METHODS FOR EXPANDING KAPPA MYELOMA ANTIGEN CHIMERIC ANTIGEN RECEPTORS EXPRESSING CELLS

CROSS REFERENCE

[0001] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62/592,621, filed November 30, 2017, and U.S. Provisional Application Serial No. 62/657,145, filed April 13, 2018, each of which is hereby incorporated by reference in its entirety for all purposes.

STATEMENT REGARDING SEQUENCE LISTING

[0002] The sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is HMLX_003_02WO_SeqList_ST25.txt. The text file is about 76 KB, was created on November 30, 2018, and is being submitted electronically via EFS-Web.

BACKGROUND OF THE INVENTION

[0003] Multiple myeloma (MM) is a malignancy of bone marrow plasma cells, which despite recent advances in therapy, remains incurable. Its clinical course is characterized by an initial response to therapy, followed by repeated relapse with eventual resistance to all forms of treatment. It is also associated with significant morbidity and disability both due to the disease itself and toxicity from available treatments.

[0004] Multiple myeloma is characterized by malignant plasma cells, which secrete either a kappa or lambda light chain restricted monoclonal paraprotein. Kappa restriction occurs in 60% of myeloma patients and the expression of kappa myeloma antigen (KMA) is highly restricted to multiple myeloma and B-cell malignancies. KappaMab is a KMA-specific monoclonal antibody, which has demonstrated safety and efficacy in phase I and II clinical trials.

[0005] Treatment with monoclonal antibodies alone is not curative with incomplete eradication of the tumor leading to eventual relapse. This may be due to inadequate penetration of antibody into the tumor (via passive diffusion), heterogeneity of antigen expression on tumor cells or resistance of tumor cells to mechanisms of antibody dependent cytotoxicity. Thus, there is a need for effective therapies with low toxicity, which can provide long-term disease cure.

[0006] Chimeric Antigen Receptor bearing T cells (CAR T-cells) represent a possible solution to this problem. CAR T-cells incorporate the antigen-binding domain of monoclonal antibodies with one or more intracellular signaling domain(s) of T cells to produce a localized, tumor specific immune response. CAR T-cells have several advantages over monoclonal antibodies: they actively migrate into the tumor, proliferate in response to antigen bearing tumor cells, secrete factors that recruit other arms of the immune response and can survive long term to provide ongoing protection from relapse. Another benefit of a CAR-T cell over an antibody therapeutic targeting the same antigen is that the CAR T-cell may also be further modified to enhance safety and function. For example, a T cell can be modified to include expression of a homing receptor, which enhances T cell specificity and the ability of the T cell(s) to infiltrate cancer cells or tumors or they may include an“off switch” that can function to eliminate cells when toxicity occurs. Furthermore, and importantly for the treatment of multiple myeloma and its related disorders, the T cell may be modified to express additional biologically active or pharmaceutically active molecules that may enhance the anti-tumor response, such as, for example, tumor suppressive cytokines.

[0007] While methods currently exist for selecting and expanding CAR T-cells, improved methods are needed that stimulate CAR T-cells and allow for the generation of CAR T-cells with greater functional responses than methods that currently exist. The constructs and methods provided herein address this need.

SUMMARY OF THE INVENTION

[0008] In one aspect, provided herein is a method for producing an antigen presenting cell (APC) comprising introducing an expression vector encoding a chimeric construct comprising a kappa myeloma antigen (KMA) sequence fused to a nucleotide sequence encoding a reporter protein into a cell line. In some cases, the expression vector is a transposable vector expression system. In some cases, the expression vector is a PiggyBac transposon expression vector. In some cases, the expression vector is a PiggyBat transposon expression vector. In some cases, the introducing comprises electroporation. In some cases, the KMA sequence is a switch and/or constant region of a kappa free light chain. In some cases, the chimeric construct further comprises a linker and a CD28 transmembrane domain. In some cases, the linker is a glycine-serine linker. In some cases, the glycine-serine linker is a 15-20 amino acid linker. In some cases, the 15 amino acid linker comprises (Gly4Ser)3. In some cases, the reporter protein is mCherry. In some cases, the cell line is an immortalized cancer cell line. In some cases, the immortalized cancer cell line is a K562 cell line or HEK293 cell line.

[0009] In another aspect, provided herein is a chimeric construct comprising a kappa myeloma antigen (KMA) sequence fused to a nucleotide sequence encoding a reporter protein. In some cases, the chimeric construct is present in a transposable vector expression system. In some cases, the transposable vector expression system uses a PiggyBac transposon expression vector. In some cases, the transposable vector expression system uses a PiggyBat transposon expression vector. In some cases, the KMA sequence is a switch and/or constant region of a kappa free light chain. In some cases, the chimeric construct further comprises a linker and a CD28 transmembrane domain. In some cases, the linker is a glycine-serine linker. In some cases, the glycine-serine linker is a 15-20 amino acid linker. In some cases, the 15 amino acid linker comprises (Gly4Ser)3. In some cases, the reporter protein is mCherry.

[0010] In yet another aspect, provided herein is a genetically modified cell-line engineered to express a chimeric construct comprising a kappa myeloma antigen (KMA) sequence fused to a nucleotide sequence encoding a reporter protein. In some cases, the chimeric construct is present in a transposable vector expression system. In some cases, the transposable vector expression system uses a PiggyBac transposon expression vector. In some cases, the transposable vector expression system uses a PiggyBat transposon expression vector. In some cases, the KMA sequence is a switch and/or constant region of a kappa free light chain. In some cases, the chimeric construct further comprises a linker and a CD28 transmembrane domain. In some cases, the linker is a glycine-serine linker. In some cases, the glycine-serine linker is a 15-20 amino acid linker. In some cases, the 15 amino acid linker comprises (Gly4Ser)3. In some cases, the reporter protein is mCherry. In some cases, the cell-line is a K562 cell line.

[0011] In still another aspect, provided herein is a method for enriching and expanding genetically engineered T-cells comprising chimeric antigen receptor (CAR) constructs by growing the genetically engineered T-cells comprising CAR constructs in the presence of a genetically modified cell-line engineered to express a chimeric construct comprising a kappa myeloma antigen (KMA) sequence fused to a nucleotide sequence encoding a reporter protein. In some cases, the chimeric construct is present in a transposable vector expression system. In some cases, the transposable vector expression system uses a PiggyBac transposon expression vector. In some cases, the transposable vector expression system uses a PiggyBat transposon expression vector. In some cases, the KMA sequence is a switch and/or constant region of a kappa free light chain. In some cases, the chimeric construct further comprises a linker and a CD28 transmembrane domain. In some cases, the linker is a glycine-serine linker. In some cases, the glycine-serine linker is a 15-20 amino acid linker. In some cases, the 15 amino acid linker comprises (Gly4Ser)3. In some cases, the reporter protein is mCherry. In some cases, the cell-line is a K562 cell line. In some cases, the CAR constructs comprise one or more intracellular signaling domains and an extracellular antigen binding domain, wherein the extracellular antigen binding domain specifically recognizes kappa myeloma antigen (KMA). In some cases, the one or more intracellular signaling domains comprises one or more co-stimulatory endodomains. In some cases, the one or more co stimulatory endodomains is one or more of a CD28 domain, a CD3z domain, a 4-1BB domain or OX-40 domain or combinations thereof. In some cases, the co-stimulatory endodomains are a CD3z domain and a CD28 domain. In some cases, the co-stimulatory endodomains are a CD3z domain and an OX-40 domain. In some cases, the method further comprises an OX-40 domain. In some cases, the co-stimulatory endodomains are a CD3z domain and a 4-1BB domain. In some cases, the method further comprises a 4-1BB domain. In some cases, the method further comprises an OX-40 domain. In some cases, the extracellular binding domain comprises a single chain variable fragment (scFv) that specifically recognizes KMA. In some cases, the scFv comprises the complementarity determining regions (CDRs) derived from a KappaMab monoclonal antibody. In some cases, the scFv comprises the VL chain and VH chain from a KappaMab. In some cases, the VL chain and VH chain from KappaMab are attached via a glycine-serine linker. In some cases, the glycine-serine linker is a 15-20 amino acid linker. In some cases, the 15 amino acid linker comprises (Gly4Ser)3. In some cases, the scFv is attached to the one or more intracellular signaling domains via a spacer. In some cases, the spacer is an immunoglobulin constant region or a CD8alpha chain. In some cases, the immunoglobulin constant region comprises one or more of an IgG hinge domain, an IgG CH2 domain and an IgG CH3 domain. In some cases, the immunoglobulin constant region comprises an immunoglobulin hinge domain. In some cases, the immunoglobulin constant region further comprises an IgG CH3 domain. In some cases, the immunoglobulin constant region further comprises an IgG CH2 domain. In some cases, the spacer is attached to the scFv via a glycine-serine linker. In some cases, the glycine-serine linker is a 15-20 amino acid linker. In some cases, the 15 amino acid linker comprises (Gly4Ser)3..

[0012] In another aspect, provided herein is a construct comprising kappa myeloma antigen (KMA) sequence fused to a solid substrate. In some cases, the solid substrate is a bead. In some cases, the KMA sequence is a switch and/or constant region of a kappa free light chain.

[0013] In still another aspect, provided herein is a method for enriching and expanding genetically engineered T-cells comprising chimeric antigen receptor (CAR) constructs by growing the genetically engineered T-cells comprising CAR constructs in the presence of a construct comprising kappa myeloma antigen (KMA) sequence fused to a solid substrate. In some cases, the solid substrate is a bead. In some cases, the KMA sequence is a switch and/or constant region of a kappa free light chain.

[0014] In a still further aspect, provided herein is a method for enriching and expanding genetically engineered T-cells comprising chimeric antigen receptor (CAR) constructs by growing the genetically engineered T-cells comprising CAR constructs in the presence of soluble kappa myeloma antigen (KMA). In some cases, the soluble KMA consists of a switch and/or constant region of a kappa free light chain. In some cases, the CAR constructs comprise one or more intracellular signaling domains and an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain specifically recognizes kappa myeloma antigen (KMA). In some cases, the one or more intracellular signaling domains comprises one or more co-stimulatory endodomains. In some cases, the one or more co stimulatory endodomains is one or more of a CD28 domain, a CD3z domain, a 4-1BB domain or OX-40 domain or combinations thereof. In some cases, the co-stimulatory endodomains are a CD3z domain and a CD28 domain. In some cases, the co-stimulatory endodomains are a CD3z domain and an OX-40 domain. In some cases, the method further comprises an OX-40 domain. In some cases, the co-stimulatory endodomains are a CD3z domain and a 4-1BB domain. In some cases, the method further comprises a 4-1BB domain. In some cases, the method further comprises an OX-40 domain. In some cases, the extracellular binding domain comprises a single chain variable fragment (scFv) that specifically recognizes KMA. In some cases, the scFv comprises the complementarity determining regions (CDRs) derived from a KappaMab monoclonal antibody. In some cases, the scFv comprises the VL chain and VH chain from a KappaMab. In some cases, the VL chain and VH chain from KappaMab are attached via a glycine-serine linker. In some cases, the glycine-serine linker is a 15-20 amino acid linker. In some cases, the 15 amino acid linker comprises (Gly4Ser)3. In some cases, the scFv is attached to the one or more intracellular signaling domains via a spacer. In some cases, the spacer is an immunoglobulin constant region or a CD8alpha chain. In some cases, the immunoglobulin constant region comprises one or more of an IgG hinge domain, an IgG CH2 domain and an IgG CH3 domain. In some cases, the immunoglobulin constant region comprises an immunoglobulin hinge domain. In some cases, the immunoglobulin constant region further comprises an IgG CH3 domain. In some cases, the immunoglobulin constant region further comprises an IgG CH2 domain. In some cases, the spacer is attached to the scFv via a glycine-serine linker. In some cases, the glycine-serine linker is a 15-20 amino acid linker. In some cases, the 15 amino acid linker comprises (Gly4Ser)3..

[0015] These above-characterized aspects, as well as other aspects, of the present invention are exemplified in a number of illustrated implementations and applications, some of which are shown in the figures and characterized in the claims section that follows. However, the above summary is not intended to describe each illustrated embodiment or every implementation of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention is obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0017] Figures 1A-1B shows the structural relationship of CARs to Immunoglobulin (IgG) and the T-cell receptor (TCR) Figure 1A shows a single chain variable fragment (scFv) consisting of the parent antibody’s light chain variable region (VL) joined to the heavy chain variable region (VH) by a polypeptide linker confers antigen specificity to the CAR. A flexible hinge connects the scFv to the transmembrane and the intracellular signaling domain of a co-stimulatory molecule such as CD28, 4-1 BB or OX-40 followed by CD3 zeta. Figure 1B shows T-cells transduced with the CAR are activated on encountering tumor cells bearing the target antigen (Ag) leading to tumor cell lysis.

[0018] Figure 2 shows the structural determinants of chimeric antigen receptor function.

[0019] Figure 3 shows KMA expression on primary myeloma cells.

[0020] Figures 4A-4C shows KMA.CAR-28z function. Figure 4A is flow cytometry analysis of KMA expression on various cell lines; Figure 4B is interferon-gamma (IFNy) expression of KMA.CAR-28z transduced (upper plots) and non-transduced (lower plots) CD8 ⁺ T cells. Figure 4C shows the specific lysis of KMA positive and negative cell lines by KMA.CAR-28z transduced T cells.

[0021] Figure 5A shows RPMI-Rag mice injected with 5 x l0 ⁵ -5 x 10 ⁶ myeloma cells Figure 5B shows infiltration of the bone marrow and spleen with CDl38 ⁺ RPMI9226 cells Figure 5C shows elevated levels of serum human lambda light chain on progressive disease Figure 5D shows CDl38 ⁺ /cytoplasmic lambda light chain positive cells in the bone marrow Figure 5E shows RPMI-Rag mice as a therapeutic model.

[0022] Figures 6A-6C shows the optimization of KMA. CAR Figure 6A show the initial KMA.CAR-28z construct; Figure 6B shows constructs with Ig heavy chain hinge and CH3 or hinge alone Figure 6C shows constructs combining optimal hinge region (opti) with combinations of various costimulatory molecule endodomains and CD3 zeta.

[0023] Figure 7 shows the IL-12 and SANT7 vectors.

[0024] Figures 8A-8B shows KM. CAR T-cell expansion and CAR expression with constructs described in Example 3. Figure 8A shows expansion of total cells in CAR T-cell cultures with (left) and without (right) the addition of the KMA expressing JJN3 cell line. Figure 8B CAR expression as measured by GFP in cultures with (top plots) and without (bottom plots) the KMA expressing JJN3 cell line. hCH2CH3= KM.CAR_hCH2CH3_28z T- cells; hCH2CH3mut= KM. CAR hCH2CH3mut_28TM_4lBBz T-cells; h= KM. C AR_h_28TM_41 BBz T-cells; CD8a= KM.CAR_8a_28TM_4lBBz T-cells.

[0025] Figure 9 shows the structure of the activation inducible transposon cassette. IR= inverted repeats; Ins= Insulator flanking the two ends of the gene insert; NFATpro= activation inducible promoter; BGHpA= bovine growth hormone polyadenylation signal; EFla= human elongation factor- 1 alpha promoter; RQR8= marker; SV40= simian virus late polyadenylation signal.

[0026] Figure 10 shows expression of eGFP under activation induced promoter control. Transduced PBMCs stimulated with PMA and Ionomycin (right plot) were assessed for co- expression of RQR8 (x-axis) and eGFP (y-axis) and compared to unstimulated controls (left plot). Transduced cells did not express eGFP in the absence of stimulation. Fifty percent of transduced cells expressed eGFP with stimulation.

[0027] Figure 11 shows the structure of the activation inducible transposon cassette with CAR and biological. IR= inverted repeats; Ins= Insulator flanking the two ends of the gene insert; NFATpro= activation inducible promoter; BGHpA= bovine growth hormone polyadenylation signal; EFla= human elongation factor-l alpha promoter; SV40= simian virus late polyadenylation signal.

[0028] Figures 12A-12B shows KMA-specific interferon-gamma production and cytotoxicity of KM.CAR_hCH2CH3_28z T-cells (Figure 12A) or

KM.CAR_h_28TM_4lBBz T-cells (Figure 12 B) standard chromium release assay with KMA+ and KMA- cell lines. KMA positive cell lines used included JJN3, Pfeiffer, NCI- H929, while KMA negative cell lines included Nalm-6 and Molt.

[0029] Figure 13 shows KMA-mCherry construct used for generating kappa myeloma antigen-antigen presenting cells (KMA APCs) used for stimulating KMA-CAR T-cells as provided herein. The KMA component of the KMA-mCherry construct comprises the kappa myeloma light chain portion of KMA. The construct further comprises a flexible linker ((G4S)3) and CD28 Tm domain fused to mCherry reporter in a PiggyBac transposon vector.

[0030] Figure 14 shows generation of KMA-mCherry expressing APCs from K562 cells.

[0031] Figure 15 shows post-bulk sorting performed during generation of KMA APCs.

[0032] Figure 16 shows general scheme for producing, stimulating and harvesting KM. CAR T-cells.

[0033] Figure 17 shows results of enrichment of KM. CAR T-cells using biotin-KMA selection prior to expansion of the T-cells with PBMCs.

[0034] Figure 18 shows results of stimulating CAR T-cells expressing the CAR-

KM8a28TM4lBBz construct with the KMA-mCherry construct (KMAmCh) without pre selection of CD3+ T-cells.

[0035] Figure 19 shows results of stimulating CAR T-cells expressing the CAR-

KMhCH2CH3mutant28z construct with the KMA-mCherry construct (KMAmCh) without pre-selection of CD3+ T-cells. [0036] Figure 20 shows results of stimulating CAR T-cells expressing the CAR- KMhCH2CH3mutant28z construct with the KMA-mCherry construct (KMAmCh) following pre-selection of CD3+ cells. Pre-selection of CD3+ cells can serve to remove or substantially reduce the number of natural killer (NK) cells.

[0037] Figure 21 shows phenotypic characterization of T-cell cultures electroporated with KM. CAR constructs provided herein. Expression of CAR was detected by staining with biotinylated KMA followed by Streptavidin-PE. % of CAR+ T-cells is plotted (n=3). Flow cytometry dot plots from a representative day 8 culture are shown.

[0038] Figure 22 shows the percent recovery of CD3+ T-cells l-day post-electroporation with KM.CAR constructs described in Example 8.

[0039] Figure 23 shows expansion of T-cells electroporated with KM.CAR constructs in Example 8 after various days post-electroporation when stimulated with KMA-mCherry APCs. Average expansion of CAR T-cells from 3 donor PBMCs over 3 weeks showed a range between 150 to 500 fold expansion upon exposure to KMA antigen.

[0040] Figure 24 shows expression of KM.CAR in CD3+ T-cells from cultures of T-cells electroporated with KM.CAR constructs in Example 8 after various days post-electroporation when stimulated with KMA-mCherry APCs. Expression of CAR was detected by staining with biotinylated KMA followed by Streptavidin-PE. % of CAR+ T-cells is plotted (n=3).

[0041] Figures 25-28 A-B show phenotypic characterization of KM.CAR T-cell cultures from T-cells electroporated with KM.CAR constructs in Example 8 when stimulated with KMA-mCherry APCs. Figure 25 shows distribution of CD4 and CD8 CAR T-cells. Average CD4/CD8 distribution within the final CAR T-cell culture showing a preponderance of CD8+ T-cells over CD4+ cells in a majority of cultures (n=3).

[0042] Figures 26 and 27 show distribution of effector memory cells. Distribution of naive, central and effector memory, and 45RA-effector cells is shown after staining of CAR T-cell cultures (d22) with CD45RA and CD62L n=3.

[0043] Figure 28A shows low levels of the PD1, TIM3 and LAG3 exhaustion markers, while Figure 28B shows a cytokine array. Expression of exhaustion markers PD-l, TIM-3 and LAG3 on CAR+CD3+ cells was determined by flow cytometry n=3 (G) Cytokine membrane array- Release of cytokines from a KMA CD8a28z and CD8a4lBBz CAR T-cell culture alone or after co-culture overnight with KMA+cell lines JJN3 and KMA mCherry K562. Cytokines from target cells alone is also indicated. Normalized pixel density representing levels of cytokines is shown in the heat map.

[0044] Figures 29-34 show functional characterization of KM.CAR T-cell cultures from T- cells electroporated with KM.CAR constructs in Example 8 when stimulated with KMA- mCherry APCs. Figures 29-31 show percentage of CAR T-cells expressing IFNg (Figure 31) or IFNg (Figures 29-30) and TNF-alpha in CD3+ (Figure 29) or CD8+ (Figure 30) cells following electroporation with KM.CAR constructs from Example 8. Figures 32-34 show percentage of specific lysis of KMA-expressing cells or non-KMA expressing cells by T-cells expressing KM.CAR constructs from Example 8.

[0045] Figure 35 shows stimulation of KM.CAR T-cells with kappa myeloma antigen (KMA) coated plates.

[0046] Figure 36 shows stimulation of KM.CAR T-cells with kappa myeloma antigen (KMA) coated plates.

[0047] Figure 37 shows stimulation of KM.CAR T-cells with kappa myeloma antigen (KMA) coated biotin beads.

[0048] Figure 38 shows stimulation of KM.CAR T-cells with kappa myeloma antigen (KMA) coated biotin beads.

[0049] Figure 39 shows the distribution of KMA+ cells in KMA-mCherry K562 cell lines as compared to the KMA expressing JJN3 cell line. KMA staining was performed using an anti- KMA antibody (referred to as KM03 ab, K1-21 or MDX-1097).

[0050] Figure 40 shows the second generation KMA CAR constructs used in Example 8 with the various spacer regions- CH2CH3 (from IgGl) or mutant CH2CH3 (mutated at the Fey binding region) or from CD8a.

[0051] Figure 41 shows the general scheme for generating and expanding CAR T-cells as described in Example 8. CD3+ cells were isolated by MACS separation and co

electroporated with CAR piggyBac transposon DNA and piggyBac transposase DNA using the Neon system (Invitrogen). Transfected CAR T-cells were preferentially expanded by stimulation at days 1, 8 and 15 post-transfection with irradiated PBMC, irradiated KMA expressing K562 cell line (aAPC; see Example 6) and IL-15. Phenotyping and functional characterization of CAR T-cells were carried out at end of culture.

-to- [0052] Figure 42 shows a representative CD8a28z KMA CAR T-cell culture specifically releasing IFNy and TNFa when co-cultured with KMA mCherryK562. This release was not inhibited when free k-light chains at 20, 200, 2000mg/l were added to the co-culture. Free K- light chains by themselves (FLC 20,200, 2000 lanes) did not elicit a response similar to the negative control when no target cells were added. PMA served as a positive control for the cytokine release assay.

[0053] Figure 43 shows expansion of CAR in 3 independent donor derived PBMC- CESI, ROHA and GEYU

[0054] Figure 44 shows the expression of KMA in T-cells from the donors CESI, ROHA and GEYU as described in Example 10 that were exposed to CD8a_28z KM. CAR/piggy Bat constructs from T-cell cultures. The T-cells were engineered to also intrinsically express GFP and the KMA expression was examined throughout the culture period using flow cytometry staining for CAR T cells with biotinylated KMA+secondary streptavidin-PE along with antibodies against CD3, CD4 and CD8. The bottom graph shows consolidated data (n=3) from the three donor T-cell cultures.

[0055] Figure 45 shows the CD4/CD8 proportion of T-cells in the T-cell cultures from the donors CESI, ROHA and GEYU as described in Example 10 throughout the culturing period. The CD4/CD8 proportion was determined by flow cytometry using antibodies against CD4 and CD 8 as well as antibodies against KMA and CD3.

[0056] Figure 46 shows flow cytometry analysis of CD8a_28z KM. CAR T-cells at the end of culturing (i.e., 22 days post-electroporation with the CD8a_28z KM. CAR construct) stained with antibodies against CD62L and CD45RA along with staining with antibodies against CD3 and KMA. The data is consolidated data from the three donor KM. CAR T-cell cultures described in Example 10. Naive defined by CD62L+CD45RA+; Tcm (Central memory) defined by CD62L hi CD45Ra lo; Tern (effector memory) defined by CD62L lo CD45Ra lo; and Teffectors defined by CD45RA hi CD62L lo.

[0057] Figure 47 shows flow cytometry analysis of CD8a_28z KM.CAR T-cells from donors CESI, ROHA and GEYU T-cell cultures stained with antibodies targeting the exhaustion markers PD-l, TIM-3 and Lag-3. The graph on the right shows consolidated n=three data from the three donor T-cell cultures. [0058] Figure 48 shows an analysis on the release of intracellular cytokines INFgamma and TNF alpha from CD8a_28z KM.CAR T-cells upon co-culture with KMA+ specific targets KMAmCherry and JJN3 cell lines or KMA negative cell line LP-l; PMA treatment served as a positive control. The cytokine release data is consolidated data (n=3) from the three donor KM.CAR T-cell cultures described in Example 10.

DETAILED DESCRIPTION OF THE INVENTION

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and material similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following definitions will be used. It will also be understood that the terminology used herein is not meant to be limiting but rather is used herein for the purpose of describing particular embodiments.

[0060] The articles“a” and“an” are used herein to refer to one or more than one (i.e. to at least one or to one or more) of the grammatical object of the article.

[0061] The term “expression vector” as used herein refers to a vector comprising a recombinant nucleic acid sequence comprising at least one expression control sequence operatively linked to the nucleic acid sequence to be expressed. An expression vector comprises all necessary cis acting elements required for expression. Examples of expression vectors include, but are not limited to, plasmids, cosmids, and viruses that encode the recombinant polynucleotide to be expressed. In some embodiments, the expression vector comprises transposable elements that are capable of integrating into the genome, for example, the PiggyBac expression system. In some embodiments, the expression vector is a viral vector that allows for integration of the expression vector contents into the host genome, for example retroviral and lentiviral vectors.

[0062] By “chimeric antigen receptor” or“CAR” is meant an engineered receptor that includes an extracellular antigen-binding domain and an intracellular signaling domain. While the most common type of CAR comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a transmembrane and intracellular domain of a T cell co-receptor, such as the CD3 z chain, the invention described herein is not limited to these domains. Rather, as used herein“chimeric antigen receptor” or“CAR” refers to any receptor engineered to express and extracellular antigen-binding domain fused or linked to any intracellular signaling molecule.

[0063] As used herein the term“CAR-T cell” refers to a T lymphocyte that has been genetically engineered to express a CAR. The definition of CAR T-cells encompasses all classes and subclasses of T-lymphocytes including CD4 ⁺ , CD8 ⁺ T cells as well as effector T cells, memory T cells, regulatory T cells, and the like. The T lymphocytes that are genetically modified may be“derived” or“obtained” from the subject who will receive the treatment using the genetically modified T cells or they may“derived” or“obtained” from a different subject.

[0064] By“intracellular signaling domain” is meant the portion of the CAR that is found or is engineered to be found inside the T cell. The“intracellular signaling domain” may or may not also contain a“transmembrane domain” which anchors the CAR in the plasma membrane of a T cell. In one embodiment, the“transmembrane domain” and the“intracellular signaling domain” are derived from the same protein (e.g. CD3z). In other embodiments, the intracellular signaling domain and the transmembrane domain are derived from different proteins (e.g. the transmembrane domain of a CD3z and intracellular signaling domain of a CD28 molecule, or vice versa).

[0065] By “co-stimulatory endodomain” is meant an intracellular signaling domain or fragment thereof that is derived from a T cell costimulatory molecule. A non-limiting list of T cell costimulatory molecules include CD3, CD28, OX-40, 4-1BB, CD27, CD270, CD30 and ICOS. The co-stimulatory endodomain may or may not include a transmembrane domain from the same or different co-stimulatory endodomain.

[0066] By“extracellular antigen binding domain” is meant the portion of the CAR that specifically recognizes and binds to the antigen of interest. The“extracellular binding domain” may be derived from a monoclonal antibody. For example, the “extracellular binding domain” may include all or part of a Fab domain from a monoclonal antibody. In certain embodiments, the “extracellular binding domain” includes the complementarity determining regions of a particular monoclonal antibody. In still another embodiment, the “extracellular binding domain” is a single-chain variable fragment (scFv). [0067] By “single-chain variable fragment” or“scFv” is meant a fusion protein of the variable heavy (VH) and variable light (VL) chains of an antibody with a peptide linker between the VL and VH. The linker length and composition vary depending on the antibody portions used, but generally are between about 10 and about 25 amino acids in length. In some embodiments, the peptide linker is a glycine rich to provide for flexibility. In some embodiments, the linker also includes serine and/or threonine, which may, without being bound by theory, aid in solubility. In some embodiments, the linker is an amino acid with SEQ ID NO: 23. ScFvs are designed to retain the antigen binding specificity of the parent antibody from which their variable chains were derived despite lacking the immunoglobulin heavy chain. In some embodiments, only the complementary determining regions (CDRs) from the VH and VL are used in the scFv. In some embodiments, the entire VL and VH chains are used.

[0068] The term“antibody” as used herein refers to an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) ₂ , as well as single chain antibodies and humanized antibodies (Harlow et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al, 1988, Science 242:423-426). As used herein the term“antibody” also encompasses antibody fragments.

[0069] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0070] An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0071] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations k and l light chains refer to the two major antibody light chain isotypes. [0072] As used herein the term“complementarity determining region” or“CDR” refers to the part of the two variable chains of antibodies (heavy and light chains) that recognize and bind to the particular antigen. The CDRs are the most variable portion of the variable chains and provide the antibody with its specificity. There are three CDRs on each of the variable heavy (VH) and variable light (VL) chains and thus there are a total of six CDRs per antibody molecule.

[0073] By“KappaMab” is meant the monoclonal antibody previously termed IST-1097 or MDX-1097. Furthermore, as used herein KappaMab may refer to the full antibody sequence of the KappaMab antibody ( See e.g. U.S. Patent Nos. 7,344,715 and 7,556,803 each of which are hereby incorporated by reference in their entireties.) Additionally, the term “KappaMab” as used herein is used to encompass any polypeptide containing the CDR sequences of SEQ ID NOs: 3-8 and/or the VL sequence of SEQ ID NO: 2 and the VH sequence of SEQ ID NO: 1. The term“KappaMab” as used herein can encompass any polypeptide containing the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO: 22. In the compositions and methods of the current invention, KappaMab may include the full monoclonal antibody or any antigen-binding fragment thereof including Fab and scFv.

[0074] The term "antigen" or "Ag" as used herein is defined as a molecule that is recognized by an immune cell receptor (e.g. a T cell receptor, B cell receptor/Immunoglobulin). In some embodiments, an antigen is a molecule that elicits an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, release of cytotoxic mediators or immunostimulatory or regulatory cytokines. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0075] As used herein the term“specifically binds” or“specifically recognizes” as used in connections with an antibody, antibody fragment or CAR refers to a an antibody, antibody fragment or CAR which recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample.

[0076] By“ribosomal skip” is meant an alternative mechanism of translation in which a specific peptide prevents the ribosome of a cell from covalently linking a new inserted amino acid and instead allows it to continue translation thus resulting in a co-translational cleavage of the polyprotein. This process is induced by a“2A ribosomal skip” element or cis-acting hydrolase element (e.g. CHYSEL sequence). In some embodiments, this sequence comprises a non-conserved amino acid sequence with a strong alpha-helical propensity followed by the consensus sequence -D(V/I)ExNPG P, where x=any amino acid. The apparent cleavage occurs between the G and P. In some embodiments, the ribosomal skip element is a 2A ribosomal skip element. The 2A ribosomal skip element can be a 5’ T2A ribosomal skip element.

[0077] As used herein “immunomodulatory drug” or “IMiD” is a class of drugs that constitute thalidomide and its analogs. Thalidomide analogs include lenalidomide, pomalidomide and apremilast.

[0078] As used, herein the term“histone deacetyalse inhibitor” or“HDAC inhibitor” or “HDI” refers to a class of compounds that interferes with the function of histone deacetylase. Examples of HDIs include, but are not limited to, hyroxamic acids including, for example, trichostatin A, vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (LBH589); cyclic tripeptides, including for example, depsipeptides and tapoxin B; benzamides, including for example, entinostat (MS-275), CI994 and mocetinostat (MGCD0103); electrophilic ketones; and aliphatic compounds, such as for example, phenylbutyrate and valproic acid.

Kappa Myeloma Antigen and Antibodies

[0079] Kappa myeloma antigen or KMA is a cell membrane antigen that is found on the surface of myeloma cells. Specifically, KMA consists of free kappa light chains expressed in non-covalent association with actin on the cell membrane (Goodnow et al. (1985) J. Immunol. 135:1276). While any antibody that specifically binds to KMA may be used in accordance with the present invention, in a preferred embodiment the KappaMab monoclonal antibody will be used as a basis for the extracellular antigen binding domain of the CARs of the current invention. The monoclonal antibody designated KappaMab (formally designated IST-1097, also known as MDX-1097) binds to a conformational epitope in the switch region of human kappa free light chain that is only available when the kappa chain is not associated with a heavy chain and therefore does not bind to intact kappa-chain containing IgG, IgM, IgE or IgA (Hutchinson et al. (2011) Mol. Immunol.). Typical expression of KMA on primary myeloma cells derived from patient bone marrow biopsies is shown by KappaMab binding in Figure 3. The KappaMab antibody can comprise the VH chain of SEQ ID NO: 1 and the VL chain of SEQ ID NO: 2. More specifically the KappaMab VH chain can comprise the CDRs of SEQ ID NO: 3-5 and the VL CDRs of SEQ ID NO: 6-8. Additionally, the KappaMab can comprise a VH region of SEQ ID NO: 22 and a VL region of SEQ ID NO: 21

Chimeric Antigen Receptors

[0080] Chimeric antigen receptors (CARs) are artificial receptors consisting of the tumor antigen binding regions of monoclonal antibodies and the intracellular activating portion of the T cell receptor complex in a single polypeptide chain held together by a series of linker(s) and spacer(s) (Figures 1A-1B). Most commonly, CARs are fusion proteins of single-chain variable fragments (ScFv) fused to the CD3z transmembrane domain. However, other intracellular signaling domains such as CD28, 41 -BB and 0x40 may be used in various combinations to give the desired intracellular signal. In some embodiments, the CARs provided herein comprise an Ig Heavy Chain Leader peptide. The leader peptide can be SEQ ID NO: 20.

I. Extracellular Antigen Binding Domain

[0081] In one embodiment, the CAR of the current invention comprises an extracellular antigen-binding domain from a monoclonal antibody that is specific for one or more KMA epitopes expressed on MM cells. In one embodiment, the CAR of the current invention comprises an extracellular antigen-binding domain from KappaMab. In one embodiment, the extracellular binding domain comprises the VL CDRs of SEQ ID NOs: 6-8 and VH CDRs of SEQ ID NOs: 3-5. In a particular embodiment, the extracellular binding domain is a scFv comprising the VL (SEQ ID NO: 2) and VH (SEQ ID NO: 1) domains of KappaMab. In another embodiment, the extracellular binding domain is a scFv comprising the VL (SEQ ID NO: 21) and VH (SEQ ID NO: 22) domains of KappaMab.

II. Linker between VL and VH domains of KappaMab scFv

[0082] In a further embodiment, the KappaMab VL is linked to the KappaMab VH via a flexible linker. Specifically, the flexible linker is a glycine/serine linker of about 10-30 amino acids (for example 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids) and comprises the structure (Gly4Ser)3. In a particular embodiment, the linker is 15 amino acids in length. Linker length is an important determinant of a CAR. Without being bound by theory, shorter linkers may enhance affinity but can also lead to intracellular multimer formation thus impairing expression of the CAR whereas longer linkers tend to decrease antigen affinity by moving the VL and VH CDRs further apart in space.

III. Spacers between extracellular antigen binding domain and intracellular signaling domain

[0083] The extracellular antigen-binding domain (e.g. KappaMab scFv) is linked to the intracellular signaling domain by the use of a“spacer”. The spacer is designed to be flexible enough to allow for orientation of the antigen-binding domain in such a way as facilitates antigen recognition and binding. The spacer may derive from immunoglobulins themselves and can include the IgGl hinge region or the CH2 and/or CH3 region of an IgG. Alternatively, the hinge may comprise all or part of a CD8a chain. The length and flexibility of the spacer(s) is dependent on both the antigen recognition domain as well as the intracellular binding regions and what may be functional and/or optimal for one CAR construct may not be for another CAR. In certain instances, the spacer may be designated herein as“opti” ( See Figures 6A-6C) to signify that optimal spacer identity and length varies depending on the extracellular binding portion used and the intracellular signaling domains selected. In certain embodiment, an IgG hinge alone is used. In other embodiments, the IgG hinge is used together with all or part of IgG CH2 domain. In other embodiments, the IgG hinge is used together with all or part of an IgG CH3 domain. In other embodiments, the IgG hinge is used together with all or part of both an IgG CH2 and CH3 domain. In other embodiments, all or part of an IgG CH2 domain is used. In other embodiments, all or part of an IgG CH3 domain is used. In still other embodiments, all or part of both an IgG CH2 and CH3 domain is used. In one embodiment, the hinge, CH2 and CH3 domains used in any of the constructs provided herein comprises a C to P mutation in the hinge region at amino acid position 103 of Uniprot P01857). In one embodiment, the hinge, CH2 and CH3 domains used in any of the constructs provided herein is SEQ ID NO: 24. In another embodiment, the hinge is used together with all or part of both an IgG CH2 and CH3 domain, wherein mutations are introduced at amino acids important for CH2 interaction with Fc-receptors. These mutations may mediate improved survival post infusion by decreasing Fc interaction with CAR T-cells provided herein. An example of these mutations can be seen in the KM.CAR_hCH2CH3mut_28TM_4lBBz construct shown in Example 3 as provided herein. In a further embodiment still, a CD8a polypeptide is used. In a further embodiment, the spacer (e.g., derived from immunoglobulin domains as described herein) can be attached to the scFv via a flexible linker. Specifically, the flexible linker is a glycine/serine linker of about 10-30 amino acids (for example 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids) and comprises the structure (Gly4Ser) _x where X is 1-5. In other embodiments, the glycine/serine linker comprises (Gly4Ser)3.

IV. Intracellular Signaling Domain

[0084] The intracellular signaling domain comprises all or part of the CD3 z chain. Oϋ3z, also known as CD247, together with either the CD4 or CD8 T cell co-receptor is responsible for coupling extracellular antigen recognition to intracellular signaling cascades. In one embodiment, the CD3 z used in any of the constructs provided herein is SEQ ID NO: 26.

[0085] In addition to the including of the 6Ό3z signaling domain, the inclusion of co stimulatory molecules has been shown to enhance CAR T-cell activity in murine models and clinical trials. Several have been investigated including CD28, 4-1BB, ICOS, CD27, CD270, CD30 and OX-40. The CAR of the current invention, in addition to including the KappaMab scFv, flexible linker, optimal hinge and CD3z chain also include one or more additional costimulatory domains from CD28, 4-1BB (CD137), ICOS, CD27, CD270, CD30 and/or OX-40, for example. These co-stimulatory domains are selected based on the desired functionality of the resulting CAR T-cell. Exemplary combinations are shown, for example, in Figures 6A-6C. In addition to altering the length of the extracellular hinge, the inclusion of particular combinations of costimulatory domains (e.g. CD28, OX-40, 4-1BB) also enhances the proliferation and survival of CAR T-cells in vivo. In one embodiment, the CD28 domain used in any of the constructs provided herein is SEQ ID NO: 25.

Co-Expression of Biologically Active Molecules

[0086] The CAR T-cells of the current invention have the added benefit, when compared to the use of the KappaMAb alone to be further modifiable to contain additional biologically active molecules to enhance the anti-tumor function and/or safety of the compositions. In one embodiment, the CAR T-cells may be further genetically modified to produce antitumor cytokines, which allow for focused delivery to the tumor microenvironment/cancer cells, while avoiding systemic toxicity. Examples of additional biologically active molecules which may enhance the anti-tumor response of the CAR T-cells of the current invention include, without limitation, IL-12, the carbohydrate binding protein Galectin-3 (GAL3) or its truncated form, GAL3C, and the cytokine receptor super antagonist SANT7. In another embodiment, CAR T-cells of the current invention may also be co-transduced with a plasmid that expresses a hepatocyte growth factor (HGF) binding protein. In one embodiment, the hepatocyte growth factor protein is an antibody or fragment thereof that is able to bind to and inhibit the function of HGF.

[0087] IL-12 is a potent tumor suppressor cytokine, decreasing tumor growth and angiogenesis and enhancing the tumor specific immune response. Multiple myeloma cells retain expression of the IL-12 receptor and administration of IL-12 to myeloma bearing mice decreases tumor progression as a single agent and acts synergistically with the proteasome inhibitor bortezomib (Airoldi, et al. (2008) Blood, H2(3):750-759; Wang, et al. (2014) Anticancer Drugs, 25(3): 282-288). Expression of IL-12 by CAR T-cells dramatically enhances their ability to eradicate solid tumors but this approach has not yet been investigated in multiple myeloma (Pegram, (2012) Blood, 119(180:4133-4141 and Zhang, et al. (2011) Mol. Ther. 19(4):751-759).

[0088] SANT7 is a cytokine receptor super-antagonist. It is an analogue of IL-6 that has been genetically modified to enhance its binding to the IL-6 receptor a-subunit 70-fold, with virtually no interaction with the gpl30 signaling subunit. SANT7 induces apoptosis in IL-6 dependent myeloma cell lines in-vitro, overcomes stroma mediated resistance to dexamethasone in in-vitro and murine model and combined with NFKB inhibitors, completely overcomes resistance to apoptosis. IL-6 is a cytokine, which plays a role in the growth and survival of a variety of tumors including multiple myeloma, lung cancer, colorectal cancer, breast cancer and others. Binding of IL-6 to its receptor activates the JAK- STAT pathway, with subsequent phosphorylation of STAT3, which modulates expression of apoptosis related genes such as BCL-XL and p53, causing resistance to apoptosis. IL-6 also promotes down-regulation of the IL-12 receptor on myeloma cells, decreasing IL-l2’s tumor suppressive properties. (Airoldi, et al. (2008) Blood, H2(3):750-759). The IL-6 receptor is upregulated in myeloma and elevated systemic levels of IL-6 correlate with a poor prognosis. (Rawstron, et al. (2000) Blood 96(12) 3880-3886; Ludwig, et al. (1991) Blood, 77(l2):2794- 2795). Monoclonal antibodies to IL-6 have been developed for clinical use, however, although early clinical trials in myeloma showed measurable biological effects, the antibodies appeared to form complexes with circulating IL-6, leading to reduced clearance and potentially limiting their efficacy. (Bataille, et al. (1995) Blood, 86(2): 685-691). Recently, the chimeric IL-6 specific monoclonal antibody Siltuximab has been assessed in phase I and II clinical trials in relapsed and refractory multiple myeloma. There were no responses to Siltuximab alone, but hematological toxicity was common with more than half experiencing therapy related infections.

[0089] Galectin-3 is a carbohydrate binding protein, which may play a role in tumour adhesion and invasion. A truncated form of Galectin-3, Gal3C, acts as a dominant negative form and can inhibit myeloma cell growth and invasion. A Gal3C construct for activation inducible secretion was designed based on John et al (2003) Clin Cancer Res., 9(6):2374-83 and Mirandola et al. (2011) PLoS One, 6(7):e2l8l l. This consists of the 143 amino acid carboxy terminal, which retains its carbohydrate binding properties, but lacks the N-terminal amino acids required for ligand crosslinking. The construct also contains the CD8-alpha leader peptide to direct secretion and a 6xHis tag for detection.

[0090] In certain embodiments, in addition to expression vectors containing the CAR construct described above, T cells are further modified with one or more expression vectors comprising IL-12, SANT7 and/or GAL3C. Specifically, expression constructs expressing a single chain IL-12 comprising the IL-12 p35 subunit linked to the IL-12 p40 subunit are particularly useful in that the resulting protein is a fully bioactive IL-12 p70 heterodimer, however, expressed as a single polypeptide. In one embodiment, the single chain IL-12 construct, termed Flexi-l2, is described, for example in Anderson, et al. (1997) Hum. Gene Ther. 8(9): 1125-35 is used. The IL-12 single chain construct may be expressed in the same expression vector as the CAR construct or it may expressed in a separate expression vector and co-transduced into the T cell. Similarly, T cells transduced with the CAR construct described above may be co-transduced with an additional expression vector comprising SANT7 and/or GAL3C, alternatively, one expression vector may be used to transduce T cells with both of SANT7 and GAL3C either alone or in combination and the CAR construct described above. In another embodiment three expression vectors may be used, one expressing the CAR construct, one expressing the single chain IL-12 construct and one expressing the SANT7 construct. A similar strategy may be used to co-express GAL3C with IL-12 and/or SANT7. Alternatively, the IL-12, GAL3C and/or SANT7 construct may be expressed via a single expression vector while the CAR construct is expressed by its own expression vector. One of skill in the art will appreciate the different combinations and possibilities for expressing these molecules in the same T cell. HGF Binding Protein

[0091] Hepatocyte growth factor (HGF) and its receptor, MET have been implicated in cancer development and progression, in particular in tumor invasion and progression to metastatic disease. Multiple myeloma cells express both HGF and MET, thus creating both an autocrine and paracrine loop whereas normal plasma cells do not express HGF (Zhan et al. (2002); Borset, et al. (1996). Furthermore, HGF concentrations are significantly increased in the blood and bone marrow of plasma patients with multiple myeloma and high serum HGF levels correlate with advanced stage disease and extensive bone lesions (Seidel et al. (1998); Wader, et al. (2008); Alexandrakis, et al. (2003). Furthermore, serum biomarker analysis of patients in a phase I trial with KappaMab shows statistically significant dose related decrease in serum HGF after treatment with KappaMab compared to control. In order to enhance this reduction in serum HGF, in certain embodiments an HGF binding protein will be expressed in the CAR T-cells of the current invention. In a particular embodiment, the HGF binding protein expressed is an antibody or fragment thereof. In a particular embodiment, the anti- HGF binding protein is an antibody, a diabody, a scFv or a Fab. In one embodiment, the HGF binding protein is expressed in the same expression vector as the CAR construct. In a further embodiment, the HGF binding protein is expressed in a separate expression vector but is co-transduced with the CAR construct. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and IL-12. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and SANT7. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and GAL3C. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and IL-12 and GAL3C. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and SANT7 and GAL3C. In still another embodiment, the CAR-T cell expresses the CAR, an anti-HGF binding protein, IL-12 and SANT7. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein, IL-12, SANT7 and GAL3C.

Methods of Producing the CAR T-cells

[0092] In one aspect, methods are provided for generating CAR T-cells expressing the CAR(s) described herein and optionally one or more anti-tumoral cytokine (e.g. IL-12 and/or SANT7) and/or one or more HGF binding protein. One of skill in the art will readily understand that while preferred methods of constructing expression vectors containing the CARs and anti-tumoral cytokines/antibodies of the present invention are described herein, that any methods which are able to transduce T cells to express these constituents may be used.

[0093] In one embodiment, T cells are obtained from the blood of a subject by venous puncture, aspiration of bone marrow, steady state leukapheresis or cytokine primed leukapheresis and subsequent isolation of peripheral blood mononuclear cells including T cells using density gradient separation. In certain embodiments, after lysing red blood cells, T cells are sorted by flow cytometry or purified using antibodies to antigens expressed on T cells and magnetic beads to obtain a population of pure T cells. In a particular embodiment, T cells are sorted based on their expression of CD3 to obtain a whole T cell fraction. In another embodiment T cells are sorted based on their expression of CD4 or CD 8 to obtain a population of either CD4 ⁺ T cells or CD8 ⁺ T cells. In a particular embodiment, T cells are obtained from the subject in need of CAR T-cell therapy. In another embodiment, T cells are obtained from a donor subject who is not the intended recipient of CAR T-cell therapy.

[0094] In one embodiment, separated T cells are cultured in vivo under conditions suitable for their survival and are transduced with expression vectors containing the sequences necessary for expression of the CARs described herein and/or IL-12, SANT7, GAL3C and/or an HGF binding protein. In one embodiment, the expression vector is a transposable vector expression system. In a particular embodiment, the expression vector is a PiggyBac transposon expression plasmid or a viral vector (e.g. retroviral vector or lentiviral vector). In one embodiment, the PiggyBac transposon expression plasmid is inducible such as, for example, the PiggyBac transposon plasmid described in the Examples provided herein. In one embodiment, the PiggyBac transposon expression plasmid comprises a constitutively active promoter and/or an activation inducible promoter. The constitutively active promoter can be an elongation factor 1 alpha (EF1 alpha) promoter. The activation inducible promoter can be a (NFAT pro) promoter. In one aspect, a PiggyBac expression plasmid is used and produces permanent integration of the CAR by cutting and pasting the CAR, IL-12, SANT-7, GAL3C and/or HGF binding protein coding sequences into the T cell’s genome. In some cases, the promoter operably linked to IL-12, SANT7, GAL3C and/or HGF binding protein coding sequences in constructs containing said protein coding sequences can be an activation inducible promoter such as, for example, the NFAT promoter or a constitutively active promoter as provided herein (e.g., EF1 alpha promoter) or known in the art. In some cases, IL-12, SANT7, GAL3C and/or HGF binding protein present within a constructs/plasmid provided herein can be separated by 2A ribosomal skip elements. In a particular embodiment, the expression vectors of the current invention further comprise a detectable marker, which allows for identification of T cells that have been successfully transduced with the one or more expression vectors. In one embodiment, the detectable marker is chosen from the group consisting of a cell surface marker such as CD34 or CD20 or another surface protein, a fluorophore such as fluorescein isothiocyanate or any other fluorescent dye that emits light when excited to a higher energy state including by a laser, and an antibiotic resistance cassette such as kanamycin resistance, ampicillin resistance or any other cassette that confers resistance to an antibiotic substance contained in medium in which transduced T cells are to be cultured. In one embodiment, the detectable marker is a green fluorescence protein (GFP). The GFP can be an enhanced GFP, such as, for example, the constructs shown in the Examples provided herein. In a particular embodiment, each expression vector used (e.g. one expression vector comprising a CAR, and one comprising an IL-12, GAL3C and/or SANT-7 and one comprising an HGF binding protein) comprises a unique detectable marker. In one embodiment, the expression vectors are transduced into the T cell by a method suitable for the expression vector(s) selected. In one embodiment, the PiggyBac expression vector is transduced into T cells by electroporation.

[0095] In another embodiment, the expression vector is a PiggyBat transposon expression plasmid. In one embodiment, the PiggyBat transposon expression plasmid is inducible. In one embodiment, the PiggyBat transposon expression plasmid comprises a constitutively active promoter and/or an activation inducible promoter. The constitutively active promoter can be an elongation factor 1 alpha (EF1 alpha) promoter. The activation inducible promoter can be a (NFAT pro) promoter. In one aspect, a PiggyBat expression plasmid is used and produces permanent integration of the CAR by cutting and pasting the CAR, IL-12, SANT-7, GAL3C and/or HGF binding protein coding sequences into the T cell’s genome. In some cases, the promoter operably linked to IL-12, SANT7, GAL3C and/or HGF binding protein coding sequences in constructs containing said protein coding sequences can be an activation inducible promoter such as, for example, the NFAT promoter or a constitutively active promoter as provided herein (e.g., EF1 alpha promoter) or known in the art. In some cases, IL-12, SANT7, GAL3C and/or HGF binding protein present within a constructs/plasmid provided herein can be separated by 2A ribosomal skip elements. In a particular embodiment, the expression vectors of the current invention further comprise a detectable marker, which allows for identification of T cells that have been successfully transduced with the one or more expression vectors. In one embodiment, the detectable marker is chosen from the group consisting of a cell surface marker such as CD34 or CD20 or another surface protein, a fluorophore such as fluorescein isothiocyanate or any other fluorescent dye that emits light when excited to a higher energy state including by a laser, and an antibiotic resistance cassette such as kanamycin resistance, ampicillin resistance or any other cassette that confers resistance to an antibiotic substance contained in medium in which transduced T cells are to be cultured. In one embodiment, the detectable marker is a green fluorescence protein (GFP). The GFP can be an enhanced GFP. In a particular embodiment, each expression vector used (e.g. one expression vector comprising a CAR, and one comprising an IL-12, GAL3C and/or SANT-7 and one comprising an HGF binding protein) comprises a unique detectable marker. In one embodiment, the expression vectors are transduced into the T cell by a method suitable for the expression vector(s) selected. In one embodiment, the PiggyBat expression vector is transduced into T cells by electroporation.

Stimulation and Expansion of KM CAR T-cells

[0096] After introduction of the appropriate expression vectors (e.g., any of the KM. CAR constructs provided herein in PiggyBac or PiggyBat vectors), T cells may be cultured and expanded in vitro by co-culture with autologous peripheral blood mononuclear cells (PBMCs) and appropriate growth factors (e.g., IL-15) alone or in combination with cells expressing an antigen of interest (e.g., expressing KMA) and further screened for the presence of the one or more detectable markers. For example, a general outline for the generation, stimulation and expansion of T-cells expressing any of the KM. CAR constructs provided herein is shown in Figure 16 or 41. As shown in Figure 16 or 41, cells from a donor are thawed and CD3+ T-cells are pre-selected and grown in culture for up to two days. Subsequently, the T-cells are electroporated with a KM. CAR construct (e.g., any of the KM.CAR constructs provided herein or in WO2016/172703, which is herein incorporated by reference) and grown in culture overnight. Following electroporation and growth, the T-cells are stimulated and expanded by being cultured in the presence of PBMCs (e.g., irradiated (IR) PBMCs) plus cytokines with or without the addition of KMA. In some cases, the KMA can be found in KMA antigen presenting cells (APCs). In some cases, the KMA can be bound to a solid substrate. The solid substrate can be a planar surface such as a glass slide or microtiter plate. The solid substrate can be a bead. In some cases, the KMA can be bound to the solid substrate via a binding moiety such as, for example, biotin. In some cases, the KMA can be soluble. The KMA can be a kappa free light chain (KLC). The KLC can comprise, consist or contain the switch region (e.g., nucleic acid SEQ ID NO: 46) and/or constant region (e.g., nucleic acid SEQ ID NO: 47) of KLC. Fresh PBMCs plus cytokines and APCs, if used, can be introduced during the I ^st , 8 ^th or l5 ^th day in culture following electroporation and KM. CAR expressing T-cells can be harvested during the l5 ^th or 22 ^nd day in culture following electroporation. In one embodiment, as shown in Figure 17, the KM.CAR T-cells can be enriched from non-KMA expressing T-cells using KMA bound to a solid substrate prior to stimulation of the KM.CAR T-cells. The solid substrate can be a planar surface such as a glass slide or microtiter plate. The solid substrate can be a bead. In some cases, the KMA can be bound to the solid substrate via a binding moiety such as, for example, biotin. The KMA can be conjugated to biotin and subsequently be bound by anti biotin beads or another solid substrate. The KMA can comprise, consist or contain the switch region (e.g., nucleic acid SEQ ID NO: 46) and/or constant region (e.g., nucleic acid SEQ ID NO: 47) of KLC.

[0097] In one embodiment, the KMA expressing antigen presenting cell (APC) used to stimulate KM.CAR T-cells is a cell line known in the art such as, for example, JJN3 or Pfeiffer cell lines. In another embodiment, the KMA expressing APC is a cell line engineered to express a KMA construct -reporter protein as provided herein. In one embodiment, the cell line is a K562 cell line. In another embodiment, the cell line is an HEK293 cell line. The KMA construct can comprise sequence encoding KMA or a portion thereof as well as sequence encoding a reporter protein. In one embodiment, the KMA is a kappa light chain of KMA. The kappa light chain of KMA can be a kappa light chain known in the art and/or as provided herein. The KMA can comprise, consist or contain the switch region (e.g., nucleic acid SEQ ID NO: 46) and/or constant region (e.g., nucleic acid SEQ ID NO: 47) of the kappa free light chain. In one embodiment, the KMA-reporter protein construct is the KMA- mCherry construct of SEQ ID NO: 44. The KMA-reporter construct can further comprise one or more co-stimulatory domains such as, for example, the KMA-mCherry construct of SEQ ID NO: 45.

[0098] In another embodiment, KM.CAR expressing T-cells may be stimulated and expanded by culturing the KM.CAR expressing T-cells in media with or without appropriate growth factors (e.g., IL-15) in the presence of KMA-coated solid substrates. The KMA- coated solid substrates can be KMA-coated plates. The KMA-coated solid substrates can be KMA coated beads. The KMA can be a kappa light chain as known in the art and/or as provided herein such as the Kappa light chain switch domain (e.g., nucleic acid SEQ ID NO: 47) and/or kappa light chain constant domain (e.g., nucleic acid SEQ ID NO: 48). An example of this method is described in Example 9.

[0099] T cells expressing the appropriate detectable markers for the expression vectors chosen may then be sorted and purified for use in the methods of the current invention.

Generation of KMA-expressing Antigen Presenting Cells (APCs).

[00100] In one embodiment, methods are provided for generating cells or cell lines that express a KMA antigen. These cells or cell lines can be referred to as KMA expressing antigen-presenting cells (APCs). The KMA antigen can be the entire KMA or portions thereof. In one embodiment, the KMA antigen can be the KMA light chain. The KMA light chain can comprise, consist essentially of or consist of the KMA light chain switch region (e.g., nucleic acid SEQ ID NO: 46) and/or constant region (e.g., nucleic acid SEQ ID NO: 47). The KMA antigen can be present in a chimeric construct. The chimeric construct can comprise a reporter protein. The reporter protein can be GFP or mCherry. The chimeric construct can further comprise one or more glycine/serine linkers comprising the structure (Gly4Ser) _x where X is 1-5. The chimeric construct can further comprise a transmembrane domain provided herein, such as, for example, a CD28 transmembrane domain. In one embodiment, the KMA construct is a KMA-mCherry chimeric construct of SEQ ID NO: 44. In another embodiment, the KMA-chimeric construct can further comprise one or more co stimulatory domains such as, for example, the KMA-mCherry construct of SEQ ID NO. 45. In one embodiment, the KMA-chimeric construct is cloned into an expression vector. The expression vector can be a transposable vector expression system. In one embodiment, the expression vector is a PiggyBac transposon expression plasmid or a viral vector (e.g. retroviral vector or lentiviral vector). In another embodiment, the expression vector is a PiggyBat transposon expression plasmid or a viral vector. Following cloning of the KMA construct into an expression vector, said vector is introduced (e.g., via electroporation) into a cell line. In one embodiment, the cell line is a K562 cell line. In another embodiment, the cell line is an HEK293 cell line. Following introduction into the cell line, the cells expressing the KMA-reporter protein construct can be isolated, purified and expanded in appropriate growth media. Isolation and/or purification of cells expressing the KMA-reporter protein construct can be via flow cytometry. Following several passages, cell lines reproducibly producing predominantly KMA-reporter protein expressing cells in culture can be selected for the methods described herein for expanding and stimulating KM.CAR T-cells.

[00101] In one embodiment, for use in the constructs and methods provided herein, the KMA light chain switch region has the nucleic acid sequence of CTGGAAATTAAACGC (SEQ ID NO: 46)

[00102] In one embodiment, for use in the constructs and methods provided herein, the

KMA light chain constant region has the nucleic acid sequence of

ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC

TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA

GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC

ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTG

AGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG

GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO:

47)

Methods of treating KMA-Expressing Malignancies

[00103] In one aspect, methods are provided for treating subjects in need thereof with the CAR T-cells provided herein. In a particular aspect, the subject in need thereof is a human subject who has been diagnosed with or is suspected of having a malignancy that expresses KMA, for example a B cell malignancy expressing KMA. In certain embodiments, a patient has or is suspected of having multiple myeloma (MM), Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), or amyloidosis. Methods for diagnosing B cell malignancies expressing KMA, for example, multiple myeloma (MM) Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), and amyloidosis are known in the art, and as such are not described in detail herein. The CAR T- cells may be used alone or in combination with other therapeutically effective agents for the treatment of multiple myeloma (MM) Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), amyloidosis or another B cell malignancy expressing KMA. In certain aspects, the CAR T-cells of the current invention are administered in a pharmaceutical formulation suitable for intravenous delivery. [00104] In certain aspects, the CAR T-cells of the current invention are administered before, during or after one or more immunomodulatory drugs. In a particular aspect, the one or more immunomodulatory drugs is thalidomide or a thalidomide analog such as, for example, lenolidomide or pomalidomide.

[00105] In certain aspects of the invention, the CAR T-cells of the current invention act synergistically when administered with one or more immunomodulatory drugs.

[00106] In a further embodiment, the CAR T-cells of the current invention are administered before, during or after treatment with one or more histone deacetylase inhibitors such as panobinostat, vorinostat, trichostatin A, depsipeptides, phenylbutyrate, valproic acid, belinostat, LAQ824, entinostat, CI944 or mocetinostat.

[00107] In certain aspects of the invention, the CAR T-cells of the current invention act synergistically when administered in combination with one or more histone deacetylase inhibitors.

[00108] In certain aspects of the invention, the CAR T-cells of the current invention act synergistically when administered in combination with intermediate or high dose chemotherapy and following administration of autologous or allogenic human blood stem cells.

[00109] In one embodiment, the CAR T-cells of the current invention are administered before, during or after an allogenic stem cell transplant. In still another embodiment, the CAR T-cells of the current invention are administered before during or after an allogenic stem cell transplant. Without being bound by theory, the CAR T-cells of the present invention, when administered in combination with an autologous or allogeneic stem cell transplant prevent the appearance of minimal residual disease that may occur by incomplete ablation of the bone marrow prior to stem cell transplant or by reemergence of malignant B cell clones expressing KMA.

[00110] All patents, patent applications, and publications cited herein are expressly incorporated by reference in their entirety for all purposes.

EXAMPLES

[00111] The invention is further described in detail by reference to the following experimental examples. These examples are provided for the purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations, which become evident as a result of the teaching provided herein.

[00112] Without further description, it is believed that one of ordinary skill in the art can using the preceding description and following examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLE 1: Generation of KM A.CAR-28z

[00113] Based on the nucleotide sequence coding for the variable regions of KappaMab (SEQ ID NOS: 9 and 10), a scFv was designed and cloned into a CAR construct containing an immunoglobulin heavy chain hinge, a CD28 co-stimulatory domain and the CD3-zeta endodomain (Figure 6A). The construct was designed in Clone Manage 9 (Sci-Ed Software) using the genetic sequence of the antibody variable regions provided by Haemal ogix Pty Ltd. The amino acid sequence from 5’ to 3’ of portions of this construct(i.e., KM.CAR-hCH2CH3-28z; Figure 6A) are as follows:

[00114] The Ig heavy chain leader peptide (Uniprot P01764) is

MEFGLSWLFLVAILKGVQCSR (SEQ ID NO: 20).

[00115] The KappaMab antibody light chain variable region is

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSTSYRYS GVPDRFT GS GS GTDFTLTISNV Q S EDL AEYFCQQ YN S YP YTF GGGTKLEIK (SEQ ID NO: 21).

[00116] The heavy chain variable region is

EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANG NTKYDPKFQGKATIIADTSSNTAYLQLSSLTSEDTAVYYCARGVYHDYDGDYWGQG TTLTVSSYVTVSS (SEQ ID NO: 22).

[00117] The (G ₄ S) ₃ flexible linker is GGGGS GGGGS GGGGS (SEQ ID NO: 23).

[00118] The hinge, CH2 and CH3 domains of IgGl constant region with a C>P mutation in the hinge region at amino acid position 103 (Uniprot P01857) is YVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKE YKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGKKDPK (SEQ ID NO: 24).

[00119] The transmembrane and intracellular domains of CD28 (Uniprot P 10747) is FWVLVVV GGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QP YAPPRDFAAYRS (SEQ ID NO: 25).

[00120] The intracellular domain of human CD3 zeta (Uniprot P20963) is RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALP PR (SEQ ID NO: 26).

[00121] The full length amino acid sequence as follows is

MEFGLSWLFLVAILKGVQCSRDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVA

WYQQKPGQSPKALIYSTSYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQY

NS YP YTF GGGTKLEIKGGGGS GGGGS GGGGS EVQLQQ S GAEL VKPGAS VKL S CT AS

GFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATIIADTSSNTAYLQ

LSSLTSEDTAVYYCARGVYHDYDGDYWGQGTTLTVSSYVTVSSQDPAEPKSPDKTH

TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

VEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTIS

KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDP

KF WVLVVV GGVLACY SLLVTV AFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY Q

PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

EMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATK

DTYDALHMQALPPR (SEQ ID NO: 27).

[00122] This amino acid sequence (SEQ ID NO: 27) is encoded by the following DNA sequence:

[00123] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCACA

TCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACT AATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACT

CGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGG

GACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTAT

TTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTG

GAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT

GAGGTGC AGCT GC AGC AGT C AGGGGC GGAGCTT GTGAAGCC AGGGGC CT C AGT C

AAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGG

TGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGA

ATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAATAGCAG

ACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACA

CTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG

GGGCCAAGGGACCACGCTCACCGTCTCCTCCTACGTCACCGTCTCTTCACAGGAT

CCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCA

CCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA

CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA

CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA

TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG

CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA

GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA

AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCT

GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA

CATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCA

CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT

GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA

GGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAA

AGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGC

TT GCT AGT AAC AGTGGCCTTT ATT ATTTT CT GGGT GAGGAGT AAGAGGAGC AGGC

TCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAA

GCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTG

AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTC

TATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA

CGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGA

AGGC CTGT AC AAT GA ACT GC AGAAAGATAAGAT GGCGGAGGC CT AC AGT GAGAT TGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCC CCCTCGC (SEQ ID NO: 28).

[00124] In terms of constructing this construct, a gene sequence consisting of a 5’ EcoRI restriction enzyme site, a 5’ Kozak sequence, Leader Peptide, single chain variable fragment and a portion of the IgGl constant region incorporating an Alel restriction enzyme site, was synthesized by GeneArt (ThermoFisher Scientific), sequence verified and then cloned into a PiggyBac transposon expression plasmid. This was then introduced into donor T-cells from two normal donors by co-electroporation with the PiggyBac Transposase plasmid to mediate stable integration. The PiggyBac transposon/transposase system produces permanent integration of the CAR by cutting and pasting the gene of interest into the target cell genome. The PiggyBac expression system was chosen because it is capable of producing high levels of permanent genetic modification at a fraction of the cost of retroviral vectors. However, one of skill in the art will understand that other expression systems, including retroviral vectors could also be used in accordance with the current invention. For example, one of skill in the art may also use the PiggyBat expression system as described herein.

[00125] The KM.CAR-hCH2CH3-28z expressing T-cells were expanded according to our optimized protocols by co-culturing with autologous peripheral blood mononuclear (PBMC) feeder cells supplemented with 200 IU or 200 ng/ml of interleukin- 15 (IL-15). After culturing for 3 weeks with replacement of PBMCs on a weekly basis and replenishment of IL-15 two to three times per week, T-cells were harvested and assessed for phenotype and CAR expression by flow cytometry, KMA-specific function by interferon gamma intracellular cytokine flow cytometry on stimulation with KMA+ and KMA- cell lines (Figure 4A) and cytotoxicity of the same cell lines in a chromium release assay.

[00126] At the end of 3 weeks, the cultures were predominantly CAR expressing CD3 ⁺ T-cells (55% and 70% of live cells), expressed interferon-gamma in response to KMA ⁺ myeloma and B-cell lines (Figure 4B) and demonstrated KMA-specific cytotoxicity (Figure 4C).

Example 2: Establishing a human myeloma xenograft murine model

[00127] A human myeloma to mouse xenotransplant model of multiple myeloma was established. RPMI8226 or alternative myeloma cell lines were inoculated i.v. into Rag2-/- yc-/- (BALB/c) mice to form the Rag MM model (Figure 5A-5D). The Rag2-/-yc-/- (BALB/c) mice lack mouse lymphocytes (T, B and NK cells) and are receptive hosts for human xenograft studies. This model has been used successfully to test novel therapeutics such as bortezomib in combination with a novel antibody (Figure 5E). We will use this MM model to test and further optimize the KMA.CAR T cells.

Example 3: Optimized KMA.CAR Constructs

[00128] Based on the construct described in Example 1, 6 CAR constructs containing the KM scFv described in Example 1 with variable length spacer regions and co-stimulatory endodomains (e.g., CD28 or 4-1BB (CD 137-Uniport Q07011)) with the CD3 zeta endodomain were constructed (Figure 2 & Figures 6B-6D). Varying the spacer length altered the distance between the T-cell and the target cell with a shorter spacer potentially enhancing target cell lysis. In all constructs, the CD28 transmembrane domain was used to ensure stable T-cell surface expression of the KMA.CARs. In all cases where components of the IgGl heavy chain constant region was used as a spacer, a second (GrS), flexible linker was placed between the scFv and the spacer region. These CARs were synthesized commercially by genscript and cloned into a pVAXlPB PiggyBac transposon plasmid for further testing.

[00129] 3 of the 6 KM. CAR constructs contained a CD28 Costimulatory Endodomain and were as follows:

[00130] The first construct of this group was the KM.CAR_hCH3_28z construct, which contains only the hinge and CH3 domains of IgGl heavy chain constant region as the spacer and whose nucleic acid sequence is as follows:

[00131] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCA

CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGG

GTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACT

GATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGC

AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAG

ACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGG

AGGGGGGACCAAGCTGGAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGG

TCGGGTGGCGGCGGATCT GAGGTGCAGCTGCAGCAGTCAGGGGCGGAGCTTGTGAA GCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACACC

TATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATT

GATCCTGCGAATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATA A

TAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG A

CACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG G

GGCCAAGGGACCACGCTCACCGTCTCCTCCGGTGGAGGCGGGTCTGGGGGCGGAG

GTTCAGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACAAAACTCACACATGC

CCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAT

GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT

CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA

CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA

TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTC

TCCCTGTCTCCGGGTAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCT

TGCTA TA GCTTGCTA GTAA CA GTGGCCTTTA TTA TTTTCTG G GTGA GGA GTAA GA G

GAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCC

A CCCGCAA GCA TTA CCA GCCCTA TGCCCCA CCA CGCGA CTTCGCA GCCTA TCGCTC

CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACC

AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGA G

ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG

GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA T

GAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA

CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEP

ID NO: 29).

[00132] From 5’ to 3’, this construct (SEQ ID NO: 29) has a leader peptide, a KappaMab light chain variable region, a linker a KappaMab heavy chain variable

region, a second linker, an IgGl hinge & CH3 constant region domains a CD28

transmembrane and intracellular domains, and a CD3 zeta intracellular domain. A diagram of this construct is shown in Figure 6B.

[00133] The second construct of this group is the KM.CAR_h_28z construct, which contains only the hinge domain of IgGl heavy chain constant region as the spacer, and whose nucleic acid sequence is as follows: [00134] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCA

CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGG

GTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACT

GATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGC

AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAG

ACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGG

AGGGGGGACCAAGCTGGAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGG

TCGGGTGGCGGCGGATCT GAGGTGCAGCTGCAGCAGTCAGGGGCGGAGCTTGTGAA

GCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACAC C

TATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATT

GA TCCTGCGAA TGGTAACACTAAA TA TGACCCGAA GTTCCA GGGCAA GGCCA CTA TAA

TAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG A

CACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG G

GGCCAAGGGACCACGCTCACCGTCTCCTCCGGTGGAGGCGGGTCTGGGGGCGGAG

GTTCAGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACAAAACTCACACATGC

CCA TTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTA TA GCTTGCTA GT

AA CA GTGGCCTTTA TTA TTTTCTGGGTGA GGA GTAA GA GGA GCA GGCTCCTGCA CA

GTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCA

GCCCTA TGCCCCA CCA CGCGA CTTCGCA GCCTA TCGCTCCA GAGTGAA GTTCA GCA

GGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCA

ATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTG

AGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC

AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA

GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT

ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 30).

[00135] From 5’ to 3’, this construct (SEQ ID NO: 30) has a leader peptide, a KappaMab light chain variable region, a (G4SE linker a KappaMab heavy chain variable region, a second (G4SE linker, an IgGl hinge constant region domain a CD28 transmembrane and intracellular domains, and a CD3 zeta intracellular domain. A diagram of this construct is shown in Figure 6B. [00136] The third construct of this group was the KM.CAR_CD8a_28z construct, which contains a CD8 alpha stalk (Uniprot P01732, amino acids 138-182) as the spacer, and whose nucleic sequence is as follows:

[00137] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCA

CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGG

GTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACT

GATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGC

AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAG

ACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGG

AGGGGGGACCAAGCTGGAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGG

TC GGGTGGC GGCGGATCT A GG Ί (X (X (X (X G Ί X GGGGC 'GGA (X Ί Ί G Ί ^' GAA

GCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACAC C

TATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATT

GATCCTGCGAATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATA A

TAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG A

CACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG G

GGCCAAGGGACCACGCTCACCGTCTCCTCCACCACGACGCCAGCGCCGCGACCA

CCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAG

GCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTT

CGCCTGTG/1 TTTTTG G GTG CTG GTG GTGGTTG GTGGA GTCCTG G CTTG CTA TA G C

TTGCTA GTAA CA GTGGCCTTTA TTA TTTTCTGGGTGA GGA GTAA GA GGA GCA GGCT

CCTGCA CA GTG A CTA CA TGAA CA TGA CTCCCCGCCGCCCCGGGCCCA CCCGCAA G

CA TTA CCA GCCCTA TGCCCCA CCA CGCGA CTTCGCA GCCTA TCGCTCCA GAGTGAA

GTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA

CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCG

GGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA

TGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGA

GCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA

AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 31).

[00138] From 5’ to 3’, this construct (SEQ ID NO: 31) has a leader peptide, a KappaMab light chain variable region, a (G4SE linker a KappaMab heavy chain variable region, a CD8 alpha stalk a CD28 transmembrane and intracellular domains, and a CD3 zeta intracellular domain.

[00139] The remaining 3 constructs of the 6 KM.CAR constructs described in this example contained a 4-lBB (CD137) Costimulatory Endodomain and were as follows:

[00140] The first construct of this group is KM.CAR_h_28TM_4lBBz, which contains only the hinge domain of IgGl heavy chain constant region as the spacer and replaces the intracellular domain of CD28 with the intracellular domain of the 4-1BB co-stimulatory molecule, and whose nucleic acid sequence is as follows:

[00141] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCA

CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGG

GTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACT

GATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGC

AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAG

ACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGG

AGGGGGGACCAAGCTGGAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGG

TCGGGTGGCGGCGGATCT GAGGTGCAGCTGCAGCAGTCAGGGGCGGAGCTTGTGAA

GCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACAC C

TATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATT

GATCCTGCGAATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATA A

TAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG A

CACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG G

GGCCAAGGGACCACGCTCACCGTCTCCTCCGGTGGAGGCGGGTCTGGGGGCGGAG

GTTCAGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACAAAACTCACACATGC

CCA TTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTA TA GCTTGCTA GT

AA CA GTGGCCTTTA TTA TTTTCTGGGTGAAA CGGGGCA GAAA GAAA CTCCTG TA TA

TA TTCAAA CAA CCA TTTA TGA GA CCA GTA CAAA CTA CTCAA GA GGAA GA TGGCTGT

A GCTGCCGA TTTCCA GAA GAA GAA GAA GGA GGA TGTGAA CTGA GA GTGAAGTTCA G

CAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCT

CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC

TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACT

GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCG GAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC

CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 32).

[00142] From 5’ to 3’, this construct (SEQ ID NO: 32) has a leader peptide, a KappaMab light chain variable region, a linker a KappaMab heavy chain variable

region, a second linker an IgG hinge constant region domain a CD28

transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain.

[00143] The second construct of this group was KM.CAR_8a_28TM_4lBBz, which contains the CD8 alpha stalk (Uniprot P01732, amino acids 138-182) as the spacer and replaces the intracellular domain of CD28 with the intracellular domain of the 4-1BB co stimulatory molecule, and whose nucleic sequence is as follows:

[00144] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCA

CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGG

GTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACT

GATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGC

AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAG

ACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGG

AGGGGGGACCAAGCTGGAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGG

TC GGGTGGC GGCGGATCTG4 GG Ί G( G( G( G( G Ί X GGGGC 'GGA G( 77 G Ί GAA

GCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACAC C

TATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATT

GATCCTGCGAATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATA A

TAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG A

CACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG G

GGCCAAGGGACCACGCTCACCGTCTCCTCCACCACGACGCCAGCGCCGCGACCA

CCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAG

GCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTT

CGCCTGTGATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGC

TTGCTA GTAA CA GTGGCCTTTA TTA TTTTCTG G GTGAAA CGGGGCA GAAA GAA A CT

CCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG

A TGGCTGTA GCTGCCGA TTTCCA GAA GAA GAA GAA GGA GGA TGTGAA CTGA GAGT GAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTA

TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGG C CGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCAC CAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEP ID NO: 33).

[00145] From 5’ to 3’, this construct (SEQ ID NO: 33) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker a KappaMab heavy chain variable region, a CD8 alpha stalk a CD28 transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain.

[00146] The third construct of this group is KM.CAR_hCH2CH3mut_28TM_4lBBz, which contains the hinge, CH2 and CH3 domains of IgGl heavy chain constant region as the spacer, with mutations introduced at amino acids important for CH2 interaction with Fc- receptors (3-6) which may mediate reduced CAR T-cell survival in-vivo (3, 6, 7) by clearance of CAR T-cells in the reticuloendothelial system. The nucleic acid sequence is as follows:

[00147] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCA

CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGG

GTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACT

GATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGC

AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAG

ACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGG

AGGGGGGACCAAGCTGGAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGG

TC GGGTGGC GGCGGATCTGA GG Ί G( G( G( G( G Ί X GGGGC 'GGA G( A 7 Ί GAA

GCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACAC C

TATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATT

GATCCTGCGAATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATA A

TAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG A

CACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG G

GGCCAAGGGACCACGCTCACCGTCTCCTCCGGTGGAGGCGGGTCTGGGGGCGGAG

GTTCAGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACAAAACTCACACATGC

CCACCGTGCCCAGCACCTCCAGTCGCGGGACCGTCAGTCTTCCTCTTCCCCC CAAAACCCAAGGACACCCTCATGATCG1WCGGACCCCTGAGGTCACATGCG

TGGTGGTGAACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG

TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG

TACG11AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC

TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA

GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA

CAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC

AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG

CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGA

GCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC

TGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA7T7TG

GGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTA TL GCTTGCTA GTAA CA GTGG

CCTTTA TTA TTTTCTG G GTGAAA CGGGGCA GAAA GAAA CTCCTGTA TA TA TTCAAA

CAA CCA TTTA TGA GA CCA GTA GAAA CTA CTCAA GA GGAA GA TGGCTGTA GCTGCCG

A TTTCCA GAA GAA GAA GAA GGA GGA TGTGAA CTGA GA G 1 ^' GAA G 1 Ί X A G( A GGA G( ,

CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG

GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG

GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG

ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGC

AAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC

GCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 34).

[00148] From 5’ to 3’, this construct (SEQ ID NO: 34) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker a KappaMab heavy chain variable resion . a second linker a mutated IgGl hinge, CH2 and CH3 constant region

domains a CD28 transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain. The mutated IgGl hinge domain has, from 5’ to 3’, E233P, L234V, L235A, G236-, S254A, D265N, and N297A mutations highlighted within the shaded boxes of this construct (SEQ ID NO: 34). Mutations at these sites (E233P, L234V, L235A, G236-, S254A, D265N, N297A) may decrease Fc interaction with CAR T-cells, allowing improved survival post-infusion.

[00149] Addition of 2A ribosomal skip element and eGFP to KM.CARs [00150] For ease of detection of T-cells expressing each of the CARs described above, an eGFP with a 5’ T2A ribosomal skip element with overlapping sequences was synthesized with the CAR- CD3 zeta endodomain and the plasmid backbone. This was then cloned by restriction enzyme digestion and ligation into the CAR containing pVAXl PB transposon plasmids to create the following-

[00151] 28z Endodomain_2A_GFP containing constructs:

[00152] 1. pVAXlPB KM.CAR_hCH2CH3_28z_2A_GFP

[00153] 2. pVAXlPB KM.CAR_hCH3_28z_2A_GFP

[00154] 3. pVAXlPB KM.CAR_h_28z_2A_GFP

[00155] 4. pVAXlPB KM.CAR_8a_28z_2A_GFP

[00156] 41BBz Endodomain_2A_GFP containing constructs:

[00157] 1. pVAXlPB KM.CAR_h_28TM_4lBBz_2A_GFP

[00158] 2. pVAXlPB KM.CAR_8a_28TM_4lBBz_2A_GFP

[00159] 3. pVAXlPB KM.CAR_hCH2CH3mut_28TM_4lBBz_2A_GFP

[00160] Generation of KM.CAR T-cells with 4-1BB costimnlatorv domain.

[00161] Comparison was made between the preliminary KM.CAR_hCH2CH3_28z and the 4-1BB containing CARs. KM.CAR T-cells were generated by electroporation using the PiggyBac system as previously described herein and in the art (2). Four million peripheral blood mononuclear cells (PBMCs) from healthy donors were electroporated with the Neon electroporation system at 2400V for 20ms, single pulse, in the presence of 5ug each of PiggyBac transposase and PiggyBac Transposon plasmids. KMA.CAR constructs tested included KM.CAR_hCH2CH3_28z_2A_GFP; KM.CAR_h_28TM_4lBBz_2A_GFP; KM. C AR_8a_28TM_41 BBz_2 A GFP ; or

KM. CAR_hCH2CH3mut_28TM_4lBBz_2A_GFP.

[00162] Electroporated PBMCs (CAR-PBMCs) were rested overnight in AIMV with 10% Fetal calf serum (AIM-V CM), harvested, washed and resuspended in AIM-V CM at lxl0 ⁶ /ml. CAR-PBMCs were cocultured with autologous irradiated PBMC feeder cells with or without irradiated KMA expressing JJN3 cells at a CAR-PBMC:JJN3 ratio of 5: 1. Interleukin- 15 (IL-15) was added at 200 IU or 200 ng/ml of every 3 days. Cells were enumerated by trypan blue exclusion and fresh irradiated stimulator/feeder cells were added every 7 days.

[00163] Assessment of KM.CAR Expression

[00164] KM.CAR expression was assessed by flow cytometry at initiation of culture (Day 1), Day 15 and Day 21 (Figures 8A-8B). KM.CAR T-cell cultures were surface stained with anti-human-CD3 antibody and CAR expression assessed by GFP expression.

[00165] KM.CAR T-cells require kappa myeloma antigen to persist in-vitro

[00166] Cultures containing the KMA expressing JJN3 cell line showed either greater total expansion, increased KM.CAR expression or both, compared to cultures with PBMC alone (Figures 8A-8B). Consistent with known interaction of the IgG constant region-CH2 domain with Fc-receptors, the KM.CAR_hCH2CH3_28z expressing T-cells were enriched in the presence of PBMC alone (28% of CD3 ⁺ T-cells), but showed greater expansion and enrichment with addition of JJN3 cells (l5-fold expansion with 38% CAR expression compared to 6-fold expansion with 29% CAR expression).

[00167] KM.CAR_hCH2CH3mut_28TM_4lBBz expressing T-cells showed only low level CAR expression (6%) and expansion (6-fold) with PBMC alone compared to co-culture with JJN3 (26% CAR expression and l7-fold expansion). The KM.CAR T-cells containing the IgGl hinge only spacer had similar expansion (5-fold with JJN3, 6 fold without JJN3) but increased CAR expression (17% with JJN3, 9% without). Only the KM.CAR T-cells containing the CD8alpha chain spacer did not show any enhanced expansion or enrichment in the presence of JJN3 cells (8-fold expansion and 5% CAR expression in the presence of JJN3, compared to 5-fold expansion and 5% CAR expression without JJN3).

[00168] Functional assessment of KM.CAR T-cells

[00169] KMA-specific interferon-gamma production and cytotoxicity of KM.CAR T- cells were assessed by intracellular cytokine flow cytometry and standard chromium release assay with KMA+ and KMA- cell lines using protocols previously described (2). KMA positive cell lines used included JJN3, Pfeiffer, NCI-H929. KMA negative cell lines included Nalm-6 and Molt (Figure 12A-12B).

[00170] For cytokine flow cytometry, 2xl0 ⁵ KM.CAR T-cells were stimulated with target cells at a ratio of 1: 1 for 5 hours. Monensin (2mM) (BD Biosciences) and Brefeldin A (1 mg/mL) (BD Biosciences) were added after 1 hour. CAR T-cells activated non-specifically with 50ng/ml phorbol myristate acetate (PMA: Sigma-Aldrich) and lug/ml ionomycin (Sigma-Aldrich) and unstimulated cells were used as positive and negative controls. CAR T- cells were then harvested, washed, surface stained for CD3, CD4 and CD8. CAR T-cells were fixed and permeabilized with cytofix and perm/wash buffer (BD Biosciences) and stained with anti-interferon gamma antibody (BD Biosciences) followed by further washing with perm/wash buffer. Stained cells were analyzed using a FACSCanto™ II flow cytometer with acquisition of at least 30,000 events.

[00171] KMA-specific cytotoxicity was assessed using a standard chromium ( ⁵¹ Cr) release assay. Target cells were labelled with Sodium chromate (Na2 ⁵¹ Cr0 ₄ ) (Perkin-Elmer, Waltham, MA, USA). KM.CAR T-cells were preincubated with the K562 cell line at a 1 : 1 ratio to absorb NK cell activity. Chromium labelled target cells were added to the KM.CAR T-cells in triplicate at effector: target ratios ranging from 40: 1 to 1.25: 1 and incubated at 37°C, 5% CC for 4 hours. Triplicate targets were lysed with 10% sodium dodecyl sulphate to determine maximal release and triplicate targets with no effectors were used to assess spontaneous release. Supernatants were aspirated and read using a MicroBeta2 Plate Counter (PerkinElmer). Percentage specific lysis was calculated using the standard formula- % Specific lysis= (test release - spontaneous release) / (maximal release - spontaneous release) x 100]

Example 4: Generation of PiggyBac transposon plasmid with the activation inducible promoter

[00172] A single transposon cassette containing a constitutively active promoter (EF1 alpha) and an activation inducible promoter (NFATpro) was designed and cloned. The activation inducible gene expression cassette was produced by designing the NFATpro using Clone Manage 9 (Sci-Ed Software), based on Fiering et al(8). This includes 6 copies of the 30 base pair DNA sequence (response element-RE) bound by the Nuclear Factor of Activated T- cells (NFAT-RE)- GGAGGAAAAACTGTTTCATACAGAAGGCGT (SEQ ID NO: 35) followed by the minimal IL-2 promoter-

ACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCT TGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCC TG (SEQ ID NO: 36) found on chromosome 4 (NCBI Reference Sequence: NG_0l6779. l). [00173] To enable detection of activation induced gene expression, the enhanced green fluorescent protein (eGFP) DNA sequence followed by the bovine growth hormone (BGH) polyadenylation signal (9-11) was placed 3’ of the NFATpro. The DNA sequence of this gene cassette is as follows-

[00174] GGAGGAAAAACTGTTTCATACAGAAGGCGTCAATTAGGAGGAAAA

ACTGTTTCATACAGAAGGCGTCAATTAGGAGGAAAAACTGTTTCATACAGAAGG

CGTCAATTGTCCCATCGAATTAGGAGGAAAAACTGTTTCATACAGAAGGCGTCA

ATTAGGAGGAAAAACTGTTTCATACAGAAGGCGTCAATTAGGAGGAAAAACTGT

TTCATACAGAAGGCGTCAATTGTCCCGGGACATTTTGACACCCCCATAATATTT

TTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACT

CTCTTTAATCACTACTCACAGTAACCTCAACTCCTGAACTCCATGG47GG7GAG

CAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCG

ACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC

GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCC

ACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCAC

ATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGC

ACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG

GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGC

AACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATG G

CCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG A

CGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGATCCGGAG

CCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTTGAAGAAAACCCCGGTC

CTATTTAAATCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC

CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC

TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC

TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAAT

AGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGC (SEQ ID NO: 37)

[00175] From 5’ to 3’, this constructs contains the NFAT-RE. the IL-2 Minimal Promoter, the eGFP , and the BGH polyadenylation signal.

[00176] This cassette was synthesized commercially by Genscript and cloned into the pVAXlPB transposon plasmid between the 5’ cHS4 Insulator (GenBank: U78775 2)(l2) and the human elongation factor 1 promoter. To identify transduced T-cells in initial experiments, the chimeric RQR8 marker consisting of the epitope of CD34 recognized by the QBEndlO monoclonal antibody and mimotopes of the CD20-specific monoclonal antibody Rituximab(l3) was cloned into the transposon multi cloning site to produce the transposon gene insert shown in Figure 9 (pVAXlPB NFATGFP-RQR8 plasmid). Co-electroporation of the activation inducible gene cassette containing pVAXlPB NFATGFP-RQR8 transposon plasmid and the pVAXl PBase transposase plasmid leads to permanent integration of the NFATGFP-RQR8 gene insert seen in Figure 9.

Demonstration of function of the activation inducible gene containing transposon

[00177] To demonstrate the function of the pVAXlPB NFATGFP-RQR8 transposon from Example 4 (see Figure 9), 4x10 ⁶ PBMCs were electroporated in the presence of 5ug each of the transposon and transposase plasmids. Electroporated cells were rested for 24 hours and then stimulated non-specifically over-night with 50ng/ml phorbol myristate acetate (PMA: Sigma-Aldrich) and lug/ml ionomycin (Sigma-Aldrich) and compared to unstimulated controls. Transduced cells were identified by QBEndlO staining for RQR8 marker expression and activation induced gene expression (eGFP) was assessed at 19 hours. At that time point, 50% of transduced cells were seen to express eGFP (Figure 10).

Example 5: Design of KM.CAR controlled biological therapies

[00178] Expression plasmids containing IL-12 and/or the interleukin-6 receptor antagonist SANT7 and also containing the optimized chimeric antigen receptor with the expression of IL-12 and/or SANT7 under control of an activation inducible promoter (Hooijberg et al. 2000) were also constructed. The SANT-7 sequence was provided by Prof Rocco Savino and was based on mutating the wildtype IL-6 gene sequence (NCBI Reference Sequence: NM_000600.4) provided as per Savino et al 1994 and Sporeno et al 1996(14-17). The sequence was imported into Clone Manage 9 (Sci-Ed Software) and a 6xHis tag added for detection in supernatants by ELISA.

[00179] The nucleotide sequence of SANT-7 is provided with amino acid substitutions highlighted, underlined and listed below:

[00180] MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTS

SERIDKOIRi¾LDilSALRKETCNKSNMCESSKEAiAiiiiNLNLPKMAEKDGCFi iiGF

NEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMlrKiLIQFLQKKAKNLDA I

TTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLIRSLRALRIMHHHHHH (SEQ ID NO: 38). The nucleotide substitutions correspond to Y31D, G35F, L57D, E59F, N60W, Q75Y, S76K, S118R, V121D. The sequence provided also contained a Q211A substitution not listed in the published sequence.

[00181] The DNA sequence corresponding to this amino acid sequence (i.e., SEQ ID NO: 38) is as follows:

[00182] ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCT

GGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTACCCCCAGGAGAA

GATTCCAAAGATGTAGCCGCCCCACACAGACAGCCACTCACGAGCTCAGAACGA

ATTGACAAACAAATTCGGGACATCCTCGACTTTATCTCAGCCTTAAGAAAGGAGA

CATGTAACAAGAGTAACATGTGTGAGAGCTCCAAAGAGGCAGACGCATTCTGGA

ACCTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCTACAAAGGATTCA

ATGAGGAGACTTGCCTGGTGAAAATCATCACTGGTCTTCTCGAGTTTGAGGTATA

CCTAGAGTACCTCCAGAACAGATTTGAGAGTAGTGAGGAACAAGCCAGAGCTGT

GCAGATGCGCACAAAAGACCTGATCCAGTTCCTGCAGAAAAAGGCAAAGAATCT

AGATGCAATAACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGACGAAGCT

GCAGGCACAGAACCAGTGGCTGCAGGACATGACAACTCATCTCATTCTGAGATC

TTTTAAGGAGTTCCTGATCCGTAGCCTGAGGGCTCTTCGGGCTATGCATCATCAC

CATCACCACT (SEQ ID NO: 39).

[00183] A single chain interleukin- 12 (Flexi-IL-l2) construct was designed by joining the IL-12 p40 and p35 subunits (Uniprot P29459 and P29460) with a flexible (GrS), linker similar to Zhang et al and Chinnasamy et al (18, 19), which allows both subunits to be expressed as a single peptide chain that readily forms the bioactive p70 heterodimer was used. The Flexi-IL-l2 construct was synthesized and constructs containing IL-12 and SANT7 were cloned into the activation inducible transposon cassette described herein and shown in Figure 11.

[00184] Additionally, the Flexi-IL-l2 construct could be synthesized and constructs containing IL-12 and SANT7 separated by 2A ribosomal skip elements could be cloned into the PiggyBac plasmid described herein and shown in Figure 7. It should be noted that the promoter operably linked to IL-12 and/or SANT7 in constructs containing IL-12 and/or

SANT7 can be an activation inducible promoter such as, for example, the NFAT promoter or a constitutively active promoter as provided herein or known in the art. Alternatively, expression of the IL-12 and/or SANT7 could be driven by the same promoter as the CAR by inserting the IL-12 and/or SANT7 DNA sequences 3’ of the CAR sequence and separating then by 2A ribosomal skip elements.

[00185] The amino acid sequence of Flexi-IL-l2 is as follows:

[00186] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEM

VVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLS

HSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDL

TFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLP

IEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDT

WSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYY

SSSWSEWASXFCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSONLLRAVS

NML QKARQTLEFYPCTSEEIDHEDITKDK TS TVEA CLPLEL TKNESCLNSRE TSFITN

GSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVI

DELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

(SEQ ID NO: 40).

[00187] From 5’ to 3’, the Flexi-IL-l2 construct contains a leader peptide, the IL-12 p40 subunit, the Linker and the IL-12 p35 subunit.

[00188] The DNA sequence corresponding to the amino acid sequence above (i.e., SEQ ID NO: 40) is as follows:

[00189] ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCT

GGCATCTCCCCTCGTGGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAA

TTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCC

CTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCT

CTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACA

CCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAA

GGA AGAT GGA ATTT GGT C C ACT GAT ATTTT A A AGGAC C AGA A AGA AC C C A A A A A

TAAGACCTTTCTAAGATGCGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGG

TGGCTGACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGAGGC

TCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAG

TCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGT

GCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTC

ACAAGCTCAAGTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCA AACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGG

TGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCCACATTCCTACTTCTC

CCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAG

AGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAAATGCCAGCAT

TAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGAATGGGCATCT

GTGCCCTGCAGTGGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGC

AGCAGAAACCTCCCCGTGGCCACTCCAGACCCAGGAATGTTCCCATGCCTTCACC

ACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAGACAAA

CTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCACAAA

AGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGA

GAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCC

TCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTT

GAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCC

TAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATG

CAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAAC

CGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATT

CGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCC (SEQ ID NO:

41).

[00190] Additionally, expression plasmids containing the truncated dominant negative form of Galectin-3, GAL3C is also constructed. The construct contains a CD8- alpha leader peptide to direct secretion as well as a 6xHis tag for detection. The amino acid sequence of GAL3C is listed here:

[00191] MEFGLSWLFLVAILKGVOCSRHHHHHHGAPAGPLIVPYNLPLPGGV

VPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGR EERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDID LTSASYTMI (SEQ ID NO: 42)

[00192] The corresponding DNA sequence for the GAL 3C construct is provided here:

[00193] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGACATCATCACCATCACCACGGCGCCCCTGCTGGGCCACTG

ATTGTGCCTTATAACCTGCCTTTGCCTGGGGGAGTGGTGCCTCGCATGCTGATAA

CAATTCTGGGCACGGTGAAGCCCAATGCAAACAGAATTGCTTTAGATTTCCAAAG

AGGGAATGATGTTGCCTTCCACTTTAACCCACGCTTCAATGAGAACAACAGGAG AGTCATTGTTTGCAATACAAAGCTGGATAATAACTGGGGAAGGGAAGAAAGACA GTCGGTTTTCCCATTTGAAAGTGGGAAACCATTCAAAATACAAGTACTGGTTGAA CCTGACCACTTCAAGGTTGCAGTGAATGATGCTCACTTGTTGCAGTACAATCATC GGGTTAAAAAACTCAATGAAATCAGCAAACTGGGAATTTCTGGTGACATAGACC TCACCAGTGCTTCATATACCATGATA (SEQ ID NO: 43)

[00194] The CAR and ‘biologicals’ transposon plasmids will be nucleofected to generate CAR T-cells expressing either IL-12 alone, SANT7 alone, GAL3C alone or both IL- 12 and SANT7 or both of IL12 and GAL3C or both of SANT7 and GAL3C or all three of IL- 12. SANT7 and GAL3C. Cells successfully transduced with‘biologicals’ constructs may be identified by selectable marker expression for example by flow cytometry. Levels of IL-12, SANT7 and or GAL3C will be measured intracellularly by cytokine flow cytometry and in supernatants of CAR T-cell cultures by ELISA using commercial kits and reagents and compared to control T-cells expressing CAR alone. CAR T-cells will be assessed for function by cytokine flow cytometry and cytotoxicity assays as above as well as co-culture assays with myeloma cell lines to assess inhibition of tumour growth. Experiments will be performed in triplicate and the 2 optimal CAR constructs identified will be chosen to be assessed in a murine model with and without IL-12, GAL3C and/or SANT7 expression.

[00195] Based on the previously established RPMI-Rag human myeloma murine xenograft model, RPMI-Rag-Luc (KMA-) and JJN3-Rag-Luc (KMA+) models will be developed to assess the function of our CAR T-cells in-vivo. JJN3 and RPMI8226 cells will be transfected with Luc-l and then inoculated i.v. into Rag2-/-yc-/- (BALB/c) mice to form the JJN3- Rag-Luc and RPMI-Rag-Luc MM models. Engraftment and disease levels will be monitored by optical imaging following IP injection with luciferin and correlated with levels of levels of serum human kappa (JJN3) and lambda (RPMI) light chain. Optimal time for inoculation with candidate CAR T-cells will be established using Optical Imaging prior to the development of hind limb paralysis, usually from weeks 5-8. Cohorts of 6 JJN3-Rag-Luc and RPMI-Rag-Luc mice will be inoculated IV with increasing doses of CAR T-Cells (with and without IL-12/SANT7 expression) to establish the therapeutic dose starting at lxlO ⁶ total cells. Mice will be imaged on day 0, +1, +3, +8 and weekly thereafter until the development disease progression as determined by the development of hind limb paralysis, increasing serum free light chains (SFLC) or other institutional guidelines. Marrow and extramedullary tumors will be collected and examined histologically for distribution of MM cells and CAR T-cells. Efficacy will be determined by imaging response and survival compared with controls.

[00196] References

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Adoptive cellular immunotherapy with CDl9-specific T cells. Klin Padiatr. 2005;2l7(6):35l- 6

[00198] 2. Ramanayake S, Bilmon I, Bishop D, Dubosq MC, Blyth E, Clancy L, et al. Low-cost generation of Good Manufacturing Practice-grade CDl9-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials. Cytotherapy. 2015.

[00199] 3. Hombach A, Hombach AA, Abken H. Adoptive immunotherapy with genetically engineered T cells: modification of the IgGl Fc 'spacer' domain in the extracellular moiety of chimeric antigen receptors avoids 'off-target' activation and unintended initiation of an innate immune response. Gene Ther. 2010;17(10): 1206-13.

[00200] 4. Shields RL, Namenuk AK, Hong K, Meng YG, Rae J, Briggs J, et al.

High resolution mapping of the binding site on human IgGl for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgGl variants with improved binding to the Fc gamma R. J Biol Chem. 200l;276(9):659l-604.

[00201] 5. Armour KL, van de Winkel JG, Williamson LM, Clark MR.

Differential binding to human FcgammaRIIa and FcgammaRIIb receptors by human IgG wildtype and mutant antibodies. Mol Immunol. 2003;40(9):585-93.

[00202] 6. Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L,

Rader C, et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer immunology research. 20l5;3(2): 125-35.

[00203] 7. Clemenceau B, Valsesia-Wittmann S, Jallas AC, Vivien R, Rousseau

R, Marabelle A, et al. In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD 16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer. J Immunol Res. 20l5;20l5:482089.

[00204] 8. Fiering S, Northrop JP, Nolan GP, Mattila PS, Crabtree GR,

Herzenberg LA. Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev. 1990;4(10): 1823-34.

[00205] 9. Miller WL, Martial JA, Baxter JD. Molecular cloning of DNA complementary to bovine growth hormone mRNA. J Biol Chem. 1980;255(16):7521-4.

[00206] 10. Miller WL, Thirion JP, Martial JA. Cloning of DNA complementary to bovine prolactin mRNA. Endocrinology. 1980;107(3):851-3.

[00207] 11. Goodwin EC, Rottman FM. The 3'-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation. J Biol Chem. 1992;267(23): 16330-4.

[00208] 12. Chung JH, Bell AC, Felsenfeld G. Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci U S A. l997;94(2):575-80.

[00209] 13. Philip B, Thomas S, Marin V, Jathoul A, Kopec A, Linch DC, et al. A

Highly Compact Epitope-Based Marker-Suicide Gene for More Convenient and Safer T-Cell Adoptive Immunotherapy. ASH Annual Meeting Abstracts. 20l0;l 16(21): 1473-

[00210] 14. Demartis A, Bemassola F, Savino R, Melino G, Ciliberto G.

Interleukin 6 receptor superantagonists are potent inducers of human multiple myeloma cell death. Cancer Res. 1996;56(18):4213-8.

[00211] 15. Savino R, Ciapponi L, Lahm A, Demartis A, Cabibbo A, Toniatti C, et al. Rational design of a receptor super-antagonist of human interleukin-6. EMBO J. 1994;13(24):5863-70.

[00212] 16. Savino R, Lahm A, Salvati AL, Ciapponi L, Sporeno E, Altamura S, et al. Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gpl 30 activation. EMBO J. 1994;13(6): 1357-67.

[00213] 17. Sporeno E, Savino R, Ciapponi L, Paonessa G, Cabibbo A, Lahm A, et al. Human interleukin-6 receptor super-antagonists with high potency and wide spectrum on multiple myeloma cells. Blood. 1996;87(11):4510-9.

[00214] 18. Zhang L, Kerkar SP, Yu Z, Zheng Z, Yang S, Restifo NP, et al.

Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther. 20l l;l9(4):75l-9. [00215] 19. Chinnasamy D, Yu Z, Kerkar SP, Zhang L, Morgan RA, Restifo NP, et al. Local delivery of interleukin- 12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice. Clin Cancer Res. 2012;18(6): 1672-83.

Example 6: Development of stable KMA expressing antigen presenting cells

[00216] Aim: To generate stable antigen presenting cell lines for expanding CAR T- cells such as T-cells expressing any of the CAR’s provided herein.

[00217] Methods/Results: A chimeric construct of Kappa Myeloma Antigen (KMA) fused to mCherry was designed as shown in Figure 13 to enable the creation of stable antigen presenting cell lines expressing a fusion KMA-mCherry protein. More specifically, the KMA-mCherry construct (i.e., nucleic acid SEQ ID NO: 44) shown in Figure 13 comprises, from 5’ to 3’, a Gaussia leader peptide, KMA (i.e., Kappa light chain (KLC) switch region; KLC constant region) a (048)3 flexible linker as described herein, CD28TM (i.e., CD28 extracellular domain, CD28 transmembrane domain: CD28 intracellular domain) a G4S linker, and an mCherry reporter.

[00218] The nucleotide sequence of the KMA-mCherry construct is as follows:

[00219] ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGA

GGCCCTGGAAATTAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCC

ATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC

TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG

GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAG

CCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTA

CGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAA

CAGGGGAGAGTGT GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGG

TATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGG

AGCAGGCTCCTGCACAGTGACGGTGGztGGCGGGTGTATGGTCAGCAAGGGAG

AGGAAGATAATATGGCGATCATCAAAGAGTTTATGAGATTTAAGGTGCACAT

GGAAGGAAGCGTTAATGGTCATGAGTTTGAAATCGAAGGCGAAGGCGAAGG

AAGACCGTATGAAGGCACACAGACGGCTAAACTTAAGGTCACAAAAGGCGG

ACCGCTTCCATTCGCGTGGGATATTCTTTCACCGCAATTTATGTATGGTTCT

AAAGCCTATGTGAAACATCCTGCGGATATTCCTGACTACCTTAAACTGTCTT TCCCGGAAGGATTTAAATGGGAACGCGTCATGAACTTCGAAGATGGCGGCG

TTGTTACGGTGACGCAGGATTCATCACTGCAAGATGGAGAATTTATTTATAA

AGTTAAACTGCGCGGCACAAACTTTCCGTCAGACGGACCTGTCATGCAGAA

GAAAACGATGGGCTGGGAAGCCAGCAGCGAGAGAATGTACCCGGAGGACG

GAGCACTTAAAGGCGAAATCAAGCAACGCCTGAAGCTGAAAGATGGAGGCC

ATTATGATGCCGAGGTCAAGACGACATACAAAGCTAAGAAACCGGTACAAT

TACCTGGAGCATACAACGTCAATATCAAGCTGGATATTACGTCACATAATGA

AGACTATACGATTGTAGAGCAATATGAAAGAGCAGAGGGAAGACACTCTAC

AGGTGGAATGGACGAATTATACAAA (SEP ID NO: 44)

[00220] Said chimeric construct was cloned into a PiggyBac transposon vector and introduced into K562 cells as provided herein. Examples of KMA-mCherry expressing K562 cells can be seen in Figure 14. K562 cells exposed to the KMA-mCherry transposon vectors were subjected to flow cytometry in order to isolate KMA-mCherry expressing K562 cells (see Figure 15). Isolated KMA-mCherry expressing K562 cells were then grown and passaged in culture in order to identify stable KMA-mCherry expressing K562 cells for use in subsequent experiments as provided herein. As shown in Figure 39, KMA-mCherry K562 APCs showed a higher percentage of KMA+ cells than other KMA expressing cell lines (i.e., JJN3 cells).

Example 7: Development of stable KMA expressing antigen presenting cells (APCs) expressing co-stimulatory molecules

[00221] Aim: To generate stable KMA antigen presenting cell lines expressing co stimulatory molecules for expanding CAR T-cells such as T-cells expressing any of the CAR’s provided herein.

[00222] Methods/Results: A chimeric construct of Kappa Myeloma Antigen (KMA) fused to mCherry as well as co-stimulatory molecules will be generated and used for the creation of stable antigen presenting cell lines expressing a fusion KMA-mCherry protein as well as co-stimulatory molecules. More specifically, a KMA-mCherry-co-stimulatory domain chimeric construct (i.e., nucleic acid SEQ ID NO: 45) comprising, from 5’ to 3’, the KMA- mCherry construct from Example 6 (i.e., SEQ ID NO: 44) a G5GT2A linker, a CD86 extracellular domain, a CD8a transmembrane domain: a G5GP2A linker, an QX40L co- stimulatory molecule a G5GE2A linker and a 4-1BBL costimulatory molecule_will be generated. [00223] The nucleotide sequence of the KMA-mCherry-co-stimulatory domain chimeric construct will be as follows:

[00224] ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGA

GGCCCTGGAAATTAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA

TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT

TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGG

GT AACTCC C AGGAGAGT GT C AC AGAGC AGGAC AGC AAGGAC AGC AC CT AC AGCC

TCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG

CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA

GGGGAGAGTGTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGAT

CTCCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTAT

AGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCA

GGCTCCTGCACAGTGACGGTGGAGGCGGGTCTATGGTCAGCAAGGGAGAGGAAG

ATAATATGGCGATCATCAAAGAGTTTATGAGATTTAAGGTGCACATGGAAGGAA

GCGTTAATGGTCATGAGTTTGAAATCGAAGGCGAAGGCGAAGGAAGACCGTATG

AAGGCACACAGACGGCTAAACTTAAGGTCACAAAAGGCGGACCGCTTCCATTCG

CGTGGGATATTCTTTCACCGCAATTTATGTATGGTTCTAAAGCCTATGTGAAACA

TCCTGCGGATATTCCTGACTACCTTAAACTGTCTTTCCCGGAAGGATTTAAATGG

GAACGCGTCATGAACTTCGAAGATGGCGGCGTTGTTACGGTGACGCAGGATTCA

TCACTGCAAGATGGAGAATTTATTTATAAAGTTAAACTGCGCGGCACAAACTTTC

CGTCAGACGGACCTGTCATGCAGAAGAAAACGATGGGCTGGGAAGCCAGCAGCG

AGAGAATGTACCCGGAGGACGGAGCACTTAAAGGCGAAATCAAGCAACGCCTG

AAGCTGAAAGATGGAGGCCATTATGATGCCGAGGTCAAGACGACATACAAAGCT

AAGAAACCGGTACAATTACCTGGAGCATACAACGTCAATATCAAGCTGGATATT

AC GT C AC AT AAT GAAGACT AT AC GATT GTAGAGC AATAT GAAAGAGC AGAGGGA

AG AC ACT CT AC AGGT GG A AT GG ACG A ATT AT ACAAAGGAAG( 'GGA GA GGG( GG

GGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGGACTGA

GTAACATTCTCTTTGTGATGGCCTTCCTGCTCTCTGGTGCTGCTCCTCTGAA

GATTCAAGCTTATTTCAATGAGACTGCAGACCTGCCATGCCAATTTGCAAAC

TCTCAAAACCAAAGCCTGAGTGAGCTAGTAGTATTTTGGCAGGACCAGGAA

AACTTGGTTCTGAATGAGGTATACTTAGGCAAAGAGAAATTTGACAGTGTTC

ATTCCAAGTATATGGGCCGCACAAGTTTTGATTCGGACAGTTGGACCCTGAG ACTTCACAATCTTCAGATCAAGGACAAGGGCTTGTATCAATGTATCATCCAT

CACAAAAAGCCCACAGGAATGATTCGCATCCACCAGATGAATTCTGAACTGT

CAGTGCTTGCTAACTTCAGTCAACCTGAAATAGTACCAATTTCTAATATAAC

AGAAAATGTGTACATAAATTTGACCTGCTCATCTATACACGGTTACCCAGAA

CCTAAGAAGATGAGTGTTTTGCTAAGAACCAAGAATTCAACTATCGAGTATG

ATGGTATTATGCAGAAATCTCAAGATAATGTCACAGAACTGTACGACGTTTC

CATCAGCTTGTCTGTTTCATTCCCTGATGTTACGAGCAATATGACCATCTTC

TGTATTCTGGAAACTGACAAGACGCGGCTTTTATCTTCACCTTTCTCTATAG

AGCTTGAGGACCCTCAGCCTCCCCCAGACCACATCTACATCTGGGCGCCCTTG

GCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCGG i 7 ( 'GGA (]( ( '

GAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGGA

AAGGGTCCAACCCCTGGAAGAGAATGTGGGAAATGCAGCCAGGCCAAGATTCGA

GAGGAACAAGCTATTGCTGGTGGCCTCTGTAATTCAGGGACTGGGGCTGCTCCTG

TGCTTCACCTACATCTGCCTGCACTTCTCTGCTCTTCAGGTATCACATCGGTATCC

TCGAATTCAAAGTATCAAAGTACAATTTACCGAATATAAGAAGGAGAAAGGTTT

CATCCTCACTTCCCAAAAGGAGGATGAAATCATGAAGGTGCAGAACAACTCAGT

CATCATCAACTGTGATGGGTTTTATCTCATCTCCCTGAAGGGCTACTTCTCCCAGG

AAGTCAACATTAGCCTTCATTACCAGAAGGATGAGGAGCCCCTCTTCCAACTGAA

GAAGGTCAGGTCTGTCAACTCCTTGATGGTGGCCTCTCTGACTTACAAAGACAAA

GTCTACTTGAATGTGACCACTGACAATACCTCCCTGGATGACTTCCATGTGAATG

GCGGAGAACTGATTCTTATCCATCAAAATCCTGGTGAATTCTGTGTCCTTGGC4G

CGGGCAGTGCACAAACTACGCACTTCTTAAGCTGGCAGGCGACGTGGAATCCAATCC T

GG^CCCATGGAATACGCCTCTGACGCTTCACTGGACCCCGAAGCCCCGTGGC

CTCCCGCGCCCCGCGCTCGCGCCTGCCGCGTACTGCCTTGGGCCCTGGTCG

CGGGGCTGCTGCTGCTGCTGCTGCTCGCTGCCGCCTGCGCCGTCTTCCTCG

CCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCA

GCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCC

TCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTC

TGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCG

TGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTG

GTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGC

GTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAG

CCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTG CCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGC TTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAG GCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA CTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCG GAA (SEQ ID NO: 45).

[00225] Said chimeric construct will be generated and cloned into a PiggyBac transposon vector and introduced into K562 cells as provided herein. KMA-mCherry-co- stimulatory domain expressing K562 cells will be sorted and purified as provided herein. Isolated, stable KMA-mCherry-co-stimulatory domain expressing K562 cells will then be used in subsequent experiments to expand CAR T-cells as provided herein.

Example 8: Development of highly specific CAR T-cells targeting the Kappa Myeloma Antigen for the treatment of multiple myeloma.

[00226] Introduction:

[00227] Multiple myeloma (MM) is an incurable malignancy of differentiated plasma cells, which can be accompanied by severe bone lesions, cytopenia and hypercalcemia. The majority of MM patients produce excess monoclonal immunoglobulin (M-protein) and/or isotype-restricted free light chains (FLC). The chimeric monoclonal antibody KappaMab (previously called MDX-1097) can bind to a unique conformational epitope on the Kappa Myeloma Antigen (KMA). MDX-1097 is currently being assessed in a Phase lib clinical trial for the treatment of kappa light-chain restricted (K-type) MM (trial ID- ACTRN 12616001164482).

[00228] KMA consists of membrane bound kFLC that is not associated with immunoglobulin heavy chain but is non-covalently associated with sphingomyelin in the cell membrane. KMA is present on malignant B cells such as kappa-restricted MM, some kappa- type lymphomas and B cells associated with Waldenstroms macroglobulinemia. This antigen is not present on normal B cells, immune cells or normal human tissue. Recently, the clinical success of synthetic Chimeric Antigen Receptors (CARs) in immunotherapy for hematological malignancies has prompted the development of CAR T-cells against various cancer antigens. For example, there have been recent reports of phenomenal success with CARs targeting BCMA as well as other preclinical studies reported targeting CS1/SLAMF7, CD44v6, CD138, CD38 and CD70. The restricted expression of KMA makes it an ideal target and the unique specificity of KappaMab suggests that this antibody may be an excellent candidate for the development of a treatment for myeloma patients.

[00229] Aims: To develop highly specific and effective CAR T-cells expressing Chimeric Antigen Receptors targeting the Kappa Myeloma Antigen.

[00230] Methods: KappaMab heavy and light chain variable region genes were linked together to form a single chain Fv fragment (scFv). In some cases, the scFv gene construct was fused with the co-stimulatory domains from either CD28 or CD137 (41BB), along with the activation domain of the CD3 zeta subunit to construct several second generation CARs in the PiggyBac transposon vector (see Figure 40). The second generation CARs used in this example included the following KM-CARs (see also Figure 21):

[00231] hCh2Ch3 28z (also referred to herein as KM.CAR-hCH2CH3-28z (nucleic acid SEQ ID NO: 28; amino acid SEQ ID NO: 27)

[00232] hCh2Ch3 mutant 28z (also referred to herein as CAR.KMhCH2CH3mutant28z.

[00233] The nucleic acid sequence is as follows:

[00234] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCA

CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGG

GTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACT

GATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGC

AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAG

ACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGG

AGGGGGGACCAAGCTGGAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGG

TC GGGTGGC GGCGGATCTGri GG Ί (X (X 7 (X (X G Ί X GGGGC ,GA (X 77 1 GAA

GCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACAC C

TATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATT

GATCCTGCGAATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATA A

TAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG A

CACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG G

GGCCAAGGGACCACGCTCACCGTCTCCTCCGGTGGAGGCGGGTCTGGGGGCGGAG

GTTCAGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACAAAACTCACACATGC CCACCGTGCCCAGCACCTCCAGTCGCGGGACCGTCAGTCTTCCTCTTCCCCC

CAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCG

TGGTGGTGAACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG

TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG

TACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC

TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA

GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA

CAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC

AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG

CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGA

GCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC

TGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA7T7TG

GGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTA TL GCTTGCTA GTAA CA GTGG

CCTTTATTATTTTCTGGGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGT

ACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGT

ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA

GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGG

CCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC

CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG G

CCCTGCCCCCTCGC (SEQ ID NO: 48).

[00235] From 5’ to 3’, this construct (SEQ ID NO: 48) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker a KappaMab heavy chain variable resion. a second (G4SE linker a mutated IgGl hinge. CH2 and CH3 constant region domains a CD28 transmembrane domain, and a CD3 zeta intracellular domain. The mutated IgGl hinge domain has, from 5’ to 3’, E233P, L234V, L235A, G236-, S254A, D265N, and N297A mutations highlighted within the shaded boxes of this construct (SEQ ID NO: 48). Mutations at these sites (E233P, L234V, L235A, G236-, S254A, D265N, N297A) may decrease Fc interaction with CAR T-cells, allowing improved survival post-infusion.

[00236] CD8a_28z (also referred to herein as KM.CAR_CD8a_28z (SEQ ID NO: 31))

[00237] hCh2Ch3_4lBBz (also referred to herein as

KM. C AR_hCH2Ch3_28TM_41 BBz (SEQ ID NO: 49)) [00238] The nucleic acid sequence is as follows:

[00239] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGG

TGTCCAGTGCTCTAGAGACATCGTCATGACCCAGTCTCAAAAATTCATGTCCA

CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGG

GTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACT

GATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGC

AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAG

ACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGG

AGGGGGGACCAAGCTGGAAATAAAGGGTGGCGGTGGCTCGGGCGGTGGTGGG

TC GGGTGGC GGCGGATCT A GG Ί (X (X (X (X G Ί X Ά GGGGC XiGA (X Ί Ί G Ί ^' GAA

GCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACAC C

TATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATT

GATCCTGCGAATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATA A

TAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG A

CACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGACTACTG G

GGCCAAGGGACCACGCTCACCGTCTCCTCCGGTGGAGGCGGGTCTGGGGGCGGAG

GTTCAGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACAAAACTCACACATGC

CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTC

CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT

GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT

ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG

CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG

GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC

CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA

CCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG

GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG

GAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCC

GTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA

AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG

CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA7T

TTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTA TA GCTTGCTA GTAA CA G

TGGCC TTAHAm TCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATT CAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG

CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGC

AGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC

AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCT

GAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG

CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG

AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC

TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 49).

[00240] From 5’ to 3’, this construct (SEQ ID NO: 49) has a leader peptide, a KappaMab light chain variable region, a (G4SE linker a KappaMab heavy chain variable resion . a second (G4SE linker an IgGl hinge, CH2 and CH3 constant region domains a CD28 transmembrane domain, a 4-1BB intracellular domain and a CD3 zeta intracellular domain.

[00241] hCh2Ch3 mutant 4lBBz (also referred to herein as KM. CAR_hCH2Ch3mut_28TM_4l BBz (SEQ ID NO: 34))

[00242] CD8a4lBBz (also referred to herein as CAR.KM8a28TM4lBBz (SEQ ID NO: 33))

[00243] Initially, cells from normal venesection donors were electroporated with the CAR DNA constructs (i.e., Figure 18: CAR.KM8a28TM4lBBz (SEQ ID NO: 33) and Figure 19: CAR.KMhCH2CH3mutant28z (SEQ ID NO: 48)) without pre-selection for CD3+ T-cells. CAR+T-cells were enriched and expanded, using irradiated autologous PBMCs with the addition of 200IU of IL-15 every 48 hours in the presence of KMA+ cells (i.e., the irradiated KMA-mCherry cell line (see Example 6 above)). As shown in Figures 18 and 19, large percentages of non-CD3+ T-cells were present in the cultures 15 days post electroporation. As shown in Figure 20 (T-cells were electroporated with CAR.KMhCH2CH3mutant28z (SEQ ID NO: 48)), pre-selection of CD3+ T-cells prior to electroporation and subsequent stimulation in the presence of APCs drastically reduced the percentage of non-CD3+ T-cells present in the cultures 15 days post-electroporation.

[00244] Given the results of the experiments whose results are shown in Figures 18- 20, the remaining experiments were conducted using the general scheme outlined in Figure 41. In particular, pre-selected CD3+ T-cells from normal venesection donors were electroporated with the CAR DNA constructs described above. CAR+T-cells were enriched and expanded, using irradiated autologous PBMCs with the addition of 200IU of IL-15 every 48 hours in either the presence of KMA+ cells (i.e., the irradiated KMA-mCherry cell line (see Example 6 above); JJN3 cells; Pfeiffer cells) or KMA- cells (i.e., NALM6 cells). As shown in Figure 22, recovery of the T-cells following electroporation was similar for each of the constructs 1 day post-electroporation. After a three week expansion, the CAR T-cells were phenotyped (see Figures 23-28A-B) and analyzed for functional specificity (see Figures 29-34). The potency of the CAR T-cells was confirmed in cytotoxicity assays, using ⁵¹ Cr labelled cells (see Figures 32-34). Further, as shown in Figure 42, CAR T-cell function as indicated by IFN-gamma and TNF-alpha release, was tested in the presence of free kappa- light chains by adding 20, 200, 2000 mg/l to CAR T-cell/KMA-containing APC co-cultures. Free kappa-light chains by themselves were also added in CAR T-cell only cultures.

[00245] Results: Pre-clinical in vitro data indicated the successful development of a KM. CAR T-cell that can specifically target KMA. Enrichment and expansion of KM. CAR T-cells expressing any of the KM. CARs described above was enhanced in the presence of KMA expressing cell lines, especially the KMA-mCherry expressing APCs generated as described in Example 6 above. The KM. CAR T-cell containing the spacer region from the CD8 molecule was the most efficacious amongst all the KM.CAR T-cells generated. The CD28 co-stimulatory domain containing KM.CAR T-cells expressed and expanded better than the ones containing the 41BB co-stimulatory domain. All the KM.CAR T-cells were effective in clearing target cells that expressed antigen at high levels, but the KM.CAR T- cells with the CD8 spacer region performed best against target cells even with moderate antigen expression. Overall, even though there was donor variability, there was a preponderance of effector memory cells at the end of culture which indicated a potential for long term survival and function in vivo (see Figures 26 and 27). Most of the KM.CAR T- cells showed negligible expression of the exhaustion marker PD-l and increased expression of cytokines following co-culture with KMA containing cell lines (see Figure 28A-B). Release of IFN-g (also referred to as IFNg) and TNF-alpha upon co-culture with KMA+ cells or KMA- cells indicated target specific activation (see Figures 29-31). Further, as shown in Figure 42, CAR T-cell function as indicated by IFN-gamma and TNFalpha release, was not inhibited when free kappa-light chains were added to the CAR T-cell/KMA-containing APC co-cultures. [00246] Conclusions: Myeloma demonstrated phenomenal response rates to CAR therapy. The in vitro data indicates that the KM.CAR T-cell is likely to be effective in kappa restricted myeloma patients. CAR T-cells targeting KMA are highly specific and effective. KMA CAR T-cells containing the CD8a spacer expanded the best in vitro and were the most efficacious against KMA+ cells. 28z co-stimulatory domain containing CAR T-cells expanded better in vitro than the 41BB co-stimulatory domain containing CAR T-cells.

Approximately 30% of the CAR+T-cells were memory T-cells with negligent expression of PD-l and expected to persist and function in vivo; however, expression of TIM-3 and LAG-3 was significant. CAR T-cell function is not inhibited by the presence of free kappa light chains

Example 9: Expansion of KM.CAR T-cells using KMA-coated solid substrates

[00247] Aim: To expand/stimulate T-cells expressing KM.CAR constructs using KMA-coated solid substrates.

Experiment 1: Stimulating KM.CAR T-cells with KMA-coated plates

[00248] Methods/Results: Initially, 200 ul of biotin-KMA (bKMA)/PBS mixture was added to each well of a titer plate coated with anti-biotin moieties such that a portion of wells received 0.5 ug, 1 ug, 2 ug, 4 ug, 5 ug or 10 ug of bKMA. Following a 48 hour incubation at 4 degrees Celsius and two PBS washes, a KM.CAR T cells expressing the CD8a 28z construct (SEQ ID NO: 31) were plated at 1 x 10 ⁶ in 500 ul media in the bKMA coated plates and cultured with IL-15 for 48 hours. After 48 hours, the KM.CAR T-cells were moved to fresh wells without bKMA coating and growth was continued with IL-15 for eight days. As shown in Figure 35, after 8 days in culture, the KM.CAR T-cells incubated with 5 ug of bKMA produced the highest percentage of CD3+ cells. That being said the percentage of CD3+ cells reached only about 10%. Subsequently, 1 x 10 ⁶ of T-cells expressing the various KM.CARs shown in Figure 36 (see Example 8), were each cultured in 500 ul of media comprising IL-15 in wells coated with 5 ug KMA. Following 48 hours of culturing the T- cells were each moved to fresh well without bKMA coating and cultured for additional 13 days in the presence of IL-15. As shown in Figure 36, most of the T-cells did not last for the entire experiment.

Experiment 2: Stimulating KM.CAR T-cells with KMA-coated beads [00249] Methods/Results: KMA coated beads were generated by incubating 5 ug of biotin-KMA (bKMA) with 1 x 10 ⁸ anti-biotin coated MACSi beads for 2 hours as described in the Miltenyi kit. In parallel, as generally shown in Figure 16, KM. CAR T cells were generated by electroporating T-cells with a CD8a 28z construct (SEQ ID NO. 31) and subsequently growing the transformants overnight in growth media. Following overnight growth, the KM.CAR T-cells were divided into 3 groups for stimulation: Group 1: KM.CAR T-cells were incubated with irradiated (IR) PBMCs plus IL-15 + KMA-mCherry APCs (see Examples above); Group 2: KM.CAR T-cells were incubated with IR PBMCs plus IL-15 plus bKMA-coated beads; Group 3: KM.CAR T-cells were incubated with bKMA-coated beads plus TransAct (T) plus IL-2. For group 1, fresh IR PBMCs, IL-15 and APCs were introduced 1 day and 8 days after electroporation. For group 2, fresh IR PBMCs, IL-15 and bKMA- beads were introduced 1 day and 8 days after electroporation. For Group 3, 40 ul of TransAct (CD3/CD28 activation beads) plus IL-2 were added to about 1 million KM.CAR T- cells per well per TransAct kit recommendation. The TransAct beads were removed and the T-cells were cultured with just the cytokines for the remainder of the experiment. As shown in Figures 37 and 38, KM.CAR T-cells showed the greatest level of KM.CAR expression and expansion when stimulated using APCs.

Example 10: Demonstration of the Utility of the PiggyBat Transposon Expression System

Introduction:

[00250] The transposon piggyBat is a member of the piggyBac superfamily of DNA transposons present in the Myotis lucifugus genome. In contrast to the piggyBac transposon, piggyBat has been shown to have a similar integration profile by lower integration activity in human cell lines (HCT116 & HeLa; see Ray et al. Genome Research 2008 18: 717-728 and Mitra et al., PNAS 2013 110:234-239). The goal of this experiment was to determine the effectiveness of the piggyBat transposon in human derived T-cells.

Methods/Results:

[00251] The CD8a_28z (also referred to herein as KM.CAR_CD8a_28z (SEQ ID NO: 31)) construct previously described herein was cloned into a PiggyBat transposon. T-cells from three human donors designated CESI, ROHA and GEYU in Figures 43-48 were thawed and grown in culture media for two days. On day 0 (i.e., following growth for 2 days), about 5 million T-cells from each of the three donors were subjected to electroporation with the 5 micrograms of piggyBat transposase and 5 micrograms of transposon plasmid comprising the CD28a_28z KM. CAR. It should also be noted that T-cells from each donor were engineered to intrinsically express GFP after a 2A self-cleaving peptide using the method described previously herein (see Example 3).

[00252] On day 1 post-electroporation, the number of viable cells was determined using trypan blue exclusion and counting manually with a haemocytometer. Subsequently, for each donor, a culture comprising KM. CAR T-cells from said donor plus irradiated auto PBMCs, irradiated KMAmCherry APCs (as described in Example 6) and IL-15 at 200 IU/ml were set up. It should be noted that the ratio of irradiated auto PBMCs to donor KM CAR T- cells was 2: 1, while the ratio of donor KM.CAR T-cells to irradiated KMAmCherry APCs was 0.2 to 1. IL-15 was added at 200 IU/ml to each of the donor KM.CAR T-cell cultures every 48 hours. Further, the number of viable cells from each donor KM.CAR T-cell culture was determined using trypan blue staining as described herein on days 8 and 15 post electroporation and the cultures were subsequently stimulated. Additionally, on days 8, 15 and 22 post-electroporation, 200,000 cells from each donor KM.CAR T-cell culture were subjected to flow cytometry analysis following staining of the T-cells with biotinylated KMA+secondary streptavidin -PE along with Ab to detect CD3, CD4, CD8 in order to determine CD8a_28z expression (see Figure 44) and CD4/CD8 distribution (see Figure 45).

[00253] On day 22 post-electroporation, each of donor KM.CAR T-cells cultures were stopped and phenotypic analysis of the cultures was conducted. The analysis included determining the memory phenotype (see Figure 46) via flow cytometry by staining cells with antibodies against CD62L and CD45RA along with staining for CD3, KMA. The analysis also included determining the levels of exhaustion markers in the cultures by flow cytometry on cells stained cells for PD-l, TIM-3 and Lag-3 (see Figure 47) and assessing functional specificity by examining the release of cytokines upon co-culture of donor KM.CAR T-cells from each donor with KMA+ specific cells lines such as KMAmCherry and JJN3 cell lines.

[00254] As shown in Figure 43, PiggyBat KM.CAR CD8a_28z expressing T-cells from each donor showed the piggyBat system can be used to successfully expand KM.CAR T-cells similar to the piggyBac system showed previously herein. In particular, as shown in Figure 43, CESI and ROHA were very similar in their expansion/expression profile, showing about 700 fold expansion at day 22 post electroporation, while GEYU stands out with >3000 fold expansion at day 22 post-electroporation. Figure 44 showed that GFP expression was slightly higher in each donor T-cell line than KMA and that expression of KMA CAR (i.e., CD8a_28z) started from 5-15% on day 8 and reached about 60-80% by day 22 post electroporation. These results were similar to what was seen using piggyBac-KM.CAR constructs as provided herein. Figure 45 showed that while the T-cells from donor GEYU had more CD8+ cells from the start and expanded to -90% at end of culture, the T-cell cultures from the other two donors had a predominance of CD4+ cells. The memory phenotype analysis consolidated for the three donor T-cell cultures as shown in Figure 46 showed that less than 50% of CAR T cells were terminal effectors. Further, significant naive and memory cells were found at the end of culturing, which indicated potential for in vivo persistence and expansion. The consolidated data from the 3 donor T-cells cultures shown Figure 47 revealed that similar to what was seen previously in piggyBac KM.CAR T-cell cultures, very little PD-l was expressed in the donor T-cell cultures and thus said T-cells have the potential to be functional. Finally, Figure 48 showed that like KM.CAR piggyBat cultures, the piggyBat KM.CAR T-cells showed functional specificity.