Chimeric Antigen Receptors (CARs) with Mutations in the Fc Spacer Region and Methods of Use Thereof

Abstract

Adoptive immunotherapy using T cells genetically redirected via expression of a chimeric antigen receptor (CAR) is a promising approach for cancer treatment. However, this immunotherapy partly depends on the optimal molecular design of the CAR, which involves an extracellular ligand-binding domain linked to an intracellular signaling domain via a spacer and / or transmembrane sequence.

Description

[0001] This application is a divisional application of the patent application with the filing date of March 14, 2014, the priority date of January 13, 2014, the application number of 201480076612.7, and the invention title of "Chimeric Antigen Receptors (CARs) Having Mutations in the Fc Spacer Region and Methods of Use Thereof".

[0002] Priority Claims

[0003] This application claims the priority of U.S. Provisional Patent Application No. 61 / 926,881, filed on January 13, 2014, which is incorporated herein by reference in its entirety (including the drawings).

[0004] Statement of Government Interests

[0005] This invention was made with government support under grants P50 CA107399 and P01CA030206 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. Background of the Invention

[0006] Adoptive immunotherapy using T cells expressing chimeric antigen receptors (CARs) is a promising cancer treatment because these cells can directly recognize and kill antigen-expressing tumor cells in a manner independent of human leukocyte antigen (HLA). However, in addition to the careful selection of the target tumor-associated antigen, this treatment method highly depends on the optimal molecular design of the CAR.

[0007] Although CARs containing TAA-specific scFvs have been shown to exhibit significant anti-tumor efficacy in various systems, which generate intracellular signals via cytoplasmic co-stimulatory (e.g., CD28 or 4-1BB) domains fused to CD3-zeta (Brentjens et al. 2013; Brentjens et al. 2011; Grupp et al. 2013; Kalos et al. 2011; Kochenderfer et al. 2012), host immunological rejection and clearance remain challenges for effective cancer treatment.

[0008] Certain modifications in CAR designs have been used to prevent FcR-mediated clearance of therapeutic cells. For example, hinge / spacer sequences that do not derive from the Ig Fc domain can be used, such as those from CD8α or CD28 (Brentjens et al. 2007; Kalos et al. 2011; Imai et al. 2004; Kochenderfer et al. 2009). Although these spacer sequences can reduce FcR binding, their length cannot confer optimal potency to CAR T cells when targeting certain antigens. For example, when using CAR T cells to target 5T4, NCAM, and MUC1, optimal potency requires a longer linker region (i.e., longer than those derived from CD8α or CD28) (Wilkie et al. 2008; Guest et al. 2005). Thus, there is a desire to design CARs that address these challenges while maintaining their potency in killing cancer cells. SUMMARY OF THE INVENTION

[0009] According to some embodiments, provided is a recombinant chimeric antigen receptor (CAR) having impaired binding to an Fc receptor (FcR). Such CARs can include, but are not limited to, an antigen recognition domain, a spacer domain derived from a modified immunoglobulin Fc region, and an intracellular signaling domain, wherein the modified immunoglobulin Fc region has one or more mutations in its CH2 region that result in impaired binding to the FcR.

[0010] In another embodiment, provided is a population of human immune cells transduced with a viral vector comprising an expression cassette including a CAR gene. In some aspects, the CAR gene comprises a nucleotide sequence encoding an antigen recognition domain, a spacer domain derived from a modified immunoglobulin Fc region, and an intracellular signaling domain, wherein the modified immunoglobulin Fc region has one or more mutations in its CH2 region that result in impaired binding to the FcR, and wherein the population of human immune cells expresses the CAR gene.

[0011] In another embodiment, provided is a method of treating cancer in a subject. Such methods include administering to the subject a population of human immune cells transduced with a CAR gene. In some aspects, the CAR gene comprises a nucleotide sequence encoding an antigen recognition domain that targets a cancer-associated antigen specific for the cancer, a spacer domain derived from a modified immunoglobulin Fc region, and an intracellular signaling domain, wherein the modified immunoglobulin Fc region has one or more mutations in its CH2 region that result in impaired binding to the FcR.

[0012] Designing CARs with spacer domains (such as those described herein) that have reduced or impaired binding to FcRs helps prevent FcR-expressing cells from recognizing and destroying, or inadvertently activating, CAR-expressing immunotherapeutic cells in vivo. Thus, such CARs help prevent immune rejection and clearance of the cells, with the intent of providing a therapeutic benefit to the patient.

[0013] The present invention includes the following:

[0014] 1. A recombinant chimeric antigen receptor (CAR) having impaired binding to an Fc receptor (FcR), comprising:

[0015] An antigen recognition domain;

[0016] A spacer domain derived from a modified immunoglobulin Fc region that has one or more mutations in its CH2 region that result in impaired binding to FcR; and

[0017] An intracellular signaling domain.

[0018] 2. The method of embodiment 1, wherein the antigen recognition domain is a scFv.

[0019] 3. The method of embodiment 1, wherein the antigen recognition domain targets a cancer-associated antigen selected from: 5T4, 8H9, αvβ6 integrin, alpha-fetoprotein (AFP), B7-H6, carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD52, CD123, CD171, carcinoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1 / EGFR, ErbB2 / HER2 / neu / EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-alpha, G250 / CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor alpha2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, murine CMV-infected cells, mucin-1 (MUC1), mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligand, neural cell adhesion molecule (NCAM), NY-ESO-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptor.

[0020] 4. The method of embodiment 1, wherein the modified immunoglobulin Fc region is a modified IgG1, IgG2, IgG3, or IgG4 Fc region.

[0021] 5. The method of embodiment 1, wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more amino acid substitutions selected from the S228P amino acid substitution, the L235E amino acid substitution, the N297Q amino acid substitution, or a combination thereof.

[0022] 6. The method of embodiment 1, wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more deletions.

[0023] 7. The method of embodiment 1, further comprising a transmembrane domain.

[0024] 8. The method of embodiment 1, wherein the intracellular signaling domain is a T cell receptor (TCR) zeta chain signaling domain.

[0025] 9. The method of embodiment 8, further comprising one or more co-stimulatory intracellular signaling domains derived from CD28, inducible co-stimulator (ICOS), OX40, CD27, DAP10, 4-1BB, p56lck or 2B4.

[0026] 10. The method of embodiment 1, wherein the CAR is encoded by a nucleic acid sequence inserted in a viral vector.

[0027] 11. A population of human immune cells transduced with a viral vector, the viral vector comprising an expression cassette comprising a CAR gene, the gene comprising a nucleotide sequence encoding:

[0028] an antigen recognition domain;

[0029] a spacer domain derived from a modified immunoglobulin Fc region, the modified immunoglobulin Fc region having one or more mutations in its CH2 region that result in impaired binding to FcR; and

[0030] an intracellular signaling domain,

[0031] wherein the population of human immune cells expresses the CAR gene.

[0032] 12. The method of embodiment 11, wherein the antigen recognition domain targets a cancer-associated antigen selected from: 5T4, 8H9, αvβ6 integrin, alpha-fetoprotein (AFP), B7-H6, carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD52, CD123, CD171, carcinoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1 / EGFR, ErbB2 / HER2 / neu / EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-alpha, G250 / CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor alpha2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, murine CMV-infected cells, mucin-1 (MUC1), mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligand, neural cell adhesion molecule (NCAM), NY-ESO-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptor.

[0033] 13. The method of embodiment 11, wherein the modified immunoglobulin Fc region is a modified IgG1, IgG2, IgG3, or IgG4 Fc region.

[0034] 14. The method of embodiment 11, wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more amino acid substitutions selected from S228P amino acid substitution, L235E amino acid substitution, N297Q amino acid substitution, or a combination thereof.

[0035] 15. The method of embodiment 11, wherein the one or more mutations of the modified immunoglobulin Fc region comprise one or more deletions.

[0036] 16. The method of embodiment 11, wherein the intracellular signaling domain is a T cell receptor (TCR) zeta chain signaling domain.

[0037] 17. The method of embodiment 16, further comprising one or more co-stimulatory intracellular signaling domains derived from CD28, inducible co-stimulator (ICOS), OX40, CD27, DAP10, 4-1BB, p56lck, or 2B4.

[0038] 18. A method of treating cancer in a subject, comprising administering to the subject a population of human immune cells transduced with a CAR gene, wherein the CAR gene comprises a nucleotide sequence encoding:

[0039] An antigen recognition domain that targets a cancer-associated antigen specific for the cancer;

[0040] A spacer domain derived from a modified immunoglobulin Fc region, the modified immunoglobulin Fc region having one or more mutations in its CH2 region that result in impaired binding to FcR; and

[0041] An intracellular signaling domain.

[0042] 19. The method of embodiment 18, wherein the impaired binding to FcR results in improved persistence of the human immune cells compared to human immune cells transduced with a CAR gene comprising a nucleotide sequence encoding a spacer domain derived from an unmodified immunoglobulin Fc region.

[0043] 20. The method of embodiment 18, further comprising co-administering the population of human immune cells transduced with the CAR gene in combination with one or more anti-cancer therapies selected from stem cell transplantation, radiation therapy, surgical resection, chemotherapeutic agents, immunotherapeutic agents, targeted therapeutic agents, or combinations thereof.

[0044] 21. A recombinant chimeric antigen receptor (CAR) having impaired binding to an Fc receptor (FcR), comprising:

[0045] An antigen recognition domain comprising a scFv;

[0046] A spacer domain derived from a modified immunoglobulin Fc region, the modified immunoglobulin Fc region having one or more mutations in its CH2 region that result in impaired binding to FcR, wherein the one or more mutations are selected from S228P amino acid substitution, L235E amino acid substitution, N297Q amino acid substitution, or combinations thereof; and

[0047] An intracellular signaling domain. Brief Description of the Drawings

[0048] Figure 1 Shows that, according to one embodiment, T cells expressing a CD19-specific CAR do not effectively engraft in NSG mice. Figure 1a shows schematic diagrams of CD19R / EGFRt (top) and EGFRt (bottom) expression constructs for gene-modifying T cells for engraftment studies. The CD19-specific CD28-costimulatory CAR (CD19R), self-cleavable T2A, huEGFRt, and drug-resistant DHFR FS and IMPDH2 IY sequence portions of the genes, as well as the elongation factor 1 promoter sequence (EF-1p), GM-CSF receptor alpha chain signal sequence (GMCSFRss), and 3-nucleotide stop codon. Figure 1 b is a flow cytometry analysis of T cells administered to NSG mice for engraftment studies. T CM -derived cells were kept untransduced (non-Txd), or transduced with lentiviral vectors containing the CD19R / EGFRt (CD19R) or EGFRt / DHFRFS / IMPDH2IY (EGFRt) constructs described in (A), and immunomagnetically selected for EGFRt expression. Cells were then expanded in vitro for 19 days and surface phenotypes analyzed. Using quadrants created based on negative control staining, the percentage of cells stained with antibodies against CD4 (top) or CD8 (bottom) relative to an antibody specific for EGFRt is indicated in each histogram. In Figure 1 c, 10 7 T CM -derived cells described in (B) were administered i.v. to NSG mice in the presence of irradiated NS0-IL15 supports. Peripheral blood leukocytes harvested on days 7 and 14 from each group (n = 3 - 5 mice) were stained with anti-human CD45 conjugated to FITC, and biotinylated cetuximab, followed by streptavidin conjugated to PE. Using quadrants created based on negative control staining, the percentage of lymphocyte-gated huCD45+ and huCD45+EGFRt+ cells is indicated in each histogram. Data represent 4 different experiments performed with T CM -derived cells from multiple donors.

[0049] Figure 2 shows that, according to one embodiment, CD19-specific CAR-expressing T cells bind soluble FcγR1. The same T cells described in Figure 1 were stained with biotinylated soluble human Fc gamma receptor 1 at the indicated volume titers, followed by streptavidin conjugated to PE (SA-PE, gray histograms). For cells expressing CD19R, the percentage of immunoreactive cells is indicated in each histogram and gated based on M1 set to detect <1% of cells stained only with SA-PE (black line).

[0050] Figure 3 Shows that, according to one embodiment, the mutated IgG4 spacer does not affect the CD19 - specific effector function of CAR - expressing T cells. Figure 3 a shows a schematic diagram of the parental CD19 - specific CAR (CD19R), the CD19 - specific CAR with two point mutations L235E and N297Q in the CH2 portion of the IgG4 spacer (CD19R(EQ)), and the CD19 - specific CAR with a truncated IgG4 spacer lacking the entire CH2 domain (CD19Rch2Δ). Also depicted are the ligand - binding scFv domain derived from the FMC63 mAb, the transmembrane and cytoplasmic signaling domains derived from huCD28, and the cytoplasmic signaling domain of huCD3ζ. In Figure 3 b, transgenic expression was analyzed in T CM - derived, EGFRt - enriched and expanded cells that express the parental CD19R, the EGFRt marker alone, CD19R with a single IgG4 point mutation at amino acid 235 (CD19R(L235E)) or amino acid 297 (CD19R(N297Q)), the double - mutant CD19R(EQ), or the CH2 - deleted CD19Rch2Δ. The percentage of cells stained with antibodies specific for the CAR containing Fc (top) or EGFRt (bottom) is indicated in each histogram, and is based on an M1 gating set to detect < 1% of the cells stained only with SA - PE (black line). In Figure 3 c, Figure 3 the same cells used in b were used as effector cells in a 4 - hour chromium release assay against 51 51Cr - labeled CD19 + LCL or SupB15 targets. LCL expressing the CD3 agonist OKT3 (LCL - OKT3) and CD19 - negative K562 cells were used as positive and negative control targets, respectively. The mean percentage chromium release ± S.D. of triplicate wells at the indicated E:T ratios is described.

[0051] Figure 4Shows that, according to one embodiment, CARs with mutated IgG4 spacers exhibit inhibited FcγR binding. TCM-derived, EGFRt-enriched, expanded cell lines expressing the sole EGFRt marker, parental CD19R, single-point mutant CD19R (L235E) or CD19R (N297Q), double-point mutant CD19R (EQ), or CH2-deleted CD19Rch2Δ were stained with the following biotinylated reagents: anti-Fc antibody (to detect CAR), cetuximab (to detect EGFRt), or the indicated human (Hu) or murine (Mu) soluble Fc receptors (FcγR1, R2a, or R2b); followed by staining with streptavidin conjugated to PE (SA-PE, gray histograms). The percentage of immunoreactive cells is indicated in each histogram and is based on M1 gating set to detect ≤1% of cells stained only with SA-PE (black line).

[0052] Figure 5 Shows that, according to one embodiment, T cells expressing CARs with mutated IgG4 spacers exhibit enhanced in vivo engraftment. Using irradiated NS0-IL15 supports, 10 7 T cells expressing parental CD19R, the sole EGFRt marker, single-point mutant CD19R (L235E) or CD19R (N297Q), double-point mutant CD19R (EQ), or CH2-deleted CD19Rch2Δ (see phenotype Figure 3 b) were injected i.v. into NSG mice on day 0. Peripheral blood leukocytes harvested on days 7 and 14 from each group (n = 5 mice) were stained with anti-human CD45 conjugated to PerCP and biotinylated cetuximab, followed by staining with streptavidin conjugated to PE. In CM Figure 5 a, the mean percentage ± S.E.M. of CD45+EGFRt+ cells in the live lymphocyte gated population is indicated. *, p < 0.034 when compared to mice given cells expressing CD19R using unpaired Student t-test. Figure 5 b shows representative histograms (i.e., the middle 3 of 5 mice per group) delineated by quadrants created based on control staining. The percentage of huCD45+EGFRt+ cells is indicated in each histogram.

[0053] Figure 6 Shows that, according to some embodiments, T CM cells derived from those expressing CARs with mutated IgG4 spacers exhibit enhanced therapeutic efficacy. On day 0, 1.5 x 10 6 ffLuc + ​LCL cells were administered i.v. to NSG mice, and then on day 3, 5 x 10 6 CAR+ T cells expressing a single EGFRt marker, parental CD19R, double-point mutant CD19R (EQ), or CH2-deleted CD19Rch2Δ CM (a total of 10 7 cells) were infused i.v. into NSG mice. Then, LCL tumor growth was monitored by Xenogen imaging. Figure 6 a shows flow cytometry analysis depicting the CAR profile of the input T CM derived cells (used on day 23 after bead stimulation and lentitransduction). The percentage of immunoreactive cells is indicated in each histogram and is based on M1 gating set to detect <1% of cells stained only with SA-PE (black line). Figure 6 b shows the mean flux level (±S.E.M.) depicting luciferase activity for each group (n = 6). Figure 6 c shows representative bioluminescence images of NSG mice on day 21 for each group. Figure 6 d shows the mean percentage (+S.E.M.) of CD45 + EGFRt + cells in the live lymphocyte gated population of peripheral blood on day 21. *, p < 0.035 when compared to mice given cells expressing CD19R using an unpaired Student t-test. Figure 6 e shows the Kaplan Meier survival analysis for each group. Statistical survival analysis between groups was performed using the log-rank (Mantel-COX) test; *, p = 0.0009 when compared to mice receiving T cells expressing parental CD19R.

[0054] Figure 7 It is shown that, according to one embodiment, T cells expressing a large amount of CD19R (EQ) exhibit enhanced therapeutic efficacy. On day 0, 1.5 x 10 6 ffLuc + LCL cells were administered i.v. to NSG mice, and then on day 2, 5 x 10 6 CAR + T cells expressing parental CD19R or double-point mutant CD19R (EQ) were infused i.v. into NSG mice. Then, LCL tumor growth was monitored by Xenogen imaging. Figure 7a shows flow cytometry analysis of CAR (top), EGFRt vs CD3 (middle), and CD4 vs CD8 (bottom) profiles of input T cells (used on day 21 after bead stimulation and slow transduction). The percentage of immunoreactive cells depicted in each histogram was determined by histogram subtraction (top), or based on quadrants drawn according to mock-transduced cell staining and isotype control staining (middle, bottom). Figure 7 b shows representative bioluminescence images of NSG mice on day 2, day 11, and day 23 for each group. Figure 7 c shows the mean flux levels (±S.E.) of luciferase activity depicted for each group (n = 3). Figure 7 d shows the Kaplan Meier survival analysis for each group. Statistical survival analysis between groups was performed using the log-rank (Mantel-COX) test; *, p = 0.0295 when compared to mice receiving T cells expressing parental CD19R.

[0055] Figure 8 It is shown that according to some embodiments, non-enriched TCM-derived cells expressing CAR with a mutant IgG4 spacer exhibit enhanced in vivo engraftment. Using irradiated NS0-IL15 supports, 10 7 T-derived cells expressing either a single EGFRt marker, parental CD19R, or double point mutant CD19R (EQ) CM were infused i.v. into NSG mice on day 0. Peripheral blood leukocytes harvested on day 7 and day 14 from each group (n = 4 - 6 mice) were stained with anti-human CD45 conjugated to PerCP, and biotinylated cetuximab followed by streptavidin conjugated to PE. Figure 8 A shows flow cytometry analysis depicting the CAR profile of input TCM-derived cells (used on day 26 after bead stimulation and slow transduction). The percentage of cells stained with antibodies specific for CAR containing Fc (top) or EGFRt (bottom) is indicated in each histogram, and M1 gating was set to detect ≤1% of cells stained only with SA-PE (black line). Figure 8 B shows the mean percentage ±S.E.M. of CD45+EGFRt+ cells in the live lymphocyte gated population. *, p = 0.004 and **, p = 0.057 when comparing mice infused with TCM-derived cells expressing parental CD19R vs CD19R (EQ) using an unpaired Student t test. Figure 8 C shows representative histograms (i.e., the middle 2 of 4 - 6 mice per group) depicting quadrants created based on control staining. The percentage of huCD45+EGFRt+ cells is indicated in each quadrant. DETAILED DESCRIPTION OF THE INVENTION

[0056] The following examples are intended to illustrate various embodiments of the present invention. Accordingly, the specific embodiments discussed should not be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that various equivalent schemes, variations, and modifications can be made without departing from the scope of the present invention, and it should be understood that such equivalent embodiments are included herein. In addition, all references cited herein are hereby incorporated by reference in their entirety as if fully set forth herein.

[0057] Chimeric Antigen Receptor

[0058] According to the embodiments described herein, recombinant chimeric antigen receptors (CARs) that target cancer-associated antigens and methods of using the same are provided. As described in the embodiments below, a CAR can comprise a series of protein or peptide domains, including but not limited to one or more of the following: an antigen-binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain, and an intracellular co-stimulatory domain.

[0059] In some embodiments, a gene encoding a CAR is provided, wherein the gene comprises a nucleotide or nucleic acid sequence that includes a series of regions encoding amino acid sequences corresponding to the protein or peptide domains of the CAR described herein. Due to the known degeneracy of the genetic code, any amino acid sequence disclosed herein also indicates all degenerate nucleic acid codons corresponding to each amino acid in the sequence. Accordingly, it should be understood that embodiments describing the CAR and its domains can be provided in terms of a gene comprising a nucleic acid sequence and the amino acid sequence encoded by the gene.

[0060] In one embodiment, a CAR can comprise, but is not limited to, an antigen-binding domain, a spacer domain, optionally at least one intracellular signaling domain, and optionally at least one intracellular co-stimulatory domain.

[0061] In other embodiments, a CAR can comprise, but is not limited to, an antigen-binding domain, a spacer domain, and at least one intracellular signaling domain.

[0062] In other embodiments, a CAR can comprise, but is not limited to, an antigen-binding domain, a spacer domain, at least one intracellular signaling domain, and at least one intracellular co-stimulatory domain.

[0063] Antigen-binding domain

[0064] The CAR antigen-binding domain can comprise a nucleotide sequence that, when expressed as a peptide or polypeptide, binds to an epitope of a cancer-associated antigen. In some embodiments, the cancer-associated antigen can be any antigen expressed or overexpressed by cancer cells (e.g., tumor cells, neoplastic cells, malignant cells, or any other cancerous cells), and can be a protein, peptide, carbohydrate, glycoprotein, ganglioside, proteoglycan, or any combination or complex thereof. In some aspects, the cancer-associated antigen is a tumor-specific antigen (TSA) that can be expressed only on cancer cells or tumor cells, while in other aspects, the cancer-associated antigen is a tumor-associated antigen (TAA) that can be expressed on both tumor cells and normal cells. In other aspects, the cancer-associated antigen can be the product of a mutant oncogene or tumor suppressor gene, or the product of another mutant gene (e.g., an overexpressed or aberrantly expressed cellular protein, a tumor antigen generated by an oncogenic virus, a carcinoembryonic antigen, an altered cell surface glycolipid or glycoprotein, or a cell type-specific differentiation antigen).

[0065] According to the embodiments described herein, cancer-associated antigens that can be targeted by the CAR antigen-binding domains described herein include, but are not limited to, 5T4, 8H9, α vβ6 integrin, alpha-fetoprotein (AFP), B7-H6, carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD52, CD123, CD171, carcinoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1 / EGFR, ErbB2 / HER2 / neu / EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-alpha, G250 / CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma associated antigen (HMW-MAA), IL-13 receptor alpha2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma associated antigen (MAGE-A1), mesothelin, murine CMV-infected cells, mucin-1 (MUC1), mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligand, neural cell adhesion molecule (NCAM), NY-ESO-1, carcinoma embryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE, tumor associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors. In some embodiments, the antigen-binding domain that is part of the CAR described herein targets CD19 or CD123.

[0066] The antigen-binding domain can be any targeting moiety that targets an antigen associated with cancer. In some embodiments, the antigen-binding domain is an antibody or a functional fragment of an antibody. An antibody refers to an immunoglobulin molecule that specifically binds to an antigen or epitope, or immunoreacts with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, as well as functional antibody fragments, including but not limited to fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term “antibody or its functional fragment” also includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv). Unless otherwise stated, the term “antibody” should be understood to encompass its functional antibody fragments. In one embodiment, the antigen-binding domain is a scFv having a heavy chain (V H ) and a light chain (V L ). In other embodiments, the antigen-binding domain is a scFv that targets CD19 or CD123. In such embodiments, the scFv targeting CD19 can have the following amino acid sequence:

[0067]

[0068] And, the scFv targeting CD123 can have one of the following amino acid sequences:

[0069]

[0070] Spacer domain

[0071] The spacer domain (also referred to as the "hinge region" or "spacer / hinge region") can be derived from or contain an immunoglobulin Fc region, such as at least a portion of an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the spacer domain contains at least a portion of an IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains. In some embodiments, the spacer domain can also contain at least a portion of the corresponding immunoglobulin hinge region. In some embodiments, the spacer domain is derived from or contains a modified immunoglobulin Fc region, such as at least a portion of a modified IgG1 Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region. The modified immunoglobulin Fc region can have one or more mutations (e.g., point mutations, insertions, deletions, duplications) that result in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the spacer domain to an Fc receptor (FcR). In some aspects, the modified immunoglobulin Fc region can be designed to have one or more mutations that result in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the spacer domain to one or more FcRs, including but not limited to FcγRI, FcγR2A, FcγR2B1, FcγR2B2, FcγR3A, FcγR3B, FcεRI, FcεR2, FcαRI, Fcα / μR, or FcRn.

[0072] Some amino acid sequences within the Fc CH2 domain have been identified as having a role in antibody-FcR interactions (Strohl, 2009). FcRs, such as FcγRI, are integral membrane proteins located on immune cells, including natural killer (NK) cells and macrophages, which then use this Fc-targeting ability to carry out various immune functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis.

[0073] Impairing binding to FcRs by the spacer domain prevents cells expressing FcRs from recognizing and destroying, or inadvertently activating, CAR-expressing immunotherapeutic cells in the body, thereby helping to prevent immunological rejection and clearance of the cells with the intent of providing a therapeutic benefit to the patient. The mutations described herein also contribute to reducing off-target effects of the CAR, thereby increasing its specificity and potency.

[0074] As used herein, "amino acid modification" or "amino acid substitution" or "substitution" refers to amino acid substitution, insertion, and / or deletion in a protein or peptide sequence. As used herein, "amino acid substitution" or "substitution" means replacing an amino acid at a specific position in a parental peptide or protein sequence with another amino acid. For example, substitution S228P refers to a variant protein or peptide in which serine at position 228 is replaced with proline.

[0075] Amino acid substitutions can be made by mutation such that a specific codon in the nucleic acid sequence encoding the protein or peptide is changed to a codon encoding a different amino acid. Such mutations are generally made with the fewest possible nucleotide changes. Substitution mutations of this type can be made so as to change the amino acid in the resulting protein in a non-conservative manner (i.e., by changing a codon from an amino acid belonging to an amino acid grouping having a specific size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing a codon from an amino acid belonging to an amino acid grouping having a specific size or characteristic to an amino acid belonging to the same grouping). Such conservative changes generally result in smaller changes in the structure and function of the resulting protein.

[0076] Examples of various groupings of amino acids are as follows:

[0077] Amino acids with non-polar R groups: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine.

[0078] Amino acids with uncharged polar R groups: glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine.

[0079] Amino acids with charged polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid.

[0080] Basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (at pH 6.0).

[0081] Another grouping can be those amino acids having a phenyl group: phenylalanine, tryptophan, tyrosine.

[0082] Another grouping can be based on molecular weight (i.e., the size of the R group), as shown below:

[0083]

[0084] In certain embodiments, the spacer domain is derived from a modified IgG1, IgG2, IgG3, or IgG4 Fc region that comprises one or more amino acid residues substituted with amino acid residues different from those present in the unmodified hinge. The one or more substituted amino acid residues are selected from, but not limited to, one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, 339, or combinations thereof.

[0085] In some embodiments, the spacer domain is derived from a modified IgG1, IgG2, IgG3, or IgG4 Fc region that comprises one or more of the following amino acid residue substitutions, but not limited to: C220S, C226S, S228P, C229S, P230S, E233P, V234A, L234V, L234F, L234A, L235A, L235E, G236A, G237A, P238S, S239D, F243L, P247I, S267E, H268Q, S280H, K290S, K290E, K290N, R292P, N297A, N297Q, S298A, S298G, S298D, S298V, T299A, Y300L, V305I, V309L, E318A, K326A, K326W, K326E, L328F, A330L, A330S, A331S, P331S, I332E, E333A, E333S, E333S, K334A, A339D, A339Q, P396L, or combinations thereof.

[0086] In some embodiments, the spacer domain is derived from an IgG Fc region that has one or more modifications made to its CH2-CH3 region, wherein the unmodified IgG CH2-CH3 region corresponds to one of the following amino acid sequences:

[0087]

[0088] In some embodiments, the spacer domain is derived from an IgG Fc region that has one or more modifications made to its hinge region, wherein the unmodified IgG hinge region corresponds to one of the following amino acid sequences:

[0089] In some embodiments, the spacer domain is derived from an IgG4 Fc region having the following amino acid sequence: Pos.219 ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY Pos.279 VDGVEVHNAKTKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK Pos.339 AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL Pos.399 DSDGSFFLYSRLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK(SEQ ID NO:15)

[0090] In certain embodiments, the spacer domain is derived from a modified IgG4 Fc that comprises one or more amino acid residues substituted with amino acid residues different from the amino acid residues present in the unmodified IgG4 Fc region. The one or more substituted amino acid residues are selected from, but not limited to, one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, 339, 396, or combinations thereof.

[0091] In some embodiments, the spacer domain is derived from a modified IgG4 Fc region that comprises one or more of the following amino acid residue substitutions: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 247I, 267E, 268Q, 280H, 290S, 290E, 290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V, 299A, 300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S, 331S, 332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L, or combinations thereof, wherein the amino acid substitution in the unmodified IgG4 Fc region is the amino acid identified above at the designated position.

[0092] In some embodiments, the spacer domain is derived from a modified IgG4 Fc region that includes, but is not limited to, two or more (i.e., "double mutations"), three or more (i.e., "triple mutations"), four or more, five or more, or more than five of the following amino acid residue substitutions: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 247I, 267E, 268Q, 280H, 290S, 290E, 290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V, 299A, 300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S, 331S, 332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L, or combinations thereof, wherein the amino acids in the unmodified IgG4 Fc region are replaced with the amino acids identified above at the designated positions.

[0093] In some embodiments, the spacer domain is derived from a modified IgG4 Fc region that includes, but is not limited to, the substitution of proline (P) for serine (S) at position 228 (S228P), the substitution of leucine (L) for glutamate (E) at position 235 (L235E), the substitution of asparagine (N) for glutamine (Q) at position 297 (N297Q), or combinations thereof. In certain embodiments, the modified IgG4 Fc region has a single mutation as indicated in the following amino acid sequences (mutations are bolded and underlined):

[0094]

[0095] In other embodiments, the spacer domain is derived from a modified IgG4 Fc region that is double mutated to include the L235E substitution and the N297Q substitution ("EQ"). In another embodiment, the modified IgG4 Fc region is triple mutated to include the S228P substitution, the L235E substitution, and the N297Q substitution ("S228P+L235E+N297Q"). In certain embodiments, the modified IgG4 Fc and / or hinge region can include a nucleotide sequence that encodes an amino acid sequence selected from the group (mutations are bolded and underlined):

[0096]

[0097] In certain embodiments, the spacer domain is derived from a modified immunoglobulin Fc region that contains one or more deletions of all or a part of its CH2 domain. In one embodiment, the spacer domain is derived from a modified IgG4 Fc region that contains one or more deletions of all or a part of its CH2 domain ("ch2Δ"). In one aspect of such embodiments, the spacer domain can comprise a nucleotide sequence encoding the following amino acid sequence:

[0098]

[0099] In some embodiments, the spacer domain can be modified to replace a spacer that lacks the ability to bind to FcR, such as the hinge region of CD8a, with an immunoglobulin Fc region. Alternatively, the Fc spacer region of the hinge can be deleted. Such replacements would reduce or eliminate Fc binding.

[0100] As used herein, the term "position" refers to a position in the sequence of a protein. Positions can be numbered sequentially or according to an established format, such as Kabat positions or EU positions or the EU index in Kabat. For all positions discussed herein, the numbering is according to the EU index or EU numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, which is hereby incorporated by reference in its entirety). The EU index or the EU index in Kabat or the EU numbering scheme refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, which is hereby incorporated by reference in its entirety). Although Kabat positions are also well known in the art, for a given position, it can vary from the EU position. For example, the S228P and L235E substitutions described above refer to EU positions. However, these substitutions can also correspond to Kabat positions 241 (S241P) and 248 (L248E).

[0101] Transmembrane and signaling domains

[0102] The intracellular signaling domain can comprise any suitable T cell receptor (TCR) complex signaling domain, or a portion thereof. In some embodiments, the intracellular signaling domain is derived from the CD3 complex. In some embodiments, the intracellular signaling domain is the TCR zeta chain (ζ-chain) signaling domain. In certain embodiments, the ζ-chain signaling domain can comprise a nucleotide sequence encoding the following amino acid sequence:

[0103]

[0104] The intracellular signaling domain can be associated with any suitable co-stimulatory domain, including but not limited to the 4-1BB co-stimulatory domain, the OX-40 co-stimulatory domain, the CD27 co-stimulatory domain, the CD28 co-stimulatory domain, the DAP10 co-stimulatory domain, the inducible co-stimulator (ICOS) domain, or the 2B4 co-stimulatory domain. According to the embodiments described herein, the CAR can comprise at least one co-stimulatory signaling domain. In one aspect, the CAR has a single co-stimulatory signaling domain, or it can comprise two or more co-stimulatory signaling domains, such as those described above. In another aspect, the co-stimulatory domain can consist of a single co-stimulatory domain, such as those co-stimulatory domains described above, or alternatively can consist of two or more portions of two or more co-stimulatory domains. Or, in some embodiments, the CAR does not comprise a co-stimulatory signaling domain. In one embodiment, the CAR comprises a co-stimulatory signaling domain that is the CD28 co-stimulatory domain. In this embodiment, such a modified CD28 co-stimulatory domain can have one or more amino acid substitutions or modifications, including but not limited to the substitution of leucine-leucine (LL) to glycine-glycine (GG). In certain embodiments, the modified co-stimulatory signaling domain region can comprise a nucleotide sequence encoding an amino acid sequence selected from the following:

[0105] RSKRSRGGHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S(SEQ ID NO:22)

[0106] One or more signaling domains can comprise a transmembrane domain selected from the group consisting of the CD28 transmembrane domain, the CD3 transmembrane domain, or any other suitable transmembrane domain known in the art. In some embodiments, the transmembrane domain is the CD28 transmembrane domain. In certain embodiments, the modified co-stimulatory signaling domain region can comprise a nucleotide sequence encoding an amino acid sequence selected from the following:

[0107] MFWVLVVVGG VLACYSLLVT VAFIIFWV(SEQ ID NO:23)

[0108] Expression of the CAR gene and transduction of T cells

[0109] In some embodiments, the CAR gene is part of an expression cassette. In some embodiments, outside of the CAR gene, the expression cassette may further comprise an accessory gene. When expressed by a T cell, the accessory gene can serve as a selection marker for the transduced T cell, a tracking marker in vitro, or a suicide gene for the transduced T cell.

[0110] In some embodiments, the accessory gene is a truncated EGFR gene (EGFRt). EGFRt can be used as a non-immunogenic selection tool (e.g., immunomagnetic selection using biotinylated cetuximab in combination with anti-biotin microbeads to enrich T cells that have been lentivirally transduced with a construct containing EGFRt), a tracking marker (e.g., flow cytometry analysis to track T cell engraftment), and a suicide gene (e.g., via the cetuximab / mediated antibody-dependent cell cytotoxicity (ADCC) pathway). Examples of truncated EGFR (EGFRt) genes that can be used according to the embodiments described herein are described in International Application No. PCT / US2010 / 055329, the subject matter of which is incorporated herein by reference as if fully set forth herein. In other embodiments, the accessory gene is a truncated CD19 gene (CD19t).

[0111] In another embodiment, the accessory gene is an inducible suicide gene. A suicide gene is a recombinant gene that causes a cell expressing the gene to undergo programmed cell death or antibody-mediated clearance at a desired time. In one embodiment, an inducible suicide gene that can be used as an accessory gene is the inducible caspase 9 gene (see Straathof et al. (2005). An inducible caspase 9 safety switch for T-cell therapy. Blood. June 1;105(11):4247–4254, the subject matter of which is incorporated herein by reference as if fully set forth herein).

[0112] In some embodiments, the expression cassette comprising the CAR gene described above can be inserted into a vector for delivery to target cells via transduction or transfection. Any suitable vector can be used, such as a bacterial vector, a viral vector, or a plasmid. In some embodiments, the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, a poxviral vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the vector can transduce a population of healthy immune cells, such as T cells. The successfully transduced or transfected target cells express one or more genes that are part of the expression cassette.

[0113] Thus, immune cell populations, such as T cells, can be transduced with a CAR gene, such as the CAR genes described above. The transduced T cells can be from a donor or can be from a subject having cancer and in need of cancer treatment. In some embodiments, the transduced T cells are used in adoptive immunotherapy treatment to treat cancer (residues in bold / underline denote substitutions). In some embodiments, the transduced T cells express a CAR gene encoding an amino acid sequence selected from SEQ ID NO: 24-27:

[0114] CD19R(L235E)28Z(SEQ ID NO:24):

[0115]

[0116] CD19R(N297Q)28Z(SEQ ID NO:25):

[0117]

[0118] CD19R(EQ)28Z(SEQ ID NO:26):

[0119]

[0120] CD19RCH2ΔCD28 Z(SEQ ID NO:27):

[0121]

[0122] In addition, one or more T cell populations can be part of a pharmaceutically acceptable composition for delivery for administration to a subject. In addition to the CAR transduced T cells, the pharmaceutically effective composition can comprise one or more pharmaceutically effective carriers. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that participates in carrying or transporting a treatment of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. Such carriers can include, for example, liquid, solid, or semi-solid fillers, solvents, surfactants, diluents, excipients, adjuvants, binders, buffers, solubilizing agents, solvents, encapsulating materials, chelating agents, dispersants, preservatives, lubricants, disintegrants, thickening agents, emulsifying agents, antimicrobial agents, antioxidants, stabilizers, colorants, or some combination thereof.

[0123] Each component of the carrier is "pharmaceutically acceptable" because it must be compatible with the other ingredients of the composition and must be suitable for contact with any tissue, organ, or part of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that outweighs its therapeutic benefit.

[0124] Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) natural polymers, such as gelatin, collagen, fibrin, fibrinogen, laminin, decorin, hyaluronic acid, alginate, and chitosan; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) diols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as trimethylene carbonate, ethyl oleate, and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid (or alginate); (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohols, such as ethanol and propanol; (20) phosphate buffer solutions; (21) thermoplastics, such as polylactic acid, polyglycolic acid; (22) polyesters, such as polycaprolactone; (23) self-assembling peptides; and (24) other non-toxic compatible substances employed in pharmaceutical formulations, such as acetone.

[0125] The pharmaceutical composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0126] In one embodiment, the pharmaceutically acceptable carrier is an aqueous carrier, such as buffered saline, etc. In certain embodiments, the pharmaceutically acceptable carrier is a polar solvent, such as acetone and alcohols.

[0127] The concentration of CAR-transduced T cells in these formulations can vary widely and will be selected based on the specific mode of administration chosen and the needs of the biological system, primarily based on fluid volume, viscosity, organ size, body weight, etc.

[0128] In certain embodiments, a population of T cells transduced with a CAR gene (i.e., CAR-transduced T cells) can be cultured in cell culture, such as those described herein for use in methods for targeting and killing cancer or tumor cells. In certain aspects of this embodiment, methods can be used in vitro or in a research setting to investigate the role of a particular cancer-associated antigen in cancer etiology, or to evaluate the targeting ability of a new CAR construct.

[0129] Treatment of cancer with CAR-transduced T cells

[0130] According to some embodiments, a CAR gene and a population of T cells transduced with the CAR gene, such as those described above, can be used in a method for treating cancer in a subject. Such methods can include the step of administering to the subject a therapeutically effective amount of at least one population of T cells transduced with at least one CAR gene. In these embodiments, the CAR-transduced T cell population expresses one or more CAR genes, such as those CAR genes described above. In certain embodiments, the T cells are transduced with and express a single mutant gene construct, such as the CD19R (L235E) or CD19R (N297Q) construct described herein, a double mutant gene construct having both the L235E and N297Q mutations (e.g., CD19R (EQ)) as described herein, or a deletion gene construct (e.g., CD19Rch2Δ) as described herein. When such cells are administered via adoptive immunotherapy treatment, the transduced T cells specifically target and lyse cells (i.e., cancer cells) expressing the cancer-associated antigen in vivo, thereby delivering their therapeutic effect of eliminating cancer cells.

[0131] Cancers that can be treated using a transduced T cell population can include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T cell lymphoma, embryonal tumors, central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, nasal glioma, Ewing sarcoma family of tumors extracranial germ cell tumor, extragonadal germ cell tumor extrahepatic bile duct cancer, eye cancer fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST) - see soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary midline tract carcinoma involving NUTGene), oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloma, multiple, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, malignant cancer of the paranasal sinuses and nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumors of intermediate differentiation, pineoblastoma, and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous carcinoma of the neck, gastric cancer, supratentorial primitive neuroectodermal tumor, T cell lymphoma, skin, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0132] One or more T cell populations transduced with one or more CAR genes that can be used according to the methods described herein can be administered alone or as part of a pharmaceutical composition by any suitable route of administration. The route of administration can refer to any route of administration known in the art, including but not limited to intracranial, parenteral, or percutaneous. "Parenteral" refers to a route of administration generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intralung, intraspinal, intrasternal, intrathecal, intratumoral, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. In certain embodiments, the transduced T cells are administered intravenously or intrathecally.

[0133] As used herein, the term "effective amount" refers to the amount of an agent, compound, treatment, or therapy that produces a desired effect. For example, a cell population can be contacted with an effective amount of an agent, compound, treatment, or therapy to study its in vitro effects (e.g., cell culture) or to produce a desired ex vivo or in vivo therapeutic effect. An effective amount of an agent, compound, treatment, or therapy can be used to produce a therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect. In such cases, the effective amount of the compound is a "therapeutically effective amount", "therapeutically effective concentration", or "therapeutically effective dose". The precise effective or therapeutically effective amount is the amount of the composition that produces the most effective result in terms of therapeutic efficacy in a given subject or cell population. This amount can vary with a variety of factors, including but not limited to the characteristics of the compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), or the physiological condition of the cells, the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. In addition, the effective or therapeutically effective amount can vary depending on whether the compound is administered alone or in combination with another compound, drug, therapy, or other treatment method or modality. Those skilled in the clinical and pharmacological arts will be able to determine the effective or therapeutically effective amount via routine experimentation, i.e., by monitoring the response of the cells or subject to the administration of the compound and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy, 21 st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005, which is incorporated herein by reference as if fully set forth herein. An agent, compound, treatment, or therapy that can be used in an effective or therapeutically effective amount in accordance with the embodiments described herein to produce a desired effect can include, but is not limited to, a CAR gene, an expression cassette comprising the CAR gene, a vector for delivering the expression cassette comprising the CAR gene to a target cell such as a T cell, and a population of T cells transduced with the CAR gene.

[0134] "Treatment / processing" of a condition can refer to preventing the condition, slowing the onset or rate of formation of the condition, reducing the risk of forming the condition, preventing or delaying the formation of symptoms associated with the condition, reducing or ending the symptoms associated with the condition, producing a complete or partial regression of the condition, or some combination thereof. Treatment / processing can also mean prophylactic or preventive treatment / processing of the condition.

[0135] As used herein, the term "subject" refers to a human or an animal, including all mammals such as primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows. In some embodiments, the subject is a human.

[0136] In certain embodiments, a method for treating cancer can include the step of administering a first population of T cells transduced with a first CAR gene in combination with a second population of T cells transduced with a second CAR gene, each in a therapeutically effective amount.

[0137] In other embodiments, the CAR-transduced T cells can be administered in combination with one or more additional anti-cancer therapies. As used herein, "in combination" or "in combination with" means that two or more agents, drugs, therapeutic agents, procedures, treatment regimens, treatment modalities, or combinations thereof are used in any order during the treatment of the same cancer in the same subject. This includes simultaneous administration, as well as sequential administration at time intervals up to several days apart. Such combination therapies can also include more than a single administration of any one or more of the agents, drugs, therapeutic agents, procedures, treatment regimens, and treatment modalities. In addition, two or more agents, drugs, therapeutic agents, procedures, treatment regimens, treatment modalities, or combinations thereof can be administered by the same or different routes of administration.

[0138] Additional anti-cancer therapies that can be used according to the methods described herein can include one or more anti-cancer procedures, treatment modalities, anti-cancer therapeutic agents, or combinations thereof. In some embodiments, the CAR-transduced T cells can be administered in combination with one or more anti-cancer procedures or treatment modalities, including but not limited to stem cell transplantation (e.g., using allogeneic stem cells, autologous stem cell bone marrow grafts or peripheral blood stem cell grafts; or non-myeloablative transplants), radiotherapy, or surgical resection. In other embodiments, the CAR-transduced T cells can be administered in combination with one or more anti-cancer therapeutic agents or drugs that can be used to treat cancer, including but not limited to chemotherapeutic agents and other anti-cancer drugs, immunotherapeutic agents, targeted therapeutic agents, or combinations thereof.

[0139] Chemotherapeutic agents and other anti-cancer drugs that can be co-administered with CAR-transduced T cells according to the embodiments described herein include, but are not limited to, all-trans retinoic acid (ATRA), arsenic trioxide, anthracyclines and their pharmaceutically acceptable salts (e.g., doxorubicin hydrochloride, daunorubicin hydrochloride, idarubicin, mitoxantrone), alkylating agents (e.g., cyclophosphamide, laromustine), anti-metabolite analogs (cytarabine, 6-thioguanine, 6-mercaptopurine, methotrexate), demethylating agents (e.g., decitabine, 5-azacytidine), nucleic acid synthesis inhibitors (e.g., hydroxyurea), topoisomerase inhibitors (e.g., etoposide), vinca alkaloids (e.g., vincristine sulfate), or combinations thereof (e.g., "ADE", which is a combination therapy comprising a combination of cytarabine (Ara-C), daunorubicin hydrochloride and etoposide).

[0140] Immunotherapeutic agents that can be co-administered with CAR-transduced T cells according to the embodiments described herein include, but are not limited to, immunomodulators (e.g., STAT3 inhibitors, lenalidomide) and therapeutic monoclonal antibodies. Therapeutic monoclonal antibodies can be designed to target one or more additional cancer-associated antigens.

[0141] Targeted therapeutic agents that can be co-administered with CAR-transduced T cells according to the embodiments described herein include, but are not limited to, tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, sunitinib), farnesyl transferase inhibitors (e.g., tipifarnib), FLT inhibitors, and c-Kit (or CD117) inhibitors (imatinib, dasatinib, nilotinib).

[0142] The following examples are intended to illustrate various embodiments of the invention. Accordingly, the specific embodiments discussed should not be construed as limiting the scope of the invention. For example, while the examples below relate to embodiments of a CAR targeting CD19, it should be appreciated that CARs can be generated to target any antigen. It will be apparent to those skilled in the art that various equivalent schemes, variations and modifications can be made without departing from the scope of the invention, and such equivalent embodiments are to be included herein. In addition, all references mentioned in the disclosure are incorporated by reference in their entirety as if fully set forth herein. Examples

[0143] Example 1: Incorporating mutations in the IgG4 Fc spacer region of a chimeric antigen receptor (CAR) avoids Fc receptor-mediated recognition and clearance of CAR T cells, resulting in improved T cell persistence and anti-tumor efficacy.

[0144] To determine whether cellular FcR-mediated interactions play a role in the immunological rejection and clearance, or even inadvertent activation, of adoptively transferred CAR-expressing T cells, mutations were made at one or both sites (L235E and / or N297Q) in the CH2 region of the IgG4 Fc spacer of a CD19-specific CAR – referred to herein as CD19R(L235E), CD19R(N297Q), or CD19R(EQ) – as well as a CD19-specific CAR with a CH2 deletion in its IgG4 Fc spacer – referred to herein as CD19Rch2Δ. T cells expressing these mutant CARs were then compared to T cells expressing a non-mutant CAR (CD19R) or only a truncated EGFR molecule (EGFRt) as a tracking marker for in vitro FcγR binding and CAR-mediated cytolytic activity, as well as in vivo engraftment and therapeutic efficacy (Wang et al. 2011). The results provide evidence that the elimination of cellular FcγR interactions improves the persistence and anti-tumor response of adoptively transferred CAR-expressing T cells.

[0145] Materials and methods

[0146] DNA constructs and lentiviral vectors. The CD19R28Z-T2A-EGFRt_epHIV7 lentiviral construct contains a) a chimeric antigen receptor (CAR) sequence consisting of the V H and V L gene segments of the CD19-specific FMC63 mAb, the transmembrane and cytoplasmic signaling domains of the co-stimulatory molecule CD28 containing the gg mutation that enhances chimeric receptor expression and function (Nguyen et al., 2003), and the cytoplasmic domain of the CD3ζ chain (Kowolik et al. 2006); b) a ribosome skipping T2A sequence (Szymczak et al., 2004) and c) a truncated EGFR sequence (W ang et al. 2011a). EGFRt-T2A-DHFR FS -T2A-IMPDH2 IY_epHIV7 lentiviral vectors. Site-directed mutagenesis of the codon-optimized CD19R28Z_pGA plasmid, which had been synthesized by Geneart, was performed using the QuikChange II XL kit (Agilent Technologies, Santa Clara, CA) to generate the CD19R(L235E)28Z-T2A-EGFRt_epHIV7, CD19R(N297Q)28Z-T2A-EGFRt_epHIV7, and CD19R(EQ)28Z-T2A-EGFRt_epHIV7 vectors, which were digested with NheI / RsrII and ligated to similarly digested CD19R28Z-T2A-EGFRt_epHIV7. The CD19Rch2Δ28Z-T2A-EGFRt_epHIV7 vector was generated from the codon-optimized CD19R-HL-CH3(CO)_pMK-RQ plasmid, which had been synthesized by Geneart, digested with NheI / RsrII, and ligated to similarly digested CD19R28Z-T2A-EGFRt_epHIV7.

[0147] Cell lines and maintenance. As described (Wang, 2011b), human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood obtained from discarded kits containing residual blood components of healthy donors undergoing apheresis at the City of Hope National Medical Center (COHNMC). Since this is de-identified discarded blood material, informed consent was waived under approval by the COHNMC Institutional Review Board (IRB protocol #09025) and the COHNMC Office of Human Subjects Protection. Then, T CM isolation (using CD14 and CD45RA depletion, followed by CD62L selection), anti-CD3 / CD28 bead stimulation, and lentivirus-mediated transduction were performed as previously described (Wang et al., 2012). In some cases, transduced T cells were immunomagnetically enriched for EGFRt expression as previously described (Wang et al., 2011a).

[0148] EBV-transformed lymphoblastoid cell lines (LCLs) and LCLs expressing OKT3 (LCL-OKT3) (Wang et al. 2011b) or ffLuc were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT), 2 mM L-glutamine (Irvine Scientific), and 25 mM HEPES (Irvine Scientific). + LCL cells. ffLuc+ LCLs were generated by transduction with the lentiviral vector eGFP-ffluc_epHIV7 at an MOI of 20 in the presence of 5 μg / mL polybrene in 500 μL of medium, and subsequent purification by sorting of GFP+ cells.

[0149] Mouse myeloma cells secreting human homeostatic IL-15 cytokine (NSO-IL15) were generated as previously described (Wang et al. 2011b).

[0150] SupB15 and K562 leukemia cell lines (ATCC) were cultured in the corresponding ATCC-recommended medium.

[0151] Antibodies and flow cytometry. Isotype controls, anti-CD3, anti-CD4, anti-CD8, anti-CD45, and streptavidin conjugated with fluorescent dyes were obtained from BD Biosciences (San Jose, CA). Biotinylated anti-Fc was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Generation of biotinylated cetuximab has been previously described (Wang et al. 2011a). Biotinylated huFcγR1, muFcγR1, huFcγR2a, huFcγR2b, and muFcγR2b were obtained from Sino Biological, Inc. (Beijing, P.R. China). The percentage of immunofluorescent cells was analyzed by the FACScalibur system (BD Biosciences), and the percentage of cells in the analyzed region was calculated using FCS Express V3 (De Novo Software, CA, USA).

[0152] In vivo T cell engraftment and therapy. All mouse experiments were approved by the COHNMC Institutional Animal Care and Use Committee. For engraftment studies, 6- to 10-week-old NOD / ScidIL-2RγC null (NSG) mice were injected intravenously (i.v.) on day 0 with 10 7 specified TCM derived cells, and intraperitoneally (i.p.) injected three times a week with 2 x 10 7 irradiated NS0-IL15 to provide systemic delivery of human IL-15 in vivo. Peripheral blood was harvested by retro-orbital bleeding, red blood cells were lysed, and the cell suspension was analyzed by flow cytometry. For the treatment study, 1.5 x 10 6 ffLuc + LCL cells were administered i.v. to 6-8 week-old NSG mice, and then 5 x 10 6 designated CAR+T CM derived cells were administered i.v. on day 3. Luciferase activity was measured by Xenogen imaging as previously described (Kahlon et al 2004).

[0153] Chromium release assay. A 4-hour 51 Cr-release assay was performed using the designated effector / target cell ratios as previously described (Stastny et al 2007).

[0154] Results

[0155] CD19R+ T cells cannot engraft in NSG mice. Central memory T cells (T CM ), a subset of T cells, have been characterized as having excellent engraftment potential and thus therapeutic efficacy after adoptive transfer (Wang et al 2011b). Further evidence has shown that CAR expression on T CM -derived cells appears to be associated with reduced in vivo persistence in an in vivo xenograft model using NSG mice. Studies described herein indicate that such a reduction in persistence was demonstrated in experiments comparing non-transduced T CM -derived cells with (i) T CM -derived cells lentivirally transduced to express both a CD19-specific CAR (CD19R) and a truncated EGFR (EGFRt) as a tracking marker, and (ii) T CM -derived cells lentivirally transduced to express only the EGFRt tracking marker on the cell surface ([[]] Figure 1 ). Examination of peripheral blood collected 7 and 14 days after i.v. administration of the cells to mice and staining with anti-human CD45 mAb allowed detection of non-transduced T CM -derived cells ([[]] Figure 1 c). However, after co-staining with the EGFRt tracking marker to detect gene-modified cells, it was evident that despite similar levels of transduction and / or EGFRt expression of the input cells ([[]] Figure 1b, 78 - 79% positive), significantly fewer cells engrafted in the peripheral blood of mice receiving CD19R / EGFRt + TCM compared to those receiving EGFRt + TCM( Figure 1 c, p < 0.0001, unpaired Student t - test was used to compare the percentage of CD45 / EGFRt + cells in each group on day 7 or day 14). Although low levels of T cells were detected in mice treated with CD19R / EGFRt + TCM, all persistent T cells were CAR - negative on days 7 and 14. This impaired in - vivo persistence was not related to lentiviral transduction of T cells as it was specific for cells transduced to express the CAR transgene but not the EGFRt transgene. Additionally, the lack of CD19 antigen in these NSG mice, and the fact that similar phenomena have been seen with T cells expressing CARs with different antigen specificities (data not shown) suggest that the lack of engraftment / persistence in the peripheral blood is antigen - independent.

[0156] HuFcγR binds to CD19R + T cells. The CD19R construct contains a CD19 - specific scFv derived from the murine monoclonal antibody FMC63, a human IgG4 Fc linker, a human CD28 transmembrane and cytoplasmic domain, and a human CD3 - zeta cytoplasmic domain. Since the CAR construct contains a portion of the human IgG4 Fc region, the propensity of FcR - mediated innate immune responses to selectively eliminate CD19R / EGFRt + cells, but not EGFRt + cells, was investigated. Indeed, binding assays using soluble human FcγR1 revealed that, in contrast to untransduced or T CM -derived cells expressing only EGFRt, those expressing CD19R exhibited binding of FcγR1 molecules at higher dilutions (lower titers). Figure 2 ) Notably, although NSG mice are immunodeficient, they are known to still have neutrophils and monocytes expressing FcR (Ishikawa et al. 2005; Ito et al. 2002), thus providing a potential rationale for the lack of CAR + T cell persistence observed in previous engraftment studies.

[0157] Generation of CD19R mutants. To further test the significance of the potential FcR - mediated effects on the population of CAR - expressing T CM cells, the CD19 - specific CAR was mutated at amino acids within the IgG4 CH2 domain that can participate in FcR binding (L235E and / or N297Q)( Figure 3 a). A CD19 - specific CAR with a deletion of the IgG4 CH2 domain (i.e., deletion of the domain containing residues 235 and 297) was also generated( Figure 3a). The resulting single mutants CD19R(L235E) and CD19R(N297Q), double mutant CD19R(EQ) (having both L235E and N297Q mutations), and the deleted CD19Rch2Δ sequence were incorporated into different lentiviral constructs, where the T2A ribosome skipping sequence was used in a design similar to the design described in Figure 1 a, and they were each co-expressed with EGFRt from a single transcript. After lentiviral transduction, immunomagnetic enrichment of cells expressing EGFRt, and single-round rapid amplification, each T CM -derived line was 92 - 99% positive for the expected transgene ( Figure 3 b), demonstrating that the mutations had no adverse effect on CAR expression. Additionally, in a 4-hour 51 Cr release assay, none of these mutations altered the CD19-specific cytolytic potential of these T CM -derived cells ( Figure 3 c).

[0158] Binding of huFcγR to CARs with mutated IgG4 spacers was impaired. To determine the potency by which different mutations / deletions in the CAR affected FcR binding, flow cytometry analysis was performed using various human and murine biotinylated soluble FcγRs, and PE-streptavidin (SA-PE) to detect the binding of FcγR to different cell populations. T cells expressing non-mutated CD19R were bound by human FcγR1, FcγR2a, and FcγR2b, as well as murine FcγR1 and FcγR2b ( Figure 4 ). In comparison, T cells expressing only EGFRt were not bound by these FcγRs, and T cells expressing the CD19R(N297Q), CD19R(L235E), or CD19R(EQ) mutants or the CD19Rch2Δ deletion all showed significantly reduced binding to these FcγRs.

[0159] T cells with CD19R mutants exhibited improved in vivo engraftment and persistence. To determine whether CD19R mutations or deletions that helped prevent FcγR binding would translate into increased in vivo persistence after adoptive transfer, 10 7 T cells expressing the parental CD19R, the EGFRt marker alone, CD19R(L235E), CD19R(N297Q), CD19R(EQ), or CD19Rch2Δ were infused i.v. into NSG mice. At 1 and 2 weeks, CD45 + EGFRt + cell engraftment was determined in peripheral blood ( Figure 5)。Implantable EGFRt+ cells can be detected when T cells express the single mutant CD19R (L235E) or CD19R (N297Q). In addition, the expression of the double point mutant CD19R (EQ) or CD19Rch2Δ with CH2 deletion rescued T cell engraftment, as the levels of CD45 / EGFRt+ cells observed in mice of these groups were similar to those seen when expressing EGFRt alone. Such rescued engraftment and persistence of the genetically modified cells were also observed using TCM-derived cells without EGFRt enrichment prior to adoptive transfer ( Figure 8 )。

[0160] T cells with CD19R mutants exhibited improved therapeutic efficacy. Based on the engraftment findings, the effects of CD19R (EQ) or CD19Rch2Δ on the CM antitumor efficacy of T-derived cells were compared. LCL is a tumor cell line expressing CD19, which was transduced to express firefly luciferase (ffLuc), allowing bioluminescent monitoring of in vivo tumor growth. Three days after i.v. administration of ffLuc+LCL to NSG mice, the mice were treated i.v. with PBS as a control or 5 x 10 6 T cells expressing non-mutant CD19R, the EGFRt marker alone, the double point mutant CD19R (EQ), or CD19Rch2Δ with CH2 deletion. The expression of CD19R (EQ) or CD19Rch2Δ on the CM T-derived cells led to significant control of tumor growth ( Figure 6 )。This efficacy was correlated with the presence / persistence of the genetically modified cells in peripheral blood on day 21 ( Figure 6 d). In fact, while all mice in the PBS, CD19R, and EGFRt control groups had to be euthanized on day 21, all mice in the CD19R (EQ) and CD19Rch2Δ groups survived 100 days ( Figure 6 e). Although these engraftment and efficacy studies focused on the TCM subgroup of T cells, these findings suggest that the positive benefit of IgG4 mutation in eliminating FcR interaction is independent of the engineered T cell population. In fact, the expression of CD19R (EQ) in a large number of PBMC-derived T cells, rather than in the TCM-derived lines, also led to improved antitumor efficacy and extended survival (p = 0.0295) ( Figure 7 )。

[0161] Discussion

[0162] Clinically, the in vivo therapeutic efficacy of adoptive T cell strategies is directly linked to engraftment and persistence after adoptive transfer (Heslop et al. 2003; Brenner & Heslop 2010). A variety of methods have been proposed to improve the persistence of transferred T cells, including host lymphodepletion prior to cell transfer (Gattinoni et al. 2005), cytokine support after cell transfer (recently reviewed in (Overwijk & Schluns 2009), and transfer using optimal T cell populations (Berger et al. 2008; Hinrichs et al. 2011; Yang et al. 2013; Gattinoni et al. 2011; Cieri et al. 2013). The studies described above provide further evidence that chimeric antigen receptor (CAR) design plays a major role in directing the engraftment and persistence of therapeutic cells. Previously, CAR design has been utilized to benefit engraftment and persistence of therapeutic cells by including costimulatory signaling domains in second- and third-generation CARs (see Cartellieri et al. 2010). However, as also suggested by the data above, the sequence used to link the ligand-binding domain of the CAR to the signaling domain (termed the spacer, hinge, and / or linker) has a previously unappreciated importance for in vivo therapeutic outcomes in murine models of malignant disease. Specifically, the use of an Ig Fc spacer was found to potentially inhibit the engraftment and / or persistence of CAR-expressing cells in the NSG mouse model in a manner associated with FcγR binding. Then, prevention of FcγR binding by point mutation or deletion of relevant sequences within the CAR Fc domain could restore the in vivo persistence of adoptively transferred cells to that of non-CAR-expressing cells. Then, the increased in vivo persistence mediated by spacer-optimized CARs translated into significantly improved CAR-directed antitumor therapy in in vivo murine models.

[0163] The immunological clearance of adoptively transferred T cells is not a new problem. For example, cellular immune rejection responses against the HyTK and NeoR selectable genes have been shown to coordinate with CAR expression (Berger et al. 2006; Jensen et al. 2010). However, the studies described above highlight the importance of FcR-mediated responses against CAR-expressing T cells for in vivo T cell persistence and antitumor efficacy. Thus, the studies also show a "dilemma" in avoiding such forms of immunogenicity, namely, incorporating mutations in CAR design to prevent FcγR recognition.

[0164] Based on these results, the mutations described herein can be extrapolated to humans, and thus the persistence and therapeutic efficacy of T cells expressing CARs containing IgG-spacers should be enhanced in humans. Any differences in CAR T cell engraftment and in vivo anti-tumor efficacy may be influenced by the nature of the murine NSG model system. Human IgG4 has been shown to efficiently bind murine FcRs to mediate potent antibody-dependent cell-mediated cytotoxicity (Isaacs et al., Steplewski et al., 1988). In contrast, human FcRs have the strongest affinity for IgG1 and IgG3 and reduced affinity for IgG4 (Schroeder & Cavacini, 2010; Nirula et al., 2011). Additionally, given that NSG mice lack serum antibodies, the FcRs expressed by their innate immune cells are unoccupied and thus have a greater potential to bind the IgG-Fc spacer within the CAR. Except in cases of hypogammaglobulinemia, immunocompetent humans have high serum IgG levels of approximately 10 mg / mL (Stoop et al., 1969), which can potentially compete for the recognition of IgG-containing CARs. Indeed, several groups have administered CAR T cells carrying IgG-Fc to humans, and in some cases, low levels of CAR T cells have been detectable by quantitative PCR for up to 6 weeks (Savoldo et al., 2011) and even 1 year (Till et al., 2012) after administration. Incorporation of the mutations described herein may further improve the persistence of these CAR T cells in humans.

[0165] Overall, the studies reported herein provide evidence that CARs containing Ig Fc spacer components should incorporate modifications that prevent FcR-mediated cellular recognition in vivo. Such modifications can involve point mutations that alter the amino acid sequence or sequence deletions, such as those seen with the CD19R(EQ) and CD19Rch2Δ constructs described herein. Such modifications would not only prevent the ability of FcR-expressing cells to recognize CAR-expressing immunotherapeutic cell products in vivo, but they can also prevent the inadvertent activation of transferred T cells and / or host immune responses (Hombach et al., 2010), which can contribute to various unwanted side effects of this immunotherapeutic strategy.

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[0221] Zhong, XS, Matsushita, M, Plotkin, J, Riviere, I, and Sadelain, M (2010). Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI3kinase / AKT / Bcl-XL activation and CD8+ T cell-mediated tumor eradication. Mol Ther 18:413-420. Sequence Listing <110> City of Hope <120> Chimeric Antigen Receptors (CARs) with Mutations in the Fc Spacer Region and Methods of Use Thereof <130> 40056-0009CN2 <140> PCT / US2014 / 028961 <141> 2014-03-14 <150> US 61 / 926,881 <151> 2014-01-13 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 107 <212> PRT <213> Artificial sequence <220> <223> CD19 receptor variable light chain antigen-binding domain <400> 1 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 100 105 <210> 2 <211> 120 <212> PRT <213> Artificial sequence <220> <223> CD19 receptor variable heavy chain antigen-binding domain <400> 2 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> CD123 Variable Heavy Chain Antigen Binding Domain <400> 3 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Ser Phe Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ser Ala Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu His Ile Asn Asp Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Ser Gly Gly Tyr Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 4 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> CD123 Variable Heavy Chain Antigen Binding Domain <400> 4 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 5 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> CD123 Variable Light Chain Antigen Binding Domain <400> 5 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CD123 variable light chain antigen binding domain <400> 6 Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Unmodified IgG1 CH1-CH2 Region <400> 7 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Ile Ser Lys 115 120 125 Ala Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 8 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Unmodified IgG1 CH1-CH2 Region <400> 8 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Met Leu Asp Lys Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 9 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Unmodified IgG1 CH1-CH2 Region <400> 9 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 10 <211> 217 <212> PRT <213> Artificial sequence <220> <223> Unmodified IgG1 CH1-CH2 region <400> 10 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 11 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Unmodified IgG1 hinge region <400> 11 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Unmodified IgG2 hinge region <400> 12 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 1 5 10 <210> 13 <211> 62 <212> PRT <213> Artificial sequence <220> <223> Unmodified IgG3 hinge region <400> 13 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55 60 <210> 14 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Unmodified IgG4 hinge region <400> 14 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10 <210> 15 <211> 229 <212> PRT <213> Artificial sequence <220> <223> IgG4 Fc region <400> 15 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 16 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> Modified IgG4 Fc Region - L235E Mutation <400> 16 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 17 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> Engineered IgG4 Fc Region - N297Q Mutation <400> 17 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 18 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> Modified IgG4 Fc Region - EQ Mutation <400> 18 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 19 <211> 229 <212> PRT <213> Artificial sequence <220> <223> Modified IgG4 Fc region - S228P+L235E+N297Q mutation <400> 19 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 20 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> IgG4 Spacer Domain - ch2delta Mutation / Deletion <400> 20 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser 1 5 10 15 Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 20 25 30 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 35 40 45 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 50 55 60 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 65 70 75 80 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 85 90 95 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 100 105 110 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 115 120 125 Lys <210> 21 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> T cell receptor zeta - chain signaling domain <400> 21 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 22 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Modified costimulatory signaling domain region <400> 22 Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 23 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Modified costimulatory signaling domain region <400> 23 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 1 5 10 15 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 24 <211> 680 <212> PRT <213> Artificial sequence <220> <223> Anti-CD19 CAR (CD19R(L235E)28Z) <400> 24 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser 275 280 285 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 290 295 300 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 305 310 315 320 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 370 375 380 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 385 390 395 400 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 405 410 415 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 420 425 430 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 435 440 445 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 450 455 460 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 465 470 475 480 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 485 490 495 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 500 505 510 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 515 520 525 Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 530 535 540 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 545 550 555 560 Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala 565 570 575 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 580 585 590 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 595 600 605 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 610 615 620 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 625 630 635 640 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 645 650 655 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 660 665 670 His Met Gln Ala Leu Pro Pro Arg 675 680 <210> 25 <211> 680 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD19 CAR (CD19R(N297Q)28Z) <400> 25 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 275 280 285 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 290 295 300 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 305 310 315 320 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 370 375 380 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 385 390 395 400 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 405 410 415 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 420 425 430 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 435 440 445 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 450 455 460 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 465 470 475 480 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 485 490 495 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 500 505 510 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 515 520 525 Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 530 535 540 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 545 550 555 560 Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala 565 570 575 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 580 585 590 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 595 600 605 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 610 615 620 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 625 630 635 640 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 645 650 655 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 660 665 670 His Met Gln Ala Leu Pro Pro Arg 675 680 <210> 26 <211> 680 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD19 CAR (CD19R(EQ)28Z) <400> 26 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser 275 280 285 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 290 295 300 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 305 310 315 320 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 370 375 380 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 385 390 395 400 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 405 410 415 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 420 425 430 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 435 440 445 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 450 455 460 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 465 470 475 480 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 485 490 495 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 500 505 510 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 515 520 525 Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 530 535 540 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 545 550 555 560 Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala 565 570 575 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 580 585 590 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 595 600 605 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 610 615 620 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 625 630 635 640 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 645 650 655 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 660 665 670 His Met Gln Ala Leu Pro Pro Arg 675 680 <210> 27 <211> 580 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD19 CAR (CD19RCH2?CD28Z) <400> 27 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser 275 280 285 Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 290 295 300 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 305 310 315 320 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345 350 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 355 360 365 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 370 375 380 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val 385 390 395 400 Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr 405 410 415 Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly 420 425 430 His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 435 440 445 Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 450 455 460 Ser Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 465 470 475 480 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 485 490 495 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 500 505 510 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 515 520 525 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 530 535 540 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 545 550 555 560 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 565 570 575 Leu Pro Pro Arg 580

Claims

1. A chimeric antigen receptor having impaired binding to an Fc receptor, comprising: an antigen recognition domain; a spacer domain of SEQ ID NO:19; a transmembrane domain; an intracellular signaling domain, wherein the intracellular signaling domain is a T cell receptor zeta chain signaling domain; and one or more co-stimulatory intracellular signaling domains derived from CD28, inducible co-stimulator, OX40, CD27, DAP10, 4-1BB, p56lck or 2B4.

2. The chimeric antigen receptor of claim 1, wherein the antigen recognition domain is a scFv.

3. The chimeric antigen receptor of claim 1 or 2, wherein the antigen recognition domain targets CD19.

4. The chimeric antigen receptor of any one of claims 1-2, wherein the chimeric antigen receptor is encoded by a nucleic acid sequence inserted into a viral vector.

5. The chimeric antigen receptor of claim 3, wherein the chimeric antigen receptor is encoded by a nucleic acid sequence inserted into a viral vector.

6. An isolated population of human immune cells transduced with a viral vector, the viral vector comprising an expression cassette comprising a chimeric antigen receptor gene, the gene comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-3, wherein the population of human immune cells expresses the chimeric antigen receptor gene.

7. An isolated population of human immune cells that expresses the chimeric antigen receptor of any one of claims 1-3.

8. An isolated population of human immune cells transduced with a chimeric antigen receptor gene for use in a method of treating cancer in a subject, wherein the chimeric antigen receptor gene comprises a nucleotide sequence encoding: an antigen recognition domain that targets a cancer-associated antigen specific to the cancer; a spacer domain of SEQ ID NO:19; a transmembrane domain; an intracellular signaling domain, wherein the intracellular signaling domain is a T cell receptor zeta chain signaling domain; and one or more co-stimulatory intracellular signaling domains derived from CD28, inducible co-stimulator, OX40, CD27, DAP10, 4-1BB, p56lck or 2B4.

9. The isolated population of human immune cells transduced with a chimeric antigen receptor gene of claim 8, wherein the antigen recognition domain is a scFv.

10. The isolated population of human immune cells transduced with a chimeric antigen receptor gene of claim 8 or 9, wherein the antigen recognition domain targets CD19.

11. The isolated population of human immune cells transduced with a chimeric antigen receptor gene of claim 8 or 9, wherein impaired binding to an Fc receptor results in improved persistence of the human immune cells compared to human immune cells transduced with a chimeric antigen receptor gene comprising a nucleotide sequence encoding a spacer domain derived from an unmodified immunoglobulin Fc region.

12. The isolated population of human immune cells transduced with a chimeric antigen receptor gene of claim 8 or 9, the method further comprising co-administering the isolated population of human immune cells transduced with the chimeric antigen receptor gene in combination with one or more anti-cancer therapies selected from stem cell transplantation, radiotherapy, surgical resection, chemotherapeutic agents, immunotherapeutic agents or combinations thereof.

13. An isolated population of human immune cells transduced with a chimeric antigen receptor gene according to claim 8 or 9, the method further comprising co-administering the isolated population of human immune cells transduced with the chimeric antigen receptor gene in combination with one or more anti-cancer therapies selected from stem cell transplantation, radiotherapy, surgical resection, targeted therapeutic agents, or combinations thereof.

14. Use of an isolated population of human immune cells transduced with a chimeric antigen receptor gene in the preparation of a medicament for treating cancer in a subject, wherein the chimeric antigen receptor gene comprises a nucleotide sequence encoding: an antigen recognition domain that targets a cancer-associated antigen specific to the cancer; a spacer domain of SEQ ID NO:19; a transmembrane domain; an intracellular signaling domain, wherein the intracellular signaling domain is a T cell receptor zeta chain signaling domain; and one or more co-stimulatory intracellular signaling domains derived from CD28, inducible co-stimulator, OX40, CD27, DAP10, 4-1BB, p56lck, or 2B4, and wherein the cancer is leukemia or B cell lymphoma.

15. Use according to claim 14, wherein the antigen recognition domain is a scFv.

16. Use according to claim 14, wherein the antigen recognition domain targets CD19.

17. Use according to claim 15, wherein the antigen recognition domain targets CD19.

18. Use according to any one of claims 14-17, wherein the leukemia is acute lymphoblastic leukemia or chronic myeloid leukemia.

19. Use according to any one of claims 14-17, wherein impaired binding to Fc receptors results in improved persistence of the human immune cells as compared to human immune cells transduced with a chimeric antigen receptor gene comprising a nucleotide sequence encoding a spacer domain derived from an unmodified immunoglobulin Fc region.

20. Use according to any one of claims 14-17, wherein the medicament is further combined with one or more anti-cancer therapies selected from stem cell transplantation, radiotherapy, surgical resection, chemotherapeutic agents, immunotherapeutic agents, or combinations thereof.

21. Use according to any one of claims 14-17, wherein the medicament is further combined with one or more anti-cancer therapies selected from stem cell transplantation, radiotherapy, surgical resection, targeted therapeutic agents, or combinations thereof.

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