Chimeric antigen receptors and methods for reducing toxicity

Abstract

Nucleic acid molecules that include a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleotide sequence encoding a protease sensitive scFv, wherein the chimeric antigen receptor comprises: an scFv targeting a tumor antigen, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain; and the protease-sensitive scFv and the scFv target the same tumor antigen are described.

Claims

1. A nucleic acid molecule comprising: (a) a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising amino acids 23 to 665 of SEQ ID NO: 31 and (b) a nucleotide sequence encoding a protease sensitive scFv comprising, from amino to carboxy terminus, a VH domain comprising SEQ ID NO: 33 and a VL domain SEQ ID NO:34 joined by a protease-sensitive linker that is sensitive to MMP-2 or MMP-9.

2. An expression vector comprising the nucleic acid molecule of claim 1.

3. A viral vector comprising the nucleic acid molecule of claim 1.

4. A population of human T cells transduced by a vector comprising the nucleic acid molecule of claim 1.

5. A method of treating cancer in a patient comprising administering a population of autologous or allogeneic human T cells transduced by a vector comprising the nucleic acid molecule of claim 1.

6. The method of claim 5, wherein MMP-2 or MMP-9 is present in the tumor microenvironment.

7. The nucleic acid molecule of claim 1, wherein the protease sensitive scFv comprises the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1: CD44v6R-41BBZ-T2A-huEGFRt construct contained in SIN lentiviral vector. Diagram of the CD44v6R:41BB:zeta-T2A-EGFRt construct, where the CD44v6-specific ScFv, IgG4 Fc hinge, CD4 transmembrane, 41BB and CD3z cytoplasmic signaling domains of the CD44v6R:41BB:zeta CAR, as well as the T2A ribosome skip and truncated huEGFR sequences are indicated. This vector, which expresses the CAR used in the studies described below, does not express a soluble scFv targeted to CD44v6.

(2) FIG. 2 Purification and expansion of CD8.sup.+T.sub.CM derived CD44v6CAR/EGFRt T cells. (A) CD8.sup.+ central memory T cells (CD62L.sup.+CD45RO.sup.+CD8.sup.+; CD8.sup.+T.sub.CM) were isolated from a healthy donor and transduced with lentivirus encoding CD44v6CAR/EGFRt after CD3/CD28 beads activation. (A) Gene modified cells were immunomagnetically purified after labeling with biotinylated Erbitux followed by anti-biotin microbeads and expanded in rapid expansion medium (REM) containing OKT3 and feeder cells in the presence of IL-2 (50 U/ml) and IL-15 (1 ng/ml). (B) Fold expansion of purified CD44v6CAR T cells after each cycle of REM stimulation.

(3) FIG. 3 Phenotype of ex vivo expanded CD44v6CAR/EGFRt CD8.sup.+ T cells Gene modified cells were immunomagnetically purified after labeling with biotinylated Erbitux followed by anti-biotin microbeads and expanded in rapid expansion medium (REM) containing OKT3 and feeder cells in the presence of IL-2 (50 U/ml) and IL-15 (1 ng/ml). After 2 cycles of in vitro expansion, T cells were labeled with antibodies against EGFRt (Erbitux), CD62L, CD28 and CD27. % Positive cells (closed histogram) over isotype controls (open histogram) are presented.

(4) FIG. 4: CD44v6 expression on leukemic cells. AML (THP-1 and KG1a) and B cell lymphoma (LCL) cells were labeled with PE-conjugated CD44v6 Ab (clone 2F10) (closed histogram) and isotype control Ab (open histogram) and analyzed by flow cytometry. % Positive cells over isotype controls are presented.

(5) FIG. 5: Cytolytic activity of CD44v6CAR/EGFRt CD8.sup.+ T cells After 2 cycles of in vitro expansion, CD44v6CAR/EGFRt CD8.sup.+ T cells were incubated for 4 hrs with 51Cr-labeled THP-1 or LCL cells, or OKT3-expressing LCL (LCLOKT3) cells as positive targets and CD44v6 negative KG1a cells as negative targets at 25:1 E:T ratios. Mean percent cytotoxicity of triplicate wells that was normalized over LCL OKT3 is depicted.

(6) FIG. 6: Anti-lymphoma effects of adoptively transferred CD8.sup.+T.sub.CM derived CD44v6CAR/EGFRt T cells. 2×10.sup.6 CD19.sup.+ffluc.sup.+ lymphoblastoid cell lines (LCL) cells were injected (i.v) into NSG mice on day-3. Subsequently, 5×10.sup.6 CD8.sup.+T.sub.CM derived CD44v6CAR T cells were intravenously infused into the tumor bearing mice on day 0. Recipient mice received intraperitoneal injection of irradiated human IL15 secreting NSO cells to support human T cell persistence. Tumor signals were monitored by biophotonic imaging. (A) Representative images on day 14 are presented. (B) Mean±SEM of phonton/sec from multiple mice are depicted.

(7) FIG. 7: Anti-AML effects of adoptively transferred CD8.sup.+T.sub.CM derived CD44v6CAR/EGFRt T cells 1.5×10.sup.6 GFPffluc.sup.+AML cells (THP-1) were injected (i.v) into NSG mice on day −3. 5×10.sup.6 CD8.sup.+T.sub.CM derived CD44v6CAR T cells were intravenously infused into the tumor bearing mice on day 0. Mice received no T cells or CD8.sup.+T.sub.CM derived irrelevant CAR (CD19CAR) T cells from the same donor were used as negative controls. All recipient mice received intraperitoneal injection of irradiated human IL15 secreting NSO cells to support human T cell persistence. (A) Tumor signals were monitored by biophotonic imaging. (B) Means±SEM of phonton/sec from multiple mice are depicted.

(8) FIG. 8: CD8.sup.+T.sub.CM derived CD44v6 CAR/EGFRt. T cells interfere with human leukemia initiation in immunodeficient mice 20×10.sup.6 CD44v6CAR T cells or irrelevant CAR T cells (CD19CAR) derived from CD8.sup.+T.sub.CM of the same donor were adoptively transferred (i.v) into NSG mice. 2 weeks post T cell infusion, 1.5×10.sup.6 GFPffluc.sup.+ THP-1 cells expressing CD44v6 were inoculated (i.v) into the mice. (A) Tumor signals were monitored by biophotonic imaging 7 and 14 days post tumor infusion. (B) % Human T cells in peripheral blood are depicted

(9) FIG. 9: Amino acid sequence of a CD44v6 CAR. The amino acid sequence presented is the amino acid sequence of a CD44v6 CAR, including the signal sequence, together with the sequence of the truncated EGFR sequence used for monitoring CAR expression and the T2A ribosomal skip sequence that allows the CAR to be co-expressed, but not fused to the truncated EGFR sequence (SEQ ID NO:35). The immature CAR includes: GMCSFR signal peptide, a CD44v6 scFv, an IgG4 derived sequence that acts as a spacer, a CD4 transmembrane domain, a 4-IBB co-stimulatory domain that includes a LL to GG sequence alteration, a three Gly sequence, a CD3 Zeta stimulatory domain. The transcript also encodes a T2A ribosomal sequence and a truncated EGFR sequence that are not part of the CAR protein sequence. The mature CAR is identical to the immature CAR, but lacks the GMCSF signal peptide. The various domains are indicated.

(10) FIG. 10: Amino acid sequences of CD44v6 scFv. (A) CD44v6 scFv with MMP-2 sensitive linker (SEQ ID NO:38). (B) CD44v6 scFv with MMP-9 sensitive linker (SEQ ID NO: 39). (C) CD44v6 scFv VH domain (SEQ ID NO:33). (D) CD44v6 scFv VL domain (SEQ ID NO:34).

DETAILED DESCRIPTION

(11) CD44v6 Targeted CAR

(12) The CD44v6-targeted CAR described herein include a CD44v6-targeting scFv (e.g., an (e.g., an scFv comprising the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG GSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRG TLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSINYIYW YQQKPGQAPRLLIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQW SSNPLTFGGGTKVEIK (SEQ ID NO:1) or comprising the heavy chain sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG GSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRG TLVTVSS (SEQ ID NO:33) and the light chain sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG GSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRG TLVTVSS (SEQ ID NO:34).

(13) Useful CD44v6 CAR consist of or comprises the amino acid sequence of SEQ ID NO:29 (mature CAR lacking a signal sequence, T2A skip sequence and EGFRt) or the CD44v6 CAR consists of or comprises the amino acid sequence of SEQ ID NO: 30 (immature CAR having a GMCSFRa signal sequence, but lacking T2A skip sequence and EGFRt). The CAR and can be expressed in a form that includes a signal sequence, e.g., a human GM-CSF receptor alpha signal sequence (MLLLVTSLLLCELPHPAFLLIP; SEQ ID NO:26). The CAR can be expressed with additional sequences that are useful for monitoring expression, for example a T2A skip sequence and a truncated EGFRt. Thus, the CAR can comprise or consist of the amino acid sequence of any of SEQ ID Nos: 29-30 or can comprise or consist of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID Nos: 29-30. The CAR can comprise or consist of the amino acid sequence of any of SEQ ID Nos: 29-30 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes).

(14) Spacer Region

(15) The CAR described herein can include a spacer located between the CD44v6 targeting domain (i.e., a gp120-targeted ScFv or variant thereof) and the transmembrane domain. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 1 below provides various spacers that can be used in the CARs described herein.

(16) TABLE-US-00001 TABLE 1  Examples of Spacers Name Length Sequence a3   3 aa AAA linker  10 aa GGGSSGGGSG (SEQ ID NO: 2) IgG4 hinge (S.fwdarw.P)  12 aa ESKYGPPCPPCP (SEQ ID NO: 3) (S228P) IgG4 hinge  12 aa ESKYGPPCPSCP (SEQ ID NO: 4) IgG4 hinge (S228P)+  22 aa ESKYGPPCPPCPGGGSSGGGSG (SEQ ID NO: 5) linker CD28 hinge  39 aa IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP  (SEQ ID NO: 6) CD8 hinge-48aa  48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACD (SEQ ID NO: 7) CD8 hinge-45aa 45aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACD (SEQ ID NO: 8) IgG4(HL-CH3) 129 aa ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEE (includes 5228P  MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP in hinge) VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK (SEQ ID NO: 9) IgG4(L235E, N297Q) 229 aa ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQF QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10) IgG4(5228P, L235E, 229 aa ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTP N297Q) EVTCVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQF QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 11) IgG4(CH3) 107 aa GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK  (SEQ ID NO: 12)

(17) Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

(18) The hinge/linker region can also comprise a IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO:4) or ESKYGPPCPPCP (SEQ ID NO:3).

(19) The hinge/linger region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO:3) followed by the linker sequence GGGSSGGGSG (SEQ ID NO:2) followed by IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:12). Thus, the entire linker/spacer region can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MEIEALHNHYTQKSLSLSLGK (SEQ ID NO:11). In some cases, the spacer has 1, 2, 3, 4, or 5 single amino acid changes (e.g., conservative changes) compared to SEQ ID NO:11. In some cases, the IgG4 Fc hinge/linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).

(20) Transmembrane Domain

(21) A variety of transmembrane domains can be used in the. Table 2 includes examples of suitable transmembrane domains. Where a spacer region is present, the transmembrane domain is located carboxy terminal to the spacer region.

(22) TABLE-US-00002 TABLE 2  Examples of Transmembrane Domains Name Accession Length Sequence CD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL  (SEQ ID NO: 13) CD28 NM_006139 27aa FWVLVVVGGVLACYSLLVTVA FIIFWV (SEQ ID NO: 14) CD28(M) NM_006139 28aa MFWVLVVVGGVLACYSLLVTV AFIIFWV (SEQ ID NO: 15) CD4 M35160 22aa MALIVLGGVAGLLLFIGLGIFF  (SEQ ID NO: 16) CD8tm NM_001768 21aa IYIWAPLAGTCGVLLLSLVIT  (SEQ ID NO: 17) CD8tm2 NM_001768 23aa IYIWAPLAGTCGVLLLSLVITLY  (SEQ ID NO: 18) CD8tm3 NM_001768 24aa IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 19) 41BB NM_001561 27aa IISFFLALTSTALLFLLFFLTLRF  SVV (SEQ ID NO: 20)
Costimulatory Domain

(23) The costimulatory domain can be any domain that is suitable for use with a CD3ζ signaling domain. In some cases, the costimulatory domain is a CD28 costimulatory domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:23; LL to GG amino acid change double underlined). In some cases, the CD28 co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative and preferably not in the underlined GG sequence) compared to SEQ ID NO:23. In some cases the co-signaling domain is a 4-1BB co-signaling domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:24). In some cases, the 4-1BB co-signaling domain has 1, 2, 3, 4 or 5 amino acid changes (preferably conservative) compared to SEQ ID NO:24.

(24) The costimulatory domain(s) are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable costimulatory domains together with the sequence of the CD3ζ signaling domain.

(25) TABLE-US-00003 TABLE 3  CD3ζ Domain and Examples of Costimulatory Domains Name Accession Length Sequence CD3ζ  J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLVNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR (SEQ ID NO: 21) CD28 NM_006139 42aa RSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRS  (SEQ ID NO: 22) CD28gg* NM_006139 42aa RSKRSRGGHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRS  (SEQ ID NO: 23) 41BB NM_001561  42 aa KRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCEL  (SEQ ID NO: 24) OX40  42 aa ALYLLRRDQRLPPDAHKPPGGGS FRTPIQEEQADAHSTLAKI  (SEQ ID NO: 25)

(26) In various embodiments: the costimulatory domain is selected from the group consisting of: a costimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications in present. In some embodiments there are two costimulatory domains, for example a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions. The costimulatory domain is amino terminal to the CD3ζ signaling domain and in some cases a short linker consisting of 2-10, e.g., 3 amino acids (e.g., GGG) is positioned between the costimulatory domain and the CD3ζ signaling domain.

(27) CD3ζ Signaling Domain

(28) The CD3ζ Signaling domain can be any domain that is suitable for use with a CD3ζ signaling domain. In some cases, the CD3ζ signaling domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO:21). In some cases, the CD3ζ signaling has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:21.

(29) Truncated EGFR

(30) The CD3ζ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL PVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGR TKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSG QKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKC NLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIA TGMVGALLLLLVVALGIGLFM (SEQ ID NO:28). In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:28.

(31) CD44v6 Targeted CAR

(32) FIG. 9 depicts the amino acid sequence of a CD44v6 targeted CAR together with a truncated EGFR that can be used as a marker, which is joined to the CAR sequence via a T2A skip sequence. The truncated EGFR is expressed by cells that express the CAR and in this way serves as a marker and as a means to target CAR expressing cells. The sequence also includes a GMSCFRa signal sequence, that like the EGFRt sequence that are not present in the mature CAR as expressed on T cells. described herein. Useful CAR include hose that comprise or consist of the amino acid sequence of any of SEQ ID Nos: 29-30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID Nos: 29-30 or the amino acid sequence of any of SEQ ID Nos: 29-30 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes). In various embodiments: the population of human T cells are CD8+ cells.

(33) An amino acid modification refers to an amino acid substitution, insertion, and/or deletion in a protein or peptide sequence. An “amino acid substitution” or “substitution” refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

(34) The CAR can include a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to the mature amino acid sequence depicted in FIG. 9 (SEQ ID Nos: 29-40), either including or excluding the GMCSFRa signal sequence and either including or excluding the T2A ribosomal skip sequence and the truncated EGFRt).

(35) In some cases, the CD44v6 CAR can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated EGFR (EGFRt), which lacks the cytoplasmic signaling tail. In this arrangement, co-expression of EGFRt provides an inert, non-immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking of the therapeutic T cells in vivo following adoptive transfer. Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is an important hurdle for the success of T cell immunotherapy. The EGFRt incorporated in the CD44v6CAR lentiviral vector can act as suicide gene to ablate the CAR+T cells in cases of treatment-related toxicity.

(36) The CAR described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte cell line, and most preferably an autologous T lymphocyte cell line.

(37) Various T cell subsets isolated from the patient can be transduced with a vector for CAR expression. Central memory T cells are one useful T cell subset. Central memory T cell can be isolated from peripheral blood mononuclear cells (PBMC) by selecting for CD45RO+/CD62L+ cells, using, for example, the CliniMACS® device to immunomagnetically select cells expressing the desired receptors. The cells enriched for central memory T cells can be activated with anti-CD3/CD28, transduced with, for example, a lentiviral vector that directs the expression of an CD44v6 CAR as well as a non-immunogenic surface marker for in vivo detection, ablation, and potential ex vivo selection. The activated/genetically modified CD44v6 central memory T cells can be expanded in vitro with IL-2/IL-15 and then cryopreserved.

Example 1: Preparation and Characterization of CD44v6 CAR

(38) CD44v6R-41BBZ-T2A-huEGFRt construct contained in SIN lentiviral vector. A lentiviral construct was prepared using the approach described in Wang et al. (Blood 118:1255, 2011). FIG. 1 is a diagram of the CD44v6R:41BB:zeta-T2A-EGFRt construct, where the CD44v6-specific ScFv, IgG4 Fc hinge, CD4 transmembrane, 41BB and CD3z cytoplasmic signaling domains of the CD44v6R:41BB:zeta CAR, as well as the T2A ribosome skip and truncated huEGFR sequences are indicated. This vector, which expresses the CAR used in the studies described below, does not express a soluble scFv targeted to CD44v6.

(39) Purification and expansion of CD8.sup.+T.sub.CM derived CD44v6CAR/EGFRt T cells. CD8.sup.+ central memory T cells (CD62L.sup.+CD45RO.sup.+CD8.sup.+; CD8.sup.+T.sub.CM) were isolated from a healthy donor and transduced with lentivirus encoding CD44v6CAR/EGFRt after CD3/CD28 beads activation using the approach described in Wang et al. (Blood 117:1888, 2011). Gene modified cells were immunomagnetically purified after labeling with biotinylated Erbitux followed by anti-biotin microbeads and expanded in rapid expansion medium (REM) containing OKT3 and feeder cells in the presence of IL-2 (50 U/ml) and IL-15 (1 ng/ml) (FIG. 2A). FIG. 2B shows the fold expansion of purified CD44v6CAR T cells after each cycle of REM stimulation.

(40) Phenotype of ex vivo expanded CD44v6CAR/EGFRt CD8.sup.+ T cells. Gene modified cells were immunomagnetically purified after labeling with biotinylated Erbitux followed by anti-biotin microbeads and expanded in rapid expansion medium (REM) containing OKT3 and feeder cells in the presence of IL-2 (50 U/ml) and IL-15 (1 ng/ml). After 2 cycles of in vitro expansion, T cells were labeled with antibodies against EGFRt (Erbitux), CD62L, CD28 and CD27. The results of this analysis are presented in FIG. 3 where the % Positive cells (closed histogram) over isotype controls (open histogram) are reported.

(41) CD44v6 expression on leukemic cells. AML (THP-1 and KG1a) and B cell lymphoma (LCL) cells were labeled with PE-conjugated CD44v6 Ab (clone 2F10) and isotype control Ab and analyzed by flow cytometry. The results of this analysis are presented in FIG. 4 where the % Positive cells over isotype controls are presented.

(42) Cytolytic activity of CD44v6CAR/EGFRt CD8.sup.+ T cells. After 2 cycles of in vitro expansion, CD44v6CAR/EGFRt CD8.sup.+ cells were incubated for 4 hrs with 51Cr-labeled THP-1 or LCL cells, or OKT3-expressing LCL (LCLOKT3) cells as positive targets and CD44v6 negative KG1a cells as negative targets at 25:1 E:T ratios. The results of this analysis are presented in FIG. 5.

(43) Anti-lymphoma effects of adoptively transferred CD8.sup.+T.sub.CM derived CD44v6CAR/EGFRt T cells. The examine the anti-lymphoma effect of adoptively transferred CD8+TCM derived CD44v6CAR/EGFRt T cells, 2×10.sup.6 CD19.sup.+ffluc.sup.+ lymphoblastoid cell lines (LCL) cells were injected (i.v) into NSG mice on day-3. Subsequently, 5×10.sup.6 CD8.sup.+T.sub.CM derived CD44v6CAR T cells were intravenously infused into the tumor bearing mice on day 0. Recipient mice received intraperitoneal injection of irradiated human IL15 secreting NSO cells to support human T cell persistence. Tumor signals were monitored by biophotonic imaging. The results of this analysis are presented in FIG. 6.

(44) Anti-AML effects of adoptively transferred CD8.sup.+T.sub.CM derived CD44v6CAR/EGFRt T cells. For this study, 1.5×10.sup.6 GFPffluc.sup.+ AML cells (THP-1) were injected (i.v) into NSG mice on day-3.5×10.sup.6 CD8.sup.+T.sub.CM derived CD44v6CAR T cells were intravenously infused into the tumor bearing mice on day 0. Mice received no T cells or CD8.sup.+T.sub.CM derived irrelevant CAR (CD19CAR) T cells from the same donor were used as negative controls. All recipient mice received intraperitoneal injection of irradiated human IL15 secreting NSO cells to support human T cell persistence. The results are presented in FIG. 7.

(45) CD8.sup.+T.sub.CM derived CD44v6 CAR/EGFRt T cells interfere with human leukemia initiation in immunodeficient mice. In this study, 20×10.sup.6 CD44v6CAR T cells or irrelevant CAR T cells (CD19CAR) derived from CD8.sup.+T.sub.CM of the same donor were adoptively transferred (i.v) into NSG mice. 2 weeks post T cell infusion, 1.5×10.sup.6 GFPffluc.sup.+ THP-1 cells expressing CD44v6 were inoculated (i.v) into the mice. The results of this analysis are presented in FIG. 8.

(46) Summary. The results of these studies suggest that targeting CD44v6 with CD44v6 CAR CD8+T.sub.CM cells can lead to potent anti-tumor activity upon adoptive transfer in a murine model and that CD44v6 CAR T cells are capable of interfering leukemia initiation by inhibiting leukemic stem cell homing and proliferation.

Example 2: Generation of Protease Sensitive scFv

(47) A selected scFv (e.g., the scFv used in the selected CAR) can be converted to a protease sensitive scFv by replacing the linker between the variable heavy chain and variable light chain portions with a linker that is sensitive to the selected protease, for example, MMP-2 or MMP-9, both of which are overexpressed and accumulated in various tumors. Yeast surface display, a genotype-phenotype linkage strategy for facile screening of protein libraries can be used to select scFv linkers that are appropriately susceptible to cleavage by MMP-2 and/or MMP-9 and allow for proper heavy and light chain association (Gai et al. 2007 Curr Opin Struct Biol 17:467-473). Because MMP-2 and MMP-9 are known to be promiscuous (Prudova et al. 2010 Mol Cell Proteomics 9:894-911), a library of candidate cleavage sites (residues G-P-X-X-X-X-X-A, 3.3×10.sup.7 variants) can be screened as the central portion of the scFv linker. Sequential fluorescence-activated cell sorting (FACS) experiments are employed to: (1) select linker candidates that yield scFvs with equivalent binding activity to the parent scFv, (2) select linker candidates that are cleaved by physiological concentrations of recombinant active MMP-2 and/or MMP-9, and (3) select linker candidates with equivalent serum stability to the parental linker. Successful linker candidates are screened for immunogenicity using the EpiSweep software package (Choi et al. 2017 Methods in Molecular Biology 1529: 375, 2017), with mutations made as needed to eliminate T cell epitopes. Once appropriate lead linker candidates are selected, the scFv can undergo affinity maturation using error-prone PCR (Zaccolo et al. 1996 Mol Biol 255:589-60324) followed by FACS to select for scFvs with improved binding affinity, an approach that has been employed successfully for scFvs in the past (Tillotson et al. 2013 Protein Eng Des Sel 26:101-112). Lead scFvs are cloned into a bicistronic construct encoding for the EF1α promoter, the soluble protease-susceptible scFv, a T2A ribosome skip sequence, and the parental CAR. Off-tumor toxicities of the CAR are evaluated in vitro and in appropriate murine models.