Anti-ROR1 antibodies and engineered cells targeting ROR1

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 365,230, filed May 24, 2022, which is incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH none.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on May 22, 2023, is named CBI047.30_SL.xml, and is 55,553 bytes in size.

[0004] The present invention relates to the field of immunology, and more particularly to antibodies, T cell receptors, and immune cells that target ROR1, which are useful in the field of adoptive cellular immunotherapy for tumors. [Background technology]

[0005] Immunotherapy has emerged as a highly promising approach for the treatment of cancer. T cells or T lymphocytes, the armies of the immune system, constantly search for foreign antigens and distinguish abnormal (cancer or infected) cells from normal cells. Genetic modification of T cells or natural killer (NK) cells with CAR (chimeric antigen receptor) constructs is the most common approach to engineer tumor-specific T cells and NK cells. CAR-T cells and CAR-NK cells targeting tumor-associated antigens (TAAs) can be infused into patients (referred to as adoptive cell transfer or ACT), making them an efficient immunotherapy approach (see Grupp, et al., (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 368, 1509-1518; and Maus, et al., (2013) T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res 1, 26-31). The advantage of CAR-T (and CAR-NK) technology compared to chemotherapy or antibodies is that the engineered cells can proliferate and persist in the patient as a "living drug." Maus, et al., (2014). Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood 123, 2625-2635, and 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.

[0006] CARs typically consist of a monoclonal antibody-derived single-chain variable fragment (scFv), a hinge, a transmembrane domain, one or more intracellular co-activation domains (e.g., CD8, CD28, CD137 (4-1BB), CD27), and one or more activation domains (e.g., CD3-zeta domain) (see Figure 1 and Maus (2013) and Maus (2014) above). CAR evolution has progressed from first generation (no co-stimulatory domains) to second generation (one co-stimulatory domain) to third generation CARs (several co-stimulatory domains). CAR-T cells containing third generation CARs with multiple co-stimulatory domains have increased cytolytic activity and improved persistence, resulting in enhanced anti-tumor activity.

[0007] Natural killer (NK) cells are a type of cytotoxic lymphocyte important to the innate immune system. The role played by NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells, acting approximately 3 days after infection, and respond to tumor formation.

[0008] Tyrosine-protein kinase transmembrane receptor ROR1, also known as neurotrophic tyrosine kinase, receptor-related 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. ROR1 is a 937-amino acid protein, with amino acids 30–406 comprising the extracellular domain. The ROR1 gene encodes a receptor tyrosine kinase-like orphan receptor that regulates neurite outgrowth in the central nervous system. ROR1 is a glycosylated type I membrane protein belonging to the ROR subfamily of cell surface receptors. ROR1 is a receptor for the ligand WNT5A, which can activate the downstream NF-kappaB signaling pathway and cause suppression 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 sphere formation. ROR1 has been shown to be overexpressed in both hematologic and solid tumors, making it a useful target for CAR-T therapy.

[0009] Low expression of ROR1 has been shown in most normal human tissues, including adipose and soft tissues, bone marrow and immune system, endocrine tissues, female tissues, gastrointestinal tract, kidney and bladder, liver and gallbladder, lung, muscle, male tissues, and skin. Summary of the Invention

[0010] In some embodiments, the present invention provides a V H and V having an amino acid sequence at least 90% identical to SEQ ID NO:3. L and (iii) a humanized mouse amino acid sequence. In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv). In some embodiments, the scFv is a V comprising SEQ ID NO: 17. H and V comprising SEQ ID NO: 18 L and a linker. In some embodiments, the scFv comprises a V consisting of SEQ ID NO: 17. H and V consisting of SEQ ID NO: 18 L and a linker. In some embodiments, the scFv is encoded by a nucleic acid comprising SEQ ID NO: 38. In some embodiments, the scFv has V H and V L Complementarity determining regions (CDRs) are included in V H CDR1 of V contains the sequence TYA, H CDR2 of V comprises SEQ ID NO: 41, H CDR3 of V comprises SEQ ID NO: 42, L CDR1 of V comprises SEQ ID NO: 43, L CDR2 of V contains the sequence RAN L CDR3 of comprises SEQ ID NO:45.

[0011] In some embodiments, the invention is a chimeric antigen receptor (CAR) comprising an scFv and further comprising a transmembrane domain, at least one costimulatory domain, and an activation domain. In some embodiments, the costimulatory domain is CD28 or 4-1BB. In some embodiments, the activation domain is CD3 zeta. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CAR further comprises a signaling peptide and a hinge domain. In some embodiments, the signaling peptide and hinge domain are a CD8 signaling peptide and a CD8 hinge domain. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the CAR consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the CAR is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 39.

[0012] In some embodiments, the invention is an engineered immune cell that expresses a CAR of SEQ ID NO: 19. In some embodiments, the cell is selected from a CAR-T cell and a CAR-NK (natural killer) cell.

[0013] In some embodiments, the invention is a composition comprising an engineered immune cell expressing a CAR of SEQ ID NO: 19 and an excipient. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows first, second, and third generation chimeric antigen receptors (CARs) known in the art. [Figure 2] FIG. 1 shows the structure of anti-ROR1 CAR. [Figure 3] 1 is a Western blot showing binding of anti-ROR1 antibodies to human ROR1 antigen. [Figure 4] 1 shows FACS data demonstrating staining of different cell lines with anti-ROR1 antibodies using different cancer cell lines. [Figure 5A]RTCA assay showing dose-dependent 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. [Figure 5B] RTCA assay showing dose-dependent 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. [Figure 6] 1 shows the measurement of IFN-γ 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). [Figure 7] 1 shows an RTCA assay demonstrating the dose-dependent in vitro cytotoxicity of anti-ROR1 CAR-T cells bearing humanized scFvs (PMC1182, PMC1183, and PMC1194 expressing PMC857, PMC858, and PMC862 anti-ROR1 CARs, respectively) against SKOV3 cells. [Figure 8] 1 shows the measurement of IFN-γ secretion by anti-ROR1 CAR-T cells harboring 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

[0015] definition As used herein, "antibody" refers to an antigen-binding protein of the immune system. Naturally occurring antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant (CH) region. The heavy chain constant region is composed of three domains: CHI, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant CL region. The light chain constant region is composed of one domain, CL. VH and VL contain complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0016] As used herein, the term "human antibody" 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.

[0017] The term "humanized antibody" refers to an antibody 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.

[0018] As used herein, "antigen-binding fragment" refers to a protein fragment, including a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and an scFv, that has antigen-binding activity.

[0019] As used herein, a "chimeric antigen receptor (CAR)" is a receptor protein engineered to confer new capabilities to T cells to target specific proteins. The receptor is chimeric because it combines both antigen-binding and T-cell activation functions in a single receptor. A CAR is a fusion protein that includes an extracellular antigen-binding domain, a transmembrane domain, and at least one intracellular domain.

[0020] As used herein, "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to a specific antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits a signal that causes activation or inhibition of a biological process in a cell.

[0021] As used herein, "domain" means a region within a polypeptide that folds into a particular structure independently of other regions.

[0022] As used herein, "single-chain variable fragment (scFv)" refers to a single-chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Typical examples of scFvs include antigen-binding polypeptides formed by recombinant DNA technology in which the 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 scFvs are known to those skilled in the art.

[0023] As used herein, "tumor antigen" means a biological molecule that has antigenicity that is characteristic of a tumor.

[0024] The present inventors have used hybridoma technology to generate anti-ROR1 monoclonal antibodies that specifically target the human ROR1 antigen. The present inventors have generated anti-ROR1 CAR-T cells that target cancer cells that overexpress the ROR1 tumor antigen. The anti-ROR1 CAR-T cells of the present invention have high cytotoxic activity against several cancer cell lines and in vivo anti-tumor activity. Anti-ROR1 CAR-NK cells expressing the same CAR are also contemplated.

[0025] In some embodiments, the present invention provides a V H and V having the amino acid sequence of SEQ ID NO:3 L The present invention also includes a monoclonal mouse anti-human ROR1 antibody having the amino acid sequence of SEQ ID NO: 1, or an antigen-binding fragment thereof, comprising:

[0026] In some embodiments, the present invention provides a V H and V having the amino acid sequence of SEQ ID NO:6 L The present invention also includes a monoclonal mouse anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising:

[0027] In some embodiments, the present invention provides a V H and V having the amino acid sequence of SEQ ID NO: 10. L The present invention also includes a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising:

[0028] In some embodiments, the present invention provides a V H and V having the amino acid sequence of SEQ ID NO: 14 L The present invention also includes a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising:

[0029] In some embodiments, the present invention provides a V H and V having the amino acid sequence of SEQ ID NO: 18 LThe present invention also includes a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising:

[0030] In some embodiments, monoclonal anti-human ROR1 antibodies are raised against the extracellular domain of a purified recombinant fragment of human ROR1.

[0031] In some embodiments, the invention comprises a single-chain variable fragment (scFv) derived from any of the monoclonal mouse anti-human ROR1 antibodies disclosed herein, or the humanized versions thereof disclosed herein.

[0032] 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 costimulatory domain, and (iv) an activation domain.

[0033] Figure 1 shows the structures of a first-generation CAR lacking a costimulatory domain, a second-generation CAR with one costimulatory domain (CD28 or 4-1BB), and a third-generation CAR with two or more costimulatory 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).

[0034] Figure 2 shows the structure of an anti-ROR1 CAR of the present invention. Second generation CARs were used with either a CD28 or 4-1BB costimulatory domain. (A CAR with a CD28 costimulatory domain is shown.) In Figure 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 costimulatory 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 scFv configuration is V H -Linker-V L In some embodiments, the configuration is V L -Linker-V H is.

[0035] The costimulatory domain can be selected from the group consisting of CD28, 4-1BB (CD137), GITR, ICOS-1, CD27, OX-40, and DAP10 costimulatory domains. In some embodiments, the costimulatory domain is CD28.

[0036] In some embodiments, the activation domain is CD3 zeta (CD3 Z or CD3-zeta), which is encoded by the CD247 gene.

[0037] Transmembrane domains may be derived from naturally occurring polypeptides or may be artificially designed. Transmembrane domains derived from naturally occurring polypeptides can be obtained from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of T cell receptor α or β chain, CD3-zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR. Artificially designed transmembrane domains are polypeptides containing primarily hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0038] 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 polypeptide, e.g., having a length of 2 to 10 amino acids. Peptide linkers generally comprise about 5 to about 40 amino acids. The linker may 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 to 40 amino acids from the CH1, CH2, or CH3 domain of an immunoglobulin heavy chain. In some embodiments, the linker comprises the sequence (G x S y ) n

[0023] Additional linker examples and sequences are disclosed in U.S. Patent No. 5,525,491 (Serine-rich peptide linkers), U.S. Patent No. 5,482,858 (Polypeptide linkers for the production of biosynthetic proteins), and publication WO2014 / 087010 (Improved polypeptides against IgE).

[0039] In some embodiments, the present invention comprises one or more nucleic acids encoding anti-ROR1 CARs. Nucleic acids encoding CARs can be prepared from the amino acid sequence of a particular CAR by conventional methods. Nucleic acid sequences encoding the amino acid sequence can be obtained using tools publicly provided by the National Center for Biotechnology Information (NCBI), for example, from the NCBI RefSeq ID or accession number in GenBank for the amino acid sequence of each domain. Nucleic acids of the present invention can be prepared using standard molecular biological or chemical procedures. In some embodiments, portions of the nucleic acid are synthesized based on the nucleotide sequence. In some embodiments, nucleic acids of the present invention are prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0040] In some embodiments, a nucleic acid encoding a 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 oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), an adenoviral vector, an adeno-associated viral (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 replication-deficient viral vector is used so that it does not replicate autonomously in infected cells.

[0041] In some embodiments, retroviral particles are prepared using a packaging cell line. In such embodiments, a suitable packaging cell line is selected based on the LTR sequence and the packaging signal sequence carried by the viral vector. Examples of 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 HEK293 or HEK293t cell lines, which have high transfection efficiency. Those skilled in the art are aware of many commercially available retroviral vectors and packaging cell lines.

[0042] CAR-T cells (or CAR-NK cells) bind to a specific antigen via the CAR, thereby transmitting a signal to the cell and activating it. Activation of CAR-expressing cells varies depending on the cell type and the type of intracellular domain of the CAR. Cell activation can be confirmed, for example, based on cytokine release, any improvement in cell proliferation rate, any change in cell surface molecules, etc. Furthermore, the release of cytotoxic cytokines (IFNγ, TNFα, etc.) from activated CAR-T cells (or CAR-NK cells) causes the destruction of target cells expressing the antigen, which can be detected or measured. In addition, the release of cytokines or changes in cell surface molecules result in detectable or measurable stimulation of other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0043] In some embodiments, cells expressing a CAR are used as a therapeutic agent for a disease. The therapeutic agent comprises cells expressing a CAR as an active ingredient and may further comprise a suitable excipient.

[0044] In one embodiment, the present 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. The anti-ROR1 CAR-T cells or CAR-NK cells exhibit 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.

[0045] In some embodiments, the present invention provides a humanized VL1 antibody of a mouse monoclonal anti-human ROR1 antibody. H and V L , humanized V H and V L and CAR-T cells (or CAR-NK cells) harboring a CAR comprising a humanized anti-ROR1 scFv that targets ROR1-positive cells. Without being bound to one particular theory, the inventors believe that at least one advantage of humanizing the mouse anti-ROR1 scFv is that the immune response against CAR-T (CAR-NK) cells in humans is potentially reduced.

[0046] In some embodiments, an anti-ROR1 antibody or antigen-binding fragment or derivative thereof (such as an scFv) comprises complementarity-determining regions (CDRs). Each of the light and heavy chains of the antibody comprises three CDRs. In some embodiments, the CDRs are identified using a crystal structure of an antigen-antibody complex. In some embodiments, the CDRs are identified using an in vitro method such as phage display. In some embodiments, the CDRs are identified using in silico methods, such as 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., USHHS, NIH). 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 portion of a CDR is identified as the overlap between the sequence identified by the IMGT tool and the sequence identified by the Kabat tool.

[0047] In some embodiments, the anti-ROR1 scFv is V H In some embodiments, the anti-ROR1 scFv comprises a TYA sequence in CDR1 of V H In some embodiments, the anti-ROR1 scFv comprises SEQ ID NO: 41 in CDR2 of V H In some embodiments, the anti-ROR1 scFv comprises SEQ ID NO: 42 in the CDR3 of V L In some embodiments, the anti-ROR1 scFv comprises SEQ ID NO: 43 in CDR1 of V L In some embodiments, the anti-ROR1 scFv comprises the sequence R A N in CDR2 of V L The CDR3 of the antibody contains SEQ ID NO: 45.

[0048] Some anti-ROR1 scFvs have H The CDR1 sequence of H CDR2 of SEQ ID NO: 41, V H CDR3 of V contains SEQ ID NO: 42, L CDR1 of SEQ ID NO: 43, V L CDR2 sequences of RAN, V L and further comprising SEQ ID NO: 45 in CDR3. [Table 1]

[0049] In some embodiments, the anti-ROR1 scFv comprises a light chain (V L ) and the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain (V H ) contains CDR1, CDR2, and CDR3, and V H The CDR1 sequence of H CDR2 of SEQ ID NO: 41, V H CDR3 of SEQ ID NO: 42 、 V L CDR1 of SEQ ID NO: 43, V L The CDR2 sequences of R, and V L In some embodiments, the anti-ROR1 scFv comprises SEQ ID NO: 45 in its CDR3. H CDR1 of V consists of the sequence TYA, H CDR2 of V consists of SEQ ID NO: 41, H CDR3 of V consists of SEQ ID NO: 42, L CDR1 of V consists of SEQ ID NO: 43, L CDR2 of V consists of the sequence RAN L CDR3 of the present invention consists of SEQ ID NO:45.

[0050] The humanized anti-ROR1 antibodies and scFvs derived therefrom disclosed herein can be used in the following immunotherapy applications: toxin-drug conjugated antibodies, monoclonal therapeutic antibodies, bispecific antibodies, and CAR-T cell (or CAR-NK cell)-based immunotherapy.

[0051] Anti-ROR1 CAR-T cells (or CAR-NK cells) generated using the anti-ROR1 antibodies disclosed herein can be effectively used to target the ROR1 antigen in ROR1-positive cells and tumors. Anti-ROR1 CAR-T cells (or CAR-NK cells) can be used clinically against chemotherapy-resistant, aggressive tumor-forming tumor cells, tumors, and cancer stem cells.

[0052] Anti-ROR1 CAR-T cells (or CAR-NK cells) can be combined with different therapeutic agents: checkpoint inhibitors; targeted therapies, small molecule inhibitors, antibodies, etc. For example, anti-ROR1 CAR-T cells (or CAR-NK cells) can be combined with CAR-T (or CAR-NK) cells that target other tumor antigens or antigens present in the tumor microenvironment (e.g., VEGFR-1-3, PDL-1, CD80). Bispecific antibodies and scFvs (e.g., bispecific for ROR1 and CD3), as well as cells expressing antibodies and scFvs, can be used to improve the activity of ROR1-targeted therapies.

[0053] The anti-ROR1 antibodies and derivatives thereof disclosed herein can be modified by site-directed mutagenesis, e.g., error-prone PCR for affinity tuning, and selected by affinity maturation. Co-activation domain modifications: CD28, 4-1BB, and others can be used to increase the efficacy of CARs generated from the antibodies (and their derivatives) disclosed herein. Tag-conjugated anti-ROR1 scFv can be used for CAR production. First-, second-, and third-generation CAR constructs can be made with the same anti-ROR1 scFv disclosed herein.

[0054] The anti-ROR1 CARs disclosed herein can be used to generate CAR-T cells, CAR-NK cells, and other types of cells, such as T cells, NK cells, macrophages, and other anti-ROR1 CAR-expressing hematopoietic cell-derived iPSCs (induced pluripotent stem cells) that can target ROR1-positive cancers. The present invention provides T cells, or NK cells, or macrophages, or hematopoietic cells that have been modified to express an anti-ROR1 CAR.

[0055] The CAR-expressing cells disclosed herein can be autologous and allogeneic cells.

[0056] The following examples further illustrate the present invention. These examples are intended to be merely illustrative of the invention and should not be construed as limiting. [Example]

[0057] We generated an anti-ROR1 CAR construct and cloned it into a lentiviral vector. The CAR construct contains an anti-ROR1 ScFv-CD28-CD3 zeta insert (or a similar insert with a 41BB costimulatory domain instead of the CD28 domain). The CMV, EF1, or MNDU3 promoter can be used to drive expression of the CAR construct. Lentivirus was generated in HEK293t cells, and titers were established by RT-PCR. Equal amounts of lentivirus were then used to transduce T cells, as described in the Examples.

[0058] Example: Anti-ROR1 scFv was used to detect ROR1 protein by Western blotting, and anti-ROR1 antibody was used to detect ROR1 by FACS staining. In this example, a mouse monoclonal anti-ROR1 antibody was generated using standard hybridoma technology. The mouse anti-ROR1 antibody (IgG1 type) detected extracellular ROR1 protein by ELISA (data not shown). This hybridoma clone, 2H6, was sequenced and identified as V. H and V LThe scFv was generated using the method described above (see further Example 2). Western blot analysis showed that the anti-ROR1 scFv binding the ROR1 extracellular domain was fused to a human Fc (hFc) protein (Figure 3, left panel). The ROR1-human Fc fusion protein was detected with an antibody against the human Fc domain. The right panel of Figure 3 shows the ROR1 antigen detected with anti-ROR1 scFv mouse Fc fusion as the primary antibody and anti-mouse IgG-HRP as the secondary antibody.

[0059] Furthermore, we performed fluorescence-activated cell sorting (FACS) experiments and demonstrated that the mouse anti-ROR1 monoclonal antibody detected elevated ROR1 expression in several cancer cell lines, including hepatocellular carcinoma (HepG2), breast cancer (MDA231), colon cancer (HT-29), and ovarian cancer (SKOV-3). Normal keratinocytes were used as a negative control (Figure 4). (MFI: medium fluorescence intensity relative to the isotype).

[0060] Example 2. Anti-ROR1 V H , V L , and sequencing of the CAR construct In this example, the anti-ROR1 antibody, hybridoma clone 2H6, was sequenced. H , V L、 The sequences of the anti-ROR1 scFv are shown below. H -Linker-V L and the linker has the sequence (G4S)3 (SEQ ID NO: 46). In the following sequence, the sequence is V H and the underscore is V L The nucleotide sequence of is shown, with the linker sequence in italics.

[0061] Anti-ROR1 scFv (mouse clone 2H6) nucleotide sequence (SEQ ID NO: 20): [ka]

[0062] Anti-ROR1 scFv (mouse clone 2H6) amino acid sequence: (SEQ ID NO: 1): [ka]

[0063] Anti-ROR1 scFv (mouse clone 2H6)V H Amino acid sequence (SEQ ID NO:2): VKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFGNSVYYAMDYWGQGTSVTVSS

[0064] Anti-ROR1 scFv (mouse clone 2H6)V L Amino acid sequence (SEQ ID NO: 3): DIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKR

[0065] Example 3. Anti-ROR1-CAR sequence with mouse anti-ROR1 scFv In this example, a CAR was designed with an scFv derived from the murine anti-ROR1 antibody 2H6. The scheme of the anti-ROR1 CAR construct is shown in Figure 2. The lentiviral vector Lenti CMV-MCS-EF1a-puro was used for cloning the CAR sequence. The CD3 zeta CAR construct was under the control of the CMV promoter. For the 4-1BB CAR construct, a different lentiviral vector with the MNDU3 promoter was used to obtain a higher percentage of CAR-expressing cells.

[0066] A. CD28 as a costimulatory domain The CAR comprises the following structure: anti-ROR1 scFv-CD8 hinge-CD28 TM-CD28 costimulatory domain CD3 zeta activation domain (Figure 2). This structure further comprises a human CD8 signaling peptide. The anti-ROR1 scFv is represented by structure V H-Linker(G4S)3-V L ("(G4S)3", disclosed as SEQ ID NO: 46).

[0067] CD8 signaling peptide nucleotide sequence (SEQ ID NO:21): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0068] CD8 signaling peptide amino acid sequence (SEQ ID NO: 22): MALPVTALLLPLALLLHAARP

[0069] Nhe I restriction site: GCTAGC

[0070] XhoI restriction site: CTCGAG

[0071] CD8 hinge nucleotide sequence (SEQ ID NO: 24): AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGAT

[0072] CD8 hinge amino acid sequence (SEQ ID NO: 25): KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD

[0073] CD28 TM / activation nucleotide sequence (SEQ ID NO: 26): TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

[0074] CD28 TM / activation amino acid sequence (SEQ ID NO: 27): FWVLVVVGGVLACYSLLVTVAFIIFWV / RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0075] CD3 zeta nucleotide sequence (SEQ ID NO:28): AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0076] CD3 zeta amino acid sequence (SEQ ID NO:29): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0077] EcoRI restriction site: GAATTC

[0078] Anti-ROR1 CAR (mouse) nucleotide sequence (SEQ ID NO: 30):

[0079] Anti-ROR1 CAR (mouse) amino acid sequence (SEQ ID NO: 4): MALPVTALLLPLALLLHAARPASVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFG NSVYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQ YDEFPYTFGGGTKLEIKRLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0080] We also generated a CAR with a 4-1BB costimulatory domain instead of the CD28 activation domain. This construct, PMC1195, was cloned into a vector containing the KanR gene. The ROR1 scFv was inserted between the Nhe I and Xho I sites in the sequence (underlined). CAR expression was under the control of the MNDU3 promoter.

[0081] The nucleotide sequence of the codon-optimized CAR (anti-ROR1 scFv-4-1BB-CD3zeta) is shown below. The scFv is inserted between the Nhe I and Xho I sites (underlined). 4-1BB is in italics, followed by the CD3-zeta domain.

[0082] Anti-ROR1 CAR (4-1BB in place of CD28 activation domain) nucleotide sequence (SEQ ID NO:31): [ka]

[0083] B.4-1BB as a costimulatory domain Additionally, the structure (human CD8 signaling peptide-alternative (see below) anti-ROR1 scFv (V H -Linker(G4S)3-V L ("(G4S)3" disclosed as SEQ ID NO: 46), CD8 hinge, CD28 transmembrane domain, 4-1BB costimulatory domain, CD3 zeta activation domain). In the alternative scFv, each segment of the sequence is represented by a V H , V L The same as the segment in Example 3(A) except for V H is represented by SEQ ID NO: 5 (the first amino acid is E, which is not present in SEQ ID NO: 2). L is represented by SEQ ID NO: 6 (with the terminal R removed compared to SEQ ID NO: 3).

[0084] Alternative anti-ROR1 scFv amino acid sequence (SEQ ID NO: 23): [ka]

[0085] Anti-ROR1 CAR Alternative V H Amino acid sequence (SEQ ID NO: 5): EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFGNSVYYAMDYWGQGTSVTVSS

[0086] Compared to SEQ ID NO:2, SEQ ID NO:5 has an extra E at the N-terminus.

[0087] Anti-ROR1 CAR Alternative VL Acid sequence (SEQ ID NO: 6): DIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK

[0088] Compared to SEQ ID NO:3, SEQ ID NO:6 lacks an R at the C-terminus.

[0089] 4-1BB domain nucleotide sequence: (SEQ ID NO:32): AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0090] 4-1BB amino acid sequence (SEQ ID NO:33): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0091] Anti-ROR1 CAR (alternative scFv and 4-1BB) amino acid sequence (SEQ ID NO: 7): (V H is underlined, the linker (G4S)3 (SEQ ID NO: 46) is in italics, and V L is underlined, and the 4-1BB domain is underlined in italics.)

[0092] [ka]

[0093] Example 4. ROR1 CAR with humanized ROR1 scFv In this example, mouse anti-ROR1 V H (SEQ ID NO: 5) and mouse V L(SEQ ID NO: 6) was humanized to generate several humanized scFvs. Using the 4-1BB domain and CD3 zeta domain, a CAR with three scFvs having the same structure as in Example 3(B) was generated. Several humanized scFvs were tested, and three scFvs were selected based on their best performance in the functional assays described below. The humanized scFvs were inserted between the Nhe I and Xho I sites of the CAR sequence.

[0094] Three CARs with humanized anti-ROR1 scFv are shown below: PMC857, PMC858, and PMC862.

[0095] A. PMC857 scFv and CAR Humanized anti-ROR1 scFv PMC857 nucleotide sequence: (SEQ ID NO: 34): GAA GTA CAG CTT GTT GAA TCA GGT GGT GGT CTT ATT CAG CCA GGA GGC TCC TTG CGA CTG AGC TGT GCC GCT TCT GGG TTC ACC TTT AGC ACT TAC GCA ATG AGT TGG GTC CGA CAA GCC CCA GGT AAG GGA TTG GAA TGG GTA AGT TCC ATT TCC AGC GGA GGG AAC ACT TAT TAC GCC GAT TCT GTG AAA GGA CGC TTT ACT ATA TCC CGA GAC AAT AGT AAA AAC ACA TTG TAT TTG CAA ATG AAC TCT TTG AGG GCC GAG GAC ACT GCC GTC TAC TAT TGT GCC CGC GAC AGC TAT TAT TTC GGC AAC TCT GTG TAT TAC GCG ATG GAT TAC TGG GGT GCC GGC ACA ACT GTC ACC GTT TCA TCT GGC GGA GGA GGC AGT GGC GGA GGG GGC TCA GGC GGT GGT GGA AGT GAT ATT CAA ATG ACC CAA TCA CCC TCT TCA TTG TCT GCA AGC GTA GGT GAC CGA GTC ACG ATA ACC TGC AAA GCC TCT CAA GAT ATT AAT TCA TAC TTT TCT TGG TTT CAA CAA AAA CCG GGA AAG GCG CCT AAG TCA TTG ATT TAC CGC GCG AAC CGG TTG GTA TCA GGA GTA CCG TCA AGA TTC TCA GGG AGT GGG TCA GGC ACA GAT TTC ACA CTC ACT ATT TCT TCC TTG CAA CCT GAA GAC TTC GCA ACC TAT TAT TGC TTG CAG TAT GAT GAG TTT CCG TAC ACT TTC GGG GGG GGT ACA AGG CTG GAG ATC AAA

[0096] Humanized anti-ROR1 scFv PMC857 amino acid sequence: (SEQ ID NO: 8): EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0097] Humanized anti-ROR1 scFv PMC857 V H Amino acid sequence (SEQ ID NO:9): EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTV

[0098] Humanized anti-ROR1 scFv(PMC857)V L Amino acid sequence (SEQ ID NO: 10): DIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0099] Humanized anti-ROR1 CAR scFv PMC857 nucleotide sequence: (SEQ ID NO: 35): <h2 style=";text-align:left;direction:ltr">ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCTAGC GAA GTA CAG CTT GTT GAA TCA GGT GGT GGT CTT ATT CAG CCA GGA GGC TCC TTG CGA CTG AGC TGT GCC GCT TCT GGG TTC ACC TTT AGC ACT TAC GCA ATG AGT TGG GTC CGA CAA GCC CCA GGT AAG GGA TTG GAA TGG GTA AGT TCC ATT TCC AGC GGA GGG AAC ACT TAT TAC GCC GAT TCT GTG AAA GGA CGC TTT ACT ATA TCC CGA GAC AAT AGT AAA AAC ACA TTG TAT TTG CAA ATG AAC TCT TTG AGG GCC GAG GAC ACT GCC GTC TAC TAT TGT GCC CGC GAC AGC TAT TAT TTC GGC AAC TCT GTG TAT TAC GCG ATG GAT TAC TGG GGT GCC GGC ACA ACT GTC ACC GTT TCA TCT GGC GGA GGA GGC AGT GGC GGA GGG GGC TCA GGC GGT GGT GGA AGT GAT ATT CAA ATG ACC CAA TCA CCC TCT TCA TTG TCT GCA AGC GTA GGT GAC CGA GTC ACG ATA ACC TGC AAA GCC TCT CAA GAT ATT AAT TCA TAC TTT TCT TGG TTT CAA CAA AAA CCG GGA AAG GCG CCT AAG TCA TTG ATT TAC CGC GCG AAC CGG TTG GTA TCA GGA GTA CCG TCA AGA TTC TCA GGG AGT GGG TCA GGC ACA GAT TTC ACA CTC ACT ATT TCT TCC TTG CAA CCT GAA GAC TTC GCA ACC TAT TAT TGC TTG CAG TAT GAT GAG TTTCCG TACACTTTCGGGGGGGGTACAAGGCTGGAGATCAAACTCGAGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTCTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

[0100] Amino acid sequence of humanized anti-ROR1 CAR (scFv PMC857): (SEQ ID NO: 11): MALPVTALLLPLALLLHAARPASEVQLVESGGGLIQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYF GNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLLVSGVPSRFSGSGGTDFTLTISSLQPEDFATYYCL QYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0101] B. PMC858 scFv and CAR Humanized anti-ROR1 scFv PMC858 nucleotide sequence (SEQ ID NO: 36): CAG GTA CAA TTG GTA GAG TCC GGC GGA GGG GTT GTT CAG CCA GGA CGG TCC TTG CGG TTG TCT TGT GCT GCG TCA GGA TTC ACA TTC TCA ACG TAC GCG ATG TCT TGG GTG CGC CAA GCT CCC GGT AAA GGG CTG GAA TGG GTG GCC TCA ATC TCA TCT GGA GGG AAC ACT TAC TAC CCT GAT AGT GTT AAA GGT CGC TTT ACT ATC TCA AGG GAC AAT AGC AAG AAT ACC TTG TAT CTG CAA ATG AAC TCA CTT AGA GCA GAG GAC ACA GCG GTA TAT TAC TGT GCT AGA GAC TCA TAT TAT TTC GGC AAC TCC GTT TAT TAC GCG ATG GAT TAC TGG GGC GCA GGG ACT ACG GTA ACT GTA TCT TCT GGT GGT GGA GGG TCT GGG GGC GGG GGT AGT GGC GGC GGT GGC AGT GAC ATC CAG ATG ACA CAG TCT CCG TCT TCA TTG AGT GCA AGC GTC GGC GAT CGG GTT ACC ATT ACG TGT AAG GCA AGT CAG GAC ATC AAC AGT TAT TTT TCA TGG TTT CAA CAA AAG CCT GGA AAA GCG CCG AAA TCA CTC ATT TAC CGA GCT AAT AGG CTT GTC TCT GGC GTT CCG TCT CGC TTC AGT GGA AGT GGG AGC GGT ACT GAT TTT ACC CTC ACC ATA TCA AGC CTT CAA CCG GAG GAT TTT GCC ACG TAC TAT TGT CTC CAG TAC GAT GAA TTT CCA TAT ACG TTT GGC GGC GGG ACT CGC TTG GAG ATT AAA

[0102] Humanized anti-ROR1 scFv PMC858 amino acid sequence (SEQ ID NO: 12): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0103] Humanized anti-ROR1 scFv PMC858 V H Amino acid sequence: (SEQ ID NO: 13): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSS

[0104] Humanized anti-ROR1 scFv PMC858 V L Amino acid sequence (SEQ ID NO: 14): DIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0105] Humanized anti-ROR1 CAR (scFv PMC858) nucleotide sequence (SEQ ID NO: 37):

[0106] Humanized anti-ROR1 CAR (scFv PMC858) amino acid sequence (SEQ ID NO: 15): MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYF GNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCL QYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0107] C. PMC862 scFv and CAR Humanized anti-ROR1 scFv PMC862 nucleotide sequence (SEQ ID NO: 38): CAG GTA CAA CTG GTG GAA TCC GGC GGG GGA GTA GTA CAG CCC GGA CGA TCT CTT CGA CTC TCA TGT GCA GCG TCC GGG TTC ACT TTT TCT ACC TAC GCA ATG TCA TGG GTA CGA CAG GCG CCG GGC AAA GGC CTC GAA TGG GTT GCA TCC ATT TCA TCA GGA GGT AAT ACA TAT TAT CCT GAT TCA GTC AAG GGC CGA TTC ACG ATT AGT CGA GAT AAT AGC AAG AAC ACT CTC TAC TTG CAG ATG AAC TCC CTG CGG GCT GAG GAC ACG GCC GTG TAT TAT TGC GCT CGC GAT AGT TAT TAC TTC GGC AAT TCC GTA TAT TAT GCG ATG GAC TAT TGG GGC GCC GGT ACT ACC GTG ACT GTT TCC TCT GGT GGG GGT GGG TCC GGG GGC GGT GGT TCA GGT GGA GGC GGA TCC GAC ATT CAA ATG ACC CAG TCT CCC TCA AGT TTG TCT GCA TCT GTT GGC GAT AGA GTT ACA ATA ACA TGC AAA GCC AGT CAA GAC ATC AAC TCA TAC TTC TCC TGG TAT CAA CAA AAG CCA GGA AAA GCT CCG AAA CTG TTG ATC TAC CGG GCC AAC CGG CTG GTC ACT GGC GTG CCA TCC CGG TTC AGT GGC AGC GGA AGC GGA ACA GAT TTC ACG TTT ACC ATC TCT AGC CTC CAA CCG GAG GAC ATC GCA ACA TAC TAT TGC CTT CAG TAT GAT GAG TTT CCC TAC ACT TTC GGT GGC GGC ACC CGA CTT GAG ATC AAA

[0108] Humanized anti-ROR1 scFv PMC862 amino acid sequence (SEQ ID NO: 16): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTRLEIK

[0109] Humanized anti-ROR1 scFv PMC862 V H Amino acid sequence (SEQ ID NO: 17): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSS

[0110] Humanized anti-ROR1 scFv PMC862V L Amino acid sequence (SEQ ID NO: 18): DIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTRLEIK

[0111] Humanized anti-ROR1 CAR (scFv PMC862) nucleotide sequence (SEQ ID NO: 39):

[0112] Humanized anti-ROR1 CAR (scFv PMC862) amino acid sequence (SEQ ID NO: 19): MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYF GNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCL QYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0113] Example 5. Generating CARs using lentiviral vectors In this example, a CAR containing the three humanized scFvs from Example 4 was packaged into a lentiviral vector. Lentivirus was produced by standard procedures 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.

[0114] Example 6. Isolation of peripheral blood mononuclear cells (PBMCs) from whole blood In this example, PBMCs were isolated from whole blood for the purpose of generating CAR-T cells. Whole blood (Stanford Hospital Blood Center, Stanford, Calif.) was collected from individual or mixed donor samples (depending on the volume of blood required) in 10 mL heparin vacutainers (Becton Dickinson, San Jose, Calif.). Approximately 10 mL of whole anticoagulated blood was placed in a 50 mL conical centrifuge tube with a total volume of 20 mL of sterile phosphate-buffered saline (PBS pH 7.4, Ca). 2+ and Mg 2+- The diluted plasma was mixed with Ficoll-free solution. The layer of cells containing PBMCs found at the diluted plasma / Ficoll interface was carefully removed, avoiding the Ficoll, washed twice with PBS, and centrifuged at 200 × g for 10 minutes at room temperature. The cells were counted using a hemocytometer. PBMCs were washed once with CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies, San Diego, Calif.)) containing 5% AB serum, 1.25 μg / mL amphotericin B (Gemini Bioproducts, Woodland, Calif.), 100 U / mL penicillin, and 100 μg / mL streptomycin, and either used for experiments or frozen at 80°C.

[0115] Example 7. T cell activation from PBMCs Isolated PBMCs were incubated in 1x PBS (pH 7.4, Ca 2+ / Mg 2+ once in the absence of human interleukin-2 (huIL2) and once in the absence of 5x10 5 The cells were washed in CAR-T medium (Example 6) at a concentration of 10 cells / mL, and then cultured at 5x10 in CAR-T medium (from 1000x stock; Invitrogen, Carlsbad, Calif.) containing 300 U / mL of huIL2. 5 The beads were resuspended to a final concentration of 1000 cells / mL. PBMCs and beads (for T cell activation) were then mixed at a 1:1 bead-to-cell ratio by transferring 25uL of beads to 1mL of PBMCs and incubating at 37°C in the presence of CO for 24 hours before viral transduction.

[0116] Example 8. T cell transduction and expansion After PBMC activation, 5x10 6 5x10 lentivirus 5 10 T cells (MOI 10:1) and 2 μL / mL of Transplus (Alstem, Richmond, Calif.) medium was added to give a final dilution of 1:500. Cells were incubated for an additional 24 hours before virus addition was repeated. Cells were then grown in the presence of 300 U / mL of IL-2 for 12–14 days (total incubation time depended on the final number of CAR-T cells required). Cell numbers were analyzed every 2–3 days, at which point medium was added and the cell suspension was diluted to 1×10 6 cells / ml.

[0117] Example 9. T cell transduction and CAR validation by FACS The cells of Example 8 were washed and suspended in FACS buffer (PBS + 0.1% sodium azide and 0.4% BSA). The cells were then diluted to 1 x 10 6 Cells were divided into aliquots. Fc receptors were blocked with normal goat IgG (Life Technologies, San Diego, Calif.). Biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody was used to detect mouse anti-ROR1 scFv, and biotin-labeled normal polyclonal goat IgG antibody also served as an isotype control. Cells were incubated at 4°C for 25 minutes and washed once with FACS buffer. After staining with anti-F(ab)2 antibody, cells were stained using phycoerythrin (PE)-labeled streptavidin (BD Pharmingen, San Diego, Calif.) and allophycocyanin (APC)-labeled CD3 (eBiocience, San Diego, Calif.). For humanized anti-ROR1 scFv, anti-human F(ab)2 antibody (Life Technologies) was also used.

[0118] Example 10. Real-time cytotoxicity assay. Cytotoxicity was performed using the xCELLigence real-time cell analysis system (Agilent, San Jose, Calif.) 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).

[0119] Example 11. CAR-T cells bearing mouse anti-ROR1 scFv exhibited high cytotoxic activity against ROR1-positive cancer cells. Expression of the mouse scFv anti-ROR1 CAR was confirmed by FACS using an anti-mouse Fab antibody. CAR-T cells bearing a 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 an RTCA impedance-based assay on the xCELLigence system according to the manufacturer's conditions. In this assay, the integrity of the target cell monolayer is continuously monitored via its impedance in a weak electric field. Killing of target cells by CAR-T cells reduces the monolayer integrity and therefore its impedance. Anti-ROR1 CAR-transduced T cells were added to target cells at effector:target (E:T) ratios of 10:1, 20:1, 30:1, and 40:1 (Figure 5, panel A). CAR-T cells caused a sustained, dose-dependent decrease in target cell monolayer impedance. Thus, anti-ROR1-CD28-CD3 CAR-T cells killed ROR1-positive SKOV-3 ovarian solid tumor cells in a dose-dependent manner.

[0120] Similar high cytotoxic activity was observed with CAR-T cells bearing a CAR comprising a murine anti-ROR1 scfv, a 4-1BB costimulatory domain, and a CD3 zeta activation domain (Example 3(B)), as well as with ROR1-positive SKOV-3 target cells (Figure 5, panel B).

[0121] Example 12. Anti-ROR1-CAR T cells (mouse scFv) secrete high levels of IFNγ in the presence of ROR1-positive cancer cells. After co-incubation of ROR1-41BB-CD3-CAR-T cells with SKOV-3 cells, supernatants were collected and subjected to ELISA using a commercially available kit (ThermoFisher Scientific, Waltham, Mass.). The nonadherent HL-60 ROR1-negative cell line was used as a control. Anti-ROR1-CAR-T cells secreted significantly higher levels of IFN-γ in the presence of ROR1-positive SKOV-3 cancer cells than in the presence of ROR1-negative control cells, and compared with T cells and mock CAR-T cells used as a control (P<0.05) (Figure 6).

[0122] Example 13. Anti-ROR1-CAR T cells (humanized scFv) exhibited cytotoxicity and secreted high levels of IFN-γ in the presence of ROR1-positive cancer cells. First, CAR-T cells were tested using scFv PMC857, PMC868, or PMC862 (Example 5) in a cytotoxicity assay using ROR1-positive cells, demonstrating that the CAR-T cells were highly cytotoxic. Next, these CAR constructs (PMC857, PMC868, or PMC862) were inserted into a lentiviral vector carrying the KanR gene (preferred for clinical use) instead of the AmpR gene. The resulting CAR-T cell clones were PMC1182, 1183, and 1194, respectively. CAR expression in the CAR-T cells was approximately 30% CAR+, as detected by FACS using human Fab. RTCA assays were performed to detect high cytotoxic activity of these CAR-T cells against SKOV-3 (ROR1-positive) cells (Figure 7).

[0123] Next, cytokine secretion by CAR-T cells was evaluated. After co-incubation of CAR-T cells with SKOV-3 cells, culture supernatants were collected and ELISA was performed to detect interferon-γ in the supernatants as described in Example 12, using the non-adherent HL-60 ROR1-negative cell line as a control. Anti-ROR1 CAR-T cells secreted significantly higher levels of IFN-γ in the presence of ROR1-positive SKOV-3 cancer cells than in the presence of ROR1-negative control cells, and compared to T cells and mock CAR-T cells used as a control (P<0.05) (Figure 8).

[0124] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various modifications can be made within the scope of the invention. Accordingly, the scope of the invention should be limited not by the embodiments described herein, but by the claims set forth below.

Claims

1. V having an amino acid sequence at least 90% identical to SEQ ID NO:2 Ｈ and V having an amino acid sequence at least 90% identical to SEQ ID NO:

3. Ｌ and an anti-human ROR1 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises the V Ｈ and the V Ｌ and a complementarity determining region (CDR) in said V Ｈ wherein the CDR1 of said V comprises the sequence TYA; Ｈ wherein CDR2 of said V comprises SEQ ID NO: 41; Ｈ wherein the CDR3 of said V comprises SEQ ID NO: 42; Ｌ wherein CDR1 of said V comprises SEQ ID NO: 43; Ｌ wherein CDR2 of said V comprises the sequence RAN; Ｌ An anti-human ROR1 antibody or an antigen-binding fragment thereof, wherein CDR3 comprises SEQ ID NO:

45.

2. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1 , comprising a humanized mouse amino acid sequence.

3. The anti-human ROR1 antibody or antigen-binding fragment thereof according to claim 2, wherein the antigen-binding fragment is a single-chain variable fragment (scFv).

4. V comprising SEQ ID NO: 17 Ｈ and V comprising SEQ ID NO: 18 Ｌ and a linker.

5. V consisting of SEQ ID NO: 17 Ｈ and V consisting of SEQ ID NO: 18 Ｌ and a linker.

6. The scFv of claim 3, encoded by a nucleic acid comprising SEQ ID NO:

38.

7. A chimeric antigen receptor (CAR) comprising the scFv of claim 3, and further comprising a transmembrane domain, at least one costimulatory domain, and an activation domain.

8. The CAR according to claim 7, wherein the costimulatory domain is CD28 or 4-1BB.

9. The CAR of claim 7, wherein the activation domain is CD3 zeta.

10. The CAR of claim 7, wherein the transmembrane domain is a CD8 transmembrane domain.

11. The CAR of claim 7, comprising the amino acid sequence of SEQ ID NO:

19.

12. The CAR according to claim 11, consisting of the amino acid sequence of SEQ ID NO:

19.

13. The CAR of claim 7, encoded by a nucleic acid comprising the sequence of SEQ ID NO:

39.

14. An engineered immune cell expressing the CAR of claim 7.

15. 15. The engineered immune cell of claim 14, wherein the cell is selected from a CAR-T cell and a CAR-NK (natural killer) cell.

16. 15. A composition comprising the engineered immune cells of claim 14 and an excipient.

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