WO2023072953A1 - Cell lines for producing a retroviral vector encoding a car - Google Patents

Abstract

Cells transduced with a retroviral vector comprising a nucleic acid encoding a CD30.CAR are disclosed. Also disclosed are retroviral vector supernatants obtained from a culture of transduced cells, CAR-T cells, methods of producing CAR T-cells, and cell banks.

Description

Cell Lines for Producing a Retroviral Vector Encoding a CAR

This application claims priority from US 63/272061 filed 26 October 2021 , the contents and elements of which are herein incorporated by reference for all purposes.

Field of the Invention

The present invention relates to retroviral vector producer cell lines and particularly, although not exclusively, to CD30.CAR vector producer cell lines.

Background

The generation of Chimeric Antigen Receptor (CAR) T cells commonly involves the use of a retrovirus to induce T cells, either from the subject undergoing treatment (autologous T cells) or from a donor (allogeneic T cells), to express the Chimeric Antigen Receptor. Production of retrovirus commonly involves the transfection of nucleic acid encoding retroviral genome into producer cells (sometimes known as “helper” cell lines or “packaging” cells). Producer cells have been designed to provide all viral proteins but not to package or transmit the RNAs encoding these functions. Retroviral vectors produced by packaging cells can transduce cells but cannot replicate further (i.e. they are not replication competent retroviral (RCR) vectors).

Clinical trials currently in progress involve the use of T cells that express a CAR that targets CD30 (referred to herein as CD30.CAR T cells). These T cells are currently prepared using viral supernatant obtained from PG-13 producer cells. However, PG-13 producer cells are murine in origin, and thus there is a need to develop a producer cell line for robust long-term production of CD30.CAR retrovirus that is of human origin.

The present invention has been devised in light of the above considerations.

Summary of the Invention

In a first aspect, the present disclosure provides a HEK293Vec-Galv cell transduced with a retroviral vector comprising a nucleic acid encoding a CD30.CAR.

In a second aspect, the present disclosure provides a retroviral vector supernatant obtained from a culture of HEK293Vec-Galv cells transduced with a retroviral vector comprising a nucleic acid encoding a CD30.CAR. In a third aspect, the present disclosure provides a CD30.CAR-T cell produced using a retroviral supernatant obtained from a culture of HEK293Vec-Galv cells transduced with a retroviral vector comprising a nucleic acid encoding a CD30.CAR.

In a fourth aspect, the present disclosure provides a method comprising (a) obtaining a retroviral vector supernatant from a culture of HEK293Vec-Galv cells, wherein the HEK293Vec-Galv cells have been transduced with a retroviral vector comprising a nucleic acid encoding a CD30.CAR; (b) contacting a T- cell or T-cell precursor cell, optionally a PBMC, with the retroviral vector supernatant; and (c) expanding the T-cell or T-cell precursor cell from (b) to obtain a CD30.CAR-T cell.

In some embodiments, the retroviral supernatant from (a) is diluted before contacting with the T-cell or T- cell precursor cell in (b).

In some embodiments, the retroviral vector supernatant is diluted at least 1 :50.

In some embodiments, the CD30.CAR-T cell obtained in (c) has a vector copy number (VCN) of <5.

In some embodiments, the method further comprises (d) cryogenically storing the CD30.CAR-T cell obtained in (c).

In some embodiments, the method further comprises harvesting and washing the CD30.CAR-T cell obtained in (c).

In some embodiments, the T-cell or T-cell precursor cell is expanded in the presence of IL-7 and IL-15.

In a fifth aspect, the present disclosure provides a CD30.CAR-T cell obtained by the method according to the present disclosure.

In a sixth aspect, the present disclosure provides a method comprising (a) transducing a HEK293Vec- Galv cell with a retroviral vector comprising a nucleic acid encoding a CD30.CAR, (b) culturing the transduced HEK293Vec-Galv cells, (c) obtaining retroviral vector supernatant comprising a nucleic acid encoding a CD30.CAR from the cell culture; and (d) diluting the retroviral vector supernatant.

In some embodiments, the HEK293Vec-Galv cells are cultured for 3 days or less.

In some embodiments, the retroviral vector supernatant is diluted at least 1 :50.

In a seventh aspect, the present disclosure provides a retroviral vector supernatant obtained by the method according to the present disclosure. In some embodiments, the CAR comprises a HRS3 scFv.

In some embodiments, the CAR is encoded by SEQ ID NO: 37.

In some embodiments, the retroviral vector is derived from a gammaretrovirus. In some embodiments, the gammaretrovirus is gibbon ape leukemia virus.

In some embodiments, the retroviral vector is pSFG_CD30-CAR.

In some embodiments, the methods of the present disclosure further comprise a step of storing the supernatant at -80° C .

In an eighth aspect, the present disclosure provides a method for producing a chimeric antigen receptor T cell, the method comprising modifying an immune cell to express a chimeric antigen receptor (CAR) by exposing the virus-specific immune cell to a retroviral vector supernatant according to the present disclosure.

In some embodiments, the immune cell is a virus-specific immune cell or virus-specific T cell.

In some embodiments, the virus-specific immune cell or virus-specific T cell is specific for Epstein-Barr virus (EBV).

In a ninth aspect, the present disclosure provides a virus-specific immune cell obtained by the method according to the present disclosure, wherein the vector copy number (VCN) is <5.

In a tenth aspect, the present disclosure provides a cell bank derived from an individual not suffering from lymphoma, wherein cells in the cell bank comprise virus-specific T cells obtained by the method of the present disclosure.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures:

Figure 1. Cell growth rates for activated T cells transduced with the viruses produced from transduced HEK293Vec-Galv clones c4, c15, and c115 at neat concentrations. The un-transduced cells (ATC) were cultured at the same time for comparison. The cell number was 0.5x10⁶ on day 3.

Figure 2. Transduction rate as examined by CD30 expression by flow cytometry in three cell clone c4, c15, and c115, and from a transient producer clone, Galv is shown. The virus materials in the form of tissue culture supernatants from these cells were used under the neat, 1 :5, and 1 :25 diluted condition for the transduction in cell culture plates coated with RetroNectin. Y axis shows the CD30.CAR expressing cells in the live cell population (transduction rate). The un-transduced cells are used as the negative control.

Figure 3. Cytotoxicity of CD30.CAR transduced T cells on Farage cells. The cytolysis measured by xCelligence® shows the cytotoxicity of Farage cells which were tethered to the xCelligence® plate surface. Y axis is the cytolysis percentage of the total Farage cells compared to the control wells under each condition. For example, 70% cytolysis indicates that the biosensor detects only 30% of intact and adherent Farage cells remaining in the well, compared to the control wells.

Figure 4. Vector copy number of integrated retroviral vectors in CD30.CAR transduced T cells. The virus dilution conditions used in the transduction procedure were indicated in the figure. For the cells transduced by c115-produced virus, the 1 :25 and 1 OO samples were tested in the same assay while the sample with the undiluted virus was tested in a later ddPCR assay.

Figure 5. ATCs transduced with GMP retroviral lot from the 293 based stable clone MOB were examined for CD30.CAR expression, vector copy number and potency in terms of cytotoxicity and IFNy release. The virus dilution conditions used in the transduction procedure were indicated in the figure. The transduction rates of the final cell products are plotted in the upper panels and the cytotoxicity and IFNy release data for the same samples in the corresponding two middle panels. Vector copy number in the T cells for different dilutions of viral vector is shown in the bottom panels.

Figure 6. Study Design for Comparison of Cell Product Quality Attributes Using Two Sources of CD30.CAR Vector PG13VG and 293VG.

Figure 7. Full sequence of the pSFG_CD30-CAR.

Detailed Description of the Invention

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

The inventors have developed a retroviral vector producer cell which is capable of producing retroviral vectors suitable for the manufacture of Chimeric Antigen Receptor (CAR) T cells.

Chimeric antigen receptors

CARs comprise an antigen-binding domain linked via a transmembrane domain to a signalling domain. An optional hinge or spacer domain may provide separation between the antigen-binding domain and transmembrane domain and may act as a flexible linker. When expressed by a cell, the antigen-binding domain is provided in the extracellular space, and the signalling domain is intracellular. The antigen-binding domain mediates binding to the target antigen for which the CAR is specific. The antigen-binding domain of a CAR may be based on the antigen-binding region of an antibody which is specific for the antigen to which the CAR is targeted. For example, the antigen-binding domain of a CAR may comprise amino acid sequences for the complementarity-determining regions (CDRs) of an antibody which binds specifically to the target antigen. The antigen-binding domain of a CAR may comprise or consist of the light chain and heavy chain variable region amino acid sequences of an antibody which binds specifically to the target antigen. The antigen-binding domain may be provided as a single chain variable fragment (scFv) comprising the sequences of the light chain and heavy chain variable region amino acid sequences of an antibody. Antigen-binding domains of CARs may target antigens based on other protein:protein interactions, such as ligand:receptor binding; for example an IL-13Ra2-targeted CAR has been developed using an antigen-binding domain based on IL-13 (see e.g. Kahlon etal. 2004 Cancer Res 64(24): 9160-9166).

The transmembrane domain is provided between the antigen-binding domain and the signalling domain of the CAR. The transmembrane domain provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding domain in the extracellular space and signalling domain inside the cell. Transmembrane domains of CARs may be derived from transmembrane region sequences for cell membrane-bound proteins (e.g. CD28, CD8, etc.).

Throughout this specification, polypeptides, domains and amino acid sequences which are ‘derived from’ a reference polypeptide/domain/amino acid sequence have at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference polypeptide/domain/amino acid sequence. Polypeptides, domains and amino acid sequences which are ‘derived from’ a reference polypeptide/domain/amino acid sequence preferably retain the functional and/or structural properties of the reference polypeptide/domain/amino acid sequence.

By way of illustration, an amino acid sequence derived from the intracellular domain of CD28 may comprise an amino acid sequence having 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the intracellular domain of CD28, e.g. as shown in SEQ ID NO:26. Furthermore, an amino acid sequence derived from the intracellular domain of CD28 preferably retains the functional properties of the amino acid sequence of SEQ ID NO:26, i.e. the ability to activate CD28-mediated signalling.

The amino acid sequence of a given polypeptide or domain thereof can be retrieved from, or determined from a nucleic acid sequence retrieved from, databases known to the person skilled in the art. Such databases include GenBank, EMBL and UniProt.

The signalling domain comprises amino acid sequences required for activation of immune cell function. The CAR signalling domains may comprise the amino acid sequence of the intracellular domain of CD3- , which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing cell. Signalling domains comprising sequences of other ITAM- containing proteins have also been employed in CARs, such as domains comprising the ITAM containing region of FcyRI (Haynes etal., 2001 J Immunol 166(1): 182-187). CARs comprising a signalling domain derived from the intracellular domain of CD3- are often referred to as first generation CARs.

The signalling domains of CARs typically also comprise the signalling domain of a costimulatory protein (e.g. CD28, 4-1 BB etc.), for providing the costimulation signal necessary for enhancing immune cell activation and effector function. CARs having a signalling domain including additional costimulatory sequences are often referred to as second generation CARs. In some cases, CARs are engineered to provide for costimulation of different intracellular signalling pathways. For example, CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1 BB costimulation triggers signalling is through TNF receptor associated factor (TRAF) adaptor proteins. Signalling domains of CARs therefore sometimes contain costimulatory sequences derived from signalling domains of more than one costimulatory molecule. CARs comprising a signalling domain with multiple costimulatory sequences are often referred to as third generation CARs.

An optional hinge or spacer region may provide separation between the antigen-binding domain and the transmembrane domain and may act as a flexible linker. Such regions may be or comprise flexible domains allowing the binding moiety to orient in different directions, which may e.g. be derived from the CH1-CH2 hinge region of IgG.

Through engineering to express a CAR specific for a particular target antigen, immune cells (typically T cells, but also other immune cells such as NK cells) can be directed to kill cells expressing the target antigen. Binding of a CAR-expressing T cell (CAR-T cell) to the target antigen for which it is specific triggers intracellular signalling, and consequently activation of the T cell. The activated CAR-T cell is stimulated to divide and produce factors resulting in killing of the cell expressing the target antigen.

Antigen-binding domain

An “antigen-binding domain” refers to a domain which is capable of binding to a target antigen. The target antigen may e.g. be a peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Antigen-binding domains according to the present disclosure may be derived from an antibody/antibody fragment (e.g. Fv, scFv, Fab, single chain Fab (scFab), single domain antibodies (e.g. VhH), etc.) directed against the target antigen, or another target antigen-binding molecule (e.g. a target antigen-binding peptide or nucleic acid aptamer, ligand or other molecule).

In some embodiments, the antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen. In some embodiments, the domain capable of binding to a target antigen comprises or consists of an antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (/.e. a singledomain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto etal., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma etal., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

The antigen-binding domains of the present disclosure generally comprise a VH and a VL of an antibody capable of specific binding to the target antigen. Antibodies generally comprise six complementaritydetermining regions CDRs; three in the heavy chain variable region (VH): HC-CDR1 , HC-CDR2 and HC- CDR3, and three in the light chain variable region (VL): LC-CDR1, LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target antigen. The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VHs comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VLs comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.

VH and VL sequences may be provided in any suitable format provided that the antigen-binding domain can be linked to the other domains of the CAR. Formats contemplated in connection with the antigenbinding domain of the present disclosure include those described in Carter, Nat. Rev. Immunol (2006), 6: 343-357, such as scFv, dsFV, (scFv)2 diabody, triabody, tetrabody, Fab, minibody, and F(ab)2 formats.

In some embodiments, the antigen-binding domain comprises the CDRs of an antibody/antibody fragment which is capable of binding to the target antigen. In some embodiments, the antigen-binding domain comprises the VH region and the VL region of an antibody/antibody fragment which is capable of binding to the target antigen. A moiety comprised of the VH and a VL of an antibody may also be referred to herein as a variable fragment (Fv). The VH and VL may be provided on the same polypeptide chain, and joined via a linker sequence; such moieties are referred to as single-chain variable fragments (scFvs). Suitable linker sequences for the preparation of scFv are known to the skilled person, and may comprise serine and glycine residues.

In some embodiments, the antigen-binding domain comprises, or consists of, Fv capable of binding to the target antigen. In some embodiments, the antigen-binding domain comprises, or consists of, a scFv capable of binding to the target antigen.

In aspects and embodiments of the present disclosure, the target antigen is CD30. Accordingly, in some aspects and embodiments of the present disclosure the antigen-binding domain is a CD30-binding domain.

CD30 (also known as TNFRSF8) is the protein identified by UniProt: P28908. CD30 is a single pass, type I transmembrane glycoprotein of the tumor necrosis factor receptor superfamily. CD30 structure and function is described e.g. in van der Weyden etal., Blood Cancer Journal (2017) 7: e603 and Muta and Podack Immunol. Res. (2013) 57(1 -3): 151 -8, both of which are hereby incorporated by reference in their entirety. Alternative splicing of mRNA encoded by the human TNFRSF8 gene yields three isoforms: isoform 1 (‘long’ isoform; UniProt: P28908-1 , v1; SEQ ID NO:1), isoform 2 (‘cytoplasmic’, ‘short’ or ‘C30V’ isoform, UniProt: P28908-2; SEQ ID NO:2) in which the amino acid sequence corresponding to positions 1 to 463 of SEQ ID NO:1 are missing, and isoform 3 (UniProt: P28908-3; SEQ ID NO:3) in which the amino acid sequence corresponding to positions 1 to 111 and position 446 of SEQ ID NO:1 are missing. The N- terminal 18 amino acids of SEQ ID NO:1 form a signal peptide (SEQ ID NO:4), which is followed by a 367 amino acid extracellular domain (positions 19 to 385 of SEQ ID NO:1, shown in SEQ ID NO:5), a 21 amino acid transmembrane domain (positions 386 to 406 of SEQ ID NO:1, shown in SEQ ID NO:6), and a 189 amino acid cytoplasmic domain (positions 407 to 595 of SEQ ID NO:1, shown in SEQ ID NO:7).

In this specification “CD30” refers to CD30 from any species and includes CD30 isoforms, fragments, variants or homologues from any species. As used herein, a “fragment”, “variant” or “homologue” of a reference protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein (e.g. a reference isoform). In some embodiments, fragments, variants, isoforms and homologues of a reference protein may be characterised by ability to perform a function performed by the reference protein.

In some embodiments, the CD30 is from a mammal (e.g. a primate (rhesus, cynomolgous, or human) and/or a rodent (e.g. rat or murine) CD30). In preferred embodiments the CD30 is a human CD30. Isoforms, fragments, variants or homologues may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature CD30 isoform from a given species, e.g. human. A fragment of CD30 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 590 amino acids, and may have a maximum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 595 amino acids.

In some embodiments, the CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1 , 2 or 3.

In some embodiments, the CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.

In some embodiments, a fragment of CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5 or 19.

The CD30-binding domain of the CAR of the present disclosure preferably displays specific binding to CD30 or a fragment thereof. The CD30-binding domain of the CAR of the present disclosure preferably displays specific binding to the extracellular domain of CD30. The CD30-binding domain may be derived from an anti-CD30 antibody or other CD30-binding agent, e.g. a CD30-binding peptide or CD30-binding small molecule.

The CD30-binding domain may be derived from the antigen-binding moiety of an anti-CD30 antibody.

Anti-CD30 antibodies include HRS3 and HRS4 (described e.g. in Hornbach etal., Scand J Immunol

(1998) 48(5):497-501), HRS3 derivatives described in Schlapschy etal., Protein Engineering, Design and Selection (2004) 17(12): 847-860, BerH2 (MBL International Cat# K0145-3, RRID:AB_590975), SGN-30 (also known as cAC10, described e.g. in Forero-Torres etal., Br J Haematol (2009) 146:171-9), MDX- 060 (described e.g. in Ansell et al., J Clin Oncol (2007) 25:2764—9; also known as 5F11 , iratumumab), and MDX-1401 (described e.g. in Cardarelli etal., Clin Cancer Res. (2009) 15(10):3376-83), and anti- CD30 antibodies described in WO 2020/068764 A1 , WO 2003/059282 A2, WO 2006/089232 A2, WO 2007/084672 A2, WO 2007/044616 A2, WO 2005/001038 A2, US 2007/166309 A1 , US 2007/258987 A1 , WO 2004/010957 A2 and US 2005/009769 A1.

In some embodiments a CD30-binding domain according to the present disclosure comprises the CDRs of an anti-CD30 antibody. In some embodiments a CD30-binding domain according to the present disclosure comprises the VH and VL regions of an anti-CD30 antibody. In some embodiments a CD30- binding domain according to the present disclosure comprises a scFv comprising the VH and VL regions of an anti-CD30 antibody.

There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia etal., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter etal., Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674. The CDRs and FRs of the VH regions and VL regions of the antibodies described herein are defined according to VBASE2.

In some embodiments the antigen-binding domain of the present disclosure comprises: a VH incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:8 HC-CDR2 having the amino acid sequence of SEQ ID NO:9 HC-CDR3 having the amino acid sequence of SEQ ID NO:10, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC- CDR2, or HC-CDR3 are substituted with another amino acid; and a VL incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:11 LC-CDR2 having the amino acid sequence of SEQ ID NO:12 LC-CDR3 having the amino acid sequence of SEQ ID NO:13, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC- CDR2, or LC-CDR3 are substituted with another amino acid. In some embodiments the antigen-binding domain comprises: a VH comprising, or consisting of, an amino acid sequence having at least 80% sequence identity (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO:14; and a VL comprising, or consisting of, an amino acid sequence having at least 80% sequence identity (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO:15.

In some embodiments, a CD30-binding domain may comprise or consist of a single chain variable fragment (scFv) comprising a VH sequence and a VL sequence as described herein. The VH sequence and VL sequence may be covalently linked. In some embodiments, the VH and the VL sequences are linked by a flexible linker sequence, e.g. a flexible linker sequence as described herein. The flexible linker sequence may be joined to ends of the VH sequence and VL sequence, thereby linking the VH and VL sequences. In some embodiments the VH and VL are joined via a linker sequence comprising, or consisting of, the amino acid sequence of SEQ ID NO: 16 or 17.

In some embodiments, the CD30-binding domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.

In some embodiments the CD30-binding domain is capable of binding to CD30, e.g. in the extracellular domain of CD30. In some embodiments, the CD30-binding domain is capable of binding to the epitope of CD30 which is bound by antibody HRS3, e.g. within the region of amino acid positions 185-335 of human CD30 numbered according to SEQ ID NO:1, shown in SEQ ID NO:19 (Schlapschy etal., Protein Engineering, Design and Selection (2004) 17(12): 847-860, hereby incorporated by reference in its entirety).

Transmembrane domain

The CAR of the present disclosure comprises a transmembrane domain. A transmembrane domain refers to any three-dimensional structure formed by a sequence of amino acids which is thermodynamically stable in a biological membrane, e.g. a cell membrane. In connection with the present disclosure, the transmembrane domain may be an amino acid sequence which spans the cell membrane of a cell expressing the CAR.

The transmembrane domain may comprise or consist of a sequence of amino acids which forms a hydrophobic alpha helix or beta-barrel. The amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of a transmembrane domain of a protein comprising a transmembrane domain. Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as TMHMM (Krogh etal., 2001 J Mol Biol 305: 567-580).

In some embodiments, the amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of the transmembrane domain of a protein expressed at the cell surface. In some embodiments the protein expressed at the cell surface is a receptor or ligand, e.g. an immune receptor or ligand. In some embodiments the amino acid sequence of the transmembrane domain may be, or may be derived from, the amino acid sequence of the transmembrane domain of one of ICOS, ICOSL, CD86, CTLA-4, CD28, CD80, MHC class I a, MHC class II a, MHC class II 0, CD3E, CD36, CD3y, CD3- , TCRa TCR0, CD4, CD8a, CD80, CD40, CD40L, PD-1, PD-L1 , PD-L2, 4-1 BB, 4-1 BBL, 0X40, OX40L, GITR, GITRL, TIM-3, Galectin 9, LAG3, CD27, CD70, LIGHT, HVEM, TIM-4, TIM-1, ICAM1, LFA-1 , LFA-3, CD2, BTLA, CD160, LILRB4, LILRB2, VTCN1, CD2, CD48, 2B4, SLAM, CD30, CD30L, DR3, TL1A, CD226, CD155, CD112 and CD276. In some embodiments, the transmembrane is, or is derived from, the amino acid sequence of the transmembrane domain of CD28, CD3- , CD8a, CD80 or CD4. In some embodiments, the transmembrane is, or is derived from, the amino acid sequence of the transmembrane domain of CD28.

In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:20.

In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21.

In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:22.

Signalling domain

The chimeric antigen receptor of the present disclosure comprises a signalling domain. The signalling domain provides sequences for initiating intracellular signalling in cells expressing the CAR.

ITAM-containing sequence:

The signalling domain comprises ITAM-containing sequence. An ITAM-containing sequence comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs comprise the amino acid sequence YXXL/I (SEQ ID NO:23), wherein “X” denotes any amino acid. In ITAM-containing proteins, sequences according to SEQ ID NO:23 are often separated by 6 to 8 amino acids; YXXL/I (X)e-8 YXXL/I (SEQ ID NO:24). When phosphate groups are added to the tyrosine residue of an ITAM by tyrosine kinases, a signalling cascade is initiated within the cell. In some embodiments, the signalling domain comprises one or more copies of an amino acid sequence according to SEQ ID NO:23 or SEQ ID NO:24. In some embodiments, the signalling domain comprises at least 1 , 2, 3, 4, 5 or 6 copies of an amino acid sequence according to SEQ ID NO:23. In some embodiments, the signalling domain comprises at least 1 , 2, or 3 copies of an amino acid sequence according to SEQ ID NO:24.

In some embodiments, the signalling domain comprises an amino acid sequence which is, or which is derived from, the amino acid sequence of an ITAM-containing sequence of a protein having an ITAM- containing amino acid sequence. In some embodiments the signalling domain comprises an amino acid sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of one of CD3- , FcyRI, CD3E, CD36, CD3Y, CD79a, CD790, FCYRUA, FCYRUC, FCYRHIA, FcyRIV or DAP12. In some embodiments the signalling domain comprises an amino acid sequence which is, or which is derived from, the intracellular domain of CD3- .

In some embodiments, the signalling domain comprises an amino acid sequence which comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25.

Costimulatory sequence:

The signalling domain may additionally comprise one or more costimulatory sequences. A costimulatory sequence is an amino acid sequence which provides for costimulation of the cell expressing the CAR of the present disclosure. Costimulation promotes proliferation and survival of a CAR-expressing cell upon binding to the target antigen, and may also promote cytokine production, differentiation, cytotoxic function and memory formation by the CAR-expressing cell. Molecular mechanisms of T cell costimulation are reviewed in Chen and Flies, (2013) Nat Rev Immunol 13(4):227-242.

A costimulatory sequence may be, or may be derived from, the amino acid sequence of a costimulatory protein. In some embodiments the costimulatory sequence is an amino acid sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of a costimulatory protein.

Upon binding of the CAR to the target antigen, the costimulatory sequence provides costimulation to the cell expressing the CAR of the kind which would be provided by the costimulatory protein from which the costimulatory sequence is derived upon ligation by its cognate ligand. By way of example in the case of a CAR comprising a signalling domain comprising a costimulatory sequence derived from CD28, binding to the target antigen triggers signalling in the cell expressing the CAR of the kind that would be triggered by binding of CD80 and/or CD86 to CD28. Thus, a costimulatory sequence is capable of delivering the costimulation signal of the costimulatory protein from which the costimulatory sequence is derived. In some embodiments, the costimulatory protein may be a member of the B7-CD28 superfamily (e.g. CD28, ICOS), or a member of the TN F receptor superfamily (e.g. 4-1 BB, 0X40, CD27, DR3, GITR, CD30, HVEM). In some embodiments, the costimulatory sequence is, or is derived from, the intracellular domain of one of CD28, 4-1 BB, ICOS, CD27, 0X40, HVEM, CD2, SLAM, TIM-1, CD30, GITR, DR3, CD226 and LIGHT. In some embodiments, the costimulatory sequence is, or is derived from, the intracellular domain of CD28.

In some embodiments the signalling domain comprises more than one non-overlapping costimulatory sequences. In some embodiments the signalling domain comprises 1 , 2, 3, 4, 5 or 6 costimulatory sequences. Plural costimulatory sequences may be provided in tandem.

Whether a given amino acid sequence is capable of initiating signalling mediated by a given costimulatory protein can be investigated e.g. by analysing a correlate of signalling mediated by the costimulatory protein (e.g. expression/activity of a factor whose expression/activity is upregulated or downregulated as a consequence of signalling mediated by the costimulatory protein).

Costimulatory proteins upregulate expression of genes promoting cell growth, effector function and survival through several transduction pathways. For example, CD28 and ICOS signal through phosphatidylinositol 3 kinase (PI3K) and AKT to upregulate expression of genes promoting cell growth, effector function and survival through NF-KB, mTOR, NFAT and AP1/2. CD28 also activates AP1/2 via CDC42/RAC1 and ERK1/2 via RAS, and ICOS activates C-MAF. 4-1 BB, 0X40, and CD27 recruit TNF receptor associated factor (TRAF) and signal through MAPK pathways, as well as through PI3K.

In some embodiments the signalling domain comprises a costimulatory sequence which is, or which is derived from CD28.

In some embodiments, the signalling domain comprises a costimulatory sequence which comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:26.

Kofler etal. Mol. Then (2011) 19: 760-767 describes a variant CD28 intracellular domain in which the lek kinase binding site is mutated in order to reduce induction of IL-2 production on CAR ligation, in order to minimise regulatory T cell-mediated suppression of CAR-T cell activity. The amino acid sequence of the variant CD28 intracellular domain is shown in SEQ ID NO:27.

In some embodiments, the signalling domain comprises a costimulatory sequence which comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:28.

In some embodiments the signalling domain comprises a costimulatory sequence which is, or which is derived from 4-1 BB.

Hinge region

The CAR may further comprise a hinge region. The hinge region may be provided between the antigenbinding domain and the transmembrane domain. The hinge region may also be referred to as a spacer region. A hinge region is an amino acid sequence which provides for flexible linkage of the antigenbinding and transmembrane domains of the CAR.

The presence, absence and length of hinge regions has been shown to influence CAR function (reviewed e.g. in Dotti etal., Immunol Rev (2014) 257(1) supra).

In some embodiments, the CAR comprises a hinge region which comprises, or consists of, an amino acid sequence which is, or which is derived from, the CH1-CH2 hinge region of human lgG1 , a hinge region derived from CD8a, e.g. as described in WO 2012/031744 A1, or a hinge region derived from CD28, e.g. as described in WO 2011/041093 A1. In some embodiments, the CAR comprises a hinge region derived from the CH1-CH2 hinge region of human lgG1.

In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:29 or 30.

In some embodiments, the CAR comprises a hinge region derived from the CH1-CH2 hinge region of human lgG4.

In some embodiments, the CAR comprises a hinge region which comprises, or consists of, an amino acid sequence which is, or which is derived from, the CH2-CH3 region (/.e. the Fc region) of human IgG 1.

In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:31.

Hornbach etal., Gene Therapy (2010) 17:1206-1213 describes a variant CH2-CH3 region for reduced activation of FcyR-expressing cells such as monocytes and NK cells. The amino acid sequence of the variant CH2-CH3 region is shown in SEQ ID NO:32. In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:32.

In some embodiments, the hinge region comprises, or consists of: an amino acid sequence which is, or which is derived from, the CH1-CH2 hinge region of human lgG1, and an amino acid sequence which is, or which is derived from, the CH2-CH3 region (j.e. the Fc region) of human IgG 1.

In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:33.

Additional sequences

Signal peptide:

The CAR may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

The signal peptide may be present at the N-terminus of the CAR, and may be present in the newly synthesised CAR. The signal peptide provides for efficient trafficking of the CAR to the cell surface. Signal peptides are removed by cleavage, and thus are not comprised in the mature CAR expressed by the cell surface.

In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:34.

Linker sequences and further functional sequences:

In some embodiments the CAR comprises one or more linker sequences between the different domains (i.e. the antigen-binding domain, hinge region, transmembrane domain, signalling domain). In some embodiments the CAR comprises one or more linker sequences between subsequences of the domains (e.g. between VH and VL of an antigen-binding domain).

Linker sequences are known to the skilled person, and are described, for example in Chen etal., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen etal., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues. In some embodiments, the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-20, 1-30, 1-40 or 1-50 amino acids.

In some embodiments a linker sequence comprises, or consists, of the amino acid sequence shown in SEQ ID NO:16. In some embodiments a linker sequence comprises, or consists, of 1 , 2, 3, 4 or 5 tandem copies of the amino acid sequence shown in SEQ ID NO:16.

The CARs may additionally comprise further amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection. For example, the CAR may comprise a sequence encoding a His, (e.g. 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus. In some embodiments the CAR comprises a detectable moiety, e.g. a fluorescent, luminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.

Particular exemplary CARs

In some embodiments of the present disclosure, the CAR comprises, or consists of:

An antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:18;

A hinge region comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:33;

A transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:20; and

A signalling domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:28.

In some embodiments of the present disclosure, the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:35 or 36. In some embodiments, the CAR is selected from an embodiment of a CD30-specific CAR described in Hornbach et al. Cancer Res. (1998) 58(6): 1116-9, Hornbach et al. Gene Therapy (2000) 7:1067-1075, Hornbach etal. J Immunother. (1999) 22(6):473-80, Hornbach etal. Cancer Res. (2001) 61:1976-1982, Hornbach etal. J Immunol (2001) 167:6123-6131, Savoldo etal. Blood (2007) 110(7):2620-30, Koehler et al. Cancer Res. (2007) 67(5):2265-2273, Di Stasi etal. Blood (2009) 113(25):6392-402, Hornbach etal. Gene Therapy (2010) 17:1206-1213, Chmielewski etal. Gene Therapy (2011) 18:62-72, Kofler ef a/. Mol. Then (2011) 19(4):760-767, Gilham, Abken and Pule. Trends in Mol. Med. (2012) 18(7):377-384, Chmielewski etal. Gene Therapy (2013) 20:177-186, Hornbach etal. Mol. Then (2016) 24(8): 1423-1434, Ramos etal. J. Clin. Invest. (2017) 127(9):3462-3471, WO 2015/028444 A1 or WO 2016/008973 A1, all of which are hereby incorporated by reference in their entirety.

Production of retroviral vector supernatant using HEK293Vec-Galv cells

HEK293 cells

Human Embryonic Kidney 293 cells (HEK293 cells), also known as HEK 293, HEK-293, 293 cells or HEK cells, are immortalized cells derived from human embryonic kidney cells grown in tissue culture. HEK293 cells are particularly useful for producing exogenous proteins or viruses due to their high transfection efficiency and ability to produce high levels of proteins or viruses.

HEK293 cells or derivatives thereof (such as HEK293Vec-RD114 and HEK293Vec-Galv cells) may be used as packaging cell lines. Packaging cell lines typically stably express the viral proteins that are required for capsid production and the virion maturation of the vector, including the gag, pol and env genes. The gag gene encodes a structural precursor protein, the pol gene encodes a polymerase (reverse transcriptase), and the env gene encodes an envelope protein.

In some embodiments, a plasmid encoding a gene of interest may be transfected into the packaging cell line, which, following culture of the cells, results in the production of viral particles containing the gene of interest. In other aspects, the packaging cell line may be transduced with a viral vector comprising a gene of interest, which results in the stable production of viral particles comprising the gene of interest, thus producing a viral vector producer cell line. The resulting viral particles can then be used to transduce other cell types, such as T cells. In other embodiments, a first packaging cell line (such as HEK293Vec- RD114) may be transfected with a plasmid encoding a gene of interest, and the resulting viral particles may be used to transduce a second packaging cell line (such as HEK293Vec-Galv), thus producing a viral vector producer cell line which stably produces viral particles. The viral particles produced by the second packaging cell line or viral vector producer cell line may be used to transduce other cell types, such as T cells.

In other embodiments, a plasmid encoding a gene of interest may be co-transfected into a packaging cell line together with a plasmid encoding one or more viral proteins, in order to produce viral particles. Retroviruses

In some aspects, the viral particles produced by the viral vector producer cell line are retroviral vectors or retroviruses. In some embodiments, the retrovirus is a gamma(y)-retrovirus. Gamma-retroviruses are part of the Retroviridae family having a single-stranded RNA genome, with examples being the murine leukemia virus, the feline leukemia virus and the gibbon ape leukemia virus. Gamma-retroviruses efficiently integrate into a host genome and therefore they are useful for establishing stably-expressing cell lines.

Accordingly, in some aspects, the viral vector producer cell line is a retroviral vector producer cell line. In some embodiments, the retroviral vector producer cell line is a y-retroviral (gammaretroviral) producer cell line.

HEK293Vec-Galv cells

The retroviral vector producer cell according to the present invention may be a HEK293Vec-Galv cell. Conveniently, the HEK293Vec-Galv cell produces a retroviral vector supernatant. In some embodiments, the supernatant comprises a nucleic acid encoding a CAR, a vector encoding a CAR, a retroviral vector comprising a CAR or a virus comprising a nucleic acid encoding a CAR. In preferred embodiments, the CAR-T cells are CD30.CAR-T cells. Preferably, the CD30.CAR is encoded by a nucleic acid according to SEQ ID NO: 37.

It will be appreciated that where cells are referred to herein in the singular (i.e. “a/the cell”), pluralities/populations of such cells are also contemplated.

HEK293Vec-Galv cells are derived from Human Embryonic Kidney 293 cells (HEK293 cells).

HEK293Vec-Galv produce retroviral vectors that are pseudotyped by the gibbon ape leukemia virus envelope protein.

In some aspects, HEK293Vec-Galv cells are transfected with plasmid encoding a retrovirus. In others, the HEK293Vec-Galv cells have been transfected with plasmid encoding a retrovirus.

In some aspects, HEK293Vec-Galv cells are transduced with retroviral vector. In others, HEK293Vec- Galv cells have been transduced with retroviral vector. It will be appreciated that the term “retrovirus” can also be used to describe the retroviral vector.

Immune cells

The present disclosure concerns immune cells, in particular immune cells modified to express a chimeric antigen receptor (CAR). It will be appreciated that where cells are referred to herein in the singular (i.e. “a/the cell”), pluralities/populations of such cells are also contemplated.

The immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. A lymphocyte may be e.g. a T cell, B cell, NK cell, NKT cell or innate lymphoid cell (I LC), or a precursor thereof. The immune cell may express e.g. CD3 polypeptides (e.g. CD3y CD3E CD3 or CD36), TCR polypeptides (TCRa orTCR0), CD27, CD28, CD4 or CD8. In some embodiments, the immune cell is a T cell, e.g. a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).

Immune cells useful in the methods described herein may be obtained from any suitable source. The source may be animal or human. The source may be a non-human mammal, but is more preferably human. The source may be any gender. The source may be the patient that is to be treated with adoptive cell therapy (autologous cells). As such, the source may have been diagnosed with a disease/condition requiring treatment, may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition. In some cases, the source is a different individual to the patient that is to be treated (allogeneic cells). In such cases, the source would normally be a healthy individual, or an individual that is not known to be suffering from a disease/condition or at risk of developing/contracting such a disease/condition.

Virus-specific immune cells

An aspect of the present disclosure concerns virus-specific immune cells, in particular Epstein-Barr virus (EBV)-specific immune cells. It will be appreciated that where cells are referred to herein in the singular (i.e. “a/the cell”), pluralities/populations of such cells are also contemplated.

A “virus-specific immune cell” as used herein refers to an immune cell which is specific for a virus. A virus-specific immune cell expresses/comprises a receptor (preferably a T cell receptor) capable of recognising a peptide of an antigen of a virus (e.g. when presented by an MHO molecule). The virusspecific immune cell may express/comprise such a receptor as a result of expression of endogenous nucleic acid encoding such antigen receptor, or as a result of having been engineered to express such a receptor. The virus-specific immune cell preferably expresses/comprises a TCR specific for a peptide of an antigen of a virus.

The immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. A lymphocyte may be e.g. a T cell, B cell, NK cell, NKT cell or innate lymphoid cell (I LC), or a precursor thereof. The immune cell may express e.g. CD3 polypeptides (e.g. CD3y CD3E CD3 or CD36), TCR polypeptides (TCRa orTCR0), CD27, CD28, CD4 or CD8. In some embodiments, the immune cell is a T cell, e.g. a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).

A virus-specific T cell may display certain functional properties of a T cell in response to the viral antigen for which the T cell is specific, or in response a cell comprising/expressing the virus/antigen. In some embodiments, the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.

In some embodiments, a virus-specific T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing the virus /the viral antigen for which the T cell is specific; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with the virus/the viral antigen for which the T cell is specific, or in response to exposure to a cell comprising/expressing the virus /the viral antigen for which the T cell is specific.

Virus-specific T cells express/comprise a TCR capable of recognising a peptide of the viral antigen for which the T cell is specific when presented by the appropriate MHO molecule. Virus-specific T cells may be CD4+ T cells and/or CD8+ T cells.

The virus for which the virus-specific immune cell is specific may be any virus. For example, the virus may be a dsDNA virus (e.g. adenovirus, herpesvirus, poxvirus), ssRNA virus (e.g. parvovirus), dsRNA virus (e.g. reovirus), (+)ssRNA virus (e.g. picornavirus, togavirus), (-)ssRNA virus (e.g. orthomyxovirus, rhabdovirus), ssRNA-RT virus (e.g. retrovirus) or dsDNA-RT virus (e.g. hepadnavirus). In particular, the present disclosure contemplates viruses of the families adenoviridae, herpesviridae, papillomaviridae, polyomaviridae, poxviridae, hepadnaviridae, parvoviridae, astroviridae, caliciviridae, picornaviridae, coronaviridae, flaviviridae, togaviridae, hepeviridae, retroviridae, orthomyxoviridae, arenaviridae, bunyaviridae, filoviridae, paramyxoviridae, rhabdoviridae and reoviridae. In some embodiments the virus is selected from Epstein-Barr virus, adenovirus, Herpes simplex type 1 virus, Herpes simplex type 2 virus, Varicella-zoster virus, Human cytomegalovirus, Human herpesvirus type 8, Human papillomavirus, BK virus, JC virus, Smallpox, Hepatitis B virus, Parvovirus B19, Human Astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, Rubella virus, Hepatitis E virus, Human immunodeficiency virus, influenza virus, lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, coltivirus, and banna virus.

In some embodiments, the virus is selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).

In some embodiments, the virus-specific immune cell may be specific for a peptide/polypeptide of a virus e.g. selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV). A T cell which is specific for an antigen of a virus may be referred to herein as a virus-specific T cell (VST). A T cell which is specific for an antigen of a particular virus may be described as being specific for the relevant virus; for example, a T cell which is specific for an antigen of EBV may be referred to as an EBV-specific T cell, or “EBVST”.

Accordingly, in some embodiments the virus-specific immune cell is an Epstein-Barr virus-specific T cell (EBVST), adenovirus-specific T cell (AdVST), cytomegalovius-specific T cell (CMVST), human papilloma virus (HPVST), influenza virus-specific T cell, measles virus-specific T cell, hepatitis B virus-specific T cell (HBVST), hepatitis C virus-specific T cell (HCVST), human immunodeficiency virus-specific T cell (HI VST), lymphocytic choriomeningitis virus-specific T cell (LCMVST), or herpes simplex virus-specific T cell (HSVST).

In some preferred embodiments, the virus-specific immune cell is specific for a peptide/polypeptide of an EBV antigen. In preferred embodiments the virus-specific immune cell is an Epstein-Barr virus-specific T cell (EBVST).

EBV virology is described e.g. in Stanfield and Luftiq, FIOOORes. (2017) 6:386 and Odumade et al., Clin Microbiol Rev (2011) 24(1): 193-209, both of which are hereby incorporated by reference in their entirety.

EBV infects epithelial cells via binding of viral protein BMFR2 to f31 integrins, and binding of viral protein gH/gL with integrins av|36 and av|38. EBV infects B cells through interaction of viral glycoprotein gp350 with CD21 and/or CD35, followed by interaction of viral gp42 with MHC class II. These interactions trigger fusion of the viral envelope with the cell membrane, allowing the virus to enter the cell. Once inside, the viral capsid dissolves and the viral genome is transported to the nucleus.

EBV has two modes of replication; latent and lytic. The latent cycle does not result in production of virions, and can take place in place B cells and epithelial cells. The EBV genomic circular DNA resides in the cell nucleus as an episome and is copied by the host cell’s DNA polymerase. In latency, only a fraction of EBV's genes are expressed, in one of three different patterns known as latency programs, which produce distinct sets of viral proteins and RNAs. The latent cycle is described e.g. in Amon and Farrell, Reviews in Medical Virology (2004) 15(3): 149-56, which is hereby incorporated by reference in its entirety.

EBNA1 protein and non-coding RNA EBER are expressed in each of latency programs l-l II . Latency programs II and III further involve expression of EBNALP, LMP1, LMP2A and LMP2B proteins, and latency program III further involves expression of EBNA2, EBNA3A, EBNA3B and EBNA3C.

EBNA1 is multifunctional, and has roles in gene regulation, extrachromosomal replication, and maintenance of the EBV episomal genome through positive and negative regulation of viral promoters (Duellman et aL, J Gen Virol. (2009); 90(Pt 9): 2251-2259). EBNA2 is involved in the regulation of latent viral transcription and contributes to the immortalization of cells infected with EBV (Kempkes and Ling, Curr Top Microbiol Immunol. (2015) 391:35-59). EBNA-LP is required for transformation of native B cells, and recruits transcription factors for viral replication (Szymula et al., PLoS Pathog. (2018); 14(2):e1006890). EBNA3A, 3B and 3C interact with RBPJ to influence gene expression, contributing to survival and growth of infected cells (Wang et al., J Virol. (2016) 90(6):2906-2919). LMP1 regulates expression of genes involved in B cell activation (Chang et al., J. Biomed. Sci. (2003) 10(5): 490-504). LMP2A and LMP2B inhibit normal B cell signal transduction by mimicking the activated B cell receptor (Portis and Longnecker, Oncogene (2004) 23(53): 8619-8628). EBERs form ribonucleoprotein complexes with host cell proteins, and are proposed to have roles in cell transformation.

The latent cycle can progress according to any of latency programs I to III in B cells, and usually progresses from III to II to I. Upon infection of a resting naive B cell, EBV enters latency program III. Expression of latency III genes activates the B cell, which becomes a proliferating blast. EBV then typically progresses to latency II by restricting expression to a subset of genes, which cause differentiation of the blast to a memory B cell. Further restriction of gene expression causes EBV to enter latency I. EBNA1 expression allows EBV to replicate when the memory B cell divides. In epithelial cells, only latency II occurs.

In primary infection, EBV replicates in oropharyngeal epithelial cells and establishes Latency III, II, and I infections in B-lymphocytes. EBV latent infection of B-lymphocytes is necessary for virus persistence, subsequent replication in epithelial cells, and release of infectious virus into saliva. EBV Latency III and II infections of B-lymphocytes, Latency II infection of oral epithelial cells, and Latency II infection of NK- or T cell can result in malignancies, marked by uniform EBV genome presence and gene expression.

Latent EBV in B cells can be reactivated to switch to lytic replication. The lytic cycle results in the production of infectious virions and can take place in place B cells and epithelial cells, and is reviewed e.g. by Kenney in Chapter 25 of Arvin et aL, Human Herpesviruses: Biology, Therapy and Immunoprophylaxis; Cambridge University Press (2007), which is hereby incorporated by reference in its entirety.

Lytic replication requires the EBV genome to be linear. The latent EBV genome is episomal, and so it must be linearised for lytic reactivation. In B cells, lytic replication normally only takes place after reactivation from latency.

Immediate-early lytic gene products such as BZFL1 and BRLF1 act as transactivators, enhancing their own expression, and the expression of later lytic cycle genes.

Early lytic gene products have roles in viral replication (e.g. EBV DNA polymerase catalytic component BALF5; DNA polymerase processivity factor BMRF1, DNA binding protein BALF2, helicase BBLF4, primase BSLF1, and primase-associated protein BBLF2/3) and deoxynucleotide metabolism (e.g. thymidine kinase BXLF1 , dUTPase BORF2). Other early lytic gene products act transcription factors (e.g. BMRF1, BRRF1), have roles in RNA stability and processing (e.g. BMLF1), or are involved in immune evasion (e.g. BHRF1, which inhibits apoptosis).

Late lytic gene products are traditionally classed as those expressed after the onset of viral replication. They generally encode structural components of the virion such as nucleocapsid proteins, as well as glycoproteins which mediate EBV binding and fusion (e.g. gp350/220, gp85, gp42, gp25). Other late lytic gene products have roles in immune evasion; BCLF1 encodes a viral homologue of IL-10, and BALF1 encodes a protein with homology to the anti-apoptotic protein Bcl2.

An “EBV-specific immune cell” as used herein refers to an immune cell which is specific for Epstein-Barr virus (EBV). An EBV-specific immune cell expresses/comprises a receptor (preferably a T cell receptor) capable of recognising a peptide of an antigen of EBV (e.g. when presented by an MHC molecule). The EBV-specific immune cell preferably expresses/comprises a TOR specific for a peptide of an EBV antigen presented by MHC class I.

In some embodiments, the EBV-specific immune cell is a T cell, e.g. a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell)). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).

An EBV-specific T cell may display certain functional properties of a T cell in response to the EBV antigen for which the T cell is specific, or in response a cell comprising/expressing EBV (e.g. a cell infected with EBV) or the relevant EBV antigen. In some embodiments, the properties are functional properties associated with effector T cells, e.g. cytotoxic T lymphocytes (CTLs).

In some embodiments, an EBV-specific T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing EBV/the EBV antigen for which the T cell is specific; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with EBV/the EBV antigen for which the T cell is specific, or in response to exposure to a cell comprising/expressing EBV/the EBV antigen for which the T cell is specific.

EBV-specific T cells preferably express/comprise a TCR capable of recognising a peptide of the EBV antigen for which the T cell is specific when presented by the appropriate MHC molecule. EBV-specific T cells may be CD4+ T cells and/or CD8+ T cells.

An immune cell specific for EBV may be specific for any EBV antigen, e.g. an EBV antigen described herein. A population of immune cell specific for EBV, or a composition comprising a plurality of immune cells specific for EBV, may comprise immune cells specific for one or more EBV antigens.

In some embodiments, an EBV antigen is an EBV latent antigen, e.g. a type III latency antigen (e.g. EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1, EBNA2, EBNA3A, EBNA3B or EBNA3C), a type II latency antigen (e.g. EBNA1 , EBNA-LP, LMP1, LMP2A, LMP2B or BARF1), or a type I latency antigen, (e.g. EBNA1 or BARF1). In some embodiments, an EBV antigen is an EBV lytic antigen, e.g. an immediate-early lytic antigen (e.g. BZLF1, BRLF1 or BMRF1), an early lytic antigen (e.g. BMLF1, BMRF1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU or EBNA1-FUK), or a late lytic antigen (e.g. BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3 or gp350). CD30.CAR Vectors

Nucleic acids and vectors:

The present disclosure also provides a nucleic acid, or a plurality of nucleic acids, encoding a CAR according to the present disclosure. In some embodiments, the nucleic acid is purified or isolated, e.g. from other nucleic acid, or naturally-occurring biological material.

Also provided is a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids.

In some embodiments, the nucleic acid is purified or isolated e.g. from other nucleic acid, or naturally- occurring biological material. In some embodiments, the nucleic acid(s) comprise or consist or DNA and/or RNA.

The nucleotide sequence may be contained in a vector, e.g. an expression vector. A “vector” as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the invention.

The term “operably linked” may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into a desired peptide(s)/polypeptide(s).

Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, which is hereby incorporated by reference in its entirety. In some embodiments, the viral vector may be a lentiviral, retroviral, adenoviral, or Herpes Simplex Virus vector. In some embodiments, the lentiviral vector may be pELNS, or may be derived from pELNS. In some embodiments, the vector may be a vector encoding CRISPR/Cas9.

In some embodiments, the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

In some embodiments the nucleic acid encodes a CAR as described herein. In some embodiments the vector is multicistronic (e.g. bicistronic, tricistronic, etc.); that is, in some embodiments the vector encodes mRNA with multiple protein-coding regions. In some embodiments the vector is bicistronic. In some embodiments the vector comprises nucleic acid encoding an internal ribosome entry site (IRES). In some embodiments the vector comprises nucleic acid permitting a CAR to be translated separately from the same RNA transcript.

Constituent polypeptides of a CAR according to the present invention may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors.

The present disclosure relates generally to the retrovirus mediated gene transfer of nucleic acid that encodes a CD30.CAR. In an aspect, the disclosure provides retrovirus vectors that comprise nucleic acid that encodes a CD30.CAR. In other words, retrovirus vectors that, when expressed in a packaging cell result in the production of a retrovirus that is capable of delivering nucleic acid encoding a CD30.CAR to a target cell. The retrovirus produced may be capable of delivering and stably integrating the nucleic acid encoding the CD30.CAR into the genome of the target cell.

Plasmids:

The CD30.CAR may be encoded by a plasmid. The plasmid may be based on plasmid pSFG (described for example in Hakre et al Mol Cell. 2006 Oct 20. 24(2):301-8 which is incorporated by reference herein in its entirety). Preferably, the plasmid comprises the nucleic acid of SEQ ID NO: 37.

The retrovirus vector may be encoded by a plasmid. The plasmid may encode a gamma retrovirus. The plasmid may encode a murine leukemia virus. The plasmid may be based on plasmid pSFG (described for example in Hakre et al Mol Cell. 2006 Oct 20. 24(2):301-8 which is incorporated by reference herein in its entirety).

Plasmids useful in this disclosure may comprise nucleic acid encoding a retrovirus. The plasmid may further comprise nucleic acid encoding the CAR. The nucleic acid may be arranged within the retrovirus, such that retrovirus expressed by a producer cell comprising the plasmid comprises the nucleic acid encoding the CAR. Preferably, the plasmid comprises the nucleic acid of SEQ ID NO: 37.

The plasmid may contain additional sequences in addition to the nucleic acid encoding the retrovirus and/or CAR that contribute to expression of the retrovirus by the producer cell. For example, the plasmid may further comprise nucleic acid encoding a transcriptional regulator (for example, AmpR) and/or an origin of replication. The plasmid may comprise one or more long terminal repeat (LTR) sequences. LTR sequences may be useful for facilitating integration of the plasmid into the genome of the producer cell. The plasmid may comprise one or more restriction sites. Restriction sites may be useful for cloning the nucleic acid sequence encoding the CAR into the plasmid. The plasmid may comprise an internal ribosome entry site (IRES).

In the present case, the plasmid may comprise nucleic acid encoding a retrovirus and nucleic acid encoding a CAR. The nucleic acid encoding the CAR may be encoded within the nucleic acid encoding the retrovirus, such that nucleic acid encoding the CAR is contained within the retrovirus on expression of the nucleic acid encoding the retrovirus. In other words, the plasmid comprises nucleic acid encoding the retrovirus and nucleic acid encoding the CAR, the nucleic acid encoding the CAR arranged such that the retrovirus expressed from the nucleic acid is a retrovirus that encodes the CAR.

In some cases, the plasmid comprises the nucleic acid of SEQ ID NO: 37. In some cases, the plasmid consists of the nucleic acid of SEQ ID NO: 37. In some cases, the plasmid comprises or consists of a nucleic acid having at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the nucleic acid of SEQ ID NO: 37.

In preferred aspects, the plasmid is pSFG_CD30-CAR (SEQ ID NO: 38).

In some cases, the plasmid comprises the nucleic acid of SEQ ID NO: 38. In some cases, the plasmid consists of the nucleic acid of SEQ ID NO: 38. In some cases, the plasmid comprises or consists of a nucleic acid having at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the nucleic acid of SEQ ID NO: 38.

Retroviral vectors:

The CD30.CAR may be encoded by a viral vector. The viral vector may be a retroviral vector. The retroviral vector may be a gamma retrovirus. Preferably, the gamma retrovirus is derived from a murine leukemia virus. The gamma retrovirus may be derived from a gibbon ape leukemia virus. The retrovirus may be a retroviral vector pseudotyped by gibbon ape leukemia virus envelope protein. The retrovirus may be comprise nucleic acid derived from the genome of a murine leukemia virus.

The present disclosure also relates to methods for manufacturing a retroviral producer cell. The methods may comprise transfection and/or transduction of cells, such as HEK293 cells. In some embodiments, the cells to be transfected and/or transduced, or the cells that have been transfected and/or transduced, are HEK293Vec-RD114 cells or HEK293Vec-Galv cells. In some embodiments, the cells are transfected and/or transduced with vectors comprising a nucleic acid sequence encoding a CAR. In preferred embodiments, the CAR is a CD30.CAR.

Transfection

The methods according to the present disclosure may comprise transfection of HEK293 cells (such as HEK293Vec-RD114 cells) with a plasmid encoding a CD30.CAR. The integrity of the plasmid may be verified by DNA gel electrophoresis prior to transfection. Transfection relates to the process of introducing nucleic acids into cells using means other than viral infection and is hence a non-viral method.

Transfection may be performed by physical/mechanical methods (including electroporation, sonoporation, magnetofection, gene microinjection and laser irradiation) or chemical methods (liposomal-based or non- liposomal based). Liposomal-based transfection reagents are chemicals which enable the formation of positively charged lipid aggregates, which can then merge with the phospholipid bilayer of the cell to facilitate the entry of foreign genetic material. Examples of liposomal-based transfection reagents include, but are not limited to Oligofectamine®, Lipofectamine® and DharmaFECT®. Non-liposomal transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendrimers and non- liposomal lipids. One example of a non-liposomal transfection reagent is polyethylenimine (PEI).

Accordingly, in some embodiments the HEK293 cells are transfected using a physical/mechanical method such as electroporation. In other embodiments, the HEK293 cells are transfected a chemical method using, for example, liposomal-based or non-liposomal transfection reagents. In preferred embodiments, the HEK293 cells are transfected using PEI.

HEK293Vec-RD114 cells according to the present disclosure may be transfected with a plasmid encoding a CD30.CAR using PEI. In some embodiments, the plasmid encoding the CD30.CAR is pSFG_CD30- CAR, as set out in SEQ ID NO: 37 and Figure 7.

In some embodiments, the HEK293Vec-RD114 cells are transfected with a plasmid encoding a CD30.CAR at least 2 times. In some embodiments, the HEK293Vec-RD114 cells are transfected 2 times,

3 times, 4 times, 5 times or more.

HEK203Vec-RD114 cells according to the present disclosure may be cultured in order to allow for the production of retroviral vector. In some embodiments, the cells are cultured for 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less. Preferably, the cells are cultured for

4 days or less, or 3 days or less. Most preferably, the cells are cultured for 3 days or less. In some embodiments, the cells are cultured overnight.

Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells of cell cultures can be established and/or maintained at any suitable density, as can readily be determined by the skilled person. In some embodiments, the cells are cultured in the presence or absence of antibiotics.

A retroviral vector supernatant comprising a nucleic acid encoding a CD30.CAR may be obtained from the cell culture. The supernatant may be filtered prior to use.

Transduction

The methods generally comprise transducing a HEK293Vec-Galv cell with a retroviral vector or virus comprising a nucleic acid encoding a CD30.CAR. Transduction is a process by which nucleic acids may be introduced into a cell by a virus or a viral vector.

Accordingly, in some embodiments the nucleic acid(s) is/are comprised in a viral vector(s), or the vector(s) is/are a viral vectors). Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola-lla, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety.

Agents may be employed in the methods of the present disclosure to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

HEK293Vec-Galv cells according to the present disclosure may be subjected to more than one transduction step. In some embodiments, the HEK293Vec-Galv cells are transduced with a retroviral vector encoding an anti-CD30 CAR at least 2 times. In some embodiments, the HEK293Vec-Galv cells are transduced 2 times, 3 times, 4 times, 5 times or more.

HEK293Vec-Galv cells according to the present disclosure may be cultured in order to allow for the production of retroviral vector. In some embodiments, the cells are cultured for 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less. Preferably, the cells are cultured for 4 days or less, or 3 days or less. Most preferably, the cells are cultured for 3 days or less. Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells of cell cultures can be established and/or maintained at any suitable density, as can readily be determined by the skilled person. In some embodiments, the cells are cultured in the presence or absence of antibiotics.

A retroviral vector supernatant comprising a nucleic acid encoding a CD30.CAR may be obtained from the cell culture. Retroviral vector supernatant is obtained from cells that have been transfected with retroviral vector.

Supernatant is the liquid phase of a cell culture, and thus comprises of culture media. The supernatant preferably does not contain cells or fragments of cells such as cell membrane or cell organelles. The supernatant may be obtained by separating the liquid phase of the cell culture from the cells, and collection of the liquid phase. Separation may involve pipetting. In preferred embodiments, the separation involves filtration. Separation may involve centrifugation of the culture such that the liquid and solid phases of the culture are distinct prior to pipetting, filtration or other removal of liquid from the culture.

The supernatant may be collected and filtered prior to use e.g. through a 0.45 pm filter. In some embodiments, the supernatant is stored at about -80°C prior to use. In some cases, the supernatant may be mixed with one or more buffers, preservatives or excipients. In some embodiments, the retroviral vector supernatant is diluted prior to use. The retroviral vector supernatant may be diluted to at least 1 :50 (i.e. 1 part retroviral vector supernatant per 50 parts diluent). In some embodiments, the retroviral vector supernatant is diluted 1 :50 - 1 JOO, 1 :60 - 1 :90 or 1 :65 - 1 :75. In some embodiments, the retroviral vector supernatant is diluted 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 JOO, 1 J 10, 1 J 20, 1 J 30 or 1 J 40. The retroviral vector supernatant may be diluted cell culture medium. The cell culture medium may be CTL media. The media may consist of 45% RPMI 1640, 45% EHAA (Click’s medium) containing 10% HI FBS and 2mM L-glutamine.

In some aspects disclosed herein, there is provided a method comprising: a. culturing HEK293Vec-Galv cells that comprises nucleic acid encoding a retroviral vector and a nucleic acid encoding a CD30.CAR; b. separating supernatant from the culture; and c. collecting the supernatant.

In some aspects disclosed herein, there is provided a method comprising: a. obtaining a HEK293Vec-Galv cell, wherein the HEK293Vec-Galv cell comprises nucleic acide encoding a retroviral vector and a nucleic acid encoding a CD30.CAR; and b. culturing the HEK293Vec-Galv cell, for sufficient time under suitable conditions for the HEK293Vec-Galv cell to produce retrovirus encoding a CD30.CAR; and c. collecting supernatant from the culture, wherein the supernatant is retroviral vector supernatant.

Methods disclosed herein may involve filtering the collected supernatant to remove unwanted solid matter such as cell fragments. Methods may involve filtering the supernatant through a filter, such as a 0.45pm filter.

Methods may involve diluting or concentrating the collected supernatant, such as diluting the supernatant at least 1 :20, 1 :30, 1 :40 1 :50. 1 :60, 1 :70, 1 :80, 1 :90 or 1 JOO. Preferably, the supernatant is diluted at least 1 :50, at least 1 :60, at least 1 :70, at least 1 :80, or more.

In some methods, the collected supernatant is preserved, such as mixing the collected supernatant with a buffer, a preservative, and/or an excipient. For example, the collected supernatant may be mixed with FBS.

A CD30-specific CAR-expressing T cell or CD30.CAR-T cell may be produced by obtaining a retroviral supernatant from a culture of HEK293Vec-Galv cells, wherein the HEK293Vec-Galv cells comprise nucleic acid encoding a retroviral vector and nucleic acid encoding a CD30.CAR, contacting a T-cell or T- cell precursor cell with the retroviral vector supernatant and expanding the T-cell or T-cell precursor cell to obtain a CD30.CAR-T cell. In some embodiments, the T-cell or T-cell precursor cell is a PBMC.

The T-cell or T-cell precursor cell may be expanded in the presence of IL-7 and IL-15. The retroviral supernatant may be diluted prior to contact with the T-cell or T-cell precursor. In some embodiments, the retroviral supernatant is diluted at least 1 :50. The retroviral supernatant may be diluted in cell medium.

The method may include harvesting and washing the CD30.CAR-T cells. In some embodiments, the CD30.CAR-T cells are washed in Phosphate Buffered Saline (PBS). The CD30.CAR-T cells may be washed at least 2 times.

The CD30.CAR-T cell may have a vector copy number (VCN) of <5.

In some embodiments, the CD30.CAR T-cell is frozen or cryogenically stored.

A CD30-specific CAR-expressing T cell according to the present disclosure may display certain functional properties of a T cell in response to CD30, or in response a cell comprising/expressing CD30. In some embodiments, the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.

In some embodiments, a CD30-specific CAR-expressing T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing CD30; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with CD30, or in response to exposure to a cell comprising/expressing CD30; anti-cancer activity (e.g. cytotoxicity to cancer cells, tumor growth inhibition, reduction of metastasis, etc.) against cancer comprising cells expressing CD30.

Cell proliferation/population expansion can be investigated by analysing cell division or the number of cells over a period of time. Cell division can be analysed, for example, by in vitro analysis of incorporation of 3H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety. Proliferating cells can also be identified by analysis of incorporation of 5-ethynyl-2'-deoxyuridine (EdU) by an appropriate assay, as described e.g. in Buck et al., Biotechniques. 2008 Jun; 44(7):927-9, and Sali and Mitchison, PNAS USA 2008 Feb 19; 105(7): 2415-2420, both hereby incorporated by reference in their entirety.

As used herein, “expression” may be gene or protein expression. Gene expression encompasses transcription of DNA to RNA, and can be measured by various means known to those skilled in the art, for example by measuring levels of mRNA by quantitative real-time PCR (qRT-PCR), or by reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

Cytotoxicity and cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, hereby incorporated by reference in its entirety. Examples of in vitro assays of cytotoxicity/cell killing assays include release assays such as the 51 Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) release assay, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing by a given cell type can be analysed e.g. by co-culturing the test cells with the given cell type, and measuring the number/proportion of cells viable/dead test cells after a suitable period of time.

Cells may be evaluated for anti-cancer activity by analysis in an appropriate in vitro assays or in vivo models of the relevant cancer.

CAR-expressing immune cells and CAR-expressing virus-specific immune cells

The present disclosure relates to immune cells and virus-specific immune cells comprising/expressing chimeric antigen receptors (CARs). For conciseness, the following paragraphs refer to immune cells, but the skilled person will appreciate that the immune cell may be a virus-specific immune cell.

CAR-expressing immune cells may express or comprise a CAR according to the present disclosure. CAR-expressing immune cells may comprise or express nucleic acid encoding a CAR according to the present disclosure. It will be appreciated that a CAR-expressing cell comprises the CAR it expresses. It will also be appreciated that a cell expressing nucleic acid encoding a CAR also expresses and comprises the CAR encoded by the nucleic acid.

An immune cell comprising a CAR/nucleic acid encoding a CAR according to the present disclosure may be characterised by reference to functional properties of the cells.

In some embodiments, CD30-specific CAR-expressing immune cells of the present disclosure display one or more of the following properties:

(a) expression of one or more cytotoxic/effector factors (e.g. IFNy, granzyme, perforin, granulysin, CD107a, TNFa, FASL) in response to cells expressing CD30, in response to cells infected with EBV, and/or in response to cells presenting a peptide of an EBV antigen;

(b) cytotoxicity to cells expressing CD30;

(c) no cytotoxicity (i.e. above baseline) to cells which do not express CD30;

(d) anti-cancer activity (e.g. cytotoxicity to cancer cells, tumor growth inhibition, reduction of metastasis, etc.) against cancer comprising cells expressing CD30; and

(e) cytotoxicity to alloreactive immune cells, e.g. alloreactive immune cells expressing CD30.

Cell proliferation/population expansion can be investigated by analysing cell division or the number of cells over a period of time. Cell division can be analysed, for example, by in vitro analysis of incorporation of 3H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in its entirety. Proliferating cells can also be identified by analysis of incorporation of 5-ethynyl-2'-deoxyuridine (Edll) by an appropriate assay, as described e.g. in Buck et al., Biotechniques. 2008 Jun; 44(7):927-9, and Sali and Mitchison, PNAS USA 2008 Feb 19; 105(7): 2415-2420, both hereby incorporated by reference in their entirety. As used herein, “expression” may be gene or protein expression. Gene expression encompasses transcription of DNA to RNA, and can be measured by various means known to those skilled in the art, for example by measuring levels of mRNA by quantitative real-time PCR (qRT-PCR), or by reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

Cytotoxicity and cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601 -616, hereby incorporated by reference in its entirety. Examples of in vitro assays of cytotoxicity/cell killing assays include release assays such as the 51 Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) release assay, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing by a given cell type can be analysed e.g. by co-culturing the test cells with the given cell type, and measuring the number/proportion of cells viable/dead test cells after a suitable period of time.

Cells may be evaluated for anti-cancer activity by analysis in an appropriate in vitro assays or in vivo models of the cancer.

In some embodiments in accordance with the various aspects of the present disclosure, immune cells may comprise/express more than one (e.g. 2, 3, 4, etc.) CAR.

In some embodiments, immune cells may comprise/express more than one, non-identical CAR. Immune cells comprising/expressing more than one non-identical CAR may comprise/express CARs specific for non-identical target antigens. For example, Example 4 herein describes immune cells comprising/expressing a CD30-specific CAR and a CD19-specific CAR. Each of the non-identical target antigens may independently be a target antigen as described herein. In some embodiments each non- identical target antigen is independently a cancer cell antigen as described herein.

In some embodiments, one of the non-identical target antigens is CD30. In some embodiments, an immune cell comprising/expressing more than one, non-identical CAR comprises: a CD30-specific CAR, and a CAR specific for a target antigen other than CD30.

CAR-expressing T cells may express or comprise a CAR according to the present disclosure. CAR- expressing T cells may comprise or express nucleic acid encoding a CAR according to the present disclosure. It will be appreciated that a CAR-expressing cell comprises the CAR it expresses. It will also be appreciated that a cell expressing nucleic acid encoding a CAR also expresses and comprises the CAR encoded by the nucleic acid.

The T cell may express e.g. CD3 polypeptides (e.g. CD3y CD3E CD3 or CD36), TCR polypeptides (TCRa orTCRP), CD27, CD28, CD4 or CD8. In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell)). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).

Methods for producing CAR-expressing T cells are well known to the skilled person. They generally involve modifying T cells to express/comprise a CAR, e.g. introducing nucleic acid encoding a CAR into T cells.

T cells (may be modified to comprise/express a CAR or nucleic acid encoding a CAR described herein according to methods that are well known to the skilled person. The methods generally comprise nucleic acid transfer for permanent (stable) or transient expression of the transferred nucleic acid.

Methods for producing CAR-expressing immune cells

Aspects of the present disclosure relate to immune cells comprising/expressing CD30-specific chimeric antigen receptors (CARs), particularly, CD30-specific CAR-expressing T cells.

It will be appreciated that where cells are referred to herein in the singular (i.e. “a/the cell”), pluralities/populations of such cells are also contemplated.

Methods useful herein involve the use of retroviral vectors comprising genetic material derived from murine leukemia virus. The retroviral vector may be pseudotyped with gibbon ape leukemia virus. The methods involve obtaining a retroviral vector supernatant from a culture of HEK293Vec-Galv cells, wherein the HEK293Vec-Galv cells are transfected with a retroviral vector comprising a nucleic acid encoding a CD30.CAR. A T-cell or T-cell precursor cell is contacted with the retroviral vector supernatant. The T-cell or T-cell precursor is preferably a peripheral blood mononuclear cell (PBMC).

Methods for generating/expanding populations of CAR-expressing T cells in vitro/ex vivo are well known to the skilled person. Suitable culture conditions (i.e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), cell numbers, culture periods and methods for introducing nucleic acid encoding a CAR into cells, etc. can be determined by reference e.g. to Hornbach et al. J Immunol (2001) 167:6123-6131, Ramos et al. J. Clin. Invest. (2017) 127(9):3462-3471 and WO 2015/028444 A1, all of which are hereby incorporated by reference in their entirety.

The T-cell or T-cell precursor cell is expanded to obtain a CD30.CAR T cell following transduction with the retroviral vector supernatant. In some embodiments, the T-cell or T-cell precursor cell is expanded in the presence of cytokines or chemokines, e.g. IL-7 and/or IL-15, preferably IL-7 and IL-15.

The method may comprise harvesting and washing the CD30.CAR-T cells. The CD30.CAR-T cells may be washed once, twice, three times, four times or more. In some embodiments, the CD30.CAR-T cells are washed at least twice. In some embodiments, the CD30.CAR-T cells are washed at least 3 times.

The method may further comprise cryogenically storing the CD30.CAR-T cells prior to use, which may involve storing the CD30.CAR-T cells in freeze buffer. In some embodiments, the cells are stored at about <150°C. In some embodiments, the CD30.CAR T cells have a vector copy number (VCN) of <5. In some embodiments, the CD30.CAR T cells have a VCN of <10, <9, <8, <7, <6, <5, <4, <3, ^2 or <1. Preferably, the CD30.CAR T cells have a VCN of <6, or <5. The symbol < denotes less than or equal to.

Typical culture conditions (j.e. cell culture media, additives, temperature, gaseous atmosphere), cell numbers, culture periods, etc. can be determined by reference e.g. to Ngo etal., J Immunother. (2014) 37(4): 193-203, which is hereby incorporated by reference in its entirety.

Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells of cell cultures can be established and/or maintained at any suitable density, as can readily be determined by the skilled person. For example, cultures may be established at an initial density of ~0.5 x 10⁶ to -5 x 10⁶ cells/ml of the culture (e.g. -1 x 10⁶ cells/ml).

Cultures can be performed in any vessel suitable for the volume of the culture, e.g. in wells of a cell culture plate, cell culture flasks, a bioreactor, etc. In some embodiments cells are cultured in a bioreactor, e.g. a bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1(8): 1435-1437, which is hereby incorporated by reference in its entirety. In some embodiments cells are cultured in a GRex cell culture vessel, e.g. a GRex flask or a GRex 100 bioreactor.

In some embodiments, transduction, expansion and/or culture of HEK293Vec-Galv or CD30.CAR-T cells is performed in a biosafety cabinet.

T cells may be activated prior to introduction of nucleic acid encoding the CAR. For example, T cells within populations of PBMCs may be non-specifically activated by stimulation in vitro with agonist anti- CD3 and agonist anti-CD28 antibodies, in the presence of IL-2.

Introducing nucleic acid(s)/vector(s) into a cell may comprise transduction, e.g. retroviral transduction. Accordingly, in some embodiments the nucleic acid(s) is/are comprised in a viral vector(s), or the vector(s) is/are a viral vector(s). Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola-lla, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety.

Agents may be employed to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

In some embodiments the methods comprise centrifuging the cells into which it is desired to introduce nucleic acid encoding the CAR in the presence of cell culture medium comprising viral vector comprising the nucleic acid (referred to in the art as ‘spinfection’). In some embodiments, the methods comprises introducing a nucleic acid or vector according to the present disclosure by electroporation, e.g. as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e114, which is hereby incorporated by reference in its entirety.

The methods generally comprise introducing a nucleic acid encoding a CAR into a cell, and culturing the cell under conditions suitable for expression of the nucleic acid/CAR by the cell. In some embodiments, the methods culturing T cells into which nucleic acid encoding a CAR has been introduced in order to expand their number. In some embodiments, the methods comprise culturing T cells into which nucleic acid encoding a CAR has been introduced in the presence of IL-7 and/or IL-15 (e.g. recombinant IL-7 and/or IL-15).

In some embodiments the methods further comprise purifying/isolating CAR-expressing T cells, e.g. from other cells (e.g. cells which do not express the CAR). Methods for purifying/isolating immune cells from heterogeneous populations of cells are well known in the art, and may employ e.g. FACS- or MACS- based methods for sorting populations of cells based on the expression of markers of the immune cells. In some embodiments the methods purifying/isolating cells of a particular type, e.g. CAR-expressing CD8+ T cells, CAR-expressing CTLs).

In preferred embodiments, CD30-specific CAR-expressing T cells may be generated from T cells within populations of PBMCs by a process comprising: stimulating PBMCs with antagonist anti-CD3 and anti- CD28 antibodies, transducing the cells with a viral vector (e.g. a gamma-retroviral vector) encoding the CD30-specific CAR, and subsequently culturing the cells in the presence of IL-7 and IL-15.

In some aspects, this disclosure provides a method comprising: a. obtaining a retroviral vector supernatant according to the present disclosure; b. contacting a T-cell or T-cell precursor cell, optionally a PBMC, with the retroviral vector supernatant; and c. expanding the T-cell or T-cell precursor cell from (b) to obtain a CD30.CAR-T cell.

In some aspects, this disclosure provides a method comprising: a. obtaining a retroviral vector supernatant from a culture of HEK293Vec-Galv cells, wherein the HEK293Vec-Galv cells have been transduced with a retroviral vector comprising a nucleic acid encoding a CD30.CAR; b. contacting a T-cell or T-cell precursor cell, optionally a PBMC, with the retroviral vector supernatant; and c. expanding the T-cell or T-cell precursor cell from (b) to obtain a CD30.CAR-T cell.

Some methods according to this disclosure comprise: a. contacting a T-cell or a T-cell precursor cell, optionally a PBMC, with a retroviral vector supernatant according to the present disclosure; and b. expanding the T-cell or T-cell precursor cell from (a) to obtain a CD30.CAR-T cell. The T-cell or T-cell precursor cell may be contacted with the retroviral vector supernatant for sufficient time for the retroviral vector to transduce the T-cell or T-cell precursor. The contacting may be done under suitable conditions to allow the retroviral vector to transduce the T-cell or T-cell precursor.

Sequences

5

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The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

The term average as used herein refers to the arithmetic mean.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Examples

In the following Examples, the inventors describe the generation of HEK293Vec-Galv cells which produce a CD30.CAR retroviral vector.

Example 1 - Generation of CD30.CAR retroviral vector producing cell line

The inventors established a CD30.CAR retroviral vector producing cell line using HEK293Vec-Galv cells and characterized the supernatant generated by the cell line.

Materials and methods

Cells/cell lines used: HEK293Vec-Galv (BioVec), HEK293Vec-RD114 (BioVec), Jurkat cells, PBMCs from healthy donors.

Plasmid: pSFG_CD30-CAR, synthesized and cloned by ATUM (Newark, CA) using the backbone of pSFG-TGFbDNRII (AT1089-90) where the transgene was replaced with a de novo synthesized CD30.CAR sequence. The full sequence of the plasmid is shown in Figure 7 and SEQ ID NO: 37.

Retroviral vector: MFG-GFP(ATG-) (BioVec) was used as the positive control for the generation of the pool of producer cells. The titer of the vector is typically determined using HT1080 cells with 8 pg/mL of polybrene. However, in the initial screening of CD30.CAR clones, the titer of the control vector was determined using Jurkat cells, the same target cell line as the screened clones.

Cloning of 293VG-CD30CAR: The integrity of plasmid DNA pSFG_CD30-CAR was verified by DNA gel electrophoresis before transfection into 293Vec RD114 cells using PEI. The MFG-GFP was transfected at the same time as a positive control. After overnight incubation, the supernatants were collected and filtered through a 0.45 pm filter. The pSFG_CD30-CAR and MFG-GFP supernatants were then used to infect HEK293Vec-Galv cells at a density of 5x10^s cells/well on a 6 well plate. The cells were cultured over three days. The bulk infected cells were diluted and plated out on 96 well plates at about 30 cells/well. Viral supernatants from the bulk infected cells were titrated by infecting Jurkat cells at 2x10⁴ cells/well in a 96 well plate with 4 pg/mL polybrene to confirm the activity of the construct.

Initially, 115 clones from 96 well plates were transferred to 24 well plates. Virus supernatants (50 pL) produced from 83 clones were screened with Jurkat cells (2x10⁴ cells/well) on 96 well plates with 4 pg/mL polybrene. Positive clones were found in 72 out of 83 clones, out of which 6 clones were selected for further evaluation. The 6 clones were thawed and plated in a 12 well plate, 2 wells/clone. After three days, the confluent clones were split in T75 flasks to continue the culture with Zeocin and Puromycin. Two clones were discontinued due to slow growth rates. Cells of the remaining four clones were expanded and tested for sterility and mycoplasma. After this point, the cells were cultured without antibiotics. The clones were screened again for transgene expression using Jurkat cells with the GFP viral supernatants as control. The three best performing clones (c4, c15, c115) were subject to further analysis and characterization.

FACS analysis: The expression of CD30.CAR was detected using the antibody against Human IgG (Jackson Immunoresearch Lab: Alexa Fluor® 647-AffiniPure F(ab")2 Fragment Goat Anti-Human IgG (H+L) (# 109-606-088). Cells were gated with parental Jurkat cells uninfected labeled with the antibody.

Transduction and culture of T cells: The activities of the viral supernatants were assessed by transducing the Jurkat cells, and the CD3/CD28 activated peripheral blood lymphocytes at Biovec. The viral supernatants of the clones were evaluated by transducing Activated T cells (ATCs).

Transduction rate determination: 300,000 live cells (as determined using NC200) were stained with 3 pl of 1 :10 PBS diluted Goat Anti-Human IgG (H+L) antibodies for 30 minutes at 4°C in the dark. Cells were washed one time with 1 ml of PBS and resuspended in a final volume of 200 pl Stain Buffer (FBS) (Becton Dickinson, Mountain View, CA, USA) with 2.5% 7AAD (Becton Dickinson, Mountain View, CA, USA) added. Stained cells were acquired on the Aurora flow cytometer (Cytek Biosciences Inc).

Cytotoxicity determination: The cytotoxicity of the T cells was determined using the xCELLigence® Real Time Cell Analysis (RTCA™) technology. Briefly, 120k/well Farage (CD30+) cells in medium containing 6% FBS were tethered onto 96 well xCELLigence® E-Plate View using CD19 for 20 hours, and cell index was measured using xCELLigence® every 15 min. Proliferating Effector cells in culture were then added 20 hours later at an Effector-to-Target ratio of 1 :1. The cell index was monitored in xCELLigence® continuously. %Cytolysis at the 24-hour timepoint after adding the effector cells was reported. The E plate was washed once with 160 pL media prior to cell index monitoring.

Viral titer determination: For titer determination, viral supernatant was collected from the virus-producing cell line and centrifuged to remove cells and debris. The viral supernatant was treated with solutions from the QuickTiter Retrovirus Quantitation Kit (Cell Bio Labs, #VPK-120) - Solution A to digest free DNA and/or RNA and Solutions B1/2 to pull down intact virus as described in the manufacturer’s protocol. The pellet was resuspended in TE buffer, and viral genomic RNA was purified using the NucleoSpin RNA virus kit that is provided with the Retro-X qRT-PCR Titration kit (Takara, #631453). The RNA was treated with DNase I to remove any residual plasmid DNA that may have been carried over from the transient transfection of the packaging cells. Serial dilutions of the viral RNA sample were subjected to qRT-PCR to determine the threshold cycle (Ct) values for each dilution. The RNA genome copy number in a sample dilution was determined by finding the copy number that corresponded to its Ct value on a standard curve generated from serial dilutions of the calibrated Retro-X RNA Control Template. The starting copy number was determined by back-calculation to include all dilution steps that the viral supernatant had gone through.

Determination of vector copy numbers of the integrated transqene: The vector copy number of the integrated transgene was determined using digital droplet polymerase chain reaction(ddPCR) assay. Briefly, 2.5 - 5 million activated T-cells (ATCs) harvested from fresh cultures or cryopreserved in Gibco™ Recovery™ Cell Culture Freezing Medium (Gibco, # 1 2648010) were pelleted by centrifugation at 500 g for 5 min. Cells were washed once in 1 ml 1x Dulbecco’s Phosphate-Buffered Saline (DPBS), no calcium, no magnesium (Gibco, #14190-144) and resuspended to 200 pl in 1x DPBS. Genomic DNA was extracted from cells using the QIAamp DNA Blood Mini kit (Qiagen, #51104) or the QIAamp DSP DNA Mini kit (Qiagen, 61304), eluted in nuclease-free water (Ambion, #AM9339) and quantified using the Nanodrop One (ThermoFisher Scientific, BFT#NANO- DROP-ONE-W). A reaction mixture (25 ul) containing 5 - 100 ng of genomic DNA, CD30-CAR TaqMan assay, and ddPCR Supermix for Probes (No dUTP) (Bio-Rad, #1863023) was loaded onto a 96-well PCR plate and transferred into the Automated Droplet Generator (Bio-Rad, #1864101) according to the manufacturer’s protocol. The PCR plate containing sample droplets was heat-sealed and placed into the Bio-Rad T100 thermal cycler for amplification under the following conditions: 95°C for 10 minutes followed by 40 cycles of 94°C for 30 sec and 60°C for 1 minute, with a final heating step of 98°C for 10 minutes. The reacted products were held at 4°C till the sealed 96-well plate was transferred to the QX200 Droplet Reader (Bio-Rad, #1864003) for data acquisition. The manufacturer’s QuantaSoft software was used to determine the concentration of the CD30-CAR target amplicon in the total reaction mixture. CD30-CAR vector copy number per cell was calculated by normalizing to the mass of the human genome in the total reaction mixture.

Interferon-gamma secretion: Effector cells were thawed, and 10 mL media is added. Centrifuge Farage cell (target cells) in culture and Effector cells at 400 x g to remove spent media. Both target cells and effectors were resuspended in assay media (10% FBS RMPI). Target and Effector cells were seeded at 125k on a 24 well non-tissue culture treated plate in 1 mL assay media. The supernatants were collected after 24 hours and centrifuge at 500 x g to remove cell debris.

The supernatant was aliquoted into a 96 well V-bottom plate and stored at -80°C before assay. IFN- gamma ELISA (R&D System, SIF50) was performed using the manufacturer’s protocol. The absorbance at 450 nm was measured using a Tecan plate reader.

Results

The plasmid carrying the synthesized CD30.CAR sequence, pSFG_CD30CAR, was first used to transfect HEK293Vec-RD114 cells in the presence of polyethylenimine (PEI). The resulting culture supernatant was then used to transduce the HEK293Vec-Galv cells twice. The bulk of the transduced 293Vec-Galv cells were subcloned by limiting dilution in 96-well plates. Six clones were evaluated by transduction efficiency in Jurkat cells. Out of the six clones, three with the highest titers were selected for further evaluation. The results are summarized in Table 1. Using Jurkat cells to determine the infectious titers, the clone c115 had a slightly higher infectious titer than the others.

Table 1 - Preliminary stable clone assessment

* The titer was determined using Jurkat cells. The transduction rate (%CAR expression) was determined using activated T cells.

Three stable clone candidates (c4, c15, c115) were further evaluated for cell growth and functional attributes (transgene expression and cytotoxicity). Viral supernatants were generated by culturing the clones. The supernatants were then used to transduce Activated T cells (ATC). Figure 1 shows that the cell expansion rates for the three clones are similar (Figure 1 shows the cell growth rates for activated T cells transduced with the viruses produced from c4, c15, and c115 clones at neat concentrations. The untransduced cells (ATC) were cultured at the same time for comparison. The cell number was 0.5x10⁶ on day 3). The stable clones were then evaluated based on the following attributes: transgene expression, cytotoxicity and integrated vector copy number (VCN) of the transduced ATCs. Figure 2 demonstrates that the CD30.CAR retrovirus produced from all stable clones are able to express CD30.CAR transgene as measured by flow cytometry in the final cell product. The cytotoxicity of transduced cells towards CD30+ tumor cells was evaluated using the newly developed label free cytotoxicity assay (xCelligence®). As shown in Figure 3, cell products transduced with CD30.CAR from all three stable clones exhibited strong cytotoxicity towards the CD30+ Farage cells. The control experiment using CD30- Raji cells showed low cytotoxicity. Figure 4 provides the integrated VCN results of cells transduced with various dilutions of CD30.CAR retrovirus from the three stable clones. Using 1 OO diluted virus from these stable clones showed higher VCN than those transduced with the neat transient virus material, suggesting that the viral titers in the stable clones are significantly higher than the transiently produced virus. These results also emphasize the importance of determination of viral vector titer for the manufacturing process to control the VCN integration to a permissible level.

In summary, all 3 stable clones demonstrated similar results in various screening assays and c115 exhibited higher transduction rates in titration studies. Based on the results obtained, c115 was selected for the MCB production.

Functional analysis of the GMP production of the retroviral vector using the MCB:

The Master Cell Bank lot, MCB1902.VGT, was produced by expanding Clone C115 following the same procedure that was used for the PG-13 producer cell line previously used. SFG_CD30-CAR-293VG retroviral vector was manufactured from MCB1902.VGT. Four sub-lots were generated according to the timing of harvesting (over four days, one sub-lot a day). The sublot A was characterized by transduction rate, cytotoxicity, interferon gamma secretion and integrated vector copy number of the transduced ATCs from three donors to account for donor to donor variability. The results are shown in in Figure 5.

The functional titer results demonstrate that the viral vector has a high transduction efficiency. The retroviral vector can be significantly diluted and still achieve high transduction rates of >95% and exhibit acceptable levels of cytotoxicity for the transduced cells. Although the transduction with the undiluted virus supernatant also shows good transduction rate and cytotoxicity, the retrovirus vector copy number (VCN) per transduced cells exceeds 5. The cells transduced with highly diluted CD30.CAR all have VCN less than 5, meeting safety guidelines. The first set of titration results shown in Figure 5 suggested that a high dilution should be used for this new GMP lot. There was also a high donor to donor variability in VCN and Interferon gamma secretion.

Conclusion

A HEK293Vec-Galv-based CD30.CAR retroviral vector using a de novo synthesized CD30.CAR transgene sequence has been generated. Transduction efficiency, cytotoxicity, and integrated vector copy number of the vector in activated T cells were used to assess candidates of the stable clones. The final selected clone (c115) was further characterized using the GMP lot. The HEK293Vec-Galv-based retroviral supernatants had a high transduction rate (>90%) after 100-fold dilution. Cytotoxicity and interferon gamma secretion of the ATCs cultured ten days after transduction were not affected by the dilution levels of the retroviral supernatants used during the transduction. However, vector copy number of the integrated transgene in the ATC correlated with the amounts of the transduced retroviral vector. Assessment of the GMP lot using the ATC batches from three donors showed significant donor to donor variability in interferon gamma secretion and vector copy number of the integrated transgene. The results indicate that it is necessary to evaluate multiple donors in order to determine the dilution level for the manufacturing process, and that the vector copy number of the integrated transgene may be the most critical factor.

Example 2 - Comparative studies of supernatants obtained from HEK293Vec-Galv and PG13 cell lines

The inventors performed studies in order to compare the viral supernatants obtained from the HEK293Vec-Galv cell line obtained in Example 1 with viral supernatants obtained from a PG13 cell line, another CD30.CAR vector producer cell line.

In order to demonstrate the suitability of SFG_CD30-CAR-293VG (RV1905A) (abbreviated as 293VG vector below) in the CD30 CAR-T manufacturing process, a two-tiered study was conducted. First, 293VG (GMP lot 1905A) was subjected to a series of titration experiments to determine the optimal dilution for the transduction step. The results from the initial characterizations performed in Example 1 suggested a range of dilution levels that would generate suitable vector copy number (VCN). This range was further verified by side-by-side comparison to a PG13.SFG.CD30.CD28.zeta vector (abbreviated as PG13VG below). Secondly, a head-to-head comparability for undiluted PG13VG and 293VG at the determined dilution level was conducted using the split-lot approach, where each donor material was split and then transduced by these two vectors. Furthermore, three batches of the split-lot CD30.CAR-T cells transduced with either 293VG or PG13 VG vectors were subjected to a real time stability evaluation at about the 2-month time point to assess the stability profile of 293VG-transduced CD30.CAR-T cells.

The objective of the present study was to compare the 293VG and PG13VG vectors using split cells from four healthy donors and two lymphoma patient donors.

Materials and methods

Retroviral vector supernatants: The construction of the retroviral vector, SFG-Tessa-T6-CD30CAR- 293VG, was described in Example 1. The GMP lot 1905 was produced from a stable producer line 293VG-CD30CAR (lot MCB1902.VGT) at the GMP viral vector production facility at CAGT, BCM. Four sublots collected over four consecutive days (one sublot a day) were produced. The sublot 1905A was used for the titration and comparability study described in this report. The CD30.CAR retroviral vector from PG13 producer cell line used in RELY30 trial has been described in Ramos et al., 2017.

PBMCs: Peripheral Blood Mononuclear Cells (PBMC) that were used in the study were collected from healthy donors. The materials from healthy donors B1, B2, B3 and B4 were isolated from whole blood; healthy donors B5 and B6 were from leukapheresis. The materials from lymphoma patients, B8 and B9, were isolated from whole blood.

Transqene expression by transduction efficiency determination: The transduction efficiency was determined using Immunofluorescent Staining. The antibody Goat anti-Human lgG(H+L, (Jackson Immuno Research) is used to stain CD30 CAR. For samples B1, B2, B3, B4, B5 and B6, the procedure was followed with minor modifications at the research laboratory. For these samples, the cell suspensions were first washed with PBS and then incubated with BV421 Anti-Human IgG Fc (Biolegend). At least 1000 events were analyzed with proper gating for the lymphocyte population by light scatter.

Viability determination: Enumeration of Viable Cells. Properly diluted cell sample was mixed with trypan blue solution and loaded into one of the V-shaped troughs formed between the ridges of the hemocytometer. Viable (clear) and non-viable (blue) cells in at least two of the nine 1.0 mm squares in each chamber of the hemocytometer were counted and recorded separately. A minimal count of 50 cells is required for evaluation of cell concentration. At least 200 cells are required to determine percentage of viability.

Percent Cell Viability = (Viable Cells Counted / Total Cells Counted) x 100%

Viable Cell Concentration (Viable cells/mL) = Viable Cells Counted / no. of 1 mm Squares Counted x 104 x Total Dilution Factor Total Viable Cells = Viable Cell Concentration x Volume of Cell Suspension Phenotypinq Assay: Direct Immunofluorescent Staining with Lyophilized Monoclonal Antibodies Using FACS Lyse-Wash Assistant. Samples (cell suspension) were first washed with PBS. After resuspension, the cells were incubated with Cluster of Differentiation (CD) marker antibodies (including CD3) conjugated with various fluorescence dyes. After removing the unbound antibodies, the stained cells were analyzed using a flow cytometry. Fifty thousand events were collected and counted after gating the lymphocyte population using light scatter properties. The results for the percent CD3 positive cells are reported as a lot release criterion. The results for other markers (CD19, TCRap, TCRyS, CD45RA, CD45RO, CD4, CD8. CD56. CD16. CD127 and 7AAD) are evaluated for product characterization.

Chromium-Release Cytotoxicity Assay: The cytotoxic activity of CD30.CAR-T cell product is determined by measuring the release of radioactive ^s1Cr from CD30-expressing target cell line-HDLM2 Hodgkin lymphoma cell line. In each assay HDLM2 cells are first labeled with ^s1Cr and co-cultured with CD30.CAR-T cells at different effector-to-target ratios. The amount of dead tumor cells is determined by measuring the ^s1Cr released into culture supernatant by gamma counter. The method is described in the CAGT SOP D03.04: Determination of Cytotoxic Specificity of Cytotoxic T Lymphocytes and Natural Killer Cells. Wells containing medium only are used for calculating spontaneous lysis background, and wells containing 1% Triton-X 100 are used for determining the maximum lysis. The target cells (HDLM2) were first examined under microscope to ensure acceptable level (>60%) of viability. After cell count, 1-2 x 106 target cells were centrifuged and resuspended in medium containing 0.1 mCi ⁵¹ Cr sodium chromate. The cells were incubated for 1 hour at 37°C with periodic resuspension of pellet before the unincorporated label was removed. The target cells were counted again and loaded into 96 well V bottom plate containing the four levels of effector cells, medium only and 1 % Triton-X 100 at a cell density of 5 x 103 cells per well. At the end of incubation, the plate was centrifuged, and the supernatant from each well was transferred into microtubes arranged in the corresponding 96 well format. The radioactivity of each tube was counted in Wizard2 gamma counter.

The percentage of ⁵¹Cr release is calculated as follows:

% ⁵¹ Cr release = (Experimental release - Background release)/(Maximum release - background release) x 100

The result of the effectontarget ratio of 20:1 is reported.

Vector copy number determination by gPCR: The real-time quantitative polymerase chain reaction (qPCR) assay is based on TaqMan Gene Expression Assays (Applied Biosystems, ThermoFisher Scientific) with primers and probes specifically designed for the integrated proviral sequence. Genomic DNA extracted from CD30.CAR-T cells (using QIAamp Blood Mini Kit) was tested as the sample and plasmid carrying the CD30.CAR transgene as the standard. In this assay, 100 ng of DNA template per sample was tested per reaction (rxn) in MicroAmp Optical 96 well Reaction plate with TaqMan Assay reagents (DNA polymerase, primers, probe, buffer) in a total volume of 13.75 pL. The qPCR reaction was performed using 7900HT Real Time PCR thermocycler according to the instructions for TaqMan assays. After PCR reaction was complete, data analysis was performed with manual threshold of 0.2. With this setting, the Ct (threshold cycle) value for the standard with 300,000 copies should be about 20-21 and Ct for the standard with 3 copies about 37-39. Sample quantitation is based on interpolation of the standard dilutions of plasmid DNA carrying transgene used in T-cells transduction. The vector copy number is first calculated corresponding to 100ng/rxn, and then normalized with the percentage of transduced T cells.

Droplet Digital Polymerase Chain Reaction (ddPCR) to determine vector copy number: The same primers and probe used in the qPCR assay was used in droplet digital polymerase chain reaction (ddPCR) assay to determine integrated vector copy number (VCN). In addition, primers and probe with a different dye for RNase P were included as a reference gene in a multiplex assay. Briefly, genomic DNA was purified using QIAamp Mini spin column according to manufacturer’s instruction. The polymerase chain reaction was carried out using the QX200 droplet digital PCR system. The CD30.CAR vector copy number per cell was calculated by normalizing to the RNase P reference gene and multiplying by 2 to account for the two copies of RNase P gene per cell. Transgene vector copy number per transduced cell was determined by normalizing to the transduction rate, as measured by flow cytometry, for the respective sample. CD30.CAR Vector Comparability Study Plan: A comparability plan was designed and implemented aiming to evaluate the impact of the retrovirus producer cell line change on the final cell therapy product. Due to the autologous nature of this product and the expected donor-to-donor variability, the study involved a split lot approach for a side-by-side comparison between PG13VG and 293VG on four healthy donor materials (Figure 6). Briefly, PBMCs from each donor was cultured and activated until the transduction step. Cells from the same donor were split into two portions, one being transduced with PG13VG and another with 293VG. After transduction, both arms of transduced cells were expanded in culture with IL-7 and IL-15 and then harvested, washed, formulated and frozen. Both the final cell products and the in- process samples were collected for analytical assessment. A short term (approximately 2 month) real time stability study was also performed.

The comparability assessment of PG13VG and 293VG viral vectors is based on analytical results of in- process, release, and characterization testing of CD30.CAR T cells manufactured using either PG13VG or 293VG. Due to the high variation in the starting materials from each donor or patient, the split lot approach where each lot of starting material was split prior to the transduction step and then each portion was transduced by either PG13VG or diluted 293VG. The split lot comparability results between the PG 13 and 293VG were performed for materials from 4 healthy donors and two lymphoma patients. The study design is illustrated in Figure 6, and the test plan is described in Table 2. The study was conducted at BCM.

Table 2 - Proposed PG13 and 293VG Vector Comparability Assessment Plan

Results

Initial characterization of the CD30.CAR retroviral vector suggested that the vector lot RV1905A should be diluted prior to use in the CD30.CAR-T transduction manufacturing process (Example 1). In order to optimize the conditions for transduction of the HEK293Vec-Galv-based retroviral vector lot RV1905A (293VG), a titration study was conducted. The 293VG vector was diluted to 1 : 10, 1 :20, 1 :40 and 1 :80, and for comparison, the PG13 based RV1004 (PG13VG) vector was used without dilution in a head-to-head split lot study to transduce two healthy donor ATC samples. The results of the 293VG vector transduction of two healthy donor samples (B1 and B2) are summarized in Table 3. Across all dilutions, the cell viability and CD3+ T cells of the CAR-T transduced with 293VG were similar to that of the same donor cells transduced with PG13VG. The transduction efficiency of 293VG appeared to be slightly lower than PG13VG, but the CD30-specific cytotoxicity and the growth rate of the 293VG transduced cells were higher than the PG13VG transduced cells. The VCN of the 293VG transduced cells was higher than that of the PG13VG transduced cells.

Additional titration experiment was conducted using two more healthy split lot donor samples (B3 and B4) transduced with two dilution levels of 293VG (1:70 and 1:140) and PG13VG (neat). The same attributes tested in the initial titration were evaluated (Table 4). The results of B3 and B4 were similar to those of B1 and B2. The cell viability, transduction efficiency and CD3 positive population were similar for both 293VG and PG13VG transduced CD30 CAR-T cells. The CD30-specific cytotoxicity was increased for 293VG compared with PG13VG transduced CD30 CAR-T cells. The transduction efficiency and the integrated vector copy numbers were lower for the 1 :140 dilution than the 1 :70 dilution. The 1 :140 dilution of 293VG transduced cells also had the highest growth rate.

Amongst all these attributes, the VCN is the most sensitive one to the dilution levels of the input 293VG supernatants. To achieve a target VCN of < 5, the vector should be diluted to at least 50 folds before use in the CD30.CAR-T manufacturing process.

Table 3. First Titration Study of CD30 CAR Retroviral Vector 293VG (lot RV1905A), with the PG13VG as a control

¹ BV421 Anti-Human IgG Fc (Biolegend) was used for CD30CAR staining in the research laboratory. ² CD3-APC was used for CD3 staining. Data was analyzed by A750 flow cytometer (Beckman Coulter).³' ND = Not determined. The high vector copy number suggests the dilution of the supernatants was not sufficient for the CD30CAR-T manufacturing process; therefore, the determination of cytotoxicity was omitted. DOM = Date of Manufacturing

Table 4. Second Titration Study of CD30 CAR Retroviral Vector 293VG (lot RV1905A)

¹ BV421 Anti-Human IgG Fc (Biolegend) was used to stain CD30.CAR for transduction efficiency.² CD3-APC was used for CD3 staining. Data was analyzed by A750 flow cytometer (Beckman Coulter)

Conclusion: The comparability assessment is based on a split lot design as described in the materials and methods section.

Cytotoxicity: In general, the CD30-specific cytotoxicity of CD30.CAR T cells was increased for 293VG transduced cells compared with PG 13 transduced cells (average (mean) (PG 13); average (mean) (293VG)). Conclusion

The study evaluated the CD30CAR-T cells transduced with either the current PG13VG (Lot 1004) or the proposed 293VG (Lot RV1905). Titration and comparability studies were executed to demonstrate the comparability between the current PG13VG and the new 293VG, and the possibility of replacing the PG13VG with the 293VG. A study of viral supernatant titration was carried out to determine the dilution level of the new retroviral vector, 293VG, for use in the CAR-T manufacturing process to generate comparable results as PG13VG. The result showed that a dilution of above 1:50 should be used, and it could produce CD30.CAR T cells with similar properties as those transduced by neat PG13VG vector.

Based on the head-to-head comparability study, the 293VG produced from the 293Vec-Galv stable producer cells at greater than 1 :50 diluted 293VG was deemed acceptable for use.

References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

Ghani, K. et al. (2009). Efficient human hematopoietic cell transduction using RD114- and GALV- pseudotyped retroviral vectors produced in suspension and serum-free media gene therapy. Hum Gen Then 20:966-974

Ramos, C., et al. (2017) Clinical and immunological responses after CD30-specific chimeric antigen receptor-directed lymphocytes. J Clin Invest; 127:3462-3471.

Wang, X et al. (2015). Large-scale clinical-grade retroviral vector production in a fixed-bed bioreactor. J. Immunother. 38:127-135.

For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

Claims

53 Claims:

1. A HEK293Vec-Galv cell transduced with a retroviral vector comprising a nucleic acid encoding a CD30.CAR.

2. A retroviral vector supernatant obtained from a culture of HEK293Vec-Galv cells according to claim 1.

3. A CD30.CAR-T cell produced using the retroviral vector supernatant according to claim 2.

4. A method comprising: a. obtaining a retroviral vector supernatant from a culture of HEK293Vec-Galv cells, wherein the HEK293Vec-Galv cells have been transduced with a retroviral vector comprising a nucleic acid encoding a CD30.CAR; b. contacting a T-cell or T-cell precursor cell, optionally a PBMC, with the retroviral vector supernatant; and c. expanding the T-cell or T-cell precursor cell from (b) to obtain a CD30.CAR-T cell.

5. The method according to claim 4 wherein the retroviral vector supernatant from (a) is diluted before contacting with the T-cell or T-cell precursor cell in (b).

6. The method according to claim 5 wherein the retroviral vector supernatant is diluted at least 1 :50.

7. The method according to claim 6 wherein the CD30.CAR-T cell obtained in (c) has a vector copy number (VCN) of <5.

8. The method according to claim 7 further comprising: d. cryogenically storing the CD30.CAR-T cell obtained in (c).

9. The method according to claim 7 wherein the method further comprises harvesting and washing the CD30.CAR-T cell obtained in (c).

10. The method according to any one of claims 4 to 9 wherein the T-cell or T-cell precursor cell is expanded in the presence of IL-7 and IL-15.

11. A CD30.CAR-T cell obtained by the method according to any one of claims 4 to 10.

12. A method comprising: a. transducing a HEK293Vec-Galv cell with a retroviral vector comprising a nucleic acid encoding a CD30.CAR; b. culturing the transduced HEK293Vec-Galv cells; 54 c. obtaining retroviral vector supernatant comprising a nucleic acid encoding a CD30.CAR from the cell culture; and d. diluting the retroviral vector supernatant.

13. The method of claim 12 wherein the HEK293Vec-Galv cells are cultured for 3 days or less.

14. The method of claim 12 or claim 13 wherein the retroviral vector supernatant is diluted at least 1 :50.

15. A retroviral vector supernatant prepared by the method of any one of claims 12 to 14.

16. The HEK293Vec-Galv cell, retroviral vector supernatant, CD30.CAR-T cell, or method according to any one of the preceding claims wherein the CAR comprises an HRS3 scFv.

17. The HEK293Vec-Galv cell, retroviral vector supernatant, CD30.CAR-T cell, or method according to any one of the preceding claims wherein the CAR is encoded by SEQ ID NO: 37.

18. The HEK293Vec-Galv cell, retroviral vector supernatant, CD30.CAR-T cell, or method according to any one of the preceding claims wherein the retroviral vector is pSFG_CD30-CAR.

19. The method of claim 12 further comprising filtering the retroviral vector supernatant.

20. The method of claim 12 or claim 18 further comprising a step of storing the supernatant at -80°C prior to use.

21. A method for producing a chimeric antigen receptor T cell, the method comprising modifying an immune cell to express a chimeric antigen receptor (CAR) by exposing the virus-specific immune cell to a retroviral vector supernatant according to claim 2.

22. The method of claim 21 , wherein the immune cell is a virus-specific immune cell or a virusspecific T cell.

23. The method of claim 21 , wherein the virus-specific immune cell or virus-specific T cell is specific for Epstein-Barr virus (EBV).

24. A virus-specific immune cell obtained by the method of any one of claims 21 to 23, wherein the vector copy number is < 5.

25. A cell bank derived from an individual not suffering from lymphoma, wherein cells in the cell bank comprise virus-specific T cells obtained by the method of any one of claims 12 to 14.