NZ738636A - Method and compositions for cellular immunotherapy - Google Patents

Abstract

The invention relates to a nucleic acid encoding a chimeric receptor, the chimeric receptor comprising: (a) an extracellular domain consisting of: an extracellular ligand binding domain consisting of a single chain variable fragment (scFv) that binds to a CD19, wherein the scFv comprises a variable light chain (VL) domain comprising a CDRL1, a CDRL2, and a CDRL3 of the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a CDRH1, a CDRH2, and a CDRH3 of the amino acid sequence encoded by SEQ ID NO:3; and an extracellular polypeptide spacer that is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); (b) a transmembrane domain; and (c) an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain. Also provided is a nucleic acid encoding a chimeric receptor, the nucleic acid comprising: (a) a polynucleotide encoding a single chain variable fragment (scFv) that binds to CD19, wherein the scFv comprises: a variable light chain (VL) domain comprising a VL domain present in the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a VH domain present in the amino acid sequence encoded by SEQ ID NO:3; or the amino acid sequence encoded by SEQ ID NO:3; (b) a polynucleotide encoding a transmembrane domain; (c) a polynucleotide encoding a polypeptide spacer located between the scFv and the transmembrane domain, wherein the polypeptide spacer is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); and (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain. Also provided are vectors that include the nucleic acids and methods of using the nucleic acids and vectors.

Description

METHOD AND COMPOSITIONS FOR CELLULAR IMMUNOTHERAPY This application is a divisional of New Zealand patent application 705475, which is the national phase entry in New Zealand of PCT international application (published as WO 2014//031687), and claims the benefit of priority to U.S. patent application serial no. 61/691,117 filed on 20 August 2012, the disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention The present invention relates to the field of biomedicine and specifically methods useful for cancer therapy. In particular, embodiments of the invention relate to methods and compositions for ng out cellular immunotherapy comprising T cells modified with tumor targeting receptors.

Statement Regarding lly Sponsored Research This invention was made with government support in the form of grants from the United States Department of Health and Human es and from the Leukemia and Lymphoma Society. The United States government has n rights in the invention.

Background of the Invention The adoptive transfer of human T lymphocytes that are engineered by gene transfer to express chimeric antigen receptors (chimeric receptors) specific for surface molecules expressed on tumor cells has the ial to effectively treat advanced malignancies. Chimeric receptors are synthetic ors that include an extracellular ligand binding domain, most commonly a single chain variable fragment of a monoclonal antibody (scFv) linked to intracellular signaling components, most ly CD3ζ alone or combined with one or more costimulatory s. Much of the research in the design of chimeric receptors has focused on defining scFvs and other ligand g elements that target malignant cells without causing serious toxicity to essential normal tissues, and on defining the l composition of intracellular ing modules to activate T cell effector functions. r, it is uncertain whether the variations in chimeric receptor design that mediate superior in vitro function will translate reproducibly into improved in vivo therapeutic activity in clinical applications of chimeric receptormodified T cells.

There is a need to identify methods for determining elements of ic receptor design that are important for therapeutic activity and cell populations to genetically modify and adoptively transfer that provide ed survival and efficacy in vivo. It an object of the present invention to go someway towards g this need and/or to provide the public with a useful choice.

Summary of the Invention In a first aspect the present invention provides a nucleic acid encoding a chimeric receptor, the chimeric receptor comprising: (a) an extracellular domain consisting of: an extracellular ligand binding domain consisting of a single chain variable fragment (scFv) that binds to a CD19, wherein the scFv comprises a variable light chain (VL) domain comprising a CDRL1, a CDRL2, and a CDRL3 of the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a CDRH1, a CDRH2, and a CDRH3 of the amino acid sequence encoded by SEQ ID NO:3; and an extracellular polypeptide spacer that is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a ne, glycine, or arginine and X2 is a cysteine or a ine (SEQ ID NO:1); (b) a transmembrane domain; and (c) an ellular signaling domain that comprises a CD3ζ ing domain and a costimulatory domain.

In a second aspect the present invention provides a nucleic acid encoding a chimeric or, the nucleic acid comprising: (a) a cleotide encoding a single chain variable fragment (scFv) that binds to CD19, wherein the scFv comprises: the amino acid sequence encoded by SEQ ID NO:3; (b) a polynucleotide encoding a transmembrane domain; (c) a polynucleotide encoding a polypeptide spacer located between the scFv and the transmembrane domain, wherein the ptide spacer is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); and (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain.

In a third aspect the t invention provides an expression vector, comprising the nucleic acid of the first aspect.

In a fourth aspect the present ion provides an in vitro method for preparing an isolated host cell, comprising: ucing the nucleic acid of the first or second aspects or the expression vector of the third aspect into cells of a lymphocyte population and ing the cells in the presence of anti-CD3 and/or anti-CD28, and at least one homeostatic cytokine.

Also described are methods and compositions to confer and/or augment immune responses mediated by cellular immunotherapy, such as by adoptively erring tumor-specific, genetically modified subsets of CD8+ or CD4+ T cells alone, or in combination. The sure describes chimeric receptor nucleic acids, and vectors and host cells including such nucleic acids. The nucleic acid sequence that encodes the chimeric receptor links together a number of modular components that can be excised and replaced with other components in order to customize the ic or for efficient T cell activation and recognition of a specific target le or an epitope on the target molecule.

In embodiments described herein, a chimeric receptor c acid comprises a polynucleotide coding for a ligand binding domain, wherein the ligand is a molecule expressed on malignant or infected cells, a polynucleotide coding for a polypeptide spacer wherein the polypeptide spacer is about 200 amino acids or less, a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for intracellular signaling s. In embodiments, the ptide spacer comprises a modified IgG4 hinge region containing an amino acid sequence X1PPX2P that may be linked to other amino acid sequences including but not limited to the CH2 and CH3 or CH3 only sequences of the Ig Fc. It has been surprisingly found that the length of the spacer region that is presumed not to have signaling capability affects the in vivo efficacy of the T cells modified to express the chimeric or and needs to be customized for dual target molecules for optimal tumor or target cell recognition.

Another aspect of the sure describes an ed chimeric receptor nucleic acid comprising: a polynucleotide coding for a ligand binding domain, n the ligand is a tumor specific antigen, viral antigen, or any other molecule expressed on a target cell population that is suitable to mediate recognition and elimination by a lymphocyte; a polynucleotide coding for a polypeptide spacer wherein the polypeptide spacer is of a customized length that is specific for each targeted ligand, wherein the spacer provides for enhanced T cell proliferation and/ or cytokine production as compared to a nce chimeric receptor; a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for one or more intracellular signaling domains. In embodiments, a long spacer is employed if the epitope on the target ligand is in a membrane proximal on and a short spacer is employed if the epitope on the target ligand is in a membrane distal position. The disclosure includes expression vectors and host cells comprising the isolated chimeric receptor as described herein. r aspect of the disclosure describes a chimeric receptor polypeptide comprising a ligand binding domain, n the ligand is a tumor specific antigen, viral antigen or any other le that is sed on a target cell population and can be targeted to mediate recognition and ation by lymphocytes; a polypeptide spacer wherein the polypeptide spacer is about 10-229 amino acids; a transmembrane domain; and one or more intracellular signaling domains. In embodiments, the polypeptide spacer comprises a modified IgG hinge region containing the amino acid sequence X1PPX2P.

In another aspect, the present disclosure describes compositions to confer and/or augment immune responses mediated by cellular immunotherapy, such as by adoptively erring tumor-specific, subset specific genetically modified CD4+ T cells, wherein the CD4+ T cells confer and/or augment the ability of CD8+ T cells to n anti-tumor reactivity and increase and/or maximize tumor-specific proliferation. In embodiments, the CD4+ cells are genetically modified to express a chimeric receptor nucleic acid and/or chimeric receptor ptide as described herein.

In another aspect, described are compositions to confer and/or augment immune responses ed by cellular immunotherapy, such as by adoptively transferring tumor-specific, subset specific genetically modified CD8+ T cells. In embodiments, the CD8+ cells express a chimeric receptor nucleic acid and/or chimeric receptor polypeptide as described herein.

Also described is an adoptive cellular immunotherapy composition having a cally ed CD8+ xic T lymphocyte cell preparation to confer and/or augment immune responses, n the cytotoxic T lymphocyte cell preparation comprises CD8+ T cells that express a chimeric receptor comprising a ligand g domain for a ligand associated with the e or disorder, a customized spacer region, a transmembrane domain; and an intracellular signaling domain of a T cell or other receptors, such as a costimulatory domain, and/or a genetically modified helper T lymphocyte cell preparation, wherein the helper T lymphocyte cell preparation has CD4+ T cells that express a chimeric receptor comprising an antibody variable domain specific for the ligand associated with the disease or disorder, a customized spacer region, a transmembrane domain; and one or more intracellular signaling domains.

Also described is a method of performing ar immunotherapy in a subject having a disease or disorder by administering to the subject a genetically modified cytotoxic T cyte cell preparation that provides a cellular immune response, wherein the cytotoxic T lymphocyte cell preparation comprises CD8+ T cells that have a ic receptor comprising a polynucleotide coding for a ligand binding domain, wherein the ligand is a tumor specific n, viral antigen, or any other molecule expressed on a target cell population that is suitable to mediate recognition and ation by a lymphocyte; a polynucleotide coding for a polypeptide spacer wherein the polypeptide spacer is of a ized length that is specific for each targeted ligand, wherein the spacer provides for enhanced T cell proliferation and/or cytokine production as compared to a reference chimeric receptor; a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for one or more intracellular signaling domains. In embodiment, the ligand binding domain is an extracellular dy variable domain specific for a ligand associated with the disease or disorder. An embodiment es a genetically modified helper T lymphocyte cell preparation that wherein the helper T lymphocyte cell preparation comprises CD4+ T cells that have a chimeric receptor comprising an a polynucleotide coding for a ligand g domain, wherein the ligand is a tumor specific antigen, viral antigen, or any other molecule expressed on a target cell population that is suitable to mediate recognition and ation by a lymphocyte; a polynucleotide coding for a polypeptide spacer n the polypeptide spacer is of a customized length that is specific for each targeted ligand, wherein the spacer provides for enhanced T cell proliferation and/or cytokine production as compared to a reference ic receptor; a polynucleotide coding for a transmembrane ; and a polynucleotide coding for one or more intracellular signaling domains. In embodiments, the genetically ed CD8+ and genetically modified CD4+ cell population are coadministered. In embodiments, the T cells are autologous or allogeneic T cells.

Various modifications of the above method are possible. For example, the chimeric receptor that is expressed by the CD4+ T cell and the CD8+ T cell can be the same or ent.

Also described is a method of manufacturing an ve immunotherapy composition by obtaining a chimeric receptor modified tumor-specific CD8+ cytotoxic T lymphocyte cell preparation that elicits a cellular immune response and expresses an n-reactive chimeric receptor, wherein the modified cytotoxic T lymphocyte cell preparation ses CD8+ T cells that have a chimeric receptor comprising a ligand binding domain, wherein the ligand is a tumor specific antigen, viral antigen, or any other molecule expressed on a target cell population that is suitable to mediate ition and elimination by a lymphocyte; a polypeptide spacer wherein the polypeptide spacer is of a customized length that is specific for each targeted , wherein the spacer provides for enhanced T cell proliferation and/or cytokine production as compared to a reference chimeric receptor; a transmembrane domain; and one or more intracellular signaling domains.; and/or obtaining a modified naïve or memory CD4+ T helper cell wherein the modified helper T lymphocyte cell preparation comprises CD4+ cells that have a chimeric receptor comprising a ligand binding domain, wherein the ligand is a tumor specific antigen, viral antigen, or any other molecule expressed on a target cell population that is le to mediate recognition and elimination by a lymphocyte; a polypeptide spacer wherein the ptide spacer is of a customized length that is specific for each targeted ligand, wherein the spacer provides for enhanced T cell proliferation and/or cytokine production as compared to a reference chimeric receptor; a transmembrane domain; and one or more intracellular ing domains.

These and other embodiments of the invention are described further in the anying specification, drawings and claims.

Brief Description of the Drawings Figure 1 Library of spacer sequences. We constructed a plasmid library that contain codon optimized DNA sequences that encode extracellular ents including of the IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Any scFV sequence (VH and VL) can be cloned 5’ to the sequences encoding this library of variable spacer domains. The spacer domains are in turn linked to CD28 transmembrane and intracellular signaling domains and to CD3 zeta. A T2A sequence in the vector separates the ic receptor from a selectable marker encoding a ted human epidermal growth factor receptor (tEGFR).

Figure 2: In vitro cytotoxicity, cytokine production, and proliferation of T-cells modified to express 2A2 ROR1 chimeric receptors with modified spacer . (A) Phenotype of purified CD8+ TCM-derived cell lines modified with each of the 2A2 ROR1 chimeric receptors with long, intermediate and short spacer domain. Staining with anti-F(ab) dy that binds to an epitope in the 2A2 scFV shows surface expression of ROR1 chimeric receptors with full length or ted spacer. (B) Cytolytic activity of T-cells expressing the various 2A2 ROR1 chimeric receptors with long (●), intermediate (▲) and short spacer (♦), or a tEGFR control lentiviral vector against ROR1+ (x) and control target cells. The bar m summarizes cytotoxicity data from 3 independent ments (E:T = 30:1) normalized to tic activity by 2A2 ROR1 chimeric receptor ‘long’ = 1, and analyzed by Student’s . (C) CFSE dye dilution was used to measure proliferation of 2A2 ROR1 chimeric receptor and tEGFR control T-cells, 72 hours after stimulation with Raji/ROR1 (left panel) and primary CLL cells (right panel) without addition of exogenous cytokines. For analysis, triplicate wells were pooled and the proliferation of live (PI-), CD8+ s analyzed. Numbers above each histogram indicate the number of cell divisions the proliferating subset underwent, and the fraction of T-cells in each gate that underwent ≥4/3/2/1 cell divisions is provided next to each plot. (D) Multiplex cytokine assay of supernatants ed after 24 hours from triplicate co-cultures of 5x104 T-cells expressing the various 2A2 ROR1 chimeric receptors with Raji/ROR1 and primary CLL cells. Multiplex cytokine data from 3 independent experiments were normalized (cytokine release by 2A2 ROR1 chimeric or ‘long’ = 1) and ed by t’s t-test (right bar diagram).

Figure 3. R11 chimeric receptor requires a long spacer for recognition of ROR1+ tumor cells. The sequences encoding the scFV from the R11 monoclonal antibody that is specific for an epitope in the membrane proximal Kringle domain of the orphan tyrosine kinase or ROR1 were cloned upstream of IgG4 hinge only (short), IgG4 hinge/CH3 (intermediate), and IgG4 hinge/CH2/CH3 sequences in our chimeric receptor library containing the 4-1BB ulatory s and prepared as lentiviral vectors. A). Human CD8+ T cells were transduced and the uction efficiency with each of the short, intermediate and long chimeric ors was determined by staining for the tEGFR marker. B). Transduced T cells expressing the short (top), intermediate (middle), and long (bottom) were assayed for lysis of K562 leukemia cells alone or transfected to express ROR1. Only the T cells expressing the long spacer chimeric receptor efficiently killed ROR1+ K562 cells. C).

Transduced T cells expressing the short (top), intermediate (middle), and long (bottom) were labeled with CFSE, stimulated with K562 cells expressing ROR1 or CD19 (control) and assayed for cell proliferation over 72 hours. The T cells expressing the long spacer chimeric receptor proliferated specifically to the ROR1+ K562 cells. D). uced T cells expressing the short (top), intermediate (middle), and long (bottom) were stimulated with Raji lymphoma cells and K562 cells that expressed ROR1 or CD19 (control) and assayed for the secretion of interferon gamma into the supernatant over 24 hours. The T cells expressing the long spacer chimeric receptor erated and ed the highest levels of interferon gamma in response to ROR1 ve target cells.

Figure 4: Design of ROR1 chimeric receptors with modified spacer length and derived from the 2A2 and R12 scFV with different affinity. (A) Design of lentiviral transgene inserts ng a panel of ROR1 chimeric receptors containing the 2A2 scFV, an IgG4-Fc derived spacer of ‘Hinge-CH2-CH3’ (long spacer, 229 AA), ‘Hinge-CH3’ (intermediate, 119 AA), or ‘Hinge’ only (short, 12 AA), and a signaling module with CD3ζ and CD28. Each chimeric receptor cassette contains a truncated EGFR marker encoded downstream of a T2A element. (B) Lentiviral transgene inserts encoding ROR1-specific chimeric receptors derived from the R12 and 2A2 scFV with short IgG4-Fc ‘Hinge’ spacer (12 AA), and a signaling module containing CD28 or 4-1BB and CD3ζ respectively (total: 4 constructs).

Figure 5: Anti-tumor reactivity of T-cells modified with ROR1 ic receptors derived from mAb R12 with higher affinity than 2A2. (A) tEGFR expression on ed polyclonal CD8+ TCM-derived T-cell lines modified with each of the R12 and 2A2 ROR1 chimeric receptors with short IgG4-Fc ‘Hinge’ spacer, and CD28 or 4-1BB ulatory domain. (B) Cytotoxicity against ROR1+ and control target cells by T-cells expressing R12(28-▲; 4-1BB-∆) and 2A2 ROR1 chimeric receptors (28-●; 4-1BB○) or a tEGFR control vector (x). (C) lex cytokine assay of supernatants obtained after 24 hours from co-cultures of 5x104 T- cells sing the various ROR1 chimeric receptors with Raji/ROR1 tumor cells.

The middle/right bar diagrams show normalized lex data from 3 independent experiments ine release by ROR1 chimeric receptor 2A2 = 1) analyzed by Student’s t-test. (D) Proliferation of ROR1 chimeric receptor T-cells and tEGFR control T-cells 72 hours after stimulation with Raji/ROR1 cells and without addition of exogenous cytokines was assessed by CFSE dye dilution. Numbers above each histogram indicate the number of cell divisions the proliferating subset ent, and the fraction of T-cells in each gate that underwent ≥4/3/2/1 cell ons is provided above each plot.

Figure 6: Analysis of cytokine production and proliferation of CD4+ T- cells lines modified with a ROR1 chimeric receptor derived from mAb R12 with higher affinity than 2A2. (A-B) The 2A2 and R12 ROR1 chimeric receptors had the short spacer and a CD28 costimulatory domain. (A) Multiplex cytokine analysis from supernatants obtained 24 hours after ation of 5x104 CD4+ T- cells expressing the 2A2 and R12 ROR1 chimeric receptor with Raji/ROR1 tumor cells. (B) Proliferation of CD4+ R12 and 2A2 ROR1 chimeric receptor T-cells and tEGFR control T-cells 72 hours after stimulation with Raji/ROR1 cells and without addition of ous cytokines was assessed by CFSE dye dilution. Numbers above each ram indicate the number of cell divisions the proliferating subset underwent, and the fraction of T-cells in each gate that underwent ≥5/4/3/2/1 cell ons is provided above the rams.

Figure 7: ition of y CLL by T-cells modified with 2A2 and R12 ROR1 chimeric receptors with optimal short spacer and 4-1BB costimulatory domain or with a CD19-specific chimeric or. (A) Expression of ROR1/CD19 on primary CLL, and CD80/86 on primary CLL and Raji/ROR1 tumor cells (black dot plots) that can engage CD28 on chimeric receptor T-cells (white histograms). Staining with matched isotype control mAbs is shown as grey dot plots/histograms. (B) Cytolytic activity of T-cells expressing the 2A2(●) and R12 ROR1 chimeric receptor (■), a CD19-specific ic receptor (▲) and T-cells modified with a tEGFR control vector (x) against primary CLL (left diagram) and normal B cells (right m) analyzed by chromium release assay.

Cytotoxicity data t primary CLL from 4 independent experiments (E:T = :1) were normalized (cytolytic activity by ROR1 chimeric receptor 2A2 = 1) and analyzed by Student’s t-test (bar diagram). (C) Multiplex cytokine analysis after a 24-hour stimulation of 5x104 chimeric receptor T-cells with primary CLL cells.

Cytokine release of unstimulated chimeric receptor T-cells was below 3.6 pg/ml (detection limit) (left bar diagram). ELISA for IFN-γ tion by 5x104 2A2 and R12 ROR1 chimeric receptor T-cells after a 24-hour co-culture with primary CLL.

O.D. of 1 corresponds to approximately 250 pg/ml (right bar diagram). (D) Proliferation of CD8+ T-cells modified with the 2A2 ROR1, R12 ROR1 and a CD19 chimeric receptor, 72 hours after ation with primary CLL cells. Numbers above each histogram indicate the number of cell divisions, and the fraction of T- cells in each gate that underwent ≥3/2/1 cell divisions is provided next to each plot.

Figure 8: The function of ROR1-chimeric receptor and CD19-chimeric receptor modified CD8+ T-cells against primary CLL is augmented by chimeric receptor-modified CD4+ helper T-cells. (A) ELISA for IL-2 production from triplicate co-cultures of 5x104 CD8+ and CD4+ T-cells sing the R12 ROR1 and CD19-chimeric receptor respectively, incubated with primary CLL for 24-hours.

O.D. of 1 corresponds to approx. 800 pg/ml. (B) Proliferation of ic receptor- modified CD8+ T-cells in response to primary CLL is ed by addition of chimeric receptor-modified CD4+ T-cells. CFSE-labeled CD8+ T-cells expressing the 2A2 ROR1, R12 ROR1 and CD19-chimeric receptor respectively, were cocultured with tumor cells and with 2A2 ROR1, R12 ROR1 and himeric receptor transduced or control untranduced CD4+ T-cells (CD8+:CD4+ = 1:1).

Proliferation of the CD8+ subset was analyzed 72 hours after stimulation. s above each histogram indicate the number of cell divisions, and the on of T- cells in each gate that underwent ≥3/2/1 cell divisions is provided above each plot.

Figure 9: In vivo anti-tumor efficacy of 2A2 ROR1, R12 ROR1 and CD19 chimeric receptor T-cells. Cohorts of mice were inoculated with 0.5x106 JeKo-1/ffluc MCL via tail vein injection, and 5x106 2A2 ROR1, R12 ROR1 or CD19 chimeric receptor T-cells, or s expressing a tEGFR control vector were administered 7 days after tumor inoculation. All chimeric receptor constructs had the short IgG4 ‘Hinge-only’ spacer and a 4-1BB costimulatory domain. (A, B) Serial bioluminescence imaging of tumor in s of mice treated with T-cells expressing the 2A2 ROR1 chimeric receptor (▼), the high affinity R12 ROR1 chimeric receptor (■), a CD19-specific chimeric receptor (▲), with T-cells transduced with tEGFR alone (●), and untreated mice. Bioluminescence imaging showed tumor manifestations in the bone marrow and thorax and thus, signal intensity was measured in s of interest that encompassed the entire body and thorax of each individual mouse. (C) Kaplan-Meier analysis of survival in individual treatment and control groups. Statistical analyses were performed using the log-rank test. The data shown in A-C are representative of results obtained in 2 independent experiments.

(D) Proliferation of 2A2 ROR1, R12 ROR1 and CD19 chimeric receptor s in vivo. Tumor bearing NSG/JeKo-1 mice ed a single dose of 5x106 CFSE- labeled 2A2 ROR1, R12 ROR1 or CD19 chimeric receptor T-cells on day 7 after tumor inoculation, and 72 h later peripheral blood, bone marrow and spleen were ted from each individual mouse. The frequency and proliferation of live (PI-), CD45+ CD8+ tEGFR+ T-cells was analyzed. The frequency of 2A2 ROR1, R12 ROR1 and CD19 chimeric receptor T-cells respectively is provided on the left of each histogram as percentage of live cells, and the fraction of T-cells that underwent ≥4/3/2/1 cell divisions is provided above each plot.

Figure 10 Expression of ROR1 and NKG2D ligands on epithelial cancer cell lines. (A) sion of ROR1 on the triple ve breast cancer cell lines MDA-MB-231 and 468, and the renal cell cancer lines FARP, TREP and RWL (black histograms). Staining with matched isotype control antibody is shown as grey histograms. (B) Expression of CD80/86 and the NKG2D ligands MICA/B on MDAMB-231 and Raji/ROR1 tumor cells, and NKG2D (CD314) on 2A2 and R12 ROR1- chimeric receptor T-cells. Staining with matched isotype control mAbs is shown as grey dot plots/histograms.

Figure 11: ROR1-chimeric receptor modified T-cells recognize ROR1+ epithelial tumor cells in vitro. (A) Chromium release assay to evaluate the cytolytic activity of R12 ROR1-chimeric receptor modified T-cells (short /4- 1BB costimulatory domain, closed symbols) and tEGFR control s (open symbols) against ROR1+ breast cancer and renal cell cancer lines. (A-D) The 2A2 and R12 himeric receptors had the optimal short spacer and a 4-1BB costimulatory domain. (B) lex cytokine analysis after ation of T-cells sing the 2A2 and R12 ROR1-chimeric receptor with MDA-MB-231 and Raji/ROR1 tumor cells. (C) eration of CD8+ T-cells modified with the 2A2 and R12 ROR1-chimeric receptor 72 hours after stimulation with MDA-MB-231 tumor cells. For analysis, triplicate wells were pooled and the proliferation of live (PI-), CD8+ T-cells analyzed. Numbers above each histogram indicate the number of cell divisions the proliferating subset underwent, and the fraction of T-cells in each gate that underwent ≥4/3/2/1 cell divisions is ed next to each ram. (D) ELISA for IL-2 production by R12 ROR1-chimeric receptor s after a 24-hour co-culture with MDA-MB-231 in plain medium, and after on of an antibody cocktail blocking of the NKG2D pathway [anti-NKG2D (clone 1D11), anti-MICA/B (clone 6D4) and anti-ULBP] or matched isotype control mAbs. O.D. of 0.6 corresponds to approximately 1900 pg/ml.

Figure 12. Effect of extracellular spacer length on recognition and triggering of tumor cell lysis by CD8+ human T cells that express a HER2- specific chimeric receptor. A.) Depiction of Herceptin Fab epitope location on tumor cell membrane proximal epitope on human HER2, B.) Structural formats of Herceptin scFv CAR spacer length variants as –T2A- linked polypeptides with the carboxyl EGFRt marker transmembrane protein, C.) Western blot ion of short, medium, and long spacer Herceptin-CAR variant expression in human CD8+ CTL’s, D.) Flow cytometric detection of EGFRt by transduced human CD8+ CTL’s transduced with tin CAR variants then immunomagnetically purified by tin-biotin, anti-biotin microbeads, E.) Distinct tic function by T cells transduced to express the Herceptin CAR variants (short – S; medium – M; and long – L) t HER2+ Med411FH and D283 human medulloblastoma cell lines (D341 is a HER2- l oblastoma cell line, inset flow plots are tumor target lines stained with anti-HER2 specific mAb). Green=full IgG4 (Long Spacer,▼), Blue=IgG4hinge:CH3(Medium Spacer;▲), Red=IgG4hinge only (Short Spacer;■).

Figure 13: himeric receptor vectors and generation of CD19- chimeric receptor T cells.

(A) Design of lentiviral transgene inserts encoding a panel of CD19-specific chimeric receptors that differ in extracellular spacer length and intracellular costimulation.

Each ic receptor encoded the CD19-specific single chain variable fragment derived from the FMC63 mAb in a VL-VH orientation, an IgG4-derived spacer domain of Hinge-CH2-CH3 (long spacer, 229 AA) or Hinge only (short spacer, 12 AA), and a signaling module containing CD3ζ with CD28 or 4-1BB alone or in tandem. Each chimeric receptor cassette contains a truncated EGFR marker encoded downstream of a ble 2A element. (B, C) Polyclonal T cell lines modified with each of the CD19-chimeric receptor constructs were prepared from purified CD8+ CD45RO+ CD62L+ central memory T cells (TCM) of normal donors.

Following lentiviral transduction, transgene-positive T cells in each cell line were purified using the tEGFR marker and ed for in vitro and in vivo experiments.

(D) MFI after staining for the tEGFR marker shows equivalent transgene expression in T cells modified with each of the CD19-chimeric receptors.

Figure 14: In vitro cytotoxicity, cytokine production, and proliferation of T cells modified with distinct CD19-chimeric ors. (A) Cytolytic activity of T cells expressing the various CD19-chimeric receptors against CD19+ and control target cells. (B) Multiplex cytokine assay of supernatants ed after 24 hours from triplicate co-cultures of T cells expressing the various CD19-chimeric receptors and K562 cells transfected with CD19, and CD19+ Raji cells. (C) Comparison of cytokine production by T cells expressing the various CD19- chimeric receptors. Multiplex cytokine data from 6 independent experiments were normalized (cytokine release by CD19-chimeric receptor /CD28’ CTL = 1) and analyzed by Student’s t-test. (D) CFSE dye dilution was used to e proliferation of himeric receptor T cells 72 hours after stimulation with K562/CD19 (upper panel) and CD19+ Raji tumor cells (lower panel) without addition of exogenous cytokines. For analysis, triplicate wells were pooled and the proliferation of live (PI-), CD8+ T cells analyzed. Numbers above each histogram indicate the number of cell divisions the proliferating subset underwent, and the fraction of T cells in each gate that underwent ≥4/3/2/1 cell divisions is provided in the upper left of each plot. (E) PI ng was performed at the end of a 72-hour coculture of T cells expressing the various himeric receptors with Raji tumor cells. The tage of PI+ cells within in chimeric or T cell line (CD3+) is provided in each histogram.

Figure 15: CD19-chimeric receptor T cells with a short extracellular spacer domain eradicate Raji tumors in NOD/SCID mice. (A) Cohorts of mice were inoculated with Raji-ffluc via tail vein injection, and T cells transduced with CD19-chimeric receptors ning long and short spacer domains or with tEGFR alone were administered 2 and 9 days after tumor inoculation by tail vein injection.

Tumor progression and distribution was evaluated by serial bioluminescence imaging after injection of luciferin substrate. (B) Serial bioluminescence imaging of tumor in cohorts of mice either treated with T cells expressing CD19-chimeric receptors with short spacer (‘short/CD28’ and ‘short/4-1BB’) and long spacer (‘long/CD28’ and ‘long/4-1BB’) domains, with T cells transduced with the tEGFR control vector, or untreated. Each diagram representing cohorts of mice treated with CD19-chimeric receptor or tEGFR transduced T cells also shows the mean of tumor progression in untreated mice for comparison (red triangles). (C) Kaplan-Meier analyses of survival of untreated mice and mice treated with T cells expressing himeric receptors with short spacer (‘short/CD28’ and ‘short/4-1BB’), long spacer (‘long/CD28’ and ‘long/4-1BB’) domains, and with control tEGFR.

Statistical analyses were performed using the log-rank test. The data shown in B and C are representative of s obtained in 3 ndent experiments.

Figure 16: CD19-chimeric or T cells with a short spacer (short/4- 1BB) ate established Raji tumors in NSG mice in a dose-dependent manner. (A) Mice were inoculated with Raji-ffluc via tail vein injection and tumor engraftment confirmed by bioluminescence imaging on day 6. On day 7, mice received a single i.v. injection of various doses of T cells transduced with the CD19- ic receptor ‘short/4-1BB’ or with the tEGFR-control lentivirus. (B, C) Dose dependent anti-tumor efficacy of T cells expressing the CD19-chimeric or ‘short/4-1BB’. A control cohort of mice received a single high dose of T cells modified with tEGFR alone. (D) Persistence of himeric receptor T cells following adoptive transfer into NSG/Raji mice. Flow cytometric analysis of peripheral blood (eye bleeds) in the cohort of mice treated with 2.5x106 CD19- chimeric receptor ‘short/4-1BB’ T cells. The frequency of CD8+ tEGFR+ T cells is shown as percentage of live peripheral blood cells.

Figure 17: T cells expressing CD19-chimeric receptors with a short spacer and either CD28 or 4-1BB are more effective against ished lymphoma than those expressing CD19-chimeric receptors with a long spacer.

(A) NSG mice were inoculated with Raji-ffluc on day 0, and treated on day 7 with one dose of 2.5x106 CD19 chimeric or T cells expressing short or long spacer and either CD28 or 4-1BB costimulatory domain. (B) -Meier analyses of survival of mice in each of the ent groups. Statistical analyses were performed using the log-rank test. (C) Bioluminescence imaging of cohorts of mice d with T cells expressing CD19-chimeric receptors with short spacers (‘short/CD28’ and ‘short/4-1BB’), and long spacers (‘long/CD28 and ‘long/4-1BB’). The mean tumor burden ed in untreated mice at each time point is shown in each diagram for comparison (triangles). (D) In vivo persistence of T cells expressing CD19-chimeric receptor with short spacer domain is enhanced compared to T cells expressing CD19-chimeric receptors with long spacer domain. The frequency of CD8+ tEGFR+ T cells in the peripheral blood obtained at day 3 and 10 after transfer was ined by flow cytometry and is shown as percentage of live (PI-) peripheral blood cells.

Statistical analyses were performed by Student’s t-test. The data shown in B-D are representative for results obtained in 3 independent experiments.

Figure 18: Increasing chimeric receptor T cell dose or augmenting costimulatory signaling does not improve the anti-tumor efficacy of CD19- chimeric receptors with a long spacer domain against established lymphoma.

(A) Cytolytic activity of T cells expressing ‘long/CD28’, ‘long/4-1BB’ and ‘long/CD28_4-1BB’ CD19 chimeric receptors against CD19+ and control target cells. (B) Multiplex cytokine assay of supernatant obtained after 24 hours from triplicate co-cultures of K562/CD19 and Raji tumor cells with T cells expressing the various CD19-chimeric receptors. (C) Evaluation of proliferation of CD19-chimeric receptor T cells 72 hours after stimulation with CD19+ tumor cells (K562/CD19 – left panel; Raji – right panel) by CFSE dye dilution. For analysis, triplicate wells were pooled and the proliferation of live (PI-) CD8+ T cells analyzed. Numbers above each histogram indicate the number of cell ons the proliferating subset underwent, and the on of T cells in each gate that underwent ≥4/3/2/1 cell divisions is provided in the upper left of each plot. (D) Kaplan-Meier analyses of survival of mice treated with T cells expressing CD19-chimeric ors with short (‘short/CD28’) and long spacer domain (‘long/CD28’ and CD28_4-1BB’), or T cells ed with a tEGFR-encoding control lentiviral vector. Statistical es were performed using the log-rank test. (E) Bioluminescence imaging of cohorts of mice treated with T cells sing himeric receptors with short spacer (‘short/CD28’), and long spacers (‘long/CD28 and ‘long/CD28_4-1BB’).

Diagrams show mean tumor progression in untreated mice for comparison (red triangles). (F) In vivo persistence of T cells expressing the various CD19-chimeric receptors. The frequency of CD8+ tEGFR+ T cells in the peripheral blood ed at day 3 and 10 after transfer was determined by flow cytometry and is shown as percentage of live (PI-) peripheral blood cells. tical analyses were performed by t’s t-test.

Figure 19: CD19-chimeric receptor T cells with a long spacer domain are activated by tumor in vivo but fail to increase in cell number. (A) Expression of CD69 and CD25 on T cells modified with each CD19-chimeric receptor prior to transfer into NSG/Raji mice. (B) s of mice were inoculated with fluc tumor cells and 7 days later received CFSE-labeled CD19-chimeric receptor transduced or control T cells. Bone marrow and spleens were harvested from subgroups of mice 24 and 72 hours after T cell administration. (C, D) Multiparameter flow cytometric analysis of bone marrow mononuclear cells obtained 24 hours (C) and 72 hours (D) after T cell transfer. Dot plots show anti CD3 and anti CD45 staining after gating on PI- cells to detect viable human T cells.

The CD3- CD45+ gate ns Raji tumor cells. Expression of CD25 and CD69 on live (PI-) CD3+ CD45+ T cells is shown in the histograms. (E) Frequency of CD3+ CD45+ T cells in spleens obtained 24 and 72 hours after T cell transfer. Dot plots are gated on live PI- cytes and the tage of CD3+ CD45+ T cells is shown in each plot. (F) PI staining of bone marrow and splenocytes hours after T cell transfer into NSG/Raji mice. The numbers in the histograms indicate the percentage of PI+ cells within the CD3+ population. (G) Bioluminescence imaging of cohorts of mice treated with T cells sing CD19-chimeric receptors with short spacer (‘short/CD28’ and ‘short/4-1BB’), long spacers (‘long/CD28 and ‘long/4-1BB’), or control T cells.

Figure 20: T cells expressing CD19 chimeric receptors with 4-1BB and CD3zeta and a modified IgG4-Fc hinge exhibit or in vitro and in vivo function compared to T cells expressing CD19 chimeric ors with 4-1BB and a and a CD8 alpha hinge.A. Cytolytic activity of CD19 chimeric or modified T-cells with IgG4 Fc hinge, CD8 alpha hinge and control T cells against Cr51-labeled K562 cells transfected with CD19, Raji lymphoma cells that express CD19, and K562 control T cells.

Lysis is shown at different E/T ratios in a 4 hour Cr51 release assay. B. Interferon gamma production by 5x104 T cells expressing a CD19 chimeric receptor with an IgG4 Fc hinge or CD8 alpha hinge after a 24-hour coculture with Raji tumor cells. O.D. of 1 corresponds to ~500 pg/ml of interferon gamma. C. CFSE dye dilution assay to measure proliferation of T cells expressing a CD19 chimeric receptor with an IgG4 Fc hinge or CD8 alpha hinge and T cells that express tEGFR alone (control) after 72 hours coculture with CD19 positive Raji ma cells. Numbers above each ram indicate the number of cell divisions the proliferating cell subset underwent. The fraction of T cells in each gate that underwent >3/2/1 cell divisions is provided next to the plot. D. In vivo antitumor activity of T cells expressing a CD19 chimeric receptor with an IgG4 Fc hinge (group 1) or CD8 alpha hinge (group 2) and T cells that express tEGFR alone (group 3) in NSG mice inoculated with Raji tumor cells expressing firefly luciferase (ffluc). Mice were imaged 17 days after tumor inoculation and 10 days after T cell inoculation. The data shows greater tumor burden in mice treated with control tEGFR T cells (group 3) or with CD19 chimeric receptor CD8 alpha hinge T cells (group 2) compared with mice treated with CD19 chimeric receptor IgG4 Fc hinge T cells (group 1).

Detailed Description Unless defined otherwise, all cal and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

“About” as used herein when referring to a measurable value is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±l%, and still more preferably ±0.1 % from the specified value.

"Activation", as used herein, refers to the state of a T cell that has been sufficiently ated to induce detectable cellular proliferation, cytokine production or expression of cell surface markers such as CD69 and CD25, or able effector functions.

“Activation Induced cell death” as used herein refers to a state of a T cell that is activated but is not able to proliferate for more than 2 tions and exhibits markers of apoptosis. en" or "Ag" as used herein refers to a molecule that provokes an immune response. This immune response may e either antibody production, or the activation of specific immunologically-competent cells, or both. It is readily apparent that an antigen can be generated synthesized, produced recombinantly or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor , a cell or a biological fluid.

"Anti-tumor effect" as used herein, refers to a biological effect, which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an se in life expectancy, or a decrease of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by a decrease in recurrence or an increase in the time before ence.

“Chimeric or” as used herein refers to a synthetically designed or comprising a ligand binding domain of an antibody or other protein sequence that binds to a molecule associated with the disease or disorder and is linked via a spacer domain to one ore more intracellular ing domains of a T cell or other receptors, such as a costimulatory domain.

"Co-stimulatory domain," as the term is used herein refers to a signaling moiety that provides to T cells a signal which, in addition to the primary signal provided by for instance the CD3 zeta chain of the TCR/CD3 complex, mediates a T cell response, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, and the like. A co-stimulatory domain can include all or a portion of, but is not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, , ICOS, lymphocyte function-associated n-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that ically binds with CD83. In embodiments, the costimulatory domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate a cell response including activation, proliferation, differentiation and cytokine secretion, and the like.

"Coding for" are used herein refers to the ty of specific sequences of tides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a d sequence of amino acids. Thus, a gene codes for a protein if transcription and ation of mRNA corresponding to that gene produces the protein in a cell or other biological system. A "nucleic acid sequence coding for a polypeptide" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence.

“Cytotoxic T lymphocyte “(CTL) as used herein refers to a T cyte that ses CD8 on the surface thereof (i.e., a CD8+ T cell). In some embodiments such cells are ably "memory" T cells (TM cells) that are n- experienced.

"Central memory" T cell (or "TCM") as used herein refers to an antigen experienced CTL that expresses CD62L or CCR-7 and CD45RO on the surface thereof, and does not express or has decreased expression of CD45RA as compared to naive cells. In embodiments, central memory cells are positive for expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and have decreased expression of CD54RA as compared to naïve cells.

"Effector memory" T cell (or "TEM") as used herein refers to an antigen experienced T cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells, and does not express or has decreased expression of CD45RA as compared to naïve cell. In embodiments, effector memory cells are negative for expression of CD62L andCCR7, compared to naïve cells or l memory cells, and have variable expression of CD28 and CD45RA.

“Naïve “ T cells as used herein refers to a non antigen experienced T lymphocyte that expresses CD62L and CD45RA, and does not express CD45RO- as compared to central or effector memory cells. In some embodiments, naïve CD8+ T lymphocytes are characterized by the sion of phenotypic markers of naïve T cells including CD62L, CCR7, CD28, CD127, and .

“Effector “ “TE” T cells as used herein refers to a n experienced cytotoxic T lymphocyte cells that do not express or have decreased sion of CD62L, CCR7, CD28, and are positive for granzyme B and perforin as compared to central memory or naïve T cells. hed" and ted" as used herein to describe amounts of cell types in a mixture refers to the subjecting of the mixture of the cells to a process or step which results in an increase in the number of the "enriched" type and a decrease in the number of the "depleted" cells. Thus, depending upon the source of the original population of cells subjected to the enriching process, a mixture or composition may contain about 60, 70, 80, 90, 95, or 99 percent or more (in number or count) of the "enriched" cells and about 40, 30, 20, 10, 5 or 1 percent or less (in number or count) of the "depleted" cells.

“Epitope” as used herein refers to a part of an antigen or molecule that is recognized by the immune system ing antibodies, T cells, and/ or B cells. es usually have at least 7 amino acids and can be linear or conformational.

"Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide or nucleic acid that has been identified and separated and/or recovered from a component of its l environment. Preferably, the isolated polypeptide or nucleic acid is free of association with all components with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the ptide or nucleic acid, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

“Intracellular signaling domain” as used herein refers to all or a portion of one or more domains of a molecule (here the ic receptor molecule) that provides for activation of a lymphocyte. Intracellular domains of such molecules mediate a signal by interacting with cellular mediators to result in proliferation, entiation, activation and other effector functions. In embodiments, such molecules include all or portions of CD28, CD3, 4-1BB, and combinations thereof.

“Ligand” as used herein refers to a substance that binds specifically to another substance to form a complex. Example of ligands include epitopes on antigens, molecules that bind to receptors, substrates, tors, hormones, and activators. “Ligand binding domain” as used herein refers to substance or portion of a substance that binds to a . Examples of ligand binding domains include antigen g portions of dies, extracellular domains of receptors, and active sites of enzymes.

"Operably linked" as used herein refers to functional linkage between a regulatory sequence and a heterologous nucleic acid ce resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid ce is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

"Percent (%) amino acid sequence identity" with respect to the chimeric or polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence for each of the ligand binding domain, spacer, transmembrane domain, and/or the lymphocyte activating domain, after aligning the sequences and introducing gaps, if necessary, to achieve the m percent sequence ty, and not considering any vative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for ce, using publicly ble computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) re. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For example, % amino acid ce identity values generated using the WU-BLAST-2 computer program [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] uses several search parameters, most of which are set to the default values. Those that are not set to default values (i.e., the adjustable parameters) are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11 and scoring matrix=BLOSUM62. A % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the each or all of the polypeptide amino acid sequence of the reference chimeric or sequence provided in Table 2 and the comparison amino acid ce of interest as determined by WUBLAST-2 by (b) the total number of amino acid residues of the ptide of interest.

"Chimeric receptor variant polynucleotide" or "chimeric receptor variant nucleic acid sequence" as used herein refers to a polypeptide-encoding nucleic acid molecule as defined below having at least about 80% nucleic acid sequence identity with the polynucleotide acid ce shown in Table 1 or a specifically derived fragment thereof, such as polynucleotide coding for an antigen binding domain, a cleotide encoding a spacer domain, a polynucleotide coding for a transmembrane domain and/ or a polynucleotide coding for a lymphocyte atory domain. Ordinarily, a ic receptor variant of polynucleotide or fragment thereof will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence ty, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% c acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more ably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% c acid ce ty, more ably at least about 96% nucleic acid ce identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence as shown in Table or a derived fragment thereof. Variants do not encompass the native nucleotide sequence.

In this regard, due to the degeneracy of the genetic code, one of ordinary skill in the art will ately recognize that a large number of chimeric receptor variant polynucleotides having at least about 80% nucleic acid sequence identity to the nucleotide sequence of Table 1 will encode a polypeptide having an amino acid sequence which is identical to the amino acid sequence of Table 2.

"Substantially purified" refers to a molecule that is essentially free of other molecule types or a cell that is essentially free of other cell types. A substantially ed cell also refers to a cell, which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells.

“Not substantially found” when used in reference the presence of a tumor n or other molecules on normal cells refers to the percentage of a normal cell type that has the antigen or le, and / or the density of the n on the cells.

In embodiments, not substantially found means that the antigen or molecule is found on less than 50% of normal cell type and/or at a 50% less y as compared to the amount of cells or antigen found on a tumor cell or other ed cell.

"T cells" or "T lymphocytes" as used herein may be from any ian, preferably primate, species, including monkeys, dogs, and humans. In some embodiments the T cells are allogeneic (from the same species but different donor) as the recipient subject; in some embodiments the T cells are autologous (the donor and the recipient are the same); in some embodiments the T cells arc eic (the donor and the recipients are different but are identical .

Modes of the Disclosure Described herein are chimeric receptor nucleic acids, and vectors and host cells including such nucleic acids. The chimeric receptor nucleic acid comprises a number of modular components that can be excised and replaced with other components in order to customize the chimeric receptor for a ic target molecule. The disclosure describes that one of the modular components is the spacer component. It has been singly found that the length of the spacer region that is presumed not to have signaling capability affects the in vivo efficacy of the T cells modified to express the chimeric receptor and needs to be customized for individual target les for enhanced therapeutic activity.

In one aspect, methods and c acid constructs are described to design a chimeric receptor that has improved tumor recognition, sed T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric or. In embodiments, a library of nucleic acids is described, wherein each nucleic acid codes for a spacer region that differs from the others in sequence and . Each of the nucleic acids can then be used to form a chimeric receptor nucleic acid construct that can be tested in vivo (in an animal model) and/or in vitro so that a spacer can be selected that provides for improved tumor recognition, increased T cell proliferation and/or cytokine tion in response to the ligand.

In embodiments, a chimeric receptor c acid comprises a polynucleotide coding for a ligand binding domain, wherein the ligand is a tumor or viral specific antigen or molecule, a polynucleotide coding for a customized polypeptide , wherein the spacer provides for enhanced T cell proliferation; a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for one or more intracellular signaling domains. In embodiments, a long spacer is employed if the e of the target molecule is membrane proximal on the target cell and a short spacer is employed if the epitope of the target molecule is ne distal on the target cell.

The design of a chimeric receptor can be customized depending on the type of tumor or virus, the target antigen or molecule present on the tumor, the affinity of the antibody for the target molecule, the flexibility needed for the antigen binding domain, and/or the intracellular signaling domain. In ments, a number of chimeric receptor ucts are tested in vitro and in in vivo models to determine the ability of T cells modified with the receptor to kill tumor cells in immunodeficient mice and to proliferate and persist after adoptive transfer. In embodiments, a chimeric receptor is ed that provides for capability of at least 30% of the cells to proliferate through at least two generations in vitro and/or within 72 hours after introduction in vivo. In embodiments, a chimeric receptor is not selected that results in greater than 50% of the cells undergoing activation induced cell death (AICD) within 72 hours in vivo in immunodeficient mice, and fails to eradicate tumor cells.

Depending on whether the target molecule is t on a subject’s tumor cells, the chimeric receptor es a ligand binding domain that specifically binds to that target molecule. In embodiments, a subject’s tumor cells are characterized for cell surface tumor molecules. The target molecule may be selected based on a determination of its presence on a particular subject’s tumor cells. In embodiments, a target molecule is selected that is a cell surface molecule found predominantly on tumor cells and not found on normal tissues to any substantial degree. In ments, an antibody is selected to bind to an epitope on the targeted cell surface molecule. In some cases, the epitope is characterized with respect to its proximity to the cell membrane. An epitope is terized as proximal to the membrane when it is predicted or known by ural analysis to reside closer to the target cell membrane than alternative epitopes that are predicted or known by structural analysis to reside a greater distance from the target cell membrane. In embodiments, the affinity of the antibody from which the scFV is constructed is compared by binding assays, and antibodies with different affinities are examined in ic receptor formats expressed in T cells to determine which affinity confers optimal tumor recognition, based on superior cytotoxicity of target cells, and/or T cell cytokine tion and eration.

In addition, the spacer region of the chimeric receptor may be varied to optimize T cell recognition of the ligand on the target cell. In embodiments, when an antibody binds to an e on the target cell that is very proximal to the membrane, a spacer is selected that is longer than about 15 amino acids. For example, in embodiments, if the epitope or portion thereof on the target antigen is in the first 100 amino acids of the linear sequence of the ellular domain adjacent to the transmembrane domain, a long spacer region may be selected. In embodiments, when an antibody binds to an epitope on the target cell that is distal to the ne, a spacer is selected that is about 119 or 15 amino acids or less. For example, in embodiments, when the epitope or n thereof is found in the 150 amino acids of the linear sequence of the extracellular domain from the terminus, a short or inetermediate spacer may be utilized. In embodiments, a spacer comprises an amino acid ce X1PPX2P.

A variety of combinations of primary and costimulatory intracellular signaling domain may be employed to enhance the in vivo cy of the chimeric receptor. In embodiments, different constructs of the chimeric receptor can be tested in an in vivo animal model to determine efficacy for tumor killing. In embodiments, a costimulatory intracellular signaling domain is selected from the group consisting of CD28 and modified versions thereof, 4-1BB and modified versions thereof and combinations f. Other costimulatory domains, such as OX40 may be incorporated.

CD8+ central memory T cells have an intrinsic programming that allows them to persist for ed periods after stration, which makes them a preferred subset of CD8+ T cells for immunotherapy. In embodiments, CD19 specific chimeric receptor modified cytotoxic T cells prepared from sort purified CD8+ central memory T cells are stered in the presence or absence of CD4+ CD19 ic chimeric receptor -modified T cells. In embodiments, specific CD4+ T cells exert anti-tumor reactivity and provide help to tumor-specific CD8+ T cells in vitro and in vivo. In a specific embodiment, tumor-specific CD4+ T cells or CD4+ T cells selected from the naïve or the central memory subsets are ed alone or in combination with CD8+ TCM.

Nucleic Acids, Vectors, and polypeptides Also described is a chimeric receptor nucleic acid useful for transforming or transducing lymphocytes for use in ve immunotherapy. In embodiments, the nucleic acid contains a number of modular components that e for easy substitution of elements of the nucleic acid. While not meant to limit the scope of the disclosure, it is believed that the chimeric receptor for each tumor antigen is desirably customized in terms of components in order to provide for in vivo efficacy and efficient expression in mammalian cells. For example, in a specific embodiment, for efficacy of a chimeric or sing a scFV that binds to a ROR1 epitope located in the membrane distal Ig/Frizzled domain, a spacer that is about 15 amino acids or less is ed. In another specific embodiment, for efficacy of a chimeric receptor comprising a scFV that binds to a ROR1 epitope located in the membrane proximal Kringle domain, a spacer that is longer than 15 amino acids is employed.

In another embodiment, for efficacy of a chimeric receptor comprising a scFV that binds to CD19, a spacer that is 15 amino acids or less is employed.

In embodiments, an isolated chimeric receptor nucleic acid comprises a polynucleotide coding for a ligand binding domain, wherein the target molecule is a tumor specific antigen, a polynucleotide coding for a polypeptide spacer wherein the polypeptide spacer is about 229 amino acids or less; a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for an intracellular signaling domain. In embodiments, an expression vector comprises a chimeric nucleic acid as described herein. Polypeptides encoded by all of or a portion of the chimeric or c acids are also included herein.

Ligand binding domain In embodiments, the chimeric receptor nucleic acid comprises a polynucleotide coding for a ligand binding domain. In embodiments, the ligand binding domain specifically binds to a tumor or viral specific antigen. In ments, the ligand binding domain is an antibody or fragment thereof. A nucleic acid sequence coding for an antibody or antibody fragment can readily be ined. In a specific embodiment, the polynucleotide codes for a single chain Fv that specifically binds CD19. In other specific embodiments, the polynucleotide codes for a single chain Fv that specifically binds ROR1. The sequences of these antibodies are known to or can readily be determined by those of skill in the art.

Tumor antigens are proteins that are ed by tumor cells that elicit an immune response. The selection of the ligand binding domain bed herein will depend on the type of cancer to be treated, and may target tumor antigens or other tumor cell surface molecules. A tumor sample from a subject may be characterized for the presence of n biomarkers or cell surface markers. For example, breast cancer cells from a subject may be ve or negative for each of Her2Neu, Estrogen or, and/or the Progesterone receptor. A tumor n or cell surface molecule is selected that is found on the individual subject’s tumor cells. Tumor antigens and cell surface molecules are well known in the art and include, for example, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, terone receptor, ephrinB2, CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, mesothelin, c-Met, GD-2, and MAGE A3 TCR. In embodiments a target molecule is a cell surface molecule that is found on tumor cells and is not substantially found on normal tissues, or restricted in its expression to tal normal tissues.

Other target molecules include but are not limited to antigens derived from infectious pathogens such as HIV (human immunodeficiency virus), HBV itis B virus), HPV (human papilloma virus) and Hepatitis C virus.

In one ment, the target le on the tumor comprises one or more es associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target ns for T cell receptor or chimeric receptor mediated recognition. Other target molecules belong to the group of cell transformation-related molecules such as the oncogene HER-2/Neu/ErbB2. In embodiments, the tumor n is selectively expressed or pressed on the tumor cells as compared to control cells of the same tissue type. In other embodiments, the tumor antigen is a cell surface polypeptide.

Once a tumor cell surface molecule that might be targeted with a chimeric receptor is identified, an epitope of the target molecule is selected and characterized.

In embodiments, an epitope is selected that is proximal to the tumor cell membrane.

In other embodiments, an epitope is selected that is distal to the tumor cell membrane. An epitope is characterized as al to the membrane when it is predicted or known by structural analysis to reside closer to the target cell membrane than alternative epitopes that are predicted or known by structural analysis to reside a greater distance from the target cell ne. dies that specifically bind a tumor cell surface molecule can be prepared using methods of obtaining monoclonal antibodies, methods of phage y, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies. Phage display libraries of lly or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target molecule. Phage display libraries of human antibodies are also available. In embodiments, antibodies specifically bind to a tumor cell surface molecule and do not cross react with nonspecific components such as bovine serum albumin or other unrelated antigens.

Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and/or determined.

Antibodies or antigen binding fragments include all or a portion of polyclonal antibodies, a monoclonal antibody, a human antibody, a humanized dy, a synthetic antibody, a ic dy, a bispecific antibody, a minibody, and a linear antibody. dy fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody and can readily be prepared. Examples of antibody fragments include Fab, Fab', 2, and Fv fragments; diabodies; linear antibodies; -chain antibody molecules; and multispecific antibodies formed from antibody fragments.

In embodiments, a number of different antibodies that bind to a particular tumor cell surface molecules can be isolated and characterized. In embodiments, the antibodies are characterized based on epitope specificity of the targeted molecule. In addition, in some cases, antibodies that bind to the same epitope can be selected based on the affinity of the antibody for that epitope. In embodiments, an antibody has an affinity of at least 1 mM, and ably <50 nM. In embodiments, an dy is selected that has a higher affinity for the epitope as compared to other antibodies. For example, an antibody is selected that has at least a 2 fold, at least a 5 fold, at least a 10 fold, at least a 20 fold, at least a 30 fold, at least a 40 fold, or at least a 50 fold greater affinity than a reference antibody that binds to the same epitope.

In embodiments, target les are selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, mesothelin, Her2, c-Met, PSMA, GD-2, MAGE A3 TCR and combinations thereof.

In specific embodiments, the target antigen is CD19. A number of antibodies ic for CD19 are known to those of skill in the art and can be readily characterized for sequence, epitope binding, and affinity. In a ic ment, the chimeric receptor construct includes a scFV sequence from FMC63 dy. In other embodiments, the scFV is a human or zed ScFv comprising a variable light chain comprising a CDRL1 sequence of RASQDISKYLN, CDRL2 sequence of SRLHSGV, and a CDRL3 sequence of GNTLPYTFG. In other embodiments, the scFV is a human or humanized ScFv sing a variable heavy chain comprising CDRH1 sequence of DYGVS , CDRH2 sequence of VIWGSETTYYNSALKS, and a CDRH3 sequence of YAMDYWG. The disclosure also contemplates variable regions that have at least 90% amino acid sequence identity to that of the scFv for FMC63 and that have at least the same affinity for CD19. In embodiments, the chimeric receptor has a short or intermediate spacer of 119 amino acids or less, or 12 amino acids or less. In a specific embodiment, the spacer is 12 amino acid or less and has a sequence of SEQ ID NO:4.

In embodiments, CDR regions are found within antibody regions as numbered by Kabat as follows: for the light chain; CDRL1 amino acids 24- 34;CDRL2 amino acids 50-56; CDRL3 at amino acids 89-97; for the heavy chain at CDRH1 at amino acids 31-35; CDRH2 at amino acids 50-65; and for CDRH3 at amino acids 95-102. CDR s in antibodies can be readily determined.

In specific embodiments, the target antigen is ROR1. A number of antibodies specific for ROR1 are known to those of skill in the art and can be readily terized for sequence, epitope binding, and ty. In a specific ment, the chimeric receptor construct includes a scFV ce from R12 antibody. In other embodiments, the scFV is a human or humanized ScFv comprising a variable light chain comprising a CDRL1 sequence of ASGFDFSAYYM, CDRL2 sequence of TIYPSSG, and a CDRL3 sequence of ADRATYFCA. In other embodiments, the scFV is a human or humanized ScFv comprising a variable heavy chain comprising CDRH1 sequence of DTIDWY, CDRH2 sequence of YTKRPGVPDR, and a CDRH3 sequence of YIGGYVFG. The sure also contemplates variable regions that have at least 90% amino acid sequence identity to that of the scFv for R12 and that have at least the same affinity for ROR1. In embodiments, the chimeric receptor has a short or ediate spacer of 119 amino acids or less, or 12 amino acids or less. In a specific embodiment, the spacer is 12 amino acid or less and has a sequence of SEQ ID NO:4.

In specific embodiments, the target antigen is ROR1. A number of antibodies specific for ROR1 are known to those of skill in the art and can be readily characterized for ce, epitope binding, and affinity. In a specific embodiment, the chimeric receptor construct includes a scFV sequence from R11 antibody. In other embodiments, the scFV is a human or humanized ScFv comprising a variable light chain sing a CDRL1 sequence of SGSDINDYPIS, CDRL2 sequence of INSGGST, and a CDRL3 sequence of YFCARGYS. In other embodiments, the scFV is a human or humanized ScFv comprising a variable heavy chain comprising CDRH1 ce of SNLAW, CDRH2 sequence of RASNLASGVPSRFSGS, and a CDRH3 sequence of NVSYRTSF. The disclosure also contemplates variable regions that have at least 90% amino acid sequence identity to that of the scFv for R11 and that have at least the same affinity for ROR1. In embodiments, the chimeric receptor has a long spacer of 229 amino acids or less. In a specific embodiment, the spacer is 229 amino acids and has a sequence of SEQ ID NO:50.

In specific embodiments, the target antigen is Her2. A number of antibodies specific for Her2 are known to those of skill in the art and can be readily characterized for sequence, epitope binding, and affinity. In a ic embodiment, the ic receptor construct includes a scFV sequence from Herceptin antibody.

In other embodiments, the scFV is a human or humanized ScFv comprising a variable light chain comprising a CDRL1 sequence, CDRL2 sequence and a CDRL3 sequence of the Herceptin dy. In other embodiments, the scFV is a human or humanized ScFv comprising a variable heavy chain comprising CDRH1 sequence, CDRH2, and a CDRH3 sequence of Herceptin. The CDR ces can readily be determined from the amino acid sequence of Herceptin. The disclosure also contemplates variable regions that have at least 90% amino acid sequence identity to that of the scFv for tin and that have at least the same ty for Her2. In embodiments, the chimeric receptor has a long spacer of 229 amino acids or less. In a ic embodiment, the spacer is 229 amino acids and has a sequence of SEQ ID NO:50.

In embodiments, a polynucleotide coding for a ligand binding domain is operably linked to a polynucleotide coding for a spacer region. In embodiments, the polynucleotide coding for a ligand binding domain may also have one or more restriction enzyme sites at the 5’ and/or 3’ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide with another polynucleotide coding for a ligand g domain coding for a different antigen or that has different binding characteristics. For example, a restriction site, NheI, is encoded upstream of the leader sequence; and a 3’ RsrII located within the hinge region allows subcloning of any desirable scFv into a chimeric receptor vector. In embodiments, the polynucleotide is codon optimized for expression in mammalian cells.

In embodiments, the cleotide coding for a ligand binding domain is operably linked to a signal peptide. In ments the signal peptide is a signal peptide for granulocyte colony stimulating factor. Polynucleotides coding for other signal peptides such as CD8 alpha can be utilized.

In embodiments, the cleotide coding for a ligand binding domain is operably linked to a promoter. A promoter is selected that provides for expression of the ic antigen receptor in a ian cell. In a specific embodiment the er is the elongation growth factor promoter (EF-1). Another example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV 40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HlV) long terminal repeat (LTR) promoter, MuMoLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early er, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Inducible promoters are also contemplated. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone er, and a tetracycline er.

A specific embodiment of a polynucleotide coding for a ligand binding domain is shown in Table 1 as the scFv from an antibody that specifically binds CD19, such as FMC63. A polynucleotide encoding for a flexible linker ing the amino acids GSTSGSGKPGSGEGSTKG (SEQ ID NO:36)separates the VH and VL chains in the scFV. The amino acid sequence of the scFv including the linker is shown in Table 2.(SEQ ID NO:11) Other argeting antibodies such as SJ25C1 and HD37 are known. (SJ25C1: Bejcek et al. Cancer Res 2005, PMID 7538901; HD37: Pezutto et al. JI 1987, PMID 2437199).

Spacer In ments, the chimeric receptor nucleic acid comprises a polynucleotide coding for a spacer . It has been surprisingly found that the length of the spacer region that is presumed not to have signaling capability affects the in vivo efficacy of the T cells modified to express the chimeric receptor and needs to be customized for individual target molecules for optimal tumor or target cell recognition. In embodiments, the chimeric receptor nucleic acid comprises a polynucleotide coding for a customizable spacer region selected from a library of polynucleotides coding for spacer regions. In embodiments, a spacer length is ed based upon the location of the epitope, affinity of the antibody for the epitope, and/or the ability of the T cells expressing the chimeric receptor to proliferate in vitro and/or in vivo in response to antigen recognition.

Typically a spacer region is found n the ligand binding domain and the transmembrane domain of the chimeric receptor. In embodiments, a spacer region provides for flexibility of the ligand binding domain, allows for high expression levels in lymphocytes. A CD19-specific chimeric receptor having a spacer domain of about 229 amino acids had less antitumor activity than a CD19- specific chimeric receptor with a short spacer region comprised of the modified IgG4 hinge only. Other chimeric ors, such as those constructed from the R12 or 2A2 scFvs also require a short spacer for optimal triggering of T cell effector functions, while a chimeric receptor constructed with the R11 ROR1 scFv requires a long spacer domain of about 229 amino acids for tumor recognition.

In embodiments, a spacer region has at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the nts of any of the listed ranges. In embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less.

In some embodiments, the spacer region is derived from a hinge region of an globulin like le. In embodiments, a spacer region comprises all or a portion of the hinge region from a human IgG1, human IgG2, a human IgG3, or a human IgG4, and may contain one or more amino acid substitutions. Exemplary sequences of the hinge regions are provided in Table 8. In embodiments, a portion of the hinge region es the upper hinge amino acids found between the le heavy chain and the core, and the core hinge amino acids ing a polyproline region. Typically, the upper hinge region has about 3 to 10 amino acids. In some cases, the spacer region comprises an amino acid sequence of X1PPX2P(SEQ ID NO:1). In embodiments, X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine.

In embodiments, hinge region sequences can be modified in one or more amino acids in order to avoid undesirable structural interactions such as dimerization. In a ic ment, the spacer region comprises a portion of a modified human hinge region from IgG4, for example, as shown in Table 2 or Table 8(SEQ ID NO:21). A representative of a polynucleotide coding for a portion of a modified IgG4 hinge region is provided in Table 1. (SEQ ID NO:4)In embodiments, a hinge region can have at least about 90%, 92%, 95%, or 100% sequence identity with a hinge region amino acid sequence identified in Table 2 or Table 8. In a specific embodiment, a portion of a human hinge region from IgG4 has an amino acid substitution in the core amino acids from CPSP to CPPC.

In some ments, all or a portion of the hinge region is combined with one or more domains of a constant region of an immunoglobulin. For example, a portion of a hinge region can be combined with all or a portion of a CH2 or CH3 domain or variant thereof. In embodiments, the spacer region does not include the 47-48 amino acid hinge region sequence from CD8apha or the spacer region consisting of an extracellular portion of the CD28 molecule.

In embodiments, a short spacer region has about 12 amino acids or less and comprises all or a portion of a IgG4 hinge region ce or variant thereof, an intermediate spacer region has about 119 amino acids or less and comprises all or a portion of a IgG4 hinge region sequence and a CH3 region or variant thereof, and a long spacer has about 229 amino acids or less and ses all or a portion of a IgG4 hinge region ce , a CH2 region, and a CH3 region or variant thereof.

A polynucleotide coding for a spacer region can be readily prepared by synthetic or recombinant methods from the amino acid sequence. In embodiments, a polynucleotide coding for a spacer region is operably linked to a polynucleotide coding for a transmembrane . In embodiments, the polynucleotide coding for the spacer region may also have one or more restriction enzyme sites at the 5’ and/or 3’ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide with r cleotide coding for a different spacer region. In embodiments, the polynucleotide coding for the spacer region is codon optimized for expression in mammalian cells.

In embodiments, a library of polynucleotides, each coding for different spacer region is described. In an embodiment, the spacer region is selected from the group consisting of a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 or portion f, a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in ation with all or a portion of a CH2 region or variant thereof, a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a n of a CH3 region or variant thereof, and a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a portion of a CH2 region or variant thereof, and a CH3 region or variant thereof. In embodiments, a short spacer region is a modified IgG4 hinge sequence(SEQ ID NO:4) having 12 amino acids or less, an intermediate sequence is a IgG4 hinge sequence with a CH3 sequence having 119 amino acids or less(SEQ ID NO:49); or a IgG4 hinge sequence with a CH2 and CH3 region having 229 amino acids or less (SEQ ID NO:50) In embodiments, a method of selecting a spacer region for a chimeric receptor is described herein. Surprisingly some chimeric or constructs, although effective to activate T cells and direct their killing of tumor cells in vitro, were not effective in vivo. In addition, the side effect profile of the chimeric receptor modified T cells can be such as to result in more cells oing activation induced cell death or causing an increase in in vivo cytokines. In embodiments, a method comprises providing a plurality of chimeric receptor nucleic acids, wherein the chimeric receptor nucleic acids differ only in the spacer region; ucing each of the chimeric or nucleic acids into a separate T lymphocyte population; expanding each separate lymphocyte population in vitro, and introducing each lymphocyte population into an animal bearing a tumor to determine the anti-tumor efficacy of each of the chimeric receptors when expressed in T cells, and selecting a chimeric receptor that provides anti-tumor efficacy as compared to each of the other separate lymphocyte populations modified with each of the other chimeric receptors.

Animal models of different tumors are known. Anti-tumor efficacy can be measured by identifying a decrease in tumor , by determining animal death, persistence of the cally modified T cells in vivo, activation of genetically modified T cells (for example, by detecting an increase in sion of CD25 and/CD69), and/or proliferation of cally modified T cells in vivo. In an embodiment, a chimeric receptor is selected that provides for the best anti-tumor efficacy in vivo as determined by one or more of these ters. Lack of antitumor cy can be determined by lack of persistence of the genetically modified lymphocytes in vivo, animal death, an increase in apoptosis as measured by an increase in induction of caspase -3, and/or a decrease in proliferation of cally modified lymphocytes.

In other embodiments, a method for selecting a spacer comprises selecting an epitope of a target le and characterizing the location of the epitope with respect to the cell membrane, selecting a spacer region that is long or short depending on the location of the epitope with respect to the cell membrane, selecting an antibody or fragment thereof that has an affinity for the epitope that is higher or lower as compared to a reference antibody, and determining whether the chimeric receptor construct provides for enhanced T cell proliferation or cytokine production in vitro and/or in vivo.

In some ments, if the target epitope or portion thereof is d proximal to the membrane it is located in the first 100 amino acids of the linear sequence of the extracellular domain adjacent to the transmembrane domain. If the epitope is located proximal to the membrane, a long spacer (e.g. 229 amino acids or less and greater than 119 amino acids) is selected. In some embodiments, if the target epitope is located distal to the membrane, it is located in the first 150 amino acids of the linear sequence of the extracellular domain terminus. If the e is located distal to the membrane, an intermediate or short spacer is selected (e.g. 119 amino acids or less or 12-15 amino acids or less). Alternatively, r the epitope is proximal or distal to the membrane can be determined by modeling of the three dimensional structure or based on analysis of the crystal structure, In some embodiments, a chimeric receptor is selected that provides for at least 30% of the cells erating through two generations in vitro and/or in vivo.

In other embodiments a ic receptor is not selected if it results in at least 50% of the cells undergoing activation induced cell death in 72 hours. In embodiments, a short spacer (e.g. 15 amino acids or less) is selected if the epitope is distal to the membrane. In embodiments, a long spacer (e.g. 229 amino acid or less and greater than 119 amino acids) is selected if the epitope is al to the membrane.

In embodiments, providing a plurality of chimeric receptor nucleic acids, wherein the chimeric receptor nucleic acids differ only in the spacer region comprises providing a ic receptor uct comprising a cleotide coding for a ligand binding domain, n the ligand is a tumor specific antigen, viral antigen, or any other molecule sed on a target cell population that is suitable to mediate recognition and elimination by a lymphocyte; a polynucleotide coding for a first polypeptide spacer having a defined restriction site at the 5’ and 3’ end of the coding sequence for the first polypeptide ; a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for one or more ellular ing s.

In ments, a method further comprises providing one or more polynucleotides, each encoding a different spacer region. In embodiments, the ent spacer regions are selected from the group consisting of a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 or variant thereof or portion thereof, a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a portion of a CH2 region or variant thereof, a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a portion of a CH3 region or variant thereof, and a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a portion of a CH2 region or variant thereof and a CH3 region or variant thereof. In ments, CH2 or CH3 regions may be modified by one or more deletions or amino acid substitutions in order to provide for expression in lymphocytes and/or in order to minimize interactions with other molecules. In ments, a portion of a hinge region ses at least the upper amino acids and the core sequence. In embodiments, a hinge region comprises the sequence X1PPX2P.

In embodiments, a method further comprises replacing the polynucleotide coding for the spacer region with a polynucleotide encoding a different spacer region to form a chimeric receptor nucleic acid with a different spacer region. The method can be repeated to form any number of chimeric or nucleic acids, each differing in the spacer region. In embodiments, the chimeric receptor nucleic acids differ from one r only in the spacer region.

Transmembrane domain In embodiments, the chimeric receptor nucleic acid comprises a polynucleotide coding for a transmembrane domain. The transmembrane domain provides for anchoring of the chimeric receptor in the membrane.

In an embodiment, the embrane domain that naturally is associated with one of the domains in the chimeric receptor is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

The embrane domain may be derived either from a natural or a synthetic source. When the source is l, the domain may be d from any membrane-bound or transmembrane protein. Transmembrane regions comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In a specific embodiment, the transmembrane domain comprises the amino acid sequence of the CD28 transmembrane domain as shown in Table 2. A representative polynucleotide sequence coding for the CD28 transmembrane domain is shown in Table 1(SEQ ID NO:5).

A transmembrane domain may be synthetic or a variant of a naturally occurring transmembrane domain. In embodiments, synthetic or variant transmembrane s comprise predominantly hydrophobic residues such as leucine and . In embodiments, a transmembrane domain can have at least about 80%, 85%, 90%, 95%, or 100% amino acid sequence identity with a transmembrane domain as shown in Table 2 or Table 6. Variant transmembrane domains preferably have a hydrophobic score of at least 50 as calculated by Kyte Doolittle.

A cleotide coding for a transmembrane domain can be readily prepared by synthetic or recombinant methods. In embodiments, a polynucleotide coding for a transmembrane domain is operably linked to a polynucleotide coding for a intracellular signaling . In embodiments, the polynucleotide coding for a transmembrane domain may also have one or more restriction enzyme sites at the 5’ and/or 3’ ends of the coding sequence in order to e for easy excision and replacement of the polynucleotide coding for a transmembrane domain with another polynucleotide coding for a different transmembrane domain. In embodiments, the cleotide coding for a transmembrane domain is codon optimized for expression in ian cells.

Intracellular signaling domain In embodiments, the chimeric receptor nucleic acid comprises a polynucleotide coding for an intracellular signaling domain. The intracellular signaling domain provides for activation of one function of the transduced cell expressing the chimeric receptor upon binding to the ligand expressed on tumor cells. In embodiments, the ellular signaling domain contains one or more intracellular signaling s. In embodiments, the intracellular signaling domain is a n of and/or a variant of an intracellular signaling domain that es for tion of at least one function of the transduced cell.

Examples of intracellular signaling domains for use in a chimeric receptor of the sure include the cytoplasmic sequences of the CD3 zeta chain, and/or ptors that act in concert to te signal transduction following ic receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional lity. T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation and provide a T cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigenindependent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences that act in a stimulatory manner may n signaling motifs which are known as or tyrosine-based activation motifs or ITAMs. es of ITAM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In embodiments, the primary signaling intracellular domain can have at least about 80%, 85%, 90%, or 95% sequence identity to CD3zeta having a sequence provided in Table 2. In embodiments variants, of CD3 zeta retain at least one, two, three or all ITAM regions as shown in Table 7.

In a preferred embodiment, the intracellular signaling domain of the ic receptor can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s). For example, the intracellular signaling domain of the chimeric receptor can comprise a CD3zeta chain and a costimulatory signaling region.

The costimulatory signaling region refers to a portion of the chimeric receptor comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for a response of lymphocytes to an antigen. Examples of such les include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. In embodiments, the costimulatory signaling domain can have at least about 80%, 85%, 90%, or 95% amino acid sequence identity to the intracellular domain of CD28 as shown in Table or to 4-1BB having a sequence provided in Table 2. In an embodiment, a variant of the CD28 intracellular domain comprises an amino acid substitution at positions 186-187, wherein LL is substituted with GG.

The intracellular signaling sequences of the chimeric receptor may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. In one ment, the intracellular signaling domains comprises all or a portion of the signaling domain of ta or variant thereof and all or a portion of the signaling domain of CD28 or a variant thereof. In another embodiment, the intracellular signaling domain comprises all or a portion of the signaling domain of ta or variant thereof and all or a portion of the signaling domain of 4-lBB or variant thereof. In yet another ment, the intracellular signaling domain comprises all or a portion of the signaling domain of CD3-zeta or variant f, all or a portion of the ing domain of CD28 or variant thereof, and all or a portion of the signaling domain of 4-lBB or variant thereof. In a specific embodiment, the amino acid sequence of the intracellular signaling domain comprising a variant of CD3zeta and a portion of the 4-1BB intracellular signaling domain is provided in Table 2. A representative nucleic acid sequence is provided in Table 1(SEQ ID NO:6; SEQ ID NO:7).

In an embodiment, a cleotide coding for an intracellular ing domain comprises a 4-1BB ellular domain linked to a portion of a a domain. In other embodiments, a 4-1BB intracellular domain and a CD28 intracellular domain are linked to a portion of a CD3 zeta domain.

A polynucleotide coding for an intracellular signaling domain can be readily prepared by synthetic or recombinant methods from the amino acid sequence. In embodiments, the polynucleotide coding for an ellular signaling domain may also have one or more restriction enzyme sites at the 5’ and/or 3’ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide coding for an intracellular signaling domain with another polynucleotide coding for a ent ellular signaling domain. In embodiments, the polynucleotide coding for an intracellular signaling domain is codon optimized for expression in mammalian cells.

Marker sequences In embodiments, the chimeric receptor nucleic acid ally further comprises a polynucleotide sequence coding for a marker sequence. A marker sequence can provide for selection of transduced cells, and identification of transduced cells. In embodiments, the marker ce is operably linked to a polynucleotide sequence coding for a linker sequence. In embodiments, the linker sequence is a cleavable linker sequence.

A number of different marker sequences can be employed. Typically a marker ce has a functional characteristic that allows for selection of transduced cells and/or detection of uced cells. In embodiments, the marker sequence is compatible with transduction of human lymphocytes.

The positive selectable marker may be a gene, which upon being introduced into the host cell, expresses a dominant phenotype permitting ve selection of cells carrying the gene. Genes of this type are known in the art, and include, inter alia, hygromycin-B otransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside otransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the ofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene.

In an embodiment, a chimeric receptor nucleic acid further comprises a polynucleotide coding for a marker sequence. In an embodiment, the marker ce is a truncated epidermal growth factor receptor as shown in Table 2. An exemplary polynucleotide for the truncated epidermal growth factor receptor is shown in Table 1. (SEQ ID NO:9)In embodiments, the polynucleotide coding for the marker sequence is operably linked to a polynucleotide coding for a linker ce.

In a specific embodiment, the linker sequence is a cleavable linker sequence T2A as shown in Table 2. An exemplary polynucleotide sequence coding for the T2A linker is provided in Table 1.(SEQ ID NO:8) A polynucleotide coding for marker sequence can be readily prepared by synthetic or recombinant methods from the amino acid sequence. In embodiments a polynucleotide coding for a marker sequence is operably linked to a polynucleotide coding for an intracellular signaling domain. In embodiments, the polynucleotide coding for a marker sequence may also have one or more restriction enzyme sites at the 5’ and/or 3’ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide coding for a marker sequence with another polynucleotide coding for a different marker sequence. In embodiments, the polynucleotide coding for a marker sequence is codon optimized for expression in ian cells.

Vectors, Cells and Methods of transducing cells Selection and Sorting of T lymphocyte populations The compositions described herein provide for CD4+ and/or CD8+ T cytes. T lymphocytes can be ted in accordance with known techniques and enriched or depleted by known techniques such as ty binding to antibodies such as flow cytometry and/or immunomagnetic selection. After enrichment and/or depletion steps, in vitro expansion of the d T cytes can be d out in accordance with known techniques (including but not limited to those described in US Patent No. 6,040,177 to Riddell et al.), or variations thereof that will be apparent to those skilled in the art. In ments, the T cells are autologous T cells ed from the patient.

For example, the desired T cell population or subpopulation may be expanded by adding an initial T cyte tion to a culture medium in vitro, and then adding to the culture medium feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and ting the culture (e.g. for a time sufficient to expand the numbers of T cells). The non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. The order of addition of the T cells and feeder cells to the culture media can be reversed if desired. The culture can typically be incubated under conditions of temperature and the like that are suitable for the growth of T lymphocytes. For the growth of human T lymphocytes, for example, the temperature will lly be at least about 25 degrees Celsius, preferably at least about 30 degrees, more preferably about 37 degrees.

The T cytes expanded include CD8+ cytotoxic T lymphocytes (CTL) and CD4+ helper T lymphocytes that may be specific for an antigen present on a human tumor or a pathogen.

Optionally, the expansion method may further comprise the step of adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells may be provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). ally, the expansion method may further comprise the step of adding IL-2 and/or IL-15 to the culture medium (e.g., wherein the concentration of IL-2 is at least about 10 units/m1).

After ion of T lymphocytes both cytotoxic and helper T cytes can be sorted into naïve, , and effector T cell subpopulations either before or after expansion.

CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further sorted into naïve, central memory, and effector memory cells by fying cell surface ns that are associated with each of those types of CD8+ cells. In embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some ments, the sion of phenotypic markers of central memory TCM e CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and are negative or low for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector TE are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some embodiments, naïve CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naïve T cells including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

Whether a cell or cell population is positive for a particular cell surface marker can be determined by flow cytometry using staining with a specific antibody for the surface marker and an isotype matched control antibody. A cell population negative for a marker refers to the absence of significant staining of the cell population with the specific dy above the isotype control, positive refers to uniform staining of the cell tion above the isotype control. In some embodiments, a decrease in expression of one or s refers to loss of 1 log10 in the mean fluorescence intensity and/or decrease of percentage of cells that exhibit the marker of at least about 20% of the cells, 25% of-the cells, 30% of the cells, 35% of the cells, 40% of the cells, 45% of the cells, 50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells, 90% of the cell, 95% of the cells, and 100% of the cells and any % between 20 and 100% when compared to a reference cell population. In some embodiments, a cell population positive for one or markers refers to a percentage of cells that exhibit the marker of at least about 50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells, 90% of the cell, 95% of the cells, and 100% of the cells and any % between 50 and 100% when compared to a reference cell population.

CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naïve CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, l memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

In ments, populations of CD4+ and CD8+ that are antigen specific can be obtained by stimulating naïve or antigen ic T lymphocytes with antigen.

For example, antigen-specific T cell lines or clones can be ted to Cytomegalovirus antigens by isolating T cells from ed subjects and ating the cells in vitro with the same antigen. Naïve T cells may also be used. Any number of antigens from tumor cells may be ed as targets to elicit T cell responses. In some embodiments, the adoptive cellular immunotherapy compositions are useful in the treatment of a disease or disorder including a solid tumor, logic malignancy, breast cancer or melanoma.

Modification of T lymphocyte tions In some embodiments it may be desired to introduce functional genes into the T cells to be used in immunotherapy in accordance with the present disclosure.

For example, the introduced gene or genes may improve the efficacy of therapy by promoting the viability and/or function of transferred T cells; or they may provide a genetic marker to permit selection and/or evaluation of in vivo survival or migration; or they may orate functions that improve the safety of immunotherapy, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and l et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. This can be carried out in accordance with known ques (see, e.g., US Patent No. 177 to Riddell et al. at columns 14-17) or variations thereof that will be apparent to those skilled in the art based upon the present disclosure.

In embodiments, T cells are modified with chimeric receptors as described herein. In some embodiments, the T cells are obtained from the subject to be d, in other embodiments, the lymphocytes are obtained from allogeneic human donors, preferably healthy human donors.

In some embodiments, chimeric receptors comprise a ligand binding domain that ically binds to a tumor cell e le, a polypeptide spacer region, a transmembrane domain and an intracellular signaling domain as described herein.

In embodiments, the ligand binding domain is a single-chain antibody fragment (scFv) that is derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb). Costimulatory signals can also be provided through the chimeric or by fusing the costimulatory domain of CD28 and/or 4-1BB to the CD3ζ chain. Chimeric receptors are specific for cell surface molecules independent from HLA, thus overcoming the limitations of TCR-recognition including striction and low levels of HLA-expression on tumor cells.

Chimeric receptors can be constructed with a specificity for any cell surface marker by utilizing antigen binding fragments or antibody variable domains of, for example, dy molecules. The antigen binding molecules can be linked to one or more cell signaling s. In embodiments, cell signaling modules include CD3 transmembrane , CD3 intracellular signaling domains, and CD28 transmembrane domains. In embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 zeta intracellular domain. In some embodiments, a chimeric receptor can also e a transduction marker such as tEGFR.

In ments, the same or a different chimeric receptor can be introduced into each of population of CD4+ and CD8+ T lymphocytes. In embodiments, the chimeric receptor in each of these populations has a ligand binding domain that specifically binds to the same ligand on the tumor or infected cell. The cellular ing modules can differ. In embodiments, the intracellular signaling domain of the CD8+ cytotoxic T cells is the same as the intracellular signaling domain of the CD4+ helper T cells. In other embodiments, the intracellular ing domain of the CD8+ cytotoxic T cells is different than the intracellular signaling domain of the CD4+ helper T cells.

In embodiments each of the CD4 or CD8 T lymphocytes can be sorted in to naïve, central memory, effector memory or effector cells prior to transduction as described herein. In alternative embodiments, each of the CD4 or CD8 T lymphocytes can be sorted in to naïve, central memory, effector memory, or effector cells after uction.

Various transduction techniques have been developed which utilize recombinant infectious virus particles for gene ry. This represents a currently preferred approach to the transduction of T lymphocytes described herein. The viral vectors which have been used in this way include virus vectors derived from simian virus 40, adenoviruses, adeno-associated virus (AAV), lentiviral vectors, and retroviruses. Thus, gene transfer and expression s are numerous but essentially function to introduce and express genetic material in mammalian cells.

Several of the above techniques have been used to transduce hematopoietic or lymphoid cells, including m ate transfection, last fusion, electroporation, and infection with recombinant adenovirus, adeno-associated virus and irus vectors. Primary T lymphocytes have been successfully uced by electroporation and by retroviral or lentiviral infection.

Retroviral and lentiviral s provide a highly efficient method for gene transfer into eukaryotic cells. Moreover, retroviral or lentiviral integration takes place in a controlled fashion and results in the stable integration of one or a few copies of the new genetic information per cell.

It is contemplated that overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to the treated individual. Therefore, it is within the scope of the t disclosure to include gene segments that cause the T cells described herein to be susceptible to negative selection in vivo. By ''negative selection" is meant that the infused cell can be eliminated as a result of a change in the in vivo condition of the dual. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes are known in the art, and include, inter alia the ing: the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers ganciclovir sensitivity; the cellular hypoxanthine ribosyltransferase (HPRT) gene, the ar adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase, In some embodiments it may be useful to e in the T cells a ve marker that enables the selection of cells of the negative selectable phenotype in vitro. The positive selectable marker may be a gene that upon being introduced into the host cell expresses a dominant phenotype permitting positive selection of cells carrying the gene. Genes of this type are known in the art, and include, inter alia, ycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the ofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene.

A variety of methods can be employed for transducing T lymphocytes, as is well known in the art. In embodiments, transduction is carried out using iral vectors.

In embodiments, CD4+ and CD8+ cells each can separately be modified with an expression vector encoding a chimeric receptor to form defined populations. In embodiments, these cells are then further sorted into subpopulations of naïve, central memory and effector cells as described above by sorting for cell surface antigens unique to each of those cell populations. In addition, CD4+ or CD8+ cell populations may be selected by their cytokine profile or proliferative activities. For example, CD4+ T lymphocytes that have enhanced production of cytokines such as IL-2, IL-4, IL-10, TNFα, and IFNγ as compared to sham transduced cells or transduced CD8+ cells when ated with antigen can be selected. In other ments, naïve or central memory CD4+ T cells that have enhanced production of IL-2 and/or TNFα are selected. Likewise, CD8+ cells that have enhanced IFNγ production are selected as compared to sham transduced CD8+ cells.

In embodiments, CD4+ and CD8+cells that proliferate in response to antigen or tumor targets are selected. For example, CD4+ cells that erate vigorously when stimulated with antigen or tumor targets as compared to sham transduced cells, or CD8+ uced cells are selected. In some ments, CD4+ and CD8+ cells are selected that are cytotoxic for antigen bearing cells. In embodiments, CD4+ are ed to be weakly cytotoxic as compared to CD8+ cells.

In a red embodiment, transduced lymphocytes, such as CD8+ central memory cells, are selected that provide for tumor cell killing in vivo using an animal model established for the particular type of cancer. Such animal models are known to those of skill in the art and exclude human beings. As described herein, not all chimeric receptor constructs transduced into lymphocytes confer the ability to kill tumor cells in vivo e the ability to become activated and kill tumor cells in vitro. In particular, for some target molecules T cells having chimeric receptor constructs with a long spacer region were less effective at killing tumor cells in vivo as ed to T cells having a chimeric receptor with short spacer region. For other target molecules, T cells having chimeric receptor constructs with a short spacer region were less effective at g tumor cells in vivo as compared to T cells having ic receptors with a long spacer region.

In yet other embodiments, transduced chimeric receptor expressing T cells are selected that can persist in vivo using an animal model established for the particular type of cancer. In embodiments, transduced chimeric receptor CD8+ central memory cells with a short spacer region have been shown to persist in vivo after introduction into the animal for about 3 day or more, 10 days or more, 20 days or more, 30 days or more, 40 days or more, or 50 days or more.

The disclosure contemplates that combinations of CD4+ and CD8+ T cells will be utilized in the compositions. In one embodiment, combinations of chimeric receptor transduced CD4+ cells can be combined with chimeric receptor transduced CD8+ cells of the same ligand specificity or combined with CD8+ T cells that are specific for a distinct tumor ligand. In other embodiments, chimeric receptor transduced CD8+ cells are combined with chimeric receptor transduced CD4+ cells specific for a ent ligand expressed on the tumor. In yet another embodiment, chimeric receptor modified CD4+ and CD8+ cells are combined. In embodiments CD8+ and CD4+ cells can be combined in different ratios for e, a 1:1 ratio of CD8+ and CD4+, a ratio of 10:1 of CD8+ to CD4+, or a ratio of 100:1 of CD8+ to CD4+. In embodiments, the combined tion is tested for cell proliferation in vitro and/or in vivo, and the ratio of cells that provides for proliferation of cells is selected.

As described herein, the disclosure contemplates that CD4+ and CD8+ cells can be r separated into subpopulations, such as naïve, central memory, and effector memory cell populations. As described herein, in some embodiments, naïve CD4+ cells are CD45RO-, +, CD62L+, CD4+ positive T cells. In some ments, central memory CD4+ cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+ cells are CD62L negative and CD45RO positive. Each of these populations may be ndently modified with a chimeric receptor.

As described herein, in embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some ments, the expression of phenotypic markers of central memory T cells (TCM) include CD62L, CCR7, CD28, CD3, and CD127 and are ve or low for granzyme B. In some embodiments, central memory T cells are +, CD62L+, CD8+ T cells. In some embodiments, or T cells (TE) are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some embodiments, naïve CD8+ T lymphocytes are characterized by CD8+, CD62L+, CD45RO+, CCR7+, CD28+ CD127+, and CD45RO+. Each of these populations may be independently modified with a chimeric receptor .

After transduction and/or selection for ic receptor g cells, the cell populations are preferably expanded in vitro until a sufficient number of cells are obtained to provide for at least one infusion into a human subject, typically around 104 cells/kg to 109 cells/kg In embodiments, the transduced cells are cultured in the presence of n bearing cells, anti CD3, anti CD28, and IL 2, IL-7, IL 15, IL-21 and combinations f.

Each of the subpopulations of CD4+ and CD8+ cells can be combined with one another. In a specific embodiment, modified naïve or central memory CD4+ cells are combined with modified central memory CD8+ T cells to provide a synergistic cytotoxic effect on antigen bearing cells, such as tumor cells.

Compositions Also described is an adoptive cellular immunotherapy composition comprising a genetically modified T lymphocyte cell preparation as described herein.

In embodiments, the T lymphocyte cell preparation comprises CD4 + T cells that have a chimeric receptor sing an extracellular antibody variable domain ic for a ligand associated with the disease or disorder, a izable spacer , a transmembrane domain, and an intracellular signaling domain of a T cell receptor or other receptors as described herein. In other embodiments, an adoptive cellular immunotherapy composition r comprises a chimeric receptor modified specific CD8+ cytotoxic T lymphocyte cell preparation that provides a cellular immune response, wherein the cytotoxic T lymphocyte cell preparation comprises CD8+ T cells that have a chimeric receptor sing an ellular single chain antibody specific for a ligand associated with the disease or disorder, a customizable spacer region, a transmembrane domain, and an intracellular signaling domain of a T cell receptor as described herein. In embodiments, the chimeric receptor modified T cell population of the disclosure can persist in vivo for at least about 3 days or longer.

In some embodiments, an adoptive cellular immunotherapy composition comprises a chimeric or modified tumor-specific CD8+ cytotoxic T lymphocyte cell preparation that provides a cellular immune response, wherein the cytotoxic T lymphocyte cell preparation comprises CD8+ T cells that have a chimeric receptor comprising an extracellular single chain antibody specific for a ligand associated with the disease or disorder, a customizable spacer region, a transmembrane domain, and an intracellular signaling domain of a T cell receptor, in combination with an antigen-reactive chimeric receptor modified naïve CD4+ T helper cell derived from CD45RO- CD62L+ CD4+ T cells, and a pharmaceutically acceptable carrier.

In other embodiments, an adoptive cellular immunotherapy composition comprises an antigen specific CD8+ cytotoxic T lymphocyte cell preparation that provides a cellular immune response derived from the patient combined with an antigen-reactive chimeric receptor modified naïve CD4+ T helper cell that ts the CD8+ immune response, wherein the helper T lymphocyte cell preparation comprises CD4 + T cells that have a chimeric receptor comprising an extracellular antibody variable domain specific for the antigen associated with the disease or disorder, a customizable spacer region, a embrane domain, and an intracellular signaling domain of a T cell or.

In a further embodiment, an adoptive cellular immunotherapy ition comprises an antigen-reactive chimeric receptor modified naïve CD4+ T helper cell that augments the CD8+ immune response, wherein the helper T lymphocyte cell preparation comprises CD4 + T cells that have a ic receptor comprising an extracellular antibody variable domain specific for a ligand associated with a disease or disorder, a customizable spacer region, a embrane domain, and an ellular signaling domain of a T cell receptor.

In embodiments, the CD4+ T helper cyte cell is selected from the group ting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, or bulk CD4+ T cells. In some embodiments, CD4+ helper cyte cell is a naïve CD4+ T cell, wherein the naïve CD4+ T cell comprises a CD45RO-, CD45RA+, CD62L+ CD4+ T cell. In embodiments, the CD8+ T cytotoxic lymphocyte cell is selected from the group ting of naïve CD8+ T cells, central memory CD8+ T cells, or memory CD8+ T cells or bulk CD8+ T cells. In some embodiments, the CD8+ cytotoxic T lymphocyte cell is a central memory T cell wherein the central memory T cell ses a CD45RO+, CD62L+, CD8+ T cell. In yet other embodiments, the CD8+ cytotoxic T lymphocyte cell is a central memory T cell and the CD4+ helper T lymphocyte cell is a naïve or central memory CD4+ T cell.

Methods Also described are methods of making adoptive immunotherapy compositions and uses or methods of using these compositions for performing cellular therapy in a subject having a disease or disorder. In ments, the chimeric receptor modified T cells as described herein are able to persist in vivo for at least 3 days, or at least 10 days. In embodiments, the chimeric receptor ed T cells as described herein can proliferate in vivo through at least 2, or at least 3 generations as determined by CFSE dye dilution. Proliferation and persistence of the chimeric receptor modified T cells can be determined by using an animal model of the disease or disorder and administering the cells and determining persistence and/ or proliferative capacity of the transferred cells. In other ments, proliferation and activation can be tested in vitro by going through multiple cycles of activation with antigen bearing cells.

In embodiments, a method of manufacturing the compositions comprises obtaining a modified naïve CD4+ T helper cell, wherein the modified helper T lymphocyte cell preparation comprises CD4+ T cells that have a chimeric receptor comprising a ligand binding domain specific for a tumor cell surface molecule, a customized spacer domain, a transmembrane domain, and an intracellular signaling domain as described herein.

In r embodiment, a method further comprises obtaining a modified CD8+ cytotoxic T cell, wherein the modified cytotoxic T lymphocyte cell preparation ses CD8+ cells that have a chimeric or comprising a ligand binding domain specific for a tumor cell surface molecule, a customized spacer domain, a transmembrane domain, and an ellular signaling domain as described herein.

In another embodiment, a method comprises ing a modified CD8+ cytotoxic T cell, wherein the modified cytotoxic T lymphocyte cell preparation ses CD8+ T cells that have a chimeric or comprising a ligand binding domain specific for a tumor cell surface le, a customized spacer domain, a transmembrane domain, and an intracellular signaling domain as described herein, and further comprising combining the modified CD8+ cytotoxic T cells with a CD4+ helper cell lymphocyte cell preparation.

The preparation of the CD4+ and CD8+ cells that are modified with a chimeric receptor has been described above as well as in the examples. Antigen specific T lymphocytes can be obtained from a t having the disease or disorder or can be prepared by in vitro stimulation of T lymphocytes in the presence of antigen. Subpopulations of CD4+ and CD8+ T lymphocytes that are not selected for antigen specificity can also be isolated as described herein and combined in the s of manufacturing. In embodiments, the ation of cell populations can be evaluated for uniformity of cell surface makers, the ability to proliferate through at least two generations, to have a uniform cell differentiation status. Quality control can be performed by coculturing an cell line expressing the target ligand with chimeric receptor modified T cells to determine if the chimeric receptor modified T cells recognize the cell line using cytotoxicity, eration, or cytokine production assays that are known in the field. Cell differentiation status and cell surface markers on the chimeric receptor modified T cells can be determined by flow cytometry. In embodiments, the markers and cell differentiation status on the CD8+ cells include CD3, CD8, CD62L, CD28, CD27, CD69, CD25, PD-1, CTLA-4, CD45RO, and CD45RA. In embodiments, the markers and the cell differentiation status on the CD4+ cells e CD3, CD4, CD62L, CD28, CD27, CD69, CD25, PD-1, CTLA-4 CD45RO, and CD45RA.

In ments, a method of selecting a spacer region for a chimeric receptor is described herein. Surprisingly some chimeric receptor ucts, although effective to activate T cells in vitro, were not effective in vivo. In embodiments, a method comprises providing a plurality of ic receptor nucleic acids, wherein the chimeric receptor nucleic acids differ only in the spacer region; introducing each of the chimeric receptor nucleic acids into a separate T lymphocyte population; expanding each separate lymphocyte tion in vitro, and introducing each lymphocyte population into an animal bearing a tumor to determine the anti-tumor efficacy of each of the ic receptor modified T cells, and selecting a ic receptor that provides anti-tumor efficacy as compared to each of the other separate lymphocyte populations modified with each of the other chimeric or modified T cells.

Animal models of different tumors are known. Anti-tumor efficacy can be measured by fying a decrease in tumor volume, by determining animal death, persistence of the genetically ed T cells in vivo, activation of genetically modified T cells (for example, by detecting an increase in expression of CD25 and/CD69), and/or proliferation of genetically modified T cells in vivo. In an embodiment, a ic receptor is selected that provides for the best anti-tumor efficacy in vivo as determined by one or more of these parameters. Lack of antitumor efficacy can be determined by lack of tence of the genetically ed lymphocytes in vivo, animal death, an increase in sis as measured by an increase in induction of caspase -3, and/or a decrease in eration of cally modified lymphocytes.

In embodiments, providing a plurality of chimeric receptor nucleic acids, wherein the chimeric receptor c acids differ only in the spacer region comprises providing a chimeric or construct comprising a polynucleotide coding for a ligand binding domain, wherein the ligand is a tumor specific antigen, viral antigen, or any other molecule expressed on a target cell population that is suitable to mediate recognition and elimination by a lymphocyte; a polynucleotide coding for a first polypeptide spacer having a defined restriction site at the 5’ and 3’ end of the coding sequence for the first polypeptide spacer; a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for an intracellular signaling domain.

Also described are methods of performing cellular immunotherapy in a subject having a e or disorder comprising: administering a composition of lymphocytes expressing a chimeric receptor as described herein. In other embodiments, a method comprises administering to the subject a genetically modified cytotoxic T lymphocyte cell preparation that provides a cellular immune response, wherein the cytotoxic T lymphocyte cell preparation comprises CD8 + T cells that have a chimeric or comprising a ligand binding domain specific for a tumor cell surface molecule, a customized spacer domain, a transmembrane domain, and an intracellular signaling domain as described herein, and a cally modified helper T lymphocyte cell preparation that elicits direct tumor recognition and augments the cally modified cytotoxic T lymphocyte cell preparations y to mediate a cellular immune response, wherein the helper T lymphocyte cell preparation comprises CD4+ T cells that have a chimeric receptor comprising a ligand binding domain specific for a tumor cell surface molecule, a customized spacer domain, a transmembrane , and an intracellular signaling domain as described herein.

While not limiting the scope of the disclosure, it is ed by selecting the chimeric receptor modified T cell population that can persist and proliferate in vivo prior to administration may result in the ability to use a lower dose of T cells and provide more uniform therapeutic activity. In embodiments, the dose of T cells can be reduced at least 10%, 20%, or 30% or greater. ion in the dose of T cells may be beneficial to reduce the risk or tumor lysis syndrome and cytokine storm.

In another embodiment, a method of performing cellular immunotherapy in t having a disease or disorder comprises: administering to the t a genetically modified helper T lymphocyte cell preparation, wherein the modified helper T lymphocyte cell preparation comprises CD4+ T cells that have a chimeric receptor comprising a ligand binding domain specific for a tumor cell surface molecule, a customized spacer domain, a transmembrane domain, and an intracellular signaling domain as described herein. In an embodiments, the method further comprises administering to the t a genetically ed cytotoxic T lymphocyte cell preparation, wherein the modified cytotoxic T cyte cell preparation comprises CD8+ cells that have a chimeric receptor comprising a ligand binding domain ic for a tumor cell surface molecule, a customized spacer , a transmembrane domain, and an intracellular signaling domain as described herein.

Another embodiment describes a method of performing cellular immunotherapy in a subject having a disease or disorder comprising: analyzing a biological sample of the subject for the presence of a target molecule associated with the disease or disorder and administering the ve immunotherapy compositions described herein, wherein the chimeric receptor ically binds to the target In some embodiments, the CD4+ T helper lymphocyte cell is selected prior to introduction of the chimeric or from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells or bulk CD4+ T cells. In a specific embodiment, CD4+ helper lymphocyte cell is a naïve CD4+ T cell, wherein the naïve CD4+ T cell comprises a CD45RO-, CD45RA+, CD62L+ CD4+ T cell. In yet other embodiments, the CD8+ T cytotoxic cyte cell is selected prior to introduction of the chimeric receptor from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells or bulk CD8+ T cells. In a specific embodiment, the CD8+ cytotoxic T lymphocyte cell is a central memory T cell wherein the central memory T cell comprises a CD45RO+, CD62L+, CD8+ T cell. In a specific embodiment, the CD8+ cytotoxic T lymphocyte cell is a central memory T cell and the CD4+ helper T lymphocyte cell is a naïve CD4+ T cell.

In embodiments, the CD8+ T cell and the CD4+ T cell are both genetically modified with a chimeric receptor comprising an antibody heavy chain domain that specifically binds a tumor-specific cell surface molecule. In other embodiments, the ellular signaling domain of the CD8 xic T cells is the same as the ellular signaling domain of the CD4 helper T cells. In yet other embodiments, the ellular signaling domain of the CD8 cytotoxic T cells is different than the ellular signaling domain of the CD4 helper T cells.

Subjects that can be treated by the methods described herein are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes. The subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric ts.

The methods are useful in the ent of, for example, hematologic malignancy, melanoma, breast cancer, and other epithelial ancies or solid tumors. In some embodiments, the molecule associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, Her2, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen.

Subjects that can be treated include ts afflicted with cancer, including but not limited to colon, lung, liver, breast, renal, prostate, ovarian, skin (including melanoma), bone, and brain cancer, etc. In some embodiments the tumor associated antigens or molecules are known, such as melanoma, breast cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma, and prostate cancer. In other embodiments the tumor associated molecules can be ed with genetically modified T cells expressing an engineered chimeric receptor. Examples e but are not limited to B cell lymphoma, breast cancer, prostate cancer, and leukemia.

Cells prepared as described above can be utilized in methods and compositions for adoptive therapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure.

In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and trating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a ent-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum n, fetal bovine serum or other human serum components.

A treatment effective amount of cells in the composition is at least 2 cell subsets (for example, 1 CD8+ central memory T cell subset and 1 CD4+ helper T cell subset) or is more typically greater than 102 cells, and up to 106, up to and including 108 or 109 cells and can be more than 1010 cells. The number of cells will depend upon the te use for which the composition is intended as will the type of cells included therein. For example, if cells that are ic for a particular antigen are desired, then the tion will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells. For uses described , the cells are generally in a volume of a liter or less, can be 500 mls or less, even 250 mls or 100 mls or less. Hence the density of the desired cells is typically greater than 104 cells/m1 and generally is r than 107 cells/ml, generally 108 cells/ml or greater.

The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 106, 107, 108, 108, 109, 1010 or 1011 cells.

In some embodiments, the lymphocytes described herein may be used to confer immunity to individuals. By "immunity" is meant a lessening of one or more physical symptoms associated with a response to infection by a pathogen, or to a tumor, to which the lymphocyte response is ed. The amount of cells administered is usually in the range present in normal duals with immunity to the pathogen. Thus, the cells are usually administered by infusion, with each infusion in a range of from 2 cells, up to at least 106 to 3x1010 cells, preferably in the range of at least 107 to 109 cells. The T cells may be administered by a single infusion, or by multiple ons over a range of time. However, since different individuals are expected to vary in responsiveness, the type and amount of cells infused, as well as the number of infusions and the time range over which multiple infusions are given are determined by the attending physician, and can be determined by e examination. The generation of sufficient levels of T lymphocytes (including cytotoxic T cytes and/or helper T lymphocytes) is readily achievable using the rapid expansion method described herein, as ified herein. See, e.g., US Patent No. 6,040,177 to Riddell et al. at column 17.

In embodiments, the composition as described herein are administered intravenously, intraperitoneally, intratumorly, into the bone marrow, into the lymph node, and /or into cerebrospinal fluid. In embodiments, the chimeric receptor engineered compositions are delivered to the site of the tumor. Alternatively, the compositions as described herein can be combined with a compound that s the cells to the tumor or the immune system compartments and avoid sites such as the lung.

In embodiments, the compositions as described herein are administered with chemotherapeutic agents and/or immunosuppressants. In an ment, a patient is first treated with a chemotherapeutic agent that inhibits or destroys other immune cells followed by the compositions described herein. In some cases, herapy may be avoided entirely.

The present invention is illustrated further in the examples set forth below.

EXPERIMENTAL Example I. Customizing spacer domain length and scFv affinity for optimal recognition of ROR1 with chimeric receptor modified T cells We constructed chimeric receptors specific for the ROR1 molecule that is expressed on a large number of human ancies ing chronic lymphocytic leukemia, mantle cell lymphoma, acute lymphoblastic leukemia, and breast, lung prostate, pancreas and ovarian cancer. The ROR1 ic receptors were designed from ROR1 specific scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of ent lengths. The ability of s expressing each ROR-1 specific chimeric receptor to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma ted into immunodeficient mice was analyzed. als and Methods Human subjects Peripheral blood mononuclear cells (PBMC) were obtained from y donors and patients after written informed consent on research protocols approved by the Institutional Review Board of the Fred Hutchinson Cancer ch Center (FHCRC).

Cell lines The K562, Raji, JeKo-1, MDA-MB-231, MDA-MB-468, and 293T cell lines were obtained from the American Type Culture tion. Dr. Edus H. Warren ) kindly provided the renal cell cancer lines FARP, TREP and RWL.

K562/ROR1 and Raji/ROR1 were generated by lentiviral uction with the fulllength ROR1-gene. To derive JeKo-1/ffluc, native JeKo-1 cells were transduced with a lentiviral vector encoding the y luciferase (ffluc)-gene upstream of a T2A sequence and eGFP. The transduced JeKo-1 cells were sorted for eGFP expression, and expanded for in vivo experiments.

Immunophenotyping PBMC and cell lines were stained with the following conjugated mAbs: CD3, CD4, CD5, CD8, CD19, CD28, CD45RO, CD62L, CD314 (NKG2D), MICA/B and matched isotype controls (BD Biosciences). Propidium iodide (PI) staining was performed for live/dead cell discrimination. Cell surface expression of ROR1 was analyzed using a polyclonal goat anti-human-ROR1 antibody (R&D Systems).

Surface expression of 2A2 ROR1chimeric receptor was analyzed using a polyclonal goat anti-mouse-IgG antibody (Fab-specific) (Jackson Research).

Flow analyses were done on a FACSCanto®, sort-purifications on a iaII® (Becton Dickinson) and data analyzed using FlowJo® software (Treestar).

Vector construction and preparation of chimeric receptor encoding lentivirus ROR1-specific and pecific chimeric receptors were constructed using VL and VH chain segments of the 2A2, R12, and R11 mAbs (ROR1) and FMC63 mAb (CD19). (Variable region sequences for R11 and R12 are ed in Yang et al, Plos One 6(6):e21018, June 15, 2011) Each scFV was linked by a (G4S)3(SEQ ID NO:12) peptide to a spacer domain derived from IgG4-Fc (Uniprot Database: P01861,SEQ ID NO:13) comprising either ‘Hinge-CH2-CH3’ (229 AA, SEQ ID NO:), ‘Hinge-CH3’ (119 AA,SEQ ID NO:) or ‘Hinge’ only (12 AA,SEQ. ID NO:4) sequences (Figure 1). All spacers contained a SP substitution within the ‘Hinge’ domain located at position 108 of the native IgG4-Fc protein, and were linked to the 27 AA transmembrane domain of human CD28 (Uniprot: P10747, SEQ ID NO:14) and to a signaling module comprising either (i) the 41 AA cytoplasmic domain of human CD28 with an LLGG substitution located at positions 186-187 of the native CD28 protein (SEQ ID NO:14)or (ii) the 42 AA cytoplasmic domain of human 4-1BB (Uniprot: Q07011, SEQ ID , each of which was linked to the 112 AA asmic domain of isoform 3 of human CD3ζ (Uniprot: , SEQ ID NO;16). The construct encoded a T2A ribosomal skip element (SEQ ID NO:8))and a tEGFR sequence (SEQ ID NO:9) downstream of the chimeric receptor.

Codon-optimized nucleotide sequences encoding each transgene were synthesized (Life Technologies) and cloned into the epHIV7 lentiviral vector ROR1-chimeric receptor, CD19-chimeric receptor or tEGFR-encoding lentiviruses were produced in 293T cells using the packaging vectors pCHGP-2, pCMV-Rev2 and pCMV-G, and Calphos® transfection reagent (Clontech).

Generation of T-cell lines expressing ROR1 and CD19-chimeric receptors CD8+ CD45RO+ CD62L+ central memory T-cells (TCM) or bulk CD4+ T- cells were sorted from PBMC of normal donors, activated with anti-CD3/CD28 beads (Life Technologies), and uced on day 3 after activation by centrifugation at 800 g for 45 min at 32ºC with lentiviral supernatant (MOI = 3) supplemented with 1 μg/mL polybrene (Millipore). s were expanded in RPMI with 10% human serum, 2 mM L-glutamine and 1% penicillin-streptomycin (CTL medium), mented with recombinant human IL-2 to a final concentration of 50 U/mL. The tEGFR+ subset of each T-cell line was enriched by immunomagnetic selection with -conjugated anti-EGFR mAb ne s) and streptavidin-beads (Miltenyi). himeric receptor and tEGFR control T-cells were ed using a rapid expansion protocol (Riddell SR, Greenberg PD,The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells J Immunol s. 1990;128(2):189-201. Epub 1990/04/17.), and CD19-chimeric receptor modified T-cells were expanded by stimulation with irradiated (8,000 rad) B-LCL at a T-cell:LCL ratio of 1:7. T-cells were cultured in CTL medium with 50 U/mL IL-2.

Cytotoxicity, cytokine ion and proliferation assays Target cells were labeled with 51Cr (PerkinElmer), washed and incubated in triplicate at 1-2x103 cells/well with effector chimeric receptor modified T-cells at various effector to target (E:T) ratios. Supernatants were harvested for ting after a 4-hour incubation and specific lysis calculated using the standard formula.

For analysis of cytokine secretion, 5x104 s were plated in triplicate with target cells at an E:T ratio of 1:1 (primary CLL), 2:1 (Raji/ROR1; ), 4:1 (K562/ROR1, K562/CD19 and K562) or 10:1 (MDA-MB-231), and IFN-γ, TNF-α and IL-2 measured by ELISA or multiplex cytokine immunoassay (Luminex) in supernatant removed after 24-h tion. In experiments blocking NKG2D signaling, anti-NKG2D (clone 1D11), anti-MICA/B (clone 6D4, all from BD) and anti-ULBP (kindly provided by Dr. Veronika Groh, FHCRC) were used at saturating concentrations. For analysis of proliferation, T-cells were d with 0.2 μM yfluorescein succinimidyl ester (CFSE, ogen), washed and plated in triplicate with stimulator cells in medium t exogenous cytokines. After 72-h incubation, cells were labeled with anti-CD8 mAb and PI, and ed by flow cytometry to assess cell division of live CD8+ T-cells.

Experiments in NOD/SCID/γc-/- (NSG) mice The Institutional Animal Chimeric receptor and Use Committee approved all mouse experiments. Six- to 8-week old female -Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were obtained from the Jackson Laboratory or bred in-house. Mice were injected with 0.5x106 JeKo-1/ffluc tumor cells via tail vein and received a subsequent tail vein injection of chimeric receptor-modified or control T-cells.

For bioluminescence imaging of tumor growth, mice received intraperitoneal injections of luciferin substrate (Caliper Life Sciences) resuspended in PBS (15 µg/g body weight). Mice were anesthetized with isoflurane and imaged using an Xenogen IVIS Imaging System (Caliper) 10, 12 and 14 minutes after the injection of luciferin in small binning mode at an ition time of 1 s to 1 min to obtain unsaturated images. Luciferase activity was analyzed using Living Image Software (Caliper) and the photon flux analyzed within regions of interest that assed the entire body or the thorax of each individual mouse.

Statistical analyses Statistical analyses were performed using Prism Software (GraphPad®).

Student’s t-test was performed as a ded paired test with a confidence interval of 95% and s with a p-value of p<0.05 were considered icant. Statistical analysis of survival were done by log-rank testing and results with a e of p<0.05 considered significant.

Results Truncating the spacer domain of the 2A2 ROR1-chimeric receptor confers superior recognition of ROR1+ tumors We previously reported the design of a ROR1-specific chimeric receptor using the 2A2 scFV, which binds to an epitope in the NH2-terminal, membrane distal Ig-like/Frizzled portion of ROR1-1. The initial 2A2 ROR1-chimeric or had a long 229 AA spacer that included the ‘Hinge-CH2-CH3’ region of IgG4-Fc, and incorporated CD28 costimulatory and CD3ζ signaling domains (Hudecek M et al. Blood, 2010). This chimeric receptor conferred specific recognition of ROR1+ tumors, but we hypothesized that because of the ne distal location of the ROR1 epitope, truncating the spacer domain might enhance tumor recognition and T-cell signaling. Therefore, we constructed 2 additional chimeric ors in which the IgG4-Fc spacer domain was sequentially deleted to derive ‘Hinge-CH3’ (119 AA, intermediate), and ‘Hinge-only’ (12 AA, short) variants. Each of the new receptors contained the cal 2A2 scFV, and CD28 and CD3ζ ing modules.

The transgene cassette included a truncated EGFR (tEGFR) to serve as a transduction, selection and in vivo tracking marker for chimeric receptor-modified We transduced purified CD8+ TCM with the 2A2 himeric receptors containing full length or truncated IgG4-Fc spacers, and with a tEGFR control . The mean transduction efficiency was 15% (range 9-22%), and transgenepositive T-cells were ed to uniform purity (>90%) on day 10 by selection for tEGFR expression, and expanded (Figure 2A). Surface expression of each of the chimeric receptors was confirmed by staining with F(ab)-specific antibodies (Figure 2A).

Analysis of the in vitro function of CD8+ T-cells modified to express each of the 2A2 ROR1-chimeric receptors demonstrated that each receptor conferred specific lysis of JeKo-1 MCL and primary CLL cells that naturally express ROR1, and of K562 cells that had been transduced with ROR1, but did not confer recognition of control ROR1- targets e 2B). T-cells expressing the short ‘Hinge-only’ 2A2 ROR1-chimeric or had maximum cytolytic activity, and a hierarchy (short>intermediate>>long) of tumor lysis was y evident against all ROR1+ tumor targets e 2B), illustrating the importance of spacer domain length on the recognition of ROR1+ tumor cells.

Anti-tumor cy of adoptive T-cell therapy correlates with proliferation and al of transferred T-cells, which could be altered by signaling through the chimeric receptor. We used CFSE dilution assays to analyze proliferation of T-cells modified with each of the 2A2 ROR1-chimeric receptors after engagement of Raji/ROR1 or CLL, and found that the short spacer construct promoted the greatest T-cell proliferation following stimulation (Figure 2C). To ensure that the enhanced proliferation was not associated with greater activation induced cell death (AICD), we also analyzed the proportion of 2A2 ROR 1 chimeric receptor modified T-cells that stained with propidium iodide (PI) after stimulation with Raji/ROR1 and JeKo- 1 tumor cells. We ed a much lower ncy of PI+ CD8+ T-cells in the T-cell line modified with the short (Raji/ROR1: 17.2%/JeKo-1: 20.2%) compared to the intermediate /42.4%) and long (44.5%/48.5%) spacers.

Quantitative analysis of cytokine production in response to stimulation with Raji/ROR1 and y CLL cells showed production of IFN-γ, TNF-α and IL-2 by T-cells expressing each of the 2A2 ROR1 chimeric receptors. As observed in cytotoxicity assays, the short spacer construct was superior in mediating cytokine secretion after tumor recognition (Figure 2D). Thus, this analysis shows that truncating the extracellular IgG4-Fc spacer domain of the 2A2 ROR1-chimeric receptor leads to a significant increase in cytotoxicity, proliferation and in vitro effector functions after tumor ition.

The R11 scFv that is specific for a membrane proximal epitope in the ROR1 Kringle domain requires a long extracellular spacer domain.

We transduced ed CD8+ T cells with ROR1-chimeric receptors containing the R11 scFv that is specific for the Kringle domain of ROR1 and containing full length or truncated c spacers (CH3 and hinge only). The transduction ency with each of the short (IgG4 hinge only), intermediate (IgG4 hinge/CH3), and long (IgG4 hinge/CH2/CH3) vectors was comparable (45-51%) as measured by EGFR expression. (Figure 3A). T cells transduced with each of the vectors were assayed for cytolysis (Figure 3 B), eration (Figure 3C), and cytokine production (Figure 3D) in response to ia or lymphoma cells that did or did not express ROR1. As shown, only T cells uced with the R11 chimeric receptor containing a long spacer sequence were able to efficiently recognize ROR1+ tumors and mediate effector functions.

ROR1 chimeric receptors derived from a mAb R12 with higher affinity than 2A2 mediate superior anti-tumor reactivity We next examined whether increasing the ty of the scFV used to construct the ROR1 chimeric receptor might influence tumor recognition and T-cell function. We generated ROR1-specific ic receptors from the mAb R12 that like 2A2, binds to an epitope in the NH2-terminal Ig/Frizzled domain of ROR1 but with ld higher monovalent binding affinity.

R12 ROR1 ic receptors were constructed with both long and short IgG4-Fc spacers to determine whether the optimal spacer design for this higher affinity scFV differed from that for a lower affinity scFV. We found that similar to 2A2, the short spacer R12 ROR1 chimeric receptor conferred improved cytolytic activity, cytokine secretion and proliferation (data not shown), suggesting that the shorter spacer length provides superior spatial engagement of the T-cell and ROR1+ target cell for T-cell tion.

We then designed R12 and 2A2 ROR1 ic receptors that contained an optimal (short) extracellular spacer, and either a CD28 or 4-1BB ulatory domain in tandem with CD3ζ (4 constructs) for comparison (Figure 4A.B). These himeric receptor constructs were expressed in purified CD8+ TCM of healthy donors, and we confirmed equivalent transgene expression by tEGFR ng (Figure 5A). T-cells modified with each of the 2A2 and R12 himeric receptors specifically lysed K562/ROR1 and Raji/ROR1 tumor cells with approximately equivalent efficiency (Figure 5B). However, analysis of cytokine production showed that the high affinity R12 ROR1 chimeric receptors that contained CD28 or 4-1BB conferred significantly higher IFN-γ, TNF-α and IL-2 production compared to the corresponding 2A2 constructs e 5C). We found that T-cells expressing chimeric receptors with a CD28 costimulatory domain produced more IFN-γ, TNF-α and IL-2 compared to those with 4-1BB.

Experiments to analyze the proliferation of ROR1 chimeric receptor T-cells showed a higher percentage of proliferating T-cells and a higher number of cell divisions in T-cells expressing the high affinity R12 ROR1 chimeric receptors with CD28 and 4-1BB domain compared to s expressing the respective 2A2 counterparts (Figure 4D). There was more vigorous proliferation in T-cells that sed chimeric receptors with a CD28 domain, consistent with higher IL-2 production induced by these receptors. There was a lower frequency of AICD as ed by PI staining in T-cell lines modified with R12 ed to 2A2 ROR1- chimeric receptors after stimulation with OR1 and JeKo-1 tumor cells respectively (R12: .9% vs. 2A2: 65%). T-cell lines that expressed chimeric receptors with a CD28 domain also had lower AICD compared to 4-1BB in response to Raji/ROR1 and JeKo-1 tumor cells respectively (R12: 16.4%/18.4% vs. 2A2 38.1%/39.6%).

To determine if the enhanced function observed with R12 ROR1 ic receptors in CD8+ T-cells ed to CD4+ T-cells, we transduced bulk CD4+ T- cells with the 2A2 and R12 ROR1 chimeric ors containing the short spacer and CD28 costimulatory domain. In response to Raji/ROR1+ tumor cells, CD4+ T- cells that expressed the high affinity R12 scFV produced higher levels of IFN-γ, TNF-α, IL-2, IL-4, and IL-10, and underwent greater proliferation than CD4+ s that expressed 2A2 (Figure 5A,B). Both cytokine production and proliferation was superior in CD4+ compared to CD8+ T-cells modified with the same ROR1 chimeric receptors. In summary, our data demonstrate that tailoring both the length of the non-signaling extracellular chimeric receptor spacer domain and scFV affinity are independent parameters that affect the on of ROR1-chimeric receptor T-cells.

D8+ T-cells modified with a high affinity ROR1 chimeric receptor have comparable activity to a CD19 chimeric receptor against primary CLL in vitro ROR1 and CD19 are both uniformly expressed on all primary CLL (Figure 6A), however the absolute number of ROR1-molecules per tumor cell is estimated to be 10-fold lower than that of CD19, which has been successfully targeted in clinical trials with CD19 chimeric receptor T-cells. We compared recognition of y CLL by CD8+ T-cells expressing the optimized R12 and 2A2 ROR1 chimeric receptors, and a CD19 chimeric receptor derived from the FMC63 scFV.

We used purified CD8+ TCM for chimeric receptor-modification to provide a m cell t and each chimeric receptor contained a short IgG4-Fc only ’ spacer and 4-1BB costimulatory domain. We confirmed our CD19 chimeric receptor (IgG4 Hinge) was at least as and more effective in recognizing CD19+ tumors as a CD19 chimeric receptor with CD8a Hinge spacer and 4-1BB costimulatory domain that is being used in ongoing clinical trials. (Figure 20). T cells expressing CD19 chimeric receptors with 4-1BB and CD3zeta and a ed IgG4-Fc hinge exhibit superior in vitro and in vivo function compared to T cells expressing CD19 chimeric receptors with 4-1BB and CD3zeta and a CD8 alpha hinge. In Figure 20D, in vivo antitumor activity of T cells expressing a CD19 chimeric receptor with an IgG4 Fc hinge (group 1) or CD8 alpha hinge (group 2) and T cells that express tEGFR alone (group 3) in NSG mice inoculated with Raji tumor cells expressing firefly luciferase (ffluc) were compared. Mice were imaged 17 days after tumor inoculation and 10 days after T cell inoculation. The data shows greater tumor burden in mice treated with control tEGFR T cells (group 3) or with CD19 chimeric receptor CD8 alpha hinge T cells (group 2) compared with mice d with CD19 chimeric receptor IgG4 Fc hinge T cells (group 1).

The cytolytic activity of R12 ROR1 chimeric receptor s t y tumor cells from multiple CLL patients (n=4) was higher compared to T- cells modified with the lower affinity 2A2 ROR1 chimeric receptor, and equivalent to the lysis observed with CD19 chimeric receptor T-cells (Figure 6B). Multiplex cytokine analysis showed nearly lent production of IFN-γ and TNF-α, but less IL-2 production by CD8+ T-cells expressing the R12 ROR1 compared with those expressing the CD19-chimeric receptor after co-culture with primary CLL (Figure 6C). 2A2 ROR1 chimeric or T-cells produced lower amounts of all cytokines than R12 ROR1 chimeric receptor s as noted previously. Cytokine production by all of the ic receptor-transduced T-cells after stimulation with CLL was substantially less than with Raji/ROR1, which unlike CLL ses both CD80 and CD86 that can engage CD28 expressed on chimeric receptor T-cells (Figure 6A, C).

We observed less proliferation of T-cells expressing the R12 and 2A2 ROR1 chimeric receptor compared to the CD19 chimeric receptor after ation with CLL (CD19>R12>2A2) (Figure 6D). We hypothesized that proliferation of CD8+ ROR1 chimeric receptor T-cells in response to CLL may be augmented in the presence of chimeric receptor-modified CD4+ T-cells because of their higher secretion of IL-2 compared to CD8+ TCM (Figure 4A; Figure 8A). To test this possibility, we med in vitro co-culture experiments where CD4+ and CD8 TCM were separately modified with the R12 ROR1, 2A2 ROR1 and CD19 chimeric receptors respectively, enriched for chimeric receptor expression, and combined at a 1:1 ratio to ensure lent proportions of CD8+ and CD4+ T-cells modified with each of the vectors. These cells were CFSE-labeled and stimulated with primary CLL. We observed a dramatic increase in proliferation of CD8+ R12 ROR1 chimeric receptor s after addition of chimeric receptor-transduced, but not untransduced CD4+ T-cells (Figure 8B). Notably, when provided with CD4-help, we observed equivalent proliferation of R12 ROR1 and CD19 chimeric receptor CD8+ T-cells in response to CLL, whereas proliferation of CD8+ s expressing the lower affinity 2A2 ROR1 chimeric or remained less. Collectively, our data show that the high ty R12 ROR1 chimeric receptor confers superior reactivity compared to 2A2 against primary CLL cells in vitro.

ROR1-chimeric receptor T-cells mediate in vivo anti-tumor activity in a mouse model of systemic mantle cell lymphoma It ed uncertain whether the superior in vitro ty of T-cells modified with the higher ty R12 chimeric receptor would translate into improved anti-tumor activity in vivo, and how targeting ROR1 would compare to targeting CD19. To address these questions, we inoculated cohorts of immunodeficient NSG mice with the human MCL line JeKo-1/ffluc by tail vein injection, and seven days later when tumor was disseminated, d the mice with a single intravenous dose of R12 ROR1, 2A2 ROR1 or CD19 chimeric receptor CD8+ T-cells. l mice were treated with tEGFR T-cells or untreated. All ic receptors had the optimal short spacer and the 4-1BB costimulatory domain.

Untreated NSG/JeKo-1 mice developed a rapidly progressive systemic lymphoma necessitating euthanasia approximately 4 weeks after tumor inoculation (Figure 9AC We observed tumor regression and improved survival in all mice treated with R12 ROR1, 2A2 ROR1 and CD19 chimeric receptor T-cells. Mice treated with R12 ROR1 chimeric or T-cells had a superior anti-tumor response and survival compared to mice treated with 2A2 ROR1 chimeric receptor T-cells (p<0.01), and comparable anti-tumor activity to mice treated with CD19 chimeric receptor T-cells (Figure 9A-C).

We analyzed the frequency of ic receptor T-cells in the peripheral blood following adoptive er and detected higher numbers of tEGFR+ T-cells in mice treated with the R12 ROR1 chimeric receptor compared to the 2A2 ROR1 chimeric receptor, suggesting more vigorous proliferation in vivo ed tumor control. To confirm this, we administered CFSE-labeled CD19 chimeric receptor, R12 and 2A2 ROR1 chimeric receptor T-cells to cohorts of NSG mice bearing JeKo-1/ffluc, and analyzed T-cell proliferation in the peripheral blood, bone marrow and spleen 72 hours after transfer. A higher percentage of the R12 and CD19 chimeric receptor T-cells erated and underwent a greater number of cell divisions compared to 2A2 ROR1 chimeric receptor T-cells e 9D). The JeKo- 1 tumor eventually recurred in all mice treated with ROR1 or CD19 chimeric receptor T-cells (Figure 9A-C). Tumor recurrence was not a result of the selection of ROR1 or CD19 loss variants, as recurrent tumors were positive for both molecules.

For comparison, we analyzed anti-tumor cy of CD19 chimeric or T-cells in NSG mice engrafted with Raji tumors and observed complete tumor ation, indicating the recurrence of JeKo-1 reflects difficulty eradicating this tumor (data not . In summary, this data is the first to show that ROR1 chimeric receptor T-cells have anti-tumor efficacy in vivo, and suggest that for B- cell malignancies, an optimized ROR1 chimeric receptor such as R12 may be effective and spare normal CD19+ B-cells that lack ROR1 expression.

T-cells expressing the R12 ROR1 chimeric receptor have superior reactivity compared to 2A2 t ROR1+ epithelial tumor cells ROR1 has been detected on many epithelial tumors, although it is unknown whether ROR1 sion is sufficient for recognition by ROR1 chimeric receptor T-cells. Using flow cytometry, we confirmed ROR1 expression on breast cancer lines MDA-MB-231 and 468, and on the renal cell carcinoma lines FARP, TREP, and RWL (Figure 10A). We then analyzed tumor ition by CD8+ T-cells uced with the R12 ROR1 chimeric receptors with the optimal short spacer and 4-1BB domain, and observed ent recognition of MDA-MB-231, MDA-MB- 468, FARP, TREP and RWL e 11A). We analyzed cytokine secretion and proliferation of T-cells modified with the R12 and 2A2 ROR1-chimeric receptors after co-culture with MDA-MB-231, and observed greater cytokine tion and proliferation with the R12 ROR1 chimeric receptor (Figure 11 B, C). Similar to what we ed with ROR1+ B cell malignancies, the or activation of R12 ROR1 ic receptor T cells after stimulation with MDA-MB-231 was not associated with increased AICD (R12: 9.8% vs. 2A2: .

Discussion ROR1 has attracted st as a potential target for cancer immunotherapy due to its expression on the surface of many B-lymphoid and epithelial cancers, including subsets of lung, colorectal and renal cell cancer. We previously showed that CLL and MCL were specifically recognized by T-cells modified to express a ROR1-specific chimeric receptor (Hudecek M, et al. Blood. 2010;116(22):4532-41.

Epub 2010/08/13). The design and function of ROR1-chimeric receptors has been improved through modification of the ellular spacer domain and deriving the chimeric or from a scFV of higher affinity, and demonstrate that T-cells modified with designed ROR1 chimeric receptors have in vivo activity against ROR1+ B-cell lymphoma and in vitro activity against a wide range of epithelial tumors.

We compared the function of T-cells modified with ROR1 chimeric receptors derived from the 2A2 mAb that contained either the original long IgG4-Fc ‘Hinge-CH2-CH3’ spacer that we have shown enables high level cell surface expression, or truncated intermediate ‘Hinge-CH3’ and short ‘Hinge-only’ spacer ts. We preserved the 12 AA Hinge domain in our short spacer construct based on prior data that a flexible spacer was required for separating the scFV from the T- cell membrane and allowing antigen recognition on tumor cells ( Fitzer-Attas CJ, et al.,Harnessing Syk family tyrosine kinases as signaling domains for chimeric single chain of the variable domain receptors: optimal design for T cell activation. J Immunol. 1998;160(1):145-54. Epub 1998/04/29.) Our studies with the 2A2 ROR1 chimeric receptor show that T-cell cytokine secretion and proliferation after tumor cell recognition are superior with the intermediate and short spacer constructs compared to the long spacer uct. ng with anti-F(ab) Abs showed lent chimeric receptor expression of all three receptors, demonstrating the improved T-cell on with the short spacer chimeric receptor was not due to differences in chimeric or density. This data supports the principle that the design of ellular spacers should be tailored for each target molecule and epitope.

The affinity of the scFV selected for ing a ic receptor is an additional parameter that could affect T-cell recognition. We generated and characterized a panel of ROR1-specific mAbs of different affinities and selected the R12 mAb, which recognizes an epitope in the Ig-like/Frizzled region as 2A2. R12 has a higher affinity for rotein due to a much slower dissociation. The R12 chimeric receptor, like the 2A2 chimeric receptor conferred optimal T-cell recognition and function when designed with a short extracellular . A direct comparison of proliferation and cytokine production after tumor engagement by T- cells modified with the 2A2 and R12 chimeric receptors demonstrated that the R12 chimeric receptor derived from the higher ty mAb was superior. We were concerned that the slower dissociation of R12 from ROR1 could prolong T-cell tion and confer an increased susceptibility to AICD. However, we detected a lower rate of AICD in T-cells ed with the R12 ROR1-chimeric receptor compared to 2A2, trating that the increased affinity of R12 had no detrimental effect on T-cell survival in our nical models.

ROR1 has a potential advantage over CD19 as a target for CLL and MCL since it is not expressed on normal mature naïve and memory B-cells. However, there is a lower number of ROR1 molecules on B-cell tumors compared with CD19 and it is uncertain if an optimized ROR1 chimeric receptor would be as effective as a CD19 chimeric receptor similar in design to those being used in the clinic.

Unfortunately, B-cell tumor xenograft models used previously in NSG mice to evaluate the function of CD19 ic receptor T-cells including Raji, Daudi and Nalm-6, are not derived from CLL or MCL and do not constitutively express ROR1.

Thus, to compare targeting CD19 and ROR1 in vivo, we used the JeKo-1 MCL cell line, which naturally expresses both CD19 and ROR1 and engrafts in NSG mice. To make our model clinically relevant, we inoculated JeKo-1 ma cells intravenously to generate systemic , and treated mice with T-cell products of uniform consistency once tumors were established. We found that T-cells expressing the high affinity R12 chimeric receptor conferred equivalent anti-tumor activity in vivo as CD19 chimeric receptor T-cells. Consistent with our in vitro analysis, the R12 ROR1 chimeric receptor also mediated superior activity in vivo compared to the optimal 2A2 ROR1-chimeric receptor. These s should be interpreted cautiously since murine tumor models may not predict the efficacy of ve therapy in clinical settings. However, the s suggest that ROR1 warrants consideration as an alternative to CD19, or to provide an onal target to minimize the potential for CD19 loss variants to emerge.

ROR1 appears to play a decisive role in survival of some epithelial tumors.

Thus, an advantage of targeting ROR1 is that a single chimeric receptor may be useful to treat patients with a large number of hematopoietic and non-hematopoietic tumors.

Our data shows for the first time that T-cells that express a designed ROR1 ic receptor efficiently recognize epithelial cancers in vitro. Cytokine secretion and T-cell proliferation induced by ROR1+ breast cancer cells were higher than that induced by leukemia cells, despite the absence of the CD80/86 costimulatory ligand.

The studies reported here demonstrate that the design of the extracellular spacer domain and chimeric receptor affinity are parameters that can be modulated to enhance the recognition of ROR1+ hematologic and epithelial tumors in vitro and in vivo by ROR1-chimeric receptor modified T-cells. The development of ROR1- chimeric receptors with enhanced tumor reactivity provides the unity for clinical applications in a variety of human s. e 2 Effect of extracellular spacer domain length on triggering of tumor cell lysis with a Her2-specific chimeric receptor that recognizes an epitope d proximal to the tumor cell membrane.

The effect of CAR spacer length on recognition and triggering of tumor cell recognition by CD8+ human T cytes that expressed a HER2-specific chimeric receptor was examined using similar methods to those described above for ROR1. HER2-specific chimeric receptors were constructed using VL and VH chain segments of a pecific mAb that recognized a membrane proximal epitope on HER2 (Figure 12A), and the scFVs were linked to IgG4 hinge/CH2/CH3, IgG4 hinge/CH3, and IgG4 hinge only extracellular spacer domains and to the CD28 transmembrane domain, 4-1BB and CD3 zeta signaling domains (Figure 12B).

Primary CD8+ T cells were transduced with each of the HER2 chimeric receptors and ed for expression of the EGFR transducton marker (Figure 12D).

Expression of the HER2 chimeric receptors and the size of each receptor was confirmed by Western Blot (Figure 12C). The T cells were then expanded with anti CD3 mAb and feeder cells and ed for their y to recognize HER2+ tumor cells. As observed with the R11 ROR 1 specific chimeric or, the HER2 chimeric receptor that contained a long extracellular spacer domain conferred superior T cell recognition of HER2+ tumor cells (Figure 12E).

Discussion This example of the effect of extracellular spacer length on ic receptor modified T cell recognition of tumor cells used a chimeric receptor sing a scFv built from the VH+L sequences of the Herceptin chimeric mAb. Studies by Cho et al (Nature 421:756, 2003) localized to epitope location of Herceptin to a ne proximal location on the HER2 (ERRB2) extracellular domain (Figure 12A). Based on our understanding of the structure of human IgG4 hinge:Fc variants (Figure 12B), we esize that a membrane al location of the targeting epitope on an ellular tumor cell antigen would best recognized by effector T cells that express a chimeric receptor encoding a long spacer. Our data demonstrating a gradient of cytolytic activity from near back ground activity by T cells expressing a short spacer Herceptin chimeric receptor, to intermediate activity by T cells expressing a medium length spacer chimeric receptor, and maximal lysis by T cells that expressed the long spacer chimeric receptor. Thus, the extracellular spacer has definitive effects on tumor recognition by T cells, and this data provides further support for the need to tailor chimeric receptor design based on epitope location of tumor sed target molecules.

Example 3 – Customizing spacer length and sequence for optimal recognition and in vivo efficacy of CD19 with chimeric receptor modified T cells.

Materials and Methods Human subjects Blood samples were obtained from healthy donors who provided written informed consent to participate in research protocols approved by the utional Review Board of the Fred Hutchinson Cancer Research Center ). Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll- Hypaque (Sigma, St.Louis, MO), and eserved in RPMI, 20% human serum and 10% dimethyl sulfoxide.

Cell lines The K562, Raji, JeKo-1, and 293T cell lines were obtained from the American Type Culture Collection (Manassas, VA) and cultured as ed. A lentivirus encoding the ffluc-gene upstream of a T2A sequence and eGFP was produced in 293T cells and used to transduce Raji and JeKo-1 tumor cells. Raji, and JeKo-1 cells were expanded after lentiviral transduction and the eGFP positive subset sort-purified.

Immunophenotyping PBMC and T-cell lines were stained with one or more of the following conjugated onal antibodies: CD3, CD4, CD8, CD25, CD45RA, CD45RO, CD62L, CD69 and matched isotype controls (BD Biosciences). Staining with propidium iodide (PI, BD Biosciences) was performed for live/dead cell mination as directed by the manufacturer. Flow analyses were done on a FACSCanto, sort-purifications on a FACSAriaII (Becton Dickinson) and data ed using FlowJo software (Treestar).

Vector construction and preparation of CD19 chimeric receptor encoding lentivirus CD19 ic chimeric receptors were constructed using: (1) the VL and VH chain segments of the CD19-specific mAb FMC63 (SEQ ID NO:3), linked by a (G4S)3 linker (SEQ ID NO:12)peptide (VL-linker-VH); (2) a spacer domain derived from IgG4-Fc (Uniprot Database: P01861, (SEQ ID NO:13)) comprising either the Hinge-CH2- CH3 portion (229 AA, (SEQ ID NO:)) or Hinge only (12 AA; (SEQ ID NO:4)). Both spacers contained a S → P substitution within the Hinge domain located at position 108 of the native IgG4-Fc protein; the 27 AA transmembrane domain of human CD28 (Uniprot Database: P10747, (SEQ ID ); (4) a signaling module comprising either (i) the 41 AA cytoplasmic domain of human CD28 with an LL → GG substitution located at position 186-187 of the native CD28 protein (SEQ ID NO:14) ; and/or (ii) the 42 AA cytoplasmic domain of human 4- 1BB (Uniprot Database: Q07011, (SEQ ID ); linked to (iii) the 112 AA cytoplasmic domain of m 3 of human CD3ζ (Uniprot Database: P20963, (SEQ ID NO:16)); the self ng T2A sequence (SEQ ID NO:8); and (6) a truncated epidermal growth factor receptor (EGFR)sequence (SEQ ID NO:9).

Codon-optimized nucleotide sequences encoding each trans gene were synthesized (LifeTechnologies, Carlsbad, CA) and cloned into the epHIV7 lentiviral vector using NheI and Not1 restriction sites. The epHIV7 lentiviral vector had been derived from the pHIV7 vector by replacing the cytomegalovirus promoter of pHIV7 with an EF-1 promoter.

CD19 chimeric receptor or tEGFR-encoding lentivirus was produced in 293T cells co-transfected with the lentiviral vector and the packaging s pCHGP-2, ev2 and pCMV-G using Calphos transfection reagent (Clontech). Medium was changed 16 h after transfection, and lentivirus collected after 24, 48 and 72 h.

Generation of T -cell lines expressing the CD19 chimeric receptors Sort-purified CD8+ CD45RA- CD45RO+ CD62L + l memory T -cells (TCM) of normal donors were activated with anti-CD3/ CD28 beads (Life Technologies) ing to the cturer's instructions, and transduced with lentiviral supernatant (MOI = 3) supplemented with 1 μg/mL polybrene (Millipore) on day 3 after tion by centrifugation at 2,100 rpm for 45 min at 32°C. T cells were expanded in RPMI, 10% human serum, 2 mM L-glutamine and 1 % penicillinstreptomycin (CTL medium), supplemented with recombinant human (rh) lL-2 to a final concentration of 50 U/mL every 48 h. After expansion, an aliquot of each transduced T cell line was stained with biotin-conjugated anti-EGFR (epithelial growth factor receptor) antibody and streptavidin-beads (Miltenyi), and tEGFR+ T cells isolated by immunomagnetic selection.

The tEGFR+ T-cell subset was then stimulated with irradiated (8,000 rad) TM EBV-LCL at a T cell: LCL ratio of 1 :7, and expanded for 8 days in CTL medium with addition of 50 U/mL rh IL-2 every 48 h.

Chromium release, cytokine secretion and CFSE eration assays Target cells were labeled with 51Cr (PerkinElmer) overnight, washed and incubated in triplicate at 1-2x l03 cells/well with effector T cells at various effector to target (E:T) . Supernatants were harvested for γ counting after a 4-hour tion and specific lysis calculated using the standard formula. For analyses of cytokine secretion, target and effector cells were plated in triplicate wells at an E:T ratio of 2: 1 (Raji) or 4: 1 (K562/CDI9 and K562), and INF-γ, TNF-α, IL-2, IL-4, IL-6 and IL-10 measured by multiplex ne assay (Luminex) in atant removed after a 24-hour incubation.

For analysis of proliferation, T cells were labeled with 0.2 μM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), washed and plated in triplicate wells with stimulator cells at a ratio of 2: 1 (Raji) or 4: 1 (K562/CD19 and K562) in CTL medium without exogenous cytokines. After 72 h of incubation, cells were labeled with anti-CD3 mAb and propidium iodide (PI) to exclude dead cells from analysis. Samples were analyzed by flow cytometry and cell division of live CD3+ s assessed by CFSE dilution.

Experiments in NOD/SCID and NOD/SCID/γc-/- (NSG) mice All mouse experiments were approved by the FRCRC Institutional Animal Chimeric receptore and Use Committee. Six- to 8-week old female I7- Prkdcscid /J (NOD/SCID) and NOD.Cg-Prkdcscid Il2rgtmlWjl/SzJ (NSG) mice were obtained from the Jackson Laboratory or bred in-house (FRCRC. Mice were injected intravenously (i. v.) with 0.5 x l06 Raji-ffluc tumor cells via tail vein injection, and received injections of chimeric receptor-modified T cells, control T cells, or PBS via tail vein injection as indicated.

For inescence imaging, mice received intraperitoneal (i.p.) injections of freshly prepared luciferin substrate er Life Sciences, MA) resuspended in PBS (15 μg/g body weight) and were then anesthetized with isoflurane in an induction r. After induction of deep esia, mice were imaged using an Xenogen IVIS In Vivo Imaging System (Caliper Life es, MA) at 10, 12 and 14 minutes post i.p. injection of luciferin at an acquisition time of 1 second to 1 minute in small binning mode to obtain unsaturated images. Luciferase activity was analyzed using Living Image re (Caliper Life Sciences, MA) and the photon flux analyzed within regions of interest that encompassed the entire body of each individual mouse.

Statistical analyses Statistical analyses were performed using Prism Software (GraphPad, CA).

Student's t-test was med as a two-sided test with a confidence interval of 95% and results considered significant with a p-value of p<0.05. Statistical analysis of survival were done by nk testing and results considered significant with a pvalue of p<0.05.

Results Preparation of polyclonal CD8+ TCM-derived cell lines that express CD19 chimeric receptors with long and short extracellular spacers We constructed individual lentiviral vectors encoding a panel of codon- optimized CD 19 ic receptor genes to examine the influence of extracellular spacer length on the in vitro function and in vivo antitumor activity of CD19 ic receptor-modified T cells. Each chimeric receptor was comprised of a single chain variable fragment corresponding to the sequence of the CDl9-specific mAb FMC63 (scFv: VL-VH), a spacer derived from IgG4-Fc including either the 'Hinge-CH2-CH3' domain (229 AA, long spacer) or the 'Hinge' domain only (12 AA, short spacer), and a signaling module of CD3ζ with membrane proximal CD28 or 4-1 BB costimulatory s, either alone or in tandem (Figure 13A). The ene cassette included a truncated EGFR (tEGFR) downstream from the chimeric receptor gene and separated by a cleavable T2A element, to serve as a transduction, selection and in vivo tracking marker for chimeric receptor-modified T cells.

We ed a CD8+ CD45RO+ CD62L+ central memory T cell (TCM) cell population by cell sorting from the blood of normal donors for transduction and expansion, because of the superior ability of TCM to persist in vivo after adoptive er. CD8+ T cells were stimulated with anti CD3/28 beads, transduced with each of the lentiviral vectors, and expanded in culture for 18 days before being used for in vitro and in vivo experiments. (Figure 13B) Similar transduction efficiencies were ed with each of the lentiviral s (mean 25%) and transgene-positive T cells were enriched to uniform purity by immunomagnetic selection using a biotinylated anti-EGFR mAb and streptavidin beads. Following tEGFR-enrichment, each of the CD19 chimeric or T cell lines were expanded by a single stimulation with CD19+B-LCL, without apparent differences in in vitro growth kinetics between T cell lines expressing the various CD 19 chimeric or constructs. After expansion, the tEGFR marker was expressed at lent levels on >90% of the T cells transduced with each of the vectors (Figure 13C).

CD19 chimeric receptors with long and short extracellular spacer domain confer specific anti-tumor reactivity in vitro We compared the or function of rived T cell lines modified to express CD19 chimeric receptors with CD28 and 4-1BB costimulatory signaling moieties, and either a short ('short/CD28';'short/4-1BB') or long ('long/CD28'; 'long/4-1BB') extracellular spacer domain tively. T cells expressing each of the 4 CD19 chimeric receptor constructs conferred specific cytolytic activity against CD19+ Raji and JeKo-l lymphoma cells, and against K562 cells that had been stably transfected with CD19, but not native CD19- K562 cells (Figure 14A). Quantitative analyses of cytokine production in response to stimulation with K562/CD19 or Raji tumor cells by multiplex cytokine assay (Luminex) showed production of IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 by T cells expressing each of the CD19 chimeric receptors (Figure 14B). T cells sing CD19 chimeric receptors with a CD28 costimulatory domain produced significantly higher levels of IFN-γ, TNF-α, IL-2 and IL-10 compared to the corresponding constructs with a 4-1BB costimulatory domain e 14B, C). There was significantly higher IFN-y production and significantly less IL-4 production by T cells expressing the CD19 'long/CD28' chimeric receptor compared with those expressing the 'short/CD28' chimeric receptor. Amongst the CD19 chimeric receptors with 4-1BB costimulatory signaling module, we detected significantly higher levels of IFN-γ, TNF -α, IL-2, IL-4, and IL-10 secretion in T cells expressing the construct with the short spacer domain (Figure 14B, C).

We used CFSE dye dilution to analyze proliferation of T cells modified with each of the CD 19 chimeric receptors after engagement of CD 19+ tumor cells.

Specific and vigorous proliferation of each of the CD19 chimeric receptor T cell lines was observed 72 hours following stimulation with either D19 or Raji.

The average number of cell divisions was higher for CD19 chimeric or T cells with a CD28 costimulatory domain compared to those with 4-1BB, consistent with greater IL-2 production by T cells expressing a CD28 containing chimeric or (Figure 14B-D). We also analyzed the tion of chimeric receptor T cells that underwent activation d cell death after stimulation with K562/CD19 and Raji tumor cells at the end of the 72-hours by ning the e with CD3+ and PI.

We detected a higher frequency of CD3+ CD8+ PI+ T cells in the CD 19 chimeric or cell line 'long/4-1 BB', but few PI+ cells were observed with the other CD19 chimeric receptors. (Figure 14E).

This analysis of in vitro or functions was consistent with prior studies that have compared CD28 and 4-1BB costimulatory domains, and did not reveal differences in T cell function that would suggest that a particular CD19 chimeric receptor construct from this panel would lack anti-tumor efficacy in vivo.

T cells expressing CDI9 chimeric receptors with short extracellular spacer domains but not long extracellular spacer domains eradicate Raji tumors in immunodeficient mouse models We next evaluated the in vivo antitumor efficacy of T cells ed with each of the CD19 chimeric receptors in immunodeficient (NOD/SCID) mice engrafted with firefly luciferase transfected Raji cells (Raji-ffluc), which enables sequential quantitative analyses of tumor burden and distribution using bioluminescence imaging. NOD/SCID mice inoculated with 6 Raji-ffluc cells via tail vein ion developed disseminated ma, which if ted led to hind limb paralysis after approximately 3.5 weeks, necessitating euthanasia. Tumor bearing mice were treated with 2 doses of CD8+ TCM-derived T cells modified with each of the CD19 chimeric receptors or with a tEGFR l vector administered on day 2 and day 9 after tumor inoculation (Figure 15A).

Surprisingly, only T cells modified to s CD19 chimeric receptors with short extracellular spacer domain ('short/CD28' and 'short/4-1BB') eradicated Raji tumors in this model, whereas mice treated with T cells expressing CD19 chimeric receptors with long spacer ('long/CD28' and 'long/4-1BB') developed systemic lymphoma and hind limb paralysis with nearly identical kinetics as untreated mice or mice treated with control tEGFR+ T cells (Figure 15B, C). The striking difference in mor activity between CD19 chimeric receptors with short and long spacer domains was highly significant and reproducible in multiple experiments with chimeric receptor T cell lines generated from 3 different normal donors.

The NOD/SCID ma model may be suboptimal for predicting antitumor activity in a al setting because of the short interval n tumor inoculation and T cell administration and the greater resistance to engraftment of human cells compared to more immunodeficient mouse strains such as ID/γc-/- (NSG). Thus, we evaluated antitumor activity of adoptive therapy in a more clinically relevant model in which Raji-ffluc lymphoma was ished in NSG mice, and the CD19 chimeric receptor T cells were administered after 7 days when the tumor was readily detectable in the bone marrow by bioluminescence imaging (Figure 16A). We performed initial dose titration experiments to determine the minimal dose of T cells uced with the CD19 'short/4-1BB' chimeric receptor that was required for ation of established Raji tumors. A single dose of 2.5x106 T cells expressing CD19-chimeric receptor 'short/4-1BB' promoted complete regression of established Raji tumors and resulted in long-term tumor-free survival in 100% of mice (Figure 16B,C). At the 2.5x106 dose level, the T-cells were easily ed in the peripheral blood of NSG mice for at least 3 weeks following adoptive transfer and tumor eradication. Thus, this model enabled comparative studies both of antitumor activity and persistence of T cells modified with each of the CD19-chimeric receptors in our panel (Figure 16D).

We then treated cohorts of NSG mice that were ted with Raji lymphoma with PBS alone, with a single dose of 2.5x106 T cells expressing each of the CD19 chimeric receptors or with T cells modified with a tEGFR encoding l vector (Figure 17A). In this model of established lymphoma, T cells sing CD19 ic receptors with a short extracellular spacer domain and either 4- 1BB or CD28 costimulatory domains (‘short/CD28' and 'short/4-1BB') mediated complete tumor regression over 7-10 days and all mice survived tumor free for >56 days. By contrast, mice treated with T cells modified to express CD19 chimeric receptors with a long spacer domain /CD28' and 4-1BB') exhibited tumor progression and had to be sacrificed at a similar time as mice that had received control tEGFR T cells (Figure 17B, C). The lack of in vivo antitumor activity of the chimeric receptor constructs with long spacers was unexpected given the ability of T cells expressing these constructs to lyse tumor cells in vitro, and the enhanced IL-2 production and proliferation after engagement of T cells expressing the 'long/CD28' CD19 chimeric receptor compared to the 4-1BB constructs.

To provide insight into the basis for the lack of efficacy, we performed sequential flow try on peripheral blood samples of mice at als after the T cell infusion. All mice treated with T cells expressing the 'short/CD28' and 'short/4-1BB' CD19 chimeric receptors had significantly higher levels of erred T cells in the blood at all time points after adoptive transfer, ed to mice treated with T cells that expressed corresponding CD19 chimeric receptors with long ellular spacer (p<0.01) (Figure 17D). We did not observe significant differences in T-cell persistence in the peripheral blood of mice that had received T cells expressing CD19 chimeric receptors with CD28 or 4-1BB co-stimulatory domains and short spacer domains (Figure 17D).

The in vivo anti-tumor efficacy of CD19 chimeric receptors with long spacers is not improved by increasing T cell dose or ing an additional costimulatory domain The lack of in vivo anti-tumor efficacy and the lower level of persisting chimeric receptor T cells in mice treated with T cells modified with CD19 chimeric receptors with long spacer domains suggested that efficacy might be ed by increasing the chimeric receptor T cell dose or by including both CD28 and 4- IBB domains into the chimeric or to augment costimulatory signaling. To evaluate this possibility we modified CD8+ TCM with 'long/CD28', 'short CD28', and 'long/CD28_ 4-1BB' CD19 chimeric receptor vectors and confirmed that the long/CD28_ 4-1BB' CD19 chimeric receptor conferred ic lysis and cytokine production in vitro after recognition of CD19+ target cells (Figure 18A-C).

Consistent with previous studies of CD19 chimeric ors, the level of cytokine production and proliferation in vitro in T cells expressing the CD28_ 4-IBB' CDI9 chimeric or was or compared to the identical construct with CD28 alone, and superior to T cells expressing the 'long 4-IBB' CD19 chimeric receptor (Figure 18B, C).

Groups of NSG mice with established Raji tumors were then treated with a high dose of T cells (10 x106 ) T cells expressing the CD28' CD19 chimeric receptor, the 'long/CD28_ 4-IBB' CDI9 chimeric or, the 'short/CD28' CD19- chimeric receptor, and tEGFR alone. Tumor burden was measured by bioluminescence imaging and serial flow cytometric analyses of peripheral blood samples performed to determine the frequency of transferred T cells. Consistent with the results of our prior experiments using much lower doses of T cells, Raji tumors were completely eradicated in mice treated with T cells expressing the 'short/CD28' CD19-chimeric receptor. However, even with a 4-fold higher T cell dose, treatment with T cells expressing the 'long/CD28' CD19 chimeric receptor or the 'long/CD28_ 4-1BB' CD19 chimeric receptor did not provide a nible mor effect (Figure 18D,E).

Thus, increasing the chimeric receptor T cell dose and adding a 4-1BB ulatory domain to CD19 chimeric receptors failed to overcome the ve impact of the longer spacer domain on antitumor activity in vivo. Thus, in this model, anti-tumor reactivity of CD19 chimeric receptors is dictated to a great extent by the length of the ellular spacer domain, and not by the intracellular costimulatory signaling modules.

T cells modified with CD19 chimeric ors that possess long extracellular spacers o activation induced cell death in vivo We sought to determine potential mechanisms underlying the inferior in vivo antitumor activity of T cells that express CD19 chimeric receptors with long spacer domains. Because lower s of transferred T cells modified to express CD19 ic receptors with long spacer domains were present in the blood, we considered the possibility that the T cells were not efficiently activated by tumor cells in vivo or conversely, that they underwent activation induced T cell death in vivo. Therefore, we labeled CD19 chimeric receptor modified and corresponding control T cells with CFSE and administered these T cells to tumor g NSG/Raji mice to examine activation, proliferation and survival of T cells modified with each of the CD19 chimeric receptor constructs at tumor sites in vivo (Figure 19A). At the end of their in vitro expansion and immediately prior to CFSE labeling and infusion into NSG mice bearing established Raji tumors, T cells transduced with each of the CD19 chimeric receptors expressed low levels of the activation markers CD69 and CD25 (Figure 19B).

Bone marrow was obtained from subgroups of mice 24 and 72 hours after the T cell infusion to examine the frequency, activation and proliferation of transferred T cells. At 24 hours, tumor cells (CD45+ CD3-) were present in the bone marrow in all treatment groups and a large on of chimeric receptor T cells, but not control T cells, had upregulated CD69 and CD25. There was no measurable dilution of CFSE in the transferred chimeric receptor T cells. (Figure 19C) Both CD69 and CD25 were expressed in a higher proportion of T cells modified with 'long spacer' CD19 chimeric receptors, suggesting these cells may have received a er stimulus compared to T cells with 'short spacer' CD19 chimeric ors (Figure __C). Despite evidence of T cell activation at 24 hours there were significantly lower numbers of chimeric receptor T cells in the bone marrow of mice treated with T cells modified with the CD28 and 4-IBB 'long spacer' ucts compared to those modified with the CD28 and 4-IBB 'short spacer' constructs, or with the control tEGFR vector (Figure 19C, E).

At 72 hours after T cell er, T cells expressing the 'short/CD28' and 'short/4-lBB' CD19 chimeric receptors had increased 3 to > 10 fold in frequency in the bone marrow and spleen, and had undergone several cell divisions (Figure 19D,E). Control tEGFR+ T cells remained present in the bone marrow and spleen at 72 hours at a level similar to that observed at 24 hours, and had not divided as measured by CFSE dilution. By contrast, the numbers of T cells expressing the CD28' and 'long/4-IBB' CD19 chimeric receptors had not sed in the bone marrow and . (Figure 19D, E) Consistent with lower cell numbers, analysis of CFSE staining in viable PI- 'long/CD28' and 'long/4-IBB' CDl9 ic receptor T cells demonstrated these cells had undergone a much lower number of cell ons compared with 'short/CD28' and 'short/4-IBB' CDl9 chimeric receptor T cells.

(Figure 19D)When the flow data was analyzed to include PI+ T cells, we detected a much higher frequency of PI+ CD3+ T cells in bone marrow and spleen of mice that received CD19 chimeric receptor T cells with 'long spacer' s, demonstrating that a significant proportion of T cells, despite being activated by tumor in vivo had undergone cell death (Figure 19F). Consistent with the bioluminescence imaging, CD45+ CD3- Raji tumor cells were present in greater numbers in the bone marrow of mice treated with T cells sing CD19 ic receptors with long spacer domains or expressing tEGFR only compared to mice treated with CD19 chimeric receptors with short spacer domains (Figure 19D,E, G). tively, the data provides evidence that CD19 chimeric ors with long extracellular spacer domain, despite mediating equivalent or superior effector function in vitro and recognizing tumor in vivo, induce a high level of activation induced cell death in vivo and fail to eradicate established lymphoma.

Discussion Chimeric receptors are artificial receptors that include an extracellular antigen-binding scFv, a spacer domain that provides separation of the scFv from the cell ne and an intracellular signaling module that mediates T cell activation.

Chimeric receptors that contain a scFv derived from the CD19-specific FMC63 mAb studied here, have advanced to testing in clinical trials in patients with B-cell malignancies. Antitumor activity and T cell persistence have varied ntially in different trials. Each of these clinical trials differed in potentially critical variables, including ent gene transfer vectors, cell culture methodologies, and conditioning regimens prior to CD19 chimeric receptor T cell transfer.

We examined the possibility that the extracellular spacer domain of CD19 chimeric receptors may be an important determinant of umor activity in vivo, independent of the costimulatory signaling provided by the chimeric receptor. We derived spacer domains from IgG4-Fc, which enables high levels of chimeric receptor cell surface sion and is less likely to provoke ition by innate immune cells compared to other IgG isotypes. We used the IgG4 ‘Hinge-CH2-CH3’ in the design of the long (229 AA) spacer constructs and the IgG4 ‘Hinge’ domain in our short (12 AA) spacer chimeric receptors. To compare the individual chimeric receptor constructs, we used ed (>90%) chimeric receptor positive CD8+ TCM– derived T cells to remove differences in the cellular composition and transduction ncy as a potential source of bias in the analysis of in vitro and in vivo function. CD8+ TCM have been shown to have superior traits for adoptive immunotherapy, compared with other more prevalent T cell subsets in blood that persist poorly and are ineffective in tumor therapy. The CD19 chimeric or T cells were generated using a standardized culture protocol that is similar to that used to derive chimeric receptor T cells for clinical trials. Our data show that CD19 chimeric receptors with a short IgG4 ‘Hinge’ spacer conferred potent anti-tumor reactivity in vitro and in vivo, whereas corresponding CD19 chimeric receptors with a long spacer of IgG4 -CH2-CH3’, despite equivalent or superior reactivity in vitro, failed to confer icant anti-tumor s in murine lymphoma models.

Surprisingly, the length of the spacer domain proved to be a decisive element for in vivo antitumor activity, and the lack of efficacy of the ‘long ’ chimeric receptor could not be overcome by increasing the T cell dose.

We also observed major differences in cytokine secretion and proliferation in vitro between T cells expressing CD19 chimeric receptors ning CD28 and 4- 1BB costimulatory domains, with CD28 augmenting secretion of IFN-γ, IL-2, and TNF-α compared with 4-1BB. CD19 chimeric receptors that sed a tandem CD28_4-1BB also produced higher levels of these cytokines compared to chimeric receptors encoding 4-1BB only. However, our data shows that these differences in in vitro function were not predictive of in vivo anti-tumor efficacy, since CD19 ic receptors with either CD28 or 4-1BB costimulatory domain and a short spacer were similarly ive at eradicating ed established Raji tumors in NSG mice. In contrast, CD19 chimeric receptors with suboptimal spacer length and CD28, 4-1BB, or both costimulatory domains, despite conferring similar in vitro function as the cal chimeric receptor construct with a short spacer domain, lacked significant anti-tumor activity in vivo, demonstrating the contribution of spacer length to in vivo function of chimeric receptor T cells.

Our studies provide insight into the mechanism responsible for the lack of in vivo efficacy of CD19 chimeric ors with long spacer domains. T cells expressing CD19 chimeric receptors with both long and short spacer domains could be detected in the bone marrow and spleen after adoptive transfer into NSG mice bearing established Raji lymphoma, and the majority were activated as demonstrated by upregulation of CD25 and CD69. However, T cells modified to express a CD19 ic receptor with a long spacer domain exhibited a steep decline in cell number, in st to the marked in vivo expansion of T cells expressing CD19 chimeric receptors with a short spacer domain. The decline in T cell number was a consequence of much higher levels of cell death in the first 72 hours after adoptive transfer compared with T cells with short spacer domains, and control T cells that did not express a CD19 chimeric or. Collectively, these data indicate that recognition of tumor cells in vivo resulted in death of T cells expressing CD19- chimeric receptors with long spacer domains. A similar mechanism may explain the short duration and low levels of T cell persistence in the clinical trials that ed long spacer CD19-chimeric ors (14).

The studies ed here are the first to show that the spacer domains of CD19 chimeric receptors that lack intrinsic signaling properties have dramatic effects on in vivo antitumor activity independent of costimulatory signaling, and identify the importance of analyzing the optimal composition of this region in the design of chimeric receptors for clinical applications.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When reting statements in this specification, and claims which include the term “comprising”, it is to be understood that other features that are additional to the es prefaced by this term in each statement or claim may also be present. d terms such as “comprise” and “comprised” are to be interpreted in similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of ation, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person d in the art and may assist in putting into practice the invention as defined in the claims of this ation.The foregoing is illustrative of the present invention, and is not to be ued as limiting f. The invention is defined by the following claims, with equivalents of the claims to be included therein. All references and documents referred to herein are hereby incorporated by reference.

Table 1 Sequence of anti-CD19 short spacer chimeric receptor GMCSFRss-CD19scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt Atgctgctgctggtgaccagcctgctgctgtgcgagctgccccaccccgcctttctgctgatcccc (GMCSFRss) (SEQ ID NO:2) Gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgggtgaccatcagctgccggg ccagccaggacatcagcaagtacctgaactggtatcagcagaagcccgacggcaccgtcaagctgctgatctac cacaccagccggctgcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgactacagcctgac catctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggc ggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcacc aagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgca gcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatg gctgggcgtgatctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaag gacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgc caagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc (CD19scFv) (SEQ ID NO:3) Gaatctaagtacggaccgccctgccccccttgccct (IgG4hinge) (SEQ ID NO:4) Atgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatctt ttgggtg m-)(SEQ ID NO:5) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagagg aagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (41BB) (SEQ ID NO:6) Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctga gcagaagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagc ctcggcggaagaacccccaggaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcg agatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtatcagggcctgtccaccgcca ccaaggatacctacgacgccctgcacatgcaggccctgcccccaagg (CD3Zeta)- (SEQ ID NO:7) ggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg (T2A) (SEQ ID NO:9) Atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtg taacggaataggtattggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgc acctccatcagtggcgatctccacatcctgccggtggcatttaggggtgactccttcacacatactcctcctctggat ccacaggaactggatattctgaaaaccgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaac aggacggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaagcaacatggtcagttttctctt gcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggagatgtgataattt caggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaacc aaaattataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccga ctggggcccggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggac aagtgcaaccttctggagggtgagccaagggagtttgtggagaactctgagtgcatacagtgccacccagagtg cctgcctcaggccatgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcccactacattga cggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaacaccctggtctggaagtacgca gacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggccaggtcttgaaggctgt ccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtggccc tggggatcggcctcttcatgtga (EGFRt) (SEQ ID NO:9) Table 2 GMCSFRss DNA: CTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC AA: M L L L V T S L L L C E L P H P A CD19scFv DNA: TTTCTGCTGATCCCC:GACATCCAGATGACCCAGACCACCTCCAGCCTGAGC AA: F L L I P D I Q M T Q T T S S L S DNA: GCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATC AA: A S L G D R V T I S C R A S Q D I DNA: AGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTG AA: S K Y L N W Y Q Q K P D G T V K L DNA: CTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGC AA: L I Y H T S R L H S G V P S R F S DNA: GGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAG AA: G S G S G T D Y S L T I S N L E Q DNA: GAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACC AA: E D I A T Y F C Q Q G N T L P Y T DNA: TTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACCTCCGGCAGCGGC AA: F G G G T K L E I T G S T S G S G DNA: AAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAA AA: K P G S G E G S T K G E V K L Q E DNA: AGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACC AA: S G P G L V A P S Q S L S V T C T DNA: GTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCC AA: V S G V S L P D Y G V S W I R Q P DNA: CCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACC AA: P R K G L E W L G V I W G S E T T 40 DNA: TACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGC AA: Y Y N S A L K S R L T I I K D N S DNA: AAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCC AA: K S Q V F L K M N S L Q T D D T A DNA: TACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGAC AA: I Y Y C A K H Y Y Y G G S Y A M D IgG4hinge 50 DNA: TACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC:GAGAGCAAGTACGGA AA: Y W G Q G T S V T V S S E S K Y G CD28tm DNA: CCGCCCTGCCCCCCTTGCCCT:ATGTTCTGGGTGCTGGTGGTGGTCGGAGGC 55 AA: P P C P P C P M F W V L V V V G G DNA: GTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGG AA: V L A C Y S L L V T V A F I I F W 41BB DNA: GTG:AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATG AA: V K R G R K K L L Y I F K Q P F M DNA: AGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCA AA: R P V Q T T Q E E D G C S C R F P CD3Zeta DNA: GAAGAAGGAGGATGTGAACTGCGGGTGAAG:TTCAGCAGAAGCGCC AA: E E E E G G C E L R V K F S R S A DNA: GACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAAC AA: D A P A Y Q Q G Q N Q L Y N E L N DNA: CTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGAC AA: L G R R E E Y D V L D K R R G R D DNA: CCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTAT AA: P E M G G K P R R K N P Q E G L Y DNA: AACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATG AA: N E L Q K D K M A E A Y S E I G M DNA: AAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTG AA: K G E R R R G K G H D G L Y Q G L DNA: TCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCC AA: S T A T K D T Y D A L H M Q A L P DNA: CCAAGG:CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGT AA: P R L E G G G E G R G S L L T C G EGFRt DNA: GACGTGGAGGAGAATCCCGGCCCTAGG:ATGCTTCTCCTGGTGACAAGCCTT AA: D V E E N P G P R M L L L V T S L DNA: CTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACGCAAAGTG AA: L L C E L P H P A F L L I P R K V DNA: TGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCT AA: C N G I G I G E F K D S L S I N A DNA: ACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCAC 45 AA: T N I K H F K N C T S I S G D L H DNA: ATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTG AA: I L P V A F R G D S F T H T P P L DNA: GATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTT AA: D P Q E L D I L K T V K E I T G F DNA: TTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAG AA: L L I Q A W P E N R T D L H A F E DNA: AACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTT AA: N L E I I R G R T K Q H G Q F S L DNA: GCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAG AA: A V V S L N I T S L G L R S L K E DNA: ATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCA AA: I S D G D V I I S G N K N L C Y A DNA: AATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAA AA: N T I N W K K L F G T S G Q K T K DNA: ATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGC AA: I I S N R G E N S C K A T G Q V C DNA: CATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGC AA: H A L C S P E G C W G P E P R D C DNA: GTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAAC AA: V S C R N V S R G R E C V D K C N DNA: CTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAG AA: L L E G E P R E F V E N S E C I Q DNA: TGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGG AA: C H P E C L P Q A M N I T C T G R DNA: GGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGC AA: G P D N C I Q C A H Y I D G P H C DNA: GTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGG AA: V K T C P A G V M G E N N T L V W DNA: AAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACC AA: K Y A D A G H V C H L C H P N C T DNA: TACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAG 45 AA: Y G C T G P G L E G C P T N G P K DNA: TCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTG AA: I P S I A T G M V G A L L L L L V 50 DNA: GTGGCCCTGGGGATCGGCCTCTTCATGTGA (SEQ ID NO:10) AA: V A L G I G L F M * (SEQ ID NO:11) Table 3 ZXR-014 Nucleotide and amino acid sequences (map of sections) GMCSFRss: nt2084-2149 CD19scFv: nt2150-2884 Igg4Hinge: nt2885-2920 CD28tm: nt2921-3004 41BB: nt3005-3130 Zeta: nt3131-3466 T2A: nt3467-3538 EGFRt: nt3539-4612 Primers for sequencing: Oligo name Sequence Region oJ02649 AGAATAGACCGAGATAGGGT pre-U5(SEQ ID NO:22) oJ02648 CCGTACCTTTAAGACCAATGACTTAC delU3(SEQ ID NO:23) oJ02650 TTGAGAGTTTTCGCCCCG mid-Ampr(SEQ ID NO:24) oJ02651 AATAGACAGATCGCTGAGATAGGT post-Ampr(SEQ ID NO:25) oJ02652 CAGGTATCCGGTAAGCGG CoE1 Q ID NO:26) oJ02653 CGACCAGCAACCATAGTCC SV40(SEQ ID NO:27) oJ02654 TAGCGGTTTGACTCACGG CMV(SEQ ID NO:28) AGCTAGAACGATTC psi(SEQ ID NO:29) oJ02656 ATTGTCTGGTATAGTGCAGCAG RRE(SEQ ID NO:30) oJ02657 TCGCAACGGGTTTGCC EF1p(SEQ ID NO:31) oJ02658 AGGAAGATATCGCCACCTACT CD19Rop(SEQ ID NO:32) 1 CGGGTGAAGTTCAGCAGAAG Zeta(SEQ ID NO:33) oJ02735 ACTGTGTTTGCTGACGCAAC WPRE(SEQ ID NO:34) oJ02715 CTCCTGGTGACAAG EGFRt(SEQ ID NO:35) Table 4 Uniprot P0861 IgG4-Fc(SEQ ID NO:13) 20 30 40 50 60 ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 70 80 90 100 110 120 GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPSCP GPSV 130 140 150 160 170 180 FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY 190 200 210 220 230 240 RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK QVYT LPPSQEEMTK 250 260 270 280 290 300 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL WQEG 310 320 NVFSCSVMHE ALHNHYTQKS LSLSLGK 1-98 CH1 99-110 Hinge 111-220 CH2 221-327 CH3 Position 108 S→P Table 5 Uniprot P10747 EQ ID NO:14) 20 30 40 50 60 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD 70 80 90 100 110 120 SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 130 140 150 160 170 180 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 190 200 210 220 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA AYRS 1-18 signal peptide 19-152 extracellular domain 153-179 transmembrane domain 180-220 intracellular domain Position 186-187 LL→GG Table 6 Uniprot Q07011 4-1BB(SEQ ID NO:15) 20 30 40 50 60 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 70 80 90 100 110 120 QCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 130 140 150 160 170 180 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 190 200 210 220 230 240 PGHSPQIISF FLALTSTALL TLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL 1-23 signal e 24-186 extracellular domain 187-213 transmembrane domain 214-255 intracellular domain Table 7 Uniprot P20963 human CD3ζ isoform 3 (SEQ ID NO:16) 20 30 40 50 60 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 70 80 90 100 110 120 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QEGL YNELQKDKMA 130 140 150 160 GMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR 1-21 signal peptide 22-30 extracellular 31-51 transmembrane 52-164 intracellular domain 61-89 ITAM1 100-128 ITAM2 131-159 ITAM3 Table 8 Exemplary Hinge region Sequences Human IgG1 EPKSCDKTHTCPPCP (SEQ ID NO:17) Human IgG2 ERKCCVECPPCP (SEQ ID NO:18) Human IgG3 ELKTPLGDTHTCPRCP (EPKSCDTPPPCPRCP)3 (SEQ ID NO:19) Human IgG4 ESKYGPPCPSCP (SEQ ID NO:20) Modified Human IgG4 ESKYGPPCPPCP (SEQ ID NO:21) Modified Human IgG4 YGPPCPPCP (SEQ ID NO:51) Modified Human IgG4 KYGPPCPPCP (SEQ ID NO:52) ed Human IgG4 EVVKYGPPCPPCP (SEQ ID NO:53) Table 9 R12 long spacer CAR: PJ_R12-CH2-CH3-41BB-Z-T2A-tEGFR (SEQ ID NO:37) GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA ACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA CACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC CCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT GTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC 40 CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT TACAGATCCAAGCTGTGACCGGCGCCTACG GCTAGC GAATTCCTCGAGGCC ACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC 45 TTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACA CCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCC TACTACATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCC ACCATCTACCCCAGCAGCGGCAAGACCTACTACGCCACCTGGGTGAACGGACG GTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACA GCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCG ACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTA GCGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTG GTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATC ACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCA GGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCT ACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCC TACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTAC TGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACC GTGACCGGCGAGTCTAAG IgG4 spacer TA CGGACCGCCCTGCCCCCCTTGCCCT GCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCAGGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGT GCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGG TGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGC AAGGCCAAG GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG AGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG 4-1BB ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CD3 zeta AAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC 40 CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA GATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG CAGGCCCTGCCCCCAAGG 45 CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA GGAGAATCCCGGCCCTAGG tEGFR ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT AGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT 40 ACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT GCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT 45 CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA TAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA AGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT 40 GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT 45 TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG Table 10 Leader _R12- Hinge-CH2-CH3- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:38) Leader MLLLVTSLLLCELPHPAFLLIP R12 scFv QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSG KTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNI WGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTD TIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDE ADYYCGADYIGGYVFGGGTQLTVTG Hinge Spacer PCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK CD28 MFWVLVVVGGVLACYSLLVTVAFIIFWV 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3 zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR LEGGGEGRGSLLTCGDVEENPGPR tEGFR MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL LLLVVALGIGLFM Table 11 R12 intermediate spacer CAR: PJ_R12-CH3-41BB-Z-T2A-tEGFR (SEQ ID NO:39) GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA GAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC AGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG CGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT GTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC 40 CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT TACAGATCCAAGCTGTGACCGGCGCCTACG GCTAGCGAATTCCTCGAGGCC R12 ScFv 45 ACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC TTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACA CCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCC TACTACATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCC ACCATCTACCCCAGCAGCGGCAAGACCTACTACGCCACCTGGGTGAACGGACG GTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACA GCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCG ACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTA GAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTG GTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATC ACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCA GGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCT ACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCC GACCGCTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTAC TGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACC GTGACCGGCGAGTCTAAG Hinge Spacer TA CGGACCGCCCTGCCCCCCTTGCCCT GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG AGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG 4-1BB ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG GGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG CAGGCCCTGCCCCCAAGG CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA GGAGAATCCCGGCCCTAGG 40 tEGFR ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA 45 CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC TCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA ATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC TGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT 40 CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC 45 ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA CTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT ATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA 40 TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA GGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT 45 CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG Table 12 Leader _R12- Hinge- CH3- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:40) Leader MLLLVTSLLLCELPHPAFLLIP R12 scFV QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSG KTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNI WGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTD TIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDE ADYYCGADYIGGYVFGGGTQLTVTG Hinge Spacer ESKYGPPCPPCP GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQE SVMHEALHNHYTQKSLSLSLGK CD28tm MFWVLVVVGGVLACYSLLVTVAFIIFWV 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3 zeta SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR LEGGGEGRGSLLTCGDVEENPGPR tEGFR MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL LLLVVALGIGLFM Table 13 R12 short spacer CAR: PJ_R12-Hinge-41BB-Z-T2A-tEGFR (SEQ ID NO:41) GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA GGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA GAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG CTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC ACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT 40 GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT TACAGATCCAAGCTGTGACCGGCGCCTACG GCTAGF R12 scFV ACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC 45 TTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACA CCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCC TACTACATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCC ACCATCTACCCCAGCAGCGGCAAGACCTACTACGCCACCTGGGTGAACGGACG GTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACA GCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCG ACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTA GCGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTG GTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATC ACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCA GCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCT ACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCC GACCGCTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTAC TGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACC GGCGAGTCTAAG Hinge/Spacer TA CGGACCGCCCTGCCCCCCTTGCCCT 4-1BB ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CD3 zeta CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG CAGGCCCTGCCCCCAAGG GGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA GGAGAATCCCGGCCCTAGG tEGFR ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA CTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC 40 TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG 45 GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT AGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT GCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG 40 AGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT 45 CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG TTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG TTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA CGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC 40 AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA 45 TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG Table 14 Leader _R12 - CD28tm/41BB-Z-T2A-tEGFR(SEQ ID NO:42) Leader SLLLCELPHPAFLLIP scFv R12 QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSG KTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNI WGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTD TIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDE ADYIGGYVFGGGTQLTVTG Hinge/spacer ESKYGPPCPPCP CD28tm MFWVLVVVGGVLACYSLLVTVAFIIFWV 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR LEGGGEGRGSLLTCGDVEENPGPR tEGFR MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL LLLVVALGIGLFM Table 15 R11 long spacer CAR: PJ_R11-CH2-CH3-41BB-Z-T2A-tEGFR (SEQ ID NO:43) GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA TCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG CTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA CATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC 40 CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT TCCAAGCTGTGACCGGCGCCTACG GCTAGC scFv R12 45 GAATTCGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCC CACCCCGCCTTTCTGCTGATCCCCCAGAGCGTGAAAGAGTCCGAGGGCGACCTG GTCACACCAGCCGGCAACCTGACCCTGACCTGTACCGCCAGCGGCAGCGACATC AACGACTACCCCATCTCTTGGGTCCGCCAGGCTCCTGGCAAGGGACTGGAATGG ATCGGCTTCATCAACAGCGGCGGCAGCACTTGGTACGCCAGCTGGGTCAAAGGC CGGTTCACCATCAGCCGGACCAGCACCACCGTGGACCTGAAGATGACAAGCCT GACCACCGACGACACCGCCACCTACTTTTGCGCCAGAGGCTACAGCACCTACTA CGGCGACTTCAACATCTGGGGCCCTGGCACCCTGGTCACAATCTCTAGCGGCGG AGGCGGCAGCGGAGGTGGAGGAAGTGGCGGCGGAGGATCCGAGCTGGTCATGA CCCAGACCCCCAGCAGCACATCTGGCGCCGTGGGCGGCACCGTGACCATCAATT GCCAGGCCAGCCAGAGCATCGACAGCAACCTGGCCTGGTTCCAGCAGAAGCCC CCCCCCACCCTGCTGATCTACAGAGCCTCCAACCTGGCCAGCGGCGTG CCAAGCAGATTCAGCGGCAGCAGATCTGGCACCGAGTACACCCTGACCATCTCC GGCGTGCAGAGAGAGGACGCCGCTACCTATTACTGCCTGGGCGGCGTGGGCAA CGTGTCCTACAGAACCAGCTTCGGCGGAGGTACTGAGGTGGTCGTCAAA Hinge/Spacer TA CGGACCGCCCTGCCCCCCTTGCCCT GCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG CTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCAGGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGT GCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGG TGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGC AAGGCCAAG GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG AGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG 4-1BB ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA GTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC 40 CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG CAGGCCCTGCCCCCAAGG 45 CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA GGAGAATCCCGGCCCTAGG tEGFR ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC CAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA TCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT GACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT GGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT 40 CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT 45 AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA GCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG GGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC 40 ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT 45 TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA ACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG Table 16 Leader _R11- Hinge-CH2-CH3- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:44) Leader MLLLVTSLLLCELPHPAFLLIP R11 scFv QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFINSGGSTW YASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYYGDFNIWGPGTLVT ISSGGGGSGGGGSGGGGSELVMTQTPSSTSGAVGGTVTINCQASQSIDSNLAWFQQ KPGQPPTLLIYRASNLASGVPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGNV SYRTSFGGGTEVVVK Hinge/Spacer ESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY HN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK CD28tm MFWVLVVVGGVLACYSLLVTVAFIIFWV 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR LEGGGEGRGSLLTCGDVEENPGPR tEGFR MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI GDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL LLLVVALGIGLFM Table 17 R11 intermediate spacer CAR: PJ_R11-CH3-41BB-Z-T2A-tEGFR (SEQ ID NO:45) GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC AGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA GGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA ACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC CTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC 40 CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT TACAGATCCAAGCTGTGACCGGCGCCTACG GCTAGC R11 scFV 45 GAATTCGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCC CACCCCGCCTTTCTGCTGATCCCCCAGAGCGTGAAAGAGTCCGAGGGCGACCTG GTCACACCAGCCGGCAACCTGACCCTGACCTGTACCGCCAGCGGCAGCGACATC TACCCCATCTCTTGGGTCCGCCAGGCTCCTGGCAAGGGACTGGAATGG ATCGGCTTCATCAACAGCGGCGGCAGCACTTGGTACGCCAGCTGGGTCAAAGGC CGGTTCACCATCAGCCGGACCAGCACCACCGTGGACCTGAAGATGACAAGCCT GACCACCGACGACACCGCCACCTACTTTTGCGCCAGAGGCTACAGCACCTACTA CGGCGACTTCAACATCTGGGGCCCTGGCACCCTGGTCACAATCTCTAGCGGCGG AGGCGGCAGCGGAGGTGGAGGAAGTGGCGGCGGAGGATCCGAGCTGGTCATGA CCCCCAGCAGCACATCTGGCGCCGTGGGCGGCACCGTGACCATCAATT GCCAGGCCAGCCAGAGCATCGACAGCAACCTGGCCTGGTTCCAGCAGAAGCCC GGCCAGCCCCCCACCCTGCTGATCTACAGAGCCTCCAACCTGGCCAGCGGCGTG CCAAGCAGATTCAGCGGCAGCAGATCTGGCACCGAGTACACCCTGACCATCTCC GGCGTGCAGAGAGAGGACGCCGCTACCTATTACTGCCTGGGCGGCGTGGGCAA CTACAGAACCAGCTTCGGCGGAGGTACTGAGGTGGTCGTCAAA Hinge/spacer TAGGACCGCCCTGC CCCCCTTGCCCT GCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG GACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG AGCCAGGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGT GCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGG TGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGC AAGGCCAAG CH3 GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG TGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG 4-1BB ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CD3zeta CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG 40 ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG CAGGCCCTGCCCCCAAGG 45 T2A CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA GGAGAATCCCGGCCCTAGG tEGFR ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT CCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT ACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT GGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT 40 CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT 45 AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA TGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG TTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG GGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC 40 ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT TTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT 45 TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG Table 18 Leader _R11- CH3- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:46) Leader MLLLVTSLLLCELPHPAFLLIP scFV R11 QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFINSGGSTW YASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYYGDFNIWGPGTLVT ISSGGGGSGGGGSGGGGSELVMTQTPSSTSGAVGGTVTINCQASQSIDSNLAWFQQ KPGQPPTLLIYRASNLASGVPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGNV SYRTSFGGGTEVVVK Hinge/spacer ESKYGPPCPPCP GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK CD28tm VVGGVLACYSLLVTVAFIIFWV 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR LEGGGEGRGSLLTCGDVEENPGPRM tEGFR LLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL PVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTK QHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKT KIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEG EPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMG ENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLL LLVVALGIGLFM Table 19 R11 short spacer CAR: PJ_R11- -T2A-tEGFR(SEQ ID NO:47) GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA ACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC 40 CCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT TACAGATCCAAGCTGTGACCGGCGCCTACG GCTAGC scFV R11 45 GAATTCGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCC CACCCCGCCTTTCTGCTGATCCCCCAGAGCGTGAAAGAGTCCGAGGGCGACCTG GTCACACCAGCCGGCAACCTGACCCTGACCTGTACCGCCAGCGGCAGCGACATC AACGACTACCCCATCTCTTGGGTCCGCCAGGCTCCTGGCAAGGGACTGGAATGG ATCGGCTTCATCAACAGCGGCGGCAGCACTTGGTACGCCAGCTGGGTCAAAGGC ACCATCAGCCGGACCAGCACCACCGTGGACCTGAAGATGACAAGCCT GACCACCGACGACACCGCCACCTACTTTTGCGCCAGAGGCTACAGCACCTACTA CGGCGACTTCAACATCTGGGGCCCTGGCACCCTGGTCACAATCTCTAGCGGCGG AGGCGGCAGCGGAGGTGGAGGAAGTGGCGGCGGAGGATCCGAGCTGGTCATGA CCCAGACCCCCAGCAGCACATCTGGCGCCGTGGGCGGCACCGTGACCATCAATT CCAGCCAGAGCATCGACAGCAACCTGGCCTGGTTCCAGCAGAAGCCC GGCCAGCCCCCCACCCTGCTGATCTACAGAGCCTCCAACCTGGCCAGCGGCGTG CCAAGCAGATTCAGCGGCAGCAGATCTGGCACCGAGTACACCCTGACCATCTCC GGCGTGCAGAGAGAGGACGCCGCTACCTATTACTGCCTGGGCGGCGTGGGCAA CGTGTCCTACAGAACCAGCTTCGGCGGAGGTACTGAGGTGGTCGTCAAA Hinge/spacer TACGGACCGCCCTGCCCCCCTTGCCCT GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG AGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG 4-1BB ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG GTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CD3zeta CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG CAGGCCCTGCCCCCAAGG CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA GGAGAATCCCGGCCCTAGG tEGFR 40 ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA ATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA 45 CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA ATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG CAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC TTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA 40 TGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG 45 AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA TAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA AGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA 40 ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG TTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC 45 TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG Table 20 Leader _R11- Hinge- /41BB-Z-T2A-tEGFR (SEQ ID NO:48) Leader MLLLVTSLLLCELPHPAFLLIP ScFv R11 QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFINSGGSTW YASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYYGDFNIWGPGTLVT ISSGGGGSGGGGSGGGGSELVMTQTPSSTSGAVGGTVTINCQASQSIDSNLAWFQQ KPGQPPTLLIYRASNLASGVPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGNV SYRTSFGGGTEVVVK Spacer/Hinge ESKYGPPCPPCP CD28tm MFWVLVVVGGVLACYSLLVTVAFIIFWV 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR LEGGGEGRGSLLTCGDVEENPGPR tEGFR MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI GDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL LLLVVALGIGLFM Table 21 Intermediate Spacer (SEQ ID NO:49) Hinge/spacer ESKYGPPCPPCP GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK Long spacer (SEQ ID NO:50) Hinge PCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK Table 22 Her2 uct-short spacer (SEQ Id No:54 ) GMCSFss-Her2scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt Leader Atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatccca Her2scFV gatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtcaccatcacctgccgtgccagtcaggatgtg aatactgctgtagcctggtatcaacagaaaccaggaaaagctccgaaactactgatttactcggcatccttcctctactctggagtccct tctcgcttctctggttccagatctgggacggatttcactctgaccatcagcagtctgcagccggaagacttcgcaacttattactgtcag caacattatactactcctcccacgttcggacagggtaccaaggtggagatcaaaggcagtactagcggcggtggctccgggggcg gatccggtgggggcggcagcagcgaggttcagctggtggagtctggcggtggcctggtgcagccagggggctcactccgtttgtc ctgtgcagcttctggcttcaacattaaagacacctatatacactgggtgcgtcaggccccgggtaagggcctggaatgggttgcaag gatttatcctacgaatggttatactagatatgccgatagcgtcaagggccgtttcactataagcgcagacacatccaaaaacacagcct acctgcagatgaacagcctgcgtgctgaggacactgccgtctattattgttctagatggggaggggacggcttctatgctatggacta ctggggtcaaggaaccctggtcaccgtctcgagt Hinge spacer Gagagcaagtacggaccgccctgccccccttgccct CD28tm atgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcttttgggtg 4-1BB Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagc tgccgatttccagaagaagaagaaggaggatgtgaactg CD3 zeta Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcag aagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccag gaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggg gccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccc caagg Ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg tEGFR atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggt attggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcc tgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatc acagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggacc aagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggaga tgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaa ttataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggccc ggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagcc 40 gtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacgg ggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaa acaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggcca ggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtg gccctggggatcggcctcttcatgtga Table 23 Her2 construct-intermediate spacer (SEQ Id No:55 ) Leader ctcctggtgacaagccttctgctctgtgagttaccacaccca Fv Gcattcctcctgatcccagatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtcaccatcacctgc cgtgccagtcaggatgtgaatactgctgtagcctggtatcaacagaaaccaggaaaagctccgaaactactgatttactcggcatcct tcctctactctggagtcccttctcgcttctctggttccagatctgggacggatttcactctgaccatcagcagtctgcagccggaagactt cgcaacttattactgtcagcaacattatactactcctcccacgttcggacagggtaccaaggtggagatcaaaggcagtactagcggc ggtggctccgggggcggatccggtgggggcggcagcagcgaggttcagctggtggagtctggcggtggcctggtgcagccagg gggctcactccgtttgtcctgtgcagcttctggcttcaacattaaagacacctatatacactgggtgcgtcaggccccgggtaagggc ctggaatgggttgcaaggatttatcctacgaatggttatactagatatgccgatagcgtcaagggccgtttcactataagcgcagacac atccaaaaacacagcctacctgcagatgaacagcctgcgtgctgaggacactgccgtctattattgttctagatggggaggggacgg cttctatgctatggactactggggtcaaggaaccctggtcaccgtctcgagt Hinge spacer GagagcaagtacggaccgccctgccccccttgccctGgccagcctagagaaccccaggtgtacaccctgcctcccagccagga agagatgaccaagaaccaggtgtccctgacctgcctggtcaaaggcttctaccccagcgatatcgccgtggaatgggagagcaac ggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggcagcttcttcctgtactcccggctgaccgtgg acaagagccggtggcaggaaggcaacgtcttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccc tgagcctgagcctgggcaag CD28tm Atgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcttttgggtg 4-1BB Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagc tgccgatttccagaagaagaagaaggaggatgtgaactg CD3 zeta Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcag aagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccag gaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggg gcaagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccc caagg Ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg tEGFR atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggt attggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcc tgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatc acagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggacc aagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggaga 40 aatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaa ttataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggccc ggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagcc aagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacgg ggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaa 45 acaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggcca ggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtg gccctggggatcggcctcttcatgtga Table 24 Her2 construct-long spacer (SEQ Id No:56 ) leader Atgcttctcctggtgacaagccttctgctctgtgagttaccacaccca Her2scFV gcattcctcctgatcccagatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtcaccatcacctgcc gtgccagtcaggatgtgaatactgctgtagcctggtatcaacagaaaccaggaaaagctccgaaactactgatttactcggcatcctt cctctactctggagtcccttctcgcttctctggttccagatctgggacggatttcactctgaccatcagcagtctgcagccggaagactt cgcaacttattactgtcagcaacattatactactcctcccacgttcggacagggtaccaaggtggagatcaaaggcagtactagcggc ggtggctccgggggcggatccggtgggggcggcagcagcgaggttcagctggtggagtctggcggtggcctggtgcagccagg gggctcactccgtttgtcctgtgcagcttctggcttcaacattaaagacacctatatacactgggtgcgtcaggccccgggtaagggc ctggaatgggttgcaaggatttatcctacgaatggttatactagatatgccgatagcgtcaagggccgtttcactataagcgcagacac atccaaaaacacagcctacctgcagatgaacagcctgcgtgctgaggacactgccgtctattattgttctagatggggaggggacgg cttctatgctatggactactggggtcaaggaaccctggtcaccgtctcgagt long spacer gagagcaagtacggaccgccctgccccccttgccctgcccccgagttcctgggcggacccagcgtgttcctgttcccccccaagcc caaggacaccctgatgatcagccggacccccgaggtgacctgcgtggtggtggacgtgagccaggaagatcccgaggtccagtt caattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctaccgggtggt gtctgtgctgaccgtgctgcaccaggactggctgaacggcaaagaatacaagtgcaaggtgtccaacaagggcctgcccagcagc atcgaaaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacaccctgcctccctcccaggaagagatgacc aagaaccaggtgtccctgacctgcctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcct gagaacaactacaagaccacccctcccgtgctggacagcgacggcagcttcttcctgtacagccggctgaccgtggacaagagcc ggtggcaggaaggcaacgtctttagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtc cctgggcaag CD28tm tgggtgctggtggtggtgggcggggtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtg 4-1BB aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagct gccgatttccagaagaagaagaaggaggatgtgaactg CD3zeta Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcag aagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccag gaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggg gcaagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccc caagg Ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg tEGFR atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggt 40 attggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcc tgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatc acagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggacc aagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggaga tgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaa 45 gcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggccc ggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagcc aagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacgg ggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaa ccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggcca ggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtg gccctggggatcggcctcttcatgtga

Claims (45)

WHAT WE CLAIM IS:

1. A nucleic acid encoding a chimeric receptor, the chimeric receptor comprising: (a) an extracellular domain consisting of: 5 an extracellular ligand g domain consisting of a single chain variable nt (scFv) that binds to a CD19, wherein the scFv comprises a variable light chain (VL) domain comprising a CDRL1, a CDRL2, and a CDRL3 of the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a CDRH1, a CDRH2, and a CDRH3 of the amino acid sequence 10 encoded by SEQ ID NO:3; and an extracellular polypeptide spacer that is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a ne or a threonine (SEQ ID NO:1); (b) a transmembrane ; and 15 (c) an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain.

2. A nucleic acid encoding a chimeric receptor, the nucleic acid comprising: (a) a polynucleotide encoding a single chain variable nt (scFv) 20 that binds to CD19, wherein the scFv comprises: the amino acid sequence encoded by SEQ ID NO:3; (b) a polynucleotide encoding a transmembrane domain; (c) a polynucleotide encoding a polypeptide spacer d between the scFv and the transmembrane domain, wherein the polypeptide spacer is about 15 25 amino acids or less in length and comprises an amino acid ce of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); and (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain.

3. The nucleic acid of claim 1 or 2, wherein the polypeptide spacer comprises a human IgG1, IgG2, or IgG4 hinge region or a modified version of a human IgG1, IgG2, or IgG4 hinge region.

4. The nucleic acid of any one of claims 1-3, n the polypeptide 5 spacer comprises the amino acid sequence set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

5. The c acid of any one of claims 1-3, wherein the polypeptide spacer comprises a modified human IgG4 hinge region. 10

6. The nucleic acid of any one of claims 1-5, wherein the polypeptide spacer comprises the amino acid sequence set forth in SEQ ID NO:51.

7. The nucleic acid of any one of claims 1-6, wherein the polypeptide spacer is 10 to 15 amino acids in length.

8. The nucleic acid of any one of claims 1-7, wherein the ptide 15 spacer is about 12 amino acids in .

9. The nucleic acid of any one of claims 1-8, wherein the polypeptide spacer consists of the amino acid sequence set forth in SEQ ID NO:21.

10. The nucleic acid of any one of claims 1 and 3-9, wherein the VL domain comprises a VL domain present in the amino acid sequence encoded by SEQ ID 20 NO:3; and theVH domain comprises a VH domain present in the amino acid sequence encoded by SEQ ID NO:3.

11. The nucleic acid of any one of claims 1 and 3-10, n theVL domain comprises a CDRL1 sequence of RASQDISKYLN, a CDRL2 sequence of SRLHSGV, and a CDRL3 sequence of TFG, and theVH domain comprises a CDRH1 sequence of DYGVS, a CDRH2 sequence of VIWGSETTYYNSALKS, and a CDRH3 sequence of YAMDYWG.

12. The nucleic acid of any one of claims 1 and 3-11, wherein the VH 5 domain and theVL domain are separated by a peptide linker.

13. The nucleic acid of claim 12, wherein the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:36.

14. The nucleic acid of claim 12 or 13, wherein the scFv has a VL-linker-VH orientation. 10

15. The nucleic acid of any one of claims 1-14, n the transmembrane domain comprises a transmembrane domain of a CD8 or of a CD28.

16. The nucleic acid of any one of claims 1-15, wherein the transmembrane domain comprises a transmembrane domain of a CD28 comprising the amino acid sequence encoded by SEQ ID NO:5, or the amino acid sequence 15 MFWVLVVVGGVLACYSLLVTVAFIIFWV.

17. The nucleic acid of any one of claims 1-16, wherein the mulatory domain comprises: the signaling domain of a 4-1BB or a modified version thereof; or the ing domain of a CD28 or a modified version thereof. 20

18. The nucleic acid of claim 17, wherein the 4-1BB signaling domain comprises amino acids 214-255 of SEQ ID NO:15 or ses the amino acid sequence encoded by SEQ ID NO:6.

19. The nucleic acid of claim 17, wherein the CD28 signaling domain comprises amino acids 180-220 of SEQ ID NO:14 or comprises a modified version thereof comprising an LL → GG substitution d at positions 186-187 of SEQ ID NO:14. 5

20. The nucleic acid of any one of claims 1-19, wherein the CD3ζ signaling domain comprises the amino acid ce encoded by SEQ ID NO:7.

21. The nucleic acid of any one of claims 1, 3-18 and 20, wherein: the VL domain comprises the VL domain present in the amino acid sequence encoded by SEQ ID NO:3; and the VH domain comprises the VH domain present in 10 the amino acid sequence d by SEQ ID NO:3; the ptide spacer comprises the amino acid sequence set forth in SEQ ID NO:51; the transmembrane domain comprises the amino acid sequence encoded by SEQ ID NO:5; and 15 the intracellular signaling domain comprises the amino acid sequence encoded by SEQ ID NO:6 and the amino acid sequence encoded by SEQ ID NO:7.

22. The nucleic acid of any one of claims 1, 3-18, 20 and 21, wherein: the VL domain comprises the VL domain present in the amino acid sequence encoded by SEQ ID NO:3; and the VH domain comprises the VH domain present in 20 the amino acid sequence encoded by SEQ ID NO:3; the polypeptide spacer ts of the amino acid sequence encoded by SEQ ID NO:4 or consists of the amino acid ce set forth inf SEQ ID NO:21; the transmembrane domain comprises the amino acid sequence encoded by SEQ ID NO:5; and 25 the intracellular signaling domain comprises the amino acid sequence encoded by SEQ ID NO:6 and the amino acid sequence encoded by SEQ ID NO:7.

23. The nucleic acid of any one of claims 1, 3, 7, 8, 10, 18 and 20, wherein: the scFv has a ker-VH orientation; the polypeptide spacer comprises a modified IgG4 hinge region; 5 the transmembrane domain comprises the amino acid sequence encoded by SEQ ID NO:5; and the intracellular signaling domain comprises the amino acid sequence encoded by SEQ ID NO:6 and the amino acid sequence encoded by SEQ ID NO:7.

24. The nucleic acid of any one of claims 1 and 3-23, wherein the scFv is 10 encoded by the sequence set forth in SEQ ID NO:3.

25. The nucleic acid of any one of claims 1-18 and 20-24, wherein the nucleic acid ses: (a) the sequence set forth in SEQ ID NO:3 encoding the scFv; (b) the sequence set forth in SEQ ID NO:4 encoding the polypeptide 15 spacer; (c) the sequence set forth in SEQ ID NO:5 encoding the embrane domain; (d) the sequence set forth in SEQ ID NO:6 encoding the costimulatory domain; and 20 (e) the sequence set forth in SEQ ID NO:7 encoding the CD3ζ signaling domain.

26. The nucleic acid of any one of claims 1-18 and 20-25, wherein the nucleic acid encodes a chimeric receptor encoded by a nucleic acid sing the ce set forth in SEQ ID NO:10. 25

27. An expression vector, comprising the nucleic acid of any one of claims 1-26.

28. The sion vector of claim 27, further comprising a polynucleotide encoding a marker sequence, wherein the polynucleotide encoding the marker sequence is ly linked in frame with the nucleic acid encoding the chimeric receptor. 5

29. The expression vector of claim 28, wherein the nucleic acid encoding the ic receptor and the polynucleotide encoding the marker sequence are separated by a polynucleotide encoding a ble linker.

30. The sion vector of claim 28 or 29, wherein the marker sequence is a truncated EGFR sequence. 10

31. The expression vector of claim 30, wherein the truncated EGFR sequence is encoded by the polynucleotide of SEQ ID NO:9.

32. The expression vector of any one of claims 29-31, wherein the cleavable linker comprises a T2A peptide.

33. The expression vector of claim 32, wherein the T2A peptide is 15 encoded by the polynucleotide of SEQ ID NO:8.

34. An expression vector encoding the amino acid sequence MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKY LNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIAT YFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLV 20 APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ 25 KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR LEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGI GIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDIL TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKA 5 TGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVEN SECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGEN NTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGA LLLLLVVALGIGLFM.

35. An in vitro method for preparing an isolated host cell, comprising: 10 introducing the nucleic acid of any one of claims 1-26 or the expression vector of any one of claims 27-34 into cells of a lymphocyte population and ing the cells in the presence of D3 and/or anti-CD28, and at least one homeostatic cytokine.

36. The method of claim 35, wherein the lymphocyte population 15 comprises a lymphocyte that is CD45RA-, CD45RO+, and CD62L+.

37. The method of claim 35 or 36, wherein the lymphocyte population comprises a T cell.

38. The method of claim 37, wherein the T cell is a CD8+ T cell or a CD4+ T cell. 20

39. The method of claim 38, wherein the CD8+ T cell is selected from the group consisting of a naïve CD8+ T cell, a central memory CD8+ T cell, an effector memory CD8+ T cell, and a bulk CD8+ T cell.

40. The method of claim 39, wherein the CD8+ T cell is a central memory CD8+ T cell, wherein the central memory CD8+ T cell is + and 25 CD62L+.

41. The method of claim 38, wherein the CD4+ T cell is selected from the group consisting of a naïve CD4+ T cell, a central memory CD4+ T cell, an effector memory CD4+ T cell, and a bulk CD4+ T cell.

42. The method of claim 41, wherein the CD4+ T cell is a naïve CD4+ T 5 cell, wherein the naïve CD4+ T cell is CD45RA+, CD62L+, and CD45RO-.

43. A nucleic acid encoding a chimeric receptor as claimed in any one of claims 1-26 substantially as herein bed and with reference to any e thereof.

44. An sion vector as claimed in any one of claims 27-34 10 substantially as herein described and with reference to any example thereof.

45. An in vitro method as claimed in any one of claims 35-42 substantially as herein described and with reference to any example thereof. PCT/U