WO2016025884A1 - Glycosyl-phosphatidylinositol (gpi)-linked gdnf family alpha-receptor 4 (gfralpha4)-specific antibody and uses thereof - Google Patents

Abstract

The present invention relates to compositions and methods for diagnosing and treating diseases, disorders or conditions associated with the expression of the Glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4).

Description

TITLE

Glycosyl-phosphatidylinositol (GPI)-linked GDNF family alpha-receptor 4

(GFRalpha4)-Specific Antibody And Uses Thereof

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application Serial No. 62/037,434, filed August 14, 2014, the content of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Thyroid cancer is one of the few cancers that has increased in incidence over recent years with the incidence of new cases rising on average 5.5% each year from 2002- 2011. It is the most common endocrine cancer with an expected incidence of -60,000 new cases this year and about 2,000 deaths. Papillary and follicular thyroid carcinomas comprise 80-90% of all thyroid cancers while medullary and anaplastic comprise 5-10% and 1-2% respectively (Pacini et al, Ann Oncol 23 (suppl 7), 2012; Howlader et al, SEER Cancer Statistics Review, 1975-201 1, National Cancer Institute). While thyroid cancer has a good prognosis overall, this is not necessarily the case for the medullary and anaplastic forms if they are not treated early before they spread beyond the thyroid gland.

Medullary Thyroid Cancer (MTC) is a type of thyroid cancer that develops from the parafollicular cells of the thyroid that are not related with the main function of the thyroid gland, i.e. production and secretion of thyroid hormone.

Rather, these cells are involved in the production of calcitonin, a calcium-regulatory hormone apparently unimportant to humans for maintaining calcium homeostasis. Approximately 25% of MTC is genetic in nature caused by a mutation in the proto- oncogene receptor tyrosine kinase RET (Pacini et al, Clin Oncol, 22(6):475-85, 2010; Roy et al, Oncologist, 18(10): 1093-100, 2013). MTC can also coexist with tumors of the parathyroid gland and adrenal gland (pheochromocytoma) in a syndrome known as multiple endocrine neoplasia type 2 (MEN2). Calcitonin doubling time (CDT) can be used as a prognostic marker; e.g. when the CDT is <6 months, 5-year survival is <25%. Surgery and radiation therapy are used for MTC, though risk of recurrence remains high due to the fact that 50% of patients have metastasis to regional lymph nodes at the time of diagnosis. Tyrosine kinase inhibitors such as vandetanib

(Caprelsa) and cabozantinib (Cometriq) were approved by the FDA in April, 201 1 and November, 2012, respectively, for treatment of late-stage metastatic MTC, though only 10-30% of patients show clear evidence of response.

The GDNF family of neurotrophic factors includes four members: glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin, and persephin (PSPN). GDNF family ligands signal through receptors consisting of a GPI-linked GFRa subunit and the transmembrane receptor tyrosine kinase RET. In order to activate the transmembrane receptor tyrosine kinase RET, each of the GDNF family neurotrophic factors binds preferentially to one of the glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptors (GFRal-4) (Airaksinen et al, Mol Cell Neurosci.; 13(5):313-25, 1999). GDNF signals via GFRal, neurturin via GFRa2 , artemin via GFRa3: however, the mammalian GFRa receptor for persephin (PSPN) and the biological role of GFRa4 has so far remained unclear. In adult humans, GFRa4 is restricted to normal and malignant thyroid medullary cells (Lindahl et al, J. Biol. Chem. 276:9344-51, 2001), although it may be expressed elsewhere during fetal development. GFRal, GFRa2, and GFRa3 appear to be expressed in non-thyroid tissues of the human body that may include brain.

Thus, the relative specific expressions of GFRa4 on the cell surface of malignant parafollicular cells of the thyroid tissues make it an attractive target for MTC tumor diagnosis and therapy. Although generic anti-GFRa4 antibodies were previously identified (WO2001062795A1 - Patent Application Number 10/203639), GFRa4-specific T bodies, particularly the GFRa4-specific scFv as targeting moieties, remain unexplored.

There is a need in the art for the development of therapies to treat medullary thyroid carcinoma. The present invention addresses this need.

SUMMARY OF THE INVENTION

As described below, the present invention includes compositions and methods for diagnosing and treating diseases, disorders or conditions associated with the expression of the Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a- receptor 4 (GFRa4).

In one aspect, the invention includes a composition comprising an anti- Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (anti-GFRa4) binding domain, wherein the anti-GFRa4 binding domain is a GFRa4 antibody, or fragment thereof.

In yet another aspect, the invention includes a isolated polynucleotide encoding an anti-GFRa4 antibody or a fragment thereof comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20 and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 12 and 28.

In still another aspect, the invention includes an isolated polypeptide encoding an anti-GFRa4 antibody or a fragment thereof comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20 and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 12 and 28.

In another aspect, the invention includes a method of imaging or visualizing a sample taken from a normal or malignant thyroid medullary cell, the method comprising contacting the sample with a labeled anti-GFRa4 binding domain.

In yet another aspect, the invention includes a method of diagnosing a condition in a mammal associated with the expression of GFRa4 in a cell, the method comprising a) contacting the cell with an anti-GFRa4 antibody fragment comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20 and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 12 and 28; and b) detecting the presence of GFRa4 in the cell, wherein the presence of GFRa4 in the cell is an indication that the mammal has the condition.

In another aspect, the invention includes a method of diagnosing, or determining risk of thyroid cancer in a mammal, the method comprising : a) contacting a sample with an anti-GFRa4 antibody fragment comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20, and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 12 and 28; and b) detecting the presence of GFRa4 in the sample wherein the presence of GFRa4 is an indication that the mammal has or is at risk of having thyroid cancer, wherein the sample is not derived from thyroid tissue.

In still another aspect, the invention includes a method of inhibiting growth of a GFRa4-expressing tumor cell, the method comprising contacting the tumor cell with an anti-GFRa4 antibody or a fragment thereof comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20 and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NO: 12 and 28.

In various embodiments of the above aspects or any other aspect of the invention delineated herein, the antibody or fragment thereof is a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody. In another embodiment, the anti-GFRa4 binding domain or the antibody or fragment thereof is encoded by a nucleic acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 35, 37, 39 and 40.

In another embodiment, the anti-GFRa4 binding domain is an antibody, or an antigen-binding fragment thereof, such as a fragment antigen-binding (Fab) or a single-chain variable fragment (scFv) or single-domain antibody. In yet another embodiment, the anti-GFRa4 binding domain binds specifically to a thyroid cell antigen present in a tumor microenvironment, such as a thyroid cell antigen is present on a medullary thyroid carcinoma (MTC) cell. In another embodiment, the thyroid cell antigen is a GFRa4 cell-surface receptor comprising at least one isoform selected from the group consisting of GFRa4a and GFRa4b.

In another embodiment, the nucleic acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 3 and 19, and the nucleic acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 1 1 and 27.

In yet another embodiment, the mammal is a human. In another embodiment, the cell or tumor cell is a medullary thyroid carcinoma cell.

In another embodiment, the anti-GFRa4 antibody or a fragment thereof binds specifically to the tumor cell, such as binds specifically to the GFRa4 on the tumor cell. In yet another embodiment, the GFRa4 comprises at least one isoform selected from the group consisting of GFRa4a and GFRa4b.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and

instrumentalities of the embodiments shown in the drawings.

Figure 1 displays the amino acid sequence of the fragment of GFRa4 isoform a expressed as a human Fc bivalent fusion protein used for selection of anti- GFRa4 antibodies and for stimulating CAR-transduced Jurkat reporter cells. The fragment comprises Asn24 - Ser245 of the native full-length protein (UniProt accession Q9GZZ7-2), a Factor Xa cleavage site, a human IgGl Fc domain fragment, and a 6 x His sequence for purification.

Figure 2 displays the amino acid sequence of the fragment of GFRa4 isoform b expressed as a human Fc bivalent fusion protein used for selection of anti-

GFRa4 antibodies and for stimulating CAR-transduced Jurkat reporter cells. The fragment comprises Asn24 - Val274 of the native full-length protein (UniProt accession Q9GZZ7-1), a tobacco etch virus (TEV) protease cleavage site and linker, and a human IgGl Fc domain fragment.

Figure 3 is a graph depicting the reactivity of recombinant human/rabbit chimeric Fabs against the GFRal, GFRa2, GFRa3, GFRa4a and GFRa4b, and demonstrates that 2 antibodies to GFRa4, P4-6 and P4-10, isolated by antibody phage display, crossreact with GFRa4a and GFRa4b, but do not show binding to GFRal, GFRa2, and GFRa3 above the background binding seen with only secondary antibody reagent (HRP-anti human Fab, "no Fab") or binding with an irrelevant recombinant human/rabbit chimeric lambda light chain-containing Fab ("control Fab") .

Figure 4, comprising Figures 4A-B, is a sequence alignment showing how the P4-6 scFv construct with restriction sites and linker was pieced together from P4-6 VH and VL segments. Figure 4A shows the nucleotide sequence alignments and

Figure 4B shows amino acid alignments.

Figure 5, comprising Figures 5A-B, is a sequence alignment showing how the P4-10 scFv construct with restriction sites and linker was pieced together from P4-10 VH and VL segments. Figure 5 A shows the nucleotide sequence alignments and Figure 5B shows amino acid alignments.

Figure 6 is a sequence alignment comparing the nucleotide bases between the original P4-6 scFv construct and the human codon optimized P4-6 scFv construct used for CAR construction.

Figure 7 is a sequence alignment comparing the nucleotide bases between the original P4-10 scFv construct and the human codon optimized P4-10 scFv construct used for CAR construction.

Figure 8 illustrates the map of the P4-6 CAR GS linker BBz plasmid. Map indicates the position of anti-GFRa4 scFv P4-6, glycine/serine rich linker, CD8 transmembrane domain, and the cytoplasmic fragment of the 4- IBB domain. In addition, the drawing depicts the positions of other components necessary for lentiviral construction as described in WO/2012/079000.

Figure 9 illustrates the map of the P4-10 CAR GS linker BBz plasmid. Map indicates the position of anti-GFRa4 scFv P4-10, glycine/serine rich linker, CD8 transmembrane domain, and the cytoplasmic fragment of the 4- IBB domain. In addition, the drawing depicts the positions of other components necessary for lentiviral construction.

Figure 10 illustrates the map of the P4-6 CD8 linker BBz plasmid. Map indicates the position of anti-GFRa4 scFv P4-6, CD8 hinge linker, CD8 transmembrane domain, and the cytoplasmic fragment of the 4- IBB domain. In addition, the drawing depicts the positions of other components necessary for lentiviral construction.

Figure 1 1 illustrates the map of the P4-10 CD8 linker BBz plasmid. Map indicates the position of anti-GFRa4 scFv P4-10, CD8 hinge linker, CD8 transmembrane domain, and the cytoplasmic fragment of the 4- IBB domain. In addition, the drawing depicts the positions of other components necessary for lentiviral construction.

Figure 12 is an image of the results of a flow cytometry experiment demonstrating the expression of the P4-6 and P4-10 scFv T bodies (CARs) on the T- cell surface. T bodies were detected with biotinylated donkey anti-rabbit IgG followed by phycoerythrin-conjugated streptavidin. Numbers on each panel adjacent to flow gate represent % cells positive for T body.

Figure 13 is an image of a series of flow cytometric measurements demonstrating the specificity of anti-GFRa4 CARs. Reporter Jurkat cells expressing GFP under an NFAT-responsive promoter were transduced with P4-6(gs) or P4-

10(gs) CAR's and incubated with various immobilized Fc-fusion proteins or cell lines. Figure shows that Jurkat cells are activated by immobilized GFRa4a protein, but not by its homologs GFRal, GFRa2, and GFRa3. Figure also shows that TT cells as well as MZ-CRC-1 cells (both MTC cell lines) also activate the Jurkat cells, but not K562 cells expressing mesothelin. Jurkat cells expressing the mesothelin-specific CAR (SS1KIRS2) were activated by K562 mesothelin-expressing cells, but not by TT or MZ-CRC-1 cells or by the immobilized GFRa proteins, including GFRa4a. Wells coated with the anti-CD3 antibody OKT3 represent positive control. Numerical values in figure above GFP-positive cell gate represent percentage of total Jurkat reporter cells in the positive gate.

Figure 14 is a graph demonstrating that T cells expressing P4-6(gs) and P4-10(gs) scFv T bodies (P4-6bbz and P4-10bbz, respectively) are capable of killing a calcitonin-secreting MTC cell line cells (TT cells) when these are incubated in vitro at several effector to target ratios. FMCbbz cells, a CD 19/mesothelin-specific CAR-T cell, serves as a negative control along with non-transduced (NTD) T cells.

Figure 15 is a control experiment showing that CD19/mesothelin- specific CAR-T cells (FMCbbz) lysed CD19/mesothelin-expressing K562 cells (K562-CD19meso) while the P4-6 and P4-10 CAR-T cells do not.

Figure 16 is an image of a series of flow cytometric measurements demonstrating the specificity of GFRa4-expressing CAR-T cells.

Figure 17 is an image of a series of flow cytometric measurements demonstrating the expression of GFRa4-specific CAR-T protein in CD4-positive and CD4-negative T cells from multiple healthy donors.

Figure 18 is a panel of two graphs demonstrating specific lysis of

GFRa4-expressing cells by anti- GFRa4-specific CAR-transduced T cells. Human T cells from two healthy donors transfected with either the FMC63bbz anti-CD 19 CAR or the P4-6bbz GFRa4-specific CAR were mixed at the indicated effector to target ratios with K562 cells (ATCC) expressing either GFRa4 isoform b (top graph) or human CD19 (bottom graph) pre-loaded with ⁵¹Cr.

Figure 19 is a panel of three graphs showing specific lysis of GFRa4- expressing tumor cells by T cells expressing anti-GFRa4-CARs with different cytoplasmic signaling domains.

Figure 20 is a panel of two graphs demonstrating that T cells expressing a GFRa4-specific P4-6bbz and P4-10bbz CARs show GFRa4-dependent secretion of the cytokines IFN-γ and IL-2.

Figure 21 is a panel of two graphs demonstrating that cells expressing a GFRa4-specific p4-10-28z CAR show GFRa4-dependent secretion of the cytokines IFN-γ and IL-2. Figure 22, comprising Figures 22A and 22B, is a panel of two graphs demonstrating that T cells expressing a GFRa4 CAR reduce the size of medullary thyroid carcinoma-derived TT cell tumors in vivo when TT cells were implanted sub- cutaneously and T-cells were injected intra-tumorally. Figure 22A shows the mean with standard error of the mean of tumor volume over time. Arrows indicate times of T cell injection. Figure 22B shows tumor size of individual mice at day 38 for each group (P=0.0008 by Mann- Whitney test). Mean and standard error of the mean are indicated for each group.

Figure 23, comprising Figures 23 A and 23B, is a panel of two graphs showing reduction in medullary thyroid carcinoma cell tumor size in mice treated intravenously with T cells expressing a GFRa4-specific CAR. Figure 23A shows the mean with standard error of the mean of tumor volume over time. The arrow indicates time of T cell injection. Figure 23B shows tumor size of individual mice at day 27 for each group (P=0.0093 by Mann- Whitney test).

Figure 24, comprising Figures 24A-24C, is a panel of two graphs showing reduction in medullary thyroid carcinoma cell tumor burden in mice treated intravenously with GFRa4 CAR-T cells. Each line in Figures 24A and B shows the bioluminescence intensity (BLI) of an individual mouse over time. Figure 24C shows the mean with standard deviation of BLI over time.

Figure 25 shows images demonstrating that GFRa4 RNA is expressed by medullary thyroid carcinoma

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of

±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "antibody," as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Tetramers may be naturally occurring or reconstructed from single chain antibodies or antibody fragments. Antibodies also include dimers that may be naturally occurring or constructed from single chain antibodies or antibody fragments. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989,

In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al, 1988, Science 242:423-426).

The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.

Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies, such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231 :25- 38), composed of either a VL or a VH domain which exhibit sufficient affinity for the target, and multispecific antibodies formed from antibody fragments. The antibody fragment also includes a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody. An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, κ and λ light chains refer to the two major antibody light chain isotypes.

By the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

The term "antigen" or "Ag" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. The term "anti-tumor effect" as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.

As used herein, the term "autologous" is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

"Allogeneic" refers to a graft derived from a different animal of the same species.

"Xenogeneic" refers to a graft derived from an animal of a different species.

The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. In certain embodiments, the cancer is medullary thyroid carcinoma.

As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.

Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for the ability to bind GFRa4 using the functional assays described herein.

"Co-stimulatory ligand", as the term is used herein, includes a molecule on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L,

CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also

encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4- IBB, OX40,

CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

A "co-stimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co- stimulatory response by the T cell, such as, but not limited to, proliferation. Co- stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

The phrase "disease associated with expression of GFRa" as used herein includes but is not limited to, a disease associated with expression of GFRa or condition associated with cells which express GFRa including, e.g., a proliferative disease, such as a cancer or malignancy or a precancerous condition; or a noncancer related indication associated with cells which express GFRa. In one aspect, a cancer associated with expression of GFRa is medullary thyroid cancer (MTC). Further disease associated with expression of GFRa expression include, but are not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of GFRa. Non-cancer related indications associated with expression of GFRa may also be included.

The term "dysregulated" when used in the context of the level of expression or activity of GFRa4 refers to the level of expression or activity that is different from the expression level or activity of GFRa4 in an otherwise identical healthy animal, organism, tissue, cell or component thereof. The term "dysregulated" also refers to the altered regulation of the level of expression and activity of GFRa4 compared to the regulation in an otherwise identical healthy animal, organism, tissue, cell or component thereof.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA

corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

"Effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.

As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system. As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

"Fully human" refers to an immunoglobulin, such as an antibody, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody.

"Homologous" as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab¾ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, Nature, 321 : 522-525, 1986; Reichmann et al, Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol, 2: 593- 596, 1992.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

"Identity" as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

A "lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non- dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

As used herein, the terms "GDNF family receptor alpha 4"and "GFRa4," are used interchangeably, and include variants, isoforms and species homologs of human GFRa4. Isoforms of GFRa4 include GFRa4a and GFRa4b. Accordingly, human antibodies of this disclosure may, in certain cases, cross-react with GFRa4 from species other than human. In certain embodiments, the antibodies may be completely specific for one or more human GFRa4 proteins and may not exhibit species or other types of non-human cross-reactivity. The complete amino acid sequence of an exemplary human GFRa4 has Genbank/NCBI accession number: NM_022139.

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

"Parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

A "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell. An example of a "cell surface receptor" is human GFRa4.

"Single chain antibodies" refer to antibodies formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to each other using an engineered span of amino acids to recapitulate the Fv region of an antibody as a single polypeptide. Various methods of generating single chain antibodies are known, including those described in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242: 1038-1041.

The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A "subject" or "patient," as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In one embodiment, the subject is human.

As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

The term "therapeutic" as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The phrase "under transcriptional control" or "operatively linked" as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, poly lysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

By the term "specifically binds," as used herein, is meant an antibody, or a ligand, which recognizes and binds with a cognate binding partner (e.g., a stimulatory and/or costimulatory molecule present on a T cell) protein present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample.

By the term "stimulation," is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β, and/or reorganization of cytoskeletal structures, and the like.

A "stimulatory molecule," as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell and/or on a tumor cell.

A "stimulatory ligand," as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) or a tumor cell, can specifically bind with a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention provides isolated antibodies that bind specifically to GFRa4. In certain embodiments, the antibodies of the invention comprise particular structural features such as CDR regions comprising particular amino acid sequences. The invention also provides methods of making such antibodies.

In one embodiment, a peptide containing amino acids (SEQ ID NOs: 1 or 2) of human GFRa4 was used to screen a phage display library to isolate single- chain variable fragment (scFv) against GFRa4. In a particular embodiment, the isolated scFv targets GFRa4 isoform "a" (SEQ ID NOs: 1). In another embodiment, the isolated scFv targets GFRa4 isoform "b" (SEQ ID NOs: 2).

In one embodiment, the scFv antibodies of the invention can be used for diagnosing the presence of GFRa4 in a biological sample. In one embodiment, the scFv antibodies of the invention can be used for diagnosing the presence of GFRa4 in a tumor cell.

In one embodiment, the scFv antibodies of the invention can be used for therapy against a disease, disorder or condition associated with normal or dysregulated expression of GFRa4. The level of expression of GFRa4 on the surface of medullary cancer cells may be considered the same as on normal thyroid C-cells. That said, should a different cell type (e.g. adrenal cells, neuronal cells) exhibit a pathology that is the result of dysregulated GFRa4, the present invention may be useful in targeting these cells to relieve the pathology.

In one embodiment, the scFv antibodies of the invention can be used for cancer therapy against cancers associated with normal or dysregulated expression of GFRa4. In another embodiment, the scFv antibodies of the invention can be used for cancer therapy against thyroid cancers. In yet another embodiment, the scFv antibodies of the invention can be used for cancer therapy against Medullary Thyroid Cancer (MTC).

The present invention relates generally to the treatment of a patient having a cancer associated with the expression of GFRa4, or at risk of having a cancer associated with the expression of GFRa4, using cellular infusion. In one embodiment, lymphocyte infusion, such as an autologous lymphocyte infusion is used in the treatment. In another embodiment, the cancer associated with expression of GFRa4 is a thyroid cancer. In yet another embodiment, the cancer associated with expression of GFRa4 is MTC.

In one embodiment, peripheral blood mononuclear cells (PBMCs) are collected from a patient in need of treatment and T cells therefrom are engineered and expanded using the methods described herein and then infused back into the patient. In another embodiment, autologous or heterologous NK cells or NK cell lines are engineered and expanded using the methods described herein and then infused back into the patient. The invention should not be limited to a particular cell or cell type. Rather, any cell or cell type can be engineered and expanded using the methods described herein and then infused back into the patient.

In one embodiment, the scFv antibodies of the invention can be cloned into vectors that allow expression in cis with cellular cytotoxins. The combination of the scFv antibodies with cellular cytotoxins can be used for transarterial infusion into patients in need thereof.

The antibodies of the invention can be incorporated into an immunoconjugate, a chimeric antigen receptor (CAR), a pharmaceutical composition, and the like. In one embodiment, the immunoconjugates of the invention may be therapeutic agents, for example, cytotoxins or radioactive isotopes. Accordingly, the present invention provides compositions and methods for treating, among other diseases, cancer or any malignancy or autoimmune disease in which expression of GFRa4 is expressed on the cell surface.

The present invention also relates generally to the use of T cells engineered to express a Chimeric Antigen Receptor (CAR). CARs combine an antigen recognition domain of a specific antibody with an intracellular signaling molecule. For example, the intracellular signaling molecule can include but is not limited to CD3-zeta chain, 4- IBB and CD28 signaling modules and combinations thereof. In one embodiment, the antigen recognition domain binds to GFRa4. In some instances, the antigen recognition domain comprises an anti-GFRa4.

Accordingly, the invention provides an anti-GFRa4-CAR engineered into a T cell and methods of their use for adoptive therapy. In one embodiment, the invention includes autologous cells that are transfected with a vector comprising an anti-GFRa4 CAR transgene. In another embodiment, the vector is a retroviral vector. In yet another embodiment, the vector is a self-inactivating lentiviral vector as described elsewhere herein.

In one embodiment, the anti-GFRa 4-CAR T cells of the invention can be generated by introducing a lentiviral vector comprising a GFRa4 binding domain, a glycine-serine linker and transmembrane domain, and a CD3zeta signaling domain into the cells. In another embodiment, the anti-GFRa 4-CAR T cells of the invention can be generated by introducing a lentiviral vector comprising a GFRa4 binding domain, CD8a hinge and transmembrane domain, and a CD3zeta signaling domain into the cells. In some instances, the vector further comprises the signaling domain of 4- IBB, CD28, or a combination of both. This is because the present invention is partly based on the discovery that CAR-mediated T-cell responses can be further enhanced with the addition of costimulatory domains. For example, inclusion of the CD28 signaling domain significantly increased anti-tumor activity and in vivo persistence of CAR T cells compared to an otherwise identical CAR T cell not engineered to express CD28.

In one embodiment, the CAR-modified T cells of the invention are expected to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

Antibodies

Anti-GFRa4 Antibodies

In one aspect, the invention includes a composition comprising an anti- Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (anti-GFRa4) binding domain, wherein the anti-GFRa4 binding domain is a GFRa4 antibody, or fragment thereof. The antibodies of the invention are characterized by particular functional features or properties of the antibodies. For example, the antibodies specifically bind to human GFRa4. The antibodies of the invention bind to GFRa4 with high affinity. The antibodies of the invention specifically recognize naturally expressed hGFRa4 protein on a cell and do not cross-react to other surface molecules. In one embodiment, the antibodies of the invention binds specifically to an isoform of a GFRa4 cell-surface receptor, such as GFRa4a and GFRa4b. In one embodiment, the antibodies of the invention are human antibodies designated as P4-6 or P4-10. The VH amino acid sequences of P4-6 or P4- 10 are shown in SEQ ID NOs: 4 and 20, respectively (Table 1). The VL amino acid sequences of P4-6 or P4-10 are shown in SEQ ID NOs: 12 and 28, respectively (Table 1).

In one embodiment, the antibody includes heavy chain variable regions (Table 1) having CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in SEQ ID NOs: in any of the following (a) to (b):

(a) SEQ ID NOs: 6, 8 and 10 (P4-6),

(b) SEQ ID NOs: 22, 24 and 26 (P4-10),

In one embodiment, the antibody includes light chain variable regions (Table 1) having CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in SEQ ID NOs: in any of the following (c) to (d):

(c) SEQ ID NOs: 14, 16 and 18 (P4-6),

(d) SEQ ID NOs: 30, 32 and 34 (P4-10),

Given that each of these antibodies binds to GFRa4, the VH and VL sequences can be "mixed and matched" to create other anti-GFRa4 binding molecules of the invention. GFRa4 binding of such "mixed and matched" antibodies can be tested using the binding assays described herein, in the art, for example, in the Examples section (e.g., ELISAs). When VH and VL chains are mixed and matched, a VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence. Likewise, a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence. It will be readily apparent to the ordinary skilled artisan that novel VH and VL sequences can be created by substituting one or more VH and/or VL CDR region sequences with structurally similar sequences from the CDR sequences disclosed herein.

In one embodiment, the invention includes antibodies that comprise the heavy chain and light chain (CDRls, CDR2s, and CDR3s) of P4-6 and P4-10, or combinations thereof.

In one embodiment, the antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are identical to the amino acid sequences of the antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-GFRa4 antibodies of the invention. For example, the invention includes an isolated antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 20, (b) the light chain variable region comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 28.

In certain embodiments, an antibody of the invention comprises a heavy chain variable region comprising CDRl and CDR2 sequences and a light chain variable region comprising CDRl and CDR2 sequences, wherein one or more of these

CDR sequences comprise specified amino acid sequences based on the antibodies described herein (e.g., P4-6 and P4-10), or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-GFRa4 antibodies of the invention. Accordingly, the invention provides an isolated antibody (s-g scFv), or antigen binding portion thereof, comprising a heavy chain variable region comprising CDRl, CDR2, and CDR3 sequences and a light chain variable region comprising CDRl, CDR2, and CDR3 sequences, wherein: (a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 10 and 26, and conservative modifications thereof; (b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequence of SEQ ID NOs: 18 and 34, and conservative modifications thereof.

In another embodiment, the invention includes antibodies that bind to the same epitope on human GFRa4 as any of the GFRa4 antibodies of the invention (i.e., antibodies that have the ability to cross-compete for binding to GFRa4 with any of the antibodies of the invention). In one embodiment, the reference antibody for cross-competition studies can be one of the antibodies described herein (e.g., P4-6 and P4-10). For example, Biacore analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition with the antibodies of the current invention. The ability of a test antibody to inhibit the binding of, for example, P4-6 and P4-10, to human GFRa4 demonstrates that the test antibody can compete with P4-6 and P4-10 for binding to human GFRa4 and thus is considered to bind to the same epitope on human GFRa4 as P4-6 and P4-10. In one embodiment, the GFRa4 antibody, or fragment thereof, or anti-GFRa4 binding domain binds specifically to a thyroid cell antigen present in a tumor microenvironment, such as a thyroid cell antigen present on a medullary thyroid carcinoma (MTC) cell. In another embodiment, the GFRa4 antibody, or fragment thereof, or anti-GFRa4 binding domain binds specifically to a tumor cell, such as a medullary thyroid carcinoma cell.

An antibody of the invention is prepared using an antibody having one or more of the VH and/or VL sequences disclosed herein as a starting material to engineer a modified antibody, which modified antibody may have altered properties as compared with the starting antibody. An antibody can be engineered by modifying one or more amino acids within one or both variable regions (i.e., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody. Humanized antibodies

For in vivo use of antibodies in humans, it may be preferable to use human antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See, also, U.S. Pat. Nos. 4,444,887 and 4,716,1 11 ; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. A human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human D A.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. The modified embryonic stem cells are expanded and micro injected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Anti-GFRa4 antibodies directed against the human GFRa4 antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies, including, but not limited to, IgGl (gamma 1) and IgG3. For an overview of this technology for producing human antibodies, see,

Lonberg and Huszar (Int. Rev. Immunol, 13:65-93 (1995)). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923;

5,625, 126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. For a specific discussion of transfer of a human germ-line immunoglobulin gene array in germ-line mutant mice that will result in the production of human antibodies upon antigen challenge see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann et al, Year in Immunol, 7:33 (1993); and Duchosal et al, Nature, 355:258 (1992).

Human antibodies can also be derived from phage-display libraries

(Hoogenboom et al, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581-597 (1991); Vaughan et al, Nature Biotech., 14:309 (1996)). Phage display technology (McCafferty et al, Nature, 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of unimmunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al, J. Mol. Biol, 222:581-597 (1991), or Griffith et al, EMBO J., 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905, each of which is incorporated herein by reference in its entirety.

Human antibodies may also be generated by in vitro activated B cells (see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety). Human antibodies may also be generated in vitro using hybridoma techniques such as, but not limited to, that described by Roder et al.

(Methods Enzymol, 121 : 140-167 (1986)).

Alternatively, in some embodiments, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human. For instance, in the present invention, the GFRa4 antibody comprises a rabbit scFv. In one embodiment, the antigen binding domain portion is humanized.

A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European

Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530, 101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP 592, 106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al, 1994, Protein Engineering, 7(6):805-814; and Roguska et al, 1994, PNAS, 91 :969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No.

US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al, J. Immunol, 169: 1119-25 (2002), Caldas et al, Protein Eng., 13(5):353-60 (2000), Morea et al, Methods, 20(3):267-79 (2000), Baca et al, J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al, Protein Eng., 9(10):895-904 (1996), Couto et al, Cancer Res., 55 (23 Supp):5973s-5977s (1995),

Couto et al, Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al, J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Pat. No. 5,585,089; and Riechmann et al, 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)

A humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human.

Humanization of antibodies is well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference herein in their entirety). In such humanized chimeric antibodies, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies can also be achieved by veneering or resurfacing (EP 592, 106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al, Protein Engineering, 7(6):805-814 (1994); and Roguska et al, PNAS, 91 :969-973 (1994)) or chain shuffling (U.S. Pat. No.

5,565,332), the contents of which are incorporated herein by reference herein in their entirety.

In some instances, a human scFv may also be derived from a yeast display library.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al, J. Immunol, 151 :2296 (1993); Chothia et al, J. Mol. Biol, 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al, J. Immunol., 151 :2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).

Antibodies can be humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three- dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

A humanized antibody retains a similar antigenic specificity as the original antibody, i.e., in the rabbit scFv of the present invention, the ability to bind human GFRa4. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody for human GFRa4 may be increased using methods of "directed evolution," as described by Wu et al, J. Mol. Biol, 294: 151 (1999), the contents of which are incorporated herein by reference herein in their entirety.

Rabbit Antibody

Notwithstanding the above, it is contemplated that the rabbit antibody disclosed herein may be equally useful as a therapeutic antibody in the methods of the invention without humanization. Antigen binding moiety

The choice of moiety depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

In the context of the present invention, "tumor antigen" refers to antigens that are common to specific thyroid disorders. In certain aspects, the thyroid antigens of the present invention are derived from, cancers including but not limited to thyroid cancer, papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, anaplastic thyroid cancer and the like. In one embodiment, the cancer is a medullary thyroid carcinoma (MTC).

In one embodiment, the tumor antigen of the present invention comprises one or more antigenic cancer epitopes immunologically recognized by tumor infiltrating lymphocytes (TIL) derived from a cancer tumor of a mammal. The antigen binding domain can be any domain that binds to the antigen including but not limited to monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies and fragments thereof. Thus, in one embodiment, the antigen binding domain portion comprises a rabbit antibody or a fragment thereof.

CAR Composition

The present invention encompasses a recombinant DNA construct comprising sequences of the antibody of the invention that specifically binds to human GFRa4, wherein the sequence of the antibody or a fragment thereof is operably linked to the nucleic acid sequence of an intracellular domain. The intracellular domain or otherwise the cytoplasmic domain comprises, a costimulatory signaling region and/or a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that are required for an efficient response of lymphocytes to antigen.

The present invention therefore encompasses a recombinant DNA construct comprising sequences of a fully human CAR, wherein the sequence comprises the nucleic acid sequence of a GFRa4 binding domain operably linked to the nucleic acid sequence of an intracellular domain. An exemplary intracellular domain that can be used in the CAR includes but is not limited to the intracellular domain of CD3-zeta, CD28, 4- IBB, CD27, and the like. In some instances, the CAR can comprise any combination of CD3-zeta, CD28, 4- IBB, CD27, and the like.

Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term "spacer domain" generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.

The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than as cloned molecules. In one embodiment, the CAR of the invention comprises a target-specific binding element otherwise referred to as an antigen binding moiety as described elsewhere herein. Examples of cell surface markers that may act as ligands for the antigen moiety domain in the CAR of the invention include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.

In one embodiment, the CAR-mediated T-cell response can be directed to an antigen of interest by way of engineering a desired antigen into the CAR.

In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen binding domain of the CAR to comprise a human antibody or a fragment thereof.

In one embodiment, the antigen binding moiety of the CAR includes a nucleic acid sequence encoding an antibody as described elsewhere herein.

In one embodiment, the antigen binding moiety portion of the CAR targets GFRa4, such as a human GFRa4.

Transmembrane domain

With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some instances, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge.

In one embodiment, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between any domain, such as the transmembrane domain and the cytoplasmic signaling domain of the CAR, or at the beginning or end of any domain, and any combination thereof. A glycine-serine doublet provides a particularly suitable linker.

Cytoplasmic domain

The cytoplasmic domain or otherwise the intracellular signaling domain of the CAR of the invention is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in.

The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

Some examples of intracellular signaling domains for use in the CAR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen- dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.

Examples of IT AM containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma , CD3 delta , CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the invention comprises a cytoplasmic signaling sequence derived from CD3-zeta.

In one embodiment, the cytoplasmic domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-lBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-

1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. Thus, while the invention in exemplified primarily with CD28 and 4- IBB as the co-stimulatory signaling element, other costimulatory elements are within the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a particularly suitable linker. In one embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB.

Vectors

The present invention also provides vectors in which a DNA of the present invention is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

In brief summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the

CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, volumes 1 -3 (3^rd ed., Cold Spring Harbor Press, NY 2001), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

An example of a promoter is the EF 1 alpha promoter. An additional example includes the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al, 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, volumes 1-3 (3^rd ed., Cold Spring Harbor Press, NY 2001).

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes,

nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g. , an artificial membrane vesicle).

In the case where a non- viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories

(Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes. Sources of T cells

Prior to expansion and genetic modification, a source of T cells is obtained from a subject. The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Again, surprisingly, initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow- through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA , and CD45RO cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3x28)-conjugated beads, such as

DY ABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain embodiments, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.

For example, to enrich for CD4⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD l ib, CD 16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4⁺, CD25⁺, CD62L^hi, GITR⁺, and FoxP3⁺. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8⁺ T cells that normally have weaker CD28 expression. In a related embodiment, it may be desirable to use lower

concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4⁺ T cells express higher levels of CD28 and are more efficiently captured than CD8⁺ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5 X 10⁶/ml. In other embodiments, the concentration used can be from about 1 X 10⁵/ml to 1 X 10⁶/ml, and any integer value in between.

In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10°C or at room temperature.

T cells for stimulation can also be frozen after a washing step.

Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10%

Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time.

In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents,

chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, Cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al, Cell 66:807-815, 1991 ; Henderson et al, Immun. 73 :316-321, 1991; Bierer et al, Curr. Opin. Immun. 5:763- 773, 1993). In a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Activation and Expansion of T Cells

T cells are activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7, 144,575; 7,067,318; 7, 172,869; 7,232,566;

7, 175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 ; and U.S. Patent Application Publication No. 20060121005.

Generally, the T cells of the invention are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co- stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4⁺

T cells or CD8⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et ah, Transplant Proc. 30(8):3975-3977, 1998; Haanen et ah, J. Exp. Med. 190(9): 13191328, 1999; Garland et ah, J. Immunol Meth. 227(l-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and the co- stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., "cis," or to separate beads, i.e., "trans." By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co- immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4⁺ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3 :CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1. In one particular embodiment, a 1 : 100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1 :50 CD3 :CD28 ratio of antibody bound to beads is used.

In another embodiment, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used. In one preferred embodiment, a 1 : 10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3 : 1 CD3 :CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1 :500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1 with one preferred ratio being at least 1 : 1 particles per T cell. In one embodiment, a ratio of particles to cells of 1 : 1 or less is used. In one particular embodiment, a preferred particle: cell ratio is 1 :5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1 : 1 to 10: 1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1 : 1 to 1 : 10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1 : 1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 :5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1 : 10 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 : 10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size and on cell size and type.

In further embodiments of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells. In one embodiment the cells (for example, 10⁴ to 10⁹ T cells) and beads (for example, DY ABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1 : 1) are combined in a buffer, preferably PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75,

80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments,

concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF , and TNF-a. or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2).

T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (T_H, CD4⁺) that is greater than the cytotoxic or suppressor T cell population (T_c, CD8⁺). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of T_H cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of T_c cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately predominantly of TH cells may be

advantageous. Similarly, if an antigen-specific subset of T_c cells has been isolated it may be beneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated

T cell product for specific purposes. Therapeutic Application

In one aspect, the invention pertains to a method of inhibiting growth of a GFRa4-expressing tumor cell, comprising contacting the tumor cell with at least one antibody or a fragment thereof of the invention such that growth of the tumor cell is inhibited. In one embodiment, the tumor cell is a medullary thyroid carcinoma cell.

Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by the GFRa4 antibody of this invention may be an active or a passive immune response. The GFRa4 antibody of the invention may be used in some type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one embodiment, the mammal is a human.

With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a CAR to the cells or iii) cryopreservation of the cells.

The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5, 199,942, incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art, therefore the present invention is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Pat. No. 5, 199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

The GFRa4 antibody of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.

In one embodiment, the invention pertains to a method of inhibiting growth of a GFRa4-expressing tumor cell, comprising contacting the tumor cell with an anti-GFRa4 CAR T cell of the present invention such that growth of the tumor cell is inhibited.

In another aspect, the invention pertains to a method of treating cancer in a subject. The method comprises administering to the subject an antibody or a fragment of the invention or an anti-GFRa4 CAR T cell of the present invention such that the cancer is treated in the subject. Particularly cancers for treatment are thyroid cancer, papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer. More specifically, the cancer for treatment is a medullary thyroid cancer.

The present invention includes a type of cellular therapy where T cells are genetically modified to express a chimeric antigen receptor (CAR) and the CAR T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, CAR-modified T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control (unless using mRNA electroporation introduction of CAR). In various embodiments, the T cells administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.

Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by the CAR-modified T cells may be an active or a passive immune response. In another embodiment, the CAR transduced T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing GFRa4, resist soluble GFRa4 inhibition, mediate bystander killing and mediate regression of an established human tumor. For example, antigen-less tumor cells within a heterogeneous field of GFRa4-expressing tumor may be susceptible to indirect destruction by GFRa4-redirected T cells that has previously reacted against adjacent antigen-positive cancer cells.

The CAR-modified T cells of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one embodiment, the mammal is a human. Ex vivo procedures are well known in the art as discussed more fully above. Briefly, cells are isolated from a mammal (such as a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed elsewhere herein, or by electroporating the CAR mRNA disclosed elsewhere herein. The CAR-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cell can be autologous with respect to the recipient.

Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

Generally, the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the CAR-modified T cells of the invention are used in the treatment of diseases, disorders and conditions associated with the normal or dysregulated expression of GFRa4. In certain embodiments, the cells of the invention are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of GFRa4. Thus, the present invention provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of GFRa4 comprising administering to a subject in need thereof, a therapeutically effective amount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.

Pharmaceutical compositions

Pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine;

antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one embodiment, the compositions of the present invention are formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

When "an immunologically effective amount", "an anti-tumor effective amount", "an tumor-inhibiting effective amount", or "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, for example 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

In certain embodiments, it may be desired to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom according to the present invention, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from lOcc to 400cc. In certain embodiments, T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or lOOcc. Not to be bound by theory, using this multiple blood draw/multiple reinfusion protocol, may select out certain populations of T cells.

The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.

In certain embodiments of the present invention, cells activated and expanded using the methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation,

immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al, Cell 66:807-815, 1991 ; Henderson et al, Immun. 73 :316-321, 1991; Bierer et al, Curr. Opin. Immun. 5:763- 773, 1993). In a further embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.

For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. The dose for CAMPATH, for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days. The daily dose includes 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used

(described in U.S. Patent No. 6, 120,766).

Diagnostic Methods

In another aspect, the present invention provides a method of diagnosing a disease such as cancer by detecting GFRa4 protein in a test sample with the use of the antibody of the present invention. Because GFRa4 is present in normal thyroid tissue this method is useful when the patient expresses GFRa4 in either non- thyroid tissue or has already undergone a thyroidectomy.

The detection used herein includes quantitative detection and non- quantitative detection. The non-quantitative detection include, for example, determination of merely whether or not GFRa4 protein is present, determination of whether or not a specific amount or more of GFRa4 protein is present, determination for comparison of the amount of GFRa4 protein with that of another sample (e.g., a control sample). The quantitative detection includes determination of the

concentration of GFRa4 protein, determination of the amount of GFRa4 protein.

The test sample is not particularly limited as long as it is a sample that may contain GFRa4 protein. Specific examples of the test sample may include biopsy from the thyroid, biopsy from the medullary thyroid, blood, serum and/or plasma. In addition, a sample obtained from the test sample such as culture solution of cells collected from the body of the living organism is also included in the test sample of the present invention.

The cancer to be diagnosed may be limited to medullary thyroid cancer (MTC). Other thyroid cancers could potentially be diagnosed such as papillary thyroid cancer, follicular thyroid cancer, and anaplastic thyroid cancer. GFRa4 to be detected is not particularly limited, and may be either full-length GFRa4 (i.e.GFRa4 isoform "a" and/or GFRa4 isoform "b") or a fragment thereof. In the case where a fragment of GFRa4 is detected, it may be either the N- terminal fragment or the C-terminal fragment.

The method of detecting GFRa4 protein contained in a test sample is not particularly limited, however, detection is performed by an immunological method with the use of an anti-GFRa4 antibody. Examples of the immunological method include, for example, a radioimmunoassay, an enzyme immunoassay, a fluorescence immunoassay, a luminescence immunoassay, immunoprecipitation, a turbidimetric immunoassay. One immunological method is an enzyme immunoassay, and particularly an enzyme-linked immunosorbent assay (ELISA) (e.g., a sandwich ELISA). The above-mentioned immunological method such as an ELISA can be carried out by a method known to those skilled in the art.

A general detection method with the use of an anti-GFRa4 antibody comprises immobilizing an anti-GFRa4 antibody on a support, adding a test sample thereto, incubating the support to allow the anti-GFRa4 antibody and GFRa4 protein to bind to each other, washing the support, and detecting the GFRa4 protein binding to the support via the anti-GFRa4 antibody to detect GFRa4 protein in a test sample.

The binding between the anti-GFRa4 antibody and the GFRa4 protein is generally carried out in a buffer. Buffers used in the invention include, for example, a phosphate buffer, a Tris buffer. Incubation is carried out under the conditions generally employed in the art, for example, at 4°C to room temperature for 1 hour to 24 hours. The washing after incubation can be carried out by any method as long as it does not inhibit the binding between the GFRa4 protein and the anti-GFRa4 antibody, using for example a buffer containing a surfactant such as Tween 20.

In the method of detecting GFRa4 protein of the present invention, a control sample may be provided in addition to a test sample to be tested for GFRa4 protein. The control samples include a negative control sample that does not contain GFRa4 protein and a positive control sample that contains GFRa4 protein. In this case, it is possible to detect GFRa4 protein in the test sample by comparing the result obtained with the negative control sample that does not contain GFRa4 protein with the result obtained with the positive control sample that contains GFRa4 protein. It is also possible to quantitatively detect GFRa4 protein contained in the test sample by obtaining the detection results of the control samples and the test sample as numerical values, and comparing these numerical values.

One method for detecting GFRa4 protein binding to the support via an anti-GFRa4 antibody is a method that employs an anti-GFRa4 antibody labeled with a detectable label. For example, GFRa4 protein may be detected by contacting the test sample with an anti-GFRa4 antibody immobilized on the support, washing the support, and then detecting GFRa4 with the use of the labeled antibody that specifically binds to GFRa4 protein.

The labeling of an anti-GFRa4 antibody can be carried out by any method known in the art. Examples of the detectable label known to those skilled in the art include a fluorescent dye, an enzyme, a coenzyme, a chemiluminescent substance or a radioactive substance. Specific examples may include radioisotopes

32 14 125 3 131

( P, C, I, H, I and the like), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, beta-galactosidase, beta- glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase, microperoxidase, biotin and the like. In the case where biotin is used as a detectable label, it is preferred that a biotin-labeled antibody is added, and then avidin conjugated to an enzyme such as alkaline phosphatase is further added.

Specifically, a solution containing an anti-GFRa4 antibody is added to a support such as a plate to allow the anti-GFRa4 antibody to be immobilized. After washing, the plate is blocked with, for example, BSA in order to prevent the nonspecific binding of a protein. The plate is washed again, and then the test sample is added to the plate. After being incubated, the plate is washed, and then the labeled anti-GFRa4 antibody is added. After being incubated appropriately, the plate is washed, and then the labeled anti-GFRa4 antibody remaining on the plate is detected

(e.g. GFRa4 ELISA kit, mybiosource.com Product_id=939378).The detection of the protein can be carried out by a method known to those skilled in the art. For example, in the case where the antibody is labeled with a radioactive substance, the protein may be detected by liquid scintillation or the RIA method. In the case where the antibody is labeled with an enzyme, the protein may be detected by adding a substrate and detecting an enzymatic change of the substrate such as color development with an absorbance reader. In the case where the antibody is labeled with a fluorescent substance, the protein may be detected with the use of a fluorometer. Another embodiment of the method of detecting GFRa4 protein of the present invention is a method using an anti-GFRa4 antibody labeled with biotin and avidin. Specifically, a solution containing an anti-GFRa4 antibody is added to a support such as a plate to allow the anti-GFRa4 antibody to be immobilized thereon. After washing, the plate is blocked with, for example, BSA in order to prevent the nonspecific binding of a protein. The plate is washed again, and then the test sample is added to the plate. After being incubated, the plate is washed, and then the biotin- labeled anti-GFRa4 antibody is added. After being incubated appropriately, the plate is washed, and then avidin conjugated to an enzyme such as alkaline phosphatase or peroxidase is added. After being incubated, the plate is washed, and then a substrate of the enzyme conjugated to avidin is added. Then, GFRa4 protein is detected by means of the enzymatic change of the substrate as an indicator.

Another embodiment of the method of detecting GFRa4 protein of the present invention is a method using a primary antibody that specifically binds to GFRa4 protein and a secondary antibody that specifically binds to the primary antibody. For example, the test sample is brought into contact with an anti-GFRa4 antibody immobilized on the support, the support is incubated and washed, and the bound GFRa4 protein after washing is detected with a primary anti-GFRa4 antibody and a secondary antibody that specifically binds to the primary antibody. In this case, the secondary antibody is preferably labeled with a detectable label.

Specifically, a solution containing an anti-GFRa4 antibody is added to a support such as a plate to allow the anti-GFRa4 antibody to be immobilized thereon. After washing, the plate is blocked with, for example, BSA in order to prevent the nonspecific binding of a protein. The plate is washed again, and then the test sample is added to the plate. After being incubated, the plate is washed, and then a primary anti-GFRa4 antibody is added. After being incubated appropriately, the plate is washed, and then a secondary antibody that specifically binds to the primary antibody is added. After being incubated appropriately, the plate is washed, and then the secondary antibody remaining on the plate is detected. The detection of the secondary antibody can be carried out by the above-mentioned method.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

The following experiments were designed to develop and validate GFRa4-specific T bodies. The results of the experiments are now described.

Example 1 : Isolation of 2 unique human/rabbit chimeric Fab antibodies to GFRot4

Antibody phage display was performed utilizing a naive chimeric human/rabbit Fab library and solid phase antibody selection against immobilized human GFRa4. For construction of the library, rabbit spleen and bone marrow for the preparation of B-cell RNA was provided by Pocono Rabbit Farm & Laboratory (PRF&L, Canadensis, PA) and R & R Research (Stanwood, WA). A total of nine rabbits (ages 3-4 months) were used. Five of these rabbits were of the New Zealand

White (NZW) strain, with three obtained from PRF&L and two obtained from R & R Research. Four b9 wild-type rabbits were also used and obtained from a separate R & R Research colony (Popkov et al, J. Mol. Biol. 325, 325-335, 2003). Total RNA was prepared from spleen and bone marrow from each rabbit and RT-PCR amplification of rabbit V_K, Υχ, and VH encoding sequences was performed using established protocols (Rader, et al., Methods Mol. Biol. 525, 101-128, 2009). Rabbit (rb) V_K/human (hu) C_K/rbVH and rb huC?yrbVH segments, respectively, were assembled in one fusion step based on 3 -fragment overlap extension PCR as described. VL derived from b9 rabbits were also assembled with VH from NZW rabbits. The Fab- encoding fragments (less the heavy chain CHI constant domain) were digested with

Sfil and ligated at 16°C for 24 h with 5/zI-digested phage display vector pC3C that provided the CHI domain to complete the Fab construct (Hofer et al, J. Immunol. Methods 318, 75-87, 2007). Subsequently, 15 μg of purified pC3C- rbV_K/hC_K/rbVH/hCHi ligated products were transformed into E. coti strain SR320 by 30 separate electroporations (each using 0.5 μg DNA in 50 μΐ electrocompetent cells) and yielded 7.5 x 10⁹ independent transformants for the γ/κ-light chain sub-library. For the γ Ζλ-light chain sub-library, 4.8 x 10⁹ independent transformants were obtained using the same procedure. Using VCSM13 helper phage (Stratagene, La Jolla, CA), the phagemid libraries were converted to phage particle libraries and stored at -80°C. The day prior to selecting anti-GFRa4 antibodies from the κ and λ libraries, reamplification of phagemids in XLl-Blue strain of E. coli (Stratagene) was performed and equal volumes of each library were combined.

Library selections against human Fc-fusion constructs of immobilized

GFRa4 isoforms a (GFRa4a) and b (GFRa4b) were performed in separate experiments and carried out as described in Rader and colleagues (Rader et al, Selection from antibody libraries in Phage Display: A Laboratory Manual (Chapter 10), eds. Barbas, C.F., Burton, D.R., Scott, J.K., and Silverman, G.J., 10.1-10.20; 2001) with the following modifications. For each round of panning, 8 wells of an

ELISA plate (1/2-area wells, Costar #3690, Corning Life Sciences, Tewksbury, MA) were each coated overnight at 4°C with 50 μΐ of a 10 μg/ml PBS solution of either GFRa4a (R&D Systems, Inc.) or GFRa4b (LakePharma, Inc.) and blocked with 2% nonfat dry milk in PBS (MPBS) for 1 hour at 37°C. In order to target the capture of GFRa4-specific antibodies, phage were initially incubated with a mixture of soluble human GFRal, GFRa2, and GFRa3 (R&D Systems, Inc., 6 μg/ml final

concentration) in MPBS and blocked for 1 hour at room temperature. Addition of phage (with GFRa's 1, 2, and 3) to antigen-coated wells, incubation, washing, low pH buffer elution of bound phage, and overnight phage amplification were performed as described (Steinberger et al, Analysis of Selected Antibodies in Phage Display: A

Laboratory Manual (Chapter 11), eds Barbas, C.F., Burton, D.R., Scott, J.K., and Silverman, G.J., 11.1-11.24; 2001).

GFRalpha4 isoform "a" (GFRa4a) was purchased from R&D Systems (Minneapolis, MN) and comprises a portion of GFRa 4a (Asn24 - Ser245, UniProt accession Q9GZZ7-2), followed by a Factor Xa cleavage site/linker, a portion of optimized human IgGl Fc domain (Pro 100 - Lys330), and 6 His residues for purification (Figure 1). GFRot4a construct Amino acid sequence (SEQ ID NO: 1)

NRCVDAAEACTADARCQRLRSEYVAQCLGRAAQGGCPRARCRRALRRFFAR

GPPALTHALLFCPCAGPACAERRRQTFVPSCAFSGPGPAPPSCLEPLNFCERSR

VCRPRLLAFQVSCTPAPSAPDGCLLDQGARCLRAYAGLVGTAVTPNYVDNV

SARVAPWCDCGASGNRREDCEAFRGLFTRNRCLDGAIQAFASGWPPVLLDQ

LNPQGDPEHSLLQVSIEGRMDPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST

YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSF

FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

GFRalpha4 isoform "b" (GFRa4b) was purchased from LakePharma, Inc. (Belmont, CA) and comprises a portion of GFRa 4b (Asn24 - Val274, UniProt #Q9GZZ7-1), followed by a TEV cleavage site linker, and a portion of human IgGl Fc domain (Aspl04 - Lys330) (Figure 2).

GFRa4b construct Amino acid sequence (SEP ID NO: 2)

NRCVDAAEACTADARCQRLRSEYVAQCLGRAAQGGCPRARCRRALRRFFAR GPPALTHALLFCPCAGPACAERRRQTFVPSCAFSGPGPAPPSCLEPLNFCERSR VCRCARAAAGPWRGWGRGLSPAHRPPAAQASPPGLSGLVHPSAQRPRRLPA GPGRPLPARLRGPRGVPAGTAVTPNYVDNVSARVAPWCDCGASGNRREDCE AFRGLFTRNRCLDGAIQAFASGWPPVLLDQLNPQGDPEHSLLQVGGGENLYF QGGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

After 4 rounds of selection of the phage display library on either GFRa4a or GFRa4b, phage ELISAs were performed to assess enrichment in the capture of anti-GFRa4-binding phage (Steinberger et al, Analysis of Selected Antibodies in Phage Display: A Laboratory Manual (Chapter 11), eds Barbas, C.F., Burton, D.R., Scott, J.K., and Silverman, G.J. 11.1-11.24; 2001). Significant enrichment was observed beginning in panning round 3 with only background reactivity to wells coated with GFRal, GFRa2, or GFRa3. Individual monoclonal phage preparations were prepared from rounds 3 and 4 of selection for the GFRa4a and GFRa4b libraries, and phage ELISAs were performed to identify positive clones. For the GFRa4a library, 18 of 19 randomly selected phage clones were positive against wells coated with GFRa4a. Nucleotide sequencing of antibody heavy and light chains of positive clones revealed 2 unique antibodies designated P4-6 and P4- 10. Subsequently, these 2 phage antibodies were also found to bind to GFRa4b even though they were the result of a panning experiment selecting against GFRa4a. For the GFRa4b-panned library, 8 of 8 randomly selected phage clones were positive against GFRa4b, and all of these clones cross-reacted with GFRa4a. Nucleotide sequencing of theses clones showed them to all be the same antibody and to be identical to antibody P4-10 originally identified in the GFRa4a selection experiment.

Rabbit P4-6

a) Rabbit P4-6 Heavy chain variable region (Nucleic acid sequence, SEQ ID NO: 3) -

- VH/D/JH:

GAGCAGCTGAAGGAGTCCGGGGGAGGTCTCTTCAAGCCAACGGATACCCT GACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTTACTATGGAGTGAA CTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGATCGGAACCATTG GTGGTAGTGGTGACACATACTACGCGAGCTGGGCGAAGAGCCGATCCACC ATCATCAGAAACACCAACGAGAACACGGTGACTCTGAAAATGACCAGTCT GACAGCCGCGGACACGGCCACCTATTTCTGTGTGAGATATGCTAATATTG GTTATGAGTACTTTAACGTCTGGGGTCCAGGCACCCTGGTCACCGTCTCTT CA

b) Rabbit P4-6 Heavy chain variable region (Amino acid sequence, SEQ ID NO: 4)— VH/D/JH:

EQLKESGGGLFKPTDTLTLTCTVSGFSLSYYGVNWVRQAPGNGLEWIGTIGG SGDTYYASWAKSRSTIIRNTNENTVTLKMTSLTAADTATYFCVRYANIGYEY FNVWGPGTLVTVSS

c) Rabbit P4-6 HC CDR1 (Nucleic acid sequence, SEQ ID NO: 5):

GGATTCTCCCTCAGTTACTATGGA d) Rabbit P4-6 HC CDR1 (Amino acid sequence, SEQ ID NO: 6):

GFSLSYYG

e) Rabbit P4-6 HC CDR2 (Nucleic acid sequence, SEQ ID NO: 7):

ATTGGTGGTAGTGGTGACACA

f) Rabbit P4-6 HC CDR2 (Amino acid sequence, SEQ ID NO: 8):

IGGSGDT

g) Rabbit P4-6 HC CDR3 (Nucleic acid sequence, SEQ ID NO: 9):

GTGAGATATGCTAATATTGGTTATGAGTACTTTAACGTC

h) Rabbit P4-6 HC CDR3 (Amino acid sequence, SEQ ID NO: 10):

VRYANIGYEYFNV

i) Rabbit P4-6 Light chain variable region— VL/JL (Nucleic acid sequence, SEQ ID

NO: 11): CAGTTTGTGCTGACTCAGTCGCCCTCTGCATCTGCTGCCCTGGGAGCCTCG GCCAAGCTCACCTGCACCCTGAGCAGTGCCCACAAGACCTACACCATTGA CTGGTATCAGCAGCAGAAAGGGAAGGCCCCTCGCTACCTGATACAAGTTA AGAGTGATGGAACCTACACCAAGGCGACCGGGGTCCCTGATCGCTTCTCG GGCTCCAGCTCTGGGGCTGACCGCTACCTGATCATCCCCAGCGTCCAGGC TGATGACGAAGCCGACTACTATTGTGGTACAGATTATACCGGTGGGTATG TGTTCGGCGGGGGGACCCAGCTGACCGTCACA

j) Rabbit P4-6 Light chain variable region— VL/JL (Amino acid sequence, SEQ ID NO: 12):

QFVLTQSPSASAALGASAKLTCTLSSAHKTYTIDWYQQQKGKAPRYLIQVKS

DGTYTKATGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGTDYTGGYVFGG

GTQLTVT

k) Rabbit P4-6 LC CDR1 (Nucleic acid sequence, SEQ ID NO: 13):

AGTGCCCACAAGACCTACACC

1) Rabbit P4-6 LC CDR1 (Amino acid sequence, SEQ ID NO: 14):

SAHKTYT m) Rabbit P4-6 LC CDR2 (Nucleic acid sequence, SEQ ID NO: 15):

GTTAAGAGTGATGGAACCTAC

n) Rabbit P4-6 LC CDR2 (Amino acid sequence, SEQ ID NO: 16):

VKSDGTY

o) Rabbit P4-6 LC CDR3 (Nucleic acid sequence, SEQ ID NO: 17):

GGTACAGATTATACCGGTGGGTATGTG

p) Rabbit P4-6 LC CDR3 (Amino acid sequence, SEQ ID NO: 18):

GTDYTGGYV

Rabbit P4-10

a) Heavy chain variable region (Nucleic acid sequence, SEQ ID NO: 19)—

VH/D/JH:

CAGTCAGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCT GACACTCACCTGCACGGTCTCTGGATTCTCCCTCAGTAGACATGCACTGAC CTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGATCGGAGCCATTG ATAACGCTGGTACCACATACTACGCGAGCTGGGCGAAAAGCCGCTCCACC ATCACCAGAAACACCGACCTGCACACGGTGACTCTGAAAATGACCAGTCT GACAGCCTCGGACACGGCTACCTATTTCTGTGCGAGAGTCTTTTATGATAT TAATAGTGGTTATTATCTGGACGGCATGGACCTCTGGGGCCCAGGGACCC TCGTCACCGTCTCTTCA

b) Heavy chain variable region (Amino acid sequence, SEQ ID NO: 20)— VH/D/JH: QSVKESEGGLFKPTDTLTLTCTVSGFSLSRHALTWVRQAPGNGLEWIGAIDNA GTTYYASWAKSRSTITRNTDLHTVTLKMTSLTASDTATYFCARVFYDINSGY YLDGMDLWGPGTLVTVSS c) HC CDR1 (Nucleic acid sequence, SEQ ID NO: 21):

GGATTCTCCCTCAGTAGACATGCA

d) HC CDR1 (Amino acid sequence, SEQ ID NO: 22):

GFSLSRHA

e) HC CDR2 (Nucleic acid sequence, SEQ ID NO: 23):

ATTGATAACGCTGGTACCACA f) HC CDR2 (Amino acid sequence, SEQ ID NO: 24):

IDNAGTT

g) HC CDR3 (Nucleic acid sequence, SEQ ID NO: 25):

GCGAGAGTCTTTTATGATATTAATAGTGGTTATTATCTGGACGGCATGGAC CTC

h) HC CDR3 (Amino acid sequence, SEQ ID NO: 26):

ARVFYDINSGYYLDGMDL i) Light chain variable region— VL/JL (Nucleic acid sequence, SEQ ID NO: 27):

CAGTTTGTGCTGACTCAGTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCT GCCAAGCTCACCTGCACCCTGAGCAGTGCCCACAAGACCTACACCATTGA CTGGTATCAGCAGCAGCAAGGGGAGGCCCCTCGGTACCTGATGCAAGTTA AGAGTGATGGAAGCTACACCAAGGGGACCGGGGTCCCTGATCGCTTCTCG GGCTCCAGCTCTGGGGCTGACCGCTACTTGATCATCCCCAGCGTCCAGGCT GATGACGAAGCCGGCTACGTTTGTGGTGCAGATGATAACGGTGGGTATGT GTTCGGCGGAGGGACCCAGCTGACCGTCACA

j) Light chain variable region— VL/JL (Amino acid sequence, SEQ ID NO: 28): QFVLTQSPSVSAALGASAKLTCTLSSAHKTYTIDWYQQQQGEAPRYLMQVKS DGSYTKGTGVPDRFSGSSSGADRYLIIPSVQADDEAGYVCGADDNGGYVFGG GTQLTVT k) LC CDR1 (Nucleic acid sequence, SEQ ID NO: 29):

AGTGCCCACAAGACCTACACC

1) LC CDR1 (Amino acid sequence, SEQ ID NO: 30):

SAHKTYT

m) LC CDR2 (Nucleic acid sequence, SEQ ID NO: 31):

GTTAAGAGTGATGGAAGCTAC

n) LC CDR2 (Amino acid sequence, SEQ ID NO: 32):

VKSDGSY o) LC CDR3 (Nucleic acid sequence, SEQ ID NO: 33):

GGTGCAGATGATAACGGTGGGTATGTG

p) LC CDR3 (Amino acid sequence, SEQ ID NO: 34): GADDNGGYV

Table 1 : Summary of the sequence identifiers for anti-GFRa4 scFV

S I Q I D NO: Description

SEQ ID NO: 3 P4-6; heavy chain (nucleic acid)

SEQ ID NO: 4 P4-6; heavy chain (amino acid)

SEQ ID NO: 5 P4-6; CDRl heavy chain (nucleic acid)

SEQ ID NO: 6 P4-6; CDRl heavy chain (amino acid)

SEQ ID NO: 7 P4-6; CDR2 heavy chain (nucleic acid)

SEQ ID NO: 8 P4-6; CDR2 heavy chain (amino acid)

SEQ ID NO: 9 P4-6; CDR3 heavy chain (nucleic acid)

SEQ ID NO: 10 P4-6; CDR3 heavy chain (amino acid)

SEQ ID NO: 1 1 P4-6; light chain (nucleic acid)

SEQ ID NO: 12 P4-6; light chain (amino acid)

SEQ ID NO: 13 P4-6; CDRl light chain (nucleic acid)

SEQ ID NO: 14 P4-6; CDRl light chain (amino acid)

SEQ ID NO: 15 P4-6; CDR2 light chain (nucleic acid)

SEQ ID NO: 16 P4-6; CDR2 light chain (amino acid)

SEQ ID NO: 17 P4-6; CDR3 light chain (nucleic acid)

SEQ ID NO: 18 P4-6; CDR3 light chain (amino acid)

SEQ ID NO: 19 P4-10; heavy chain (nucleic acid)

SEQ ID NO: 20 P4-10; heavy chain (amino acid)

SEQ ID NO: 21 P4-10; CDRl heavy chain (nucleic acid)

SEQ ID NO: 22 P4-10; CDRl heavy chain (amino acid)

SEQ ID NO: 23 P4-10; CDR2 heavy chain (nucleic acid)

SEQ ID NO: 24 P4-10; CDR2 heavy chain (amino acid)

SEQ ID NO: 25 P4-10; CDR3 heavy chain (nucleic acid)

SEQ ID NO: 26 P4-10; CDR3 heavy chain (amino acid)

SEQ ID NO: 27 P4-10; light chain (nucleic acid)

SEQ ID NO: 28 P4-10; light chain (amino acid)

SEQ ID NO: 29 P4-10; CDRl light chain (nucleic acid)

SEQ ID NO: 30 P4-10; CDRl light chain (amino acid)

SEQ ID NO: 3 1 P4-10; CDR2 light chain (nucleic acid) SEQ ID NO: 32 P4-10; CDR2 light chain (amino acid)

SEQ ID NO: 33 P4-10; CDR3 light chain (nucleic acid)

SEQ ID NO: 34 P4-10; CDR3 light chain (amino acid)

To verify that human/rabbit Fabs P4-6 and P4-10 retain their binding to both isoforms of GFRa4 but do not cross-react with GFRal, GFRa2, and GFRa3 when expressed as soluble Fabs (i.e. unlinked to phage particles), Fabs were expressed in E. coli without helper phage rescue and harvested from the periplasmic space as described (Elia et al, Production and purification of Fab and scFv in Phage Display: A Laboratory Manual (Chapter 12), eds Barbas, C.F., Burton, D.R., Scott, J.K., and Silverman, G.J.12.1-12.26; 2001) (Figure 3).

In sum, this example demonstrates the isolation of 2 novel and unique human/rabbit chimeric monoclonal Fab antibodies (P4-6 and P4-10) to be used for development of potential humoral or cellular therapies for the treatment of medullary thyroid carcinoma.

Example 2— Expression of P4-6 and P4-10 scFv's as CARs

ScFv constructs for P4-6 and P4-10 based on the rabbit Fab VH/VL nucleotide sequences were designed in the orientation Vn-linker-V_L with the linker comprising nucleotides to encode a 15-amino acid glycine/serine rich peptide and with 5' and 3' BamHl and Nhel restriction sites, respectively (SEQ ID NOs: 35-38). Figure 4 shows nucleotide (Figure 4A) and amino acid (Figure 4B) alignments of the individual VH and VL segments to the scFv P4-6 construct. Figure 5 shows nucleotide

(Figure 5A) and amino acid alignments (Figure 5B) of the individual VH and VL segments to the scFv P4-10 construct. Optimization for human codon usage (except for restriction sites) was performed by Genewiz, Inc. (South Plainfield, NJ).

Described herein are the nucleotide sequences of optimized P4-6 (SEQ ID NO: 39) and P4-10 (SEQ ID NO: 40) scFv constructs . Figures 6 and 7 compare original and optimized nucleotide sequences for P4-6 and P4-10.

ScFv constructs for P4-6 and P4-10 were restriction digested with BamHl and Nhel and ligated into the corresponding restriction sites of plasmid vectors that provide a 10-amino acid glycine/serine (GS)-rich linker

(GGGGSGGGGS) at the carboxy terminus of the scFv, followed by the transmembrane domain of human CD8, a 4-1BB domain and CD3zeta domain sequentially (plasmids pTRPE p4-6(GS linker)BBz, and pTRPE p4-10(GS linker)BBz, Figures 8 and 9 respectively). The resulting vectors encode CARs termed P4-6gs (SEQ ID NO: 41) and P4-10gs (SEQ ID NO: 42). Additionally, scFv constructs for P4-6 and P4-10 were similarly ligated into vectors identical except that the 10-amino acid GS-rich linker was replaced with a 47-amino acid peptide derived from the human CD8a hinge region (plasmids pTRPE p4-6(CD8 hinge)BBz, and pTRPE p4-10(CD8 hinge)BBz, Figures 10 and 11 respectively). The resulting vectors encode CARs termed P4-6cd8 (SEQ ID NO: 43) and P4-10cd8 (SEQ ID NO: 44).

To generate lentiviral supernatants, LentiX-293T cells (Clontech, Inc.,

Mountain View, CA) were seeded on Day 0 and transfected using Lipofectamine 2000 (Life Technologies, Grand Island, NY) on Day 1 as described (Milone et al, Molec. Ther., 17, 1453-1464, 2009). For each construct, the plasmids used were pVSV-G(VSV glycoprotein expression plasmid), pRSV.REV (Rev expression plasmid), pMDLg/p-l.RRE (Gag/Pol expression plasmid), and the CAR transfer vector (pTRPE). Lentiviral-supernatants were filtered through 0.45um pore size filters and concentrated by centrifugation at 12,000 x g at 4°C for 12-18 hours.

Reporter Jurkat T cells with a stably integrated NFAT promoter driven EGFP construct (Lin et al, J. Cell Biol, 162, 673-682., 2003; Hooijberg et al, Blood, 96, 459-466, 2003) were transduced with lentiviral supernatant to express the P4-6- and P4-10-containing CARs or a mesothelin-specific CAR control (SS1-KIRS2, Dr. V. Bhoj, unpublished) at a MOI of approximately 5. Primary T-cells were isolated, expanded and transduced as previously described (Milone et al, Molec. Ther., 17, 1453-1464, 2009).

Expression of P4-6 and P4-10 scFv's on the extracellular portion of the

CARs is illustrated for the GS linker set of scFv's along with non-transduced cells ("NTD") in Figure 12 by flow cytometry (FACSCalibur (BD Biosciences, Franklin Lakes, NJ)) of cells stained with biotin-labeled F(ab')2 fragment donkey anti-rabbit IgG (H+L) followed by streptavidin-conjugated-phycoerythrin (Jackson

ImmunoResearch, West Grove, PA).

Below are the nucleotide and amino acid sequences of P4-6 and P4-10 single chain antibody (scFv) constructs derived from the V_H and VL sequences of recombinant human/rabbit Fabs. The scFv constructs position the heavy and light chain variable regions in the order VH-linker-VL with the linker comprising nucleotides to encode a 15-amino acid glycine/serine rich peptide. Each nucleotide sequence begins and ends with a restriction site (BamHl and Nhel, respectively) for subsequent cloning into CAR plasmids. The nucleotide sequences depicted here are before human codon optimization. They include: Nucleotide sequence of P4-6 scFv construct (SEQ ID NO: 35); Amino acid sequence of P4-6 scFv construct (SEQ ID NO: 36); Nucleotide sequence of P4-10 scFv construct (SEQ ID NO: 37); and Amino acid sequence of P4-10 scFv construct with restriction sites (SEQ ID NO: 38). P4-6 scFv (SEQ ID NO: 35)

P4-6 scFv construct with restriction sites (original rabbit codon usage):

GGATCCGAGCAGCTGAAGGAGTCCGGGGGAGGTCTCTTCAAGCCAACGG ATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTTACTATG GAGTGAACTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGATCGG AACCATTGGTGGTAGTGGTGACACATACTACGCGAGCTGGGCGAAGAGCC GATCCACCATCATCAGAAACACCAACGAGAACACGGTGACTCTGAAAATG ACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGTGAGATATGC TAATATTGGTTATGAGTACTTTAACGTCTGGGGTCCAGGCACCCTGGTCAC CGTCTCTTCAGGTGGAGGCGGTTCAGGCGGCGGTGGCTCTAGCGGTGGTG GATCGCAGTTTGTGCTGACTCAGTCGCCCTCTGCATCTGCTGCCCTGGGAG CCTCGGCCAAGCTCACCTGCACCCTGAGCAGTGCCCACAAGACCTACACC ATTGACTGGTATCAGCAGCAGAAAGGGAAGGCCCCTCGCTACCTGATACA AGTTAAGAGTGATGGAACCTACACCAAGGCGACCGGGGTCCCTGATCGCT TCTCGGGCTCCAGCTCTGGGGCTGACCGCTACCTGATCATCCCCAGCGTCC AGGCTGATGACGAAGCCGACTACTATTGTGGTACAGATTATACCGGTGGG TATGTGTTCGGCGGGGGGACCCAGCTGACCGTCACAGCTAGC

B- Amino acid sequence of P4-6 scFv (SEQ ID NO: 36)

P4-6 scFv construct with restriction sites (aa):

GSEQLKESGGGLFKPTDTLTLTCTVSGFSLSYYGVNWVRQAPGNGLEWIGTI GGSGDTYYASWAKSRSTIIRNTNENTVTLKMTSLTAADTATYFCVRYANIGY EYFNVWGPGTLVTVSSGGGGSGGGGSSGGGSQFVLTQSPSASAALGASAKLT CTLSSAHKTYTIDWYQQQKGKAPRYLIQVKSDGTYTKATGVPDRFSGSSSGA DRYLIIPSVQADDEADYYCGTDYTGGYVFGGGTQLTVTAS

C- Nucleotide sequence of P4-10 scFv (SEP ID NO: 37)

P4-10 scFv construct with restriction sites (original rabbit codon usage):

GGATCCCAGTCAGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGA TACCCTGACACTCACCTGCACGGTCTCTGGATTCTCCCTCAGTAGACATGC ACTGACCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGATCGGAG CCATTGATAACGCTGGTACCACATACTACGCGAGCTGGGCGAAAAGCCGC TCCACCATCACCAGAAACACCGACCTGCACACGGTGACTCTGAAAATGAC CAGTCTGACAGCCTCGGACACGGCTACCTATTTCTGTGCGAGAGTCTTTTA TGATATTAATAGTGGTTATTATCTGGACGGCATGGACCTCTGGGGCCCAG GGACCCTCGTCACCGTCTCTTCAGGTGGAGGCGGTTCAGGCGGCGGTGGC TCTAGCGGTGGTGGATCGCAGTTTGTGCTGACTCAGTCGCCCTCTGTGTCT GCCGCCCTGGGAGCCTCTGCCAAGCTCACCTGCACCCTGAGCAGTGCCCA CAAGACCTACACCATTGACTGGTATCAGCAGCAGCAAGGGGAGGCCCCTC GGTACCTGATGCAAGTTAAGAGTGATGGAAGCTACACCAAGGGGACCGG GGTCCCTGATCGCTTCTCGGGCTCCAGCTCTGGGGCTGACCGCTACTTGAT CATCCCCAGCGTCCAGGCTGATGACGAAGCCGGCTACGTTTGTGGTGCAG ATGATAACGGTGGGTATGTGTTCGGCGGAGGGACCCAGCTGACCGTCACA GCTAGC

D- Amino acid sequence of P4-10 scFv (SEP ID NO: 38)

P4-10 scFv construct with restriction sites (aa):

GSQSVKESEGGLFKPTDTLTLTCTVSGFSLSRHALTWVRQAPGNGLEWIGAID

NAGTTYYASWAKSRSTITRNTDLHTVTLKMTSLTASDTATYFCARVFYDINS

GYYLDGMDLWGPGTLVTVSSGGGGSGGGGSSGGGSQFVLTQSPSVSAALGA

SAKLTCTLSSAHKTYTIDWYQQQQGEAPRYLMQVKSDGSYTKGTGVPDRFS

GSSSGADRYLIIPSVQADDEAGYVCGADDNGGYVFGGGTQLTVTAS

Below are the nucleotide and amino acid sequences of the P4-6 (SEQ ID NO: 39) and P4-10 (SEQ ID NO: 40) scFv constructs with codons optimized for human expression (except for BamHl and Nhel restriction sites). Further shown are the representative nucleotide sequences of the vector P4-6(gs) encoding CAR (SEQ ID NO: 41); the nucleotide sequence of the vector P4-10(gs) encoding CAR (SEQ ID NO: 42); the nucleotide sequence of the vector P4-6cd8 encoding CAR (SEQ ID NO:

43) ; and the nucleotide sequence of the vector P4-10cd8 encoding CAR (SEQ ID NO:

44) .

P4-6 scFv construct with restriction sites codon optimized for human expression

(SEQ ID NO: 39):

GGATCCGAGCAGCTGAAGGAGTCCGGCGGAGGCCTGTTTAAGCCCACCGA

CACCCTGACACTGACCTGCACAGTGTCCGGCTTCAGCCTGAGCTACTATG

GCGTGAACTGGGTGAGACAGGCCCCTGGCAACGGACTGGAGTGGATCGG CACCATTGGCGGCAGCGGAGACACCTACTACGCCAGCTGGGCCAAGTCCA GGAGCACCATCATCAGAAACACCAACGAGAACACCGTGACCCTGAAGAT GACCTCCCTGACAGCCGCCGACACCGCCACCTACTTCTGCGTGAGGTACG CCAACATCGGCTACGAGTACTTCAACGTGTGGGGCCCTGGCACCCTGGTG ACAGTGTCCAGCGGCGGAGGAGGAAGCGGCGGCGGCGGCTCCAGCGGAG GCGGCAGCCAGTTTGTGCTGACCCAGAGCCCTAGCGCTTCCGCCGCCCTG GGCGCCAGCGCCAAGCTCACCTGTACCCTGAGCAGCGCCCACAAGACCTA TACCATCGACTGGTACCAGCAGCAGAAGGGCAAGGCCCCCAGGTACCTGA TCCAGGTGAAGTCCGACGGCACCTACACCAAAGCCACCGGCGTGCCCGAC AGATTTAGCGGCAGCAGCTCCGGCGCCGACAGGTATCTGATCATCCCTTC

CGTGCAGGCCGACGACGAGGCCGACTACTACTGCGGAACCGACTACACCG

GCGGATACGTGTTCGGAGGCGGCACCCAGCTGACCGTGACCGCTAGC

P4-10 scFv construct with restriction sites codon optimized for human expression

(SEP ID NO: 40):

GGATCCCAGTCCGTGAAGGAGAGCGAGGGCGGCCTGTTCAAGCCCACCG ACACCCTGACCCTGACCTGCACAGTGAGCGGCTTCAGCCTGTCCAGACAC GCCCTGACATGGGTGAGACAGGCCCCTGGCAACGGCCTGGAATGGATCGG CGCCATCGACAACGCCGGCACCACCTACTACGCCAGCTGGGCCAAGTCCA GGTCCACCATCACCAGGAACACCGACCTCCACACCGTGACCCTGAAGATG ACAAGCCTGACCGCCTCCGACACCGCCACCTACTTCTGCGCCAGGGTGTT CTACGACATCAACAGCGGCTACTACCTGGATGGCATGGACCTGTGGGGAC CTGGCACACTGGTGACCGTGAGCAGCGGAGGCGGCGGCAGCGGCGGCGG CGGCAGCAGCGGCGGCGGAAGCCAGTTCGTGCTGACACAGAGCCCTAGC GTGAGCGCCGCCCTGGGAGCCTCCGCTAAACTGACCTGCACCCTGAGCAG CGCCCACAAGACCTACACCATCGACTGGTACCAACAGCAGCAGGGCGAG GCCCCCAGGTATCTGATGCAGGTGAAGTCCGACGGCAGCTACACCAAAGG CACCGGCGTGCCTGACAGGTTCAGCGGCAGCTCCAGCGGAGCCGACAGGT ACCTGATCATCCCCTCCGTGCAGGCCGACGACGAGGCTGGCTACGTGTGT GGCGCCGACGACAATGGCGGCTACGTGTTCGGAGGCGGCACCCAGCTGAC CGTGACAGCTAGC

Vector P4-6gs encoding CAR (SEP ID NO: 41)

GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTT GAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACT CGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACG ATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCA TGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGC AAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATA GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCG TAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGA CAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCC AGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCT CCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCG ACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGA TTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGC TGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGT AACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCA TGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCA ACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCA GAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGT TAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGT CTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTG TGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGG GAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCA CGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAG CGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGG GAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCT AGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTA GACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAA CTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGG ATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGC AAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGAC CTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATA TAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAG AGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGT TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATG ACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCA GAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCA CAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGA TACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACT CATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCT GGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATT AACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCA GCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTC ATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTT TCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACC CACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAG AAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC TCGACGGTATCGATTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTA CACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGA TATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCAT ACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACA GACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTG GGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAG GAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGT AAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCA TCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGA AAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAA AAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAG AGATCCAGTTTGGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTG GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTC GGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAA GTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGT ATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCG CCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTT ACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTAC GTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAG GCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCC TGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTC TCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTG CGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGC ACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCG TCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAG AATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCC TCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCG GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGG GAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTT TTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTT TCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGA TGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCT CAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG AGCTAGAGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTAT TTTAAAAGGTGTCCAGTGCGGATCCGAGCAGCTGAAGGAGTCCGGGGGAG GTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGAT TCTCCCTCAGTTACTATGGAGTGAACTGGGTCCGCCAGGCTCCAGGGAAC GGGCTGGAATGGATCGGAACCATTGGTGGTAGTGGTGACACATACTACGC GAGCTGGGCGAAGAGCCGATCCACCATCATCAGAAACACCAACGAGAAC ACGGTGACTCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTA TTTCTGTGTGAGATATGCTAATATTGGTTATGAGTACTTTAACGTCTGGGG TCCAGGCACCCTGGTCACCGTCTCTTCAGGTGGAGGCGGTTCAGGCGGCG GTGGCTCTAGCGGTGGTGGATCGCAGTTTGTGCTGACTCAGTCGCCCTCTG CATCTGCTGCCCTGGGAGCCTCGGCCAAGCTCACCTGCACCCTGAGCAGT GCCCACAAGACCTACACCATTGACTGGTATCAGCAGCAGAAAGGGAAGG CCCCTCGCTACCTGATACAAGTTAAGAGTGATGGAACCTACACCAAGGCG ACCGGGGTCCCTGATCGCTTCTCGGGCTCCAGCTCTGGGGCTGACCGCTAC CTGATCATCCCCAGCGTCCAGGCTGATGACGAAGCCGACTACTATTGTGG TACAGATTATACCGGTGGGTATGTGTTCGGCGGGGGGACCCAGCTGACCG TCACAGCTAGCGGTGGCGGAGGTTCTGGAGGTGGAGGTTCCTCCGGAATC TACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGTCCCTG GTCATCACCCTGTACTGCAAGCGGGGCAGAAAGAAGCTGCTGTACATCTT CAAGCAGCCCTTCATGCGGCCTGTGCAGACCACACAGGAAGAGGACGGCT GTAGCTGTAGATTCCCCGAGGAAGAGGAAGGCGGCTGCGAGCTGAGAGT GAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACC AGCTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGACGTGCT GGACAAGAGAAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCAGACGG AAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGG CCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGAAGAGGCAA GGGCCATGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCT ACGACGCCCTGCACATGCAGGCCCTGCCTCCAAGATGAGTCGACAATCAA CCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTA TTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCT GTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTG CACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTG TCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGG GCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGC TGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACG TCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGG CTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCT CCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAAGACC AATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGG GGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTT GCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCT GGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATC CCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATG TCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAG TGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATA GCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTG GTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCC GCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAAT TTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCA GAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATT CGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAAC GTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCA CATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCG CCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTA GCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCT ACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTC TCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATT AGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCC CTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTG GAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTT TGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA ACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCG GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAA TATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTG AAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTT TTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAA AGTAAAAGATGCTGAAGATCAGTTGG

Vector P4-10gs encoding CAR (SEP ID NO: 42)

GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTT GAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACT CGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACG ATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCA TGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGC AAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATA GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCG TAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGA CAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCC AGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCT CCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCG ACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGA TTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGC TGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGT AACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCA TGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCA ACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCA GAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGT TAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGT CTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTG TGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGG GAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCA CGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAG CGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGG GAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCT AGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTA GACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAA CTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGG ATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGC AAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGAC CTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATA TAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAG AGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGT TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATG ACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCA GAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCA CAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGA TACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACT CATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCT GGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATT AACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCA GCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTC ATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTT TCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACC CACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAG AAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC TCGACGGTATCGATTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTA CACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGA TATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCAT ACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACA GACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTG GGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAG GAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGT AAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCA TCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGA AAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAA AAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAG AGATCCAGTTTGGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTG GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTC GGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAA GTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGT ATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCG CCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTT ACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTAC GTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAG GCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCC TGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTC TCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTG CGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGC ACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCG TCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAG AATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCC TCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCG GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGG GAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTT TTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTT TCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGA TGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCT CAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG AGCTAGAGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTAT TTTAAAAGGTGTCCAGTGCGGATCCCAGTCAGTGAAGGAGTCCGAGGGAG GTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACGGTCTCTGGAT TCTCCCTCAGTAGACATGCACTGACCTGGGTCCGCCAGGCTCCAGGG AACGGGCTGGAATGGATCGGAGCCATTGATAACGCTGGTACCACATACTA CGCGAGCTGGGCGAAAAGCCGCTCCACCATCACCAGAAACACCGACCTGC ACACGGTGACTCTGAAAATGACCAGTCTGACAGCCTCGGACACGGCTACC TATTTCTGTGCGAGAGTCTTTTATGATATTAATAGTGGTTATTATCTGGAC GGCATGGACCTCTGGGGCCCAGGGACCCTCGTCACCGTCTCTTCAGGTGG AGGCGGTTCAGGCGGCGGTGGCTCTAGCGGTGGTGGATCGCAGTTTGTGC TGACTCAGTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCTGCCAAGCTCA CCTGCACCCTGAGCAGTGCCCACAAGACCTACACCATTGACTGGTATCAG CAGCAGCAAGGGGAGGCCCCTCGGTACCTGATGCAAGTTAAGAGTGATGG AAGCTACACCAAGGGGACCGGGGTCCCTGATCGCTTCTCGGGCTCCAGCT CTGGGGCTGACCGCTACTTGATCATCCCCAGCGTCCAGGCTGATGACGAA GCCGGCTACGTTTGTGGTGCAGATGATAACGGTGGGTATGTGTTCGGCGG AGGGACCCAGCTGACCGTCACAGCTAGCGGTGGCGGAGGTTCTGGAGGTG GAGGTTCCTCCGGAATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGC GTGCTGCTGCTGTCCCTGGTCATCACCCTGTACTGCAAGCGGGGCAGAAA GAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCTGTGCAGACCA CACAGGAAGAGGACGGCTGTAGCTGTAGATTCCCCGAGGAAGAGGAAGG CGGCTGCGAGCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCT ATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACG GGAGGAATACGACGTGCTGGACAAGAGAAGAGGCCGGGACCCTGAGATG GGCGGCAAGCCCAGACGGAAGAACCCCCAGGAAGGCCTGTATAACGAAC TGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGG CGAGCGGAGAAGAGGCAAGGGCCATGACGGCCTGTACCAGGGCCTGAGC ACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCTCC AAGATGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGA CTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTT AATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCC TTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTC AGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGG TTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCC CCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTG GACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGA AGCTGACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGC GCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTC CTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTC GCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTC GAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTA GCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAA CGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAG ATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCC TCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTG TGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAAT CTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCA AAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAA TGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTT TTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCA TGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCT CCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC TCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTA GCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTC ACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCC AACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG AAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC GAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGT TACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTT CGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT CTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTC GACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCC CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAG TGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTC TTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGA GCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTAC AATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTA TTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGA TAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTT CCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG

Vector P4-6cd8 encoding CAR (SEP ID NO: 43)

GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTT

GAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT

CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACT

CGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT

CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG

CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACG ATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCA TGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGC AAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATA GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCG TAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGA CAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCC AGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCT CCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCG ACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGA TTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGC TGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGT AACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCA TGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCA ACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCA GAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGT TAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGT CTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTG TGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGG GAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCA CGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAG CGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGG GAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCT AGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTA GACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAA CTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGG ATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGC AAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGAC CTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATA TAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAG AGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGT TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATG ACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCA GAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCA CAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGA TACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACT CATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCT GGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATT AACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCA GCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTC ATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTT TCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACC CACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAG AAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC TCGACGGTATCGATTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTA CACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGA TATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCAT ACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACA GACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTG GGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAG GAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGT AAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCA TCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGA AAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAA AAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAG AGATCCAGTTTGGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTG GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTC GGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAA GTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGT ATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCG CCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTT ACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTAC GTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAG GCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCC TGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTC TCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTG CGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGC ACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCG TCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAG AATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCC TCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCG GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGG GAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTT TTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTT TCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGA TGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCT CAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG AGCTAGAGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTAT TTTAAAAGGTGTCCAGTGCGGATCCGAGCAGCTGAAGGAGTCCGGGGGAG GTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGAT TCTCCCTCAGTTACTATGGAGTGAACTGGGTCCGCCAGGCTCCAGGGAAC GGGCTGGAATGGATCGGAACCATTGGTGGTAGTGGTGACACATACTACGC GAGCTGGGCGAAGAGCCGATCCACCATCATCAGAAACACCAACGAGAAC ACGGTGACTCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTA TTTCTGTGTGAGATATGCTAATATTGGTTATGAGTACTTTAACGTCTGGGG TCCAGGCACCCTGGTCACCGTCTCTTCAGGTGGAGGCGGTTCAGGCGGCG GTGGCTCTAGCGGTGGTGGATCGCAGTTTGTGCTGACTCAGTCGCCCTCTG CATCTGCTGCCCTGGGAGCCTCGGCCAAGCTCACCTGCACCCTGAGCAGT GCCCACAAGACCTACACCATTGACTGGTATCAGCAGCAGAAAGGGAAGG CCCCTCGCTACCTGATACAAGTTAAGAGTGATGGAACCTACACCAAGGCG ACCGGGGTCCCTGATCGCTTCTCGGGCTCCAGCTCTGGGGCTGACCGCTAC CTGATCATCCCCAGCGTCCAGGCTGATGACGAAGCCGACTACTATTGTGG TACAGATTATACCGGTGGGTATGTGTTCGGCGGGGGGACCCAGCTGACCG TCACAGCTAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGC GGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTCCG GAATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGT CCCTGGTCATCACCCTGTACTGCAAGCGGGGCAGAAAGAAGCTGCTGTAC ATCTTCAAGCAGCCCTTCATGCGGCCTGTGCAGACCACACAGGAAGAGGA CGGCTGTAGCTGTAGATTCCCCGAGGAAGAGGAAGGCGGCTGCGAGCTGA GAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGCCAG AACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGACG TGCTGGACAAGAGAAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCAG ACGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAG ATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGAAGAG GCAAGGGCCATGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGA CACCTACGACGCCCTGCACATGCAGGCCCTGCCTCCAAGATGAGTCGACA ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACT ATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCA TGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGG TTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTG GTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACC ACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGG CGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTG TTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCT TGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGC TACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTG CCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGG ATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAA GACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAA AAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCT TTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTG AGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAG ATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTC ATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGA GAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCA ATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTT GTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTAT CCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATT CCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGCTAGGGACGTACCCA ATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTAC AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCA GCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGA TCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCT GTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACC GCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCT TTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCC CTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTC GCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAA CTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGA TTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAAT TTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTT TCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTC AAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAAT ATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTC CCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT GAAAGTAAAAGATGCTGAAGATCAGTTGG

Vector P4-10cd8 encoding CAR (SEP ID NO: 44):

GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTT GAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACT CGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACG ATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCA TGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGC AAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATA GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCG TAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGA CAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCC AGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCT CCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCG ACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGA TTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGC TGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGT AACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCA TGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCA ACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCA GAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGT TAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGT CTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTG TGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGG GAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCA CGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAG CGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGG GAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCT AGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTA GACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAA CTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGG ATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGC AAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGAC CTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATA TAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAG AGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGT TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATG ACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCA GAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCA CAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGA TACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACT CATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCT GGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATT AACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCA GCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTC ATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTT TCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACC CACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAG AAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC TCGACGGTATCGATTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTA CACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGA TATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCAT ACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACA GACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTG GGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAG GAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGT AAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCA TCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGA AAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAA AAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAG AGATCCAGTTTGGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTG GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTC GGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAA GTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGT ATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCG CCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTT ACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTAC GTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAG GCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCC TGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTC TCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTG CGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGC ACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCG TCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAG AATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCC TCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCG GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGG GAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTT TTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTT TCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGA TGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCT CAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG AGCTAGAGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTAT TTTAAAAGGTGTCCAGTGCGGATCCCAGTCAGTGAAGGAGTCCGAGGGAG GTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACGGTCTCTGGAT TCTCCCTCAGTAGACATGCACTGACCTGGGTCCGCCAGGCTCCAGGGAAC GGGCTGGAATGGATCGGAGCCATTGATAACGCTGGTACCACATACTACGC GAGCTGGGCGAAAAGCCGCTCCACCATCACCAGAAACACCGACCTGCAC ACGGTGACTCTGAAAATGACCAGTCTGACAGCCTCGGACACGGCTACCTA TTTCTGTGCGAGAGTCTTTTATGATATTAATAGTGGTTATTATCTGGACGG CATGGACCTCTGGGGCCCAGGGACCCTCGTCACCGTCTCTTCAGGTGGAG GCGGTTCAGGCGGCGGTGGCTCTAGCGGTGGTGGATCGCAGTTTGTGCTG ACTCAGTCGCCCTCTGTGTCTGCCGCCCTGGGAGCCTCTGCCAAGCTCACC TGCACCCTGAGCAGTGCCCACAAGACCTACACCATTGACTGGTATCAGCA GCAGCAAGGGGAGGCCCCTCGGTACCTGATGCAAGTTAAGAGTGATGGA AGCTACACCAAGGGGACCGGGGTCCCTGATCGCTTCTCGGGCTCCAGCTC TGGGGCTGACCGCTACTTGATCATCCCCAGCGTCCAGGCTGATGACGAAG CCGGCTACGTTTGTGGTGCAGATGATAACGGTGGGTATGTGTTCGGCGGA GGGACCCAGCTGACCGTCACAGCTAGCACCACGACGCCAGCGCCGCGACC ACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAG AGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGA CTTCGCCTGTGATTCCGGAATCTACATCTGGGCCCCTCTGGCCGGCACCTG TGGCGTGCTGCTGCTGTCCCTGGTCATCACCCTGTACTGCAAGCGGGGCAG AAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCTGTGCAGA CCACACAGGAAGAGGACGGCTGTAGCTGTAGATTCCCCGAGGAAGAGGA AGGCGGCTGCGAGCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCT GCCTATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCA GACGGGAGGAATACGACGTGCTGGACAAGAGAAGAGGCCGGGACCCTGA GATGGGCGGCAAGCCCAGACGGAAGAACCCCCAGGAAGGCCTGTATAAC GAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGA AGGGCGAGCGGAGAAGAGGCAAGGGCCATGACGGCCTGTACCAGGGCCT GAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGC CTCCAAGATGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGA TTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTG CTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTC CTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTT GTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCAC TGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTT CCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTG CTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGG GGAAGCTGACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTC TGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACC TTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCT TCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAAT TCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTT AGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCA ACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCA GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGC CTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGT GTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAA ATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTG CAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTAT AATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATT TTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAT CATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATT CTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCG CCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCC TAGCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCT CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCC AACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG AAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC GAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGT TACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTT CGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT CTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTC GACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCC CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAG TGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTC TTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGA GCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTAC AATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTA TTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGA TAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTT CCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG Example 3— Reporter T cells expressing CART-P4-6 and CART -P4- 10 are specifically activated by GFRot4

Reporter Jurkat cells expressing GFP under an NFAT-responsive promoter (Lin et al, J. Cell Biol, 162, 673-682., 2003; Hooijberg et al, Blood, 96, 459-466, 2003) transduced with P4-6gs and P4-10gs CARs were incubated with GFRa4-expressing cells and control cells, as well as with immobilized Fc-fusion proteins in order to assess the ability of GFRa4 to specifically activate reporter cells and trigger GFP expression. Fc-fusion proteins GFRal, GFRa2, GFRa3 and GFRa4a were captured in tissue culture wells by first coating wells overnight with mouse anti- human-Fc (lOug/ml) followed by 3 washes with PBS, blocking with 5% BSA/PBS for 1 hour, washing with PBS 3 times and then incubating overnight again with each of the GFRa-Fc fusion proteins in 1% BSA/PBS (5ug/ml). OKT3 antibody was directly coated on wells by overnight incubation (lOug/ml). Wells were then washed 3 times with PBS before Jurkat reporter cells containing no CAR, a mesothelin-specific CAR (SS1-KIRS2), or P4-6 and P4-10 GS linker CARs. Reporter Jurkats were incubated in the GFRa-Fc fusion proteins and OKT3-coated wells overnight. Reporter Jurkats were also co-incubated with K562 cells expressing mesothelin (Carpenito et al, Proc. Natl. Acad. Sci (USA), 106, 3360-3365; 2009), and medullary thyroid cancer cell lines TT (ATCC CRL-1803, Manassas, VA) and MZ-CRC-1 (Plaza-Menacho et al, Cancer Res., 65, 1729-1737, 2005) cells at a 1: 1 ratio overnight. After overnight incubation, cells were analyzed by flow cytometry. Jurkat cells were gated based on forward- and side-light scatter characteristics and GFP expression was measured. As shown in Figure 13, the Jurkat cells are activated by immobilized GFRa4a protein, but not by its homologs GFRal, GFRa2, and GFRa3. TT cells as well as MZ-CRC-1 cells also activated the Jurkat cells (TT > MZ-CRC-1), but not K562 cells expressing mesothelin. Jurkat cells expressing the mesothelin-specific CAR (SS1KIRS2) were activated by K562 mesothelin-expressing cells, but not by TT or MZ-CRC-1 cells or by the immobilized GFRa proteins, including GFRa4a. Wells coated with the anti- CD3 antibody OKT3 served as a positive control. Numerical values in figure above

GFP-positive cell gate represent % of total Jurkat reporter cells in the positive gate.

To further show specificity, Jurkat cells stably expressing the coding sequence for green fluorescence protein (GFP) under the control of a promoter containing four NFAT/AP 1 binding sites from the IL-2 promoter (NFAT-GFP Jurkat) were transduced by lentiviral vector to express a CD19-specific chimeric antigen receptor (FMC63bbz) or the GFRa4-specific CAR, P4-6bbz, or were left non- transduced (NTD). The Jurkat cells were then mixed in a 1 : 1 ratio with wild-type Nalm6 cells (an acute lymphoblastic leukemia cell line that expresses CD 19), Nalm6 cells engineered to express GFRa4 isoform b, or wild-type TT cells. As a positive control for reporter activation, NFAT-GFP Jurkat cells were also plated into wells of polystyrene microtiter plates that were pre-coated overnight with the anti-CD3 agonist antibody, clone OKT3 (lOug/ml), which stimulates GFP expression through the endogenous TCR/CD3 complex. After overnight incubation, GFP expression in the cells was analyzed by flow cytometry. Numbers in each plot indicate the percentage of GFP positive Jurkat cells (Figure 16). Results show specificity of the GFRa4- directed P4-6bbz CAR: wild-type Nalm6 cells only stimulated GFP expression in FMC63bbz cells; Nalm6 cells co-expressing GFRa4 activate both FMC63bbz and the GFRa4-directed P4-6bbz cells; and wild-type TT cells that express GFRa4 but lack CD 19 only induce GFP in GFRa4-directed P4-6bbz cells.

Example 4— Expression of GFRa4-specific CAR-T protein in CD4-positive and CD4-negative T cells from multiple healthy donors

Human T cells from two healthy donors were activated with anti-CD3 and anti-CD28 coated paramagnetic beads (DYNABEADS^® Human T-Activator CD3/CD28, Life Technologies). One day following activation, cells were transduced with lentiviral vector encoding either the CD19-specific FMC63bbz CAR or the GFRa4-specific P4-6bbz CAR, or were left non-transduced (NTD). Cells were expanded and on day 7, were stained with anti-CD4-PerCP and either biotinylated- donkey anti-rabbit (DaR, Figure 17, top two panels for each donor) or biotinylated- goat anti-mouse (GaM, Figure 17, bottom two panels for each donor) followed by a secondary stain with streptavidin-APC after thorough washing. Cells were fixed in 2% paraformaldehyde prior to analysis by flow cytometry. Results show similar expression of the CARs on the surface of CD4-positive and CD4-negative T cells indicating that there are no differences in transduction efficiency for either CAR- encoding lentiviral vector in the CD4-positive and CD4-negative, presumably CD8+, T cell subsets.

Example 5— Primary T cells expressing CART-P4-6 and CART-P4-10 kill a thyroid medullary cancer cell line in vitro

Cytotoxicity of target cells by P4-6 and P4-10 CARs was evaluated using a ⁵¹Cr release-assay. Target TT cells were labeled with ⁵¹Cr (sodium dichromate salt), washed and co-cultured with effector CAR T cells at effectontarget ratios of 30: 1, 10: 1, and 3: 1. Ten thousand target cells were co-cultured with the appropriate number of effector T cells in each well. Supernatants were collected after overnight co-culture and placed into 96-well Lumaplates (Perkin Elmer, Inc., Walthan MA). The amount of ⁵¹Cr released from the labeled target cells was measured on a liquid scintillation counter (MicroBeta Trilux, Perkin Elmer). Target cells incubated in medium alone or with 1% SDS were used to determine spontaneous (S) or maximum (M) ⁵¹Cr release. Percentage of specific lysis was calculated as follow: [(cpm experimental release- cpm S release)/ (cpm M release- cpm S release)] x 100. As shown in Figure 14, T cells transduced to express P4-6 and P4-10 GS linker CARs lysed TT cells while non-transduced T cells ( TD) and CD 19/mesothelin-specific CAR-T cells (FMCbbz) did not. As shown in control Figure 15, FMCbbz cells lysed CD19/mesothelin-expressing K562 cells (K562-CD19meso) while the P4-6 and P4-10 CAR-T cells do not.

Example 6— Specific lysis of GFRot4-expressing cells by anti- GFRa4-specific CAR- transduced T cells

Human T cells from two healthy donors trans fected with either the FMC63bbz anti-CD 19 CAR or the P4-6bbz GFRa4-specific CAR (Example 4) were mixed at the indicated effector to target ratios with K562 cells (ATCC) expressing either GFRa4 isoform b (Figure 18, top panel) or human CD 19 (Figure 18, bottom panel) pre-loaded with 51Cr. K562 cell lines expressing GFRa4b or CD 19 were generated by lentiviral vector-mediated transduction. Lentiviral vectors expressing these proteins were generated by cloning of cDNA from PMBC or synthesized DNA (Genewiz, South Plainfield, NJ) through PCR and standard molecular biology techniques. All plasmids were confirmed by sequencing. Expression of the antigens on the surface of the transduced K562 cells was confirmed by flow cytometry. The procedures for generation of high-titer lentiviral vectors have been previously described (Parry,R.V JI 2003). Briefly, 293T cells grown in RPMI with 10% FBS were co-transfected with lentiviral vector plasmids along with the pMDL.g/p, pRSV.rev and pVSVg packaging plasmids using Lipofectamine 2000 (Life

Technologies, Carlsbad, CA) transfection reagent. Vector containing supernatants were harvested at 24 and 48 hours after transfection, and concentrated by

centrifugation at 12,000 rpm for 2 hrs. Concentrated vector was stored at -70o C until use. Lysis of target cells was measured as described in Example 5. CAR expression percentages were as follows: Donor 1 P4-6bbz were 77% CAR+; Donor 1 FMC63bbz were 69% CAR+; Donor 2 P4-6bbz were 53% CAR+; Donor 2 FMC63bbz were 57%

CAR+. The results demonstrate a requirement for GFRa4-expression on cells for cytotoxicity by T cells expressing the GFRa4-specific P4-6bbz CAR.

Example 7— Specific lysis of GFRot4-expressing tumor cells by T cells expressing anti-GFRa4-CARs with different cytoplasmic signaling domains

Human T cells were activated with anti-CD3 and anti-CD28 coated paramagnetic beads (DYNABEADS^® Human T-Activator CD3/CD28, Life

Technologies). One day following activation, cells were transduced with lentiviral vector encoding CARs constructed with either the CD 19-specific scFv FMC63 or the GFRa4-specific scFvs, P4-6 or P4-10. Non-transduced T cells were used as a negative control (NTD). For each scFv, CARs were further constructed to contain the 4-lbb and CD3-zeta cytoplasmic domains (FMC63bbz, P4-6bbz or P4-10bbz, Figure 19), the CD28 and CD3-zeta cytoplasmic domains (FMC6328z, P4-6-28z or P4-10-28z, Figure 19) or a KIR2DS2 transmembrane and cytoplasmic domain with human DAP 12 co-delivered using the T2A ribosomal skipping sequence from the Thosea asigna virus (19KIRS2, P4-6-KIRS2 or P4-10-KIRS2, Figure 19). The transduced T cells were mixed at the indicated effector to targets ratios (E:T) with ⁵¹Cr-labeled TT- CD19 cells, a medullary thyroid carcinoma cell line that expresses endogenous GFRa4 and was engineered to also express human CD19. Engineering of TT cells to express CD 19 was carried out as described above for K562 cells. After a 4-hour co- incubation, culture supernatants were harvested and percent of target cells lysis (percent lysis) was calculated as in the previous examples. CAR expression for each of the CAR bearing T cells was in the range of 61% to 79% with the exception of FMC63-28z that were approximately 9% CAR+. As shown in Figure 19, results show the ability of both P4-6 and P4-10 GFRa4-specific CAR-expressing T cells to lyse TT target cells utilizing several different signaling configurations. Error bars indicate standard deviations. Example 8— T cells expressing a GFRa4-specific P4-6bbz and P4-10bbz CARs show

GFRa4-dependent secretion of the cytokines IFN-γ and IL-2

Human T cells were activated with anti-CD3 and anti-CD28 coated paramagnetic beads (DY ABEADS^® Human T-Activator CD3/CD28, Life

Technologies). One day following activation, cells were transduced with lentiviral vector encoding either the CD 19-specific FMC63bbz CAR, the GFRa4-specific P4-

6bbz or P4-10bbz CARs, or were left non-transduced ( TD). Expanded T cells were co-cultured with TT-CD19 cells or K562 cells engineered to express GFRa4 isoform b by lentiviral transduction at a T cell to target cell ratio of 1 : 1. After overnight incubation, culture supernatants were harvested and analyzed by ELISA for interferon-gamma (IFN-γ) and interleukin-2 (IL-2). Results demonstrate secretion of cytokines by T cells expressing the GFRa4-specific P4-6bbz and P4-10bbz CARs when incubated with target cells expressing GFRa4, but not with target cells lacking GFRa4 expression (Figure 20). Error bars indicate standard deviations. Example 9— T cells expressing a GFRa4-specific p4-10-28z CAR show GFRa4- dependent secretion of the cytokines IFN-γ and IL-2

Human T cells were activated with anti-CD3 and anti-CD28 coated paramagnetic beads (DYNABEADS^® Human T-Activator CD3/CD28, Life

Technologies). One day following activation, cells were transduced with lentiviral vector encoding either the CD19-specific FMC63-28z CAR, the GFRa4-specific p4-

10-28z CAR, or were left non-transduced (NTD). Expanded T cells were co- incubated with wild-type TT cells or K562 cells engineered to express CD 19 by lentiviral transduction at a T cell to target cell ratio of 1 : 1. After overnight co-culture, culture supernatants were harvested and analyzed by ELISA for interferon-gamma (IFN-γ) and interleukin-2 (IL-2). Results show secretion of cytokines by T cells expressing P4-10-28z CAR only in the presence of target cells expressing GFRa4 (Figure 21). Error bars indicate standard deviations. Example 10 - CART-P4-10 kill MTC cells in mouse model

In vivo experiment on a mouse model demonstrated that CART-P4-10 have the capacity of killing MTC cells and treating cancer. On day O, 5xl0⁶ TT cells were implanted sub-cutaneous ly in the flank of NOD-SCID-yc^"7" (NSG) mice. On day 24, lxlO⁷ non-transduced ( TD, n=6) or P4-10bbz-transduced (n=9) human T cells were injected intra-tumorally. On day 31, 6xl0⁶ non-transduced or P4-10bbz- transduced T cells from the same donor were again injected intra-tumorally. Tumor volume was measured by caliper measurement over time. Figure 22A shows the mean with standard error of the mean of tumor volume over time. Arrows indicate times of T cell injection. Figure 22B shows tumor size of individual mice at day 38 for each group (P=0.0008 by Mann- Whitney test). Mean and standard error of the mean are indicated for each group. Results show the continued growth of tumors in mice treated intra-tumorally with non-transduced T cells compared with a reduction in tumor volume in mice treated intra-tumorally with T cells transduced with the GFRa4- specific P4-10bbz CAR.

On day 0, 5xl0⁶ TT cells engineered to express click-beetle-green luceriferase were implanted sub-cutaneously in the flank of NSG mice. Lentiviral transduction of TT cells with click-beetle green luciferase was performed by using a vector encoding GFP followed by the T2A ribosomal skipping sequence from the Thosea asigna virus, followed by click-beetle green luciferase, all under the regulation of the EF-1 alpha promoter. The use of luciferase expressing TT cells permitted imaging by bioluminescence. On day 9, lxlO⁷ non-transduced (NTD, n=7) or P4-10bbz-transduced (n=8) human T cells were injected intravenously. Tumor volume was measured with calipers over time. Figure 23A shows the mean with standard error of the mean of tumor volume over time. The arrow indicates time of T cell injection. Figure 23B shows tumor size of individual mice at day 27 for each group (P=0.0093 by Mann- Whitney test). Mean and standard error of the mean are indicated for each group. Statistical analysis was performed for day 27 as this was the last time point containing all mice as some were then euthanized due to graft-versus- host effects. Results show continued growth of tumors in mice treated intravenously with non-transduced T cells and reduction in tumor volume in mice treated intravenously with T cells transduced with P4-10bbz CAR-T construct.

The mice shown herein that had been injected with TT cells engineered to expressed luciferase were imaged using an IVIS Spectrum bioluminescence imaging system (Perkin Elmer) following the intravenous injection of luciferin to determine the bioluminescence intensity (BLI) of the sub-cutaneous tumors in each mouse. Each line in Figures 24A and 24B shows the BLI of an individual mouse over time. Figure 24C shows the mean with standard deviation of BLI over time. These results show the reduction in tumor burden in mice treated intravenously with GFRa4-specific P4- 1 Obbz CAR T cells, but not with the NTD negative control T cells.

Example 11 - GFRot4 RNA is expressed by medullary thyroid carcinoma

Formalin-fixed, paraffin-embedded (FFPE) sections from a surgically resected, medullary thyroid carcinoma (MTC), from normal thyroid tissue, or from a cell pellet of the TT cell line (obtained from ATCC) were analyzed by RNA in situ hybridization using proprietary RNAscope technology (Advanced Cell Diagnostics, Hayward, CA). Sections were probed using a negative control probe targeting the bacterial RNA for the bacterial gene, DapB, a positive control probe for the RNA derived from the human PPIB gene, or a probe targeting the GFRa4 gene that binds to

RNA sequences shared by both isoforms a and b of human GFRa4 (Figure 25). The results show specific hybridization of the GFRa4 probe to malignant cells within the resected MTC tissue and TT cells, but no hybridization to normal thyroid follicular epithelial cells. Hybridization to normal parafollicular C-cells was observed but not shown.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is:

1. A composition comprising an anti-Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (anti-GFRa4) binding domain, wherein the anti- GFRa4 binding domain is a GFRa4 antibody, or fragment thereof.

2. The composition of claim 1, wherein the antibody or fragment thereof is a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody.

3. The composition of claim 1, wherein the antibody or fragment thereof is encoded by a nucleic acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 35, 37, 39 and 40.

4. The composition of claim 1 , wherein the anti-GFRa4 binding domain is a fragment antigen-binding (Fab) or a single-chain variable fragment (scFv) or single-domain antibody.

5. The composition of claim 1, wherein the anti-GFRa4 binding domain binds specifically to a thyroid cell antigen present in a tumor microenvironment.

6. The composition of claim 5, wherein the thyroid cell antigen is present on a medullary thyroid carcinoma (MTC) cell.

7. The composition of claim 5, wherein the thyroid cell antigen is a GFRa4 cell- surface receptor comprising at least one isoform selected from the group consisting of GFRa4a and GFRa4b.

8. A method of imaging or visualizing a sample taken from a normal or

malignant thyroid medullary cell, the method comprising contacting the sample with a labeled anti-GFRa4 binding domain.

9. The method of claim 8, wherein the anti-GFRa4 binding domain is an

antibody, or an antigen-binding fragment thereof.

10. The method of claim 9, wherein the antigen-binding fragment is a Fab or a scFv or single-domain antibody.

11. The method of claim 9, wherein the antibody or antigen-binding fragment is a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody.

12. The method of claim 8, wherein the anti-GFRa4 binding domain is encoded by a nucleic acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 35, 37, 39 and 40.

13. An isolated polynucleotide encoding an anti-GFRa4 antibody or a fragment thereof comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20 and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 12 and 28.

14. The isolated polynucleotide of claim 14, wherein the nucleic acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 3 and 19, and the nucleic acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 1 1 and 27.

15. An isolated polypeptide encoding an anti-GFRa4 antibody or a fragment thereof comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20 and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 12 and 28.

16. A method of diagnosing a condition in a mammal associated with the

expression of GFRa4 in a cell, the method comprising a) contacting the cell with an anti-GFRa4 antibody fragment comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20 and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 12 and 28; and b) detecting the presence of GFRa4 in the cell, wherein the presence of GFRa4 in the cell is an indication that the mammal has the condition.

17. The method of claim 16, wherein the cell is a medullary thyroid carcinoma cell.

18. A method of diagnosing, or determining risk of thyroid cancer in a mammal, the method comprising : a) contacting a sample with an anti-GFRa4 antibody fragment comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20, and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NOs: 12 and 28; and b) detecting the presence of GFRa4 in the sample wherein the presence of GFRa4 is an indication that the mammal has or is at risk of having thyroid cancer, wherein the sample is not derived from thyroid tissue.

19. The method of claims 16 and 18, wherein the mammal is a human.

20. A method of inhibiting growth of a GFRa4-expressing tumor cell, the method comprising contacting the tumor cell with an anti-GFRa4 antibody or a fragment thereof comprising a heavy chain and light chain, wherein the amino acid sequence of the heavy chain is selected from the group consisting of SEQ ID NOs: 4 and 20 and the amino acid sequence of the light chain is selected from the group consisting of SEQ ID NO: 12 and 28.

21. The method of claim 20, wherein the tumor cell is a medullary thyroid

carcinoma cell.

22. The method of claim 20, wherein the anti-GFRa4 antibody or a fragment thereof binds specifically to the tumor cell.

23. The method of claim 20, wherein the anti-GFRa4 antibody or a fragment thereof binds specifically to the GFRa4 on the tumor cell.

24. The method of claim 23, wherein the GFRa4 comprises at least one isoform selected from the group consisting of GFRa4a and GFRa4b.