Anti-ROR1 antibody 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 in its entirety.

[0002] Description of Federally Sponsored Research None.

[0003] Sequence Listing This application is electronically filed in XML format and includes a sequence listing that is incorporated herein by reference in its entirety. The XML copy was created on May 22, 2023, and 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 specifically, to antibodies, T - cell receptors, and immune cells targeting ROR1, which are useful in the field of adoptive cell immunotherapy for tumors.

Background Art

[0005] Immunotherapy has emerged as a very promising approach for the treatment of cancer. T cells or T lymphocytes, which are the army of the immune system, constantly search for foreign antigens and distinguish abnormalities (cancer or infected cells) from normal cells. Genetic modification of T cells or natural killer (NK) cells using chimeric antigen receptor (CAR) constructs is the most common approach for engineering 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), which has become 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 as "biological drugs" in patients. 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 single-chain variable fragment (scFv) derived from a monoclonal antibody, a hinge, a transmembrane domain, and one or more intracellular co-activation domains (e.g., CD8, CD28, CD137 (4-1BB), CD27), and one or more activation domains (e.g., the CD3-zeta domain) in the N-C direction (see Figure 1 and Maus (2013) and Maus (2014) supra). The evolution of CARs has progressed from first-generation (no co-stimulatory domain) to second-generation (one co-stimulatory domain) to third-generation CARs (multiple 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 for 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 three days after infection, and respond to tumor formation.

[0008] Receptor tyrosine 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 containing 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 kappa B signaling pathway and cause suppression of WNT-mediated signaling. In addition, ROR1 has recently been shown to be expressed on ovarian cancer stem cells, promoting migration, invasion, and cancer stem cell sphere formation. ROR1 has been shown to be overexpressed in both hematologic malignancies and solid tumors and is a useful target for CAR-T therapy.

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

Summary of the Invention

[0010] In some embodiments, the present invention provides a V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 H and a V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 L and is an anti-human ROR1 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-human ROR1 antibody or an antigen-binding fragment thereof comprises 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 comprises a V comprising SEQ ID NO: 17 H and a 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 a 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 comprises complementarity-determining regions (CDRs) in V H and V L wherein the CDR1 of V H comprises the sequence TYA, the CDR2 of V H comprises SEQ ID NO: 41, the CDR3 of V H comprises SEQ ID NO: 42, the CDR1 of V L comprises SEQ ID NO: 43, the CDR2 of V L comprises the sequence RAN, and the CDR3 of V L comprises SEQ ID NO: 45.

[0011] In some embodiments, the invention is a chimeric antigen receptor (CAR) comprising a scFv and further comprising a transmembrane domain, at least one co-stimulatory domain, and an activation domain. In some embodiments, the co-stimulatory domain is CD28 or 4-1BB. In some embodiments, the activation domain is CD3 zeta. In some embodiments, the transmembrane domain is the CD8 transmembrane domain. In some embodiments, the CAR further comprises a signaling peptide and a hinge domain. In some embodiments, the signaling peptide and the hinge domain are the CD8 signaling peptide and the 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 expressing the CAR of SEQ ID NO: 19. In some embodiments, the cell is selected from CAR-T cells and CAR-NK (natural killer) cells.

[0013] In some embodiments, the invention is a composition comprising an engineered immune expressing the CAR of SEQ ID NO: 19 and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5A

Figure 5B

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0015] Definitions As used herein, "antibody" refers to an antigen-binding protein of the immune system. Naturally occurring antibodies are glycoproteins that include 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, CH1, 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 include complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL includes three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: 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. Further, when the antibody includes a constant region, the constant region is also derived from a human immunoglobulin sequence.

[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, are grafted onto human framework sequences. Modifications of additional framework regions may be made within the human framework sequences.

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

[0019] As used herein, a "chimeric antigen receptor (CAR)" is a receptor protein engineered to give T cells the new ability 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, an "extracellular domain capable of binding an antigen" means any oligopeptide or polypeptide capable of binding a specific antigen. An "intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits signals that cause activation or inhibition of biological processes in a cell.

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

[0022] As used herein, a "single-chain variable fragment (scFv)" means a single-chain polypeptide derived from an antibody that retains the ability to bind an antigen. Typical examples of scFvs include antigen-binding polypeptides formed by recombinant DNA technology in which the Fv regions of immunoglobulin heavy (H) chain and light (L) chain fragments are linked via a spacer or linker sequence. Various methods for engineering scFvs are known to those of skill in the art.

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

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

[0025] In some embodiments, the present invention provides a monoclonal mouse anti-human ROR1 antibody having the amino acid sequence of SEQ ID NO: 1, or an antigen-binding fragment thereof, comprising V having the amino acid sequence of SEQ ID NO: 2 H and V having the amino acid sequence of SEQ ID NO: 3 L .

[0026] In some embodiments, the present invention provides a monoclonal mouse anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising V having the amino acid sequence of SEQ ID NO: 5 H and V having the amino acid sequence of SEQ ID NO: 6 L .

[0027] In some embodiments, the present invention provides a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising V having the amino acid sequence of SEQ ID NO: 9 H and V having the amino acid sequence of SEQ ID NO: 10 L .

[0028] In some embodiments, the present invention provides a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising V having the amino acid sequence of SEQ ID NO: 13 H and V having the amino acid sequence of SEQ ID NO: 14 L .

[0029] In some embodiments, the present invention provides a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising V having the amino acid sequence of SEQ ID NO: 17 H and V having the amino acid sequence of SEQ ID NO: 18 LComprising a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof.

[0030] In some embodiments, the monoclonal anti-human ROR1 antibody is generated against the extracellular region of a purified recombinant fragment of human ROR1.

[0031] In some embodiments, the invention comprises a single-chain variable fragment (scFv) derived from either the monoclonal mouse anti-human ROR1 antibody disclosed herein or its humanized version 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 co-stimulatory domain, and (iv) an activation domain.

[0033] Figure 1 shows the structures of first-generation CARs lacking co-stimulatory domains, second-generation CARs having one co-stimulatory domain (CD28 or 4-1BB), and third-generation CARs having two or more co-stimulatory domains (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 the anti-ROR1 CAR of the present invention. A second-generation CAR was used with either the CD28 or 4-1BB co-stimulatory domain. (A CAR having a CD28 co-stimulatory domain is shown). In Figure 2, "scFv" is a single-chain variable fragment, "CD8 h" is the CD8 hinge, "CD28 TM" is the CD28 transmembrane domain, "CD28 cs" is the CD-28 co-stimulatory domain, "CD3-zeta" is the CD3 zeta activation domain, "VH" is the heavy-chain variable region, "L" is the linker, and "VL" is the light-chain variable region. The arrangement of the scFv is shown as V H -linker-V L as shown. In some embodiments, the arrangement is V L -linker-V H is.

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

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

[0037] The transmembrane domain may be derived from a natural polypeptide or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding protein or transmembrane protein. In some embodiments, the transmembrane domain is the transmembrane domain of a protein selected from the group consisting of the 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. An artificially designed transmembrane domain is a polypeptide mainly containing hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0038] In some embodiments, the CAR includes a linker between the transmembrane domain and the intracellular domain. In some embodiments, the linker is an oligopeptide or polypeptide, for example, having a length of 2 to 10 amino acids. The peptide linker generally contains about 5 to about 40 amino acids. The linker may be a naturally occurring sequence or a engineered sequence. For example, in some embodiments, the linker is derived from an immunoglobulin selected from human proteins such as IgG, IgA, IgD, IgE, or IgM. In some embodiments, the linker contains 5 to 40 amino acids from the CH1, CH2, or CH3 domain of the immunoglobulin heavy chain. In some embodiments, the linker is a glycine and serine rich linker having the sequence (G x S y ) n . Examples and sequences of additional linkers are disclosed in U.S. Patent No. 5,525,491 (serine-rich peptide linker), U.S. Patent No. 5,482,858 (polypeptide linker for the production of biosynthetic proteins), and publication WO2014 / 087010 (improved polypeptides against IgE).

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

[0040] In some embodiments, the nucleic acid encoding the CAR of the 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 virus (AAV) vector, a simian virus vector, a vaccinia virus vector, a Sendai virus vector, an Epstein-Barr virus (EBV) vector, or a herpes simplex virus (HSV) vector. In some embodiments, a viral vector lacking replication ability is used so that it does not self-replicate 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 a HEK293 cell line or a HEK293t cell line having high transfection efficiency. Those skilled in the art are aware of many commercially available types of retroviral vectors and packaging cell lines.

[0042] CAR-T cells (or CAR-NK cells) bind to a specific antigen via the CAR, whereby a signal is transmitted to the cell and the cell is activated. The activation of cells expressing the CAR varies depending on the cell type and the type of intracellular domain of the CAR. The activation of cells can be confirmed, for example, based on the release of cytokines, any improvement in the 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 an antigen that can be detected or measured. In addition, the release of cytokines or the change in cell surface molecules results in a detectable or measurable stimulation of other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

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

[0044] In one embodiment, the present invention includes anti-ROR1 scFv-CD28-CD3 zeta-CAR-T (anti-ROR1 CAR-T) cells or anti-ROR1 CAR-NK cells against cancer cells that overexpress ROR1. The anti-ROR1-CAR-T cells or CAR-NK cells exhibit higher cytotoxic activity against ROR1-positive cancer cells compared to non-transduced (CAR-free) T cells (or CAR NK cells without CAR) 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 includes a humanized scFv comprising humanized V H and V L , humanized V H and V L , and CAR-T cells (or CAR-NK cells) having a CAR internally that includes a humanized anti-ROR1 scFv targeting ROR1-positive cells. Without being bound by 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, the anti-ROR1 antibody or its antigen-binding fragment or derivative (such as scFv) contains complementarity-determining regions (CDRs). Each of the light and heavy chains of the antibody contains three CDRs. In some embodiments, the CDRs are identified using the crystal structure of the antigen-antibody complex. In some embodiments, the CDRs are identified using in vitro methods 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., U.S.H.H.S., N.I.H.). In some embodiments, the CDRs are identified using the IMGT tool. In some embodiments, the CDRs are identified using the Kabat tool. In some embodiments, the minimal portion of the CDRs 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 contains the TYA sequence in CDR1 of V H In some embodiments, the anti-ROR1 scFv contains SEQ ID NO: 41 in CDR2 of V H In some embodiments, the anti-ROR1 scFv contains SEQ ID NO: 42 in CDR3 of V H In some embodiments, the anti-ROR1 scFv contains SEQ ID NO: 43 in CDR1 of V L In some embodiments, the anti-ROR1 scFv contains the sequence RAN in CDR2 of V L In some embodiments, the anti-ROR1 scFv contains SEQ ID NO: 45 in CDR3 of V L

[0048] Some anti-ROR1 scFvs have the sequence TYA in the CDR1 of V H , the sequence of SEQ ID NO: 41 in the CDR2 of V H , and the sequence of SEQ ID NO: 42 in the CDR3 of V H . Some anti-ROR1 scFvs further include the sequence of SEQ ID NO: 43 in the CDR1 of V L , the sequence RAN in the CDR2 of V L , and the sequence of SEQ ID NO: 45 in the CDR3 of V L .

Table 1

[0049] In some embodiments, the anti-ROR1 scFv includes complementarity determining regions CDR1, CDR2, and CDR3 in the light chain (V L ), and CDR1, CDR2, and CDR3 in the heavy chain (V H ). The CDR1 of V H has the sequence TYA, the CDR2 of V H has the sequence of SEQ ID NO: 41, and the CDR3 of V H has the sequence of SEQ ID NO: 42 、 V L has the sequence of SEQ ID NO: 43 in its CDR1, the sequence RAN in its CDR2, and V L has the sequence of SEQ ID NO: 45 in its CDR3. In some embodiments, in the anti-ROR1 scFv, the CDR1 of V L consists of the sequence TYA, the CDR2 of V H consists of the sequence of SEQ ID NO: 41, the CDR3 of V H consists of the sequence of SEQ ID NO: 42, the CDR1 of V H consists of the sequence of SEQ ID NO: 43, the CDR2 of V L consists of the sequence RAN, and the CDR3 of V L consists of the sequence of SEQ ID NO: 45, and the CDR3 of V L consists of the sequence 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 conjugate antibodies, monoclonal therapeutic antibodies, bispecific antibodies, and CAR-T cell (or CAR-NK cell)-based immunotherapy.

[0051] The 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. The anti-ROR1 CAR-T cells (or CAR-NK cells) are resistant to chemotherapy and can be clinically used against tumor cells, tumors, and cancer stem cells that form invasive tumors.

[0052] The anti-ROR1 CAR-T cells (or CAR-NK cells) can be used in combination with different therapeutic agents: checkpoint inhibitors; targeted therapies, small molecule inhibitors, antibodies, etc. For example, the anti-ROR1 CAR-T cells (or CAR-NK cells) can be used in combination 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 their derivatives disclosed herein can be modified by site-directed mutagenesis, e.g., error-prone PCR for affinity tuning, and selected by affinity maturation. Modification of co-activation domains: 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 scFvs can be used for CAR generation. First, second, and third generation CAR constructs can be made with the same anti-ROR1 scFvs disclosed herein.

[0054] The anti-ROR1 CAR disclosed in this specification 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 modified to express an anti-ROR1 CAR.

[0055] The CAR-expressing cells disclosed in this specification can be autologous cells and allogeneic cells.

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

Example

[0057] The inventors prepared an anti-ROR1 CAR construct and cloned the construct into a lentiviral vector. The CAR construct contains an anti-ROR1 ScFv-CD28-CD3 zeta insert (or a similar insert having a 41BB co-stimulatory domain instead of the CD28 domain). A CMV, EF1 or MNDU3 promoter can be used to drive the expression of the CAR construct. Lentivirus was generated in HEK293t cells and the titer was established by RT-PCR. Then, an equal amount of lentivirus was used for transduction of T cells as described in the examples.

[0058] Example. The anti-ROR1 scFv detected the ROR1 protein by Western blot, and the anti-ROR1 antibody detected ROR1 by FACS staining. In this example, a mouse monoclonal anti-ROR1 antibody was prepared 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, V H and V LscFv was generated using [reference to Example 2]. Western blot was performed, showing that the anti-ROR1 scFv bound to the extracellular domain of ROR1 was fused to the 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 the anti-ROR1 scFv mouse Fc fusion as the primary antibody and anti-mouse IgG-HRP as the secondary antibody.

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

[0060] Example 2. Sequencing of anti-ROR1 V H 、V L 、and CAR constructs In this example, the anti-ROR1 antibody, hybridoma clone 2H6, was sequenced. The sequences of V H 、V L、 and scFv are shown below. The structure of the anti-ROR1 scFv is V H -linker-V L where the linker has the sequence (G 4 S) 3 (SEQ ID NO: 46). In the following sequences, the sequences start with V H and the underline indicates the nucleotide sequence of V L and the linker sequence is in italics.

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

Chemical formula

[0062] Amino acid sequence of anti-ROR1 scFv (mouse clone 2H6): (SEQ ID NO: 1):

Chemical formula

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

[0066] A. CD28 as a co-stimulatory domain The CAR comprises the following structure: anti-ROR1 scFv-CD8 hinge-CD28 TM-CD28 co-stimulatory domain-CD3 zeta activation domain (Figure 2). This structure further comprises a human CD8 signaling peptide. The anti-ROR1 scFv has structure V H -linker (G 4 S) 3 -V L (disclosed as SEQ ID NO: 46 as "(G 4 S) 3 ").

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

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

[0069] NheI 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): AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[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): MALPVTALLLPLALLLHAARPASVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFGNSVYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKRLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0080] In addition, a CAR having a 4-1BB co-stimulatory domain instead of the CD28 activation domain was also prepared. This construct PMC1195 was cloned into a vector having a KanR gene. The ROR1 scFv was inserted between the Nhe I site and the Xho I site in the sequence (underlined part). 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 site and the Xho I site (underlined part). 4-1BB is in italics, followed by the CD3-zeta domain.

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

Chemical formula

[0083] B.4-1BB as a co-stimulatory domain Also, a CAR having a structure (human CD8 signaling peptide - alternative (see below) anti-ROR1 scFv (V H - linker (G 4 S) 3 -V L (disclosed as SEQ ID NO: 46 as "(G 4 S) 3 "), CD8 hinge, CD28 transmembrane domain, 4-1BB co-stimulatory domain, CD3 zeta activation domain) was also constructed. In the alternative scFv, each segment of the sequence is the same as the segment in Example 3(A) except for V H and V L . V H is represented by SEQ ID NO: 5 (the first amino acid is E and is not present in SEQ ID NO: 2). V L is represented by SEQ ID NO: 6 (the terminal R is removed compared to SEQ ID NO: 3).

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

Chemical formula

[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 replaces V L Amino acid sequence (SEQ ID NO: 6): DIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK

[0088] Compared with SEQ ID NO: 3, SEQ ID NO: 6 lacks 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 (replacement scFv and 4-1BB) amino acid sequence (SEQ ID NO: 7): (V H is the underlined part, linker (G 4 S) 3 (SEQ ID NO: 46) is in italics, V L is the underlined part, and the 4-1BB domain is the underlined part in italics.)

[0092]

Chemical Structure

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

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

[0095] A. PMC857 scFv and CAR Humanized anti-ROR1 scFv PMC857 nucleotide sequence: (Accession 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] Amino acid sequence of humanized anti-ROR1 scFv PMC857: (SEQ ID NO: 8): EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

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

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

[0099] Nucleotide sequence of humanized anti-ROR1 CAR scFv PMC857: (SEQ ID NO: 35): 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): MALPVTALLLPLALLLHAARPASEVQLVESGGGLIQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[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] Amino acid sequence of humanized anti-ROR1 scFv PMC858 (SEQ ID NO: 12): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[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): DIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0105] Nucleotide sequence of humanized anti-ROR1 CAR (scFv PMC858) (SEQ ID NO: 37):

[0106] Amino acid sequence of humanized anti-ROR1 CAR (scFv PMC858) (SEQ ID NO: 15): MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0107] C. PMC862 scFv and CAR Nucleotide sequence of humanized anti-ROR1 scFv PMC862 (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] Amino acid sequence of humanized anti-ROR1 scFv PMC862 (SEQ ID NO: 16): QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTRLEIK

[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] Nucleotide sequence of humanized anti-ROR1 CAR (scFv PMC862) (SEQ ID NO: 39):

[0112] Amino acid sequence of humanized anti-ROR1 CAR (scFv PMC862) (SEQ ID NO: 19): MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0113] Example 5. Preparation of CAR using a lentiviral vector In this example, a CAR containing the three humanized scFvs of Example 4 was packaged into a lentiviral vector. Lentiviruses were 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, Cal.) was collected from individual or mixed donor samples (depending on the amount of blood required) in 10 mL heparin vacutainers (Becton Dickinson, San Jose, Cal.). Approximately 10 ml of whole anticoagulated blood was transferred to 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 100 μg / mL Ficoll-free solution. The layer of cells including PBMCs found at the diluted plasma / Ficoll interface was very carefully removed, avoiding the Ficoll, washed twice with PBS, and centrifuged at 200×g for 10 min at room temperature. The cells were counted in a hemocytometer. The PBMCs were washed once with CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies, San Diego, Cal.)) containing 5% AB serum and 1.25 ug / mL amphotericin B (Gemini Bioproducts, Woodland, Cal.), 100 U / mL penicillin, and 100 ug / mL streptomycin, and used for experiments or frozen at 80° C.

[0115] Example 7. T cell activation from PBMCs The isolated PBMCs were then resuspended in 1x PBS (pH 7.4, Ca 2+ / Mg 2+ without human interleukin-2 (huIL2) and 5x10 5 The cells were then 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, Cal.) containing 300 U / mL huIL2. 5 The beads were then resuspended to a final concentration of cells / mL. 25uL of beads were then transferred to 1mL of PBMCs and incubated for 30 min at 4°C for 24 hours prior to viral transduction. 2 PBMCs and beads (for T cell activation) were mixed at a 1:1 bead-to-cell ratio by incubating for 24 hours at 37°C in the presence of .

[0116] Example 8. Transduction and Proliferation of T Cells After activation of PBMCs, 5x10 6 of lentivirus was added to 5x10 5 T cells (MOI 10:1), and 2 μL / mL of Transplus (Alstem, Richmond, Cal.) was added to the medium to a final dilution ratio of 1:500. The cells were incubated for an additional 24 hours before repeating the addition of the virus. The cells were then grown for 12 - 14 days in the presence of 300 U / mL of IL-2 (the total incubation time was dependent on the final number of CAR-T cells required). The cell number was analyzed every 2 - 3 days, and at that time the medium was added and the cell suspension was diluted to 1×10 6 cells / ml.

[0117] Example 9. Transduction of T Cells and CAR Verification by FACS The cells from Example 8 were washed and resuspended in FACS buffer (PBS + 0.1% sodium azide and 0.4% BSA). The cells were then divided into 1×10 6 cell aliquots. The Fc receptor was blocked with normal goat IgG (Life Technologies, San Diego, Cal.). Biotin-labeled polyclonal goat anti-mouse F(ab) 2 antibody was used to detect the mouse anti-ROR1 scFv, and the biotin-labeled normal polyclonal goat IgG antibody also functioned as an isotype control. The cells were incubated at 4°C for 25 minutes and washed once with FACS buffer. After staining the cells with the anti-F(ab) 2 antibody, the cells were stained with phycoerythrin (PE)-labeled streptavidin (BD Pharmingen, San Diego, Cal.) and allophycocyanin (APC)-labeled CD3 (eBiocience, San Diego, Cal.). For the 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 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) using the xCELLigence real-time cell analysis system (Agilent, San Jose, Cal.).

[0119] Example 11. CAR-T cells with mouse anti-ROR1 scFv expressed 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 with a CAR containing a mouse anti-ROR1 scfv, CD28 co-stimulatory 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 integrity of the monolayer and thus 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 continuous 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 also observed with CAR-T cells having a CAR containing a mouse anti-ROR1 scfv, 4-1BB co-stimulatory domain, and CD3 zeta activation domain (Example 3(B)), and 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-incubating ROR1-41BB-CD3-CAR-T cells with SKOV-3 cells, the supernatant was collected and ELISA was performed using a commercial kit (ThermoFisher Scientific, Waltham, Mass.). As a control, a non-adherent HL-60 ROR1-negative cell line was used. 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 also compared to T cells and mock CAR-T cells used as controls (P < 0.05). (Figure 6).

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

[0123] Next, cytokine secretion by CAR-T cells was evaluated. After co-incubating CAR-T cells with SKOV-3 cells, the culture supernatant was collected and ELISA was performed to detect interferon-γ in the supernatant 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 also compared to T cells and mock CAR-T cells used as controls (P<0.05). (Figure 8).

[0124] Although the present invention has been described in detail with reference to specific examples, it will be apparent to those skilled in the art that various modifications can be made within the scope of the present invention. Therefore, the scope of the present invention should be limited by the following claims rather than by the examples described herein.

Claims

1. V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 Ｈ and V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 Ｌ and an anti-human ROR1 antibody or an antigen-binding fragment thereof.

2. The anti-human ROR1 antibody or antigen-binding fragment thereof according to 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 containing SEQ ID NO: 17 Ｈ and V containing SEQ ID NO: 18 Ｌ and a linker, the scFv according to claim 3.

5. V consisting of SEQ ID NO: 17 Ｈ and V consisting of SEQ ID NO: 18 Ｌ and a linker, the scFv according to claim 4.

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

38.

7. said V Ｈ and said V Ｌ comprise complementarity-determining regions (CDRs), wherein CDR1 of said V Ｈ comprises the sequence TYA, CDR2 of said V Ｈ comprises SEQ ID NO: 41, CDR3 of said V Ｈ comprises SEQ ID NO: 42, CDR1 of said V Ｌ comprises SEQ ID NO: 43, CDR2 of said V Ｌ comprises the sequence RAN, and CDR3 of said V Ｌ comprises SEQ ID NO: 45, the scFv according to claim 3.

8. A chimeric antigen receptor (CAR) comprising the scFv according to claim 3, further comprising a transmembrane domain, at least one co-stimulatory domain, and an activation domain.

9. The CAR according to claim 8, wherein the co-stimulatory domain is CD28 or 4-1BB.

10. The CAR according to claim 8, wherein the activation domain is CD3 zeta.

11. The CAR according to claim 8, wherein the transmembrane domain is the CD8 transmembrane domain.

12. The CAR according to claim 8, further comprising a signal transduction peptide and a hinge domain.

13. The CAR according to claim 12, wherein the signal transduction peptide and the hinge domain are the CD8 signal transduction peptide and the CD8 hinge domain.

14. The CAR according to claim 8, comprising the amino acid sequence of SEQ ID NO:

19.

15. The CAR according to claim 14, consisting of the amino acid sequence of SEQ ID NO:

19.

16. The CAR according to claim 8, encoded by a nucleic acid comprising the sequence of SEQ ID NO:

39.

17. An engineered immune cell expressing the CAR according to claim 8.

18. The engineered immune cell according to claim 17, wherein the cell is selected from CAR-T cells and CAR-NK (natural killer) cells.

19. A composition comprising the engineered immune cell according to claim 17 and an excipient.

Images