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 CD3 ζ signaling domain; and the protease-sensitive scFv and the scFv target the same tumor antigen are described.

Description

Chimeric Antigen Receptors and Methods for Reducing T oxicityCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Application Serial No. 63 / 607,426, filed on December 7, 2023. The entire contents of the foregoing are incorporated herein by reference.BACKGROUNDCancer initiating / stem cells (CSCs) (also known as tumor stem cells (TSCs)) are a distinct and highly motile and aggressive subpopulation of cells that reside within tumors, with the capacity to proliferate, differentiate and seed tumors (1). Initially identified in acute myeloid leukemia (AML), CSCs have also been identified in solid tumors. TSCs are resistant to radio / chemotherapy and evidence suggests that they may be responsible for disease relapse. Complete elimination and prevention of tumor recurrence and growth relies on the eradication of CSCs, which remains challenging for the treatment of cancers, including hematopoietic malignancies. The CD44 transmembrane receptor (Uniprot Pl 6070) is a cell adhesion glycoprotein that binds to components of the extracellular matrix (ECM) such as osteopontin, hyaluronan, collagen, laminin and fibronectin. CD44 has many biological functions including cellular growth, survival, and differentiation. Evidence also suggests that it is involved in tumor migration and metastasis. CD44 is widely expressed in a variety of cancers, and is a biomarker for CSCs. A splice variant of CD44, CD44 variant 6 (CD44v6), is overexpressed on malignant hematopoietic cells including AML, B cell malignancies and multiple myeloma (MM), especially in late stage diseases. CD44v6 plays an important role in proliferation, homing, and metastasis of tumor stem cells (Heider et al. (2004) Cancer Immunol Immunother 53:567).While CD44v6 appears to be an excellent target for CAR T cell therapy, its expression in human keratincytes and other tissues, as well as in monocytes, presents a challenge to CAR T cell or targeted antibody mediated therapy due to on-target, off-tumor toxicity. Previous efforts using antibody drug conjugates employing bivatuzumab (a CD44v6specific antibody) resulted in skin toxicity and fatalities from toxicity related events. To address on-target off-tumor toxicity, CAR-T cells that incorporate a suicide gene to target CD44v6 in AML and MM have been developed and examined in preclinical studies (2).SUMMARYDescribed herein are nucleic acid molecules comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) targeted to CD44v6. Also described are nucleic acid molecules comprising a nucleotide sequence encoding a protease sensitive scFv fusion protein targeted to CD44v6.The chimeric antigen receptor comprises: an scFv targeting a CD44v6 antigen (e.g., an scFv that binds human cd44 exon v6 such as an scFv derived from 2F10, VFF4, VFF7, VFF18 (BIWA 1), U36, V6B3, HB-256 or Var 3.1), a spacer domain, a transmembrane domain, a co-stimulatory domain, and a CD3^ signaling domain (“CD44v6 CAR”). The protease sensitive scFv fusion protein comprises protease-sensitive scFv targeting a CD44v6 antigen and an Fc domain. This scFv fusion protein is referred to as PSscFv-Fc. The svFv of the CD44v6 CAR and the scFv of the PSscFv-Fc target the same CD44v6 epitope. The svFv of the CD44v6 CAR and the scFv of the PSscFv-Fc can share the same VH CDR sequence and the same VL CDR sequence. In some cases, the scFv within the CD44v6 CAR has VH and VL sequences that are identical to that of the PSscFv-Fc. The scFv portion of the PSscFv-Fc includes a protease sensitive amino acid sequence (e.g., an MMP-2 or MMP-9 sensitive amino acid sequence) between the VH and VL sequences, e.g., within a linker that joins the VH and VL sequences. This makes the PSscFv-Fc subject to degradation when exposed to an appropriate protease.Described herein are methods for using T cells expressing the CD44v6 CAR (“CD44v6 CAR T cells”) to treat a variety of cancers while reducing on-target, off-tumor toxicity. The methods entail the use of CD44v6 CAR T cells that secrete a PSscFv-Fc. The PSscFv-Fc can block CD44v6 CAR binding in healthy tissue by competing with CD44v6 CAR T cells for binding to CD44v6 expressed on the surface of cells.However, in the presence of certain proteases present in tumor tissue (e.g., MMP-2 or MMP-9), the PSscFv-Fc is degraded, allowing CD44v6 CAR T cells to bind CD44v6 on tumor cells, leading to activation of the CD44v6 CAR T cells and killing of thetumor cell. Thus, described herein is a population of T cells expressing both a CD44v6 CAR and a fusion protein that includes a protease sensitive CD44v6 scFv.In various embodiments: the protease sensitive scFv is sensitive to MMP-2 or MMP-9; the protease sensitive scFv comprises a VH domain and a VL domain joined by a proteasesensitive linker; the scFv and the protease-sensitive scFv have the same CDR sequences; the nucleic acid molecule comprises a nucleotide sequence encoding a T2A skip sequence; the nucleotide sequence encoding a T2A skip sequence is located between the nucleotide sequence encoding the chimeric antigen receptor and the nucleotide sequence encoding a protease sensitive; the tumor antigen is CD44v6; the scFv of the CAR comprises the amino acid sequence of SEQ ID NO:1 or comprises VH domain comprising SEQ ID NO: 33 and a VL domain comprising SEQ ID NO: 34; and the VH domain of the protease sensitive scFv comprises SEQ ID NO: 33 and the VL domain of the protease sensitive scFv comprises SEQ ID NO: 34.In various embodiments, the CAR comprises: a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-5 single amino acid substitutions, a CD8 transmembrane domain or variant thereof having 1-5 single amino acid substitutions, a CD28 transmembrane domain or a variant thereof having 1-5 single amino acid substitutions; a costimulatory domain selected from: a 4-IBB costimulatory domain or a variant thereof having 1-5 single amino acid substitutions and an CD28 costimulatory domain or a variant thereof having 1-5 single amino acid substitutions; a CD3^ signaling domain of a variant thereof having 1-5 single amino acid substitutions; and a spacer region having 10-150 amino acids located between the scFv and the transmembrane domain.In various embodiments: the costimulatory domain is selected from the group consisting of: a 4-IBB costimulatory domain and variants thereof having 1-5 single amino acid substitutions; the transmembrane domain is a CD4 transmembrane domain or variant thereof having 1-5 single amino acid substitutions; the transmembrane domain is a CD4 transmembrane domain; the CAR comprises two different costimulatory domains selected from the group consisting of: a CD28 costimulatory domain or a variant thereof having 1 -5 single amino acid substitutions, a 4-IBB costimulatory domain or a variant thereof having 1-5 single amino acid substitutions and an 0X40 costimulatory domain or a variant thereof having 1-5 single amino acid substitutions; the chimeric antigen receptor comprises a CD44v6-binding scFv having the amino acid sequence of SEQ ID NO: 1 or a variant thereof having 1-2 single amino acid substitutions; a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-2 single amino acid substitutions, a CD8 transmembrane domain or variant thereof having 1-2 single amino acid substitutions, a CD28 transmembrane domain or a variant thereof having 1-2 single amino acid substitutions, and a CD3^ transmembrane domain or a variant thereof having 1-2 single amino acid substitutions; a 4-IBB costimulatory domain; or a variant thereof having 1-2 single amino acid substitutions; and CD3^ signaling domain of a variant thereof having 1-2 single amino acid substitutions; and a spacer region having 10- 150 amino acids located between the scFv and the transmembrane domain; the spacer region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-12 or a variant thereof having 1-5 single amino acid substitutions; the spacer comprises an IgG hinge region; the spacer comprises 10-50 amino acids; the 4- IBB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof having 1-5 single amino acid substitutions; the CD3^ signaling domain comprises the amino acid sequence of SEQ ID NO:21; a linker of 3 to 15 amino acids is located between the costimulatory domain and the CD3^ signaling domain or variant thereof; and the CAR comprises the amino acid sequence of SEQ ID NO: 29 or 30 or a variant thereof having 1-5 single amino acid substitutions.Also described here is an expression vector and a viral vector comprising the nucleic acid molecules described herein. Also described is a population of human T cells transduced by a vector comprising a nucleic acid molecule described herein. Also described is a method of treating cancer in a patient comprising administering a population of autologous or allogeneic human T cells transduced by a vector comprising a nucleic acid molecule described herein. In embodiments of the method: the population of human T cells comprise central memory T cells; MMP-2 or MMP-9 is present in the tumor microenvironment; the patient is suffering from a leukemia or lymphoma; and the patient is suffering from AML or multiple myeloma.The approach used for CD44v6 CAR can be used with any target for treating cancer where the tumor microenvironment has proteases such as MMP-2 orMMP-9.In various embodiments: the chimeric antigen receptor comprises: a CD44v6 scFv (e.g., an scFv comprising the amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG GSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRG TLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSINYIYW YQQKPGQAPRLLIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQW SSNPLTFGGGTKVEIK (SEQ ID NO:1) with up to 10 single amino acid substitutions outside the CDRs; a spacer region; a CD28 transmembrane domain; a CD28 co-signaling domain; and a CD3 signaling domain; the chimeric antigen receptor comprises: a CD44v6 scFv; a spacer region; a CD4 transmembrane domain; a 4- IBB co-signaling domain; and a CD3 signaling domain; the chimeric antigen receptor comprises: a CD44v6 scFv; a spacer region comprising an amino acid sequence selected from SEQ ID Nos:2-12; a CD4 transmembrane domain; a 4-1BB co-signaling domain; and a CD3 signaling domain; the chimeric antigen receptor comprises: a CD44v6 scFv; a spacer region comprising an amino acid sequence selected from SEQ ID Nos: 212; a CD28 transmembrane domain; a CD28 co-signaling domain; and a CD3 signaling domain; the chimeric antigen receptor comprises: a CD44v6 scFv; a spacer a spacer comprising an amino acid sequence selected from SEQ ID Nos: 2-12; CD4 transmembrane domain; a 4- 1BB co-signaling domain; and CD3 signaling domain; the chimeric antigen receptor comprises: a spacer a spacer comprising an amino acid sequence selected from SEQ ID Nos :212; a CD28 transmembrane domain; a CD28 co-signaling domain; and a CD3^ signaling domain; the chimeric antigen receptor comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from: SEQ ID NOs: 29-30; the chimeric antigen receptor comprises an amino acid sequence identical to an amino acid sequence selected from: SEQ ID NOs: 29-30; the chimeric antigen receptor comprises an amino acid sequence identical to an amino acid sequence selected from: SEQ ID NOs: 29-30, each with no more than 5 single amino acid substitutions; at least 20%, 30%, or 40% of the transduced human T cells are central memory T cells; at least 30% of the transducedhuman T cells are CD4+ and CD62L+ or CD8+ and CD62L+; the population of human T cells are autologous to the patient; and the population of human T cells are allogenic to the patient.The scFv present in the CD44v6 CAR includes a VH domain followed by a linker and a VL domain. The VH domain can comprise the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG GSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRG TLVTVSS (SEQ ID NO: 33). The VH domain can include a VH CDR1 comprising SYDMS (SEQ ID NO: 35); a VH CDR2 comprising HSSGGSYTYYLDSIKG (SEQ ID NO: 36); and a VH CDR3 comprising QGLDY (SEQ ID NO: 37).The VL domain can comprise the sequence:EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVP ARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK(SEQ ID NO: 34). The VH domain can include a VL CDR1 comprising SASSSINYIY (SEQ ID NO: 38); a VL CDR2 comprising LTSNLAS (SEQ ID NO: 39); and a VL CDR3 comprising LQWSSNPLT (SEQ ID NO: 40).Protease sensitive CD44v6 scFv - Fc fusionThe protease sensitive scFv portion of the CD44v6 scFv - Fc fusion can comprise the sequence of SEQ ID NO: 1 wherein the linker (GGGGSGGGGSGGGG; SEQ ID NO: 41) is replaced by a protease sensitive linker, for example, a linker sensitive to MMP-2 (e.g., GSTSGSKGPLGLAGATKG; SEQ ID NO: 42) or MMP-9 (e g., GSTSGSKGPKGLKGATKG; SEQ ID NO: 43), a linker of 15 - 20 ammo acids comprising the sequence PLGLAG (SEQ ID NO: 44); or a linker of 15 - 20 amino acids comprising the sequence PKGLKG (SEQ ID NO:45).For example, The scFv of the protease sensitive scFv can include a VH domain comprising the sequence:EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS (SEQ ID NO: 33). The VH domain of the protease sensitive scFv can include a VH CDR1 comprising SYDMS (SEQ ID NO: 35); a VH CDR2 comprising TISSGGSYTYYLDSIKG (SEQ ID NO: 36); and a VH CDR3 comprising QGLDY (SEQ ID NO: 37). The VH domain is followed by a linker comprising any of SEQ ID NOs: 42-45. The scFv of the protease sensitive scFv can include a VL domain comprising the sequence:EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPARFS GSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK(SEQ ID NO: 34). The VL domain of the protease sensitive scFv can include a VL CDR1 comprising SASSSINYIY (SEQ ID NO: 38); a VL CDR2 comprising LTSNLAS (SEQ ID NO: 39); and a VL CDR3 comprising LQWSSNPLT (SEQ ID NO: 40). For example, the protease-sensitive scFv can comprise the sequence:EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTY YLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSSGSTS GSKGPLGLAGATKGEIVLTOSPATLSLSPGERATLSCSASSSINYIYWYOQKPGOAPRLLIY LTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYCL QWSSNPLTFGGGTKVEIK (SEQ ID NO: 50).The Fc domain of the protease sensitive scFv can comprise any of SEQ ID NOs: 9-12 or any of SEQ ID NOS: 10 and 11.Preferably the Fc domain of the protease sensitive scFv comprises or consists of the sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAI<TI<PREEQFQSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<G LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 11)The PSscFv-Fc can include the sequence (Fc sequence underlined):MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWV RQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCA RQGLDYWGRGTLVTVSSGSTSGSKGPLGLAGATKGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYCL OWSSNPLTFGGGTKVEIKESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSOEDPEVOFNWYVDGVEVHNAI<TI<PREEOFOSTYRVVSVLTVLHQDWLNGI<EY KCKVSNKGLPSSIEKTISKAKGOPREPQVYTLPPSOEEMTKNOVSLTCLVKGFYPSDIAVE WESNGOPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 46; with signal sequence (shown in bold)) orEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTY YLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSSGSTS GSKGPLGLAGATKGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIY LTSNLAS GVPARFSGSGS GTDFTLTIS SLEPEDF AVYCLQWS SNPLTFGGGTKVEIKESKYG PPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSOEDPEVOFNWYVDGVEV HNAKTKPREEQFOSTYRVVSVLTVLHODWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSOEEMTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47; without signal sequence) or a sequence that is at least 90%, 95%, 97% or 99% identical to SEQ ID NO: 46 or 47 and includes: a VH domain comprising a VH CDR1 comprising SYDMS (SEQ ID NO: 36); a VH CDR2 comprising TISSGGSYTYYLDSIKG (SEQ ID NO: 36); and a VH CDR3 comprising QGLDY (SEQ ID NO: 37); and a VH domain comprising a VL CDR1 comprising SASSSINYIY (SEQ ID NO: 38); a VL CDR2 comprising LTSNLAS (SEQ ID NO: 39); and a VL CDR3 comprising LQWSSNPLT (SEQ ID NO: 40).DESCRIPTION OF DRAWINGSFIG 1: Generation of soluble CD44v6scFv. A) Schematic representation of engineered soluble CD44v6scFv displayed on the surface of yeast. B) Yeast displaying the protease- susceptible CD44v6 scFv were digested with varying concentration of MMP-2 and varying duration. Digestion efficiency was quantified as percentage of MYC epitope labeling intensity relative to undigested samples. Data are shown as mean ± standard deviation of three trials except when otherwise indicated. C) Engineered soluble scFv with PLGLAG linker and original GS linker bound targets with KD values of 5nM and 30nM respectively.FIG 2: Generation of soluble CD44v6scFv. A) Schema of engineered CD44v6CAR construct with and without inserted soluble CD44v6scFv-Fc. B) Transduction efficiencies of CD44v6CAR (CD44v6CAR only) and sCD44v6CAR (CD44v6CAR + CD44v6scFv- Fc) constructs in primary human T cells. C) Soluble CD44v6scFv from supernatant of transduced T cells incubated with MM.1 S cells expressing CD44v6. Blue, red and gray histograms represent cells only, co-culture with recombinant MMP2 and co-culture without MMP2 respectively. D) Western blot of clarified supernatant showing scFv-Fc at 52kDa size.FIG 3: Effector activity of engineered sCD44v6CAR T cells. A) Long-term killing activity of sCD44v6C AR T cells against MM.1 S tumor lines after 24- and 72 hours respectively. Co-culture was setup in the presence or absence of recombinant MMP2. B) Real-time xCelligence killing assays comparing soluble scFv-producing CD44v6CAR T cells to control CD44v6 CAR T cells against primary human keratinocytes. C) Keratinocyte killing is demonstrated by addition of MMP2 to coculture assay. (n=2 of biological replicates each with 2 technical replicates).FIG 4: Amino acid sequence of a CD44v6 CAR. The amino acid sequence presented (SEQ ID NO: 30) is the amino acid sequence of a CD44v6 CAR, including the signal sequence. 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 costimulatory domain, a three Gly linker sequence, a CD3 Zeta stimulatory domain. The mature CAR (SEQ ID NO: 29) is identical to the immature CAR, but lacks the GMCSF signal peptide. The various domains are indicated.FIG 5: Amino acid sequences of various protease sensitive CD44v6 scFv. (A) CD44v6 scFv with MMP-2 sensitive linker (SEQ ID NO: 50). (B) CD44v6 scFv with MMP-9 sensitive linker (SEQ ID NO: 51). (C) CD44v6 scFv VHdomain (SEQ ID NO:33). (D) CD44v6 scFv VLdomain (SEQ ID NO: 34).FIG 6: Amino acid sequence of a CD44v6 CAR as co-expressed with a protease sensitive CD44v6scFc. The amino acid sequence presented (SEQ ID NO:31) is the amino acid sequence of a CD44v6 CAR, including the signal sequence, together with the protease sensitive CD44v6scFc and the T2A ribosomal skip sequence that allows the CAR to be co-expressed, but not fused to, the protease sensitive CD44v6scFc. The immature CAR includes: GMCSFR signal peptide, a CD44v6 scFv, an IgG4 derived FC sequence that acts as a spacer, a CD4 transmembrane domain, a 4-IBB co-stimulatory domain, a three Gly linker sequence, a CD3 Zeta stimulatory domain. Also present is a T2A ribosomal sequence followed by TR to provide a cloning site, a signal sequence and then CD44v6scFc (including VH, protease sensitive linker VL and a variant Fc domain). The various domains are indicated. SEQ ID NO: 32 is identical to SEQ ID NO: 31, but lacks the signal sequence that precedes the CAR.FIG 7: Amino acid sequence of a CD44v6 CAR as co-expressed with a protease sensitive CD44v6scFc. The amino acid sequence presented (SEQ ID NO:48) is the amino acid sequence of a CD44v6 CAR, including the signal sequence, together with the protease sensitive CD44v6scFc and the T2A ribosomal skip sequence that allows the CAR to be coexpressed, but not fused to, the protease sensitive CD44v6scFc. The immature CAR includes: GMCSFR signal peptide, a CD44v6 scFv, an IgG4 derived FC sequence that acts as a spacer, a CD4 transmembrane domain, a 4-IBB co-stimulatory domain, a three Gly linker sequence, a CD3 Zeta stimulatory domain. Also present is a T2A ribosomal sequence, a signal sequence and then CD44v6scFc (including VH, protease sensitive linker VL and a variant Fc domain). The various domains are indicated. This sequence lacks TR sequence (present in SEQ IS NOs: 31 and 32) between the T2A sequence following the CAR sequence and the signal sequence preceding the protease CD44v6 scFv - Fc fusion. SEQ ID NO: 49 is identical to SEQ ID NO: 48, but lacks the signal sequence that precedes the CAR.DETAILED DESCRIPTIONCD44v6 Targeted CARThe CD44v6-targeted CAR described herein include a CD44v6-targeting scFv comprising the amino acid sequenceEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG 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 sequenceEIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVP ARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK (SEQ ID NO: 34). The VH domain can include: a VH CDR1 comprising SYD S (SEQ ID NO: 35); a. VH CDR2 comprising TfSSG<ISYTY¥LDSIKG (SEQ ID NO: 36); and a VH CDR3 comprising QGLD Y (SEQ ID NO: 37). The VL domain can include: a VL CDR1 comprising SASSSINYIY (SEQ ID NO: 38); a VL CDR2 comprising L TSNL.AS (SEQ ID NO: 39); and a VL CDR3 comprising LQWSSNPLT (SEQ ID NO: 40).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).Spacer RegionThe CAR described herein can include a spacer located between the CD44v6 targeting domain (i.e., a gpl20-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. Table 1: Examples of Spacers

[0025] Some spacer regions include all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI 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.

[0026] 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).

[0027] 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 MHEALHNHYTQKSLSLSLGK (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 NOT E 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).Transmembrane Domain

[0028] 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.Table 2: Examples of Transmembrane DomainsCostimulatory Domain

[0029] The costimulatory domain can be any domain that is suitable for use with a CD3^ 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: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRS (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 thatincludes 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.

[0030] The costimulatory domain(s) are located between the transmembrane domain and the CD3^ signaling domain. Table 3 includes examples of suitable costimulatory domains together with the sequence of the CD3^ signaling domain.Table 3: ( 1)31 Domain and Examples of Costimulatory Domains

[0031] 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 0X40 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 4-1BBco-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 CD3^ signaling domain and in some cases a short linker consisting of 2 to 10, e.g., 3 amino acids (e.g., GGG) is positioned between the costimulatory domain and the CD3^ signaling domain.CD3C Signaling Domain

[0032] The CD3^ Signaling domain can be any domain that is suitable for use with a CD3^ signaling domain. In some cases, the CD3^ 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 CD3^ signaling has 1, 2, 3, 4 or 5 amino acid changes (preferably conservative) compared to SEQ ID NO:21.Truncated EGFR

[0033] The CD3^ 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 TKQHGQFSLAWSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSG QKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKC NLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIA TGMVGALLLLLWALGIGLFM (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.FIG 4 depicts the amino acid sequence of a CD44v6 targeted CAR together with a signal sequence (SEQ ID NO: 29). is not present in the mature CAR as expressed on T cells.SEQ ID NO: 29 is the mature CAR without the signal sequence. Useful CAR include those 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.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.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.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 toimmunomagnetically 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.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.Example 1: Preparation and Characterization of CD44v6 CARAs described in WO 2019 / 148006, a lentiviral construct was prepared using the approach described in Wang et al. (Blood 118: 1255, 2011). The construct expresses a CAR that includes a CD44v6-specific ScFv, an IgG4 Fc hinge, a CD4 transmembrane domain, a 4 IBB co-stimulatory domain, a GGG linker, and a CD3z cytoplasmic signaling domain inframe with a T2A ribosome skip and truncated huEGFR sequence (FIG. 5). This vector, which expresses the CAR used in the studies described in this example, does not express a soluble scFv targeted to CD44v6.CD8+central memory T cells (CD62L+CD45RO+C D8+; CD8+TCM) 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 (50U / ml) and IL-15 (Ing / ml).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 (50U / ml) and IL- 15 (Ing / ml). After 2 cycles of in vitro expansion, T cells were labeled with antibodies against EGFRt (Erbitux), CD62L, CD28 and CD27. AML (THP-1 and KGla) and B cell lymphoma (LCL) cells were labeled with PE-conjugated CD44v6 Ab (clone 2F10 an isotype control and analyzed by flow cytometry.After 2 cycles of in vitro expansion, CD44v6CAR / EGFRt CD8+cells were incubated for 4 hrs with 51Cr- labled THP-1 or LCL cells, or OKT3 -expressing LCL ( LCLOKT3) cells as positive targets and CD44v6 negative KGla cells as negative targets at 25:1 E:T ratios. As shown in WO 2019 / 148006, there was good cell killing activity against LCL OKT3 and THP-1 cells. KGla cells were not killed.To examine the anti-lymphoma effect of adoptively transferred CD8+TCM derived CD44v6CAR / EGFRt T cells, 2x 10^ CD19+ffluc+lymphoblastoid cell lines (LCL) cells were injected (i.v) into NSG mice on day -3. Subsequently, 5xl06CD8+TCM 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. As shown in WO 2019 / 148006, CD8+TCM derived CD44v6CAR / EGFRt T cellsexhibited good anti-tumor activity.In another study described in WO 2019 / 148006, 1.5x10^ GFPffluc+AML cells (THP-1) were injected (i.v) into NSG mice on day -3.5xl06CD8+TCM derived CD44v6CAR T cells were intravenously infused into the tumor bearing mice on day 0. Mice received no T cells or CD8+TCM 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 CD8+TCM derived CD44v6CAR / EGFRt cells exhibited good anti-tumor activity, while the CD 19 CAR T cells did not.In another study described in WO 2019 / 148006, 20 xlO^ CD44v6CAR T cells or irrelevant CAR T cells (CD19CAR) derived from CD8+TCM of the same donor were adoptively transferred (i.v) into NSG mice. 2 weeks post T cell infusion, 1.5xl06GFPffluc+THP-1 cells expressing CD44v6 were inoculated (i.v) into the mice. This study demonstrated that CD8+TCM derived CD44v6 CAR / EGFRt T cells interfere with human leukemia initiation in immunodeficient mice.The results of these studies suggest that targeting CD44v6 with CD44v6 CAR CD8+ TCM 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 scFvA selected scFv (e.g., an scFv having the VL and VH domains of the 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.Specifically, a protease sensitive scFv was designed having a VH domain having thesequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTY YLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS (SEQ ID NO 33); a protease sensitive linker having the sequence: GSTSGSKGPLGLAGATKG SEQ ID NO: 42); a VL domain having the sequence:EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPARFS GSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK (SEQ ID NO: 34); and a Fc domain having the sequence:ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYV DGVEVHNAI<TI<PREEQFQSTYRVVSVLTVLHQDWLNGI<EYI<C I<VSNI<GLPSSIEI<TISI< AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 11). Overall, the mature protease-sensitive scFv-Fc fusion has the sequence:EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTY YLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSSGSTS GSKGPLGLAGATKGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLI YLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIKESK YGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAI<TI<PREEQFQSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<GLPSSIEI<TISI<AI< GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47).This protease sensitive scFv was expressed with the CD44v6 CAR in a lentiviral vector. The CD44v6 CAR (SEQ ID NO: 29) was preceded by a GMCSFRa signal peptide (MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 26). The CD44v6 CAR was followed by a T2A ribosomal skip sequence, a second GMCSFRa signal peptide (MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 29) and then the protease sensitive scFv. The overall sequence encoded was: MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQ APGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQ GLDYWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSINY lYWYQQKPGQAPRLLIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSSQEDPEVQFNWYVDGVEVHNAI<TI<PREEQFNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKGLPS SIEKTISK AKGQPREPQ VYTLPPS QEEMTKNQ VSLTCLVKGF YPS DI AVE WES NG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGC ELGGGRVI<FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDI<RRGRDPEMGGI<PR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPRLEGGGEGRGSLLTCGDVEENPGPRTRMLLLVTSLLLCELPHPAFLLIPEVQLVESG GGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSSGSTSGSKGPLGLA GATKGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGV PARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIKESKYGPPCPPCPA PEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47).This construct is referred to as sCD44v6CAR. The underlined TR between the T2A skip sequence and the signal sequence that precedes the protease sensitive scFV-Fc fusion provides a restriction site and can be excluded.In an initial study, a variant of the protease sensitive scFv lacking an Fc domain was displayed on the surface of yeast flanked by HA and MYC epitopes on the N-terminal and C-terminal ends, respectively (FIG 1A).Kinetic analysis with varying MMP-2 concentration and digestion time revealed moderate proteolytic susceptibility of the scFv, with digestion efficiency increasing with both MMP-2 concentration and time (FIG IB). Interestingly, the engineered scFv also demonstrated high affinity binding to CD44v6-expressing tumors (MM line MM.1 S) as determined in titration assays (FIG 1C). For improved protein stability and characterization, the soluble protease-sensitive scFv was modified to include IgGFc domain at the C-terminus of VL domain. Following successful characterization, the engineered scFv-Fc was cloned into the backbone of CD44v6CAR construct (sCD44v6CAR) and packaged into a lentivirus. This construct expresses the CD44v6CAR and the protease sensitive CD44v6 scFv-Fc fusion protein (FIG. 6)Human T cells transduced with the newly developed, lentiviral CAR construct (sCD44v6CAR) showed -45% CAR expression as examined by flow cytometry (Figure 2). To determine whether T cells secreted soluble scFv, the culture supernatant was harvested and incubated with CD44v6- expressing MM. IS line in the presence or absence of recombinant MMP2. In parallel, expression of soluble scFv in the fPLC clarified supernatant was confirmed by Western blot (FIG 2D). Accordingly, we observed more than 90% binding of soluble scFv to MM.1 S by flow and less than 50% binding following the addition of lOOnM MMP2, highlighting proteolytic susceptibility of our scFv. Next, the effector activity of sCD44v6CAR T cells following coculture with MM.1 S was assessed. The sCD44v6CAR T cells demonstrated significantly lower tumor cell killing after 24- and 72-hours of coculture, when compared to control T cells (CD44v6CAR T cells) (FIG 3A). However, increased tumor killing was observed following addition of lOOnM MMP2 to the coculture, suggesting that soluble scFv successfully blocked CAR T cell binding to tumor targets prior to proteolytic cleavage (addition of MMP2). Similarly, coculture of sCD44v6CAR T cells with primary human keratinocytes in xCelligence assays also demonstrated similar cell lysis pattern, with significantly lower keratinocyte killing compared to controls (FIG 3B). The addition of recombinant MMP-2 in co-culture assays cleaved soluble scFvs, enabling CAR-mediated keratinocyte lysis. Taken together, the data confirms our proof-of-concept hypothesis and highlights the protective capacity of newly engineered sCD44v6CAR T cells, with its potential to neutralize off-target toxicity and improve anti-MM tumor activity.What is claimed is:

Claims

1. A nucleic acid molecule comprising: a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an scFv targeting CD44v6, a spacer domain, a transmembrane domain, a co-stimulatory domain, and a CD3^ signaling domain; and a nucleotide sequence encoding a fusion protein comprising a protease-sensitive scFv targeting CD44v6 and an Fc domain.

2. The nucleic acid molecule of claim 1, wherein the protease-sensitive scFv is sensitive to MMP-2 or MMP-9.

3. The nucleic acid molecule of claim 1, wherein the protease-sensitive scFv comprises a VH domain and a VL domain joined by a protease-sensitive linker.

4. The nucleic acid molecule of claim 1, wherein: the scFv targeting CD44v6 and the protease-sensitive scFv targeting CD44v6 have the same CDR sequences or the scFV targeting CD44v6 and the protease-sensitive scFv targeting CD44v6 have the same VH and VL sequences.

5. The nucleic acid molecule of claim 1, wherein the scFv targeting CD44v6 and the protease-sensitive scFv targeting CD44v6 comprise: a VH domain comprising the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG GSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVS S (SEQ ID NO:33) and a VL domain comprising the sequence:EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPARFSG SGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK (SEQ ID NO: 34).

6. The nucleic acid molecule of claim 1, wherein scFv targeting CD44v6 and the proteasesensitive scFv targeting CD44v6 comprise: a VH domain comprising a VH CDR1 comprising SYDMS (SEQ ID NO: 5), a VH CDR2 comprising TISSGGSYTYYLDSIKG (SEQ ID NO: 36), and a VH CDR3 comprising QGLDY (SEQ ID NO: 37); and a VL domain comprising a VL CDR1 comprising SASSSINYIY (SEQ ID NO: 38), a VL CDR2 comprising LTSNLAS (SEQ ID NO: 39), and a VL CDR3 comprising LQWSSNPLT (SEQ ID NO' 40) .

7. The nucleic acid molecule of claim 1, wherein the Fc domain comprises the sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: H).

8. The nucleic acid molecule of claim 1, wherein the fusion protein comprising a proteasesensitive scFv and an Fc domain comprises the sequence:EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTY YLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSSGSTS GSKGPLGLAGATKGEIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIY LTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYCLQWSSNPLTFGGGTKVEIKESKYG PPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47).

9. The nucleic acid molecule of claim 1, wherein the CAR and the fusion protein are joined by a translation skip sequence.

10. The nucleic acid molecule of claim 1 , wherein the VH domain of both the scFv and the protease sensitive scFv comprise a VH domain sequence at least 96%, 97%, 98% or 99% identical SEQ ID NO: 33 and comprising a VH CDR1 comprising SYDMS (SEQ ID NO: 35), a VH CDR2 comprising TISSGGSYTYYLDSIKG (SEQ ID NO: 36), and a VH CDR3 comprising QGLDY (SEQ ID NO: 37); and the VL domain sequence at least 96%, 97%, 98% or 99% identical of SEQ ID NO:34 and comprising a VL CDR1 comprising SASSSINYIY (SEQ ID NO: 38), a VL CDR2 comprising LTSNLAS (SEQ ID NO: 39), and a VL CDR3 comprising LQWSSNPLT (SEQ ID11. The nucleic acid molecule of claim 1, wherein the CAR comprises: a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-5 single amino acid substitutions, a CD8 transmembrane domain or variant thereof having 1-5 single amino acidsubstitutions, a CD28 transmembrane domain or a variant thereof having 11-5 single amino acid substitutions; a costimulatory domain selected from: a 4-IBB costimulatory domain or a variant thereof having 1-5 single amino acid substitutions and an CD28 costimulatory domain or a variant thereof having 1-5 single amino acid substitutions; a CD3 signaling domain or a variant thereof having 1-5 single amino acid substitutions; and a spacer domain having 20-150 amino acids located between the scFv and the transmembrane domain.

12. The nucleic acid molecule of claim 1, wherein the costimulatory domain is selected from the group consisting of: a 4-IBB costimulatory domain and variants thereof having 1-5 single amino acid substitutions.

13. The nucleic acid molecule of claim 1, wherein the transmembrane domain is a CD4 transmembrane domain or variant thereof having 1-5 single amino acid substitutions.

14. The nucleic acid molecule of claim 1 , wherein the transmembrane domain is a CD4 transmembrane domain.

15. The nucleic acid molecule of claim 6, wherein the VH domain comprises the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG GSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVS S (SEQ ID NO:33) or a variant thereof with 1-5 single amino acid substitutions and the VL domain comprising the sequence:EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPARFSG SGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK (SEQ ID NO: 34) or a variant thereof with 1-5 single amino acid substitutions.

16. The nucleic acid molecule of claim 1, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 29 or 30 or a variant thereof having 1-5 single amino acid substitutions.

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

18. A viral vector comprising the nucleic acid molecule of claim 1.19 A population of human T cells comprising the nucleic acid molecule of claim 1.

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

21. The method of claim 20, wherein MMP-2 or MMP-9 is present in the tumor microenvironment.

22. The method of claim 21, wherein the patient is suffering from a leukemia or lymphoma.

23. The method of claim 21, wherein the patient is suffering from AML or multiple myeloma.

24. A nucleic acid molecule comprising: a nucleotide sequence encoding a fusion protein comprising a protease-sensitive scFv targeting CD44v6 and an Fc domain.

25. The nucleic acid molecule of claim 24, wherein the protease-sensitive scFv is sensitive to MMP-2 or MMP-9.

26. The nucleic acid molecule of claim 24, wherein the protease-sensitive scFv comprises a VH domain and a VL domain joined by a protease-sensitive linker.

27. The nucleic acid molecule of claim 26, wherein the protease-sensitive scFv targeting CD44v6 comprise: a VH domain comprising the sequence:EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSG GSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS (SEQ ID NO:33) and a VL domain comprising the sequence:EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK (SEQ ID NO: 34).

28. The nucleic acid molecule of claim 26, wherein the protease-sensitive scFv targeting CD44v6 comprise: a VH domain comprising a VH CDR1 comprising SYDMS (SEQ ID NO: 35), a VH CDR2 comprising TISSGGSYTYYLDSIKG (SEQ ID NO’ 36), and a VH CDR3 comprising QGLDY (SEQ ID NO: 37); and a VL domain comprising a VL CDR1 comprising SASSSINYIY (SEQ ID NO: 38), a VL CDR2 comprising LTSNLAS (SEQ ID NO: 39), and a VL CDR3 comprising LQWSSNPLT (SEQ ID NO: 40) .

29. The nucleic acid molecule of claim 26, wherein the Fc domain comprises the sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: H)30. The nucleic acid molecule of claim 26, wherein the fusion protein comprising a proteasesensitive scFv and an Fc domain comprises the sequence:MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQ APGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQ GLDYWGRGTLVTVSSGSTSGSKGPLGLAGATKGEIVLTQSPATLSLSPGERATLSCSASSSI NYIYWYQQKPGQAPRLLIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYCLQWS SNPLTFGGGTKVEIKESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAI<TI<PREEQFQSTYRVVSVLTVLHQDWLNGI<EYI<CI< VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK (SEQ ID NO: 46).

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