WO2021202863A1 - Anticorps de ror-1 humain et lymphocytes car-t anti-ror-1 - Google Patents

Anticorps de ror-1 humain et lymphocytes car-t anti-ror-1 Download PDF

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WO2021202863A1
WO2021202863A1 PCT/US2021/025355 US2021025355W WO2021202863A1 WO 2021202863 A1 WO2021202863 A1 WO 2021202863A1 US 2021025355 W US2021025355 W US 2021025355W WO 2021202863 A1 WO2021202863 A1 WO 2021202863A1
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car
ror
cells
scfv
seq
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Lijun Wu
Vita Golubovskaya
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Promab Biotechnologies, Inc.
Forevertek Biotechnology Co., Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/50—Colon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes

Definitions

  • the present invention relates to human ROR-1 -specific antibody and anti-ROR-1- CAR-T cells, which are useful in the field of adoptive immunity gene therapy for tumors.
  • T cells or T lymphocytes the armed forces of our immune system, constantly look for foreign antigens and discriminate abnormal (cancer or infected cells) from normal cells.
  • Genetically modifying T cells with CAR (Chimeric antigen receptor) constructs is the most common approach to design tumor-specific T cells.
  • CAR-T cells targeting tumor-associated antigens (TAA) can be infused into patients (called adoptive cell transfer or ACT) representing an efficient immunotherapy approach [1, 2].
  • adoptive cell transfer or ACT representing an efficient immunotherapy approach [1, 2].
  • the advantage of CAR-T technology compared with chemotherapy or antibody is that reprogrammed engineered T cells can proliferate and persist in the patient (“a living drug”)[3], [4], [1],
  • CARs usually consist of a monoclonal antibody-derived single-chain variable fragment (scFv) at the N-terminal part, hinge, transmembrane domain and a number of intracellular co-activation domains: (i) CD28, (ii) CD137 (4-1BB), CD27 or other costimulatory domains, in tandem with an activation CD3-zeta domain.
  • scFv monoclonal antibody-derived single-chain variable fragment
  • NK cells Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system.
  • the role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells, acting at around 3 days after infection, and respond to tumor formation.
  • Figure 1 show the structures of CAR.
  • the left panel shows the structure of the first generation (no costimulatory domains).
  • the middle panel shows the structure of the second generation (one co-stimulatory domain CD28 or 4-BB).
  • the right panel shows the third generation of CAR (two or several co-stimulatory domains) are shown.
  • the Figure is from Golubovskaya, Wu, Cancers, 2016 [4],
  • Tyrosine-protein kinase transmembrane receptor ROR1 also known as neurotrophic tyrosine kinase, receptor-related 1 (NTRKR1), is an enzyme that in humans is encoded by the ROR1 gene.
  • ROR1 is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family.
  • ROR-1 is 937 amino-acid protein, about 104 kb with 30-406 aa extracellular domain. ROR-1 has low expression in most tissues which is advantageous to use it as a target for CAR-T cell therapy.
  • the ROR-1 gene encodes a receptor tyrosine kinase-like orphan receptor that modulates neurite growth in the central nervous system.
  • the encoded protein is a glycosylated type I membrane protein that belongs to the ROR subfamily of cell surface receptors.
  • ROR-1 is receptor for ligand WNT5A which activate downstream NF kappa B signaling pathway and may result in the inhibition of WNT-mediated signaling.
  • ROR1 has recently been shown to be expressed on ovarian cancer stem cells and promote migration, invasion and cancer stem cell spheroid formation.
  • ROR-1 is shown to be overexpressed in both hematological cancers and solid tumors that makes this target useful for CAR-T therapy.
  • ROR-1 Low expression of ROR-1 has been shown in most of normal human tissues such as adipose and soft tissue, bone marrow and immune system, endocrine tissues, female tissue, gastrointestinal tract, kidney and urinary bladder, liver and gallbladder, lung, muscle tissues, male tissues, and skin.
  • FIG. 1 The structures of ROR-1 CAR constructs. Abbreviations: ScFv, single chain variable fragment; h-hinge; TM-transmembrane; Figure 3. Binding of ROR-1 done B2 with Human ROR-1 antigen. Different dilutions are shown on X-axis; binding is shown on Y-axis, OD450 nM.
  • FIG. 4 FACS with goat anti-human Fab’i antibody to detect ROR-1 -CAR positive cdls. Left pand: isotype staining, right pand: goat anti-human Fab'a human FAB staining. T cdls and ROR-l-CAR-T cdls are shown.
  • RTCA assay demonstrates killing activity of ROR-l-CAR-T cdls in vitro.
  • Figure 5. The cytotaxidty assay was performed using ROR1 -CAR-T cdls from 3 donors with ACEA XCdligence system at different Effector to Target cdl ratio 3:1 (right pands) and 10:1 (left pands). Cytotaxidty was calculated as (X-Y)*100/X, where X is the average impedance of the target cdl monolayer in the absence of effector cdls and Y is the average impedance of the target cdl monolayer in the presence of effector cdls.
  • Figure 6B IFN-gamma secretion after RTC A assay with ROR1-CAR-T cdls from 3 different donors (#000, 155, and 160) using target HCT116, HT29 and Lovo colon cancer cdls.
  • the p values for CAR-T cdls vs T cdls are ⁇ 0.0001 by 2-way ANOVA with
  • Figures 7A-7D ROR-l-CAR-T cdls significantly decrease Lovo-l xenograft tumor growth.
  • Figure 7A shows tumor growth curve.
  • Figure 7B and 7C shows tumor size and tumor weight of ex vivo tumors.
  • Figure 7D shows mouse body wdght *p ⁇ 0.05 ROR-1- CD28-CD3 CAR-T cdls versus Mock CAR-T cdls.
  • figure 8. Flow cytometric analysis of CAR expression, figure 9. Quantitation of cytotoxicity in the RTCA assay. For each cdl line, bars from left to right show: T cdls, then PMC167, FMC869, and FMC870 CAR-T cdls. figure 10.
  • a "chimeric antigen receptor (CAR)” is a receptor protein that has been engineered to give T cells the new ability to target a specific protein.
  • the receptor is chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor.
  • CAR is a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain.
  • the "chimeric antigen receptor (CAR)” is sometimes called a “chimeric receptor", a "T-body”, or a “chimeric immune receptor (CIR).”
  • extracellular domain capable of binding to an antigen means any oligopeptide or polypeptide that can bind to a certain antigen.
  • intracellular domain means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.
  • a “domain” means one region in a polypeptide which is folded into a particular structure independently of other regions.
  • scFv single chain variable fragment
  • An example of the scFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence.
  • H chain immunoglobulin heavy chain
  • L chain light chain
  • tumor antigen means a biological molecule having antigenicity, expression of which causes cancer.
  • the inventors have generated human ROR-1 monoclonal antibody using phage display library specifically targeting human ROR-1 antigen.
  • the inventors have produced ROR-1 -CAR- T cells to target cancer cells overexpressing 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 is directed to a human anti-human ROR-1 antibody or an antigen-binding fragment therefore, comprising VH having the amino acid of SEQ ID NO: 3 and VL having the amino acid of SEQ ID NO: 4.
  • the monoclonal anti-human ROR-1 antibody is generated against the extracellular region of the purified recombinant fragment of human ROR-1.
  • the monoclonal anti-human ROR-1 antibody is a singlechain variable fragment (scFv).
  • the present invention is also directed to a chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) against ROR-1, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain.
  • a chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) against ROR-1, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain.
  • FIG. 2 shows one structure of ROR-1 CAR construct.
  • the second -generation CAR is shown with either CD28 or 4 IBB co-stimulatory domains.
  • ScFv can be VH-linker-VL or VL-linker-VH.
  • the co-stimulatory domain is selected from the group consisting of CD28, 4- IBB, GITR, ICOS-1, CD27, OX-40 and DAP10.
  • a preferred the co-stimulatory domain is CD28.
  • a preferred activating domain is CD3 zeta (CD3 Z or CD3Q
  • the transmembrane domain may be derived from a natural polypeptide, or may be artificially designed.
  • the transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein.
  • a transmembrane domain of a T cell receptor a or ⁇ chain, a CD3 zeta chain, CD28, CD3e., CD45, CD4, CDS, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD 137, ICOS, CD 154, or a GITR can be used.
  • the artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. It is preferable that a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.
  • a short oligopeptide linker or a 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.
  • a linker sequence having a glycine-serine continuous sequence can be used.
  • the present invention provides a nucleic acid encoding the ROR-1 CARs.
  • the nucleic acid encoding the CAR can be prepared from an amino acid sequence of the specified CAR by a conventional method.
  • a base sequence encoding an amino acid sequence can be obtained from the aforementioned NCBI RefSeq IDs or accession numbers of GenBenk for an amino acid sequence of each domain, and the nucleic acid of the present invention can be prepared using a standard molecular biological and/or chemical procedure.
  • a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • 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.
  • a virus vector such as a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV vector can be used.
  • a virus vector lacking the replicating ability so as not to self- replicate in an infected cell is preferably used.
  • a suitable packaging cell based on a LTR sequence and a packaging signal sequence possessed by the vector can be selected for preparing a retrovirus particle using the packaging cell.
  • the packaging cell include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm- 12, and Psi-Crip.
  • a retrovirus particle can also be prepared using a 293 cell or a 293 T cell having high transfection efficiency.
  • Many kinds of retrovirus vectors produced based on retroviruses and packaging cells that can be used for packaging of the retrovirus vectors are widely commercially available from many companies.
  • a CAR-T cell binds to a specific antigen via the CAR, thereby a signal is transmitted into the cell, and as a result, the cell is activated.
  • the activation of the cell expressing the CAR is varied depending on the kind of a host cell and an intracellular domain of the CAR, and can be confirmed based on, for example, release of a cytokine, improvement of a cell proliferation rate, change in a cell surface molecule, or the like as an index.
  • release of a cytotoxic cytokine a tumor necrosis factor, lymphotoxin, etc.
  • release of a cytokine or change in a cell surface molecule stimulates other immune cells, for example, a B cell, a dendritic cell, a NK cell, and a macrophage.
  • the cell expressing the CAR can be used as a therapeutic agent for a disease.
  • the therapeutic agent comprises the cell expressing the CAR as an active ingredient, and it may further comprise a suitable excipient.
  • the 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 cancer, and other cancer).
  • the inventors have provided data demonstrating efficient expression of ROR- 1 in different types of cancer.
  • ROR-1 -CAR-T cells express higher cytotoxic activity against ROR-1 -positive cancer cells than against non-transduced T cells and Mock-CAR-T cells.
  • the advantage of this antibody versus mouse monoclonal or humanized antibodies is that it is a full human sequence that will not generate an immune response against antibody sequence in patients.
  • the present human monoclonal anti-human ROR-1 antibody detects ROR-1 in ROR- 1 -positive cancer cells.
  • the present ROR-1 antibody can be used for immunotherapy applications: toxin/drug- conjugated antibody, monoclonal therapeutic antibody, humanization of ROR-1 antibody, and CAR-T cell immunotherapy.
  • ROR-l-CAR-T cells using the present ROR-1 antibody can be effectively used to target ROR-1 antigen in ROR-1 -positive cell lines.
  • ROR-l-CAR-T can be used in combination with different therapeutic agents: checkpoint inhibitors; targeted therapies, small molecule inhibitors, antibodies.
  • ROR-1 antibody can be modified with site-directed mutagenesis for affinity tuning.
  • ROR-l-CAR-T cells can be used clinically for ROR-1 -positive cells.
  • Third generation CAR-T or other co-activation signaling domains can be used for the same ROR-1 -scFv inside CAR.
  • ROR-1 with other CAR targeting other tumor antigens or tumor microenvironment (VEGFR-1 -3), PDL-1, CD80 or bi-scFv-CAR can be used to enhance activity of monotherapy ROR-l-CAR.
  • Bi-specific antibodies with ROR-1 and CD3 or other antigens can be generated for therapy.
  • ROR-l-CAR-T cells can be used against cancer stem cells that are resistant against chemotherapy and form aggressive tumors.
  • ROR-l-CAR can be used for generating other types of cells such as CAR-natural killer (NK) cells, iPS (induced pluripotent)-NK or iPS-T cells, ROR-1 -CAR-macrophages, and other ROR-l-CAR hematopoietic cells, which can target ROR-1 -positive cancers.
  • the present invention provides T cells, or NK cells, or macrophages, or hematopoietic cells, modified to express the ROR-l-CAR.
  • ROR1-CAR can be used both for autologous cells and allogenic hematopoietic cells.
  • the following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.
  • the inventors generated ROR-1 CAR constructs inside lenti viral vector cloned into the lentiviral vector with either EF1 or MNDU3 promoters to drive expression of CAR.
  • Three CAR constructs were prepared.
  • the CAR constructs were inside lentiviral vector and had either Amp resistance gene or kanamycin resistance gene as a marker.
  • CAR PMC 167 used EF1 promoter and Amp resistance gene, and had CD28 domain.
  • CAR PMC869 used MNDU3 promoter and AMP resistance gene, and had 4-1BB domain.
  • CAR PMC870 used MNDU3 promoter and kanamycin resistance gene, and had 4-1 BB domain.
  • the lenti viruses were generated in 293 T cells and titer was established by RT-PCR. Then equal dose of lentiviruses was used for transduction of T cells, as described in Examples.
  • Example 1. ROR-1 antibody detects ROR-1 protein by ELISA and FACS staining
  • the human antibody ROR-1, clone B2 detected extracellular ROR-1 protein by ELISA in a dose-dependent manner ( Figure 3).
  • the antibody is IgGl type and detects ROR-1 extracellular domain.
  • the antibody detected ROR-1 in both hematological cancer cell lines such as Raji, Molt-4, RPMI 8226, lymphoblastic leukemia CEM, leukemia HL-60, and in solid cancer cell lines such as cervical Hela, breast MCF-7, and ovarian SKOV-3 cancers (FACS data not shown).
  • ROR-1 antibody clone B2.
  • the sequence of VH and VL and ScFv are shown below.
  • the structure of ROR-1 scFv is: VH-linker-VL.
  • Linker is G4Sx3.
  • the bold shows the nucleotide sequence; the underlined shows the nucleotide sequence of V L ; in between (shown in italics font) is the nucleotide sequence encoding a linker.
  • ROR-1 scFv nucleotide sequence (SEQ ID NO: 1)
  • ROR-1 scFv amino acid sequence (SEQ ID NO: 2) QVQLQESGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHS GSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGHSSGWYRRYF DLWGRGTLVTVSSGGGGjtGGGG ⁇ GGGG ⁇ EIVLTOSPATLSLSPGERATLSCRASOSV
  • VL EIVLTOSPATLSLSPGERATLSCRASOSVSSYLAWYOOKPGOAPRLLIYDASNRATGIP
  • the linker sequence is 3xG4S (SEQ ID NO: 5): G G G G S G G GG S G G GG S Example 3A.
  • ROR-l-CAR Sequences CD28 as Co-Stimulating Domain
  • ROR-l-CAR construct PMC 167) is shown on Figure 2.
  • Lentiviral vector Lenti CMV-MCS-EF 1 a-puro was used for cloning of ROR-l-CAR sequence.
  • the following nucleotide sequence and amino acid sequences show ROR-1 ScFv - CDS hinge-TM28-CD28-CD3 zeta of the present invention.
  • the structure includes Human CDS signaling peptide, human ROR-1 scFv (Vu-Linker 3x(G4S) -VL), human CDS hinge, human CD28 transmembrane, human CD28 co-stimulatory, CD3 zeta domains ( Figure 2).
  • ROR-1 scFv Vn-Linker -VL>CD8 hinge CD28 TM.
  • CD28-CD3-zeta CD28-CD3-zeta:
  • Amino-acid sequence (SEQ ID NO: 7): MALPVTALLLPLALLLHAARP
  • Nucleotide Sequence (SEQ ID NO: 10): TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA CAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAG TGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAG CCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC Amino-acid sequence (SEQ ID NO: 11):
  • Example 3 A Similar to Example 3 A, we also generated CAR with 41BB costimulatory domain.
  • Lentiviral vector Lenti CMV -MC S -MNDU 3 -puro was used for cloning of ROR-l-CAR sequence.
  • the following nucleotide sequence and amino acid sequences show ROR-1 ScFv - CDS hinge-IM28-4-lBB-CD3 zeta of the present invention.
  • the structure includes Human CDS signaling peptide, human ROR-1 scFv (Vn-Linker 3x(G4S) -V L ), human CDS hinge, human CD28 transmembrane, co-stimulating domains human 4- IBB, CD3 zeta ( Figure 2).
  • the only difference between the CAR sequences of Examples 3 A and 3B is the costimulating domain: Example 3 A has CD28 sequence, and Example 3B has 4- IBB sequence.
  • the lenti viruses were produced by the standard procedure using 293 cells as described in
  • PBMC peripheral blood mononuclear cells
  • Isolated PBMC were washed with once lxPBS (pH7.4), no Ca 2+ /Mg 2-h ), and in CAR- T media (AIM V-AlbuMAX(BSA) (Life Technologies), with 5% AB serum and 1.25 pg/mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U/mL penicillin, and 100 pg/mL streptomycin), in the absence of human interleukin-2 (huIL-2)(Invitrogen), at a concentration of 5 x 10 s cells/mL, then resuspended to a final concentration of 5x10 s cells/mL in CAR-T medium with 300U/mL huIL2 (from a lOOOx stock; Invitrogen).
  • PBMC and beads were then mixed at a 1 : 1 bead-to-cell ratio, by transferring 25 uL of beads to 1 mL of PBMC, and incubated at 37°C in the presence of C02 for 24 hours before viral transduction. Addition of beads activated T cells before addition of virus.
  • PBMC peripheral blood mononuclear cells
  • 5xl0 6 lentivirus were added to T cells atMOI 10:1, and 2 pL/mL of media of Transplus (Alstem, Richmond, CA) (a final dilution of 1:500). Cells were incubated for an additional 24 hours before repeating addition of virus. Cells were then grown in the presence of 300 U/mL of 1L-2 for a period of 12-14 days (total incubation time was dependent on the final umber of CAR-T cells required). Cells concentrations were analyzed every 2-3 days, with media being added at that time to dilute the cell suspension to lxlO 6 cells/ml.
  • FACS buffer Phosphate-buffered saline (PBS) plus 0.1% sodium azide and 0.4% BSA. Cells were then divided at lxlO 6 aliquots.
  • the cytotoxicity was performed using ACEA machine according to manufacturer’s protocol as described [6],
  • ROR-l-CAR cells (PMC 167) were effectively expanded in vitro (not shown). Mock control with ScFv from intracellular protein were generated and used as a negative control in cytotoxicity and cytokine assay.
  • the ROR-1-CAR+ cells were detected by FACS with biotinylated goat anti-human Fab’2 and then stained with phycoerythrin-conjugated streptavidin; the staining was higher in ROR-l-CAR-T cells than in T cells with no CAR+ expression ( Figure 4). Thus, transduction of lentiviral ROR-1 resulted in expression of ROR- 1-CARscFv.
  • Example 11 ROR-l-CAR-T cells expressed high cytotoxic activity against ROR-1- positive cancer cells.
  • the cytotoxicity assay was performed using RTCA impedance-based assay according to manufacturer’s conditions.
  • ROR-1-CD28-CD3 CAR-T cells PMC167
  • killed ROR-1 positive SKOV-3 ovarian solid tumor cells not shown
  • HCT116, HT29, Lovo-1 colon cancer cells Figure 5
  • the integrity of the target cell monolayer was continually monitored via its impedance in a weak electrical field; killing of the target cells by the CAR-T cells decreased the monolayer’s integrity and, therefore, its impedance decreased.
  • PMC167-transduced cells were added to the target cells at effectontarget (E:T) ratios of 10:1 and 3 :1; at both ratios, the PMC167 cells caused a sustained decrease in target cell monolayer impedance.
  • E:T effectontarget
  • Calculation of cytotoxicity at the end of the assay indicated that the PMC167-transduced cells were significantly more cytotoxic than the control T cells at both E:T ratios ( Figure 5).
  • Approximately 80% of the target cells were killed by the PMC167-transduced cells at the 10:1 ratio, despite the relatively low frequency of CAR-T cells in the PMC167 cultures.
  • Example 14 ROR1-CAR T cells were cytotoxic.
  • Figure 8 shows cytometric analysis of CAR expression.
  • Non-transduced T cells and PMC 167, PMC 869 and PMC870 CAR-T cells were stained on day 10 with biotinylated goat anti-human Fab ’2 and then with phy coerythrin-conj ugated streptavidin (X-axis). The quantitation of staining frequency is shown on Y-axis.
  • Figure 9 shows quantitation 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.
  • X is the average impedance of the target cell monolayer in the absence of effector cells
  • Y is the average impedance of the target cell monolayer in the presence of effector cells.
  • the p value for CAR-T cells vs T cells is ⁇ 0.0001 by 2-way ANOVA with Sidak’s post-hoc test.
  • Figure 10 shows IFN-gamma production in response to RORl + tumor cells.
  • Medium was collected from the 10:1 RTCA wells, centrifuged to remove cells and analyzed by ELISA for the levels of IFN-7.
  • the p values for each of the CAR-T cell cultures vs T cells are ⁇ 0.0001 by 2-way ANOVA with Sidak’s post-hoc test.. All 3 CAR-T cell cultures produced significantly higher levels of IFN- ⁇ than the control T cells in response to each of the target cell lines at each E:T ratio. References
  • T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res 1, 26-31.

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Abstract

La présente invention concerne un anticorps anti-ROR-1 humain, ou un fragment variable monocaténaire (scFv), comprenant VH ayant la séquence d'acides aminés de SEQ ID No : 3 Et VL ayant la séquence d'acides aminés de SEQ ID No : 4. La présente invention concerne en outre un récepteur antigénique chimérique (CAR) ROR-1 comprenant, de l'extrémité N-terminale à l'extrémité C-terminale : (i) un fragment variable monocaténaire (scFv) de la présente invention, (ii) un domaine transmembranaire, (iii) au moins un domaine costimulateur, et (iv) un domaine d'activation. L'anticorps monoclonal de la présente invention présente une liaison sélective et à haute affinité à l'égard de ROR-1.
PCT/US2021/025355 2020-04-02 2021-04-01 Anticorps de ror-1 humain et lymphocytes car-t anti-ror-1 WO2021202863A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023020423A1 (fr) * 2021-08-19 2023-02-23 Shandong Boan Biotechnology Co., Ltd. Cellules car t double ror1/cd19 ou car ror1 pour traiter des tumeurs
WO2023077026A1 (fr) 2021-10-28 2023-05-04 Lyell Immunopharma, Inc. Procédés de culture de cellules exprimant une protéine de liaison à ror1
WO2024064952A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a surexprimant c-jun
WO2024064958A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
WO2024077174A1 (fr) 2022-10-05 2024-04-11 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120058906A1 (en) * 2008-11-07 2012-03-08 Vaughn Smider Combinatorial antibody libraries and uses thereof
US20170121414A1 (en) * 2015-11-03 2017-05-04 Janssen Biotech, Inc. Subcutaneous Formulations of Anti-CD38 Antibodies and Their Uses
US20180371081A1 (en) * 2009-09-25 2018-12-27 Xoma (Us) Llc Novel Modulators
CN110590951A (zh) * 2019-09-27 2019-12-20 安萌得医药科技(上海)有限公司 一种ror1单克隆抗体、制备方法及其应用
US20200291120A1 (en) * 2019-02-11 2020-09-17 Zumutor Biologics Inc. Anti-clec2d antibodies and methods of use thereof
US20210047419A1 (en) * 2015-01-20 2021-02-18 Igm Biosciences, Inc. Tumor necrosis factor (tnf) superfamily receptor binding molecules and uses thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120058906A1 (en) * 2008-11-07 2012-03-08 Vaughn Smider Combinatorial antibody libraries and uses thereof
US20180371081A1 (en) * 2009-09-25 2018-12-27 Xoma (Us) Llc Novel Modulators
US20210047419A1 (en) * 2015-01-20 2021-02-18 Igm Biosciences, Inc. Tumor necrosis factor (tnf) superfamily receptor binding molecules and uses thereof
US20170121414A1 (en) * 2015-11-03 2017-05-04 Janssen Biotech, Inc. Subcutaneous Formulations of Anti-CD38 Antibodies and Their Uses
US20200291120A1 (en) * 2019-02-11 2020-09-17 Zumutor Biologics Inc. Anti-clec2d antibodies and methods of use thereof
CN110590951A (zh) * 2019-09-27 2019-12-20 安萌得医药科技(上海)有限公司 一种ror1单克隆抗体、制备方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
M. HUDECEK, M.-T. LUPO-STANGHELLINI, P. L. KOSASIH, D. SOMMERMEYER, M. C. JENSEN, C. RADER, S. R. RIDDELL: "Receptor Affinity and Extracellular Domain Modifications Affect Tumor Recognition by ROR1-Specific Chimeric Antigen Receptor T Cells", CLINICAL CANCER RESEARCH, ASSOCIATION FOR CANCER RESEARCH, vol. 19, no. 12, 15 June 2013 (2013-06-15), pages 3153 - 3164, XP055177780, ISSN: 10780432, DOI: 10.1158/1078-0432.CCR-13-0330 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023020423A1 (fr) * 2021-08-19 2023-02-23 Shandong Boan Biotechnology Co., Ltd. Cellules car t double ror1/cd19 ou car ror1 pour traiter des tumeurs
WO2023077026A1 (fr) 2021-10-28 2023-05-04 Lyell Immunopharma, Inc. Procédés de culture de cellules exprimant une protéine de liaison à ror1
WO2024064952A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a surexprimant c-jun
WO2024064958A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
WO2024077174A1 (fr) 2022-10-05 2024-04-11 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a

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