B cell maturation complex CAR T constructs and primers

Abstract

The present invention provides probe and primer sets and related methods and kits for generating B cell maturation antigen chimeric antigen receptor (CAR) T cells. The present invention also provides probe and primer sets and related methods and kits for performing quantitative polymerase chain reaction to quantify incorporation of a B cell maturation antigen CAR transgene into a CAR T drug product.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 62 / 894,663, filed August 30, 2019, the entire contents of which are incorporated herein by reference.

[0002] (Incorporating material by reference in an ASCII text file) This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on August 28, 2020, is named JBI6148WOPCT1_SL.txt, and is 8175 bytes in size. [Background technology]

[0003] T cell therapy utilizes genetically modified and isolated T cells to enhance their specificity for certain tumor-associated antigens. Genetic modification can involve the expression of chimeric antigen receptors (CARs) or exogenous T cell receptors to provide new antigen specificity on the T cells. T cells expressing chimeric antigen receptors (CAR-T cells) can induce tumor immune reactivity. B cell maturation antigen (BCMA) is a molecule expressed on the surface of mature B cells and malignant plasma cells, and is a target molecule in the treatment of cancer, such as multiple myeloma. There is a need for better cancer therapies utilizing CAR T cells, particularly CAR T cells specific for the BCMA tumor-associated antigen. Summary of the Invention [Means for solving the problem]

[0004] The present invention relates to probes and primers for polymerase chain reaction (PCR), e.g., quantitative PCR. The present invention also relates to kits and methods utilizing the probes and primers described herein for quantifying transgene integration into chimeric antigen receptor (CAR) T cells.

[0005] In a first aspect, the present invention provides a probe and primer set comprising a probe comprising a nucleotide sequence of SEQ ID NO: 10, at least one label attached to the probe, a first primer comprising a nucleic acid sequence of SEQ ID NO: 11, and a second primer comprising a nucleic acid sequence of SEQ ID NO: 12 (see Example 1, Table 1).

[0006] In another aspect, the present invention provides a probe and primer set comprising a probe comprising a nucleotide sequence of SEQ ID NO:1, at least one label attached to the probe, a first primer comprising a nucleic acid sequence of SEQ ID NO:2, and a second primer comprising a nucleic acid sequence of SEQ ID NO:3 (see Example 1, Table 1).

[0007] In another aspect, the present invention provides a probe and primer set (see Table 1), comprising a probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, and combinations thereof; at least one label attached to the probe; a first primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, and combinations thereof; and a second primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and combinations thereof.

[0008] In some embodiments, at least one label comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0009] In another aspect, the invention provides a kit for quantifying transgene integration into CAR T cells, the kit comprising a probe comprising the nucleotide sequence of SEQ ID NO: 10, at least one label attached to the probe, a first primer comprising the nucleic acid sequence of SEQ ID NO: 11, and a second primer comprising the nucleic acid sequence of nucleic acid sequence 12.

[0010] In another aspect, the invention provides a kit for quantifying transgene integration into CAR T cells, the kit comprising a probe comprising the nucleotide sequence of SEQ ID NO:1, at least one label attached to the probe, a first primer comprising the nucleic acid sequence of SEQ ID NO:2, and a second primer comprising the nucleic acid sequence of nucleic acid sequence 3.

[0011] In a further aspect, the invention provides a kit for quantifying transgene integration into CAR T cells, comprising a probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, and combinations thereof, at least one label attached to the probe, a first primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, and combinations thereof, and a second primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and combinations thereof.

[0012] In some embodiments of the kits of the invention, at least one label attached to a probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0013] In one embodiment, the kit of the invention comprises an array comprising the probes, hi some embodiments, the array is a multiwell array.

[0014] In one embodiment, the kit further comprises a human albumin (hALB) probe comprising the nucleic acid sequence of SEQ ID NO: 22 (see Example 2, Table 2, probes from hALB Set 1), at least one label attached to the hALB probe, a first hALB primer comprising the nucleic acid sequence of SEQ ID NO: 23 (forward primer from Table 2, hALB Set 1), and a second hALB primer comprising the nucleic acid sequence of SEQ ID NO: 24 (reverse primer from Table 2, hALB Set 1). In certain embodiments, the at least one label attached to the hALB probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0015] In another embodiment, the kit further comprises a human albumin (hALB) probe comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, and combinations thereof, at least one label attached to the hALB probe, a first hALB primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, and combinations thereof, and a second hALB primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, and combinations thereof (Table 2). In certain embodiments, the at least one label attached to the hALB probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0016] In another aspect, the present invention provides a method for quantitating transgene integration into CAR T cells, comprising: amplifying nucleic acid from the CAR T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the CAR T cells using a first hALB primer comprising the nucleic acid sequence of SEQ ID NO:23 and a second hALB primer comprising the nucleic acid sequence of SEQ ID NO:24, thereby generating an amplified hALB nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and a hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 via a reference signal from at least one label attached to the hALB probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0017] In another aspect, the present invention provides a method for quantitating transgene integration into CAR T cells, comprising: amplifying nucleic acid from the CAR T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:2 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:3, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the CAR T cells using a first hALB primer comprising the nucleic acid sequence of SEQ ID NO:23 and a second hALB primer comprising the nucleic acid sequence of SEQ ID NO:24, thereby generating an amplified hALB nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO:1 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and a hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 via a reference signal from at least one label attached to the hALB probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0018] In another aspect, the present invention provides a method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from a CAR T cell with a first CAR primer, a second CAR primer, a first hALB primer, and a second hALB primer, where the first CAR primer comprises the nucleic acid sequence of SEQ ID NO:11, the second CAR primer comprises the nucleic acid sequence of SEQ ID NO:12, the first hALB primer comprises the nucleic acid sequence of SEQ ID NO:23, and the second hALB primer comprises the nucleic acid sequence of SEQ ID NO:24; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying a hALB nucleic acid using a first hALB primer and a second hALB primer, thereby generating an amplified hALB nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and the hALB probe via a reference signal from at least one label attached to the hALB probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0019] In another aspect, the present invention provides a method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from a CAR T cell with a first CAR primer, a second CAR primer, a first hALB primer, and a second hALB primer, where the first CAR primer comprises the nucleic acid sequence of SEQ ID NO:2, the second CAR primer comprises the nucleic acid sequence of SEQ ID NO:3, the first hALB primer comprises the nucleic acid sequence of SEQ ID NO:23, and the second hALB primer comprises the nucleic acid sequence of SEQ ID NO:24; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying a hALB nucleic acid using a first hALB primer and a second hALB primer, thereby generating an amplified hALB nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO:1 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and the hALB probe via a reference signal from at least one label attached to the hALB probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0020] In a further aspect, the present invention provides a method for quantifying transgene integration into CAR T cells, comprising: contacting nucleic acid from the CAR T cell with a first CAR primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, and combinations thereof; contacting nucleic acid from the CAR T cell with a second CAR primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and combinations thereof; contacting nucleic acid from the CAR T cells with a first hALB primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, and combinations thereof; contacting nucleic acid from the CAR T cells with a second hALB primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, and combinations thereof; contacting the nucleic acid from the CAR T cell with a CAR probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, and combinations thereof; contacting the nucleic acid from the CAR T cells with a hALB probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, and combinations thereof; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid molecule; amplifying a hALB nucleic acid using a first hALB primer and a second hALB primer, thereby generating an amplified hALB nucleic acid molecule; Detecting hybridization between the amplified CAR nucleic acid molecule and the CAR probe via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid molecule and the hALB probe via a reference signal from at least one label attached to the hALB probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0021] In a further aspect, the invention provides a method of generating a CAR T cell, comprising: Introducing a CAR transgene into a T cell to obtain a T cell having the transgene integrated therein, and determining the integration of the CAR transgene, amplifying nucleic acid from the transgene-integrated T cell with a first CAR primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, and combinations thereof, and a second CAR primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and combinations thereof, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the transgene-integrated T cell using a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, and combinations thereof via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; Quantifying the copy number of the transgene by comparing the target signal to a reference signal; ), and and obtaining a CAR T cell comprising at least one copy of the integrated CAR transgene.

[0022] In a still further aspect, the invention provides a method of generating a CAR T cell, comprising: Introducing a CAR transgene into a T cell to obtain a T cell having the transgene integrated therein, and determining the integration of the CAR transgene, contacting nucleic acid from a T cell having integrated a transgene with a first CAR primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, and combinations thereof; and contacting nucleic acid from a T cell having integrated a transgene with a second CAR primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and combinations thereof; contacting nucleic acid from a T cell having integrated a transgene with a first hALB primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, and combinations thereof; and contacting nucleic acid from a T cell having integrated a transgene with a second hALB primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, and combinations thereof; contacting nucleic acid from a T cell incorporating the transgene with a CAR probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, and combinations thereof; contacting nucleic acid from a T cell incorporating the transgene with a hALB probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, and combinations thereof; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid molecule; amplifying a hALB nucleic acid using a first hALB primer and a second hALB primer, thereby generating an amplified hALB nucleic acid molecule; Detecting hybridization between the amplified CAR nucleic acid molecule and the CAR probe via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid molecule and the hALB probe via a reference signal from at least one label attached to the hALB probe; Quantifying the copy number of the transgene by comparing the target signal to a reference signal; ), and and obtaining a CAR T cell comprising at least one copy of the integrated CAR transgene.

[0023] Aspects of the invention also provide CAR T cells generated by the methods described herein.

[0024] In certain embodiments, the step of detecting hybridization between the amplified CAR nucleic acid molecule and the CAR probe comprises detecting a change in a target signal from at least one label attached to the CAR probe during or after hybridization relative to a target signal from at least one label attached to the CAR probe before hybridization.

[0025] In certain embodiments, the step of detecting hybridization between the amplified hALB nucleic acid molecule and the hALB probe comprises detecting a change in a target signal from at least one label attached to the hALB probe during or after hybridization relative to a target signal from at least one label attached to the hALB probe before hybridization.

[0026] In certain embodiments, at least one of the amplification steps comprises a polymerase chain reaction (PCR), such as real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), ligase chain reaction, or transcription-mediated amplification (TMA).

[0027] In certain embodiments, the amplified nucleic acid is an amplicon.

[0028] In some embodiments, at least one label attached to the CAR probe comprises a fluorophore. In some embodiments, at least one label attached to the hALB probe comprises a fluorophore. [Brief description of the drawings]

[0029] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1] 1 shows a gel image from a singleplex primer / probe screening assay. [Diagram 2] A gel image of a multiplex primer / probe assay is shown. [Diagram 3] A gel image of transgene (FP) Set 1 and (RP) Set 2 multiplexed with hALB Set 1 is shown. [Figure 4A] Amplification curves for transgene (FP) Set 1 and (RP) Set 2 and the standard curve for hALB Set 1 are shown. [Figure 4B] Amplification curves for transgene (FP) Set 1 and (RP) Set 2 and the standard curve for hALB Set 1 are shown. [Figure 4C] Amplification curves for transgene (FP) Set 1 and (RP) Set 2 and the standard curve for hALB Set 1 are shown. [Figure 4D] Amplification curves for transgene (FP) Set 1 and (RP) Set 2 and the standard curve for hALB Set 1 are shown. [Figure 5A] Fresh vs. frozen standard curves (transgene targets) are shown. [Figure 5B] Fresh vs. frozen standard curves (transgene targets) are shown. [Figure 6] Circular vs. linear standard curves (transgene target) are shown. [Figure 7] Characterized vs. typical transgene qPCR standard curves are shown. [Figure 8] Characterized transgene standard linearity plots are shown. [Figure 9] An exemplary qPCR plate layout is shown. [Figure 10] An exemplary control quantitative qPCR plate layout is shown. [Figure 11] 1 shows an exemplary transgene linearity plot. [Figure 12] 1 shows an exemplary hALB linearity plot. [Figure 13-1] The nucleotide sequence of the human serum albumin (hALB) gene, GenBank accession M12523.1, is shown. [Figure 13-2] The nucleotide sequence of the human serum albumin (hALB) gene, GenBank accession M12523.1, is shown. [Figure 13-3] The nucleotide sequence of the human serum albumin (hALB) gene, GenBank accession M12523.1, is shown. [Figure 13-4] The nucleotide sequence of the human serum albumin (hALB) gene, GenBank accession M12523.1, is shown. [Figure 13-5] The nucleotide sequence of the human serum albumin (hALB) gene, GenBank accession M12523.1, is shown. [Figure 13-6] The nucleotide sequence of the human serum albumin (hALB) gene, GenBank accession M12523.1, is shown. [Figure 14] SEQ ID NO:11 is disclosed.

[0030] The foregoing will be apparent from the following more particular description of exemplary embodiments, as illustrated in the accompanying drawings, in which like reference characters represent the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] A description of exemplary embodiments follows below.

[0032] Some aspects of the present invention are described below with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present invention. However, one of ordinary skill in the art will readily recognize that the present invention can be practiced without one or more of the specific details, or can be practiced with other methods, protocols, reagents, cell lines, and animals. The present invention is not limited by the order of acts or events illustrated, as some acts may occur in different orders and / or in parallel with other acts or events. Furthermore, not all illustrated acts, steps, or events are required to practice a methodology in accordance with the present invention. Many of the techniques and procedures described or referenced herein are well understood by those of ordinary skill in the art and are commonly employed using conventional techniques.

[0033] Unless otherwise defined, all technical terms, designations, and other scientific or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is generally understood in the art. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.

[0034] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the articles "a," "an," and "the" are to be understood as including plural referents unless the context clearly indicates otherwise.

[0035] The term "comprising", or variations such as "comprises" or "comprising", as used herein, may be used to indicate the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0036] The present invention relates to kits and methods for quantifying transgene integration into chimeric antigen receptor (CAR) T cells. Additionally, a panel of probes and primers is provided for performing a polymerase chain reaction (PCR), e.g., quantitative PCR, to quantify transgene integration into CAR T cells.

[0037] In some embodiments, the transgene qPCR method and kit described by the present invention includes a multiplex quantitative polymerase chain reaction (qPCR) assay designed to quantify the BCMA CAR transgene plasmid integrated into the CAR T drug product. Both (1) the BCMA CAR transgene plasmid (transgene) and (2) the human albumin (hALB) reference gene are amplified in this qPCR method. The primer and probe set for the transgene target amplifies the junction between the CD137 and CD3z regions of the plasmid to ensure that only the BCMA CAR transgene plasmid is present and integrated into the CAR T drug product is detected.

[0038] Chimeric Antigen Receptor Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides (scFv) that contain the antigen-binding domain of an antibody linked to a T cell signaling domain. As used herein, the terms "T cell", "T-cell" and "T lymphocyte" are used interchangeably. CARs feature the ability to redirect the specificity and reactivity of T cells toward selected targets in a non-MHC-restricted manner, utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition allows T cells expressing CARs to recognize antigens independent of antigen processing, thus bypassing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0039] The CARs described herein provide recombinant polypeptide constructs that include at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a "cytoplasmic signaling domain") that includes a functional signaling domain derived from a stimulatory molecule as defined below. T cells expressing CARs are referred to herein as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably herein. The cells can be genetically modified to stably express an antibody binding domain on their surface and confer novel antigen specificity that is MHC-independent.

[0040] In some cases, T cells are genetically engineered to stably express a CAR that combines the antigen recognition domain of a specific antibody with the intracellular domain of the CD3-zeta chain or the FcγRI protein into a single chimeric protein. In one embodiment, the stimulatory molecule is the zeta chain associated with the T cell receptor complex.

[0041] "Intracellular signaling domain", as the term is used herein, refers to the intracellular portion of a molecule. It is a functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity through a defined signaling pathway by generating a second messenger, or by functioning as an effector by responding to such a messenger. The intracellular signaling domain generates a signal that promotes immune effector functions of the CAR-containing cell, e.g., CAR T cells. Examples of immune effector functions, e.g., in CAR T cells, include cytolytic activity and helper activity, including secretion of cytokines.

[0042] In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Examples of primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent simulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Examples of costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CAR T, the primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0043] The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3-zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12. In a specific embodiment, the signaling sequence is CD3-zeta.

[0044] The term "zeta", or alternatively "zeta chain", "CD3-zeta", or "TCR-zeta" is defined as the protein provided under GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species, e.g., murine, rabbit, primate, mouse, rodent, monkey, ape, etc., and "zeta stimulatory domain", or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit an initial signal required for T cell activation. In one aspect, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., that is a functional ortholog thereof.

[0045] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell (such as, but not limited to, proliferation). A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (also referred to herein as "CD137"). In a specific embodiment, the costimulatory molecule is 4-1BB (CD137).

[0046] The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and the ligand that specifically binds to CD83.

[0047] The intracellular signaling domain can comprise the entire intracellular portion (i.e., full length) or the entire native intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof.

[0048] The term "4-1BB" refers to a member of the tumor necrosis factor receptor (TNFR) superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., a mammal (mouse, rodent, monkey, ape, etc.), and a "4-1BB costimulatory domain" is defined as amino acid residues 214 to 255 of GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., a mammal (mouse, rodent, monkey, ape, etc.).

[0049] In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined herein. In one embodiment, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, CD3-zeta, and / or CD28. CD28 is a T cell marker important in T cell costimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and functions as a costimulatory immune checkpoint molecule. 4-1BB delivers a strong costimulatory signal to T cells, promoting differentiation and improving long-term survival of T lymphocytes. CD3-zeta generates signals upon association with TCR and contains immunoreceptor tyrosine-based activation motifs (ITAMs).

[0050] In one embodiment, the CAR comprises an intracellular hinge domain comprising CD8 and an intracellular T cell receptor signaling domain comprising CD28, 4-1BB, CD3-zeta, and combinations thereof.

[0051] In certain embodiments, the CAR comprises the CD8a transmembrane, CD137, and CD3z coding regions.

[0052] The present disclosure further provides primers, probes, and associated kits useful for quantifying variant plasmids incorporated into CAR T products, such as functional variants of the CARs, nucleic acids, polypeptides, and proteins described herein. As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, such as substitutions, insertions, or deletions. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity with a parent CAR, polypeptide, or protein, and these functional variants retain the biological activity of the CAR, polypeptide, or protein of which they are variants. Functional variants include, for example, variants of a CAR, polypeptide, or protein (parent CAR, polypeptide, or protein) described herein that retain the ability to recognize target cells to a similar extent, to the same extent, or to a greater extent than the parent CAR, polypeptide, or protein. With respect to a parent CAR, polypeptide, or protein, a functional variant can be, for example, at least about 30%, about 40%, about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent CAR, polypeptide, or protein.

[0053] A functional variant may, for example, comprise the amino acid sequence of a parent CAR, polypeptide, or protein with at least one conservative amino acid substitution. In another embodiment, a functional variant may comprise the amino acid sequence of a parent CAR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution cannot suppress or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can improve the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent CAR, polypeptide, or protein.

[0054] The amino acid substitutions of CAR may be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions that replace one amino acid with a particular physical and / or chemical property with another amino acid with the same or similar chemical or physical property. For example, conservative amino acid substitutions may be an acidic amino acid (e.g., Asp or Glu) substituted with another acidic amino acid, an amino acid with a non-polar side chain substituted with another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid (Lys, Arg, etc.) substituted with another basic amino acid, an amino acid with a polar side chain substituted with another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0055] A CAR, polypeptide, or protein can consist essentially of a particular amino acid sequence described herein, such that other components, e.g., other amino acids, do not substantially alter the biological activity of the CAR, polypeptide, or protein.

[0056] Examples of modified nucleotides that can be used to generate recombinant nucleic acids utilized to generate the polypeptides utilized in the methods / kits described herein include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N-acetyl-2-pyridine ... 6 -Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyladenine, uracil-5-oxyacetate (v), wybutoxosine, pseudouracil, queosine, beta-D-galactosylqueosine, inosine, N6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0057] The nucleic acids of the invention can include any isolated or purified nucleotide sequence encoding a CAR, a polypeptide or protein, or any functional portion or variant thereof. Alternatively, the nucleotide sequence can include a nucleotide sequence that is degenerate to any of the sequences, or a combination of degenerate sequences.

[0058] Some embodiments of the present invention also utilize isolated or purified nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0059] Nucleotide sequences that hybridize under stringent conditions may hybridize under high stringency conditions.

[0060] As described herein, "high stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of the nucleic acids described herein) in an amount detectably greater than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences or polynucleotides with only a few interspersed mismatches from random sequences that end up having several small regions (e.g., 3-12 bases) that match the nucleotide sequence. Such small regions of complementation melt more easily than full-length complements of 14-17 or more bases, making them easily distinguishable by high stringency hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for hybridization to the CAR nucleic acids described herein. It is generally understood that conditions can be made more stringent by the addition of increasing amounts of formamide.

[0061] As used herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows for the expression of an mRNA, protein, polypeptide, or peptide by a host cell, where the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors described herein are not naturally occurring in their entirety. However, portions of the vector may be naturally occurring. The recombinant expression vectors described may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Non-naturally occurring or modified nucleotides or internucleotide linkages do not inhibit transcription or replication of the vector.

[0062] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) may be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector, for example a gamma retroviral vector.

[0063] In one embodiment, recombinant expression vectors are prepared using standard recombinant DNA techniques as described, for example, in Ausubel FM, Brent R, Kingston RE et al. (eds) (1999) Short Protocols in Molecular Biology, 4th edn. New York: Wiley Green MR and Sambrook J. (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor, New York. Circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, SV40, 2μ plasmid, lambda, bovine papilloma virus, and the like.

[0064] In certain embodiments, expression vectors utilized by the present disclosure are linearized to prepare working stocks of plasmids for generating standards and controls.

[0065] The recombinant expression vector may, as appropriate, include regulatory sequences, such as transcriptional and translational initiation and termination codons, specific for the type of host (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, considering whether the vector is DNA- or RNA-based.

[0066] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation to provide prototrophy in auxotrophic hosts, etc. Suitable marker genes for the described expression vectors include, for example, the neomycin / G418 resistance gene, the histidinol x resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0067] The recombinant expression vector may include a native or canonical promoter operably linked to a nucleotide sequence encoding a CAR, polypeptide, or protein (including functional portions and functional variants thereof), or to a nucleotide sequence that is complementary to or hybridizes to a nucleotide sequence encoding a CAR, polypeptide, or protein. Selection of a promoter, e.g., strong, weak, tissue-specific, inducible, and developmental specific, is within the skill of one of ordinary skill in the art. Similarly, combining a nucleotide sequence with a promoter is also within the skill of one of ordinary skill in the art. The promoter may be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, a RSV promoter, an SV40 promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0068] Recombinant expression vectors can be designed for either transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0069] Additionally, the recombinant expression vector can be made to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene can be a gene that confers sensitivity to an agent (e.g., a drug) on ​​a cell expressing the gene, causing the cell to die when the cell is contacted or exposed to the agent. Suicide genes are known in the art and include, for example, Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0070] Conjugates, e.g., bioconjugates, comprising CAR, polypeptides, or proteins (including any functional portion or variant thereof), host cells, nucleic acids, recombinant expression vectors, host cell populations, or antibodies, or any antigen-binding portion thereof, are included within the scope of the present invention. Conjugates, as well as methods for synthesizing conjugates, are generally known in the art (see, e.g., Hudecz, F., Methods Mol. Biol. 298:209-223 (2005) and Kirin et al., Inorg Chem. 44(15):5405-5415 (2005)).

[0071] In certain embodiments, the recombinant expression vector utilized in the embodiments of the present invention is a vector comprising various components of the B-cell maturation antigen (BCMA) chimeric antigen receptor. The plasmid is an 8,518 base pair (bp) plasmid containing sequences encoding various components of the BCMA chimeric antigen receptor as disclosed by SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 in WO 2017 / 025038(A1), the entire contents of which are incorporated herein by reference. In one aspect, the plasmid encodes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, the extracellular antigen binding domain binding to the BCMA antigen. As used herein, the terms "B cell", "B-cell", and "B lymphocyte" are used interchangeably. In one embodiment, the plasmid comprises the nucleic acid sequence of any one of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1). In some embodiments, the plasmid comprises a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1).

[0072] The term "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a specified sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a specified sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, may be referred to as encoding the protein or other product of that gene or cDNA.

[0073] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns to the extent that in some cases the nucleotide sequence encoding the protein may contain introns.

[0074] In one embodiment, the present disclosure provides an expression vector comprising any of the nucleic acid sequences of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1).

[0075] The term "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids incorporating a recombinant polynucleotide, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0076] Methods for generating CAR T cells and quantifying transgene incorporation into CAR T cells In one aspect, the present invention provides a method for quantifying transgene integration into a CAR T cell, comprising: contacting nucleic acid from the CAR T cell with a first CAR primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, and combinations thereof; contacting nucleic acid from the CAR T cell with a second CAR primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and combinations thereof; contacting nucleic acid from the CAR T cells with a first hALB primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, and combinations thereof; contacting nucleic acid from the CAR T cells with a second hALB primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, and combinations thereof; contacting the nucleic acid from the CAR T cell with a CAR probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, and combinations thereof; contacting the nucleic acid from the CAR T cells with a hALB probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, and combinations thereof; amplifying the CAR amplicon using a first CAR primer and a second CAR primer, thereby generating an amplified CAR nucleic acid molecule; amplifying the hALB amplicon using a first hALB primer and a second hALB primer, thereby generating an amplified hALB nucleic acid molecule; Detecting hybridization between the amplified CAR nucleic acid molecule and the CAR probe via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid molecule and the hALB probe via a reference signal from at least one label attached to the hALB probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0077] In some embodiments, the contacting step of the CAR primer is performed in a separate reaction from the hALB primer. In other embodiments, the contacting steps are performed in the same reaction (i.e., multiplexed).

[0078] In some embodiments, the contacting step of the CAR probe is performed in a separate reaction from the hALB probe. In other embodiments, the contacting steps are performed in the same reaction (i.e., multiplexed).

[0079] In some embodiments, the amplification step of the CAR amplicon is performed in a separate reaction from the hALB amplicon. In other embodiments, the amplification steps are performed in the same reaction (i.e., multiplexed).

[0080] In some embodiments, the step of detecting hybridization of the CAR nucleic acid and the CAR probe is performed in a separate reaction from the hALB nucleic acid and the hALB probe. In other embodiments, the detection step is performed in the same reaction (i.e., multiplexed).

[0081] In some embodiments, the method involves amplifying a CAR nucleic acid with a first CAR primer of about 20 to about 40 nucleotides in length. In some embodiments, the first CAR primer is capable of hybridizing under high stringency conditions to a CAR nucleic acid sequence set forth as any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1).

[0082] A primer, ie, a nucleotide sequence that hybridizes under stringent conditions, is capable of hybridizing under high stringency conditions.

[0083] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, and SEQ ID NO:20. In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, and SEQ ID NO:20. In certain embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, and SEQ ID NO:20.

[0084] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 2. In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 2. In certain embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 2.

[0085] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 5. In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 5. In certain embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 5.

[0086] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 8. In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 8. In certain embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 8.

[0087] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO:11.

[0088] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 11. In certain embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 11.

[0089] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 14. In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 14. In certain embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 14.

[0090] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 17. In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 17. In certain embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 17.

[0091] In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 20. In some embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 20. In certain embodiments, the first CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 20.

[0092] In some embodiments, the method involves amplifying the CAR nucleic acid with a second CAR primer of about 20 to about 40 nucleotides in length. In some embodiments, the second CAR primer is capable of hybridizing under high stringency conditions to a CAR nucleic acid sequence set forth as any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1).

[0093] In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, and SEQ ID NO:21. In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, and SEQ ID NO:21. In certain embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, and SEQ ID NO:21.

[0094] In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 3. In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 3. In certain embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 3.

[0095] In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 6. In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 6. In certain embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 6.

[0096] In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 9. In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 9. In certain embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 9.

[0097] In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 12. In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 12. In certain embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 12.

[0098] In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 15. In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 15. In certain embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 15.

[0099] In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 18. In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 18. In certain embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 18.

[0100] In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 21. In some embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 21. In certain embodiments, the second CAR primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 21.

[0101] In some embodiments, the method involves hybridizing a CAR nucleic acid molecule to a CAR-specific probe of about 20 to about 40 nucleotides in length and detecting hybridization between the CAR nucleic acid and the probe. In some embodiments, the probe is detectably labeled. In some embodiments, the CAR-specific probe is capable of hybridizing under high stringency conditions to a CAR nucleic acid sequence set forth as any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1).

[0102] In some embodiments, detecting hybridization can be carried out by conventional molecular biology techniques known in the art, such as, but not limited to, gel electrophoresis, Southern blot, and the like.

[0103] In some embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, and SEQ ID NO:19.

[0104] In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, and SEQ ID NO:19. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, and SEQ ID NO:19.

[0105] In some embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 1. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 1. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 1.

[0106] In some embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 4. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 4. In certain embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 4.

[0107] In some embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 7. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 7. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 7.

[0108] In some embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 10. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 10. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 10.

[0109] In some embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 13. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 13. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 13.

[0110] In some embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 16. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 16. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 16.

[0111] In some embodiments, the CAR-specific probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 19. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 19. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 19.

[0112] In some embodiments, the method involves amplifying a hALB nucleic acid using a first hALB primer about 20 to about 40 nucleotides in length. In some embodiments, the first hALB primer is capable of hybridizing under high stringency conditions to the hALB nucleic acid sequence set forth as SEQ ID NO:31 (FIG. 13).

[0113] In some embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:26, and SEQ ID NO:29. In some embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:26, and SEQ ID NO:29. In certain embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:26, and SEQ ID NO:29.

[0114] In some embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 23. In some embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 23. In certain embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 23.

[0115] In some embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 26. In some embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 26. In certain embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 26.

[0116] In some embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 29. In some embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 29. In certain embodiments, the first hALB primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 29.

[0117] In some embodiments, the method involves amplifying a hALB nucleic acid using a second hALB primer about 20 to about 40 nucleotides in length. In some embodiments, the second hALB primer is capable of hybridizing under high stringency conditions to the hALB nucleic acid sequence set forth as SEQ ID NO:31.

[0118] In some embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, and SEQ ID NO:30. In some embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, and SEQ ID NO:30. In certain embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, and SEQ ID NO:30.

[0119] In some embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 24. In some embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 24. In certain embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 24.

[0120] In some embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 27. In some embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 27. In certain embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 27.

[0121] In some embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 30. In some embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 30. In certain embodiments, the second hALB primer comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 30.

[0122] In some embodiments, the method involves hybridizing a hALB nucleic acid molecule to a hALB-specific probe of about 20 to about 40 nucleotides in length and detecting hybridization between the hALB nucleic acid and the probe. In some embodiments, the probe is detectably labeled. In some embodiments, the hALB-specific probe is capable of hybridizing under high stringency conditions to the hALB nucleic acid sequence set forth as SEQ ID NO:31.

[0123] In some embodiments, detecting hybridization can be carried out by conventional molecular biology techniques known in the art, such as, but not limited to, gel electrophoresis, Southern blot, and the like.

[0124] In some embodiments, the probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28.

[0125] In some embodiments, the probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 22. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 22. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 22.

[0126] In some embodiments, the probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 25. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 25. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 25.

[0127] In some embodiments, the probe comprises a nucleic acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 28. In some embodiments, the probe comprises a nucleic acid sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleic acid sequence set forth as SEQ ID NO: 28. In certain embodiments, the probe comprises a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 28.

[0128] In certain aspects, the present invention provides a method for quantifying transgene integration into CAR T cells, comprising: contacting nucleic acid from the CAR T cell with a first CAR primer comprising the nucleic acid sequence of SEQ ID NO: 11; and contacting nucleic acid from the CAR T cell with a second CAR primer comprising the nucleic acid sequence of SEQ ID NO: 12; contacting nucleic acid from the CAR T cells with a first hALB primer comprising the nucleic acid sequence of SEQ ID NO:23; and contacting nucleic acid from the CAR T cells with a second hALB primer comprising the nucleic acid sequence of SEQ ID NO:24; contacting nucleic acid from a CAR T cell with a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10; contacting nucleic acid from the CAR T cells with a hALB probe comprising the nucleotide sequence of SEQ ID NO:22; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid molecule; amplifying a hALB nucleic acid using a first hALB primer and a second hALB primer, thereby generating an amplified hALB nucleic acid molecule; Detecting hybridization between the amplified CAR nucleic acid molecule and the CAR probe via a target signal from at least one label attached to the CAR probe; The method includes detecting hybridization between an amplified hALB nucleic acid molecule and a hALB probe via a reference signal from at least one label attached to the hALB probe, and quantifying the copy number of the transgene by comparing the target signal to the reference signal.

[0129] In certain embodiments, the step of detecting hybridization between the amplified CAR nucleic acid molecule and the CAR probe comprises detecting a change in a target signal from at least one label attached to the CAR probe during or after hybridization relative to a target signal from at least one label attached to the CAR probe before hybridization.

[0130] In certain embodiments, the step of detecting hybridization between the amplified hALB nucleic acid molecule and the hALB probe comprises detecting a change in a target signal from at least one label attached to the hALB probe during or after hybridization relative to a target signal from at least one label attached to the hALB probe before hybridization.

[0131] In another aspect, the present invention provides a method for quantitating transgene integration into CAR T cells, comprising: amplifying nucleic acid from the CAR T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the CAR T cell using a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0132] In yet another aspect, the present invention provides a method of generating a CAR T cell, comprising: introducing the CAR transgene into a T cell to obtain a T cell incorporating the transgene; 2. Determine the integration of the CAR transgene (wherein the determination is amplifying nucleic acid from the transgene-integrated T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the transgene-integrated T cell using a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; Quantifying the copy number of the transgene by comparing the target signal to a reference signal; ), and and obtaining a CAR T cell comprising at least one copy of the integrated CAR transgene.

[0133] Aspects of the invention also provide CAR T cells generated by the methods described herein.

[0134] "Reference gene" refers to an internal reaction control that has a sequence different from the target gene. For a gene to be considered as a reference, it must meet several important criteria (Chervoneva I, Li Y, Schulz S, Croker S, Wilson C, Waldman SA, Hyslop T. Selection of optimal reference genes for normalization in quantitative RT-PCR. BMC Bioinforma. 2010;11:253. doi:10.1186 / 1471-2105-11-253.). Most importantly, its expression level must not be affected by experimental factors. It must also show minimal variation in expression between tissues and physiological states of the organism. It is desirable to choose a reference gene that shows a similar threshold cycle as the gene of interest. The reference gene must show variation resulting from imperfections in the techniques and preparation procedures used, which ensures that any variation in the amount of genetic material is related to the same extent as the study subjects and controls. An example of a "reference gene" that meets the above criteria is a basic metabolic gene called a housekeeping gene, which, by definition, is involved in a process essential for cell survival. A housekeeping gene that is useful as a reference gene must also be expressed at a stable and unregulated level (Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E. Housekeeping genes as internal standards: Use and limits. J Biotechnol. 1999; 75: 291-295. doi: 10.1016 / S0168-1656 (99) 00163-7). Housekeeping genes useful as "reference genes" in the methods, kits, and primers / probes of the invention include, but are not limited to, LDHA, NONO, PGK1, PPIH, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29.

[0135] In another aspect, the present invention provides a method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from a CAR T cell with a first CAR primer, a second CAR primer, a first reference gene primer, and a second reference gene primer, where the first CAR primer comprises the nucleic acid sequence of SEQ ID NO:11 and the second CAR primer comprises the nucleic acid sequence of SEQ ID NO:12; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying the reference gene nucleic acid using the first reference gene primer and the second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0136] In some embodiments, the detection of hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from at least one label attached to the CAR probe during or after hybridization, relative to a target signal from a label attached to the CAR probe before hybridization. In some embodiments, the detection of hybridization between the amplified reference gene nucleic acid molecule and the reference gene probe comprises detecting a change in a target signal from at least one label attached to the reference gene probe during or after hybridization, relative to a target signal from a label attached to the reference gene probe before hybridization. In some embodiments, the at least one label attached to the reference gene probe comprises a fluorophore.

[0137] In some embodiments, detecting hybridization can be carried out using conventional molecular biology techniques known in the art, such as, but not limited to, gel electrophoresis, Southern blot, and the like.

[0138] In certain embodiments, at least one of the amplification steps comprises a polymerase chain reaction (PCR), such as real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), ligase chain reaction, or transcription-mediated amplification (TMA).

[0139] PCR is well known to those skilled in the art. PCR is a method widely used in molecular biology to make many copies of a specific DNA segment. Using PCR, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of copies of that specific DNA segment. Most PCR methods rely on thermal cycling. Thermal cycling exposes reactants to repeated cycles of heating and cooling to enable different temperature-dependent reactions - DNA melting and enzyme-driven DNA replication. PCR uses two main reagents - primers (short single-stranded nucleotide fragments known as oligonucleotides whose sequence is complementary to the target DNA region) and DNA polymerase. In the first step of PCR, the two strands of the DNA double helix are physically separated at high temperature by DNA melting. In the second step, the temperature is lowered, allowing the primers to bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase, which enzymatically assembles new DNA strands from the available free nucleotides in the reaction mixture. As PCR proceeds, the DNA generated uses itself as a template for replication, resulting in exponential amplification of the original DNA template. Typically, PCR applications use a thermostable DNA polymerase such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus.

[0140] Quantitative PCR or real-time PCR (qPCR), as known to those skilled in the art, is a technique that allows to estimate the amount of a given sequence present in a sample and is often applied to quantitatively determine the level of gene expression. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA products after each round of PCR amplification. qPCR measures the concentration while the synthesis process is taking place, allowing the quantification and detection of specific DNA sequences in real time.

[0141] Reverse transcriptase polymerase chain reaction (RT-PCR), as known to those skilled in the art, is a laboratory technique that combines reverse transcription of RNA into DNA (complementary DNA or cDNA) using PCR with the amplification of a specific DNA target. It is generally used to measure the amount of a specific RNA. This is accomplished by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). The combination of RT-PCR and qPCR is routinely used to analyze gene expression and quantification of RNA.

[0142] As known to those skilled in the art, ligase chain reaction (LCR) is a method of DNA amplification. Ligase chain reaction (LCR) is an amplification process that differs from PCR in that it involves a thermostable ligase to join two probes or other molecules together, which can then be amplified by standard PCR cycling.

[0143] Transcription-mediated amplification (TMA), as known to those skilled in the art, is an isothermal single-tube nucleic acid amplification system that utilizes two enzymes, RNA polymerase and reverse transcriptase. In contrast to PCR and LCR, the TMA method involves RNA transcription (via RNA polymerase) and DNA synthesis (via reverse transcriptase) to generate an RNA amplicon (the source or product of amplification) from a target nucleic acid.

[0144] In some embodiments, the methods described by this disclosure utilize other quantitative PCR methods known in the art, such as, but not limited to, digital PCR (dPCR).

[0145] In some embodiments, at least one label attached to the CAR probe comprises a fluorophore. In some embodiments, at least one label attached to the hALB probe comprises a fluorophore. The term "fluorophore" as used herein refers to any fluorescent compound or protein that can be used to quantify and detect the nucleotide sequence to which the probe hybridizes.

[0146] The present disclosure also relates to primers capable of hybridizing to and amplifying a CAR nucleic acid, e.g., a nucleic acid sequence spanning the CD137 / CD3z junction of a CAR construct. The primers described can be utilized in the methods described herein. In some embodiments, these primers are about 20 to about 40 nucleotides in length and can hybridize under ultra-high stringency conditions to a CAR nucleic acid sequence set forth as any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1).

[0147] In some embodiments, these primers comprise a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, or SEQ ID NO:20. In some embodiments, these primers further comprise a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:21.

[0148] The present disclosure also relates to probes that can hybridize to and distinguish between various CAR nucleic acid sequences, for example, various nucleic acid sequences spanning the CD137 / CD3z junction of a CAR construct. The probes described can be utilized in the methods described herein. In some embodiments, these probes are about 20 to about 40 nucleotides in length and can hybridize under ultra-high stringency conditions to the CAR nucleic acid sequences set forth as any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1). In some embodiments, these probes comprise a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 19.

[0149] In one aspect, the invention provides a probe and primer set comprising a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10, at least one label attached to the probe, a first CAR primer comprising the nucleic acid sequence of SEQ ID NO: 11, and a second CAR primer comprising the nucleic acid sequence of nucleic acid sequence 12. In one embodiment, the probe and primer set further comprises a hALB probe comprising the nucleotide sequence of SEQ ID NO: 22, at least one label attached to the probe, a first hALB primer comprising the nucleic acid sequence of SEQ ID NO: 23, and a second hALB primer comprising the nucleic acid sequence of nucleic acid sequence 24.

[0150] In some embodiments, at least one label comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof, In some embodiments, the labels may be made to emit light through photochemical, chemical, and electrochemical means.

[0151] The present invention also provides a kit for quantifying transgene integration into CAR T cells. The term "kit" as used herein refers to a combination of reagents and other materials. It is contemplated that the kit may include reagents such as buffers, protein stabilizing reagents, signal generating systems (e.g., fluorescent signal generating systems), antibodies, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not intended to be limited to a particular combination of reagents and / or other materials. In one embodiment, the kit further includes instructions for using the reagents. The kit may be packaged in any suitable manner, typically with the elements in a single container or in various containers as needed, in addition to a sheet of instructions for performing the test. In some embodiments, the kit also includes a positive control sample. The kit may be produced in a variety of ways known in the art.

[0152] In one aspect, a kit for quantifying transgene integration into CAR T cells comprises a probe comprising the nucleotide sequence of SEQ ID NO: 10, at least one label attached to the probe, a first primer comprising the nucleic acid sequence of SEQ ID NO: 11, and a second primer comprising the nucleic acid sequence of nucleic acid sequence 12. In one embodiment, the kit further comprises a hALB probe comprising the nucleotide sequence of SEQ ID NO: 22, at least one label attached to the probe, a first hALB primer comprising the nucleic acid sequence of SEQ ID NO: 23, and a second hALB primer comprising the nucleic acid sequence of nucleic acid sequence 24.

[0153] In some embodiments of the kits of the invention, at least one label attached to a probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0154] In one embodiment, the kit of the invention comprises an array comprising the probes, hi some embodiments, the array is a multiwell array.

[0155] In certain embodiments, at least one label attached to the hALB probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0156] As used in the aspects described by the present invention, the term "label" refers to a moiety capable of generating a detectable signal, i.e., a moiety that can be detected in small amounts by a detection means that generates a signal. Examples of suitable such means include spectroscopic or photochemical means, such as fluorescence or luminescence, or biochemical, immunochemical, or chemical means, such as a change in physical, biochemical, immunochemical, or chemical properties upon contact with a detector analyte compound or upon reaction with a polypeptide or polypeptide / enzyme mixture to form a detectable complex. Thus, as used herein, the term "label" is intended to include both moieties that can be directly detected, such as radioisotopes or fluorescent dyes, as well as reactive moieties that are indirectly detected via a reaction that forms a detectable product, such as an enzyme that reacts with a substrate to form a product that can be detected spectrophotometrically. It should be noted that the labeling reagent may contain a radioactive labeling moiety, such as a radioisotope. In one embodiment, the hybridization probes herein are labeled with non-radioactive substances to avoid the disadvantages associated with radioanalysis.

[0157] For use of the target detection scheme in the methods and kits described by this invention, the nucleotide bases are labeled by covalently attaching compounds such that a fluorescent or chemiluminescent signal is generated following incorporation of the dNTP into the extending DNA primer / template.

[0158] Examples of fluorescent compounds for labeling dNTPs include, but are not limited to, fluorescein, rhodamine, and BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). See "Handbook of Molecular Probes and Fluorescent Chemicals" available from Molecular Probes, Inc. (Eugene, Oreg.). Examples of chemiluminescent compounds that can be used include, but are not limited to, luminol and dioxetanones (see Gundennan and McCapra, "Chemiluminescence in Organic Chemistry", Springer-Verlag, Berlin Heidleberg, 1987).

[0159] Fluorescently or chemiluminescently labeled dNTPs are added separately to a DNA template system containing template DNA annealed to a primer, DNA polymerase, and appropriate buffer conditions. After a reaction interval, excess dNTPs are removed and the system is probed to detect whether fluorescently or chemiluminescently tagged nucleotides have been incorporated into the DNA template. Detection of the incorporated nucleotides can be accomplished using a variety of methods that depend on the type of tag utilized.

[0160] In the case of fluorescently tagged dNTPs, the DNA template system may be illuminated with optical radiation at a wavelength that is strongly absorbed by the tag body, and the fluorescence from the tag is detected, for example, using a photodetector with an optical filter that eliminates any scattered light at the excitation wavelength.

[0161] In a further embodiment utilizing fluorescence detection in the kits and methods described herein, the fluorescent tag is attached to the dNTP by a photocleavable or chemically cleavable linker, and the tag is cleaved after the extension reaction and transferred from the template system to a detection cell, where the presence and amount of the tag is determined by light excitation at a suitable wavelength and detection of fluorescence. In this embodiment, due to the presence of multiple fluorescent tags directly adjacent to each other on the extended primer strand complementary to the single base repeat region in the template, the possibility of fluorescence quenching is minimized and the accuracy with which the repeat number can be determined is optimized. Furthermore, by exciting the fluorescence in a separate chamber, the possibility of photolytic damage to the DNA primer / template system is minimized.

[0162] In one embodiment, the probe contains a 5' 6-FAM™ (fluorescein) label. 6-AM™ is a single isomer derivative of fluorescein. FAM™ is a fluorescent dye linkage for oligonucleotides and is compatible with most fluorescence detection instruments. It is protonated and has reduced fluorescence below pH 7 and is typically used in the pH range of 7.5-8.5. FAM™ can be attached to either the 5' or 3' end of the oligo.

[0163] In one embodiment, the probe contains a 5'HEX™ (hexachlorofluorescein) label. Hexachlorofluorescein is a chemical analog of fluorescein utilized in multiplex assays with FAM™. HEX™ can only be added to the 5' end of an oligonucleotide.

[0164] The present disclosure also contemplates the use of any other label known in the art for use in labeling the probes described herein, for example, but not limited to, VIC®, TET™, JOE™, NED™, PET®, ROX™, TAMRA™, TET™, Texas Red®, ATTO™ 532, Cy3, Tye™ 563, Tye™ 665, TEX615™, Cy5, ZEN™, Iowa Black® FQ, Iowa Black® RQ, DABYCL, and Yakima Yellow™.

[0165] In one embodiment, the probe comprises a fluorescent quencher label. The quencher label can be used as a dual quencher in the reactions disclosed herein. In one embodiment, the probe comprises an IowaBlack® FQ quencher. IowaBlack® FQ has a broad absorbance spectrum ranging from 420-620 nm with a peak absorbance at 531 nm. This quencher is utilized with fluorescein and other fluorescent dyes that emit in the green to pink spectral range. The present disclosure contemplates the use of any fluorescent quencher label known in the art, for example, but not limited to, ZEN™, Black Hole Quencher® (BHQ-1, BHQ-2, BHQ-3, etc.).

[0166] The transgene qPCR method and kit described by the present invention includes a multiplex quantitative polymerase chain reaction (qPCR) assay designed to quantify the BCMA CAR transgene plasmid integrated into the CAR T drug product. In this qPCR method, two targets are amplified: (1) the BCMA CAR transgene plasmid (transgene) and (2) the human albumin (hALB) reference gene. The primer and probe set for the transgene target amplifies the junction between the CD137 and CD3z regions of the plasmid to ensure that only the BCMA CAR transgene plasmid is present and integrated into the CAR T drug product is detected. The copy number result of the hALB reference gene is used to calculate the reportable result of vector copy number (VCN) per cell for each sample tested in the qPCR reaction.

[0167] Exemplary embodiments are described below.

[0168] Embodiment 1. A probe and primer set comprising a probe comprising a nucleotide sequence of SEQ ID NO:10, at least one label attached to the probe, a first primer comprising a nucleic acid sequence of SEQ ID NO:11, and a second primer comprising a nucleic acid sequence of nucleic acid sequence 12.

[0169] Embodiment 2. The probe and primer set of embodiment 1, wherein at least one label comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0170] Embodiment 3. A kit for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising a probe comprising the nucleotide sequence of SEQ ID NO:10, at least one label attached to the probe, a first primer comprising the nucleic acid sequence of SEQ ID NO:11, and a second primer comprising the nucleic acid sequence of nucleic acid sequence 12.

[0171] Embodiment 4. The kit of embodiment 3, wherein at least one label attached to the probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0172] Embodiment 5. The kit of embodiment 3, comprising an array comprising the probes.

[0173] Embodiment 6. The kit of embodiment 5, wherein the array is a multi-well plate.

[0174] Embodiment 7. The kit of embodiment 3, further comprising a human albumin (hALB) probe comprising the nucleic acid sequence of SEQ ID NO:22, at least one label attached to the hALB probe, a first hALB primer comprising the nucleic acid sequence of SEQ ID NO:23, and a second hALB primer comprising the nucleic acid sequence of SEQ ID NO:24.

[0175] Embodiment 8. The kit of embodiment 7, wherein at least one label attached to the hALB probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

[0176] Embodiment 9. The kit of embodiment 3, further comprising a reference gene probe, at least one label attached to the reference gene probe, a first reference gene primer, and a second reference gene primer.

[0177] Embodiment 10. A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: amplifying nucleic acid from the CAR T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the CAR T cells using a first hALB primer comprising the nucleic acid sequence of SEQ ID NO:23 and a second hALB primer comprising the nucleic acid sequence of SEQ ID NO:24, thereby generating an amplified hALB nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and a hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 via a reference signal from at least one label attached to the hALB probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0178] Embodiment 11. A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from a CAR T cell with a first CAR primer, a second CAR primer, a first hALB primer, and a second hALB primer, where the first CAR primer comprises the nucleic acid sequence of SEQ ID NO:11, the second CAR primer comprises the nucleic acid sequence of SEQ ID NO:12, the first hALB primer comprises the nucleic acid sequence of SEQ ID NO:23, and the second hALB primer comprises the nucleic acid sequence of SEQ ID NO:24; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying a hALB nucleic acid using a first hALB primer and a second hALB primer, thereby generating an amplified hALB nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and the hALB probe via a reference signal from at least one label attached to the hALB probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0179] Embodiment 12. The method of embodiment 10 or 11, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from at least one label attached to the CAR probe during or after hybridization relative to a target signal from a label attached to the CAR probe before hybridization.

[0180] Embodiment 13. The method of embodiment 10 or 11, wherein detecting hybridization between the amplified hALB nucleic acid molecule and the hALB probe comprises detecting a change in a target signal from at least one label attached to the hALB probe during or after hybridization relative to a target signal from a label attached to the hALB probe before hybridization.

[0181] Embodiment 14. The method of embodiment 10 or 11, wherein the amplification comprises polymerase chain reaction (PCR).

[0182] Embodiment 15. The method of embodiment 14, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

[0183] Embodiment 16 The method of embodiment 10, wherein at least one label attached to the CAR probe comprises a fluorophore.

[0184] Embodiment 17 The method of embodiment 10, wherein at least one label attached to the hALB probe comprises a fluorophore.

[0185] Embodiment 18. A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: amplifying nucleic acid from the CAR T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the CAR T cell using a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0186] Embodiment 19. A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from a CAR T cell with a first CAR primer, a second CAR primer, a first reference gene primer, and a second reference gene primer, where the first CAR primer comprises the nucleic acid sequence of SEQ ID NO:11 and the second CAR primer comprises the nucleic acid sequence of SEQ ID NO:12; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying the reference gene nucleic acid using the first reference gene primer and the second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal to a reference signal.

[0187] Embodiment 20. The method of embodiment 18 or 19, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from at least one label attached to the CAR probe during or after hybridization relative to a target signal from a label attached to the CAR probe before hybridization.

[0188] Embodiment 21. The method according to embodiment 18 or 19, wherein detecting hybridization between the amplified Reference Gene nucleic acid molecule and the Reference Gene probe comprises detecting a change in a target signal from at least one label attached to the Reference Gene probe during or after hybridization, relative to a target signal from a label attached to the Reference Gene probe before hybridization.

[0189] Embodiment 22. The method of embodiment 18 or 19, wherein the amplification comprises polymerase chain reaction (PCR).

[0190] Embodiment 23. The method of embodiment 22, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

[0191] Embodiment 24 The method of embodiment 18, wherein at least one label attached to the CAR probe comprises a fluorophore.

[0192] Embodiment 25 The method of embodiment 18, wherein at least one label attached to the reference gene probe comprises a fluorophore.

[0193] Embodiment 26. A method for generating chimeric antigen receptor (CAR) T cells, comprising: introducing the CAR transgene into a T cell to obtain a T cell incorporating the transgene; Determining integration of the CAR transgene, wherein the determination comprises: amplifying nucleic acid from the transgene-integrated T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the transgene-integrated T cell using a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; quantitating the copy number of the transgene by comparing the target signal to a reference signal; and obtaining a CAR T cell comprising at least one copy of the integrated CAR transgene.

[0194] Embodiment 27. The method of embodiment 26, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from at least one label attached to the CAR probe during or after hybridization relative to a target signal from a label attached to the CAR probe before hybridization.

[0195] Embodiment 28. The method according to embodiment 26, wherein detecting hybridization between the amplified Reference Gene nucleic acid molecule and the Reference Gene probe comprises detecting a change in a target signal from at least one label attached to the Reference Gene probe during or after hybridization, relative to a target signal from a label attached to the Reference Gene probe before hybridization.

[0196] Embodiment 29. The method of embodiment 26, wherein the amplification comprises polymerase chain reaction (PCR).

[0197] Embodiment 30. The method of embodiment 29, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

[0198] Embodiment 31 The method of embodiment 26, wherein at least one label attached to the CAR probe comprises a fluorophore.

[0199] Embodiment 32 The method of embodiment 26, wherein at least one label attached to the reference gene probe comprises a fluorophore.

[0200] Embodiment 33. A CAR T cell generated by the method according to any one of embodiments 26 to 32.

[0201] The following examples are provided to further illustrate some of the embodiments disclosed herein and are intended to be illustrative and not limiting of the embodiments of the present disclosure.

[0202] Example 1: Development of a transgene qPCR method Summary of method: The exemplary transgene qPCR method described is a multiplex quantitative polymerase chain reaction (qPCR) assay designed to quantify the BCMA CAR transgene plasmid (referred to in the examples as "pLLV-LICAR2SIN plasmid") integrated into the CAR T drug product. This qPCR method amplifies: (1) the transgene pLLV-LICAR2SIN plasmid (transgene) (a transgene having a nucleotide sequence comprising any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from WO 2017 / 025038(A1)) and (2) the human albumin (hALB) reference gene. To ensure detection of only the pLLV-LICAR2SIN plasmid that is present and integrated into the CAR T drug product, the primer and probe set for the transgene target amplifies the junction between the CD137 and CD3z regions of the plasmid. The copy number results of the hALB reference gene are used to calculate a reportable result of vector copy number (VCN) per cell for each sample tested in the qPCR reaction. The VNC / cell sample results of the transgene qPCR method are reported for the safety, purity, and identity of the CAR T drug product sample.

[0203] Transgene primer and probe design: The BCMA CAR transgene plasmid, referred to as "pLLV-LICAR2SIN plasmid", is an 8,518 base pair (bp) plasmid that contains sequences encoding various different components of the B cell maturation antigen (BCMA) chimeric antigen receptor (CAR). The transgene targeting of the qPCR was required to detect only the pLLV-LICAR2SIN plasmid that was present and integrated into the CAR T drug product, and the targeted region had to belong specifically to the BCMA CAR construct. To ensure the specificity of the transgene qPCR targeting, primer and probe pairs were designed to target at least one junction between at least two regions of the pLLV-LICAR2SIN plasmid that encode the CAR construct. First, a suitable region of the pLLV-LICAR2SIN plasmid had to be identified.

[0204] The longest base pair (bp) coding region integrated into the genome of the CAR T drug product and specific to the CAR construct belongs to the two variable heavy chain parts of the BCMA CAR construct. These two regions are separated by a short linker sequence. The nucleotide sequence regions of the plasmid corresponding to the two variable heavy chain parts and the linker were entered into the National Center for Biotechnology Information (NCBI) Nucleotide Basic Local Alignment Search Tool (BLAST) site (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to determine whether these regions could be suitable targets for transgene qPCR. However, the BLAST results yielded multiple hits for various immunoglobulin variable regions across many different species, including human (homo sapiens). Therefore, it was determined that the coding regions of the two variable heavy chain components of the CAR were not suitable targets for transgene qPCR.

[0205] The junction between the CD137 and CD3z regions of the pLLV-LICAR2SIN plasmid is included in the region of the plasmid that is integrated into the genome of the CAR T drug product and is a component of the BCMA CAR. The size of the CD3z coding region of the pLLV-LICAR2SIN plasmid makes it the second longest bp coding region of the CAR segment of the plasmid, making it a more suitable region for targeting because a larger number of potentially suitable primer and probe pairs may be found from the larger coding region. The CD3z coding region is directly adjacent to the CD137 coding region of the plasmid. On the other side of the CD137 coding region is a plasmid backbone sequence that is not specific to the BCMA CAR construct. Therefore, the junction between CD137 and CD3z was the only suitable option for targeting when the larger CD3z coding region is included in the primer and probe design.

[0206] The PrimerQuest® Tool from Integrated DNA Technologies, Inc. (IDT) (Coralville, Iowa) (https: / / www.idtdna.com / Primerquest / Home / Index) was used to design suitable primer and probe pairs for testing in the development of the qPCR method. The nucleotide sequence of the pLLV-LICAR2SIN plasmid corresponding to the CD137 and CD3z coding regions was entered into the PrimerQuest Tool. The optimal primer melting temperature (Tm) was set at 60° C., and the nucleotides corresponding to the junction between CD137 and CD3z were entered into the “overlapping junction list” to ensure that either the forward or reverse primer overlapped this junction. This resulted in four primer and probe pairs (see Table 1). Two pairs have a forward primer that spans the CD137 / CD3z junction, and two pairs have a reverse primer that spans the junction. The assay design parameters were then adjusted to restrict the probe design to span the CD137 / CD3z junction. This resulted in three additional primer and probe pairs (see Table 1), bringing the total to seven primer and probe pairs suitable for testing to develop a qPCR method. All seven primer / probe pairs were entered into the NCBI BLAST site to check for possible cross-reactivity in the human genome. None of the BLAST results for any of the seven pairs indicated any potential cross-reactivity.

[0207] [Table 1] Note: FP = junction-spanning forward primer; RP = junction-spanning reverse primer; PRB = junction-spanning probe.

[0208] Three hALB primer and probe sets were used and tested in the development of the qPCR method (see Table 2). One set was chosen from a published paper (S Charrier et al. Lentiviral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients. Gene Therapy (2007) 14,415-428.), a second set was chosen from a CRO digital PCR method assay that was ultimately not used, and a third set was designed using the PrimerQuest Tool and the hALB gene region targeted by both the published paper and the CCHMChALB primer and probe set. All three primer / probe pairs were entered into the NCBI BLAST site to check for possible cross-reactivity with any non-hALB products in the human genome. None of the BLAST results for any of the three pairs indicated any possible cross-reactivity.

[0209] [Table 2] Note: Set 1 = CCHMC hALB set, Set 2 = hALB set from published paper, Set 3 = internally designed hALB set

[0210] Screening primers and probes: Seven different transgene primer and probe sets and three different hALB primer and probe sets were screened by performing singleplex qPCR reactions with CAR T drug product, mock T cell DNA as sample plus pLLV-LICAR2SIN plasmid, and mock T cell DNA. Mock T cell DNA was taken from T cells that underwent the same selection and amplification process as CAR T product before transduction with lentivector. The qPCR products of CAR T cells, mock T cells, mock T cell DNA plus pLLV-LICAR2SIN plasmid, and "no template control" (NTC) samples were then run on an agarose gel. Any primer / probe set that did not result in a single band of the expected PCR product size was excluded from further transgene qPCR method development. All qPCR products, including NTC samples, also showed the presence of a primer dimer band of approximately 25 bp, which is an expected by-product of the qPCR reaction. The expected target bands were generated only from the transgene (FP) set 1 and transgene (RP) set 2 primer / probe sets, with only two <50 bp bands visible on the gel. One of the <50 bp bands was likely the expected primer dimer. The second <50 bp band was not clearly visible in the NTC samples, and it was unclear what this additional lower molecular weight band might be. See Figure 1 for an example gel image from a singleplex primer / probe screening assay. Additional unexpected bands seen in the qPCR products of CAR T, mock T cell, and mock T cell DNA plus plasmid samples eliminated only the hALB set 3 primer / probe set from further qPCR method development. Additionally, all three hALB primer / probe sets produced two <50 bp bands visible on the gel. One of the <50 bp bands was likely the expected primer dimer. A second <50 bp band was not clearly seen in the NTC samples and the possible origin of this additional lower molecular weight band was uncertain.However, given that this was the same low molecular weight binding pattern seen with all transgene primer / probe sets and that optimizing the amount of primer or probe in the reaction and testing different annealing temperatures did not eliminate the bands, this additional band was expected to be the result of the presence of uracil-DNA glycosylase (UNG) or some other unrelated qPCR by-product in the qPCR master mix.

[0211] The next step in the screening process for transgene primer / probe sets was to run the two acceptable sets (FP Set 1 and RP Set 2) in a multiplex qPCR reaction with the two acceptable hALB primer / probe sets (Set 1 and Set 2) using a standard curve. A standard curve was generated by spiking known concentrations of pLLV-LICAR2SIN plasmid into mock T cell DNA and making five 5-fold serial dilutions of mock T cell samples spiked with this plasmid using low EDTA TE buffer as the diluent. Each point of the standard curve was generated and frozen as a single-use aliquot. Initially, both transgene primer / probe sets were tested in a multiplex qPCR with hALB Set 2. Additionally, it was run with CAR T DNA and mock T cell DNA samples to obtain specificity of the multiplex reaction. The criteria that the standard curves were expected to meet for both transgene and hALB targets to be acceptable for further transgene qPCR method development were as follows: (1) R 2 ≥ 0.98 and (2) qPCR efficiency 90-110%. CAR T DNA was required to measurably amplify in both the transgene and hALB targets, while mock T cell DNA samples were required to have measurable amplification only in the hALB target and no amount of amplification in the transgene target to meet the requirements for assay specificity.

[0212] Multiplex reactions of transgene (FP) Set 1 and hALB Set 2 yielded acceptable R values ​​of >0.98 for both transgene and hALB targets. 2 Results were obtained, but none of the target standard curves yielded acceptable qPCR efficiencies. Multiplex reactions of transgene (RP) Set 2 and hALB Set 2 also yielded acceptable R values ​​of >0.98 for both transgene and hALB targets. 2Results were obtained. Also, the transgene standard curve, but not the hALB standard curve, yielded acceptable qPCR efficiency. Both the transgene (FP) set 1 / hALB set 2 and transgene (RP) set 2 / hALB set 2 multiplex reactions yielded acceptable specificity results, with the CAR T DNA sample measurably amplified at both targets, and the mock T cell DNA measurably amplified only at the hALB target, with no amplification at the transgene target. The multiplex qPCR products were also run on a gel to determine if there were any off-target bands when the two primer / probe sets were multiplexed (see FIG. 2 for an exemplary gel image). No unexpected bands were seen in the gel results for any of the multiplex reactions. It was decided to test the standard curves in singleplex reactions to determine if multiplexing the reactions potentially affected qPCR efficiency. Given that only the efficiency of the transgene (RP) Set 2 reaction was within the acceptable range in the multiplex reaction, only the transgene (RP) Set 2 and hALB Set 2 primer / probe sets were tested in singleplex. The transgene (RP) Set 2 singleplex reaction resulted in low qPCR efficiency, which was outside the acceptable range. The hALB Set 2 singleplex reaction resulted in high qPCR efficiency, which was within the acceptable range. Attempting to optimize the primer / probe concentration of both target oligo sets, or attempting higher annealing temperatures, did not improve the efficiency of either target standard curve in the multiplex reaction. Both acceptable transgene primer / probe sets were then run in a multiplex reaction with the hALB Set 1 primer / probe.

[0213] First, we attempted a multiplex reaction of transgene (FP) set 1 and hALB set 1, and only the hALB standard curve showed a R 2 The R of the transgene target standard curve was >0.98. 2The R was <0.97. Both the transgene and hALB target standard curves yielded efficiencies outside the acceptable range, with the transgene target having a lower efficiency than that seen for the multiplex reaction with the primers / probes of hALB Set 2. The singleplex reaction with hALB Set 1 had an R 2 ≥ 0.98, yielding efficiencies similar to those seen in the multiplex reactions. A new standard curve was generated using a 5-point, 4-fold dilution scheme, which contained a smaller amount of pLLV-LICAR2SIN plasmid and mock T-cell DNA in standard #1. This was done in an attempt to improve qPCR efficiency by potentially diluting out any possible PCR inhibitors that may be present in the mock T-cell DNA stock, as well as reducing the amount of mock T-cell DNA required to generate larger lots of standards. Both the acceptable transgene primer / probe set and the hALB Set 1 primer / probe set were then tested in a multiplex reaction using this new standard curve. The R of both the transgene and hALB standard curves were 2 The efficiency and efficiencies were well within acceptable limits for both the transgene (FP) set 1 / hALB set 1 multiplex reaction and the transgene (RP) set 2 / hALB set 1 multiplex reaction using the new standard curve. The multiplex qPCR reaction products were also run on a gel to confirm that no off-target bands were detected (see FIG. 3). No off-target bands were detected for any of the multiplex reactions. Although the efficiency was similar between the multiplex reactions of the two transgene primer / probe sets, the shape of the transgene target amplification curve for the transgene (RP) set 2 / hALB set 1 multiplex reaction was a more typical sigmoidal curve with a clear upper plateau than the transgene (FP) set 1 / hALB set 1 multiplex reaction (see FIG. 4). Therefore, the transgene (RP) set 2 and hALB set 1 primer / probe sets were selected for further transgene qPCR method development.

[0214] Repeatability of troubleshooting efficiency: R 2 To determine whether acceptable results for efficiency and reproducibility were obtained, the transgene (RP) Set 2 / hALB Set 1 multiplex reaction using the novel standard curve scheme was repeated. However, standard curves for both transgene and hALB targets were only 88% and 89%, respectively, for the replicate assays. Attempts to optimize primer / probe concentrations for both targets slightly improved efficiency for both targets, while attempts to increase annealing temperature, as well as PCR enhancers DMSO, TMAC, and betaine, did not. At the same time, the reproducibility of efficiency results was also examined, and it was asked whether the VCN / cell range covered by the 4-fold standard curve could be increased to lower the potential LOQ of the assay. Thus, a multiplex reaction was run using five frozen 4-fold standard point samples, as well as a 5-point 5-fold standard curve was created by diluting frozen standard #1. The two standard curves were then run side-by-side in a multiplex qPCR reaction. The 4-fold standard curves yielded efficiencies of 94% and 91% for the transgene and hALB targets, respectively. The 5x standard curve yielded efficiencies of 102% and 99% for the transgene and hALB targets, respectively. The increased efficiency seen with the 5x standard curve versus the 4x standard curve was likely due to greater variability at the lower standard curve points when they were frozen versus diluting frozen standard #1 immediately prior to running the curve in the assay to make fresh standards #2-5.

[0215] The 4x and 5x standard curves were repeated to see if the 5x "fresh" standard curve still showed an improvement in efficiency versus the 4x "frozen" standard curve points. In replicate assays, the 4x "frozen" standard curve yielded efficiencies of 94% and 88% for the transgene and hALB targets, respectively. The 5x "fresh" standard curve yielded efficiencies of 102% and 99% for the transgene and hALB targets, respectively. To further confirm the reproducibility of these "fresh" vs. "frozen" standard curve results, additional runs were performed (see Figure 5). It was determined that the primary source of variability issues seen in the efficiency of the standard curves was due to the use of frozen standard curve points. Therefore, it was decided to make only standard #1 and freeze it as a single-use aliquot. This frozen standard #1 will be used to make fresh standards #2-5 immediately prior to running any of the assays in the future. It was also decided to use the 5x standard curve in the future to increase the VCN / cell range of the assay.

[0216] Troubleshooting VCN / Cell Discrepancies with DDPCR The assays were run and data collected while at least the first six batches of material were released. The VCN / cell assay run targets the RU5 promoter region of the pLLV-LICAR2SIN plasmid backbone [Inventor: Are the further details required to describe these regions specific enough for the skilled artisan?] Since it is a regulatory requirement that the VCN / cell qPCR assay targets the transgene portion of the CAR plasmid for cell therapy, the transgene qPCR method is intended to replace this backbone method. Therefore, it was a requirement that the results of the method VCN / cell be comparable to those of the transgene qPCR method. Genomic DNA from CAR T was tested with the transgene qPCR method and compared to the results of the LB_12 samples. Transgene standards and controls were run with ddPCR to determine if the assigned transgene and hALB copy values ​​were correct. The LB_12 DNA samples were also run with ddPCR to determine the true VCN / cell value.

[0217] The ddPCR reactions used primers / probes for transgene (RP) set 2 and hALB set 1 in BioRad Supermix for Probes ddPCR master mix. The thermocycling conditions used were those recommended in the Supermix kit. EcoRI was added to the master mix since enzymatic digestion of DNA is recommended for the most accurate ddPCR results. EcoRI was confirmed to cut the pLLV-LICAR2SIN plasmid only once and did not cut the amplified regions of either the transgene or the hALB target. The ddPCR results confirmed that the transgene standard and control copies of transgene and hALB were correct, but the LB_12 results were more comparable to the RU5 VCN / cell results. The VCN / cell results for the LB_12 qPCR results were determined to be inaccurate by ddPCR, but it was unknown how correct the transgene standard and control could be. Therefore, we used an enzyme that cut the pLLV-LICAR2SIN plasmid multiple times, considering that the smaller DNA pieces in the LB_12 gDNA may give more accurate results. Two additional enzymes, one cutting twice and one cutting three times, were tried, but the results of the VCN / cell ddPCR did not change. Therefore, we enzymatically digested several CAR T DNA samples and two transgene controls, cleaned up the reactions, and ran the DNA in transgene qPCR along with undigested standards, controls, and CAR T samples. The VCN / cell results for the digested controls were about 3.8-fold higher than the undigested VCN / cell results, while the VCN / cell results for the digested CAR T samples were about 1.3-fold lower than the undigested CAR T samples. These results raised the question of whether it was necessary to linearize the pLLV-LICAR2SIN plasmid to obtain accurate sample VCN / cell results.

[0218] An aliquot of pLLV-LICAR2SIN plasmid was linearized by digesting the plasmid using EcoRI enzyme. The enzyme digestion was cleaned up and the linearized plasmid was quantified. This linearized plasmid was diluted to the transgene copy value of the 5-fold transgene standard curve and run in the transgene qPCR assay. LB_12CAR TDNA was also run in the assay and the VCN / cell results were calculated from both the circularized (undigested) and linearized standard curve results. The Ct values ​​of the linearized standard curve points were approximately 2-fold lower than the undigested standard curve points (see Figure 6). Furthermore, the LB_12VCN / cell results calculated from the linearized standard curve were comparable to those obtained with ddPCR and by the additional RU5 qPCR method, whereas the VCN / cell results calculated from the circularized (undigested) standard curve were approximately 4-fold higher. This confirmed the need to linearize the pLLV-LICAR2SIN plasmid to obtain accurate VCN / cell results. Two lots of linearized plasmid standards and controls were generated: one large lot to be used as a GMP lot for clinical batch release testing and any other GMP testing, and one smaller lot to be used for analyst training and any non-GMP activity.

[0219] Linearity for a given amount of DNA typical of a sample: Sample DNA was diluted to a concentration of 0.02ug / uL and 5uL per 100ng total DNA per reaction was loaded into the qPCR assay. Samples with a stock concentration of <0.02ug / uL can be run in the assay as is, but the tolerance for hALB copies should be adjusted based on the amount of DNA loaded into the reaction. To obtain standard curves for both the transgene and hALB targets, a standard curve was created using a starting mock T cell DNA concentration of 0.05ug / uL and diluted in low EDTA TE buffer (see Table 3). To ensure that the linearity of the assay remained within acceptable limits when the standard curve was created with a constant amount of mock T cell gDNA typical of the samples, a characterization standard curve was created using a mock T cell DNA concentration of 0.02ug / uL and serially diluted using mock T cell DNA at 0.02ug / uL (see Table 4). This standard curve was then run side-by-side with the typical standard curve to confirm the linearity of the assay (see Figure 7). The log copies observed versus the log copies expected were also plotted to obtain the R 2 It was confirmed that a linear response of ≧0.98 was obtained (see FIG. 8).

[0220] Characterization standard curve results showed that transgene copies could still be accurately quantified at DNA concentrations consistent with typical sample concentrations (0.02 ug / uL).

[0221] [Table 3]

[0222] [Table 4]

[0223] Method Qualification: The transgene qPCR method was qualified according to the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. Three assays were performed to complete the method qualification. The assays passed the acceptance criteria for all method qualification parameters specified in the method qualification protocol. Table 5 summarizes the method qualification parameters, acceptance criteria, and qualification results (see Example 2).

[0224] [Table 5]

[0225] Example 2: Transgene qPCR 1.0 Purpose 1.1 This example describes an exemplary procedure for performing a quantitative real-time PCR (qPCR) assay to quantify the LiCAR plasmid incorporated into the CAR T product. The assay is designed as a multiplex qPCR targeting the junction between the CD137 and CD3z regions of the LiCAR plasmid as well as human albumin (reference gene).

[0226] 2.0 Scope 2.1 This method is applicable to CAR T cells after harvest, immediately prior to dose prescription, to determine: 2.1.1 Vector copy number 2.1.2 Transduction efficiency 2.1.3 Identity of LiCAR expression

[0227] 3.0 Definitions and Abbreviations 3.1 LOQ (Limit of Quantification) 3.2 NTC (No Template Control) 3.3 Ct (cycle threshold) 3.4 CV (Coefficient of Variation) 3.5 SD (standard deviation) 3.6 hALB (human albumin) 3.7 BCMA (B cell maturation antigen) 3.8 VCN (Vector Copy Number)

[0228] 4.0 Equipment 4.1 Centrifuge capable of spinning 96-well PCR plates (e.g., Beckman Coulter, Allegra X-14R with SX4750 rotor and swing set for 96-well plates) 4.2 QuantStudio 6 Real-Time PCR System 4.3 Freezer capable of reaching -70℃ 4.4 Freezer capable of -20℃ 4.5 Calibrated 8 or 12 channel pipette (20, 50uL, or other suitable size), e.g., Rainin pipette 4.6 Calibrated single channel pipettes (20, 100, 200, 1000uL or other suitable size), e.g. Rainin pipettes 4.7 Refrigerator or cold room capable of maintaining 2-8°C 4.8 QuantStudio PCR Software v1.3 or higher 4.9 Vortex mixer 4.10 Biosafety Cabinets

[0229] 5.0 Materials Note: Materials designated as "for example" may be substituted by similar materials without prior qualification. For materials designated as "or equivalent," the equivalent of the substitute must be demonstrated before samples can be used for testing. 5.1 DNase / RNase free water, e.g., Invitrogen catalogue no. 10977015. 5.2 TaqPath ProAmp Master Mix, ThermoFisher catalog number A 30866, or equivalent. 5.3 Qualified lots of BCMA transgene and ALB primers and probes, custom sequences through IDT or equivalent. 5.4 Qualified Lots of BCMA Transgene Standard #1 5.5 Qualified Lots of BCMA Transgene Medium and Low Controls 5.6 1x Low EDTA TE Buffer pH 8.0, RNase / DNase free, e.g., Quality Biological catalogue number 351-324-721. 5.7 0.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf Cat. No. 022431005 5.8 1.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf Cat. No. 022431021 5.9 2 mL centrifuge tubes, sterile, RNase / DNase-free, e.g., Eppendorf Cat. No. 022431048 5.10 5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf Cat. No. 0030119460 5.11 Pipette tips, sterile, filtered, (20, 200, 1000uL, or other suitable sizes), e.g., Rainin Catalog Nos. 30389226, 30389240, 30398213 5.12 96-well PCR plates, Applied Biosystems, catalog numbers 4483343, 4483354, 4483349, 4483350, 4483395, or equivalent 5.13 Micro Amp Optical Adhesive Film, Applied Biosystems, Catalog No. 4311971, or equivalent 5.14 Reagent reservoirs, sterile, RNase / DNase free, e.g., VistaLabs Cat. No. 3054-1002

[0230] 6.0 Notes 6.1 Wear appropriate PPE when working in the laboratory. 6.2 Follow detailed facility guidelines for working with hazardous chemicals. Refer to manufacturer's SDS for further details. 6.3 All pipetting steps should be performed using aseptic technique in a BSC. To minimize the risk of contamination of either the materials or reagents, it is recommended that the analyst wear disposable sleeve covers during all steps of the procedure.

[0231] 7.0 Procedure 7.1 Obtain an aliquot of each of the following: 7.1.1 BCMA transgene forward primer 10 μM working stock 7.1.2 BCMA transgene reverse primer 10 μM working stock 7.1.3 BCMA transgene probe 10 μM working stock 7.1.4 hALB forward primer 10 μM working stock 7.1.5 hALB reverse primer 10 μM working stock 7.1.6 hALB probe 10 μM working stock 7.1.7 Qualified BCMA Transgene qPCR Standard #1 7.1.8 Qualified BCMA transgene qPCR 2.00 copies / cell medium control 7.1.9 Qualified BCMA transgene qPCR low control 0.20 copies / cell 7.2 Prepare a multiplex master mix for the appropriate number of reactions according to Table 6. Additional excess reactions can be added by including more samples than will actually be run in the assay. For example, if you are running 10 samples but want extra reactions beyond the 10 already included in the calculations in the table below, run 11 samples (i.e., N=11). Each additional sample adds three reaction volumes.

[0232] [Table 6] *The formula is the volumes of components needed for a single 25 uL reaction multiplied by the sum of 24 standard / control wells + 10 excess reactions (34) x 3 x the number of samples (3 reaction wells per sample). 7.3 Vortex the master mix tube briefly and set aside. 7.4 Prepare standards #2-5 using low EDTA TE buffer according to Table 7. Be sure to mix each dilution briefly before moving on to make the next dilution.

[0233] [Table 7] 7.5 Vortex the master mix solution briefly again and pipette the mix into the reagent reservoir. 7.6 Using a multichannel pipette, pipette 20 µL of master mix into appropriate wells of a 96-well PCR plate (see Figure 9). 7.7 Using a single channel pipette, load 5 uL of standard, control, and sample DNA into the appropriate wells of a 96-well PCR plate according to the plate layout in Figure 9. Load 5 uL of low EDTA TE buffer into the NTC wells. 7.8 Seal the plate with optical adhesive film and centrifuge briefly at approximately 300 x g. 7.9 Load the PCR plate into the PCR instrument. 7.10 Open "Assay Template.edt", select "Save As", enter an appropriate name for your qPCR experiment, and save as an .eds file. Do not overwrite the template file. 7.11 Enter the name of the experiment in the Name field. 7.12 Ensure that the experimental characteristics are as specified below and that the thermal cycling conditions (Run Method tab) are set correctly according to those specified in Table 8 using a reaction volume of 25 uL. 7.12.1 Instrument Type: QuantStudio 6 Flex System 7.12.2 Block type: 96 well (0.2 mL) 7.12.3 Experiment Type: Standard Curve 7.12.4 Detection Reagents: TaqMan Reagents 7.12.5 Equipment characteristics: Standard

[0234] [Table 8] 7.13 Select Run and click on the instrument serial number to run the assay.

[0235] 8.0 Data Analysis 8.1 Use the Auto Baseline and Auto Ct features of the software to analyze the data by clicking Analyze. Then click Save to save the analysis. 8.2 Print the report PDF and include this with your assay documentation. 8.3 Calculate VCN / cell for each sample and positive control in triplicate as follows:

[0236]

number

[0237] 9.0 Assay Acceptance Criteria 9.1 Assay Acceptance Criteria 9.1.1 R of both the transgene and the hALB standard curve 2 The value must be ≥ 0.97. 9.1.2 The slope of the standard curve should be between -3.585 and -3.104 (equivalent to a PCR efficiency of 90.08% to 109.97%) 9.1.3 No Ct replicates can be "unconfirmed" for any standard. 9.1.4 All Ct replicates of the NTC must be “unconfirmed” for both the transgene and the hALB target. 9.1.5 The average Ct of standard #1 must be <23.0 for transgene targets and <22.0 for hALB targets. 9.1.6 The Ct SD of each standard must be ≦0.60 for both the transgene and hALB targets. 9.1.7 The mean hALB copies for both the medium and low controls should be 30,303.030 copies ± 30% (expected range: 21,212.121 to 39,393.939 copies). 9.1.8 2.00 VCN / cell medium control and The mean VCN / cell result for the low control of 0.20 VCN / cell should be ±35% of the target VCN / cell value for each control. 9.1.9 The CV% for VCN / cell replicates of the medium and low positive controls should be ≤20%. 9.1.10 If any of the above criteria are not met, the assay is invalid. 9.2 Sample Acceptance Criteria 9.2.1 The average hALB copies for each sample should be 30,303.030 copies ± 30% (expected range: 21,212.121 - 39,393.939 copies). 9.2.1.1 If the sample gDNA concentration is <0.02ug / uL, calculate the expected copies of hALB for that sample from the amount of DNA actually loaded into the reaction. Example: Sample gDNA concentration is 0.01ug / uL. (5uL)(0.01ug / uL) = 0.05ug = 50ng sample gDNA per reaction (50ng DNA) (1 copy ALB / 0.0033ng gDNA) = 15,152 copies of ALB. Expected range: 10,606-19,697 copies of ALB. 9.2.2 Triplicate hALB target copy values ​​for samples must be within the Ct range covered by the hALB standard curve. The Ct range is defined as the lowest Ct value of the triplicates for standard #1 and the highest Ct value of the triplicates for standard #5. 9.2.3 Triplicate transgene target copy values ​​for samples must exceed the transgene LOQ copies of 303.030. 9.2.3.1 If one or more replicates of a sample for the transgene target are below the transgene LOQ of 303.030 copies, the sample should be reported as below the LOQ. 9.2.4 If one or more replicates of a sample for a transgene target are lower than the lowest transgene Ct value of standard #1, then the sample is above the standard curve range and the sample should be reported as not quantifiable. For example, if a sample has transgene Ct values ​​of 20.1, 19.9, and 20.2, but the lowest transgene Ct value obtained with standard #1 is only 20.0, then the replicates of the 19.9 sample cannot be accurately quantified, therefore the sample is above the standard curve range and the sample should be reported as not quantifiable. Notify management and study director if a sample is determined to be not quantifiable. 9.2.5 CV% of sample VCN / cell replicates must be ≤20%. Note: For samples with replicates below the LOQ or determined to be not quantifiable, do not utilize CV%. 9.2.6 For any sample where all triplicate transgene copy values ​​are above the LOQ and all of the above acceptance criteria are met, report the mean VCN / cell value to two decimal places (e.g., 2.02 VCN / cell). 9.2.7 Any sample that does not meet all of the above acceptance criteria is invalid.

[0238] Genomic DNA isolation, qualification, and dilution 1.0 Purpose 1.1 An exemplary procedure for isolating and quantifying gDNA from CAR T samples or mock T cell suspensions or frozen cell pellets is described.

[0239] 2.0 Scope 2.1 Describe the procedure for isolating gDNA from: 2.1.1 CAR T cell samples post-harvest supplied as frozen cell pellets or fresh cell suspensions. 2.1.2 Mock T cells supplied as frozen cell pellets or fresh cell suspension.

[0240] 3.0 Equipment 3.1 Centrifuge capable of spinning 1.5 mL and 5 mL microcentrifuge tubes (e.g., Beckman Coulter, Allegra X-14R with SX4750 rotor with adapter for 1.5 mL microcentrifuge tubes) 3.2 Qubit4 Fluorometer, Invitrogen Catalog No. Q33226 3.3 Freezer capable of -70℃ 3.4 Freezer capable of -20℃ 3.5 Calibrated single channel pipettes (20, 100, 200, 1000uL or other suitable size), e.g. Rainin pipettes 3.6 Heat block capable of 55°C and suitable for 1.5 mL and 2 mL microcentrifuge tubes 3.7 Refrigerator or thermostat capable of maintaining 2-8℃ 3.8 Vortex mixer 3.9 Biosafety Cabinets

[0241] 4.0 Materials Note: Materials designated as "for example" may be substituted by similar materials without prior qualification. For materials designated as "or equivalent", the equivalent of the substitute must be demonstrated before it can be used to test samples. 4.1 DNase / RNase free water, e.g., Invitrogen catalogue no. 10977015. 4.2 RPMI 1640 medium containing L-glutamine and 25 mM HEPES, e.g., Corning catalog number 10-041-CV 4.3 1x Low EDTA TE Buffer pH 8.0, RNase / DNase free, molecular biology grade, e.g., Quality Biological catalogue number 351-324-721. 4.4 200 proof (96-100%) ethanol molecular biology grade, e.g., Decon Labs catalog number 3616EA 4.5 10x PBS molecular biology grade, e.g., Affymetrix Cat. No. 75889 4.6 PureLink Genomic DNA Mini Kit, Invitrogen Catalog No. K182001 4.7 Qubit™ Assay Tubes, Invitrogen™ Catalog No. Q32856 (Invitrogen, Carlsbad, CA). 4.9 Qubit dsDNA BR Assay Kit, Invitrogen Catalog No. Q328350 4.9 0.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf Cat. No. 022431005 4.10 1.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalog number 022431021 4.11 2 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalog number 022431048 4.12 5 mL centrifuge tubes, sterile, RNase / DNase-free, e.g., Eppendorf Cat. No. 0030119460 4.13 15 mL conical tube, sterile, e.g., Corning catalog no. 431470 4.14 Pipette tips, sterile, filtered, (20, 200, 1000uL, or other suitable sizes), e.g., Rainin Catalog Nos. 30389226, 30389240, 30398213

[0242] 5.0 Notes 5.1 Wear appropriate PPE when working in the laboratory. 5.2 Follow detailed facility guidelines for working with hazardous chemicals. Refer to manufacturer's SDS for further details. 5.2 All pipetting steps should be performed using aseptic technique in a BSC. To minimize the risk of contamination of any materials or reagents, it is recommended that the analyst wear disposable sleeve covers during all steps of the procedure.

[0243] 6.0 Procedure 6.1 When opening a new PureLink Genomic DNA Mini Kit, be sure to add ethanol to the Wash Buffer 1 and Wash Buffer 2 bottles according to the instructions on the label of each bottle. Mix the bottles thoroughly after adding the ethanol and mark each label where ethanol was added. Include your initials and the date along with the markings for ethanol addition. When all components are stored at room temperature, the kit is stable for up to one year. 6.2 Set the heat block to 55°C and allow it to reach the correct temperature before starting DNA extraction. 6.3 Use cell pellets to perform DNA extraction. Either fresh cell pellets or cell pellets stored frozen at -70°C may be used. A minimum of 2 x 10 cells per column 6 It is recommended that 10 cells are extracted, but in any case a maximum of 4 x 10 cells per column. 6 Viable cells can be extracted. Note: The manufacturer's specifications state that a maximum of 5 x 10 6 Although it is stated that 4 x 10 cells can be extracted, this method uses the viable cell count to determine the cell number to perform DNA isolation. Therefore, 20% buffer is provided to allow for the presence of dead cells that are also present in the cell suspension. 4 x 10 per column in the specification 6 If the viability percentage is below 80%, the maximum number of viable cells extracted per column needs to be reduced to compensate for the >20% dead cells present in the cell suspension. 6.4 Preparation of cell pellets from fresh cell suspensions. 6.4.1 At least a 150 uL aliquot of the cell suspension is required for counting with the NC-200. The aliquot should be diluted with NC-200 (5.0 x 10 4 ~5.0×10 6 Stock cell suspensions may be diluted in RPMI medium as necessary to stay within the dynamic range of RT-PCR (cells / mL). 6.4.2 Count the cells using the counting protocol. 6.4.3 Use the viable cell count to determine the volume of cells needed to achieve the desired number of extracted cells per PureLink column and dispense the cell suspension into 1.5 mL or 2 mL microcentrifuge tubes. For example, the number of viable cells from NC-200 was 2 × 10 7 The desired number of cells extracted per PureLink column is 4 x 10 cells / mL with a viability of 88%. 6 There are 10 surviving cells:

[0244]

number

[0245]

number

[0246]

number

[0247]

number

[0248] Preparation and qualification of new oligo lots 1.0 Purpose 1.1 An exemplary procedure for generating and qualifying a new lot of oligos for transgene qPCR is described.

[0249] 2.0 Equipment 2.1 Centrifuge capable of spinning 1.5 mL microcentrifuge tubes (e.g., Beckman Coulter, Allegra X-14R with SX4750 rotor and swing set for 96-well plates and adapters for 1.5 mL microcentrifuge tubes) 2.2 QuantStudio 6 Real-Time PCR System 2.3 Freezer capable of storing -20℃ 2.4 Calibrated 8 or 12 channel pipette (20, 50uL, or other suitable size), e.g., Rainin pipette 2.5 Calibrated single channel pipettes (20, 100, 200, 1000uL or other suitable size), e.g. Rainin pipettes 2.6 Refrigerator or low temperature room capable of maintaining 2-8℃ 2.7 QuantStudio PCR Software v1.3 or higher 2.8 A heat block capable of 55°C and suitable for 1.5 mL microcentrifuge tubes. 2.9 Vortex mixer

[0250] 3.0 Material Note: Materials designated as "for example" may be substituted by similar materials without prior qualification. For materials designated as "or equivalent", the equivalent of the substitute must be demonstrated before it can be used to test samples. 3.1 DNase / RNase free water, e.g., Invitrogen catalogue no. 10977015. 3.2 TaqPath ProAmp Master Mix, ThermoFisher catalog number A 30866, or equivalent. 3.3 Primers and probes for BCMA transgene and hALB lyophilized stocks, custom sequences through IDT or equivalent. 3.4 Qualified lots of BCMA transgene and ALB primers and probes, custom sequences through IDT or equivalent. 3.5 Qualified Lots of BCMA Transgene Standard #1 3.6 Qualified Lots of BCMA Transgene Medium and Low Controls 3.7 1x Low EDTA TE Buffer pH 8.0, RNase / DNase free, e.g., Quality Biological catalogue number 351-324-721. 3.8 0.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., VWR Screw-Cap Microcentrifuge Tubes Catalog No. 89004-286 or Eppendorf Catalog No. 022431005 3.9 1.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf Cat. No. 022431021 3.10 2 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalog number 022431048 3.11 5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalogue no. 0030119460 3.12 Pipette tips, sterile, filtered, (20, 200, 1000uL, or other suitable sizes), e.g., Rainin Catalog Nos. 30389226, 30389240, 30398213 3.13 96-well PCR plates, Applied Biosystems, Catalog Numbers 4483343, 4483354, 4483349, 4483350, 4483395, or equivalent 3.14 Micro Amp Optical Adhesive Film, Applied Biosystems, Catalog No. 4311971, or equivalent 3.15 Reagent reservoirs, sterile, RNase / DNase free, e.g., VistaLabs Cat. No. 3054-1002

[0251] 4.0 Notes 4.1 Wear appropriate PPE when working in the laboratory. 4.2 Follow detailed facility guidelines for working with hazardous chemicals. Refer to manufacturer's SDS for further details. 4.3 All pipetting steps should be performed using aseptic technique in a BSC. To minimize the risk of contamination of any materials or reagents, it is recommended that the analyst wear disposable sleeve covers during all steps of the procedure.

[0252] 6.0 Procedure NOTE: Oligos are qualified as a multiplex set (BCMA transgene and hALB). The last aliquot of any of the six oligos in the set is used before the lot is depleted of oligos. Oligos from one qualified set are mixed and not matched with oligos from another qualified set. Any remaining oligos from an already qualified set that are depleted should be discarded. 6.1 Order BCMA transgene oligos and hALB oligos (see Table 9 for sequences).

[0253] [Table 9] 6.2 Oligos are supplied lyophilized by the vendor with a specification sheet for each oligo. Store lyophilized oligos at -20°C until ready for reconstitution. Lyophilized oligos can be stored at -20°C for up to 12 months. Note: On average, it takes at least 2 weeks to receive oligos from the manufacturer. Therefore, at least one set of back-up lyophilized oligos should be kept on hold at all times to minimize impact to sample testing in case of a problem with any of the oligos from a new or qualified lot. 6.3 Reconstitution of oligos 6.3.1 Remove lyophilized oligo stocks from -20°C and centrifuge at 10,000 x g for 30 seconds to ensure all oligo stock reaches the bottom of the tube before opening the tube. Do not open the tubes prior to centrifugation to prevent loss of any oligos that may be dislodged from the bottom of the tube during transport. 6.3.2 Reconstitute each oligo to a 100 uM stock using low EDTA TE buffer as follows. 6.3.2.1 Find the nmol amount of each oligo on the individual oligo specification sheet. 6.3.2.2 Multiply the nmol amount by 10 to get the volume of TE buffer that needs to be added to the oligo tube to make a 100uM stock. 6.3.2.3 Add the volume of TE buffer calculated in step 11.3.2.2 for each oligo to the respective oligo tube. 6.3.2.4 Vortex each oligo tube briefly to thoroughly resuspend the lyophilized oligos in TE buffer. 6.3.2.5 Examine the primer tube to ensure that all lyophilized oligo is completely dissolved in the TE buffer. If it appears that there may be some particles of oligo that are not completely dissolved in the TE buffer, the tube may be heated to 55°C for 1-5 minutes to aid in resuspension. After heating, vortex the tube again briefly. 6.3.3 Dilute 100uM stock to oligo working stock concentration 6.3.3.1 Dilute 100uM primer stocks to 10uM working stocks using low EDTA TE buffer 6.3.3.2 Dilute the 100 uM probe stock to a 10 uM working stock using low EDTA TE buffer

[0254] [Table 10] 6.3.3.3 Prepare 20 uL aliquots of all primers and 35 uL aliquots of all probes in 0.5 mL screw cap tubes. The aliquot volumes specified are sufficient for a half PCR plate assay. Primers and probes may be dispensed in larger volumes to accommodate up to a full plate assay if necessary. 6.3.3.4 All aliquots should be labeled with the minimum information. 6.3.3.4.1 Oligo Nomenclature 6.3.3.4.2 uM concentration 6.3.3.4.3 Lot Number 6.3.3.4.4 Expiration date (1 year from date of reconstitution) 6.3.3.4.5 Store all working stocks at -20°C. 6.3.4 Qualification of New Lots of Oligos 6.3.4.1 Run both the currently qualified lot of oligos and any new lots of oligos in a minimum of three independent transgene qPCR assays using qualified lots of Standard #1, Mid, and Low controls following the protocols as follows: 6.3.4.1.1 Set up two master mixes for each of the three assays, one master mix will be made using the currently qualified lot of oligos and the second master mix will be made using a new lot of oligos. 6.3.4.1.2 Load the qPCR plate according to the plate map. 6.3.4.1.3 Open the "Oligo Qualification.edt" template, select "Save As", enter an appropriate name for your qPCR experiment, and save as an .eds file. Do not overwrite the template file. Load and run qPCR plate according to 6.3.4.1.4 protocol. 6.3.4.2 In the Setup section, select Assign. Highlight wells D1 through E12, right-click, and select Omit. This omits the new oligolot reactions from the analysis. Select the entire plate and click Analyze. 6.3.4.3 Print a PDF report of the data and indicate that it is an analysis of a qualified oligo lot. 6.3.4.3.1 All assay acceptance criteria outlined in DSTMD-24448 must be met. If any of the assay acceptance criteria are not met, the assay is invalid and must be repeated. Record any invalid assays on the Reagent Qualification Report. 6.3.4.4 In the Setup section, select Assign. Highlight wells D1 through E12, right-click, and select Include. Highlight wells A1 through B12, right-click, and select Omit. This will omit the qualified oligo lot reactions from the analysis. Select the entire plate and click Analyze. 6.3.4.5 Print a PDF report of the data and indicate that it is an analysis of a new oligonucleotide. 6.3.4.5.1 All results from the analysis of new oligolots must meet all assay acceptance criteria described herein. 6.3.4.5.2 Calculate the % difference in the mean Ct values ​​for each standard in the qualified and new oligolot reactions as follows:

[0255]

number

[0256] 2. Preparation of Linearized LiCAR Plasmid Working Stocks 1.0 Purpose 1.1 This example describes an exemplary procedure for linearizing the LiCAR plasmid and converting plasmid concentrations in mg / mL to copies / uL to create working stocks of linearized plasmid for use in generating standards and controls for transgene qPCR methods.

[0257] 2.0 Equipment 2.1 1.5 mL and 5 mL microcentrifuge tubes can be spun (e.g., Beckman Coulter, Allegra X-14R with a SX4750 rotor with an adapter for 1.5 mL microcentrifuge tubes). 2.2 Electrophoresis equipment, such as the Lonza FlashGel DNA system and FlashGel Dock, or the Invitrogen E-Gel electrophoresis equipment 2.3 Freezer capable of -70℃ 2.4 Refrigerator or low temperature room capable of maintaining 2-8℃ 2.5 Calibrated single channel pipettes (20, 100, 200, 1000uL or other suitable size), e.g. Rainin pipettes 2.6 Qubit4 Fluorometer, Invitrogen Cat. No. Q33226 2.7 A heat block capable of 37°C and suitable for 1.5 mL microcentrifuge tubes. 2.8 Biosafety Cabinets 2.9 Vortex mixer

[0258] 3.0 Material Note: Materials designated as "for example" may be substituted by similar materials without prior qualification. For materials designated as "or equivalent", the equivalent of the substitute must be demonstrated before it can be used to test samples. 3.1 pLLV-LiCAR2SIN LiCAR plasmid stock 3.2 DNase / RNase free water, e.g., Invitrogen Cat. No. 10977015 3.3 EcoRI HF enzyme, New England Biolabs Catalog No. R3101S, or equivalent 3.4 GeneJet Gel Extraction and DNA Cleanup Kit, ThermoFisher, Cat. No. K0831, or equivalent 3.5 Precast electrophoresis gels, such as Lonza FlashGel DNA Cassettes or Invitrogen E-Gel, suitable for resolution of high molecular weight bands (e.g., FlashGel DNA Cassettes, 1.2% 12+1 single layer, Lonza Cat. No. 57023) 3.6 Molecular weight DNA ladder suitable for determination of band sizes of at least 8000 kb (e.g., E-Gel 1Kb Plus DNA ladder, Invitrogen catalogue no. 10488090)1.1 1.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalogue no. 0224310211.2 2 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalogue no. 022431048 3.7 5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf Cat. No. 0030119460 3.8 1x Low EDTA TE Buffer pH 8.0, RNase / DNase free, e.g., Quality Biological catalogue number 351-324-721. 3.9 0.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., VWR Screw-Cap Microcentrifuge Tubes Catalog No. 89004-286 or Eppendorf Catalog No. 022431005 3.10 Pipette tips, sterile, filtered, (20, 200, 1000uL, or other suitable sizes), e.g., Rainin Catalog Nos. 30389226, 30389240, 30398213

[0259] 4.0 Notes 4.1 Wear appropriate PPE when working in the laboratory. 4.2 Follow detailed facility guidelines for working with hazardous chemicals. Refer to manufacturer's SDS for further details. 4.3 All pipetting steps should be performed using aseptic technique in a BSC. To minimize the risk of contamination of any materials or reagents, it is recommended that the analyst wear disposable sleeve covers during all steps of the procedure.

[0260] 5.0 Procedure 5.1 Before starting the plasmid digestion, pre-heat the heat block to 37°C. 5.2 Thaw a vial of LiCAR plasmid stock. 5.3 Determine the concentration of LiCAR DNA using a Qubit4 fluorometer and the procedure described previously herein. It may be necessary to dilute the plasmid stock to bring it within the range of the Qubit dsDNA BR kit (2-1000 ng DNA). 5.4 Dilute the required amount of plasmid in water required to digest 0.09ug / uL of plasmid DNA. For example, dilute a plasmid stock concentration of 1.24 ug / uL to 0.09 ug / uL by adding 3.63 uL of plasmid DNA to 46.37 uL of DNase / RNase free water. 5.5 Prepare the EcoRI HF enzymatic digestion reaction mixture at room temperature, adding each reagent in the order shown in Table 11.

[0261] [Table 11] NOTE: Multiple 50 uL enzymatic digestion reactions may be run repeatedly, if necessary, to obtain a sufficient amount of linearized DNA to prepare working stocks of LiCAR plasmids to generate standards and controls. 5.6 Prepare at least 5 uL of undigested plasmid DNA to serve as an intact plasmid DNA control on the gel. 5.7 Gently mix the digestion tube by pipette mixing or gently tapping the tube (do not vortex the reaction). Briefly spin down the reaction at 300 x g for 5 seconds. 5.8 Incubate the tubes in a 37°C heat block for 15 minutes. 5.9 Purify the digested plasmid DNA using the GeneJet Gel Extraction and DNA Cleanup Mini Kit. NOTE: The volume used for DNA purification should not exceed 200uL and the total amount of purified plasmid DNA should not exceed 10ug. If the total volume is greater than 200uL, split the volume equally between two or more tubes before proceeding with the purification steps below. 5.10 When opening a new GenJet kit, add 96-100% ethanol to the wash buffer bottles according to the instructions for each bottle before starting the DNA cleanup procedure. Verify the addition of ethanol by noting the date and initials of addition on the label. Store the wash buffer with ethanol at room temperature. Also check all solutions in the kit for any salting out prior to use. Dissolve any precipitates by warming the solutions to 37 °C, then equilibrate to room temperature before use. NOTE: DNA purification columns should be stored at 2-8 °C when the kit arrives and when the columns are not in use. Be sure to close the bag containing the DNA purification columns after each use. 5.11 Adjust the volume of the reaction mixture to 200 uL with DNase / RNase free water. For example, add 150 uL of water to 50 uL of digested DNA mixture. 5.12 Add 100 μL of Binding Buffer. Mix thoroughly by pipetting. 5.13 Add 300 μL of ethanol (96-100%) and mix by pipetting. 5.14 Transfer the mixture to a DNA purification microcolumn and collection tube. Centrifuge the column at 14,000 x g for 1 minute. Discard the flow-through. Place the DNA purification microcolumn back into the collection tube. Alternatively, place the tube into a 2 mL microcentrifuge tube and discard the collection tube. 5.15 Add 700 µL of wash buffer (supplemented with ethanol) to the column and centrifuge at 14,000 x g for 1 min. Discard the flow-through and place the column back into the collection tube. Alternatively, the tube may be placed into a 2 mL microcentrifuge tube and the collection tube discarded. 5.16 Perform another wash step by adding 700 µL of wash buffer to the column and centrifuging at 14,000 x g for 1 min. Discard the flow-through and place the column back into the same collection tube. Alternatively, the tube may be placed into a 2 mL microcentrifuge tube and the collection tube discarded. 5.17 Centrifuge the column for an additional minute at 14,000 x g to completely remove any residual wash buffer. 5.18 Transfer the column to a clean 1.5 mL microcentrifuge tube and add 10 uL of Elution Buffer (supplied with the GeneJet kit) to the center of the column. Do not touch or puncture the silica membrane with the pipette tip. 5.19 Elute the DNA by centrifugation at 14,000 x g for 1 minute. 5.20 Verify digestion of the VSV-G plasmid by gel electrophoresis of intact circular (undigested DNA) and linearized DNA. To preserve the greatest amount of linearized plasmid possible, it is recommended to make dilutions of the linearized plasmid stock to be used for gel electrophoresis. 5.21 Determine the concentration of purified linear VSV-G plasmid DNA using a Qubit4 fluorometer and the procedures described previously herein. 5.22 Convert the linear plasmid concentration determined in step 5.21 to copies / uL of LiCAR plasmid as follows:

[0262]

number

[0263]

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[0264] Preparation and qualification of new lots of standards 1.0 Purpose 1.1 An exemplary procedure for generating and qualifying a new lot of standard for transgene qPCR is described.

[0265] 2.0 Equipment 2.1 Centrifuge capable of spinning 1.5 mL microcentrifuge tubes (e.g., Beckman Coulter, Allegra X-14R with SX4750 rotor and swing set for 96-well plates and adapters for 1.5 mL microcentrifuge tubes) 2.2 QuantStudio 6 Real-Time PCR System 2.3 Freezer capable of storing -20℃ 2.4 Freezer capable of -70℃ 2.5 Calibrated 8 or 12 channel pipette (20, 50uL, or other suitable size), e.g. Rainin pipette 2.6 Calibrated single channel pipettes (20, 100, 200, 1000uL or other suitable size), e.g. Rainin pipettes 2.7 Refrigerator or thermostat capable of maintaining a temperature of 2 to 8°C 2.8 QuantStudio PCR Software v1.3 or higher 2.9 A heat block capable of 55°C and suitable for 1.5 mL microcentrifuge tubes. 2.10 Vortex mixer 2.11 Biosafety Cabinets

[0266] 3.0 Material Note: Materials designated as "for example" may be substituted by similar materials without prior qualification. For materials designated as "or equivalent", the equivalent of the substitute must be demonstrated before it can be used to test samples. 3.1 DNase / RNase free water, e.g., Invitrogen catalogue no. 10977015. 3.2 TaqPath ProAmp Master Mix, ThermoFisher catalog number A 30866, or equivalent. 3.3 Mock T cell pellet with 2 x 106 - 4 x 106 cells per pellet. 3.4 Qualified lots of BCMA transgene and ALB primers and probes, custom sequences through IDT or equivalent. 3.5 Qualified Lots of BCMA Transgene Standard #1 3.6 Qualified Lots of BCMA Transgene Medium and Low Controls 3.7 Working Stock Aliquot of pLLV-LICAR2SIN 3.8 1x Low EDTA TE Buffer pH 8.0, RNase / DNase free, e.g., Quality Biological catalogue number 351-324-721. 3.9 0.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., VWR Screw-Cap Microcentrifuge Tubes Catalog No. 89004-286 or Eppendorf Catalog No. 022431005 3.10 1.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalog number 022431021 3.11 2 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalog number 022431048 3.12 5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf Cat. No. 0030119460 3.13 Pipette tips, sterile, filtered, (20, 200, 1000uL, or other suitable sizes), e.g., Rainin Catalog Nos. 30389226, 30389240, 30398213 3.14 96-well PCR plates, Applied Biosystems, Catalog Numbers 4483343, 4483354, 4483349, 4483350, 4483395, or equivalent 3.15 Micro Amp Optical Adhesive Film, Applied Biosystems, Catalog No. 4311971, or equivalent 3.16 Reagent reservoirs, sterile, RNase / DNase free, e.g., VistaLabs Cat. No. 3054-1002

[0267] 4.0 Notes 4.1 Wear appropriate PPE when working in the laboratory. 4.2 Follow detailed facility guidelines for working with hazardous chemicals. Refer to manufacturer's SDS for further details. 4.3 All pipetting steps should be performed using aseptic technique in a BSC. To minimize the risk of contamination of any materials or reagents, it is recommended that the analyst wear disposable sleeve covers during all steps of the procedure.

[0268] 5.0 Procedure 5.1 Isolate gDNA from mock T cell pellets following the protocol previously described herein. Mock T cells should be representative of the CAR T manufacturing process but not transduced with the lentivector. 5.2 Isolate the required amount of gDNA using several silica DNA extraction columns. Combine the DNA eluted from all columns to create a single stock of mock T cell gDNA prior to quantification. 5.3 Once quantified, ensure that enough gDNA is isolated to generate a sufficient number of aliquots to accommodate at least 4 months of testing. 5.3.1 If enough gDNA is not obtained from the initial gDNA isolation to generate a lot large enough to continue for at least 4 months, an additional mock T cell pellet can be isolated and combined with the initial mock T cell gDNA stock, and the combined stock quantified using Qubit. 5.4 Standard #1 consists of mock T cell DNA at a concentration of 0.05ug / uL spiked with pLLV-LICAR2SIN plasmid (LiCAR plasmid) such that 5uL of standard #1 contains 121,212.1212 copies of LiCAR plasmid. 5.4.1 Determine the total volume of Standard #1 that can be made from the mock T cell DNA stock as follows:

[0269]

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[0270]

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[0271]

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[0272]

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[0273]

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[0274]

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[0275]

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[0276]

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[0277] Creation and qualification of new lots of medium and low controls 1.0 Purpose 1.1 An exemplary procedure for generating and qualifying a new lot of standard for transgene qPCR is described.

[0278] 2.0 Equipment 2.1 Centrifuge capable of spinning 1.5 mL microcentrifuge tubes (e.g., Beckman Coulter, Allegra X-14R with SX4750 rotor and swing set for 96-well plates and adapters for 1.5 mL microcentrifuge tubes) 2.2 QuantStudio 6 Real-Time PCR System 2.3 Freezer capable of storing -20℃ 2.4 Freezer capable of -70℃ 2.5 Calibrated 8 or 12 channel pipette (20, 50uL, or other suitable size), e.g. Rainin pipette 2.6 Calibrated single channel pipettes (20, 100, 200, 1000uL or other suitable size), e.g. Rainin pipettes 2.7 Refrigerator or thermostat capable of maintaining a temperature of 2 to 8°C 2.8 QuantStudio PCR Software v1.3 or higher 2.9 A heat block capable of 55°C and suitable for 1.5 mL microcentrifuge tubes. 2.10 Vortex mixer 2.11 Biosafety Cabinets

[0279] 3.0 Material Note: Materials designated as "for example" may be substituted by similar materials without prior qualification. For materials designated as "or equivalent", the equivalent of the substitute must be demonstrated before it can be used to test samples. 3.1 DNase / RNase free water, e.g., Invitrogen catalogue no. 10977015. 3.2 TaqPath ProAmp Master Mix, ThermoFisher catalog number A 30866, or equivalent. 3.3 2×106 to 4×10 per pellet 6 Mock T cell pellet with 100 ng / mL cells. 3.4 Qualified lots of BCMA transgene and ALB primers and probes, custom sequences through IDT or equivalent. 3.5 Qualified Lots of BCMA Transgene Standard #1 3.6 Qualified Lots of BCMA Transgene Medium and Low Controls 3.7 Working Stock Aliquot of pLLV-LICAR2SIN 3.8 1x Low EDTA TE Buffer pH 8.0, RNase / DNase free, e.g., Quality Biological catalogue number 351-324-721. 3.9 0.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., VWR Screw-Cap Microcentrifuge Tubes Catalog No. 89004-286 or Eppendorf Catalog No. 022431005 3.10 1.5 mL centrifuge tubes, sterile, RNase / DNase free, e.g., Eppendorf catalog number 022431021 3.11 2 mL centrifuge tubes, sterile, RNase / DNase-free, e.g., Eppendorf catalog number 022431048 3.12 5 mL centrifuge tubes, sterile, RNase / DNase-free, e.g., Eppendorf Cat. No. 0030119460 3.13 Pipette tips, sterile, filtered, (20, 200, 1000uL, or other suitable sizes), e.g., Rainin Catalog Nos. 30389226, 30389240, 30398213 3.14 96-well PCR plates, Applied Biosystems, Catalog Numbers 4483343, 4483354, 4483349, 4483350, 4483395, or equivalent 3.15 Micro Amp Optical Adhesive Film, Applied Biosystems, Catalog No. 4311971, or equivalent 3.16 Reagent reservoirs, sterile, RNase / DNase free, e.g., VistaLabs Cat. No. 3054-1002

[0280] 4.0 Notes 4.1 Wear appropriate PPE when working in the laboratory. 4.2 Follow detailed facility guidelines for working with hazardous chemicals. Refer to manufacturer's SDS for further details. 4.3 All pipetting steps should be performed using aseptic technique in a BSC. To minimize the risk of contamination of any materials or reagents, it is recommended that the analyst wear disposable sleeve covers during all steps of the procedure.

[0281] 5.0 Procedure 5.1 Isolate gDNA from mock T cell pellets following the protocol previously described herein. Mock T cells are representative of the CAR T manufacturing process but should not be transduced with the lentivector. 5.2 Isolate the required amount of gDNA using several silica DNA extraction columns. Combine the DNA eluted from all columns to create a single stock of mock T cell gDNA prior to quantification. 5.3 After quantification, be sure to isolate enough gDNA to make a sufficient number of aliquots to cover at least 4 months of testing. The low control is a 1:10 dilution of the medium control using 0.02ug / uL of mock T cell gDNA as the diluent. Low and medium controls do not need to be made together from the same mock T cell gDNA stock, but if they are, a portion of the isolated mock T cell DNA stock should be saved to make the low control. The example shown in the following steps is an example of how to make medium and low controls using the same stock of mock T cell gDNA. 5.3.1 If enough gDNA is not obtained from the initial gDNA isolation to generate both control lots large enough to continue for at least 4 months, an additional Mock T cell pellet may be isolated and combined with the initial Mock T cell gDNA stock, and the combined stock quantified using Qubit. 5.4 The BCMA transgene medium control consisted of mock T cell DNA at a concentration of 0.02ug / uL spiked with pLLV-LICAR2SIN plasmid (also referred to herein as "LiCAR plasmid") such that the 5uL medium control contained 30,303.030 copies of LiCAR plasmid. This equates to a vector copy number of 2.00 copies / cell in the 5uL control. 5.4.1 Determine the total volume of medium control that can be made from mock T cell DNA stock as follows:

[0282]

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[0283]

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[0284]

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[0285]

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[0286]

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[0287]

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[0288]

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[0290]

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[0291]

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[0292]

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[0293]

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[0294] Example 3: Qualification of the transgene qPCR method 1.0 Purpose 1.1 The purpose of this example is to describe the results obtained during the execution of a transgene qPCR method qualification protocol.

[0295] 2.0 Scope 2.1 The qPCR assay for quantifying the LiCAR plasmid incorporated into the CAR T product was qualified by examining the following qualification parameters: specificity, accuracy, linearity, precision (reproducibility and intermediate precision), range, and LOQ.

[0296] 3.0 Definitions and Abbreviations 3.1 qPCR Quantitative Polymerase Chain Reaction 3.2 hALB Human albumin 3.3 VCN Vector Copy Number 3.4 CV Coefficient of variation 3.5 SD standard deviation 3.6 Ct cycle threshold 3.7 LOQ Limit of Quantification 3.8 Development of BMD bioassay methods 3.9 QC Quality Control

[0297] 4.0 Testing Approach 4.1 The linearity of the assay was qualified by testing a 5-point standard curve generated by performing 5-fold serial dilutions of Transgene qPCR Standard #1 and plotting the standard curve results as Log10 amounts versus Ct. 4.2 Assay precision, i.e. both reproducibility and intermediate precision, was qualified by testing a five-point standard curve and mid and low assay controls. 4.3 Assay accuracy was qualified by testing medium and low assay controls. 4.4 Assay specificity was demonstrated by testing mock T cell DNA samples and representative CAR T day 10 harvested DNA samples. 4.5 The LOQ of the assay was determined by testing LOQ samples of 0.02 VCN / cell and 0.014 VCN / cell. 4.6 The three qualification assays were performed by two analysts, with one of the three assays performed on a separate day.

[0298] 5.0 Materials 5.1 For a complete list of materials required to perform the transgene qPCR assay, please refer to the qualification protocol described herein. 5.2 Assay Standards and Controls: 5.2.1 BCMA Transgene Standard #1, Lot No. LM-RP3-00533A. 5.2.2 BCMA Transgene Medium Control, Lot No. LM-RP3-00533B. 5.2.3 BCMA Transgene Low Control, Lot No. LM-RP3-00533C. 5.3 Assay Specificity Samples: 5.3.1 Mock T cell DNA, Lot No. LM-RP3-00533. 5.3.2 CAR T DNA, LM-RP3-00541. 5.4 Assay LOQ Sample: 5.4.1 LOQ samples of 0.02VCN / cell and 0.014VCN / cell, lot number LM-RP3-00533. 5.4.2 Transgene Method Oligo Set, Lot No. LM-RP3-00460

[0299] 6.0 Overview 6.1 This example outlines the results obtained during the execution of the transgene qPCR method qualification protocol. 6.2 The method demonstrated acceptable accuracy, precision, specificity, and linearity as summarized in Table 12 (10.2-10.5). In addition, the assay range and LOQ were identified for both the transgene and hALB targets. Therefore, the method is qualified for testing of CAR T drug product material prior to formulation with cryopreservation media.

[0300] [Table 12]

[0301] [Table 13]

[0302] 7.0 Procedure 7.1 Method Qualification All assays were performed as described in the protocol. Only assays that met the assay acceptance criteria specified in the method were included in the evaluation of the method qualification acceptance criteria.

[0303] 8.0 Results and Discussion 8.1 Linearity and precision of the standard curve (reproducibility and intermediate precision) 8.1.1 The results of the five-point standard curves for all validated qualification assays for both the transgene and hALB targets are summarized in Tables 13-14 and Figures 11-12. The R2 values ​​of the Log10 vs. Ct value plots for both the transgene and hALB targets are 1.00. The reproducibility of each standard ranged from 0.06-0.54% for the transgene targets and from 0.03-0.41% for the hALB targets. The intermediate precision ranged from 0.26-0.47% for the transgene targets and from 0.17-0.55% for the hALB targets.

[0304] [Table 14]

[0305] [Table 15] 8.2 Accuracy and Precision of QC Controls (Reproducibility and Intermediate Precision) 8.2.1 The results for the medium control of 2.00 VCN / cell and the low control of 0.20 VCN / cell are summarized in Table 15. Recovery of the mean VCN / cell results for the medium controls ranges from 93-95%. The recovery of the mean VCN / cell results for the low control ranges from 79-84%. The reproducibility of both the medium and low controls ranges from 4-6%. The intermediate precision of the medium and low controls is 4% and 6%, respectively.

[0306] [Table 16] 8.3 Specificity 8.3.1 Results of Mock T cell DNA and CAR T DNA samples run to assess assay specificity are summarized in Table 16. All replicates of Mock T cell DNA samples were "indeterminate" for the transgene target. All replicates of CAR T DNA had quantifiable transgene target copy results. In addition, both Mock T cell DNA and CAR T DNA samples met the hALB sample acceptance criteria of 30,303.030 hALB copies expected per 100 ng of DNA ± 30%.

[0307] [Table 17] 8.4 Range and LOQ 8.4.1 Both the ttransgene and hALB target met all the acceptance criteria for accuracy, linearity, and intermediate precision. Therefore, the ranges of both the transgene and hALB target are defined as the copy ranges of the 5-point standard curve. The range of the transgene is 193.939 - 121212.121 copies. The range of hALB is 121.212 - 75757.576 copies. Furthermore, the LOQ of the hALB target is defined as 121.212 copies. 8.4.2 The results of the 0.014VCN / cell and 0.02VCN / cell LOQ samples are summarized in Table 17. At least one of the triplicate Ct values ​​of the 0.014VCN / cell LOQ sample was not within the Ct range of the transgene standard curve in each valid qualification assay. Therefore, the transgene copy value could not be determined precisely and the LOQ criteria could not be evaluated. However, the 0.02VCN / cell LOQ sample yielded recovery rates ranging from 73 to 80%. The 0.02VCN / cell LOQ sample also yielded mean transgene copy values ​​and mean VCN / cell result CV%s ranging from 1 to 9% and 2 to 11%, respectively. Thus, the LOQ of the transgene target is defined as the expected transgene copy value of the 0.02VCN / cell LOQ sample of 303.030 copies.

[0308] [Table 18]

[0309] The teachings of all patents, published patents, and references cited herein are incorporated by reference in their entirety.

[0310] Although exemplary embodiments have been particularly shown and described, those skilled in the art will understand that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims. The present invention may include the following aspects. [1] A probe and primer set, comprising: a probe comprising a nucleotide sequence of SEQ ID NO: 10; at least one label bound to the probe; a first primer comprising a nucleic acid sequence of SEQ ID NO: 11; and a second primer comprising a nucleic acid sequence of nucleic acid sequence 12. The probe and primer set comprising: [2] The probe and primer set described in [1] above, wherein the at least one label comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof. [3] A kit for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising a probe comprising the nucleotide sequence of SEQ ID NO: 10, at least one label bound to the probe, a first primer comprising the nucleic acid sequence of SEQ ID NO: 11, and a second primer comprising the nucleic acid sequence of nucleic acid sequence 12. The kit comprising: [4] The kit described in [3] above, wherein the at least one label attached to the probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof. [5] The kit according to [3] above, comprising an array containing the probe. [6] The kit according to [5] above, wherein the array is a multi-well plate. [7] The kit described in [3] above, further comprising a human albumin (hALB) probe comprising the nucleic acid sequence of SEQ ID NO: 22, at least one label bound to the hALB probe, a first hALB primer comprising the nucleic acid sequence of SEQ ID NO: 23, and a second hALB primer comprising the nucleic acid sequence of SEQ ID NO: 24. [8] The kit described in [7] above, wherein the at least one label attached to the hALB probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof. [9] The kit according to [3] above, further comprising a reference gene probe, at least one label bound to the reference gene probe, a first reference gene primer, and a second reference gene primer.

[10] A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: amplifying nucleic acid from the CAR T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the CAR T cells using a first hALB primer comprising the nucleic acid sequence of SEQ ID NO:23 and a second hALB primer comprising the nucleic acid sequence of SEQ ID NO:24, thereby generating amplified hALB nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and a hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 via a reference signal from at least one label attached to the hALB probe; quantitating transgene copy number by comparing said target signal to said reference signal; The method comprising:

[11] A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from the CAR T cell with a first CAR primer, a second CAR primer, a first hALB primer, and a second hALB primer, wherein the first CAR primer comprises the nucleic acid sequence of SEQ ID NO:11, the second CAR primer comprises the nucleic acid sequence of SEQ ID NO:12, the first hALB primer comprises the nucleic acid sequence of SEQ ID NO:23, and the second hALB primer comprises the nucleic acid sequence of SEQ ID NO:24; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying a hALB nucleic acid using said first hALB primer and said second hALB primer, thereby generating an amplified hALB nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; detecting hybridization between the amplified hALB nucleic acid and the hALB probe via a reference signal from at least one label attached to the hALB probe; quantitating transgene copy number by comparing said target signal to said reference signal; The method comprising:

[12] The method according to

[10] or

[11] above, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from at least one label bound to the CAR probe during or after hybridization relative to a target signal from the label bound to the CAR probe before hybridization.

[13] The method according to

[10] or

[11] above, wherein detecting hybridization between the amplified hALB nucleic acid molecule and the hALB probe comprises detecting a change in a target signal from at least one label bound to the hALB probe during or after hybridization relative to a target signal from the label bound to the hALB probe before hybridization.

[14] The method according to

[10] or

[11] above, wherein the amplification comprises polymerase chain reaction (PCR).

[15] The method according to

[14] above, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

[16] The method according to

[10] above, wherein at least one label attached to the CAR probe comprises a fluorophore.

[17] The method according to

[10] above, wherein at least one label attached to the hALB probe comprises a fluorophore.

[18] A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: amplifying nucleic acid from the CAR T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying the nucleic acid from the CAR T cell using a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; quantitating transgene copy number by comparing said target signal to said reference signal; The method comprising:

[19] A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from the CAR T cell with a first CAR primer, a second CAR primer, a first reference gene primer, and a second reference gene primer, wherein the first CAR primer comprises the nucleic acid sequence of SEQ ID NO:11 and the second CAR primer comprises the nucleic acid sequence of SEQ ID NO:12; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying a reference gene nucleic acid using the first reference gene primer and the second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; quantitating transgene copy number by comparing said target signal to said reference signal; The method comprising:

[20] The method according to

[18] or

[19] above, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from at least one label bound to the CAR probe during or after hybridization relative to a target signal from the label bound to the CAR probe before hybridization.

[21] The method according to

[18] or

[19] above, wherein detecting hybridization between the amplified reference gene nucleic acid molecule and the reference gene probe comprises detecting a change in a target signal from at least one label bound to the reference gene probe during or after hybridization relative to a target signal from the label bound to the reference gene probe before hybridization.

[22] The method according to

[18] or

[19] above, wherein the amplification comprises polymerase chain reaction (PCR).

[23] The method according to

[22] above, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

[24] The method according to

[18] above, wherein at least one label attached to the CAR probe comprises a fluorophore.

[25] The method according to

[18] above, wherein at least one label attached to the reference gene probe comprises a fluorophore.

[26] A method for generating chimeric antigen receptor (CAR) T cells, comprising introducing a CAR transgene into a T cell to obtain a T cell incorporating the transgene; Determining integration of the CAR transgene, wherein the determination comprises: amplifying nucleic acid from the transgene-integrated T cell using a first CAR primer comprising the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer comprising the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the transgene-integrated T cell using a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; Detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; quantitating transgene copy number by comparing said target signal to said reference signal; ), and Obtaining a CAR T cell containing at least one copy of the integrated CAR transgene. The method comprising:

[27] The method according to

[26] above, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from at least one label bound to the CAR probe during or after hybridization relative to a target signal from the label bound to the CAR probe before hybridization.

[28] The method according to

[26] above, wherein detecting hybridization between the amplified reference gene nucleic acid molecule and the reference gene probe comprises detecting a change in a target signal from at least one label bound to the reference gene probe during or after hybridization relative to a target signal from the label bound to the reference gene probe before hybridization.

[29] The method according to

[26] above, wherein the amplification comprises polymerase chain reaction (PCR).

[30] The method according to

[29] above, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

[31] The method according to

[26] above, wherein at least one label attached to the CAR probe comprises a fluorophore.

[32] The method according to

[26] above, wherein at least one label attached to the reference gene probe comprises a fluorophore.

[33] CAR T cells generated by the method described in any one of

[26] to

[32] above.

Claims

1. A probe and primer set comprising a probe comprising a nucleotide sequence of SEQ ID NO:10, at least one label attached to the probe, a first primer comprising a nucleic acid sequence of SEQ ID NO:11 or a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:11, and a second primer comprising a nucleic acid sequence of SEQ ID NO:12 or a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:

12. The probe and primer set comprising:

2. 2. The probe and primer set of claim 1, wherein the at least one label comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

3. 1. A kit for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising a probe comprising the nucleotide sequence of SEQ ID NO: 10, at least one label attached to said probe, a first primer consisting of a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 11, and a second primer consisting of a nucleic acid sequence of SEQ ID NO: 12 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:

12. The kit comprising:

4. 4. The kit of claim 3, wherein the at least one label attached to the probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

5. The kit of claim 3 comprising an array comprising the probes.

6. The kit of claim 5 , wherein the array is a multi-well plate.

7. 4. The kit of claim 3, further comprising a human albumin (hALB) probe comprising the nucleic acid sequence of SEQ ID NO:22, at least one label bound to the hALB probe, a first hALB primer consisting of the nucleic acid sequence of SEQ ID NO:23 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:23, and a second hALB primer consisting of the nucleic acid sequence of SEQ ID NO:24 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:

24.

8. The kit of claim 7, wherein the at least one label attached to the hALB probe comprises a radioisotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical, or a combination thereof.

9. 4. The kit of claim 3, further comprising a reference gene probe, at least one label attached to the reference gene probe, a first reference gene primer, and a second reference gene primer.

10. 1. A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: amplifying nucleic acid from said CAR T cell with a first CAR primer consisting of a nucleic acid sequence of SEQ ID NO:11, or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:11, and a second CAR primer consisting of a nucleic acid sequence of SEQ ID NO:12, or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from said CAR T cells with a first hALB primer consisting of a nucleic acid sequence of SEQ ID NO:23, or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:23, and a second hALB primer consisting of a nucleic acid sequence of SEQ ID NO:24, or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:24, thereby generating an amplified hALB nucleic acid; detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and a hALB probe comprising the nucleotide sequence of SEQ ID NO:22 via a reference signal from at least one label attached to the hALB probe; quantitating transgene copy number by comparing said target signal to said reference signal; The method comprising:

11. 1. A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from the CAR T cell with a first CAR primer, a second CAR primer, a first hALB primer, and a second hALB primer, wherein the first CAR primer consists of a nucleic acid sequence of SEQ ID NO:11 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:11, the second CAR primer consists of a nucleic acid sequence of SEQ ID NO:12 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:12, the first hALB primer consists of a nucleic acid sequence of SEQ ID NO:23 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:23, and the second hALB primer consists of a nucleic acid sequence of SEQ ID NO:24 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:24; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying a hALB nucleic acid using said first hALB primer and said second hALB primer, thereby generating an amplified hALB nucleic acid; detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; Detecting hybridization between the amplified hALB nucleic acid and the hALB probe via a reference signal from at least one label attached to the hALB probe; quantitating transgene copy number by comparing said target signal to said reference signal; The method comprising:

12. 12. The method of claim 10 or 11, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from the at least one label attached to the CAR probe during or after hybridization relative to a target signal from the label attached to the CAR probe before hybridization.

13. The method of claim 10 or 11, wherein detecting hybridization between the amplified hALB nucleic acid molecule and the hALB probe comprises detecting a change in a target signal from at least one label bound to the hALB probe during or after hybridization relative to a target signal from the label bound to the hALB probe before hybridization.

14. The method of claim 10 or 11, wherein the amplification comprises polymerase chain reaction (PCR).

15. 15. The method of claim 14, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

16. 11. The method of claim 10, wherein at least one label attached to the CAR probe comprises a fluorophore.

17. The method of claim 10, wherein at least one label attached to the hALB probe comprises a fluorophore.

18. 1. A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: amplifying nucleic acid from said CAR T cell with a first CAR primer consisting of a nucleic acid sequence of SEQ ID NO:11, or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:11, and a second CAR primer consisting of a nucleic acid sequence of SEQ ID NO:12, or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying the nucleic acid from the CAR T cell with a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; quantitating transgene copy number by comparing said target signal to said reference signal; The method comprising:

19. 1. A method for quantifying transgene integration into chimeric antigen receptor (CAR) T cells, comprising: contacting nucleic acid from said CAR T cell with a first CAR primer, a second CAR primer, a first reference gene primer, and a second reference gene primer, wherein said first CAR primer consists of a nucleic acid sequence of SEQ ID NO:11 or a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:11 and said second CAR primer consists of a nucleic acid sequence of SEQ ID NO:12 or a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:12; amplifying the CAR nucleic acid using the first CAR primer and the second CAR primer, thereby generating an amplified CAR nucleic acid; amplifying a reference gene nucleic acid using the first reference gene primer and the second reference gene primer, thereby generating an amplified reference gene nucleic acid; detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; quantitating transgene copy number by comparing said target signal to said reference signal; The method comprising:

20. 20. The method of claim 18 or 19, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from the at least one label attached to the CAR probe during or after hybridization relative to a target signal from the label attached to the CAR probe before hybridization.

21. 20. The method of claim 18 or 19, wherein detecting hybridization between the amplified reference gene nucleic acid molecule and the reference gene probe comprises detecting a change in a target signal from the at least one label bound to the reference gene probe during or after hybridization relative to a target signal from the label bound to the reference gene probe before hybridization.

22. 20. The method of claim 18 or 19, wherein the amplification comprises polymerase chain reaction (PCR).

23. 23. The method of claim 22, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

24. 20. The method of claim 18, wherein at least one label attached to the CAR probe comprises a fluorophore.

25. 20. The method of claim 18, wherein at least one label attached to the reference gene probe comprises a fluorophore.

26. A method of generating a chimeric antigen receptor (CAR) T cell, comprising: introducing a CAR transgene into a T cell to obtain a T cell having an integrated transgene; Determining integration of the CAR transgene, wherein the determination comprises: amplifying nucleic acid from the transgene-integrated T cell with a first CAR primer consisting of a nucleic acid sequence of SEQ ID NO:11 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:11 and a second CAR primer consisting of a nucleic acid sequence of SEQ ID NO:12 or a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:12, thereby generating an amplified CAR nucleic acid; amplifying nucleic acid from the transgene-integrated T cell using a first reference gene primer and a second reference gene primer, thereby generating an amplified reference gene nucleic acid; detecting hybridization between the amplified CAR nucleic acid and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 via a target signal from at least one label attached to the CAR probe; detecting hybridization between the amplified reference gene nucleic acid and the reference gene probe via a reference signal from at least one label bound to the reference gene probe; quantitating transgene copy number by comparing said target signal to said reference signal; ), and Obtaining a CAR T cell comprising at least one copy of an integrated CAR transgene. The method comprising:

27. 27. The method of claim 26, wherein detecting hybridization between the amplified CAR nucleic acid and the CAR probe comprises detecting a change in a target signal from the at least one label attached to the CAR probe during or after hybridization relative to a target signal from the label attached to the CAR probe before hybridization.

28. 27. The method of claim 26, wherein detecting hybridization between the amplified Reference Gene nucleic acid molecule and the Reference Gene probe comprises detecting a change in a target signal from the at least one label bound to the Reference Gene probe during or after hybridization relative to a target signal from the label bound to the Reference Gene probe before hybridization.

29. 27. The method of claim 26, wherein the amplification comprises polymerase chain reaction (PCR).

30. 30. The method of claim 29, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (rtRT-PCR), digital PCR (dPCR), ligase chain reaction, or transcription-mediated amplification (TMA).

31. 27. The method of claim 26, wherein at least one label attached to the CAR probe comprises a fluorophore.

32. 27. The method of claim 26, wherein at least one label attached to the reference gene probe comprises a fluorophore.

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