Anti-ROR1 antibodies and ROR1-targeting engineered cells

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 365,230, filed on May 24, 2022, which is incorporated herein by reference.

[0003] Statement Regarding Federally Sponsored Research

[0004] none.

[0005] Sequence Listing

[0006] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on May 22, 2023 is named CBI047.30_SL.xml and is 55,553 bytes in size. Technical Field

[0007] The present invention relates to the field of immunology, and more specifically, to antibodies, T cell receptors and immune cells targeting ROR1 that can be used in the field of adoptive cell immunotherapy of tumors. Background Art

[0008] Immunotherapy is becoming a very promising approach for treating cancer. T cells or T lymphocytes, the armed forces of the immune system, constantly search for foreign antigens and distinguish abnormal cells (cancer cells or infected cells) from normal cells. Genetic modification of T cells or natural killer (NK) cells using CAR (chimeric antigen receptor) constructs is the most common approach used 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 (known as adoptive cell transfer or ACT), which is a highly effective immunotherapy method, see Grupp et al., (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. New England Journal of Medicine (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 over chemotherapy or antibodies is that the engineered cells can proliferate and persist in the patient as "active drugs". 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 TCells, Memory, Effector Functions, and CAR-T Immunotherapy. Cancers (Basel). 2016 Mar 15;8(3).pii:E36.

[0009] In the NC orientation, CAR is usually composed of a monoclonal antibody-derived single-chain variable fragment (scFv), 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., CD3-ζ domains), see Figure 1 and Maus (2013) and Maus (2014) above. The evolution of CAR has entered from the first generation of CAR (without a co-stimulatory domain) to the second generation of CAR (with one co-stimulatory domain) to the third generation of CAR (with several co-stimulatory domains). CAR-T cells of the third generation of CAR with multiple co-stimulatory domains have increased cytolytic activity and improved persistence, which enhances anti-tumor activity.

[0010] Natural Killer (NK) cells are a type of cytotoxic lymphocyte that is critical to the innate immune system. NK cells play a role similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells, act about 3 days after infection, and respond to tumor formation.

[0011] The 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 protein consisting of 937 amino acids, of which amino acids 30-406 include the extracellular domain. The ROR1 gene encodes a receptor tyrosine kinase-like orphan receptor that regulates neurite growth 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 activates the downstream NFκB signaling pathway and can produce inhibition of WNT-mediated signaling. In addition, it has recently been shown that ROR1 is expressed on ovarian cancer stem cells and promotes migration, invasion, and cancer stem cell sphere formation. ROR1 has been shown to be overexpressed in hematological cancers and solid tumors, making it a useful target for CAR-T therapy.

[0012] ROR1 shows low expression in most normal human tissues (such as adipose tissue 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, etc.). Summary of the invention

[0013] In some embodiments, the present invention is an anti-human ROR1 antibody or an antigen-binding fragment thereof, which comprises a V sequence 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 In some embodiments, the anti-human ROR1 antibody or 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 , V comprising SEQ ID NO: 18 L In some embodiments, the scFv has a V consisting of SEQ ID NO: 17 H , V consisting of SEQ ID NO: 18 L In some embodiments, the scFv is encoded by a nucleic acid comprising SEQ ID NO: 38. In some embodiments, the scFv comprises a H and the V L The complementarity determining region (CDR) in H The CDR1 includes the sequence TYA, the V H The CDR2 comprises SEQ ID NO: 41, the V H The CDR3 comprises SEQ ID NO:42, wherein the V L The CDR1 comprises SEQ ID NO:43, the V L The CDR2 includes the sequence RAN, and the V L The CDR3 includes SEQ ID NO:45.

[0014] In some embodiments, the present invention is a chimeric antigen receptor (CAR), which includes the scFv and further includes: a transmembrane domain, at least one co-stimulatory domain and an activation domain. In some embodiments, the co-stimulatory domain is CD28 and / or 4-1BB. In some embodiments, the activation domain is CD3ζ. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CAR further includes a signaling peptide and a hinge domain. In some embodiments, the signaling peptide and the hinge domain are CD8 signaling peptides and CD8 hinge domains. In some embodiments, the CAR includes an amino acid sequence of SEQ ID NO: 19. In some embodiments, the CAR is composed of an amino acid sequence of SEQ ID NO: 19. In some embodiments, the CAR is encoded by a nucleic acid comprising a sequence of SEQ ID NO: 39. In some embodiments, the present invention is an engineered immune cell expressing a CAR of SEQ ID NO: 19. In some embodiments, the cell is selected from CAR-T cells and CAR-NK (natural killer) cells.

[0015] In some embodiments, the present invention is a composition comprising an engineered immune cell expressing a CAR of SEQ ID NO: 19 and an excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a diagram showing first, second, and third generation chimeric antigen receptors (CARs) known in the art.

[0017] Figure 2 is a diagram showing the structure of anti-ROR1 CAR.

[0018] Figure 3 is a Western blot demonstrating that the anti-ROR1 antibody binds to the human ROR1 antigen.

[0019] Figure 4 Shown are FACS data demonstrating staining of different cell lines with anti-ROR1 antibodies using different cancer cell lines.

[0020] FIG. 5 shows an RTCA assay demonstrating 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.

[0021] Figure 6Shown are the results of 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).

[0022] Figure 7 Shown is a RTCA assay demonstrating dose-dependent in vitro cytotoxicity of anti-ROR1 CAR-T cells with humanized scFvs (PMC1182, PMC1183, and PMC1194 expressing PMC857, PMC858, and PMC862 anti-ROR1 CARs, respectively) against SKOV3 cells.

[0023] Figure 8 Shown are the results of measuring IFN-γ secretion by anti-ROR1 CAR-T cells with humanized scFvs (PMC1182, PMC1183, and PMC1194 expressing PMC857, PMC858, and PMC862 anti-ROR1 CARs, respectively) in the presence of SKOV-3 cells or control HL-60 cells. DETAILED DESCRIPTION

[0024] definition

[0025] As used herein, "antibody" refers to the 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 comprises a heavy chain variable region (VH) and a heavy chain constant (CH) region. The heavy chain constant region comprises three domains, namely CHI, CH2 and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant CL region. The light chain constant region comprises one domain CL. VH and VL comprise complementary determining regions (CDRs) interspersed with more conservative regions known as framework regions (FRs). Each VH and VL comprises three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0026] 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. In addition, if the antibody contains a constant region, the constant region is also derived from human immunoglobulin sequences.

[0027] The term "humanized antibody" refers to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0028] As used herein, “antigen-binding fragment” refers to a protein fragment including a Fab fragment, a Fab′ fragment, a F(ab′) 2 fragment and a scFv having antigen-binding activity.

[0029] As used herein, "chimeric antigen receptor (CAR)" is a receptor protein that has been engineered to produce T cells (a new ability to target specific proteins). The receptor is chimeric because it combines both antigen binding function and T cell activation function in a single receptor. CAR is a fusion protein including an extracellular antigen binding domain, a transmembrane domain, and at least one intracellular domain.

[0030] As used herein, "extracellular domain capable of binding to an antigen" means any oligopeptide or polypeptide that can bind to an antigen. "Intracellular domain" means any oligopeptide or polypeptide known to act as a domain that transmits signals to cause activation or inhibition of biological processes in cells.

[0031] As used herein, "domain" means a region of a polypeptide that folds into a specific structure independently of other regions.

[0032] As used herein, "single-chain variable fragment (scFv)" means a single-chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Typical examples of scFv include antigen-binding polypeptides formed by recombinant DNA technology and in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are connected by spacer or linker sequences. Various methods for engineering scFv are known to those skilled in the art.

[0033] As used herein, "tumor antigen" refers to a biological molecule with antigenicity that is characteristic of a tumor.

[0034] The inventors have produced anti-ROR1 monoclonal antibodies that specifically target the human ROR1 antigen using hybridoma technology. The inventors have produced anti-ROR1 CAR-T cells for targeting cancer cells that overexpress the ROR1 tumor antigen. The anti-ROR1 CAR-T cells of the present invention have high cytotoxic activity and in vivo anti-tumor activity against several cancer cell lines. Anti-ROR1 CAR-NK cells expressing the same CAR are also envisioned.

[0035] In some embodiments, the present invention includes a monoclonal mouse anti-human ROR1 antibody or an antigen-binding fragment thereof having an amino acid sequence of SEQ ID NO: 1, wherein the antibody includes a V having an amino acid sequence of SEQ ID NO: 2. H and V having the amino acid sequence of SEQ ID NO: 3 L .

[0036] In some embodiments, the present invention includes a monoclonal mouse anti-human ROR1 antibody or an antigen-binding fragment thereof, which includes a V having an amino acid sequence of SEQ ID NO: 5. H and V having the amino acid sequence of SEQ ID NO: 6 L .

[0037] In some embodiments, the present invention includes a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, which includes a V having an amino acid sequence of SEQ ID NO: 9. H and V having the amino acid sequence of SEQ ID NO: 10 L .

[0038] In some embodiments, the present invention includes a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, which includes a V having an amino acid sequence of SEQ ID NO: 13. H and V having the amino acid sequence of SEQ ID NO: 14 L .

[0039] In some embodiments, the present invention includes a monoclonal humanized anti-human ROR1 antibody or an antigen-binding fragment thereof, which includes a V having an amino acid sequence of SEQ ID NO: 17. H and V having the amino acid sequence of SEQ ID NO: 18 L .

[0040] In some embodiments, monoclonal anti-human ROR1 antibodies are generated against the extracellular region of a purified recombinant fragment of human ROR1.

[0041] In some embodiments, the present invention includes a single chain variable fragment (scFv) derived from a monoclonal mouse anti-human ROR1 antibody disclosed herein, or any humanized version thereof also disclosed herein.

[0042] In some embodiments, the present invention includes a chimeric antigen receptor (CAR) fusion protein comprising, from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) to ROR1 disclosed herein, (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activation domain.

[0043] Figure 1 shows the structures of first generation CARs lacking a costimulatory domain, second generation CARs with one costimulatory domain (CD28 or 4-1BB), and third generation CARs with two or more costimulatory domains (adapted from Goluboskaya et al. (2016) Different subsets of T cells, memory, effector function, and CAR-T immunotherapy. Cancer (Basel). 2016 Mar 15;8(3).pii:E36).

[0044] Figure 2 The structure of the anti-ROR1 CAR of the present invention is shown. A second generation CAR with a CD28 or 4-1BB co-stimulatory domain was used. (A CAR with a CD28 co-stimulatory domain is shown.) Figure 2 In the figure, "scFv" is a single chain variable fragment; "CD8h" is a CD8 hinge; "CD28 TM" is a CD28 transmembrane domain; "CD28 cs" is a CD-28 co-stimulatory domain, "CD3-ζ" is a CD3ζ activation domain, "VH" is a heavy chain variable region, "L" is a linker, and "VL" is a light chain variable region. The arrangement of scFv is shown as V H -Connector-V L In some embodiments, the arrangement is V L -Connector-V H .

[0045] The co-stimulatory domain may be selected from the group consisting of: CD28, 4-1BB (CD137), GITR, ICOS-1, CD27, OX-40 and DAP10 co-stimulatory domains. In some embodiments, the co-stimulatory domain is CD28.

[0046] In some embodiments, the activation domain is CD3 zeta (CD3 zeta or CD3-zeta) encoded by the CD247 gene.

[0047] The membrane-spanning domain can be derived from a natural polypeptide, or can be artificially designed. The membrane-spanning domain derived from a natural polypeptide can be obtained from any membrane-bound protein or membrane-spanning protein. In certain embodiments, the membrane-spanning domain is a membrane-spanning domain of a protein selected from the group consisting of: T cell receptor α or β chain, CD3-ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 or GITR. The artificially designed membrane-spanning domain is a polypeptide mainly including hydrophobic residues such as leucine and valine. In certain embodiments, a triplet of phenylalanine, tryptophan and valine is present at each end of the synthetic membrane-spanning domain.

[0048] In some embodiments, 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 includes about 5 to about 40 amino acids. The linker can be a naturally occurring sequence or an engineered sequence. For example, in some embodiments, the linker is derived from a human protein, for example, an immunoglobulin selected from IgG, IgA, IgD, IgE or IgM. In some embodiments, the linker includes 5-40 amino acids from the CH1, CH2 or CH3 domains of an immunoglobulin heavy chain. In some embodiments, the linker is a linker having a sequence (G x S y ) n Additional examples of linkers and sequences are disclosed in U.S. Pat. No. 5,525,491: Serine-rich peptide linkers, U.S. Pat. No. 5,482,858: Polypeptide linkers for production of biosynthetic proteins, and publication WO2014087010: Improved polypeptides directed against IgE.

[0049] In some embodiments, the present invention includes one or more nucleic acids encoding anti-ROR1 CAR. The nucleic acid encoding CAR can be prepared by a conventional method by specifying the amino acid sequence of CAR. The tools provided to the public by the National Center for Biotechnology Information (NCBI) can be used to obtain, for example, the nucleotide sequence encoding the amino acid sequence is obtained from the NCBI RefSeq ID or GenBank accession number for the amino acid sequence of each domain. The nucleic acid of the present invention can be prepared using standard molecular biology or chemical procedures. In some embodiments, based on the nucleotide sequence, the part of the synthetic nucleic acid. In some embodiments, the nucleic acid of the present invention is prepared by combining DNA fragments obtained from a cDNA library using a polymerase chain reaction (PCR).

[0050] In some embodiments, the nucleic acid encoding the CAR of the present invention is inserted into a vector, and the vector is introduced into a cell. In some embodiments, the vector is a viral vector, such as a retroviral vector (including an oncoretroviral vector, a lentiviral vector, and a pseudotype vector), an adenoviral vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector, or a Sendai virus vector (Sendai virus vector), an Epstein-Barr virus (Epstein-Barr virus, EBV) vector, and a herpes simplex virus (HSV) vector. In some embodiments, a viral vector that lacks replication ability so that it cannot self-replicate in infected cells is used.

[0051] In some embodiments, retroviral particles are prepared using packaging cell lines. In such embodiments, a suitable packaging cell line based on the packaging signal sequence possessed by the LTR sequence and the viral vector is selected. 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 cell lines or HEK293t cell lines with high transfection efficiency. One of skill in the art understands many types of retroviral vectors and packaging cell lines that are commercially available.

[0052] CAR-T cells (or CAR-NK cells) are combined with specific antigens by CAR, thereby transmitting signals to cells, and activating cells. The activation of cells expressing CAR varies depending on the cell type and the type of intracellular domain of CAR. The activation of cells can be confirmed based on, for example, the release of cytokines, any increase in cell proliferation rate, changes in any cell surface molecules, etc. In addition, cytotoxic cytokines (IFNγ, TNFα, etc.) are released from activated CAR-T cells (or CAR-NK cells) to cause the destruction of target cells expressing antigens, and the destruction can be detected or measured. In addition, the release of cytokines or changes in cell surface molecules can lead to detectable or measurable stimulation of other immune cells (e.g., B cells, dendritic cells, NK cells, and macrophages).

[0053] In some embodiments, cells expressing CAR are used as therapeutic agents for diseases. The therapeutic agent includes cells expressing CAR as active ingredients, and it may further include a suitable excipient.

[0054] In one embodiment, the present invention includes anti-ROR1scFv-CD28-CD3ζ-CAR-T (anti-ROR1 CAR-T) cells or anti-ROR1 CAR-NK cells for cancer cells overexpressing ROR1. Compared to untransduced (CAR-free) T cells (or CAR-free NK cells) and simulated CAR-T cells (or simulated CAR-NK cells), anti-ROR1-CAR-T cells or CAR-NK cells express higher cytotoxic activity against ROR1-positive cancer cells. The mouse monoclonal anti-human ROR1 antibody disclosed herein detects ROR1 in ROR1-positive cancer cells.

[0055] In some embodiments, the present invention includes a humanized V H and V L , including humanized V H and V L Humanized scFv and CAR-T cells (or CAR-NK cells) carrying CARs including humanized anti-ROR1 scFv targeting ROR1-positive cells. Without being bound by a particular theory, the inventors believe that at least one advantage of humanizing mouse anti-ROR1 scFv is that it potentially reduces the immune response to CAR-T (CAR-NK) cells in the human body.

[0056] In some embodiments, the anti-ROR1 antibody or its antigen-binding fragment or derivative (such as scFv) comprises a complementary determining region (CDR). Each of the light chain and heavy chain 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 an in silico method, such as IMGT (Lefranc et al., (2009) International Immunogenetics Information System ( The international immunogenetics information system), "Nucl. Acids Res." 37: D1006) and Kabat (Kabat et al., (1987) Sequences of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services National Institutes of Health (USHHS, NIH)). In some embodiments, CDRs are identified using the IMGT system. In some embodiments, CDRs are identified using the Kabat tool. In some embodiments, the smallest portion of a CDR is identified as the overlap of a sequence identified by the IMGT tool with a sequence identified by the Kabat tool.

[0057] In some embodiments, the anti-ROR1 scFv comprises V H In some embodiments, the anti-ROR1 scFv comprises V H In some embodiments, the anti-ROR1 scFv comprises V H In some embodiments, the anti-ROR1 scFv comprises V L In some embodiments, the anti-ROR1 scFv comprises V L In some embodiments, the anti-ROR1 scFv comprises V L SEQ ID NO:45 in CDR3 of.

[0058] In some anti-ROR1 scFvs, the anti-ROR1 comprises V H The sequences TYA, and V H SEQ ID NO: 41 and V in CDR2 of H SEQ ID NO: 42 in CDR3 of L SEQ ID NO: 43 in CDR1, and V L The sequences in CDR2 of L SEQ ID NO:45 in CDR3 of.

[0059]

[0060]

[0061] In some embodiments, the anti-ROR1 scFv comprises a complementarity determining region, a light chain (V L ) in the CDR1, CDR2 and CDR3 and the heavy chain (V H ) in CDR1, CDR2 and CDR3, and including V H The sequences TYA, V H SEQ ID NO: 41, V H SEQ ID NO: 42, V L SEQ ID NO: 43, V L The sequences in CDR2 of L SEQ ID NO: 45 in CDR3 of ROR1. In some embodiments, in the anti-ROR1 scFv, V H The CDR1 of H The CDR2 of the invention consists of SEQ ID NO:41, V H The CDR3 of the invention consists of SEQ ID NO: 42, V L The CDR1 of the invention consists of SEQ ID NO: 43, V L The CDR2 consists of the sequence RAN, and V L The CDR3 of the polypeptide consists of SEQ ID NO:45.

[0062] The humanized anti-ROR1 antibodies disclosed herein and scFvs derived therefrom can be used for immunotherapy applications: toxin-drug conjugated antibodies, monoclonal therapeutic antibodies, bispecific antibodies, and CAR-T cell (or CAR-NK cell) based immunotherapy.

[0063] Anti-ROR1 CAR-T cells (or CAR-NK cells) generated using the anti-ROR1 antibodies disclosed herein can be effectively used to target ROR1 antigens in ROR1-positive cells and tumors. Anti-ROR1 CAR-T cells (or CAR-NK cells) can be used clinically against tumor cells, tumors, and cancer stem cells that are resistant to chemotherapy and form aggressive tumors.

[0064] Anti-ROR1 CAR-T cells (or CAR-NK cells) can be used in combination with different therapeutic agents: checkpoint inhibitors; targeted therapy; small molecule inhibitors; antibodies, etc. For example, anti-ROR1 CAR-T cells (or CAR-NK cells) can be used in combination with CAR-T (or CAR-NK) cells targeting other tumor antigens or antigens present in the tumor microenvironment (e.g., VEGFR-1-3, PDL-1, CD80). Bispecific antibodies and scFv (e.g., bispecific for ROR1 and CD3) and cells expressing antibodies and scFvs can be used to enhance the activity of ROR1 targeted therapy.

[0065] The anti-ROR1 antibodies and derivatives thereof disclosed herein can be modified by site-directed mutagenesis (e.g., by error-prone PCR) for affinity regulation and selected by affinity maturation. Modification of coactivation domains: CD28, 4-1BB, etc. can be used to improve the efficacy of CARs produced by the antibodies (and derivatives thereof) disclosed herein. CARs can be produced using tag-conjugated anti-ROR1 scFvs. First, second- and third-generation CAR constructs can be prepared using the same anti-ROR1 scFv disclosed herein.

[0066] The anti-ROR1 CAR disclosed herein can be used to produce CAR-T cells, CAR-NK cells and other types of cells, such as T cells, NK cells, macrophages and other iPSCs (induced pluripotent stem cells) from which hematopoietic cells expressing anti-ROR1 CARs can be targeted to ROR1 positive cancers. The present invention provides T cells, or NK cells, or macrophages or hematopoietic cells modified to express anti-ROR1 CARs.

[0067] The CAR-expressing cells disclosed herein can be autologous cells and allogeneic cells.

[0068] The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting the present invention.

[0069] Examples

[0070] The inventors have produced an anti-ROR1 CAR construct and cloned the construct into a lentiviral vector. The CAR construct contains an anti-ROR1 ScFv-CD28-CD3ζ insert (or a similar insert with a 41BB costimulatory domain instead of a CD28 domain). CMV, EF1 or MNDU3 promoters can be used to drive the expression of the CAR construct. Slow viruses are produced in HEK293t cells and their titers are established by RT-PCR. Equal doses of slow viruses are then used to transduce T cells as described in the examples.

[0071] Example 1: Anti-ROR1 scFv detects ROR1 protein by Western blot, and anti-ROR1 antibody detects ROR1 by FACS staining

[0072] In this example, mouse monoclonal anti-ROR1 antibodies were generated using standard hybridoma technology. Mouse anti-ROR1 antibodies (IgG1 type) detected extracellular ROR1 protein by ELISA (data not shown). This hybridoma clone 2H6 was sequenced and cloned using V H and V L scFv was generated (see further Example 2). Western blot was performed which demonstrated that the anti-ROR1 scFv bound the extracellular domain of ROR1 fused to the human Fc (hFc) protein ( Figure 3 , left panel). ROR1-human Fc fusion protein was detected using an antibody against the human Fc domain. Figure 3 , The right panel shows ROR1 antigen detected using anti-ROR1 scFv-mouse Fc fusion as the primary antibody and anti-mouse IgG-HRP as the secondary antibody.

[0073] Fluorescence activated cell sorting (FACS) experiments were further performed, which demonstrated that mouse anti-ROR1 monoclonal antibodies detected elevated expression of ROR1 in several cancer cell lines such as liver cancer (HepG2), breast cancer (MDA231), colon cancer (HT-29), and ovarian cancer (SKOV-3). Normal keratinocytes were used as negative controls ( Figure 4 ). (MFI: median fluorescence intensity compared to the isotype).

[0074] Example 2: Against ROR1 V H 、V L and CAR constructs for sequencing

[0075] In this example, an anti-ROR1 antibody (i.e., hybridoma clone 2H6) was sequenced. H 、V L The sequences of the scFv are shown below. The structure of the anti-ROR1 scFv is: H -Connector-V L , wherein the linker has the sequence (G4S)3 (SEQ ID NO: 46). In the sequence below, the sequence starts at V H ; The underline shows V L nucleotide sequence of ; the linker sequence is in italics.

[0076] Anti-ROR1 scFv (mouse clone 2H6) nucleotide sequence (SEQ ID NO: 20):

[0077]

[0078] Anti-ROR1 scFv (mouse clone 2H6) amino acid sequence (SEQ ID NO: 1):

[0079]

[0080] Anti-ROR1 scFv (mouse clone 2H6) V H Amino acid sequence (SEQ ID NO: 2): VKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFGNSVYYAMDYWGQGTSVTVSS

[0081] Anti-ROR1 scFv (mouse clone 2H6) V L Amino acid sequence (SEQ ID NO: 3): DIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKR

[0082] Example 3: Anti-ROR1-CAR sequence with mouse anti-ROR1 scFv

[0083] In this example, a CAR with a 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 to clone the CAR sequence. The CD3ζCAR construct was under the control of the CMV promoter. For the 4-1BB CAR construct, another lentiviral vector with the MNDU3 promoter was used to obtain a higher percentage of cells expressing the CAR.

[0084] A. CD28 as a co-stimulatory domain

[0085] CAR includes the following structures: anti-ROR1 ScFv-CD8 hinge-CD28 TM-CD28 co-stimulatory domain CD3ζ activation domain ( Figure 2 ). The structure further comprises a human CD8 signaling peptide. The anti-ROR1 scFv has structure V H -Connector (G4S) 3-V L (such as "(G4S)3" disclosed in SEQ ID NO:46).

[0086] CD8 signaling peptide nucleotide sequence (SEQ ID NO: 21):

[0087] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0088] CD8 signaling peptide amino acid sequence (SEQ ID NO: 22):

[0089] MALPVTALLLPLALLLHAARP

[0090] Nhe I restriction site: GCTAGC

[0091] XhoI restriction site: CTCGAG

[0092] CD8 hinge nucleotide sequence (SEQ ID NO: 24):

[0093] AAGCCCACCACGACGCCAGCGCCGCCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGAT

[0094] CD8 hinge amino acid sequence (SEQ ID NO: 25):

[0095] KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD

[0096] CD28 TM / activating nucleotide sequence (SEQ ID NO:26):

[0097] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCCACCACGCGACTTCGCAGCCTATCGCTCC

[0098] CD28 TM / activating amino acid sequence (SEQ ID NO:27):

[0099] FWVLVVVGGVLACYSLLVTVAFIIFWV / RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0100] CD3ζ nucleotide sequence (SEQ ID NO:28):

[0101] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0102] CD3ζ amino acid sequence (SEQ ID NO:29): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0103] EcoRI restriction site: GAATTC

[0104] Anti - ROR1 CAR (mouse) nucleotide sequence (SEQ ID NO 30):

[0105]

[0106] Anti-ROR1 CAR (mouse) amino acid sequence (SEQ ID NO: 4):

[0107] MALPVTALLLPLALLLHAARPASVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFG NSVYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQ YDEFPYTFGGGTKLEIKRLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0108] A CAR with a 4-1BB co-stimulatory domain instead of the CD28 activation domain was also produced. This construct PMC1195 was cloned into a vector with the KanR gene. The ROR1 scFv was inserted between the Nhe I site and the Xho I site in the sequence (underlined). CAR expression is under the control of the MNDU3 promoter.

[0109] The nucleotide sequence of the codon-optimized CAR (anti-ROR1 scFv-4-1BB-CD3ζ) is shown below. The scFv is inserted between the Nhe I site and the Xho I site (underlined). 4-1BB is italicized, followed by the CD3-ζ domain.

[0110] Anti-ROR1 CAR (4-1BB replaces CD28 activation domain) nucleotide sequence (SEQ ID NO: 31):

[0111]

[0112]

[0113] B. 4-1BB as a co-stimulatory domain

[0114] A scFv (V H -Connector (G4S) 3-V L (SEQ ID NO: 46 disclosed "(G4S)3")), CD8 hinge, CD28 transmembrane domain, 4-1BB costimulatory domain, CD3ζ activation domain) CAR. In alternative scFv, in addition to V H 、V L Except for this, each fragment of the sequence is identical to the fragment in Example 3(A). H Represented by SEQ ID NO: 5 (the first amino acid is E, which is not present in SEQ ID NO: 2). L Represented by SEQ ID NO: 6 (compared to SEQ ID NO: 3, the terminal R is removed).

[0115] Alternative anti-ROR1 scFv amino acid sequence (SEQ ID NO:23):

[0116]

[0117] Anti-ROR1 CAR alternative V H Amino acid sequence (SEQ ID NO:5):

[0118] EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTISRDNARHILYLQMSSLRSEDTAMYYCARDSYYFGNSVYYAMDYWGQGTSVTVSS

[0119] Compared to SEQ ID NO: 2, SEQ ID NO: 5 has an additional R at the N-terminal end.

[0120] Anti-ROR1 CAR alternative V L Acid sequence (SEQ ID NO:6):

[0121] DIKMTQSPSSMYASLGERVTITCKASQDINSYFSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK

[0122] Compared to SEQ ID NO:3, SEQ ID NO:6 lacks R at the C-terminal end.

[0123] 4-1BB domain nucleotide sequence: (SEQ ID NO: 32):

[0124] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0125] 4-1BB amino acid sequence (SEQ ID NO:33):

[0126] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0127] Anti-ROR1 CAR (alternative scFv and 4-1BB) amino acid sequence (SEQ ID NO: 7): (V H is underlined, linker (G4S) 3 (SEQ ID NO: 46) is italicized, V L is underlined; the 4-1BB domain is underlined in italics. )

[0128]

[0129] Example 4: ROR1 CAR with humanized ROR1 scFv

[0130] In this example, mouse anti-ROR1 V H (SEQ ID NO: 5) and mouse V L (SEQ ID NO:6) was humanized and several humanized scFvs were produced.Cars with 4-1BB domains and CD3 ζ domains of scFvs with three structures identical to those of Example 3 (B) were produced. Several humanized scFvs were tested, and three scFvs were selected based on the best performance in the functional assays shown below. Humanized scFvs were inserted between the Nhe I site and the Xho I site in the CAR sequence.

[0131] Three CARs with humanized anti-ROR1 scFv: PMC857, PMC858, and PMC862 are shown below.

[0132] _A. PMC857 scFv and CAR

[0133] 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 CTGGAG ATC AAA

[0134] Humanized anti-ROR1 scFv PMC857 amino acid sequence: (SEQ ID NO: 8):

[0135] EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0136] Humanized anti-ROR1 scFv PMC857 V H Amino acid sequence (SEQ ID NO:9):

[0137] EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTV

[0138] Humanized anti-ROR1 scFv (PMC857) V L Amino acid sequence (SEQ ID NO: 10):

[0139] DIQMTQSPSSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0140] <h2 style=";text-align:left;direction:ltr">ROR1 CAR scFv PMC857 is available for download: (SEQ ID NO:35) : ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCTAGC GAA GTA CAG CTTGTT GAA TCA GGT GGT GGT CTT ATT CAG CCA GGA GGC TCC TTG CGA CTG AGC TGT GCCGCT TCT GGG TTC ACC TTT AGC ACT TAC GCA ATG AGT TGG GTC CGA CAA GCC CCA GGTAAG GGA TTG GAA TGG GTA AGT TCC ATT TCC AGC GGA GGG AAC ACT TAT TAC GCC GATTCT GTG AAA GGA CGC TTT ACT ATA TCC CGA GAC AAT AGT AAA AAC ACA TTG TAT TTGCAA ATG AAC TCT TTG AGG GCC GAG GAC ACT GCC GTC TAC TAT TGT GCC CGC GAC AGCTAT TAT TTC GGC AAC TCT GTG TAT TAC GCG ATG GAT TAC TGG GGT GCC GGC ACA ACTGTC ACC GTT TCA TCT GGC GGA GGA GGC AGT GGC GGA GGG GGC TCA GGC GGT GGT GGAAGT GAT ATT CAA ATG ACC CAA TCA CCC TCT TCA TTG TCT GCA AGC GTA GGT GAC CGAGTC ACG ATA ACC TGC AAA GCC TCT CAA GAT ATT AAT TCA TAC TTT TCT TGG TTT CAACAA AAA CCG GGA AAG GCG CCT AAG TCA TTG ATT TAC CGC GCG AAC CGG TTG GTA TCAGGA GTA CCG TCA AGA TTC TCA GGG AGT GGG TCA GGC ACA GAT TTC ACA CTC ACT ATTTCT TCC TTG CAA CCT GAA GAC TTC GCA ACC TATTAT TGC TTG CAG TAT GAT GAG TTTCCG

[0141] TACACTTTCGGGGGGGGTACAAGGCTGGAGATCAAACTCGAGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTCTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

[0142] Amino acid sequence of humanized anti-ROR1 CAR (scFv PMC857): (SEQ ID NO:11):

[0143] MALPVTALLLPLALLLHAARPASEVQLVESGGGLIQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSSISSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0144] B.PMC858 scFv and CAR

[0145] Humanized anti - ROR1 scFv PMC858 nucleotide sequence (SEQ ID NO:36):

[0146] CAG GTA CAA TTG GTA GAG TCC GGC GGA GGG GTT GTT CAG CCA GGA CGG TCCTTG CGG TTG TCT TGT GCT GCG TCA GGA TTC ACA TTC TCA ACG TAC GCG ATG TCT TGGGTG CGC CAA GCT CCC GGT AAA GGG CTG GAA TGG GTG GCC TCA ATC TCA TCT GGA GGGAAC ACT TAC TAC CCT GAT AGT GTT AAA GGT CGC TTT ACT ATC TCA AGG GAC AAT AGCAAG AAT ACC TTG TAT CTG CAA ATG AAC TCA CTT AGA GCA GAG GAC ACA GCG GTA TATTAC TGT GCT AGA GAC TCA TAT TAT TTC GGC AAC TCC GTT TAT TAC GCG ATG GAT TACTGG GGC GCA GGG ACT ACG GTA ACT GTA TCT TCT GGT GGT GGA GGG TCT GGG GGC GGGGGT AGT GGC GGC GGT GGC AGT GAC ATC CAG ATG ACA CAG TCT CCG TCT TCA TTG AGTGCA AGC GTC GGC GAT CGG GTT ACC ATT ACG TGT AAG GCA AGT CAG GAC ATC AAC AGTTAT TTT TCA TGG TTT CAA CAA AAG CCT GGA AAA GCG CCG AAA TCA CTC ATT TAC CGAGCT AAT AGG CTT GTC TCT GGC GTT CCG TCT CGC TTC AGT GGA AGT GGG AGC GGT ACTGAT TTT ACC CTC ACC ATA TCA AGC CTT CAA CCG GAG GAT TTT GCC ACG TAC TAT TGTCTC CAG TAC GAT GAA TTT CCA TAT ACG TTT GGC GGC GGG ACT CGC TTG GAG ATT AAA

[0147] Humanized anti-ROR1 scFv PMC858 amino acid sequence (SEQ ID NO: 12):

[0148] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0149] Humanized anti-ROR1 scFv PMC858 V H Amino acid sequence: (SEQ ID NO: 13):

[0150] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSS

[0151] Humanized anti-ROR1 scFv PMC858 V L Amino acid sequence (SEQ ID NO: 14):

[0152] DIQMTQSPSSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIK

[0153] Humanized anti-ROR1 CAR (scFv PMC858) nucleotide sequence (SEQ ID NO: 37):

[0154]

[0155] Humanized anti-ROR1 CAR (scFv PMC858) Amino Acid Sequence (SEQ ID NO:15): MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDINSYFSWFQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0156] C. PMC862 scFv and CAR

[0157] Humanized anti-ROR1 scFv PMC862 Nucleotide Sequence (SEQ ID NO:38):

[0158] CAG GTA CAA CTG GTG GAA TCC GGC GGG GGA GTA GTA CAG CCC GGA CGA TCTCTT CGA CTC TCA TGT GCA GCG TCC GGG TTC ACT TTT TCT ACC TAC GCA ATG TCA TGGGTA CGA CAG GCG CCG GGC AAA GGC CTC GAA TGG GTT GCA TCC ATT TCA TCA GGA GGTAAT ACA TAT TAT CCT GAT TCA GTC AAG GGC CGA TTC ACG ATT AGT CGA GAT AAT AGCAAG AAC ACT CTC TAC TTG CAG ATG AAC TCC CTG CGG GCT GAG GAC ACG GCC GTG TATTAT TGC GCT CGC GAT AGT TAT TAC TTC GGC AAT TCC GTA TAT TAT GCG ATG GAC TATTGG GGC GCC GGT ACT ACC GTG ACT GTT TCC TCT GGT GGG GGT GGG TCC GGG GGC GGTGGT TCA GGT GGA GGC GGA TCC GAC ATT CAA ATG ACC CAG TCT CCC TCA AGT TTG TCTGCA TCT GTT GGC GAT AGA GTT ACA ATA ACA TGC AAA GCC AGT CAA GAC ATC AAC TCATAC TTC TCC TGG TAT CAA CAA AAG CCA GGA AAA GCT CCG AAA CTG TTG ATC TAC CGGGCC AAC CGG CTG GTC ACT GGC GTG CCA TCC CGG TTC AGT GGC AGC GGA AGC GGA ACAGAT TTC ACG TTT ACC ATC TCT AGC CTC CAA CCG GAG GAC ATC GCA ACA TAC TAT TGCCTT CAG TAT GAT GAG TTT CCC TAC ACT TTC GGT GGC GGC ACC CGA CTT GAG ATC AAA

[0159] Humanized anti-ROR1 scFv PMC862 amino acid sequence (SEQ ID NO: 16):

[0160] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTRLEIK

[0161] Humanized anti-ROR1 scFv PMC862 V H Amino acid sequence (SEQ ID NO: 17):

[0162] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYFGNSVYYAMDYWGAGTTVTVSS

[0163] Humanized anti-ROR1 scFv PMC862 V L Amino acid sequence (SEQ ID NO: 18):

[0164] DIQMTQSPSSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTRLEIK

[0165] Humanized anti-ROR1 CAR (scFv PMC862) nucleotide sequence (SEQ ID NO: 39):

[0166]

[0167] Humanized anti-ROR1 CAR (scFv PMC862) amino acid sequence (SEQ ID NO: 19):

[0168] MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYF GNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCL QYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0169] Example 5: Production of CAR using lentiviral vectors

[0170] In this example, a CAR containing the three humanized scFvs of Example 4 was packaged into a lentiviral vector. Lentivirus was produced using HEK293 cells by standard procedures as described in: Goluboskaya et al. (2016) Different subsets of T cells, memory, effector function and CAR-T immunotherapy. Cancer (Basel). 2016 Mar 15;8(3).pii:E36.

[0171] Example 6: Isolation of peripheral blood mononuclear cells (PBMC) from whole blood

[0172] In this example, PBMCs are separated from whole blood for the purpose of producing CAR-T cells. Whole blood (Stanford Hospital Blood Center, Stanford, Calif.) is collected from individual or mixed donor samples (depending on the amount of blood required) and collected in 10 mL heparin vacuum containers (Becton Dickinson, San Jose, Calif.). About 10 ml of anticoagulated whole blood is mixed with sterile phosphate buffered saline (PBS pH 7.4, Ca-free) in a total volume of 20 ml. 2+ and Mg 2+- ) are mixed in a 50ml conical centrifuge tube. The cell layer containing PBMCs seen at the diluted plasma / Ficoll interface is removed very carefully to avoid any Ficoll, washed twice with PBS, and centrifuged at room temperature for 10 minutes at 200x g. Cells are counted with a hemocytometer. PBMCs are washed once with CAR-T culture medium (AIM V-AlbuMAX (BSA) (Life Technologies, San Diego, Cal., San Diego, Cal.) with 5% AB serum and 1.25ug / mL amphotericin B (Gemini Bioproducts, Woodland, Cal., Woodland, Cal., California), 100U / mL penicillin and 100ug / mL streptomycin, and used for experiments or frozen at -80°C.

[0173] Example 7: T cell activation by PBMCs

[0174] The isolated PBMCs were washed with 1x PBS (pH 7.4, without Ca 2+ / Mg 2+ ) and in the absence of human interleukin-2 (huIL2) CAR-T culture medium (Example 6) at 5 x 10 5 The cells were washed once at a concentration of 10 cells / mL and then resuspended to 5 x 10 cells / mL in CAR-T medium with 300 U / mL huIL2 (from 1000x stock; Invitrogen, Carlsbad, Cal.). 5 PBMCs and beads (for T cell activation) were then mixed at a 1:1 bead-to-cell ratio by transferring 25 uL of beads into 1 mL of PBMCs and incubated at 37°C in the presence of CO2 for 24 hours prior to viral transduction.

[0175] Example 8: T cell transduction and expansion

[0176] After PBMC activation, 5x 10 6 Add lentivirus to 5x 10 5 T cells (MOI 10: 1) were added and 2 μL / mL Transplus medium (Alstem, Richmond, Cal.) was added to a final dilution of 1:500. The cells were incubated for another 24 hours, after which the virus addition was repeated. The cells were then grown in the presence of 300 U / mL IL-2 for a period of 12-14 days (the total incubation time depended on the final number of CAR-T cells required). The number of cells was analyzed every 2-3 days, at which time medium was added to dilute the cell suspension to 1 x 10 6 cells / ml.

[0177] Example 9: Transduction of T cells and validation of CAR by FACS

[0178] The cells from Example 8 were washed and suspended in FACS buffer (PBS plus 0.1% sodium azide and 0.4% BSA). The cells were then split into 1 x 10 6 Aliquots of cells. Fc receptors were blocked with normal goat IgG (Life Technologies, San Diego, California). A biotinylated polyclonal goat anti-mouse F(ab)2 antibody was used to detect mouse anti-ROR1 scFv; a biotinylated normal polyclonal goat IgG antibody also served as an isotype control. The cells were incubated at 4°C for 25 minutes and washed once with FACS buffer. After staining the cells with anti-F(ab)2 antibodies, streptavidin labeled with phycoerythrin (PE) (BD Pharmingen, San Diego, Cal.) and CD3 labeled with allophycocyanin (APC) (eBiocience, San Diego, Cal.) were used to stain the cells. For humanized anti-ROR1 scFv, an anti-human F(ab)2 antibody (Life Technologies) was also used.

[0179] Example 10: Real-time cytotoxicity assay.

[0180] Cytotoxicity was performed using the xCELLigence Real-Time Cell Analysis System (Agilent, San Diego, CA) according to the manufacturer's protocol described below: Berahovich et al., (2018) CAR-T cells based on Novel BCMA monoclonal antibody block multiple myeloma Cell growth. Cancer (Basel) (9).

[0181] Example 11: CAR-T cells with mouse anti-ROR1 scFv exhibited high cytotoxic activity against ROR1-positive cancer cells.

[0182] The expression of mouse-scFv anti-ROR1 CAR was confirmed by FACS with anti-mouse Fab antibody. CAR-T cells with mouse anti-ROR1 scfv, CD28 costimulatory domain and CD3ζ activation domain (see Example 3 (A)) were used in this cytotoxicity assay. According to the manufacturer's conditions, cytotoxicity assays were performed using RTCA impedance-based assays on the xCELLigence system. In this assay, the integrity of the target cell monolayer is continuously monitored by the impedance of the target cell monolayer in a weak electric field. CAR-T cells kill target cells to reduce the integrity of the monolayer, and thus reduce their impedance. Anti-ROR1 CAR transduced T cells are added to target cells with effectors of 10: 1, 20: 1, 30: 1 and 40: 1: target (E: T) ratios (Figure 5, Figure A). CAR-T cells cause a sustained dose-dependent reduction in the impedance of the target cell monolayer. Thus, anti-ROR1-CD28-CD3 CAR-T cells kill ROR1 positive SKOV-3 ovarian solid tumor cells in a dose-dependent manner.

[0183] Similar high cytotoxic activity was also observed in the case of CAR-T cells with a CAR containing a mouse anti-ROR1 scfv, a 4-1BB co-stimulatory domain, and CD3ζ activation (Example 3(B)) and ROR1-positive SKOV-3 target cells (Figure 5, panel B).

[0184] Example 12: Anti-ROR1-CAR T cells (mouse scFv) secrete high levels of IFN-γ in the presence of ROR1-positive cancer cells.

[0185] After co-incubation of ROR1-41BB-CD3-CAR-T cells with SKOV-3 cells, supernatants were collected and subjected to ELISA 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 compared to the presence of ROR1-negative control cells, and compared to T cells used as control and mock CAR-T cells (P<0.05). ( Figure 6 ).

[0186] Example 13: Anti-ROR1-CAR T cells (humanized scFv) exhibit cytotoxicity and secrete high levels of IFN-γ in the presence of ROR1-positive cancer cells.

[0187] First, CAR-T cells were tested with scFv PMC857, PMC868 or PMC862 (Example 5) in a cytotoxicity assay using ROR1-positive cells, and it was shown that CAR-T cells were highly cytotoxic. Next, these CAR constructs (PMC857, PMC868 or PMC862) were inserted into a lentiviral vector with the KanR gene (preferred for clinical use) instead of the AmpR gene. The CAR-T cell clones were cloned as clones PMC1182, 1183, and 1194, respectively. CAR expression in CAR-T cells was approximately 30% CAR+, as detected by FACS with human Fab. RTCA assays were performed, and these CAR-T cells with high cytotoxic activity against SKOV-3 (ROR1-positive) cells were detected ( Figure 7 ).

[0188] Next, cytokine secretion by CAR-T cells was evaluated. After co-incubation of CAR-T cells with SKOV-3 cells, the culture supernatant was collected and ELISA was performed to detect interferon-γ in the supernatant using the non-adherent HL-60ROR1-negative cell line as a control as described in Example 12. Compared with the presence of ROR1-negative control cells, anti-ROR1-CAR-T cells secreted significantly higher levels of IFN-γ in the presence of ROR1-positive SKOV-3 cancer cells, and compared with T cells used as controls and mock CAR-T cells (P<0.05). ( Figure 8 ).

[0189] 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 may be made within the scope of the present invention. Therefore, the scope of the present invention should not be limited to the examples described herein, but should be limited by the following claims.

Claims

1. An anti-human ROR1 antibody or an antigen-binding fragment thereof, comprising a 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 . 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. The scFv according to claim 3, comprising a V comprising SEQ ID NO: 17 H , V comprising SEQ ID NO: 18 L and connectors.

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

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

38.

7. The scFv according to claim 3, comprising a H and the V L The complementarity determining region (CDR) in H The CDR1 includes the sequence TYA, the V H The CDR2 comprises SEQ ID NO:41, the V H The CDR3 comprises SEQ ID NO: 42, wherein the V L The CDR1 comprises SEQ ID NO:43, the V L The CDR2 includes the sequence RAN, and the V L The CDR3 includes SEQ ID NO:

45.

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

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

10. The CAR of claim 8, wherein the activation domain is CD3ζ.

11. The CAR of claim 8, wherein the transmembrane domain is a CD8 transmembrane domain.

12. The CAR of claim 8, further comprising a signaling peptide and a hinge domain.

13. The CAR of claim 12, wherein the signaling peptide and the hinge domain are a CD8 signaling peptide and a 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, which consists of the amino acid sequence of SEQ ID NO:

19.

16. The CAR of 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 of claim 17, wherein the claim 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.

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