CN115443140A - Human ROR-1 antibodies and anti-ROR-1-CAR-T cells - Google Patents

Abstract

The present invention relates to human anti-human ROR-1 antibodies or single chain variable fragments (scFv) comprising V having amino acid SEQ ID NO 3 _H And V with amino acid SEQ ID NO 4 _L . The invention also relates to a ROR-1 Chimeric Antigen Receptor (CAR) comprising from N-terminus to C-terminus: (ii) 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. The monoclonal antibodies of the invention exhibit selectivity and high affinity for binding to ROR-1.

Description

Human ROR-1 antibody and anti-ROR-1-CAR-T cells

Reference sequence listing, table or computer program

The sequence listing is submitted together with this specification as an ASCII formatted text file through EFS-Web at the same time, the file name is Sequence Listing.txt, the date of creation is March 31, 2021, and the size is 20.5 kilobytes. The sequence listing submitted via EFS-Web is part of the specification and is hereby incorporated by reference in its entirety.

technical field

The invention relates to a human ROR-1 specific antibody and anti-ROR-1-CAR-T cells that can be used in the field of tumor adoptive immune gene therapy.

Background technique

Immunotherapy has emerged as a very promising approach to treating cancer. T cells, or T lymphocytes, serve as the armies of our immune system, constantly seeking out foreign antigens and distinguishing abnormalities (cancerous or infected cells) from normal cells. Genetic modification of T cells with CAR (chimeric antigen receptor) constructs is the most common approach to engineer tumor-specific T cells. CAR-T cells targeting tumor-associated antigens (TAAs) can be infused into patients representing an effective immunotherapy approach (termed adoptive cell transfer or ACT) [1,2]. The advantage of CAR-T technology over chemotherapy or antibodies is that the reprogrammed engineered T cells proliferate and persist in the patient (“living drug”) [3], [4], [1].

CARS typically consist of monoclonal antibody-derived single-chain variable fragments (scFv) located in the N-terminal portion, hinge, transmembrane domain, and many intracellular coactivation domains: (i) CD28, (ii) CD137 (4- 1BB), CD27 or other co-stimulatory domains, co-exist with activating CD3-zeta domains. (Fig. 1) [2,3]. CARs have evolved from first-generation (no costimulatory domain) to second-generation (with one costimulatory domain) to third-generation CARs (with several costimulatory domains). Generation of CARs with multiple co-stimulatory domains (so-called third-generation CARs) has resulted in increased cytolytic CAR-T cell activity, and improved CAR-T cell persistence has resulted in enhanced antitumor activity.

Natural killer cells or NK cells are a type of cytotoxic lymphocytes that are critical to the innate immune system. NK cells play a similar role to cytotoxic T cells in vertebrate adaptive immune responses. NK cells provide a rapid response to virus-infected cells, functioning about 3 days after infection, and respond to tumor formation.

Figure 1 shows the structure of CAR. The left panel shows the structure of the first generation (no co-stimulatory domain). The middle panel shows the structure of the second generation (one co-stimulatory domain CD28 or 4-BB). The right panel shows third-generation CARs (two or several co-stimulatory domains). This picture is from Golubovskaya, Wu, "Cancer", 2016[4].

ROR-1 antigen.

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

ROR-1 is a 937 amino acid protein, approximately 104 kb, with an extracellular domain of 30-406 amino acids. ROR-1 has low expression in most tissues, which facilitates its use as a target for CAR-T cell therapy. The ROR-1 gene encodes a receptor tyrosine kinase-like orphan receptor that regulates neurite outgrowth in the central nervous system. The encoded protein is a glycosylated type I membrane protein belonging to the ROR subfamily of cell surface receptors. ROR-1 is the receptor for the ligand WNT5A, which activates the downstream NF-kB signaling pathway and can lead to inhibition of WNT-mediated signaling. Furthermore, recent studies have shown that ROR1 is expressed on ovarian cancer stem cells and promotes migration, invasion, and cancer stem cell spheroid formation. ROR-1 is overexpressed in both hematological and solid tumors, making this target useful for CAR-T therapy.

ROR-1 is expressed in most normal human tissues such as adipose and soft tissues, bone marrow and immune system, endocrine tissues, female tissues, gastrointestinal tract, kidney and bladder, liver and gallbladder, lung, muscle tissue, male tissues and skin for low expression.

Description of drawings

Figure 1 shows the structure of CAR.

Figure 2 shows the structure of the ROR-1 CAR construct. Abbreviations: ScFv, single chain variable fragment; h hinge; TM transmembrane;

Figure 3 shows the binding of ROR-1 clone B2 to human ROR-1 antigen. Different dilutions are shown on the X-axis; binding is shown on the Y-axis, OD450 nM.

Figure 4 shows the detection of ROR-1-CAR positive cells by FACS using goat anti-human _Fab'2 antibody. Left panel: isotype staining, right panel: goat anti-human Fab' ₂ human FAB staining. T cells and ROR-1-CAR-T cells are shown.

Figure 5 shows that the RTCA assay confirmed the killing activity of ROR-1-CAR-T cells in vitro. Figure 5. Cytotoxicity assays were performed using ROR1-CAR-T cells from 3 donors at different effector-to-target cell ratios of 3:1 (right panel) and 10:1 (left panel). Cytotoxicity was calculated as (X-Y)*100/X, where X is the average impedance of the target cell monolayer in the absence of effector cells and Y is the average impedance of the target cell monolayer in the presence of effector cells. For each donor, the p-value for CAR-T cells versus T cells was <0.005 at each E:T ratio by two-way ANOVA using Sidak's post hoc test. Left bars are T cells; right bars are CAR-T cells from each donor (#000, #155, and #166).

6A-6B show the IFN-γ secretion of ROR-1-CAR-T on ROR-1 positive cancer cells. Figure 6A: The target cell line SKOV-3 ovarian ROR-1 positive cancer cells and HT29 colon cancer cells are both ROR-1 positive, and the target cell line is used for IFN-γ ELISA assay. Figure 6B: IFN-γ secretion after RTCA assay using ROR1-CAR-T cells from 3 different donors (#000, 155 and 160) with target HCT116, HT29 and Lovo colon cancer cells. The p-value for CAR-T cells versus T cells was <0.0001 by two-way ANOVA using Sidak's post hoc test. Left bars are T cells; right bars are CAR-T cells per donor.

Figures 7A-7D show that ROR-1-CAR-T cells significantly reduced Lovo-1 xenograft tumor growth. Figure 7A shows tumor growth curves. Figure 7B and Figure 7C show tumor size and tumor weight of ex vivo tumors. Figure 7D shows mouse body weights. *p<0.05 The graph of ROR-1-CD28-CD3 CAR-T cells and Mock CAR-T cells.

Figure 8 shows flow cytometry analysis of CAR expression.

Figure 9 shows the quantification of cytotoxicity in the RTCA assay. For each cell line, bar graphs from left to right are shown: T cells, then PMC167, PMC869, and PMC870 CAR-T cells.

Figure 10 shows IFN-γ production in the RTCA assay. For each cell line, the bar graphs from left to right are shown: T cells, then PMC167, PMC869, PMC870 CAR-T cells.

detailed description

definition

As used herein, a "chimeric antigen receptor (CAR)" is a receptor protein that has been engineered to give T cells a new ability to target a specific protein. The receptors are chimeric in that they combine antigen binding and T cell activation functions into a single receptor. A CAR is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain and at least one intracellular domain. A "chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)."

"An extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding an antigen. "Intracellular domain"refers to any oligopeptide or polypeptide known to function as a domain that, by transmitting a signal, causes the activation or inhibition of a biological process in a cell.

As used herein, "domain" refers to a region in a polypeptide that folds into a specific structure independently of other regions.

As used herein, "single-chain variable fragment (scFv)" refers to a single-chain polypeptide derived from an antibody that retains the ability to bind an antigen. Examples of scFv include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of the heavy (H chain) and light (L chain) chain fragments of an immunoglobulin are linked by a spacer sequence. Various methods for engineering scFv are known to those skilled in the art.

As used herein, "tumor antigen" refers to an antigenic biomolecule, the expression of which causes cancer.

The present inventors have generated human ROR-1 monoclonal antibodies using a phage display library specifically targeting human ROR-1 antigen. The present inventors have generated ROR-1-CAR-T cells to target cancer cells overexpressing the ROR-1 tumor antigen. The ROR-1-CAR-T cells of the present invention have high cytotoxic activity against several cancer cell lines and anti-tumor activity in vivo.

The present invention relates to a human anti-human ROR-1 antibody or an antigen-binding fragment thereof, comprising a V _H having the amino acid of SEQ ID NO:3 and a V _L having the amino acid of SEQ ID NO:4. A monoclonal anti-human ROR-1 antibody is raised against the extracellular region of a purified recombinant fragment of human ROR-1. In one embodiment, the monoclonal anti-human ROR-1 antibody is a single chain variable fragment (scFv).

The present invention also relates to a chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus: (i) single chain variable fragment (scFv) against ROR-1, (ii) transmembrane domain, (iii ) at least one co-stimulatory domain, and (iv) an activation domain.

Figure 2 shows a structure of the ROR-1 CAR construct. Second-generation CARs were shown to have CD28 or 41BB co-stimulatory domains. Abbreviations: scFv, single chain variable fragment; h hinge; TM transmembrane; A activator domain of CAR.

In the ROR-1 CAR construct, the ScFv can be VH-linker-VL or VL-linker-VH.

In one embodiment, the co-stimulatory domain is selected from the group consisting of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAP10. A preferred co-stimulatory domain is CD28.

A preferred activation domain is CD3 zeta (CD3 Z or CD3z)

Transmembrane domains can be derived from natural polypeptides or artificially designed. Transmembrane domains derived from native polypeptides can be obtained from any membrane-bound or transmembrane protein. For example, the transmembrane domain of the T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR. Artificially designed transmembrane domains are polypeptides that primarily contain hydrophobic residues such as leucine and valine. A triplet of phenylalanine, tryptophan and valine is preferably found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, for example a linker having a length of 2 to 10 amino acids, can be arranged between the transmembrane domain and the intracellular domain. In one embodiment, a linker sequence with a glycine-serine contiguous sequence can be used.

The present invention provides nucleic acid encoding ROR-1 CAR. A nucleic acid encoding a CAR can be prepared from the amino acid sequence of a specific CAR by conventional methods. The base sequence encoding the amino acid sequence can be obtained from the NCBI RefSeq ID or GenBenk accession number of the amino acid sequence of each domain mentioned above, and the nucleic acid of the present invention can be prepared using standard molecular biology and/or chemical procedures. For example, based on the base sequence, nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

A nucleic acid encoding the CAR of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, retroviral vectors (including oncoretroviral vectors, lentiviral vectors and pseudotyped vectors), adenoviral vectors, adeno-associated viral (AAV) vectors, simian virus vectors, vaccinia virus vectors or Sendai virus vectors, Epstein - Viral vectors such as Barr virus (EBV) vectors and HSV vectors. Preference is given to using viral vectors which lack the ability to replicate in infected cells so that they cannot replicate themselves.

For example, if a retroviral vector is used, packaging cells can be used to prepare retroviral particles and suitable packaging cells can be selected based on the LTR sequence and the packaging signal sequence possessed by the vector. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. Retroviral particles can also be prepared using 293 cells or 293T cells, which have high transfection efficiency. Retroviral vectors that can be used for packaging Retroviral vectors are produced based on retroviruses and packaging cells. Many types of retroviral vectors are widely commercially available from a number of companies.

CAR-T cells bind to specific antigens through CAR, thereby transmitting signals into the cells, and the cells are activated as a result. Activation of CAR-expressing cells varies depending on the type of host cell and the intracellular domain of CAR, and can be confirmed based on, for example, release of cytokines, improvement in cell proliferation rate, changes in cell surface molecules, etc. as indicators. For example, the release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells destroys the target cells expressing the antigen. In addition, release of cytokines or changes in cell surface molecules stimulate other immune cells such as B cells, dendritic cells, NK cells and macrophages.

Cells expressing CARs can be used as therapeutic agents for diseases. The therapeutic agent contains cells expressing CAR as an active ingredient, and it may further contain suitable excipients.

The present inventors have generated ROR-1-ScFv-CD28-CD3-CAR-T (ROR-1-CAR-T) cells against solid tumor cancer cells overexpressing ROR-1 (ovarian, cervical and other cancers) . The inventors have presented data demonstrating that ROR-1 is efficiently expressed in different types of cancer. The cytotoxic activity expressed by ROR-1-CAR-T cells against ROR-1-positive cancer cells was higher than that expressed against untransduced T cells and Mock-CAR-T cells.

The advantage of this antibody over mouse monoclonal or humanized antibodies is that it is a fully human sequence that does not generate an immune response against the antibody sequence in the patient.

The human monoclonal anti-human ROR-1 antibodies of the present invention detect ROR-1 in ROR-1 positive cancer cells.

The ROR-1 antibody of the present invention can be used in immunotherapeutic applications: toxin/drug conjugated antibody, monoclonal therapeutic antibody, ROR-1 antibody humanization and CAR-T cell immunotherapy.

ROR-1-CAR-T cells using the ROR-1 antibody of the present invention can be effectively used to target the ROR-1 antigen in ROR-1 positive cell lines.

ROR-1-CAR-T can be used in combination with the following different therapeutic agents: checkpoint inhibitors; targeted therapy, small molecule inhibitors, antibodies.

ROR-1 antibodies can be modified using site-directed mutagenesis for affinity modulation.

ROR-1-CAR-T cells can be used clinically for ROR-1 positive cells.

Modification of the coactivation domain: CD28, 4-1BB, etc. can be used to increase its efficacy. Tag-conjugated ROR-1 scFv can be used for CAR generation

Third-generation CAR-T or other co-activation signaling domains can be used for the same ROR-1-scFv within the CAR.

ROR-1 can be combined with other CARs targeting other tumor antigens or tumor microenvironment (VEGFR-1-3), PDL-1, CD80, or bi-scFv-CAR to enhance the efficacy of monotherapy ROR-1-CAR. active. Bispecific antibodies with ROR-1 and CD3 or other antigens can be generated for therapy.

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

ROR-1-CAR can be used to generate other types of cells, such as CAR-natural killer (NK) cells, iPS (induced pluripotency)-NK or iPS-T cells, ROR-1-CAR-macrophages and other ROR - 1-CAR hematopoietic cells that can target ROR-1-positive cancers. The present invention provides T cells, or NK cells, or macrophages, or hematopoietic cells modified to express ROR-1-CAR.

ROR1-CAR can be used in autologous and allogeneic hematopoietic cells.

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

Example

The inventors generated the ROR-1 CAR construct within a lentiviral vector that was cloned into a lentiviral vector with EF1 or MNDU3 promoters to drive the expression of the CAR. The lentiviral CAR construct contained either a ROR-1ScFv-CD28-CD3zeta insert or a ROR-1ScFv-41BB-CD3zeta insert. Three CAR constructs were made. The CAR construct is located within a lentiviral vector and has an Amp resistance gene or a kanamycin resistance gene as a marker. CAR PMC167 utilizes the EF1 promoter and Amp resistance gene, and has a CD28 domain. CAR PMC869 uses the MNDU3 promoter and AMP resistance gene and has a 4-1BB domain. CAR PMC870 uses the MNDU3 promoter and kanamycin resistance gene and has a 4-1BB domain.

Lentiviruses were produced in 293T cells and titered by RT-PCR. T cells were then transduced using equal doses of lentivirus as described in the Examples.

Example 1. ROR-1 antibody detects ROR-1 protein by ELISA and FACS staining

We generated human ROR-1 antibodies using standard phage display screening of phage display libraries. Phage display technology is a standard technique and is described in Dal Ferro [5]. Human antibody ROR-1 clone B2 detected extracellular ROR-1 protein in a dose-dependent manner by ELISA (Figure 3). The antibody is of the IgG1 type and detects the ROR-1 extracellular domain. By FACS staining, the antibody was effective in Raji, Molt-4, RPMI8226, lymphoblastic leukemia CEM, leukemia HL-60 and other blood cancer cell lines and cervical Hela, breast MCF-7 and ovarian SKOV-3 cancers (FACS data not shown), etc. ROR-1 has been detected in solid cancer cell lines.

Our results show that ROR-1 antibody detects ROR-1 antigen in hematological and solid cancers by ELISA and FACS.

Example 2. Human ROR-1 VH and VL sequences

We sequenced ROR-1 antibody clone B2. The sequences of VH, VL and ScFv are shown below. The structure of ROR-1 scFv is: VH-linker-VL. The connector is G4Sx3. The nucleotide sequence is shown in bold; the nucleotide sequence of the _VL is underlined; in the middle (shown in italics) is the nucleotide sequence encoding the linker.

ROR-1 scFv nucleotide sequence: (SEQ ID NO: 1)

ROR-1 scFv amino acid sequence: (SEQ ID NO: 2)

In the scFv protein, the amino acid sequence of VH (SEQ ID NO.3) is highlighted in bold:

In the scFv protein, the amino sequence of VL (SEQ ID NO.4) is underlined:

EIVLTQSPATLSSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

The linker sequence is 3xG4S (SEQ ID NO:5):

G G G G S G G G G S G G G G S

Example 3A. ROR-1-CAR sequence (CD28 as co-stimulatory domain)

The scheme of the ROR-1-CAR construct (PMC 167) is shown in Figure 2. The lentiviral vector Lenti CMV-MCS-EF1a-puro was used to clone the ROR-1-CAR sequence.

The following nucleotide and amino acid sequences show the ROR-1 ScFv-CD8 hinge-TM28-CD28-CD3 zeta of the present invention. The structure includes human CD8 signal peptide, human ROR-1 scFv (V _H -Linker 3x(G4S)-V _L ), human CD8 hinge, human CD28 transmembrane, human CD28 co-stimulator, and CD3 zeta domain (Figure 2).

ROR-1 scFv(V _H -Linker-V _L )-CD8 hinge-CD28 TM-CD28-CD3-zeta:

<CD8 leader sequence>

Nucleotide sequence (SEQ ID NO:6):

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

Amino acid sequence (SEQ ID NO:7):

MALPVTALLLPLALLLLHAARP

<Nhe I restriction site>

Gctagc

<Nhe I restriction site amino acid sequence>

AS

<ROR-1scFV>

See Example 2 for the nucleotide and amino acid sequences.

<XhoI restriction site>

CTCGAG

<XhoI restriction site amino acid sequence>

LE

<CD8 hinge>

Nucleotide sequence (SEQ ID NO:8):

AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATaagccc

Amino acid sequence (SEQ ID NO:9):

KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKP

<CD28TM/Activation>

Nucleotide sequence (SEQ ID NO: 10):

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

Amino acid sequence (SEQ ID NO: 11):

FWVLVVVGGVLACYSLLVTVAFIIFWV/RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

<CD3 zeta>

Nucleotide sequence (SEQ ID NO: 12):

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAG

Amino acid sequence (SEQ ID NO: 13):

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

<EcoRI restriction site>

gaattc

Amino acid sequence of ROR-1-CAR protein (SEQ ID NO: 14):

Example 3B. ROR-1-CAR sequence (4-1BB as co-stimulatory domain)

Similar to Example 3A, we also generated a CAR with a 41BB co-stimulatory domain. The lentiviral vector LentiCMV-MCS-MNDU3-puro was used to clone the ROR-1-CAR sequence. CAR PMC869 is located in a vector with the AmpR gene, and CAR PMC870 is located in a vector with the KanR gene. Both CARs were constructed under the MNDU3 promoter.

The following nucleotide and amino acid sequences show the ROR-1ScFv-CD8 hinge-TM28-4-1BB-CD3 zeta of the present invention. The structure includes human CD8 signal peptide/human ROR-1 scFv (V _H -Linker 3x(G4S)-V _L ), human CD8 hinge, human CD28 transmembrane, co-stimulatory domain human 4-1BB, CD3 zeta (Figure 2) . The only difference between the CAR sequences of Examples 3A and 3B is the co-stimulatory domain: Example 3A has the CD28 sequence and Example 3B has the 4-1BB sequence.

ROR-1-CAR nucleotide sequence with underlined 4-1BB (SEQ ID NO: 15)

ROR-1-CAR amino acid sequence with 4-1BB (SEQ ID NO: 16), VH, bold, linker in italics, VL underlined; 41BB domain in bold and underlined.

Nucleotide sequence 4-1BB (SEQ ID NO: 17)

aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg

Amino acid sequence 4-1BB (SEQ ID NO: 18)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Example 4. CAR lentivirus production.

Lentiviruses were generated by standard procedures using 293 cells, as described in [4].

Example 5. Isolation of Peripheral Blood Mononuclear Cells (PBMC) from Whole Blood

Whole blood (Stanford Hospital Blood Center, Stanford, CA) was collected from individual or pooled donors (depending on blood volume required) into 10 mL heparinized vacuum containers (Becton Dickinson). In a 50 ml conical centrifuge tube (PBS, pH7/4, Ca2+/Mg2+ free), mix about 10 ml of whole anticoagulated blood with sterile phosphate-buffered saline (PBS) buffer for a total volume of 20 ml. The cell layer containing peripheral blood mononuclear cells (PBMC) observed at the diluted plasma/Ficoll interface was very carefully removed, avoiding any Ficoll, washed twice with PBS, and centrifuged at 200 xg for 10 minutes at room temperature. Count the cells with a hemocytometer. With CAR-T medium (AIM V-AlbuMAX (BSA) (LifeTechnologies), containing 5% AB serum and 1.25ug/mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100U/mL penicillin and 100ug/mL chain Mycin) to wash PBMCs once and use for experiments or freeze at -80°C.

Example 6. T cell activation from PBMCT

In CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies) without human interleukin-2 (huIL-2) (Invitrogen) containing 5% AB serum and 1.25 μg/mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100U/mL penicillin and 100μg/mL streptomycin), the separated PBMC were washed once with 1xPBS (pH7.4, no Ca ²⁺ /Mg ²⁺ ) at a concentration of 5×105 cells /mL, and then suspended in CAR-T medium containing 300U/mLhuIL2 (from 1000x stock solution; Invitrogen) to a final concentration of 5x105 cells/mL. Then, mix PBMC and beads at a 1:1 bead-to-cell ratio by transferring 25 μL of beads into 1 mL of PBMC, and incubate at 37 °C in the presence of CO for 24 h prior to viral transduction. Add beads to activate T cells before adding virus.

Example 7. T cell transduction and expansion

After PBMC activation, ^5x106 lentivirus was added to T cells at an MOI of 10:1 and 2 μL/mL of Transplus medium (Alstem, Richmond, CA) (final dilution 1:500). Cells were incubated for an additional 24 hours before repeated virus additions. Cells were then grown for a period of 12-14 days in the presence of 300 U/mL IL-2 (total incubation time depends on the desired final number of CAR-T cells). Analyze cell concentration every 2-3 days, at which time culture medium is added to dilute the cell suspension to ^1x106 cells/ml.

Example 8. Transduction Validation by FACS

Cells were washed and suspended in FACS buffer (phosphate buffered saline (PBS) plus 0.1% sodium azide and 0.4% BSA). Then, divide the cells into ^1x106 aliquots.

FC receptors were blocked with normal goat IgG (Life Technologies). ROR-1 scFv was detected with a biotinylated polyclonal goat anti-human F(ab)2 antibody (Life Technologies); a biotinylated normal polyclonal goat IgG antibody (Life Technologies) was used as an isotype control. Cells were incubated at 4 °C for 25 min and washed once with FACS buffer. After staining cells with anti-F(ab)2 antibody, phycoerythrin (PE)-labeled streptavidin (BDPharmingen, San Diego, CA) and allophycocyanin (APC)-labeled CD3 (eBioscience, San Diego, CA) Diego, CA) stained cells.

Example 9. Cytotoxicity Assay (Real Time Cytotoxicity Assay).

Cytotoxicity assays were performed using the ACEA machine according to the manufacturer's protocol as described in [6].

Example 10. ROR-1-CAR-T cells express human ROR-1 ScFv

ROR-1-CAR cells (PMC167) were efficiently expanded in vitro (not shown). Mock controls were generated using ScFvs from intracellular proteins and used as negative controls in cytotoxicity and cytokine assays. ROR-1-CAR+ cells were detected by FACS with biotinylated goat anti-human _Fab'2 , followed by staining with phycoerythrin-conjugated streptavidin; staining in ROR-1-CAR-T cells was higher than There were no CAR+ expressing T cells (Figure 4). Thus, transduction of lentiviral ROR-1 resulted in the expression of ROR-1-CARscFv.

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

Cytotoxicity assays were performed using an RTCA impedance-based assay according to the manufacturer's conditions. ROR-1-CD28-CD3 CAR-T cells (PMC167) killed ROR-1-positive SKOV-3 ovarian solid tumor cells (not shown) and HCT116, HT29, Lovo-1 colon cancer cells (Fig. 5). In this assay, the integrity of the target cell monolayer is continuously monitored by its impedance in a weak electric field; killing of target cells by CAR-T cells reduces the integrity of the monolayer, and thus its impedance. PMC167-transduced cells were added to target cells at an effector-to-target ratio (E:T) of 10:1 and 3:1, respectively. In these two ratios, PMC167 cells caused a continuous decrease in the impedance of the target cell monolayer. Calculating cytotoxicity at the end of the assay (approximately 25 h after CAR-T addition) showed that PMC167-transduced cells were significantly more cytotoxic than control T cells at both E:T ratios (Figure 5). Although the frequency of CAR-T cells in PMC167 cultures was relatively low, PMC167-transduced cells killed about 80% of target cells at a ratio of 10:1.

Example 12. ROR-1-CAR secretes high levels of IFN-γ ROR-1 positive cancer cells.

After co-incubating CAR-T cells (PMC167) with SKOV-3 cells and HT-29 cells in Fig. 6A and Lovo, HCT116 and HT29 colon tumor cell lines in Fig. Protocol ELISA was performed with Fisher kit. The level of IFN-γ secretion by ROR-1-CAR-T on ROR-1-positive cancer cells was significantly higher than that of T cells and Mock-CAR-T cells (Figure 6A and Figure 6B).

Example 13. ROR-1-28-CD3-CAR-T cells significantly reduced Lovo-1 tumor growth in vivo.

We injected Lovo-1 colon cancer cells into NSG mice, followed by three intravenous injections of ROR-1-CAR-T cells on days 27, 40, and 47. ROR-1-CAR-T cells (PMC167) significantly reduced Lovo-1 xenograft tumor growth (Fig. 7A). Tumor size (Fig. 7B) and tumor weight (Fig. 7C) were significantly reduced relative to Mock-CAR-T cells. ROR-1-CAR-T cells did not reduce the body weight of mice (Fig. 7D), indicating that CAR-T cells were not toxic.

Example 14. ROR1-CAR T cells are cytotoxic.

Figure 8 shows cytometric analysis of CAR expression. Untransduced T cells and PMC167, PMC869, and PMC870 CAR-T cells were stained with biotinylated goat anti-human Fab'2 at day 10, followed by phycoerythrin-conjugated streptavidin (X-axis )dyeing. Quantification of staining frequency is shown on the Y-axis.

Figure 9 shows the quantification of cytotoxicity in the RTCA assay. Cytotoxicity was calculated at the end of the assay as (X-Y)*100/X, where X is the average impedance of the target cell monolayer in the absence of effector cells and Y is the average impedance of the target cell monolayer in the presence of effector cells impedance. For the target cell lines, the p-value for CAR-T cells versus T cells was <0.0001 at each E:T ratio by two-way ANOVA using Sidak's post hoc test.

Figure 10 shows the production of IFN-γ in response to ROR1+ tumor cells. Media was harvested from 10:1 RTCA wells, cells were removed by centrifugation and analyzed for IFN-γ levels by ELISA. The p-value for each CAR-T cell culture versus T cells was <0.0001 by two-way ANOVA using Sidak's post hoc test. At different E:T ratios, the three CAR-T cell cultures produced significantly higher levels of interferon-γ than control T cells.

references

1. Grupp, S.A., Kalos, M., Barrett, D., Aplenc, R., Porter, D.L., Rheingold, S.R., Teachey, D.T., Chew, A., Hauck, B., Wright, J.F., et al. (2013). Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 368,1509-1518.

2.Maus,M.V.,Haas,A.R.,Beatty,G.L.,Albelda,S.M.,Levine,B.L.,Liu,X.,Zhao,Y.,Kalos,M.,and June,C.H.(2013).T cells expressing chimericantigen receptors can cause anaphylaxis in humans. Cancer Immunol Res 1,26-31.

3. Maus, M.V., Grupp, S.A., Porter, D.L., and June, C.H. (2014). Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood 123, 2625-2635.

4. Goluboskaya V, Wu L. Different Subsets of T Cells, Memory, EffectorFunctions, and CAR-T Immunotherapy. Cancers (Basel). 2016 Mar 15; 8 (3).pii:E36.doi:10.3390/cancers8030036.Review.

5. Dal Ferro M, Rizzo S, Rizzo E, Marano F, Luisi I, Tarasiuk O, Sblattero D. Phage Display Technology for Human Monoclonal Antibodies. Methods Enzymol 121, 332-340.

6. Berahovich R, Zhou H, Xu S, Wei Y, Guan J, Guan J, Harto H, Fu S, Yang K, Zhu S, Li L, Wu L, Golubovskaya V. CAR-T cells based on Novel BCMA monoclonal antibody block multiple myeloma Cell growth. Cancers (Basel), 10 (9), 2018.

SEQUENCE LISTING

<110> PROMAB BIOTECHNOLOGIES, INC.

FOREVERTEK BIOTECHNOLOGY CO., LTD

<120> HUMAN ROR-1 ANTIBODY AND ANTI-ROR-1-CAR-T CELLS

<130> 119995-8026.WO01

<150> 63/004,214

<151> 2020-04-02

<160> 18

<170> PatentIn version 3.5

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<211> 729

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caggtgcagc tgcaggagtc gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aggccatagc 300

agtggctggt atcgaaggta cttcgatctc tggggccgtg gcaccctggt caccgtctca 360

agcggtggcg gtggttctgg tggcggtggt tctggtggcg gtggttctga aattgtgctg 420

actcagtctc cagccaccct gtctttgtct ccaggggaaa gagccaccct ctcctgcagg 480

gccagtcaga gtgttagcag ctacttagcc tggtaccaac agaaacctgg ccaggctccc 540

aggctcctca tctatgatgc atccaacagg gccactggca tcccagccag gttcagtggc 600

agtgggtctg ggacagactt cactctcacc atcagcagcc tagagcctga agattttgca 660

gtttaattact gtcagcagcg tagcaactgg cctccgacgt tcggccaagg gaccaaggtg 720

gaaatcaaa 729

<210> 2

<211> 243

<212> PRT

<213> Homo sapiens

<400> 2

Gln Val Gln Leu Gln Glu Ser Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly His Ser Ser Gly Trp Tyr Arg Arg Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro

130 135 140

Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

145 150 155 160

Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro

165 170 175

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr

180 185 190

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

195 200 205

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

210 215 220

Gln Gln Arg Ser Asn Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val

225 230 235 240

Glu Ile Lys

<210> 3

<211> 121

<212> PRT

<213> Homo sapiens

<400> 3

Gln Val Gln Leu Gln Glu Ser Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly His Ser Ser Gly Trp Tyr Arg Arg Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 4

<211> 107

<212> PRT

<213> Homo sapiens

<400> 4

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 5

<211> 15

<212> PRT

<213> Artificial Sequence

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<223> Synthetic

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Gly Gly GLy Gly Ser Gly Gly GLy Gly Ser Gly Gly GLy Gly Ser 15

<210> 6

<211> 63

<212>DNA

<213> Homo sapiens

<400> 6

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 7

<211> 21

<212> PRT

<213> Homo sapiens

<400> 7

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 8

<211> 147

<212>DNA

<213> Homo sapiens

<400> 8

aagccccacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 60

cccctgtccc tgcgcccaga ggcgagccgg ccagcggcgg ggggcgcagt gcacacgagg 120

gggctggact tcgccagtga taagccc 147

<210> 9

<211> 49

<212> PRT

<213> Homo sapiens

<400> 9

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys

35 40 45

Pro

<210> 10

<211> 204

<212>DNA

<213> Homo sapiens

<400> 10

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180

cgcgacttcg cagcctatcg ctcc 204

<210> 11

<211> 68

<212> PRT

<213> Homo sapiens

<400> 11

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

20 25 30

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

35 40 45

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

50 55 60

Ala Tyr Arg Ser

65

<210> 12

<211> 342

<212>DNA

<213> Homo sapiens

<400> 12

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctaat ag 342

<210> 13

<211> 112

<212> PRT

<213> Homo sapiens

<400> 13

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 14

<211> 498

<212> PRT

<213> Homo sapiens

<400> 14

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ala Ser Gln Val Gln Leu Gln Glu Ser Gly Ala

20 25 30

Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr

35 40 45

Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro

50 55 60

Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr

65 70 75 80

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr

85 90 95

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Gly His Ser Ser Gly Trp Tyr Arg

115 120 125

Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

145 150 155 160

Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu

165 170 175

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu

180 185 190

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

195 200 205

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

225 230 235 240

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Thr

245 250 255

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Leu Glu Lys Pro Thr Thr

260 265 270

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

275 280 285

Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala

290 295 300

Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Phe Trp Val

305 310 315 320

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

325 330 335

Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu

340 345 350

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

355 360 365

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

370 375 380

Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

385 390 395 400

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

405 410 415

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

420 425 430

Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

435 440 445

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

450 455 460

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

465 470 475 480

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

485 490 495

Pro Arg

<210> 15

<211> 1500

<212>DNA

<213> Homo sapiens

<400> 15

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggctagcc aggtgcagct gcaggagtcg ggcgcaggac tgttgaagcc ttcggagacc 120

ctgtccctca cctgcgctgt ctatggtggg tccttcagtg gttactactg gagctggatc 180

cgccagcccc cagggaaggg gctggagtgg attgggaaa tcaatcatag tggaagcacc 240

aactacaacc cgtccctcaa gagtcgagtc accatatcag tagacacgtc caagaaccag 300

ttctccctga agctgagctc tgtgaccgcc gcggacacgg ctgtgtatta ctgtgcgaga 360

ggccatagca gtggctggta tcgaaggtac ttcgatctct ggggccgtgg caccctggtc 420

accgtctcaa gcggtggcgg tggttctggt ggcggtggtt ctggtggcgg tggttctgaa 480

attgtgctga ctcagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 540

tcctgcaggg ccagtcagag tgttagcagc tacttagcct ggtaccaaca gaaacctggc 600

caggctccca ggctcctcat ctatgatgca tccaacaggg ccactggcat cccagccagg 660

ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 720

gattttgcag tttaattactg tcagcagcgt agcaactggc ctccgacgtt cggccaaggg 780

accaaggtgg aaatcaaact cgagaagccc accacgacgc cagcgccgcg accaccaaca 840

ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgag ccggccagcg 900

gcggggggcg cagtgcacac gagggggctg gacttcgcca gtgataagcc cttttgggtg 960

ctggtggtgg ttggtggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt 1020

attttctggg tgaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1200

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgcagag aaggaagaac 1320

cctcaggaag gcctctacaa tgaactgcag aaagataaga tggcggaggc cctacagtgag 1380

atgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1440

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctaa 1500

<210> 16

<211> 499

<212> PRT

<213> Homo sapiens

<400> 16

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ala Ser Gln Val Gln Leu Gln Glu Ser Gly Ala

20 25 30

Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr

35 40 45

Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro

50 55 60

Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr

65 70 75 80

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr

85 90 95

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Gly His Ser Ser Gly Trp Tyr Arg

115 120 125

Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

145 150 155 160

Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu

165 170 175

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu

180 185 190

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

195 200 205

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

225 230 235 240

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Thr

245 250 255

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Leu Glu Lys Pro Thr Thr

260 265 270

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

275 280 285

Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala

290 295 300

Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Phe Trp Val

305 310 315 320

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

325 330 335

Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu

340 345 350

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

355 360 365

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

370 375 380

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

385 390 395 400

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

405 410 415

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

420 425 430

Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

435 440 445

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

450 455 460

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

465 470 475 480

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

485 490 495

Pro Pro Arg

<210> 17

<211> 126

<212>DNA

<213> Homo sapiens

<400> 17

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 18

<211> 42

<212> PRT

<213> Homo sapiens

<400> 18

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

Claims (14)

CLAIMS 1. A human anti-human ROR-1 antibody comprising a V _H having said amino acid of SEQ ID NO:3 and a V _L having said amino acid of SEQ ID NO:4. 2. A single chain variable fragment (scFv) comprising a _VH having said amino acid of SEQ ID NO:3 and a _VL having said amino acid of SEQ ID NO:4. 3. The scFv according to claim 3, further comprising a linker between _VH and _VL . 4. The scFv according to claim 3, wherein it has the amino acid sequence of SEQ ID NO:2. 5. A chimeric antigen receptor (CAR), comprising from N-terminal to C-terminal: (i) the scFv of claim 2, (ii) transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) Activation domain. 6. The CAR of claim 5, wherein the scFv further comprises a linker between _VH and _VL . 7. The CAR of claim 5, wherein the co-stimulatory domain is CD28. 8. The CAR of claim 5, wherein the co-stimulatory domain is 4-1BB. 9. The CAR of claim 5, wherein the activation domain is CD3 zeta. 10. The CAR according to claim 5, wherein it has the amino acid sequence of SEQ ID NO: 14. 11. The CAR according to claim 5, wherein it has the amino acid sequence of SEQ ID NO: 16. 12. A nucleic acid encoding the CAR of claim 5. 13. A T cell modified to express the CAR of claim 5. 14. A natural killer cell modified to express the CAR of claim 5.

Images