Ror-1 antibodies and anti-ror-1-car-t cells

Abstract

The present invention relates to monoclonal mouse or humanized ROR-1 antibodies or antigen-binding fragments thereof, including single-chain variable fragments (scFv). The present invention also relates 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 costimulatory domain, and (iv) an activating domain.

Description

Technical Field

[0001] This invention relates to mouse and humanized ROR-1 specific monoclonal antibodies and scFv, as well as ROR-1-CAR-T cells, which can be used in the field of adoptive immunogene therapy for tumors. Background Technology

[0002] Immunotherapy is emerging as a promising approach to cancer treatment. T cells, or T lymphocytes, are the armed forces of our immune system, constantly seeking out foreign antigens and distinguishing abnormal cells (cancer or infected cells) from normal cells. Genetic modification of T cells using CAR (chimeric antigen receptor) constructs is the most common method for designing tumor-specific T cells. CAR-T cells targeting tumor-associated antigens (TAAs) can be infused into patients (called adoptive cell transfer or ACT) and are an effective immunotherapy approach [1, 2]. The advantage of CAR-T technology over chemotherapy or antibodies is that the reprogrammed engineered T cells can proliferate and persist in the patient’s body (“live drug”) [3], [4], [1].

[0003] CARs typically consist of a monoclonal antibody-derived single-chain variable fragment (scFv) located at the N-terminus, a hinge, a transmembrane domain, and numerous intracellular co-stimulatory domains: (i) CD28, (ii) CD137 (4-1BB), CD27, or other domains, tandemly linked to the activation domain CD3-zeta [2,3,4]. CARs have evolved from first-generation (without a co-stimulatory domain) to second-generation (with one co-stimulatory domain) and then to third-generation CARs (with multiple co-stimulatory domains). CARs with multiple co-stimulatory domains (so-called third-generation CARs) can enhance the cytolytic activity of CAR-T cells, improve the persistence of CAR-T cells, and thus enhance their antitumor activity.

[0004] Natural killer (NK) cells are cytotoxic lymphocytes that are crucial to the innate immune system. NK cells function similarly to cytotoxic T cells in the adaptive immune response of vertebrates. NK cells respond rapidly to virus-infected cells, becoming active approximately 3 days after infection, and also respond to tumor formation.

[0005] ROR-1 antigen ROR1, also known as neurotrophic tyrosine kinase transmembrane receptor, receptor-associated 1 (NTRKR1), is an enzyme encoded by the ROR1 gene in humans. ROR1 is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family.

[0006] ROR-1 is a protein with 937 amino acids and an extracellular domain of 30-406 amino acids. ROR-1 is expressed at low levels in most tissues, which is advantageous for its use as a target for CAR-T cell therapy. Figure 1 The ROR-1 gene encodes a receptor tyrosine kinase-like orphan receptor that regulates neurite growth in the central nervous system. The encoded protein is a glycosylated type I membrane protein belonging to the ROR subfamily of cell surface receptors. ROR-1 is a receptor for the ligand WNT5A, activating the downstream NF-κB signaling pathway and potentially inhibiting WNT-mediated signaling. Furthermore, ROR1 has recently been shown to be expressed in ovarian cancer stem cells and promote migration, invasion, and cancer stem cell spheroidization. ROR-1 is overexpressed in both hematologic malignancies and solid tumors, making this target potentially applicable to CAR-T therapy.

[0007] ROR-1 is expressed at low levels in most normal human tissues, such as adipose and soft tissues, bone marrow and immune system, endocrine tissues, female tissues, gastrointestinal tract, kidneys and bladder, liver and gallbladder, lungs, muscle tissues, male tissues and skin. Attached Figure Description

[0008] Figure 1 The structure of the ROR-1 CAR construct is shown. The second-generation CAR employs a 41BB co-stimulatory domain and a CD3 zeta activation domain. Abbreviations: ScFv, single-stranded variable fragment; h-type hinge; TM-transmembrane; 4-1BB co-stimulatory and CD3 zeta activation domain.

[0009] Figure 2 The results show that FACS detection of ROR-1 antibody binding to human ROR-1 antigen in Lovo cells, but not binding in CRISPR / Cas9-treated Lovo KO cells, was performed. Lovo ROR-1 positive and Lovo CRISPR / Cas-9 KO cell lines were assayed using the ROR1 8A2A3 antibody. Mouse anti-ROR1 antibody was used as the primary antibody, and goat anti-mouse PE antibody was used as the secondary antibody.

[0010] Figure 3 The image shows the FACS detection of ROR1 ScFv-positive CAR-T cell expression. FACS was performed using mouse F(ab)2 antibody (left panel) or recombinant extracellular ROR1 domain biotinylated protein (right panel).

[0011] Figure 4A and 4BThe RTCA assay demonstrates the in vitro cytotoxic activity of mouse ROR1-4-1BB-CD3 CAR-T cells against the ROR1-positive CHO-ROR1 cell line. Cytotoxicity assays were performed using the ACEA XCelligence system, with the ratio of ROR1-4-1BB-CD3 CAR-T cells as effector cells to target cells (CHO-ROR1 and CHO cells) at 10:1.

[0012] Figures 5A-5C The study demonstrates how ROR1-CAR-T cells kill ROR-1 positive cancer cell lines. ROR1-CAR-T cells were used as target cell lines for RTCA assays, including pancreatic BXPC3 (A), colorectal cancer Lovo (B), and gastrointestinal (GI) OVCAR-5 (C).

[0013] Figure 6 This study demonstrates the IFN-γ secretion of ROR-1-41BB-CD3 CAR-T cells against ROR-1-positive target cells. CHO-ROR-1-positive and control CHO target cell lines were used for IFN-γ ELISA assay. p<0.05, Student's t-test, ROR1-CAR-T cells against CHO-ROR1-positive target cells compared to those against CHO target cells.

[0014] Figures 7A-7C The study demonstrates IFN-γ secretion by ROR-1CAR-T cells in response to 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 targeting ROR1-positive target cells compared with T cells targeting ROR1-positive target cells.

[0015] Figure 8A This study showed that ROR1-CAR-T cells significantly reduced the growth of OVCAR-5 xenograft tumors in vivo. p<0.05. Student's t-test was used to compare ROR-1 CAR-T cells with T cells. Figure 8B The results showed that ROR-1 CAR-T cells did not significantly affect the body weight of mice.

[0016] Figure 9 The expression of humanized CARs is shown using FACS. FACS shows ROR1 CAR-T cells binding to the ROR1 antigen. FACS was performed using anti-mouse F(ab)2, anti-human F(ab)2 antibodies, and recombinant biotinylated ROR1 extracellular domain protein.

[0017] Figures 10A-10CThe results show that human PMC2509 CAR-T cells killed several cancer cell lines as detected by RTCA. 10A RTCA was performed using OVCAR-5 target cells at an E:T ratio of 10:1. 10B RTCA was performed on PC3 target cells at an E:T ratio of 20:1. 10C RTCA was performed using CHO-ROR1 (bottom panel) and CHO cells (top panel) at an E:T ratio of 10:1.

[0018] Figures 11A-11C This illustrates the secretion of IFN-γ by hROR1-CAR-T cells on different target cells. A. OVCAR-5 target cells; B. PC3 target cells; A, B, p<0.05 Student's t-test for CAR-T cells compared to T cells. C. CHO and CHO-ROR1 target cells. p<0.05 Student's two-tailed t-test compared to CAR-T cells and CHO-ROR1 target cells, and CAR-T cells and CHO target cells.

[0019] Figure 12A This study demonstrates the in vivo efficacy of humanized ROR-1-CAR-T cells in an OVCAR-5 xenograft model. N=6 tumors / mouse per group. Tumor volume change was compared (p<0.02) with CAR T cells compared to T cells using a Student's t-test. Figure 12B The results showed no significant change in mouse body weight. Detailed Implementation

[0020] As used in this article, “antigen-binding fragment” refers to Fab fragment, Fab' fragment, F(ab')2 fragment, and scFv, which have antigen-binding activity.

[0021] As used herein, a "chimeric antigen receptor (CAR)" is a receptor protein engineered to give T cells the novel ability to target specific proteins. This receptor is chimeric because it combines antigen binding and T cell activation functions into a single receptor. A CAR is a fusion protein containing an extracellular domain capable of binding antigens, a transmembrane domain, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)."

[0022] Extracellular domains that can bind to antigens refer to any oligopeptide or polypeptide that can bind to a certain antigen. Intracellular domains refer to any oligopeptide or polypeptide that is known to act as a signaling domain to induce activation or inhibition of biological processes in the cell.

[0023] As used in this article, a "domain" refers to a region in a polypeptide that folds into a specific structure independently of other regions.

[0024] As used herein, “single-chain variable fragment (scFv)” means a single-chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Examples of scFvs include antibody polypeptides formed by recombinant DNA technology, wherein the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) segments are linked by spacer sequences. Various methods for engineering scFvs are known to those skilled in the art.

[0025] As used in this article, "tumor antigen" refers to a biomolecule that has antigenicity and whose expression can cause cancer.

[0026] The inventors have prepared a ROR-1 monoclonal antibody specifically targeting the human ROR-1 antigen using hybridoma technology. The monoclonal anti-human ROR-1 antibody is generated targeting the extracellular region of a purified recombinant human ROR-1 fragment.

[0027] On one hand, the present invention relates to a monoclonal mouse anti-human ROR-1 antibody or an antigen-binding fragment thereof, comprising (i) a 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) a VL having the amino acid sequence of SEQ ID NO:8.

[0028] In one embodiment, the monoclonal mouse anti-human ROR-1 antibody or its antigen-binding fragment comprises VH having the amino acid sequence SEQ ID NO: 3 and VL having the amino acid sequence SEQ ID NO: 8.

[0029] In one embodiment, the monoclonal anti-human ROR-1 antibody is a single-chain variable fragment (scFv). The scFv can be VH-connector-VL or VL-connector-VH. On the other hand, the present invention relates to a humanized anti-human ROR-1 antibody or its antigen-binding fragment thereof, which is achieved by using the CDRs of the VH and VL of a mouse anti-human ROR-1 antibody and transplanting them into a human frame. In some cases, the amino acids at the CDR and frame boundaries can be changed to another amino acid. The humanized anti-human ROR-1 antibody or its antigen-binding fragment comprises (i) 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.

[0030] In one embodiment, the humanized anti-human ROR-1 antibody or its antigen-binding fragment comprises a humanized VH having the amino acid sequence of SEQ ID NO: 10 and a humanized VL having the amino acid sequence of SEQ ID NO: 11.

[0031] In one embodiment, the humanized anti-human ROR-1 antibody is a single-chain variable fragment (scFv). The scFv can be VH-connector-VL or VL-connector-VH.

[0032] The present invention also relates to a chimeric antigen receptor fusion protein comprising, from its N-terminus to its C-terminus: (i) a single-stranded variable fragment (scFv) targeting ROR-1, (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activation domain. One embodiment of CAR is, for example... Figure 1 As shown. CAR-T cells targeting ROR1-positive target cells.

[0033] 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. The preferred co-stimulatory domain is CD28.

[0034] The preferred activation domain is CD3 zeta (CD3 Z or CD3ζ).

[0035] The transmembrane domain can be derived from a natural peptide or can be artificially designed. Transmembrane domains derived from natural peptides can be obtained from any membrane-binding or transmembrane protein. For example, transmembrane domains of T cell receptor α or β chains, CD3zeta chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR can be used. Artificially designed transmembrane domains are peptides primarily containing hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or peptide linker, such as a linker with 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.

[0036] This invention provides isolated nucleic acids encoding ROR-1 CARs. The nucleic acids encoding CARs can be prepared using conventional methods. The base sequences encoding the amino acid sequences can be obtained from the NCBI RefSeq IDs or GenBank accession numbers of the amino acid sequences of each domain, and the nucleic acids of this invention can be prepared using standard molecular biology and / or chemical methods. For example, nucleic acids can be synthesized based on the base sequences, and the nucleic acids of this invention can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0037] The nucleic acid encoding the CAR of this invention can be inserted into a vector, and the vector can be introduced into cells. For example, viral vectors such as retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus (AAV) vectors can be used. Viral vectors that lack replication ability and therefore cannot self-replicate in infected cells are preferred.

[0038] For example, when using retroviral vectors, suitable packaging cells based on the LTR sequence and the packaging signal sequence possessed by the vector can be selected to prepare retroviral particles using these packaging cells. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86, GP+envAm-12, and Psi-Crip. 293 cells or 293T cells with high transfection efficiency can also be used to prepare retroviral particles. A variety of retroviral vectors produced based on retroviruses and packaging cells are widely available from many companies.

[0039] CAR-T cells activate and kill target cells by binding to specific antigens present on target cells via CARs. Cell activation via CAR-T varies depending on the host cell type and the intracellular domains of the CAR. Activation can be confirmed based on factors such as the release of cytokines, increased cell proliferation, and changes in cell surface molecules. For example, activated CAR-T cells release cytotoxic cytokines (IFN-γ, granzyme B, tumor necrosis factor, etc.) leading to the destruction of target cells expressing antigens. Furthermore, the release of cytokines or changes in cell surface molecules stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0040] Cells expressing CARs can be used as therapeutic agents for diseases. Therapeutic agents contain CAR-expressing cells as active ingredients, and may further contain suitable excipients.

[0041] The inventors have prepared ROR-1-CAR-T cells to target cancer cells that overexpress the ROR-1 tumor antigen. The ROR-1-CAR-T cells of this invention exhibit high cytotoxic activity and in vivo antitumor activity against various cancer cell lines.

[0042] The inventors have prepared mouse and human ROR-1-ScFv-4-1BB-CD3-CAR-T (ROR-1-CAR-T) cells targeting solid tumor cancer cells overexpressing ROR-1. The ROR-1-CAR-T cells exhibit higher cytotoxic activity against ROR-1-positive cancer cells than non-transduced T cells and mock-CAR-T cells.

[0043] The present invention provides mouse and human anti-human ROR-1 antibodies for detecting ROR-1 in ROR-1 positive cancer cells.

[0044] One of the advantages of humanized ROR-1 scFv CAR-T cells is that humanized scFv may elicit a less pronounced immune response in humans compared to mouse scFv CAR-T cells.

[0045] Mouse and humanized ROR-1 antibodies can be used for immunotherapy applications: toxin / drug conjugates, monoclonal therapeutic antibodies, bispecific antibodies, and CAR-T cell immunotherapy.

[0046] ROR-1-CAR-T cells using the ROR-1 antibody of the present invention can effectively target the ROR-1 antigen in ROR-1 positive cell lines.

[0047] ROR-1-CAR-T can be used in combination with different therapeutic agents: checkpoint inhibitors; targeted therapies, small molecule inhibitors, and antibodies.

[0048] ROR-1 antibodies can be optimized through site-directed mutagenesis or through affinity maturation modification via error-prone PCR to adjust affinity.

[0049] ROR-1-CAR-T cells can be used clinically to combat ROR-1 positive cells.

[0050] Modification of co-stimulatory domains: CD28, 4-1BB, etc., can be used to improve efficacy. Tag-conjugated ROR-1scFv can be used for CAR generation.

[0051] Third-generation CAR-T or other co-stimulatory domains can be used with the same ROR-1-scFv within the CAR.

[0052] ROR-1 can be combined with other CARs that target other tumor antigens or the tumor microenvironment (VEGFR-1-3), PDL-1, CD80, or bi-scFv-CAR to enhance the activity of monotherapy ROR-1-CAR.

[0053] Bispecific antibodies with ROR-1 and CD3 or other antigens can be generated for treatment.

[0054] ROR-1-CAR-T cells can be used to combat cancer stem cells that are resistant to chemotherapy and form aggressive tumors.

[0055] ROR-1-CAR can be used to generate other cell types, 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. This invention provides T cells modified to express ROR-1-CAR.

[0056] ROR1-CAR can be used to generate both autologous CAR-positive cells and allogeneic hematopoietic cells.

[0057] The following examples further illustrate the present invention. These examples are intended to illustrate the invention only and should not be construed as limiting.

[0058] Example Example 1. Detection of ROR1 with mouse ROR1 antibody by FACS staining.

[0059] We generated mouse monoclonal ROR-1 antibodies using standard hybridoma technology. The ROR-1 content of the mouse ROR1 monoclonal antibody was detected by FACS using Lovo ROR1-positive cells and ROR1 knockout (KO) Lovo cells. Figure 2 ).

[0060] We sequenced the hybridoma clone 8A2A3 and used VH and VL to generate ScFv (as shown in Example 2A).

[0061] Example 2A. Sequencing of mouse ROR-1 VH, VL and scFv We sequenced the mouse ROR-1 antibody and clone 8A2A3; the sequences of VH, VL, and ScFv are shown below. The structure of ROR-1 scFv is: VH-connector-VL.

[0062] The first sequence is the signal peptide, followed by the nucleotide sequence of VH in bold; the nucleotide sequence of VL is shown in underline; and in between (in italics) are the nucleotide sequences encoding the linker. ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCTAGCGAAGTTAAGTTGGTAGAATCAGGCGGCGGATTGGTTAAGCCCGGTGGATCACTCAAGCTGAGCTGCGCTGCCTCTGGTTTTACTTTTAGCTCATACGCTATGAGTTGGGGGCGACAGACTCCTGAAAAACGGTTGGAATGGGTCGCATCC ATCAATACAGGTGGCGGCACCTACTACCCAGATTCAGTCAAAGGGCGGTTCACTATATCACGGGATAACGCCAGAAACATTCTGTACTTGCAAATGAATTCCCTTCGATCTGAGGACACAGCAATGTATTACTGTGCCCGGGAAGGTTACTATTACGGGGGCAGTAGCTATTACGCAATGGATTATTGGGGGCCTGGAACTAGCGTAACTGTAAGTTCT GGCGGCGGTG GCAGCGGAGGTGGTGGGTCCGGCGGCGGCGGCTCA GATATAAAGATGACTCAGAGCCCAAGCTCCATGTATGTCTC CCTTGGCGAGCGCGTAACAATAACTTGCAAAGCATCACAGGATATAAATTCTTACCTCTCATGGTTCCAACAGAAG CCTGGCAAGAGTCCAAAGACTTTGATTTATAGGGCTAACAGGTTGGTAGATGGTGTTCCCTCAAGATTCAGCGGAT CAGGTAGCGGACAAGATTACTCATTGACAATCAGTAGCCTGGAGTACGAGGATATGGGAATATACTATTGTCTGCA ATACGATGAGTTCCCTTATACTTTCGGTGGCGGGACCAAGCTTGAAATAAAACGC (SEQ ID NO: 1) Mouse ROR-1 scFv protein sequence (VH is bold, CDR regions based on the Kabat system are underlined, linkers are in italics, and VL is underlined): EVKLVESGGGLVKPGGSLKLSCAASGFTFS SYAMS WGRQTPEKRLEWVA SINTGGGTYYPDSVKG RFTISRDNARNILYLQMNSLRSEDTAMYYCAR EGYYYGGSSYYAMDY WGPGTSVTVSS GGGGSGGGGSGGGGS DIKMTQ SPSSMYVSLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYED MGIYYCLQYDEFPYTFGGGTKLEIKR (SEQ ID NO: 2) Mouse ROR1 VH (SEQ ID NO: 3) EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWGRQTPEKRLEWVASINTGGGTYYPDSVKGRFTISRDNARNILYLQMNSLRSEDTAMYYCAREGYYYGGSSYYAMDYWGPGTSVTVSS CDR area based on Kabat system VH CDR1 SYAMS (SEQ ID NO: 4) VH CDR2 SINTGGGTYYPDSVKG (SEQ ID NO: 5) VH CDR3 EGYYYGGSSYYAMDY (SEQ ID NO: 6) connector GGGGSGGGGSGGGGS (SEQ ID NO: 7) ROR1 VL (The CDR area based on the Kabat system is shown in bold and underlined): DIKMTQSPSSMYVSLGERVTITC KASQDINSYLS WFQQKPGKSPKTLIY RANRLVD GVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC LQYDEFPYT FGGGTKLEIKR (SEQ ID NO: 8) VL Example 2B. Sequencing of human ROR-1 VH, VL and scFv We used the mouse CDR shown in Example 2A and transplanted it into the human frame region to prepare the human ROR-1 antibody. The structure of the human ROR-1 scFv is: VH-connector-VL.

[0063] We then sequenced the human ROR-1 antibody; the sequences of scFv, VH, and VL are shown below.

[0064] ROR-1 scFv (VH in bold, connector in italics, VL), CDR with underline EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVA SINTGGGTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EGYYYGGSSYYAMDY WGPGTSVTVSS GGGGS GGGGS GGGGS DIQMTQSPSSLSASVGDRVTITC KASQDINSYLS WYQQKPGKAPKLLIY RANRLVS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYDEFPYT FGAGTKLELK (SEQ ID NO: 9) Humanized VH (CDRs are underlined) EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVA SINTGGGTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EGYYYGGSSYYAMDY WGPGTSVTVSS(SEQ ID NO: 10) Humanized VL (CDRs are underlined) DIQMTQSPSSLSASVGDRVTITC KASQDINSYLS WYQQKPGKAPKLLIY RANRLVS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYDEFPYT FGAGTKLELK (SEQ ID NO: 11) Example 3. ROR-1-CAR sequence using mouse ROR scFv The scheme of the mouse ROR-1-CAR construct is as Figure 2 shown. The mouse ROR1 CAR is under the MNDU3 promoter.

[0065] The CAR structure includes a human CD8 signal peptide, ROR-1 scFv (VH-linker-VL), CD8 hinge, CD28 transmembrane, 4-1BB-costimulatory domain, CD3 zeta activation domain ( Figure 2 ).

[0066] CD8 leader sequence - ROR-1 scFv (VH-linker-VL) - CD8 hinge - CD28 TM - 4-1BB - CD3-zeta: <CD8 leader sequence> Nucleotide sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQID NO: 12) Amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 13) <Nhe I restriction enzyme cleavage site> Nucleotide sequence GCTAGC Amino acid sequence AS <ROR-1 scFV> Bold is VH, underlined is VL, and italic is the linker in between Amino acid sequence GAAGTTAAGTTGGTAGAATCAGGCGGCGGATTGGTTAAGCCCGGTGGATCACTCAAGCTGAGCTGCGCTGCCTCTGGTTTTACTTTTAGCTCATACGCTATGAGTTGGGGGCGACAGACTCCTGAAAAACGGTTGGAATGGGTCGCATCCATCAATACAGGTGGCGGCACCTACTACCCAGATTCAGTCAAAGGGCGGTTCACTATATCACGGGATAACGCCAGAAACATTCTGTACTTGCAAATGAATTCCCTTCGATCTGAGGACACAGCAATGTATTACTGTGCCCGGGAAGGTTACTATTACGGGGGCAGTAGCTATTACGCAATGGATTATTGGGGGCCTGGAACTAGCGTAACTGTAAGTTCT GGC GGCGGTGGCAGCGGAGGTGGTGGGTCCGGCGGCGGCGGCTCA GATATAAAGATGACTCAGAGCCCAAGCTCCATGT ATGTCTCCCTTGGCGAGCGCGTAACAATAACTTGCAAAGCATCACAGGATATAAATTCTTACCTCTCATGGTTCCA ACAGAAGCCTGGCAAGAGTCCAAAGACTTTGATTTATAGGGCTAACAGGTTGGTAGATGGTGTTCCCTCAAGATTC AGCGGATCAGGTAGCGGACAAGATTACTCATTGACAATCAGTAGCCTGGAGTACGAGGATATGGGAATATACTATT GTCTGCAATACGATGAGTTCCCTTATACTTTCGGTGGCGGGACCAAGCTTGAAATAAAACGC (SEQ ID NO:14) Amino acid sequence: See Example 2B <XhoI restriction enzyme cleavage site> Nucleotide sequence CTCGAG Amino acid sequence LE <CD8 hinge> Nucleotide sequence AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGAT(SEQ ID NO: 15) Amino acid sequence KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD (SEQ ID NO: 16) Spacer region Nucleotide sequence Aagccc Amino acid sequence KP <CD28 TM> Nucleotide sequence TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTAT TATTTTCTGGGTG (SEQ ID NO: 17) Amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 18) <4-1BB costimulatory domain> Nucleotide sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCA [[ID=3​​​​​​​​​​​​​​​​​​​​​​​​​ gaattc Nucleotide sequence of mouse ROR-1 CAR ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCTAGCGAAGTTAAGTTGGTAGAATCAGGCGGCGGATTGGTTAAGCCCGGTGGATCACTCAAGCTGAGCTGCGCTGCCTCTGGTTTTACTTTTAGCTCATACGCTATGAGTTGGGGGCGACAGACTCCTGAAAAACGGTTGGAATGGGTCGCATCCATCAATACAGGTGGCGGCACCTACTACCCAGATTCAGTCAAAGGGCGGTTCACTATATCACGGGATAACGCCAGAAACATTCTGTACTTGCAAATGAATTCCCTTCGATCTGAGGACACAGCAATGTATTACTGTGCCCGGGAAGGTTACTATTACGGGGGCAGTAGCTATTACGCAATGGATTATTGGGGGCCTGGAACTAGCGTAACTGTAAGTTCTGGCGGCGGTGGCAGCGGAGGTGGTGGGTCCGGCGGCGGCGGCTCAGATATAAAGATGACTCAGAGCCCAAGCTCCATGTATGTCTCCCTTGGCGAGCGCGTAACAATAACTTGCAAAGCATCACAGGATATAAATTCTTACCTCTCATGGTTCCAACAGAAGCCTGGCAAGAGTCCAAAGACTTTGATTTATAGGGCTAACAGGTTGGTAGATGGTGTTCCCTCAAGATTCAGCGGATCAGGTAGCGGACAAGATTACTCATTGACAATCAGTAGCCTGGAGTACGAGGATATGGGAATATACTATTGTCTGCAATACGATGAGTTCCCTTATACTTTCGGTGGCGGGACCAAGCTTGAAATAAAACGCCTCGAG AAGCCCACCACGACG CCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGAT AAGCCC T TTTGGGTGCTGGTGGTGGT TGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAG AAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCA GCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTCTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCT TTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 23) Amino acid sequence of mouse ROR-1-CAR MALPPVTALLLPLALLLHAARPASEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWGRQTPEKRLEWVASINTGGGTYYPDSVKGRFTISRDNARNILYLQMNSLRSEDTAMYYCAREGYYYGGSSYYAMDYWGPGTSVTVSS GGGGSGGGGSGGGGS DIKMTQSPSSMYVSLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVP SRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKR LE KPTTTPAPRPPTPAPTIASQPLSLR PEASRPAAGGAVHTRGLDFASD KP FWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 24) Example 4. ROR-1-CAR containing human ROR-1 scFv The structure of human ROR-1-CAR includes: human CD8 signal peptide, human ROR-1 scFv (VH-linker-VL), CD8 hinge, CD28 transmembrane domain, 4-1BB-co-stimulatory domain, and CD3 zeta activation domain. Figure 2 The CAR sequence is similar to that described in Example 3, except that it has a humanized ROR-1 scFv (see Example 2B).

[0067] Human ROR-1 CAR: CD8 leader sequence - ROR-1 scFv (V H -Connector-V L )-CD8 hinge-CD28 TM-4-1BB-CD3-zeta. nucleotide sequence amino acid sequence MALPVTALLLPLALLLHAARPASEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASINTGGGTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGYYYG GSSYYAMDYWGPGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCKASQDINSYLSWYQQKPGKAPKLLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQY DEFPYTFGAGTKLELKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 26) Example 5. Preparation of CAR lentivirus.

[0068] Lentiviral cells were prepared using 293 cells via a standard procedure, as described in [4].

[0069] The ROR-1 CAR construct containing ROR-1 ScFv-4-1BB-CD3zeta was cloned into a lentiviral vector. The CAR construct contains the MNDU3 promoter, which drives the expression of the CAR construct.

[0070] Lentiviral cells were prepared in 293 T cells, and the titer was determined by FACS. T cells were then transduced with an equal dose of lentivirus and T cells.

[0071] Example 6. Isolation of peripheral blood mononuclear cells (PBMCs) from whole blood. Whole blood (Stanford Hospital Blood Center, Stanford, California) is collected from individual or pooled donors (depending on the required blood volume) into 10 mL heparinized vacuum blood collection tubes (Becton Dickinson). Approximately 10 mL of anticoagulated whole blood is then mixed with sterile phosphate-buffered saline (PBS) in a 50 mL conical centrifuge tube (PBS, pH 7.4, Ca-free). 2+ / Mg 2 + Mix in a total volume of 20 mL. The cell layer containing peripheral blood mononuclear cells (PBMCs) seen at the diluted plasma / Ficoll interface was carefully removed, avoiding any Ficoll, washed twice with PBS, and centrifuged at 200 x g for 10 minutes at room temperature. Cells were counted using a hemocytometer. PBMCs were washed once with CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies) containing 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 frozen at -80°C.

[0072] Example 7. T cell activation from PBMCs PBMCs isolated in CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies) containing 5% AB serum and 1.25 μg / mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U / mL penicillin and 100 μg / mL streptomycin) without human interleukin-2 (huIL-2) (Invitrogen) were treated with 1xPBS (pH 7.4, Ca2+-free). 2+ / Mg 2+ Wash once, concentration 5×10 5 Cells / mL were then resuspended in CAR-T medium containing 300 U / mL huIL2 (from 1000x stock solution; Invitrogen) to a final concentration of 5 x 10⁻⁶ cells / mL. 5 Cells / mL. Then, 25 µL of CD3-CD28 magnetic beads were transferred to 1 mL of PBMC, mixed at a 1:1 bead-to-cell ratio, and incubated at 37°C for 24 h in CO2 presence before viral transduction. Magnetic beads were added to activate T cells before adding the virus.

[0073] Example 8. T cell transduction and expansion After activating PBMC, 5x10 6 Lentiviral cells were added to T cells at an MOI (multiple of infection) of 10:1 and 2 µL / mL Transplus (Alstem, Richmond, CA) medium (final dilution 1:500). Cells were cultured for another 24 hours before repeating the viral addition. Cells were then grown for 12–14 days in the presence of 300 U / mL IL-2 (total incubation time depends on the desired final number of CAR-T cells). Cell counts were analyzed every 2–3 days, with medium added to dilute the cell suspension to 1 x 10⁻⁶ cells / mL. 6 Cells / ml

[0074] Example 9. Validation of transduction by FACS Cells were washed and resuspended in FACS buffer (phosphate-buffered saline (PBS) with 0.1% sodium azide and 0.4% BSA). Cells were then divided into 1x10⁶ cells. 6 Divide into equal portions.

[0075] The Fc receptor was blocked by normal goat IgG (Life Technologies). Mouse ROR-1 scFv was detected using a biotinylated polyclonal goat anti-mouse F(ab)2 antibody (Life Technologies); a biotinylated normal polyclonal goat IgG antibody (Life Technologies) was used as an isotype control. Cells were incubated at 4°C for 25 min and washed once with FACS buffer. After staining with anti-F(ab)2 antibody, cells were stained with phycoerythrin (PE)-labeled streptavidin (BD Pharmingen, San Diego, CA) and allophycocyanin (APC)-labeled CD3 (eBiocience, San Diego, CA).

[0076] Example 10. Cytotoxicity assay Cytotoxicity was determined in real time using an ACEA machine according to the manufacturer’s protocol [6].

[0077] Example 11. Mouse ROR-1-CAR-T cells express high cytotoxic activity against ROR-1 positive cancer cells.

[0078] Mouse ROR1 ScFv expression was measured by FACS using anti-mouse FAB antibody ( Figure 3 (left image) and biotinylated ROR1 recombinant extracellular domain ( Figure 3(See right figure) confirms this. More than 50% of the extracellular domain protein was detected using mouse FAB or recombinant ROR1, confirming ROR1-ScFV CAR expression in expanded CAR-T cells.

[0079] To test the specific cytotoxicity of CAR-T cells, RTCA-based impedance measurements were performed using CHO-ROR1 and control CHO cells, according to the Agilent / ACEA manufacturer's specifications. In this measurement, the integrity of the target cell monolayer was continuously monitored by impedance in a weak electric field; CAR-T cell killing of target cells reduces the integrity of the monolayer, thereby decreasing its impedance. ROR1-CAR-transduced T cells were added to the target cells at a 10:1 effector cell:target cell (E:T) ratio (Figure 4). ROR-1 CAR-T cells resulted in a significant decrease in the impedance of the target cell CHO-ROR1 monolayer (…). Figure 4A This is more pronounced compared to T cells. Compared to T cells in ROR-1 negative CHO target cells, there was less killing ( Figure 4B Therefore, ROR-1-4-1BB-CD3CAR-T cells specifically kill ROR-1 positive target cells.

[0080] High cytotoxic activity was also observed in ROR1-41BB-CD3 CAR-T cells in ROR1-positive cancer target cells: pancreatic cancer BxPC3, colon cancer Lovo, and gastrointestinal (GI) OVCAR-5 cells. Figure 5A -C). ROR1-CAR-T cells also kill other cancer cell lines: HCT116, HT29, SW480, SW620 and lung cancer A549 (not shown).

[0081] Example 12. Mouse ROR-1-CAR secretes high levels of IFN-γ in response to ROR-1 positive cancer cells.

[0082] After co-incubating ROR1-41BB-CD3-CAR-T cells with target cells, we collected the supernatant and performed ELISA using the Fisher kit according to the manufacturer's protocol. Figure 6 The results showed that ROR-1-CAR-T cells secreted significantly higher levels of IFN-γ against ROR-1-positive CHO target cells than against ROR-1-negative CHO target cells. Figure 6 It also showed that ROR1-CAR-T cells secreted more IFN-γ than T cells secreted IFN-γ.

[0083] Figure 7A-7CThe results showed that ROR-1 CAR-T cells secreted IFN-γ against ROR-1 positive BxPC3 target (7A), Lovo (7B), and OVCAR-5 (7C) cancer cell lines.

[0084] Example 13. Mouse ROR1-CAR-T cells effectively blocked the growth of OVCAR-5 colorectal xenograft tumors in vivo.

[0085] 2x10 6 One OVCAR-5 cell was subcutaneously injected into both sides of NSG mice, followed by five intravenous injections of ROR-1-CAR-T cells (1 x 10⁶ cells per mouse) on days 2, 9, 16, 23, and 30 of each week. 7 ROR1-CAR-T cells significantly reduced xenograft tumor growth (p<0.05). Figure 8A The mice did not experience significant changes in body weight, indicating that there was no toxicity. Figure 8B ).

[0086] The results showed that ROR1-CAR-T cells were highly effective both in vitro and in vivo.

[0087] Example 14. In vitro efficacy of humanized ROR1-CAR-T cells Humanized PMC2509 CARs were prepared using humanized ROR-1 scFv (sequence details in Example 4). CAR-T cells were prepared using lentiviral PMC2509 CAR. The binding of ROR1 scFv to recombinant ROR1 protein in CAR-T cells was detected using FACS. Figure 9 Untransduced T cells were used as a negative control. Figure 9 ).

[0088] The in vitro efficacy of human ROR1-CAR-T cells was then tested using RTCA assays. Human ROR1-CAR-T cells effectively killed OVCAR-5 (… Figure 10A ) and PC3 cells ( Figure 10B Compared to T cells, PMC2509-CAR-T cells also killed more CHO-ROR1 cells. Figure 10C Compared to T cells, it caused less killing of ROR-negative CHO cells. Figure 10C ).

[0089] Compared to T cells, ROR1 is more effective against OVCAR-5 cells ( Figure 11A ) and PC3 ( Figure 11BPMC2509 ROR1-CAR-T cells secreted significantly more IFN-γ than CHO-ROR1 cells (p<0.05, Student's t-test). Figure 11C ).

[0090] Example 15. In vivo efficacy of humanized ROR1-CAR-T cells.

[0091] To demonstrate the in vivo efficacy of hROR1-CAR-T cells (PC2509), 4 x 10^6 OVCAR-5 cells were subcutaneously injected into NSG mice, followed by intravenous injection of 1 x 10^7 CAR-T cells on days 2, 9, 16, 23, and 30. In the NSG mouse model, PC2509-CAR-T cells significantly reduced OVCAR-5 tumor growth in vivo. Figure 12A The absence of significant changes in mouse body weight indicates that ROR1-CAR-T cells are not toxic. Figure 12B ).

[0092] References 1. Grupp, SA, Kalos, M., Barrett, D., Aplenc, R., Porter, DL, Rheingold, SR, Teachey, DT, Chew, A., Hauck, B., Wright, JF, et al. (2013). Chimeric antigen receptor-modified T cells for acute lymphoidleukemia. N Engl J Med 368 , 1509-1518. 2. Maus, MV, Haas, AR, Beatty, GL, Albelda, SM, Levine, BL, Liu, X., Zhao, Y., Kalos, M., and June, CH (2013). T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer ImmunolRes 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 hematologicmalignancies. 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 BCMAmonoclonal antibody block multiple myeloma Cell growth. Cancers (Basel), 10(9), 2018.

Claims

1. A human anti-human ROR-1 antibody or an antigen-binding fragment thereof, comprising: (a) 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:

11.

2. The human anti-human ROR-1 antibody or its antigen-binding fragment according to claim 1, wherein VH has the amino acid sequence of SEQ ID NO:

10.

3. The human anti-human ROR-1 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the fragment is a single-chain variable fragment (scFv).

4. The scFv according to claim 3, further comprising a connector between VH and VL.

5. The scFv according to claim 4, having 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) 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 its antigen-binding fragment according to claim 6, wherein VH has the amino acid sequence of SEQ ID NO:

3.

8. The mouse anti-human ROR-1 antibody or its antigen-binding fragment according to claim 6 or 7, wherein the fragment is a single-chain variable fragment (scFv).

9. The scFv according to claim 8, further comprising a connector between VH and VL.

10. The scFv according to claim 9, having the amino acid sequence of SEQ ID NO:

2.

11. A chimeric antigen receptor (CAR) comprising, from the N-terminus to the C-terminus: (i) The scFv of any one of claims 3-5 and 8-10. (ii) Transmembrane domains, (iii) at least one co-stimulatory domain, and (iv) Activate the structural domain.

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

13. The CAR of claim 11, wherein the activation domain is CD3zeta.

14. The CAR according to claim 13, having the amino acid sequence of SEQ ID NO: 24 or 26.

15. An isolated nucleic acid encoding the CAR according to 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.

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