INTRACELLULAR DOMAIN AS CO-STIMULATORY DOMAIN REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with the specification as an ASCII formatted text file via EFS-Web with a file name of Sequence Listing.txt with a creation date of August 30, 2017, and a size of 38.7 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.

FIELD OF THE INVENTION

The present invention relates to chimeric antigen receptors (C ARs) having GITR intracellular domain as a co-stimulatory domain, which effectively attack tumor cells overexpressing a tumor antigen such as EGFR, mesothelin, or CD 19, and not against cancer cells that do not express such a tumor antigen.

BACKGROUND OF THE INVENTION

Immunotherapy is emerging as a highly promising approach for the treatment of cancer. T cells or T lymphocytes, the armed forces of our immune system, constantly look for foreign antigens and discriminate abnormal (cancer or infected cells) from normal cells. Genetically modifying T cells with CARs is the most common approach to design tumor- specific T cells. CAR-T cells targeting tumor-associated antigens (TAA) can be infused into patients (called adoptive cell transfer or ACT) representing an efficient immunotherapy approach [1, 2]. The advantage of CAR-T technology compared with chemotherapy or antibody is that reprogrammed engineered T cells can proliferate and persist in the patient ("a living drug")[3], [4].

CARs

CARs (Chimeric antigen receptors) usually consist of a monoclonal antibody-derived single-chain variable fragment (scFv) linked by a hinge and transmembrane domain to a variable number of intracellular signaling domains: (i) a single, cellular activating, CD3-zeta domain; or (ii) CD28 or CD137 (4-1BB) as a co- stimulatory domains, in tandem with a CD3- zeta domain. (FIG. 1). The CD27 signaling domain has also been used in the place of either the CD28 or CD137 domain)[l, 2]. The evolution of CARs went from first generation (with no costimulation domains) to second generation (with one co-stimulation domain) to third generation CAR (with several costimulation domains). Generating CARs with multiple costimulatory domains (the so-called 3 ^rd generation CAR) have led to increased cytolytic activity, and significantly improved persistence of CAR-T cells that demonstrate augmented antitumor activity. The CAR structure is shown in FIG. 1.

CAR-T cells are effectively used in clinical trials against hematological cancer targets [6]. Recently, CAR-T cells were used against solid tumors against mesothelin, EGFR, Her-2 or other targets [5, 7]. Affinity-tuned Her-2 and EGFR showed high specificity against cancer cells versus normal cells, providing higher safety for CAR-T therapy [5].

EGFR

EGFR is an epidermal growth factor receptor that is overexpressed in many types of cancer [3]. EGFR is one of the four receptor tyrosine kinases of Erb family such as

ErbB2/HER-2, ErbB3/HER3 and ErbB4/HER4 [3]. EGFR plays important function in proliferation, growth regulation, angiogenesis, survival and metastasis.

EFGR contains 4 extracellular domains, transmembrane domain, and intracellular domain containing tyrosine kinase domain and carboxy-terminal tail (FIG. 2). Many types of cancers such as glioblastoma and others have deletion of amino-acids 5-273 resulting in expression of EGFRvIII form [4]. Both forms of EGFR wild type and EGFRvIII are critical for tumor survival signaling.

EGFR is involved in regulation of MAP kinase, PI3K, AKT, STAT signaling pathways. There are many tyrosine kinase inhibitors were developed, and recently immunotherapy approaches to target EGFR signaling were developed [5].

Mesothelin

Mesothelin is a tumor surface antigen that is highly overexpressed in many types of tumors [8], including ovarian tumors [9]. Mesothelin-CD28-CD3 zeta CAR-T cells have been shown to kill ovarian tumors [10].

CD19

Cluster of differentiation 19 (CD 19) is a protein encoded by the CD 19 gene, and is a B-lymphocyte antigen found on the surface of B-cells. CD19 is highly expressed in many types of hematologic cancers [2, 6, 11, 12]. CD19-CD28-CD3 zeta and CD19-4-1BB-CD3 zeta CAR-T cells have been shown to kill hematological cancers [13]. Many CD19-CAR-T cell clinical trials demonstrated promising results in clinic with this type of therapy[14].

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. The structure of CAR. On the left panel, the structure of first generation (no costimulation domains) is shown. In the middle panel, a second generation (one co- stimulation domain CD28) is shown. On the right panel, a third generation (two co- stimulation domains of CD28 and 4- IBB) is shown [7].

FIG. 2. The structure of EGFR and EGFRvIII proteins.

FIG. 3. The structures of EGFR-CAR, mesothelin-C AR and CD 19-CAR constructs .

The present invention uses GITR as co-stimulatory domain.

FIG. 4. Expansion of EGFR-GITR-CD3 zeta CAR-T cells in vitro.

FIG. 5A-5D. FAB staining demonstrates expression of EGFR scFv in transduced T cells.

FIGs. 6A-6E. EGFR-GITR-CD3 zeta CAR-T cells were highly cytotoxic against

EGFR-positive cancer cells but were not against EGFR-negative cancer cells. FIG. 6A-6D show the cytotoxic activities of EGFR-CAR with different co-stimulatory domains in different cancer cell lines with high expression of EGFR. FIG. 6E shows no cytotoxic activity of EGFR-CAR in MCF-7 cells, that were EGFR- 1 -negative. E:T, Effector to Target cell ratio.

FIG. 7. Mesothelin-GITR-CD3 zeta CAR-T cells kill mesothelin-positive ovarian and pancreatic cancer cells.

FIG. 8. Mesothelin-GITR CAR-T cells produce >2 fold less IFN-gamma cytokine than mesothelin-28-CD3 zeta in BxPC3 cells.

FIG. 9. CD 19-GITR-CD3 zeta CAR-T cells kill CD19-positive Hela-CD19 cells.

CAR-T to target cells ratio is 10:1. CD19-GITR-Z denotes CD 19-GITR-CD3 zeta. (1) and (2) show different CD 19 constructs that differ in their TM domain. CD19-GITR-Z (1) has CD8 transmembrane domain and CD19-GITR-Z (2) has CD28 TM domain. Both constructs have about the same cytolytic activity against Cdl9-positive cancer cells.

FIG. 10. CD 19-GITR-CD3 zeta CAR-T cells decrease tumor growth in Raji- luciferase+ xenograft NSG mouse model. Three mice were treated with CAR-T cells, 3 mice with lxPBS, and one mice with T cells. DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, "adoptive cell therapy" (ACT) is a treatment that uses a cancer patient's own T lymphocytes with anti-tumor activity, expanded in vitro and reinfused into the patient with cancer.

As used herein, "affinity" is the strength of binding of a single molecule to its ligand. Affinity is typically measured and reported by the equilibrium dissociation constant (K _D or Kd), which is used to evaluate and rank order strengths of bimolecular interactions.

As used herein, a "chimeric antigen receptor (CAR)" means a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain. The "chimeric antigen receptor (CAR)" is sometimes called a "chimeric receptor", a "T-body", or a "chimeric immune receptor (CIR)." The "extracellular domain capable of binding to an antigen" means any oligopeptide or polypeptide that can bind to a certain antigen. The "intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.

As used herein, a "domain" means one region in a polypeptide which is folded into a particular structure independently of other regions.

GITR (glucocorticoid-induced TNFR-related protein) is a surface receptor molecule that is known to induce T lymphocyte survival and inhibit suppressive activity of regulatory T cells [15]. GITR is a member of TNF family receptors. GITR is also known as tumor necrosis factor receptor superfamily member 18 (TNFRSF18), activation- inducible TNFR family receptor (AITR) and is a protein encoded by the TNFRSF18 gene in humans. GITR has 241 amino acids and its NCBI Reference Sequence Number is NP 004186.1. The intracellular domain of GITR (SEQ ID NO: 12) is used in the present invention, which is the amino acid residues 184-241 of NCBI Reference Sequence No. NP_004186.1).

As used herein, a "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the scFv includes an antibody polypeptide which is formed by a recombinant

DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for preparing an scFv are known to a person skilled in the art.

As used herein, a "tumor antigen" means a biological molecule having antigenicity, expression of which causes cancer. Description

The inventors have discovered that GITR intracellular domain can replace other traditional co-stimulatory domains such as CD28 and 4- IBB in CAR and provides advantages in clinics. The inventors have demonstrated that CARs having GITR intracellular domain as a co- stimulatory domain activate CAR-T cells to express ScFv of EGFR, mesothelin, or CD 19, and kills EGFR-, mesothelin-, or CD19-positive cells. GITR intracellular domain as a co- stimulatory domain provides advantages over CD28 or 4- IBB, because GITR can provide dual functions of (i) inducing T cell effector function and activating T cells, and (ii) suppressing inhibitory T regulatory cells that block immune response. GITR intracellular domain - containing CAR T cells can decrease the production of cytokines, which results in less cytokine release syndrome (CRS).

The present invention provides CAR-T cells that target a tumor antigen which is highly overexpressed in many types of cancer such as breast cancer, pancreatic cancer, glioblastoma, ovarian cancer, and hematologic cancers (leukemia, lymphoma, multiple myeloma). The inventors use (i) an antibody that specifically recognizes a tumor antigen- expressing cancer cells to prepare scFv, and (ii) GITR intracellular domain as a co- stimulatory domain, to generate GITR-CAR-T cells. The inventors have demonstrated that several GITR-CD3 zeta-CAR-T cells of the present invention have high cytotoxic activity against several cancer cells with high tumor antigen expression and have no activity in tumor antigen-negative cells.

The present invention provides a chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) having activity against a tumor antigen, (ii) a transmembrane domain, (iii) a GITR intracellular domain as a co-stimulatory domain, and (iv) an activating domain.

In one embodiment, the human tumor antigen is selected from the group consisting of: EGFR, mesothelin, CD19, CD20, BCMA, CD22, CD38, CD138, VEGFR-2, CD4, CD5, CD30, CD22, CD24, CD25, CD28, CD30, CD33, CD47, CD52, CD56, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, CD133, GPC3; PMSA, CD3, CEACAM6, c-Met,

EGFRvIII, ErbB2/HER-2, ErbB3/HER3, ErbB4/HER-4, EphA2,10a, IGFIR, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gpl30, Lewis A, Lewis Y, NGFR, MCAM, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE- A, NY-ESO-1, PSMA, RANK, ROR1, ROR-2, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robol, a, Frizzled, OX40, CD79b, and Notch- 1-4. Preferred tumor antigens are human EGFR, human mesothelin, and human CD 19.

The CAR of the present invention comprises a single chain variable fragment (scFv) that binds specifically to the tumor antigen of interest. The heavy chain (H chain) and light chain (L chain) fragments of an antibody are linked via a linker sequence. For example, a linker can be 5-20 amino acids. The scFv structure can be VL-linker-VH, or VH-linker-VL, from N-terminus to C-terminus.

The CAR of the present invention comprises a transmembrane domain which spans the membrane. The transmembrane domain may be derived from a natural polypeptide, or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a transmembrane domain of a T cell receptor a or β chain, CD28, CD3-epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD 154, or a GITR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. It is preferable that a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. In preferred embodiments, the transmembrane domain is derived from CD28 or CD8, which give good receptor stability.

The CAR of the present invention comprises a GITR intracellular domain as a co- stinulatory domain.

The endodomain (the activating domain) is the signal-transmission portion of the CAR. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3 zeta (CD3 Z or CD3ζ ), which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound.

The CAR fusion protein may comprise a FLAG tag located at N-terminus to scFv, or C-terminus to scFv, or between VH and VL. The FLAG tag needs to be in extracellular domain, and not in the intracellular domain. In addition to FLAG tag, other tags may be used in the construct. FLAG tag is a preferred tag because it does not cause immunogenicity and has decreased level of cytokine secretion.

The CAR of the present invention may comprise a signal peptide N-terminal to the scFv so that when the CAR is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. As an example, the signal peptide may derive from human CD8 or GM-CSF, or a variant thereof having 1 or 2 amino acid mutations provided that the signal peptide still functions to cause cell surface expression of the CAR.

The CAR of the present invention may comprise a spacer sequence as a hinge to connect scFv with the transmembrane domain and spatially separate antigen binding domain from the endodomain. A flexible spacer allows to the binding domain to orient in different directions to enable its binding to a tumor antigen. The spacer sequence may, for example, comprise an IgGl Fc region, an IgGl hinge or a CD8 stalk, or a combination thereof. A human CD28 or CD8 stalk is preferred.

FIG. 3 shows the structure of EGFR, mesothelin, or CD 19 CAR constructs. The second generation of CAR-T constructs are used. The co-stimulatory domains are CD28 (comparison), 4- IBB (comparison), or GITR intracellular domain (this invention). CD 19- GITR-CD3 zeta are designed with CD8 and CD28 trans-membrane domain.

The present invention provides a nucleic acid encoding the CAR described above. The nucleic acid encoding the CAR can be prepared from an amino acid sequence of the specified CAR by a conventional method. A base sequence encoding an amino acid sequence can be obtained from the aforementioned NCBI RefSeq IDs or accession numbers of GenBenk for an amino acid sequence of each domain, and the nucleic acid of the present invention can be prepared using a standard molecular biological and/or chemical procedure. For example, based on the base sequence, a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR).

The nucleic acid encoding the CAR of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, a virus vector such as a retrovirus vector (including a lentivirus vector, an oncoretrovirus vector, and a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a Sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV vector can be used. As the virus vector, a virus vector lacking the replicating ability so as not to self-replicate in an infected cell is preferably used.

For example, when a retrovirus vector is used, the process of the present invention can be carried out by selecting a suitable packaging cell based on a LTR sequence and a packaging signal sequence possessed by the vector and preparing a retrovirus particle using the packaging cell. Examples of the packaging cell include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. A retrovirus particle can also be prepared using a 293 cell or a 293 T cell having high transfection efficiency. Many kinds of retrovirus vectors produced based on retroviruses and packaging cells that can be used for packaging of the retrovirus vectors are widely commercially available from many companies.

The present invention provides T cells modified to express the chimeric antigen receptor fusion protein as described above. CAR-T cells of the present invention bind to a specific antigen via the CAR, thereby a signal is transmitted into the cell, and as a result, the cell is activated. The activation of the cell expressing the CAR is varied depending on the kind of a host cell and an intracellular domain of the CAR, and can be confirmed based on, for example, release of a cytokine, improvement of a cell proliferation rate, change in a cell surface molecule, or the like as an index.

T cells modified to express the CAR can be used as a therapeutic agent for a disease. The therapeutic agent comprises the T cells expressing the CAR as an active ingredient, and may further comprise a suitable excipient. Examples of the excipient include

pharmaceutically acceptable excipients known to a person skilled in the art.

In one embodiment, the EGFR scFv useful in the present invention has a low affinity against human EFGR, i.e., it has a dissociation constant (K _D ) > 50 nM, or >80 nM, or >100 nM, or >150 nM, and preferably > 200 nM or 250 nM. In one embodiment, the scFv is derived from CIO or P3-5 (see References 5 and 8) or has at least 90% sequence identity, as that of CIO or P3-5. Preferably, the scFv has at least 92%, 95%, 98%, or 99% sequence identity, as that of CIO or P3-5. CAR-T cells with low affinity scFv to EGFR exhibits robust antitumor efficacy similar to high-affinity antibody cells, but spared normal cells expressing physiologic EGFR levels, and thus they increase the therapeutic index.

In one embodiment, the mesothelin scFv useful in the present invention is derived from the anti-mesothelin antibody reported by Lanitis et al [17]. In one embodiment, the CD 19 scFv useful in the present invention is derived from the anti-CD 19 antibody reported by Kochenderfer et al [18].

GITR intracellular domain belongs to TNFR superfamily (TNFRSF) protein family and is a co-stimulatory domain for activating T cells. The inventors have discovered the following advantages of using GITR for CAR-T therapy. GITR-GITRL interaction mediates effective anti-tumor immune responses, by promoting expansion and activation of effector T cell populations and by suppressing T regulatory cells that suppress immune activity. The dual function of GITR to activate effector cells and to inhibit the repressing functions of T regulatory cells makes it effective for CAR-T immunotherapy.

This invention demonstrates construction of lentiviral EGFR-GITR-CD3 zeta containing vector and EGFR-GITR-CAR-T cells; lentiviral mesothelin-GITR-CD3 zeta containing vector and mesothelin-GITR-CAR-T cells; lentiviral CD19-GITR-CD3 zeta containing vector and CD 19-GITR-CD3 zeta CAR-T cells.

The present invention provides a CAR fusion protein having the amino acid sequence of SEQ ID NO: 2, 25, or 27, or a sequence having at least 95%, or 97%, or 99% sequence identity thereof.

The present invention provides a nucleic acid encoding a CAR fusion protein, the nucleic acid has the sequence of SEQ ID NO: 1, 24, or 26, or a sequence having at least 95%, or 97%, or 99% sequence identity thereof.

The present invention provides an adoptive cell therapy method for treating cancer, comprising the step of administering EGFR-GITR-CD3 zeta CAR-T cells to a subject suffering from cancer.

The present invention provides an adoptive cell therapy method for treating cancer, comprising the step of administering mesothelin-GITR-CD3 zeta CAR-T cells to a subject suffering from cancer.

The present invention provides an adoptive cell therapy method for treating cancer, comprising the step of administering CD19-GITR-CD3 zeta CAR-T cells to a subject suffering from cancer.

In one embodiment, low affinity EGFR antibody and a GITR intracellular domain as a co-stimulatory domain are used to prepare the EFGR-GITR CAR-T cells construct. EGFR scFv (e.g., from low affinity antibody CIO or P3-5 [16]) is cloned into Xba I and EcoR I sites of lentiviral vector. The CAR construct contains CD8 signaling peptide, EGFR scFv: VH- (variable heavy chain)-linker 3-VL( variable light chain) from low affinity EGFR antibody, CD8 hinge, CD28 transmembrane domain, GITR intracellular domain and CD3 zeta activation domains.

The inventors have generated EGFR-scFv-GITR-CD3 zeta-CAR-T cells against EGFR-positive cancer cell lines such as breast, ovarian, pancreatic, brain cancer and others. The inventors have provided data demonstrating efficient expansion of the EGFR-GITR CAR-T cells in culture. EGFR-GITR-CD3 zeta CAR-T can target both EGFR-positive and EGFR-vIII-positive cancer cells.

The inventors have generated mesothelin-scFv-GITR-CD3 zeta-CAR-T cells against mesothelin-positive cancer cell lines such as ovarian and pancreatic cells. The inventors have demonstrated efficient expansion of the mesothelin-GITR CAR-T cells in culture.

Mesothelin-GITR-CD3 zeta CAR-T cells were positive against Mesothelin-positive CAR-T cells.

The inventors have generated CD19-ScFv-GITR-CD3 zeta-CAR-T cells against CD19-positive cancer cell lines such as cervical cancer cells and hemotologic cancers. The inventors have demonstrated efficient expansion of the CD19-GITR CAR-T cells in culture. CD 19-GITR-CD3 zeta CAR-T cells were positive against CD19-positive CAR-T cells.

EGFR-GITR-CD3 zeta CAR-T, mesothelin-GITR-CD3 zeta CAR-T, and CD 19- GITR-CD3 zeta CAR-T can be used in combination with chemotherapy such as checkpoint inhibitors, targeted therapies, small molecule inhibitors, and antibodies.

Third generation CAR or other co-stimulatory signaling domains can be used with

EGFR-GITR-CD3 zeta CAR, mesothelin-GITR-CD3 zeta CAR, and CD 19-GITR-CD3 zeta CAR.

Combination of EGFR-GITR-CD3 zeta CAR-T, mesothelin-GITR-CD3 zeta CAR-T, or CD19-GITR-CD3 zeta CAR-T with CAR-T targeting other tumor antigens or tumor microenvironment (VEGFR-1-3) can be used to enhance activity of a monotherapy.

Bi-scFv-GITR CAR can be used to enhance activity of a single antibody scFv-GITR CAR, scFv-mesothelin CAR or scFv-CD19 CAR.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

EXAMPLES

The inventors generated EGFR, Mesothelin (Meso), CD 19 CAR constructs inside lentiviral vector cloned into Xba I and EcoR I sites of lentiviral vector. pCD510-FMC63-28z lentiviral CAR construct containing the CD8 signal peptide-EGFRscFv (or Meso scFv, or CD 19 scFv)-CD8 hinge-CD28 transmembrane intracellular domain-GITR domain-CD3 zeta insert - between the Xba I and EcoRI cloning sites.

The lentiviruses were generated in 293T cells and titer was established by RT-PCR. Then an equal dose of lentiviruses was used for transduction of T cells, as described in

Examples Section. We used CAR lentiviruses to transduce T cells and also used control non- transduced T cells to test cytotoxic activities of CAR-T cells against EGFR, mesothelin, or CD19-positive cell lines. Example 1. Cell Lines

A 1847, SKOV-3, ovarian cells and BxPC3 pancreatic cancer cells were cultured in DMEM (GE Healthcare, Chicago, IL) containing 10% FBS (AmCell, Mountain View, CA). Human peripheral blood mononuclear cells (PBMC) were isolated by density sedimentation over Ficoll-Paque {GE Healthcare). HEK293FT cells were a gift from AlStem (Richmond, CA) and were cultured in DMEM containing 10% FBS. All cell lines were authenticated by flow cytometry in our laboratory, using cell-specific surface markers.

Example 2. EGFR, Mesothelin, CD19 CAR Constructs

The ScFvs of Mesothelin P4 human antibody (Lanitis, et al. (2012), Mol Ther 20, 633-643), EGFR CIO human antibody ((Liu et al (2015) Cancer Res 75, 3596-3607), or mouse CD 19 FMC063 (Kochenderfer et al, 2009, JImmunother 32, 689-702. [13] were inserted into a second-generation CAR cassette flanked by Nhe I and Xho I restriction sites between a signaling peptide from human CD8, and a hinge region from CD8 alpha, then followed by transmembrane domain and costimulatory domains from (i) CD28, or (ii) GITR domain, and the CD3 zeta activation domain as shown in FIG. 3. DNAs encoding the CARs were synthesized and subcloned into a third-generation lentiviral vector, Lenti CMV-MCS- EFla-puro by Syno Biological (Beijing, China). All CAR lentiviral constructs were sequenced in both directions to confirm CAR sequence and used for lentivirus production. Example 3. Generation of CAR-Encoding Lentivirus

Ten million growth-arrested HEK293FT cells {Thermo Fisher) were seeded into T75 flasks and cultured overnight, then transfected with the pPACKHl Lenti vector Packaging mix {System Biosciences, Palo Alto, CA) and 10 μg of each lentiviral vector using the CalPhos Transfection Kit (Takara, Mountain View, CA). The next day the medium was replaced with a fresh medium, and 48 h later the lentivirus-containing medium was collected. The medium was cleared of cell debris by centrifugation at 2100 g for 30 min. The virus particles were collected by centrifugation at 112,000 g for 100 min, suspended in AIM V medium, aliquoted and frozen at -80 °C. The titers of the virus (expressed in pfu/ml) were determined by quantitative RT-PCR using the Lenti-X qRT-PCR kit (Takara) according to the manufacturer's protocol and the 7900HT thermal cycler (Thermo Fisher).

Example 4. Generation and Expansion of CAR-T Cells

PBMCs were suspended at 1 x 10 ⁶ cells/ml in AIM V-AlbuMAX medium (Thermo Fisher) containing 10% FBS and 300 U/ml IL-2 (Thermo Fisher), mixed with an equal number (1 :1 ratio) of CD3/CD28 Dynabeads (Thermo Fisher), and cultured in non-treated 24-well plates (0.5 ml per well). At 24 and 48 hours, lentivirus was added to the cultures at a multiplicity of infection (MOI) of 5, along with 1 μΐ of TransPlus transduction enhancer (AlStem). As the T cells proliferated over the next two weeks, the cells were counted every 2- 3 days and fresh medium with 300 U/ml IL-2 was added to the cultures to maintain the cell density at 1-3 x 10 ⁶ cells/ml.

Example 5. Flow Cytometry

To measure CAR expression, 0.5 million cells were suspended in 100 μΐ of buffer (PBS containing 0.5% BSA) and incubated on ice with 1 μΐ of human serum (Jackson

Immunoresearch, West Grove, PA) for 10 min. Then 1 μΐ of allophycocyanin (APC)-labeled anti-CD3 (eBioscience, San Diego, CA), 2 μΐ of 7-aminoactinomycin D (7-AAD, BioLegend, San Diego, CA), and 2 μΐ of biotin- labeled polyclonal goat anti-human-F(ab) ₂ antibodies (Life Technologies) that detect EGFR scFv, Meso scFv, or CD 19 scFv, or biotin- labeled normal polyclonal goat IgG antibodies (Life Technologies) were to detect CAR expression. The cells were rinsed with 3 ml of buffer, then suspended in buffer and acquired on a FACSCalibur (BD Biosciences). Cells were analyzed first for light scatter versus 7-AAD staining, then the 7-AAD ^" live gated cells were plotted for CD3 staining versus F(as)2 staining or isotype control staining.

Example 6. Real-Time Cytotoxicity Assay (RTCA)

Adherent target cells were seeded into 96- well E-plates (Acea Biosciences, San Diego, CA) at 1 x 10 ⁴ cells per well and monitored in culture overnight with the impedance- based real-time cell analysis (RTCA) iCELLigence system (Acea Biosciences). The next day, the medium was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± 1 x 10 ⁵ effector cells (CAR-T cells or non-transduced T cells), in triplicate. The cells in the E-plates were monitored for another 2 days with the RTCA system, and impedance was plotted over time. Cytolysis was calculated as (impedance of target cells without effector cells - impedance of target cells with effector cells) xlOO/ impedance of target cells without effector cells.

Example 7. Cytokine Secretion Assay

The target cells were cultured with the effector cells (CAR-T cells or non- transduced T cells) at a 1:1 ratio (1 x 10 ⁴ cells each) in U-bottom 96-well plates with 200 μΐ of of AIM V- AlbuMAX medium containing 10% FBS, in triplicate. After 16 h the top 150 μΐ of medium was transferred to V-bottom 96-well plates and centrifuged at 300 g for 5 min to pellet any residual cells. The top 120 μΐ of supernatant was transferred to a new 96-well plate and analyzed by ELISA for human IFN-gamma and IL-2 levels using kits from Thermo Fisher according to the manufacturer's protocol.

Example 8. EGFR-GITR-CD3 zeta CAR Sequences.

The CAR construct of this example: Human CD8 signaling peptide, human EGFR scFv derived from CIO low affinity EGFR antibody [5, 8] (V _H -Linker-V _L ), CD8 hinge, CD28 transmembrane, co- stimulatory domain (GITR), CD3 zeta activation domain (see FIG. 3). CIO EGFR antibody has dissociation constant K _D of 265 nM for A431 cells.

The nucleotide sequence of lentiviral vector with EGFR-GITR-CD3 zeta CAR (FIG. 3) inside EcoRl and Xhol site is shown in SEQ ID NO: 1. The EGFR scFv is flanked with Nhel and Xhol sites and can be substituted by other scFv such as MESO scFv or CD 19 scFv.

SEQ ID NO: 1:

tctagagccgccaccATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTG CTGCTCCACGCCGCCAG GCCGgctagc

gaagtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcagcagcgtgaaa gtg agctgcaaagcgagcggcggcacctttagcagctatgcgattagctgggtgcgccaggcg ccgggccagggcctggaatggatgggcggcattattccgatttttggcaccgcgaactat gcgcagaaatttcagggccgcgtgaccattaccgcggatgaaagcaccagcaccgcgtat atggaactgagcagcctgcgcagcgaagataccgcggtgtattattgcgcgcgcgaagaa ggcccgtattgcagcagcaccagctgctatggcgcgtttgatatttggggccagggcacc ctggtgaccgtgagcagc

6S GGC∞ SGWCT 6S GGC∞ SGWCT GG GGCGGTGG CT cagagcgtgctgacccaggatccggcggtgagcgtggcgctgggccagaccgtgaaaatt acctgccagggcgatagcctgcgcagctattttgcgagctggtatcagcagaaaccgggc caggcgccgaccctggtgatgtatgcgcgcaacgatcgcccggcgggcgtgccggatcgc tttagcggcagcaaaagcggcaccagcgcgagcctggcgattagcggcctgcagagcgaa gatgaagcggattattattgcgcggcgtgggatgatagcctgaacggctatctgtttggc gcgggcaccaaactgaccgtgctg

ctcgagAAGCCCACCACG¾CGCCAGCGCCGCG¾CCACCA¾CACCGGCGCCCACC ¾TCGCGTCGCAGCCCCTGTC CCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGA CTTCGCCAGTGATa agcccfctttgggfcgctggtggtggttggtggagtcctggctfcgctatagcttgctag taacagfcggccttfcatt atttt ctgggtg

CAGCTTGGACTGCACATCTGGCAGCTGAGGAGTCAGTGCATGTGGCCCCGA GAGACCCAGCTGCTGCTGGAGGTGCCGCCGTCGACCGAAGACGCCAGAAGCTGCCAGTTC CCCGAGGAAGAGCGGGGCGAGCGATCGGCAGAGGAGAAGGGGCGGCTGGGAGACCTGTGG GTG

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG CTCTATAACGAGCTCAA TCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT GGGGGGAAAGCCGC AGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG AGGCCTACAGTGAG AT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT ACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAtaggaattc

SEQ ID NO: 2 below is the amino acid sequence of SEQ ID NO: 1. MA L P VTA L L L P L AL L L HAARPA SEVQLVQSGAEVKKPGSSV KVSCKASGGTFSSYAISWVPvOAPGOGLEWMGGIIPIFGTAN YAOKFOGRVTITADESTSTAYMELSSLRSEDTAVYYCAREE GPYCSSTSCYGAFDIWGOGTLVTVSSGGGGSGGGGSGGGGS OSVLTODPAVSVALGOTVKITCOGDSLRSYFASWYOOKPGO APTLVMYARNDRPAGVPDRFSGSKSGTSASLAISGLOSEDE ADYYCAAWDDSLNGYLFGAGTKLTVLL EKPTTTPAPRPPT PAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWV LVVVGGVLACYSLLVTVAFIIFWVQLGLHIWQLRSQCMWP RETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDL WYR VKFSRSADAPA YQOGONOL YNELNL GRREEYDVLDKRRG RDPEMGGKP ORRKNP OEGL YNEL OKDKMAEA YSEIGMKGER RRGKGHDGL YQGLSTA TKD TYDAL HMQALPPR

The scheme of EGFR-GITR CAR construct is shown below, which shows the sub-domain nucleotide sequences and amino acid sequences of SEQ ID NOs: 1 and 2, respectively.

<huCD8 signal peptide>

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG CCAGGCCG (SEQ ID NO: 3)

MALPVTALLLPLALLLHAARP (SEQ ID NO: 4)

<NheI restriction site>

GCTAGC Amino-acids: AS

< EGFR scFV>

Nucleotide sequence: SEQ ID NO: 5

gaagtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcagcagcgtgaaa gtg

agctgcaaagcgagcggcggcacctttagcagctatgcgattagctgggtgcgccag gcg

ccgggccagggcctggaatggatgggcggcattattccgatttttggcaccgcgaac tat

gcgcagaaatttcagggccgcgtgaccattaccgcggatgaaagcaccagcaccgcg tat

atggaactgagcagcctgcgcagcgaagataccgcggtgtattattgcgcgcgcgaa gaa

ggcccgtattgcagcagcaccagctgctatggcgcgtttgatatttggggccagggc acc

ctggtgaccgtgagcagc

GGTGGCGGTGGTTCT GGTGGCGGTGGTTCT GGTGGCGGTGGTTCT cagagcgtgctgacccaggatccggcggtgagcgtggcgctgggccagaccgtgaaaatt

acctgccagggcgatagcctgcgcagctattttgcgagctggtatcagcagaaaccg ggc

caggcgccgaccctggtgatgtatgcgcgcaacgatcgcccggcgggcgtgccggat cgc

tttagcggcagcaaaagcggcaccagcgcgagcctggcgattagcggcctgcagagc gaa

gatgaagcggattattattgcgcggcgtgggatgatagcctgaacggctatctgttt ggc

gcgggcaccaaactgaccgtgctg

Amino-acid sequence: SEQ ID NO: 6, which is the underlined sequence in SEQ ID NO: 2 EVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAP GOGLEWMGGIIPIFGTANYAOKFQGRVTITADESTSTAYMEL SSLRSEDTAVYYCAREEGPYCSSTSCYGAFDIWGOGTLVTV SSGGGGSGGGGSGGGGSOSVLTODPAVSVALGOTVKITCOG DSLRSYFASWYOOKPGOAPTLVMYARNDRPAGVPDRFSGSK SGTSASLAISGLQSEDEADYYCAAWDDSLNGYLFGAGTKLT VL

<XhoI restriction site>

CTCGAG

Amino-acids: LE <CD8 hinge>

AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCG TCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCA GTGCACACGAGGGGGCTGGACTTCGCCAGTGATaagccc (SEQ ID NO: 7) Amino-acid sequence: SEQ ID NO: 8, which is the larger font, bold sequence in SEQ ID NO: 2

KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAV HTRGLDFASDKP

<CD28 transmembrane domain (CD28 TM)>

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAG TGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 9) Amino-acid sequence:

FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 10) <GITR intracellular domain, gitr>

CAGCTTGGACTGCACATCTGGCAGCTGAGGAGTCAGTGCATGTGGCCCCGAGAG ACCCAGCTGCTGCTGGAGGTGCCGCCGTCGACCGAAGACGCCAGAAGCTGCCAG TTCCCCGAGGAAGAGCGGGGCGAGCGATCGGCAGAGGAGAAGGGGCGGCTGGG AGACCTGTGGGTG ( SEQ ID NO: 11)

Amino-acid sequence: SEQ ID NO: 12, which is the bold sequence in SEQ ID NO: 2

QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPE

EERGERSAEEKGRLGDLWV <CD3 zeta activation domain>

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCC TCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGCTAATAG (SEQ ID NO: 13)

Amino-acid sequence: SEQ ID NO: 14, which is in italics, larger font, underlined in SEQ ID NO: 2

R VKFSRSADAPA YQ O G ON O L YNELNL GRREEYDVLDKRRGRD P EMet G GKP RRKNP OEGL YNEL OKDKMAEA Y S E I G M K G E RRR G KG HP GLYQGLSTA TKD T YD A L H M O A L P P R Example 9. The Sequence of EGFR-GITR-CD3 zeta-CAR

This construct includes human CD8 signaling peptide, human EGFR scFv (VH- Linker-VL) derived from P3-5 [5, 8], CD8 hinge, CD28 transmembrane, GITR co- stimulatory domain, CD3 zeta activation domain (FIG.3). P3-5 has K _D =88 nM for 431 cells. Linker is 3x(GGGGS, SEQ ID NO: 15).

The sequences are similar to those described in Example 8 except the human EGFR scFv is derived from P3-5.

V _H P3-5

The underlined and bold amino-acids are different from CIO Amino-acid sequence.

EVQLVQSGAEVKKPGSSVKSCKASGGTFSSYAISWVRQAPGQGLEWVG GIIPIFGTANYAQKFQGRVKITADESASTAYMELSSLRSEDTAVYYCAR EEGPYCSSTSCYGAFDIWGQGTLVTVSS (SEQ ID NO: 16) Reverse translate using wwv^bioiTrfom¾atcs^)rgstns2/rev^ran .htxni to a 375 base sequence of most likely codons.

gaagtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcagcagcgtgaaa agc

tgcaaagcgagcggcggcacctttagcagctatgcgattagctgggtgcgccaggcg ccg

ggccagggcctggaatgggtgggcggcattattccgatttttggcaccgcgaactat gcg

cagaaatttcagggccgcgtgaaaattaccgcggatgaaagcgcgagcaccgcgtat atg

gaactgagcagcctgcgcagcgaagataccgcggtgtattattgcgcgcgcgaagaa ggc

ccgtattgcagcagcaccagctgctatggcgcgtttgatatttggggccagggcacc ctg

gtgaccgtgagcagc (SEQ ID NO: 17)

V _L P3-5

The underlined amino-acids are different from CIO Amino-acid sequence.

QSVLTQDPAVSVALGQTVKITCQGDSLRSYLASWYQQKPGQAPTLVTYARNDRPA GVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYLFGAGTKLTVL (SEQ ID NO: 18)

Reverse translation of amino-acid sequence to a 324 base sequence of most likely codons. cagagcgtgctgacccaggatccggcggtgagcgtggcgctgggccagaccgtgaaaatt

acctgccagggcgatagcctgcgcagctatctggcgagctggtatcagcagaaaccg ggc

caggcgccgaccctggtgacctatgcgcgcaacgatcgcccggcgggcgtgccggat cgc

tttagcggcagcaaaagcggcaccagcgcgagcctggcgattagcggcctgcagagc gaa

gatgaagcggattattattgcgcggcgtgggatgatagcctgaacggctatctgttt ggc

gcgggcaccaaactgaccgtgctg (SEQ ID NO: 19)

Example 10. The Sequence of EGFR-CD28-CD3 zeta CAR

This construct includes human CD8 signaling peptide, human EGFR scFv (VH- Linker-VL), CD8 hinge, CD28 transmembrane, CD28 co- stimulatory domain, CD3 zeta activation domain (see FIG.3). Linker is 3x(GGGGS, SEQ ID NO: 15).

The sequences are similar to those described in Example 8 except CD28 is used as a co- stimulatory domain.

<CD28 co-stimulatory domain sequence>

Nucleotide sequence:

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCG CAGCCTATCGCTCC (SEQ ID NO: 20)

Amino acid sequence:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

(SEQ ID NO: 21) Example 11. EGFR-4-1BB-CD3 zeta CAR

This construct includes human CD8 signaling peptide, human EGFR scFv (VH- Linker-VL), CD8 hinge, CD28 transmembrane, 4- IBB co-stimulatory domain, CD3 zeta activation domain (see FIG. 3). Linker is 3x(GGGGS, SEQ ID NO: 15).

The sequences are similar to those described in Example 8 except 4- IBB is used as a co-stimulatory domain.

4-1BB co-stimulatory domain sequence:

Nucleotide sequence:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAACTG (SEQ ID NO: 22)

Amino-acid sequence

K R G R K K L L Y I F K Q P F Met R P V Q T T Q E E D G C S C R F P E E E E G G C E L (SEQ ID NO: 23)

Example 12. Mesothelin-GITR-CD3 zeta CAR sequence

Instead of EGFR ScFV in SEQ ID NOs: 1 and 2 as shown in Example 8, mesothelin

(human P4 Ab) ScFv was inserted into Xho I and Nhe I of SEQ ID NO: 1.

The Mesothelin-GITR CAR nucleotide sequence is shown in SEQ ID NO: 24 below. The first two highlighted GCTAGC and CTCGAG indicate Xho I and Nhe I sites of mesothelin insertion, respectively. The third highlighted CAGCTT indicates GITR start. ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG CCAGGCCG^^^BGAAGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAA CCGGGCAGCAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCGGCACCTTTAGCAGC TATGCGATTAGCTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGC GGCATTATTCCGATTTTTGGCACCGCGAACTATGCGCAGAAATTTCAGGGCCGCG TGACCATTACCGCGGATGAAAGCACCAGCACCGCGTATATGGAACTGAGCAGCC TGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCGAAGAAGGCCCGTATT GCAGCAGCACCAGCTGCTATGGCGCGTTTGATATTTGGGGCCAGGGCACCCTGGT

GACCGTGAGCAGCuG iGC.sCOG iGC.s i fC TGGl GiiCGGl U I HJ! GGTUGGCJGTUG

TTCTC AGAGCGTGCTGACCC AGGATCCGGCGGTGAGCGTGGCGCTGGGCC AGAC CGTGAAAATTACCTGCCAGGGCGATAGCCTGCGCAGCTATTTTGCGAGCTGGTAT CAGCAGAAACCGGGCCAGGCGCCGACCCTGGTGATGTATGCGCGCAACGATCGC CCGGCGGGCGTGCCGGATCGCTTTAGCGGCAGCAAAAGCGGCACCAGCGCGAGC CTGGCGATTAGCGGCCTGCAGAGCGAAGATGAAGCGGATTATTATTGCGCGGCG TGGGATGATAGCCTGAACGGCTATCTGTTTGGCGCGGGCACCAAACTGACCGTGC TG^^^HAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCA CCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGG GGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGG TGCTGGTGGTGGTTGGTGGAG \€C rCGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGCAGCTTGGACTGCACATCTGGCAGCTGAGGAGTCAG I GCA I G I GGCCCCGAGAGACCCAGC I GC I GC I GGAGG I GCCGCCG I CGACCGAA GACGCCAGAAGCTGCCAGTTCCCCGAGGAAGAGCGGGGCGAGCGATCGGCAGA GGAGAAGGGGCGGCTGGGAGACCTGTGGGTGAGAGTGAAGTTCAGCAGGAGCG CM \ ACGCCC ^' CCGCG ^' i AC. C AC iC AC XK XX ^* AGA ACC AC =C; C\ Λ1 AAC( i Λί ΧΊ€ΛΛΊ C TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTG AGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAAT GAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGG CGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGC CACCAACKjACACCTACGACGCCCm:ACATGCAGCKXCTC XXCCTCC¾TAA

The amino acid sequence of mesothelin-GITR CAR is shown as SEQ ID NO: 25.

MALPVTALLLPLALLLHAARPASEVQLVQSGAEVKKPGSSV KVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANY AQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREEG PYCSSTSCYGAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSQ SVLTQDPAVSVALGQTVKITCQGDSLRSYFASWYQQKPGQA PTLVMYARNDRPAGVPDRFSGSKSGTSASLAISGLQSEDEAD YYCAAWDDSLNGYLFGAGTKLTVLLEKPTTTPAPRPPTPAP TIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVV GGVLACYSLLVTVAFIIFWVQLGLHIWQLRSQCMWPRETQL LLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWVRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR

Example 13. CD19-GITR-CD3 zeta CAR sequence

Instead of EGFR ScFV shown in Example 8 in SEQ ID No 1 and 2, CD 19 ScFv was inserted into Xho I and Nhe I sites in SEQ ID NO: 1.

The CD19-GITR-Zeta CAR nucleotide sequence is shown in SEQ ID NO: 26 below. The first two highlighted GCTAGC and CTCGAG indicate Xho I and Nhe I sites of CD 19 ScFv insertion. The third highlighted CAGCTT indicates GITR start.

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG

gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtc accatcagttgcagggcaagtcaggacatta gtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctacc atacatcaagattacactcaggagtcccatc aaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagca agaagatattgccacttacttttgccaacag ggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacc tctggatccggcaagcccggatctggc gagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccc tcacagagcctgtccgtcacatgca ctgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaa agggtctggagtggctgggagtaatatgg ggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggac aactccaagagccaagttttcttaaaaatg aacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggt ggtagctatgctatggactactggggtcaag gaacctcagtcaccgtctcctcagcggccgca ^¾AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCAT CGCGTCGCAGCCCCmrCCCmCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGG CGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATaagcccttttgggtgctggtggi ggitgg t a tcct gcitgctatagciigctagiaacagiggccttiattaittictgggtg

CAGCTTGGACTGCACATCTGGCAGCTGAGGAGTCAGTGCATGTGGCCCCGA

GAGACCCAGCTGCTGCTGGAGGTGCCGCCGTCGACCGAAGACGCCAGAAGCTGC

CAGTTC

CCCGAGGAAGAGCGGGGCGAGCGATCGGCAGAGGAGAAGGGGCGGCTGGGAGA CCTGTGG GTG

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAA CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA

CAGTGAGATTGGGATGAAAGGCGAGI^GCX XIAGGGGCA

TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCA

GGCCCTGCCCCCTCGCTAA

The amino acid sequence of CD19-GITR zeta CAR is shown as SEQ ID NO: 27. CD19 scFv is bolded, GITR is underlined.

M A L P V T A L L L P L A L L L H A A R P A S D I Q M T Q T T S S L S A S L G D R V TISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVP SRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPS QSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC AKHYYYGGSYAMDYWGQGTSVTVSSAAALEKPTTTPAPRP PTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWV LVVVGGVLACYSLLVTVAFIIFWVOLGLHIWOLRSOCMWPR ETOLLLEVPPSTEDARSCOFPEEERGERSAEEKGRLGDLWVR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR We also used CD19-Flag ScFv and GM-CSF signaling peptide instead of CD8 signaling peptide to generate CD19-Flag-GITR-CD3 zeta CAR, the activity of this CAR was similar to the above CD19-GITR-CD3 zeta construct.

We also tested CD19-GITR-CD3 zeta with CD8 trans-membrane domain

(I YIW APL AGTCG VLLLSL VITL YC . SEQ ID NO: 28) instead of CD28 trans-membrane domain and both constructs (see FIG.3) had similar activities. The nucleotide and amino acids sequences of CD19-GITR-CD3 zeta with CD8 transmembrane domain is shown below.

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG CCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGG AGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAA TTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCA AGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGAT TATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCC AACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAA C AGGTGGCGGTGGC AGCGGCGGTGGTGGTTCCGGAGGCGGCGGTTCTGAGGTGA AACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCA CATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCA GCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCAC ATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAG AGCC AAGTTTTCTTAAAAATGAACAGTCTGC AAACTGATGAC AC AGCC ATTTACT ACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCA AGGAACCTCAGTCACCGTCTCCTCA

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCTGTGATATC TACATC TGGGCGCCCC FGGCCGGGA

CTTGTGC KnrCTTCTCCT

CACATCTGGCAGCTGAGGAGTCAGTGCATGTGGCCCCGAGAGACCCAGCTGCTG CTGGAGGTGCCGCCGTCGACCGAAGACGCCAGAAGCTGCCAGTTCCCCGAGGAA GAGCGGGGCGAGCGATCGGCAGAGGAGAAGGGGCGGCTGGGAGACCTGTGGGT GAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGA ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGG ACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCT TTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCTAA (SEQ ID NO: 29)

The amino-acid sequence of CD19-GITR-CD3 zeta CAR with CD8 trans-membrane domain is shown below, CD8 transmembrane domain is underlined, GITR domain is shown in bold.

MALP VTALLLPLALLL HA A RPO1QMTQTTS SLS ASLGDRVTIS CRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLE ITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVS GVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSR LTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAM DYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCO LGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEE RGERSAEEKGRLGDLWVRVKFSRSADAPAYKQGQNQLYN ELNLGRREEYDVLDKRRGRDPE Met GGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO: 30)

Example 14. The EGFR-GITR-CD3 zeta CAR-T cells demonstrate efficient expansion in culture

The EGFR-GITR CAR-T cells were effectively expanded in vitro (FIG.4). EGFR- GITR CAR-T cells were expanded more than 60-fold during 20 days in culture. The EGFR- 4-lBB-CD3z-CAR-T cells slowed their growth after 16 days. The CD19-CD28-CD3 zeta CAR-T and Non-CD 19-CAR-T cells and non-transduced T cells were also effectively expanded in vitro .

The results in FIG.4 show that EGFR-GITR-CD3 zeta CAR cells expanded better than EGFR 4-1BB-CD3 zeta and EGFR 28-CD3 zeta CAR-T cells.

Example 15. Transduction of T cells with EGFR-CAR lentiviral constructs

demonstrates expression of EGFR ScFv

To detect transduction the expression of human scFv from CIO EGFR antibody, CAR-T cells were stained with anti-human FAB antibody. In FIGs.5A-5D, Fab staining demonstrates the expression of EGFR scFv in transduced T cells. Fab staining with anti- human Fab antibody in EGFR-CAR-T cells was higher than that in non-transduced T cells. The results in FIG.5 show efficient transduction with lentiviral CAR. The expression of EGFR scFv was 22-32% higher than that in control non-transduced cells (9.3%).

Example 16. EGFR-GITR-CD3 zeta CAR expressed high cytotoxic activity against EGFR-positive cancer cells and had and no cytotoxic activity against EGFR-negative cancer cells.

The real-time cytotoxicity assay demonstrates high cytotoxic activity of EGFR-GITR- CD3 zeta-CAR cells against EGFR-positive cancer cells (FIG.6). The cytotoxic activity of EGFR-CAR was cancer-type dependent. For example, in U87 glioblastoma cells, EGFR- GITR-CD3z-CAR-T cells had better cytotoxic activity than EGFR-CD28-CD3 zeta-CAR-T cells (FIG.6A). The EGFR-GITR-CD3 zeta CAR-T activity was dose dependent; the activity increased at 20:1 compared to 10:1 ratio.

In SKOV-3 ovarian cancer cells, activities of EGFR-GITR-CD3 zeta CAR-T and EGFR-CD28-CD3 zeta CAR-T cells were the same, but higher than the activities of EGFR-4- 1BB-CD3 zeta and Mock-control CAR-T cells (FIG.6B). GITR and CD28 co- stimulatory domains had higher cytotoxic activity than 4-lBB-containing CAR-T cells in SKOV-3 ovarian cancer cells (FIG. 6 B).

In another ovarian cancer cell line, A 1847, EGFR-GITR-CD3 zeta activity was better than EGFR-CD28-CD3 zeta at 30:1 E:T (Effector: target cell ratio) (FIG. 6C).

In pancreatic cancer cells BxPC3, cytotoxic activity of EGFR-CD28-CD3 zeta was higher than EGFR-GITR-CD3 zeta and EGFR-41BB-CD3 zeta (FIG. 6D). There was no cytotoxic activity in EGFR-negative cells, MCF-7 cells (FIG. 6E).

Example 17. Mesothelin-GITR-CD3 zeta CAR expressed high cytotoxic activity against mesothelin-positive cancer cells.

We generated mesothelin-GITR-CD3 zeta CAR-T cells with mesothelin-GITR-CD3 zeta CAR constructs (FIG. 3) and tested their cytolytic activity in on A1847, SKOV-3 ovarian cancer and BxPC3 pancreatic cancer cells.

Lentiviral vectors with CMV promoters were used for generation of mesothelin- GITR-CD3 zeta and mesothelin-CD28-CD3 zeta (2nd generation) CAR-T cells. Lentiviral vector with EF1 promoter was used for generation of mesothelin-28-41BB-CD3 (3rd generation) CAR-T cells. Mesothelin-28-CD3 zeta CAR T cells are labelled as Meso-28- CAR-T 2nd cells. Mesothelin-GITR-CD3 zeta CAR T cells are labeled as Meso-GITR CAR- T cells. Mesothelin-28-4-lBB-CD3 zeta CAR T cells are labeled as Meso-28-CAR-T cells 3d generation.

Mesothelin-GITR-CD3 zeta CAR and mesothelin -CD28-CD3 zeta CAR killed mesothelin -positive cancer cells (FIG. 7) and had better activities than mesothelin-28-4- 1BB-CD3 zeta (third generation) CAR-T cells. FIG. 7, bottom panel shows that in SKOV-3 cells, mesothelin-GITR-CAR-T cells killed faster than mesothelin-CD28-CD3 zeta CAR T cells and mesothelin-28-4-lBB-CD3 zeta CAR-T cells.

Example 18. Mesothelin-GITR CAR-T cells had the same cytotoxic activity against cancer cells and produced less IFN-gamma cytokine than mesothelin-CD28 CAR-T cells.

We tested mesothelin-GITR-CD3 zeta CAR-T cells and mesothelin-CD28-CD3 zeta; both had about the same cytotoxic activity against BxPC3 cancer cells, for their production of cytokine IFN-gamma. Mesothelin-GITR-CD3 zeta CAR T cells secreted 2-fold less IFN- gamma than that secreted by mesothelin-CD28-CD3 zeta CAR T cells (FIG. 8), which suggests that mesothelin-GITR-CD3 zeta CAR-T cells may have advantages in clinic due to less cytokine release syndrome in patients.

Example 19. CD19-GITR-CD3 zeta CAR expressed high cytotoxic activity against CD19-positive cancer cells.

We generated CD19-GITR-CD3 zeta with CD8 transmembrane domain and CD28 transmembrane domain CAR-T cells and compared their cytolytic activities against Hela- CD19+ positive cells. Both CD19-GITR CAR-T cells had >50% killing activity (FIG. 9). The CD19-CD28-CD3 zeta had 100% killing activity (not shown). The less activity of CD19- GITR CAR-T cells can result in less toxicity against normal cells in clinic and can have advantages in future clinical trials.

Example 20. EGFR-GITR-CD3 zeta CAR blocked tumor growth of Raji leukemia cells in mice.

We compared the in vivo killing activity of CD19-GITR-CD3 zeta CAR-T cells in Raji xenograft mouse model. Raji-luciferase+ cells were subcutaneously injected into NSG mice. At day 12, CD 19-GITR-C AR-T cells (1x10' cells/mice) were injected intravenously when tumors reached >50 mm ³ . CD19-GITR-CD3 zeta CAR-T cells decreased

bioluminescent intensity (BLI) versus lxPBS control-treated mice or mice with injected T cells control (FIG. 10). The results demonstrate high in vivo efficacy of CD 19-GITR-CD3 zeta CAR-T cells. All three mice treated with Cdl9-GITR-Cd3 CAR-T cells decreased BLI in contrast to control groups that had either increased or unchanged signals (FIG. 10). References

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