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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 09/10/2025 has been entered.
Claim Status
Claims 1-17, 31, 34, and 36 have been cancelled; claims 18, 22, 25-30, and 35 have been amended; and claims 37-38 have been newly added, as requested in the amendment filed on 09/10/2025. Following the amendment, claims 18-30, 32-33, 35, and 37-38 are pending in the instant application.
Claims 18-30, 32-33, 35, and 37-38 are under examination in the instant office action.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Claims 18-30, 32-33, 35, and 37-38 have an effective filing date of October 28, 2020 corresponding to PRO 63/106,728.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 09/10/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections - Withdrawn
With regard to the objection to claim 18, it is noted that Applicant has amended the claim to more clearly indicate that the antibodies are labeled with different fluorochromes based on their specificity (e.g., anti-CD19 antibodies are labeled with one fluorochrome, anti-CD34 antibodies are labeled with a different fluorochrome, etc.). As such, the objection to claim 18 is withdrawn.
With regard to the objection to claims 22-26, it is noted that Applicant has amended claim 22 to more clearly indicate what the solution of three lyophilized antibodies comprises (i.e., must comprise anti-CD34 and anti-CD14 antibodies and one antibody specific for an additional marker). As such, the objection to claims 22-26 is withdrawn.
With regard to the objection to claims 27-30, it is noted that Applicant has amended the claims to indicate that the recited percentages are representative of a percentage of the total number of antibodies in a recited cocktail. As such, the objection to claims 27-30 is withdrawn.
Claim Rejections - 35 USC § 112 - Withdrawn
With regard to the rejection of claims 18-30, 32-33, and 35 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, due to lack of antecedent basis, it is noted that Applicant has amended claim 18 to recite “the fluorescently labeled antibodies”, which has proper antecedent basis. As such, the rejection of claims 18-30, 32-33, and 35 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, is withdrawn.
Response to Arguments
Applicant’s arguments, see Pages 8-9 of Remarks, filed 09/10/2025, with respect to the rejection(s) of claim(s) 18-30, 32-33, and 35 under 35 U.S.C. 103 in view of WO 2018/073267 A1, Kalos et. al., and/or Finak et. al. regarding the amended claims reciting additional limitations of genetically modifying a population of cells and/or subsequently administering the population of cells have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of newly found art reference CN 110157680 A, which renders obvious the additional limitation of amended claim 18 and as detailed below.
Beyond the argument regarding the amended claims and the additionally presented claim limitations, Applicant further argues that Kalos is directed to answering the question of whether “T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia” and uses an anti-CAR idiotype antibody to assess establishment post-treatment of a subject, while currently amended claim 18 addresses a different problem which is to identify a sub-population of cells of a population of cells prior to treatment and that, as such, a skilled artisan would not read past the title and would neither look to Auiti nor Kalos individually or together. While it is noted that the context in which the Kalos reference is utilized is different from the method of the instant claims, especially as amended, it is noted that the teachings of Kalos still read on flow cytometric methods as pertain to T cells and CAR T cells, and therefore reads on the instant claim limitations. As such, the Kalos reference is still considered to be analogous art to the instant invention, and is still relied upon in the new grounds of rejection presented below.
Claim Rejections - 35 USC § 103 - New
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 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.
Claim(s) 18-21, 32-33, 35, and 37-38 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2018/073267 A1 (previously cited on PTO-892; herein after referred to as "Auiti") in view of non-patent literature by Kalos et. al., Leukemia, 2011, 3(95), 1-11 (previously cited on PTO-892; herein after referred to as "Kalos") and CN 110157680 A (machine translation of the description provided; herein after referred to as "Huang").
With regard to claims 18 and 37-38, Auiti teaches a panel of fluorochrome-labelled antibodies that can be used to identify hematopoietic cell subtypes in an isolated sample, determine the relative frequency of and/or quantify the number of cells within hematopoietic cell subtypes in a sample utilizing a combination of cell surface markers that allows a blood sample to be labeled without division into sub-samples or prior isolation of a cell subpopulation of interest, wherein coverage of the various hematopoietic cell subtypes present in blood is provided and it is possible to discriminate 23 different hematopoietic cell subtypes (Page 3, Lines 20-27; emphasis added). Auiti further teaches the use of (i) an anti-CD3 antibody, (ii) an anti-CD56 antibody, (iii) an anti-CD14 antibody, (iv) an anti-CD38 antibody, (v) an anti-CD45 antibody, (vi) an anti-CD90 antibody, (vii) an anti-CD135 antibody, (viii) an anti-CD10 antibody, (ix) an anti-CD11c antibody, (x) an anti-CD19 antibody, (xi) an anti-CD34 antibody, (xii) an anti-CD45RA antibody, (xiii) an anti-CD7 antibody, (xiv) an anti-CD71 antibody, (xv) an anti-CD41 /CD61 complex antibody or an anti-CD41 antibody and/or an anti-CD61 antibody (xvi) an anti-CD33 antibody and/or an anti-CD66b antibody, for identifying hematopoietic cell subtypes in an isolated sample, determining the relative frequency of hematopoietic cell subtypes in an isolated sample and/or quantifying the number of cells within hematopoietic cell subtypes in an isolated sample, wherein each of (i) to (xvi) is labelled with a different fluorochrome, wherein when (xvi) is an anti-CD33 antibody and an anti-CD66b antibody, the anti-CD33 antibody and anti-CD66b are labelled with the same fluorochrome, and wherein when (xv) is an anti-CD41 antibody and an anti-CD61 antibody, the anti-CD41 antibody and the anti-CD61 antibody are labelled with the same fluorochrome (Page 4, Lines 1-15; emphasis added). Auiti also teaches an embodiment wherein the invention is used for identifying, determining the relative frequency of and/or quantifying the number of cells within one or more hematopoietic cell subtypes selected from the group consisting of immature polymorphonuclear leukocytes (iPMN), polymorphonuclear leukocytes (PMN), monocytes, dendritic cells (DC)/DC progenitors, myeloblasts, T-cells, natural killer T cells (NKT), natural killer cells (NK), B-cells, pre-B cells, pro-B cells, pro-lymphocytes, pro-erythroblasts, erythroblasts, hematopoietic stem cells (HSC), multipotent progenitors (MPP), multipotent lymphoid progenitors (MLP), early T progenitors (ETP) cells, pre-B/natural killer cells (PREB/NK), common myeloid progenitors (CMP), granuclocyte/macrophage progenitors (GMP), and megakaryocyte/erythrocyte progenitors (MEP) (Page 5, Lines 6-15; emphasis added), wherein the immunophenotypes of such subtypes are disclosed in Table 1, and teaches that hematopoietic cell subtypes may be identified and classified on the basis of their differential expression of cell surface markers, such as receptor proteins or ligands that are exposed on the cell membrane (Page 21, Lines 26-28, Table 1). In one embodiment, one use of the invention is for identifying, determining the relative frequency of and/or quantifying the number of cells within one or more T cell subpopulations (Page 6, Lines 5-7; emphasis added). Auiti further teaches that the term "fluorochrome" refers to a fluorescent molecule that can re-emit light following excitation with absorbed light that has a specific wavelength and that the term "fluorochrome" may be used interchangeably with the terms "fluorophore", "fluorescent label", "fluorescent dye", "fluorescent tag", "fluorescent marker", "fluorescent probe" and "fluorescent reporter" (Page 24, Lines 15-18). A person skilled in the art is able to routinely prepare antibodies labelled with fluorochromes without undue experimentation; antibodies labelled with fluorochromes may be prepared according to any method known in the art and/or commercially available antibodies labelled with a fluorochrome may be employed for the present invention (Page 29, Lines 8-11). Auiti also teaches that fluorochrome labeled hematopoietic cells may be detected by any suitable method known in the art (Page 36, Lines 6-7) including flow cytometry wherein the flow cytometer is equipped with multiple light sources of different wavelengths and multiple detectors to allow for the simultaneous detection of up to 17 different fluorescent signals (Page 36, Lines 16-18). The invention is useful for diagnosing such blood disorders since it allows the relative frequency of hematopoietic cells in a sample isolated from PB or BM sample to be determined; blood disorders may also be diagnosed using the present invention by quantification of the number of cells within hematopoietic cell subtypes in a sample wherein absolute cell quantification allows the testing of whether each cell compartment is within the normal range of cellularity (e.g., by comparison to the cellularity observed in a corresponding healthy control sample) (Page 55, Lines 25-32; emphasis added). Example 10 (Pages 79-86) details phenotypic analyses of bone marrow and peripheral blood from healthy donors as well as samples from Wiskott-Aldrich Syndrome (WAS), adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCIO), Metachromatic leukodystrophy (MLD), and leukemic patients. Peripheral blood and bone marrow aspirate were collected using vacutainers containing 30 K2EDTA 5.4 mg (Becton Dickinson (BO) Vacutainer®; REF no. 8019839). Cell staining, data acquisition and analyses were performed according to the protocol of Example 1 wherein after RBC lysis, the samples were labelled with fluorescent antibodies against CD3, CD56, CD14, CD61/41, CD135, CD34, CD45RA (Biolegend) and CD33, CD66b, CD38, CD45, CD90, CD10, CD11c, CD19, CD7 and CD71 (i.e., a fluorescently labeled antibody cocktail) wherein titration assays were performed to assess the best antibody concentration (Id.). Auiti also discloses a kit for identifying, determining the relative frequency of and/or quantifying the number of cells within lymphoid cell subtypes wherein the kit comprises one or more fluorescently labelled antibodies which may be individually packaged and labelled or the one or more antibodies of the kit may be packaged as a mixture (i.e., antibody cocktail) wherein the mixture of antibodies may comprise two or more of said antibodies (Page 43, Lines 1-20. Thus, a population of cells was provided and exposed to (i.e., contacted with) an antibody cocktail to create a mixture. To isolate the different hematopoietic cell subtypes for morphological validation, the inventors set up five different sorting strategies: Sorting 1 was performed to isolate T, NKT, NK and all CD19+ cells; Sorting 2 was performed to isolate mature B cells, Pre-B and Pro-B precursors; Sorting 3 was performed to isolate PMN, iPMN, monocytes and DCs; Sorting 4 was performed to isolate erythroblasts and myeloblasts. Sorting 5 was performed to isolate pro-erythroblasts and lymphoid-committed progenitors (pro-lymphocytes) (Id.; Figure 2A). As disclosed above, the hematopoietic cell subtypes could then be detected (analyzed/identified) via flow cytometry based on observed antibody staining profiles which can be compared to control cells from healthy individuals. While Auiti teaches analysis of T cell sub-populations, Auiti does not explicitly teach providing a population of cells from an apheresis, wherein the population of cells primarily comprises T cells, nor does Auiti teach/suggest genetic modification of such a population of cells. This deficiency is remedied by Kalos.
Kalos teaches that a central question in CAR-mediated cancer immunotherapy is whether optimized cell manufacturing and costimulation domains will enhance the persistence of genetically modified T cells and permit the establishment of CAR+ memory T cells in patients, and that previous studies have not demonstrated robust expansion, prolonged persistence, or functional expression of CARs on T cells after infusion (Page 4, Column 2, Paragraph 2). Flow cytometric analysis of samples from both blood and bone marrow 169 days after infusion revealed the presence of CAR19-expressing cells in UPN 03 as well as an absence of B cells (Id.; Fig. S1, A and B). The authors also used polychromatic flow cytometry to perform detailed studies and further characterize the expression, phenotype, and function of CART19 cells in UPN 03 using an anti-CAR idiotype antibody (MDA-647) and the gating strategy shown in Fig. S3; the authors observed differences in the expression of memory and activation markers in both CD4+ and CD8+ T cells based on CAR19 expression (Page 4, Column 2, Paragraph 3). The clinical trial (NCT01029366) was conducted as diagramed in Fig. 1; patients with CD19+ hematologic malignancy (i.e., chronic lymphocytic leukemia) with persistent disease after at least two previous treatment regimens and who were not eligible for allogeneic stem cell transplantation were eligible for the study and after tumor restaging, PB T cells for CART19 manufacturing were collected by apheresis and the subjects were given a single course of chemotherapy during the week before infusion, as specified in Table 1 (Page 9, Column 2, Material and Methods: Protocol Design; emphasis added). CART19 cells were administered by intravenous infusion with a 3-day split-dose regimen (10, 30, and 60%) at the dose indicated in Table 1, and, if available, a second dose was administered on day 10; only UPN 02 had sufficient cells for a second infusion and subjects were assessed for toxicity and response at frequent intervals for at least 6 months (Id.). Thus, Kalos teaches the collection of cell samples from cancer patients via apheresis and the analysis of specific cell subpopulations from said samples via flow cytometry, including monitoring of CAR T cell expression in samples after CAR T cell infusion (i.e., samples collected after CAR T cell infusion contained CAR T cells; flow cytometric analysis allowed for specific monitoring of response to T cell therapy). While Kalos suggests using flow cytometry in applications involving genetically modified cells (e.g., CAR T cells), said applications are after the administration of the genetically modified cells, not before. This deficiency is remedied by Huang.
Huang discloses a cell culture method for improving the efficacy and persistence of chimeric antigen receptor T cells involving the following steps (see Pages 2-3 of machine translation; emphasis added):
Preparation of peripheral blood mononuclear cells
Peripheral blood samples were taken, and heparin was anticoagulated. Peripheral blood mononuclear cells were isolated and separated by human lymphocyte separation solution.
CD3(+) T cell enrichment and T cell activation
After using anti-CD3/CD28 magnetic beads, fully mixed with peripheral blood mononuclear cells and bound to CD3(+) T cells, magnetic plates were enriched for CD3(+) T cells, and simultaneously bound to the surface of magnetic beads. The anti-CD3/CD28 antibody activates CD3(+) T cells.
Transfection of T cells with CAR lentivirus targeting CD19
The prepared lentivirus carrying the CD19-targeting CAR was transfected with anti-CD3/CD28 magnetic bead-activated CD3(+) T cells according to MOI=10.
Addition of tyrosine kinase inhibitor dasatinib to expand cultured CAR-T cells
GFP+CAR-T cells were cultured for 3-5 days, and the expression of CAR molecules was detected by flow. After confirming the successful preparation of CAR-T cells, CAR-T cells were cultured in two groups for 9 days: respectively. CAR-T cells were cultured by adding a culture solution of the tyrosine kinase inhibitor dasatinib. Divided into experimental group and control group 2, dasatinib 30nmol/L experimental group (RPMI1640+10%FBS+IL-2200U/ml+dasatinib 30nM), control group: equal volume DMSO control group (RPMI 1640 + 10% FBS + IL-2200 U / ml + equal volume of DMSO).
CAR-T cell subset detection
On the 9th day of culture, the labeled fluorescent antibodies CD45RO and CD62L were sampled, and the CAR-T cell subset was detected by flow cytometry; the CAR-T cells cultured in the step (4) were able to maintain the initial T cells and central memory T. In the cell stage, the differentiation of CAR-T cells into the downstream and terminal stages is effectively inhibited.
Detection of surface molecules related to CAR-T cell depletion
On the 9th day of culture, the labeled fluorescent antibodies PD1, TIM3 and LAG3 were sampled, and the surface molecules related to CAR-T cell depletion were detected by flow cytometry; the CAR-T cells cultured in the step (4) showed low inhibition of T cell depletion-related inhibition. The bodies PD1, TIM3, and LAG3 effectively inhibited the depletion tendency of CAR-T cells.
Assessing the efficacy and persistence of dasatinib-treated CAR-T cells
The acute lymphocytic leukemia (ALL)-NSG mouse model was prepared and grouped as follows: 1 control group (tail vein injection of DMSO-treated CAR-T cells), and 2 experimental groups (tail vein injection of dasatinib 30 nM cultured CAR-T cells); The mice were imaged weekly using a small animal live imager. The tumor load differences were compared between the two groups. The percentage of CAR-T cells was detected by flow cytometry weekly. The survival time of each group was recorded and the survival curve was drawn. The CAR-T cells cultured in the step (4) can be used to treat acute lymphoblastic leukemia mice, and a better therapeutic effect and survival time can be obtained.
Thus, Huang discloses obtaining a sample of peripheral blood mononuclear cells, enriching said sample for T cells and activating said T cells, genetically modifying said T cells and expanding the population, identifying T cell subpopulations/detecting surface molecules using flow cytometry and fluorescently labeled antibodies, and administering said genetically modified T cells (i.e., CAR T cells) to an ALL mouse model and monitoring efficacy using flow cytometry. As such, Huang teaches the genetic modification of T cells and detecting/analyzing said T cells using flow cytometry prior to administration to a subject.
Auiti, Kalos, and Huang are considered to be analogous to the present invention as they are in the same field of flow cytometry and/or cellular subpopulation identification and/or immunophenotyping. Thus, it would have been obvious to one of ordinary skill in the art to utilize a population of cells collected by apheresis (i.e., samples primarily comprising T cells), suggested by Kalos, and genetically modifying said T cells to produce a therapeutic population of T cells, as disclosed by Huang, and subsequently using flow cytometry for more specific T cell population/subpopulation analysis, as suggested by Huang, using the method rendered obvious by Auiti, which comprises contacting a population of cells (including T cells) with at least two fluorochrome-labeled antibodies (e.g., anti-CD34 and anti-CD14 antibodies) and analyzing the resultant mixture via flow cytometry to identify specific sub-populations. The method could be further modified such that the cell population comprising genetically modified cells and an identified subpopulation could then be administered to a subject for therapeutic purposes, as suggested by Huang and Kalos. Combining prior art elements according to known methods would be expected to yield predictable results with a reasonable expectation of success. One of ordinary skill in the art would recognize that apheresis samples primarily comprise T cells and that such a sample could be further analyzed for different sub-populations, including after the genetic modification of said T cells, based on specific markers which can be targeted/labeled via fluorochrome-labeled antibodies that can be analyzed via flow cytometry, as is routine in the art as demonstrated by Auiti, Kalos, and Huang. It would have been obvious to one of ordinary skill in the art to combine the teachings of Auiti, Kalos, and Huang to arrive at a flow cytometric method that allows for analysis of T cell populations, including genetically modified T cell populations, and subpopulations thereof in order to (i) monitor genetically modified T cell production and identify subpopulations particularly beneficial for therapeutic applications, and./or (ii) monitor patient response to therapy (e.g., after administration of a genetically modified T cell population) or monitor a disease/condition with the motivation of monitoring therapeutic cell population production, identifying therapeutic populations/subpopulations, and administering said therapeutic populations/subpopulations for the treatment of a disease/condition which can subsequently be monitored for efficacy to facilitate more effective treatments.
With regard to claims 19-20, Auiti teaches a panel of fluorochrome-labelled antibodies that can be used to identify hematopoietic cell subtypes in an isolated sample, determine the relative frequency of and/or quantify the number of cells within hematopoietic cell subtypes in a sample utilizing a combination of cell surface markers that allows a blood sample to be labeled without division into sub-samples or prior isolation of a cell subpopulation of interest, wherein coverage of the various hematopoietic cell subtypes present in blood is provided and it is possible to discriminate 23 different hematopoietic cell subtypes (Page 3, Lines 20-27; emphasis added). In one embodiment, one use of the invention is for identifying, determining the relative frequency of and/or quantifying the number of cells within one or more T cell subpopulations (Page 6, Lines 5-7; emphasis added). Auiti thus teaches quantification of cell populations/subpopulations. Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention as evidenced by the references.
With regard to claim 21, Auiti teaches an embodiment wherein the invention is used for identifying, determining the relative frequency of and/or quantifying the number of cells within one or more hematopoietic cell subtypes selected from the group consisting of immature polymorphonuclear leukocytes (iPMN), polymorphonuclear leukocytes (PMN), monocytes, dendritic cells (DC)/DC progenitors, myeloblasts, T-cells, natural killer T cells (NKT), natural killer cells (NK), B-cells, pre-B cells, pro-B cells, pro-lymphocytes, pro-erythroblasts, erythroblasts, hematopoietic stem cells (HSC), multipotent progenitors (MPP), multipotent lymphoid progenitors (MLP), early T progenitors (ETP) cells, pre-B/natural killer cells (PREB/NK), common myeloid progenitors (CMP), granuclocyte/macrophage progenitors (GMP), and megakaryocyte/erythrocyte progenitors (MEP) (Page 5, Lines 6-15; emphasis added). Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention as evidenced by the references.
With regard to claims 32-33, Auiti also teaches that fluorochrome labeled hematopoietic cells may be detected by any suitable method known in the art (Page 36, Lines 6-7) including flow cytometry wherein the flow cytometer is equipped with multiple light sources of different wavelengths and multiple detectors to allow for the simultaneous detection of up to 17 different fluorescent signals (Page 36, Lines 16-18). The invention is useful for diagnosing such blood disorders since it allows the relative frequency of hematopoietic cells in a sample isolated from PB or BM sample to be determined; blood disorders may also be diagnosed using the present invention by quantification of the number of cells within hematopoietic cell subtypes in a sample wherein absolute cell quantification allows the testing of whether each cell compartment is within the normal range of cellularity (e.g., by comparison to the cellularity observed in a corresponding healthy control sample) (Page 55, Lines 25-32; emphasis added). Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention as evidenced by the references.
With regard to claim 35, Kalos teaches flow cytometric analysis of samples from both blood and bone marrow 169 days after infusion revealed the presence of CAR19-expressing cells in UPN 03 as well as an absence of B cells (Page 4, Column 2, Paragraph 2; Fig. S1, A and B; emphasis added). The authors also used polychromatic flow cytometry to perform detailed studies and further characterize the expression, phenotype, and function of CART19 cells in UPN 03 using an anti-CAR idiotype antibody (MDA-647) and the gating strategy shown in Fig. S3; the authors observed differences in the expression of memory and activation markers in both CD4+ and CD8+ T cells based on CAR19 expression (Page 4, Column 2, Paragraph 3; emphasis added). Additionally, Huang teaches CAR-T cell subset detection wherein, on the 9th day of culture, labeled fluorescent antibodies CD45RO and CD62L were sampled, and the CAR-T cell subset was detected by flow cytometry; it was determined that the CAR-T cells cultured in the previous steps were able to maintain the initial T cells and central memory T (Page 2 of machine translation). Huang also teaches the detection of surface molecules related to CAR-T cell depletion wherein, on the 9th day of culture, labeled fluorescent antibodies PD1, TIM3 and LAG3 were sampled, and the surface molecules related to CAR-T cell depletion were detected by flow cytometry; the CAR-T cells cultured in the previous steps showed low inhibition of T cell depletion-related inhibition (Id.). Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention as evidenced by the references.
Claim(s) 22-30 is/are rejected under 35 U.S.C. 103 as being unpatentable over WO 2018/073267 A1 (previously cited on PTO-892; herein after referred to as "Auiti"), Kalos et. al., (previously cited on PTO-892; herein after referred to as "Kalos"), and CN 110157680 A (machine translation of the description provided; herein after referred to as "Huang"), as applied to claims 18-21, 32-33, and 35 above, and in further view of non-patent literature by Finak et. al., Scientific Reports, 2016, 6, 1-11 (previously cited on PTO-892; herein after referred to as "Finak").
With regard to claim 22, the method of claim 21 is rendered obvious by Auiti, Kalos, and Huang. However, Auiti does not explicitly disclose that the antibodies/antibody cocktails of the invention are lyophilized and combined into a solution prior to contacting the population of cells as required by instant claim 22. This deficiency is remedied by Finak in view of Auiti.
The Finak reference is drawn to the standardization of flow cytometry immunophenotyping analysis. More specifically, Finak teaches that experts have developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood which were produced as pre-configured, lyophilized, reagents in 96-well plates (Page 1, Paragraph 1; emphasis added). The Human Immune Phenotyping Consortium (HIPIC) promoted standardization of flow cytometry immunophenotyping in clinical studies, so that data could be compared across sites and studies; as part of these efforts, the HIPC immunophenotyping panel was developed wherein the HIPC panels consist of five eight-color antibody cocktails (i.e., antibody cocktails comprising eight fluorescently labelled antibodies), designed to phenotype major immune cell subsets in peripheral blood mononuclear cells (T cells, Treg, Th1/2/17, B cells, and NK/dendritic cells/monocytes) (Page 2, Paragraph 2). These panels were designed to standardize routine immunophenotyping in humans while still being compatible with widely available clinical flow cytometers, and although they were not designed to represent the full complexity of cutting-edge research, the cocktails were designed to be easily expanded with additional colors to serve that purpose (Id.). Finak further teaches that following development and testing of the HIPC panels, lyophilized reagent cocktails in 96-well plates were developed; the use of lyophilized reagent cocktails is a proven method for improving standardization in that it protects against errors of reagent addition or mis-titration, provides improved reagent stability, and simplifies assay setup (Page 2, Paragraph 4). It is noted that the experimental protocols described in Finak do not require reconstituting the antibody panels prior to contacting the panels with cells, but rather reconstituted cells were added directly to the lyophilized panels. However, it is noted that Auiti suggests that the antibodies/antibody cocktails of the invention were prepared as solutions prior to exposure to cells; the invention provides a composition for identifying, determining the relative frequency of and/or quantifying the number of cells within hematopoietic cell subtypes various antibodies (detailed above) wherein the composition may further comprise buffers, salts, counter ions, diluents, and/or excipients (Pages 43-45; emphasis added).
Auiti, Kalos, Huang, and Finak are considered to be analogous to the present invention as they are in the same field of flow cytometry and/or cellular subpopulation identification and/or immunophenotyping. Thus, it would have been obvious to one of ordinary skill in the art to modify the method rendered obvious by Auiti, Kalos, and Huang such that the methods utilizes lyophilized antibody panels/cocktails, as suggested by Finak, wherein said antibody panels/cocktails could be reconstituted into a solution prior to contacting said antibodies with a sample cell population, as suggested by Auiti, because combining prior art elements according to known methods would be expected to yield predictable results with a reasonable expectation of success. Finak teaches that pre-configured lyophilized reagents/panels for use in flow cytometry is a standard practice in the art, and thus it would have been within the purview of one having ordinary skill in the art to pre-configure an antibody cocktail for a cell population and/or subpopulation thereof for use in the method rendered obvious by Auiti, Kalos, and Huang.
With regard to claims 23-25, Auiti teaches a panel of fluorochrome-labelled antibodies that can be used to identify hematopoietic cell subtypes in an isolated sample, determine the relative frequency of and/or quantify the number of cells within hematopoietic cell subtypes in a sample utilizing a combination of cell surface markers that allows a blood sample to be labeled without division into sub-samples or prior isolation of a cell subpopulation of interest, wherein coverage of the various hematopoietic cell subtypes present in blood is provided and it is possible to discriminate 23 different hematopoietic cell subtypes (Page 3, Lines 20-27). Auiti also teaches an embodiment wherein the invention is used for identifying, determining the relative frequency of and/or quantifying the number of cells within one or more hematopoietic cell subtypes selected from the group consisting of immature polymorphonuclear leukocytes (iPMN), polymorphonuclear leukocytes (PMN), monocytes, dendritic cells (DC)/DC progenitors, myeloblasts, T-cells, natural killer T cells (NKT), natural killer cells (NK), B-cells, pre-B cells, pro-B cells, pro-lymphocytes, pro-erythroblasts, erythroblasts, hematopoietic stem cells (HSC), multipotent progenitors (MPP), multipotent lymphoid progenitors (MLP), early T progenitors (ETP) cells, pre-B/natural killer cells (PREB/NK), common myeloid progenitors (CMP), granuclocyte/macrophage progenitors (GMP), and megakaryocyte/erythrocyte progenitors (MEP) (Page 5, Lines 6-15), wherein the immunophenotypes of such subtypes are disclosed in Table 1, and teaches that hematopoietic cell subtypes may be identified and classified on the basis of their differential expression of cell surface markers, such as receptor proteins or ligands that are exposed on the cell membrane (Page 21, Lines 26-28, Table 1; emphasis added). To isolate the different hematopoietic cell subtypes for morphological validation, the inventors set up five different sorting strategies: Sorting 1 was performed to isolate T, NKT, NK and all CD19+ cells; Sorting 2 was performed to isolate mature B cells, Pre-B and Pro-B precursors (early progenitor cells); Sorting 3 was performed to isolate PMN, iPMN, monocytes and DCs; Sorting 4 was performed to isolate erythroblasts and myeloblasts. Sorting 5 was performed to isolate pro-erythroblasts and lymphoid-committed progenitors (pro-lymphocytes) (Id.; Figure 2A; emphasis added). Thus, Figure 2A discloses how cells were sorted into specific subpopulations based on antibody binding; Sorting 5, for example, isolated pro-lymphocytes (i.e., early progenitors) based on positive binding of CD45, CD33/CD66b, CD3, CD19, CD56, CD34, CD71, CD41/61, CD10, and CD7antibodies (i.e., cell subpopulations were bound to at least three fluorescently labelled antibodies in an antibody cocktail; emphasis added). Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention as evidenced by the references.
With regard to claim 26, Auiti teaches a panel of fluorochrome-labelled antibodies that can be used to identify hematopoietic cell subtypes in an isolated sample, determine the relative frequency of and/or quantify the number of cells within hematopoietic cell subtypes in a sample utilizing a combination of cell surface markers that allows a blood sample to be labeled without division into sub-samples or prior isolation of a cell subpopulation of interest, wherein coverage of the various hematopoietic cell subtypes present in blood is provided and it is possible to discriminate 23 different hematopoietic cell subtypes (Page 3, Lines 20-27). Auiti further teaches that the term "fluorochrome" refers to a fluorescent molecule that can re-emit light following excitation with absorbed light that has a specific wavelength and that the term "fluorochrome" may be used interchangeably with the terms "fluorophore", "fluorescent label", "fluorescent dye", "fluorescent tag", "fluorescent marker", "fluorescent probe" and "fluorescent reporter" (Page 24, Lines 15-18). A person skilled in the art is able to routinely prepare antibodies labelled with fluorochromes without undue experimentation; antibodies labelled with fluorochromes may be prepared according to any method known in the art and/or commercially available antibodies labelled with a fluorochrome may be employed for the present invention (Page 29, Lines 8-11). Auiti teaches small molecule fluorochromes which include fluoresceins (i.e., FITC) (Page 25, Lines 14-19; emphasis added) as well as allophycocyanin (APC), allophycocyanin cyanine 7 (APC-Cy7), allophycocyanin R700 (APC-5 R700), phycoerythrin (PE), phycoerythrin cyanine 5 (PE-Cy5), phycoerythrin cyanine 7 (PECy7), peridinin chlorophyll protein cyanine 5.5 (PerCP-Cy5.5), Brilliant Blue 515 (BB515), Brilliant Violet 421 (BV421 ), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 650 (BV650), Brilliant Violet 711 (BV711 ), Brilliant Violet 785 (BV785), Brilliant Ultraviolet 395 (BUV395), and Brilliant Ultraviolet 737 (BUV 737) (Page 28, Lines 3-9; emphasis added). It is noted that Finak teaches FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-H7, V450, and V500 fluorochromes for the HIPC antibody panels (Page 3, Table 1; emphasis added). Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention as evidenced by the references.
With regard to claims 27-30, it is noted that, as detailed above, Auiti and Finak both teach antibody panels comprising multiple antibodies (e.g., three or more). Auiti further teaches that a titration assay is required to define the best fluorochrome-labelled antibody concentrations (Page 60, Lines 16-17); Example 10 discloses that samples were labelled with fluorescent antibodies against CD3, CD56, CD14, CD61/41, CD135, CD34, CD45RA and CD33, CD66b, CD38, CD45, CD90, CD10, CD11c, CD19, CD7 and CD71 wherein titration assays were performed to assess the best antibody concentration (Page 80-81). Thus, as evidenced by the reference, it is noted that working concentrations/ratios for antibodies of the cytometric panels are recognized as an antibody/cytometric panel variable which achieves a recognized result and as set forth in MPEP 2144.05: “A particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation.” In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977). It is a common objective in the art to optimize result effective variables, so as achieve optimal effect and maximal benefit. See In re Boesch, 617 F.2d 272, 276, 205 USPQ 215, 219 (CCPA 1980) (“[D]iscovery of an optimum value of a result effective variable in a known process is ordinarily within the skill of the art.” (citations omitted)). Therefore, any optimization of antibody concentrations/ratios for cytometric panels would be seen as routine optimization. One of ordinary skill in the art would be motivated to optimize flow cytometry antibody panels for more accurate/optimized cellular analysis. Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention as evidenced by the references.
Conclusion
Claims 18-30, 32-33, 35, and 37-38 are pending. Claims 18-30, 32-33, 35, and 37-38 are rejected. No claims are allowed.
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/ALYSSA RAE STONEBRAKER/Examiner, Art Unit 1642
/NELSON B MOSELEY II/Primary Examiner, Art Unit 1642