Anti-ROR1 antibodies and ROR1-targeted engineered cells

Abstract

The present invention relates to a monoclonal mouse or humanized ROR1 antibody or single chain variable fragment (scFv). The present 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 present invention, (ii) a transmembrane domain, (iii) at least one costimulatory domain, and (iv) an activating domain.

Description

[0001] Cross-reference to related applications

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

[0003] Statement on Federally Funded Research

[0004] none.

[0005] sequence list

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

[0007] This invention relates to the field of immunology, and more specifically, to antibodies, T-cell receptors, and ROR1-targeting immune cells for use in adoptive cell immunotherapy for tumors. Background Technology

[0008] Immunotherapy is emerging as a very promising approach for treating cancer. T cells, or T lymphocytes (the armed forces of the immune system), constantly seek out foreign antigens and distinguish abnormal cells (cancer cells or infected cells) from normal cells. Genetically modifying T cells or natural killer (NK) cells using CAR (chimeric antigen receptor) constructs is the most common method for designing tumor-specific T cells and NK cells. CAR-T and CAR-NK cells targeting tumor-associated antigens (TAAs) can be infused into patients (a process known as adoptive cell transfer or ACT), a highly effective immunotherapy approach. See Grupp et al., (2013), Chimeric antigen receptor-modified T cells for acute lymphoblastic leukemia, *New England Journal of Medicine*, 368, 1509-1518; and Maus et al., (2013), “T cells expressing chimeric antigen receptors can cause anaphylaxis in humans,” *Cancer Immunol Research*, 1, 26-31. The advantage of CAR-T (and CAR-NK) technology over chemotherapy or antibodies is that engineered cells can proliferate and persist in the patient's body 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 T Cells, Memory, Effector Functions, and CAR-T Immunotherapy. Cancer (Basel). 15 March 2016; 8(3). pii:E36.

[0009] In terms of NC orientation, CARs typically consist of a monoclonal antibody-derived single-chain variable fragment (scFv), a hinge, a transmembrane domain, and one or more intracellular co-activating domains (e.g., CD8, CD28, CD137(4-1BB), CD27) and one or more activation domains (e.g., CD3-ζ domain), see Figure 1 and Maus (2013) and Maus (2014) as above. CAR evolution progressed from first-generation CARs (without co-stimulatory domains) to second-generation CARs (with one co-stimulatory domain), and then to third-generation CARs (with several co-stimulatory domains). CAR-T cells from third-generation CARs with multiple co-stimulatory domains exhibit enhanced cytolytic activity and improved persistence, leading to enhanced antitumor activity.

[0010] Natural killer (NK) cells are a type of cytotoxic lymphocyte that is crucial to the innate immune system. NK cells play a role similar to that of cytotoxic T cells in the adaptive immune response in vertebrates. NK cells provide a rapid response to viral infections, becoming active approximately 3 days after infection, and also respond to tumor formation.

[0011] The tyrosine-protein kinase transmembrane receptor ROR1 (also known as neurotrophic tyrosine kinase receptor-associated 1 (NTRKR1)) is an enzyme encoded by the ROR1 gene in the human body. ROR1 is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. ROR1 is a protein composed of 937 amino acids, of which amino acids 30-406 comprise extracellular domains. The ROR1 gene encodes receptor tyrosine kinase-like orphan receptors, which regulate 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 signal transduction. Furthermore, recent studies have shown that ROR1 is expressed in ovarian cancer stem cells and promotes migration, invasion, and cancer stem cell spheroid formation. ROR1 has been shown to be overexpressed in hematologic malignancies and solid tumors, making it a potentially useful target for CAR-T therapy.

[0012] ROR1 is shown to be expressed at low levels 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, comprising V having an amino acid sequence having at least 90% identical to SEQ ID NO:2.H and V having at least 90% of the same amino acid sequence as SEQ ID NO:3 L In some embodiments, the anti-human ROR1 antibody or its antigen-binding fragment 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 V containing SEQ ID NO:17. H V containing SEQ ID NO:18 L And a connector. In some embodiments, the scFv has a V consisting of SEQ ID NO:17. H V consisting of SEQ ID NO:18 L And a connector. In some embodiments, the scFv is encoded by a nucleic acid including SEQ ID NO:38. In some embodiments, the scFv includes a component located in the V H and the V L The complementary determination region (CDR) in which V H CDR1 includes the sequence TYA, the V H CDR2 includes SEQ ID NO:41, the V H CDR3 includes SEQ ID NO:42, the V L CDR1 includes SEQ ID NO:43, the V L CDR2 includes the sequence RAN, and the V L CDR3 includes SEQ ID NO:45.

[0014] In some embodiments, the present invention is a chimeric antigen receptor (CAR) comprising the 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 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 comprises a signal transduction peptide and a hinge domain. In some embodiments, the signal transduction peptide and the hinge domain are a CD8 signal transduction peptide and a CD8 hinge domain. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:19. In some embodiments, the CAR consists of the amino acid sequence of SEQ ID NO:19. In some embodiments, the CAR is encoded by a nucleic acid comprising the sequence of SEQ ID NO:39. In some embodiments, the present 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.

[0015] In some embodiments, the present invention is a composition comprising engineered immune cells expressing CAR of SEQ ID NO:19 and excipients. Attached Figure Description

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

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

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

[0019] Figure 4 FACS data are shown, illustrating the staining of different cell lines with anti-ROR1 antibodies using different cancer cell lines.

[0020] Figure 5 shows the RTCA assay, which demonstrated dose-dependent cytotoxicity of anti-ROR1 CAR-T cells (ROR1-CD28-CD in Figure A and ROR1-4-1BB-CD3 in Figure B) against the ROR1-expressing cell line SKOV-3.

[0021] Figure 6The results show the measurement of IFN-γ secretion by anti-ROR1 CAR-T cells (ROR1-4-1BB-CD3) in the presence of SKOV-3 cells or control HL-60 cells (ROR1 negative cells).

[0022] Figure 7 The RTCA assay demonstrates dose-dependent in vitro cytotoxicity of anti-ROR1CAR-T cells against SKOV3 cells with humanized scFv (PMC1182, PMC1183, and PMC1194 expressing PMC857, PMC858, and PMC862, respectively, against ROR1CAR).

[0023] Figure 8 The results show the measurement of IFN-γ secretion by anti-ROR1 CAR-T cells with humanized scFv (PMC1182, PMC1183, and PMC1194 expressing PMC857, PMC858, and PMC862, respectively, against ROR1 CAR) in the presence of SKOV-3 cells or control HL-60 cells. Detailed Implementation

[0024] definition

[0025] As used herein, “antibody” refers to an antigen-binding protein of the immune system. Naturally occurring antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant (CH) region. The heavy chain constant region contains three domains: CHI, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant CL region. The light chain constant region contains one domain, CL. Both 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 from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0026] As used herein, the term "human antibody" is intended to encompass antibodies having variable regions, in which both the framework region and the CDR region are derived from human gene sequences. Furthermore, if the antibody contains constant regions, those constant regions are also derived from human immunoglobulin sequences.

[0027] The term "humanized antibody" refers to an antibody in which the CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human frame sequence. Further frame region modifications can be performed within the human frame sequence.

[0028] As used in this article, "antigen-binding fragment" refers to a protein fragment containing Fab fragment, Fab' fragment, F(ab')2 fragment, and scFv, which have antigen-binding activity.

[0029] As used in this article, a "chimeric antigen receptor (CAR)" is a receptor protein engineered to generate T cells (a novel ability to target specific proteins). The receptor is chimeric because it combines antigen-binding and T-cell activation functions in a single receptor. A CAR is a fusion protein comprising 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" refers to any oligopeptide or polypeptide that can bind to an antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to act as a signaling domain to induce activation or inhibition of biological processes in the cell.

[0031] As used in this article, "domain" refers to a region in 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 scFvs comprise antigen-binding polypeptides formed using recombinant DNA technology, wherein the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) segments are linked by spacer or adapter sequences. Various methods for engineering scFvs are known to those skilled in the art.

[0033] As used in this article, "tumor antigen" refers to a biomolecule that has antigenicity, which is a characteristic of tumors.

[0034] The inventors used hybridoma technology to produce an anti-ROR1 monoclonal antibody that specifically targets the human ROR1 antigen. The inventors have also produced anti-ROR1 CAR-T cells for targeting cancer cells overexpressing the ROR1 tumor antigen. The anti-ROR1 CAR-T cells of this invention exhibit high cytotoxic activity against several cancer cell lines and in vivo antitumor activity. 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 having the amino acid sequence of SEQ ID NO:1 or an antigen-binding fragment thereof, said antibody comprising V having the amino acid sequence of SEQ ID NO:2. H and V with 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, comprising V having the amino acid sequence of SEQ ID NO:5. H and V with 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, comprising V having the amino acid sequence of SEQ ID NO:9. H and V with 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, comprising V having the amino acid sequence of SEQ ID NO:13. H and V with 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, comprising V having the amino acid sequence of SEQ ID NO:17. H and V with the amino acid sequence of SEQ ID NO:18 L .

[0040] In some embodiments, a monoclonal anti-human ROR1 antibody was generated targeting the extracellular region of a purified recombinant fragment of human ROR1.

[0041] In some embodiments, the 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 from the N-terminus to the C-terminus, comprising: (i) a single-stranded variable fragment (scFv) targeting 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 a first-generation CAR lacking a costimulatory domain, a second-generation CAR with one costimulatory domain (CD28 or 4-1BB), and a third-generation CAR with two or more costimulatory domains (adapted from Goluboskaya et al., (2016) Different subsets of T cells, memory, effector function and CAR-T immunotherapy. Cancer (Basel). 15 March 2016; 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 is used. (A CAR with a CD28 co-stimulatory domain is shown.) Figure 2 In this diagram, "scFv" stands for single-chain variable segment; "CD8h" is the CD8 hinge; "CD28TM" is the CD28 transmembrane domain; "CD28 cs" is the CD-28 co-stimulatory domain; "CD3-ζ" is the CD3ζ activation domain; "VH" is the heavy chain variable region; "L" is the linker; and "VL" is the 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 costimulatory domain can be selected from the group consisting of: CD28, 4-1BB (CD137), GITR, ICOS-1, CD27, OX-40, and DAP10 costimulatory domains. In some embodiments, the costimulatory domain is CD28.

[0046] In some embodiments, the activation domain is CD3ζ (CD3 Z or CD3-ζ) encoded by the CD247 gene.

[0047] Transmembrane domains can be derived from natural peptides or can be artificially designed. Transmembrane domains derived from natural peptides can be obtained from any membrane-binding protein or transmembrane protein. In some embodiments, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of: T cell receptor α or β chain, CD3-ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR. Artificially designed transmembrane domains are peptides that primarily comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is present at each end of the synthetic transmembrane domain.

[0048] In some embodiments, the CAR includes a linker located between a transmembrane domain and an intracellular domain. In some embodiments, the linker is an oligopeptide or polypeptide, for example, having a length of 2 to 10 amino acids. Peptide linkers typically comprise 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, such as an immunoglobulin selected from IgG, IgA, IgD, IgE, or IgM. In some embodiments, the linker comprises 5-40 amino acids from the CH1, CH2, or CH3 domains of the immunoglobulin heavy chain. In some embodiments, the linker has a sequence (G x S y ) n The linkers are rich in glycine and serine. Other examples and sequences of linkers are disclosed in U.S. Patent No. 5,525,491: Serine-rich peptide linkers, U.S. Patent No. 5,482,858: Polypeptide linkers for production of biosynthetic proteins, and WO2014087010: Improved polypeptides directed against IgE.

[0049] In some embodiments, the present invention comprises one or more nucleic acids encoding an anti-ROR1 CAR. The nucleic acid encoding the CAR can be prepared by conventional methods from the amino acid sequence of a specified CAR. It can be obtained using tools publicly available from the National Center for Biotechnology Information (NCBI), for example, from the NCBI RefSeq ID or GenBank accession number for the amino acid sequence for each domain. The nucleic acid of the present 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 acid of the present invention is prepared by combining DNA fragments obtained from a cDNA library using 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 cells. In some embodiments, the vector is a viral vector, such as a retroviral vector (including oncogenic retroviral vectors, lentiviral vectors, and pseudotyped vectors), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or Sendai virus vector, an Epstein-Barr virus (EBV) vector, and a herpes simplex virus (HSV) vector. In some embodiments, a viral vector lacking replication capacity is used so that it cannot self-replicate in infected cells.

[0051] In some embodiments, the retroviral particles are prepared using packaging cell lines. In such embodiments, a suitable packaging cell line is selected based on the LTR sequence and the packaging signal sequence possessed 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, the retroviral particles are prepared using the HEK293 cell line or the HEK293t cell line, which have high transfection efficiency. Those skilled in the art will recognize the many types of commercially available retroviral vectors and packaging cell lines.

[0052] CAR-T cells (or CAR-NK cells) bind to specific antigens via CARs, thereby transmitting signals into the cell and activating it. The activation of CAR-expressing cells varies depending on the cell type of the CAR and the type of intracellular domains. Cell activation can be confirmed based on, for example, the release of cytokines, any increase in cell proliferation rate, or changes in any cell surface molecules. Furthermore, the release of cytotoxic cytokines (IFNγ, TNFα, etc.) from activated CAR-T cells (or CAR-NK cells) causes destruction of the antigen-expressing target cells, which can be detected or measured. Additionally, the release of cytokines or changes in cell surface molecules can lead to detectable or measurable stimulation of other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0053] In some embodiments, CAR-expressing cells are used as a therapeutic agent for a disease. The therapeutic agent comprises CAR-expressing cells as an active ingredient, and may further comprise suitable excipients.

[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 targeting ROR1-overexpressing cancer cells. Compared to untransduced (CAR-free) T cells (or CAR-free NK cells) and CAR-T / NK cell mimics, 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 is used to detect ROR1 in ROR1-positive cancer cells.

[0055] In some embodiments, the present invention includes humanized V of mouse monoclonal anti-human ROR1 antibody. H and V L Including humanized V H and V L The humanized scFv and CAR-T cells (or CAR-NK cells) carrying a humanized anti-ROR1 scFv targeting ROR1-positive cells. Without being bound by any 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) includes a complementarity-determining region (CDR). Each of the light and heavy chains of the antibody includes 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)). International Immunogenetic Information System ( The International Immunogenetics Information System (IIISIS), Nucleic Acids Research (Nucl. Acids Res.) 37:D1006, and Kabat (Kabat et al., (1987) Sequences of Proteins of Immunological Interest), 4th Edition, National Institutes of Health, U.S. Department of Health and Human Services (USHHS, NIH)). In some embodiments, the CDR is identified using the IMGT system. In some embodiments, the CDR is identified using the Kabat tool. In some embodiments, the smallest portion of the CDR is identified as an overlap between the sequence identified by the IMGT tool and the sequence identified by the Kabat tool.

[0057] In some embodiments, ROR1 scFv includes V H The sequence TYA in CDR1. In some embodiments, anti-ROR1 scFv includes V H SEQ ID NO:41 in CDR2. In some embodiments, anti-ROR1 scFv includes V H SEQ ID NO:42 in CDR3. In some embodiments, anti-ROR1 scFv includes V L SEQ ID NO:43 in CDR1. In some embodiments, anti-ROR1 scFv includes V L The sequence RAN in CDR2. In some embodiments, anti-ROR1 scFv includes V L SEQ ID NO:45 in CDR3.

[0058] In some anti-ROR1 scFv, anti-ROR1 includes V H The sequences TYA and V in CDR1 H SEQ ID NO:41 and V in CDR2 H SEQ ID NO:42 in CDR3, and further includes V L SEQ ID NO:43 and V in CDR1 L The sequence RAN and V in CDR2 L SEQ ID NO:45 in CDR3.

[0059]

[0060]

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

[0062] The humanized anti-ROR1 antibody and its derived scFv disclosed in this article 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 antibody disclosed herein can be effectively used to target the ROR1 antigen in ROR1-positive cells and tumors. Anti-ROR1 CAR-T cells (or CAR-NK cells) can be used clinically against 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 various therapeutic agents: checkpoint inhibitors; targeted therapies; small molecule inhibitors; antibodies, etc. For example, anti-ROR1 CAR-T cells (or CAR-NK cells) can be combined with CAR-T (or CAR-NK) cells that target other tumor antigens or antigens present in the tumor microenvironment (e.g., VEGFR-1-3, PDL-1, CD80). Bispecific antibodies and scFv (e.g., bispecific to ROR1 and CD3) and cells expressing both antibodies and scFv can be used to enhance the activity of ROR1-targeted therapies.

[0065] The anti-ROR1 antibodies and their derivatives disclosed herein can be modified for affinity regulation via site-directed mutagenesis (e.g., by error-prone PCR) and selected via affinity maturation. Modification of the co-activating domain: CD28, 4-1BB, etc., can be used to enhance the efficacy of CARs generated from the antibodies (and their derivatives) disclosed herein. CARs can be generated using tag-conjugated anti-ROR1 scFv. Firstly, 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 generate CAR-T cells, CAR-NK cells, and other cell types, such as iPSCs (induced pluripotent stem cells) derived from T cells, NK cells, macrophages, and other hematopoietic cells expressing anti-ROR1 CAR that can target ROR1-positive cancers. This invention provides T cells, NK cells, macrophages, or hematopoietic cells modified to express anti-ROR1 CAR.

[0067] The cells expressing CAR disclosed in this article can be autologous cells or allogeneic cells.

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

[0069] Example

[0070] The inventors generated an anti-ROR1 CAR construct and cloned it into a lentiviral vector. The CAR construct contained an anti-ROR1 ScFv-CD28-CD3ζ insert (or a similar insert with a 41BB co-stimulatory domain instead of the CD28 domain). Expression of the CAR construct could be driven using CMV, EF1, or MNDU3 promoters. Lentiviral cells were generated in HEK293t cells, and their titer was established by RT-PCR. Equivalent doses of lentivirus were then used to transduce T cells as described in the examples.

[0071] Example 1: Anti-ROR1 scFv was used to detect ROR1 protein by protein blotting, and anti-ROR1 antibody was used to detect ROR1 by FACS staining.

[0072] In this example, a mouse monoclonal anti-ROR1 antibody was generated using standard hybridoma technology. The mouse anti-ROR1 antibody (IgG1 type) was detected for extracellular ROR1 protein via ELISA (data not shown). This hybridoma clone 2H6 was sequenced and analyzed using V... H and V L scFv was generated (see Example 2 for further details). Protein imprinting was performed, demonstrating that the ROR1 extracellular domain binding to anti-ROR1 scFv is fused with human Fc (hFc) protein. Figure 3 (Left figure). Detection of ROR1-human Fc fusion protein using an antibody targeting the human Fc domain. Figure 3 The right figure shows the ROR1 antigen detected using anti-ROR1 scFv-mouse Fc fusion as the first antibody and anti-mouse IgG-HRP as the second antibody.

[0073] Further fluorescence-activated cell sorting (FACS) experiments demonstrated that the mouse anti-ROR1 monoclonal antibody detected elevated ROR1 expression in several cancer cell lines, such as liver cancer (HepG2), breast cancer (MDA231), colon cancer (HT-29), and ovarian cancer (SKOV-3). Normal keratinized cells were used as a negative control. Figure 4 (MFI: Medium fluorescence intensity compared to the same type).

[0074] Example 2: Countering ROR1 V H V L Sequencing with CAR constructs

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

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

[0077]

[0078] The amino acid sequence of anti-ROR1 scFv (mouse clone 2H6) (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 an scFv derived from mouse anti-ROR1 antibody 2H6 was designed. The scheme for the anti-ROR1 CAR construct is shown in... Figure 2 The CAR sequence was cloned using the lentiviral vector Lenti CMV-MCS-EF1a-puro. The CD3ζCAR construct was under the control of the CMV promoter. For the 4-1BB CAR construct, a different lentiviral vector with the MNDU3 promoter was used to obtain a higher percentage of cells expressing CAR.

[0084] A. CD28 as a costimulatory domain

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

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

[0087] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0088] CD8 signal transduction 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™ / activating nucleotide sequence (SEQ ID NO:26):

[0097] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCCACCACGCGACTTCGCAGCCTATCGCTCC

[0098] CD28™ / Activated 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 replacing the CD28 activation domain was also generated. This construct, PMC1195, was cloned into a vector containing the KanR gene. The ROR1 scFv was inserted between the Nhe I and Xho I sites in the sequence (underlined). CAR expression is controlled by the MNDU3 promoter.

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

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

[0111]

[0112]

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

[0114] A structure with human CD8 signaling peptide-alternative (see below) anti-ROR1 scFv(V) was also constructed. H -Connector (G4S) 3-V L (The CAR of "(G4S)3" disclosed in SEQ ID NO:46), CD8 hinge, CD28 transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ activation domain. In alternative scFv, except V H V L Furthermore, each segment of the sequence is identical to the segment in Example 3(A). V H Represented by SEQ ID NO:5 (the first amino acid is E, which is not present in SEQ ID NO:2). V L Represented by SEQ ID NO:6 (the terminal R is removed compared to SEQ ID NO:3).

[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 the R at the end of the C.

[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 The underlined part is "Connector (G4S)3 (SEQ ID NO:46)" is in italics, and "V" is in V. L (The text is underlined; the 4-1BB structural domain is in italics with an underline.)

[0128]

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

[0130] In this example, the mouse anti-ROR1 V H (SEQ ID NO:5) and mouse V L (SEQ ID NO:6) was humanized, resulting in several humanized scFvs. CARs with 4-1BB and CD3ζ domains were generated, each containing scFvs with the same structure as those in Example 3(B). Several humanized scFvs were tested, and three scFvs were selected based on the best performance in the functional assays shown below. The humanized scFvs were inserted between the Nhe I and Xho I sites 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] Humanized anti-ROR1 CAR scFv PMC857 nucleotide sequence: (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] The humanized anti-ROR1 CAR (scFv PMC862) amino acid sequence (SEQ ID NO:19):

[0168] MALPVTALLLPLALLLHAARPASQVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGNTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSYYF GNSVYYAMDYWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCKASQDINSYFSWYQQKPGKAPKLLIYRANRLVTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCL QYDEFPYTFGGGTRLEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0169] Example 5: Generating CAR using lentiviral vectors

[0170] In this example, CARs containing the three humanized scFvs from Example 4 were packaged into lentiviral vectors. The lentiviruses were generated using HEK293 cells via a standard procedure as described below: Goluboskaya et al., (2016) Different subsets of T cells, memory, effector function and CAR-T immunotherapy. Cancer (Basel). 15 Mar 2016; 8(3). pii:E36.

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

[0172] In this example, PBMCs were isolated from whole blood for the purpose of generating CAR-T cells. Whole blood was collected from individual or mixed donor samples (depending on the amount of blood required) (Stanford Hospital Blood Center, Stanford, California) in 10 mL heparin vacuum containers (Becton Dickinson, San Jose, California). Approximately 10 mL of anticoagulated whole blood was mixed with sterile phosphate-buffered saline (PBS, pH 7.4, Ca2+) to a total volume of 20 mL. 2+ and Mg 2+- Mix in 50 ml conical centrifuge tubes. Very carefully remove the cell layer containing PBMCs seen at the diluted plasma / Ficoll interface, avoiding any Ficoll, wash twice with PBS, and centrifuge at 200 x g for 10 minutes at room temperature. Count the cells using a hemocytometer. Wash the PBMCs once with CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies, San Diego, Cal.)) with 5% AB serum and 1.25 μg / mL amphotericin B (Gemini Bioproducts, Woodland, Cal.) , 100 U / mL penicillin, and 100 μg / mL streptomycin, and use for experiments or freeze at -80°C.

[0173] Example 7: T cell activation by PBMC

[0174] The isolated PBMCs were treated with 1x PBS (pH 7.4, Ca-free). 2+ / Mg 2+ ) and in CAR-T medium (Example 6) in the absence of human interleukin-2 (huIL2) at 5 x 10 5 Wash once at a concentration of 10 cells / mL, then resuspend in CAR-T medium at 5x 10⁻¹⁰ cells / mL (from 1000x stock solution; Invitrogen, Carlsbad, Cal.) 5 The final concentration was 1 cell / mL. The PBMCs and beads (for T cell activation) were then mixed at a 1:1 bead-cell ratio by transferring 25 μL of beads to 1 mL of PBMCs and incubating at 37°C in the presence of CO2 for 24 hours before viral transduction.

[0175] Example 8: T cell transduction and expansion

[0176] After PBMC is activated, 5x10 6 Add one lentivirus to 5 x 10 5 Add 100 T cells (MOI 10:1) and 2 μL / mL Transplus medium (Alstem, Richmond, Cal., California) to achieve a final dilution of 1:500. Incubate the cells for another 24 hours, then repeat the virus addition. Then allow the cells to grow in the presence of 300 U / mL IL-2 for a period of 12–14 days (total incubation time depends on the desired final number of CAR-T cells). Analyze cell counts every 2–3 days, adding medium at that time to dilute the cell suspension to 1 x 10⁻⁶ cells / mL. 6 Cells / ml

[0177] Example 9: Transducing T cells and validating CAR via FACS

[0178] Cells from Example 8 were washed and resuspended in FACS buffer (PBS with 0.1% sodium azide and 0.4% BSA). The cells were then divided into 1 x 10⁶ cells / mL. 6 Aliquots of cells were prepared. Fc receptors were blocked using normal goat IgG (Lifetech, San Diego, CA). Mouse anti-ROR1 scFv was detected using a biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody; a biotin-labeled normal polyclonal goat IgG antibody also served as an isotype control. Cells were incubated at 4°C for 25 minutes and washed once with FACS buffer. After staining with anti-F(ab)2 antibody, cells were stained with phycoerythrin (PE)-labeled streptavidin (BD Pharmingen, San Diego, CA) and allophycocyanin (APC)-labeled CD3 (eBiocience, San Diego, CA) for further staining. For humanized anti-ROR1 scFv, anti-human F(ab)2 antibody (Lifetech, CA) was also used.

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

[0180] Cytotoxicity was performed using the xCELLigence real-time cell analysis system (Agilent Technologies, 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 showed high cytotoxic activity against ROR1-positive cancer cells.

[0182] The expression of mouse-scFv anti-ROR1 CAR was confirmed by FACS using an anti-mouse Fab antibody. CAR-T cells containing mouse anti-ROR1 scfv, a CD28 co-stimulatory domain, and a CD3ζ activation domain (see Example 3(A)) were used in this cytotoxicity assay. The cytotoxicity assay was performed on the xCELLigence system using an RTCA-based impedance assay, according to the manufacturer's specifications. In this assay, the integrity of the target cell monolayer was continuously monitored by the impedance of the target cell monolayer in a weak electric field. CAR-T cell killing of target cells reduced 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 (Fig. 5, Fig. A). CAR-T cells resulted in a sustained dose-dependent decrease in the impedance of the target cell monolayer. Thus, anti-ROR1-CD28-CD3 CAR-T cells killed ROR1-positive SKOV-3 ovarian solid tumor cells in a dose-dependent manner.

[0183] Similar high cytotoxic activity was also observed in CAR-T cells containing CARs with mouse anti-ROR1 scfv, 4-1BB co-stimulatory domains and CD3ζ activation (Example 3(B)) and ROR1-positive SKOV-3 target cells (Fig. 5, Fig. 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-incubating ROR1-41BB-CD3-CAR-T cells with SKOV-3 cells, the supernatant was collected and ELISA was performed using a commercial kit (Thermo Fisher Scientific, Waltham, Mass.). As a control, a non-adherent HL-60 ROR1-negative cell line was used. Compared to 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 this was compared with T cells and CAR-T cells used as controls (P<0.05). Figure 6 ).

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

[0187] First, CAR-T cells were tested in a cytotoxicity assay using ROR1-positive cells with scFv PMC857, PMC868, or PMC862 (Example 5), demonstrating high cytotoxicity. Next, these CAR constructs (PMC857, PMC868, or PMC862) were inserted into lentiviral vectors containing the KanR gene (preferred for clinical use) but not the AmpR gene. The CAR-T cell clones were named PMC1182, 1183, and 1194, respectively. CAR expression in the CAR-T cells was approximately 30% CAR+, as detected by FACS using human Fab. RTCA assays were performed, and these CAR-T cells showed high cytotoxic activity against SKOV-3 (ROR1-positive) cells. Figure 7 ).

[0188] Next, cytokine secretion from CAR-T cells was evaluated. After co-incubating CAR-T cells with SKOV-3 cells, the culture supernatant was collected, and ELISA was performed using a non-adherent HL-60 ROR1-negative cell line as a control, as described in Example 12, to detect interferon-γ in the supernatant. Compared to 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 this was compared with T cells and simulated CAR-T cells used as controls (P<0.05). Figure 8 ).

[0189] Although the 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 invention. Therefore, the scope of the invention should not be limited to the examples described herein, but rather to the following claims.

Claims

1. An anti-human ROR1 antibody or antigen-binding fragment thereof, comprising a V H and a V L having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, the antibody or antigen-binding fragment comprising complementarity determining regions (CDRs) located in the V H and the V L , wherein CDR1 of the V H consists of the sequence TYA, CDR2 of the V H consists of SEQ ID NO: 41, CDR3 of the V H consists of SEQ ID NO: 42, CDR1 of the V L consists of SEQ ID NO: 43, CDR2 of the V L consists of the sequence RAN, and CDR3 of the V L consists of SEQ ID NO:

45.

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

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

4. The scFv of claim 3, comprising V H comprising SEQ ID NO: 18 L and a linker.

5. The scFv of claim 4, comprising a V H comprised of SEQ ID NO: 18 L and a linker.

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

38.

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

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

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

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

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

19.

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

19.

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

39.

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

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

16. A composition for immunotherapy, comprising the engineered immune cell of claim 14 and an excipient.

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