ANTI-ROR1 ANTIBODY AND ROR1-TARGETING ENGINEERED CELLS

Abstract

The present invention is directed to a monoclonal mouse or humanized ROR1 antibody, or a single-chain variable fragment (scFv). The present invention is also directed to a mouse or humanized ROR1 chimeric antigen receptor (CAR) comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) of the present invention, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain.

Claims

1-19. (canceled)

20. A method of making an engineered immune cell expressing an anti-human ROR1 chimeric antigen receptor (CAR), the method comprising expressing in an immune cell a CAR comprising an antibody or antigen-binding fragment comprising a heavy chain V.sub.H and a light chain V.sub.L, each of the V.sub.H and the V.sub.L comprising complementarity determining regions (CDRs), wherein a CDR1 of the V.sub.H comprises the sequence TYA, a CDR2 of the V.sub.H comprises SEQ ID NO: 41, a CDR3 of the V.sub.H comprises SEQ ID NO: 42, a CDR1 of the V.sub.L comprises SEQ ID NO: 43, a CDR2 of the V.sub.L comprises the sequence RAN, and a CDR3 of the V.sub.L comprises SEQ ID NO: 45.

21. The method of claim 20, wherein the V.sub.H comprises SEQ ID NO: 17, and the V.sub.L comprises SEQ ID NO: 18.

22. The method of claim 20, wherein the CAR comprises a single-chain variable fragment (scFv) consisting of the V.sub.H, the V.sub.L, and a linker.

23. The method of claim 22, wherein the scFv comprises or consists essentially of SEQ ID NO: 16.

24. The method of claim 22, wherein the CAR comprises or consists essentially of the amino acid sequence of SEQ ID NO: 19 without the signaling peptide consisting of SEQ ID NO: 22.

25. The method of claim 20, wherein the cell is isolated from the peripheral blood mononuclear cells (PBMC) fraction of the human whole blood.

26. The method of claim 20, wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell and a macrophage.

27. The method of claim 20, wherein the cell is allogeneic.

28. The method of claim 20, wherein the expressing comprises contacting the cell with a construct comprising a vector and a coding sequence for the CAR.

29. The method of claim 28, wherein the coding sequence for the CAR is codon-optimized for expression in human cells.

30. The method of claim 28, wherein the coding sequence for the CAR comprises or consists essentially of SEQ ID NO: 39.

31. The method of claim 28, wherein the vector is a viral vector selected from the group consisting of an AAV vector and a lentiviral vector.

32. The method of claim 20, further comprising assessing the expression of the CAR in the cell.

33. The method of claim 32, wherein the expression of the CAR is assessed by fluorescence-activated cell sorting (FACS).

34. The method of claim 32, wherein the expression of the CAR is assessed by detecting binding of the cells to ROR1-expressing cells.

35. The method of claim 32, wherein the expression of the CAR is assessed by detecting cytokines secreted by the cells in the presence of ROR1-expressing cells.

36. The method of claim 32, wherein the expression of the CAR is assessed by detecting cytotoxic activity of the cells directed against ROR1-expressing cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a diagram showing the first, second, and third generation chimeric antigen receptors (CARs) known in the art.

[0013] FIG. 2 is a diagram showing the structure of the anti-ROR1 CAR.

[0014] FIG. 3 is a Western blot demonstrating binding of the anti-ROR1 antibody to human ROR1 antigen.

[0015] FIG. 4 shows FACS data demonstrating staining of different cell lines with the anti-ROR1 antibody using different cancer cell lines.

[0016] FIG. 5 shows an RTCA assay demonstrating dose-dependent in cytotoxicity of anti-ROR1 CAR-T cells (ROR1-CD28-CD in panel A, and ROR1-4-1BB-CD3 in panel B) against the ROR1-expressing cell line SKOV-3.

[0017] FIG. 6 shows measurements of IFN-gamma secretion by anti-ROR1 CAR-T cells (ROR1-4-1BB-CD3) in the presence of SKOV-3 cells or control HL-60 cells (ROR1-negative cells).

[0018] FIG. 7. shows an RTCA assay demonstrating dose-dependent in vitro cytotoxicity of the anti-ROR1 CAR-T cells with humanized scFvs (PMC1182, PMC1183 and PMC1194 expressing PMC857, PMC858, and PMC862 anti-ROR1 CARs respectively) against SKOV3 cells.

[0019] FIG. 8. shows measurements of IFN-gamma secretion by the anti-ROR1 CAR-T cells with humanized scFvs (PMC1182, PMC1183 and PMC1194 expressing PMC857, PMC858, and PMC862 anti-ROR1 CARs respectively) in the presence of SKOV-3 cells or control HL-60 cells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0020] As used herein, an antibody refers to antigen binding proteins of the immune system. A naturally occurring antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant CL region. The light chain constant region is comprised of one domain, CL. The VH and VL comprise complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0021] The term human antibody, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human gene sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human immunoglobulin sequences.

[0022] The term humanized antibody refers to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0023] As used herein, an antigen-binding fragment refers to a protein fragment including Fab fragment, Fab fragment, F(ab)2 fragment, and scFv with antigen-binding activity.

[0024] As used herein, a chimeric antigen receptor (CAR) is a receptor protein that has been engineered to give T cells a new ability to target a specific protein. The receptor is chimeric because it combines both antigen-binding and T-cell activating functions in a single receptor. CAR is a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and at least one intracellular domain.

[0025] As used herein, the extracellular domain capable of binding to an antigen means any oligopeptide or polypeptide that can bind to a certain antigen. The intracellular domain means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.

[0026] As used herein, a domain means one region in a polypeptide which is folded into a particular structure independently of other regions.

[0027] As used herein, a single chain variable fragment (scFv) means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. A typical example of an scFv includes an antigen-binding polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer or linker sequence. Various methods for engineering an scFv are known to a person skilled in the art.

[0028] As used herein, a tumor antigen means a biological molecule having antigenicity, which is a characteristic of a tumor.

[0029] The inventors have generated an anti-ROR1 monoclonal antibody that specifically targets the human ROR1 antigen using hybridoma technology. The inventors have produced anti-ROR1 CAR-T cells to target cancer cells overexpressing the ROR1 tumor antigen. The anti-ROR1 CAR-T cells of the present invention have high cytotoxic activity against several cancer cell lines and anti-tumor activity in vivo. Anti-ROR1 CAR-NK cells expressing the same CAR are also contemplated.

[0030] In some embodiments, the present invention comprises a monoclonal mouse anti-human ROR1 antibody having the amino acid sequence of SEQ ID NO: 1, or an antigen-binding fragment thereof, comprising a V.sub.H having the amino acid sequence of SEQ ID NO: 2 and a V.sub.L having the amino acid sequence of SEQ ID NO: 3.

[0031] In some embodiments, the present invention comprises a monoclonal mouse anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising a V.sub.H having the amino acid sequence of SEQ ID NO: 5 and a V.sub.L having the amino acid sequence of SEQ ID NO: 6.

[0032] In some embodiments, the present invention comprises a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising a V.sub.H having the amino acid sequence of SEQ ID NO: 9 and a V.sub.L having the amino acid sequence of SEQ ID NO: 10.

[0033] In some embodiments, the present invention comprises a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising a V.sub.H having the amino acid sequence of SEQ ID NO: 13 and a V.sub.L having the amino acid sequence of SEQ ID NO: 14.

[0034] In some embodiments, the present invention comprises a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising a V.sub.H having the amino acid sequence of SEQ ID NO: 17 and a V.sub.L having the amino acid sequence of SEQ ID NO: 18.

[0035] In some embodiments, the monoclonal anti-human ROR1 antibody is generated against the extracellular region of the purified recombinant fragment of human ROR1.

[0036] In some embodiments, the invention comprises single-chain variable fragments (scFv) derived from the monoclonal mouse anti-human ROR1 antibody disclosed herein or any of the humanized versions thereof also disclosed herein.

[0037] In some embodiments, the invention comprises a chimeric antigen receptor (CAR) fusion protein comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) against ROR1 disclosed herein, (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activating domain.

[0038] FIG. 1 illustrates the structure of a first-generation CAR lacking the costimulatory domains, the second-generation CAR with one co-stimulatory domain (CD28 or 4-1BB), and the third-generation CAR having two or more co-stimulatory domains (adapted from Goluboskaya et al., (2016) Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy. Cancers (Basel). 2016 Mar. 15; 8 (3). pii: E36).

[0039] FIG. 2 illustrates the structure of the anti-ROR1 CAR of the present invention. The second-generation CAR was used with either the CD28 or the 4-1BB co-stimulatory domain. (A CAR with the CD28 co-stimulatory domain is shown.) In FIG. 2, scFv is a single chain variable fragment; CD8 h is a CD8 hinge; CD28 TM is a CD28 transmembrane domain; CD28 cs is a CD-28 co-stimulatory domain, CD3-zeta is a CD3 zeta activation domain, VH is a heavy chain variable region, L is a linker and VL is a light chain variable region. The arrangement of the scFv is shown as V.sub.H-linker-V.sub.L. In some embodiments, the arrangement is V.sub.L-linker-V.sub.H.

[0040] The co-stimulatory domain can be selected from the group consisting of CD28, 4-1BB (CD137), GITR, ICOS-1, CD27, OX-40 and DAP10 co-stimulatory domains. In some embodiments, the co-stimulatory domain is CD28.

[0041] In some embodiments, the activating domain is CD3 zeta (CD3 Z or CD3-zeta, encoded by the CD247 gene.

[0042] The transmembrane domain may be derived from a natural polypeptide or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. In some embodiments, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of a T cell receptor - or -chain, a CD3-zeta chain, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.

[0043] In some embodiments, the CAR comprises a linker between the transmembrane domain and the intracellular domain. In some embodiments, the linker is an oligopeptide or a polypeptide, for example, has a length of 2 to 10 amino acids. A peptide linker generally comprises from about 5 to about 40 amino acids. The linker can be a naturally occurring sequence or an engineered sequence. For example, in some embodiments, the linker is derived from a human protein, e.g., an immunoglobulin selected from IgG, IgA, IgD, IgE, or IgM. In some embodiments, the linker comprises 5-40 amino acids from the CH1, CH2, or CH3 domain of an immunoglobulin heavy chain. In some embodiments, the linker is a glycine and serine rich linker having the sequence (G.sub.xS.sub.y).sub.n. Additional linker examples and sequences are disclosed in the U.S. Pat. No. 5,525,491 Serine-rich peptide linkers, U.S. Pat. No. 5,482,858 Polypeptide linkers for production of biosynthetic proteins, and a publication WO2014087010 Improved polypeptides directed against IgE.

[0044] In some embodiments, the invention comprises one or more nucleic acids encoding the anti-ROR1 CARs. The nucleic acid encoding the CAR can be prepared from an amino-acid sequence of the specified CAR by a conventional method. A nucleotide sequence encoding an amino acid sequence can be obtained using the tools provided to the public by the National Center for Biotechnology Information (NCBI), e.g., from NCBI RefSeq IDs or accession numbers of GenBank for an amino acid sequence of each domain. The nucleic acid of the present invention can be prepared using a standard molecular biological or chemical procedure. In some embodiments, based on the nucleotide sequence, portions of the nucleic acid are synthesized. In some embodiments, the nucleic acid of the present invention is prepared by combining DNA fragments which are obtained from a cDNA library using the polymerase chain reaction (PCR).

[0045] In some embodiments, a nucleic acid encoding the CAR of the present invention is inserted into a vector, and the vector is introduced into a cell. In some embodiments, the vector is a viral vector such as a retroviral vector (including an oncoretroviral vector, a lentiviral vector, and a pseudo-type vector), an adenoviral vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector, a Sendai virus vector, an Epstein-Barr virus (EBV) vector, or a herpes simplex virus (HSV) vector. In some embodiments, a viral vector lacking the replicating ability so as not to self-replicate in an infected cell is used.

[0046] In some embodiments, retroviral particles are prepared using a packaging cell line. In such embodiments, a suitable packaging cell line based on the LTR sequence, and the packaging signal sequence possessed by the viral vector is selected. Examples of the packaging cell lines include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86, GP+envAm-12, and Psi-CRIP. In some embodiments, retroviral particles are prepared using the HEK293 cell line or the HEK293t cell line having high transfection efficiency. One of skill in the art is aware of many kinds of retroviral vectors and packaging cell lines that are commercially available.

[0047] A CAR-T cell (or a CAR-NK cell) binds to a specific antigen via the CAR, whereby a signal is transmitted into the cell, and the cell is activated. The activation of the cell expressing the CAR is varied depending on the kind of the cell type and the intracellular domain of the CAR. Activation of the cell can be confirmed based on, for example, release of a cytokine, any improvement of a cell proliferation rate, a change in any cell-surface molecule, or the like. Furthermore, the release of cytotoxic cytokines (IFN, TNF, etc.) from the activated CAR-T cell (or CAR-NK cell) causes destruction of a target cell expressing an antigen which can be detected or measured. In addition, release of a cytokine or change in a cell-surface molecule results in detectable or measurable stimulation of other immune cells, for example, B cells, dendritic cells, NK cells, and macrophages.

[0048] In some embodiments, the cell expressing the CAR is used as a therapeutic agent for a disease. The therapeutic agent comprises the cell expressing the CAR as an active ingredient, and it may further comprise a suitable excipient.

[0049] In one embodiment, the invention comprises anti-ROR1 scFv-CD28-CD3 zeta-CAR-T (anti-ROR1 CAR-T) cells or anti-ROR1 CAR-NK cells against cancer cells overexpressing ROR1. Anti-ROR1-CAR-T cells or CAR-NK cells express higher cytotoxic activity against ROR1-positive cancer cells compared to non-transduced (no CAR) T cells (or no CAR NK cells) and mock CAR-T cells (or mock CAR-NK cells). The mouse monoclonal anti-human ROR1 antibody disclosed herein detects ROR1 in ROR1-positive cancer cells.

[0050] In some embodiments, the invention comprises humanized V.sub.H and V.sub.L of the mouse monoclonal anti-human ROR1 antibody, a humanized scFv comprising the humanized V.sub.H and V.sub.L, and CAR-T cells (or CAR-NK cells) harboring a CAR comprising the humanized anti-ROR1 scFv targeting ROR1-positive cells. Without being bound by one particular theory, the inventors perceive at least one advantage of humanizing the mouse anti-ROR1 scFv is potentially reduced immune response to the CAR-T (CAR-NK) cells in humans.

[0051] In some embodiments, the anti-ROR1 antibody or antigen binding fragment or derivative thereof (such as an scFv) comprises complementarity determining regions (CDRs). Each of the light chain and the heavy chain of an antibody comprises three CDRs. In some embodiments, CDRs are identified using crystal structure of an antigen-antibody complex. In some embodiments, CDRs are identified using in vitro methods such as phage display. In some embodiments, CDRs are identified using in silico methods, for example, IMGT (Lefranc et al., (2009) IMGT, The international immunogenetics information system, Nucl. Acids Res. 37:D1006), and Kabat (Kabat et al., (1987) Sequences of Proteins of Immunological Interest, 4th ed., U.S. H.H.S., N.I.H.). In some embodiments, the CDRs are identified using the IMGT tool. In some embodiments, the CDRs are identified using the Kabat tool. In some embodiments, the minimal portions of the CDRs are identified as an overlap of the sequences identified by the IMGT tool and the sequences identified by the Kabat tool.

[0052] In some embodiments, the anti-ROR1 scFv comprises the sequence TYA in the CDR1 of the V.sub.H. In some embodiments, the anti-ROR1 scFv comprises SEQ ID NO: 41 in the CDR2 of the V.sub.H. In some embodiments, the anti-ROR1 scFv comprises SEQ ID NO: 42 in the CDR3 of the V.sub.H. In some embodiments, the anti-ROR1 scFv comprises SEQ ID NO: 43 in the CDR1 of the V.sub.L. In some embodiments, the anti-ROR1 scFv comprises the sequence RAN in the CDR2 of the V.sub.L. In some embodiments, the anti-ROR1 scFv comprises SEQ ID NO: 45 in the CDR3 of the V.sub.L.

[0053] In some the anti-ROR1 scFv anti-ROR1 comprises the sequence TYA in the CDR1 of the V.sub.H, and SEQ ID NO: 41 in the CDR2 of the V.sub.H, and SEQ ID NO: 42 in the CDR3 of the V.sub.H and further comprises SEQ ID NO: 43 in the CDR1 of the V.sub.L, and the sequence RAN in the CDR2 of the V.sub.L, and SEQ ID NO: 45 in the CDR3 of the V.sub.L.

TABLE-US-00001 CDR1heavychain TYA CDR2heavychain SEQIDNO:41 SSGGNT CDR3heavychain SEQIDNO:42 DSYYFGNSVYYAMDY CDR1lightchain SEQIDNO:43 QDINSY CDR2lightchain RAN CDR3lightchain SEQIDNO:45 LQYDEFPYT

[0054] In some embodiments, the anti-ROR1 scFv comprises complementarity determining regions CDR1, CDR2, and CDR3 in the light chain (V.sub.L), and CDR1, CDR2, and CDR3 in the heavy chain (V.sub.H) and comprises: the sequence TYA in the CDR1 of the V.sub.H, SEQ ID NO: 41 in the CDR2 of the V.sub.H, SEQ ID NO: 42 in the CDR3 of the V.sub.H, SEQ ID NO: 43 in the CDR1 of the V.sub.L, the sequence RAN in the CDR2 of the V.sub.L, and SEQ ID NO: 45 in the CDR3 of the V.sub.L. In some embodiments, in the anti-ROR1 scFv, the CDR1 of the V.sub.H consists of the sequence TYA, the CDR2 of the V.sub.H consists of SEQ ID NO: 41, the CDR3 of the V.sub.H consists of SEQ ID NO: 42, the CDR1 of the V.sub.L consists of SEQ ID NO: 43, the CDR2 of the V.sub.L consists of the sequence RAN, and the CDR3 of the V.sub.L consists of SEQ ID NO: 45.

[0055] The humanized anti-ROR1 antibody and the scFv derived therefrom that are disclosed herein can be used for immunotherapy applications: toxin-drug conjugated antibody, monoclonal therapeutic antibody, bispecific antibody, and CAR-T cell (or CAR-NK cell) based immunotherapy.

[0056] The anti-ROR1 CAR-T cells (or CAR-NK cells) generated using the anti-ROR1 antibody disclosed herein can be effectively used to target the ROR1 antigen in ROR1-positive cells and tumors. The anti-ROR1CAR-T cells (or CAR-NK cells) can be used clinically against tumor cells, tumors, and cancer stem cells that are resistant to chemotherapy and form aggressive tumors.

[0057] The anti-ROR1 CAR-T cells (or CAR-NK cells) can be used in combination with different therapeutic agents: checkpoint inhibitors; targeted therapies, small molecule inhibitors, antibodies and the like. For example, anti-ROR1 CAR-T cells (or CAR-NK cells) can be used in combination with CAR-T (or CAR-NK) cells targeting other tumor antigens or antigens present in the tumor microenvironment (e.g., VEGFR-1-3, PDL-1, CD80). Bi-specific antibodies and scFvs (e.g., bi-specific against ROR1 and CD3), and cells expressing the antibodies and the scFvs can be used to enhance activity of ROR1-targeting therapy.

[0058] The anti-ROR1 antibody and its derivatives disclosed herein can be modified with site-directed mutagenesis, e.g., with error-prone PCR for affinity tuning and selected by affinity maturation. Modifications of co-activation domains: CD28, 4-1BB and others can be used to increase the efficacy of the CAR generated from the antibody (and its derivatives) disclosed herein. Tag-conjugated anti-ROR1 scFv can be used for CAR generation. First, second and third generation CAR constructs can be made with the same anti-ROR1 scFv disclosed herein.

[0059] The anti-ROR1 CAR disclosed herein can be used for generating CAR-T cells, CAR-NK cells and other types of cells such as iPSCs (induced pluripotency stem cells) from which T cells, NK cells, macrophages and other anti-ROR1 CAR-expressing hematopoietic cells, which can target ROR1-positive cancers. The present invention provides T cells, or NK cells, or macrophages, or hematopoietic cells, modified to express the anti-ROR1 CAR.

[0060] The CAR-expressing cells disclosed herein can be autologous cells and allogenic cells.

[0061] The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

EXAMPLES

[0062] The inventors generated anti-ROR1 CAR constructs and cloned the constructs into lentiviral vectors. The CAR construct contains the anti-ROR1 ScFv-CD28-CD3zeta insert (or similar insert with 41BB co-stimulatory domain instead of CD28 domain). CMV, EF1 or MNDU3 promoter can be used to drive expression of CAR construct. The lentiviruses were generated in HEK293t cells and titer was established by RT-PCR. Then equal dose of lentiviruses was used for transduction of T cells, as described in Examples.

Example 1. Anti-ROR1 scFv Detected the ROR1 Protein by Western Blotting, and the Anti-ROR1 Antibody Detected ROR1 by FACS Staining

[0063] In this example, we generated a mouse monoclonal anti-ROR1 antibody using standard hybridoma technology. The mouse anti-ROR1 antibody (IgG1 type) detected extracellular ROR1 protein by ELISA (data not shown). We sequenced this hybridoma clone 2H6 and generated an scFv using V.sub.H and V.sub.L (see further in Example 2). We performed a Western blot demonstrating that the anti-ROR1 scFv bound ROR1 extracellular domain fused to human Fc (hFc) protein (FIG. 3, left panel). The ROR1-human Fc fusion protein was detected with an antibody directed against the human Fc domain. FIG. 3, right panel shows ROR1 antigen detected with the anti-ROR1 scFv-mouse Fc fusion as a primary antibody and anti-mouse IgG-HRP as a secondary antibody.

[0064] We further performed a fluorescence activated cell sorting (FACS) experiment demonstrating that the mouse anti-ROR1 monoclonal antibody detected elevated expression of ROR1 in several cancer cell lines such as hepatocellular carcinoma (HepG2), breast cancer (MDA231), colon cancer (HT-29), and ovarian cancer (SKOV-3). Normal keratinocytes were used as a negative control (FIG. 4). (MFI: medium fluorescence intensity compared to the isotype).

Example 2. Sequencing of Anti-ROR1 V.SUB.H., V.SUB.L., and CAR Constructs

[0065] In this example we sequenced the anti-ROR1 antibody, hybridoma clone 2H6. The sequences of V.sub.H, V.sub.L, and the scFv are shown below. The structure of the anti-ROR1 scFv is: V.sub.H-linker-V.sub.L with the linker having the sequence (G.sub.4S).sub.3 (SEQ ID NO: 46). In the sequences below the sequence starts with the V.sub.H; the underline shows the nucleotide sequence of V.sub.L; the linker sequence is in italics.

TABLE-US-00002 Anti-ROR1scFv(mouseclone2H6)nucleotidesequence (SEQIDNO:20): GTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCC CTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGCCATGTCTTG GGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTAGTGG TGGTAACACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGA TAATGCCAGGCACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACAC GGCCATGTATTACTGTGCAAGAGATTCTTATTACTTCGGTAATAGCGTTTACTAT GCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGCGGTG GTTCTGGTGGCGGTGGTTCTGGTGGCGGTGGTTCTGACATCAAGATGACCCAGTCT CCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGA GTCAGGACATTAATAGCTATTTTAGCTGGTTCCAGCAAAAACCAGGGAAATCTCC TAAGACCCTGATCTATCGTGCAAATAGATTGGTAGATGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGCAGGATTATTCTCTCACCATCAGCAGCCTGGAGTATG AAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGG AGGGGGGACCAAACTGGAAATAAAACGG Anti-ROR1scFv(mouseclone2H6)aminoacidsequence: (SEQIDNO:1): VKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASI SSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFGNSVY YAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCK ASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRESGSGSGQDYSLTISSLEYED MGIYYCLQYDEFPYTFGGGTKLEIKR Anti-ROR1scFv(mouseclone2H6)V.sub.Haminoacidsequence (SEQIDNO:2): VKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASI SSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFGNSVY YAMDYWGQGTSVTVSS Anti-ROR1scFv(mouseclone2H6)V.sub.Laminoacidsequence (SEQIDNO:3): DIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRA NRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKR

Example 3. Anti-ROR1-CAR Sequences with Mouse Anti-ROR1 scFv

[0066] In this example we designed a CAR with the scFv derived from the mouse anti-ROR1 antibody 2H6. The scheme of the anti-ROR1 CAR construct is shown in FIG. 2. The lentiviral vector Lenti CMV-MCS-EF1a-puro was used for cloning of the CAR sequence. The CD3 zeta CAR construct was under the control of the CMV promoter. For 4-1BB CAR construct we used another lentiviral vector with MNDU3 promoter to get higher percent of CAR expressing cells.

A. CD28 as a Co-stimulating Domain

[0067] The CAR comprises the following structure: anti-ROR1 ScFv-CD8 hinge-CD28 TM-CD28 co-stimulatory domains CD3 zeta activation domain (FIG. 2). The structure further includes the human CD8 signaling peptide. The anti-ROR1 scFv has the structure V.sub.H-Linker (G.sub.4S).sub.3-V.sub.L ((G.sub.4S).sub.3 disclosed as SEQ ID NO: 46).

TABLE-US-00003 CD8signalingpeptidenucleotidesequence(SEQIDNO:21): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC ACGCCGCCAGGCCG CD8signalingpeptideamino-acidsequence(SEQIDNO:22): MALPVTALLLPLALLLHAARP NheIrestrictionsite:GCTAGC XhoIrestrictionsite:CTCGAG CD8hingenucleotidesequence(SEQIDNO:24): AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACC ATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGG GGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGAT CD8hingeaminoacidsequence(SEQIDNO:25): KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD CD28TM/activationnucleotidesequence(SEQIDNO:26): TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCT GCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCA TTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC CD28TM/activationaminoacidsequence(SEQIDNO:27): FWVLVVVGGVLACYSLLVTVAFIIFWV/RSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRS CD3zetanucleotidesequence(SEQIDNO:28): AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGG CCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAA GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCG GAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT TCACATGCAGGCCCTGCCCCCTCGC CD3zetaaminoacidsequence(SEQIDNO:29): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP QRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR EcoRIrestrictionsite:GAATTC Anti-ROR1CAR(mouse)nucleotidesequence(SEQIDNO:30): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC ACGCCGCCAGGCCGGCTAGCGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGA AGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAC CTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGC ATCCATTAGTAGTGGTGGTAACACCTACTATCCAGACAGTGTGAAGGGCCGATTC ACCATCTCCAGAGATAATGCCAGGCACATCCTGTACCTGCAAATGAGCAGTCTG AGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGATTCTTATTACTTCGGTA ATAGCGTTTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTC CTCAGGTGGCGGTGGTTCTGGTGGCGGTGGTTCTGGTGGCGGTGGTTCTGACATC AAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTA TCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTTAGCTGGTTCCAGCAAAA ACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAATAGATTGGTAGATGG GGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAGGATTATTCTCTCACCATC AGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGT TTCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAACGGCTCGAGAAGC CCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGC AGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGC ACACGAGGGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGGTGCTGGTGGTGG TTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC TGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACT CCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGC GACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGAC ACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA Anti-ROR1CAR(mouse)aminoacidsequence(SEQIDNO:4): MALPVTALLLPLALLLHAARPASVKLVESGGGLVKPGGSLKLSCAASGFT FSTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSL RSEDTAMYYCARDSYYFGNSVYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSD IKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVP SRESGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKRLEKPTTTPAP RPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSL LVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0068] We also generated a CAR with the 4-1BB co-stimulatory domain in place of the CD28 activation domain. This construct PMC1195 was cloned in a vector with the KanR gene. The ROR1 scFv was inserted between Nhe I and Xho I sites in the sequence (underlined). The CAR expression was under the control of the MNDU3 promoter.

[0069] The nucleotide sequence of the codon-optimized CAR (anti-ROR1 scFv-4-1BB-CD3 zeta) is shown below. The scFv is inserted between Nhe I and Xho I sites (underlined). 4-1BB is in italics followed by the CD3-zeta domain.

TABLE-US-00004 Anti-ROR1CAR(4-1BBinplaceofCD28activationdomain) nucleotidesequence(SEQIDNO:31): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC ACGCCGCCAGGCCGGCTAGCGAAGTGAAGCTTGTCGAATCCGGCGGTGGATTGG TTAAACCAGGCGGAAGTTTGAAACTGAGTTGTGCTGCTTCTGGTTTTACCTTTAG CACATACGCTATGTCCTGGGTTAGGCAGACGCCGGAGAAACGATTGGAGTGGTA GCATCTATTTCTTCTGGCGGCAATACTTATTACCCTGACAGCGTGAAAGGCCGGT TCACTATTTCTCGAGATAATGCGCGGCACATACTCTATCTCCAGATGTCTTCTCTC CGCTCAGAGGATACAGCGATGTACTATTGTGCAAGGGATAGTTACTATTTCGGA AACTCTGTGTATTATGCAATGGATTACTGGGGTCAGGGAACTTCAGTCACAGTAA GCTCAGGTGGGGGAGGAAGCGGCGGTGGCGGCTCAGGGGGAGGTGGATCTGAT ATTAAAATGACTCAGTCTCCATCAAGCATGTACGCCTCTCTGGGAGAGCGAGTTA CTATTACCTGTAAAGCATCACAAGATATTAACTCTTATTTTAGTTGGTTTCAACA AAAGCCTGGAAAATCACCTAAAACTTTGATTTATAGAGCCAATAGGCTTGTGGA TGGTGTACCTAGTCGGTTTAGCGGCTCAGGGTCAGGCCAAGACTATTCTTTGACC ATCTCTTCTCTGGAGTATGAGGACATGGGAATCTATTACTGTCTTCAGTACGATG AGTTCCCCTATACGTTTGGTGGAGGCACTAAATTGGAGATTAAACTCGAGAAGC CCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGC AGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGC ACACGAGGGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGGTGCTGGTGGTGG TTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC TGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGA AGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA CCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGC CGCAGAGAAGGAAGAACCCTCAGGAAGGCCTCTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTA CGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

B. 4-1BB as a Co-Stimulating Domain

[0070] We also constructed a CAR with the structure (human CD8 signaling peptide-alternative (see below) anti-ROR1 scFv (V.sub.H-Linker (G.sub.4S).sub.3-V.sub.L ((G.sub.4S).sub.3 disclosed as SEQ ID NO: 46)), CD8 hinge, CD28 transmembrane domain, 4-1BB co-stimulatory domain, CD3 zeta activation domain). In the alternative scFv, each segment of the sequence is the same as that in Example 3 (A) except V.sub.H, V.sub.L. V.sub.H is represented by SEQ ID NO: 5 (the first amino acid was E, not present in SEQ ID NO: 2). V.sub.L is represented by SEQ ID NO: 6 (the terminal R is removed compared to SEQ ID NO: 3.

TABLE-US-00005 Alternativeanti-ROR1scFvaminoacidsequence (SEQIDNO:23): EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVAS ISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSY YFGNSVYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMY ASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRF SGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK Anti-ROR1CARalternativeV.sub.Haminoacidsequence (SEQIDNO:5): EVKLVESGGGLVKPGGSLKLSCAASGFTESTYAMSWVRQTPEKRLEWVAS ISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSY YFGNSVYYAMDYWGQGTSVTVSS

[0071] Compared with SEQ ID NO: 2, SEQ ID NO: 5 has an extra E on the N-terminal end.

TABLE-US-00006 Anti-ROR1CARalternativeV.sub.Lacidsequence (SEQIDNO:6): DIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYR ANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGG GTKLEIK

[0072] Compared with SEQ ID NO: 3, SEQ ID NO: 6 is lacking the R at the C-terminal end.

TABLE-US-00007 4-1BBdomainnucleotidesequence:(SEQIDNO:32): AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAG ACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAG AAGAAGAAGAAGGAGGATGTGAACTG 4-1BBaminoacidsequence(SEQIDNO:33): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Anti-ROR1CAR(alternativescFvand4-1BB)amino acidsequence(SEQIDNO:7): (V.sub.Hisunderlined,thelinker(G.sub.4S).sub.3(SEQID NO:46)isinitalics,V.sub.Lisunderlined;4-1BB domainisinunderlineditalics.) MALPVTALLLPLALLLHAARPASEVKLVESGGGLVKPGGSLKLSCAASGF TESTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHI LYLQMSSLRSEDTAMYYCARDSYYFGNSVYYAMDYWGQGTSVTVSSGGGG SGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQK PGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCL QYDEFPYTFGGGTKLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASR PAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R.

Example 4. ROR1 CAR with Humanized ROR1 scFv

[0073] In this example we humanized the mouse anti-ROR1 V.sub.H (SEQ ID NO: 5) and mouse V.sub.L (SEQ ID NO: 6) and generated several humanized scFv. We generated CARs with the three scFvs having the same structure as Example 3 (B) with the 4-1BB domain and the CD3 zeta domain. We tested several humanized scFv and selected three scFv based on best performance in functional assays shown below. The humanized scFv were inserted between Nhe I and Xho I sites in CAR sequence.

[0074] The three CARs with humanized anti-ROR1 scFv: PMC857, PMC858 and PMC862 are shown below.

TABLE-US-00008 A.PMC857scFvandCAR Humanizedanti-ROR1scFvPMC857nucleotidesequence:(SEQIDNO:34): GAAGTACAGCTTGTTGAATCAGGTGGTGGTCTTATTCAGCCAGGA GGCTCCTTGCGACTGAGCTGTGCCGCTTCTGGGTTCACCTTTAGCACTTAC GCAATGAGTTGGGTCCGACAAGCCCCAGGTAAGGGATTGGAATGGGTA AGTTCCATTTCCAGCGGAGGGAACACTTATTACGCCGATTCTGTGAAA GGACGCTTTACTATATCCCGAGACAATAGTAAAAACACATTGTATTTG CAAATGAACTCTTTGAGGGCCGAGGACACTGCCGTCTACTATTGTGCC CGCGACAGCTATTATTTCGGCAACTCTGTGTATTACGCGATGGATTAC TGGGGTGCCGGCACAACTGTCACCGTTTCATCTGGCGGAGGAGGCAGT GGCGGAGGGGGCTCAGGCGGTGGTGGAAGTGATATTCAAATGACCCAA TCACCCTCTTCATTGTCTGCAAGCGTAGGTGACCGAGTCACGATAACC TGCAAAGCCTCTCAAGATATTAATTCATACTTTTCTTGGTTTCAACAAAAA CCGGGAAAGGCGCCTAAGTCATTGATTTACCGCGCGAACCGGTTGGTA TCAGGAGTACCGTCAAGATTCTCAGGGAGTGGGTCAGGCACAGATTTC ACACTCACTATTTCTTCCTTGCAACCTGAAGACTTCGCAACCTATTATTGC TTGCAGTATGATGAGTTTCCGTACACTTTCGGGGGGGGTACAAGGCTG GAGATCAAA Humanizedanti-ROR1scFvPMC857aminoacidsequence:(SEQIDNO:8): EVQLVESGGGLIQPGGSLRLSCAASGFTESTYAMSWVRQAPGKGLEWVSSISS GGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYY AMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAS QDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCLQYDEFPYTFGGGTRLEIK Humanizedanti-ROR1scFvPMC857V.sub.Haminoacidsequence(SEQIDNO:9): EVQLVESGGGLIQPGGSLRLSCAASGFTESTYAMSWVRQAPGKGLEWVSSISS GGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYY AMDYWGAGTTVTV Humanizedanti-ROR1scFv(PMC857)V.sub.Laminoacidsequence(SEQIDNO:10): DIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRL VSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK Humanizedanti-ROR1CARscFvPMC857nucleotidesequence:(SEQIDNO:35): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACG CCGCCAGGCCGGCTAGCGAAGTACAGCTTGTTGAATCAGGTGGTGGTCTT ATTCAGCCAGGAGGCTCCTTGCGACTGAGCTGTGCCGCTTCTGGGTTC ACCTTTAGCACTTACGCAATGAGTTGGGTCCGACAAGCCCCAGGTAAG GGATTGGAATGGGTAAGTTCCATTTCCAGCGGAGGGAACACTTATTAC GCCGATTCTGTGAAAGGACGCTTTACTATATCCCGAGACAATAGTAAA AACACATTGTATTTGCAAATGAACTCTTTGAGGGCCGAGGACACTGCC GTCTACTATTGTGCCCGCGACAGCTATTATTTCGGCAACTCTGTGTATTAC GCGATGGATTACTGGGGTGCCGGCACAACTGTCACCGTTTCATCTGGC GGAGGAGGCAGTGGCGGAGGGGGCTCAGGCGGTGGTGGAAGTGATATT CAAATGACCCAATCACCCTCTTCATTGTCTGCAAGCGTAGGTGACCGA GTCACGATAACCTGCAAAGCCTCTCAAGATATTAATTCATACTTTTCTTGG TTTCAACAAAAACCGGGAAAGGCGCCTAAGTCATTGATTTACCGCGCG AACCGGTTGGTATCAGGAGTACCGTCAAGATTCTCAGGGAGTGGGTCA GGCACAGATTTCACACTCACTATTTCTTCCTTGCAACCTGAAGACTTCGCA ACCTATTATTGCTTGCAGTATGATGAGTTTCCG TACACTTTCGGGGGGGGTACAAGGCTGGAGATCAAACTCGAGAAGCCCACCACG ACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG TCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAG GGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAA ACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGT ACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGA AGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGT ACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAG GAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAA GCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTCTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA Humanizedanti-ROR1CAR(scFvPMC857)aminoacidsequence:(SEQIDNO:11): MALPVTALLLPLALLLHAARPASEVQLVESGGGLIQPGGSLRLSCAASGFTEST YAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIKLEKPTTTPAPRP PTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLV TVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR B.PMC858scFvandCAR Humanizedanti-ROR1scFvPMC858nucleotidesequence(SEQIDNO:36): CAGGTACAATTGGTAGAGTCCGGCGGAGGGGTTGTTCAGCCAGGA CGGTCCTTGCGGTTGTCTTGTGCTGCGTCAGGATTCACATTCTCAACGTA CGCGATGTCTTGGGTGCGCCAAGCTCCCGGTAAAGGGCTGGAATGGGT GGCCTCAATCTCATCTGGAGGGAACACTTACTACCCTGATAGTGTTAAA GGTCGCTTTACTATCTCAAGGGACAATAGCAAGAATACCTTGTATCTGC AAATGAACTCACTTAGAGCAGAGGACACAGCGGTATATTACTGTGCTA GAGACTCATATTATTTCGGCAACTCCGTTTATTACGCGATGGATTACTG GGGCGCAGGGACTACGGTAACTGTATCTTCTGGTGGTGGAGGGTCTGG GGGCGGGGGTAGTGGCGGCGGTGGCAGTGACATCCAGATGACACAGTC TCCGTCTTCATTGAGTGCAAGCGTCGGCGATCGGGTTACCATTACGTGT AAGGCAAGTCAGGACATCAACAGTTATTTTTCATGGTTTCAACAAAAGC CTGGAAAAGCGCCGAAATCACTCATTTACCGAGCTAATAGGCTTGTCTC TGGCGTTCCGTCTCGCTTCAGTGGAAGTGGGAGCGGTACTGATTTTACC CTCACCATATCAAGCCTTCAACCGGAGGATTTTGCCACGTACTATTGTC TCCAGTACGATGAATTTCCATATACGTTTGGCGGCGGGACTCGCTTGGA GATTAAA Humanizedanti-ROR1scFvPMC858aminoacidsequence(SEQIDNO:12): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIS SGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYY AMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAS QDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCLQYDEFPYTFGGGTRLEIK Humanizedanti-ROR1scFvPMC858V.sub.Haminoacidsequence:(SEQIDNO:13): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIS SGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYY AMDYWGAGTTVTVSS Humanizedanti-ROR1scFvPMC858V.sub.Laminoacidsequence(SEQIDNO:14): DIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRL VSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK Humanizedanti-ROR1CAR(scFvPMC858)nucleotidesequence(SEQIDNO:37): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACG CCGCCAGGCCGGCTAGCCAGGTACAATTGGTAGAGTCCGGCGGAGGGGTTGTTC AGCCAGGACGGTCCTTGCGGTTGTCTTGTGCTGCGTCAGGATTCACATTCTCAAC GTACGCGATGTCTTGGGTGCGCCAAGCTCCCGGTAAAGGGCTGGAATGGGTGGC CTCAATCTCATCTGGAGGGAACACTTACTACCCTGATAGTGTTAAAGGTCGCTTT ACTATCTCAAGGGACAATAGCAAGAATACCTTGTATCTGCAAATGAACTCACTTA GAGCAGAGGACACAGCGGTATATTACTGTGCTAGAGACTCATATTATTTCGGCA ACTCCGTTTATTACGCGATGGATTACTGGGGCGCAGGGACTACGGTAACTGTATC TTCTGGTGGTGGAGGGTCTGGGGGCGGGGGTAGTGGCGGCGGTGGCAGTGACAT CCAGATGACACAGTCTCCGTCTTCATTGAGTGCAAGCGTCGGCGATCGGGTTACC ATTACGTGTAAGGCAAGTCAGGACATCAACAGTTATTTTTCATGGTTTCAACAAA AGCCTGGAAAAGCGCCGAAATCACTCATTTACCGAGCTAATAGGCTTGTCTCTGG CGTTCCGTCTCGCTTCAGTGGAAGTGGGAGCGGTACTGATTTTACCCTCACCATA TCAAGCCTTCAACCGGAGGATTTTGCCACGTACTATTGTCTCCAGTACGATGAAT TTCCATATACGTTTGGCGGCGGGACTCGCTTGGAGATTAAACTCGAGAAGCCCAC CACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC CCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACAC GAGGGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT GAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACC AGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGA AGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCG CGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGA AAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTCTACAATGAACTGCAGAA AGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACA CCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA Humanizedanti-ROR1CAR(scFvPMC858)aminoacidsequence(SEQIDNO:15): MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFS TYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIKLEKPTTTPAPRP PTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLV TVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR C.PMC862scFvandCAR Humanizedanti-ROR1scFvPMC862nucleotidesequence(SEQIDNO:38): CAGGTACAACTGGTGGAATCCGGCGGGGGAGTAGTACAGCCCGGA CGATCTCTTCGACTCTCATGTGCAGCGTCCGGGTTCACTTTTTCTACCTAC GCAATGTCATGGGTACGACAGGCGCCGGGCAAAGGCCTCGAATGGGTT GCATCCATTTCATCAGGAGGTAATACATATTATCCTGATTCAGTCAAG GGCCGATTCACGATTAGTCGAGATAATAGCAAGAACACTCTCTACTTG CAGATGAACTCCCTGCGGGCTGAGGACACGGCCGTGTATTATTGCGCT CGCGATAGTTATTACTTCGGCAATTCCGTATATTATGCGATGGACTAT TGGGGCGCCGGTACTACCGTGACTGTTTCCTCTGGTGGGGGTGGGTCC GGGGGCGGTGGTTCAGGTGGAGGCGGATCCGACATTCAAATGACCCAG TCTCCCTCAAGTTTGTCTGCATCTGTTGGCGATAGAGTTACAATAACATGC AAAGCCAGTCAAGACATCAACTCATACTTCTCCTGGTATCAACAAAAG CCAGGAAAAGCTCCGAAACTGTTGATCTACCGGGCCAACCGGCTGGTC ACTGGCGTGCCATCCCGGTTCAGTGGCAGCGGAAGCGGAACAGATTTC ACGTTTACCATCTCTAGCCTCCAACCGGAGGACATCGCAACATACTAT TGCCTTCAGTATGATGAGTTTCCCTACACTTTCGGTGGCGGCACCCGACTT GAGATCAAA Humanizedanti-ROR1scFvPMC862aminoacidsequence(SEQIDNO:16): QVQLVESGGGVVQPGRSLRLSCAASGFTESTYAMSWVRQAPGKGLEWVASIS SGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYY AMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAS QDINSYFSWYQQKPGKAPKLLIYRANRLVTGVPSRFSGSGSGTDFTFTISSLQPEDIAT YYCLQYDEFPYTFGGGTRLEIK Humanizedanti-ROR1scFvPMC862V.sub.Haminoacidsequence(SEQIDNO:17): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIS SGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYY AMDYWGAGTTVTVSS Humanizedanti-ROR1scFvPMC862V.sub.Laminoacidsequence(SEQIDNO:18): DIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRL VTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTRLEIK Humanizedanti-ROR1CAR(scFvPMC862)nucleotidesequence(SEQIDNO:39): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACG CCGCCAGGCCGGCTAGCCAGGTACAACTGGTGGAATCCGGCGGGGGAGTAGTAC AGCCCGGACGATCTCTTCGACTCTCATGTGCAGCGTCCGGGTTCACTTTTTCTAC CTACGCAATGTCATGGGTACGACAGGCGCCGGGCAAAGGCCTCGAATGGGTTGC ATCCATTTCATCAGGAGGTAATACATATTATCCTGATTCAGTCAAGGGCCGATTC ACGATTAGTCGAGATAATAGCAAGAACACTCTCTACTTGCAGATGAACTCCCTGC GGGCTGAGGACACGGCCGTGTATTATTGCGCTCGCGATAGTTATTACTTCGGCAA TTCCGTATATTATGCGATGGACTATTGGGGCGCCGGTACTACCGTGACTGTTTCC TCTGGTGGGGGGGGTCCGGGGGCGGTGGTTCAGGTGGAGGCGGATCCGACATT CAAATGACCCAGTCTCCCTCAAGTTTGTCTGCATCTGTTGGCGATAGAGTTACAA TAACATGCAAAGCCAGTCAAGACATCAACTCATACTTCTCCTGGTATCAACAAA AGCCAGGAAAAGCTCCGAAACTGTTGATCTACCGGGCCAACCGGCTGGTCACTG GCGTGCCATCCCGGTTCAGTGGCAGCGGAAGCGGAACAGATTTCACGTTTACCA TCTCTAGCCTCCAACCGGAGGACATCGCAACATACTATTGCCTTCAGTATGATGA GTTTCCCTACACTTTCGGTGGCGGCACCCGACTTGAGATCAAACTCGAGAAGCCC ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGGTGCTGGTGGTGGTTG GTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG GTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAA GAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCC CGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAG AGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTCTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCG GAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGA CACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA Humanizedanti-ROR1CAR(scFvPMC862)aminoacidsequence(SEQIDNO:19): MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFS TYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLVTGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTRLEIKLEKPTTTPAPRP PTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLV TVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 5. Producing the CAR Using Lentiviral Vectors

[0075] In this example, CARs containing the three humanized scFvs of Example 4 were packaged into lentiviral vectors. The lentiviruses were produced by the standard procedure using HEK293 cells as described in Goluboskaya et al., (2016) Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy. Cancers (Basel). 2016 Mar. 15; 8 (3). pii: E36.

Example 6. Peripheral Blood Mononuclear Cell (PBMC) Isolation from Whole Blood

[0076] In this example PBMCs were isolated from whole blood for the purpose of producing CAR-T cells. Whole blood (Stanford Hospital Blood Center, Stanford, Cal.) was collected from individuals or mixed-donor samples (depending on the amount of blood required) in 10 mL heparin vacutainers (Becton Dickinson, San Jose, Cal.). Approximately 10 ml of whole anti-coagulation-treated blood was mixed with sterile phosphate buffered saline (PBS pH 7.4, Ca.sup.2+ and Mg.sup.2+-free) for a total volume of 20 ml in a 50 ml conical centrifuge tube. The layer of cells containing PBMCs seen at the diluted plasma/Ficoll interface was removed very carefully, avoiding any Ficoll, washed twice with PBS, and centrifuged at 200g for 10 min at room temperature. Cells were counted with a hemocytometer. The PBMCs were washed once with CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies, San Diego, Cal.) with 5% AB serum and 1.25 ug/mL amphotericin B (Gemini Bioproducts, Woodland, Cal.), 100 U/mL penicillin, and 100 g/mL streptomycin and used for experiments or frozen at 80 C.

Example 7. T-Cell Activation from PBMC

[0077] Isolated PBMC were washed with once 1PBS (pH7.4, no Ca.sup.2+/Mg.sup.2+), and in CAR-T medium (Example 6), in the absence of human interleukin-2 (huIL2) at a concentration of 510.sup.5 cells/mL, then resuspended to a final concentration of 510.sup.5 cells/mL in CAR-T medium with 300 U/mL huIL2 (from a 1000 stock; Invitrogen, Carlsbad, Cal.). PBMC and beads (for T cell activation) were then mixed at a 1:1 bead-to-cell ratio, by transferring 25 L of beads to 1 mL of PBMC and incubated at 37 C. in the presence of CO.sub.2 for 24 hr before viral transduction.

Example 8. T-Cell Transduction and Expansion

[0078] Following activation of PBMC, 510.sup.6 lentiviruses were added to 510.sup.5T cells (MOI 10:1), and 2 L/mL of media of Transplus (Alstem, Richmond, Cal.) to final dilution of 1:500. The cells were incubated for an additional 24 hours before repeating the addition of virus. The cells were then grown in the presence of 300 U/mL of IL-2 for a period of 12-14 days (total incubation time was dependent on the final umber of CAR-T cells required). Cell numbers were analyzed every 2-3 days, with media being added at that time to dilute the cell suspension to 110.sup.6 cells/ml.

Example 9. Transduction of T Cells and CAR Verification by FACS

[0079] The cells from Example 8 were washed and suspended in FACS buffer (PBS plus 0.1% sodium azide and 0.4% BSA). Cells were then divided into 110.sup.6 cell aliquots. Fc receptors were blocked with normal goat IgG (Life Technologies, San Diego, Cal.). Biotin-labeled polyclonal goat anti-mouse F(ab).sub.2 antibodies were used to detect mouse anti-ROR1 scFv; biotin-labeled normal polyclonal goat IgG antibodies also served as an isotype control. The cells were incubated at 4 C. for 25 minutes and washed once with FACS buffer. After staining the cells with anti-F(ab).sub.2 antibody, phycoerythrin (PE)-labeled streptavidin (BD Pharmingen, San Diego, Cal.) and allophycocyanin (APC)-labeled CD3 (eBiocience, San Diego, Cal.) were used to stain the cells. For humanized anti-ROR1 scFv we also used anti-human F(ab).sub.2 antibodies (Life Technologies).

Example 10. Real-Time Cytotoxicity Assay

[0080] The cytotoxicity was performed using xCELLigence real-time cell analysis system (Agilent, San Jose, Cal.), according to the manufacturer's protocol as described in Berahovich et al., (2018) CAR-T cells based on Novel BCMA monoclonal antibody block multiple myeloma Cell growth. Cancers (Basel) (9).

Example 11. CAR-T Cells with Mouse Anti-ROR1 scFv Expressed High Cytotoxic Activity Against ROR1-Positive Cancer Cells

[0081] Expression of mouse-scFv anti-ROR1 CAR was confirmed by FACS with anti-mouse Fab antibodies. The CAR-T cells with the CAR containing the mouse anti-ROR1 scfv, CD28 costimulatory domain and CD3 zeta activation domain (see Example 3 (A)) were used in this cytotoxicity assay. The cytotoxicity assay was performed using RTCA impedance-based assay on the xCELLigence system according to manufacturer's conditions. In this assay, the integrity of the target cell monolayer is continually monitored via its impedance in a weak electrical field. Killing of the target cells by the CAR-T cells decreases the monolayer's integrity and, therefore, its impedance. The anti-ROR1 CAR-transduced T cells were added to the target cells at effector: target (E: T) ratios of 10:1, 20:1, 30:1, and 40:1 (FIG. 5, panel A). The CAR-T cells caused a sustained dose-dependent decrease in target cell monolayer impedance. Thus, anti-ROR1-CD28-CD3 CAR-T cells killed the ROR1 positive SKOV-3 ovarian solid tumor cells in a dose-dependent manner.

[0082] Similar high cytotoxic activity was observed also with CAR-T cells with the CAR containing the mouse anti-ROR1 scfv, 4-1BB costimulatory domain and CD3 zeta activation (Example 3 (B)) and ROR1-positive SKOV-3 target cells (FIG. 5, panel B).

Example 12. Anti-ROR1-CAR T Cells (Mouse scFv) Secrete High Level of IFN-Gamma in the Presence of ROR1-Positive Cancer Cells

[0083] After co-incubation of ROR1-41BB-CD3-CAR-T cells with SKOV-3 cells, we collected the supernatant and performed ELISA with a commercial kit (ThermoFisher Scientific, Waltham, Mass.). As a control we used non-adherent HL-60 ROR1-negative cell line. The anti-ROR1-CAR-T cells secreted significantly higher level of IFN-gamma in the presence of ROR1-positive SKOV-3 cancer cells than in the presence of ROR1-negative control cells, and in comparison to T cells and mock CAR-T cells used as control (P<0.05). (FIG. 6).

Example 13. Anti-ROR1-CAR T Cells (Humanized scFv) Exhibited Cytotoxicity and Secreted High Level of IFN-Gamma in the Presence of ROR1-Positive Cancer Cells

[0084] First we tested CAR-T cells with scFvs PMC857, PMC868, or PMC862 (Example 5) in a cytotoxic assay with ROR1-positive cells and showed that the CAR-T cells were highly cytotoxic. Next, we inserted these CAR constructs (PMC857, PMC868, or PMC862) into lentiviral vectors with KanR gene (preferred for clinical usage) instead of AmpR gene. The CAR-T cell clones became clones PMC1182, 1183 and 1194, respectively. The CAR expression in CAR-T cells was about 30% CAR+ as detected by FACS with human Fab. We performed RTCA assay and detected high cytotoxic activity of these CAR-T cells against SKOV-3 (ROR1-positive) cells (FIG. 7).

[0085] Next we assessed cytokine secretion by the CAR-T cells. After co-incubation of the CAR-T cells with SKOV-3 cells, we collected the culture supernatant and performed ELISA to detect Interferon-Gamma in the supernatant as described in Example 12 using the non-adherent HL-60 ROR1-negative cell line as a control. The anti-ROR1-CAR-T cells secreted significantly higher level of IFN-gamma in the presence of ROR1-positive SKOV-3 cancer cells than in the presence of ROR1-negative control cells, and in comparison to T cells and mock CAR-T cells used as control (P<0.05). (FIG. 8).

[0086] While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.