An improved system for antigen targeting

Abstract

This invention relates to recombinant mammalian cells and their use in treating cancer. The present invention specifically relates to the co-expression of an adapter CAR and a conventional CAR in CAR T cells and the use of these cells in treating lymphoma. The invention also relates to pharmaceutical compositions comprising such recombinant mammalian cells, as well as their uses in cancer therapy.

Description

[0001] An Improved System for Antigen Targeting

[0002] FIELD OF THE INVENTION

[0003] This invention relates to recombinant mammalian cells and their use in treating cancer. The present invention specifically relates to the co-expression of an adapter CAR and a conventional CAR in CAR T cells and the use of these cells in treating lymphoma. The invention also relates to pharmaceutical compositions comprising such recombinant mammalian cells, as well as their uses in cancer therapy.

[0004] BACKGROUND

[0005] In recent years, chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B-lymphoid and plasma cell-derived malignancies in both pediatric and adult patients [1, 2], Especially, CAR T cells targeting CD19 have shown unprecedented response rates in clinical studies and real-world evidence [3]; however, relapses and treatment failures still occur. A major underlying mechanism is loss or downregulation of the target antigen on the tumor cell surface upon CAR T cell selective pressure [4], Indeed, antigen loss accounts for approximately a third of all relapses [5-7], highlighting the urgent need for strategies like the redirection of CAR T cells to alternative, co-expressed tumor targets in the event of antigen loss.

[0006] To overcome antigen escape, multi-antigen targeting using CAR T cells is an active field of research. This often involves the use of dual or tandem CAR designs, enabling CAR T cells to bind two different, predefined antigens either through the expression of two distinct CAR constructs or a single CAR with two binding moieties, respectively [8], The drawback of these approaches however is that the targeted antigens are predetermined and fixed. A more modular and flexible approach for multi-antigen targeting involves adapter CAR T cells [9], Instead of binding directly to the tumor antigen via the extracellular binding domain, adapter CAR T cells bind to a specific tag or region on an adapter molecule. The adapter molecule, in turn, binds the tumor antigen, thereby redirecting the CAR T cells to the target. By switching the adapter from one antigen specificity to another or by adding more than one adapter simultaneously, adapter CAR T cells can achieve modular antigen switching or multi-antigen targeting without the need to change the specificity of the CAR itself [10-15],

[0007] Here, the inventors established the previously described P329G adapter CAR platform [15, 16] for use in nonHodgkin lymphoma (NHL) and combine expression with conventional CAR constructs to improve multiantigen targeting of NHL. P329G CAR T cells are specific for the Fc-silencing P329G mutation of engineered human Ig G 1 , which act as the tumor specific adapter molecule and have shown potent activity against EGFR, MSLN and Her2 in preclinical models of mesothelioma, pancreatic and breast cancer

[0015] , However, when the inventors employed P329G CAR T cells in combination with CD19 and CD20-specific adapter antibodies to target NHL cells both in vitro and in vivo, they found them to be less efficient than conventional CAR T cells. The inventors aimed to optimize NHL dual targeting by combining efficient antitumor reactivity of conventional CAR T cells with flexible antigen targeting of P329G CAR T cells. To this end, the inventors generated dual CAR T cells co-expressing a conventional CAR and the P329G CAR. These novel dual CAR T cells demonstrated dual reactivity, functioning as both conventional and adapter CAR T cells. Dual CAR T cells showed improved NHL multi-antigen targeting compared to P329G CAR T cells and were able to eradicate antigen-heterogeneous lymphoma in vitro and in vivo, preventing the outgrowth of antigen-negative tumor cells. Moreover, conventional and adapter dual CAR T cells (which are also referred to herein as ConvAD CAR T cells or CAD CAR T cells), which co-express a conventional antigen-specific CAR and the P329G adapter CAR that leverages Fc-mutated antibodies for redirection, outperformed bispecific CAR T cells by eradicating heterogeneous lymphoma populations in the event of dual antigen loss, thereby providing a potent tool to counter antigen escape.

[0008] An additional benefit of co-expressing an adapter CAR is the potential ability to co-target both, tumor cells and immunosuppressive or tumor-promoting cells within the tumor microenvironment, thereby counteracting immune-evasive mechanisms of bystander cells. Moreover, careful titration strategies of the adapter could create therapeutic windows that allow targeting antigens expressed at low levels on healthy tissues, thereby reducing toxicity. These characteristics make ConvAD CAR T cells an equally promising option for solid tumors, where the influence of the tumor microenvironment, as well as antigen selection and heterogeneity are also major challenges for CAR T cell therapy.

[0009] Clinical experience in B cell malignancies has demonstrated that dual antigen targeting is an effective strategy to reduce the risk of antigen-negative tumor escape. However, clinical studies with CD19 / CD20 bispecific CAR T, CD19 / CD22 bispecific CAR T, or CD19 and CD22 sequential CAR T treatment have reported relapses that are associated with loss of both targets in some patients. Using in vitro and in vivo models, the inventors reflected these clinical observations by demonstrating that conventional bispecific CAR T cells are unable to control tumors that lost both targeted antigens. Crucially, they demonstrate that ConvAD CAR T cells can provide a distinct advantage over bispecific CAR T cells in such scenarios, as the secondary targets engaged by the AD CAR are not pre-determined. Through flexible triple antigen targeting, ConvAD CAR T cells achieved efficient tumor control of dual-antigen-negative tumors. While most ConvAD CAR-treated mice exhibited eventual tumor relapse, the inventors importantly did not observe triple antigen loss in 6 of 7 mice. Absence of detectable CAR T cells in the BM suggests that tumor progression was more likely driven by CAR T cell contraction rather than antigen loss. The single mouse that did experience triple antigen loss indicates that, in some cases, multispecific targeting needs to extend beyond three antigens. In conclusion, the inventors introduce the ConvAD CAR T cell platform as an effective and flexible multi-antigen-targeting strategy for lymphoma, to enhance CAR T cell therapy and reduce the risk of antigen-negative relapse.

[0010] The present invention comprises the following preferred embodiments:

[0011] 1 . A recombinant mammalian cell co-expressing at least:

[0012] (a) a CAR comprising a targeting domain binding to a cancer antigen expressed on the surface of cancer cells and

[0013] (b) a CAR comprising a targeting domain binding to an adapter molecule.

[0014] 2. The recombinant mammalian cell according to item 1 , wherein said recombinant mammalian cell is a human cell, preferably a human immune cell.

[0015] 3. The recombinant mammalian cell according to item 2, wherein the human immune cell is a lymphocyte.

[0016] 4. The recombinant mammalian cell according to item 3, wherein the lymphocyte is a B lymphocyte, a T lymphocyte, or a NK cell.

[0017] 5. The recombinant mammalian cell according to item 4, wherein the lymphocyte is a T lymphocyte, preferably wherein the lymphocyte is a CD4-positive T cell or a CD8-positive T cell.

[0018] 6. The recombinant mammalian cell according to any one of items 1-5, wherein the adapter molecule binds to cells of said cancer and is preferably a monoclonal antibody or antigen-binding fragment thereof binding to a cancer antigen of the cancer.

[0019] 7. The recombinant mammalian cell according to item 6, wherein the monoclonal antibody or antigenbinding fragment thereof carries a P329G mutation according to EU numbering and the targeting domain of the CAR according to (b) binds to antibodies carrying a P329G mutation according to EU numbering and preferably comprises an scFv containing, in an N- to C-terminal order: i) an anti-P329G variable heavy chain amino acid sequence according to SEQ ID NO: 4 or an anti-P329G variable heavy chain having the same CDRs as the amino acid sequence according to SEQ ID NO: 4, ii) an scFv linker preferably consisting of the amino acid sequence according to SEQ ID NO: 6, and ii) an anti-P329G variable light chain amino acid sequence according to SEQ ID NO: 8 or an anti-P329G variable light chain having the same CDRs as the amino acid sequence according to SEQ ID NO: 8. The recombinant mammalian cell of item 6 or 7, wherein the monoclonal antibody or antigen-binding fragment thereof binding to a cancer antigen of the cancer binds to the cancer antigen on the surface of cancer cells of the cancer. The recombinant mammalian cell of any one of items 1 -8, wherein the cancer antigen expressed on the surface of cancer cells, which is bound by the CAR according to (a), is selected from the group consisting of CD19, CD20, and R0R1. The recombinant mammalian cell of any one of items 1-9, wherein the CAR comprising the targeting domain binding to an adapter molecule has an lgG3-derived spacer between the transmembrane domain and the targeting domain, wherein the spacer preferably consists of SEQ ID NO: 57. A nucleic acid or a set of nucleic acids encoding the CARs as defined in any one of items 1-10. An expression vector comprising the nucleic acid of item 11 , or a set of expression vectors comprising the set of nucleic acids of item 11 . A method for producing a recombinant mammalian cell according to any one of items 1-10, the method comprising the steps of:

[0020] (I) providing a mammalian cell;

[0021] (II) introducing into said mammalian cell of step (I) the nucleic acid or set of nucleic acids according to item 11 or the expression vector or the set of expression vectors according to item 12; and

[0022] (III) co-expressing said nucleic acid or said set of nucleic acids according to item 11 or said expression vector or said set of expression vectors according item 12, thereby obtaining said recombinant mammalian cell. A pharmaceutical composition comprising the recombinant mammalian cell according to any one of items 1-10, the composition preferably further comprising one or more adapter molecules as defined in any one of items 1-8 and optionally further comprising a pharmaceutically acceptable carrier and / or excipient. A recombinant mammalian cell according to any one of items 1 -10 or a pharmaceutical composition according to item 14 for use as a medicament. A recombinant mammalian cell according to any one of items 1 -10 or a pharmaceutical composition according to item 14 for use in a method of treating a cancer. A recombinant mammalian cell or pharmaceutical composition for use according to item 16, wherein the cancer is lymphoma or a solid cancer. A recombinant mammalian cell or pharmaceutical composition for use according to item 17, wherein the cancer is non-Hodgkin lymphoma. A recombinant mammalian cell or pharmaceutical composition for use according to any one of items 15-18, wherein the recombinant mammalian cell or pharmaceutical composition is to be coadministered with one or more adapter molecules as defined in any one of items 6-8. A recombinant mammalian cell or pharmaceutical composition for use according to any one of items 15-19, wherein the one or more adapter molecules is / are administered before, after, or simultaneously with the recombinant mammalian cell. A kit comprising

[0023] (A) a recombinant mammalian cell and

[0024] (B) an adapter molecule, wherein the recombinant mammalian cell according to (A) and the adapter molecule according to (B) are as defined in any one of items 1-10. A kit comprising

[0025] (A) a nucleic acid, set of nucleic acids, an expression vector, or a set of expression vectors encoding the CARs as defined in any one of items 1-10 and

[0026] (B) the adapter molecule of the CARs as defined in any one of items 1-10. The recombinant mammalian cell or kit or pharmaceutical composition according to any one of the preceding items, wherein said recombinant mammalian cell is notan embryonic cell and has not been obtained by a process for modifying the germ line genetic identity of a human being. The kit or pharmaceutical composition according to any one of the preceding items, wherein the adapter molecule of the kit according to (A) or the one or more adapter molecules of the pharmaceutical composition is a single adapter molecule which binds to cells of said cancer and is a monoclonal antibody or antigen-binding fragment thereof, preferably according to item 7, that binds to a cancer antigen of the cancer which is preferably selected from CD19, CD20, and ROR1. The kit or pharmaceutical composition according to any one of items 1-23, wherein the kit comprises at least two adapter molecules according to (A) or the pharmaceutical composition comprises at least two adapter molecules, wherein the adapter molecules bind to cells of said cancer and are monoclonal antibodies or antigen-binding fragments thereof, preferably according to item 7, that bind to cancer antigens of the cancer which are preferably selected from CD19, CD20, and ROR1. The kit or pharmaceutical composition according to any one of items 1-23, wherein the kit comprises three adapter molecules according to (A) or the pharmaceutical composition comprises three adapter molecules, wherein the adapter molecules bind to cells of said cancer and are monoclonal antibodies or antigen-binding fragments thereof, preferably according to item 7, that bind to cancer antigens of the cancer which are preferably selected from CD19, CD20, and ROR1. The kit or pharmaceutical composition according to any one of items 24-26, wherein the monoclonal antibodies or antigen-binding fragments thereof bind to cancer antigens which are different from the cancer antigen expressed on the surface of cancer cells which is bound by the CAR according to (a). The method according to any one of the preceding items, wherein all the steps of the method are carried out in vitro. 29. The method according to any one of the preceding items, wherein said method does not comprise a method for treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body.

[0027] 30. The method according to any one of the preceding items, wherein said method does not comprise a process for modifying the germ line genetic identity of a human being.

[0028] Brief Description of the Drawings

[0029] Figure 1 shows the optimization and in vitro evaluation of P329G CAR T cells for NHL. (A) P329G CAR variants with different extracellular spacers. (B) Representative EGFRt expression on CAR T cells non-virally transduced with P329G CAR variants. (C) Cytolytic activity of CAR T cells with different spacer designs against NHL tumor cell lines in presence of increasing P329G Fc-mutated adapter IgG concentrations after 6 h. E:T ratio 5:1. (D) EC50 calculated from regressions in C. (E) Dose-response cytotoxicity assay of P329G CAR T cells against NHL cell lines after 24 h in combination with increasing combinations of a-CD19 (FMC63) and a-CD20 (GA101) adapter. E:T ratio 4:1. Data shown in C-E show the mean ± SEM of n=3 independent donors. Statistical analysis: (C) unpaired t-test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0030] Figure 2 shows the in vitro comparison of P329G CAR vs. conventional CAR T cells targeting CD19 or CD20. (A) Cytolytic activity of P329G CAR T cells + 1 nM a-CD19 or a-CD20 IgG and conventional CAR T cells against NHL tumor cell lines at E:T ratio 1 :1. (B) CD107a and intracellular IFN-y expression of CAR T cells upon 5 h co-culture with NHL cell lines at E:T ratio 1 :2. Adapter concentration 1 nM. (C) Scheme of constant antigen exposure (CAE) assay. (D) 24 h Cytotoxicity of P329G CAR T cells + 1 nM a-CD20 IgG and conventional CD20 CAR T cells before and after four cycles of stimulation. (E) Combined expression of CD223, CD279 and CD366 on CAR T cells during CAE assay. (F) Gene expression of CAR T cells after cycle 4 of CAE determined by Nanostring analysis. All data represented as mean ± SEM of n=3 independent donors. Statistical analysis: (A, C-E) unpaired t-test, (B) one way ANOVA with Dunnett's multiple comparisons test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0031] Figure 3 shows the bi-functional conventional and adapter dual (CAD) CAR T cells. (A) CAD CAR T cells coexpress a conventional CAR and the P329G CAR. (B) Construct design of the CD19 (FMC63) CAR and P329G CAR. (C) Representative EGFRt and LNGFRt expression of P329G CAR, CD19 CAR and CD19 CAD CAR T cells. (D) Left and middle: 24 h cytotoxicity assay of CD19 CAR T and CD19 CAD CAR T cells in absence of adapter. Right: 24 h cytotoxicity assay of P329G CAR T cells and CD19 CAD CAR T cells in presence of increasing concentrations of a-CD20 IgG against CD19- K562 expressing CD20. E:T ratio 4:1. Data represented as mean ± SEM of n=3 independent donors. (E) Scheme of Daudi xenograft model. NSG mice were engrafted with 5 x 105Daudi ffluc i.v. for 7 days and subsequently transfused with a total of 5 x 106CAR T cells (1 :1 mix of CD4+and CD8+). PBS or 5 g of a-CD20 (GA101) adapter was injected i.p. twice weekly. (F) In vivo tumor bioluminescence imaging blotted over time for individual mice. (G) Kaplan-Meier curves for mice reaching end point criteria. (H) Abundance of human T cells defined as hCD45+GFP- in bone marrow and kidney metastasis of mice analyzed at endpoint. (I) Surface expression of PD-1, TIM-3 and LAG- 3 on T cells isolated from bone marrow of mice at endpoint. (J) In vivo tumor bioluminescence imaging blotted over time for individual mice and (K) Kaplan-Meier curves for reaching endpoint criteria of mice treated with CD19 CAR T or CD19 CAD CAR T + a-CD20 IgG. Control groups in J and K are duplicated from F and G, respectively. Data in (H, I) represented as mean ± SEM. Statistical analysis: (H, I) unpaired t-test, (G, K) Logrank (Mantel-Cox) test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0032] Figure 4 shows CAD CAR T cells perform efficient dual targeting of NHL cells in vitro. (A) Co-culture of CAR T cells and Raji or JeKo-1 cells. In case of eradication of tumor cells after 1 day, CAR T cells were re-stimu- lated with CD19KO Raji or CD19KO JeKo-1, respectively. BLI signal normalized to dO and to d1 restim upon restimulation. E:T ratio 3:1, Adapter concentration 1 nM each. (B) Tumor cell lysis and percentage of IFN- y+TNF-a+CD19 CAD CAR T cells and P329G CAR T cells after 6 h of co-culture with increasing concentrations of adapter. E:T ratio 2:1. (C) Cytolytic activity after 24 h of CD19 CAD CAR T cells and P329G CAR T cells performing mono- or dual-targeting of tumor cells. E:T ratio 1 :2, adapter concentration 1 nM each. All data represented as mean ± SEM of n=3 independent donors. Statistical analysis: (B.C) unpaired t-test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 .

[0033] Figure 5 shows multi antigen targeting with R0R1 CAD CAR T cells. (A) R0R1 (R12) and P329G CAR construct used for generating R0R1 CAD CAR T cells. (B) Representative EGFRt and LNGFRt expression of P329G CAR, R0R1 CAR or R0R1 CAD CAR T cells. (C) 24 h cytotoxicity assay of R0R1 CAR T and R0R1 CAD CAR T cells in absence of adapter. (D) 24 h cytotoxicity assay of P329G CAR T cells and R0R1 CAD CAR T cells in presence of increasing concentrations of a-CD19 or a-CD20 IgG against R0R1- Daudi cells. E:T ratio 4:1. (E) Cytolytic activity of R0R1 CAD CAR T cells in presence or absence of 1 nM a-CD19 or a-CD20 IgG. E:T 1 :1. (F) CD107a and intracellular IFN-y expression of CAR T cells upon 5 h co-culture with NHL cell lines. E:T 1 :2, adapter concentration 1 nM. (G) Avidity measurements of CD8+R0R1 CAR T cells and R0R1 CAD CAR T cells to Raji tumor cells using the zMovi avidity analyzer. Data of n=1 donor. (H) R0R1 expression on a mixture of Daudi and Daudi R0R1 cells. (I) 24 h cytotoxicity assay of R0R1 CAR T cells and R0R1 CAD CAR T cells against a Daudi population with mixed R0R1 expression. E:T ratio 3:1, adapter concentration 1 nM. Data in (C-F) and (I) represented as mean ± SEM of n=3 independent donors. Statistical analysis: one way ANOVA with Dunnett's multiple comparisons test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 .

[0034] Figure 6 shows ROR1 CAD CAR T cells eradicate NHL tumor cells with heterogeneous antigen expression in vivo. (A) Treatment scheme of Daudi xenograft model with heterogeneous antigen expression. NSG mice were engrafted i.v. with 0.5 x 106Daudi ffluc (60% Daudi ROR1, 40% Daudi WT) cells for 3 days and subsequently transfused with 1x107CAR T cells (1 :1 mix of CD4+and CD8+). PBS or 5 g a-CD20 IgG were administered i.p. twice weekly. (B) Kaplan-Meier curves for mice reaching end point criteria. (C) In vivo tumor bioluminescence imaging blotted over time for individual mice. (D) Representative bioluminescence images. (E) BLI tumor signal at day 28 post T cell transfer. (F) ROR1 and CD20 expression on tumor cells isolated from bone marrow at endpoint. Dashed line indicates expression at input. Statistical analysis: (B) Log-rank (Mantel-Cox) test. (E, F) one way ANOVA with Dunnett's multiple comparisons test; *p<0.05, **p<0.01 , ***p<0.001, ****p<0.0001.

[0035] Figure 7: (A) CD19, CD20 and ROR1 expression on NHL tumor cell lines and K562 as negative control. (B) Flow cytometric estimation of phycoerythrin (PE) molecules per cell of NHL tumor cells stained with PE-la- beled antibodies against CD19, CD20 and ROR1 . (C) Activation assay of P329G CAR T cells with increasing amounts of soluble adapter antibodies. Coated anti-CD3 antibody (OKT3 (c)) served as positive control. Data represented as mean ± SEM of n=3 independent donors. Statistical analysis: unpaired t-test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0036] Figure 8 shows the bi-functional conventional and adapter dual (CAD) CAR T cells in vivo. (A) CD19 and CD20 expression of Daudi ffluc cells prior to engraftment in NSG mice. (B) Left: percentage of T cells (defined as hCD45+, GFP ) in peripheral blood of treated mice. Right: Percentage of T cells in bone marrow isolated when mice reached endpoint. (C) Expression of PD-1, TIM-3 and LAG-3 on CAR T cells prior to infusion into tumor bearing mice. (D) Expression of CD19 and CD20 on Daudi tumor cells in bone marrow or kidney metastasis at endpoint. Data represented as mean ± SEM of individual mice.

[0037] Figure 9: (A) IFN-y production after 24 h of CD19 CAD CAR T cells and P329G CAR T cells performing mono- or dual-targeting of NHL tumor cells. E:T ratio 1 :2. (B) Cytolytic activity of P329G CAR T cells with 4- 1 BB or CD28 co-stimulatory domains in combination with increasing concentrations of a-CD19 (FMC63) or a-CD20 (GA101) adapter antibodies. E:T ratio 4:1. (C) proliferation and (D) percentage of viable CFSE- labeled CAR T cells upon 72 h co-incubation with indicated tumor cells. E:T 1 :1, adapter concentration 1 nM. (E) fold expansion and expression of CD223, CD279 and CD366 of ConvROR1AD CAR T cells stimulated every 3 or 4 days with irradiated JeKo-1 tumor cells. E:T 7:1, initial CD4:CD8 ratio 1 :1, adapter concentration 1 nM. All data represented as mean ± SEM of n=3 independent donors. Statistical analysis: (B) unpaired t-test; (C,D) one way ANOVA with Dunnett's multiple comparisons test; (E) one way ANOVA with Sidak’s multiple comparisons test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0038] Figure 10 shows ROR1 CAD CAR T cells in vivo. (A) Expression of ROR1 and CD20 on the mixed population of Daudi ROR1 ffluc and Daudi ffluc angrafted into NSG mice. (B) Percentage of T cells (defined as hCD45+, GFPj in peripheral blood of treated mice. Data represented as mean ± SEM of individual mice. (C) Flow analysis data of the single ConvROR1AD20CAR T cell-treated mouse experiencing tumor outgrowth after initial clearance of heterogeneous Daudi tumor. Flow cytometry plots depicting tumor abundance and antigen expression in bone marrow at endpoint of experiment. Grey histograms depict isotype controls. This mouse did not reach endpoint criteria during the observational period.

[0039] Figure 11. Triple-targeting ConvAD CAR T cells overcome dual-antigen-negative lymphoma tumors that are resistant to conventional bispecific CAR T cells.

[0040] (A) ConvROR1CAR (R12), Conv19CAR (FMC63) and AD CAR constructs used for generating bispecific ConvROR1 / Conv19CAR and ConvROR1AD CAR T cells. (B) 6 h cytotoxicity assay of CAR T cells against JeKo- 1 ffluc cells with single or double knock-out of ROR1 and CD19. E:T 2:1, adapter concentration 1 nM each. (C) Sequential co-culture of CAR T cells and JeKo-1 or Daudi ROR1 cells. In case of eradication of tumor cells, CAR T cells were first re-stimulated with indicated ROR1- tumor on day 1 and upon repeated eradication with ROR1 CD19- tumor cells on day 2. BLI signal was normalized to day 0, and upon each re-stimulation on day 1 and day 2. E:T ratio 5:1 , adapter concentration 1 nM each. (D) 24 h cytotoxicity assay of CAR T cells against a JeKo-1 ffluc-GFP population with heterogeneous ROR1 and CD19 expression. ROR1+CD19+CD20+JeKo-1 were labeled with CellTrace™ violet (CTV), ROR1KOCD19+CD20+JeKo-1 were labeled with eFluor™ 670 (eF670) and mixed 1 :1 :1 with non-labeled ROR1KOCD19KOCD20+JeKo-1. E:T ratio 2:1, adapter concentration 1 nM each. (E) Representative flow cytometry blots for CTV and eF670 for n=1 donor. (F) Treatment scheme of JeKo-1 MCL xenograft model with heterogeneous antigen expression. NSG mice were engrafted i.v. with 0.5 x 106JeKo-1 ffluc (1 :1 :1 mix of JeKo-1, JeKo-1 ROR1KOand JeKo-1 ROR1KOCD19KO) cells on day -7 and subsequently transfused with 10x106CAR T cells (1 :1 mix of CD4+and CD8+) on day 0. PBS or 5 g each adapter were administered i.p. twice weekly (b.i.w.). (G) Antigen expression of JeKo-1 ffluc mixture before i.v. injection (day -7). (H) In vivo tumor bioluminescence signal over time for individual mice. (I) Tumor BLI signal of each treatment group at peak of response. (J) Kaplan-Meier curves for mice reaching end point criteria. (K) R0R1, CD19 and CD20 expression on tumor cells isolated from BM at endpoint. (L) Tan19 / 2° CAR (FMC63 / Leu16), Conv19CAR (FMC63), Conv20CAR (Leu16) and AD CAR constructs used for generating bispecific Conv19 / Conv20CAR, bispecific Tan19 / 2° CAR, and Conv19AD CAR T cells. (M) 6 h cytotoxicity assay of CAR T cells against JeKo-1 ffluc cells with single CD19K0or double CD19KOCD20KO. For triple targeting, a-CD20 (GA101) and a-R0R1 (R12) adapter were used. E:T 4:1, adapter concentration 1 nM each. (N) 6 h cytotoxicity assay of CAR T cells against K562 ffluc expressing CD19, CD20 or R0R1. E:T 4:1, adapter concentration 1 nM each. (0) Co-culture of CAR T cells and JeKo-1 cells. In case of eradication of tumor cells, CAR T cells were re-stimulated with JeKo-1 CD19KOCD20KOon day 1. BLI signal normalized to day 0 and upon restimulation on day 1 . E:T ratio 5: 1 , adapter concentration 1 nM each. Data in (B-D) and (M- N) represented as mean ± SEM of n=3 independent donors. Data in (I, K) represented as mean ± SEM of individual mice. Statistical analysis: (B, D, K, M-0) one way ANOVA with Dunnett's multiple comparisons test; (J) Log-rank (Mantel-Cox) test; (I) unpaired t-test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0041] Figure 12. Analysis of CAR T cells and tumor cell lines for comparison of ConvAD CAR T cells and bispecific CAR T cells. (A) Expression of transduction markers EGFRt, LNGFRt and Her2t on CD8+CAR T cells used for comparing ConvROR1AD CAR T cells to bispecific dual ConvROR1 / Conv19CAR T cells. (B) Delta median fluorescence intensity (AMFI) of CAR T cells stained with anti-FMC63 mAb, recombinant hR0R1-Fc and subsequent labeling with anti-Fc mAb, or AF647-labeled adapter IgG to determine surface expression of respective CAR binders. Normalized to unstained controls. (C) Expression of CD19, CD20 and R0R1 of JeKo-1 ffluc and respective KO versions. Grey histograms depict isotype controls. (D) Percentage of human T cells (characterized as GFP hCD45+) in peripheral blood of mice at indicated time points. (E) Percentage of human T cells in BM of treated mice analyzed at endpoint. (F) Tumor antigen expression in the bone marrow of a single ConvROR1AD19+2°-treated mouse that experienced R0R1 CD19 CD20- relapse after initial complete response. Grey histograms represent isotypes. This mouse did not reach endpoint criteria during the observation time of 84 days. (G) Expression of transduction markers EGFRt, LNGFRt and Her2t on CD8+CAR T cells used for comparing Conv19AD CAR T cells to bispecific dual Conv19 / Conv20and Tan19+2° CAR T cells. Data in (A, B, F) represented as mean ± SEM of n=3 donors. Data in (D, E) represented as mean ± SEM of treated mice. Statistical analysis: (D, E) one way ANOVA with Dunnett's multiple comparisons test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 . DETAILED DESCRIPTION OF THE INVENTION

[0042] Unless otherwise defined below, the terms used in the present invention shall be understood in accordance with their common meaning known to the person skilled in the art. Literature references referred to herein may be cited by using the full reference, or by using an abbreviation such as a number, for instance, “

[0015] ”, and by specifying the corresponding full reference in the “References” section. All literature reference referred to herein are incorporated by reference for all purposes in their entirety.

[0043] Definitions and General Techniques

[0044] Where, in the context of the present invention, reference is made to a method comprising particular method steps (e.g., steps (I), (II), and (III)), it is to be understood that the steps are to be carried out in their indicated order, i.e., in the order in which they have been listed.

[0045] A “recombinant mammalian cell” according to the invention can be any cell as defined herein. Preferably, a recombinant mammalian cell is an isolated cell. Recombinant mammalian cells according to the invention can be produced in accordance with known pharmaceutical standards. For instance, they can be formulated for administration to humans.

[0046] As used in connection with the invention, the meaning of the terms “co-expressing” or “co-express” in relation to the mammalian cells of the invention is in accordance with the common meaning of these terms and encompasses particularly the expression of two elements (e.g., proteins such as the CARs (a) and (b) as referred to herein) in the same mammalian cell.

[0047] In accordance with the invention, the term “chimeric antigen receptor (CAR)” has the meaning known in the art. A “CAR” according to the invention can be in any possible form.

[0048] The term “binding” as used herein refers to the capability to form a complex with a molecule that is to be bound (e.g. CD19, CD20, or R0R1 or the adapter molecule referred to herein). Binding typically occurs non- covalently by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces and is typically reversible. Various methods and assays to determine binding capability are known in the art. Binding is usually specific binding. Binding is usually a binding with high affinity, wherein the affinity as measured in KD values is preferably is less than 1 pM, more preferably less than 100 nM, even more preferably less than 10 nM, even more preferably less than 1 nM, even more preferably less than 100 pM, even more preferably less than 10 pM, even more preferably less than 1 pM.

[0049] As used herein, the meaning of the phrase “binds to antibodies carrying a P329G mutation according to EU numbering” in connection with a targeting domain of a CAR in accordance with the invention will be understood in accordance with the common meaning of the terms as known in the art. The meaning of this phrase preferably encompasses targeting domains which bind to various different antibodies (e.g., targeting domains which bind to antibodies to different cancer antigens) carrying the P329G mutation, i.e., targeting domains which are not specific for a particular antigen-binding domain of the antibodies carrying the P329G mutation.

[0050] Targeting domains of CARs are known in the art and typically comprise scFvs binding to the respective antigen, e.g., an scFv binding to the adapter molecule (in case of the CAR referred to in claim 1 (b) and claims 2-10) and / or an scFv binding to the cancer antigen expressed on the surface of cancer cells (in case of the CAR referred to in claim 1 (a) and claims 2-10).

[0051] “Human immune cells” as used in the invention are not particularly limited and include, for example, T cells, B cells, NK cells, NKT cells, Peripheral Blood Mononuclear Cell (PMBCs) macrophages and stem cells. In a preferred embodiment, the T cells are CD8+ T cells or CD4+ T cells.

[0052] A “monoclonal antibody” is an antibody from an essentially homogenous population of antibodies, wherein the antibodies are substantially identical in sequence (i.e., identical except for minor fraction of antibodies containing naturally occurring sequence modifications such as amino acid modifications at their N- and C- termini). Unlike polyclonal antibodies which contain a mixture of different antibodies directed to either a single epitope or to numerous different epitopes, monoclonal antibodies are directed to the same epitope and are therefore highly specific. The term “monoclonal antibody” includes (but is not limited to) antibodies which are obtained from a monoclonal cell population derived from a single cell clone, as for instance the antibodies generated by the hybridoma method described in Kohler and Milstein (Nature, 1975 Aug 7;256(5517):495-7) or Harlow and Lane (“Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988). A monoclonal antibody may also be obtained from other suitable methods, including phage display techniques such as those described in Clackson et al. (Nature. 1991 Aug 15;352(6336):624- 8) or Marks et al. (J Mol Biol. 1991 Dec 5;222(3):581 -97). A monoclonal antibody may be an antibody that has been optimized for antigen-binding properties such as decreased Kd values, optimized association and dissociation kinetics by methods known in the art. For instance, Kd values may be optimized by display methods including phage display, resulting in affinity-matured monoclonal antibodies. The term “monoclonal antibody” is not limited to antibody sequences from particular species of origin or from one single species of origin. Thus, the meaning of the term “monoclonal antibody” encompasses chimeric monoclonal antibodies such as humanized monoclonal antibodies and human antibodies. The nomenclature of the antibodies, antibody fragments and mutations thereof follow the terms as known in the art. The annotation and numbering of the CDRs of antibodies is preferably performed in accordance with the Kabat nomenclature or the EU nomenclature.

[0053] In accordance with the methods for obtaining a recombinant mammalian cell of the invention, the step of introducing the nucleic acid or set of nucleic acids or expression vector or set of expression vectors into an mammalian cell may be performed by using any appropriate standard techniques as known in the art, for example, electroporation, electro-injection, microinjection, calcium phosphate co-precipitation, a calcium chloride / rubidium chloride method, retroviral and lentiviral infection, DEAE-dextran, a cationic liposome method, polyethylene glycol-mediated uptake, gene guns, etc., but is not limited thereto.

[0054] The term “expression vector” or “vector” is known in the art and encompasses, for instance, a plasmid, a viral vector such as a retroviral vector, a lentiviral vector, an adeno-virus vector, an adeno-associated virus vector, or a transposon vector. It is understood that a vector as used in connection with the present invention is a vector which is suitable for therapeutic applications in humans.

[0055] Methods of Treatments and Uses of Lymphocytes in such Treatments

[0056] Terms such as “treatment of cancer” or “treating cancer” according to the present invention refer preferably to a therapeutic treatment. An assessment of whether or not a therapeutic treatment works can, for instance, be made by assessing whether the treatment inhibits cancer growth in the treated patient or patients. Preferably, the inhibition is statistically significant as assessed by appropriate statistical tests which are known in the art. Inhibition of cancer growth may be assessed by comparing cancer growth in a group of patients treated in accordance with the present invention to a control group of untreated patients, or by comparing a group of patients that receive a standard cancer treatment of the art plus a treatment according to the invention with a control group of patients that only receive a standard cancer treatment of the art. Such studies for assessing the inhibition of cancer growth are designed in accordance with accepted standards for clinical studies, e.g. double-blinded, randomized studies with sufficient statistical power. The term “treating cancer” includes an inhibition of cancer growth where the cancer growth is inhibited partially (i.e. where the cancer growth in the patient is delayed compared to the control group of patients), an inhibition where the cancer growth is inhibited completely (i.e. where the cancer growth in the patient is stopped), and an inhibition where cancer growth is reversed (i.e the cancer shrinks). An assessment of whether or not a therapeutic treatment works can be made based on known clinical indicators of cancer progression. A treatment of cancer according to the present invention does not exclude that additional or secondary therapeutic benefits also occur in patients. However, it is understood that the primary treatment for which protection is sought is for treating the cancer itself, and any secondary or additional effects only reflect optional, additional advantages of the treatment of cancer growth. The treatment of cancer according to the invention can be a first-line therapy, a second-line therapy, a third-line therapy, or a fourth-line therapy. The treatment can also be a therapy that is beyond fourth-line therapy. The meaning of these terms is known in the art and in accordance with the terminology that is commonly used by the US National Cancer Institute.

[0057] A pharmaceutically acceptable carrier, including any suitable diluent or, can be used herein as known in the art. As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, European Pharmacopia or other generally recognized pharmacopia for use in mammals, and more particularly in humans. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. It will be understood that the formulation will be appropriately adapted to suit the mode of administration and the ingredients of the pharmaceutical composition or formulation (e.g., the recombinant mammalian cell of the invention).

[0058] Compositions and formulations in accordance with the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions and formulations. For instance, the compositions and formulations are prepared in a way that they can be stored and administered appropriately, e.g., by using pharmaceutically acceptable components such as carriers, excipients or stabilizers. Such pharmaceutically acceptable components are not toxic in the amounts used when administering the pharmaceutical composition or formulation to a patient. The pharmaceutical acceptable components added to the pharmaceutical compositions or formulations may depend on the ingredients present in the composition or formulation, the particular intended use of the pharmaceutical compositions and the route of administration.

[0059] In a preferred embodiment in accordance with the invention, the pharmaceutical composition or formulation is suitable for administration to humans, preferably the formulation is sterile and / or non-pyrogenic.

[0060] In accordance with the present invention, each occurrence of the term “comprising” or “comprises” may optionally be substituted with the term “consisting of” or “consists of”.

[0061] Sequences Preferred amino acid and DNA sequences in accordance with the invention can be independently selected from the following sequences. The sequences are represented in an N-terminal to C-terminal order; and they are represented in the one-letter amino acid code. Note that asterisks in the amino acid sequences shown below denote the end of the respective amino acid sequence (which is effected by a stop codon in the coding sequence).

[0062] Preferred embodiments of CARs according to claim 1 (b) including CARs which bind to antibodies carrying a P329G mutation (“P329G CARs”) are exemplified in 1.-6. below:

[0063] Note that the signaling peptides which are specified for the CARs below are merely optional (i.e., they may be present or absent). If present, the signaling peptides can be the signaling peptides which are specified for the CARs below, or they can be any signaling peptide suitable for the expression of CARs in the recombinant mammalian cells of the invention. Suitable signaling peptides are known in the art.

[0064] Also note that the transduction markers which are specified for the CARs below are merely optional (i.e., they may be present or absent). If present, the transduction markers can be the transduction markers which are specified for the CARs below, or they can be any transduction marker polypeptide suitable for use in the expression of CARs in the recombinant mammalian cells of the invention. Suitable transduction markers are known in the art.

[0065] Nucleotide and amino acids sequences used in connection with the invention, which represent preferred sequences in accordance with the invention, are as follows:

[0066] 1 . aP329G_lgG4short_CD28tm_41 BB_CD3z_EGFRt

[0067] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 1): ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA TCCCT

[0068] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2): MLLLVTSLLLCELPHPAFLLIP

[0069] Nucleotide sequence of anti-P329G scFv Variable heavy chain (SEQ ID NO: 3): ATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAAGCT

[0070] GAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGGCAGG

[0071] CCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCATCAACT

[0072] ACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACACCCTGT

[0073] ACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAGGCCCT

[0074] ACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0075] Amino Acid sequence of anti-P329G scFv Variable heavy chain (SEQ ID NO: 4):

[0076] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0077] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSA

[0078] Nucleotide sequence of G4S(4): linker (SEQ ID NO: 5):

[0079] GGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGGGCGGA

[0080] TOT

[0081] Amino Acid sequence of G4S(4): linker (SEQ ID NO: 6):

[0082] GGGGSGGGGSGGGGSGGGGS

[0083] Nucleotide sequence of anti-P329G scFv variable light chain (SEQ ID NO: 7):

[0084] CAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACCCT

[0085] GACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGG

[0086] AGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGGCCCCCGGC

[0087] GTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACCATCACCGG

[0088] CGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCACTGGGT

[0089] GTTCGGCTGTGGCACCAAGCTGACCGTGCTG

[0090] Amino Acid sequence of anti-P329G scFv variable light chain (SEQ ID NO: 8):

[0091] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPAR

[0092] FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHVWFGCGTKLTVL

[0093] Nucleotide sequence of lgG4 hinge (short) (SEQ ID NO: 9):

[0094] GAGTCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT

[0095] Amino Acid sequence of lgG4 hinge (short) (SEQ ID NO: 10):

[0096] ESKYGPPCPPCP Nucleotide sequence of CD28 transmembrane domain (SEQ ID NO: 11):

[0097] ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACC

[0098] GTGGCCTTCATCATCTTTTGGGTG

[0099] Amino Acid sequence of CD28 transmembrane domain (SEQ ID NO: 12): MFVWLVWGGVLACYSLLVTVAFIIFVW

[0100] Nucleotide sequence of 4-1 BB domain (SEQ ID NO: 13):

[0101] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAAC

[0102] TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA CTG

[0103] Amino Acid sequence of 4-1 BB domain (SEQ ID NO: 14):

[0104] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0105] Nucleotide sequence of CD3z domain (SEQ ID NO: 15):

[0106] CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCT

[0107] GTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAG

[0108] GCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTAT

[0109] AACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGA

[0110] GCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGG

[0111] ATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG

[0112] Amino Acid sequence of CD3z domain (SEQ ID NO: 16):

[0113] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[0114] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0115] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 17):

[0116] CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAA TCCCGGCCCTAGG

[0117] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18): LEGGGEGRGSLLTCGDVEENPGPR (not synthesized by ribosome) Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 19):

[0118] ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGAT CCCA

[0119] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2): MLLLVTSLLLCELPHPAFLLIP

[0120] Nucleotide sequence of EGFRt transduction marker (SEQ ID NO: 21):

[0121] CGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTAC

[0122] GAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTG

[0123] GCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCT

[0124] GAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACG

[0125] GACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGT

[0126] TTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATA

[0127] AGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTG

[0128] GAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAAC

[0129] AGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGG

[0130] CCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGG

[0131] ACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATAC

[0132] AGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAG

[0133] ACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGG

[0134] CAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGT

[0135] GCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTC

[0136] CAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGC

[0137] TGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0138] Amino Acid sequence of EGFRt transduction marker (SEQ ID NO: 22):

[0139] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI

[0140] TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKN

[0141] LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR

[0142] GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT

[0143] CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LVVALGIGLFM* Complete nucleotide sequence of aP329G_lgG4short_CD28tm_41 BB_CD3z_EGFRt (Underlined is CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 20):

[0144] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0145] TCCCTATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTG

[0146] AAGCTGAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGG

[0147] CAGGCCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCAT

[0148] CAACTACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACAC

[0149] CCTGTACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAG

[0150] GCCCTACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGA

[0151] GCGCCGGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGG

[0152] GCGGATCTCAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACC

[0153] GTGACCCTGACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTG

[0154] GGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGG

[0155] CCCCCGGCGTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACC

[0156] ATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAAC

[0157] CACTGGGTGTTCGGCTGTGGCACCAAGCTGACCGTGCTGGAGTCTAAGTACGGACCGCCC

[0158] TGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTAC

[0159] AGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCC

[0160] TGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGT

[0161] AGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGA

[0162] AGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTG

[0163] GGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGG

[0164] CGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAA

[0165] GATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGC

[0166] CACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCAC

[0167] ATGCAGGCCCTGCCCCCAAGGCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAAC

[0168] ATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCT

[0169] GCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATA

[0170] GGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAAC

[0171] TGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCA

[0172] CACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACA

[0173] GGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACC

[0174] TAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCT GAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTT

[0175] CAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCC GGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAG

[0176] GTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGT

[0177] CTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGG

[0178] TGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCC

[0179] TCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCA

[0180] CTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAA

[0181] CACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTG

[0182] CACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCC

[0183] GTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGA TCGGCCTCTTCATGTGA

[0184] Complete CAR amino acid sequence of aP329G_lgG4short_CD28tm_41 BB_CD3z_EGFRt

[0185] (Beginning of scFv to end of CD3z) (SEQ ID NO: 23):

[0186] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0187] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSAGGGGSGG

[0188] GGSGGGGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGL

[0189] IGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGCGTKLTVLESKY

[0190] GPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGC

[0191] SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG

[0192] KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL

[0193] PPR

[0194] 2. aP329G_lgG4long_CD28tm_41 BB_CD3z_EGFRt

[0195] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 1):

[0196] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0197] TCCCT

[0198] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2):

[0199] MLLLVTSLLLCELPHPAFLLIP

[0200] Nucleotide sequence of anti-P329G scFv Variable heavy chain (SEQ ID NO: 3):

[0201] ATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAAGCT

[0202] GAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGGCAGG

[0203] CCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCATCAACT

[0204] ACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACACCCTGT

[0205] ACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAGGCCCT

[0206] ACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0207] Amino Acid sequence of anti-P329G scFv Variable heavy chain (SEQ ID NO: 4):

[0208] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0209] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSA

[0210] Nucleotide sequence of G4S(4) linker (SEQ ID NO: 5):

[0211] GGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGGGCGGA

[0212] TOT

[0213] Amino Acid sequence of G4S(4) linker (SEQ ID NO: 6):

[0214] GGGGSGGGGSGGGGSGGGGS

[0215] Nucleotide sequence of anti-P329G scFv variable light chain (SEQ ID NO: 7):

[0216] CAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACCCT

[0217] GACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGG

[0218] AGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGGCCCCCGGC

[0219] GTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACCATCACCGG CGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCACTGGGT GTTCGGCTGTGGCACCAAGCTGACCGTGCTG

[0220] Amino Acid sequence of anti-P329G scFv variable light chain (SEQ ID NO: 8):

[0221] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPAR

[0222] FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHVWFGCGTKLTVL

[0223] Nucleotide sequence of lgG4 hinge (long) (SEQ ID NO: 24):

[0224] GAGTCTAAGTACGGACCGCCTTGTCCTCCTTGTCCAGCTCCTCCTGTGGCCGGACCTAGC

[0225] GTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTG

[0226] ACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTG

[0227] GACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCCAGAGCAC

[0228] CTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTA

[0229] CAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGC

[0230] CAAGGGCCAGCCTCGCGAGCCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGAC

[0231] CAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCAGCGACATTGCCGT

[0232] GGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGA

[0233] CAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGA

[0234] AGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA GTCCCTGTCTCTGAGCCTGGGCAAG

[0235] Amino Acid sequence of lgG4 hinge (long) (SEQ ID NO: 25):

[0236] ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGV

[0237] EVHNAKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE

[0238] PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0239] RLTVDKS RWQ EG N VFSCS VM H EALH N H YTQ KS LS LS LG K

[0240] Nucleotide sequence of CD28 transmembrane domain (SEQ ID NO: 26):

[0241] ATGTTCTGGGTGCTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTACAGCCTGCTCGTGACC

[0242] GTGGCCTTCATCATCTTTTGGGTC

[0243] Amino Acid sequence of CD28 transmembrane domain (SEQ ID NO: 12):

[0244] MFVWLVWGGVLACYSLLVTVAFIIFVW

[0245] Nucleotide sequence of 4-1 BB domain (SEQ ID NO: 27): AAGCGGGGCAGAAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAG

[0246] ACCACACAGGAAGAGGACGGCTGCTCCTGCCGGTTCCCCGAGGAAGAAGAAGGCGGCTG CGAGCTG

[0247] Amino Acid sequence of 4-1 BB domain (SEQ ID NO: 14):

[0248] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0249] Nucleotide sequence of CD3z domain (SEQ ID NO: 28):

[0250] AGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTG

[0251] TACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGATAAGCGGAGAGG

[0252] CCGGGACCCTGAGATGGGCGGCAAGCCTAGAAGAAAGAACCCCCAGGAAGGCCTGTATAA

[0253] CGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGC

[0254] GGAGAAGAGGCAAGGGCCACGATGGCCTGTACCAGGGACTGAGCACCGCCACCAAGGAT

[0255] ACCTATGACGCACTGCACATGCAGGCCCTGCCCCCCAGA

[0256] Amino Acid sequence of CD3z domain (SEQ ID NO: 16):

[0257] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[0258] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0259] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 29):

[0260] CTCGAGGGCGGAGGCGAAGGCAGAGGATCTCTGCTGACATGCGGCGACGTGGAAGAGAA CCCTGGCCCCAGA

[0261] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18):

[0262] LEGGGEGRGSLLTCGDVEENPGPR (not synthesized by ribosome)

[0263] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 30):

[0264] ATGCTGCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCTGCCTTTCTGCTG ATCCCC

[0265] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2):

[0266] MLLLVTSLLLCELPHPAFLLIP

[0267] Nucleotide sequence of EGFRt transduction marker (SEQ ID NO: 31): CGGAAAGTGTGCAACGGCATCGGCATCGGAGAGTTCAAGGACAGCCTGTCCATCAACGCC

[0268] ACCAACATCAAGCACTTCAAGAATTGCACCAGCATCAGCGGCGACCTGCACATCCTGCCAG

[0269] TGGCCTTTAGAGGCGACAGCTTCACCCACACCCCCCCACTGGATCCACAGGAACTGGATAT

[0270] TCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGG

[0271] ACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTC

[0272] AGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAG

[0273] ATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAAC

[0274] TGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAA

[0275] ACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGG

[0276] GGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGT

[0277] GGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCAT

[0278] ACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACC

[0279] AGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCC

[0280] GGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT

[0281] GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTG

[0282] TCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTT GCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0283] Amino Acid sequence of EGFRt transduction marker (SEQ ID NO: 22):

[0284] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI

[0285] TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKN

[0286] LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR

[0287] GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT

[0288] CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LVVALGIGLFM*

[0289] Complete nucleotide sequence of aP329G_lgG4long_CD28tm_41 BB_CD3z_EGFRt (Underlined is CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 32):

[0290] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0291] TCCCTATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTG

[0292] AAGCTGAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGG

[0293] CAGGCCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCAT

[0294] CAACTACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACAC

[0295] CCTGTACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAG

[0296] GCCCTACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGA GCGCCGGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGG

[0297] GCGGATCTCAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACC

[0298] GTGACCCTGACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTG

[0299] GGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGG

[0300] CCCCCGGCGTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACC

[0301] ATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAAC

[0302] CACTGGGTGTTCGGCTGTGGCACCAAGCTGACCGTGCTGGAGTCTAAGTACGGACCGCCT

[0303] TGTCCTCCTTGTCCAGCTCCTCCTGTGGCCGGACCTAGCGTGTTCCTGTTCCCCCCAAAGC

[0304] CCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGT

[0305] CCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACG

[0306] CCAAGACCAAGCCCAGAGAGGAACAGTTCCAGAGCACCTACCGGGTGGTGTCCGTGCTGA

[0307] CAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGG

[0308] GCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGCGAGCCC

[0309] CAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACC

[0310] TGTCTCGTGAAGGGCTTCTACCCCAGCGACATTGCCGTGGAATGGGAGAGCAACGGCCAG

[0311] CCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGT

[0312] ACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGAAGGCAACGTGTTCAGCTGCAGC

[0313] GTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCTGGGC

[0314] AAGATGTTCTGGGTGCTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTACAGCCTGCTCGTG

[0315] ACCGTGGCCTTCATCATCTTTTGGGTCAAGCGGGGCAGAAAGAAGCTGCTGTATATCTTCA

[0316] AGCAGCCCTTCATGCGGCCCGTGCAGACCACACAGGAAGAGGACGGCTGCTCCTGCCGG

[0317] TTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGAGTGAAGTTCAGCAGAAGCGCCGA

[0318] CGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACG

[0319] GGAAGAGTACGACGTGCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGC

[0320] CTAGAAGAAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCG

[0321] AGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGAGAAGAGGCAAGGGCCACGATGGC

[0322] CTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCACTGCACATGCAGGCC

[0323] CTGCCCCCCAGACTCGAGGGCGGAGGCGAAGGCAGAGGATCTCTGCTGACATGCGGCGA

[0324] CGTGGAAGAGAACCCTGGCCCCAGAATGCTGCTGCTCGTGACAAGCCTGCTGCTGTGCGA

[0325] GCTGCCCCACCCTGCCTTTCTGCTGATCCCCCGGAAAGTGTGCAACGGCATCGGCATCGG

[0326] AGAGTTCAAGGACAGCCTGTCCATCAACGCCACCAACATCAAGCACTTCAAGAATTGCACC

[0327] AGCATCAGCGGCGACCTGCACATCCTGCCAGTGGCCTTTAGAGGCGACAGCTTCACCCAC

[0328] ACCCCCCCACTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGT

[0329] TTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGA

[0330] AATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAAC ATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGG

[0331] AAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTC

[0332] AGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTG

[0333] CCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTT

[0334] GCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAG

[0335] CCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAG

[0336] GCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTAC

[0337] ATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACC

[0338] CTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACC

[0339] TACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCC

[0340] ATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGG CCTCTTCATGTGA

[0341] Complete CAR amino acid sequence of aP329G_lgG4long_CD28tm_41 BB_CD3z_EGFRt

[0342] (Beginning of scFv to end of CD3z) (SEQ ID NO: 33):

[0343] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0344] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSAGGGGSGG

[0345] GGSGGGGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGL

[0346] IGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGCGTKLTVLESKY

[0347] GPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHN

[0348] AKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT

[0349] LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD

[0350] KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVWGGVLACYSLLVTVAFIIFWVK

[0351] RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY

[0352] NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR

[0353] 3. aP329G_lgG3_CD28tm_41 BB_CD3z_EGFRt

[0354] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 1):

[0355] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0356] TCCCT

[0357] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2):

[0358] MLLLVTSLLLCELPHPAFLLIP

[0359] Nucleotide sequence of anti-P329G scFv Variable heavy chain (SEQ ID NO: 3):

[0360] ATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAAGCT

[0361] GAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGGCAGG

[0362] CCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCATCAACT

[0363] ACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACACCCTGT

[0364] ACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAGGCCCT

[0365] ACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0366] Amino Acid sequence of anti-P329G scFv Variable heavy chain (SEQ ID NO: 4):

[0367] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0368] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSA

[0369] Nucleotide sequence of G4S(4) linker (SEQ ID NO: 5):

[0370] GGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGGGCGGA

[0371] TOT

[0372] Amino Acid sequence of G4S(4) linker (SEQ ID NO: 6):

[0373] GGGGSGGGGSGGGGSGGGGS

[0374] Nucleotide sequence of anti-P329G scFv variable light chain (SEQ ID NO: 7):

[0375] CAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACCCT

[0376] GACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGG

[0377] AGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGGCCCCCGGC

[0378] GTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACCATCACCGG CGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCACTGGGT GTTCGGCTGTGGCACCAAGCTGACCGTGCTG

[0379] Amino Acid sequence of anti-P329G scFv variable light chain (SEQ ID NO: 8):

[0380] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPAR

[0381] FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHVWFGCGTKLTVL

[0382] Nucleotide sequence of lgG3 hinge (SEQ ID NO: 34):

[0383] GAACTGAAAACCCCCCTGGGCGACACCACCCACACCTGTCCTAGATGTCCG

[0384] Amino Acid sequence of lgG3 hinge (SEQ ID NO: 35):

[0385] ELKTPLGDTTHTCPRCP

[0386] Nucleotide sequence of CD28 transmembrane domain (SEQ ID NO: 36):

[0387] ATGTTCTGGGTGCTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTATAGCCTGCTCGTGACC GTGGCCTTCATCATCTTTTGGGTC

[0388] Amino Acid sequence of CD28 transmembrane domain (SEQ ID NO: 12): MFVWLVWGGVLACYSLLVTVAFIIFVW

[0389] Nucleotide sequence of 4-1 BB domain (SEQ ID NO: 37):

[0390] AAGCGGGGCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAG

[0391] ACCACCCAGGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTG CGAGCTG

[0392] Amino Acid sequence of 4-1 BB domain (SEQ ID NO: 14):

[0393] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0394] Nucleotide sequence of CD3z domain (SEQ ID NO: 38):

[0395] AGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTA

[0396] TACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGAGAAGAGG

[0397] CCGGGACCCTGAGATGGGCGGAAAGCCCAGAAGAAAGAACCCCCAGGAAGGCCTGTATAA

[0398] CGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGC

[0399] GGCGGAGAGGCAAGGGCCACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGAC

[0400] ACCTATGACGCCCTGCACATGCAGGCCCTGCCCCCTAGA Amino Acid sequence of CD3z domain (SEQ ID NO: 16):

[0401] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[0402] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0403] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 39):

[0404] CTCGAGGGCGGAGGCGAAGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAA CCCAGGCCCCAGA

[0405] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18):

[0406] LEGGGEGRGSLLTCGDVEENPGPR (not synthesized by ribosome)

[0407] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 40):

[0408] ATGCTGCTGCTCGTGACCAGCCTGCTGCTGTGTGAACTGCCTCATCCTGCTTTTCTGCTGA TTCCT

[0409] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2):

[0410] MLLLVTSLLLCELPHPAFLLIP

[0411] Nucleotide sequence of EGFRt transduction marker (SEQ ID NO: 41):

[0412] CGGAAAGTGTGCAACGGCATCGGCATCGGAGAGTTCAAGGACTCCCTGAGCATCAACGCC

[0413] ACCAACATCAAGCACTTCAAGAACTGCACCAGCATCAGCGGCGACCTGCACATCCTGCCTG

[0414] TGGCCTTTAGAGGCGACAGCTTCACCCACACACCCCCCCTGGATCCACAGGAACTGGATAT

[0415] TCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGG

[0416] ACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTC

[0417] AGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAG

[0418] ATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAAC

[0419] TGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAA

[0420] ACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGG

[0421] GGCCCGGAGCCAAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGT

[0422] GGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCAT

[0423] ACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACC

[0424] AGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCC

[0425] GGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT

[0426] GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTG TCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTT GCTGCTGGTGGTGGCCCTAGGGATCGGCCTCTTCATGTGA

[0427] Amino Acid sequence of EGFRt transduction marker (SEQ ID NO: 22):

[0428] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI

[0429] TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKN

[0430] LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR

[0431] GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT

[0432] CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LVVALGIGLFM*

[0433] Complete nucleotide sequence of aP329G_lgG3_CD28tm_41 BB_CD3z_EGFRt (Underlined

[0434] CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 42):

[0435] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0436] TCCCTATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTG

[0437] AAGCTGAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGG

[0438] CAGGCCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCAT

[0439] CAACTACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACAC

[0440] CCTGTACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAG

[0441] GCCCTACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGA

[0442] GCGCCGGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGG

[0443] GCGGATCTCAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACC

[0444] GTGACCCTGACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTG

[0445] GGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGG

[0446] CCCCCGGCGTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACC

[0447] ATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAAC

[0448] CACTGGGTGTTCGGCTGTGGCACCAAGCTGACCGTGCTGGAACTGAAAACCCCCCTGGGC

[0449] GACACCACCCACACCTGTCCTAGATGTCCGATGTTCTGGGTGCTGGTGGTCGTGGGCGGA

[0450] GTGCTGGCCTGTTATAGCCTGCTCGTGACCGTGGCCTTCATCATCTTTTGGGTCAAGCGGG

[0451] GCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCA

[0452] GGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGA

[0453] GAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTAT

[0454] ACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGAGAAGAGGC

[0455] CGGGACCCTGAGATGGGCGGAAAGCCCAGAAGAAAGAACCCCCAGGAAGGCCTGTATAAC

[0456] GAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCG GCGGAGAGGCAAGGGCCACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGACA

[0457] CCTATGACGCCCTGCACATGCAGGCCCTGCCCCCTAGACTCGAGGGCGGAGGCGAAGGC

[0458] AGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCCCAGAATGCTGCT

[0459] GCTCGTGACCAGCCTGCTGCTGTGTGAACTGCCTCATCCTGCTTTTCTGCTGATTCCTCGG

[0460] AAAGTGTGCAACGGCATCGGCATCGGAGAGTTCAAGGACTCCCTGAGCATCAACGCCACC

[0461] AACATCAAGCACTTCAAGAACTGCACCAGCATCAGCGGCGACCTGCACATCCTGCCTGTGG

[0462] CCTTTAGAGGCGACAGCTTCACCCACACACCCCCCCTGGATCCACAGGAACTGGATATTCT

[0463] GAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACG

[0464] GACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGT

[0465] TTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATA

[0466] AGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTG

[0467] GAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAAC

[0468] AGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGG

[0469] CCCGGAGCCAAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGG

[0470] ACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATAC

[0471] AGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAG

[0472] ACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGG

[0473] CAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGT

[0474] GCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTC

[0475] CAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGC

[0476] TGCTGGTGGTGGCCCTAGGGATCGGCCTCTTCATGTGA

[0477] Complete CAR amino acid sequence of aP329G_lgG3_CD28tm_41 BB_CD3z_EGFRt

[0478] (Beginning of scFv to end of CD3z) (SEQ ID NO: 43):

[0479] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0480] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSAGGGGSGG

[0481] GGSGGGGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGL

[0482] IGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGCGTKLTVLELKTP

[0483] LGDTTHTCPRCPMFWVLWVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQE

[0484] EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

[0485] EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR 4. aP329G_CD8a_41 BB_CD3z_EGFRt

[0486] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 1):

[0487] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0488] TCCCT

[0489] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2):

[0490] MLLLVTSLLLCELPHPAFLLIP

[0491] Nucleotide sequence of anti-P329G scFv Variable heavy chain (SEQ ID NO: 3):

[0492] ATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAAGCT

[0493] GAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGGCAGG

[0494] CCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCATCAACT

[0495] ACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACACCCTGT

[0496] ACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAGGCCCT

[0497] ACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0498] Amino Acid sequence of anti-P329G scFv Variable heavy chain (SEQ ID NO: 4):

[0499] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0500] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSA

[0501] Nucleotide sequence of G4S(4) linker (SEQ ID NO: 5):

[0502] GGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGGGCGGA

[0503] TOT

[0504] Amino Acid sequence of G4S(4) linker (SEQ ID NO: 6):

[0505] GGGGSGGGGSGGGGSGGGGS

[0506] Nucleotide sequence of anti-P329G scFv variable light chain (SEQ ID NO: 7):

[0507] CAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACCCT

[0508] GACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGG

[0509] AGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGGCCCCCGGC

[0510] GTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACCATCACCGG CGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCACTGGGT GTTCGGCTGTGGCACCAAGCTGACCGTGCTG

[0511] Amino Acid sequence of anti-P329G scFv variable light chain (SEQ ID NO: 8):

[0512] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPAR

[0513] FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHVWFGCGTKLTVL

[0514] Nucleotide sequence of CD8a hinge (SEQ ID NO: 44):

[0515] GCCAAGCCTACCACCACCCCTGCCCCTAGACCTCCAACACCCGCCCCAACAATCGCCAGC

[0516] CAGCCTCTGTCTCTGAGTCCTGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACC

[0517] AGAGGACTGGATTTCGCCTGCGAC

[0518] Amino Acid sequence of CD8a hinge (SEQ ID NO: 45):

[0519] AKPTTTPAPRPPTPAPTIASQPLSLSPEACRPAAGGAVHTRGLDFACD

[0520] Nucleotide sequence of CD8a transmembrane domain (SEQ ID NO: 46):

[0521] ATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATC ACC

[0522] Amino Acid sequence of CD8a transmembrane domain (SEQ ID NO: 47):

[0523] IYIWAPLAGTCGVLLLSLVIT

[0524] Nucleotide sequence of 4-1 BB domain (SEQ ID NO: 13):

[0525] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAAC

[0526] TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA CTG

[0527] Amino Acid sequence of 4-1 BB domain (SEQ ID NO: 14):

[0528] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0529] Nucleotide sequence of CD3z domain (SEQ ID NO: 15):

[0530] CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCT

[0531] GTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAG

[0532] GCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTAT

[0533] AACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGA GCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGG

[0534] ATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG

[0535] Amino Acid sequence of CD3z domain (SEQ ID NO: 16):

[0536] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[0537] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0538] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 17):

[0539] CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAA TCCCGGCCCTAGG

[0540] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18): (not synthesized by ribosome)

[0541] LEGGGEGRGSLLTCGDVEENPGPR

[0542] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 19):

[0543] ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGAT COCA

[0544] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2):

[0545] MLLLVTSLLLCELPHPAFLLIP

[0546] Nucleotide sequence of EGFRt transduction marker (SEQ ID NO: 21):

[0547] CGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTAC

[0548] GAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTG

[0549] GCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCT

[0550] GAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACG

[0551] GACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGT

[0552] TTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATA

[0553] AGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTG

[0554] GAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAAC

[0555] AGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGG

[0556] CCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGG

[0557] ACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATAC

[0558] AGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAG ACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGG

[0559] CAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGT

[0560] GCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTC

[0561] CAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGC TGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0562] Amino Acid sequence of EGFRt transduction marker (SEQ ID NO: 22):

[0563] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI

[0564] TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKN

[0565] LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR

[0566] GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT

[0567] CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LVVALGIGLFM*

[0568] Complete nucleotide sequence of aP329G_CD8a_41 BB_CD3z_EGFRt (Underlined is CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 48):

[0569] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0570] TCCCTATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTG

[0571] AAGCTGAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGG

[0572] CAGGCCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCAT

[0573] CAACTACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACAC

[0574] CCTGTACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAG

[0575] GCCCTACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGA

[0576] GCGCCGGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGG

[0577] GCGGATCTCAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACC

[0578] GTGACCCTGACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTG

[0579] GGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGG

[0580] CCCCCGGCGTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACC

[0581] ATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAAC

[0582] CACTGGGTGTTCGGCTGTGGCACCAAGCTGACCGTGCTGGCCAAGCCTACCACCACCCCT

[0583] GCCCCTAGACCTCCAACACCCGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTGAGTCCT

[0584] GAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGC

[0585] GACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTG

[0586] ATCACCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGT

[0587] ACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA TGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCA

[0588] GAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAA

[0589] GCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAA

[0590] GGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATG

[0591] AAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCG

[0592] CCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGCTCGAGGGC

[0593] GGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCC

[0594] TAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTC

[0595] CTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCAT

[0596] AAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCC

[0597] TGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACT

[0598] GGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAA

[0599] ACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAAC

[0600] ATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTC

[0601] AAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATAC

[0602] AATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAG

[0603] GTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCT

[0604] GCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAA

[0605] TGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAG

[0606] TGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGG

[0607] GGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCT

[0608] GCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGC

[0609] CATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAA

[0610] GGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTC

[0611] CTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0612] Complete CAR amino acid sequence of aP329G_CD8a_41 BB_CD3z_EGFRt (Beginning of scFv to end of CD3z) (SEQ ID NO: 49):

[0613] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0614] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSAGGGGSGG

[0615] GGSGGGGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGL

[0616] IGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGCGTKLTVLAKPTT

[0617] TPAPRPPTPAPTIASQPLSLSPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKR

[0618] GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR

[0619] 5. aP329G_lgG3_CD28tm_CD28_CD3z_LNGFRt

[0620] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 1):

[0621] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0622] TCCCT

[0623] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2): MLLLVTSLLLCELPHPAFLLIP

[0624] Nucleotide sequence of anti-P329G scFv variable heavy chain (SEQ ID NO: 3):

[0625] ATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAAGCT

[0626] GAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGGCAGG

[0627] CCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCATCAACT

[0628] ACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACACCCTGT

[0629] ACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAGGCCCT

[0630] ACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0631] Amino Acid sequence of anti-P329G scFv variable heavy chain (SEQ ID NO: 4):

[0632] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0633] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSA

[0634] Nucleotide sequence of G4S(4) linker (SEQ ID NO: 5):

[0635] GGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGGGCGGA

[0636] TOT

[0637] Amino Acid sequence of G4S(4) linker (SEQ ID NO: 6):

[0638] GGGGSGGGGSGGGGSGGGGS

[0639] Nucleotide sequence of anti-P329G scFv variable light chain (SEQ ID NO: 7):

[0640] CAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACCCT

[0641] GACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGG

[0642] AGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGGCCCCCGGC GTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACCATCACCGG CGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCACTGGGT

[0643] GTTCGGCTGTGGCACCAAGCTGACCGTGCTG

[0644] Amino Acid sequence of anti-P329G scFv variable light chain (SEQ ID NO: 8):

[0645] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPAR

[0646] FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHVWFGCGTKLTVL

[0647] Nucleotide sequence of lgG3 hinge (SEQ ID NO: 34):

[0648] GAACTGAAAACCCCCCTGGGCGACACCACCCACACCTGTCCTAGATGTCCG

[0649] Amino Acid sequence of lgG3 hinge (SEQ ID NO: 35):

[0650] ELKTPLGDTTHTCPRCP

[0651] Nucleotide sequence of CD28 transmembrane domain (SEQ ID NO: 36):

[0652] ATGTTCTGGGTGCTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTATAGCCTGCTCGTGACC GTGGCCTTCATCATCTTTTGGGTC

[0653] Amino Acid sequence of CD28 transmembrane domain (SEQ ID NO: 12): MFVWLVWGGVLACYSLLVTVAFIIFVW

[0654] Nucleotide sequence of CD28 cytoplasmatic domain (SEQ ID NO: 50):

[0655] CGCAGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCTAGACGGCC

[0656] TGGCCCCACCAGAAAGCACTACCAGCCCTACGCCCCTCCCCGGGACTTTGCCGCCTACAG

[0657] AAGC

[0658] Amino Acid sequence of CD28 cytoplasmatic domain (SEQ ID NO: 51):

[0659] RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0660] Nucleotide sequence of CD3z domain (SEQ ID NO: 38):

[0661] AGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTA

[0662] TACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGAGAAGAGG

[0663] CCGGGACCCTGAGATGGGCGGAAAGCCCAGAAGAAAGAACCCCCAGGAAGGCCTGTATAA

[0664] CGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGC GGCGGAGAGGCAAGGGCCACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGAC

[0665] ACCTATGACGCCCTGCACATGCAGGCCCTGCCCCCTAGA

[0666] Amino Acid sequence of CD3z domain (SEQ ID NO: 16):

[0667] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[0668] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0669] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 39):

[0670] CTCGAGGGCGGAGGCGAAGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAA CCCAGGCCCCAGA

[0671] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18): (not synthesized by ribosome)

[0672] LEGGGEGRGSLLTCGDVEENPGPR

[0673] Nucleotide sequence of LNGFR transduction marker (SEQ ID NO: 52):

[0674] ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCT

[0675] TCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAG

[0676] CGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCA

[0677] ACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGA

[0678] CCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGC

[0679] GTGGAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGAC

[0680] TGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCC

[0681] AGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCC

[0682] AACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCG

[0683] CGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACAC

[0684] GGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCA

[0685] CCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGC

[0686] AGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCC

[0687] ATCCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGA

[0688] Amino Acid sequence of LNGFR transduction marker (SEQ ID NO: 53):

[0689] MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQT

[0690] VCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA

[0691] CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWAD AECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTTD

[0692] NLIPVYCSILAAWVGLVAYIAFKR*

[0693] Complete nucleotide sequence of aP329G_lgG3_CD28tm_CD28_CD3z_LNGFRt (Underlined is

[0694] CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 54):

[0695] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0696] TCCCTATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTG

[0697] AAGCTGAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGG

[0698] CAGGCCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCAT

[0699] CAACTACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACAC

[0700] CCTGTACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAG

[0701] GCCCTACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGA

[0702] GCGCCGGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGG

[0703] GCGGATCTCAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACC

[0704] GTGACCCTGACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTG

[0705] GGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGG

[0706] CCCCCGGCGTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACC

[0707] ATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAAC

[0708] CACTGGGTGTTCGGCTGTGGCACCAAGCTGACCGTGCTGGAACTGAAAACCCCCCTGGGC

[0709] GACACCACCCACACCTGTCCTAGATGTCCGATGTTCTGGGTGCTGGTGGTCGTGGGCGGA

[0710] GTGCTGGCCTGTTATAGCCTGCTCGTGACCGTGGCCTTCATCATCTTTTGGGTCCGCAGCA

[0711] AGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCTAGACGGCCTGGCCCC

[0712] ACCAGAAAGCACTACCAGCCCTACGCCCCTCCCCGGGACTTTGCCGCCTACAGAAGCAGA

[0713] GTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTATAC

[0714] AACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGAGAAGAGGCCG

[0715] GGACCCTGAGATGGGCGGAAAGCCCAGAAGAAAGAACCCCCAGGAAGGCCTGTATAACGA

[0716] ACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGC

[0717] GGAGAGGCAAGGGCCACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGACACC

[0718] TATGACGCCCTGCACATGCAGGCCCTGCCCCCTAGACTCGAGGGCGGAGGCGAAGGCAG

[0719] AGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCCCAGACATATGGGGG

[0720] CAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGG

[0721] GTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAG

[0722] TGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGAC

[0723] CGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGC

[0724] CGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAG GCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCG CTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACA AGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCAC

[0725] GTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTG

[0726] CACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCA

[0727] CACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCA GAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCC CAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTG

[0728] GCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGA

[0729] Complete CAR amino acid sequence of aP329G_lgG3_CD28tm_CD28_CD3z_LNGFRt

[0730] (Beginning of scFv to end of CD3z) (SEQ ID NO: 55):

[0731] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0732] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSAGGGGSGG

[0733] GGSGGGGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGL

[0734] IGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGCGTKLTVLELKTP

[0735] LGDTTHTCPRCPMFWVLWVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPT

[0736] RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR

[0737] 6. aP329G_lgG3_CD28tm_41 BB_CD3z_LNGFRt

[0738] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 1):

[0739] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA TCCCT

[0740] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2):

[0741] MLLLVTSLLLCELPHPAFLLIP

[0742] Nucleotide sequence of anti-P329G scFv variable heavy chain (SEQ ID NO: 3):

[0743] ATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAAGCT

[0744] GAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGGCAGG CCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCATCAACT ACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACACCCTGT ACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAGGCCCT ACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0745] Amino Acid sequence of anti-P329G scFv variable heavy chain (SEQ ID NO: 4):

[0746] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0747] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSA

[0748] Nucleotide sequence of G4S(4) linker (SEQ ID NO: 5):

[0749] GGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGGGCGGA TOT

[0750] Amino Acid sequence of G4S(4) linker (SEQ ID NO: 6):

[0751] GGGGSGGGGSGGGGSGGGGS

[0752] Nucleotide sequence of anti-P329G scFv variable light chain (SEQ ID NO: 7):

[0753] CAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACCCT

[0754] GACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGG

[0755] AGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGGCCCCCGGC

[0756] GTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACCATCACCGG

[0757] CGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCACTGGGT

[0758] GTTCGGCTGTGGCACCAAGCTGACCGTGCTG

[0759] Amino Acid sequence of anti-P329G scFv variable light chain (SEQ ID NO: 8):

[0760] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPAR

[0761] FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHVWFGCGTKLTVL

[0762] Nucleotide sequence of lgG3 hinge (SEQ ID NO: 34):

[0763] GAACTGAAAACCCCCCTGGGCGACACCACCCACACCTGTCCTAGATGTCCG

[0764] Amino Acid sequence of lgG3 hinge (SEQ ID NO: 35):

[0765] ELKTPLGDTTHTCPRCP

[0766] Nucleotide sequence of CD28 transmembrane domain (SEQ ID NO: 36):

[0767] ATGTTCTGGGTGCTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTATAGCCTGCTCGTGACC GTGGCCTTCATCATCTTTTGGGTC Amino Acid sequence of CD28 transmembrane domain (SEQ ID NO: 12): MFVWLVWGGVLACYSLLVTVAFIIFVW

[0768] Nucleotide sequence of 4-1 BB domain (SEQ ID NO: 37):

[0769] AAGCGGGGCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAG

[0770] ACCACCCAGGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTG CGAGCTG

[0771] Amino Acid sequence of 4-1 BB domain (SEQ ID NO: 14):

[0772] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0773] Nucleotide sequence of CD3z domain (SEQ ID NO: 38):

[0774] AGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTA

[0775] TACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGAGAAGAGG

[0776] CCGGGACCCTGAGATGGGCGGAAAGCCCAGAAGAAAGAACCCCCAGGAAGGCCTGTATAA

[0777] CGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGC

[0778] GGCGGAGAGGCAAGGGCCACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGAC

[0779] ACCTATGACGCCCTGCACATGCAGGCCCTGCCCCCTAGA

[0780] Amino Acid sequence of CD3z domain (SEQ ID NO: 16):

[0781] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[0782] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0783] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 39):

[0784] CTCGAGGGCGGAGGCGAAGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAA CCCAGGCCCCAGA

[0785] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18): (not synthesized by ribosome)

[0786] LEGGGEGRGSLLTCGDVEENPGPR

[0787] Nucleotide sequence of LNGFR transduction marker (SEQ ID NO: 52):

[0788] ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCT

[0789] TCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAG CGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCA

[0790] ACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGA

[0791] CCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGC

[0792] GTGGAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGAC

[0793] TGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCC

[0794] AGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCC

[0795] AACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCG

[0796] CGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACAC

[0797] GGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCA

[0798] CCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGC

[0799] AGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCC

[0800] ATCCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGA

[0801] Amino Acid sequence of LNGFR transduction marker (SEQ ID NO: 53):

[0802] MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQT

[0803] VCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA

[0804] CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWAD

[0805] AECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTTD

[0806] NLIPVYCSILAAWVGLVAYIAFKR*

[0807] Complete nucleotide sequence of aP329G_lgG3_CD28tm_41 BB_CD3z_LNGFRt (Underlined is

[0808] CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 56):

[0809] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[0810] TCCCTATGGTGAAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTG

[0811] AAGCTGAGCTGCGCCGCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAACTGGGTGAGG

[0812] CAGGCCCCCGGCAAGTGTCTGGAGTGGATCGGCGAGATCACCCCCGACAGCAGCACCAT

[0813] CAACTACACCCCCAGCCTGAAGGACAAGTTCATCATCAGCAGGGACAACGCCAAGAACAC

[0814] CCTGTACCTGCAGATGATCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGTGAG

[0815] GCCCTACGACTACGGCGCCTGGTTCGCCAGCTGGGGCCAGGGCACCCTGGTGACCGTGA

[0816] GCGCCGGAGGGGGCGGAAGTGGTGGCGGGGGAAGCGGCGGGGGTGGCAGCGGAGGGG

[0817] GCGGATCTCAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACC

[0818] GTGACCCTGACCTGCAGGAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTG

[0819] GGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGAGGG

[0820] CCCCCGGCGTGCCCGCCAGGTTCTCCGGAAGCCTGATCGGCGACAAGGCCGCCCTGACC

[0821] ATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAAC CACTGGGTGTTCGGCTGTGGCACCAAGCTGACCGTGCTGGAACTGAAAACCCCCCTGGGC

[0822] GACACCACCCACACCTGTCCTAGATGTCCGATGTTCTGGGTGCTGGTGGTCGTGGGCGGA

[0823] GTGCTGGCCTGTTATAGCCTGCTCGTGACCGTGGCCTTCATCATCTTTTGGGTCAAGCGGG

[0824] GCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCA

[0825] GGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGA

[0826] GAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTAT

[0827] ACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGAGAAGAGGC

[0828] CGGGACCCTGAGATGGGCGGAAAGCCCAGAAGAAAGAACCCCCAGGAAGGCCTGTATAAC

[0829] GAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCG

[0830] GCGGAGAGGCAAGGGCCACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGACA

[0831] CCTATGACGCCCTGCACATGCAGGCCCTGCCCCCTAGACTCGAGGGCGGAGGCGAAGGC

[0832] AGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCCCAGACATATGGG

[0833] GGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGG

[0834] GGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGT

[0835] GAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCA

[0836] GACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCG

[0837] AGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTG

[0838] GAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGG

[0839] GCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGG

[0840] ACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAAC

[0841] CACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGA

[0842] GTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGT

[0843] CCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCT

[0844] CCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGC

[0845] TCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCC

[0846] TGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGA

[0847] Complete CAR amino acid sequence of aP329G_lgG3_CD28tm_41 BB_CD3z_LNGFRt

[0848] (Beginning of scFv to end of CD3z) (SEQ ID NO: 57):

[0849] MVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKCLEWIGEITPDSSTINYTPS

[0850] LKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCVRPYDYGAWFASWGQGTLVTVSAGGGGSGG

[0851] GGSGGGGSGGGGSQAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGL

[0852] IGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGCGTKLTVLELKTP

[0853] LGDTTHTCPRCPMFWVLWVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQE

[0854] EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR

[0855] Preferred embodiments of CARs according to claim 1 (a) including conventional CARs are exemplified in 7.-11. below:

[0856] 7. aCD19(FMC63)_lgG4short_CD28tm_41 BB_CD3z_EGFRt

[0857] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 58):

[0858] ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTG ATCCCC

[0859] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2):

[0860] MLLLVTSLLLCELPHPAFLLIP

[0861] Nucleotide sequence of anti-CD19 (FMC63) scFv variable light chain (SEQ ID NO: 59):

[0862] GACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGAC

[0863] CATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCC

[0864] CGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCA

[0865] GCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAAC

[0866] AGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGG CGGAACAAAGCTGGAAATCACC

[0867] Amino Acid sequence of anti-CD19 (FMC63) scFv variable light chain (SEQ ID NO: 60):

[0868] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSG

[0869] SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0870] Nucleotide sequence of Whitlow Linker (SEQ ID NO: 61):

[0871] GGCAGCACCTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGC

[0872] Amino Acid sequence of Whitlow Linker (SEQ ID NO: 62):

[0873] GSTSGSGKPGSGEGSTKG

[0874] Nucleotide sequence of anti-CD19 (FMC63) scFv variable heavy chain (SEQ ID NO: 63): GAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGT

[0875] GACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGC

[0876] CCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC

[0877] AACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTC

[0878] CTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACT

[0879] ACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGC

[0880] AGC

[0881] Amino Acid sequence of anti-CD19 (FMC63) scFv variable heavy chain (SEQ ID NO: 64):

[0882] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL

[0883] KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0884] Nucleotide sequence of lgG4 hinge short (SEQ ID NO: 65):

[0885] GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT

[0886] Amino Acid sequence of lgG4 hinge short (SEQ ID NO: 10):

[0887] ESKYGPPCPPCP

[0888] Nucleotide sequence of CD28 transmembrane domain (SEQ ID NO: 11):

[0889] ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACC

[0890] GTGGCCTTCATCATCTTTTGGGTG

[0891] Amino Acid sequence of CD28 transmembrane domain (SEQ ID NO: 12):

[0892] MFVWLVWGGVLACYSLLVTVAFIIFVW

[0893] Nucleotide sequence of 4-1 BB domain (SEQ ID NO: 13):

[0894] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAAC

[0895] TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA

[0896] CTG

[0897] Amino Acid sequence of 4-1 BB domain (SEQ ID NO: 14):

[0898] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0899] Nucleotide sequence of CD3z domain (SEQ ID NO: 15): CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCT

[0900] GTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAG

[0901] GCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTAT

[0902] AACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGA

[0903] GCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGG

[0904] ATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG

[0905] Amino Acid sequence of CD3z domain (SEQ ID NO: 16):

[0906] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[0907] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0908] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 17):

[0909] CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAA TCCCGGCCCTAGG

[0910] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18):

[0911] LEGGGEGRGSLLTCGDVEENPGPR (not synthesized by ribosome)

[0912] Nucleotide sequence of GM-CSF signaling peptide (SEQ ID NO: 19):

[0913] ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGAT COCA

[0914] Amino Acid sequence of GM-CSF signaling peptide (SEQ ID NO: 2):

[0915] MLLLVTSLLLCELPHPAFLLIP

[0916] Nucleotide sequence of EGFRt transduction marker (SEQ ID NO: 21):

[0917] CGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTAC

[0918] GAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTG

[0919] GCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCT

[0920] GAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACG

[0921] GACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGT

[0922] TTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATA

[0923] AGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTG

[0924] GAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAAC

[0925] AGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGG CCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGG

[0926] ACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATAC

[0927] AGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAG

[0928] ACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGG

[0929] CAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGT

[0930] GCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTC

[0931] CAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGC TGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0932] Amino Acid sequence of EGFRt transduction marker (SEQ ID NO: 22):

[0933] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI

[0934] TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKN

[0935] LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR

[0936] GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT

[0937] CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LVVALGIGLFM*

[0938] Complete nucleotide sequence of aCD19(FMC63)_lgG4short_CD28tm_41 BB_CD3z_EGFRt

[0939] (Underlined is CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 66):

[0940] ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTG

[0941] ATCCCCGACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCG

[0942] GGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCA

[0943] GAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCG

[0944] TGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACC

[0945] TGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTT

[0946] TGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACCTCCGGCAGCGGCAAGCCTGGCA

[0947] GCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGT

[0948] GGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACT

[0949] ACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATC

[0950] TGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAG

[0951] GACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCC

[0952] ATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGC

[0953] CAGGGCACCAGCGTGACCGTGAGCAGCGAATCTAAGTACGGACCGCCCTGCCCCCCTTGC

[0954] CCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTC

[0955] ACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAA ACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTT

[0956] CCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGC

[0957] CCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGA

[0958] AGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTC

[0959] GGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGG

[0960] CCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCT

[0961] GTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCT

[0962] GCCCCCAAGGCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACG

[0963] TGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTT

[0964] ACCACACCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAA

[0965] TTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATC

[0966] AGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTC

[0967] CTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCT

[0968] GATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATA

[0969] CGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACAT

[0970] CCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAA

[0971] AAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAA

[0972] CCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATG

[0973] CCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGG

[0974] AATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGG

[0975] GAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATG

[0976] AACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACG

[0977] GCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCT

[0978] GGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGAT

[0979] GCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCA

[0980] CTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCA

[0981] TGTGA

[0982] Complete CAR amino acid sequence of aCD19(FMC63)_lgG4short_CD28tm_41 BB_CD3z_EGFRt (Beginning of scFv to end of CD3z)

[0983] (SEQ ID NO: 67):

[0984] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSG

[0985] SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVK

[0986] LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSR

[0987] LTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCP PCPMFVWLVVVGGVLACYSLLVTVAFIIFVWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

[0988] NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0989] 8. aCD20(GA1 01 )_lgG4long_CD28tm_41 BB_CD3z_EGFRt

[0990] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 1):

[0991] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA TCCCT

[0992] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2): MLLLVTSLLLCELPHPAFLLIP

[0993] Nucleotide sequence of anti-CD20 (GA101) scFv variable heavy chain (SEQ ID NO: 68):

[0994] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGT

[0995] GAGCTGCAAGGCCAGCGGCTACGCCTTCAGCTACAGCTGGATCAACTGGGTGAGGCAGG

[0996] CCCCCGGCCAGTGCCTGGAGTGGATGGGCAGGATCTTCCCCGGCGACGGCGACACCGAC

[0997] TACAACGGCAAGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCC

[0998] TACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGAAC

[0999] GTGTTCGACGGCTACTGGCTGGTGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAG C

[1000] Amino Acid sequence of anti-CD20 (GA101) scFv variable heavy chain (SEQ ID NO: 69): QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQCLEWMGRIFPGDGDTDYN

[1001] GKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS

[1002] Nucleotide sequence of G4S(3) Linker (SEQ ID NO: 70):

[1003] GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC

[1004] Amino Acid sequence of G4S(3) Linker (SEQ ID NO: 71):

[1005] GGGGSGGGGSGGGGS

[1006] Nucleotide sequence of anti-CD20 (GA101) scFv variable light chain (SEQ ID NO: 72):

[1007] GACATCGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGACCCCCGGCGAGCCCGCCAG

[1008] CATCAGCTGCAGGAGCAGCAAGAGCCTGCTGCACAGCAACGGCATCACCTACCTGTACTG

[1009] GTACCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCAGATGAGCAACCTGGT GAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGA

[1010] TCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGAACCTGGAGCTG CCCTACACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG

[1011] Amino Acid sequence of anti-CD20 (GA101) scFv variable light chain (SEQ ID NO: 73):

[1012] DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPD

[1013] RFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGCGTKVEIK

[1014] Nucleotide sequence of lgG4 hinge long (SEQ ID NO: 24):

[1015] GAGTCTAAGTACGGACCGCCTTGTCCTCCTTGTCCAGCTCCTCCTGTGGCCGGACCTAGC

[1016] GTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTG

[1017] ACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTG

[1018] GACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCCAGAGCAC

[1019] CTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTA

[1020] CAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGC

[1021] CAAGGGCCAGCCTCGCGAGCCCCAGGTGTACACACTGCCTCCAAGCCAGGAAGAGATGAC

[1022] CAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCAGCGACATTGCCGT

[1023] GGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGA

[1024] CAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAGGA

[1025] AGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA GTCCCTGTCTCTGAGCCTGGGCAAG

[1026] Amino Acid sequence of lgG4 hinge long (SEQ ID NO: 25):

[1027] ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGV

[1028] EVHNAKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE

[1029] PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[1030] RLTVDKS RWQ EG N VFSCS VM H EALH N H YTQ KS LS LS LG K

[1031] Nucleotide sequence of CD28 transmembrane domain (SEQ ID NO: 26):

[1032] ATGTTCTGGGTGCTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTACAGCCTGCTCGTGACC

[1033] GTGGCCTTCATCATCTTTTGGGTC

[1034] Amino Acid sequence of CD28 transmembrane domain (SEQ ID NO: 12):

[1035] MFVWLVWGGVLACYSLLVTVAFIIFVW Nucleotide sequence of 4-1 BB domain (SEQ ID NO: 27):

[1036] AAGCGGGGCAGAAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAG

[1037] ACCACACAGGAAGAGGACGGCTGCTCCTGCCGGTTCCCCGAGGAAGAAGAAGGCGGCTG CGAGCTG

[1038] Amino Acid sequence of4-1 BB domain (SEQ ID NO: 14):

[1039] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[1040] Nucleotide sequence of CD3z domain (SEQ ID NO: 28):

[1041] AGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGCTG

[1042] TACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGATAAGCGGAGAGG

[1043] CCGGGACCCTGAGATGGGCGGCAAGCCTAGAAGAAAGAACCCCCAGGAAGGCCTGTATAA

[1044] CGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGC

[1045] GGAGAAGAGGCAAGGGCCACGATGGCCTGTACCAGGGACTGAGCACCGCCACCAAGGAT

[1046] ACCTATGACGCACTGCACATGCAGGCCCTGCCCCCCAGA

[1047] Amino Acid sequence of CD3z domain (SEQ ID NO: 16):

[1048] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[1049] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[1050] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 29):

[1051] CTCGAGGGCGGAGGCGAAGGCAGAGGATCTCTGCTGACATGCGGCGACGTGGAAGAGAA CCCTGGCCCCAGA

[1052] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18): (not synthesized by ribosome)

[1053] LEGGGEGRGSLLTCGDVEENPGPR

[1054] Nucleotide sequence of GM-CSF signaling peptide (SEQ ID NO: 30):

[1055] ATGCTGCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCTGCCTTTCTGCTG ATCCCC

[1056] Amino Acid sequence of GM-CSF signaling peptide (SEQ ID NO: 2):

[1057] MLLLVTSLLLCELPHPAFLLIP Nucleotide sequence of EGFRt transduction marker (SEQ ID NO: 74):

[1058] CGGAAAGTGTGCAACGGCATCGGCATCGGAGAGTTCAAGGACAGCCTGTCCATCAACGCC

[1059] ACCAACATCAAGCACTTCAAGAATTGCACCAGCATCAGCGGCGACCTGCACATCCTGCCAG

[1060] TGGCCTTTAGAGGCGACAGCTTCACCCACACCCCCCCACTGGATCCACAGGAACTGGATAT

[1061] TCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGG

[1062] ACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTC

[1063] AGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAG

[1064] ATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAAC

[1065] TGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAA

[1066] ACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGG

[1067] GGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGT

[1068] GGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCAT

[1069] ACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACC

[1070] AGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCC

[1071] GGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT

[1072] GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTG

[1073] TCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTT GCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[1074] Amino Acid sequence of EGFRt transduction marker (SEQ ID NO: 22):

[1075] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI

[1076] TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKN

[1077] LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR

[1078] GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT

[1079] CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LVVALGIGLFM*

[1080] Complete nucleotide sequence of aCD20(GA101)_lgG4long_CD28tm_41 BB_CD3z_EGFRt

[1081] (Underlined is CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 75):

[1082] ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGA

[1083] TCCCTCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTG

[1084] AAGGTGAGCTGCAAGGCCAGCGGCTACGCCTTCAGCTACAGCTGGATCAACTGGGTGAGG

[1085] CAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCAGGATCTTCCCCGGCGACGGCGACAC

[1086] CGACTACAACGGCAAGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCAC

[1087] CGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCA GGAACGTGTTCGACGGCTACTGGCTGGTGTACTGGGGCCAGGGCACCCTGGTGACCGTG

[1088] AGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATC

[1089] GTGATGACCCAGACCCCCCTGAGCCTGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAG

[1090] CTGCAGGAGCAGCAAGAGCCTGCTGCACAGCAACGGCATCACCTACCTGTACTGGTACCT

[1091] GCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCAGATGAGCAACCTGGTGAGCG

[1092] GCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGC

[1093] AGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGAACCTGGAGCTGCCCTA

[1094] CACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGAGTCTAAGTACGGACCGCCTTGTCC

[1095] TCCTTGTCCAGCTCCTCCTGTGGCCGGACCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAG

[1096] GACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAG

[1097] GAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAG

[1098] ACCAAGCCCAGAGAGGAACAGTTCCAGAGCACCTACCGGGTGGTGTCCGTGCTGACAGTG

[1099] CTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTG

[1100] CCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGCGAGCCCCAGGT

[1101] GTACACACTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCT

[1102] CGTGAAGGGCTTCTACCCCAGCGACATTGCCGTGGAATGGGAGAGCAACGGCCAGCCCG

[1103] AGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAG

[1104] CAGACTGACCGTGGACAAGAGCCGGTGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGAT

[1105] GCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCTGGGCAAGAT

[1106] GTTCTGGGTGCTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTACAGCCTGCTCGTGACCGT

[1107] GGCCTTCATCATCTTTTGGGTCAAGCGGGGCAGAAAGAAGCTGCTGTATATCTTCAAGCAG

[1108] CCCTTCATGCGGCCCGTGCAGACCACACAGGAAGAGGACGGCTGCTCCTGCCGGTTCCCC

[1109] GAGGAAGAAGAAGGCGGCTGCGAGCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCC

[1110] TGCCTATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGA

[1111] GTACGACGTGCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTAGAA

[1112] GAAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCT

[1113] ACAGCGAGATCGGAATGAAGGGCGAGCGGAGAAGAGGCAAGGGCCACGATGGCCTGTAC

[1114] CAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCACTGCACATGCAGGCCCTGCCC

[1115] CCCAGACTCGAGGGCGGAGGCGAAGGCAGAGGATCTCTGCTGACATGCGGCGACGTGGA

[1116] AGAGAACCCTGGCCCCAGAATGCTGCTGCTCGTGACAAGCCTGCTGCTGTGCGAGCTGCC

[1117] CCACCCTGCCTTTCTGCTGATCCCCCGGAAAGTGTGCAACGGCATCGGCATCGGAGAGTT

[1118] CAAGGACAGCCTGTCCATCAACGCCACCAACATCAAGCACTTCAAGAATTGCACCAGCATC

[1119] AGCGGCGACCTGCACATCCTGCCAGTGGCCTTTAGAGGCGACAGCTTCACCCACACCCCC

[1120] CCACTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGC

[1121] TGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCAT ACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACA TCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACA AAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAA

[1122] ACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATG CCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGG AATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGG GAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATG AACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACG GCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCT GGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGAT GCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCA CTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCA TGTGA

[1123] Complete CAR amino acid sequence of aCD20(GA101)_lgG4long_CD28tm_41 BB_CD3z_EGFRt (Beginning of scFv to end of CD3z) (SEQ ID NO: 76):

[1124] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQCLEWMGRIFPGDGDTDYN GKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSGGGG SGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLI YQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGCGTKVEIKESKY

[1125] GPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD

[1126] KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVWGGVLACYSLLVTVAFIIFWVK RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR 9. aR0R1 (R12)_lgG4short_CD28tm_4-1 BB_EGFRt

[1127] Nucleotide sequence of GMCSF signaling peptide (SEQ ID NO: 77):

[1128] ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTG

[1129] ATCCCC

[1130] Amino Acid sequence of GMCSF signaling peptide (SEQ ID NO: 2): MLLLVTSLLLCELPHPAFLLIP

[1131] Nucleotide sequence of anti-ROR1 (R12) scFv variable heavy chain (SEQ ID NO: 78):

[1132] CAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACACCTGGCGGCAGCCTGACCCT

[1133] GAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTCCGCCAGGC

[1134] CCCTGGCAAGGGACTGGAATGGATCGCCACCATCTACCCCAGCAGCGGCAAGACCTACTA

[1135] CGCCACCTGGGTGAACGGACGGTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGA

[1136] CCTGCAGATGAACAGCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAG

[1137] CTACGCCGACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTC

[1138] TAGC

[1139] Amino Acid sequence of anti-ROR1 (R12) scFv variable heavy chain (SEQ ID NO: 79): QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATW

[1140] VNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISS

[1141] Nucleotide sequence of G4S(3) Linker (SEQ ID NO: 80):

[1142] GGCGGAGGCGGATCCGGAGGCGGAGGAAGTGGCGGCGGAGGATCT

[1143] Amino Acid sequence of G4S(3) Linker (SEQ ID NO: 71):

[1144] GGGGSGGGGSGGGGS

[1145] Nucleotide sequence of anti-ROR1 (R12) scFv variable light chain (SEQ ID NO: 81):

[1146] GAGCTGGTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATC

[1147] ACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCAGCTGCAG

[1148] GGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCTACACCAAGAGGCC

[1149] AGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCCGACCGCTACCTGATCATCCC

[1150] CAGCGTGCAGGCCGATGACGAGGCCGATTACTACTGTGGCGCCGACTACATCGGCGGCTA

[1151] CGTGTTCGGCGGAGGCACCCAGCTGACCGTGACCGGC Amino Acid sequence of anti-ROR1 (R12) scFv variable light chain (SEQ ID NO: 82):

[1152] ELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVP

[1153] DRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG

[1154] Nucleotide sequence of lgG4 hinge short (SEQ ID NO: 65): GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT

[1155] Amino Acid sequence of lgG4 hinge short (SEQ ID NO: 10):

[1156] ESKYGPPCPPCP

[1157] Nucleotide sequence of CD28 transmembrane domain (SEQ ID NO: 11):

[1158] ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACC GTGGCCTTCATCATCTTTTGGGTG

[1159] Amino Acid sequence of CD28 transmembrane domain (SEQ ID NO: 12): MFVWLVWGGVLACYSLLVTVAFIIFVW

[1160] Nucleotide sequence of 4-1 BB domain (SEQ ID NO: 13):

[1161] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAAC

[1162] TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA CTG

[1163] Amino Acid sequence of4-1 BB domain (SEQ ID NO: 14):

[1164] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[1165] Nucleotide sequence of CD3z domain (SEQ ID NO: 15):

[1166] CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCT

[1167] GTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAG

[1168] GCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTAT

[1169] AACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGA

[1170] GCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGG

[1171] ATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG

[1172] Amino Acid sequence of CD3z domain (SEQ ID NO: 16): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[1173] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[1174] Nucleotide sequence of T2A ribosomal skipping sequence (SEQ ID NO: 17):

[1175] CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAA TCCCGGCCCTAGG

[1176] Amino Acid sequence of T2A ribosomal skipping sequence (SEQ ID NO: 18): (not synthesized by ribosome)

[1177] LEGGGEGRGSLLTCGDVEENPGPR

[1178] Nucleotide sequence of GM-CSF signaling peptide (SEQ ID NO: 19):

[1179] ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGAT COCA

[1180] Amino Acid sequence of GM-CSF signaling peptide (SEQ ID NO: 2):

[1181] MLLLVTSLLLCELPHPAFLLIP

[1182] Nucleotide sequence of EGFRt transduction marker (SEQ ID NO: 21):

[1183] CGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTAC

[1184] GAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTG

[1185] GCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCT

[1186] GAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACG

[1187] GACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGT

[1188] TTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATA

[1189] AGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTG

[1190] GAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAAC

[1191] AGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGG

[1192] CCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGG

[1193] ACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATAC

[1194] AGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAG

[1195] ACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGG

[1196] CAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGT

[1197] GCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTC CAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGC

[1198] TGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[1199] Amino Acid sequence of EGFRt transduction marker (SEQ ID NO: 22):

[1200] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI

[1201] TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKN

[1202] LCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR

[1203] GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT

[1204] CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LVVALGIGLFM*

[1205] Complete nucleotide sequence of aROR1(R12)_lgG4short_CD28tm_4-1 BB_EGFRt (Underlined is CAR sequence from beginning of scFv to the end of CD3z) (SEQ ID NO: 83):

[1206] ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTG

[1207] ATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACACCTGGCGGCAGCCT

[1208] GACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTCCG

[1209] CCAGGCCCCTGGCAAGGGACTGGAATGGATCGCCACCATCTACCCCAGCAGCGGCAAGA

[1210] CCTACTACGCCACCTGGGTGAACGGACGGTTCACCATCTCCAGCGACAACGCCCAGAACA

[1211] CCGTGGACCTGCAGATGAACAGCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCA

[1212] GAGACAGCTACGCCGACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTG

[1213] ACAATCTCTAGCGGCGGAGGCGGATCCGGAGGCGGAGGAAGTGGCGGCGGAGGATCTGA

[1214] GCTGGTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATCAC

[1215] CTGTACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCAGCTGCAGGG

[1216] CGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCTACACCAAGAGGCCAG

[1217] GCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCCGACCGCTACCTGATCATCCCCA

[1218] GCGTGCAGGCCGATGACGAGGCCGATTACTACTGTGGCGCCGACTACATCGGCGGCTAC

[1219] GTGTTCGGCGGAGGCACCCAGCTGACCGTGACCGGCGAATCTAAGTACGGACCGCCCTG

[1220] CCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAG

[1221] CCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG

[1222] TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG

[1223] CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAG

[1224] CGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGG

[1225] CAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCG GCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGA

[1226] TGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCA

[1227] CGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACAT

[1228] GCAGGCCCTGCCCCCAAGGCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACAT

[1229] GCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGC

[1230] TCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGG

[1231] TATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTG

[1232] CACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACA

[1233] CATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAG

[1234] GGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCT

[1235] AGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTG

[1236] AACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTC

[1237] AGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCG

[1238] GTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGT

[1239] CTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCT

[1240] CTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG

[1241] AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTC

[1242] AGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACT

[1243] ACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACA

[1244] CCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCA

[1245] CCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGT

[1246] CCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATC GGCCTCTTCATGTGA

[1247] Complete CAR amino acid sequence of aROR1 (R12)_lgG4short_CD28tm_4-1 BB_EGFRt

[1248] (Beginning of scFv to end of CD3z) (SEQ ID NO: 84):

[1249] QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATW

[1250] VNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGS

[1251] GGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQS

[1252] DGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTGESK

[1253] YGPPCPPCPMFWVLWVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDG

[1254] CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG

[1255] GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

[1256] ALPPR 10. a-CD20 (GA101) P329G IgG

[1257] GA101 PGLALA Heavy chain

[1258] VH (SEQ ID NO: 85):

[1259] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYN

[1260] GKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS

[1261] CH1 (SEQ ID NO: 86):

[1262] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

[1263] SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[1264] Hinge (SEQ ID NO: 87):

[1265] EPKSC

[1266] Core Hinge (SEQ ID NO: 88):

[1267] DKTHTCPPCP

[1268] CH2 (PG LALA) (SEQ ID NO: 89):

[1269] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[1270] EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK

[1271] CH3 (SEQ ID NO: 90):

[1272] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

[1273] F F LYS KLTVDKS RWQQG N VFSCS VM H EALH N H YTQ KS LS LS PG

[1274] Complete heavy chain (SEQ ID NO: 91):

[1275] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYN

[1276] GKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKG

[1277] PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW

[1278] TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT

[1279] LMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD

[1280] WLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

[1281] KSLSLSPG

[1282] GA101 PGLALA Light chain

[1283] VL (SEQ ID NO: 92):

[1284] DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPD

[1285] RFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK

[1286] CL kappa (SEQ ID NO: 93):

[1287] RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

[1288] STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

[1289] Hinge (SEQ ID NO: 94):

[1290] NRGEC

[1291] Complete light chain (SEQ ID NO: 95):

[1292] DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPD

[1293] RFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQL

[1294] KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK

[1295] HKVYACEVTHQGLSSPVTKSFNRGEC

[1296] 11. a-CD19 (FMC63) P329G IgG

[1297] FMC63 PGLALA Heavy chain

[1298] VH (SEQ ID NO: 64):

[1299] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL

[1300] KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[1301] CH1 (SEQ ID NO: 86):

[1302] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

[1303] SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[1304] Hinge (SEQ ID NO: 87):

[1305] EPKSC Core Hinge (SEQ ID NO: 88):

[1306] DKTHTCPPCP

[1307] CH2 (PG LALA) (SEQ ID NO: 89):

[1308] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[1309] EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK

[1310] CH3 (SEQ ID NO: 90):

[1311] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

[1312] F F LYS KLTVDKS RWQQG N VFSCS VM H EALH N H YTQ KS LS LS PG

[1313] Complete heavy chain (SEQ ID NO: 96):

[1314] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL

[1315] KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSASTKGP

[1316] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT

[1317] VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL

[1318] MISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW

[1319] LNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

[1320] VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG

[1321] FMC63 PGLALA Light chain

[1322] VL (SEQ ID NO: 60):

[1323] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSG

[1324] SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[1325] CL kappa (SEQ ID NO: 93):

[1326] RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

[1327] STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

[1328] Hinge (SEQ ID NO: 94):

[1329] NRGEC Complete light chain (SEQ ID NO: 97): DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSG SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

[1330] EXAMPLES

[1331] The present invention is further illustrated by the following non-limiting examples.

[1332] Example 1 - P329G CAR T cells lyse lymphoma cells in vitro

[1333] Antigen escape is a significant challenge for sustained success of targeted immunotherapies. The inventors therefore aimed to employ the P329G CAR platform to target lymphoma in a modular fashion. They therefore generated full sized P329G Fc-mutated adapter lgG1 against CD19 (clone FMC63) and CD20 (clone GA101). The inventors first aimed to optimize the P329G CAR construct design by generating P329G CAR constructs incorporating different extracellular spacer domains (Fig. 1 A). P329G CAR gene transfer into primary T cells yielded similar surface expression of EGFRt transduction marker for all P329G CAR variants (Fig. 1 B). The inventors tested dose-dependent cytolytic activity of each spacer iteration in combination with a-CD19 and a- CD20 IgG (Fig. 1 C). Based on the decreased EC50 values for P329G CAR variants with short spacer designs (Fig. 1 D), the inventors selected the lgG3-derived spacer design for further studies. The inventors investigated the ability of P329G CAR T cells to lyse NHL tumor cell lines in combination with the a-CD19 and a-CD20 IgG. For both adapter molecules, P329G CAR T cell activity was dependent on adapter concentration and antigen expression on the target cell (Fig. 1 D, Fig. 7A, B). Notably, at low IgG concentrations, the inventors observed stronger P329G CAR cytotoxicity for the a-CD20 IgG than for the a-CD19 IgG. Adapter IgG did not activate P329G CAR T cells in absence of tumor cells (Fig. 7C).

[1334] Taken together, P329G CAR T in conjunction with a-CD19 and a-CD20 adapters show functionality against lymphoma in vitro, which depends on tumor antigen expression and CAR spacer design.

[1335] Example 2 - Functional comparison of P329G CAR T and conventional CAR T cells against NHL in vitro

[1336] The inventors next aimed to compare functionality of P329G CAR T cells to conventional CAR T cells targeting the same epitopes. Cytotoxic capacity of P329G CAR T cells targeting CD19 on a panel of NHL cell lines using the a-CD19 (FMC63) IgG was significantly reduced compared to that of conventional CD19 (FMC63) CAR T cells (Fig. 2A). In combination with the a-CD20 (GA101) IgG, P329G CAR tumor cell lysis was more comparable but still reduced to conventional CD20 (GA101) CAR T cells (Fig. 2A). The inventors observed similar trends for CAR T cell degranulation and IFN-y production (Fig. 2B).

[1337] Next, the inventors tested sensitivity of P329G CAR T cells and conventional CAR T cells to activation-induced dysfunction in an in vitro assay of constant antigen exposure (CAE). To this end, they subjected P329G CAR T cells + a-CD20 IgG and CD20 CAR T cells to repeated stimulation with a high number of tumor cells (Fig. 2C). While initial cytotoxicity was similar between P329G CAR T cells and CD20 CAR T cells, the inventors observed a significantly lower lytic activity of CD20 CAR T cells after cycle 4 and therefore stronger retention of functionality in P329G CAR T cells (Fig. 2D). This effect was accompanied by elevated surface coexpression of TIM-3, LAG-3 and PD-1 (Fig. 2E), as well as increased transcriptional levels of TOX in CD20 CAR T cells (Fig. 2F). In summary, P329G CAR T cells are less active than conventional CAR T cells but retain superior cytolytic activity and a phenotype of less exhausted T cells during sequential stimulation.

[1338] Example 3 - Conventional and adapter dual (CAD) CAR T cells are bi-functional and reactive in vivo

[1339] To combine the strong anti-tumor functionality of conventional CAR T cells with the flexible antigen targeting capabilities of adapter CAR T cells, the inventors explored the approach of co-expressing a conventional CAR and an adapter CAR to generate conventional and adapter dual (CAD) CAR T cells (Fig. 3A). The inventors combined expression of the CD19 (FMC63) CAR and the P329G CAR (Fig. 3B) on primary T cells. Simultaneous gene transfer of both CAR vectors followed by EGFRt / LNGFRt enrichment yielded a pure population of CD19 CAD CAR T cells co-expressing EGFRt and LNGFRt (Fig. 3C). The inventors characterized bi-functionality of CD19 CAD CAR T cells in vitro. In absence of adapter, CD19 CAD CAR T cells lysed CD19-expressing lymphoma cells in a similar fashion to CD19 CAR T cells. Crucially, in combination with increasing concentrations of a-CD20 IgG, CD19 CAD CAR T cells were able to lyse CD19- K562 overexpressing CD20 (K562 CD20) similarly to P329G CAR T cells (Fig. 3D), thereby confirming dual reactivity as conventional and adapter CAR T cells.

[1340] The inventors next tested P329G CAR, conventional CAR and CAD CAR T cells against a xenograft model of lymphoma in vivo. To this end, the inventors treated NOD.Cg-PrkdcscidH2rgtm1 SzJ (NSG) mice systemically engrafted with CD19+CD20+Daudi cells expressing firefly luciferase (Daudi ffluc) (Fig. 8A) by infusion with CAR T. Twice weekly, mice were injected i.p. with PBS or 5 g of a-CD20 IgG. Tumor growth was monitored weekly via bioluminescent imaging over a follow-up time of 90 days (Fig. 3E). Treatment with CD20 CAR T cells and P329G CAR T cells +a-CD20 IgG induced comparable control of Daudi tumors (Fig. 3F, G). In comparison with P329G CAR T cells +a-CD20 IgG, the inventors detected a higher percentage of CD20 CAR T cells in the bone marrow and, if present, in kidney metastasis at time of sacrifice (Fig. 3H). A higher fraction of CAR T cells present in the bone marrow expressed PD-1 in CD20 CAR T treated mice as opposed to mice treated with P329G CAR T +a-CD20 IgG (Fig. 3I, Fig. 8C). Daudi tumors exposed to CD20 CAR T cells or P329G CAR T cells + a-CD20 IgG did not change surface expression of CD19 and CD20 compared to control groups (Fig. 8D). Infusion of CD19 CAD CAR T cells in combination with a-CD20 IgG induced efficient eradication of Daudi tumors to a similar level of CD19 CAR T cells, leading to survival of a majority of mice (Fig. 3 J, K). Taken together, P329G CAR T cells in combination with a-CD20 IgG perform similarly to conventional CD20 CAR T cells in a lymphoma xenograft model but display reduced enrichment at the tumor site. Moreover, bi-functional CAD CAR T cells targeting CD19 and CD20 have potent functionality against a homogeneous lymphoma population in vivo.

[1341] Example 4 - CAD CAR T cells allow efficient dual targeting of NHL

[1342] The inventors next tested the ability of CAD CAR T cells to tackle antigen negative relapse in a model of antigen-loss. They co-incubated Raji and JeKo-1 tumor cells with CD19 CAR T cells, as well as CD19 mono- or CD19 and CD20 dual-targeting P329G CAR T cells or CAD CAR T cells. Upon eradication of tumor cells, the inventors mimicked antigen loss by re-challenge with CD19KO JeKo-1 or Raji cells. While CD19KO tumor cells expanded in conditions where solely CD19 is targeted (CD19 CAR, P329G CAR + a-CD19 IgG, CD19 CAD CAR), tumor outgrowth could be efficiently controlled by dual targeting with CD19 CAD CAR T cells + a-CD20 IgG and P329G CAR T cells + a-CD19 + a-CD20 IgG (Fig. 4A). Although CD19 and CD20 dual targeting was achieved with both, P329G CAR T cells and CD19 CAD CAR T cells, functionality against CD19+CD20+tumor cells by CD19 CAD CAR T cells was not adapter-dependent (Fig 4B). Moreover, at low E:T ratio of 1 :2, the inventors found tumor lysis through dual targeting with CD19 CAD CAR T cells +a-CD20 IgG to be more efficient than dual targeting with P329G CAR T cells + a-CD19 IgG + a-CD20 IgG (Fig. 4C, Fig. 9A). In summary, dual-targeting CD19 CAD CAR T cells are able to control NHL tumor cells in a model of CD19- relapse and display superior characteristics compared to dual-targeting P329G CAR T cells.

[1343] Example 5 - R0R1 CAD CAR T cells allow multi-antigen targeting of lymphoma

[1344] To further expand the novel CAD CAR platform to additional target antigens, the inventors generated R0R1 CAD CAR T cells by co-expressing a R0R1 CAR and the P329G CAR on primary T cells (Fig. 5A, B). The inventors opted for CD28 co-stimulation in the P329G CAR construct since they observed improved P329G CAR performance compared to 41 BB co-stimulation (Fig. 9B) and aimed for synergistic co-stimulation in R0R1 CAD CAR T cells, similar to a third-generation CAR

[0017] , The inventors confirmed dual reactivity of R0R1 CAD CAR T cells in vitro (Fig. 5C, D) and explored whether simultaneous engagement of the R0R1 CAR and the P329G CAR in R0R1 CAD CAR T cells leads to improved CAR functionality compared to engaging only the R0R1 CAR. Against cell lines expressing high levels of R0R1 (JeKo-1, Daudi R0R1, Fig. 7A) addition of a-CD19 or a-CD20 IgG did not lead to elevated tumor cell lysis by R0R1 CAD CAR T cells (Fig. 5E). However, against ROR1low(Raji) or ROR- (daudi, Fig. 7A) addition of a-CD19 or a-CD20 IgG significantly increased tumor cell lysis (Fig. 5E), T cell degranulation and IFN-y production (Fig. 5F). Using the z-Movi platform, the inventors measured avidity of R0R1 CAR T and R0R1 CAD CAR T cells against Raji tumor cells. While avidity of R0R1 CAD CAR T cells in absence of adapter was similar to R0R1 CAR T cells, addition of a-CD20 IgG to R0R1 CAD CAR T cells increased avidity (Fig. 5G). Moreover, engaging both CAR receptors in ConvROR1AD CAR T cells via addition of adapter, increased proliferation in ConvROR1AD20compared to ConvROR1AD CAR T cells in tumor cells with low or no R0R1 expression, but did not significantly affect viability (Figure 9C, D). Likewise, engaging both CARs did not affect long-term expansion or expression of TIM-3, LAG-3 or PD-1 in ConvROR1AD20CAR T cells upon repeated stimulation with irradiated JeKo-1 tumor cells (Figure 9E).

[1345] The inventors next set out to test the potential of R0R1 CAD CAR T cells to target a mixed NHL tumor cell population with heterogeneous antigen expression. To this end, the inventors combined RORT Daudi and Daudi R0R1 in a 1 :3 ratio (Fig. 5H) and co-incubated them with R0R1 CAR T cells or R0R1 CAD CAR T cells with and without addition of a-CD19 or a-CD20 IgG. In absence of adapter, R0R1 CAD CAR T cells were able to lyse solely the R0R1+fraction of Daudi cells, equally to R0R1 CAR T cells. Crucially, addition of a-CD19 and especially a-CD20 IgG to R0R1 CAD CAR-T cells achieved eradication of both R0R1+and R0R1- Daudi target cells (Fig. 5I).

[1346] These data demonstrate that CAD CAR T cells can be generated with different conventional CAR constructs and that dual targeting with R0R1 CAD CAR T cells improves in vitro lysis of lymphoma cells with reduced, lack of, or heterogeneous R0R1 expression.

[1347] Example 6 - R0R1 CAD CAR T cells eradicate lymphoma in a mouse model of antigen escape

[1348] The inventors tested whether the ability of CAD CAR T cells to target a heterogeneous tumor cell population translates to the in vivo setting. They therefore engrafted NSG mice with a mixed population of 60% ROR1+and 40% ROR1- Daudi ffluc cells (Fig. 10A) and subsequently infused ROR1 CAR T cells, P329G CAR T cells or ROR1 CAD CAR T cells (Fig. 6A, Fig. 10B). Upon CAR T cell infusion, mice were injected with a- CD20 IgG or PBS twice weekly (Fig. 6A). Compared to UTD control T cells, ROR1 CAR T cells and ROR1 CAD CAR T cells + PBS only displayed minimal anti-tumor effects, without notably prolonging survival of treated mice (Fig. 6B-E). Concordantly, Daudi cells isolated at endpoint from BM of mice treated with R0R1 CAR or R0R1 CAD CAR T cells + PBS showed lack of R0R1 surface expression, but retained CD20 levels (Fig. 6F). In contrast, R0R1 and CD20 dual targeting by R0R1 CAD CAR T cells + a-CD20 IgG prevented tumor outgrowth of the heterogeneous Daudi population and led to survival of all treated mice during the follow-up time of 90 days (Fig. 6B-E). Likewise, P329G CAR T cells + a-CD20 IgG also showed significant anti-tumor efficacy, leading to survival of two thirds of mice.

[1349] The single ConvROR1AD20CAR T cell-treated mouse experiencing tumor outgrowth retained CD20 expression on isolated Daudi tumor cells (Figure 10C). Taken together, dual-targeting ConvAD CAR T cells enable tumor eradication in a xenograft model of lymphoma that is resistant to conventional CAR T cells due to heterogeneous antigen expression.

[1350] In summary, ROR1 CAD CAR T cells allow for efficient eradication of lymphoma with heterogeneous antigen expression by dual antigen targeting through the conventional and the P329G adapter CAR.

[1351] Example 7: Triple-antiqen-tarqetinq ConvAD CAR T cells outperform conventional bispecific CAR T cells in the event of dual antigen loss

[1352] In order to benchmark ConvAD CAR T cells to the clinically most advanced multi-targeted CAR T cell approach, the inventors investigated how ConvAD CAR T cells perform against conventional bispecific CAR T cells. They first generated ConvROR1AD CAR T cells and ROR1 / CD19-bispecific dual CAR T cells coexpressing a ConvROR1CAR and a Conv19CAR (Figure 11 A, Figure 12A, B). They compared cytotoxicity of dual ConvROR1 / Conv19CAR T cells to triple-targeting ConvROR1AD19+2° CAR T cells when incubated with the mantle cell lymphoma (MCL) cell line JeKo-1. In vitro, ConvROR1 / Conv19CAR T cells and ConvROR1AD19+2° CAR T cells lysed wild-type and single-antigen-deficient (ROR1K0or CD19K0) tumor cells comparably (Figure 11 B, Figure 12C). However, when CAR T cells were challenged with JeKo-1 cells lacking both antigens, only triple-targeting ConvROR1AD19+2° CAR T cells induced tumor cell lysis, mediated by additional targeting of CD20 (Figure 11 B). Likewise, ConvROR1AD19+2° CAR T cells eradicated and prevented tumor outgrowth in an in vitro antigen escape model in which CAR T cells were sequentially stimulated with ROR1+CD19+CD20+, ROR1 CD19+CD20+and ROR1 CD19 CD20+lymphoma cells, whereas bispecific ConvROR1 / Conv19failed to achieve this (Figure 11C). Moreover, compared to ConvROR1 / Conv19CAR T cells, ConvROR1AD19+2° CAR T cells showed a significantly higher lysis of total JeKo-1 tumor cells and of JeKo-1 ROR1KOCD19KOwhen coincubated with a 1 :1 :1 mixture of JeKo-1, JeKo-1 ROR1KOand JeKo-1 ROR1KOCD19KO(Figure 11 D, E). Finally, in a xenograft model of dual ROR1 / CD19 antigen escape (Figure 11 F, G), trispecific ConvROR1AD19+2° CAR T cells achieved stronger initial tumor regression (Figure 11 H, I) and significantly prolonged survival (Figure 11 J) compared to bispecific ConvROR1 / Conv19CAR T cells. At endpoint analysis, tumors of ConvROR1 / Conv19CAR T cell-treated mice lacked expression of R0R1 and CD19 (Figure 11 K). ConvROR1AD19+2° CAR-treated mice that relapsed after initial complete response exhibited absence of CAR T cells from peripheral blood and BM (Figure 12D, E), while JeKo-1 tumor cells of these mice maintained CD20 expression (Figure 11 K). One ConvROR1AD19+2° CAR-treated mouse relapsed with ROR1 CD19 CD20- tumor but did not reach cutoff criteria during the observation period (Figure 12F).

[1353] Lastly, the inventors compared ConvAD CAR T cells to bispecific CD19 / CD20 dual CAR T cells, coexpressing a Conv19and Conv20CAR, and to bispecific CD19 / CD20 tandem (Tan19+2°) CAR T cells, expressing a single CAR directed against both antigens. For this, they generated dual Conv19 / Conv20CAR T cells, Tan19 / 2° CAR T cells, and Conv19AD CAR T cells (Figure 11 L, Figure 12G). Compared to single-targeting Conv19CAR and Conv20CAR T cells, bispecific Conv19 / Conv20CAR and Tan19 / 20CAR T cells were able to lyse both, CD19+CD20- and CD19 CD20+tumor cells but equally failed to lyse CD19 CD20- tumor cells (Figure 11 M, N, Figure 12C). Crucially, through addition of an a-CD20(GA101) and an a-R0R1(R12) adapter, trispecific Conv19ADROR1+2° CAR T cells were additionally capable of lysing CD19 CD20 ROR1+tumor cells (Figure 11 M, N, Figure 12C). Moreover, after eradication of CD19+CD20+ROR1+JeKo-1 cells, Conv19ADROR1+2° CAR T cells showed preserved tumor lysis upon subsequent re-stimulation with CD19KOCD20KOROR1+JeKo-1 while Conv19 / Conv20CAR T cells and Tan19 / 2° CAR T cells failed (Figure 110). In summary, multi-antigen targeting with ConvAD CAR T cells is able to outperform conventional dual-antigen- targeting CAR T cells in the setting of dual antigen loss in vitro and in vivo. This underlines the considerable potential of ConvAD CAR T cells in malignancies at risk of antigen escape and warrants future clinical testing.

[1354] INDUSTRIAL APPLICABILITY

[1355] The recombinant mammalian cell of the invention can be manufactured industrially and sold, e.g., in the form of pharmaceutical compositions or medicaments for the treatment of diseases such as, for instance, cancer. Thus, the invention is industrially applicable.

[1356] REFERENCES

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Claims

CLAIMS1 . A recombinant mammalian cell co-expressing at least:(a) a CAR comprising a targeting domain binding to a cancer antigen expressed on the surface of cancer cells and(b) a CAR comprising a targeting domain binding to an adapter molecule.

2. The recombinant mammalian cell according to claim 1, wherein said recombinant mammalian cell is a human cell, preferably a human immune cell.

3. The recombinant mammalian cell according to claim 2, wherein the human immune cell is a lymphocyte.

4. The recombinant mammalian cell according to claim 3, wherein the lymphocyte is a B lymphocyte, a T lymphocyte, or a NK cell.

5. The recombinant mammalian cell according to claim 4, wherein the lymphocyte is a T lymphocyte, preferably wherein the lymphocyte is a CD4-positive T cell or a CD8-positive T cell.

6. The recombinant mammalian cell according to any one of claims 1-5, wherein the adapter molecule binds to cells of said cancer and is preferably a monoclonal antibody or antigen-binding fragment thereof binding to a cancer antigen of the cancer.

7. The recombinant mammalian cell according to claim 6, wherein the monoclonal antibody or antigenbinding fragment thereof carries a P329G mutation according to EU numbering and the targeting domain of the CAR according to (b) binds to antibodies carrying a P329G mutation according to EU numbering and preferably comprises an scFv containing, in an N- to C-terminal order: i) an anti-P329G variable heavy chain amino acid sequence according to SEQ ID NO: 4 or an anti-P329G variable heavy chain having the same CDRs as the amino acid sequence according to SEQ ID NO: 4, ii) an scFv linker preferably consisting of the amino acid sequence according to SEQ ID NO:6, andii) an anti-P329G variable light chain amino acid sequence according to SEQ ID NO: 8 or an anti-P329G variable light chain having the same CDRs as the amino acid sequence according to SEQ ID NO: 8.

8. The recombinant mammalian cell of claim 6 or 7, wherein the monoclonal antibody or antigen-binding fragment thereof binding to a cancer antigen of the cancer binds to the cancer antigen on the surface of cancer cells of the cancer.

9. The recombinant mammalian cell of any one of claims 1 -8, wherein the cancer antigen expressed on the surface of cancer cells, which is bound by the CAR according to (a), is selected from the group consisting of CD19, CD20, and R0R1.

10. The recombinant mammalian cell of any one of claims 1 -9, wherein the CAR comprising the targeting domain binding to an adapter molecule has an lgG3-derived spacer between the transmembrane domain and the targeting domain, wherein the spacer preferably consists of SEQ ID NO: 57.

11. A nucleic acid or a set of nucleic acids encoding the CARs as defined in any one of claims 1-10.

12. An expression vector comprising the nucleic acid of claim 11, or a set of expression vectors comprising the set of nucleic acids of claim 11 .

13. A method for producing a recombinant mammalian cell according to any one of claims 1-10, the method comprising the steps of:(I) providing a mammalian cell;(II) introducing into said mammalian cell of step (I) the nucleic acid or set of nucleic acids according to claim 11 or the expression vector or the set of expression vectors according to claim 12; and(III) co-expressing said nucleic acid or said set of nucleic acids according to claim 11 or said expression vector or said set of expression vectors according claim 12, thereby obtaining said recombinant mammalian cell.

14. A pharmaceutical composition comprising the recombinant mammalian cell according to any one of claims 1-10, the composition preferably further comprising one or more adapter molecules as defined in any one of claims 1-8 and optionally further comprising a pharmaceutically acceptable carrier and / or excipient.

15. A recombinant mammalian cell according to any one of claims 1-10 or a pharmaceutical composition according to claim 14 for use as a medicament.

16. A recombinant mammalian cell according to any one of claims 1-10 or a pharmaceutical composition according to claim 14 for use in a method of treating a cancer.

17. A recombinant mammalian cell or pharmaceutical composition for use according to claim 16, wherein the cancer is lymphoma or a solid cancer.

18. A recombinant mammalian cell or pharmaceutical composition for use according to claim 17, wherein the cancer is non-Hodgkin lymphoma.

19. A recombinant mammalian cell or pharmaceutical composition for use according to any one of claims 15-18, wherein the recombinant mammalian cell or pharmaceutical composition is to be coadministered with one or more adapter molecules as defined in any one of claims 6-8.

20. A recombinant mammalian cell or pharmaceutical composition for use according to any one of claims 15-19, wherein the one or more adapter molecules is / are administered before, after, or simultaneously with the recombinant mammalian cell.

21. A kit comprising(A) a recombinant mammalian cell and(B) an adapter molecule, wherein the recombinant mammalian cell according to (A) and the adapter molecule according to (B) are as defined in any one of claims 1-10.

22. A kit comprising(A) a nucleic acid, set of nucleic acids, an expression vector, or a set of expression vectors encoding the CARs as defined in any one of claims 1-10 and(B) the adapter molecule of the CARs as defined in any one of claims 1-10.

23. The recombinant mammalian cell or kit or pharmaceutical composition according to any one of the preceding claims, wherein said recombinant mammalian cell is not an embryonic cell and has not been obtained by a process for modifying the germ line genetic identity of a human being.

24. The kit or pharmaceutical composition according to any one of the preceding claims, wherein the adapter molecule of the kit according to (A) or the one or more adapter molecules of the pharmaceutical composition is a single adapter molecule which binds to cells of said cancer and is a monoclonal antibody or antigen-binding fragment thereof, preferably according to claim 7, that binds to a cancer antigen of the cancer which is preferably selected from CD19, CD20, and R0R1.

25. The kit or pharmaceutical composition according to any one of claims 1 -23, wherein the kit comprises at least two adapter molecules according to (A) or the pharmaceutical composition comprises at least two adapter molecules, wherein the adapter molecules bind to cells of said cancer and are monoclonal antibodies or antigen-binding fragments thereof, preferably according to claim 7, that bind to cancer antigens of the cancer which are preferably selected from CD19, CD20, and R0R1.

26. The kit or pharmaceutical composition according to any one of claims 1 -23, wherein the kit comprises three adapter molecules according to (A) or the pharmaceutical composition comprises three adapter molecules, wherein the adapter molecules bind to cells of said cancer and are monoclonal antibodies or antigen-binding fragments thereof, preferably according to claim 7, that bind to cancer antigens of the cancer which are preferably selected from CD19, CD20, and R0R1.

27. The kit or pharmaceutical composition according to any one of claims 24-26, wherein the monoclonal antibodies or antigen-binding fragments thereof bind to cancer antigens which are different from the cancer antigen expressed on the surface of cancer cells which is bound by the CAR according to (a).

28. The method according to any one of the preceding claims, wherein all the steps of the method are carried out in vitro.

29. The method according to any one of the preceding claims, wherein said method does not comprise a method for treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body.

30. The method according to any one of the preceding claims, wherein said method does not comprise a process for modifying the germ line genetic identity of a human being.

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