ROR-1 antibody and ROR-1-car-t cells

Abstract

The present invention is directed to a monoclonal mouse or humanized ROR-1 antibody, or an antigen-binding fragment thereof, including a single-chain variable fragment (scFv). The present invention is also directed to a mouse or humanized ROR-1 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.

Description

[0001] ROR-1 ANTIBODY AND ROR-1-CAR-T CELLS FIELD OF THE INVENTION The present invention relates to mouse and humanized ROR-1-specific monoclonal antibody and scFv, and ROR-1-CAR-T cells, which are useful in the field of adoptive immunity gene therapy for tumors. BACKGROUND OF THE INVENTION Immunotherapy is emerging as a highly promising approach for the treatment of cancer. T cells or T lymphocytes, the armed forces of our immune system, constantly look for foreign antigens and discriminate abnormal (cancer or infected cells) from normal cells. Genetically modifying T cells with CAR (Chimeric antigen receptor) constructs is the most common approach to design tumor-specific T cells. CAR-T cells targeting tumor-associated antigens (TAA) can be infused into patients (called adoptive cell transfer or ACT) representing an efficient immunotherapy approach [1, 2]. The advantage of CAR-T technology compared with chemotherapy or antibody is that reprogrammed engineered T cells can proliferate and persist in the patient (“a living drug”) [3], [4], [1]. CARs usually consist of a monoclonal antibody-derived single-chain variable fragment (scFv) at the N-terminal part, hinge, transmembrane domain and a number of intracellular co- stimulatory domains: (i) CD28, (ii) CD137 (4-1BB), CD27 or other, in tandem with an activation CD3-zeta domain. [2, 3,4]. The evolution of CARs went from first generation (with no costimulatory domains) to second generation (with one co-stimulation domain) to third generation CAR (with several costimulatory domains). Generating CARs with multiple costimulatory domains (the so-called 3rdgeneration CAR) has led to increased cytolytic CAR-T cell activity, improved persistence of CAR-T cells leading to its augmented antitumor activity. Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. The role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells, acting at around 3 days after infection, and responding to tumor formation.

[0002] 1 170560056.1 ROR-1 antigen. Tyrosine-protein kinase transmembrane receptor ROR1, also known as neurotrophic tyrosine kinase, receptor-related 1 (NTRKR1), is an enzyme that in humans is encoded by the ROR1 gene. ROR1 is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. ROR-1 is a 937 amino-acid protein with 30-406 aa extracellular domain. ROR-1 has low expression in most tissues which is advantageous to use it as a target for CAR-T cell therapy (FIG. 1). The ROR-1 gene encodes a receptor tyrosine kinase-like orphan receptor that modulates neurite growth in the central nervous system. The encoded protein is a glycosylated type I membrane protein that belongs to the ROR subfamily of cell surface receptors. ROR-1 is receptor for ligand WNT5A which activates downstream NF kappa B signaling pathway and may result in the inhibition of WNT-mediated signaling. In addition, ROR1 has recently been shown to be expressed on ovarian cancer stem cells and promote migration, invasion, and cancer stem cell spheroid formation. ROR-1 is shown to be overexpressed in both hematological cancers and solid tumors that makes this target useful for CAR-T therapy. Low expression of ROR-1 has been shown in most of normal human tissues such as adipose and soft tissue, bone marrow and immune system, endocrine tissues, female tissue, gastrointestinal tract, kidney and urinary bladder, liver and gallbladder, lung, muscle tissues, male tissues, and skin. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the structure of ROR-1 CAR construct. The second -generation CAR was used with 41BB co-stimulatory domain and CD3 zeta activation domain. Abbreviations: ScFv, single chain variable fragment; h-hinge; TM-transmembrane; 4-1BB co-stimulatory, and CD3 zeta activation domains. FIG. 2 shows that the binding of ROR-1 antibody with human ROR-1 antigen in Lovo cells, but not in Lovo KO by CRISPR / Cas9 cells by FACS. Lovo ROR-1-positive and Lovo CRISPR / Cas-9 KO cell lines were used for assay with ROR18A2A3 antibody. Primary mouse- anti ROR1 antibody was used, and secondary goat-anti mouse-PE antibody was used.

[0003] 2 170560056.1 FIG. 3 shows the expression of ROR1 ScFv-positive CAR-T cells by FACS. FACS was done either with mouse F(ab)2 antibody (left panel) or with recombinant extracellular ROR1 domain biotinylated protein (right panel). FIGs.4A and 4B show that RTCA assay demonstrated killing activity of mouse ROR-1-4- 1BB-CD3 CAR-T cells with ROR1-positive CHO-ROR1 cell line in vitro. The cytotoxicity assay was performed using ROR1-4-1BB -CD3 CAR-T cells with ACEA XCelligence system at Effector to Target cell ratio 10:1 using target CHO-ROR1 and CHO cells. FIGs. 5A-5C show that ROR1-CAR-T cells killed ROR-1-positive cancer cell lines. Pancreatic BXPC3 (A); colorectal cancer Lovo (B); gastrointestinal (GI) OVCAR-5 (C) target cell lines are shown in RTCA assay with ROR1-CAR-T cells. FIG. 6 shows IFN-gamma secretion by ROR-1-41BB-CD3 CAR-T against ROR-1- positive target cells. CHO-ROR-1 positive and control CHO target cell lines were used for IFN- gamma ELISA assay. p<0.05, Student’s t-test, ROR1-CAR-T cells against CHO-ROR1 positive target cells compared against CHO target cells. FIGs. 7A-7C show IFN-gamma secereted by ROR-1 CAR-T cells against ROR-1 positive cancer target BXPC-3 (7A), Lovo (7B) and OVCAR-5 (7C) cancer cell lines. *p<0.05, Student’s t-test, ROR1-CAR-T cells against ROR1 positive target cells versus T cells against ROR1 positive target cells. FIG. 8A shows ROR1-CAR-T cells significantly decrease OVCAR-5 xenograft tumor growth in vivo. p<0.05 ROR-1 CAR-T cells versus T cells, Student’s t-test. FIG. 8B shows no significant change in mouse body weight by ROR-1 CAR-T cells. FIG. 9 shows the expression of humanized CAR tested using FACS. ROR1 CAR-T cells show binding with ROR1 antigen by FACS. FACS was performed with anti-mouse F(ab)2, anti- human F(ab)2antibodies and with recombinant biotynylated ROR1 extracellular domain protein. FIGs. 10A-10C show that human PMC2509 CAR-T cells killed several cancer cell lines by RTCA assay. 10A RTCA was performed with OVCAR-5 target cells at E:T (Effector to target cell ration)=10:1. 10B. RTCA was performed with PC3 target cells at E:T (Effector to target cell ration)=20:1. 10C. RTCA was performed with CHO-ROR1 (bottom panel) and CHO cells (top panel). E:T=10:1. FIGs. 11A-11C show secretion of IFN-gamma by hROR1-CAR-T cells with different target cells. A. OVCAR-5 target cells; B-PC3 target cells; A, B, *p<0.05 CAR-T cells versus T

[0004] 3 170560056.1 cells, Student’s t-test. C. CHO and CHO-ROR1 target cells. *p<0.05 CAR-T cells with CHO- ROR1 target cells versus CAR-T cells with CHO target cells, Student’s two-tailed t-test. FIG. 12A shows in vivo efficacy of humanized ROR-1-CAR-T cells using OVCAR-5 xenograft model. N=6 tumors per group. Tumor volume, p<0.02 CAR T cells vs. T cells, Student’ t-test. FIG. 12B shows no significant change in mouse Body weight. DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, an “antigen-binding fragment” refers to Fab fragment, Fab′ fragment, F(ab′)2 fragment, and scFv with antigen-binding activity. As used herein, a "chimeric antigen receptor (CAR)" is a receptor protein that has been engineered to give T cells the new ability to target a specific protein. The receptor is chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. CAR is a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. The "chimeric antigen receptor (CAR)" is sometimes called a "chimeric receptor", a "T-body", or a "chimeric immune receptor (CIR)." The extracellular domain "capable of binding to an antigen" means any oligopeptide or polypeptide that can bind to a certain antigen. The "intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell. As used herein, a "domain" means one region in a polypeptide which is folded into a particular structure independently of other regions. As used herein, a "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the scFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for engineering an scFv are known to a person skilled in the art. As used herein, a "tumor antigen" means a biological molecule having antigenicity, expression of which causes cancer.

[0005] 4 170560056.1 The inventors have generated ROR-1 monoclonal antibody using hybridoma technology specifically targeting human ROR-1 antigen. The monoclonal anti-human ROR-1 antibody is generated against the extracellular region of the purified recombinant fragment of human ROR- 1. In one aspect, the present invention is directed to a monoclonal mouse anti-human ROR- 1 antibody or an antigen-binding fragment thereof, comprising (i) VH comprising VH-CDR1 having the amino acid sequence of SEQ ID NO: 4, VH-CDR2 having the amino acid sequence of SEQ ID NO: 5, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 6, and (ii) VL having the amino acid sequence of SEQ ID NO: 8. In one embodiment, the monoclonal mouse anti-human ROR-1 antibody or an antigen- binding fragment thereof, comprising VH having the amino acid sequence of SEQ ID NO: 3 and VL having the amino acid sequence of SEQ ID NO: 8. In one embodiment, the monoclonal anti-human ROR-1 antibody is a single-chain variable fragment (scFv). ScFv can be VH-linker-VL or VL-linker-VH. In another aspect, the present invention is directed to a humanized anti-human ROR-1 antibody or an antigen-binding fragment thereof, by using the CDRs of VH and VL of the mouse anti-human ROR-1 antibody and grafting with human frames. In some cases, an amino acid on the border of CDR and frame may be changed to another amino acid. The humanized anti- human ROR-1 antibody or an antigen-binding fragment thereof comprising (i) VH comprising VH-CDR1 having the amino acid sequence of SEQ ID NO: 4, VH-CDR2 having the amino acid sequence of SEQ ID NO: 5, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 6, and (ii) VL having the amino acid sequence of SEQ ID NO: 11. In one embodiment, the humanized anti-human ROR-1 antibody or an antigen-binding fragment thereof, comprising humanized VHhaving the amino acid sequence of SEQ ID NO: 10 and humanized VL having the amino acid sequence of SEQ ID NO: 11. In one embodiment, the humanized anti-human ROR-1 antibody is a single-chain variable fragment (scFv). ScFv can be VH-linker-VL or VL-linker-VH. The present invention is also directed to a chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) against ROR-1, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an

[0006] 5 170560056.1 activating domain. One embodiment of the CAR is shown in FIG. 1. The CAR-T cells targeting ROR1-positive target cells. In one embodiment, the co-stimulatory domain is selected from the group consisting of CD28, 4-1BB (CD137), GITR, ICOS-1, CD27, OX-40 and DAP10. A preferred co-stimulatory domain is CD28. A preferred activating domain is CD3 zeta (CD3 Z or CD3ζ) The transmembrane domain may be derived from a natural polypeptide or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a transmembrane domain of a T cell receptor α or β chain, a CD3 zeta chain, CD28, CD3ε., CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. It is preferable that a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or a polypeptide linker, for example, a linker having a length of 2 to 10 amino acids can be arranged between the transmembrane domain and the intracellular domain. In one embodiment, a linker sequence having a glycine-serine continuous sequence can be used. The present invention provides an isolated nucleic acid encoding the ROR-1 CARs. The nucleic acid encoding the CAR can be prepared by a conventional method. A base sequence encoding an amino acid sequence can be obtained from NCBI RefSeq IDs or accession numbers of GenBank for an amino acid sequence of each domain, and the nucleic acid of the present invention can be prepared using a standard molecular biological and / or chemical procedure. For example, based on the base sequence, a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR). A nucleic acid encoding the CAR of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, a virus vector such as a retrovirus vector, a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector can be used. A virus vector lacking the replicating ability so as not to self-replicate in an infected cell is preferably used.

[0007] 6 170560056.1 For example, when a retrovirus vector is used, a suitable packaging cell based on a LTR sequence, and a packaging signal sequence possessed by the vector can be selected for preparing a retrovirus particle using the packaging cell. Examples of the packaging cell include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. A retrovirus particle can also be prepared using a 293 cell or a 293T cell having high transfection efficiency. Many kinds of retrovirus vectors produced based on retroviruses and packaging cells are widely commercially available from many companies. A CAR-T cell binds to a specific antigen present on a target cell via the CAR, and as a result, the CAR-T cell is activated and target cell killed. The activation of the cell via CAR-T varies depending on the kind of a host cell and an intracellular domain of the CAR. The activation can be confirmed based on, for example, release of a cytokine, improvement of a cell proliferation rate, change in a cell surface molecule, or the like. For example, release of a cytotoxic cytokine (IFN-gamma, Granzyme B, a tumor necrosis factor, etc.) from the activated CAR-T cell causes destruction of a target cell expressing an antigen. In addition, release of a cytokine or change in a cell surface molecule stimulates other immune cells, for example, a B cell, a dendritic cell, a NK cell, and a macrophage. The cell expressing the CAR can be 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. The inventors have produced ROR-1-CAR-T cells to target cancer cells overexpressing ROR-1 tumor antigen. The ROR-1-CAR-T cells of the present invention have high cytotoxic activity against several cancer cell lines and anti-tumor activity in vivo. The inventors have generated mouse and human ROR-1-ScFv-4-1BB -CD3-CAR-T (ROR-1-CAR-T) cells against solid tumor cancer cells overexpressing ROR-1. ROR-1-CAR-T cells express higher cytotoxic activity against ROR-1-positive cancer cells than against non- transduced T cells and Mock-CAR-T cells. The present mouse and human anti-human ROR-1 antibody detects ROR-1 in ROR-1- positive cancer cells. One advantage of humanized ROR-1 scFv CAR-T cells is that humanized scFv potentially has less immune response in humans than mouse scFv CAR-T cells.

[0008] 7 170560056.1 The mouse and humanized ROR-1 antibody can be used for immunotherapy applications: toxin / drug-conjugated antibody, monoclonal therapeutic antibody, bispecific antibody and CAR- T cell immunotherapy. ROR-1-CAR-T cells using the present ROR-1 antibodies are effective to target ROR-1 antigen in ROR-1-positive cell lines. ROR-1-CAR-T can be used in combination with different therapeutic agents: checkpoint inhibitors; targeted therapies, small molecule inhibitors, antibodies. ROR-1 antibody can be modified with site-directed mutagenesis or by affinity maturation with error-prone PCR for affinity tuning. ROR-1-CAR-T cells can be used clinically against ROR-1-positive cells. Modifications of co-activation domains: CD28, 4-1BB and others can be used to increase its efficacy. Tag-conjugated ROR-1 scFv can be used for CAR generation. Third generation CAR-T or other co-activation signaling domains can be used with the same ROR-1-scFv inside CAR. Combination of ROR-1 with other CAR targeting other tumor antigens or tumor microenvironment (VEGFR-1-3), PDL-1, CD80, or bi-scFv-CAR can be used to enhance activity of monotherapy ROR-1-CAR. Bi-specific antibodies with ROR-1 and CD3 or other antigens can be generated for therapy. ROR-1-CAR-T cells can be used against cancer stem cells that are resistant against chemotherapy and form aggressive tumors. ROR-1-CAR can be used for generating other types of cells such as CAR-natural killer (NK) cells, iPS (Induced pluripotent)-NK or iPS-T cells, ROR-1-CAR-macrophages, and other ROR-1-CAR hematopoietic cells, which can target ROR-1-positive cancers. The present invention provides T cells modified to express the ROR-1-CAR. ROR1-CAR can be used both for generating autologous CAR-positive cells and allogenic hematopoietic cells. 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 limiting.

[0009] 8 170560056.1 EXAMPLES Example 1. Mouse ROR1 antibody detected ROR1 by FACS staining. We generated mouse monoclonal ROR-1 antibody using standard hybridoma technology. The mouse ROR1 monoclonal antibody was tested for ROR-1 by FACS using Lovo ROR1- positive cells and ROR1 knock-out (KO) Lovo cells (FIG. 2). We sequenced this hybridoma clone 8A2A3 and generated ScFv using VH and VL (shown in Example 2A). Example 2A. Sequencing of mouse ROR-1 VH, VL, and scFv We sequenced mouse ROR-1 antibody, clone 8A2A3; the sequences of VH and VL and ScFv are shown below. The structure of ROR-1 scFv is: VH-linker-VL. The first sequence is signaling peptide, then bold shows the nucleotide sequence of VH; the underlined shows the nucleotide sequence of VL; in between (shown in italics font) is the nucleotide sequence encoding the linker. ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC AGGCCGGCTAGCGAAGTTAAGTTGGTAGAATCAGGCGGCGGATTGGTTAAGCCC GGTGGATCACTCAAGCTGAGCTGCGCTGCCTCTGGTTTTACTTTTAGCTCATAC GCTATGAGTTGGGGGCGACAGACTCCTGAAAAACGGTTGGAATGGGTCGCATC CATCAATACAGGTGGCGGCACCTACTACCCAGATTCAGTCAAAGGGCGGTTCA CTATATCACGGGATAACGCCAGAAACATTCTGTACTTGCAAATGAATTCCCTTC GATCTGAGGACACAGCAATGTATTACTGTGCCCGGGAAGGTTACTATTACGGG GGCAGTAGCTATTACGCAATGGATTATTGGGGGCCTGGAACTAGCGTAACTGT AAGTTCTGGCGGCGGTGGCAGCGGAGGTGGTGGGTCCGGCGGCGGCGGCTCAGATA TAAAGATGACTCAGAGCCCAAGCTCCATGTATGTCTCCCTTGGCGAGCGCGTAACAA TAACTTGCAAAGCATCACAGGATATAAATTCTTACCTCTCATGGTTCCAACAGAAGC CTGGCAAGAGTCCAAAGACTTTGATTTATAGGGCTAACAGGTTGGTAGATGGTGTTC CCTCAAGATTCAGCGGATCAGGTAGCGGACAAGATTACTCATTGACAATCAGTAGC CTGGAGTACGAGGATATGGGAATATACTATTGTCTGCAATACGATGAGTTCCCTTAT ACTTTCGGTGGCGGGACCAAGCTTGAAATAAAACGC (SEQ ID NO: 1) Mouse ROR-1 scFv Protein Sequence (VH in bold with CDRs by Kabat system underlined, linker in italics, VL underlined): EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWGRQTPEKRLEWVASINTGG GTYYPDSVKGRFTISRDNARNILYLQMNSLRSEDTAMYYCAREGYYYGGSSYYAM DYWGPGTSVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYVSLGERVTITCKASQDINS YLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQY DEFPYTFGGGTKLEIKR (SEQ ID NO: 2)

[0010] 9 170560056.1 Mouse ROR1 VH (SEQ ID NO: 3) EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWGRQTPEKRLEWVASINTGGGTY YPDSVKGRFTISRDNARNILYLQMNSLRSEDTAMYYCAREGYYYGGSSYYAMDYWGP GTSVTVSS CDR regions by Kabat system: VH CDR1 SYAMS (SEQ ID NO: 4) VH CDR2 SINTGGGTYYPDSVKG (SEQ ID NO: 5) VH CDR3 EGYYYGGSSYYAMDY (SEQ ID NO: 6) Linker GGGGSGGGGSGGGGS (SEQ ID NO: 7) ROR1 VL (CDRs by Kabat system bolded and underlined): DIKMTQSPSSMYVSLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVP SRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKR (SEQ ID NO: 8) VL Example 2B. Sequencing of human ROR-1 VH, VL, AND scFv We used mouse CDR s shown in Example 2A and engraved them into human frame to make human ROR-1 antibody. The structure of human ROR-1 scFv is: VH-linker-VL. We then sequenced the human ROR-1 antibody; the sequences of scFv, VH and VL are shown below. Human ROR-1 scFv (VH in bold, Linker in Italics, VL), CDRs are underlined EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASINTGG GTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGYYYGGSSYYAM DYWGPGTSVTVSS

[0011] 10 170560056.1 GGGGS GGGGS GGGGS DIQMTQSPSSLSASVGDRVTITCKASQDINSYLSWYQQKPGKAPKLLIYRANRLVSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGAGTKLELK (SEQ ID NO: 9) Humanized VH (CDRs are underlined) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASINTGG GTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGYYYGGSSYYAM DYWGPGTSVTVSS (SEQ ID NO: 10) Humanized VL (CDRs are underlined) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLSWYQQKPGKAPKLLIYRANRLVSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGAGTKLELK (SEQ ID NO: 11) Example 3. ROR-1-CAR Sequences with mouse ROR scFv The scheme of mouse ROR-1-CAR construct is shown on FIG. 2. Mouse ROR1 CAR was under MNDU3 promoter. The CAR structure included Human CD8 signaling peptide, ROR-1 scFv (VH-Linker- VL), CD8 hinge, CD28 transmembrane, 4-1BB-co-stimulatory domains, CD3 zeta activation domain (FIG. 2). CD8 leader-ROR-1 scFv (VH-Linker -VL)-CD8 hinge-CD28 TM-4-1BB-CD3-zeta: <CD8 leader > Nucleotide Sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC AGGCCG (SEQ ID NO: 12) Amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 13) <Nhe I restriction site> Nucleotide Sequence GCTAGC Amino acid sequence AS

[0012] 11 170560056.1 <ROR-1 scFV> bold VH, underlined VL, in between linker in italics Nucleotide sequence GAAGTTAAGTTGGTAGAATCAGGCGGCGGATTGGTTAAGCCCGGTGGATCACT CAAGCTGAGCTGCGCTGCCTCTGGTTTTACTTTTAGCTCATACGCTATGAGTTG GGGGCGACAGACTCCTGAAAAACGGTTGGAATGGGTCGCATCCATCAATACAG GTGGCGGCACCTACTACCCAGATTCAGTCAAAGGGCGGTTCACTATATCACGG GATAACGCCAGAAACATTCTGTACTTGCAAATGAATTCCCTTCGATCTGAGGAC ACAGCAATGTATTACTGTGCCCGGGAAGGTTACTATTACGGGGGCAGTAGCTA TTACGCAATGGATTATTGGGGGCCTGGAACTAGCGTAACTGTAAGTTCTGGCGG CGGTGGCAGCGGAGGTGGTGGGTCCGGCGGCGGCGGCTCAGATATAAAGATGACTCA GAGCCCAAGCTCCATGTATGTCTCCCTTGGCGAGCGCGTAACAATAACTTGCAAAGC ATCACAGGATATAAATTCTTACCTCTCATGGTTCCAACAGAAGCCTGGCAAGAGTCC AAAGACTTTGATTTATAGGGCTAACAGGTTGGTAGATGGTGTTCCCTCAAGATTCAG CGGATCAGGTAGCGGACAAGATTACTCATTGACAATCAGTAGCCTGGAGTACGAGG ATATGGGAATATACTATTGTCTGCAATACGATGAGTTCCCTTATACTTTCGGTGGCG GGACCAAGCTTGAAATAAAACGC (SEQ ID NO: 14) Amino acid sequence: See Example 2B <XhoI restriction site> Nucleotide sequence CTCGAG Amino acid sequence LE <CD8 hinge> Nucleotide sequence AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTC GCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGC ACACGAGGGGGCTGGACTTCGCCAGTGAT (SEQ ID NO: 15) Amino acid sequence KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD (SEQ ID NO: 16) <spacer> Nucleotide sequence Aagccc

[0013] 12 170560056.1 Amino acid sequence KP <CD28 TM> Nucleotide sequence TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAG TGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 17) Amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 18) <4-1BB co-stimulatory domain> Nucleotide sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAA CTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTG AACTG (SEQ ID NO: 19) Amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 20) <CD3 zeta> Nucleotide sequence AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGA ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTG GACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGA AGAACCCTCAGGAAGGCCTCTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTT CACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 21) Amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 22) <EcoRI restriction site> gaattc

[0014] 13 170560056.1 Nucleotide sequence of Mouse ROR-1 CAR ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC CGCCAGGCCGGCTAGCGAAGTTAAGTTGGTAGAATCAGGCGGCGGATTGGTTA AGCCCGGTGGATCACTCAAGCTGAGCTGCGCTGCCTCTGGTTTTACTTTTAGCT CATACGCTATGAGTTGGGGGCGACAGACTCCTGAAAAACGGTTGGAATGGGTC GCATCCATCAATACAGGTGGCGGCACCTACTACCCAGATTCAGTCAAAGGGCG GTTCACTATATCACGGGATAACGCCAGAAACATTCTGTACTTGCAAATGAATTC CCTTCGATCTGAGGACACAGCAATGTATTACTGTGCCCGGGAAGGTTACTATTA CGGGGGCAGTAGCTATTACGCAATGGATTATTGGGGGCCTGGAACTAGCGTAA CTGTAAGTTCTGGCGGCGGTGGCAGCGGAGGTGGTGGGTCCGGCGGCGGCGG CTCAGATATAAAGATGACTCAGAGCCCAAGCTCCATGTATGTCTCCCTTGGCGA GCGCGTAACAATAACTTGCAAAGCATCACAGGATATAAATTCTTACCTCTCATG GTTCCAACAGAAGCCTGGCAAGAGTCCAAAGACTTTGATTTATAGGGCTAACA GGTTGGTAGATGGTGTTCCCTCAAGATTCAGCGGATCAGGTAGCGGACAAGAT TACTCATTGACAATCAGTAGCCTGGAGTACGAGGATATGGGAATATACTATTGT CTGCAATACGATGAGTTCCCTTATACTTTCGGTGGCGGGACCAAGCTTGAAATA AAACGCCTCGAGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGC CCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCG GGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGGT GCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT ATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTA TGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAG AAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGC GTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTCTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCG GAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGG ACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 23) Amino acid sequence of mouse ROR-1-CAR MALPVTALLLPLALLLHAARPASEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAM SWGRQTPEKRLEWVASINTGGGTYYPDSVKGRFTISRDNARNILYLQMNSLRSEDT AMYYCAREGYYYGGSSYYAMDYWGPGTSVTVSSGGGGSGGGGSGGGGSDIKMTQS PSSMYVSLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGS GQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKRLEKPTTTPAPRPPTPAPTIAS QPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 24)

[0015] 14 170560056.1 Example 4. ROR-1-CAR with Human ROR-1 scFv The structure of human ROR-1-CAR includes: human CD8 signaling peptide, human ROR-1 scFv (VH-Linker-VL), CD8 hinge, CD28 transmembrane, 4-1BB-co-stimulatory domains, CD3 zeta activation domain (FIG. 2). The CAR sequence is similar to that described in Example 3, except having humanized ROR-1 scFv (see Example 2B). Human ROR-1 CAR: CD8 leader-ROR-1 scFv (VH-Linker -VL)-CD8 hinge-CD28 TM-4-1BB-CD3-zeta. Nucleotide sequence atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggctagcgaagtgcagctggttgaatctg gaggtggccttgtgcagccgggaggttctctccggttgagttgcgccgcttcaggttttacgttctcttcttatgctatgtcatgggtgagacaa gcccctggtaagggattggaatgggttgcttctattaacactggcgggggaacgtattacccagattctgtgaaggggagatttacaatcagt cgagacaatgcaaagaatagcctttatcttcaaatgaatagtcttcgggccgaggacaccgcggtctattactgcgcacgagaaggttacta ctatggaggatcttcctactacgcgatggactattggggaccgggaaccagcgttaccgtatccagcggtggcggggggtctggcggggg cggaagtggagggggaggtagcgacattcagatgactcaaagtccgtcctccctgagtgcatcagtgggtgatcgagtcacaatcacgtgt aaagcgtcccaggatataaattcatatctcagttggtatcagcaaaaacctggcaaggctcctaagctccttatttacagggcaaatcgattgg tatctggcgtgccttccaggtttagtggatctgggtctgggaccgactttacgctgaccatcagttctctgcaaccagaggatttcgcaacctat tattgtctgcagtatgatgagttcccctacacatttggtgcaggtaccaagctggagcttaaactcgagaagcccaccacgacgccagcgcc gcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgagccggccagcggcggggggcgca gtgcacacgagggggctggacttcgccagtgataagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagta acagtggcctttattattttctgggtgaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactca agaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcc cccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtgg ccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctctacaatgaactgcagaaagataagatggc ggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccacc aaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa (SEQ ID NO: 25) Amino acid sequence MALPVTALLLPLALLLHAARPASEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSW VRQAPGKGLEWVASINTGGGTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CAREGYYYGGSSYYAMDYWGPGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS

[0016] 15 170560056.1 VGDRVTITCKASQDINSYLSWYQQKPGKAPKLLIYRANRLVSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCLQYDEFPYTFGAGTKLELKLEKPTTTPAPRPPTPAPTIASQPLSLRPE ASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 26) Example 5. CAR lentivirus Production. The lentiviruses were produced by standard procedures using 293 cells as described in [4]. ROR-1 CAR construct containing ROR-1 ScFv-4-1BB-CD3zeta was cloned into a lentiviral vector. The CAR construct contained the MNDU3 promoter, which was used to drive expression of CAR construct. The lentiviruses were generated in 293 T cells and titer was established by FACS. Then equal dose of lentiviruses and T cells were used for transduction of T cells. Example 6. Peripheral blood mononuclear cell (PBMC) isolation from whole blood Whole blood (Stanford Hospital Blood Center, Stanford, CA) was collected from individual or mixed donors (depending on the amount of blood required) in 10 mL Heparin vacutainers (Becton Dickinson). Approximately 10 ml of whole anti-coagulated blood was mixed with sterile phosphate-buffered saline (PBS) for a total volume of 20 mL in a 50 mL conical centrifuge tube (PBS, pH 7.4, is without Ca2+ / Mg2+). The layer of cells containing peripheral blood mononuclear cells (PBMC), seen at the diluted plasma / Ficoll interface, was removed very carefully, avoiding any Ficoll, washed twice with PBS, and centrifuged at 200xg for 10 min at room temperature. Cells were counted with a hemocytomter. The PBMC were washed once with CAR-T media (AIM V-AlbuMAX(BSA)(Life Technologies), with 5% AB serum and 1.25 ug / mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U / mL penicillin, and 100 ug / mL streptomycin) and used for experiments or were frozen at -80 C. Example 7. T-Cell Activation from PBMC Isolated PBMC were washed once with PBS (pH7.4), no Ca2+ / Mg2+, and in CAR-T media (AIM V-AlbuMAX(BSA) (Life Technologies), with 5% AB serum, 1.25 µg / mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U / mL penicillin, and 100 µg / mL streptomycin), in the absence of human interleukin-2 (huIL-2) (Invitrogen), at a concentration of

[0017] 16 170560056.1 5 x 105cells / mL, then resuspended to a final concentration of 5x105cells / mL in CAR-T medium with 300 U / mL huIL2 (from a 1000x stock; Invitrogen). PBMC and CD3-CD28 were then mixed at a 1:1 bead-to-cell ratio, by transferring 25 µL of beads to 1 mL of PBMC and incubated at 37oC in the presence of CO2for 24 hr before viral transduction. Addition of beads activated T cells before addition of virus. Example 8. T-Cell Transduction and Expansion Following activation of PBMC, 5x106lentivirus were added to T cells at MOI (multiplicity of infection) 10:1, and 2 µL / mL of media of Transplus (Alstem, Richmond, CA) (a final dilution of 1:500). Cells were incubated for an additional 24 hours before repeating addition of virus. 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 number 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 1x106cells / ml. Example 9. Transduction Verification by FACS Cells were washed and suspended in FACS buffer (Phosphate-buffered saline (PBS) plus 0.1% sodium azide and 0.4% BSA). Cells were then divided at 1x106aliquots. Fc receptors were blocked with normal goat IgG (LifeTechnologies). Biotin-labeled polyclonal goat anti-mouse F(ab)2 antibodies (Life Technologies) were used to detect mouse ROR-1 scFv; biotin-labeled normal polyclonal goat IgG antibodies (Life Technologies) served as an isotype control. Cells were incubated at 4°C for 25 minutes and washed once with FACS buffer. After staining cells with anti-F(ab)2 antibody, phycoerythrin (PE)-labeled streptavidin (BD Pharmingen, San Diego, CA) and allophycocyanin (APC)-labeled CD3 (eBiocience, San Diego, CA) were used to stain the cells. Example 10. Cytotoxicity Assay The cytotoxicity was performed by real-time cytotoxicity assay using ACEA machine according to manufacturer’s protocol as described [6].

[0018] 17 170560056.1 Example 11. Mouse ROR-1-CAR-T cells expressed high cytotoxic activity against ROR-1- positive cancer cells. The expression of mouse ROR1 ScFv was confirmed by FACS with anti-mouse FAB antibodies (FIG. 3, left panel) and with biotinylated recombinant extracellular domain of ROR1 (FIG. 3, right panel). More than 50% were detected with either mouse FAB or recombinant ROR1 extracellular domain protein, which confirms expression of ROR1-ScFV CAR in expanded CAR-T cells. To test specific cytotoxicity of CAR-T cells, RTCA impedance-based assay was performed using CHO-ROR1 and control CHO cells according to Agilent / ACEA manufacturer’s conditions. In this assay, the integrity of the target cell monolayer was continually monitored via its impedance in a weak electrical field; killing of the target cells by the CAR-T cells decreased the monolayer’s integrity and, therefore, its impedance. ROR1-CAR-transduced T cells were added to the target cells at effector: target cell (E:T) ratios of 10:1 (FIG. 4). ROR-1 CAR-T cells caused a significant decrease in target cell CHO-ROR1 monolayer impedance (FIG. 4A), which was more pronounced versus T cells. Less killing compared to T cells ware in ROR-1 negative CHO target cells (FIG. 4B). Thus, ROR-1-4-1BB-CD3 CAR-T cells specifically killed ROR-1 positive target cells. High cytotoxic activity was observed also with ROR1-41BB-CD3 CAR-T cells and ROR1- positive cancer target cells: pancreatic cancer BxPC3, colon cancer Lovo, and gastrointestinal (GI) OVCAR-5 cells (FIG. 5 A-C). ROR1-CAR-T cells also killed other cancer cell lines: HCT116, HT29, SW480, SW620 and lung cancer A549 (not shown). Example 12. Mouse ROR-1-CAR secreted high level of IFN-gamma ROR-1-positive cancer cells. After co-incubation of ROR1-41BB-CD3-CAR-T cells with target cells, we collected supernatant and performed ELISA with kit from Fisher according to manufacturer's protocol. FIG. 6 shows that ROR-1-CAR-T cells secreted a significantly higher level of IFN-gamma against ROR-1-positive CHO target cells than against ROR-1 negative CHO target cells. FIG. 6 also shows that IFN-gamma secreted by ROR1-CAR-T cells was higher than IFN-gamma secreted by T cells.

[0019] 18 170560056.1 FIGs. 7A-7C shows that IFN-gamma was secreted by ROR-1 CAR-T cells against ROR- 1 positive BxPC3 target (7A), Lovo (7B) and OVCAR-5 (7C) cancer cell lines. Example 13. Mouse ROR1-CAR-T cells effectively blocked OVCAR-5 colorectal xenograft tumor growth in vivo. 2x106OVCAR-5 cells were injected into NSG mice subcutaneously into two sides, and then ROR-1-CAR-T cells (1x107cells per mice) were injected intravenously 5 times weekly on days 2, 9, 16, 23, and 30. ROR1-CAR-T cells significantly decreased xenograft tumor growth, p<0.05 (FIG. 8A). There were no significant change of the mouse body weight suggesting absence of toxicity (FIG. 8B). The results show that ROR1-CAR-T cells had high efficacy in vitro and in vivo. Example 14. In vitro Efficacy of Humanized ROR1-CAR-T cells Humanized PMC2509 CAR was prepared with humanized ROR-1 scFv (see Example 4 for sequences). CAR-T cells were generated with lentiviral PMC2509 CAR. FACS with CAR-T cells detected binding of ROR1 recombinant protein with ROR1 scFv (FIG. 9). Non-transduced T cells were used as negative control (FIG. 9). Human ROR1-CAR-T cells were then tested by RTCA assay for in vitro efficacy. Human ROR1-CAR-T cells effectively killed OVCAR-5 (FIG. 10A) and PC3 cells (FIG. 10B). PMC2509-CAR-T cells also killed more CHO-ROR1 cells compared to T cells (FIG. 10C). The killing compared to T cells was less with ROR-negative CHO cells (FIG. 10C). ROR1 secreted more IFN-gamma with OVCAR-5 cells (FIG. 11A) and PC3 (FIG. 11B) than with T cells. PMC2509 ROR1-CAR-T cells secreted IFN-gamma significantly more (p<0.05, Student’s t-test) with CHO-ROR1 cells than with target CHO cells (FIG. 11C). Example 15. In vivo efficacy of humanized ROR1-CAR-T cells. To show in vivo efficacy of hROR1-CAR-T cells (PC2509), 4x10^6 OVCAR-5 cells were injected subcutaneously into NSG mice, and then 1x10^7 CAR-T cells were injected intravenously on days 2, 9, 16, 23 and 30. PMC2509-CAR-T cells significantly decreased OVCAR-5 tumor growth in vivo using NSG mouse model (FIG. 12A. There was no significant mouse body weight suggesting no toxicity of ROR1-CAR-T cells (FIG. 12B).

[0020] 19 170560056.1 References 1. Grupp, S.A., Kalos, M., Barrett, D., Aplenc, R., Porter, D.L., Rheingold, S.R., Teachey, D.T., Chew, A., Hauck, B., Wright, J.F., et al. (2013). Chimeric antigen receptor- modified T cells for acute lymphoid leukemia. N Engl J Med 368, 1509-1518. 2. Maus, M.V., Haas, A.R., Beatty, G.L., Albelda, S.M., Levine, B.L., Liu, X., Zhao, Y., Kalos, M., and June, C.H. (2013). T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res 1, 26-31. 3. Maus, M.V., Grupp, S.A., Porter, D.L., and June, C.H. (2014). Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood 123, 2625-2635. 4. Goluboskaya V, Wu L. Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy. Cancers (Basel). 2016 Mar 15;8(3). pii: E36. doi: 10.3390 / cancers8030036. Review. 5. Dal Ferro M, Rizzo S, Rizzo E, Marano F, Luisi I, Tarasiuk O, Sblattero D. Phage Display Technology for Human Monoclonal Antibodies. Methods Enzymol 121, 332-340. 6. Berahovich R, Zhou H, Xu S, Wei Y, Guan J, Guan J, Harto H, Fu S, Yang K, Zhu S, Li L, Wu L, Golubovskaya V. CAR-T cells based on Novel BCMA monoclonal antibody block multiple myeloma Cell growth. Cancers (Basel), 10 (9), 2018.

[0021] 20 170560056.1

Claims

WHAT IS CLAIMED IS:

1. A human anti-human ROR-1 antibody or an antigen-binding fragment thereof, comprising: (a) VH having VH-CDR1 having the amino acid sequence of SEQ ID NO: 4, VH- CDR2 having the amino acid sequence of SEQ ID NO: 5, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 6, and (b) VLhaving the amino acid sequence of SEQ ID NO:

11.

2. The human anti-human ROR-1 antibody or an antigen-binding fragment thereof of claim 1, wherein VHhaving the amino acid sequence of SEQ ID NO:

10.

3. The human anti-human ROR-1 antibody or an antigen-binding fragment thereof of claim 1 or 2, which is a single-chain variable fragment (scFv).

4. The scFv of Claim 3, further comprises a linker in between VH and VL.

5. The scFv of Claim 4, which has the amino acid sequence of SEQ ID NO:

9.

6. A mouse anti-human ROR-1 antibody or an antigen-binding fragment thereof, comprising: (a) VHhaving VH-CDR1 having the amino acid sequence of SEQ ID NO: 4, VH- CDR2 having the amino acid sequence of SEQ ID NO: 5, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 6, and (b) VL having the amino acid sequence of SEQ ID NO:

8.

7. The mouse anti-human ROR-1 antibody or an antigen-binding fragment thereof of claim 6, wherein VH having the amino acid sequence of SEQ ID NO:

3.

8. The mouse anti-human ROR-1 antibody or an antigen-binding fragment thereof of claim 6 or 7, which is a single-chain variable fragment (scFv).

9. The scFv of Claim 8, further comprises a linker in between VHand VL.

10. The scFv of Claim 9, which has the amino acid sequence of SEQ ID NO: 2.21 170560056.

111. A chimeric antigen receptor (CAR) comprising from N-terminus to C-terminus: (i) the scFv of Claim any one of Claims 3-5 and 8-10. (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain.

12. The CAR according to claim 11, wherein the co-stimulatory domain is 4-1BB or CD28.

13. The CAR according to claim 11, wherein the activation domain is CD3 zeta.

14. The CAR of claim 13, which has the amino acid sequence of SEQ ID NO: 24 or 26.

15. An isolated nucleic acid encoding the CAR of any one of claims 11-14.

16. T cells or natural killer cells modified to express the CAR of any one of claims 11-14.22 170560056.1

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