EA046825B1 - CAR T CONSTRUCT AND PRIMERS FOR B-CELL MATURATION COMPLEX - Google Patents

Description

Cross-reference to related applications

This application claims the benefit of US Provisional Patent Application Serial No. 62/894,663; filed August 30, 2019. The entire contents of the above application are incorporated herein by reference.

Including material by linking to an ascii text file

The subject application contains a sequence listing provided electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy of this listing, created on August 28, 2020, is named JBI6148WOPCT1_SL.txt and is 8175 bytes in size.

Prerequisites for creating the invention

T cell therapy uses isolated T cells that have been genetically modified to enhance their specificity for a specific tumor-associated antigen. Genetic modification may involve expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor to provide a new antigen specificity to the T cell. T cells expressing chimeric antigen receptors (CAR T cells) can induce tumor immunoreactivity. 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 cancers such as multiple myeloma. There is a need for more effective forms of cancer therapy using CAR T cells, in particular CAR T cells specific for the tumor-associated antigen BCMA.

Summary of the invention

The present invention relates to probes and primers for polymerase chain reaction (PCR), such as quantitative PCR. The present invention also provides kits and methods for using the probes and primers described herein to quantify transgene integration in chimeric antigen receptor (CAR) T cells.

In a first aspect, the invention provides sets of probes and primers comprising a probe comprising the nucleotide sequence of SEQ ID NO: 10, and at least one tag associated with the probe; a first primer containing the nucleotide sequence SEQ ID NO: 11; and a second primer containing the nucleotide sequence SEQ ID NO: 12 (see example 1, table 1).

In another aspect, the invention provides sets of probes and primers comprising a probe comprising the nucleotide sequence of SEQ ID NO: 1, and at least one tag associated with the probe; a first primer containing the nucleotide sequence of SEQ ID NO: 2; and a second primer containing the nucleotide sequence SEQ ID NO: 3 (see example 1, table 1).

In another aspect, the invention provides sets of probes and primers 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, and at least one label associated with the probe; a first primer containing a nucleotide 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 their combinations; and a second primer containing a nucleotide 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 (see Table 1).

In some embodiments, the at least one label comprises a radioactive isotope, enzyme substrate, chemiluminescent agent, fluorophore, fluorescence quencher, enzyme, chemical compound, or a combination thereof.

In another aspect, the invention provides kits for quantitative determination of transgene integration in a CAR T cell, comprising a probe comprising the nucleotide sequence of SEQ ID NO: 10, and at least one tag associated with the probe; a first primer containing the nucleotide sequence SEQ ID NO: 11; and a second primer containing the nucleotide sequence of SEQ ID NO: 12.

In another aspect, the invention provides kits for quantitative determination of transgene integration in a CAR T cell, comprising a probe comprising the nucleotide sequence of SEQ ID NO: 1, and at least one tag associated with the probe; a first primer containing the nucleotide sequence of SEQ ID NO: 2; and a second primer containing the nucleotide sequence of SEQ ID NO: 3.

In a further aspect, the invention provides kits for quantitative determination of transgene integration into a CAR T cell, 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, and at least one label associated with the probe; a first primer containing a nucleotide 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 their combinations; and a second primer containing the nucleotide sequence,

- 1 046825 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.

In some embodiments of the kits of the invention, at least one label associated with the probe contains a radioactive isotope, enzyme substrate, chemiluminescent agent, fluorophore, fluorescence quencher, enzyme, chemical compound, or a combination thereof.

In one embodiment, the kits of the invention contain a matrix that contains a probe. In some embodiments, the matrix is a multi-well plate.

In one embodiment, the kits further comprise a human albumin (hALB) probe containing the nucleotide sequence of SEQ ID NO: 22 (see Example 2, Table 2, hALB probe set 1), and at least one tag associated with the hALB probe , the first hALB primer containing the nucleotide sequence SEQ ID NO: 23 (Table 2, forward primer from hALB set 1), as well as the second hALB primer containing the nucleotide sequence SEQ ID NO: 24 (Table 2, reverse primer from hALB set 1). In certain embodiments, the at least one label associated with the hALB probe comprises a radioactive isotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical compound, or a combination thereof.

In another embodiment, the kits further comprise a human albumin (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, and at least one a tag associated with the hALB probe, a first hALB primer containing a nucleotide 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 primer containing the nucleotide 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 associated with the hALB probe comprises a radioactive isotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical compound, or a combination thereof.

In another aspect, the present invention provides methods for quantifying transgene integration in a CAR T cell, comprising:

amplifying nucleic acids from a CAR T cell with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids;

amplifying nucleic acids from the CAR T cell with a first hALB primer containing the nucleotide sequence of SEQ ID NO: 23 and a second hALB primer containing the nucleotide sequence of SEQ ID NO: 24, thereby obtaining amplified hALB nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acids and an hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 by means of a reference signal from at least one tag associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

In another aspect, the present invention provides methods for quantifying transgene integration in a CAR T cell, comprising:

amplifying nucleic acids from a CAR T cell with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 2 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 3, thereby obtaining amplified CAR nucleic acids;

amplifying nucleic acids from the CAR T cell with a first hALB primer containing the nucleotide sequence of SEQ ID NO: 23 and a second hALB primer containing the nucleotide sequence of SEQ ID NO: 24, thereby obtaining amplified hALB nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 1 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acids and an hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 by means of a reference signal from at least one tag associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

- 2 046825

In another aspect, the present invention provides methods for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising:

contacting nucleic acids from a 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 contains the nucleotide sequence of SEQ ID NO: 11, the second CAR primer contains the nucleotide sequence of SEQ ID NO: 12, the first hALB primer contains the nucleotide sequence of SEQ ID NO: 23, the second hALB primer contains the nucleotide sequence of SEQ ID NO: 24;

amplifying the CAR nucleic acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acids;

amplifying hALB nucleic acids with a first hALB primer and a second hALB primer, thereby obtaining amplified hALB nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acids and the hALB probe through a reference signal from at least one label associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

In another aspect, the present invention provides methods for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising:

contacting nucleic acids from a 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 contains the nucleotide sequence of SEQ ID NO: 2, the second CAR primer contains the nucleotide sequence of SEQ ID NO: 3, the first hALB primer contains the nucleotide sequence of SEQ ID NO: 23, the second hALB primer contains the nucleotide sequence of SEQ ID NO: 24;

amplifying the CAR nucleic acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acids;

amplifying hALB nucleic acids with a first hALB primer and a second hALB primer, thereby obtaining amplified hALB nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 1 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acids and the hALB probe through a reference signal from at least one label associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

In a further aspect, the present invention provides methods for quantifying transgene integration in a CAR T cell, comprising:

contacting nucleic acids from a CAR T cell with a first CAR primer comprising a nucleotide 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 acids from the CAR T cell with a second CAR primer containing a nucleotide 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 acids from a CAR T cell with a first hALB primer comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29 and combinations thereof, and bringing the nucleic acids from the CAR T cell into contact with a second hALB primer containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30 and combinations thereof;

bringing nucleic acids from a CAR T cell into contact with a CAR probe, wherein the CAR probe comprises 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 acids from a CAR T cell with an hALB probe, wherein the hALB probe comprises 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 acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acid molecules;

amplification of hALB nucleic acids with the first hALB primer and the second hALB primer,

- 3 046825 thereby obtaining molecules of amplified hALB nucleic acids;

detecting hybridization between the amplified CAR nucleic acid molecules and the CAR probe through a target signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acid molecules and the hALB probe by means of a reference signal from at least one label associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

In a further aspect, the present invention provides methods for producing a CAR T cell, comprising introducing a CAR transgene into a T cell to produce a transgene integrated T cell; definition of CAR transgene integration, including:

amplifying nucleic acids from a T cell into contact with an integrated transgene with a first CAR primer containing a nucleotide 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 containing a nucleotide 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 producing amplified CAR nucleic acids;

amplifying nucleic acids from the transgene-integrated T cell with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids;

detecting hybridization between amplified CAR nucleic acids and a CAR probe containing 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, by means of a target signal from at least one tag associated with a CAR probe;

detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal; and obtaining a CAR T cell containing at least one copy of the integrated CAR transgene.

In yet another further aspect, the present invention provides methods for producing a CAR T cell, comprising introducing a CAR transgene into a T cell to produce a transgene-integrated T cell; definition of CAR transgene integration, including:

bringing nucleic acids from a T cell into contact with an integrated transgene with a first CAR primer containing a nucleotide 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 acids from the T cell with an integrated transgene with a second CAR primer containing a nucleotide 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; bringing nucleic acids from the cell into contact with an integrated transgene with a first hALB primer containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29 and combinations thereof, and bringing the nucleic acids from the T cell into contact with the integrated transgene with a second primer hALB containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30 and combinations thereof;

bringing nucleic acids from a T cell into contact with an integrated transgene with a CAR probe, wherein the CAR probe comprises 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;

bringing nucleic acids from a T cell into contact with an integrated transgene with an hALB probe, wherein the hALB probe comprises 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 acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acid molecules;

amplifying hALB nucleic acids with a first hALB primer and a second hALB primer, thereby obtaining amplified hALB nucleic acid molecules;

detecting hybridization between the amplified CAR nucleic acid molecules and the CAR probe through a target signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acid molecules and the hALB probe by means of a reference signal from at least one label associated with the probe

- 4 046825 hALB;

quantifying the number of transgene copies by comparing the target signal with a reference signal; and obtaining a CAR T cell containing at least one copy of the integrated CAR transgene.

Aspects of the present invention also provide CAR T cells produced using the methods described herein.

In some embodiments, the step of detecting hybridization between the amplified CAR nucleic acid molecules and the CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the at least one label associated with with the CAR probe, prior to hybridization.

In some embodiments, the step of detecting hybridization between the amplified hALB nucleic acid molecules and the hALB probe includes detecting a change in the target signal from at least one tag associated with the hALB probe during or after hybridization relative to the target signal from the at least one tag associated with hALB probe prior to hybridization.

In some 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 (RT-PCR) real-time), ligase chain reaction or transcription-mediated amplification (TMA).

In some embodiments, the nucleic acids being amplified are amplicons.

In some embodiments, at least one label associated with the CAR probe contains a fluorophore. In some embodiments, at least one label associated with the hALB probe contains a fluorophore.

Brief description of graphic materials

The patent or application file contains at least one color graphic material. Copies of this patent or patent application publication with color graphics may be obtained upon filing of an appropriate application and payment of a fee.

In fig. Figure 1 shows a gel electrophoretic image from the results of single-plex primer/probe screening assays.

In fig. Figure 2 shows a gel electrophoretic image from the results of multiplex primer/probe screening assays.

In fig. Figure 3 shows a gel electrophoretic image of the transgene (FP) set 1 and (RP) set 2 multiplexed with hALB set 1.

In fig. 4A-D show amplification curves for transgene (FP) set 1 and (RP) set 2 and standard curves for hALB set 1.

In fig. 5A-B shows standard curves comparing intact versus frozen (target transgene).

In fig. Figure 6 shows standard curves comparing circular with linear (target transgene).

In fig. Figure 7 shows a comparison of the characterization and typical standard curves for qPCR of the transgene.

In fig. Figure 8 shows a characterizing standard line graph for the transgene.

In fig. Figure 9 shows an example of the arrangement of regions in a quantitative PCR (qPCR) plate.

In fig. Figure 10 shows an example of the location of areas in a plate for assessing qPCR control.

In fig. Figure 11 shows an example of a line graph for a transgene.

In fig. Figure 12 shows an example of a line graph for hALB.

In fig. 13 shows the nucleotide sequence of the human serum albumin gene (hALB), GenBank accession number M12523.1.

In fig. 14 is represented by SEQ ID NO: 11.

The foregoing will be apparent from the following more detailed description of the exemplary embodiments, as illustrated in the accompanying drawings, in which like reference numerals refer to the same parts in different views. Graphic materials are not necessarily drawn to scale; instead, emphasis is placed on illustrating the principles of the invention.

- 5 046825

Detailed description

Below is a description of example implementations.

Several aspects of the invention are described below with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are set forth to provide a thorough understanding of the invention. However, one of ordinary skill in the art will appreciate that the invention may be practiced without one or more specific details, or may be practiced by other methods, protocols, reagents, cell lines, and animals. The present invention is not limited to the order of actions or events shown, since some actions may occur in a different order and/or simultaneously with other actions or events. Moreover, not all illustrated acts, steps or events are necessary to implement the methodology in accordance with the present invention. Many of the techniques and procedures described or referenced herein are well understood and widely applied using traditional methodology by those skilled in the art.

Unless otherwise indicated, all art terms, symbols and other scientific terms or terminology used herein have the meanings commonly understood by those skilled in the art to which this invention relates. In some cases, terms with generally accepted meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from what is generally understood in the art. In addition, it should be understood that terms, such as those defined in commonly used dictionaries, should be interpreted to have the meaning consistent with their meaning in the context of the relevant art and/or as otherwise defined herein.

The terms used herein are used only for the purpose of describing specific embodiments of the invention and are not limiting. When used herein, the singular forms include plural references unless the context clearly requires otherwise.

The term contain or its variations such as contains or containing, when used, may be used to mean the inclusion of a specified element or whole or group of elements or wholes, but not the exclusion of any other element or whole or group of elements or wholes.

The present invention relates to kits and methods for quantifying transgene integration in chimeric antigen receptor (CAR) T cells. In addition, panels of probes and primers are provided for performing polymerase chain reaction (PCR), such as quantitative PCR, to quantify transgene integration into CAR T cells.

In some embodiments, the transgenic qPCR methods and kits described in the present invention include a multiplex quantitative polymerase chain reaction (qPCR) assay designed to quantify the transgenic BCMA CAR plasmid integrated into a CAR T drug product. In both cases (1) the BCMA CAR transgenic plasmid (transgene) and (2) the human albumin reference gene (hALB) are amplified in this qPCR method. A set of primers and probes for transgenic targets can amplify the cross-link between the CD137 and CD3z regions of the plasmid to enable detection exclusively of the transgenic BCMA CAR plasmid present and integrated into the CAR T drug product.

Chimeric antigen receptors.

A chimeric antigen receptor (CAR) is an artificially engineered fusion protein or polypeptide containing antibody antigen binding domains (scFv) linked to T cell signaling domains. In this document, the terms T cells, T cells and T lymphocytes are used interchangeably. Characteristics of CARs may include their ability to redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner using the antigen-binding properties of monoclonal antibodies. MHC-non-restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing the primary mechanism of tumor immune evasion. Moreover, when expressed in T cells, CARs do not preferentially dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

The CARs described herein provide recombinant polypeptide constructs comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a cytoplasmic signaling domain) comprising a functional signaling domain derived from a stimulatory molecule as defined below . T cells expressing CAR are referred to herein as CAR T cells, CAR T cells, or CAR-modified T cells, and these terms are used interchangeably herein. A cell can be genetically modified to stably express an anti-binding domain on its surface.

- 6 046825 body, giving a new antigenic specificity that is independent of MHC.

In some cases, a T cell is genetically modified 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 FcyRI protein into one chimeric protein. In one embodiment, the stimulatory molecule is a zeta chain associated with a T cell receptor complex.

As used herein, the term intracellular signaling domain refers to the intracellular region of the molecule. It is the functional region of the protein that acts by transmitting information within the cell to regulate cellular activity through certain signaling pathways, generating second messengers or acting as effectors in response to such messengers. The intracellular signaling domain generates a signal that induces immune effector function in a cell containing a CAR, such as a CAR T cell. Examples of immune effector function, for example in a CAR T cell, include cytolytic activity and helper activity, including cytokine secretion.

In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Examples of primary intracellular signaling domains include domains derived from molecules that are responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a co-stimulatory intracellular domain. Examples of costimulatory intracellular signaling domains include domains derived from molecules that are responsible for costimulatory signals or antigen-dependent stimulation. For example, in the case of CAR T, the primary intracellular signaling domain may contain the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may contain the cytoplasmic sequence of a coreceptor or costimulatory molecule.

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

The term zeta or alternatively zeta chain, CD3 zeta or TCR zeta is defined as the protein represented by GenBank accession number BAG36664.1, or equivalent residues derived from non-human species, e.g. mouse, rabbit, primate, mouse, rodent, monkey, ape, etc., and the zeta stimulatory domain, or alternatively the CD3-zeta stimulatory domain, or the TCR-zeta stimulatory domain is defined as amino acid residues from the cytoplasmic zeta chain domain that are sufficient for the functional transmission of the original signal, necessary for T cell activation. In one aspect, the cytoplasmic zeta domain comprises residues 52 to 164 of GenBank accession number BAG36664.1 or equivalent residues derived from non-human species, such as mouse, rodent, monkey, ape, and the like, which are their functional orthologues.

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 T cell response such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class 1 molecule, BTLA and Toll-like receptor ligands, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (also referred to as CD137 herein). In a specific embodiment, the co-stimulatory molecule is 41BB (CD137).

A costimulatory intracellular signaling domain may be an intracellular region of a costimulatory molecule. The co-stimulatory molecule can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and NK cell activating 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, a ligand that specifically binds to CD83, etc.

An intracellular signaling domain may comprise the entire (ie, full-length) intracellular region or the entire native intracellular signaling domain of the molecule from which it was derived, or a functional fragment thereof.

The term 4-1BB refers to a member of the tumor necrosis factor receptor (TNF) superfamily with the amino acid sequence represented by GenBank accession number AAA62478.2, or

- 7 046 825 equivalent residues obtained from non-human species, for example a mammal (mouse, rodent, monkey, ape, etc.); and the costimulatory domain 4-1BB is defined as amino acid residues 214-255 with GenBank accession number: AAA62478.2 or equivalent residues from a non-human species, such as a mammal (mouse, rodent, monkey, ape, etc.).

In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule, as defined herein. In one embodiment, the co-stimulatory molecule is selected from 4-1BB (ie CD137), CD27, CD3-zeta and/or CD28. CD28 is a T cell marker important for T cell costimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and acts as a co-stimulatory immune checkpoint molecule. 4-1BB transmits a powerful co-stimulatory signal to T cells, promoting differentiation and increasing long-term survival of T cells. CD3-zeta binds to the TCR to generate a signal and contains immunoreceptor tyrosine activating motifs (ITAMs).

In one embodiment, the CAR comprises an intracellular hinge domain comprising CD8 and an intracellular T cell receptor signaling domain comprising CD28, 4-1BB and CD3zeta and combinations thereof.

In a specific embodiment, the CAR comprises transmembrane CD8a, CD137, and CD3z coding regions.

The description further provides primers, probes and corresponding kits used to quantify plasmid variants integrated into CAR T products, such as functional CAR variants, nucleic acids, polypeptides and proteins described herein. As used herein, the term variant refers to a polypeptide or polynucleotide that differs from the reference polypeptide or reference 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 having substantial or significant sequence identity or similarity to a parent CAR, polypeptide or protein, wherein the functional variant retains the biological activity of the CAR, polypeptide or protein for which it is a variant. Functional variants include, for example, those variants of a CAR, polypeptide, or protein described herein (the parent CAR, polypeptide, or protein) that retain the ability to recognize target cells to a similar extent, to the same extent, or to a greater extent as parent CAR, polypeptide or protein. With respect to the parent CAR, polypeptide or protein, the functional variant may 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.

The functional variant may, for example, comprise the amino acid sequence of the parent CAR, polypeptide or protein with at least one conservative amino acid substitution. In another embodiment, the functional variants may comprise the amino acid sequence of the parent CAR, polypeptide or protein with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution may not interfere with or inhibit the biological activity of the functional variant. A non-conservative amino acid substitution may enhance the biological activity of a functional variant such that the biological activity of the functional variant is increased relative to the parent CAR, polypeptide or protein.

Amino acid substitutions in CARs may be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is replaced by another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution may be an acidic amino acid substituted with another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted with another basic amino acid (Lys, Arg, etc.), 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.

The CAR, polypeptide, or protein may consist essentially of the specified amino acid sequence or sequences described herein such that other components, such as other amino acids, do not significantly alter the biological activity of the CAR, polypeptide, or protein.

Examples of modified nucleotides that can be used to produce recombinant nucleic acids used to produce polypeptides used in the methods/kits described herein include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5 -ioduracil, hypoxanthine, xanthine, 4-acetylcytosine, 5(carboxyhydroxymethyl)uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N ⁶ -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-E -mannosylqueuosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-isopentenyladenine, uracil-5hydroxyacetic acid (v), vibutoxysine, pseudouracil, queuosine, beta-E-galactosylqueosine, inosine, N-isopentenyladenine, 1-methylguanine, 1-metinosine , 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5methyluracil, uracil-5 methyl ester -hydroxyacetic acid, 3-(3-amino-3-N2-carboxypropyl)uracil and 2,6-diaminopurine.

The nucleic acid of the invention may contain any isolated or purified nucleotide sequence that encodes any of the CARs, polypeptides, or proteins, or functional regions thereof, or functional variants thereof. Alternatively, the nucleotide sequence may comprise a sequence degenerate into any one or combination of degenerate sequences.

Some embodiments of the invention also use an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to a nucleotide sequence of any of the nucleic acids described herein, or a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence of any of the nucleic acids described in this document.

A nucleotide sequence that hybridizes under stringent conditions may hybridize under highly stringent conditions.

The highly stringent conditions described herein mean that the nucleotide sequence specifically hybridizes to the target sequence (the nucleotide sequence of any of the nucleic acids described herein) to a degree that is more detectable than nonspecific hybridization. Highly stringent conditions include conditions that distinguish between a polynucleotide with an exact complementary sequence or a polynucleotide containing only a few scattered mismatches from a random sequence that has several small regions (eg, 3-12 bases) that match the nucleotide sequence. Such small regions of complementarity melt more easily than full-length complements of 14 to 17 bases or more, and high stringency hybridization makes them easily distinguishable. Relatively high-severity conditions will include, for example, low-salt and/or high-temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent, at temperatures of about 50-70°C. Such highly stringent conditions allow for little mismatch between the nucleotide sequence and the template or target strand, if any, or are particularly suitable for hybridization with the CAR nucleic acids described herein. It is generally believed that conditions can be made more stringent by adding increasing amounts of formamide.

As used herein, the term recombinant expression vector means a genetically modified oligonucleotide or polynucleotide construct that causes expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct contains a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide and the vector is brought into contact with with a cell under conditions sufficient to permit expression of an mRNA, protein, polypeptide, or peptide within the cell. The vectors described herein are not found in nature in general; however, parts of the vectors may be of natural origin. The described recombinant expression vectors may contain nucleotides of any type, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthesized or derived in part from natural sources, and which may contain natural, non-natural or altered nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Unnatural or altered nucleotides or internucleotide linkages do not interfere with transcription or replication of the vector.

In an embodiment, the recombinant expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, MD, USA), pBluescript series (Stratagene, La Jolla, CA, USA), pET series (Novagen, Madison , Wisconsin, USA), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pETX series (Clontech, Palo Alto, California, USA). Bacteriophage vectors such as λGT10, λGT11, λEMBL4 and λΝΜ1149, λZapII (Stratagene) can be used. Examples of plant expression vectors include pBI01 pBI01.2, pBI121, pBI101.3 and pBIN19 (Clontech). Examples of animal expression vectors

- 9 046825 includes pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, such as a retroviral vector, such as a gamma retroviral vector.

In one embodiment, recombinant expression vectors were prepared using standard recombinant DNA techniques 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. Expression vector constructs that are circular or linear can be constructed to contain a replication system operating in a prokaryotic or eukaryotic host cell. Replication systems can be obtained, for example, from ColEl, SV40, 2 μm plasmid, λ, bovine papillomavirus, and the like.

In certain embodiments, expression vectors used herein are linearized to prepare working plasmid stock solutions to produce standards and controls.

The recombinant expression vector may contain regulatory sequences, such as transcription and translation start and stop codons, that are specific to the type of host (e.g., bacterium, fungus, plant or animal) into which the vector is to be introduced, as appropriate, based on whether the vector is DNA or RNA.

The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected host organisms. Marker genes include biocide resistance genes, such as resistance genes to antibiotics, heavy metals, etc., complementation in an auxotrophic host to ensure prototrophy, and the like. Suitable marker genes for the described expression vectors include, for example, neomycin/C418 resistance genes. histidinol X resistance genes, histidinol resistance genes, tetracycline resistance genes and ampicillin resistance genes.

The recombinant expression vector may contain a native or normal promoter operably linked to a nucleotide sequence encoding a CAR, polypeptide or protein (including functional regions and functional variants thereof), or to a nucleotide sequence that is complementary to or which hybridizes to a nucleotide sequence encoding a CAR, polypeptide or protein. The selection of promoters, such as strong, weak, tissue-specific, inducible and development-specific promoters is within the skill of one of ordinary skill in the art. Likewise, combining a nucleotide sequence with a promoter is also within the skill of one skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an RSV promoter, an SV40 promoter, or a promoter present in the long terminal repeat of a murine stem cell virus.

Recombinant expression vectors can be designed for either transient expression, stable expression, or both. In addition, recombinant expression vectors can be prepared for constitutive expression or for inducible expression.

Additionally, recombinant expression vectors incorporating the suicide gene can be created. As used herein, the term suicide gene refers to a gene that causes a cell expressing the suicide gene to die. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, to the cell in which the gene is expressed and causes the cell to die when the cell is brought into contact with or exposed to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deminase, purine nucleoside phosphorylase and nitroreductase.

The invention includes conjugates, for example bioconjugates, containing any CARs, polypeptides or proteins (including any functional regions or variants thereof), host cells, nucleic acids, recombinant expression vectors, host cell populations or antibodies or antigen binding regions thereof. Conjugates, as well as methods for synthesizing conjugates, are generally known in the art (see, for example, Hudez, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirin et al., Inorg Chem. 44 (15 ): 5405-5415 (2005)).

In a specific embodiment, the recombinant expression vector used in embodiments of the invention is a vector containing various components of a chimeric antigen receptor B-cell maturation antigen (BCMA). The plasmid is an 8518 base pair (bp) plasmid containing the sequence encoding various components of the BCMA chimeric antigen receptor, which is disclosed in SEQ ID NOs: 175-197, 202205, 218-227, 239, 261-264 and 271 -276 in PCT International Patent Application Publication No. WO2017/025038 A1, the contents of which are incorporated herein by reference in their entirety. In one aspect of the invention, the plasmid encodes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain binds BCMA antigen. As used herein, the terms B cells, B cells

- 10 046825 and B lymphocytes are used interchangeably. In one embodiment, the plasmid contains the nucleotide sequence of any of SEQ ID NO: 175-197, 202-205, 218-227, 239, 261-264 and 271276 from PCT International Patent Application Publication No. WO2017/025038 A1. In some embodiments, the plasmid contains 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 of SEQ ID NO: 175-197, 202-205, 218-227, 239, 261-264 and 271-276 from PCT International Patent Application Publication No. WO2017/025038 A1.

The term coding refers to the property inherent in specific nucleotide sequences in a polynucleotide, such as a gene, cDNA or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence (for example, rRNA, tRNA and mRNA), or a specific amino acid sequence, as well as the biological properties resulting from it. Thus, a gene, cDNA, or RNA encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the sequence of the mRNA, and the non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding a protein or other product of that gene or cDNA.

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 that encodes a protein or RNA may also include introns to the extent that a nucleotide sequence that encodes a protein may in some versions contain intron(s).

In one embodiment, provided herein is an expression vector comprising the nucleotide sequence of any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264 and 271-276 from PCT International Patent Application Publication No. WO2017 /025038 A1.

The term expression vector refers to a vector containing a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains a sufficient number of cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids (eg, naked or contained in liposomes) and viruses (eg, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that contain a recombinant polynucleotide.

Methods for obtaining CAR T cells and quantifying transgene integration into a CAR T cell.

In one aspect, the present invention provides methods for quantifying transgene integration in a CAR T cell, comprising:

contacting nucleic acids from a CAR T cell with a first CAR primer comprising a nucleotide 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 acids from the CAR T cell with a second CAR primer containing a nucleotide 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 acids from a CAR T cell with a first hALB primer comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29 and combinations thereof, and bringing the nucleic acids from a CAR T cell into contact with a second hALB primer containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID no: 30 and combinations thereof;

bringing nucleic acids from a CAR T cell into contact with a CAR probe, wherein the CAR probe comprises 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 acids from a CAR T cell with an hALB probe, wherein the hALB probe comprises 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 amplicons with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acid molecules;

amplifying the hALB amplicons with a first hALB primer and a second hALB primer, thereby obtaining amplified hALB nucleic acid molecules;

detecting hybridization between the amplified CAR nucleic acid molecules and the CAR probe through a target signal from at least one tag associated with the CAR probe;

detection of hybridization between molecules of amplified hALB nucleic acids and

- 11 046825 hALB probe via a reference signal from at least one label associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

In some embodiments, the contacting steps for the CAR primers are performed in a separate reaction from the hALB primers. In other embodiments, the contacting steps are performed during the same reaction (ie, multiplexed).

In some embodiments, the contacting steps for the CAR probes are performed in a separate reaction from the hALB probes. In other embodiments, the contacting steps are performed during the same reaction (ie, multiplexed).

In some embodiments, the amplification steps for the CAR amplicons are performed in a separate reaction from the hALB amplicons. In other embodiments, the amplification steps are performed in the same reaction (ie, multiplexed).

In some embodiments, the detection steps for hybridizing CAR nucleic acids and CAR probes are performed in a separate reaction from the hALB nucleic acids and hALB probes. In other embodiments, the detection steps are performed during the same reaction (ie, multiplexed).

In some embodiments, the methods include amplifying CAR nucleic acids with a first CAR primer of about 20 to about 40 nucleotides in length. In some embodiments, the first CAR primer is configured to hybridize under high stringency conditions to a CAR nucleotide sequence set forth below in any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from PCT International Patent Application Publication No. WO2017/025038 A1.

A primer, that is, a nucleotide sequence that hybridizes under stringent conditions, can hybridize under highly stringent conditions.

In some embodiments, the first CAR primer includes a nucleotide 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 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 includes a nucleotide 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 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 specific embodiments, the first CAR primer includes a nucleotide sequence that is at least about 95% identical to a nucleotide 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 includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 2. In some embodiments, the first CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 2. In particular embodiments, the first CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 2.

In some embodiments, the first CAR primer includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 5. In some embodiments, the first CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 5. In particular embodiments, the first CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 5.

In some embodiments, the first CAR primer includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 8. In some embodiments

- 12 046825 implementation of the first CAR primer includes a nucleotide 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 nucleotide the sequence that is set forth in SEQ ID NO: 8 below. In certain embodiments, the first CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is set forth in SEQ ID NO: 8 below.

In some embodiments, the first CAR primer includes a nucleotide 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 sequence, which is given below in SEQ ID NO: 11.

In some embodiments, the first CAR primer includes a nucleotide 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 sequence that is set forth in SEQ ID NO: 11 below. In certain embodiments, the first CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is set forth in SEQ ID NO: 11 below.

In some embodiments, the first CAR primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 14. In some embodiments, the first CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 14. In particular embodiments, the first CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 14.

In some embodiments, the first CAR primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 17. In some embodiments, the first CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 17. In particular embodiments, the first CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 17.

In some embodiments, the first CAR primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 20. In some embodiments, the first CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 20. In particular embodiments, the first CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 20.

In some embodiments, the methods include amplifying CAR nucleic acids with a second CAR primer of about 20 to about 40 nucleotides in length. In some embodiments, the second CAR primer is configured to hybridize under high stringency conditions to a CAR nucleotide sequence set forth below in any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271-276 from PCT International Patent Application Publication No. WO2017/025038 A1.

In some embodiments, the second CAR primer includes a nucleotide 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 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 includes a nucleotide 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 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

- 13 046825

NO: 15, SEQ ID NO: 18, and SEQ ID NO: 21. In specific embodiments, the second CAR primer includes a nucleotide sequence that is at least about 95% identical to a nucleotide 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 includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 3. In some embodiments, the second CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 3. In particular embodiments, the second CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 3.

In some embodiments, the second CAR primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 6. In some embodiments, the second CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 6. In particular embodiments, the second CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 6.

In some embodiments, the second CAR primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 9. In some embodiments, the second CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 9. In particular embodiments, the second CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 9.

In some embodiments, the second CAR primer includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 12. In some embodiments, the second CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 12. In specific embodiments, the second CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 12.

In some embodiments, the second CAR primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 15. In some embodiments, the second CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 15. In specific embodiments, the second CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 15.

In some embodiments, the second CAR primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 18. In some embodiments, the second CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 18. In certain embodiments, the second CAR primer includes a nucleotide sequence that is at least about 95% identical nucleotide sequence, which is given below in SEQ ID NO: 18.

In some embodiments, the second CAR primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 21. In some embodiments, the second CAR primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 21. In certain embodiments, the second CAR primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 21.

In some embodiments, the methods include hybridizing a CAR nucleic acid molecule with a specific CAR probe of about 20 to about 40 in length and detecting hybridization between the CAR nucleic acid and the probe. In some embodiments, a detectable label is included in the probe. In some embodiments, the specific CAR probe is configured to hybridize under high stringency conditions to a CAR nucleotide sequence set forth below in any of SEQ ID NOs: 175-197, 202-205, 218-227, 239, 261-264, and 271276 from publication international patent application PCT No. WO2017/025038 A1.

In some embodiments, the hybridization detection steps can be performed using traditional molecular biology techniques known in the art, such as, without limitation, gel electrophoresis, Southern blotting and/or the like.

In some embodiments, the specific CAR probe includes a nucleotide 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 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 some embodiments, the probe includes a nucleotide 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 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 and SEQ ID NO: 19. In particular In embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to 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 and SEQ ID NO: 19.

In some embodiments, the specific CAR probe includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 1. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical or is at least about 99% identical to the nucleotide sequence set forth in SEQ ID NO: 1. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth in SEQ below ID NO: 1.

In some embodiments, the specific CAR probe includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 4. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical or is at least about 99% identical to the nucleotide sequence set forth below in SEQ ID NO: 4. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth below in SEQ ID NO: 4.

In some embodiments, the specific CAR probe includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 7. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical or is at least about 99% identical to the nucleotide sequence that is set forth below in SEQ ID NO: 7. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 7.

In some embodiments, the specific CAR probe includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 10. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical or is at least about 99% identical to the nucleotide sequence set forth in SEQ ID NO: 10 below. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth in SEQ below ID NO: 10.

In some embodiments, the specific CAR probe includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 13. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical or is at least about 99% identical to the nucleotide sequence set forth below in SEQ ID NO: 13. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth below in SEQ ID NO: 13.

In some embodiments, the specific CAR probe includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 16. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical or is at least about 99% identical to the nucleotide sequence set forth below in SEQ ID NO: 16. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth below in SEQ ID NO: 16.

In some embodiments, the specific CAR probe includes a nucleotide 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 sequence as set forth below in SEQ ID NO: 19. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical or is at least about 99% identical to the nucleotide sequence set forth below in SEQ ID NO: 19. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth below in SEQ ID NO: 19.

In some embodiments, the methods include amplifying hALB nucleic acids with a first hALB primer of about 20 to about 40 nucleotides in length. In some embodiments, the first hALB primer is configured to hybridize under high stringency conditions to the hALB nucleotide sequence set forth below in SEQ ID NO: 31 (FIG. 13).

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

- 16046825 nucleotide sequence, which is set forth below in SEQ ID NO: 23. In some embodiments, the first hALB primer includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, is at least about 98% identical, or at least about 99% identical, to the nucleotide sequence set forth below in SEQ ID NO: 23. In certain embodiments, the first hALB primer includes a nucleotide sequence that is at least about 95% identical to nucleotide sequence, which is given below in SEQ ID NO: 23.

In some embodiments, the first hALB primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 26. In some embodiments, the first hALB primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 26. In particular embodiments, the first hALB primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 26.

In some embodiments, the first hALB primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 29. In some embodiments, the first hALB primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 29. In specific embodiments, the first hALB primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 29.

In some embodiments, the methods include amplifying hALB nucleic acids with a second hALB primer of about 20 to about 40 nucleotides in length. In some embodiments, the second hALB primer is configured to hybridize under high stringency conditions to the hALB nucleotide sequence set forth below in SEQ ID NO: 31.

In some embodiments, the second hALB primer includes a nucleotide 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 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 includes a nucleotide sequence that is at least about 96% identical to at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 30. In specific embodiments, the second hALB primer includes a nucleotide sequence that is at least about 95% identical to a nucleotide 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 includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 24. In some embodiments, the second hALB primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 24. In specific embodiments, the second hALB primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 24.

In some embodiments, the second hALB primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 27. In some embodiments, the second hALB primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 27. In certain embodiments, the second hALB primer includes a nucleotide sequence that is at least about 95% identical

- 17 046825 is identical to the nucleotide sequence shown below in SEQ ID NO: 27.

In some embodiments, the second hALB primer includes a nucleotide 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 sequence, which is set forth below in SEQ ID NO: 30. In some embodiments, the second hALB primer includes a nucleotide 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 nucleotide sequence that is set forth below in SEQ ID NO: 30. In specific embodiments, the second hALB primer includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that is given below at SEQ ID NO: 30.

In some embodiments, the methods include hybridizing a hALB nucleic acid molecule with a specific hALB probe of about 20 to about 40 in length and detecting hybridization between the hALB nucleic acid and the probe. In some embodiments, a detectable label is included in the probe. In some embodiments, the hALB specific probe is configured to hybridize under high stringency conditions to the hALB nucleotide sequence set forth below in SEQ ID NO: 31.

In some embodiments, the hybridization detection steps can be performed using traditional molecular biology techniques known in the art, such as, without limitation, gel electrophoresis, Southern blotting and/or the like.

In some embodiments, the probe includes a nucleotide 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 nucleotide 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 includes a nucleotide sequence that is at least about 96% identical to at least about 97% identical, at least about 98% identical, or at least about 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 25 and SEQ ID NO: 28. In particular In embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to a nucleotide 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 includes a nucleotide 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 nucleotide sequence which is set forth below in SEQ ID NO: 22. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or is at least about 99% identical to the nucleotide sequence set forth in SEQ ID NO: 22. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth in SEQ ID NO: 22 below. : 22.

In some embodiments, the probe includes a nucleotide 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 nucleotide sequence which is set forth below in SEQ ID NO: 25. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or is at least about 99% identical to the nucleotide sequence set forth in SEQ ID NO: 25. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth in SEQ ID NO: 25 below. : 25.

In some embodiments, the probe includes a nucleotide 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 nucleotide sequence which is set forth below in SEQ ID NO: 28. In some embodiments, the probe includes a nucleotide sequence that is at least about 96% identical, at least about 97% identical, at least about 98% identical, or is at least about 99% identical to the nucleotide sequence set forth in SEQ ID NO: 28. In particular embodiments, the probe includes a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth in SEQ ID NO: 28 below. : 28.

In a specific aspect, the present invention provides methods for quantifying transgene integration in a CAR T cell, comprising:

contacting nucleic acids from a CAR T cell with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and contacting nucleic acids from a CAR T cell with a second CAR primer containing the nucleotide sequence of SEQ ID

NO: 12;

contacting nucleic acids from the CAR T cell with a first hALB primer containing the nucleotide sequence of SEQ ID NO: 23 and contacting nucleic acids from the CAR T cell with a second hALB primer containing the nucleotide sequence of SEQ ID NO: 24;

contacting nucleic acids from a CAR T cell with a CAR probe, wherein such CAR probe comprises the nucleotide sequence of SEQ ID NO: 10;

contacting nucleic acids from the CAR T cell with an hALB probe, wherein the hALB probe comprises the nucleotide sequence of SEQ ID NO: 22;

amplifying the CAR nucleic acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acid molecules;

amplifying hALB nucleic acids with a first hALB primer and a second hALB primer, thereby obtaining amplified hALB nucleic acid molecules;

detecting hybridization between the amplified CAR nucleic acid molecules and the CAR probe through a target signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acid molecules and the hALB probe by means of a reference signal from at least one label associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

In some embodiments, detecting hybridization between the amplified CAR nucleic acid molecules and the CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the at least one label associated with the probe CAR, before hybridization.

In some embodiments, the step of detecting hybridization between the amplified hALB nucleic acid molecules and the hALB probe includes detecting a change in the target signal from at least one tag associated with the hALB probe during or after hybridization relative to the target signal from the at least one tag associated with hALB probe prior to hybridization.

In another aspect, the present invention provides methods for quantifying transgene integration in a CAR T cell, comprising:

amplifying nucleic acids from a CAR T cell with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids; amplifying nucleic acids from the CAR T cell with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids; detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

In another aspect, the present invention provides methods for producing a CAR T cell, including:

introducing a CAR transgene into a T cell to obtain a T cell with an integrated transgene;

definition of CAR transgene integration, including:

amplifying nucleic acids from a T cell with an integrated transgene with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids;

amplifying nucleic acids from the transgene-integrated T cell with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe by means of a reference signal from at least one label,

- 19 046825 associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal; and obtaining a CAR T cell containing at least one copy of the integrated transgene

CAR.

Aspects of the present invention also provide CAR T cells produced using the methods described herein.

The term reference gene refers to an internal response control with sequences other than the target gene. In order 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. Most importantly, its expression level should not be influenced by experimental factors. In addition, it should show minimal variability in its expression across tissues and under different physiological conditions of the body. It is advisable to select a reference gene with a threshold cycle that is close to the gene of interest. The reference gene must exhibit variability due to imperfections in the technology and preparatory procedures used - thereby ensuring that any change in the amount of genetic material will affect the subject and control to the same extent. Examples of reference genes that meet the above criteria include essential metabolic genes, called vital function genes, which by definition are involved in processes important for cell survival. Genes of vital functions suitable for use as reference genes must also be expressed at a stable and unregulated constant 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. Vital function genes that can be used as reference genes in the methods, kits and primers/probes of the present invention include, but are not limited to, LDHA, NONO, PGK1, PPIH, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5 , SNRPD3, VCP and VPS29.

In another aspect, the invention provides methods for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising:

contacting nucleic acids 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, wherein the first CAR primer contains the nucleotide sequence of SEQ ID NO: 11 and the second CAR primer contains the nucleotide sequence of SEQ ID NO: 11 ID NO: 12;

amplifying the CAR nucleic acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acids;

amplifying nucleic acids of the first reference gene with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

In some embodiments, detecting hybridization between amplified CAR nucleic acids and a CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the label associated with the CAR probe prior to hybridization. In some embodiments, detecting hybridization between amplified reference gene nucleic acid molecules and a reference gene probe includes detecting a change in the target signal from at least one label associated with the reference gene probe during or after hybridization relative to the target signal from the label associated with the reference probe gene, before hybridization. In some embodiments, the at least one tag associated with the reference gene probe contains a fluorophore.

In some embodiments, the hybridization detection steps may be performed using conventional molecular biology techniques known to those skilled in the art, such as, without limitation, gel electrophoresis, Southern blotting, and/or the like.

In some embodiments, at least one of the amplification steps includes a polymerase chain reaction (PCR), such as real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), ligase chain reaction (LCR) or transcription-mediated amplification (TMA).

PCR is well known to those skilled in the art. This technique is widely used in molecular biology to produce multiple copies of a specific DNA segment. With PCR, one copy (or more) of a DNA sequence is exponentially amplified to produce thousands to millions of additional copies of that particular DNA segment. Most PCR methods are based on thermal cycling. In thermal cycling, reagents undergo repeated heating and cooling cycles to carry out various temperature-dependent reactions—DNA melting and enzymatic DNA replication. PCR uses two main reagents: primers (short single-stranded nucleotide fragments known as oligonucleotides with a sequence complementary to the target DNA region) and DNA polymerase. In the first stage of PCR, the process of melting DNA at high temperatures physically separates the two strands of the DNA double helix. In the second stage, the temperature is lowered and the primers bind to complementary DNA sequences. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from the free nucleotides present in the reaction mixture. As PCR continues, the resulting DNA itself is used as a template for replication, thus exponentially amplifying the original DNA template. Typically, applications requiring PCR use a thermostable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus.

Quantitative PCR or real-time PCR (qPCR), as known to those skilled in the art, estimates the amount of a particular sequence present in a sample, a technique often used to quantify gene expression levels. Quantitative PCR is a proven DNA quantification tool that measures the accumulation of DNA product after each PCR amplification cycle. qPCR allows for real-time quantification and detection of a specific DNA sequence because it measures the concentration during the synthesis process.

Reverse transcriptase polymerase chain reaction (RT-PCR), as known to those skilled in the art, is a laboratory technique that combines the reverse transcription of RNA into DNA (complementary DNA or cDNA) and the amplification of specific target DNAs using PCR. It is usually used to measure the amount of specific RNA. This is achieved by monitoring the amplification reaction by fluorescence using a technique called real-time PCR or quantitative PCR (qPCR). A combination of RT-PCR and qPCR has traditionally been used for gene expression analysis and RNA quantification.

As is known to those skilled in the art, ligase chain reaction (LCR) is one of the methods for DNA amplification. Ligase chain reaction (LCR) is an amplification process that differs from PCR in that it uses a thermostable ligase to link together two probes or other molecules, which can then be amplified using standard PCR cycles.

Transcription-mediated amplification (TMA), as known to those skilled in the art, is an isothermal single-sample nucleic acid amplification system that uses two enzymes, RNA polymerase and reverse transcriptase. Unlike PCR and LCR, TMA involves RNA transcription (using RNA polymerase) and DNA synthesis (using reverse transcriptase) to produce an RNA amplicon (parent compound or amplification product) from the target nucleic acid.

In some embodiments, the methods presented herein use other quantitative PCR methods known to those skilled in the art, such as, but not limited to, digital PCR (dPCR).

In some embodiments, at least one label associated with the CAR probe contains a fluorophore. In some embodiments, at least one label associated with the hALB probe contains a fluorophore. As used herein, the term fluorophore refers to any fluorescent compound or protein that can be used to quantify and detect nucleotide sequences to which probes hybridize.

The present disclosure also relates to primers that are capable of hybridizing to and amplifying a CAR nucleic acid, for example a nucleotide sequence spanning the CD137/CD3z crosslink of a CAR construct. The described primers can be used in the methods described herein. In some embodiments, these primers are from about 20 to about 40 nucleotides in length and are capable of hybridizing under high stringency conditions to the CAR nucleotide sequence set forth below in any of SEQ ID NOs: 175-197, 202-205, 218-227. 239, 261-264 and 271-276 from International Patent Application Publication No. WO2017/025038 A1.

- 21 046825

In some embodiments, these primers contain a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth below in 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 nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth below in 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.

The present disclosure also relates to probes that are capable of hybridizing to and discriminating against various CAR nucleotide sequences, for example, various nucleotide sequences spanning the CD137/CD3z crosslink of the CAR construct. The described probes can be used in the methods described herein. In some embodiments, these probes are from about 20 to about 40 nucleotides in length and are capable of hybridizing under high stringency conditions to the CAR nucleotide sequence set forth below in any of SEQ ID NOs: 175-197, 202-205, 218-227. 239, 261-264 and 271-276 from PCT International Patent Application Publication No. WO2017/025038 A1. In some embodiments, these probes contain a nucleotide sequence that is at least about 95% identical to the nucleotide sequence set forth below in 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.

In one aspect, the invention provides sets of probes and primers comprising a probe comprising the nucleotide sequence of SEQ ID NO: 10, and at least one tag associated with the probe; a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11; and a second CAR primer comprising the nucleotide sequence of SEQ ID NO: 12. In one embodiment, the probe and primer sets further comprise an hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 and at least one tag associated with the probe; the first hALB primer containing the nucleotide sequence SEQ ID NO: 23; and a second primer hALB containing the nucleotide sequence of SEQ ID NO: 24.

In some embodiments, the at least one label comprises a radioactive isotope, enzyme substrate, chemiluminescent agent, fluorophore, fluorescence quencher, enzyme, chemical compound, or a combination thereof. In some embodiments, luminescence of the labels may be induced through the use of photochemical, chemical, and electrochemical means.

The present invention also provides kits for quantifying transgene integration into a CAR T cell. As used herein, the term kit 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 generation systems (e.g., fluorescence signal generation systems), antibodies, control proteins, and test containers (e.g., microtiter plates, etc.) .d.). It is not intended that the term kit be limited to a particular combination of reagents and/or other materials. In one embodiment, the kit further contains instructions for use of the reagents. The kit may be packaged in any suitable manner, typically with the components in one container or in different containers as appropriate, along with a test instructions sheet. In some embodiments, the kits also include a positive control sample. The kits can be produced in a variety of ways known to those skilled in the art.

In one aspect, kits for quantifying transgene integration into a CAR T cell comprise: a probe comprising the nucleotide sequence of SEQ ID NO: 10, and at least one tag associated with the probe; a first primer containing the nucleotide sequence SEQ ID NO: 11; and a second primer comprising the nucleotide sequence of SEQ ID NO: 12. In one embodiment, the kits further comprise an hALB probe comprising the nucleotide sequence of SEQ ID NO: 22, and at least one tag associated with the probe; the first hALB primer containing the nucleotide sequence SEQ ID NO: 23; and a second primer hALB containing the nucleotide sequence of SEQ ID NO: 24.

In some embodiments of the kits of the invention, at least one label associated with the probe contains a radioactive isotope, enzyme substrate, chemiluminescent agent, fluorophore, fluorescence quencher, enzyme, chemical compound, or a combination thereof.

In one embodiment, the kits of the invention contain a matrix that contains a probe. In some embodiments, the matrix is a multi-well plate.

In certain embodiments, the at least one label associated with the hALB probe comprises a radioactive isotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical compound, or a combination thereof.

When used in the aspects described in the invention, the term label refers to a functional group that is capable of producing a detectable signal, i.e., a signal that can be detected to a small extent by detection means that generate a signal

- 22 046825 cash. Examples of such acceptable means include spectroscopic or photochemical means, such as fluorescence or luminescence, or biochemical, immunochemical or chemical means, such as changes in physical, biochemical, immunochemical or chemical properties upon contact with the detecting analytical compound or upon reaction with a polypeptide or polypeptide mixture /enzyme to form a detectable complex. Thus, as used herein, the term label is intended to include both functional groups that can be detected directly, such as radioisotopes or fluorochromes, and reactive functional groups that are detected indirectly during the reaction that produces the product of interest, such as enzymes , which react with a substrate to form a product detectable spectrophotometrically. It is noted that the labeling reagent may contain a radiolabel functional group, such as a radioisotope. In one embodiment, the hybridization probe of the present document contains a non-radioactive label to prevent complications associated with radioactivity analysis.

To apply the label detection schemes in the methods and kits described in the invention, tags are introduced into the nucleotide bases by covalently linking a compound to generate a fluorescence or chemiluminescence signal after the introduction of dNTPs into the extended DNA primer/template. Examples of fluorescent compounds for tagging dNTPs include, but are not limited to, fluorescein, rhodamine, and BODIPY (4,4-difluoro-4-boron-3a,4a-diaza-s-indacene). See Handbook of Molecular Probes and Fluorescent Chemicals offered by Molecular Probes, Inc. (Eugene, Oregon). 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).

Fluorescently or chemiluminescently labeled dNTPs are added individually to a DNA template system containing a DNA template after annealing to a primer, DNA polymerase, in an appropriate buffer medium. After the reaction time has elapsed, excess dNTP is removed and the system is probed to ensure that the fluorescently or chemiluminescently tagged nucleotide is incorporated into the DNA template. Detection of the included nucleotide can be accomplished using various methods, which may depend on the type of label used.

For fluorescently labeled dNTPs, the DNA template system can be irradiated with optical radiation at a wavelength that the label actively absorbs. The fluorescence of the label is detected using, for example, a photodetector with an optical filter that cuts off any scattered light at the excitation wavelength.

In a further embodiment in which fluorescent detection is used in the kits and methods described herein, the fluorescent tag is coupled to the dNTP via a photocleavable or chemically cleavable linker, and the tag is cleaved after the extension reaction and removed from the matrix system into the detection cuvette, where the presence and the amount of such label is determined by optical excitation at an appropriate wavelength with fluorescence detection. In this embodiment, the likelihood of fluorescence quenching due to the presence of multiple fluorescent tags in close proximity to each other on the primer strand, which is complementary to the region of single base repeats in the template, is minimized, and it is possible to optimize the accuracy of determining the number of repeats. In addition, excitation of fluorescence in a separate cuvette minimizes the possibility of photolytic damage to the DNA primer/template system.

In one embodiment, the probe contains a 5' 6-FAM™ (fluorescein) label. 6-AM™ is the only isomeric fluorescein derivative. FAM is an oligonucleotide-linked fluorescent dye and is compatible with most fluorescence detection equipment. At pH below 7, it is protonated and exhibits reduced fluorescence intensity; Typically, it is used in the pH range of 7.5-8.5. FAM™ can bind to the 5' or 3' ends of oligonucleotides.

In one embodiment, the probe contains a 5HEX™ (hexachlorofluorescein) label. Hexachlorofluorescein is a chemically related compound of fluorescein that is used for multiplex assays with FAM™ HEX™ can only bind to the 5' end of the oligonucleotide.

The present disclosure also contemplates the use of any other labels known to those skilled in the art that can be used to label probes as described herein, such as, without limitation, VIC®, TET™, JOE™, NED™, PET®, ROX™, TAMRA™, JET™, Texas Red®, ATTO™ 532, Cy3, Tye™ 563, Tye™ 665, TEX 615™, Cy5, ZEN™, Iowa Black® FQ, Iowa Black® RQ, DABYCL and Yakima Yellow ™.

In one embodiment, the probe contains a fluorescence quencher label. The quencher tag may be used as a dual quencher in the reactions described herein. In one embodiment, the probe contains Iowa Black® FQ quencher. Iowa Black® FQ has a broad absorption spectrum ranging from 420 to 620 nm with peak absorption at 531 nm. The 23 046825 quenchant is used with fluorescein and other fluorescent dyes with emission in the spectral range from green to pink. The present disclosure contemplates the use of any fluorescent quencher tags known to those skilled in the art, such as, for example, without limitation, ZEN™, Black Hole Quencher® (BHQ-1, BHQ-2, BHQ-3, etc.).

The transgenic qPCR methods and kits described in the present invention include a multiplex quantitative polymerase chain reaction (qPCR) assay designed to quantify the transgenic BCMA CAR plasmid integrated into a CAR T drug product. There are two target molecules amplified by this qPCR method: (1) transgenic BCMA CAR plasmid (transgene) and (2) human albumin reference gene (hALB). The transgenic target primer and probe set amplifies the cross-link between the CD137 and CD3z regions of the plasmid to enable detection exclusively of the transgenic BCMA CAR plasmid present and integrated into the CAR T drug product. The hALB reference gene copy number results are used to calculate the reported vector copy number (VCN) per cell result for each sample tested in the qPCR reaction.

Below is a description of example implementations.

Option 1 implementation. A set of probes and primers comprising: a probe containing the nucleotide sequence of SEQ ID NO: 10, and at least one tag associated with the probe; a first primer containing the nucleotide sequence SEQ ID NO: 11; and a second primer containing the nucleotide sequence of SEQ ID NO: 12.

Option 2 implementation. The probe and primer set of embodiment 1, wherein at least one label comprises a radioactive isotope, enzyme substrate, chemiluminescent agent, fluorophore, fluorescence quencher, enzyme, chemical compound, or a combination thereof.

Option 3 implementation. A kit for quantitative determination of transgene integration into a T cell with chimeric antigen receptors (CARs), comprising: a probe containing the nucleotide sequence of SEQ ID NO: 10, and at least one tag associated with the probe; a first primer containing the nucleotide sequence SEQ ID NO: 11; and a second primer containing the nucleotide sequence of SEQ ID NO: 12.

Option 4 implementation. The kit of embodiment 3, wherein at least one label associated with the probe contains a radioactive isotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical compound, or a combination thereof.

Option 5 implementation. The kit of embodiment 3, comprising a matrix containing a probe.

Option 6 implementation. The kit according to embodiment 5, in which the matrix is a multi-well plate.

Option 7 implementation. The kit of embodiment 3 further comprising a human albumin (hALB) probe containing the nucleotide sequence of SEQ ID NO: 22, and at least one tag associated with the hALB probe, a first hALB primer containing the nucleotide sequence of SEQ ID NO: 23, and the second primer hALB containing the nucleotide sequence SEQ ID NO: 24.

Embodiment 8. The kit of embodiment 7, wherein at least one label associated with the hALB probe contains a radioactive isotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical compound, or a combination thereof.

Option 9 implementation. The kit of embodiment 3 further comprising a reference gene probe and at least one tag associated with the reference gene probe, a first reference gene primer and a second reference gene primer.

Embodiment 10. A method for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising:

amplifying nucleic acids from a CAR T cell with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids;

amplifying nucleic acids from the CAR T cell with a first hALB primer containing the nucleotide sequence of SEQ ID NO: 23 and a second hALB primer containing the nucleotide sequence of SEQ ID NO: 24, thereby obtaining amplified hALB nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acids and an hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 by means of a reference signal from at least one tag associated with the hALB probe; And

- 24 046825 quantitative determination of the number of transgene copies by comparing the target signal with a reference signal.

Embodiment 11. A method for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising:

contacting nucleic acids from a 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 contains the nucleotide sequence of SEQ ID NO: 11, the second CAR primer contains the nucleotide sequence of SEQ ID NO: 12, the first hALB primer contains the nucleotide sequence of SEQ ID NO: 23, the second hALB primer contains the nucleotide sequence of SEQ ID NO: 24; amplifying the CAR nucleic acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acids;

amplifying hALB nucleic acids with a first hALB primer and a second hALB primer, thereby obtaining amplified hALB nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified hALB nucleic acids and the hALB probe through a reference signal from at least one label associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

Embodiment 12. The method of embodiment 10 or 11, wherein detecting hybridization between the amplified CAR nucleic acids and the CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the label associated with with the CAR probe, prior to hybridization.

Embodiment 13. The method of embodiment 10 or 11, wherein detecting hybridization between the amplified hALB nucleic acid molecules and the hALB probe includes detecting a change in the target signal from at least one label associated with the hALB probe during or after hybridization relative to the target signal from the label associated with hALB probe, prior to hybridization.

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

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 (real-time RT-PCR), ligase chain reaction ( LCR) or transcription-mediated amplification (TMA).

Embodiment 16. The method of embodiment 10, wherein the at least one label associated with the CAR probe contains a fluorophore.

Embodiment 17. The method of embodiment 10, wherein the at least one label associated with the hALB probe contains a fluorophore.

Embodiment 18. A method for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising:

amplifying nucleic acids from a CAR T cell with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids;

amplifying nucleic acids from the CAR T cell with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

Embodiment 19. A method for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising:

contacting nucleic acids 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, wherein

- 25 046825 the first CAR primer contains the nucleotide sequence SEQ ID NO: 11, the second CAR primer contains the nucleotide sequence SEQ ID NO: 12;

amplifying the CAR nucleic acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acids;

amplifying nucleic acids of the first reference gene with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids; detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal.

Embodiment 20. The method of embodiment 18 or 19, wherein detecting hybridization between the amplified CAR nucleic acids and the CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the label associated with with the CAR probe, prior to hybridization.

Embodiment 21. The method of embodiment 18 or 19, wherein detecting hybridization between the amplified reference gene nucleic acid molecules and the reference gene probe includes detecting a change in the target signal from at least one label associated with the reference gene probe during or after hybridization relative to the target signal from label associated with the reference gene probe prior to hybridization.

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

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 (real-time RT-PCR), ligase chain reaction ( LCR) or transcription-mediated amplification (TMA).

Embodiment 24. The method of embodiment 18, wherein the at least one label associated with the CAR probe comprises a fluorophore.

Embodiment 25. The method of embodiment 18, wherein the at least one tag associated with the reference gene probe comprises a fluorophore.

Embodiment 26. A method for producing a chimeric antigen receptor (CAR) T cell, comprising introducing a CAR transgene into the T cell to produce a T cell with an integrated transgene;

definition of CAR transgene integration, including:

amplifying nucleic acids from a T cell with an integrated transgene with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids;

amplifying nucleic acids from the transgene-integrated T cell with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids;

detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe;

detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe;

quantifying the number of transgene copies by comparing the target signal with a reference signal; and obtaining a CAR T cell containing at least one copy of the integrated CAR transgene.

Embodiment 27. The method of embodiment 26, wherein detecting hybridization between the amplified CAR nucleic acids and the CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the label associated with the CAR probe , before hybridization.

Embodiment 28. The method of embodiment 26, wherein detecting hybridization between the amplified reference gene nucleic acid molecules and the reference gene probe comprises detecting a change in the target signal from at least one label associated with the reference gene probe during or after hybridization relative to the target signal from the label associated with the reference gene probe prior to hybridization.

Embodiment 29. The method of embodiment 26, wherein the amplification involves polymerase chain reaction (PCR).

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 (real-time RT-PCR), ligase chain reaction ( LCR) or transcription-mediated amplification (TMA).

Embodiment 31. The method of embodiment 26, wherein the at least one label associated with the CAR probe comprises a fluorophore.

Embodiment 32. The method of embodiment 26, wherein the at least one tag associated with the reference gene probe comprises a fluorophore.

Embodiment 33. A CAR T cell prepared by the method of any one of embodiments 26-32.

The following examples are provided to further describe some of the embodiments described herein. The examples are intended to illustrate, but not limit, the described embodiments.

Example 1. Development of a transgenic qPCR method.

General overview of the method.

The described example of a transgenic qPCR method is a multiplex quantitative polymerase chain reaction (qPCR) designed to quantify the transgenic BCMA CAR plasmid (referred to as plasmid pLLV-LICAR2SIN in the examples) integrated into a CAR T drug product. This qPCR method amplified the following: ( 1) transgenic plasmid pLLV-LICAR2SIN (transgene) (transgene with a nucleotide sequence containing any of SEQ ID NO: 175-197, 202-205, 218-227, 239, 261-264 and 271-276 from the publication of the international patent application PCT No. WO2017/025038 A1) and (2) human albumin reference gene (hALB). The transgenic target primer and probe set amplifies the cross-link between the CD137 and CD3z regions of the plasmid to exclusively detect the transgenic plasmid pLLV-LICAR2SIN present and integrated into the CAR T drug product. The hALB reference gene copy number results are used to calculate the reported vector copy number (VCN) per cell result for each sample tested in the qPCR reaction. Transgenic qPCR VNC/cell sample results are provided to monitor the safety, purity, and identity of CART drug product samples.

Construction of transgenic primers and probes.

The BCMA CAR transgenic plasmid, referred to as plasmid pLLV-LICAR2SIN, is an 8,518 nucleotide pair (bp) plasmid containing sequences encoding many different components of the B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR). The qPCR target transgene was only required to detect the presence of the pLLV-LICAR2SIN plasmid and its integration into the CAR T drug, and that the target region must belong exclusively to the BCMA CAR construct. To ensure qPCR target transgene specificity, primer and probe pairs were designed to target at least one cross-link between at least two regions of the pLLV-LICAR2SIN plasmid encoding the CAR construct. First, it was necessary to identify suitable regions in the pLLVLICAR2SIN plasmid.

The longest coding regions in nucleotide pairs (bp) that integrate into the CAR T drug genome and are specific for the CAR construct belong to the two heavy chain variable regions of the BCMA CAR construct. These two regions are separated by a short linker sequence. The region of the nucleotide sequence corresponding to the two variable regions of the heavy chain and the linker were entered into the Nucleotide Basic Local Alignment Search Tool (BLAST) program on the National Center for Biotechnology Information (NCBI) website (https://blast.ncbi.nlm.nih.gov/Blast .cgi) to determine whether these regions could be suitable targets for transgenic qPCR. However, BLAST results indicated many hits for various immunoglobulin variable regions among many species, including homo sapiens. Therefore, the coding regions for the two variable components of the CAR heavy chain were identified as inappropriate targets for the transgenic qPCR method.

The crosslink between the CD137 and CD3z regions of plasmid pLLV-LICAR2SIN is included in a region of the plasmid that is integrated into the CAR T drug genome and is a component of BCMA CAR. In terms of its length, the CD3z coding region of the pLLV-LICAR2SIN plasmid is the second longest in bp. coding region of the CAR plasmid segment, making it a more suitable target region

- 27 046825 com due to the possibility of using a larger number of potentially suitable primer and probe pairs that can be obtained from a large coding region. The CD3z coding region is immediately adjacent to the CD137 coding region of the plasmid. On the opposite side of the CD137 coding region are plasmid framework sequences that are not specific for the BCMA CAR construct. Therefore, a cross-link between CD137 and CD3z was the only possible option suitable as a target if the large CD3z coding region was to be included in primers and probes during design.

PrimerQuest® Tool from Integrated DNA Technologies, Inc. was used to design suitable primer and probe pairs for testing during qPCR method development. (IDT) (Coralville, Iowa)(https://www.idtdna.com/Primerquest/Home/Index). The nucleotide sequence of plasmid pLLV-LICAR2SIN corresponding to the CD137 and CD3z coding regions was entered into PrimerQuest Tool. The optimal melting temperature (Tm) was set to 60°C, and nucleotides corresponding to the cross-link between CD137 and CD3z were entered into the Overlap Junction List to ensure that both the forward and reverse primers would overlap the cross-link. The result was four pairs of primers and probes (see Table 1). Two pairs had a forward primer spanning the CD137/CD3z cross-link, and two pairs had a reverse primer spanning the cross-link. Design parameters were then adjusted to constrain the probe structure to span the CD137/CD3z cross-link. As a result, three additional pairs of primers and probes were obtained (see Table 1) - a total of seven pairs of primers and probes suitable for testing during the development of the qPCR method. All 7 primer/probe pairs were processed on the NCBI BLAST site to check for possible cross-reactivity in the human genome. None of the BLAST results for any of the 7 pairs indicated the possibility of cross-reactivity.

Table 1

Primer and probe pairs targeting CD137/CD3z cross-linking

Name of the set of oligonucleotides Oligonucleotides Sequence (3’-5’) PCR product length (bp) Transgene (FP) set 1 Straight GGATGTGAACTGAGAGTGAAG (SEQ ID NO: 5) 104 Back TCCTCTCTTCGTCCTAGATTG (SEQ ID NO: 6) Probe TTATAGAGCTGGTTCTGGCCCTGC (SEQ ID NO: 4) Transgene (FP) set 2 Straight TGAACTGAGAGTGAAGTTCAGCA (SEQ ID NO: 2) 93 Back CTTCGTCCTAGATTGAGCTCGT (SEQ ID NO: 3) Probe AGCAGGGCCAGAACCAGCTCTATA (SEQ ID NO: 1) Transgene (RP) set 1 Straight GGCAGAAAGAAACTCCTGTAT (SEQ ID NO: 8) 129 Back CTTCACTCTCAGTTCACATCC (SEQ ID NO: 9) Probe TCTTCTGGAAATCGGCAGCTACAGC (SEQ ID NO: 7) Transgene (RP) set 2 Straight CCAGTACAAACTACTCAAGAGG (SEQ ID NO: 11) 90 Back GCTGAACTTCACTCTCAGTT (SEQ ID NO: 12) Probe TCTTCTGGAAATCGGCAGCTACAGC (SEQ ID NO: 10) Transgene (PRB) set 1 Straight CTGCCGATTTCCAGAAGAAG (SEQ ID NO: 14) 132 Back TCCTCTCTTCGTCCTAGATTG (SEQ ID NO: 15) Probe AGAAGGAGGATGTGAACTGAGAGTGAAGT (SEQ ID NO: 13) Transgene (PRB) set 2 Straight CTGTAGCTGCCGATTTCC (SEQ ID NO: 17) 145 Back ATCGTACTCCTCTCTTCGTC (SEQ ID NO: 18) Probe AGGAGGATGTGAACTGAGAGTGAAGT (SEQ ID NO: 16) Transgene (PRB) set 3 Straight CTGCCGATTTCCAGAAGAAG (SEQ ID NO: 20) 132 Back TCCTCTCTTCGTCCTAGATTG (SEQ ID NO: 21) Probe AGGAGGATGTGAACTGAGAGTGAAGT (SEQ ID NO: 19)

Note. FP = forward primer spans the crosslink; RP = reverse primer spans the crosslink; PRB = probe covers cross-link.

Three sets of hALB primers and probes were used for testing during the development of the qPCR method (see Table 2). One set was taken from a published article (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.), the second was taken from an analysis method digital PCR carried out by KIO and was ultimately not used, and a third set was designed using the PrimerQuest Tool and a region of the hALB gene that was chosen as the target both in the published paper and for the CCHMC hALB primer and probe sets. All 3 primer/probe pairs were run on the NCBI BLAST site to test for possible cross-reactivity with any non-hALB product in the human genome. None of the BLAST results for any of the 3 pairs indicated the possibility of cross-reactivity.

- 28 046825

table 2

hALB primer and probe pairs

Name of the set of oligonucleotides Oligonucleotides Sequence (3’-5’) PCR product length (bp) hALB set 1 Straight TCATCTCTTGTGGGCTGTAATC (SEQ ID NO: 23) 123 Back TGCTGGTTTCTCTTTCACTGAC (SEQ ID NO: 24) Probe AGGGAGAGATTTGTGTGGGCATGAC (SEQ ID NO: 22) hALB set 2 Straight GCTGTCATCTCTTGTGGGCTGT (SEQ ID NO: 26) 139 Back ACTCATGGGAGCTGCTGGTTC (SEQ ID NO: 27) Probe CCTGTCATGCCCACACAAATCTCTCC (SEQ ID NO: 25) hALB set 3 Straight CTGTCATGCCCACACAAA (SEQ ID NO: 29) 95 Back ATAAGGCTATCCAAACTCATGG (SEQ ID NO: 30) Probe CCCTGGCATTGTTGTCTTTGCAGA (SEQ ID NO: 28)

Note. Set 1 = hALB CCHMC set; Set 2 = hALB set from published article; Set 3 = home-built hALB set.

Screening of primers and probes.

7 different sets of transgenic primers and probes and 3 different sets of hALB primers and probes were screened by performing conventional qPCR reactions with CAR T drug, mock T cell DNA as samples supplemented with plasmid pLLV-LICAR2SIN and mock T cell DNA. T cell mock DNA was isolated from T cells that had undergone the same selection and amplification process as the CAR T product prior to lentivector transduction. qPCR products for CAR T cell, mock T cell, mock DNA T cell spiked with pLLV-LICAR2SIN plasmid, and qPCR products of the no-template control (NTC) sample were analyzed on an agarose gel. Any primer/probe set that did not produce a single PCR product band of the expected length was excluded from further development of the transgenic qPCR method. Presence of a primer dimer band of ~25 bp. was also observed in all qPCR products, including the NTC sample, but this band is an expected derivative of the qPCR reaction. Only the primer/probe sets transgene (FP) set 1 and transgene (RP) set 2 produced the expected target band, with only two bands <50 bp observed in the gel. One of the <50 bp bands was likely due to the expected primer dimers. Second band <50 bp. NTC could not be clearly discerned in the sample, and the possible source of this additional low molecular weight band could not be determined. In fig. Figure 1 shows examples of gel electrophoretic images from a conventional primer/probe screening assay. Only the hALB primer/probe set 3 was excluded from further qPCR method development due to additional unexpected bands observed in the qPCR products for CAR T, mock T cell, and mock T cell DNA spiked with plasmid samples. For all 3 primer/probe sets, two bands <50 bp were also observed. One of the <50 bp bands was likely due to the expected primer dimers. Second band <50 bp. was not clearly visible in the NTC sample, and the possible source of this additional low molecular weight band could not be determined. However, given this uniform organization of low molecular weight bands, also observed for all primer/probe sets of the transgene, and given that optimizing the number of primers or probe in reactions and testing different annealing temperatures did not lead to the disappearance of the band, it was assumed that this the additional band is a result of the presence of uracil-DNA glycosylases (UNGs) in the main qPCR mixture or a consequence of some other minor derived qPCR products.

The next step in the transgene primer/probe set screening process was to analyze 2 acceptable sets (FP set 1 and RP set 2) in multiplex qPCR reactions with two acceptable hALB primer/probe sets (set 1 and set 2) using a standard curve. A standard curve was prepared by spiking mock T cell DNA with a known concentration of plasmid pLLV-LICAR2SIN and performing five 5-fold serial dilutions of this spiked mock T cell sample using low EDTA TE buffer as the diluent. A sample for each point on the standard curve was obtained and frozen in single-use aliquots. Both sets of transgenic primers/probes were first tested with hALB set 2 in a multiplex qPCR. In addition, CAR T DNA and a mock T cell DNA sample were analyzed to evaluate the specificity of the multiplex reaction. The following are the standard curve criteria that both transgene and hALB targets were expected to meet to be acceptable for further transgenic qPCR assay development: (1) R ² >0.98 and (2) qPCR efficiency 90-110 %. The CAR T DNA was required to demonstrate measurable amplification for both the transgene and hALB targets, whereas the mock T cell DNA sample was required to demonstrate measurable amplification for the hALB target only and no amplification for the transgene target to meet assay specificity requirements.

The multiplex response of transgene (FP) set 1 and hALB set 2 results in acceptable R ² results of >0.98 for both transgene and hALB targets, but none of the standard curves

- 29 046825 for does not result in qPCR efficiency within the acceptable range. The multiplex transgene response (RP) set 2 and hALB set 2 also demonstrated acceptable R ² results of >0.98 for both transgene and hALB targets. The transgene standard curve also resulted in qPCR efficiency within the acceptable range, but this was not observed for the hALB standard curve. Multiplex reactions of transgene (FP) set 1/hALB set 2 and transgene (RP) set 2/hALB set 2 demonstrated acceptable specificity results, with the CAR T DNA sample showing measurable amplification for both targets and mock T cell DNA measurable amplification was observed only for the hALB target, while no amplification was observed for the transgene target. The multiplex qPCR products were also run on a gel to detect the presence of off-target bands when performing a multiplex reaction of the two primer/probe sets (see example gel electrophoresis image in Figure 2). For any of the multiplex reactions, no unexpected bands were observed in the gel results. It was decided to perform a standard curve test in conventional reactions to determine whether reaction multiplexity might influence qPCR efficiency. Given that only the transgene reaction (RP) set 2 efficiency 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 the conventional reaction. Regular transgene reaction (RP) set 2 resulted in a decrease in qPCR efficiency that was outside the acceptable range. The conventional reaction for hALB set 2 resulted in improved qPCR efficiency that was within the acceptable range. Neither attempts to optimize the primer/probe concentrations of both target oligonucleotide sets nor the use of higher annealing temperatures improved the performance of any standard target curve in multiplex reactions. Multiplex reactions were then performed for both acceptable transgene primer/probe sets with hALB primer/probe set 1.

First, a multiplex reaction of transgene (FP) set 1 and hALB set 1 was performed and an R ² >0.98 was obtained for the standard curve of hALB alone. For the transgene target standard curve, R ² was <0.97. Standard curves for both the transgene and hALB targets exhibited efficiencies outside the acceptable range, and the transgene target exhibited lower efficiency than the multiplex reaction with hALB set 2 primers/probes. The conventional hALB set 1 reaction exhibited an R ² >0.98 and resulted to similar efficiency as observed in the multiplex reaction. A new 5-point standard curve was generated using a 4-fold dilution scheme and using less pLLV-LICAR2SIN plasmid and mock T cell DNA in standard #1. This approach was used as part of an attempt to improve qPCR efficiency by possibly diluting any potential inhibitors PCRs that may have been present in the original mock T cell DNA, as well as by reducing the amount of mock T cell DNA needed to produce larger batches and standards. Both the eligible primer/transgene probe sets and the hALB primer/transgene set 1 were subsequently tested in multiplex reactions using this new standard curve. The R ² and efficiency values for both transgene and hALB standard curves were well within the acceptable range for 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 crooked. The multiplex qPCR reaction products were also analyzed on a gel to ensure the absence of off-target bands (see Fig. 3). In all multiplex reactions, non-target bands were absent. Despite similar efficiencies between the multiplex reactions of the two sets of transgene primers/probes, the shape of the transgene target amplification curves for the multiplex transgene reaction (RP) set 2/hALB set 1 was more characteristic of a typical sigmoid curve with a more pronounced upper plateau than for the multiplex reaction transgene (FP) set 1/hALB set 1 (see Fig. 4). Therefore, the primer/probe sets transgene (RP) set 2 and hALB set 1 were selected for further development of the transgene qPCR method.

Eliminate performance reproducibility issues.

The multiplex transgene (RP) set 2/hALB set 1 reaction using the new standard curve design was repeated to determine the reproducibility of acceptable results for R ² and efficiencies. However, the agreement of the standard curve for both transgene and hALB targets for reanalysis was only 88% and 89%, respectively. An attempt to optimize primer/probe concentrations for both targets slightly improved performance for both targets, but experiments with increasing annealing temperatures and the use of PCR enhancers DMSO, TMAC, and betaine did not produce the same results. At the same time, the reproducibility of the efficacy results was investigated, and the question was whether the VCN/cell range covered by the 4-fold dilution standard curve could be increased to lower the possible limit of quantitation of the assay. Therefore, multiplex reactions were performed using five frozen 4-fold dilution standard points, and a standard curve was generated from five 5-fold dilutions of frozen standard No. 1. Subsequently, two standard curves were simultaneously generated in a multiplex qPCR reaction. For standard 4x curve

- 30046825 dilutions obtained efficiencies of 94% and 91% for the transgene and hALB targets, respectively. For the 5-fold dilution standard curve, efficiencies of 102% and 99% were obtained for the transgene and hALB targets, respectively. It was hypothesized that the increase in efficiency observed in the standard curve of the 5-fold dilution compared with the 4-fold dilution could be a consequence of increased variability in the low points of the standard curve when such low points of the standard curve are from frozen samples, compared with the frozen dilution. standard No. 1 to obtain fresh standards No. 2-5 immediately before obtaining the curve during the analysis.

The 4-fold and 5-fold dilution curves were repeated to ensure that the potencies still showed improvement for the 5-fold fresh standard curve compared to the 4-fold frozen standard curve points. When reanalyzed for the 4-fold dilution standard frozen curve, efficiencies of 94% and 88% were obtained for the transgene and hALB targets, respectively. For the standard fresh 5-fold dilution curve, efficiencies of 102% and 99% were obtained for the transgene and hALB targets, respectively. Additional analyzes were performed to further verify the reproducibility of these results by comparing frozen and fresh standard curves (see FIG. 5). It was determined that the primary cause of the variability problems observed for standard curve efficiencies was due to the use of frozen standard curve points. Therefore, it was decided to prepare only standard No. 1 and freeze it in single-use aliquots. This frozen standard No. 1 will subsequently be used to prepare fresh standards No. 2-5 immediately before any analysis. It was also determined that to expand the VCN/cell range, a 5-fold dilution standard curve would be used in future assays.

Eliminating the problems of VCN/cell discrepancies when performing qPCR.

Assays were performed to collect data as well as to prepare at least the first 6 batches of material. The VCN/cell assay used targets of the RU5 promoter regions of the pLLV-LICAR2SIN plasmid framework [Applicants: Is additional more detailed information needed to describe such regions, or is the data provided sufficiently specific for one skilled in the art?]. The transgenic qPCR method is intended to replace this scaffold method because there is a regulatory requirement that the target of the VCN/cell qPCR assay must be the transgenic region of the CAR plasmid for cell therapy. Therefore, it was required that the results of the VCN/cell method be comparable to the results of the transgenic qPCR method. In the transgenic qPCR method, genomic DNA from CAR T was tested and compared with the results for sample LB_12. qPCR was performed on transgenic standards and control samples to determine the accuracy of the established transgene and hALB copy number values. qPCR of the LB_12 DNA sample was also performed to determine the true VCN/cell value.

The qPCR reaction used transgene primers/probes (RP) set 2 and hALB set 1 in the BioRad Supermix for Probes qPCR master mix. The thermal cycling conditions recommended in the Supermix kit were used. As recommended, enzymatic digestion of DNA was necessary to obtain the most accurate qPCR results, so EcoRI was added to the master mixture. It was confirmed that EcoRI only cleaves the pLLVLICAR2SIN plasmid once and does not cleave the amplification site in the transgene or hALB targets. The qPCR results confirmed that the transgenic standards and controls for the transgene and hALB copies were correctly selected, but the result for LB_12 was more comparable to the result for RU5 VCN/cell. It was unclear why the transgenic standards and controls were correct while the qPCR determined that the VCN/cell results from the LB_12 qPCR were inaccurate. Therefore, enzymes that cleave the pLLV-LICAR2SIN plasmid more than once were used, in the belief that smaller DNA fragments for the LB_12 gDNA could provide more accurate results. Two additional enzymes were tried, one with a double cleavage and one with a triple cleavage, but the VCN/cell qPCR results did not change. Therefore, several CAR T DNA samples and two transgenic controls were enzymatically digested, the reaction was stopped, and the transgenic DNA qPCR was performed along with undigested standards, controls, and CAR T samples. The VCN/cell results for the digested controls were ~3.8-fold higher VCN/cell results for uncleaved samples, whereas for digested CAR T samples, VCN/cell results were ∼1.3-fold lower than those for uncleaved CAR T samples. These results questioned the need to linearize the pLLV-LICAR2SIN plasmid to obtain accurate results VCN/sample cage.

Linearization of plasmid pLLV-LICAR2SIN was carried out by digestion of the plasmid using the EcoRI enzyme. Enzymatic digestion was stopped and the linearized plasmid was quantified. The linearized plasmid was diluted to match the transgene copy number in the 5-fold dilution transgene standard curve and transgene qPCR analysis was performed. The analysis was also performed on LB_12 CAR T DNA, and VCN/cell data were calculated from the results for the circular (undigested) and linearized standard curve. The Ct values for the points of the linearized standard curve were ~2 times lower than for the points of the non-expanding

- 31 046825 captive standard curve (see Fig. 6). In addition, the VCN/cell results calculated from the linearized standard curve for LB_12 were comparable to those obtained from the cPCR as well as those obtained from the complementary RU5 qPCR method, whereas the VCN/cell results calculated from the circular (unsplit) standard curve were ~4 times higher. Thus, the need to linearize the pLLV-LICAR2SIN plasmid to obtain accurate VCN/cell results was confirmed. Two batches of standard and control samples of the linearized plasmid were obtained, one large batch for use as a GMP batch for clinical batch finished product control and any other GMP study, and one smaller batch for use in training. analysis specialists and in any non-GMP related activities.

Linearity of a constant amount of DNA inherent in a sample.

The DNA sample was diluted to a concentration of 0.02 μg/mL and qPCR analysis was performed on 5 μL for a total of 100 ng of DNA per reaction. The assay can be performed directly on samples with initial concentrations <0.02 μg/μL, but the acceptable range for hALB copies must be adjusted based on the amount of DNA introduced into the reaction. A standard curve was prepared for an initial concentration of mock T cell DNA of 0.05 μg/μl and diluted with low EDTA TE buffer to obtain a standard curve for both transgene and hALB targets (see Table 3). To ensure that assay linearity remained within an acceptable range when the standard curve was generated at a constant amount of mock T-cell gDNA specific to a given sample, a characterization standard curve was prepared for mock T-cell DNA at a concentration of 0.02 μg/μl and with successive dilutions of 0.02 μg/μl of mock T cell DNA (see Table 4). This standard curve was subsequently analyzed simultaneously with the conventional standard curve to ensure the linearity of the analysis (see Fig. 7). The logarithm of observed copies was also plotted against expected copies to ensure that the measured transgenic copy number results for the characterization standard curve resulted in a linear relationship with R2>0.98 (see FIG. 8).

The characterization standard curve demonstrated that it was still possible to accurately quantify transgene copy numbers at DNA concentrations consistent with typical sample concentrations (0.02 μg/μl).

Table 3

Transgene qPCR standard curve

Standard No. Volume of previous standard (µl) TE buffer volume (µl) Dilution factor Transgene copies Copies of hALB 1 N/A N/A N/A 121212.121 75757.576 2 5 20 5 24242.424 15151.515 3 5 20 5 4848.485 3030.303 4 5 20 5 969.697 606,061 5 5 20 5 193,939 121.212

Table 4

Transgene qPCR standard curve characterization

Standard No. Volume of previous standard (µl) Volume of mock DNA at 0.02 µg/µl (µl) Dilution factor Number of transgenic copies hALB copy number 1 N/A N/A N/A 121212.121 30303.030 2 5 20 5 24242.424 30303.030 3 5 20 5 4848.485 30303.030 4 5 20 5 969.697 30303.030 5 5 20 5 193,939 30303.030

Method certification.

Validation of the transgenic qPCR method was carried out in accordance with the International Conference on Harmonization (ICH) and MIQE guidelines (minimum information requirements for publication of data from real-time quantitative PCR experiments). To validate the method, three analyzes were performed. The analysis performed met the compliance criteria for all method validation parameters established in the method validation protocol. In table Table 5 shows the method certification parameters, compliance criteria and certification results (see example 2).

- 32 046825

Table 5

Summary of data on the certification of the transgenic multiplex qPCR method

Parameter Eligibility Criteria results Reproducibility The % CV of the three VCN/cell measurements for the moderate and weak assay controls for each valid validation assay must be < 30%. Average control (2.00 VCN/cell): 4-6% Weak control (0.20 VCN/cell): 4-6% In-lab reproducibility The % CV of VCN/cell measurements for moderate and weak assay controls for all valid validation assays must be < 30%. Average control (2.00 VCN/cell): 4% Weak control (0.20 VCN/cell): 6% Specificity (mock T cell DNA) All replicate Ct values for mock T cell DNA must be categorized as “undetectable” for the transgene target, and the average hALB copy number must be between 21,212 and 39,394 for each valid validation assay. Transgene target: For each assay, all replicate Ct values were categorized as “not detectable.” hALB target: The average hALB copy number was in the range of 28,719–29,611. Specificity (DNA CAR T) All replicate CAR T DNA measurements must demonstrate a quantifiable transgene result, and for each valid validation assay, the average hALB copy number must be between 21,212 and 39,394. Transgene target: All copy number values were quantifiable and ranged from 4592.801-5153.907. hALB target: The average hALB copy number was in the range of 31,552–33,725. Range (transgene target) Range is defined as the copy number range covered by a 5-point standard curve, provided that the transgene target satisfies all criteria for precision, linearity, and within-laboratory reproducibility. Range: 193,939 - 121,212,121 copies Range (hALB target) Range is defined as the copy number range covered by the 5-point standard curve, provided that the hALB target meets all criteria for precision, linearity, and within-laboratory reproducibility. Range: 121,212-75,757,576 copies LOQ (transgene target) The limit of quantitation (LOQ) is defined as the transgene copy number result for the sample with the lowest LOQ for which a %CV < 20% must be observed for the mean transgene copy number result and mean VCN/cell results, and a % recovery between 70-130 % for the average transgene copy number result and the average VCN/cell result for each valid validation assay. LOQ: The transgene copy number of the LOQ 0.02 VCN/cell sample is 303,030. LOQ (hALB target) The LOQ is defined as the copy number value of standard No. 5, assuming that the hALB target meets all criteria for accuracy, linearity, and within-laboratory reproducibility. LOQ: 121,212 copies

Example 2. Transgenic qPCR method.

1.0. Target.

1.1. This example presents a variant of the quantitative real-time PCR (qPCR) assay procedure for the quantification of the LiCAR plasmid integrated into the CAR T product. The assay is performed in the form of a multiplex qPCR, where the target is the cross-link between the CD137 and CD3z regions of the LiCAR plasmid , as well as human albumin (reference gene).

2.0. Application area.

2.1. This method can be applied to CAR T cells after cell collection immediately prior to dosage formulation preparation to determine the following parameters.

2.1.1. Number of copies of the vector.

2.1.2. Transduction efficiency.

- 33 046825

2.1.3. LiCAR expression identity.

3.0. Definitions and abbreviations.

3.1. LOQ (limit of quantitation).

3.2. NTC (non-matrix 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).

4.0. Equipment.

4.1. Centrifuge capable of centrifuging 96-well PCR plates (eg: Beckman Coulter, Allegra X-14R with SX4750 rotor and rotating 96-well plate holder).

4.2. QuantStudio 6 real-time PCR system.

4.3. Freezer with a temperature of -70°C.

4.4. Freezer with a temperature of -20°C.

4.5. Calibrated 8- or 12-channel pipettes (20, 50 µl or other appropriate volume), for example: Rainin pipettes.

4.6. Calibrated single-channel pipettes (20, 100, 200, 1000 µl or other appropriate volume), for example: Rainin pipettes.

4.7. A refrigerator or cold room capable of maintaining a temperature of 2-8°C.

4.8. QuantStudio PCR software ver. 1.3 or higher.

4.9. Vortex mixer.

4.10. Biological safety cabinet.

5.0. Materials.

Note. Materials with a reservation, for example, can be replaced with similar materials without prior certification. For qualified materials or equivalent, the equivalence of the alternative replacement must be confirmed prior to use for testing samples.

5.1. DNase/RNase-free water, for example: Invitrogen cat. No. 10977015.

5.2. TaqPath ProAmp master mix, ThermoFisher cat. No. A30866 or equivalent.

5.3. Certified batch of BCMA transgene and ALB primers and probes, custom generated sequences from IDT or equivalent.

5.4. Certified batch of transgene BCMA standard No. 1.

5.5. Certified batch of medium and weak control BCMA transgene.

5.6. 1X TE buffer low EDTA, pH 8.0, RNase/DNase free, e.g.: Quality Biological cat. No. 351-324-721.

5.7. Centrifuge tubes 0.5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431005.

5.8. Centrifuge tubes 1.5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431021.

5.9. Centrifuge tubes 2 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431048.

5.10. Centrifuge tubes 5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 0030119460.

5.11. Pipette tips, sterile, with filter (20, 200, 1000 µl or other appropriate volume), for example: Rainin cat. No. 30389226, 30389240, 30398213.

5.12. 96-well PCR plates, Applied Biosystems, cat. No. 4483343, 4483354, 4483349, 4483350, 4483395 or equivalent.

5.13. Micro Amp Optical Adhesive Film, Applied Biosystems, Cat. No. 4311971 or equivalent.

5.14. Containers for RNase/DNase-free reagents, for example: VistaLabs cat. No. 3054-1002.

6.0. Precautionary measures.

6.1. Wear proper PPE when working in the laboratory.

6.2. When working with hazardous chemicals, follow specific instructions for your specific work environment. For more information, see the manufacturer's safety data sheet.

6.3. All steps of pipetting sampling must be carried out using aseptic techniques in a biological safety cabinet. Analysts are advised to use disposable sleeves during all stages of the procedure to minimize the risk of contamination of any materials or reagents.

7.0. Procedure.

7.1. Prepare an aliquot of each of the following components.

7.1.1. Forward primer of the BCMA transgene, working stock solution 10 µM.

7.1.2. Reverse primer transgene BCMA working stock solution 10 µM.

- 34 046825

7.1.3. BCMA transgene probe working stock solution 10 µM.

7.1.4. Forward primer hALB working stock solution 10 µM.

7.1.5. Reverse primer hALB working stock solution 10 µM.

7.1.6. hALB probe working stock solution 10 µM.

7.1.7. Certified No. 1 BCMA transgene standard for qPCR.

7.1.8. Certified average control of 2.00 copies/cell of the BCMA transgene for qPCR.

7.1.9. Certified weak control of 0.20 copies/cell of the BCMA transgene for qPCR.

7.2. Obtain a multiplex master mixture for the appropriate number of reactions in accordance with the table. 6. Additional incremental reactions can be introduced, including more samples than will actually be analyzed. For example, if 10 samples will be analyzed, but more than 10 additional reactions are required, which are already included in the calculations in the table, you should indicate that 11 samples will be analyzed (i.e. N=11). Each additional sample will increase the volume for 3 reactions.

Table 6 Composition of the main mixture

Reagent Volume for N samples (µl)* Final concentration in 25 µl of total reaction TaqPath Pro Amp Master Mix 12.5 x (34 + 3N) IX DNase/RNase-free water 5.625 x (34 + 3N) N/A Transgene forward primer (10 µM) 0.25 x (34 + 3N) 100 nM Transgene reverse primer (10 µM) 0.25 x (34 + 3N) 100 nM FAM transgene probe (10 µM) 0.5 x (34 + 3N) 200 nM hALB forward primer (10 µM) 0.1875 x (34+ 3N) 75 nM Reverse primer hALB (10 µM) 0.1875 x (34+ 3N) 75 nM hALB HEX probe (10 µM) 0.5 x (34 + 3N) 200 nM

* The formula is the volume of component required for one reaction of 25 µl, multiplied by the sum of 24 wells of standards/controls plus 10 additional reactions (34) and 3* number of samples (3 reaction wells per sample).

7.3. Mix the test tube with the main mixture in a vortex mixer for a short time and set aside.

7.4. Prepare standards No. 2-5 using TE buffer with low EDTA content in accordance with table. 7. Be sure to stir each diluted sample briefly before moving on to the next dilution step.

Table 7

Construction of a standard curve for a 5-fold dilution using 5 points

Standard No. Volume of previous standard (µl) TE buffer volume (µl) Dilution ratio Transgene copies Copies of hALB 1 N/A N/A N/A 121212.121 75757.576 2 5 20 5 24242.424 15151.515 3 5 20 5 4848.485 3030.303 4 5 20 5 969.697 606,061 5 5 20 5 193,939 121.212

7.5. Stir the main mixture solution again for a short time in a vortex mixer and pipet the mixture into the reagent reservoir.

7.6. Using a multichannel pipette, transfer 20 μl of the master mixture into the appropriate wells of a 96-well PCR plate (see Fig. 9).

7.7. Load 5 µl of standards, controls and DNA sample into the appropriate wells of a 96-well PCR plate using a single channel pipette according to the location of the areas in the plate in Fig. 9. Load 5 µl of low EDTA TE buffer into NTC wells.

7.8. Seal the plate with optical adhesive film and briefly centrifuge at ~300xg.

7.9. Install the PCR plate in the PCR device.

7.10. Open the Assay Template.edt file, select Save As, enter an appropriate name for the qPCR experiment and save as an .eds file. Do not overwrite the template file.

7.11. Enter a name for the experiment in the name field.

7.12. Check that the Experiment Properties are set as below and the conditions

- 35 046825 thermal cycling (Run Method tab) are set correctly in accordance with those given in the table. 8 for a reaction volume of 25 µl.

7.12.1. Device type: QuantStudio 6 Flex System.

7.12.2. Plate type: 96-well (0.2 ml).

7.12.3. Experiment type: standard curve.

7.12.4. Detection reagent: TaqMan Reagents.

7.12.5. Device properties: standard.

Table 8

Thermal cycling conditions

Stage 1 Stage 2 Stage 3 (40 cycles) 50°C 95°C 95°C 60°C 2 minutes 10 min 15 sec 1 min Activation UNG Activation of polymerase Denaturation/melting Annealing/extension

7.13. Select launch and click on the serial number of the device for analysis.

8.0. Data analysis.

8.1. Use the automatic baseline correction and automatic Ct function in the data analysis software by clicking Analyze. Then click Save to save the analysis results.

8.2. Print the report in PDF format and include it in your analysis documentation.

8.3. Calculate three VCN/cell measurements for each sample and positive control as follows.

_TL ™ _T/ /Amount of transgene\ „

VCN/cell = ...................................x ......j, /

X Number of hALB ?

8.4. Calculate the mean, standard deviation, and %CV of three VCN/cell measurements for each sample and positive control.

9.0. Eligibility criteria for analysis.

9.1. Eligibility criteria for analysis.

9.1.1. The R2 value of the standard curves for the transgene and hALB should be >0.97.

9.1.2. The slope of the standard curve should be between -3.585 and -3.104 (equivalent to PCR efficiency of 90.08-109.97%).

9.1.3. No repeated measurement Ct value for any of the standards can be classified as not determined.

9.1.4. All Ct values of repeated NTC measurements should be categorized as not detectable for both transgene and hALB targets.

9.1.5. The average Ct value for standard #1 should be <23.0 for the transgene target and <22.0 for the hALB target.

9.1.6. SD values for Ct for each standard should be <0.60 for both transgene and hALB targets.

9.1.7. The average hALB copy number for moderate and weak controls should be 30,303,030 copies +/-30% (expected range: 21,212,121-39,393,939 copies).

9.1.8. The average VCN/cell result for a medium control of 2.00 VCN/cell and a weak control of 0.20 VCN/cell should be +/-35% of the target VCN/cell for each control.

9.1.9. The %CV for duplicate measurements of medium and weak positive controls VCN/cell should be <20%.

9.1.10. If any of the above criteria are not met, the analysis is invalid.

9.2. Eligibility criteria for the sample.

9.2.1. The average hALB copy number 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 hDNA sample concentration is <0.02 μg/mL, calculate the expected hALB copy number for that sample based on the amount of DNA actually introduced into the reaction.

Example. The gDNA sample concentration is 0.01 μg/ml. (5 µl)(0.01 µg/µl) =0.05 µg =50 ng gDNA sample per reaction (50 ng DNA)(1 copy ALB / 0.0033 ng gDNA) =15,152 copies ALB. Expected range: 10,606-19,697 ALB copies.

9.2.2. The hALB target copy values from three measurements for the sample must be within the Ct range covered by the hALB standard curve. The Ct range is defined as the smallest Ct value of the three measurements of standard No. 1 and the largest Ct value of the three measurements of standard No. 5.

9.2.3. Transgene target copy values across three sample dimensions must be above the LOQ co-

- 36,046,825 pius of transgene 303,030.

9.2.3.1. If 1 or more replicate measurements of a sample for a transgene target are less than the transgene's LOQ of 303,030 copies, the sample should be reported as being below the LOQ.

9.2.4. If 1 or more replicate measurements of the sample for the transgene target are less than the lowest transgene Ct value for standard #1, the sample should be reported as being above the standard curve range and the sample cannot be quantified. For example, if the transgene Ct values for a sample are 20.1, 19.9, and 20.2, but the lowest transgene Ct value achieved in standard #1 is only 20.0, then remeasuring the 19.9 sample may not be accurate quantified, and therefore a given sample must be labeled as above the range of the standard curve, the sample cannot be quantified. If it is impossible to quantify the sample, notify management and the person responsible for the study.

9.2.5. The %CV of duplicate VCN measurements/sample cell should be <20%.

Note. %CV is not estimated for samples with duplicate measurements below the LOQ or for samples that cannot be quantified.

9.2.6. For any sample that has all transgene copy number values for all three replicate measurements above the LOQ and meets all eligibility criteria above, average VCN/cell values to 2 decimal places will be reported (for example: 2.02 VCN/cell).

9.2.7. Any sample that does not meet all of the eligibility criteria listed above is invalid.

Isolation, validation and dilution of genomic DNA.

1.0. Target.

1.1 An example procedure for extracting and quantifying gDNA for CAR T samples or mock T cell suspensions or frozen cell conglomerates is described.

2.0. Application area.

2.1. A procedure is described for isolating gDNA from:

2.1.1. CAR T cell samples after collection, supplied as frozen cell conglomerates or fresh cell suspension.

2.1.2. Mock T cells supplied as frozen cell conglomerates or fresh cell suspension.

3.0. Equipment.

3.1. Centrifuge capable of centrifuging 1.5 ml and 5 ml microcentrifuge tubes (for example: Beckman Coulter, Allegra X-14R with SX4750 rotor for 1.5 ml microcentrifuge tubes).

3.2. Fluorimeter Qubit 4, Invitrogen cat. No. Q33226.

3.3. Freezer with a temperature of -70°C.

3.4. Freezer with a temperature of -20°C.

3.5. Calibrated single-channel pipettes (20, 100, 200, 1000 µl or other appropriate volume), for example: Rainin pipettes.

3.6. Thermoblock capable of maintaining a temperature of 55°C and suitable for 1.5 ml and 2 ml microcentrifuge tubes.

3.7. A refrigerator or cold room capable of maintaining a temperature of 2-8°C.

3.8. Vortex mixer.

3.9. Biological safety cabinet.

4.0. Materials.

Note. Materials with a reservation, for example, can be replaced with similar materials without prior certification. For qualified materials or equivalent, the equivalence of the alternative replacement should be confirmed prior to use for testing samples.

4.1. DNase/RNase-free water, for example: Invitrogen cat. No. 10977015.

4.2. RPMI 1640 medium with L-glutamine and 25 mM HEPES, for example: Corning cat. No. 10-041-CV.

4.3. 1X TE buffer low EDTA, pH 8.0, RNase/DNase free, for molecular biology, e.g.: Quality Biological cat. No. 351-324-721.

4.4. Ethanol 200 alcohol degrees (96-100%) for molecular biology, for example: Decon Labs cat. No. 3616EA.

4.5. 10X phosphate buffered saline (PBS) for molecular biology, e.g.: Affymetirx cat. No. 75889.

4.6. Mini kit for genomic DNA PureLink, Invitrogen cat. No. K182001.

4.7. Analytical tubes Qubit™, Invitrogen™ cat. No. Q32856 (Invitrogen, Carlsbad, California).

4.8. Qubit dsDNA BR test kit, Invitrogen cat. No. Q328350.

4.9. Centrifuge tubes 0.5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431005.

4.10. Centrifuge tubes 1.5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No.

- 37 046825

022431021.

4.11. Centrifuge tubes 2 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No.

022431048.

4.12. Centrifuge tubes 5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No.

0030119460.

4.13. 15 ml conical tubes, sterile, e.g.: Corning cat. No. 431470.

4.14. Pipette tips, sterile, with filter (20, 200, 1000 µl or other appropriate volume), for example: Rainin cat. No. 30389226, 30389240, 30398213.

5.0. Precautionary measures.

5.1. Wear proper PPE when working in the laboratory.

5.2. When working with hazardous chemicals, follow specific instructions for your specific work environment. For more information, see the manufacturer's safety data sheet.

5.2. All steps of pipetting sampling must be carried out using aseptic techniques in a biological safety cabinet. Analysts are advised to use disposable sleeves during all stages of the procedure to minimize the risk of contamination of any materials or reagents.

6.0. Procedure.

6.1. When opening a new PureLink Genomic DNA Mini Kit, be sure to add ethanol to Wash Buffer 1 and Wash Buffer 2 according to the instructions on each bottle's label. Stir the contents of the bottles well after adding ethanol and indicate on each label that ethanol has already been added. Along with the note about the addition of ethanol, indicate the initials and date of addition. When all components are stored at room temperature, the kit is stable for 1 g.

6.2. Set the thermoblock temperature to 55°C and wait until the set temperature is reached before starting DNA extraction.

6.3. DNA extraction is carried out using cell conglomerates. Fresh cell conglomerates or cell conglomerates stored frozen at -70°C can be used. A minimum extraction rate of 2x106 cells per column is recommended, but up to 4x106 viable cells per column can be extracted.

Note. Although the manufacturer's specification states that up to 5x106 cells can be extracted per column, this method uses the number of viable cells to determine the number of cells for DNA extraction. Therefore, a 20% buffer is used to allow for the presence of dead cells, which will also be contained in the cell suspension. The specified level of 4x106 viable cells per column should not be exceeded. If the percentage viability is less than 80%, the maximum number of viable cells extracted per column should be reduced to account for the >20% dead cells present in the cell suspension.

6.4. Preparation of cell conglomerates from fresh cell suspension.

6.4.1. To count cells on the NC-200, an aliquot of the cell suspension of at least 150 µl is required. An aliquot can be prepared by diluting the original cell suspension in RPMI medium to the volume required to remain within the dynamic range of NC-200 (5.0 x 104 to 5.0 x 106 cells/mL).

6.4.2. To count cells, use the counting protocol.

6.4.3. Based on the number of viable cells, determine the volume of cells required to achieve the desired number of cells for extraction on each PureLink column and withdraw appropriate aliquots of the cell suspension into 1.5 mL or 2 mL microcentrifuge tubes.

For example: the number of viable cells according to the results of the NC-200 count is 2x107 cells/ml with a viability percentage of 88%. The desired number of cells for extraction onto a PureLink column is 4x106 viable cells:

, . ,. ζ 1 ml \ _ _ (cells “· ^:Ί θΖ ml \ cells 2()eo/

Place a 200 µl aliquot of the cell suspension into 1.5 ml and 2 ml microcentrifuge tubes.

6.4.3.1. It is not recommended to use an aliquot of less than 100 µl of cells per microcentrifuge tube. If necessary, dilute cells in RPMI medium to a number of cells that will allow a minimum aliquot of 100 μl of cell suspension per tube to be collected.

6.4.4. Centrifuge the tubes at 300xg for 5 min at room temperature to form a cell conglomerate.

6.4.5. Carefully remove and discard the medium, separating the cell conglomerate(s).

6.4.6. The cell conglomerate(s) can be used immediately for DNA extraction or stored at -70°C.

6.5. DNA extraction.

6.5.1. If extracting from frozen cell conglomerates, thaw the cell conglomerates at room temperature before proceeding with the DNA extraction procedure.

- 38 046825

6.5.2. Dilute 10X PBS buffer to 1X using RNase/DNase-free water. For each cell conglomerate, use 200 µl of 1X PBS. Prepare enough 1X PBS to resuspend the total number of cells to be extracted.

6.5.3. Resuspend each cell conglomerate in 200 µl 1X PBS. Mix by pipetting to ensure complete resuspension of the cell conglomerate.

6.5.4. Add 20 µl of proteinase K to each tube and vortex to mix briefly.

6.5.5. Add 20 µl of RNase A to each tube and vortex to mix briefly.

6.5.6. Incubate the tubes for 2 minutes at room temperature.

6.5.7. Add 200 µl of PureLink Genome Lysis/Binding Buffer to each tube and shake briefly to obtain a homogeneous solution.

6.5.8. Place the tubes in a thermoblock preheated to 55°C and incubate for 10 minutes.

6.5.9. After incubation is complete, remove the tubes from the thermoblock and add 200 µl of 96-100% ethanol to each tube. Mix on a vortex mixer for a short time to form a homogeneous solution. Note. During incubation at 55°C, condensation accumulates on the lid of the tubes. When opening tubes, care should be taken not to allow the contents to splash from the cap.

6.5.10. Install one PureLink spin column in each collection tube. Label the cap of each spin column with a specific sample identifier to ensure that samples are not accidentally mixed up. For example, spin columns may be labeled with a sample lot number. Collection tubes should not be labeled because collection tubes are periodically discarded during the extraction protocol.

6.5.11. Apply the contents of each sample tube after step 5.5.9 to an appropriately labeled spin column.

6.5.12. Centrifuge the column(s) at 10,000xg for 1 min at room temperature. During centrifugation, prepare new, clean collection tubes for each sample.

6.5.13. After centrifugation, remove the centrifuge columns/collection tubes from the centrifuge. Transfer each spin column to a new, clean collection tube and discard the used collection tube along with the coloquine filtrate.

6.5.14. Apply 500 µl of Wash Buffer 1 to each spin column.

6.5.15. Centrifuge the column(s) at 10,000xg for 1 min at room temperature. During centrifugation, prepare new, clean collection tubes for each sample.

6.5.16. After centrifugation, remove the centrifuge columns/collection tubes from the centrifuge. Transfer each spin column to a new, clean collection tube and discard the used collection tube along with the column filtrate.

6.5.17. Apply 500 µl of Wash Buffer 2 to each spin column.

6.5.18. Centrifuge the column(s) at maximum speed for 3 min at room temperature. During centrifugation, prepare one 2 ml microcentrifuge tube for each sample.

6.5.19. After centrifugation, remove the centrifuge column(s)/collection tube(s) from the centrifuge. Transfer each spin column to a 2 mL microcentrifuge tube and discard the spent collection tube along with the column filtrate. Note. Do not use the collection tubes supplied in the PureLink kit for step 5.5.19. The kit does not provide additional tubes for the additional centrifugation step, so 2 ml microcentrifuge tubes must be used.

6.5.20. Centrifuge the column(s) at maximum speed for 2 min at room temperature to dry the columns. During centrifugation, prepare one 1.5 ml microcentrifuge tube for each sample. Label each tube with at least the name of the sample and the date of extraction. These are the tubes in which the DNA will be eluted.

6.5.21. After centrifugation, remove the centrifuge column(s)/tube(s) - collection(s) from the centrifuge. Transfer each spin column to a 1.5 mL microcentrifuge tube with appropriate labels and discard the 2 mL microcentrifuge tube along with the column filtrate.

6.5.22. Apply 25 µl of PureLink Genomic Elution Buffer to the columns. Make sure that the elution buffer is applied to the silica membrane, but do not touch the membrane or pierce it with the pipette tip.

6.5.23. Incubate the columns for 1 min at room temperature.

6.5.24. Centrifuge the column(s)/tube(s) at maximum speed for 1 min at

- 39 046825 room temperature for DNA elution.

6.5.25. Remove column(s)/tube(s) from the centrifuge and repeat steps 5.5.22-5.5.23 with an additional 25 µl of PureLink genomic elution buffer.

6.5.26. After incubation, centrifuge the column(s)/tube(s) at maximum speed for 1.5 min at room temperature to elute additional amounts of DNA.

6.5.27. Remove the column(s)/tube(s) from the centrifuge. Remove the column from the 1.5 mL tube and discard the column.

6.5.28. The eluted DNA can either be stored at -20°C or immediately quantitated and diluted to a working concentration. Note. DNA should not be quantified unless it is immediately diluted to a working concentration after quantitation. If samples have been quantified but cannot be immediately diluted to the qPCR working concentration, the samples should be frozen at -20°C and will need to be requantitated after thawing before diluting to the qPCR working concentration.

6.6. DNA quantification.

6.6.1 DNA quantification is performed using the Qubit dsDNA Broad Range Kit and the Qubit 4 Fluorimeter. The kit is highly selective for double-stranded DNA (dsDNA) over RNA and is designed to provide high accuracy for initial sample concentrations of 100 pg/µl-1000 ng/µl All handling of kit components must be carried out in a biosafety cabinet and under aseptic conditions to avoid contamination of any kit components.

Note. Qubit dsDNA BR reagent contains DMSO and will freeze at temperatures below room temperature. Repeated freeze-thaw cycles of Qubit reagent should be avoided and the reagent should be stored at room temperature. Qubit Buffer is formulated to be stored at room temperature and is recommended to use these storage conditions. Qubit standards should be stored at 2-8°C.

6.6.2. The Qubit 4 Fluorometer is calibrated using two standards provided in the Qubit dsDNA Broad Range Kit. Standards must be prepared and analyzed along with each set of DNA samples to be quantified. Under no circumstances should a calibration be reused from a previous analysis as the most accurate quantitation is achieved when standards and DNA samples are prepared using the same Qubit working solution.

6.6.3. Label 1 Qubit assay tube for each standard and sample to be quantified. The mark is applied only to the caps of the test tubes. Do not apply a label to the side of the tubes as this will interfere with quantitation.

Note. Only Qubit test tubes can be used with the Qubit Fluorimeter. These tubes are specially designed to provide the most accurate results.

6.6.4. Prepare Qubit working solution by diluting Qubit dsDNA BR reagent in a ratio of 1:200 in Qubit dsDNA BR buffer. Ensure that sufficient working solution is prepared to cover both the standards and all samples to be quantified. The minimum required volume is (2 (2 standards) + number of samples to be quantified +1) * 200.

For example: To quantify 8 samples, prepare enough sample solution and 2 standards plus at least one additional sample for refilling. Assume 200 µl working reagent per tube in 11 tubes (8+2+1=11 samples): (200 µl)(11 tubes) = 2200 ml Qubit working solution (11 µl Qubit reagent plus 2189 µl Qubit buffer).

6.6.5. Add 190 µl of Qubit working solution to each of the 2 standard tubes. Pre-wet the pipette tip with Qubit working solution before adding the solution to the tubes to prevent introducing bubbles into the reaction mixture.

6.6.6. 3-20 µl of DNA sample can be used for quantitation using the Qubit dsDNA Broad Range kit with a total assay tube volume of 200 µl. Add the required volume of Qubit working solution to each assay tube. For example: When using 3 µL of sample for quantitation, add 197 µL of Qubit working solution to the sample tube. Pre-wet the pipette tip with Qubit working solution before adding the solution to the tubes.

6.6.7. Add 10 µl of each standard to the appropriate standard tube. Mix the contents of the test tube with the standard in a vortex mixer for a short time.

6.6.8. Add the required amount of DNA stock to the appropriate sample tube to bring the total reaction volume to 200 µl. For example: 3 µl of DNA sample into a sample reaction tube containing 197 µl of Qubit working solution. Mix the contents

- 40 046825 press each sample tube in a vortex mixer for a short time.

6.6.9. Incubate all tubes with standard and sample for 2 minutes at room temperature.

6.6.10. First calibrate the Qubit 4 fluorometer using standards

6.6.10.1. Touch the fluorimeter screen to wake the device from standby mode.

6.6.10.2. Select the dsDNA option on the initial screen.

6.6.10.3. On the next screen select dsDNA: Broad range.

6.6.10.4. The fluorimeter screen will ask you to choose between reading new standards or using the previous calibration. You should always select Read Standards. Note. Under no circumstances should you select to read samples using previous calibration (Run samples option) as this will not provide the most accurate sample concentration.

6.6.10.5. When prompted, install sample standard #1 in Qubit. Close the sample chamber lid. Select Read Standard to read standard No. 1.

6.6.10.6. When prompted, remove standard No. 1 from the sample chamber and install standard No. 2. Close the sample chamber lid and select Read standard to read standard No. 2.

6.6.10.7. If the calibration is successful, the Qubit screen will display the calibration results after reading standard #2 is completed. If the calibration is unsuccessful, the Qubit screen will display Calibration error.

6.6.10.8. It should be ensured that the reading obtained for standard No. 2 is at least 10 times higher than the reading obtained for standard No. 1.

6.6.10.9. If a calibration error is displayed or if standard #2 is not 10 times larger than standard #1, repeat sample and standard preparation using fresh Qubit working solution. You cannot reuse a used tube in which the previous Qubit working solution was prepared. Repeat calibration with fresh standards.

6.6.10.9.1. If calibration is successful and the reading for standard #2 is at least 10 times greater than that for standard #1, continue reading samples (see steps 5.6.9.10-5.6.9.14).

6.6.10.9.2. If the calibration error reoccurs, or if standard #2 is not 10 times larger than standard #1, contact your test specialist or supervisor.

6.6.10.10. After successful calibration, select Run samples at the bottom of the Standards results screen to begin analyzing the samples.

6.6.10.11. Before the first sample is analyzed, sample volume information is displayed on the screen. Use the + or - symbols to select the sample volume used for all samples analyzed (3-20 µl). Then select the units (μg/μL) to display the sample concentration from the drop-down menu.

6.6.10.12. After selecting the correct sample volume and sample concentration units, sample 1 is installed in the sample chamber. Close the sample chamber lid and select Read tube.

6.6.10.13. The screen displays both the original calculated sample concentration and the sample concentration in the Qubit tube. The initial calculated sample concentration corresponds to the concentration in the original DNA sample.

6.6.10.13.1. If the sample concentration is outside the dial range, an Out of Range error message is displayed on the screen.

6.6.10.13.2. Click the right arrow to open the standard graph and sample results to determine if the sample results are too high or too low.

6.6.10.13.3. Samples out of range should be reanalyzed. Use a larger sample volume for sample concentrations found to be too low. Use either a smaller sample volume or a dilution of the DNA stock solution (prepared in low EDTA TE buffer) for samples whose concentrations are too high.

Re-examined samples must be analyzed against new standards. Samples and standards must be prepared using fresh Qubit working solution (do not reuse tubes from previous Qubit working solution).

6.6.10.14. Remove sample 1 from the sample chamber. If more than one sample needs to be analyzed, place the next sample in the sample chamber, close the sample chamber lid, and select Read tube.

6.6.10.15. Continue repeating 5.6.9.14 until all samples have been analyzed.

6.6.10.16. Connect Qubit to your computer using the USB cable supplied with the Qubit fluorimeter, and when the AutoPlay window appears, select Open device to view files. Continue from stage 5.6.9.17 for Qubit before making any further selection of options on the computer.

6.6.10.17. Select Data on the Sample concentration screen for the last sample read or on the Home screen to open the Export data window, which displays a list of analyzes performed on the Qubit. The data list includes listings according to the analyzes performed, reflecting the date/time of the analysis, the name of the analysis (dsDNA Broad Range) and the number of samples analyzed in each one

- 41 046825 analysis.

Note. The Qubit 4 fluorimeter stores data for up to 1000 samples.

6.6.10.18. Tap the box next to the analysis data that is to be exported. A check mark will appear in the window. Then select Export to export the entire data set to your computer.

6.6.10.19. On your computer, double-click on Internal storage and then on the Qubit 4 folder to access the exported data.

Note. The folder name looks like QubitData_day-month-year, and the date will correspond to the export date, not the date the exported data was analyzed.

6.6.10.20. Open the data folder and save the file QubitData_day-month-year_minutes-hoursseconds.csv in a secure data backup system (for example: OpenLab) or in accordance with the procedure adopted at the institution. In accordance with institutional procedures, this file must also be attached to the analysis documentation. Note. The folder also contains the QubitData_Rna_iq_day-month-year_minutes-hours-seconds.csv file, which is specific to the RNA IQ assay.

This .csv file contains values only for RNA IQ analysis and should not be saved for other purposes.

6.6.10.21. The .csv file contains the results of the analysis. The results will be presented in the reverse order of the samples read (ie from the last sample read to the first sample read). The Test Date column also indicates the time the sample was read and can be used to confirm the order of the samples, since samples analyzed first will have an earlier timestamp, while samples analyzed last will have a later time stamp.

6.6.10.22. The Original sample concentration value refers to the concentration of the original DNA solution. If the DNA stock solution is diluted as part of a Qubit assay, the Original sample concentration value will need to be multiplied by the dilution factor for the DNA stock solution dilution that is used in the Qubit assay to determine the DNA stock solution concentration. For example: in the Qubit analysis, a DNA stock solution is used in a 1:10 dilution in TE buffer with low EDTA content and 3 μl of a 1:10 dilution, the Original sample concentration value according to Qubit is 0.0561 μg/μl, then the concentration of the undiluted DNA stock solution is (0.0561 µg/µl)(10) =0.561 µg/µl.

6.7. DNA Sample Dilution for Transgenic qPCR Assay Preparation 6.7.1.1 Samples should be diluted to 0.020 µg/µl in low EDTA TE buffer. The DNA stock solution is immediately quantified. Aliquots of samples with DNA stock concentrations less than 0.020 µg/µl should be selected according to step 5.7.1.5 and immediately used without further manipulation (as is) in the qPCR assay.

6.7. 1.2. Use the following formula to determine the total volume of 0.020 µg/µl DNA dilution that can be prepared:

„ „ Volume of the initial DNA solution x Concentration of the initial DNA solution

The total volume of diluted DNA is 0.020 µg/µl ~ .............................................. ............................................7".... ........................................................ ...................

0.020 µg/µl

6.7.1.3. Then determine the amount of low EDTA TE buffer required to dilute the DNA stock solution to 0.020 µg/µl as follows:

Total volume of diluted DNA 0.02 µg/µl - Volume of original DNA solution - Volume of TE buffer

6.7.1.4. Dilute the desired volume of DNA stock solution with the calculated volume of low EDTA TE buffer calculated in 5.7.1.3 to prepare a working DNA concentration of 0.020 µg/µl. It is recommended to dilute a significant portion of the DNA stock solution to a qPCR working concentration of 0.020 μg/μL to prepare as many single aliquots of the sample as possible. However, to provide sufficient quantities for a minimum of 3 qPCR assays, a minimum of 3 single-use aliquots of 0.020 μg/μL DNA sample must be prepared.

For example: the concentration of the original DNA solution is 0.0561 μg/μl. After using 3 µl of DNA for the Qubit assay, a minimum of 47 µl of DNA stock solution remains. 40 µl of DNA stock solution will be used to dilute to a qPCR working concentration of 0.020 µg/µl.

µl 0.0561 µg/µl

Total volume of diluted DNA 0.020 µg/µl -

ITotal volume of diluted DNA 0.020 µg/µl -112.2 µl

112.2 µl - 40 µl - TE buffer volume

TE buffer volume - 72.2 µl

Dilute 40 µL DNA stock solution in 72.2 µL TE buffer low EDTA to obtain 112.2 µL total DNA sample volume of 0.020 µg/µL qPCR working stock solution.

6.7.1.5. Prepare as many single 20 µL aliquots of the 0.020 µg/µL DNA sample working stock solution as possible. Each qPCR assay uses 15 μl of DNA, so each aliquot will have ~5 μl of excess DNA. Whenever possible, it is recommended to prepare at least 3 single-use aliquots.

6.7.1.6. All aliquots must be labeled with, at a minimum, the name of the sample, the concentration of the aliquot (either 0.020 μg/μL or the concentration of the stock solution if the concentration is less than 0.020 μg/μL), and the date the aliquots were received.

6.7.1.7. All aliquots, as well as any remaining DNA sample stock solutions, should be stored at -20°C.

Preparation and certification of new batches of oligonucleotides.

1.0. Target.

1.1. An example of the procedure for obtaining and certifying new batches of oligonucleotides for the transgenic qPCR method is described.

2.0. Equipment.

2.1. Centrifuge capable of centrifuging 1.5 mL microcentrifuge tubes (for example: Beckman Coulter, Allegra X-14R with SX4750 rotor and rotating 96-well plate holder and adapters for 1.5 mL microcentrifuge tubes).

2.2. QuantStudio 6 real-time PCR system.

2.3. Freezer with a temperature of -20°C.

2.4. Calibrated 8- or 12-channel pipettes (20, 50 µl or other appropriate volume), for example: Rainin pipettes.

2.5. Calibrated single-channel pipettes (20, 100, 200, 1000 µl or other appropriate volume), for example: Rainin pipettes.

2.6. A refrigerator or cold room capable of maintaining a temperature of 2-8°C.

2.7. QuantStudio PCR software ver. 1.3 or higher.

2.8. Thermoblock capable of maintaining a temperature of 55°C and suitable for 1.5 ml microcentrifuge tubes.

2.9. Vortex mixer.

3.0. Materials.

Note. Materials with a reservation, for example, can be replaced with similar materials without prior certification. For qualified materials or equivalent, the equivalence of the alternative replacement should be confirmed prior to use for testing samples.

3.1. DNase/RNase-free water, for example: Invitrogen cat. No. 10977015.

3.2. TaqPath ProAmp master mix, ThermoFisher cat. No. A30866 or equivalent.

3.3. Lyophilized BCMA and hALB transgene primer and probe stocks, custom generated sequences from IDT or equivalent.

3.4. Certified batch of BCMA transgene and ALB primers and probes, custom generated sequences from IDT or equivalent.

3.5. Certified batch of transgene BCMA standard No. 1.

3.6. Certified batch of medium and weak control BCMA transgene.

3.7. 1X TE buffer low EDTA, pH 8.0, RNase/DNase free, e.g.: Quality Biological cat. No. 351-324-721.

3.8 Centrifuge tubes 0.5 ml, sterile, RNase/DNase-free, e.g. microcentrifuge tubes with screw cap VWR cat. No. 89004-286 or Eppendorf cat. No. 022431005.

3.9. Centrifuge tubes 1.5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431021.

3.10. Centrifuge tubes 2 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431048.

3.11. Centrifuge tubes 5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 0030119460.

3.12. Pipette tips, sterile, with filter (20, 200, 1000 µl or other appropriate volume), for example: Rainin cat. No. 30389226, 30389240, 30398213.

3.13. 96-well PCR plates, Applied Biosystems, cat. No. 4483343, 4483354, 4483349, 4483350, 4483395 or equivalent.

3.14. Micro Amp Optical Adhesive Film, Applied Biosystems, Cat. No. 4311971 or equivalent.

3.15. Containers for RNase/DNase-free reagents, for example: VistaLabs cat. No. 3054-1002.

4.0. Precautionary measures.

4.1. Wear proper PPE when working in the laboratory.

4.2. When working with hazardous chemicals, follow specific instructions for your specific work environment. For more information, see the manufacturer's safety data sheet.

4.3. All stages of pipetting sampling must be carried out using aseptic

- 43 046825 methods in a biological safety cabinet. Analysts are advised to use disposable sleeves during all stages of the procedure to minimize the risk of contamination of any materials or reagents.

6.0. Procedure.

Note. Oligonucleotides are validated as a multiplex set (BCMA and hALB transgene). A batch of oligonucleotides is considered consumed when the last aliquot of any of the 6 oligonucleotides has been used. Oligonucleotides from one validated kit should not be mixed or combined with another. Any remaining oligonucleotides from a previously validated kit that has been used up must be discarded.

6.1. Order BCMA transgene oligonucleotides and hALB oligonucleotides (see sequences in Table 9).

Table 9 Transgenic qPCR oligonucleotides

Oligonucleotide name Sequence (5’-3’) Order quantity (minimum) Cleaning DIRECT BCMA transgene CCA GTA CAA ACT ACT CAA GAG G 25 nmol DNA oligonucleotides Standard desalting REVERSE BCMA transgene GCT GAA CTT CAC TCT CAG TT 25 nmol DNA oligonucleotides Standard desalting BCMA transgene PROBE /56-FAM/TC TTC TGG A/ZE/A ATC GGC AGC TAC AGC GIABkFQ/ 250 nmol PrimeTime 5’6-FAM/ZEN/3’ IB FQ HPLC DIRECT hALB TCA TCT CTT GTG GGC TGT AAT C 25 nmol DNA oligonucleotides Standard desalting REVERSE hALB TGC TGG TTC TCT TTC ACT GAC 25 nmol DNA oligonucleotides Standard desalting hALB PROBE /5HEX/AG GGA GAG Α/ΖΕΝ/Τ TTG TGT GGG CAT GAC /3IABkFQ/ 250 nmol PrimeTime 5’6-HEX/ZEN/3’ IB FQ HPLC

6.2. Oligonucleotides will be supplied by the manufacturer in lyophilized form along with specifications for each oligonucleotide. Store lyophilized oligonucleotides at -20°C until ready for reconstitution. Lyophilized oligonucleotides can be stored at -20°C for up to 12 months. Note. On average, it takes at least 2 weeks to receive oligonucleotides from the manufacturer. Therefore, at least one reserve lyophilized set of oligonucleotides should always be kept to minimize the impact on sample testing if problems arise with a new or validated batch of samples.

6.3. Dilution of oligonucleotides.

6.3.1. Remove lyophilized oligonucleotide stocks from storage at -20°C and centrifuge at 10,000xg for 30 seconds to ensure all oligonucleotide stocks are at the bottom of the tubes before opening the tubes. Tubes should not be opened prior to centrifugation to prevent loss of any oligonucleotide that has migrated from the bottom of the tube during transport.

6.3.2. Dilute each oligonucleotide to 100 µM stock solution using low EDTA TE buffer as follows.

6.3.2.1. Determine the nanomolar amount of each oligonucleotide specified in the individual nucleotide specification.

6.3.2.2. Multiply the number of nanomoles by 10 to obtain the volume of TE buffer that must be added to the oligonucleotide tube to obtain a 100 µM stock solution.

6.3.2.3. Add the volume of TE buffer calculated in step 11.3.2.2 to each oligonucleotide in the tube containing the corresponding oligonucleotide.

6.3.2.4. Vortex the contents of each oligonucleotide tube briefly to completely resuspend the lyophilized oligonucleotide in TE buffer.

6.3.2.5. Check the primer tubes to ensure that all lyophilized oligonucleotide is completely dissolved in TE buffer. If it appears that there are any oligonucleotide particles in the mixture that are not completely dissolved in TE buffer, the tubes can be kept at 55°C for 1-5 minutes to speed up resuspension. After heating, the contents of the tubes are mixed again in a vortex mixer for a short time.

6.3.3. Diluting the stock solutions with 100 μM to the concentrations of the working oligonucleotide stock solution.

- 44 046825

6.3.3.1. Dilute 100 µM primer stock solutions to 10 µM working stock solutions using low EDTA TE buffer.

6.3.3.2. Dilute 100 µM probe stock solutions to 10 µM working stock solutions using low EDTA TE buffer.

Table 10

An example of diluting a 100 µM oligonucleotide stock solution to the concentrations of the working stock solution

Oligonucleotide name Original CONC. (µM) Required conc. (µM) Volume of 100 µM stock solution (µl) TE buffer volume (µl) Total volume (µl) Dilution factor DIRECT BCMA transgene 100 10 120 1080 1200 HY REVERSE BCMA transgene 100 10 120 1080 1200 HY BCMA transgene PROBE 100 10 120 1080 1200 HY

6.3.3.3. Prepare 20 µl aliquots for all primers and 35 µl aliquots for all probes in 0.5 ml screw cap tubes. The specified aliquot volumes are sufficient for half a PCR assay plate. If necessary, large volume aliquots of primers and probes can be prepared to fill a complete assay plate.

6.3.3.4. Label all aliquots with minimal information.

6.3.3.4.1. Name of the oligonucleotide.

6.3.3.4.2. Concentration in µM.

6.3.3.4.3. Batch no.

6.3.3.4.4. Shelf life (1 year from the date of dilution).

6.3.3.4.5. Store all working stock solutions at -20°C.

6.3.4. Certification of new batches of oligonucleotides.

6.3.4.1. The newly qualified batch of oligonucleotides and the new batch of oligonucleotides are tested in at least 3 independent transgenic qPCR assays using the qualified batch of standard #1, medium and weak controls according to the following protocol.

6.3.4.1.1. Prepare two master mixtures for each of the 3 assays, one master mixture will be prepared using the newly qualified batch of oligonucleotides, and the second master mixture will be prepared using a new batch of oligonucleotides.

6.3.4.1.2. Load the qPCR plate according to the plate diagram.

6.3.4.1.3. Open Oligo Qualification.edt template, select Save As, enter the appropriate qPCR experiment name and save as a.eds file. Do not overwrite the template file.

6.3.4.1.4. Load and analyze the qPCR plate according to the protocol.

6.3.4.2. In the Setup section, select Assign. Select wells D1-E12, right-click and select omit. This will exclude the reactions of the new batch of oligonucleotide from the analysis. Select the entire tablet and click Analyze.

6.3.4.3. Print the report data in PDF and indicate that it relates to the analysis of a certified batch of oligonucleotides.

6.3.4.3.1. All eligibility criteria described in DSTMD-24448 must be met. If any of these eligibility criteria are not met, the analysis is invalid and must be repeated. Report any unreliable analyzes in the reagent qualification report.

6.3.4.4. In the Setup section, select Assign. Select wells D1-E12, right-click and select include. Select wells A1-B12, right-click and select omit. This will allow the reactions of the certified batch of oligonucleotide to be excluded from the analysis. Select the entire tablet and click Analyze.

6.3.4.5. Print the report data in PDF and indicate that it relates to the analysis of a new batch of oligonucleotides.

6.3.4.5.1. All assay results from a new batch of oligonucleotides must satisfy all assay eligibility criteria described in this document.

6.3.4.5.2. Calculate the % difference in the average Ct values for each standard in the reactions of the certified and new batch of oligonucleotides as follows:

% difference } Abs. (Standard Xs X avg. Ct of a certified oligonucleotide - Standard Xs X avg. Ct of a new oligonucleotide) \ ( Average (Standard No. X avg. Ct of a certified oligonucleotide & Standard Xs X avg. Ct new oligonucleotide ) / :> 1OG

6.3.4.5.3. All % differences must be <1.60%.

- 45 046825

6.3.4.6. All valid validated assays must meet the Ct % difference criteria for a new batch of oligonucleotide to be validated.

6.3.4.7. If the new lot does not meet the conformity criteria, the lot fails certification and must be discarded. Prepare another new batch of oligonucleotides and certify the new batch.

Obtaining working starting costs of the linear LiCAR plasmid.

1.0. Target.

1.1. This example describes an exemplary procedure for linearizing the LiCAR plasmid and converting mg/mL plasmid concentrations to copies/μL to prepare working linear plasmid stock solutions for use in preparing transgenic qPCR standards and controls.

2.0. Equipment.

2.1. Centrifuge capable of centrifuging 1.5 ml and 5 ml microcentrifuge tubes (for example: Beckman Coulter, Allegra X-14R with SX4750 rotor with adapter for 1.5 ml microcentrifuge tubes).

2.2. An electrophoresis device, such as the Lonza FlashGel DNA System and FlashGel Dock or the Invitrogen E-Gel Electrophoresis device.

2.3. Freezer with a temperature of -70°C.

2.4. A refrigerator or cold room capable of maintaining a temperature of 2-8°C.

2.5. Calibrated single-channel pipettes (20, 100, 200, 1000 µl or other appropriate volume), for example: Rainin pipettes.

2.6. Fluorimeter Qubit 4, Invitrogen cat. No. Q33226.

2.7. Thermoblock capable of maintaining a temperature of 37°C and suitable for 1.5 ml microcentrifuge tubes.

2.8. Biological safety cabinet.

2.9. Vortex mixer.

3.0. Materials.

Note. Materials with a reservation, for example, can be replaced with similar materials without prior certification. For qualified materials or equivalent, the equivalence of the alternative replacement must be confirmed prior to use for testing samples.

3.1. Stock solution of plasmid pLLV-LICAR2SIN LiCAR.

3.2. DNase/RNase-free water, for example: Invitrogen cat. No. 10977015.

3.3. EcoRI HF Enzyme, New England Biolabs cat. No. R3101S or equivalent.

3.4. GeneJet Gel Extraction & DNA Cleanup Kit, ThermoFisher, cat. No. K0831 or equivalent.

3.5. Preformed electrophoresis gels, such as Lonza FlashGel DNA Cassette or Invitrogen E-Gels, suitable for resolving high molecular weight bands (eg: FlashGel DNA Cassettes, 1.2% 12+1 single level, Lonza cat. no. 57023).

3.6. A DNA molecular weight and length marker suitable for determining the size of bands of at least 8000 kb. (e.g.: E-Gel 1 Kb Plus DNA Ladder, Invitrogen cat. no. 10488090) 1.1 1.5 ml centrifuge tubes, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431021 1.2 2 ml centrifuge tubes, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431048

3.7. Centrifuge tubes 5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 0030119460.

3.8. 1X TE buffer low EDTA, pH 8.0, RNase/DNase free, e.g.: Quality Biological cat. No. 351-324-721.

3.9. Centrifuge tubes 0.5 ml, sterile, RNase/DNase-free, e.g. microcentrifuge tubes with screw cap VWR cat. No. 89004-286 or Eppendorf cat. No. 022431005.

3.10. Pipette tips, sterile, with filter (20, 200, 1000 µl or other appropriate volume), for example: Rainin cat. No. 30389226, 30389240, 30398213.

4.0. Precautionary measures.

4.1. Wear proper PPE when working in the laboratory.

4.2. When working with hazardous chemicals, follow specific instructions for your specific work environment. For more information, see the manufacturer's safety data sheet.

4.3. All steps of pipetting sampling must be carried out using aseptic techniques in a biological safety cabinet. Analysts are advised to use disposable sleeves during all stages of the procedure to minimize the risk of contamination of any materials or reagents.

5.0. Procedure.

5.1. Preheat the thermoblock to 37°C before starting plasmid digestion.

5.2. Thaw the vial containing the LiCAR plasmid stock solution.

5.3. Determine LiCAR DNA concentration using a Qubit 4 fluorimeter and following the procedure described herein. It may be necessary to dilute the plasmid stock solution to bring it within the range of the Qubit dsDNA BR kit (2-1000 ng DNA).

- 46 046825

5.4. Dilute the required amount of plasmid required to digest 0.09 µg/µl plasmid DNA in water.

For example: a stock solution concentration of 1.24 µg/µl is diluted to 0.09 µg/µl by adding

3.63 µl of plasmid DNA in 46.37 µl of DNase/RNase-free water.

5.5. Prepare the EcoRI HF enzymatic digestion reaction mixture at room temperature and add each reagent in the order shown in the table. eleven.

Table 11

Protocol for EcoRI HF cleavage of LiCar plasmid

Reaction Component Volume (µl) 0.09 µg/µl plasmid DNA 10 JX buffer CutSmart 5 DNase/RNase-free water 34 EcoRI-HF (20,000 units/ml) 1 Total volume 50 µl

Note. Multiple 50 µL enzymatic digestion reactions can be performed multiple times as needed to obtain sufficient linearized DNA to prepare a working LiCAR plasmid stock solution for producing standards and controls.

5.6. Save at least 5 µl of undigested plasmid DNA, which can serve as a control for intact plasmid DNA in gel electrophoresis.

5.7. Gently mix the contents of the digestion tubes by pipetting or lightly flicking the tube (do not vortex the reaction mixture). Quickly centrifuge the reaction mixture for 5 s at 300xg.

5.8. Incubate the tubes in a thermoblock at 37°C for 15 minutes.

5.9. Purify digested plasmid DNA using the GeneJet Gel DNA Extraction and Purification Mini Kit.

Note. The volume used for DNA purification should not exceed 200 µl, and the total amount of plasmid DNA purified should not exceed 10 µg. If the total volume exceeds 200 µl, divide the volume equally into 2 or more tubes before continuing with the next purification steps.

5.10. Before beginning the DNA purification procedure when opening the GenJet, add 96-100% ethanol to the wash buffer vials according to the directions on each vial. Confirm the addition of ethanol by indicating the date added and initials on the label. Store ethanol wash buffers at room temperature. Also, before use, check all solutions in the kit for possible salt precipitation. Dissolve any precipitates by heating solutions to 37°C and then equilibrating to room temperature before use.

Note. DNA purification columns should be stored at 2-8°C after kit delivery and when the columns are not in use. After each use, ensure that the DNA purification column bag is tightly closed.

5.11. Adjust the volume of the reaction mixture to 200 µl by adding DNase/RNase-free water. For example: add 150 µl of water to 50 µl of digested DNA mixture.

5.12. Add 100 µl binding buffer. Mix thoroughly by pipetting.

5.13. Add 300 µl ethanol (96-100%) and mix by pipetting.

5.14. Transfer the mixture to a DNA purification microcolumn and collection tube. Centrifuge the column for 1 min at 14000xg. Discard column filtrate. Reinstall the DNA purification microcolumn in the collection tube. In an alternative embodiment, the tube can be mounted in a 2 ml microcentrifuge tube and the collection tube can be discarded.

5.15. Add 700 µl wash buffer (with added ethanol) to the column and centrifuge for 1 min 14000xg. Discard the filtrate and return the column to the collection tube. In an alternative embodiment, the tube can be mounted in a 2 ml microcentrifuge tube and the collection tube can be discarded.

5.16. Perform another washing step by adding 700 µl of wash buffer to the column and centrifuge for 1 minute at 14,000xg. Discard the filtrate and return the column to the same collection tube. In an alternative embodiment, the tube can be mounted in a 2 ml microcentrifuge tube and the collection tube can be discarded.

5.17. Centrifuge the column for an additional 1 minute at 14,000xg to completely remove any remaining wash buffer.

5.18. Transfer the column to a clean 1.5 ml microcentrifuge tube and add 10 µl 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. Centrifuge for 1 min at 14,000xg to elute DNA.

5.20. Verify that the VSV-G plasmid is cleaved by performing gel electrophoresis of the original ring

- 47 046825 (unsplit DNA) and linearized DNA. It is recommended that the linearized plasmid stock solution be diluted for use in gel electrophoresis to retain as much linearized plasmid as possible.

5.21. Determine the concentration of purified linear VSV-G plasmid DNA using a Qubit 4 fluorometer and following the procedure described earlier herein.

5.22. Convert the linear plasmid concentration determined in step 5.21 to copies/µL of LiCAR plasmid as follows:

• / 1 g h (plasmid conc. by Qubit (µg/µl)) (--~ 1 - plasmid conc. (g/µl) \1x10 ⁶ µg/ (6.023 x 10 ²² copies/mol)(plasmid conc. ( g/µl))

------------—;---------------------~ conc. plasmids (copies/µl) (660 g/mol)(plasmid size (bp)

For example: the concentration of the LiCAR DNA solution is 1.03 μg/μl.

(1.03 µg/µl) h........~ 1.03x10 ^b g/µl \1x10 ⁶ µg/ (6.023 x 10 ²³ copies/mol)( 1.03x10 ^-6 g/µl) ( 660 g/mol)(8518 bp)

1.103 x 10 ¹¹ copies/µl

5.23. Dilute the plasmid stock solution to a working concentration suitable for preparing the standard and controls in low EDTA TE buffer.

5.24. Dilute the linear LiCAR plasmid stock solution in low EDTA TE buffer as needed to prepare a working linear LiCAR plasmid stock solution suitable for preparing standards and controls.

5.25. Prepare appropriately sized single-use aliquots of the working concentration of plasmid with a label containing minimal information.

5.25.1. Linear plasmid.

5.25.2. Plasmid concentration in copy number/μl.

5.25.3. Aliquot volume.

5.25.4. Shelf life.

5.26. Plasmids are stable for 12 months at -70°C.

Receipt and certification of new batches of standards.

1.0. Target.

1.1. An example of the procedure for obtaining and certifying new batches of standards for the transgenic qPCR method is described.

2.0. Equipment.

2.1. Centrifuge capable of centrifuging 1.5 mL microcentrifuge tubes (for example: Beckman Coulter, Allegra X-14R with SX4750 rotor and rotating 96-well plate holder and adapters for 1.5 mL microcentrifuge tubes).

2.2. QuantStudio 6 real-time PCR system.

2.3. Freezer with a temperature of -20°C.

2.4. Freezer with a temperature of -70°C.

2.5. Calibrated 8- or 12-channel pipettes (20, 50 µl or other appropriate volume), for example: Rainin pipettes.

2.6. Calibrated single-channel pipettes (20, 100, 200, 1000 µl or other appropriate volume), for example: Rainin pipettes.

2.7. A refrigerator or cold room capable of maintaining a temperature of 2-8°C.

2.8. QuantStudio PCR software ver. 1.3 or higher.

2.9. Thermoblock capable of maintaining a temperature of 55°C and suitable for 1.5 ml microcentrifuge tubes.

2.10. Vortex mixer.

2.11. Biological safety cabinet.

3.0. Materials.

Note. Materials with a reservation, for example, can be replaced with similar materials without prior certification. For qualified materials or equivalent, the equivalence of the alternative replacement should be confirmed prior to use for testing samples.

3.1. DNase/RNase-free water, for example: Invitrogen cat. No. 10977015.

3.2. TaqPath ProAmp master mix, ThermoFisher cat. No. A30866 or equivalent.

3.3. Conglomerates of mock T cells with 2x10 ⁶ -4x10 ⁶ cells in each conglomerate.

3.4. Certified batch of BCMA transgene and ALB primers and probes, custom generated sequences from IDT or equivalent.

3.5. Certified batch of transgene BCMA standard No. 1

- 48 046825

3.6. Certified batch of medium and weak control BCMA transgene.

3.7. An aliquot of pLLV-LICAR2SIN working stock solution.

3.8. 1X TE buffer low EDTA, pH 8.0, RNase/DNase free, e.g.: Quality Biological cat. No. 351-324-721.

3.9. Centrifuge tubes 0.5 ml, sterile, RNase/DNase-free, e.g. microcentrifuge tubes with screw cap VWR cat. No. 89004-286 or Eppendorf cat. No. 022431005.

3.10. Centrifuge tubes 1.5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431021.

3.11. Centrifuge tubes 2 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431048.

3.12. Centrifuge tubes 5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 0030119460.

3.13. Pipette tips, sterile, with filter (20, 200, 1000 µl or other appropriate volume), for example: Rainin cat. No. 30389226, 30389240, 30398213.

3.14. 96-well PCR plates, Applied Biosystems, cat. No. 4483343, 4483354, 4483349, 4483350, 4483395 or equivalent.

3.15. Micro Amp Optical Adhesive Film, Applied Biosystems, Cat. No. 4311971 or equivalent.

3.16. Containers for RNase/DNase-free reagents, for example: VistaLabs cat. No. 3054-1002.

4.0. Precautionary measures.

4.1. Wear proper PPE when working in the laboratory.

4.2. When working with hazardous chemicals, follow specific instructions for your specific work environment. For more information, see the manufacturer's safety data sheet.

4.3. All steps of pipetting sampling must be carried out using aseptic techniques in a biological safety cabinet. Analysts are advised to use disposable sleeves during all stages of the procedure to minimize the risk of contamination of any materials or reagents.

5.0. Procedure.

5.1. Isolated gDNA from mock T cell conglomerates according to the protocol previously described herein. Mock T cells must be produced using the CAR T production method, but without lentivector transduction.

5.2. Several silica DNA extraction columns will be used to isolate the required amount of gDNA. Combine eluted DNA from all columns to obtain one mock T cell gDNA stock solution prior to quantitation.

5.3. After quantification, ensure that sufficient amounts of gDNA have been isolated to prepare multiple aliquots to cover at least 4 months of testing.

5.3.1. If the initial gDNA isolation does not yield enough gDNA to prepare a large enough batch that can be used for at least 4 months, additional mock T cell conglomerates can be isolated, combined with the original mock T cell DNA stock solution, and quantitated the combined stock solution using Qubit.

5.4. Standard #1 is prepared from mock T cell DNA at a concentration of 0.05 μg/μL supplemented with plasmid pLLV-LICAR2SIN (LiCAR plasmid), so that 5 μL of standard #1 contains 121,212.1212 copies of LiCAR plasmid.

5.4.1 Determine the total volume of standard #1 that can be prepared from the mock T cell DNA stock solution as follows:

(gDNA volume) (gDNA concentration) _ „ ...

------- -----—-----—--- = Total volume of standard No. 1

0.05 µg/µl

For example: After DNA quantification, approximately 397.0 μl of mock T cell gDNA remains. The concentration of gDNA is 0.0853 μg/μl. 392.0 µl of gDNA will be taken to prepare standard No. 1.

1(392 µl)(0.0853 µg/µl)

-----------~~rz------------— 668.78 µl total volume of standard No. 1 0.05 µg/µl

5.4.2. Then determine the volume of low EDTA + plasmid TE buffer required to dilute gDNA to 0.05 µg/µl as follows:

Total volume of standard No. 1 - Volume of gDNA = Volume of TE + Plasmid

For example: 392.0 µl of gDNA from the gDNA stock solution will be taken to prepare a total volume of 668.8 µl of standard #1.

5.4.3. Then determine the volume of plasmid alone required to achieve 121,212,121 copies of LiCAR plasmid per 5 µl of standard #1 as follows:

- 49 046825

121,212,131 copies .................:.................................~ 24,242.4242 copies/µl

b µl (24 242.4242 copies/µl) (Total volume of standard No. 1) „_

:...................................·......... ........................................................ ......................... ;......Volume of plasmid

Concentration of working plasmid stock solution (copies/µl)

For example: The total volume of standard No. 1 is 668.8 µl. Working stock solution of LiCAR plasmid with 1.1035x106 copies/µl.

1(24.24X4242 copies/uL) (663.8 µl) ^{14.7 µl plasmids}

5.4.4. Finally, determine the volume of low EDTA TE buffer required to prepare the total volume of standard #1.

Total volume of standard No. 1 - Volume of gDNA + Volume of plasmid = Volume of TE

For example: a total volume of 668.8 μl of standard #1 of 392.0 μl of mock T cell gDNA and 14.7 μl of LiCAR plasmid.

668.8 µl - (392.8 µl -1 14.7 µl) - 262.1 µl TE

5.5. Begin the preparation of standard No. 1 by transferring a certain volume of the mock T-cell gDNA stock solution into a DNase/RNase-free tube of a suitable volume sufficient to prepare the total volume of standard No. 1 (calculated in step 5.4.1).

5.6. After step 5.5, add the volume of TE buffer with low EDTA content calculated in step 5.4.4 to the test tube.

5.7. Then add the volume of LiCAR plasmid calculated in step 5.4.3. Mix the contents of the test tube in a vortex mixer for a short time.

5.8. Prepare 20 µl single-use aliquots of standard #1 labeled with minimal information.

5.8.1. BCMA transgene standard #1.

5.8.2. Batch no.

5.8.3. Date of preparation of standard No. 1.

5.9. Store all single-use aliquots at -20°C.

5.9.1. Certification of new batches of standard No. 1.

5.9.1.1. Newly validated standard #1 and a new batch of standard #1 are tested in at least 3 independent transgenic qPCR assays using the validated batch of oligonucleotides, medium and weak controls according to the protocol previously described herein as follows.

5.9.1.1.1. One master mixture is prepared for all standard and control samples.

5.9.1.1.2. Construct two 5-point standard curves, one using the qualified batch of standard No. 1, and the second using the new batch of standard No. 1.

5.9.1.1.3. Load the qPCR plate according to the plate diagram.

5.9.1.1.4. Open Standard Qualification.edt template, select Save As, enter the appropriate qPCR experiment name and save as a.eds file. Do not overwrite the template file.

5.9.1.1.5. Load and analyze the qPCR plate according to the protocol described herein.

5.9.1.2. In the Setup section, select Assign. Select wells C1-D12, right-click and select omit. This will exclude the standard curve for the new batch of standard #1 from the analysis. Select the entire tablet and click Analyze.

5.9.1.3. Print the report data in PDF and indicate that they relate to the analysis of the certified batch of standard No. 1.

5.9.1.3.1. All eligibility criteria described in this document must be met. If any of these eligibility criteria are not met, the test is invalid and must be repeated. Report any unreliable analyzes in the reagent qualification report.

5.9.1.4. In the Setup section, select Assign. Select wells C1-D3, right-click and select include. Select wells A1-B3, right-click and select omit. This will allow the standard curve for the qualified batch of standard No. 1 to be excluded from the analysis. Select the entire tablet and click Analyze.

5.9.1.5. Print the report data in PDF and indicate that they relate to the analysis of a new batch of standard No. 1.

5.9.1.5.1. All analytical results from a new batch of Standard No. 1 must meet all analytical compliance criteria described in this document.

5.9.1.6. To meet validation requirements, the average of all three VCN/cell measurements for the intermediate and weak controls across all valid validated assays must be <20% of the expected VCN/cell results for new batches of Standard No. 1.

Receipt and certification of new batches of medium and weak controls.

1.0. Target.

- 50 046825

1.1. An example of the procedure for obtaining and certifying new batches of standards for the transgenic qPCR method is described.

2.0. Equipment.

2.1 Centrifuge capable of centrifuging 1.5 mL microcentrifuge tubes (for example: Beckman Coulter, Allegra X-14R with SX4750 rotor and rotating 96-well plate holder and adapters for 1.5 mL microcentrifuge tubes).

2.2 QuantStudio 6 real-time PCR system.

2.3 Freezer with a temperature of -20°C.

2.4 Freezer with a temperature of -70°C.

2.5 Calibrated 8- or 12-channel pipettes (20, 50 µl or other appropriate volume), for example: Rainin pipettes.

2.6 Calibrated single-channel pipettes (20, 100, 200, 1000 µl or other appropriate volume), for example: Rainin pipettes.

2.7. A refrigerator or cold room capable of maintaining a temperature of 2-8°C.

2.8 QuantStudio PCR software ver. 1.3 or higher.

2.9. Thermoblock capable of maintaining a temperature of 55°C and suitable for 1.5 ml microcentrifuge tubes.

2.10. Vortex mixer.

2.11. Biological safety cabinet.

3.0. Materials.

Note. Materials with a reservation, for example, can be replaced with similar materials without prior certification. For qualified materials or equivalent, the equivalence of the alternative replacement should be confirmed prior to use for testing samples.

3.1. DNase/RNase-free water, for example: Invitrogen cat. No. 10977015.

3.2. TaqPath ProAmp master mix, ThermoFisher cat. No. A30866 or equivalent.

3.3. Conglomerates of mock T cells with 2x 106 - 4 x 10 ⁶ cells in each conglomerate.

3.4. Certified batch of BCMA transgene and ALB primers and probes, custom generated sequences from IDT or equivalent.

3.5. Certified batch of transgene BCMA standard No. 1.

3.6. Certified batch of medium and weak control BCMA transgene.

3.7. An aliquot of pLLV-LICAR2SIN working stock solution.

3.8. 1X TE buffer low EDTA, pH 8.0, RNase/DNase free, e.g.: Quality Biological cat. No. 351-324-721.

3.9. Centrifuge tubes 0.5 ml, sterile, RNase/DNase-free, e.g. microcentrifuge tubes with screw cap VWR cat. No. 89004-286 or Eppendorf cat. No. 022431005.

3.10. Centrifuge tubes 1.5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431021.

3.11. Centrifuge tubes 2 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 022431048.

3.12. Centrifuge tubes 5 ml, sterile, RNase/DNase-free, e.g.: Eppendorf cat. No. 0030119460.

3.13. Pipette tips, sterile, with filter (20, 200, 1000 µl or other appropriate volume), for example: Rainin cat. No. 30389226, 30389240, 30398213.

3.14. 96-well PCR plates, Applied Biosystems, cat. No. 4483343, 4483354, 4483349, 4483350, 4483395 or equivalent.

3.15. Micro Amp Optical Adhesive Film, Applied Biosystems, Cat. No. 4311971 or equivalent.

3.16. Containers for RNase/DNase-free reagents, for example: VistaLabs cat. No. 3054-1002.

4.0. Precautionary measures.

4.1. Wear proper PPE when working in the laboratory.

4.2. When working with hazardous chemicals, follow specific instructions for your specific work environment. For more information, see the manufacturer's safety data sheet.

4.3. All steps of pipetting sampling must be carried out using aseptic techniques in a biological safety cabinet. Analysts are advised to use disposable sleeves during all stages of the procedure to minimize the risk of contamination of any materials or reagents.

5.0. Procedure.

5.1. Isolated gDNA from mock T cell conglomerates according to the protocol previously described herein. Mock T cells must be produced using the CAR T production method, but without lentivector transduction.

5.2. Several silica DNA extraction columns will be used to isolate the required amount of gDNA. Combine eluted DNA from all columns to obtain one mock T cell gDNA stock solution prior to quantitation.

- 51 046825

5.3. After quantification, ensure that sufficient amounts of gDNA have been isolated to prepare multiple aliquots to cover at least 4 months of testing. The weak control is a 1:10 dilution of the medium control using 0.02 μg/μl mock T cell gDNA as diluent. The weak and medium controls do not need to be prepared together from the same mock T cell gDNA stock solution, but if they are prepared at the same time, it will be necessary to retain a portion of the isolated mock T cell gDNA stock solution to prepare the weak control. The example shown in the steps below illustrates the procedure for preparing a medium and weak control from the same stock solution of mock T cell gDNA.

5.3.1. If the initial gDNA isolation does not yield enough gDNA to prepare a large enough batch of both controls that can be used for at least 4 months, additional mock T cell conglomerates can be isolated, combined with the original mock T cell DNA stock, and quantitated determination of pooled stock solution using Qubit.

5.4. The BCMA transgene medium control is prepared from 0.02 μg/μl mock T cell DNA supplemented with plasmid pLLV-LICAR2SIN (also referred to herein as the LiCAR plasmid) such that 5 μl of the medium control contains 30,303,030 copies of the LiCAR plasmid. This corresponds to a vector copy number of 2.00 copies/cell in 5 µl of control.

5.4.1. Determine the total volume of weak control that can be prepared from the mock T cell DNA stock solution as follows.

(gDNA volume)(gDNA concentration) ~

------------------------------g Total volume of average control

0.02 µg/µl

For example: After DNA quantification, approximately 547.0 μl of mock T cell gDNA remains. The concentration of gDNA is 0.0913 μg/μl. 298 µl of gDNA will be taken to prepare a medium control.

(298.0 µl)(0.0913 µg/µl)

------------—------------= 1360.4 µl Total volume of average control 0.02 µg/µl

5.4.2. Then determine the volume of low EDTA + plasmid TE buffer required to dilute gDNA to 0.02 µg/µl as follows:

Total volume of average control ····· Volume of gDNA = Volume of TE + Plasmid

For example: 298.0 µl of gDNA from the gDNA stock solution will be taken to prepare a total volume of 1360.4 µl of the average control.

1360.4 µl - 298.0 µl ™ 1062.4 µl TE 4- Plasmid

5.4.3. Then determine the volume of plasmid alone required to achieve 30,303.0303 copies of LiCAR plasmid per 5 µl of mean control as follows.

303,030 copies

----------------- 6,060.606 copies/µl µl (6,060.606 copies/µl) (Total average control volume) Plasmid working stock solution concentration (copies/µl) = Plasmid volume

For example: The total volume of the average control is 1360.4 µl. Working stock solution of LiCAR plasmid with 1.1035x106 copies/µl.

(6,060.606 copies/µl)( 13,60.4 µl)

----——g—-----------= 7.5 µl of plasmid

1.103 x 10 ⁶ (copies/µl)

5.4.4. Finally, determine the volume of low EDTA TE buffer required to prepare the total volume of the average control.

Total volume of average control - Volume of gDNA + Volume of plasmid = Volume of TE

For example: A total volume of 1360.4 µl average control of 298 µl mock T cell gDNA and 7.5 µl LiCAR plasmid.

1360.4 µl - (298 µl + 7.5 µl) - 1054.9 µl TE

5.5. Prepare a medium control by transferring a specified volume of mock T cell gDNA stock solution into a DNase/RNase-free tube of a suitable volume sufficient to prepare the total volume of the medium control (calculated in step 5.4.1).

5.6. After step 5.5, add the volume of TE buffer with low EDTA content calculated in step 5.4.4 to the test tube.

5.7. Then add the volume of LiCAR plasmid calculated in step 5.4.3. Mix the contents of the test tube in a vortex mixer for a short time.

5.8. Prepare weak control from medium control by diluting medium control 1:10 using 0.02 µg/µl mock T cell gDNA as diluent. For example: prepare 1230.0 µl of the total volume of the weak control from the medium control stock solution.

- 52 046825

1230.0 µl weak control ~

------------------------ 123.0 µl average control

An example would be to prepare weak and medium controls from one stock solution of mock T cell gDNA, and use the remainder of the medium control as a new batch of medium control.

1360.4 µl average control ~ 123.0 µl (to prepare weak control) == 1237.4 µl residual average control

5.9. Determine the amount of 0.02 µg/µl mock T cell gDNA required to prepare the required total volume of weak control. For example: The total volume of the weak control stock solution that will be prepared is 1230.0 µl and is obtained by diluting the medium control 1:10 (123.0 µl medium control).

1230.0 µl - 123.0 µl = 1107.0 µl 0.02 µg/µl gDNA

5.10. Then dilute the required volume of isolated mock T cell gDNA to 0.02 μg/μl with low EDTA TE buffer. Prepare a sufficient volume of 0.020 µg/µl gDNA (including refill) needed to prepare the weak control (step 5.9). For example: 1107.0 µl of 0.02 µg/µl mock E-cell gDNA required to prepare 1230.0 µl weak control. Dilute 0.0913 μg/μL mock T cell stock solution to 0.02 μg/μL.

(244.0 µl of gDNA stock solution)(('),09\3 µg/µl)

1113.9 µl total volume 0.02 µg/µl gDNA

113.9 µl - 244.0 µl “ 869.9 µl TE buffer volume

Dilute 244.0 µL of 0.0913 µg/µL mock T cell gDNA stock solution with 869.9 µL low EDTA TE buffer to prepare a sufficient volume of 0.02 µg/µL mock T cell gDNA to prepare 1230.0 µl weak control.

5.11. Prepare the weak control by transferring a specified volume of the mock T cell gDNA stock solution into a DNase/RNase-free tube of a suitable volume sufficient to prepare the desired total volume of the weak control.

5.12. After step 5.11, add a certain volume of TE buffer with low EDTA content, calculated in step 5.10, to the test tube after step 5.11.

5.13. Then add the specified volume of medium control required to prepare the weak control. Mix the contents of the test tube in a vortex mixer for a short time.

5.14. Prepare 20 µl single-use aliquots of medium and weak controls labeled with the following minimum information:

5.14.1. Moderate/weak control of the BCMA transgene.

5.14.2. Batch no.

5.14.3. Date of control preparation.

5.15. Store all single-use aliquots at -20°C.

5.15.1 Certification of new batches of controls.

5.15.1.1. The newly validated intermediate and weak controls and a new batch of controls are tested in at least 3 independent transgenic qPCR assays using the validated batch of oligonucleotides and standards #1 according to the protocol previously described herein, as follows:

5.15.1.1.1. One master mixture is prepared for all standard and control samples.

5.15.1.1.2. Construct one standard curve using 5 points using the certified batch of standard No. 1.

5.15.1.1.3. Load the qPCR plate according to the plate diagram in Fig. 10.

5.15.1.1.4. Open the control validation template file, select Save As, enter the appropriate qPCR experiment name and save as a.eds file. Do not overwrite the template file.

5.15.1.1.5. Load and analyze the qPCR plate according to the control validation template file.

5.15.1.2. Analyze data according to the control validation template file with a new set of controls analyzed as samples.

5.15.1.2.1 All compliance criteria described in the control validation template file must be satisfied.

5.15.1.3. Certification compliance criteria.

5.15.1.3.1. To meet validation requirements, the average of all three VCN/cell measurements for a new batch of intermediate and weak controls across all valid validated assays must be <35% of the expected VCN/cell results for new batches of controls.

5.15.1.3.2. The % CV of all three VCN/cell measurements for a new batch of medium and weak controls must be <20% for all valid validation assays.

Example 3. Transgenic qPCR validation method.

1.0. Target.

- 53 046825

1.1. The purpose of this example is to describe the results obtained during the implementation of a transgenic qPCR method validation protocol.

2.0. Application area.

2.1. The qPCR assay for quantification of the LiCAR plasmid integrated into the CAR T product was quantified based on the following validation parameters: specificity, accuracy, linearity, precision (reproducibility and intra-laboratory reproducibility), range and LOQ.

3.0. Definitions and abbreviations.

3.1. Quantitative polymerase chain reaction qPCR.

3.2. Human albumin hALB.

3.3. Number of copies of the VCN vector.

3.4. CV coefficient of variation.

3.5. SD standard deviation.

3.6. Ct cycle threshold.

3.7 LOQ limit of quantitation.

3.8. Development of methods for biological analysis of BMD.

3.9. QC quality control.

4.0. Analysis approach.

4.1. Linearity of the assay was quantified by testing a 5-point standard curve constructed from the results of a 5-fold serial dilution of transgenic qPCR standard #1 and plotting the standard curve results as the decimal logarithm of the amount versus Ct.

4.2. Assay precision, precision, and within-laboratory reproducibility were quantified by testing the 5-point standard curve and moderate and poor assay controls.

4.3. Assay precision was quantified by testing the moderate and weak assay controls.

4.4. The specificity of the assay was demonstrated by testing a mock T cell DNA sample and a representative CAR T DNA sample collected on day 10.

4.5. The LOQ of the assay was determined by testing the LOQ of samples of 0.02 VCN/cell and 0.014 VCN/cell.

4.6. Three validation assays were performed by two assayers, with one of the three assays performed on different days.

5.0. Materials.

5.1. Please review the validation protocol described in this document for a complete list of materials required to perform a transgenic qPCR assay.

5.2. Analytical standards and controls.

5.2.1. BCMA Transgene Standard No. 1, Lot No. LM-RP3-00533A.

5.2.2. Medium control BCMA transgene, lot no. LM-RP3-00533B.

5.2.3. BCMA Transgene Weak Control, Lot# LM-RP3-00533C.

5.3. Patterns of assay specificity.

5.3.1. Mock T Cell DNA, Lot No. LM-RP3-00533.

5.3.2. DNA CAR T, LM-RP3-00541.

5.4 Sample LOQ analysis.

5.4.1. Sample LOQ 0.02 VCN/cell and 0.014 VCN/cell, Lot No. LM-RP3-00533.

5.4.2. Set of oligonucleotides for the transgenic method Lot No. LM-RP3-00460.

6.0. Conclusions.

6.1. This example shows the results obtained during the implementation of the transgenic qPCR method validation protocol.

6.2. The method demonstrated acceptable accuracy, reproducibility, specificity and linearity, which are given in Table. 12 (10.2-10.5). In addition, the assay range and LOQ for transgene and hALB targets were determined. Therefore, the method is certified for testing the CAR T drug material before preparing the composition with the cryopreservation medium.

- 54 046825

Table 12

Summary information on compliance criteria and results of certification of the transgenic procedure

multiplex qPCR Parameter Eligibility Criteria results Linearity (transgene target) The R ² value of the linear regression of Logyu on the Ct values for the standard curve for all reliable validation analyzes should be > 0.97. 1.00 Reproducibility (transgene target standard curve) The % CV of Ct values across three measurements in each valid qualification analysis for each standard must be < 30%. 0.06-0.54% In-lab reproducibility (transgene target standard curve) The % CV of Ct values for all valid qualification measurements for each standard must be < 30%. 0.26-0.47% Linearity (hALB target) The R ² value of the linear regression of Log ₁₀ on the Ct values for the standard curve across all valid validation assays should be > 0.97. 1.00 Reproducibility (standard hALB target curve) The % CV of Ct values across three measurements in each valid qualification analysis for each standard must be < 30%. 0.03-0.41% In-lab reproducibility (transgene target standard curve) The % CV of Ct values for all valid qualification measurements for each standard must be < 30%. 0.17-0.55% Accuracy The % recovery for the intermediate and weak assay controls in each valid validation assay must be within 70-130% of the expected VCN/cell value for that control. Medium control (2.00 VCN/cell) 93-95% recovery Weak control (0.02 VCN/cell): 79-84% recovery Reproducibility The % CV of the three VCN/cell measurements for the moderate and weak assay controls for each valid validation assay must be < 30%. Average control (2.00 VCN/cell): 4-6% Weak control (0.02 VCN/cell): 4-6% In-laboratory %CV of measurement results Average control (2.00 reproducibility VCN/Cage for Medium and Weak Assay Control for VCN/cell): 4% Weak control (0.02

- 55 046825

all valid VCN/cell): 6% qualification analyzes should be < 30%. Specificity All Ct values of repeated transgene target: for (imitation DNA measurements for simulation of each analysis all T cells) DNA T cells must repeat Ct values belong to the category “not measurements belonged to determined" for a target of the category "not transgene, and at the same time for is determined.” each valid Target hALB: average validation analysis hALB copy number the average hALB copy number was in the range must be between 28,719-29,611. 21 221-39 394. Specificity (JNJ- All repeat values Transgene target: all 68284528 CAR-T measurements for JNJ-68284528 copy number values were DNA) CAR-T DNA must be quantifiable demonstrate quantifiable and were in the quantifiable range of 4592.801-5153.907. result for transgene, and hALB target: average this for each reliable number of copies of hALB certification analysis was within average hALB copy number 31,552-33,725. must be between 21,212-39,394. Range (target The range is defined as Range: 193.939- transgene) copy number range, 121,212,121 copies covered by standard curve at 5 points, with provided that the target of the transgene meets all criteria

regarding accuracy,

- 56 046825

linearity and in-laboratory reproducibility. Range (target The range is defined as Range 121,212- hALB) copy number range covered by a 5-point standard curve, assuming that the hALB target provides within-laboratory reproducibility. 75,757,576 copies LOQ (target LOQ is defined as LOQ: Number of copies transgene) result for the transgene copy number for the sample with the lowest LOQ for which the %CV should be observed? 20% for the result of the average transgene copy number and the results of the average sample transgene LOQ 0.02 VCN/cell is 303,030.

VCN/cell, as well as % recovery ranging from 70-130% for the average transgene copy number result and the average VCN/cell result for each valid validation assay.

LOQ (hALB Target) LOQ is defined as the LOQ value: 121,212 copies of the copy number of standard No. 5, provided that the hALB target satisfies all criteria for accuracy, linearity and within-laboratory reproducibility.

7.0. Procedure.

7.1. All analyzes were performed as described in the method validation protocol. Only assays that met the assay eligibility criteria specified in the method were included in the evaluation of method validation criteria.

8.0. Results and discussion.

8.1. Linearity and reproducibility of the standard curve (reproducibility and intra-laboratory reproducibility).

8.1.1. Results for the 5-point standard curve for all valid validation assays for transgene and hALB targets are shown in Table. 13-14 and in fig. 1-12. The R2 values of the Log10 plots against Ct values for the transgene and hALB targets are 1.00. The reproducibility for each standard ranged from 0.06–0.54% for the transgene target and from 0.03–0.41% for the hALB target. Intralaboratory reproducibility varied in the range of 0.26–0.47% for the transgene target and in the range of 0.17–0.55% for the hALB target.

- 57 046825

Table 13

Results for transgene standard curve

Analysis Ct Average Ct CtSD Ct%CV Ct%CV No. (reproducibility) (in-lab reproducibility) Standard No. 1 1 20,987 21,096 21.110 21,064 0.067 0.32 0.26 2 20,980 21,035 21,062 21,025 0.042 0.20 3 20,966 20,981 20,990 20,979 0.012 0.06 Standard No. 2 1 23,443 23,485 23,589 23,505 0.075 0.32 0.35 2 23,432 23,422 23,510 23,455 0.048 0.20 3 23,318 23,348 23,393 23,353 0.038 0.16 Standard No. 3 1 25,923 25,828 25,807 25,853 0.062 0.24 0.30 2 25,822 25,750 25,773 25,782 0.037 0.14 3 25,741 25,689 25,672 25,701 0.036 0.14 Standard No. 4 1 28,194 28,092 28.117 28,134 0.053 0.19 0.30 2 28,038 28,098 28,146 28,094 0.054 0.19 3 27,999 27,948 27,966 27,971 0.026 0.09 Standard No. 5 1 30,336 30,663 30,471 30,490 0.165 0.54 0.47 2 30,201 30,251 30,359 30,270 0.081 0.27 3 30,487 30,307 30,484 30,426 0.103 0.34 Eligibility criteria: <30 <30

- 58 046825

Table 14

Results for hALB standard curve

Analysis no. Ct average Ct CtSD Ct % CV (reproducible: Ct%CV d) (in-lab reproducibility) Standard No. 1 1 21,038 21,083 21,058 21,060 0.022 0.11 0.26 2 21,080 21,188 21,175 21,147 0.059 0.28 3 21,081 21,058 21,050 21,063 0.017 0.08 Standard No. 2 1 23,454 23.415 23,491 23,453 0.038 0.16 0.17 2 23,493 23,482 23,493 23,489 0.006 0.03 3 23,418 23,418 23,396 23,410 0.013 0.05 Standard No. 3 1 25,802 25,738 25,777 25,772 0.032 0.13 0.21 2 25,898 25,830 25,754 25,827 0.072 0.28 3 25,783 25,780 25,710 25,758 0.042 0.16 Standard No. 4 1 28,154 28,095 28,039 28,096 0.058 0.21 0.17 2 28.117 28,089 28,092 28,099 0.015 0.05 3 28,078 27,994 28,133 28,068 0.070 0.25 Standard No. 5 1 30,498 30,641 60,567 30,569 0.072 0.23 0.55 2 30,804 30,819 30,698 30,773 0.066 0.21 3 30,508 30,288 30,505 30,434 0.126 0.41 Eligibility criteria: <30 <30

8.2. Accuracy and precision of quality control samples (reproducibility and intra-laboratory reproducibility).

8.2.1. The results for the average control of 2.00 VCN/cell and weak control of 0.20 VCN/cell are shown in table. 15. The recovery percentage for the average VCN/cell results for the average control is in the range of 93-95%.

The recovery percentage for the average VCN/cell results for the weak control ranges from 79-84%. Reproducibility for medium and weak controls is in the range of 4-6%. Intralaboratory reproducibility for moderate and weak controls is 4% and 6%, respectively.

- 59 046825

Table 15

Results for medium control 2.00 VCN/cell and weak control 0.20 VCN/cell

Analysis Expected Observed Wed. SD %CV %%CV No. value value VCN/cage VCN/cage observed VCN/cell (reproducibility) recovery (in-lab value reproducibility) VCN/cage

Middle 1 2.00 1.83 1.91 0.070 4 95 4 control 1.95 1.94 2 2.00 1.80 1.90 0.081 4 95 1.92 1.96 3 2.00 1.72 1.85 0.116 6 93 1.94 1.89 Weak 1 0.20 0.16 0.17 0.010 6 84 6 control 0.16 0.18 2 0.20 0.15 0.16 0.007 4 79 0.16 0.16 3 0.20 0.16 0.17 0.010 6 84 0.17 0.18 Eligibility criteria: <30 70-130 <30

8.3. Specificity.

8.3.1. In table 16 shows the results for the mock T cell DNA and CAR T DNA samples that were tested to assess the specificity of the assay. All replicate measurements of mock T cell DNA samples were categorized as not detectable for the transgene target. All repeated CAR T DNA measurements demonstrate quantifiable transgene copy number results. In addition, the mock T cell DNA and CAR T DNA samples meet the hALB sample matching criteria for hALB copy number +/-30% of the expected copy number of 30,303.030 for 100 ng of DNA.

Table 16

Results for mock DNA T cell and DNA CAR T

Analysis C1 transgene Copies Ct hALB Copies of hALB Wed. number No. transgene copies of hALB Imitation 1 Not N/A 22,476 28,874.957 29,226.484 DNA T cells determined by Not N/A N/A 22,474 22,426 28,918,598 29,885.896 determined by Not 2 determined by Not N/A 22,505 29,269,688 29,610,689 determined by Not N/A N/A 22,491 22,467 29,537.838 30,024.545 determined by Not 3 determined by Not N/A 22,487 28,520.168 28,718,760 determined by Not N/A N/A 22,489 22,455 28,481,504 29,154.613 determined by Not determined Eligibility Criteria Not 21 212- -39 394 determined Analysis Ct of transgene Copies Ct hALB Copies of hALB Wed. number No. transgene copies of hALB JNJ-68284528 1 25,796 4,894,034 22,321 32,078.729 31,552.141 CAR-T DNA 25,831 4,776.155 22,356 31,328.813 25,830 4,779,097 22,360 31,248.881 2 25,742 4,792,772 22,353 32,438,480 33,725.105 25,783 4,656,793 22,230 35,245.887 25,803 4,592,801 22,305 33,490.949 3 25,719 4,739,884 22,373 30,857.877 32,056.623 25,597 5,153,907 22,283 33,273,906 25,742 4,666,396 22,318 32,038,086 Eligibility Criteria: Quantitatively 21212- definable result 39 394

8.4. Range and LOQ.

8.4.1. The transgene and hALB targets met all eligibility criteria for accuracy, linearity, and within-laboratory reproducibility. Therefore, the range for targets as trans-

- 60 046825 gene and hALB is defined as the copy number range of a 5-point standard curve. The transgene range is 193,939-121,212,121 copies. The range of hALB is 121,212-75,757,576 copies.

Additionally, the LOQ for the hALB target is defined as 121,212 copies.

8.4.2. The results for LOQ samples of 0.014 VCN/cell and 0.02 VCN/cell are shown in table. 17. At least one of the three Ct values measured for a sample LOQ of 0.014 VCN/cell is not within the Ct range of the transgene standard curve in each valid validation assay. Therefore, transgene copy number values cannot be accurately determined and LOQ criteria cannot be assessed. However, a sample LOQ of 0.02 VCN/cell resulted in % recovery of 73-80%. A sample LOQ of 0.02 VCN/cell also resulted in %CV for mean transgene copy number and mean VCN/cell results of 1–9% and 2–11%, respectively. Therefore, the LOQ of the transgene target is defined as the expected copy number of the transgene LOQ of the 0.02 VCN/cell sample equal to 303,030 copies.

Table 17

Results for LOQ samples 0.014 VCN/cell and 0.02 VCN/cell

Analysis no. Observable Observable Observed SD Observable % recovery Observed avg. Observed SD Observable % recovery number average COPIES %cv if- meaning VCN/ %cv VCN/ copies number transgene copies quality VCN/ cell VCN/ cell transgene copies transgene trance- cell cell transgene gene

0.014 1 N/A N/A N/A N/A N/A n/a n/a n/a n/a VCN/ 177,993 cell N/A (212,121 2 N/A N/A N/A N/A N/A n/a n/a n/a n/a COPIES N/A trance- N/A gene) 3 187,975 N/A N/A N/A N/A n/a n/a n/a n/a

n/a 188,407

Amounts reported as NA were not quantifiable because the Ct value was _______above the highest Ct value for standard #5 (i.e., outside the Ct range of the transgene standard curve)_______

Ana- Observe- Observe Observed Observe % extracted Observe Observe Observe % extracted liz no. given given required SD given values given Wed. given by SD given values NUMBER average COPIES %cv if- meaning VCN/ %cv VCN/ copies number transgene copies quality VCN/ cell VCN/ cell transgene copies transgene trance- cell cell transgene gene

0.02 1 237,648 236.475 5,320 2 78 0.016 0.0002 2 80 VCN/cage 241.111 ka 230,666 (303,030 2 212.258 212.187 2,581 1 70 0.015 0.0003 2 73 copies 214,734 transgene 209,573 ) 3 221,846 200,792 242,464 221,701 20,836 9 73 0.015 0.0016 eleven 75

Criteria <20 30-170 <20 30-170 compliance:

The contents of all patents, published applications, references cited herein are incorporated herein by reference in their entirety.

Although exemplary embodiments have been shown and described, those skilled in the art will appreciate that various changes in form and content can be made without departing from the scope of the embodiments included in the appended claims.

Sequence Listing <110> JANSSEN BYOTECH, INC.

<120> CAR T CONSTRUCT AND PRIMERS FOR B CELL MATURATION COMPLEX <130> JBI6148WOPCT1 <140>

<141>

<150> 62/894,663 <151> 2019-08-30

- 61 046825 <160> 32 <170> PatentIn, version 3.5 <210> 1 <211> 24 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note=“On the artificial sequence: Synthetic probe” <400> 1 agcaggggcca gaaccagctc tata 24 <210> 2 <211> 23 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note="Description of artificial sequence: Synthetic primer" <400> 2 tgaactgaga gtgaagttca gca 23 <210> 3 <211> 22 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note="Unification of the artificial sequence: Synthetic primer" <400> 3 cttcgtccta gattgagctc gt 22 <210> 4 <211> 24 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note=“Description of artificial sequence: Synthetic probe” <400> 4 ttatagagct ggttctggcc ctgc 24 <210> 5

- 62 046825 <211> 21 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note=“On the artificial sequence: Synthetic primer” <400> 5 ggatgtgaac tgagagtgaa g 21 <210>6 <211> 21 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note="Unification of the artificial sequence: Synthetic primer" <400> 6 tcctctcttc gtcctagatt g 21 <210>7 <211> 25 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note="Unification of the artificial sequence: Synthetic probe" <400> 7 tcttctggaa atcggcagct acagc 25 <210>8 <211> 21 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note="Description of artificial sequence: Synthetic primer" <400> 8 ggcagaaaga aactcctgta t 21 <210>9 <211> 21 <212> DNA <213> Artificial sequence <220>

<221> source

- 63 046825 <223> /po1e=“Description of artificial sequence: Synthetic primer” <400> 9 cttcactctc agttcacatc c 21 <210> 10 <211> 25 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote=“Description of the artificial sequence: Synthetic probe” <400> 10 tcttctggaa atcggcagct acagc 25 <210> 11 <211> 22 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote=“Description of the artificial sequence: Synthetic primer” <400> 11 ccagtacaaa ctactcaaga gg 22 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote="Description of the artificial sequence: Synthetic primer" <400> 12 gctgaacttc actctcagtt 20 <210> 13 <211> 29 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote="Description of the artificial sequence: Synthetic probe" <400> 13 agaaggagga tgtgaactga gagtgaagt 29

- 64 046825 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note="Description of the artificial sequence: Synthetic primer" <400> 14 ctgccgattt ccagaagaag 20 <210> 15 <211> 21 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note="Description of the artificial sequence: Synthetic primer" <400> 15 tcctctcttc gtcctagatt g 21 <210> 16 <211> 26 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note=“Description of the artificial sequence: Synthetic probe” <400> 16 aggaggatgt gaactgagag tgaagt 26 <210> 17 <211> 18 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note=“Description of artificial sequence: Synthetic primer” <400> 17 ctgtagctgc cgatttcc 18 <210> 18 <211> 20 <212> DNA <213> Artificial sequence

- 65 046825 <220>

<221> source <223> /po1e=“Description of artificial sequence: Synthetic primer” <400> 18 atcgtactcc tctcttcgtc 20 <210> 19 <211> 26 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote="On the artificial sequence: Synthetic probe" <400> 19 aggaggatgt gaactgagag tgaagt 26 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote="On the artificial sequence: Synthetic primer" <400> 20 ctgccgattt ccagaagaag 20 <210> 21 <211> 21 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote=“On the artificial sequence: Synthetic primer” <400> 21 tcctctcttc gtcctagatt g 21 <210> 22 <211> 25 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote="On the artificial sequence: Synthetic probe" <400> 22

- 66 046825 agggagagat ttgtgtgggc atgac 25 <210> 23 <211> 22 <212> DNA <213> Artificial sequence <220>

<221> source <223> /note="Announcement of the artificial sequence: Synthetic primer" <400> 23 tcatctcttg tgggctgtaa tc 22 <210> 24 <211> 21 <212> DNA <213> Artificial sequence <220>

< 221> source < 223> /note=“On the artificial sequence: Synthetic primer” < 400> 24 tgctggttct ctttcactga c 21 < 210> 25 < 211> 26 < 212> DNA < 213> Artificial sequence < 220>

< 221> source < 223> /note="Unveiling the artificial sequence: Synthetic probe" < 400> 25 cctgtcatgc ccacacaaat ctctcc 26 < 210> 26 < 211> 22 < 212> DNA < 213> Artificial sequence < 220>

<221> source <223> /note="Description of artificial sequence: Synthetic primer" <400> 26 gctgtcatct cttgtgggct gt 22 <210> 27 <211> 21 <212> DNA

- 67 046825 <213> Artificial sequence <220>

<221> source <223> /po1e=“Description of artificial sequence: Synthetic primer” <400> 27 actcatggga gctgctggtt c 21 <210> 28 <211> 24 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote=“On the artificial sequence: Synthetic probe” <400> 28 ccctggcatt gttgtctttg caga 24 <210> 29 <211> 18 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote=“On the artificial sequence: Synthetic primer” <400> 29 ctgtcatgcc cacacaaa 18 <210> 30 <211> 22 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote=“On the artificial sequence: Synthetic primer” <400> 30 ataaggctat ccaaactcat gg 22 <210> 31 <211> 16 <212> DNA <213> Artificial sequence <220>

<221> source <223> /pote="On the artificial sequence: Synthetic probe"

-

Claims (28)

<400> 31 aatcggcagc tacagc 16 <210> 32 <211> 16 <212> DNA <213> Artificial sequence <220> <221> source <223> /note=“Understanding the artificial sequence: Synthetic probe” <400> 32 tttgtgtggg catgac 16 CLAIM 1. A kit for quantitative determination of transgene integration into a T cell with chimeric antigen receptors (CAR), containing: a probe containing the nucleotide sequence of SEQ ID NO: 10, and at least one label associated with the probe; a first primer containing the nucleotide sequence SEQ ID NO: 11; and a second primer containing the nucleotide sequence of SEQ ID NO: 12. 2. The kit of claim 1, wherein at least one label associated with the probe contains a radioactive isotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical compound, or a combination thereof. 3. The kit according to claim 1, containing a matrix containing a probe. 4. The set according to claim 2, in which the matrix is a multi-well plate. 5. The kit of claim 1, further comprising a human albumin (hALB) probe containing the nucleotide sequence of SEQ ID NO: 22, and at least one tag associated with the hALB probe, a first hALB primer containing the nucleotide sequence of SEQ ID NO: 23 , and a second primer hALB containing the nucleotide sequence of SEQ ID NO: 24. 6. The kit of claim 5, wherein at least one label associated with the hALB probe contains a radioactive isotope, an enzyme substrate, a chemiluminescent agent, a fluorophore, a fluorescence quencher, an enzyme, a chemical compound, or a combination thereof. 7. The kit of claim 1, further comprising a reference gene probe and at least one tag associated with the reference gene probe, a first reference gene primer and a second reference gene primer. 8. A method for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising: amplifying nucleic acids from a CAR T cell with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids; amplifying nucleic acids from the CAR T cell with a first hALB primer containing the nucleotide sequence of SEQ ID NO: 23 and a second hALB primer containing the nucleotide sequence of SEQ ID NO: 24, thereby obtaining amplified hALB nucleic acids; detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe; detecting hybridization between the amplified hALB nucleic acids and an hALB probe comprising the nucleotide sequence of SEQ ID NO: 22 by means of a reference signal from at least one tag associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal. 9. Method for quantitative determination of transgene integration into a T cell with a chimeric antigen - 69 046825 receptor (CAR), including: contacting nucleic acids from a 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 contains the nucleotide sequence of SEQ ID NO: 11, the second CAR primer contains the nucleotide sequence of SEQ ID NO: 12, the first hALB primer contains the nucleotide sequence of SEQ ID NO: 23, the second hALB primer contains the nucleotide sequence of SEQ ID NO: 24; amplifying the CAR nucleic acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acids; amplifying hALB nucleic acids with a first hALB primer and a second hALB primer, thereby obtaining amplified hALB nucleic acids; detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe; detecting hybridization between the amplified hALB nucleic acids and the hALB probe through a reference signal from at least one label associated with the hALB probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal. 10. The method of claim 8 or 9, wherein detecting hybridization between the amplified CAR nucleic acids and the CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the label, bound to the CAR probe, prior to hybridization. 11. The method of claim 8 or 9, wherein detecting hybridization between the amplified hALB nucleic acid molecules and the hALB probe includes detecting a change in the target signal from at least one label associated with the hALB probe during or after hybridization relative to the target signal from the label bound to the hALB probe prior to hybridization. 12. The method according to claim 8 or 9, wherein the amplification includes a polymerase chain reaction (PCR). 13. The method of claim 12, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), ligase chain reaction reaction (LCR) or transcription-mediated amplification (TMA). 14. The method of claim 8, wherein at least one label associated with the CAR probe comprises a fluorophore. 15. The method of claim 8, wherein at least one label associated with the hALB probe contains a fluorophore. 16. A method for quantifying transgene integration into a chimeric antigen receptor (CAR) T cell, comprising: amplifying nucleic acids from a CAR T cell with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids; amplifying nucleic acids from the CAR T cell with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids; detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe; detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal. 17. A method for quantitatively determining the integration of a transgene into a chimeric antigen receptor (CAR) T cell, comprising: contacting nucleic acids 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, wherein the first CAR primer contains the nucleotide sequence of SEQ ID NO: 11 and the second CAR primer contains the nucleotide sequence of SEQ ID NO: 11 ID NO: 12; amplifying the CAR nucleic acids with a first CAR primer and a second CAR primer, thereby obtaining amplified CAR nucleic acids; amplifying nucleic acids of the first reference gene with a first reference gene primer and a second reference gene primer, thereby obtaining amplified nucleic acids - 70 046825 reference gene acid; detection of hybridization between amplified CAR nucleic acids and probe a CAR comprising the nucleotide sequence of SEQ ID NO: 10, by means of a target signal from at least one tag associated with a CAR probe; detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal. 18. The method of claim 16 or 17, wherein detecting hybridization between the amplified CAR nucleic acids and the CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the label, bound to the CAR probe, prior to hybridization. 19. The method of claim 16 or 17, wherein detecting hybridization between the amplified reference gene nucleic acid molecules and the reference gene probe includes detecting a change in the target signal from at least one label associated with the reference gene probe during or after hybridization relative to the target signal from the label associated with the reference gene probe prior to hybridization. 20. The method of claim 16 or 17, wherein the amplification involves polymerase chain reaction (PCR). 21. The method of claim 20, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), ligase chain reaction reaction (LCR) or transcription-mediated amplification (TMA). 22. The method of claim 16, wherein at least one label associated with the CAR probe comprises a fluorophore. 23. The method of claim 16, wherein at least one tag associated with the reference gene probe contains a fluorophore. 24. A method for producing a chimeric antigen receptor (CAR) T cell, comprising: introducing a CAR transgene into a T cell to obtain a T cell-integrated transgene; definition of CAR transgene integration, including: amplifying nucleic acids from a T cell with an integrated transgene with a first CAR primer containing the nucleotide sequence of SEQ ID NO: 11 and a second CAR primer containing the nucleotide sequence of SEQ ID NO: 12, thereby obtaining amplified CAR nucleic acids; amplifying nucleic acids from the transgene-integrated T cell with a first reference gene primer and a second reference gene primer, thereby obtaining amplified reference gene nucleic acids; detecting hybridization between the amplified CAR nucleic acids and a CAR probe comprising the nucleotide sequence of SEQ ID NO: 10 by means of a targeting signal from at least one tag associated with the CAR probe; detecting hybridization between the amplified reference gene nucleic acids and the reference gene probe through a reference signal from at least one label associated with the reference gene probe; and quantifying the copy number of the transgene by comparing the target signal with a reference signal; and obtaining a CAR T cell containing at least one copy of the integrated CAR transgene. 25. The method of claim 24, wherein detecting hybridization between the amplified CAR nucleic acids and the CAR probe includes detecting a change in the target signal from at least one label associated with the CAR probe during or after hybridization relative to the target signal from the label associated with with the CAR probe, prior to hybridization. 26. The method of claim 24, wherein detecting hybridization between the amplified reference gene nucleic acid molecules and the reference gene probe comprises detecting a change in the target signal from at least one label associated with the reference gene probe during or after hybridization relative to the target signal from label associated with the reference gene probe prior to hybridization. 27. The method of claim 24, wherein the amplification comprises a polymerase chain reaction (PCR). 28. The method of claim 27, wherein the PCR is real-time PCR, reverse transcriptase polymerase chain reaction (RT-PCR), real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), ligase chain reaction reaction (LCR) or transcription-mediated amplification (TMA). -