mutant peptide, or the target polypeptide comprises a cancer peptide, a viral peptide, an infectious disease peptide, or a non-cancer disease-associated peptide (Paragraph 16, lines 40-50). Also, Deering teaches that in some embodiments, the non-cancer disease-associated peptide is a peptide associated with a disease selected from the group consisting of diabetes, Crohn's, ulcerative colitis and IBD, and arthritis where in some embodiments, the target polypeptide activates a T cell or stimulates a T cell to proliferate. In some embodiments, the target polypeptide activates a CD4.sup.+ T cell or a CD8.sup.+ T cell or, the target polypeptide stimulates a CD4.sup.+ T cell or a CD8.sup.+ T cell to proliferate or, the target polypeptide stimulates a CD4.sup.+ T cell or a CD8.sup.+ T cell to proliferate when an APC comprising an MHC bound to the target peptide is contacted to the CD4.sup.+ T cell or the CD8.sup.+ T cell, or the T cell is from a subject with a disease or condition or, the target polynucleotide is incorporated into the genome of the APC where the genome of the APC comprises the target polynucleotide or, the APC is a CRISPR-Cas engineered APC comprising the target polynucleotide (Paragraph 16, lines 50-60). Deering also teaches that in some embodiments, the target polynucleotide further comprises a sequence encoding a reporter tag or wherein the target polypeptide is linked to a reporter tag or, the reporter tag comprises a reporter tag that can be detected in a cell optically or, the reporter tag comprises a fluorescent reporter tag or, the reporter tag comprises a fluorescent protein, e.g., GFP. In some embodiments, isolating an APC:T cell conjugate comprises isolating APC:T cell conjugates comprising the reporter tag where, APC:T cell conjugates without the reporter tag are not isolated (Paragraph 16, lines 60-65). Further, Deering teaches that in some embodiments, the method comprises selecting a target polynucleotide of an APC:T cell conjugate where in some embodiments, the selecting comprises selecting a target polynucleotide of an APC:T cell conjugate, or the APC:T cell conjugate comprises a plurality of APC:T cell conjugates and the selecting comprises selecting a target polynucleotide based on a frequency of the target polynucleotide or a barcode of a target polynucleotide in the plurality of APC:T cell conjugates where, the T cell comprises an engineered T cell or, the engineered T cell is engineered to express a TCR of interest (Paragraph 16, lines 65-70). Deering further teaches that in some embodiments, the method comprises identifying a target polypeptide that interacts with a TCR of interest expressed by the engineered T cell. In some embodiments, the target polynucleotide comprises one or more target polynucleotides encoding a library of neoantigens where in some embodiments, the method comprises identifying a neoantigen of the library of neoantigens that binds to a TCR of interest expressed by the engineered T cell and in some embodiments, the APC is a first APC, and wherein the method further comprises expressing a polypeptide from a selectively transcribed target RNA in a second antigen presenting cell (APC); contacting the second APC to a second T cell, thereby forming a second APC:T cell conjugate; isolating cellular RNA from the second APC of the second APC:T cell conjugate; and enriching for the target RNA from the cellular RNA where, the method further comprises identifying a target RNA enriched from cellular RNA isolated from an APC of an APC:T cell conjugate to encode for a polypeptide that elicits an immune response when administered to a subject with a disease or condition or, the isolated APC:T cell conjugate is isolated by flow cytometry or magnetic beads and, the method further comprises isolating polynucleotides from the APC and/or T cell of the APC:T cell conjugate where, the isolated polynucleotides comprise cellular RNA. In some embodiments, the isolated polynucleotides comprise mRNA, or , the isolated polynucleotides comprise the target polynucleotide or the method further comprises reverse transcribing a target polynucleotide from the APC of an APC:T cell conjugate (Paragraph 16, lines 70-80).
Deering also teaches that in some embodiments, the method comprises transcribing or amplifying a target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide and in some embodiments, the APC comprises a plurality of APCs and in some embodiments, the APC is a dendritic cell (DC) where, the dendritic cell is an immature dendritic cell (iDC), where in some embodiments, the T cell comprises a plurality of T cells. In some embodiments, the T cell and the APC are from the same subject (Paragraph 17, lines 1-5). Further, Deering teaches that in some embodiments, the target polynucleotide comprises a plurality of target polynucleotides where, the target polynucleotide is RNA or, the target polynucleotide is mRNA or, the target polynucleotide encodes a candidate immunogenic neoantigen or, the target polynucleotide and the amplifier polynucleotide are mixed at a molar concentration ratio of from about 1:2 to 1 : 1000 or from about 1 :20 to 1 : 1000 or from about 1 :2 to 1 : 100 before contacting with the APC (Paragraph 17, lines 5-10). Deering also teaches that in some embodiments, the method is performed in vitro or ex vivo where, the target polynucleotide and the amplifier polynucleotide are delivered into the APC by nucleofection or transfection or electroporation where, the amplifier polynucleotide is transcribed from an amplifier expression polynucleotide comprising a sequence encoding the amplifier polynucleotide and in some embodiments, the target polynucleotide is transcribed from a target expression polynucleotide comprising a sequence encoding the target polynucleotide or, the amplifier expression polynucleotide and/or the target expression polynucleotide is a vector and, the target expression polynucleotide comprises a target polynucleotide ID barcode sequence where in some embodiments, the target polynucleotide ID barcode sequence is barcoded to the sequence encoding the target polynucleotide or the target polypeptide encoded by the target polynucleotide and, the target polynucleotide ID barcode sequence of each target polynucleotide encoding a different target polypeptide is unique (Paragraph 17, lines 10-20).
Deering further teaches that the previously described method can in some embodiments be used in junction with a neoantigen therapeutic and administered as a combination therapy with two or more therapeutic agents uses agents that work by different mechanisms of action, although this is not required and can result in additive or synergetic effects and can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s) and can decrease the likelihood that resistant cancer cells will develop where, combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells (Paragraph 575, lines 1-10). Further, Deering teaches that therapeutic agents that can be administered in combination with the neoantigen therapeutic described herein include chemotherapeutic agents and thus, in some embodiments, the method or treatment involves the administration of an agent described herein in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents (Paragraph 580, lines 1-5). Specifically, Deering teaches that useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor (Paragraph 581, lines 1-5).
Additionally, Deering teaches that the present disclosure also provides non-limiting examples of applications where the methods and compositions described herein can be used and these examples include 1) functional ranking of in silico- predicted antigens to inform immunogenic antigen selection; 2) query both CD4.sup.+ T cell responses and CD8.sup.+ T cell responses to antigen specific responses to epitopes simultaneously; 3) link epitopes with cognate TCR sequences for development of T cell therapies; 4) rapid screening of low-frequency memory responses from patient peripheral blood to monitor responses to immunotherapy; 5) functional ranking predicted antigens to inform immunogenic antigen selection for any immunotherapy, including infectious disease immunotherapies (Paragraph 143, lines 1-5).
Further, Deering teaches that in both animals and humans, mutated epitopes can be potentially effective in inducing an immune response or activating T cells and in one embodiment, the potentially immunogenic epitopes of an infectious agent in a subject, such as a virus, can be determined where, the potentially immunogenic mutated epitopes of a subject with a disease, such as cancer, can be determined an in some embodiments, a potentially immunogenic neoantigen for use in the methods described herein can be a differentiation antigen expressed in a tumor and cells of the type of tissue from which they are generated or, a potentially immunogenic neoantigen for use in the methods described herein can be a cancer/germ line antigens not expressed in another differentiated tissue or, a potentially immunogenic neoantigen for use in the methods described herein can be a mutated antigen (Paragraph 148, lines 1-10). Deering further teaches that for example, a candidate immunogenic neoantigen for use in the methods described herein can comprise a missense-point mutation or a neoantigen of a fusion protein generated through tumor specific translocation of a gene segment and in some embodiments, a potentially immunogenic neoantigen for use in the methods described herein can be an over- expressed antigen, or, a potentially immunogenic neoantigen can be found in tumors where for example, a potentially immunogenic neoantigen for use in the methods described herein can include a protein whose expression is strictly regulated in cells of differentiated normal tissue. (Paragraph 148, lines 10-15).
Regarding claims 81 and 84, Deering further teaches that the previously described method can in some embodiments be used in junction with a neoantigen therapeutic and administered as a combination therapy with two or more therapeutic agents uses agents that work by different mechanisms of action, although this is not required and can result in additive or synergetic effects and can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s) and can decrease the likelihood that resistant cancer cells will develop where, combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells (Paragraph 575, lines 1-10). Further, Deering teaches that therapeutic agents that can be administered in combination with the neoantigen therapeutic described herein include chemotherapeutic agents and thus, in some embodiments, the method or treatment involves the administration of an agent described herein in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents (Paragraph 580, lines 1-5). Specifically, Deering teaches that useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor (Paragraph 581, lines 1-5).
Specifically, Deering teaches that in some embodiments, antigens are selected for administering to one or more subjects based on binding to TCRs and in some embodiments, T cells, such as T cells from a subject with a disease or condition, can be expanded, where expanded T cells that express TCRs specific to an immunogenic antigen peptide, can be administered back to a subject (Paragraph 552, liens 1-5). Further, Deering teaches that in some embodiments, suitable cells, e.g., PBMCs, are transduced or transfected with polynucleotides for expression of TCRs specific to an immunogenic antigen peptide and administered to a subject and T cells expressing TCRs specific to an immunogenic antigen peptide can be expanded and administered back to a subject where, T cells that express TCRs specific to an immunogenic antigen peptide that result in cytolytic activity when incubated with autologous diseased tissue can be expanded and administered to a subject or, T cells used in functional assays result in binding to an immunogenic antigen peptide can be expanded and administered to a subject or, TCRs that have been determined to bind to subject specific immunogenic antigen peptides can be expressed in T cells and administered to a subject (Paragraph 552, lines 5-10).
Deering teaches, as previously recited, that purification methods may be directed to isolate DNA, RNA, or both and when both DNA and RNA are isolated together during or subsequent to an extraction procedure, further steps may be employed to purify one or both separately from the other where sub-fractions of extracted nucleic acids can also be generated, for example, purification by size, sequence, or other physical or chemical characteristic and in addition to an initial nucleic acid isolation step, purification of nucleic acids can be performed after any step in the disclosed methods, such as to remove excess or unwanted reagents, reactants, or products, where a variety of methods for determining the amount and/or purity of nucleic acids in a sample are available, such as by absorbance (Paragraph 329, lines 15-20). Further, Deering teaches that, specifically relating to the purification techniques mentioned above, in some embodiments, identifying a sequence of a polynucleotide from an APC of an APC:T cell conjugate as an immunogenic neoantigen comprises comparing a barcode sequence of a first sequenced polynucleotide encoding a first neoantigen to a barcode sequence of a second sequenced polynucleotide encoding the first neoantigen where for example, recovering multiple barcodes associated with a given neoantigen sequence can provide confidence that the interaction is functionally significant or, identifying a sequence of a polynucleotide from an APC of an APC:T cell conjugate as an immunogenic neoantigen comprises comparing a barcode sequence of a first sequenced polynucleotide encoding a first neoantigen to a barcode sequence of a second sequenced polynucleotide encoding the first neoantigen; and selecting the first neoantigen as an immunogenic neoantigen when the barcode of the first sequenced polynucleotide is different than the barcode of the second sequenced polynucleotide (Paragraph 352, lines 1-15).
It would have been obvious to incorporate Deering’s incubation, recovery, and purification techniques into the method of claim 81 to isolate and enrich the desired display products, as such techniques were well-known conventional processing steps routinely used following expression and selection workflows.
Further, it would have been obvious to apply the same incubation, recovery, and purification techniques to the AIDA membrane-display embodiment of claim 84 because these are conventional post-expression processing steps that are independent of the particular display protein employed.
Regarding claims 141-148, Deering teaches methods and compositions for identifying immunogenic neoantigens and methods of using such neoantigens for the treatment of disease and for producing therapeutics for the treatment of disease (Abstract). Further, Deering teaches in some aspects, provided herein is a method for identifying a subject-specific and tumor-specific polypeptide sequence or polynucleotide sequence encoding the subject-specific and tumor-specific polypeptide sequence for preparing an immunogenic composition, the method comprising: contacting an antigen presenting cell (APC) of a plurality of APCs with a T cell of a plurality of T cells, forming one or more APC:T cell conjugates, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; isolating an APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both; and sequencing the target polynucleotide that encodes the target polypeptide from the APC of the APC:T cell conjugate; wherein the target polypeptide comprises a neoantigen sequence encoded by a nucleic acid of a tumor cell sample of a subject and that is not encoded by a nucleic acid of a non-tumor cell sample of the subject (Paragraphs 5-9). Deering also teaches that in some embodiments, the cell is an antigen presenting cell (APC), where in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; and isolating the APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both (Paragraph 10, lines 1-5).
Deering also teaches that in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide encoded by a target polynucleotide; and selectively enriching for the target polynucleotide from the APC of the APC:T cell conjugate, wherein the enriching comprises transcribing or amplifying the target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide (Paragraph 13, lines 1-5). Deering teaches that in some aspects, provided herein is a method comprising: contacting an antigen presenting cell (APC) with a T cell, thereby forming an APC:T cell conjugate, wherein the APC expresses a target polypeptide comprising at most about 100 amino acids encoded by a target polynucleotide, wherein the target polynucleotide is an in vitro transcribed target RNA, and wherein the APC and/or the T cell is from a subject with a disease or condition; and identifying target RNA enriched from cellular RNA isolated from the APC of the APC:T cell conjugate to encode for an immunogenic polypeptide (Paragraph 14, lines 1-5). And, further, Deering teaches that in some embodiments, the APC comprises an amplifier polynucleotide comprising a sequence encoding an amplifier polypeptide that transcribes the target polynucleotide and in some embodiments, the amplifier polynucleotide is linear or, the amplifier polynucleotide is an in vitro transcribed RNA, or the target polynucleotide is an in vitro transcribed RNA, or the target polynucleotide is DNA, or the target polynucleotide is linear or, the target polypeptide comprises at most about 100 amino acids, or the target polypeptide comprises at least about 20 amino acids or, the target polypeptide comprises from about 20 to about 90 amino acids or, the target polypeptide or a fragment thereof is presented on the APC (Paragraph 16, lines 1-10). Deering also teaches that in some embodiments, the method further comprises contacting the antigen presenting cell (APC) with a T cell, or in some embodiments, the method further comprises forming an APC:T cell conjugate or, the method further comprises isolating an APC:T cell conjugate from non-conjugated APCs, non-conjugated T cells, or both or, the method further comprises sequencing a target polynucleotide that encodes the target polypeptide from an APC of an APC:T cell conjugate or, the method further comprises identifying a target polynucleotide to encode for an immunogenic target polypeptide or selecting an immunogenic target polypeptide (Paragraph 16, lines 10-15). Deering also teaches that in some embodiments, the immunogenic target polypeptide elicits in an immune response when administered to a subject with a disease or condition or, the immunogenic target polypeptide elicits an immune response when administered to a subject with a disease or condition or, the immunogenic target polypeptide increases an immune response when administered to a subject with a disease or condition or, the method further comprises contacting the immunogenic target polypeptide or a nucleic acid encoding the immunogenic target polypeptide to a plurality of cells or, the plurality of cells comprises a plurality of APCs or, the method further comprises administering one or more cells of the plurality of APCs to a subject or, the method further comprises contacting a plurality of T cells to the plurality of APCs (Paragraph 16, lines 15-30). Further, Deering teaches that in some embodiments, the plurality of APCs is from a subject or, the plurality of APCs is from a cell line or, the plurality of APCs is engineered cells or, the plurality of T cells is from a subject or, the plurality of T cells is from a cell line or, the plurality of T cells is engineered cells. In some embodiments, the method further comprises expanding one or more T cells of the plurality of T cells (Paragraph 16, lines 30-40).
Further, Deering teaches that in some embodiments, the method further comprises administering one or more cells of the plurality of T cells to a subject or, the plurality of T cells and the plurality of APCs are from the same subject or, the method is performed ex vivo or, the method further comprises enriching the target polynucleotide from cellular R A from the APC of the APC:T cell conjugate or, the method further comprises selectively enriching for the target polynucleotide from an APC of an APC:T cell conjugate, wherein the enriching comprises transcribing or amplifying the target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide where, the APC is from a subject with a disease or conditions and, the target polypeptide comprises a neoantigen or a neoepitope. In some embodiments, the target polypeptide comprises an immunogenic peptide or an immunogenic epitope or, the target polypeptide comprises a mutant peptide, or the target polypeptide comprises a cancer peptide, a viral peptide, an infectious disease peptide, or a non-cancer disease-associated peptide (Paragraph 16, lines 40-50). Also, Deering teaches that in some embodiments, the non-cancer disease-associated peptide is a peptide associated with a disease selected from the group consisting of diabetes, Crohn's, ulcerative colitis and IBD, and arthritis where in some embodiments, the target polypeptide activates a T cell or stimulates a T cell to proliferate. In some embodiments, the target polypeptide activates a CD4.sup.+ T cell or a CD8.sup.+ T cell or, the target polypeptide stimulates a CD4.sup.+ T cell or a CD8.sup.+ T cell to proliferate or, the target polypeptide stimulates a CD4.sup.+ T cell or a CD8.sup.+ T cell to proliferate when an APC comprising an MHC bound to the target peptide is contacted to the CD4.sup.+ T cell or the CD8.sup.+ T cell, or the T cell is from a subject with a disease or condition or, the target polynucleotide is incorporated into the genome of the APC where the genome of the APC comprises the target polynucleotide or, the APC is a CRISPR-Cas engineered APC comprising the target polynucleotide (Paragraph 16, lines 50-60). Deering also teaches that in some embodiments, the target polynucleotide further comprises a sequence encoding a reporter tag or wherein the target polypeptide is linked to a reporter tag or, the reporter tag comprises a reporter tag that can be detected in a cell optically or, the reporter tag comprises a fluorescent reporter tag or, the reporter tag comprises a fluorescent protein, e.g., GFP. In some embodiments, isolating an APC:T cell conjugate comprises isolating APC:T cell conjugates comprising the reporter tag where, APC:T cell conjugates without the reporter tag are not isolated (Paragraph 16, lines 60-65). Further, Deering teaches that in some embodiments, the method comprises selecting a target polynucleotide of an APC:T cell conjugate where in some embodiments, the selecting comprises selecting a target polynucleotide of an APC:T cell conjugate, or the APC:T cell conjugate comprises a plurality of APC:T cell conjugates and the selecting comprises selecting a target polynucleotide based on a frequency of the target polynucleotide or a barcode of a target polynucleotide in the plurality of APC:T cell conjugates where, the T cell comprises an engineered T cell or, the engineered T cell is engineered to express a TCR of interest (Paragraph 16, lines 65-70). Deering further teaches that in some embodiments, the method comprises identifying a target polypeptide that interacts with a TCR of interest expressed by the engineered T cell. In some embodiments, the target polynucleotide comprises one or more target polynucleotides encoding a library of neoantigens where in some embodiments, the method comprises identifying a neoantigen of the library of neoantigens that binds to a TCR of interest expressed by the engineered T cell and in some embodiments, the APC is a first APC, and wherein the method further comprises expressing a polypeptide from a selectively transcribed target RNA in a second antigen presenting cell (APC); contacting the second APC to a second T cell, thereby forming a second APC:T cell conjugate; isolating cellular RNA from the second APC of the second APC:T cell conjugate; and enriching for the target RNA from the cellular RNA where, the method further comprises identifying a target RNA enriched from cellular RNA isolated from an APC of an APC:T cell conjugate to encode for a polypeptide that elicits an immune response when administered to a subject with a disease or condition or, the isolated APC:T cell conjugate is isolated by flow cytometry or magnetic beads and, the method further comprises isolating polynucleotides from the APC and/or T cell of the APC:T cell conjugate where, the isolated polynucleotides comprise cellular RNA. In some embodiments, the isolated polynucleotides comprise mRNA, or , the isolated polynucleotides comprise the target polynucleotide or the method further comprises reverse transcribing a target polynucleotide from the APC of an APC:T cell conjugate (Paragraph 16, lines 70-80).
Deering also teaches that in some embodiments, the method comprises transcribing or amplifying a target polynucleotide using a primer that hybridizes to a heterologous promoter region of the target polynucleotide and in some embodiments, the APC comprises a plurality of APCs and in some embodiments, the APC is a dendritic cell (DC) where, the dendritic cell is an immature dendritic cell (iDC), where in some embodiments, the T cell comprises a plurality of T cells. In some embodiments, the T cell and the APC are from the same subject (Paragraph 17, lines 1-5). Further, Deering teaches that in some embodiments, the target polynucleotide comprises a plurality of target polynucleotides where, the target polynucleotide is RNA or, the target polynucleotide is mRNA or, the target polynucleotide encodes a candidate immunogenic neoantigen or, the target polynucleotide and the amplifier polynucleotide are mixed at a molar concentration ratio of from about 1:2 to 1 : 1000 or from about 1 :20 to 1 : 1000 or from about 1 :2 to 1 : 100 before contacting with the APC (Paragraph 17, lines 5-10). Deering also teaches that in some embodiments, the method is performed in vitro or ex vivo where, the target polynucleotide and the amplifier polynucleotide are delivered into the APC by nucleofection or transfection or electroporation where, the amplifier polynucleotide is transcribed from an amplifier expression polynucleotide comprising a sequence encoding the amplifier polynucleotide and in some embodiments, the target polynucleotide is transcribed from a target expression polynucleotide comprising a sequence encoding the target polynucleotide or, the amplifier expression polynucleotide and/or the target expression polynucleotide is a vector and, the target expression polynucleotide comprises a target polynucleotide ID barcode sequence where in some embodiments, the target polynucleotide ID barcode sequence is barcoded to the sequence encoding the target polynucleotide or the target polypeptide encoded by the target polynucleotide and, the target polynucleotide ID barcode sequence of each target polynucleotide encoding a different target polypeptide is unique (Paragraph 17, lines 10-20).
Deering further teaches that the previously described method can in some embodiments be used in junction with a neoantigen therapeutic and administered as a combination therapy with two or more therapeutic agents uses agents that work by different mechanisms of action, although this is not required and can result in additive or synergetic effects and can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s) and can decrease the likelihood that resistant cancer cells will develop where, combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells (Paragraph 575, lines 1-10). Further, Deering teaches that therapeutic agents that can be administered in combination with the neoantigen therapeutic described herein include chemotherapeutic agents and thus, in some embodiments, the method or treatment involves the administration of an agent described herein in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents (Paragraph 580, lines 1-5). Specifically, Deering teaches that useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor (Paragraph 581, lines 1-5).
Additionally, Deering teaches that the present disclosure also provides non-limiting examples of applications where the methods and compositions described herein can be used and these examples include 1) functional ranking of in silico- predicted antigens to inform immunogenic antigen selection; 2) query both CD4.sup.+ T cell responses and CD8.sup.+ T cell responses to antigen specific responses to epitopes simultaneously; 3) link epitopes with cognate TCR sequences for development of T cell therapies; 4) rapid screening of low-frequency memory responses from patient peripheral blood to monitor responses to immunotherapy; 5) functional ranking predicted antigens to inform immunogenic antigen selection for any immunotherapy, including infectious disease immunotherapies (Paragraph 143, lines 1-5).
Further, Deering teaches that in both animals and humans, mutated epitopes can be potentially effective in inducing an immune response or activating T cells and in one embodiment, the potentially immunogenic epitopes of an infectious agent in a subject, such as a virus, can be determined where, the potentially immunogenic mutated epitopes of a subject with a disease, such as cancer, can be determined an in some embodiments, a potentially immunogenic neoantigen for use in the methods described herein can be a differentiation antigen expressed in a tumor and cells of the type of tissue from which they are generated or, a potentially immunogenic neoantigen for use in the methods described herein can be a cancer/germ line antigens not expressed in another differentiated tissue or, a potentially immunogenic neoantigen for use in the methods described herein can be a mutated antigen (Paragraph 148, lines 1-10). Deering further teaches that for example, a candidate immunogenic neoantigen for use in the methods described herein can comprise a missense-point mutation or a neoantigen of a fusion protein generated through tumor specific translocation of a gene segment and in some embodiments, a potentially immunogenic neoantigen for use in the methods described herein can be an over- expressed antigen, or, a potentially immunogenic neoantigen can be found in tumors where for example, a potentially immunogenic neoantigen for use in the methods described herein can include a protein whose expression is strictly regulated in cells of differentiated normal tissue. (Paragraph 148, lines 10-15).
As shown above, although Deering teaches a puromycin-linked RNA display workflow for identifying and enriching translated tumor-derived sequences, Deering does not teach or suggest engineering the nucleotide construct to maintain translation in an intended reading frame, designing reading constructs with a correct or intended reading frame for proper protein expression and recognizing out-of-frame translation or frameshifting as a problem in display systems and utilizing stop codons as a preventative.
Govarts teaches that phage display is a powerful technique that enables easy identification of targets for any type of ligand and targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins via means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur, and specifically in our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame (Abstract). Govarts further teaches that this event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting, where the aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting and to this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage (Abstract). Govarts further teaches that the most commonly used phage coat proteins for fusion are minor coat protein and major coat protein and when using these coat proteins, N-terminal fusion of the target is mandatory for successful phage propagation, where for the display of cDNA libraries, N-terminal fusion is not possible due to inherent stop codons present in the cDNA fragments, as represented in Figure 1; however, the free carboxyl terminus of minor coat protein 6 (p6) allows successful fusion of the cDNA without interfering with phage propagation and using the pSP6 phagemid vector which was specifically designed to enable C-terminal fusion of targets to p6, we and others have already successfully identified a variety of targets (Introduction: Paragraphs 1-2). Govarts further teaches that frameshifting is well established within the field of phage display and bacterial expression systems because previous studies were based on N-terminal fusion to p3 or p8, frameshifting had to occur in order to express the p3 and p8 coat proteins that enable phage propagation; however, C-terminal fusion to p6 does not require expression of the inserted cDNA for successful phage propagation as represented in Figure 1 and therefore, other methods must be used to detect the possible occurrence of these unusual translational events (Introduction: Paragraph 3). Specifically, Govarts teaches that a phage clone UH-FS was chosen for further study of frameshifting in the p6 display system, where UH-FS was previously isolated after affinity selecting a multiple sclerosis (MS) cDNA display library against antibodies present in MS sera and although the inserted cDNA sequence of the phage clone encodes part of the Apolipoprotein E protein (ApoE), out of frame insertion of the cDNA sequence into the pSP6 phagemid vector resulted in an early stop codon thereby preventing the display of the protein, where fusion of an E-tag to the ApoE cDNA and subcloning of the fragment in 3 reading frames in the pSP6 vector resulted in the expression of the ApoE - E-tag construct in all reading frames via measuring expression of both the ApoE polypeptide and the E-tag via ELISA revealed an increased expression both in the correct reading frame but also in the +1 reading frame, indicating the occurrence of frameshifting in the p6 display system and in addition we could demonstrate an increased antibody reactivity towards the correctly displayed ApoE polypeptide in the plasma of the MS patients used for the selection rounds, which strongly indicates the specific enrichment of UH-FS. An overview of the study is represented in Figure 2 (Introduction: Paragraph 4). Govarts also teaches specified methodology where constructed inserts in multiple reading frames, evaluating expression in the 0, +1, -1 reading frames to study the effects of frameshifting in which phage display libraries can contain inserts that are out of frame (Figure 3; Results and Discussion: Paragraph 1).
Therefore, Govarts recognizes that display systems are susceptible to expression and enrichment of out-of-frame products due to frameshifting and evaluates constructs in multiple reading frames to improve correct-frame display. Although Govarts does not expressly disclose the claimed arrangement of stop codons n both alternative reading frames, it provides the recognized problem and motivation for controlling those frames, and the claimed stop-codon arrangement would have been a predictable way to achieve that result. And because the success of display technologies depends upon expression of proteins in the intended reading frame, one of ordinary skill in the art would have been motivated to modify the constructs of Deering to favor translation only in the desired reading frame and suppress translation from alternative reading frames. Engineering stop codons into the non-desired reading frames would have represented a predictable design choice for achieving that known objective. Further, one of ordinary skill in the art would have had a reasonable expectation of success because providing stop codons in the alternative reading frames would have been obvious an obvious sequence-design technique for preventing translation of out-of-frame products while preserving translation in the intended reading frame, thereby improving the specificity and efficiency of the display library.
Kipnis teaches methods, compositions, and uses for countering the effects of aberrant meningeal lymphatic drainage and/or modulating lymphatic vessels of the central nervous system and the above methods/compositions are used for treating, preventing or ameliorating associated disease symptoms (Abstract). Specifically, Kipnis teaches recombinant expression constructs comprising nucleic acid sequences having an open reading frame, expression vectors for translation of coding sequences, and libraries of polypeptide-coding sequence fragments for screening and selection of expression proteins (Paragraphs 71-74, 92-102, 117-120).
Therefore, one of ordinary skill in the art would have been motivated to implement the display constructs of Deering using coding sequences designed for proper open reading frame expression to improve production and selection of the intended translated polypeptides, since Kipnis demonstrates that preserving the intended reading frame was a conventional and well-understood design objective in recombinant expression systems.
One of ordinary skill in the art would have been motivated to modify the RNA display constructs of Deering in view of the teachings of Govarts and Kipnis to improve fidelity of translation from the intended coding sequence while reducing or eliminating expression arising from unintended reading frames. Govarts teaches a display system that can generate unintended products through translational frameshifting and therefore identifies a known problem affecting display technologies. Kipnis reinforces that recombinant expression constructs are conventionally designed to maintain the intended open reading frame to ensure proper expression of the desired protein. Together, these teachings would have suggested improving Deering’s display constructs by engineering the nucleotide sequence to favor translation in the desired reading frame while preventing or minimizing productive translation from alternative reading frames.
Such a modification represents no more than the predictable use of known design principles to improve an existing display platform. The proposed modification would simply improve the fidelity with which Deering’s system expresses the desired translation product without changing the fundamental purpose or operation of the RNA display workflow. Rather than altering Deering’s selection methodology, the modification would increase the likelihood that translated products correspond to the intended coding sequence and reduce artifacts arising from unintended translation events.
A person of ordinary skill in the art would have had a reasonable expectation of success. Deering, Govarts and Kipnis all concern recombinant nucleic acid constructs used for protein expression or display and rely upon the same underlying principes of transcription and translation. Govarts demonstrates that maintaining the intended reading frame is an established and routine aspect of recombinant construct design. Because modifying nucleotide sequences to preserve translation in a desired reading frame was well within the ordinary level of skill, one would reasonably have expected that applying these conventional construct-design principles to Deering’s RNA display system would improve expression fidelity without requiring undue experimentation.
Furthermore, optimizing expression constructs to favor production of the intended translation product while reducing unintended constitutes routine optimization of a result-effective variable. The choice of coding sequence organization, reading frame, and translation control elements represented ordinary design considerations routinely adjusted by those skilled in recombinant molecular biology to improve expression of desired proteins. Applying those established principles to Deering’s RNA display constructs would therefore have been an obvious matter of engineering design. Accordingly, it would have been obvious to one of ordinary skill in the art at the time of the invention to modify the RNA display system of Deering in view of the teachings of Govarts and Kipnis to engineer expression constructs that preserve translation in the intended reading frame while suppressing expression from unintended reading frames, thereby improving the accuracy and reliability of the displayed translation products.
Conclusion
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684
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