USOO8802374B2

(12) United States Patent (10) Patent No.: US 8,802.374 B2 Jensen (45) Date of Patent: Aug. 12, 2014

(54) TRUNCATED EPIDERIMAL GROWTH ferred HSV-TK Modified Donor T Cells. After Allogeneic FACTOR RECEPTOR (EGFRT) FOR Hematoppoietic Cell Transplantation.” Blood 107(6):2294-2302 (2006). TRANSDUCEDT CELL SELECTION Dawson, J. P. et al., “Epidermal Receptor Dimeriza tion and Activation Require Ligand-Induced Conformational (75) Inventor: Michael C. Jensen, Bainbridge Island, Changes in the Dimer Interface.” Mol. Cell. Biol. 25(17)7734-7742 WA (US) (2005). Fehse, B., et al., “Selective Immunoaffinity-Based Enrichment of (73) Assignee: City of Hope, Duarte, CA (US) CD34+ Cells Transduced with Retroviral Vectors Containing an Intracytoplasmatically Truncated Version of the Human Low-Affin (*) Notice: Subject to any disclaimer, the term of this ity Nerve Growth Factor Receptor (deltaLNGFR) Gene.” Human Gene Ther. 8:1815-1824 (1997). patent is extended or adjusted under 35 Fehse, B., et al., “CD34 Splice Variant: An Attractive Marker for U.S.C. 154(b) by 0 days. Selection of Gene-Modified Cells.” Mol. Ther. 1(5):448-456 (2000). Gaines, P. et al., “pIRES-CD4t, A Dicistronic Expression Vector for (21) Appl. No.: 13/463,247 MACS- or FACS-Based Selection of Transfected Cells. BioTechniques 26:683-688 (1999). (22) Filed: May 3, 2012 Kowolik, C. M., et al., “CD28 Costimulation Provided Through a CD19-Specific Chimeric Antigen Receptor Enhances InVivo Persis (65) Prior Publication Data tence and Antitumor Efficacy of Adoptively Transferred T Cells.” Cancer Res.66:10995-11004 (2006). US 2012/O3O1447 A1 Nov. 29, 2012 Lange, C., et al., “CD34 Modulates the Trafficking Behavior of Hematopoietic Cells InVivo..” StemCells and Development 16:297 304 (2007). Related U.S. Application Data Lemoine, F. M., et al., “Efficient Transduction and Selection of (63) Continuation of application No. Human T-Lymphocytes with Bicistronic Thy1/HSV1-TK Retroviral Vector Produced by a Human Packaging Cell Line” J. Gene Med. PCT/US2010/055329, filed on Nov. 3, 2010. 6:374-386 (2004). Li, S., et al., “Structural Basis for Inhibition of the Epidermal Growth (60) Provisional application No. 61/257.567, filed on Nov. Factor Receptor by .” Cancer Cell 7:301-311 (2005). 3, 2009. Pelloquin, F., et al., “Human Blymphocytes Immortalization by Epstein-Barr Virus in the Presence of Cyclosporin A.” In Vitro Cell (51) Int. Cl. Devel. Biol. 22(12):689-694 (1986). CI2O I/68 (2006.01) Szymczak, A. L., et al., “Correction of Multi-Gene Deficiency In C07K I4/00 (2006.01) Vivo Using a Single 'Self-Cleaving 2A Peptide-Based Retroviral Vector.” Nat. Biotech. 22(5):589-594 (2004). (52) U.S. Cl. Tey, S.K., et al., “Inducible Caspase 9 Suicide Gene to Improve the USPC ...... 435/6.17:530/350; 530/395 Safety of Allodepleted T Cells. After Haploidentical StemCell Trans (58) Field of Classification Search plantation.” Biol. Blood Marrow Transplant. 13(8):913-924 (2007). None Yam, P. et al., “Ex Vivo Selection and Expansion of Cells Based on See application file for complete search history. Expression of a Mutated Inosine Monophosphate Dehydrogenase 2 After HIV Vector Transduction: Effects on Lymphocytes, (56) References Cited Monocytes, and CD34+ Stem Cells.” Mol. Ther. 14(2):236-244 (2006). U.S. PATENT DOCUMENTS (Continued) 5,753.462 A * 5/1998 Lok ...... 435/6.12 Primary Examiner — Joseph Woitach 6,790,614 B1 9/2004 Pippig et al. Assistant Examiner — Kimberly A Aron 2004/O126363 A1 7/2004 Jensen et al. 2005.0003484 A1 1/2005 Hirano et al...... 435/69.1 (74) Attorney, Agent, or Firm — Perkins Coie LLP; Lauren 2005.0053608 A1 3/2005 Weber et al. Sliger (57) ABSTRACT OTHER PUBLICATIONS A non-immunogenic selection epitope may be generated by Singh et al. Redirecting Specificity of T-cell Populations for CD19 removing certain amino acid sequences of the protein. For Using The Sleeping Beauty System. Cancer Research, 2008. example, a gene encoding a truncated human epidermal 68:2961-2971. growth factor receptor polypeptide (EGFRt) that lacks the Khoda et al. A 40-kDa Epidermal Growth Factor/Transforming membrane distal EGF-binding domain and the cytoplasmic Growth Factor alpha-Binding Domain Produced by Limited signaling tail, but retains an extracellular epitope recognized Proteolysis of the Extracellular Domain of The Epidermal Growth by an anti-EGFR antibody is provided. Cells may be geneti Factor Receptor. The Journal of Biological Chemistry, 1993. cally modified to express EGFRt and then purified without the 268(3): 1976-1981.* immunoactivity that would accompany the use of full-length Chakraverty, R., et al., “An Inflammatory Checkpoint Regulates EGFR immunoactivity. Through flow cytometric analysis, Recruitment of Graft-Versus-Host Reactive T Cells to Peripheral EGFRt was successfully utilized as an in vivo tracking marker Tissues.” J. Exp. Med. 203(8);2021-2031 (2006). for genetically modified human T cell engraftment in mice. Powell, D.J., et al., "Large-Scale Depletion of CD25+ RegulatoryT Furthermore, EGFRt was demonstrated to have cellular Cells from Patient Leukapheresis Samples,” J. Immunother. depletion potential through cetuximab mediated antibody 28(4):403-411 (2005). dependent cellular cytotoxicity (ADCC) pathways. Thus, United States Patent and Trademark Office, International Search Report and Written Opinion dated Apr. 7, 2011 for PCT/US 10/ EGFRt may be used as a non-immunogenic selection tool, 5.5329. tracking marker, a depletion tool or a suicide gene for geneti Berger, C., et al., “Analysis of Transgene-Specific Immune cally modified cells having therapeutic potential. Responses that Limit the In Vivo Persistence of Adoptively Trans 20 Claims, 19 Drawing Sheets US 8,802.374 B2 Page 2

(56) References Cited Wang. X., et al., “A Transgene-Encoded Cell Surface Polypeptide for Selection. In Vivo Tracking, and Ablation of Engineered Cells.” OTHER PUBLICATIONS Blood 118: 1255-1263 (2011). European Patent Office, Extended European Search Report and Opinion dated May 31, 2013 for Application No. 10829041.2. * cited by examiner U.S. Patent Aug. 12, 2014 Sheet 1 of 19 US 8,802.374 B2

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Baseline Evaluations Derivation and Cryopreservation of Autologous Tcm- ams Leukapheresis Derived CD9R Isles CARfCD8"fEGFR, Cc Salvage/Priming Product & Rituxan' w Mobilization with G-CSF W Apheresis and Salvage Therapy, HPC Cryopreservation of Mobilization and Autologous HPC Product Collection, Myeloablative Preparative Regimen, HPC W Rescue, and Supportive Care per COH Herne/BMT Myeloablative Program Standard Practice Conditioning Regimen w Infusion of CE)34-- Selected HSCs (Day ())

------b T Ceil infusion (Day +2 or +3) < 1.5 x 10" cells/m US 8,802,374 B2 1. 2 TRUNCATED EPIDERMAL GROWTH endogenous cell-Surface molecule inert, which is a desired FACTOR RECEPTOR (EGFRT) FOR property for the molecule. The non-immunogenic selection TRANSDUCEDT CELL SELECTION epitope may also be used for as a selection tool or tracking marker. PRIORITY CLAIM The modified endogenous cell-surface molecule may be, but is not limited to, any cell-surface related receptor, ligand, This application is a continuation of International Appli glycoprotein, cell adhesion molecule, antigen, integrin or cation No. PCT/US2010/055329, filed Nov. 3, 2010, which cluster of differentiation (CD) that is modified as described claims the benefit of U.S. Provisional Application No. herein. In some embodiments, the modified endogenous cell 61/257.567, filed Nov. 3, 2009, the subject matter of which is 10 Surface molecule is a truncated tyrosine kinase receptor. In incorporated by reference as if fully set forth herein. one aspect, the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor family (e.g., ErbB1, TECHNICAL FIELD ErbB2, ErbB3, ErbB4). Epidermal growth factor receptor, also known as EGFR, The present products and methods relate to the fields of 15 ErbB1 and HER1, is a cell-surface receptor for members of immunology and purification of genetically modified cells, the epidermal growth factor family of extracellular ligands. specifically to a truncated or otherwise modified receptor Alterations in EGFR activity have been implicated in certain paired with a corresponding antibody, Such as a polypeptide cancers. In a first aspect, a gene encoding an EGFR polypep derived from human epidermal growth factor receptor tide comprising human epidermal growth factor receptor (EGFR) paired with cetuximab, for use in cancer immuno (EGFR) that is constructed by removal of nucleic acid therapy. sequences that encode polypeptides including the membrane distal EGF-binding domain and the cytoplasmic signalingtail BACKGROUND (a “truncated EGFR or “EGFRt”), but retains the extracel lular membrane proximal epitope recognized by an anti Immune cell products with homogenous expression of 25 EGFR antibody. Preferably, the antibody is a known, com tumor targeting chimericantigen receptors (CARs) are desir mercially available anti-EGFR , such as able for clinical evaluation of adoptive therapy strategies to cetuximab, , or . eliminate the product-to-product variability of transgene Application of biotinylated-cetuximab to immunomag expression otherwise intrinsic to transduction and other netic selection in combination with anti-biotin microbeads genetic modification procedures without Subsequent selec 30 successfully enriches T cells that have been lentivirally trans tion. using genetically redirected immune duced with EGFRt-containing constructs from as low as 2% cells is an attractive approach for treating minimal residual of the population to greater than 90% purity without observ disease in a variety of cancer patients. However, immuno able toxicity to the cell preparation. Constitutive expression logic rejection of cell products expressing antibiotic selection of this inert EGFRt molecule does not affect T cell phenotype proteins as part of the transduction strategy has impeded this 35 or effector function as directed by the coordinately expressed strategy. A novel selection marker that is not expressed on chimeric antigen receptor (CAR), CD19R. Through flow human lymphocytes, does not contain endogenous signaling cytometric analysis, EGFRt was successfully utilized as an in or trafficking function, and is recognized by a known, pref vivo tracking marker for T cell engraftment in mice. Further erably commercially available, pharmaceutical grade anti more, EGFRt was demonstrated to have suicide gene poten body reagent that can be utilized for selection, in vivo track 40 tial through Erbitux(R) mediated antibody dependent cellular ing, and depletion of transduced cells would be a significant cytotoxicity (ADCC) pathways. Thus, EGFRt may be used as improvement in the art. a non-immunogenic selection tool, tracking marker, and Sui cide gene for transduced T cells that have immunotherapeutic SUMMARY potential. The EGFRt nucleic acid may also be detected by 45 means well known in the art. Products and methods for purification, both in vivo and ex In another embodiment, methods of discovering and vivo, of genetically modified cells are provided herein. The designing modified, truncated or altered endogenous cell genetically modified cells may be modified by transduction, surface molecules which bind to antibodies, preferably com or any other process that adds, deletes, alters, or disrupts an mercially available antibodies, as described herein are pro endogenous nucleotide sequence. The genetically modified 50 vided. The methods include modeling the protein of interest cells may be transduced T cells with altered activity, includ and truncating functional portions, while leaving the anti ing altered immunoactivity. body-binding portions intact. The resulting modified receptor According to the embodiments described herein, a non or ligand can be sorted using a labeled antibody and then immunogenic selection epitope compatible with immuno enriched such that the concentration of the modified receptor magnetic selection facilitates immunotherapy in cancer 55 or ligand is increased. patients without undesirable immunologic rejection of cell Yet another embodiment provides a method of selecting products (i.e. as seen when expressing antibiotic selection transduced T cells comprising transducing T cells with a proteins) may be generated by removing certain amino acid modified, truncated or altered endogenous cell-surface mol sequences of the protein. In some embodiments, the non ecule gene sequence (e.g., truncated EGFR) and then apply immunogenic selection epitope is a gene encoding an endog 60 ing an antibody that binds the modified ligand or receptor enous cell-surface molecule that is modified or truncated to sequence to the transduced T cells. If the modified receptor retain an extracellular epitope recognized by a known anti sequence is EGFRt, the antibody is preferably a biotinylated body or functional fragment thereof, and to remove any sig anti-EGFR monoclonal antibody. The T cells are then sorted naling or trafficking domains and/or any extracellular by adding anti-biotin microbeads and selecting the T cells domains unrecognized by the known antibody. The removal 65 using immunomagnetic separation, adding fluorochrome of the signaling or trafficking domains and/or any extracellu conjugated anti-biotin and selecting the T cells using Fluo lar domains unrecognized by the known antibody renders the rescence Activated Cell Sorting, or any other reliable method US 8,802,374 B2 3 4 of sorting the cells. The modified ligand or receptor and then lysed in the presence of phosphatase inhibitor. sequences, such as the EGFRt sequence, may be contained in Lysates run on Western blots were then probed using antibod a suitable transfer vehicle such as a lentiviral vector. ies specific for either B-actin, the cytoplasmic domain of These and other embodiments are further explained in the EGFR, or the phosphorylated tyrosine at position 1068 of drawing and detailed description herein. EGFR. FIG. 3b shows that EGF does not bind to the Surface of EGFRt expressing T cells. A431, Line A, and negative BRIEF DESCRIPTION OF THE DRAWINGS control T cells were stained with PE-conjugated anti-EGFR, or either biotinylated cetuximab or biotinylated EGF fol FIG. 1 is a molecular model of EGFR vs. EGFRt proteins lowed by PE-conjugated streptavidin (black histogram) ver based on the crystal structure files. The EGFR structure on the 10 SuS PE-conjugated isotype control Ab or Streptavidin alone left shows a full-length EGFR with the structure of the four (open histogram) by flow cytometry. Percent positive staining extracellular domains (Domains I-IV). The middle structure is indicated in each histogram. shows the truncated EGFR (EGFRt), which is missing FIGS. 4a-d illustrate that selected EGFRt" CD19RT cells Domain I, Domain II, the Juxatmembrane Domain, and the can be expanded with maintenance of effector phenotype. Tyrosine Kinase Domain as compared to an unmodified 15 FIG. 4a is a line graph showing expansion of EGFRt-selected EGFR. The EGFRt on the right shows truncated structure T cells, Lines A-E, over 12 or more days after rapid expansion bound to EribituX(R) Fab, comprised of V-C and V-C. medium (REM) stimulation was initiated on the day of The domains are separated with dotted lines. AutoMACSTM selection (day 0). (MACS is magnetic acti FIGS. 2a-d illustrate the selection of EGFRt T cells using vated cell sorting.) Expansion of T cells in rapid expansion biotinylated cetuximab (referred to in the figure as ErbituXR). medium (REM) involved the incubation of 10° T cells with 30 FIG. 2a is a schematic of the cetuximab biotinylation and ng/mL anti-CD3e (OKT3: Ortho Biotech, Raritan, N.J.), reformulation process. FIG.2b is a graph showing titration of 5x107 y-irradiated PBMCs (3500 cGy), and 107 y-irradiated biotinylated cetuximab. 10° EGFR" cells were stained with LCLs (8000 cGy) in 50 mL CM; with addition of 50 U/mL either Oug (black), 1.45 Lug (red), 0.145 lug (orange), 14.5 ng rhIL-2 and 10 ng/ml rhIL-15 (CellCienix) every 48 hours, (yellow), 1.45 ng (green), 0.145 ng (blue) or 14.5ug (purple) 25 beginning on day 1. T cells were re-stimulated in this manner of biotinylated cetuximab followed by 0.5ug PE-conjugated every 14 days. FIG. 4b shows histograms representing streptavidin and analyzed by flow cytometry. 14.5 ng or more EGFRt-selected T cells (11 to 13 days after stimulation) that of biotinylated cetuximab was deemed sufficient for future were phenotyped for surface EGFR (i.e., EGFRt, with bioti staining. FIG.2c depicts schematics of both the immunomag nylated cetuximab), Fc (i.e., CAR), and T cell markers CD4 netic (top) and the fluorescence activated cell sorting (bot 30 or CD8, (black histogram) vs. isotype control Ab (open his tom) EGFRt selection procedures. togram) by flow cytometry. Percent positive staining is indi FIG. 2d shows immunomagnetic selection of various T cell cated in each histogram. "N.D.” indicates no data. FIG. 4Care lines lentivirally transduced with CAR and EGFRt containing five lines graphs, one for each of Lines A-E, of EGFRt constructs. Schematics of the CD19CAR-T2A-EGFRt (left) selected T cells (within 11 to 15 days after REM stimulation) and CD19CAR-T2A-EGFRt-IMPDH2dm (right) constructs 35 incubated for 4 hours with 'Cr-labeled NS0, U251T, CD19t contained in lentiviral vectors are shown above the corre expressing NSO, CMV pp65-expressing U251T, CD19-ex sponding pre- and post-selection flow cytometric analyses for pressing Daudi or SupB15, or OKT3-expressing LCL cells as Surface EGFRt expression. Codon optimized sequence por targets at the indicated E:T ratios. Chromium release was tions of the CD19-specific. CD28 co-stimulatory CAR, fol measured to determine cytotoxic activity. FIG. 4d is a graph lowed by the self-cleavable T2A, EGFRt and IMPDH2dm 40 showing MPA resistance of the CD19CAREGFRt" selection markers are indicated, along with the Elongation IMPDH2dm" Line E. Control T cells that do not express Factor 1 promoter sequences (EF-1p), and the GCSFR alpha IMPDH2dm and EGFRt-selected IMPDH2dm-expressing chain signal sequences (GCSFRSS, which directs Surface Line E cells were cultured either with or without 1 uMMPA expression). Flow cytometric analysis of lentivirally trans and total cell numbers were monitored. duced T cell lines that had been stained with a biotinylated 45 FIG. 5 shows EGFRt expression can be used as a tracking cetuximab antibody and PE-conjugated anti-biotin antibody marker for in vivo T cell engraftment. Day 36 marrow (black histograms) was performed on both the input T cells harvested from a control mouse or from a mouse that had (PRE SLXN) and the positive fraction obtained from received 107 CD19CAR"EGFRt" Line Cat day 0 was stained AutoMACSTM (POS FRXN). Open histograms represent using PerCP-conjugated anti-human CD45 and biotinylated staining with PE-conjugated anti-biotin antibody alone, and 50 cetuximab ("Bio-Erb”) followed by PE-conjugated streptavi the percent positive cells are indicated in each histogram. din. Quadrants were created based on isotype control stain Selection of CD19CAREGFRt" Line A occurred 3 days ing, and percent positive staining in each quadrant is indicated after transduction of T cell blasts. Selection of CD19CAR" in each histogram. EGFRt Line B occurred after 3 REM stimulations of trans FIG. 6 is a graph showing EGFRt expression targets T cells duced CMVpp65-specific T-derived cells. Selection of 55 for cetuximab (referred to in the figure as Erbitux(R) mediated CD19CAREGFRt Line C occurred after 2 REM stimula ADCC. Cr-labeled Line A cells were pre-incubated either tions of transduced CD8 T-derived cells. Selection of with or without up to 20 g/mL of cetuximab or the CD20 CD19CAREGFRt" Line D occurred after 1 REM stimula specific mAb Rituxanas a negative control prior to addition of tion of transduced T-derived cells. Selection of human PBMC as effectors. CD19CAREGFRt IMPDH2dm Line E occurred after 1 60 FIG. 7 is the nucleotide (sense strand is SEQ ID NO: 1, REM stimulation of transduced T-derived cells. antisense strand is SEQID NO: 2) and amino acid (SEQ ID FIGS. 3a-b show that the EGFRt expressed on selected T NO: 3) sequences of GMCSFR alpha chain signal sequence cells is inert. In FIG. 3a, EGFRt expressed on T cells is not linked to EGFRt. The GMCSFRalpha chain signal sequence, phosphorylated upon co-incubation with EGF. Negative con which directs surface expression, is encoded by nucleotides trol T cells, CD19CAREGFRt" Line A cells, or A431 cells 65 1-66. EGFRt is encoded by nucleotides 67-1071. were incubated for 5 minutes with or without either 100 FIG. 8 is the nucleotide (sense strand is SEQ ID NO: 4, ng/mL EGF or cetuximab (referred to in the figure as Erbtx) antisense strand is SEQID NO: 5) and amino acid (SEQ ID US 8,802,374 B2 5 6 NO: 6) sequences of CD19R-CD28gg-Zeta(CO)-T2A-EG killer cells, neutrophils, and macrophages, recognize bound FRt. CD19R-CD28gg-Zeta(CO) is encoded by nucleotides antibody on a target cell and cause lysis of the target cell. 1-2040: T2A is encoded by nucleotides 2041-2112: GMC ADCC activity may be assessed using methods. Such as those SFR is encoded by nucleotides 2113-2178; EGFRt is encoded described in U.S. Pat. No. 5,821,337. by nucleotides 2179-3186. 5 “Effector cells are leukocytes which express one or more FIG. 9 is a graph showing CD19R-CD28gg-Zeta(CO)- constant region receptors and perform effector functions. T2A-EGFRt expression. Transduction of anti-CD3/anti To “treat a disease or a disorder, Such as cancer, means to CD28 bead stimulated primary T cell blasts with the CD19R take either therapeutic measures or preventative measures to CD28gg-Zeta(CO)-T2A-EGFRt epHIV7 lentiviral vector lessen or abate the disease or disorder. Such treatment (MOI-3) results in surface detection of both the CAR (using 10 includes prevention, alleviation of symptoms, diminishment a biotinylated anti-Fc Ab and streptavidin-PE) and the trun or stabilization of Scope, and/or remission. cated EGFR molecule (using a biotinylated cetuximab Ab The term “therapeutically effective amount” refers to an and streptavidin-PE) by flow cytometry on day 4. The white amount of a compound or molecule effective to treat a disease peak in each panel is non-transduced control T cell blasts. or disorder. FIG. 10 is a schema showing a possible process flow for 15 clinical trials for testing products of the present disclosure. “Cancer refers to cells undergoing uncontrolled cellular growth. Examples of cancer include colorectal cancer and DETAILED DESCRIPTION head and neck cancer. A “chemotherapeutic agent' is a chemical compound useful in the treatment of cancer. Certain embodiments of the invention are described in A “cytokine' is a protein released by one cell to act on detail, using specific examples, sequences, and drawings. The another cell as an intercellular mediator. enumerated embodiments are not intended to limit the inven “Non-immunogenic’ refers to a material that does not ini tion to those embodiments, as the invention is intended to tiate, provoke or enhance an immune response where the cover all alternatives, modifications, and equivalents, which immune response includes the adaptive and/or innate may be included within the scope of the present invention as 25 immune responses. defined by the claims. One skilled in the art will recognize The term “gene' means the segment of DNA involved in many methods and materials similar or equivalent to those producing a polypeptide chain; it includes regions preceding described herein, which could be used in the practice of the and following the coding region “leader and trailer as well as present invention. intervening sequences (introns) between individual coding Erbitux(R) is a registered trademark for the anti-EGFR 30 segments (exons). Some genes may be developed which lack, monoclonal antibody cetuximab and is intended to indepen in whole or in part, introns. Some leader sequences may dently include the trade name product formulation, the enhance translation of the nucleic acid into polypeptides. generic drug, and the active pharmaceutical ingredient(s) of The term "isolated” means that the material is removed the trade name product. from its original environment (e.g., the natural environmentif The term 'genetic modification” means any process that 35 it is naturally occurring). For example, a naturally-occurring adds, deletes, alters, or disrupts an endogenous nucleotide polynucleotide or polypeptidepresent in a living animal is not sequence and includes, but is not limited to viral mediated isolated, but the same polynucleotide or polypeptide, sepa gene transfer, liposome mediated transfer, transformation, rated from Some or all of the coexisting materials in the transfection and transduction, e.g., viral mediated gene trans natural system, is isolated. Such polynucleotides could be fer such as the use of vectors based on DNA viruses such as 40 part of a vector and/or Such polynucleotides or polypeptides lentivirus, adenovirus, retroviruses, adeno-associated virus could be part of a composition, and still be isolated in that and herpes virus. Such vector or composition is not part of its natural environ The term “antibody' includes monoclonal antibodies, ment. polyclonal antibodies, dimers, multimers, multispecific anti As used herein, a “vector” may be any agent capable of bodies and antibody fragments that may be human, mouse, 45 delivering or maintaining nucleic acid in a host cell, and humanized, chimeric, or derived from another species. A includes viral vectors (e.g. retroviral vectors, lentiviral vec “monoclonal antibody' is an antibody obtained from a popu tors, adenoviral vectors, or adeno-associated viral vectors), lation of Substantially homogeneous antibodies that is being plasmids, naked nucleic acids, nucleic acids complexed with directed against a specific antigenic site. polypeptide or other molecules and nucleic acids immobi “Variant” refers to polypeptides having amino acid 50 lized onto solid phase particles. The appropriate DNA sequences that differ to Some extent from a native sequence sequence may be inserted into the vector by a variety of polypeptide. Ordinarily, amino acid sequence variants will procedures. In general, the DNA sequence is inserted into an possess at least about 80% sequence identity, more prefer appropriate restriction endonuclease site(s) by procedures ably, at least about 90% homologous by sequence. The amino known in the art. Such procedures and others are deemed to be acid sequence variants may possess Substitutions, deletions, 55 within the scope of those skilled in the art. Transcription of and/or insertions at certain positions within the reference the DNA encoding the polypeptides of the present invention amino acid sequence. by higher eukaryotes is increased by inserting an enhancer “Percentage identity” or “percent identity” is defined as the sequence into the vector. Enhancers are cis-acting elements of percentage of residues in the amino acid sequence variant that DNA, usually about from 10 to 300 by that act on a promoter are identical after best aligning the sequences and introducing 60 to increase its transcription. Examples including the SV40 gaps, if necessary, to achieve the maximum percent sequence enhancer on the late side of the replication origin by 100 to identity. Methods and computer programs for the alignment 270, a cytomegalovirus early promoter enhancer, the are well known in the art. Such programs include GAP, BEST polyoma enhancer on the late side of the replication origin, FIT. FASTA, BLAST or Align 2. and adenovirus enhancers. Antibody-dependent cell-mediated cytotoxicity' and 65 “Receptor” means a polypeptide that is capable of specific ADCC refer to a cell-mediated reaction in which nonspe binding to a molecule. Whereas many receptors may typically cific cytotoxic cells that express Fc receptors. Such as natural operate on the Surface of a cell. Some receptors may bind US 8,802,374 B2 7 8 ligands when located inside the cell (and prior to transport to , , brentuximab the Surface) or may reside predominantly intra-cellularly and Vedotin, , capromab pendetide, catu bind ligand therein. maXomab, CC49, cedelizumab, celmoleukin, citatuZumab Antibody or functional fragment thereof means an bogatox, clenoliximab, clivatuzumab tetraxetan, CNTO-95, immunoglobulin molecule that specifically binds to, or is , , daclizumab, , immunologically reactive with a particular antigen or , , , efalizumab, elotu epitope, and includes both polyclonal and monoclonal anti Zumab, enlimomab pegol, epitumomab cituxetan, epratu bodies. The term antibody includes genetically engineered or Zumab, erlizumab, etaracizumab, fanolesomab, faralimo otherwise modified forms of immunoglobulins, such as intra mab, , galiximab, gavilimomab, gemtuzumab bodies, peptibodies, chimeric antibodies, fully human anti 10 oZogamicin, , gomiliximab, ibali bodies, humanized antibodies, and heteroconjugate antibod Zumab, , igovomab, , ira ies (e.g., bispecific antibodies, diabodies, triabodies, and tumumab, inolimomab, , ipili tetrabodies). The term functional antibody fragment includes mumab, keliximab, , , , antigen binding fragments of antibodies, including e.g., Fab'. , lumiliximab, , maslimomab, F(ab'). Sub.2, Fab, Fv, rigG, and scFv fragments. The term 15 , , , muromonab schv refers to a single chain Fv antibody in which the variable CD3, , natalizumab, ocrelizumab, domains of the heavy chain and of the light chain of a tradi odulimomab. , , oportuZumab tional two chain antibody have been joined to form one chain. monatox, , otelixizumab, , prilix In one embodiment, a gene encoding a modified endog imab, PRO140, , rovelizumab, ruplizumab, satu enous cell-Surface molecule that may be used as a non-im momab pendetide, Siplizumab, Sontuzumab, , munogenic selection epitope compatible with immunomag , teneliximab, teplizumab, TGN1412, netic selection is provided. Such a non-immunogenic ticilimumab (tremelimumab), , tocilizumab selection epitope may facilitate immunotherapy in cancer (Fatlizumab), toralizumab, to situmomab, tremelimumab, patients without undesirable immunologic rejection of cell tucotuZumab, vedolizumab, , visilizumab, products. The endogenous cell Surface molecule may be 25 , , Votumumab, Zanolimumab, Zirali modified or truncated to retain an extracellular epitope rec mumab, Zolimomab aritoX. ognized by a known antibody or functional fragment thereof, In some embodiments, the modified endogenous cell-sur and to remove any signaling or traffickingdomains and/or any face molecule is encoded by a modified or truncated tyrosine extracellular domains unrecognized by said known antibody. kinase receptor gene. Examples of tyrosine kinase receptors A modified endogenous cell Surface molecule which lacks a 30 that may be modified or truncated according to the embodi signaling or trafficking domain and/or any extracellular ments described herein include, but are not limited to, mem domains unrecognized by said known antibody is rendered bers of the endothelial growth factor receptor family (EGRF/ inert. ErbB1/HER1; ErbB2/HER2/neu ErbB3/HER3; ErbB4/ The modified endogenous cell-surface molecule may be, HER4), hepatocyte growth factor receptor (HGFR/c-MET) but is not limited to, any non-immunogenic cell-surface 35 and insulin-like growth factor receptor-1 (IGF-1R). Accord related receptor, glycoprotein, cell adhesion molecule, anti ing to Some embodiments, modified tyrosine kinase receptors gen, integrin or cluster of differentiation (CD) that is modified retain an extracellular epitope recognized by a known anti as described herein. Modification of such cell-surface mol body or functional fragment thereof, and lack at least a ecules is accomplished by keeping an epitope that is recog tyrosine kinase domain. A modified tyrosine kinase receptor nized by a known antibody or functional fragment thereof; 40 which lacks at least a tyrosine kinase domain renders the and removing any signaling or traffickingdomains and/or any receptor inert. extracellular domains unrecognized by a known antibody. Commercial antibodies that may be used to recognize a Removal of the signaling or trafficking domains and/or any modified tyrosine kinase receptor include, but are not limited extracellular domains unrecognized by a known antibody to AMG-102, AMG-479, BIIB022OA-5D5, CP-751,871, renders the endogenous cell-surface molecule non-immuno 45 IMC-A12, R1507, cetuximab, , , genic and/or inert. , matuZumab, necitumumab, panitumumab, per Examples of endogenous cell-Surface molecules that may tuZumab, , , , Zalutu be modified or truncated according to the embodiments mumab. described herein include, but are not limited to EpCAM, In one embodiment, the modified endogenous cell Surface VEGFR, integrins (e.g., integrins Ov?53, C4, C.IIb?33, C.437, 50 molecule is a truncated EGFR (tGFR) that lacks the mem C.5B1, Ov3, Civ), TNF receptor superfamily (e.g., TRAIL brane distal EGF-binding domain and the cytoplasmic sig R1, TRAIL-R2), PDGF Receptor, interferon receptor, folate naling tail, but retains the extracellular membrane proximal receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, epitope recognized by a known antibody or functional frag MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, or clusters ment thereof (e.g., cetuximab, matuZumab, necitumumab or of differentiation (e.g., CD2, CD3, CD4, CD5, CD11, 55 panitumumab). In another embodiment, the tEGFR is miss CD11a/LFA-1, CD15, CD18/ITGB2, CD19, CD2O, CD22, ing Domain I, Domain II, the Juxtamembrane Domain and CD23/IgE Receptor, CD25, CD28, CD30, CD33, CD38, the Tyrosine Kinase Domain as compared to an unmodified CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, EGFR (FIG. 1). CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, A gene encoding a modified endogenous cell Surface mol CD195/CCR5, CD319/SLAMF7). 60 ecule may be used as a cell selection or enrichment marker for Corresponding commercial antibodies that may be used to a genetically modified population of immune cells (e.g., T recognize a modified or truncated endogenous cell-surface cells). The gene encoding a modified endogenous cell Surface molecule include, but are not limited to, , , molecule may be coupled to a gene encoding a tumor target , , afutuZumab, , altu ing chimeric antigen receptor (CAR). These genes may be momab pentetate, , apolizumab, arci 65 inserted into a vector to transduce the population of T cells to tumomab, aselizumab, atlizumab (tocilizumab), basilix be genetically modified. After transduction, the cells that are imab, , benralizumab, besilesomab, successfully transduced and express the CAR and modified US 8,802,374 B2 10 endogenous cell-surface molecule are enriched by any Suit immunomagnetic purification of CAR-expressing transduc able purification method. Such as immunomagnetic purifica tants using cetuximab-biotin/anti-biotin microbeads. See the tion with anti-biotin microbeads or fluorochrome-conjugated CD19R-CD28gg-Zeta(CO)-T2A-EGFRt sequence attached anti-biotin for fluorescence activated cell sorting, using a as FIG. 8, SEQID NOS: 4 (nucleotide sense strand), 5 (nucle commercial antibody that recognizes the modified endog otide anti-sense Strand), and 6 (protein). Lentivector trans enous cell-surface molecule expressed by the transduced cell. duction of primary human T cells with this codon-optimized In another embodiment, a gene encoding a truncated cDNA directs the coordinated expression of the CAR and human epidermal growth factor receptor (EGFRt) that lacks EGFRt (FIG.9). the membrane distal EGF-binding domain and the cytoplas To eliminate variability between transgene expression mic signaling tail, but retains the extracellular membrane 10 products otherwise intrinsic to transduction procedures with proximal epitope recognized by the FDA-approved anti out Subsequent selection, a non-immunogenic selection EGFR monoclonal antibody (mAb) cetuximab or another epitope, EGFRt, compatible with immunomagnetic selection anti-EGFR antibody, is constructed and described herein. The using the CliniMACS device (Miltenyi Biotec, Bergisch EGFRt may be coupled with chimeric antigen receptors spe Gladbach, Germany) was developed. For example, EGFRt is cific for a tumor associated antigen. The tumor associated 15 a truncated human epidermal growth factor receptor that antigen may be CD19, CD20, or CD22, or any other tumor lacks the membrane distal EGF-binding domain and the ecto associated antigen, but is preferably CD19 (CD19CAR). The plasmic signaling tail, but retains the extracellular membrane tumor associated antigen is followed by a C-terminal 2A proximal epitope recognized by the commercial anti-EGFR cleavable linker and the coding sequence for EGFRt. The mAb cetuximab. See FIG. 1. Biotinylated-cetuximab is biotinylated-cetuximab may be used in conjunction with applied to immunomagnetic selection in combination with commercially available anti-biotin microbeads for the pur anti-biotin microbeads (Miltenyi). Human OKT3 blasts that pose of immunomagnetic purification of the tumor associated had been lentivirally transduced with CD19R-CD28gg-Zeta antigen/CAR-expressing transductants. In the instance where (CO)-T2A-EGFRt were subjected to immunomagnetic selec the tumor associated antigen is CD19 the product is tion using the Miltenyi AutoMACS device, and the frequency CD19CAR-expressing transductants. Alternatively, the bioti 25 of EGFRt-CAR+ T cells was enriched from 22% (pre-selec nylated-cetuximab may be used in conjunction with Fluoro tion) to 99% (post-selection) without observable toxicity to chrome-conjugated anti-biotin for fluorescence activated cell the cell preparation. It is also possible that, instead of or in Sorting. addition to immunomagnetic Sorting, the EGFRt can be puri In another embodiment, a modified endogenous cell-sur fied using fluorescence-based cell sorting techniques. face molecule may be used as a marker for in vivo T cell 30 Due to the absence of the EGF-binding domains and intra engraftment. For example, when the modified endogenous cellular signaling domains, EGFRt is inactive when cell-surface molecule is EGFRt, the EGFRt may be used to expressed by T cells. Importantly, the EGFRt-selected T cells track the uptake of the T cells to which it is attached in vivo maintain their desired effector phenotype—including anti without affecting cellular function of the T cells or the cells to tumor cyotoxic activity mediated by the chimeric antigen which the T cells are targeted, such as bone marrow cells in a 35 receptor that is coordinately expressed with the EGFRt—and transplant situation. The use of cetuximab conjugated to remain amenable to established expansion protocols. probes or reporter genes such as Sr39TK may be used to Overall, this EGFRt has various advantages for immuno improve the tracking potential of EGFRt-expressing cells to therapeutic cell products compared to other selection markers patients via PET imaging techniques. that have been previously reported. Specifically, unlike trun In a separate embodiment, a modified endogenous cell 40 cated CD4 and CD19, it is not endogenously expressed by surface molecule may be used to induce cell suicide. For Subpopulations of lymphocytes. Furthermore, in contrast to example, EGFRt may be used as a Suicide gene Viacetuximab truncated CD34 and low affinity nerve growth factor receptor, mediated complement and/or antibody dependent cell medi it does not have any activity that might negatively affect the ated cytotoxicity (ADCC) pathways. The fact that cetuximab immune cell product (i.e., interms of signaling or trafficking). is a therapeutic FDA-approved antibody further facilitates the 45 Lastly, it alone can be bound/recognized by a known, prefer suicide gene potential of EGFRt in the clinical setting. ably commercially available, pharmaceutical grade antibody In other embodiments, the truncated epidermal growth fac reagent, i.e., cetuximab. Together, these attributes make tor receptor (EGFRt) selection epitope or other modified cell EGFRt a superior selection marker for any transfection/trans Surface molecule is attached to other sequences. One exem duction system that can be applied to the generation of cell plar sequence is the GMCSFR alpha chain signal sequence, 50 products for adoptive immunotherapy. Thus, EGFRt is well which directs surface expression, attached to EGFRt. GMC suited to be used as a selection marker for lentivirally trans SFR is encoded by nucleotides 1-66 and EGFRt is encoded by duced T cells of immunotherapeutic relevance. nucleotides 67-1071 of SEQ ID NO: 1. See FIG. 7. Also in Also provided are methods for identifying new therapeutic FIG. 7 is the antisense strand (SEQID NO: 2) and amino acid cell products having the following criteria: a modified endog (SEQ ID NO: 3) sequences of GMCSFR alpha chain signal 55 enous cell-surface molecule, ligand or receptor that is not, as sequence linked to EGFRt. Another such sequence is a codon modified, endogenously expressed in the Subject in which it is optimized cDNA sequence encoding an anti-CD19 costimu intended to be therapeutically utilized, does not have any latory chimeric antigen receptor (CD19R-CD28gg-Zeta immunoactivity or other functional activity that would hinder (CO)), and a cleavable T2A linker. Cytotoxic T lymphocytes the functioning of the product or the subject into which the (CTLs) modified to express a CD19-specific chimericantigen 60 product is administered, and that it can be recognized by a receptor (CAR) that signals via a cytoplasmic costimulatory known antibody. (CD28) domain fused to the cytoplasmic CD3- domain Having described the invention with reference to the exhibits superior anti-tumor potency that can be attributed to embodiments and illustrative examples, those in the art may CD28-mediated survival and enhanced cytokine production. appreciate modifications to the invention as described and This construct may be further modified to incorporate a C-ter 65 illustrated that do not depart from the spirit and scope of the minal 2A cleavable linker followed by the coding sequence invention as disclosed in the specification. The examples are for a truncated human EGFR (EGFRt) for the purpose of set forth to aid in understanding the invention but are not US 8,802,374 B2 11 12 intended to, and should not be construed to limit its scope in T. CD8 cells were further selected in some cases using any way. The examples do not include detailed descriptions of AutoMACSTM (Miltenyi Biotec) per the manufacturer's pro conventional methods. Such methods are well knownto those tocol. CMV-specific cells were generated by stimulating T of ordinary skill in the art and are described in numerous cells with 5 U/ml rhIL-2 (Chiron, Emeryville, Calif.) and publications. 5 autologous irradiated viral antigen presenting cells at a 4:1 (responder:Stimulator) ratio once a week for three weeks, Example 1 using 10% human serum instead of FCS to avoid non-specific stimulation. The viral antigen presenting cells were derived Generation of EGFRt and Immunomagnetic from PBMC that had been genetically modified to express Selection of EGFRt Expressing T Cells 10 CMVpp65 antigen. PBMC were resuspended in nucleofection solution using Materials & Methods the Human T cell Nucleofector kit (Amaxa Inc., Gaithers Antibodies and Flow Cytometry berg, Md.), and 5x10" cells were aliquoted into 0.2-cm FITC-, PE- and PerCP-conjugated isotype controls, cuvettes containing 10 ug HygroR-pp 65 pEK (or pmaxGFP PerCP-conjugated anti-CD8, FITC conjugated anti-CD4. 15 from Amaxa Inc., as a transfection control) in a final Volume PE-conjugated anti-IFNY, PerCP-conjugated anti-CD45 and of 100 L/cuvette, and electroporated using the Amaxa PE-conjugated streptavidin were obtained from BD Bio Nucleofector I (Amaxa Inc.), program U-14, after which cells sciences (San Jose, Calif.). Biotinylated anti-Fc was pur were allowed to recover for 6 hours at 37° C. prior to Y-irra chased from Jackson ImmunoResearch Laboratories, Inc. diation (1200 cGy). (Westgrove, Pa.). PE-conjugated anti-Biotin was purchased The CD19CAR-T2A-EGFRt epHIV7 (p.J02104) and from Miltenyi Biotec (Auburn, Calif.). Biotinylated EGF was CD19CAR-T2A-EGFRt-T2A-IMPDH2dm epHIV7 purchased from Molecular Probes(R Invitrogen (Carlsbad, (p.J02111) lentiviral constructs contain a) the chimeric anti Calif.). PE-conjugated anti-EGFR was purchased from gen receptor (CAR) sequences consisting of the V and V, Abcam Inc. (Cambridge, Mass.). All antibodies and biotin gene segments of the CD19-specific FmC63 mAb, an IgG1 EGF were used according to the manufacturer's instructions. 25 hinge-C-C, the transmembrane and cytoplasmic signal Flow cytometric data acquisition was performed on a FACS ing domains of the costimulatory molecule CD28, and the calibur (BD Biosciences), and the percentage of cells in a cytoplasmic domain of the CD3 chain 10; b) the self-cleav region of analysis was calculated using FCS Express V3 (De ing T2A sequence 11; c) the truncated EGFR sequence (See Novo Software, Los Angeles, Calif.). FIG. 1); and d) the IMPDH2 double mutant that confers For generation of the biotinylated-cetuximab, 200 mg of 30 MPA-resistance, as indicated. Lentiviral transduction was cetuximab (Erbitux(R) was buffer exchanged (19 hours) to carried out on T cells that were stimulated with either 30 PBS (D-PBS, pH 7.5+0.1) using a MidGee Hoop Cartridge ng/mL anti-CD3e (OKT3: Ortho Biotech, Raritan, N.J.) (i.e., (UFP-30-E-H42LA) with 527 mL. The material at 2 mg/mL for Line A) or human CD3/CD28Dynal beads at a 1:10 ratio was then modified at a 20:1 ratio using Sulfo-NHS-LC-Biotin (i.e., for Lines B, C, D and E) and 25 UIL2/ml. Cells were in a reaction that was carried out for 1 hour at room tempera 35 cultured for up to 2 hours at 37° C. on RetroNectin R (50 ture and then diafiltered to remove the excess biotin. The 200 ug/ml) coated plates prior to addition of the lentivirus at an mg of biotinylated cetuximab was then buffer exchanged (18 MOI of 3 and 5 g/ml polyybrene. After 4 hours, warm hours) to PBS (D-PBS, pH 7.5+0.1) using MidCee Hoop medium was added to triple to volume, and the cells were then Cartridge (UFP-30-E-H42LA) with 533 mL. Glycerol was washed and plated in fresh media after 48 hours. added to a final concentration of 20% and then the material 40 AutoMACSTM sorting of EGFRt-expressing cells was carried was frozen in vials. out with biotinylated cetuximab and anti-biotin microbeads Cell Lines (Miltenyi Biotec) as per the manufacturers instructions. Unless otherwise indicated, all cell lines were maintained Expansion of T cells in rapid expansion medium (REM) in RPMI 1640 (Irvine Scientific, Santa Ana, Calif.) supple involved the incubation of 10° T cells with 30 ng/mL anti mented with 2 mM L-glutamine (Irvine Scientific), 25 mM 45 CD3e (OKT3: Ortho Biotech, Raritan, N.J.), 5x107 y-irradi N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid ated PBMCs (3500 cGy), and 107 y-irradiated LCLs (8000 (HEPES, Irvine Scientific), 100 U/mL penicillin, 0.1 mg/mL cGy) in 50 mL CM; with addition of 50 U/mL rhIL-2 and 10 streptomycin (Irvine Scientific), and 10% heat-inactivated ng/ml rhIL-15 (CellGenix) every 48 hours, beginning on day fetal calf serum (FCS, Hyclone, Logan, Utah), hereafter 1. T cells were re-stimulated in this manner every 14 days. referred to as culture media (CM). 50 EBV-transformed lymphoblastoid cell lines (LCLs) were To generate T cells, human peripheral blood mononuclear made from PBMC as previously described 13). LCL-OKT3 cells (PBMC) were isolated by density gradient centrifuga cells were generated by resuspending LCL in nucleofection tion over Ficoll-Paque (Pharmacia Biotech, Piscataway, N.J.) solution using the Amaxa Nucleofector kit T, adding OKT3 from heparinized peripheral blood obtained from consented 2A-Hygromycin pEK (p.J01609) plasmid at 5 g/107 cells, healthy donors participating on a City of Hope National 55 and electroporating cells using the Amaxa Nucleofector I. Medical Center Internal Review Board-approved protocol. program T-20. The resulting LCL-OKT3-2A-Hygro pEK For generation of Line A, washed PBMC were stimulated (c.J03987) were grown in CM containing 0.4 mg/ml hygro with 25 U/mL, IL-2 and a 1:1 (cell:bead) ratio of Dynabeads(R) mycin. The mouse myeloma line NSO (gift from Andrew Human Texpander CD3/CD28 (Invitrogen, Carlsbad, Calif.). Raubitschek, City of Hope National Medical Center, Duarte, For generation of the other lines, washed PBMC were first 60 Calif.) was resuspended in nucleofection solution using the autoMACSTM depleted using anti-CD45RA beads (Miltenyi Nucleofector kit T (Amaxa Inc., Gaithersberg, Md.), CD19t Biotec) per the manufacturer's protocol, and in Some cases DHFRdm-2A-IL12 pEK (p.J.01607) or GFP-IMPDH2dm also depleted with PE-conjugated anti-CD4 (BD Bio 2A-IL15 pcDNA3.1 (+) (p.J01043) plasmid was added at 5 sciences) with anti-PE beads (Miltenyi Biotec). The resulting ug/5x10° cells, and cells were electroporated using the cells then underwent autoMACSTM positive selection using 65 Amaxa Nucleofector I, program T-27. The resulting NS0 biotinylated DREG56 (anti-CD62L) and anti-biotin beads CD19t-DHFRdm-2A-IL12 pEK (c.J03935) and NS0-GFP: (Miltenyi Biotec) to produce purified CD62L"CD45RO" IMPDH2-IL15(IL2ss) pcDNA3.1 (+) (c.J02096) were grown US 8,802,374 B2 13 14 in DMEM (Irvine Scientific, Santa Ana, Calif.) supplemented selection. As shown in the FIG. 1 molecular model, the with 10% heat-inactivated FCS, 25 mM HEPES, and 2 mM EGFRt retains the ability to be bound by cetuximab, but not L-glutamine in the presence of either 0.05 uM methotrexate have any signaling capacity due to the absence of the intrac (MTX) or 6 uM mycophenolic acid (MPA). The tumorigenic ellular domains. Furthermore, it lacks the N-terminal domain strain of U251, termed U251T, was a kind gift of Dr. Walde required for EGF-binding. mar Debinski (Wake Forest, N.C.). U251T pp 65 were gen To immunomagnetically select for EGFRt-expressing erated by lentiviral transduction of U251T with pp 65-2A cells, biotinylated-cetuximab was generated (FIG.2a, b) to be eGFP-fluc epHIV7 (p.J01928) at an MOI of 1. The resulting used in conjunction with commercially available anti-biotin U251T pp 65-2A-eGFP-fluc epHIV7 were then FACS microbeads and an AutoMACSTM separator (Miltenyi Biotec) sorted for the GFP" population (c.J05058). The Daudi lym 10 (FIG.2c). Lentiviral transduction of various T cell lines with phoma line was purchased from ATCC and grown in media EGFRt-containing constructs, where the EGFRt gene was consisting of RPMI 1640 (Irvine Scientific), 2 mM separated from other genes of interest on either one or both L-Glutamine (Irvine Scientific), 10% heat-inactivated FCS ends with the self-cleaving T2A sequence, consistently (Hyclone). SupB15 acute lymphoblastic leukemia cells and resulted in surface detection of the EGFRt molecule on less A431 epidermoid carcinoma cells were purchased from 15 than 40% of the cells (FIG. 2d). Surface detection may also be ATCC. accomplished with a EGFRt-sr39TK fusion. Immunomag Protein Analysis netic selection allowed for recovery of EGFRt T cell popu Cells (up to 107) were lysed with 80 uL of 1% Triton-X lations with greater than 90% purity. T cell populations that lysis buffer containing phosphatase inhibitor cocktail II underwent this transduction and selection procedure included (Sigma-Aldrich Corp., St. Louis, Mo.) (1:20 of inhibitor to anti-CD3/anti-CD28 bead stimulated T cell blasts (for Line buffer by volume). 50 ug of protein was loaded in each lane, A), central memory (CD45RO"CD62L"T) derived T cells and Western blots were probed with antibodies from the (for Lines B, C and E), which in some cases were also pre Phospho-EGF receptor antibody sampler kit (Cell Signaling selected for CMV specificity (via the endogenous TCR; for Technology, Inc., Danvers, Mass.) followed by IRDyeTM Line B) or CD8 expression (for Line C), as well as effector 680CW or 800CW conjugated goat anti-rabbit antibodies 25 memory (CD62LCD45RO" T.) derived T cells (for line (LI-COR, Lincoln, Nebr), as well as the IRDyeTM 800 con D). These data show that EGFRt can successfully be used as jugated anti-beta-Actin antibody (LI-COR) as per the manu a selection marker for various sources of T cell transductants, facturers instructions. Blots were imaged on the Odyssey even when the original transduction efficiency was as low a Infrared Imaging System (LI-COR). 2%. Chromium-Release Assays 30 Inactivity of EGFRt on Selected T Cells The cytolytic activity of T cells was determined by 4-hour To confirm that the EGFRt is inactive, Western immunob chromium-release assay (CRA), where effector cells were lot analyses for EGFR phosphorylation were carried out on seeded into triplicate wells of V-bottom 96-well micro-plates the EGFRt-selected T cells after culture with either EGF or containing 5x10 'Cr-labeled targeT cells (Na,CrO: cetuximab. As expected, cetuximab did not induce EGFR (5mCi/mL); Amersham Pharmacia, Piscataway, N.J.) at vari 35 phosphorylation above background even in the EGFR" cell ous E:T ratios in 200 uL of CM and incubated for 4 hours at line A431 (FIG.3a). Furthermore, in contrast to that seen with 5% CO., 37°C. Plates were centrifuged, and 100 ul of super the A431 cells, no phosphorylation was seen in lysates of Line natant was removed from each well to assess chromium A after co-incubation with EGF. Indeed, using biotinylated release using a Y-counter (Packard Cobra II, Downer's Grove, EGF, flow cytometric analysis confirmed that EGF cannot Ill.). The percent specific lysis was calculated as follows: 40 bind the EGFRt-selected T cells (FIG.3b), as expected due to 100x(experimental release-spontaneous release)/(maximum the truncation in its N-terminus. These EGFRt' T cells were release-spontaneous release). Maximum release was deter also not recognized by another anti-EGFR antibody distinct mined by measuring the 'Cr content of wells containing from cetuximab. labeled targets lysed with 2% SDS. Maintenance of Effector Phenotype in Expanded EGFRt" Antibody dependent cell mediated cytotoxicity was deter 45 CD19CAR T Cells mined by chromium release as above using 5x10 Cr-la Directly after AutoMACSTM separation, the selected T beled target cells that had been pre-incubated for 90 min with cells were expanded 30-fold or greater within 12 days after up to 10 g/mL of either cetuximab or rituximab (a CD20 REM stimulation with OKT3, irradiated PBMC feeders and specific mAb), washed and then co-incubated with 5x10 LCL, IL-2 and IL-15 (FIG. 4a). Flow cytometric analysis of freshly isolated PBMC. 50 the resulting expanded EGFRt'T cells further confirmed that T Cell Engraftment and Cetuximab Mediated Suicide InVivo that they express the CD19CAR and T cell markers such as For T cell engraftment, six- to ten-week old NOD/Scid CD8, TCR, CD3, perforin, granzyme, etc. (FIG. 4b). Further IL-2RYC" mice are injected i.v. on day 0 with 107 T cells more, CD19CAR-directed cytotoxic activity of these EGFRt (Line C). 2x107 irradiated (8000 rads) NS0-GFP:IMPDH2 selected lines is evident in chromium release assays using IL15(IL2ss) pcDNA3.1 (+) (c.J02096) cells are administered 55 CD19-expressing tumor targets (FIG.4c). A direct compari i.p. 3 times a week starting on day 0 to provide a systemic son of the CD19-specific reactivity of Line E versus its non supply of human IL-15 in vivo. Bone marrow was harvested selected or parental counterpart shows that there is from euthanized animals and analyzed by flow cytometry. enhanced CD19CAR-mediated cytotoxicity upon EGFRt-se Antibody dependent cell mediated cytotoxicity assays are lection. In addition, the CMV-specific T-derived performed to determine the activity of cetuximab against 60 CD19CAR"EGFRt" Line B cells also show cytotoxic activity EGFRt T cells. through their endogenous T cell receptor against targets Results expressing CMV-pp 65 antigen. Immunomagnetic Selection of EGFRt Expressing T Cells For the CD19CAREGFRt"IMPDH2dmit Line E, the abil A truncated human EGFR (EGFRt), which contains only ity of the inosine monophosphate dehydrogenase 2 double the transmembrane domain and extracellular domains III and 65 mutant (IMPDH2dm) to confer resistance to the IMPDH2 IV of the full length EGFR, was generated as a non-immu inhibitor mycophenolic acid (MPA; a common immunosup nogenic selection epitope compatible with immunomagnetic pressant used to prevent rejection in organ transplantation) US 8,802,374 B2 15 16 was also tested. Upon culture in 1 uM MPA, the survival CD19-expressing lymphoma cells and/or the adoptively and/or proliferation of Line E cells is not inhibited (FIG. 4d). transferred T cells; FDG-PET and/or CT scans; bone marrow This is in contrast to the inhibition seen with a control T cell examination for disease specific pathologic evaluation; line that lacks expression of the IMPDH2dn gene. These data lymph node biopsy; and/or long-term follow upper the guide provide further evidence that EGFRt-mediated selection lines set forth by the FDA's Biologic Response Modifiers results in the corresponding selection of the other genes Advisory Committee that apply to gene transfer studies. FIG. present in the lentiviral construct used to transduce T cells. 10 provides a possible schematic for clinical testing of the Tracking of EGFRt" T. Cells In Vivo present products and methods. To test the potential for detecting in vivo engrafted T cells, The present invention is not to be limited in scope by the bone marrow cells collected from mice that had been 10 engrafted with CD19CAR"EGFRt" Line C was analyzed by specific embodiments disclosed in the examples which are flow cytometry using biotinylated cetuximab (FIG. 5). Con intended as illustrations of a few aspects of the invention and trol mice that did not receive T cells revealed that there was any embodiments that are functionally equivalent are within the scope of this invention. Indeed, various modifications of Some cross-reaction of the cetuximab against murine EGFR. the invention in addition to those shown and described herein Thus, it was determined that successful detection of engrafted 15 Line C cells required double staining for both human CD45 will become apparent to those skilled in the art and are and EGFRt. Cells may also analyzed using immunohis intended to fall within the scope of the appended claims. tochemistry to determine potential for Screening biopsy mate All patents, patent applications, and references cited rial. throughout the specification are expressly incorporated by Cetuximab Mediated Cytotoxicity of EGFRt" T. Cells reference. Because cetuximab is known to lyse EGFR-expressing cells via antibody dependent cell mediated cytotoxicity REFERENCES (ADCC), assays were performed to determine the ADCC activity of cetuximab against EGFRt' T cells (FIG. 6). Using 1. Berger, C, Flowers, ME, Warren, E H, and Riddell, S R Cr-labeled Line A cells as targeted and freshly isolated 25 (2006). Analysis of transgene-specific immune responses human PBMC as effectors, cetuximab was found to signifi that limit the in vivo persistence of adoptively transferred cantly mediate chromium-release above that seen when using HSV-TK-modified donor T cells after allogeneic hemato the CD20-specific humanized mAb Rituxan. poietic cell transplantation. Blood 107: 2294-302. Example of Therapeutic Use of EGFRt" T. Cells 2. Tey, S K, Dotti, G. Rooney, CM, Heslop, HE, and Brenner, Adult subjects with high-risk intermediate grade B-cell 30 M K (2007). Inducible caspase 9 suicide gene to improve lymphomas who are candidates for an autologous myeloab the safety of allodepleted T cells after haploidentical stem lative stem cell transplant procedure may receive post-trans cell transplantation. Biol Blood Marrow Transplant 13: plant immunotherapy with adoptively transferred autologous 913-24. Tcm-derived CD19R" CD8" EGFRt" T cell grafts. A leuka 3. Fehse, B. Richters, A. Putimtseva-Scharf, K. Klump, H. Li, pheresis product collected from each patient undergoes selec 35 tion of Tcm, transduction with clinical grade CD19CAR Z, Ostertag, W. etal. (2000). CD34 splice variant: an attrac T2A-EGFRt epHIV7, and then selection and expansion of tive marker for selection of gene-modified cells. Mol Ther the EGFRt cells in a closed system. After the resulting cell 1: 448-56. products have undergone quality control testing (including 4. Gaines, P. and Wojchowski, D M (1999), pIRES-CD4t, a sterility and tumor specific cytotoxicity tests), they are cryo 40 dicistronic expression vector for MACS- or FACS-based preserved. Meanwhile, following leukapheresis, study par selection of transfected cells. Biotechniques 26: 683-8. ticipants commence with standard salvage chemotherapy, 5. Fehse, B, Uhde, A, Fehse, N, Eckert, H G, Clausen, J. with mobilization for auto HSC collection with cytoreductive Ruger, R. et al. (1997). Selective immunoaffinity-based chemotherapy and G-CSF. Since the EGFRt-selected, CD19 enrichment of CD34+ cells transduced with retroviral vec specific T cells will also target normal CD20" (CD19) B 45 tors containing an intracytoplasmatically truncated version cells, the B cell numbers can first be lowered using Ritux of the human low-affinity nerve growth factor receptor imabtM to reduce the recipient’s inflammatory response upon (deltaLNGFR) gene. Hum Gene Ther8: 1815-24. receiving the genetically modified CTL and also increase 6. Lemoine, FM, Mesel-Lemoine, M, Cherai, M, Gallot, G, availability of infused T cells to immediately target lym Vie, H. Leclercq, V, et al. (2004). Efficient transduction and phoma cells. Furthermore, RituximabM may blunta humoral 50 selection of human T-lymphocytes with bicistronic Thy 1/ immune response against the genetically modified T cells. If HSV1-TK retroviral vector produced by a human packag Rituximab is not given as part of the Salvage/Priming ing cell line. J Gene Med 6:374-86. chemotherapy regimen, research participants may receive a 7. Li, S, Schmitz, KR, Jeffrey, P D, Wiltzius, JJ, Kussie, P. single intravenous infusion of RituximabtM (chimeric anti and Ferguson, KM (2005). Structural basis for inhibition CD20 antibody) at 375 mg/m within 4-weeks of the planned 55 of the epidermal growth factor receptor by cetuximab. auto-HSCT procedure. Rituximab TM infusion would be car Cancer Cell 7: 301-11. ried out per standard practice including premedication with 8. Dawson, J. P. Berger, M. B. Lin, C C, Schlessinger, J. diphenhydramine and acetaminophen and hydrocortisone. Lemmon, MA, and Ferguson, K M (2005). Epidermal On Day +2 or Day +3 after HSCT, the autologous cryopre growth factor receptor dimerization and activation require served CD19R" CD8" EGFRt" T cell product will be trans 60 ligand-induced conformational changes in the dimer inter ported, thawed and infused at the patient’s bedside. Research face. Mol Cell Biol 25: 7734-42. participants can be pre-medicated at least 30 minutes prior to 9. Lange, C. Li, Z. Fang, L, Baum, C, and Fehse, B (2007). T cell infusion with 15 mg/kg of acetaminophen P.O. (max. CD34 modulates the trafficking behavior of hematopoietic 650 mg.) and diphenhydramine 0.5-1 mg/kg I.V. (max dose cells in vivo. Stein Cells Dev 16:297-304. 50 mg). Clinical and laboratory correlative follow-up studies 65 10. Kowolik, CM, Topp, M. S. Gonzalez, S. Pfeiffer, T. can then be performed at the physicians discretion, and may Olivares, S. Gonzalez, N, et al. (2006). CD28 costimula include quantitative RT-PCR studies for the presence of tion provided through a CD19-specific chimeric antigen US 8,802,374 B2 17 18 receptor enhances in vivo persistence and antitumor effi cells based on expression of a mutated inosine monophos cacy of adoptively transferred T cells. Cancer Res 66: phate dehydrogenase 2 after HIV vector transduction: 10995-1004. effects onlymphocytes, monocytes, and CD34+ stem cells. 11. Szymczak, A L. Workman, C J, Wang, Y. Vignali, KM, Mol Ther 14: 236-44. Dilioglou, S, Vanin, EF, et al. (2004). Correction of multi- 5 gene deficiency in Vivo using a single self-cleaving 2A 13. Pelloquin, F. Lamelin, J P and Lenoir, G M (1986). peptide-based retroviral vector. Nat Biotechnol 22:589-94. Human B lymphocytes immortalization by Epstein-Barr 12.Yam, P. Jensen, M. Akkina, R, Anderson, J., Villacres, MC, virus in the presence of cyclosporin A. In Vitro Cell Dev Wu, J, et al. (2006). Ex vivo selection and expansion of Biol 22: 689-94.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 6

<21 Os SEQ I D NO 1 &211s LENGTH: 1071 212s. TYPE : DNA <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1

atgcttct co tggtgacaag ccttctgctic tgtgagttac cacacc cagc attic ct cotg 60

atcc.cacgca aagtgtgtaa cggaataggit attggtgaat ttaaag actic actic to Cata 12O

aatgctacga at attaaa.ca Cttcaaaaac tgcacct coa to agtggcga totcCacatc. 18O

Ctg.ccggtgg catttagggg tgact cott c acacatactic ct cotctgga to cacaggaa 24 O

ctggatatt c tgaaaac ct aaaggaaatc acagggittitt tgctgattica ggcttggc ct 3 OO

gaaaacagga cggacct coa tgcct ttgag aacctagaaa to at acgcgg Caggaccalag 360

caa.catggtc agttittct ct tgcagtcgtC agcctgaaca taaCat CCtt gggattacgc 42O to Cct Caagg agataagtga tggagatgtg ataattt Cag gaaacaaaaa tttgtgctat 48O

gcaaatacaa taalactggaa aaaactgttt gggacct cog gt cagaaaac Caaaattata 54 O

agcaa.ca.gag gtgaaaa.ca.g Ctgcaaggcc acaggcc agg totgc.catgc cittgtgct co 6 OO

cc.cgagggct gctggggcCC ggagccCagg gactgcgt.ct Cttgc.cggaa tgtcagc.cga 660

ggCagggaat gcgtggacaa gtgcaac citt Ctggagggtg agccaaggga gtttgttggag 72O

aactctgagt gcatacagtg ccacccagag tgcc togcctic aggc catgaa catcacctgc 78O

acaggacggg gaccagacaa ctgtatic cag tgtgcc.cact acattgacgg cc cc cactgc 84 O

gtcaagacict gcc.cggCagg agt catggga gaaaacaa.ca cc ctggtctg galagtacgca 9 OO

gacgc.cggcc atgtgtgc.ca cc tdtgc cat ccaaactgca Cctacggatg cactgggc.ca 96.O

ggit Cttgaag gctgtccaac gaatgggcct aagat.cccgt. ccatcgc.cac tgggatggtg 102O

ggggc cct cc tottgctgct Ctggggat.cg gcct citt cat 9 1071

<21 Os SEQ I D NO 2 &211s LENGTH: 1071 212s. TYPE : DNA <213> ORGANISM: Homo sapiens

<4 OOs SEQUENCE: 2 tacga agagg accactgttc ggalagacgag acact caatg taaggaggac 60

tagggtgcgt. tt Cacacatt gcctitat coa taaCCaCtta aatttctgag tgagaggt at 12O

ttacgatgct tataatttgt gaagtttittg acgtggaggit agt caccgct agaggtgtag 18O

gacggccacc gtaaatc.ccc actgaggaag tgtgtatgag gaggagacict aggtgtcCtt 24 O

gacctataag acttittggca titt cott tag tgtc.ccaaaa acgactaagt cc.galaccgga 3 OO

cttttgtcct gcctggaggt acggaaactic ttggat.ctitt agtatgcgc.c gt cctggttc 360

gttgtaccag to aaaagaga acgt.ca.gcag toggacttgt attgtaggaa cc ctaatgcg 42O US 8,802,374 B2 19 20 - Continued agggagttcC totatt cact acctictaCaC tattaaagtic Ctttgtttitt aaa.cacgata cgitt tatgtt atttgaccitt ttittgacaaa CCCtggaggc cagtc.ttittg gttittaatat 54 O tcqttgtctic cactitttgtc gacgttc.cgg tgtc.cggtc.c agacggtacg galacacgagg gggctic cc.ga cgaccc.cggg Cct cqggit Co Ctgacgcaga gaacggcctt acagt cq9ct 660 ccgt.ccctta cgcacctgtt cacgttggaa gacct cocac tcqgttc cct caaac acct c 72 O ttgagactica cg tatgtcac ggtgggtctic acggacggag tcc.gg tactt gtagtggacg tgtc.ctg.ccc ctggtotgtt gacat aggto acacgggtga tgtaactgcc gggggtgacg 84 O

Cagttctgga t cagtacc ct Cttttgttgt gggaccagac Ctt catgcgt. 9 OO

Ctgcgg.ccgg tacacacggit gga cacggta ggtttgacgt. ggatgcctac gtgaccc.ggit 96.O ccagaactitc cgacaggttg cittacccgga ttctagggca ggtagcggtg accctaccac cc.ccgggagg agaacgacga ccaccaccgg gaccc.ctago cggagaagta C 1071

<210s, SEQ ID NO 3 &211s LENGTH: 357 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 3

Met Lieu. Lieu. Lieu Val Thir Ser Lieu. Lieu. Lieu. Cys Glu Lell Pro His Pro 1. 1O 15

Ala Phe Luell Lieu. Ile Pro Arg Llys Val Cys Asn Gly Ile Gly Ile Gly 25 3O

Glu Phe Lys Asp Ser Lieu. Ser Ile Asn Ala Thr Asn Ile Llys His Phe 35 4 O 45

Asn Thir Ser Ile Ser Gly Asp Lieu. His Ile Lell Pro Wall Ala SO 55 6 O

Phe Arg Gly Asp Ser Phe Thr His Thir Pro Pro Lell Asp Pro Glin Glu 65 70 7s 8O

Lell Asp Ile Lieu Lys Thr Val Lys Glu Ile Thr Gly Phe Lieu. Lieu. Ile 85 90 95

Glin Ala Trp Pro Glu Asn Arg Thr Asp Lieu. His Ala Phe Glu Asn Lieu. 105 11 O

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Glin Phe Ser Lieu. Ala 115 12 O 125

Wall Wall Ser Lieu. Asn. Ile Thir Ser Lieu. Gly Lieu. Arg Ser Lieu Lys Glu 13 O 135 14 O

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Asn Lieu. Cys Tyr 145 150 155 160

Ala Asn Thir Ile Asn Trp Llys Llys Leu Phe Gly Thir Ser Gly Glin Lys 1.65 17O 17s

Thir Ile Ile Ser Asn Arg Gly Glu Asn. Ser Ala Thr Gly 18O 185 19 O

Glin Wall Cys His Ala Lieu. Cys Ser Pro Glu Gly Trp Gly Pro Glu 195 2O5

Pro Arg Asp Cys Val Ser Cys Arg Asn. Wal Ser Arg Gly Arg Glu. Cys 21 O 215 22O

Wall Asp Cys Asn Lieu. Lieu. Glu Gly Glu Pro Arg Glu Phe Wall Glu 225 23 O 235 24 O

Asn Ser Glu Cys Ile Glin Cys His Pro Glu. Cys Lell Pro Glin Ala Met 245 250 255

US 8,802,374 B2 27 28 - Continued ttacagtcgg citcc.gtcc ct tacgcacctg ttcacgttgg aag acct coc act cqgttcc 282O

Ctcaaacacc t cittgagact cacg tatgtc acggtgggtc t cacggacgg agt ccgg tac 288O ttgtag togga cgtgtc.ctgc CCCtggtctg ttgacat agg t cacacgggt gatgtaactg 294 O

cgcagttctg gacgggcc.gt cct cagtacc citcttttgtt gtgggacCag 3 OOO acct tcatgc gtctgcggcc ggtacacacg gtgga cacgg taggitttgac gtggatgcct 3 O 6 O acgtgaccc.g gtccagaact tcc.gacaggt tgcttaccc.g gattictaggg Cagg tag.cgg 312 O tgaccc.tacc accc.ccggga ggagaacgac gaccaccacc gggaccc.cta gcc.ggagaag 318O tacact 3186

<210s, SEQ ID NO 6 &211s LENGTH: 1061 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6

Met Lieu. Lieu. Lieu Val Thir Ser Lieu. Lieu. Lieu. Cys Glu Lell Pro His Pro 1. 1O 15

Ala Phe Luell Lieu. Ile Pro Asp Ile Gln Met Thr Glin Thir Thir Ser Ser 25

Lell Ser Ala Ser Lieu. Gly Asp Arg Wall. Thir Ile Ser Cys Arg Ala Ser 35 4 O 45

Glin Asp Ile Ser Llys Tyr Lieu. Asn Trp Tyr Glin Glin Pro Asp Gly SO 55 6 O

Thr Wall Lieu Lieu. Ile Tyr His Thir Ser Arg Lieu His Ser Gly Val 65 70

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Ser Lieu. Thir 85 90 95

Ile Ser Asn Lieu. Glu Glin Glu Asp Ile Ala Thr Phe Cys Glin Glin 105 11 O

Gly Asn Thir Leu Pro Tyr Thr Phe Gly Gly Gly Thir Lys Luell Glu Ile 115 12 O 125

Thir Gly Ser Thir Ser Gly Ser Gly Llys Pro Gly Ser Gly Glu Gly Ser 13 O 135 14 O

Thir Gly Glu Val Llys Lieu. Glin Glu Ser Gly Pro Gly Luell Wall Ala 145 150 155 160

Pro Ser Glin Ser Lieu. Ser Wall. Thir Cys Thr Val Ser Gly Wall Ser Luell 1.65 17O 17s

Pro Asp Gly Val Ser Trp Ile Arg Glin Pro Pro Arg Lys Gly Lieu. 18O 185 19 O

Glu Trp Luell Gly Val Ile Trp Gly Ser Glu Thir Thir Tyr Asn. Ser 195

Ala Luell Ser Arg Lieu. Thir Ile Ile Lys Asp Asn Ser Ser Glin 21 O 215 22O

Wall Phe Luell Llys Met Asn. Ser Lieu Gln Thr Asp Asp Thir Ala Ile Tyr 225 23 O 235 24 O

Ala Gly Gly Ser Ala Met Asp Tyr 245 250 255

Trp Gly Glin Gly. Thir Ser Val Thr Wall Ser Ser Glu Ser Lys 26 O 265 27 O

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Lell Gly Gly Pro Ser 27s 285

Wall Phe Luell Phe Pro Pro Llys Pro Lys Asp Thr Lell Met Ile Ser Arg US 8,802,374 B2 29 30 - Continued

29 O 295 3 OO

Thir Pro Glu Wall Thir Cys Wall Wall Wall Asp Wall Ser Glin Glu Asp Pro 3. OS 310 315 32O

Glu Wall Glin Phe Asn Trp Wall Asp Gly Wall Glu Wall His Asn Ala 3.25 330 335

Thir Pro Arg Glu Glu Glin Phe Asn Ser Thir Arg Wall Wall 34 O 345 35. O

Ser Wall Luell Thir Wall Lell His Glin Asp Trp Luell Asn Gly Glu 355 360 365

Cys Wall Ser Asn Lys Gly Luell Pro Ser Ser Ile Glu Thir 37 O 375

Ile Ser Ala Lys Gly Glin Pro Arg Glu Pro Glin Wall Thir Luell 385 390 395 4 OO

Pro Pro Ser Glin Glu Glu Met Thir Asn Glin Wall Ser Luell Thir 4 OS 415

Lell Wall Gly Phe Pro Ser Asp Ile Ala Wall Glu Trp Glu Ser 425 43 O

Asn Gly Glin Pro Glu Asn Asn Tyr Thir Thir Pro Pro Wall Luell Asp 435 44 O 445

Ser Asp Gly Ser Phe Phe Lell Ser Arg Luell Thir Wall Asp Ser 450 45.5 460

Arg Trp Glin Glu Gly Asn Wall Phe Ser Ser Wall Met His Glu Ala 465 470

Lell His Asn His Tyr Thir Glin Ser Luell Ser Lell Ser Luell Gly 485 490 495

Met Phe Trp Wall Lell Wall Wall Wall Gly Gly Wall Lell Ala Cys Ser SOO 505

Lell Luell Wall Thir Wall Ala Phe Ile Ile Phe Trp Wall Arg Ser Arg 515 52O 525

Ser Arg Gly Gly His Ser Asp Met Asn Met Thir Pro Arg Arg Pro 53 O 535 54 O

Gly Pro Thir Arg Lys His Glin Pro Ala Pro Pro Arg Asp Phe 5.45 550 555 560

Ala Ala Arg Ser Gly Gly Arg Wall Phe Ser Arg Ser Ala 565 st O sts

Asp Ala Pro Ala Tyr Glin Gly Glin Asn Glin Lell Asn Glu Luell 585 59 O

Asn Luell Gly Arg Arg Glu Tyr Asp Wall Luell Asp Lys Arg Arg Gly 595 6OO 605

Arg Asp Pro Glu Met Gly Pro Arg Arg Lys Asn Pro Glin Glu 610

Gly Luell Asn Glu Lell Asp Met Ala Glu Ala Ser 625 630 635 64 O

Glu Ile Gly Met Lys Gly Arg Arg Arg Gly Gly His Asp Gly 645 650 655

Lell Glin Gly Lell Ser Thir Ala Thir Lys Asp Thir Asp Ala Luell 660 665 67 O

His Met Glin Ala Lell Pro Pro Arg Luell Glu Gly Gly Gly Glu Gly Arg 675 685

Gly Ser Luell Luell Thir Gly Asp Wall Glu Glu Asn Pro Gly Pro Arg 69 O. 695 7 OO

Met Luell Luell Luell Wall Thir Ser Luell Luell Luell Glu Lell Pro His Pro 7 Os 71O 72O US 8,802,374 B2 31 32 - Continued

Ala Phe Lieu. Luell Ile Pro Arg Val Cys Asn Gly Ile Gly Ile Gly 72 73 O 73

Glu Phe Asp Ser Lieu. Ser Ile Asn Ala Thir Asn. Ile Lys His Phe 740 74. 7 O

Asn. Cys Thir Ser Ile Ser Gly Asp Lieu. His Ile Lieu. Pro Wall Ala 760 765

Phe Arg Gly Asp Ser Phe Thir His Thir Pro Pro Lell Asp Pro Glin Glu 770 775

Lell Asp Ile Luell Thir Wall Glu Ile Thr Gly Phe Luell Luell Ile 79 O 79. 8OO

Glin Ala Trp Pro Glu Asn Arg Thir Asp Lieu. His Ala Phe Glu Asn Luell 805 810 815

Glu Ile Ile Arg Gly Arg Thir Glin His Gly Glin Phe Ser Lieu Ala 825 83 O

Wall Wall Ser Luell Asn Ile Thr Ser Luell Gly Luell Arg Ser Luell Glu 835 84 O 845

Ile Ser Asp Gly Asp Wall Ile Ile Ser Gly ASn Lys Asn Luell Tyr 850 855 860

Ala Asn Thr Ile Asn Trp Lieu. Phe Gly Thir Ser Gly Gln Lys 865 87s

Thir Ile Ile Ser Asn Gly Glu Asn. Ser Ala Thir Gly 885 890 895

Glin Wall His Ala Lell Ser Pro Glu Gly Trp Gly Pro Glu 9 OO 905 91 O

Pro Arg Asp Wall Ser Arg Asn. Wal Ser Arg Gly Arg Glu 915 92 O 925

Wall Asp Asn Lieu. Lieu. Glu Gly Glu Pro Arg Glu Phe Wall Glu 93 O 935 94 O

Asn. Ser Glu Ile Glin His Pro Glu Cys Leul Pro Glin Ala Met 945 950 955 96.O

Asn Ile Thir Thir Gly Arg Gly Pro Asp Asn Cys Ile Glin Cys Ala 965 97.

His Ile Asp Gly Pro His Cys Wall Lys Thir Cys Pro Ala Gly Val 985 99 O

Met Gly Glu Asn. Asn Thir Lell Wall Trp Llys Tyr Ala Asp Ala Gly His 995 1005

Wall Cys His Lieu. Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly 1010 1015 1 O2O

Pro Gly Lieu. Glu Gly Cys Pro Thir Asin Gly Pro Llys Ile Pro Ser 1025 1O3 O 1035

Ile Ala Thir Gly Met Val Gly Ala Lieu. Lieu. Lieu. Lell Lieu. Wal Wall 104 O 1045 1 OSO

Ala Lieu. Gly Ile Gly Lieu. Phe Met 105.5 106 O

The invention claimed is: endogenous signaling or trafficking function; (ii) binds a 1. A genetically modified Epidermal Growth Factor Recep therapeuticanti-EGFR antibody; (iii) does not bind an endog tor (EGFR) gene, comprising a nucleotide sequence encoding 60 enous EGFR ligand; and (iv) acts as a marker. a truncated non-immunogenic endogenous cell Surface mol 2. The gene of claim 1, further comprising a GMCSFR ecule, said cell surface molecule comprising an EGFR alpha chain signal sequence. Domain III and an EGFR Domain IV; but lacking nucleotides all of the domains consisting of an EGFR Domain I, an EGFR 3. The gene of claim 2 comprising SEQID NO:2. Domain II, an EGFRJuxtamembrane Domain, and an EGFR 65 4. The gene of claim 2, wherein the gene encodes an amino Tyrosine Kinase Domain; wherein the truncated non-immu acid sequence comprising at least 90% identical to SEQ ID nogenic endogenous cell Surface molecule (i) does not have NO:3. US 8,802,374 B2 33 34 5. The gene of claim 2, wherein the gene encodes an amino 15. The modified EGFR gene of claim 1, wherein the acid sequence comprising SEQID NO:3. marker is used to induce cell Suicide in cells expressing the 6. The gene of claim 1, wherein the gene is part of a truncated non-immunogenic endogenous cell Surface mol construct which comprises the modified EGFR coupled via a ecule. 16. The modified EGFR gene of claim 1, wherein the C-terminal 2A cleavable linker to a chimericantigen receptor endogenous EGFR ligand is EGF. specific for a tumor associated antigen selected from CD19, a 17. The modified EGFR gene of claim 1, wherein the codon-optimized anti-CD19 costimulatory chimeric antigen therapeutic anti-EGFR antibody is cetuximab. receptor (CD19CAR), CD20 or CD22. 18. A genetically modified Epidermal Growth Factor 7. The gene of claim 6, wherein the modified EGFR is Receptor (EGFR) gene, comprising a nucleotide sequence coupled to a CD19CAR and a C-terminal 2A cleavable linker. 10 encoding a truncated non-immunogenic endogenous cell Sur 8. The gene of claim 1, comprising nucleotides 67-1071 of face molecule, said cell Surface molecule comprising an SEQID NO:2. EGFR Domain III and an EGFR Domain IV; but lacking 9. The gene of claim 1, comprising nucleotides 67-1071 of nucleotides all of the domains consisting of an EGFR Domain SEQID NO:1. I, an EGFR Domain II, an EGFR Juxtamembrane Domain, 15 and an EGFR Tyrosine Kinase Domain; wherein the trun 10. The gene of claim 2 comprising SEQID NO:1. cated non-immunogenic endogenous cell Surface molecule 11. The gene of claim 1, wherein the gene encodes an (i) does not contain endogenous signaling or trafficking func amino acid sequence comprising residues 23-357 of SEQID tion; (ii) binds a therapeutic anti-EGFR antibody; (iii) does NO:2. not bind an endogenous EGFR ligand; and (iv) acts as a 12. A modified EGFR gene coupled to a CD19CAR and a marker used to enrich cells, select cells, or induce cell Suicide C-terminal 2A cleavable linker, wherein the gene encodes an in cells expressing said cell Surface molecule. amino acid sequence comprising SEQID NO:6. 19. The modified EGFR gene of claim 18, wherein the 13. The modified EGFR gene of claim 1, wherein the endogenous EGFR ligand is EGF. marker is used to enrich cells. 20. The modified EGFR gene of claim 18, wherein the 14. The modified EGFR gene of claim 1, wherein the 25 therapeutic anti-EGFR antibody is cetuximab. marker is used to select cells. k k k k k