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WO2022152168A1 - 靶向ror1的抗体及其用途 - Google Patents

靶向ror1的抗体及其用途 Download PDF

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Publication number
WO2022152168A1
WO2022152168A1 PCT/CN2022/071621 CN2022071621W WO2022152168A1 WO 2022152168 A1 WO2022152168 A1 WO 2022152168A1 CN 2022071621 W CN2022071621 W CN 2022071621W WO 2022152168 A1 WO2022152168 A1 WO 2022152168A1
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WIPO (PCT)
Prior art keywords
seq
antibody
cdr
cells
ror1
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PCT/CN2022/071621
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English (en)
French (fr)
Inventor
周亚丽
陈功
郭婷婷
姜小燕
任江涛
贺小宏
王延宾
韩露
李国坤
张静
孙慧芳
Original Assignee
南京北恒生物科技有限公司
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Priority to EP22739046.5A priority Critical patent/EP4257610A1/en
Priority to US18/260,905 priority patent/US20240052029A1/en
Publication of WO2022152168A1 publication Critical patent/WO2022152168A1/zh

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    • 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
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Definitions

  • the present invention belongs to the field of immunotherapy. More specifically, the present invention relates to antibodies targeting ROR1 and their use in the prevention and/or treatment and/or diagnosis of diseases.
  • Receptor tyrosine kinase-like orphan receptor 1 (ROR1), a member of the receptor tyrosine kinase (RTK) family, is generally highly expressed during embryogenesis and fetal development, but not on normal adult cells.
  • ROR1 expression has been detected in many hematological and solid malignancies, such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell leukemia, hairy cell leukemia, Pancreatic cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, testicular cancer, uterine cancer, adrenal cancer, breast cancer, lung cancer, melanoma, neuroblastoma, sarcoma, kidney cancer, etc. Because of this tumor-embryo expression pattern, ROR1 has become a target for the treatment of certain cancers and tumors.
  • the present invention aims to provide an antibody targeting ROR1 and its use in disease prevention and/or treatment and/or diagnosis.
  • the present invention provides an antibody or antigen-binding fragment thereof targeting ROR1, comprising:
  • CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, CDR-L3 as shown in SEQ ID NO: 3, as shown in SEQ ID NO: 4 CDR-H1, CDR-H2 as shown in SEQ ID NO:5 and CDR-H3 as shown in SEQ ID NO:6;
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region being selected from the group consisting of SEQ ID NOs: 25, 28, 31, 34,
  • the amino acid sequences of 37, 69, 73, 77, 81, 85, 89 and 93 are at least 90% identical, or are , 85, 89, and 93 have one or several amino acid modifications (eg, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) compared to the amino acid sequences; the light chain may
  • the variable region is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 27, 30, 33, 36, 68, 72, 76, 80, 84, 88, and 92, or to an amino acid sequence selected from the group consisting of SEQ ID NO:
  • the amino acid sequences of 24, 27, 30, 33, 36, 68, 72, 76, 80, 84, 88, and 92 have one or more amino acids (e
  • the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain selected from the group consisting of SEQ ID NOs: 25, 28, 31, 34, 37, 69, 73, 77, 81, 85, 89 and 93 Variable regions and light chain variable regions selected from the group consisting of SEQ ID NOs: 24, 27, 30, 33, 36, 68, 72, 76, 80, 84, 88, and 92.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
  • the heavy chain variable region and light chain variable region have at least 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity;
  • the heavy chain variable region and light chain variable region have one or several amino acid modifications compared to the heavy chain variable region and light chain variable region of any of the groups (a)-(1). , eg, modifications of up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; preferably, the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody or antigen-binding fragment thereof of the invention has at least an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 29, 32, 35, 38, 70, 74, 78, 82, 86, 90 and 94 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or with selected from SEQ ID NO: 26, 29, 32, 35 , 38, 70, 74, 78, 82, 86, 90, and 94 have one or several amino acids (eg, up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid) modification.
  • the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the amino acid sequence of the antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of SEQ ID NOs: 26, 29, 32, 35, 38, 70, 74, 78, 82, 86, 90 and 94.
  • the antibody or antigen-binding fragment thereof of the invention is a murine, chimeric, humanized or human antibody, preferably a humanized antibody.
  • the present invention also provides nucleic acid molecules encoding the aforementioned antibodies or antigen-binding fragments thereof.
  • the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-43, 71, 75, 78, 83, 87, 91 and 95 At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity, and the encoded antibody or antigen-binding fragment thereof is capable of specificity Binds to ROR1 antigen.
  • the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 39-43, 71, 75, 78, 83, 87, 91 and 95.
  • the present invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising the anti-ROR1 antibody or antigen-binding fragment thereof as described above, and one or more antibodies with other A second antibody or antigen-binding portion thereof that specifically binds to an antigen.
  • a multispecific antibody preferably a bispecific antibody or a trispecific antibody
  • the second antibody or antigen-binding portion thereof may be in the form of any antibody or antibody fragment, such as a full-length antibody, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv, minibody, Diabodies or sdAbs.
  • the present invention also provides vectors comprising nucleic acid molecules encoding the anti-ROR1 antibodies or antigen-binding fragments or multispecific antibodies described above, and host cells expressing the anti-ROR1 antibodies or antigen-binding fragments or multispecific antibodies.
  • the present invention also provides a chimeric antigen receptor comprising the anti-ROR1 antibody or antigen-binding fragment thereof, a transmembrane domain and an intracellular signaling domain according to the present invention.
  • the chimeric antigen receptor further comprises one or more costimulatory domains.
  • the present invention also provides nucleic acid molecules encoding a chimeric antigen receptor targeting ROR1 as defined above, and vectors comprising said nucleic acid molecules.
  • the present invention also provides cells, preferably immune cells, such as T cells, NK cells, NKT cells, macrophages, dendritic cells, comprising a chimeric antigen receptor targeting ROR1 as defined above.
  • immune cells such as T cells, NK cells, NKT cells, macrophages, dendritic cells
  • the engineered immune cells further comprise a second chimeric antigen receptor targeting other tumor antigens.
  • the present invention also provides an antibody conjugate comprising an anti-ROR1 antibody or an antigen-binding fragment thereof as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, Radioisotopes, half-life extending moieties, detectable labels and drugs.
  • the half-life extending moiety is selected from: the half-life extending moiety is selected from the binding structure of albumin, the binding structure of transferrin, polyethylene glycol molecules, recombinant polyethylene glycol molecules, Human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind human serum albumin.
  • the detectable label is selected from the group consisting of fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents.
  • the drug is selected from cytotoxins and immunomodulators.
  • the present invention also provides a detection kit, which comprises the anti-ROR1 antibody or its antigen-binding fragment, multispecific antibody, antibody conjugate, chimeric antigen receptor or engineered immune system according to the present invention cell.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-ROR1 antibody or antigen-binding fragment thereof, chimeric antigen receptor, multispecific antibody, engineered immune cell or antibody conjugate of the present invention substance, and one or more pharmaceutically acceptable excipients.
  • the present invention also provides a method of treating and/or preventing and/or diagnosing a disease associated with ROR1 expression, comprising administering to a subject an anti-ROR1 antibody or antigen-binding fragment thereof, chimeric Antigen receptors, multispecific antibodies, antibody conjugates, engineered immune cells or pharmaceutical compositions.
  • antibody has the broadest meaning as understood by those skilled in the art, and includes monoclonal antibodies (including whole antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (eg, bispecific antibodies) ), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences capable of exhibiting the desired biological activity.
  • the antibodies of the invention can be of any class (eg, IgG, IgE, IgM, IgD, IgA, etc.) or subclass (eg, IgGl, IgG2, IgG2a, IgG3, IgG4, IgAl, IgA2, etc.).
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of antibodies can be achieved by fragments of full-length antibodies.
  • antibody fragments in the present invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, single chain antibody (scFv), disulfide-linked Fv (sdFv ), variable heavy (VH) or variable light (VL) domains of antibodies, linear antibodies, "diabodies” with two antigen binding sites, single domain antibodies, nanobodies, Natural ligands or functional fragments thereof, etc.
  • an "antibody” of the present invention encompasses antibody fragments or antigen-binding fragments as defined above.
  • the antibody of the invention is selected from the group consisting of IgG, Fab, Fab', F(ab') 2 , Fd, Fd', Fv, scFv, sdFv, linear antibodies and diabodies.
  • an intact antibody comprises two heavy chains and two light chains linked together by disulfide bonds, each light chain being disulfide bonded to its respective heavy chain in a "Y"-shaped structure.
  • Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs): CDR-H1, CDR-H2 and CDR-H3, the heavy chain constant
  • the region contains three constant domains: CH1, CH2 and CH3.
  • Each light chain includes a light chain variable region (VL) and a light chain constant region, wherein the light chain variable region includes three CDRs: CDR-L1, CDR-L2 and CDR-L3, and the light chain constant region includes a constant structure Domain CL.
  • the CDRs are separated by more conserved framework regions (FRs).
  • FRs conserved framework regions
  • the variable region of the heavy/light chain is responsible for antigen recognition and binding, while the constant region mediates the binding of antibodies to host tissues or factors, including various cells of the immune system (such as effector cells) and the first of the classical complement system. one component.
  • the boundaries of a given CDR or FR may vary depending on the protocol used for identification.
  • the Kabat scheme is based on structural alignment
  • the Chothia scheme is based on structural information.
  • Both Kabat and Chothia's scheme numbering is based on the most common antibody region sequence lengths, where insertions are provided by intervening letters (eg "30a") and deletions occur in some antibodies. These two schemes place certain insertions and deletions (indels) at different positions, resulting in different numbers.
  • the Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the AbM protocol is a compromise between the Kabat and Chothia definitions and is based on the protocol used by Oxford Molecular's AbM antibody modeling software.
  • CDRs of a given antibody or regions thereof (eg, variable regions thereof) encompass the CDRs defined by any of the above schemes or other known schemes.
  • CDR a particular CDR (eg, CDR3) is designated to contain a given amino acid sequence
  • FRs of a given antibody or region thereof encompass the FRs defined by any of the above schemes or other known schemes.
  • the numbering scheme used herein to define the boundaries of CDRs and FRs adopts the Chothia scheme.
  • Single-chain antibody and “scFv” are used interchangeably herein and refer to an antibody comprising an antibody heavy chain variable region (VH) and light chain variable region (VL) linked by a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker significantly affects the variable region folding and interaction of scFv. In fact, intrachain folding can be prevented if shorter linkers are used (eg between 5-10 amino acids).
  • linker size and composition see, eg, Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448; US Patent Application Publication Nos.
  • linkers are eg GTSSGSGKPGSGEGSTKG (SEQ ID NO:66), GGGGSGGGGSGGGGS (SEQ ID NO:67).
  • the scFv can comprise VH and VL linked in any order, eg, VH-linker-VL or VL-linker-VH.
  • the antibody or antigen-binding fragment thereof of the invention is a murine, chimeric, humanized or human antibody, preferably a humanized antibody.
  • chimeric antibody refers to an antibody in which a portion of each heavy and light chain amino acid sequence is homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular class, while the remainder of the chain is is homologous to a corresponding sequence in another species or belonging to another class.
  • variable regions of both light and heavy chains are derived from variable regions of antibodies from one species, while the constant regions are sequence homologous to antibodies from another species.
  • a distinct advantage of this chimeric format is that the variable regions can be conveniently produced from currently known sources using readily available B cells or hybridomas from non-human hosts, while the constant regions combined therewith are derived, for example, from human cells.
  • variable regions have the advantage of being easy to prepare and the specificity is not affected by the source, and since the constant regions are human, the antibody will be more likely to elicit a human immune response when injected than if the constant regions are from a non-human source Low.
  • a “humanized” antibody refers to an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs.
  • a "humanized form" of a non-human antibody refers to a variant of said non-human antibody that has undergone humanization to generally reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody from which the CDR residues are derived), eg, to restore or improve antibody specificity or affinity.
  • Humanized antibodies and methods of making them are well known to those skilled in the art, see eg, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).
  • Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using a "best fit" approach; framework regions derived from consensus sequences of human antibodies of a particular subset of light or heavy chain variable regions ; human mature (somatic mutation) framework regions or human germline framework regions; and framework regions obtained by screening FR libraries.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • Human antibodies of the invention may comprise amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo).
  • the present invention provides an antibody or antigen-binding fragment thereof targeting ROR1, comprising:
  • CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, CDR-L3 as shown in SEQ ID NO: 3, as shown in SEQ ID NO: 4 CDR-H1, CDR-H2 as shown in SEQ ID NO:5 and CDR-H3 as shown in SEQ ID NO:6;
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region being selected from the group consisting of SEQ ID NOs: 25, 28, 31, 34,
  • the amino acid sequences of 37, 69, 73, 77, 81, 85, 89 and 93 have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99% or 100% identical, or compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 28, 31, 34, 37, 69, 73, 77, 81, 85, 89 and 93 having one or several (e.g.
  • the light chain variable region is selected from the group consisting of SEQ ID NO: 24,
  • the amino acid sequences of 27, 30, 33, 36, 68, 72, 76, 80, 84, 88 and 92 have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical, or with a
  • the amino acid sequence of 92 has a modification of one or several amino acids (eg, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) compared to the amino acid sequence.
  • the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain selected from the group consisting of SEQ ID NOs: 25, 28, 31, 34, 37, 69, 73, 77, 81, 85, 89 and 93 Variable regions and light chain variable regions selected from the group consisting of SEQ ID NOs: 24, 27, 30, 33, 36, 68, 72, 76, 80, 84, 88, and 92.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
  • the heavy chain variable region and light chain variable region are at least 90%, at least 91% compared to the heavy chain variable region and light chain variable region of any of the groups (a)-(l). , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical;
  • the heavy chain variable region and light chain variable region have one or several amino acid modifications with the heavy chain variable region and light chain variable region of any of the groups (a)-(l), such as Modifications of up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; preferably, the modifications are conservative modifications such as conservative substitutions, additions and deletions of amino acids.
  • the antibody or antigen-binding fragment thereof of the invention has at least an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 29, 32, 35, 38, 70, 74, 78, 82, 86, 90 and 94 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or with selected from SEQ ID NO: 26, 29, 32, 35 , 38, 70, 74, 78, 82, 86, 90 and 94 have one or several amino acid modifications compared to the amino acid sequences, e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.
  • the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the amino acid sequence of the antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of SEQ ID NOs: 26, 29, 32, 35, 38, 70, 74, 78, 82, 86, 90 and 94.
  • conservative modification refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include conservative substitutions, additions and deletions of amino acids. Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues having similar side chains.
  • Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) ), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine) acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • threonine valine, isoleucine
  • aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine.
  • Conservative modifications can be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • sequence identity refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies with the exact same sequence are 100% identical.
  • sequence identity can be determined using several algorithms, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.
  • the present invention also provides a multispecific antibody (preferably a bispecific or trispecific antibody) comprising an anti-ROR1 antibody or antigen-binding fragment thereof as described above, which further comprises one or more combinations with other antigens Specific binding secondary antibody.
  • a multispecific antibody preferably a bispecific or trispecific antibody
  • multispecific refers to an antigen binding protein that has polyepitopic specificity (ie, is capable of specifically binding to two, three or more different epitopes on a biomolecule or capable of specific binds epitopes on two, three or more different biomolecules).
  • bispecific means that an antigen binding protein has two different antigen binding specificities.
  • the secondary antibody may be in any antibody or antibody fragment format, eg, a full-length antibody, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv, minibody, diabody, or sdAb.
  • the second antibody targets an antigen selected from the group consisting of BCMA, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37 , CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, angiogenic factor, VEGF, PIGF, ED-B Fibronectin, Oncogene, Oncogene Product, CD66a-d, Necrotic Antigen, Ii, IL-2, T101, TAC, IL-6, DR4, DR5, tEGFR, Her2, L1-CAM, Mesothelin, CEA, Hepatitis B Surface Antigen, Antifolate Receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, Erb
  • MART-1 gp100, carcinoembryonic antigen, VEGF-R2, CEA, prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, OGD2, CE7, WT-1, Cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, Activin, AFP, p53, Cyclin (D1), CS-1, BAFF-R , TACI, CD56, TIM-3, CD123, L1-cell adhesion molecules, cyclins (such as Cyclin A1 (CCNA1)) and/or pathogen-specific antigens, biotinylated molecules, mediated by HIV, HCV, HBV and/or molecules expressed by other pathogens; and/or neo-epitopes or neo-antigens.
  • CCNA1 Cyclin A1
  • the invention relates to nucleic acid molecules encoding the anti-ROR1 antibodies or multispecific antibodies of the invention.
  • the nucleic acid of the present invention may be RNA, DNA or cDNA.
  • the nucleic acid of the invention is a substantially isolated nucleic acid.
  • the nucleic acid molecule encoding the anti-ROR1 antibody has at least 90%, 91%, 90%, 90%, 90%, 91% affinity with the nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-43, 71, 75, 78, 83, 87, 91, and 95 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity, and the anti-ROR1 antibody it encodes is capable of specifically binding to ROR1 (i.e., with little non-target antigen binding).
  • the nucleic acid molecule encoding the anti-ROR1 antibody is selected from the group consisting of SEQ ID NOs: 39-43, 71, 75, 78, 83, 87, 91 and 95.
  • the nucleic acid of the present invention may also be in the form of, may be present in and/or be part of a vector, such as a plasmid, cosmid or YAC.
  • the vector may in particular be an expression vector, ie a vector that provides for expression of the ROR1 antibody in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system).
  • the expression vector typically comprises at least one nucleic acid molecule of the invention operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). Selection of such regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the ROR1 antibodies of the invention include, but are not limited to, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.
  • the invention also provides host cells expressing the ROR1 antibodies, multispecific antibodies of the invention and/or containing the nucleic acids or vectors of the invention.
  • Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include gram-negative bacterial strains (eg, Escherichia coli, Proteus, and Pseudomonas strains) and gram-positive bacterial strains (eg, Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains).
  • gram-negative bacterial strains eg, Escherichia coli, Proteus, and Pseudomonas strains
  • gram-positive bacterial strains eg, Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains.
  • Suitable fungal cells include cells of species of Trichoderma, Neurospora and Aspergillus; or Saccharomyces (eg Saccharomyces cerevisiae), Saccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of the genus Hansenula.
  • Saccharomyces eg Saccharomyces cerevisiae
  • Saccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cell in the art for expressing heterologous proteins may also be used in the present invention.
  • the present invention also provides recombinant receptors, eg, T cell fusion proteins, T cell antigen couplers, recombinant TCR receptors, or chimeric antigen receptors, comprising the anti-ROR1 antibodies described above.
  • the present invention also provides a chimeric antigen receptor comprising an anti-ROR1 antibody as described above.
  • T cell fusion protein refers to a recombinant polypeptide derived from components of the TCR, typically consisting of a TCR subunit and an antibody linked thereto, and expressed on the cell surface.
  • the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.
  • T cell antigen coupler includes three functional domains: 1 Tumor targeting domain, including single chain antibodies (eg, anti-ROR1 antibodies of the invention), engineered ankyrin repeat proteins (designed ankyrin repeat protein, DARPin) or other targeting groups; 2 extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor close to the TCR receptor; 3 transmembrane region and CD4 co-receptor
  • the intracellular domain where the intracellular domain is linked to the protein kinase LCK, catalyzes the immunoreceptor tyrosine activation motif (ITAM) phosphorylation of the TCR complex as an initial step in T cell activation.
  • ITAM immunoreceptor tyrosine activation motif
  • T cell receptor or "TCR” is a characteristic marker on the surface of T cells that binds non-covalently to CD3 to form a complex.
  • Antigen presenting cells present antigenic peptides to T cells through major histocompatibility complex molecules (MHCs) and bind to the TCR complex to induce a series of intracellular signaling.
  • MHCs major histocompatibility complex molecules
  • TCR consists of six peptide chains that form heterodimers respectively, which are generally classified into ⁇ type and ⁇ type. Each peptide chain includes a constant region and a variable region, where the variable region is responsible for binding specific antigens and MHC molecules.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide that generally includes a ligand binding domain (eg, an antigen binding portion of an antibody), a transmembrane domain, Optional co-stimulatory domains and intracellular signaling domains, each of which is linked by a linker.
  • CARs can exploit the antigen-binding properties of antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner.
  • the present invention provides a chimeric antigen receptor comprising an anti-ROR1 antibody or an antigen-binding fragment thereof as described above, or a multispecific antibody comprising the anti-ROR1 antibody, a transmembrane domain, and a cellular Internal signaling domain.
  • transmembrane domain refers to a polypeptide that enables expression of a chimeric antigen receptor on the surface of immune cells (eg, lymphocytes, NK cells, or NKT cells) and directs the cellular response of the immune cells against target cells structure.
  • immune cells eg, lymphocytes, NK cells, or NKT cells
  • the transmembrane domain can be natural or synthetic, and can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling when the chimeric antigen receptor binds to the target antigen.
  • Transmembrane domains particularly useful in the present invention may be derived from, for example, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments.
  • the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from the CD8 alpha chain or CD28, which is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 44 or 46 % or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% with the nucleic acid molecule shown in SEQ ID NO: 45 or 47 or 99% or 100% sequence identity.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs cells to perform specified functions.
  • the chimeric antigen receptors of the invention comprise intracellular signaling domains that may be sequences of the intracellular regions of T cell receptors and co-receptors that act together upon antigen receptor binding to elicit Signaling, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar function.
  • the intracellular signaling domain can contain a number of immunoreceptor tyrosine-based activation motifs (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motifs
  • intracellular signaling domains of the invention include, but are not limited to, intracellular regions of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d, among others.
  • the signaling domain of the CAR of the present invention may comprise the CD3 ⁇ intracellular region, which is at least 70%, preferably at least 80%, more preferably at least 90% identical to the amino acid sequence shown in SEQ ID NO: 52 or 54 %, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleic acid molecule set forth in SEQ ID NO: 53 or 55 , 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptors of the invention may further comprise a hinge region between the antibody and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an antibody. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antibody.
  • the hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region can be derived in whole or in part from a native molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • the hinge region comprises the hinge region portion of a CD8 ⁇ , CD28, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably a CD8 ⁇ , CD28 or IgG4 hinge, which is set forth in SEQ ID NO: 60, 62 or 64
  • the amino acid sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence is identical to that of SEQ ID NO: 61, 63 or 65
  • the nucleotide sequences shown have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptor may also comprise one or more costimulatory domains.
  • a costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof.
  • a "costimulatory molecule” refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, proliferation) of the T cell.
  • Costimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors.
  • Non-limiting examples of costimulatory domains of the invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, ROR1, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA) , CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70.
  • costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, ROR
  • the costimulatory domain of the CAR of the present invention is from 4-1BB, CD28 or 4-1BB+CD28.
  • the 4-1BB costimulatory domain is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:50 % sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence with the nucleic acid molecule shown in SEQ ID NO:51 identity.
  • the CD28 costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:48 Sequence identity, or its coding sequence, has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleic acid molecule set forth in SEQ ID NO:49 .
  • the CAR of the present invention may further comprise a signal peptide such that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface.
  • the core of the signal peptide may contain a long segment of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • signal peptidases At the end of the signal peptide, there is usually a segment of amino acids that is recognized and cleaved by signal peptidases.
  • the signal peptidase can cleave during or after translocation to generate the free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases.
  • Signal peptides useful in the present invention are well known to those skilled in the art, eg, signal peptides derived from B2M, CD8 ⁇ , IgG1, GM-CSFR ⁇ , and the like.
  • the signal peptide useful in the present invention is from B2M or CD8 ⁇ , which is at least 70%, preferably at least 80%, more preferably at least 90%, 95% of the amino acid sequence shown in SEQ ID NO: 56 or 58 , 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the CAR comprises an anti-ROR1 antibody or antigen-binding fragment thereof as provided herein or a multispecific antibody comprising the anti-ROR1 antibody, CD8 ⁇ or CD28 transmembrane region, CD28 and/or 4-1BB co-stimulatory domain, and CD3 ⁇ intracellular signaling domain.
  • the CAR may further comprise a signal peptide from B2M, CD8 ⁇ , IgG1 or GM-CSFR ⁇ .
  • the present invention also provides nucleic acid molecules encoding a chimeric antigen receptor targeting ROR1 as defined above, and vectors comprising said nucleic acid molecules.
  • vector is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell, in which the nucleic acid molecule can eg be replicated and/or expressed.
  • Vectors generally include targeting vectors and expression vectors.
  • a "targeting vector” is a medium that delivers an isolated nucleic acid to the interior of a cell by, for example, homologous recombination or a hybrid recombinase using a specific targeting sequence at the site.
  • An "expression vector” is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding the chimeric antigen receptor polypeptides of the invention, in suitable host cells and the translation of their mRNAs.
  • Suitable carriers for use in the present invention are known in the art and many are commercially available.
  • the vectors of the present invention include, but are not limited to, plasmids, viruses (eg, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV)), and the like ), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs).
  • the vector itself is usually a nucleic acid molecule, usually a DNA sequence containing an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
  • the VL vector typically also contains an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (eg, a multiple cloning site, MCS).
  • the vector may additionally contain a promoter, multiple elements such as polyadenylated tails (polyA), 3'UTRs, enhancers, terminators, insulators, operons, selectable markers, reporter genes, targeting sequences and/or protein purification tags.
  • polyA polyadenylated tails
  • the vector is an in vitro transcribed vector.
  • the invention also provides engineered immune cells expressing the recombinant receptors of the invention, eg, T cell fusion proteins, T cell antigen couplers, recombinant TCR receptors, or chimeric antigen receptors.
  • the recombinant receptors of the invention eg, T cell fusion proteins, T cell antigen couplers, recombinant TCR receptors, or chimeric antigen receptors.
  • the term "immune cell” refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cytotoxic activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells, and/or NKT cells.
  • the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, and the like.
  • the immune cells are T cells.
  • the T cells can be any T cells, such as T cells cultured in vitro, eg, primary T cells, or T cells from T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (eg, Th1 and Th2 cells), CD8+ T cells (eg, cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • T cells can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation.
  • Nucleic acid sequences encoding chimeric antigen receptors can be introduced into immune cells using conventional methods known in the art (eg, by transduction, transfection, transformation, etc.).
  • Transfection is the process of introducing a nucleic acid molecule or polynucleotide, including a vector, into a target cell.
  • An example is RNA transfection, the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell.
  • RNA transfection the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell.
  • the term is mainly used for non-viral methods in eukaryotic cells.
  • transduction is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides.
  • Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material.
  • Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to create liposomes that fuse with cell membranes and deposit their cargo inside.
  • Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation.
  • transformation is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells).
  • transformation is the genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by the direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means.
  • the cells or bacteria must be in a competent state.
  • techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation.
  • the engineered immune cells further comprise suppressed or silenced expression of at least one gene selected from the group consisting of CD52, GR, dCK, TCR/CD3 genes (such as TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA) and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A , CASP8, CASP10, CASP3, CASP6, CASP
  • the engineered immune cells further comprise suppressed or silenced expression of at least one gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA.
  • RNA decoys RNA decoys
  • RNA aptamers siRNA, shRNA/miRNA, trans dominant negative protein (TNP), chimeric/antibody conjugates, chemokine ligands, anti-infective cellular proteins
  • intracellular antibodies sFv
  • nucleoside analogs NRTI
  • non-nucleoside analogs NRTI
  • integrase inhibitors oligonucleotides, dinucleotides and chemicals
  • protease inhibitors to inhibit gene expression Express.
  • gene silencing can also be achieved by mediating DNA fragmentation, eg, by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system.
  • the engineered immune cells further comprise a second chimeric antigen receptor targeting other tumor antigens.
  • the other tumor antigens targeted by the second chimeric antigen receptor may be selected from, for example, BCMA, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38 , CD40, CD40L, CD46, CD52, CD54, CD80, CD126, CD138, B7, MUC-1, HM1.24, angiogenesis factor, VEGF, PIGF, ED-B fibronectin, CD66a-d, IL-2, T101, TAC, IL-6, DR4, DR5, tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP -4, EPHa2, ErbB2, ErbB3, Erb
  • a plurality of immune cells are provided, each immune cell engineered to express one or more chimeric antigen receptors.
  • one immune cell is engineered to express a chimeric antigen receptor that binds and/or targets ROR1 (eg, a CAR comprising an anti-ROR1 antibody of the invention), and another cell is engineered To express chimeric antigen receptors that bind and/or target other antigens.
  • ROR1 eg, a CAR comprising an anti-ROR1 antibody of the invention
  • immune cells may also express multispecific chimeric antigen receptors that target one or more antigens including ROR1.
  • such a multispecific chimeric antigen receptor may comprise a multispecific antibody targeting ROR1, or a combination of the anti-ROR1 antibodies of the present invention and antibodies targeting other antigens.
  • the plurality of engineered immune cells can be administered together or separately.
  • the plurality of immune cells may be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of this application.
  • the present invention provides an antibody conjugate comprising an anti-ROR1 antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, a radioisotope, a half-life prolonging structure moieties, detectable labels and drugs.
  • the present invention provides an antibody conjugate comprising an anti-ROR1 antibody as defined in the present invention and an Fc.
  • Fc is used to define the C-terminal region of an immunoglobulin heavy chain, which includes native Fc and variant Fc.
  • Native Fc refers to a molecule or sequence comprising a non-antigen-binding fragment, whether in monomeric or multimeric form, produced by digestion of an intact antibody.
  • the source of immunoglobulins for the production of native Fc is preferably derived from humans.
  • Native Fc fragments are composed of monomeric polypeptides that can be linked in dimeric or multimeric forms by covalent linkages (eg, disulfide bonds) and non-covalent linkages.
  • native Fc the disulfide-linked dimer produced by papain digestion of IgG (see Ellison et al. (1982) Nucleic Acids Res. 10:4071-9).
  • native Fc the disulfide-linked dimer produced by papain digestion of IgG (see Ellison et al. (1982) Nucleic Acids Res. 10:4071-9).
  • native Fc as used herein generally refers to monomeric, dimeric and multimeric forms.
  • “Variant Fc” refers to an amino acid sequence that differs from that of a “native” or “wild-type” Fc due to at least one "amino acid modification” as defined herein, also referred to as an "Fc variant".
  • “Fc” also includes single-chain Fc (scFc), ie, a single-chain Fc consisting of two Fc monomers linked by a polypeptide linker, which is capable of naturally folding into a functional dimeric Fc region.
  • the Fc is preferably a human immunoglobulin Fc, more preferably a human IgGl Fc.
  • the present invention provides an antibody conjugate comprising an anti-ROR1 antibody as defined in the present invention and a radioisotope.
  • radioisotopes useful in the present invention include, but are not limited to, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , 99m Tc, 123 I, 18 F and 68 Ga.
  • the present invention provides an antibody conjugate comprising an anti-ROR1 antibody as defined in the present invention and a half-life extending moiety selected from the group consisting of albumin binding structures, transferrin Binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind human serum albumin.
  • a half-life extending moiety selected from the group consisting of albumin binding structures, transferrin Binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind human serum albumin.
  • the present invention provides an antibody conjugate comprising an anti-ROR1 antibody as defined in the present invention and a detectable label.
  • detectable label herein means a compound that produces a detectable signal.
  • the detectable label can be an MRI contrast agent, a scintigraphic contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, an optical imaging contrast agent.
  • detectable labels examples include fluorophores (such as fluorescein, Alexa, or cyanine), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as Root peroxidase, glucose-6-phosphatase, ⁇ -galactosidase), antibiotics (such as kanamycin, ampicillin, chloramphenicol, tetracycline, etc.) resistance genes and contrast agents (such as nanoparticles or gadolinium).
  • fluorophores such as fluorescein, Alexa, or cyanine
  • chemiluminescent compounds such as luminol
  • bioluminescent compounds such as luciferase or alkaline phosphatase
  • enzymes such as Root peroxidase, glucose-6-phosphatase, ⁇ -galactosidase
  • antibiotics such as kanamycin, ampicillin, chloramphenicol, t
  • the present invention provides an antibody conjugate comprising an anti-ROR1 antibody as defined in the present invention and a drug, such as a cytotoxic or immunomodulatory agent (ie, an antibody drug), conjugated to the anti-ROR1 antibody conjugate).
  • a drug such as a cytotoxic or immunomodulatory agent (ie, an antibody drug), conjugated to the anti-ROR1 antibody conjugate).
  • the drug is covalently attached to the antibody and usually relies on a linker.
  • the drug is a cytotoxin.
  • the drug is an immunomodulatory agent.
  • cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, benzidine Nitrogen mustard, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, chain Zocin, mitomycin, cis-dichlorodiamineplatinum (II) (DDP), cisplatin, carboplatin, zorubicin, doxorubicin, detorubicin, caminomycin, Darubicin, epirubicin, mitoxantrone, actinomycin D, bleomycin, calicheamicin, glaremycin, atramycin (AMC), vincristine, vinblastine, Paclitaxel,
  • immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, vortexporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine , mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (such as IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18 and IL-21), colony stimulating factors (such as G-CSF and GM-CSF), interferons (such as interferon-alpha, interferon - beta and interferon-gamma), stem cell growth factors designated "S1 factor", erythropoietin and thrombopoietin, or a combination thereof.
  • TNF tumor necrosis
  • the present invention also provides a detection kit comprising the humanized antibody, multispecific antibody, antibody conjugate, engineered immune cell or chimeric antigen receptor of the present invention.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the humanized antibody, chimeric antigen receptor, multispecific antibody, engineered immune cell or antibody conjugate of the present invention, and a one or more pharmaceutically acceptable excipients.
  • the term "pharmaceutically acceptable excipient” means pharmacologically and/or physiologically compatible with the subject and the active ingredient (ie, capable of eliciting the desired therapeutic effect without causing any inconvenience desired local or systemic effect) carriers and/or excipients, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, colorants, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity modifiers . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention.
  • excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water.
  • suitable excipients depends, among other things, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • compositions according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular ointments, creams, transdermal patches, gels, powders, tablets, solutions, gaseous In the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form particularly suitable for the desired method of administration.
  • Processes known in the present invention for the manufacture of pharmaceuticals may include, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Pharmaceutical compositions comprising immune cells such as those described herein are typically provided in solution and preferably comprise a pharmaceutically acceptable buffer.
  • compositions according to the present invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated.
  • agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetate glucuronate, auristatin E, vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, act
  • the present invention also provides a method of treating and/or preventing and/or diagnosing a disease associated with ROR1 expression, comprising administering to a subject a humanized antibody, chimeric antigen receptor, Multispecific antibodies, antibody conjugates, engineered immune cells or pharmaceutical compositions.
  • diseases associated with ROR1 expression include, but are not limited to, B-cell leukemia, lymphoma, B-cell chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia ( AML), acute lymphoblastic leukemia (ALL), Burkitt lymphoma, mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC), neuroblastoma, kidney cancer, colon cancer, colorectal cancer, breast cancer Cancer, Epithelial Squamous Cell Carcinoma, Melanoma, Myeloma, Stomach Cancer, Brain Cancer, Lung Cancer, Pancreatic Cancer, Cervical Cancer, Ovarian Cancer, Liver Cancer, Bladder Cancer, Prostate Cancer, Testicular Cancer, Thyroid Cancer, Uterine Cancer, Adrenal Cancer and head and neck cancer.
  • CLL B-cell chronic lymphocytic leukemia
  • NHL non-Hodgkin's lymphoma
  • Figure 1 Shows scFv expression levels in CAR-T cells expressing ROR1 murine ScFv.
  • Figure 2 shows the killing effect of CAR-T cells expressing ROR1 murine ScFv on target cells under different effector-target ratios.
  • Figure 3 Shows the release level of IFN- ⁇ after co-culture of CAR-T cells expressing ROR1 murine ScFv with various target and non-target cells.
  • Figure 4 Shows scFv expression levels in CAR-T cells expressing ROR1 humanized ScFv.
  • Figure 5 shows the killing effect of CAR-T cells expressing ROR1 humanized ScFv on target cells Jeko-1-luci (A) and non-target cells K562-luci (B) under different effector-target ratios.
  • Figure 6 Degranulation of target cells MDA-MB-231, Jeko-1, A549 and non-target cells K562 by CAR-T cells expressing ROR1 humanized ScFv is shown.
  • Figure 7 Shows IL2(A) and IFN- ⁇ after co-culture of ROR1 humanized ScFv expressing CAR-T cells with various target cells MDA-MB-231, Jeko-1, A549 and non-target cell K562 (B) Release levels.
  • the sequence containing the extracellular region of the ROR1 protein was cloned into a pCP vector with a His tag, and the correctly sequenced plasmid was transiently infected with CHO cells. After culturing for 8-10 days, the cell culture medium was harvested, and the protein was purified by affinity chromatography to obtain The extracellular domain of human LIR1 protein as an immunogen.
  • mice 6-8 week old female Balb/c mice were used for primary and booster immunization, and the antibody titer and specificity of protein immunogens in serum were detected by ELISA and FACS. After comprehensive evaluation, mice were given the last booster immunization. Mice were sacrificed 3-4 days later, spleen cells were collected, mixed with mouse myeloma cells (SP2/0) in a certain proportion, and the proliferated hybridoma cells were identified by FACS and ELISA, and positive cell lines were screened . After multiple rounds of subcloning identification, hybridoma cells that can stably secrete ROR1 antibody were finally obtained. After purification of the antibody in the supernatant of hybridoma cells, five anti-ROR1 antibodies were obtained.
  • SP2/0 mouse myeloma cells
  • Antibodies were digested with trypsin, high-quality primary and secondary spectra were obtained through DNA data acquisition, and the spectra were de novo analyzed with peaks software to obtain preliminary amino acid sequence results; a variety of proteases were used to digest antibodies Solution, after MSE collects data, all peptide peaks and the completeness and fragmentation information of each valence state of each peptide can be obtained; the obtained more comprehensive mass spectrometry data is matched with the preliminary amino acid sequence, and finally five ROR1 antibodies are obtained.
  • the amino acid sequence of . is shown in Table 1 below.
  • the sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8 ⁇ signal peptide (SEQ ID NO: 58), anti-ROR1 antibody, CD8 ⁇ hinge region ( SEQ ID NO:60), CD8 ⁇ transmembrane region (SEQ ID NO:44), 4-1BB intracellular region (SEQ ID NO:50), CD3 ⁇ intracellular signaling domain (SEQ ID NO:52), and through Sequencing confirms the correct insertion of the target sequence.
  • PPL Public Protein/Plasmid Library
  • PPL00157-4a CD8 ⁇ signal peptide
  • anti-ROR1 antibody CD8 ⁇ hinge region
  • SEQ ID NO:44 CD8 ⁇ transmembrane region
  • 4-1BB intracellular region SEQ ID NO:50
  • CD3 ⁇ intracellular signaling domain SEQ ID NO:52
  • the amino acid sequence of anti-ROR1scFv contained in ROR1-1CAR is shown in SEQ ID NO:26; the amino acid sequence of anti-ROR1scFv contained in ROR1-2CAR is shown in SEQ ID NO:29; the amino acid sequence of anti-ROR1scFv contained in ROR1-3CAR As shown in SEQ ID NO: 32; the amino acid sequence of anti-ROR1 scFv contained in ROR1-4 CAR is shown in SEQ ID NO: 35; the amino acid sequence of anti-ROR1 scFv contained in ROR1-5 CAR is shown in SEQ ID NO: 38.
  • Opti-MEM Gibco, Item No. 31985-070
  • Opti-MEM Gibco, Item No. 31985-070
  • the packaging vector psPAX2 (Addgene, Cat. No. 12260)
  • the enveloped vector pMD2.G (Addgene, Cat. No. 12259).
  • 120 ⁇ L of X-treme GENE HP DNA Transfection Reagent (Roche, Cat. No. 06366236001) was added, mixed immediately, incubated at room temperature for 15 min, and then the plasmid/vector/transfection reagent mixture was added dropwise to the culture flask of 293T cells .
  • Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000 g, 4°C, 2.5 hours) to obtain concentrated lentiviruses.
  • T cells were activated with DynaBeads CD3/CD28CTS TM (Gibco, Cat. No. 40203D) and cultured at 37°C and 5% CO2 for an additional day. Then, the concentrated lentivirus was added and cultured for 3 days to obtain CAR-T cells expressing different ROR1 murine scFvs, and unmodified wild-type T cells were used as negative control (NT).
  • Biotin-SP Long spacer AffiniPure Goat Anti-Mouse IgG, F(ab') 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) was used as primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No.
  • MDA-MB-231 target cells carrying the fluorescein gene were plated into 96-well plates at a concentration of 1 x 104 cells/well, and then plated at an effector-to-target ratio of 10:1 or 5:1 (i.e. effector T cells and The ratio of target cells)
  • NT cells and CAR-T cells expressing ROR1 murine scFv were plated into 96-well plates for co-culture, and the fluorescence value was measured by a microplate reader after 16-18 hours.
  • the calculation formula (mean fluorescence of target cells - mean fluorescence of samples)/mean fluorescence of target cells ⁇ 100%, the killing efficiency was calculated, and the results are shown in FIG. 2 .
  • the CAR-T cells expressing ROR1 murine ScFv prepared by the present invention have significant specific killing to target cells under different effector-target ratios.
  • Target cells MDA-MB-231 cells, Hs-578T cells
  • non-target cells Jurkat cells, Huh7 cells
  • CAR-T cells or NT cells expressing ROR1 murine ScFv were added at the same ratio for co-culture, and the cell co-culture supernatant was collected 18-24 hours later.
  • the 96-well plate was coated with the capture antibody Purified anti-human IFN- ⁇ Antibody (Biolegend, Cat. No. 506502) and incubated at 4°C overnight, then the antibody solution was removed, and 250 ⁇ L of PBST containing 2% BSA (sigma, Cat. No. V900933-1kg) was added. 0.1% Tween in 1X PBS) for 2 hours at 37°C. Plates were then washed 3 times with 250 [mu]L of PBST (1XPBS with 0.1% Tween). 50 ⁇ L of cell co-culture supernatant or standards were added to each well and incubated at 37° C.
  • the cells expressing ROR1 murine ScFv prepared by the present invention did not release cytokine IFN- ⁇ after co-culture with non-target cells Jurkat and Huh27, but co-cultured with target cells MDA-MB-231 cells or Hs-578T cells.
  • the release level of IFN- ⁇ was significantly increased after culture. This shows that the CAR-T cells expressing ROR1 murine ScFv prepared in the present invention can specifically kill target cells.
  • the mouse antibody ROR1-4 was humanized, and the specific method was as follows: First, the human antibody sequence with high similarity was searched through the IG BLAST database (https://www.ncbi.nlm.nih.gov/igblast/). Then replace the FR region in the single-chain antibody with the corresponding human sequence; then replace the individual amino acid residues according to the different physical and chemical properties of the amino acid residues, and finally obtain 7 humanized single-chain antibodies, the sequences of which are shown in Table 2 Show.
  • the above vector was packaged into lentivirus and infected with activated T cells to obtain CAR-T cells containing ROR1 humanized antibody.
  • the anti-ROR1 humanized antibody in the CAR-T cells prepared by the present invention can be effectively expressed.
  • Example 6 Detection of killing effect, degranulation and cytokine release level of CAR-T cells expressing ROR1 humanized ScFv on target cells
  • Target cells Jeko-1-luci cells
  • non-target cells K562-luci cells
  • 16:1, 8:1, 4 1, 2: 1, 1: 1 effector-target ratio (that is, the ratio of effector T cells to target cells)
  • NT cells and each CAR-T cell were plated into 96-well plates for co-culture, and used after 16-18 hours.
  • the fluorescence value was measured by a microplate reader. According to the calculation formula: (mean fluorescence of target cells - mean fluorescence of samples)/mean fluorescence of target cells ⁇ 100%, the killing efficiency was calculated, and the results are shown in FIG. 5 .
  • the CAR-T cells expressing ROR1 humanized ScFv prepared by the present invention show a strong killing effect on the target cell Jeko-1-luci cells, while on the non-target cells K562-luci cells Cell killing was weaker, indicating that CAR-T cells expressing ROR1 humanized ScFv showed specific killing only to cells expressing ROR1.
  • Target cells MDA-MB-231 cells, Jeko-1 cells, A549 cells
  • non-target cells K562 cells
  • Target cells MDA-MB-231 cells, Jeko-1 cells, A549 cells
  • non-target cells K562 cells
  • 96-well plates at a concentration of 1 ⁇ 10 5 cells/well
  • 1:1 ROR1 humanized ScFv-expressing CAR-T cells, ROR1-4CAR-T-2 cells, and NT cells (negative control) were added at the ratio of 10 ⁇ L PE Mouse anti-human CD107a antibody (BD, Cat. No. 555801) to each well. , and incubated at 37°C, 5% CO 2 in the dark. After 1 h, 20 ⁇ L of Golgi Stop (BD, Cat. No.
  • the CAR-T cells expressing ROR1 humanized ScFv prepared by the present invention showed a significant increase in the three target cells MDA-MB-231 cells, Jeko-1 cells and A549 cells. specific degranulation, while no significantly elevated degranulation was observed for non-target cells.
  • Target cells MDA-MB-231 cells, Jeko-1 cells, A549 cells
  • non-target cells K562 cells
  • ROR1 humanized ScFv-expressing CAR-T cells and NT cells negative control

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Abstract

本发明提供靶向ROR1的抗体，以及包含它的多特异性抗体、嵌合抗原受体、抗体偶联物、药物组合物和试剂盒，以及它们在诊断/治疗/预防与ROR1表达相关的疾病中的用途。

Description

靶向ROR1的抗体及其用途 技术领域
本发明属于免疫治疗领域。更具体地，本发明涉及靶向ROR1的抗体，及其在预防和/或治疗和/或诊断疾病中的用途。
背景技术
受体酪氨酸激酶样孤独受体1(ROR1)是受体酪氨酸激酶(RTK)家族的成员，一般在胚胎发生期间和胎儿发育期间高度表达，而不在正常成体细胞上表达。但是，已经在许多血液和实体恶性肿瘤中检测到ROR1的表达，例如慢性淋巴细胞白血病(Chronic Lymphocytic Leukaemia,CLL)、急性淋巴细胞白血病(Acute Lymphoblastic Leukaemia,ALL)、套细胞白血病、毛细胞白血病、胰腺癌、前列腺癌、结肠癌、膀胱癌、卵巢癌、睾丸癌、子宫癌、肾上腺癌、乳腺癌、肺癌、黑素瘤、神经母细胞瘤、肉瘤、肾癌等。由于ROR1具有的这种肿瘤-胚胎表达模式，其已经成为治疗特定癌症和肿瘤的靶标。
因此，针对ROR1靶点进行药物开发、抗体开发具有重要价值和意义。本发明旨在提供一种靶向ROR1的抗体，及其在疾病预防和/或治疗和/或诊断中的用途。
发明内容
在第一个方面，本发明提供一种靶向ROR1的抗体或其抗原结合片段，其包含:
(1)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：3所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：6所示的CDR-H3；
(2)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：7所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：8所示的CDR-H2和如SEQ ID NO：9所示的CDR-H3；
(3)如SEQ ID NO：10所示的CDR-L1、如SEQ ID NO：11所示的CDR-L2、如SEQ  ID NO：12所示的CDR-L3、如SEQ ID NO：13所示的CDR-H1、如SEQ ID NO：14所示的CDR-H2和如SEQ ID NO：15所示的CDR-H3；
(4)如SEQ ID NO：16所示的CDR-L1、如SEQ ID NO：17所示的CDR-L2、如SEQ ID NO：18所示的CDR-L3、如SEQ ID NO：19所示的CDR-H1、如SEQ ID NO：20所示的CDR-H2和如SEQ ID NO：21所示的CDR-H3；或
(5)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：22所示的CDR-L2、如SEQ ID NO：7所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：23所示的CDR-H3。
在一个实施方案中，本发明的抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区与选自SEQ ID NO：25、28、31、34、37、69、73、77、81、85、89和93的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：25、28、31、34、37、69、73、77、81、85、89和93的氨基酸序列相比具有一个或几个氨基酸(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)的修饰；所述轻链可变区与选自SEQ ID NO：24、27、30、33、36、68、72、76、80、84、88和92的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：24、27、30、33、36、68、72、76、80、84、88和92的氨基酸序列相比具有一个或几个氨基酸(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。在一个优选的实施方案中，本发明的抗体或其抗原结合片段包含选自SEQ ID NO：25、28、31、34、37、69、73、77、81、85、89和93的重链可变区和选自SEQ ID NO：24、27、30、33、36、68、72、76、80、84、88和92的轻链可变区。
在一个实施方案中，本发明的抗体或其抗原结合片段包含选自以下的重链可变区和轻链可变区：
(a)如SEQ ID NO：25所示的重链可变区和如SEQ ID NO：24所示的轻链可变区；
(b)如SEQ ID NO：28所示的重链可变区和如SEQ ID NO：27所示的轻链可变区；
(c)如SEQ ID NO：31所示的重链可变区和如SEQ ID NO：30所示的轻链可变区；
(d)如SEQ ID NO：34所示的重链可变区和如SEQ ID NO：33所示的轻链可变区；和
(e)如SEQ ID NO：37所示的重链可变区和如SEQ ID NO：36所示的轻链可变区；
(f)如SEQ ID NO：69所示的重链可变区和如SEQ ID NO：68所示的轻链可变区；
(g)如SEQ ID NO：73所示的重链可变区和如SEQ ID NO：72所示的轻链可变区；
(h)如SEQ ID NO：77所示的重链可变区和如SEQ ID NO：76所示的轻链可变区；
(i)如SEQ ID NO：81所示的重链可变区和如SEQ ID NO：80所示的轻链可变区；
(j)如SEQ ID NO：85所示的重链可变区和如SEQ ID NO：84所示的轻链可变区；
(k)如SEQ ID NO：89所示的重链可变区和如SEQ ID NO：88所示的轻链可变区；
(l)如SEQ ID NO：93所示的重链可变区和如SEQ ID NO：92所示的轻链可变区。
任选地，所述重链可变区和轻链可变区与(a)-(l)任一组的重链可变区和轻链可变区相比具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％同一性；
任选地，所述重链可变区和轻链可变区与(a)-(l)任一组的重链可变区和轻链可变区相比具有一个或几个氨基酸的修饰，例如最多1、2、3、4、5、6、7、8、9或10个氨基酸的修饰；优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。
在一个实施方案中，本发明的抗体或其抗原结合片段与选自SEQ ID NO：26、29、32、35、38、70、74、78、82、86、90和94的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％同一性，或与选自SEQ ID NO：26、29、32、35、38、70、74、78、82、86、90和94的氨基酸序列相比具有一个或 几个氨基酸(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。优选地，本发明的抗体或其抗原结合片段的氨基酸序列选自SEQ ID NO：26、29、32、35、38、70、74、78、82、86、90和94。
在一个实施方案中，本发明的抗体或其抗原结合片段是鼠源抗体、嵌合抗体、人源化抗体或人抗体，优选是人源化抗体。
本发明还提供编码上述抗体或其抗原结合片段的核酸分子。因此，在一个实施方案中，编码所述抗体或其抗原结合片段的核酸分子与选自SEQ ID NO：39-43、71、75、78、83、87、91和95的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％序列同一性，并且其编码的抗体或其抗原结合片段能够特异性结合ROR1抗原。优选地，编码所述抗体或其抗原结合片段的核酸分子选自SEQ ID NO：39-43、71、75、78、83、87、91和95。
在另一个方面，本发明还提供一种多特异性抗体(优选双特异性抗体或三特异性抗体)，其包含如上所述的抗ROR1抗体或其抗原结合片段，和一个或多个与其他抗原特异性结合的第二抗体或其抗原结合部分。
在一个实施方案中，第二抗体或其抗原结合部分可以具有任何抗体或抗体片段形式，例如全长抗体、Fab、Fab'、(Fab') 2、Fv、scFv、scFv-scFv、微抗体、双抗体或sdAb。
本发明还提供包含编码上述抗ROR1抗体或其抗原结合片段或多特异性抗体的核酸分子的载体，以及表达所述抗ROR1抗体或其抗原结合片段或多特异性抗体的宿主细胞。
在另一个方面，本发明还提供一种嵌合抗原受体，其包含本发明所述的抗ROR1抗体或其抗原结合片段、跨膜结构域和胞内信号传导结构域。优选地，所述嵌合抗原受体还包含一个或多个共刺激结构域。
本发明还提供编码如上所定义的靶向ROR1的嵌合抗原受体的核酸分子，以及包含所述核酸分子的载体。
本发明还提供包含如上所定义的靶向ROR1的嵌合抗原受体的细胞，优选免疫细胞，例如T细胞、NK细胞、NKT细胞、巨噬细胞、树突细胞。在一个优选的实施方案中，所述工程化免疫细胞还包含靶向其他肿瘤抗原的第二嵌合抗原受体。
在另一个方面，本发明还提供一种抗体偶联物，其包含本发明所定义的抗ROR1抗体或其抗原结合片段和第二功能性结构，其中所述第二功能性结构选自Fc、放射性同位素、延长半衰期的结构部分、可检测标记物和药物。
在一个实施方案中，所述延长半衰期的结构部分选自：所述延长半衰期的结构部分选自白蛋白的结合结构、转铁蛋白的结合结构、聚乙二醇分子、重组聚乙二醇分子、人血清白蛋白、人血清白蛋白的片段和结合人血清白蛋白的白多肽(包括抗体)。在一个实施方案中，可检测标记物选自荧光团、化学发光化合物、生物发光化合物、酶、抗生素抗性基因和造影剂。在一个实施方案中，所述药物选自细胞毒素和免疫调节剂。
在另一个方面，本发明还提供一种检测试剂盒，其包含本发明所述的抗ROR1抗体或其抗原结合片段、多特异性抗体、抗体偶联物、嵌合抗原受体或工程化免疫细胞。
在另一个方面，本发明还提供一种药物组合物，其包含本发明所述的抗ROR1抗体或其抗原结合片段、嵌合抗原受体、多特异性抗体、工程化免疫细胞或抗体偶联物，和一种或多种药学上可接受的赋形剂。
在另一个方面，本发明还提供一种治疗和/或预防和/或诊断与ROR1表达相关的疾病的方法，包括向受试者施用如上所述的抗ROR1抗体或其抗原结合片段、嵌合抗原受体、多特异性抗体、抗体偶联物、工程化免疫细胞或药物组合物。
发明详述
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。
抗ROR1抗体或其抗原结合片段
如本文所用，术语“抗体”具有本领域技术人员所理解的最广泛的含义，并且包括单克隆抗体(包含完整抗体)、多克隆抗体、多价抗体、多特异性抗体(例如双特异性抗体)、和能够表现期望的生物活性的携带一个或多个CDR序列的抗体片段或合成多肽。本发明所述抗体可为任何种类(例如IgG、IgE、IgM、IgD、IgA等)或亚类(例如IgG1、IgG2、IgG2a、IgG3、IgG4、IgA1、IgA2等)。
如本文所用，术语“抗原结合片段”或“抗体片段”指抗体中保留特异性结合抗原的能力的一个或更多个片段。已经显示，抗体的抗原结合功能可通过全长抗体的片 段来实现。本发明中的抗体片段的实例包括但不限于：Fab、Fab'、F(ab')2、Fd片段、Fd′、Fv片段、单链抗体(scFv)、二硫键-连接的Fv(sdFv)、抗体的重链可变区(VH)或轻链可变区(VL)、线性抗体、具有两个抗原结合位点的“双体”、单结构域抗体、纳米抗体、所述抗原的天然配体或其功能性片段等。因此，除非上下文明确指出，否则本发明的“抗体”涵盖如上定义的抗体片段或抗原结合片段。因此，在一个优选的实施方案中，本发明的抗体选自IgG、Fab、Fab'、F(ab') 2、Fd、Fd′、Fv、scFv、sdFv、线性抗体和双体。
通常，完整抗体包括通过二硫键连接在一起的两条重链和两条轻链，每条轻链通过二硫键被连至各自的重链，呈“Y”形结构。每条重链包含重链可变区(VH)和重链恒定区，其中重链可变区包含三个互补决定区(CDR)：CDR-H1、CDR-H2和CDR-H3，重链恒定区包含三个恒定结构域：CH1、CH2和CH3。每条轻链包含轻链可变区(VL)和轻链恒定区，其中轻链可变区包含三个CDR：CDR-L1、CDR-L2和CDR-L3，轻链恒定区包含一个恒定结构域CL。在重链/轻链可变区中，CDR被更保守的框架区(FR)隔开。重链/轻链的可变区负责与抗原的识别和结合，恒定区则可以介导抗体与宿主组织或因子的结合，包括免疫系统的各种细胞(例如效应细胞)和经典补体系统的第一组分。
可以使用许多本领域熟知的编号方案容易地确定给定CDR或FR的精确氨基酸序列边界，这些方案包括：Kabat等人(1991),“Sequences ofProteins of Immunological Interest,”第5版Public Health Service,NationalInstitutes of Health,贝塞斯达,马里兰州(“Kabat”编号方案)；Al-Lazikani等人,(1997)JMB 273,927-948(“Chothia”编号方案)；MacCallum等人,J.Mol.Biol.262:732-745(1996),“Antibody-antigen interactions:Contact analysis and binding sitetopography,”J.Mol.Biol.262,732-745”(“Contact”编号方案)；Lefranc MP等人,“IMGTunique numbering for immunoglobulin and T cell receptor variable domains andIg superfamily V-like domains,”Dev Comp Immunol,2003年1月；27(1):55-77(“IMGT”编号方案)；Honegger A和Plückthun A,“Yet another numbering scheme forimmunoglobulin variable domains:an automatic modeling and analysis tool,”JMol Biol,2001年6月8日；309(3):657-70(“Aho”编号方案)；和Martin等人,“Modeling antibody hypervariable loops:a combined algorithm,”PNAS,1989,86(23):9268-9272(“AbM”编号方案)。
给定CDR或FR的边界可能取决于用于鉴定的方案而不同。例如，Kabat方案是基于结构比对，而Chothia方案是基于结构信息。Kabat和Chothia方案的编号都是基于最常见的抗体区域序列长度，其中通过插入字母提供插入(例如“30a”)并且在一些抗体中出现缺失。这两种方案将某些插入和缺失(indel)放置在不同的位置，从而产生不同的编号。Contact方案是基于对复杂晶体结构的分析，并且在许多方面与Chothia编号方案相似。AbM方案是介于Kabat与Chothia定义之间的折衷，其基于Oxford Molecular的AbM抗体建模软件所使用的方案。
因此，除非另有规定，否则应当理解，给定抗体或其区域(如其可变区)的“CDR”涵盖由任何上述方案或其他已知方案所定义的CDR。例如，在指定特定的CDR(例如CDR3)含有给定氨基酸序列的情况下，应理解，这样的CDR还可以具有由任何上述方案或其他已知方案所定义的相应CDR(例如CDR3)的序列。同样，除非另有规定，否则应当理解给定抗体或其区域(如其可变区)的FR涵盖由任何上述方案或其他已知方案所定义的FR。除非特别指出，否则在本文中用于界定CDR和FR的边界的编号方案采用Chothia方案。
“单链抗体”和“scFv”在本文中可互换使用，是指由抗体重链可变区(VH)和轻链可变区(VL)通过接头连接而成的抗体。可以选择接头的最佳长度和/或氨基酸组成。接头的长度会明显影响scFv的可变区折叠和相互作用情况。事实上，如果使用较短的接头(例如在5-10个氨基酸之间)，则可以防止链内折叠。关于接头的大小和组成的选择，参见例如，Hollinger等人,1993Proc Natl Acad.Sci.U.S.A.90:6444-6448；美国专利申请公布号2005/0100543、2005/0175606、2007/0014794；以及PCT公布号WO2006/020258和WO2007/024715，其全文通过引用并入本文。常用的接头例如GSTSGSGKPGSGEGSTKG(SEQ ID NO：66)、GGGGSGGGGSGGGGS(SEQ ID NO：67)。scFv可以包含以任何顺序连接的VH和VL，例如VH-接头-VL或VL-接头-VH。
在一个实施方案中，本发明的抗体或其抗原结合片段是鼠源抗体、嵌合抗体、人源化抗体或人抗体，优选是人源化抗体。
如本文所用，术语“嵌合抗体”指这样的抗体，其中每个重链和轻链氨基酸序列的一部分与来自特定物种或者属于特定类别的抗体中相应序列同源，而该链的其余区段则与另一物种或属于另一类别的相应序列同源。一般地，轻链和重链的可变区均来自 一个物种的抗体的可变区，而恒定区则与来自另一个物种的抗体序列同源。这种嵌合形式的一个明显优点是可使用易于获得的B细胞或来自非人宿主的杂交瘤从目前已知的来源方便地产生可变区，而与其组合的恒定区来自例如人细胞。所述可变区具有易于制备的优点，并且特异性不受来源的影响，而由于恒定区来自人，因此该抗体在注射时引发人免疫应答的可能性将比恒定区来自非人来源时更低。
如本文所用，“人源化”抗体是指其中所有或基本上所有CDR氨基酸残基源自非人CDR并且所有或基本上所有FR氨基酸残基源自人FR。非人抗体的“人源化形式”是指所述非人抗体的变体，其经历人源化以通常降低对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。在一些实施方案中，人源化抗体中的一些FR残基被来自非人抗体(例如，衍生CDR残基的抗体)的相应残基取代，例如以恢复或改善抗体特异性或亲和力。
人源化抗体及其制备方法是本领域技术人员熟知的，参见例如Almagro和Fransson,Front.Biosci.13:1619-1633(2008)。可用于人源化的人类框架区包括但不限于：使用“最佳拟合”方法选择的框架区；源自轻链或重链可变区的特定亚组的人类抗体的共有序列的框架区；人类成熟(体细胞突变)框架区或人类种系框架区；以及筛选FR文库得到的框架区。
如本文所用，术语“人抗体”旨在包括具有来自人生殖系免疫球蛋白序列的可变区和恒定区的抗体。本发明的人抗体可包含不是由人生殖系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机诱变或定点诱变或者通过体内体细胞突变引入的突变)。
在一个实施方案中，本发明提供一种靶向ROR1的抗体或其抗原结合片段，其包含：
(1)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：3所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：6所示的CDR-H3；
(2)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：7所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：8所示的CDR-H2和如SEQ ID NO：9所示的CDR-H3；
(3)如SEQ ID NO：10所示的CDR-L1、如SEQ ID NO：11所示的CDR-L2、如SEQ ID NO：12所示的CDR-L3、如SEQ ID NO：13所示的CDR-H1、如SEQ ID NO： 14所示的CDR-H2和如SEQ ID NO：15所示的CDR-H3；
(4)如SEQ ID NO：16所示的CDR-L1、如SEQ ID NO：17所示的CDR-L2、如SEQ ID NO：18所示的CDR-L3、如SEQ ID NO：19所示的CDR-H1、如SEQ ID NO：20所示的CDR-H2和如SEQ ID NO：21所示的CDR-H3；或
(5)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：22所示的CDR-L2、如SEQ ID NO：7所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：23所示的CDR-H3。
在一个实施方案中，本发明的抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区与选自SEQ ID NO：25、28、31、34、37、69、73、77、81、85、89和93的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与选自SEQ ID NO：25、28、31、34、37、69、73、77、81、85、89和93的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰；所述轻链可变区与选自SEQ ID NO：24、27、30、33、36、68、72、76、80、84、88和92的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与选自SEQ ID NO：24、27、30、33、36、68、72、76、80、84、88和92的氨基酸序列相比具有一个或几个氨基酸(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。在一个优选的实施方案中，本发明的抗体或其抗原结合片段包含选自SEQ ID NO：25、28、31、34、37、69、73、77、81、85、89和93的重链可变区和选自SEQ ID NO：24、27、30、33、36、68、72、76、80、84、88和92的轻链可变区。在一个实施方案中，本发明的抗体或其抗原结合片段包含选自以下的重链可变区和轻链可变区：
(a)如SEQ ID NO：25所示的重链可变区和如SEQ ID NO：24所示的轻链可变区；
(b)如SEQ ID NO：28所示的重链可变区和如SEQ ID NO：27所示的轻链可变区；
(c)如SEQ ID NO：31所示的重链可变区和如SEQ ID NO：30所示的轻链 可变区；
(d)如SEQ ID NO：34所示的重链可变区和如SEQ ID NO：33所示的轻链可变区；和
(e)如SEQ ID NO：37所示的重链可变区和如SEQ ID NO：36所示的轻链可变区；
(f)如SEQ ID NO：69所示的重链可变区和如SEQ ID NO：68所示的轻链可变区；
(g)如SEQ ID NO：73所示的重链可变区和如SEQ ID NO：72所示的轻链可变区；
(h)如SEQ ID NO：77所示的重链可变区和如SEQ ID NO：76所示的轻链可变区；
(i)如SEQ ID NO：81所示的重链可变区和如SEQ ID NO：80所示的轻链可变区；
(j)如SEQ ID NO：85所示的重链可变区和如SEQ ID NO：84所示的轻链可变区；
(k)如SEQ ID NO：89所示的重链可变区和如SEQ ID NO：88所示的轻链可变区；
(l)如SEQ ID NO：93所示的重链可变区和如SEQ ID NO：92所示的轻链可变区。
任选地，所述重链可变区和轻链可变区与(a)-(l)任一组的重链可变区和轻链可变区相比具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性；
任选地，所述重链可变区和轻链可变区与(a)-(l)任一组的重链可变区和轻链可变区具有一个或几个氨基酸的修饰，例如最多1、2、3、4、5、6、7、8、9或10个氨基酸的修饰；优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。
在一个实施方案中，本发明的抗体或其抗原结合片段与选自SEQ ID NO：26、29、32、35、38、70、74、78、82、86、90和94的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％同一性，或与选自SEQ ID NO： 26、29、32、35、38、70、74、78、82、86、90和94的氨基酸序列相比具有一个或几个氨基酸的修饰，例如最多1、2、3、4、5、6、7、8、9或10个氨基酸的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。优选地，本发明的抗体或其抗原结合片段的氨基酸序列选自SEQ ID NO：26、29、32、35、38、70、74、78、82、86、90和94。
如本文所用，术语“保守性修饰”是指不会明显影响或改变含有该氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。这些保守性修饰包括氨基酸的保守性取代、添加及缺失。修饰可以通过本领域中已知的标准技术，如定点诱变和PCR介导的诱变而引入本发明的嵌合抗原受体中。保守性氨基酸取代是氨基酸残基被具有类似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基家族已在本领域中有定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。保守性修饰可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行选择。
如本文所用，术语序列“同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员知晓，可以使用一些算法来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197)和ClustalW。
在在一个方面，本发明还提供包含如上所述的抗ROR1抗体或其抗原结合片段的多特异性抗体(优选双特异性抗体或三特异性抗体)，其还包含一个或多个与其他抗原特异性结合的第二抗体。
如本文所用，术语“多特异性”是指抗原结合蛋白具有多表位特异性(即，能够特异性结合一个生物分子上的两个、三个或更多个不同的表位或能够特异性结合两个、三个或更多个不同的生物分子上的表位)。如本文所用，术语“双特异性”表示抗原结合蛋白具有两种不同的抗原结合特异性。
在一个实施方案中，第二抗体可以具有任何抗体或抗体片段形式，例如全长抗体、Fab、Fab'、(Fab') 2、Fv、scFv、scFv-scFv、微抗体、双抗体或sdAb。
因此，在一个实施方案中，所述第二抗体靶向选自以下的抗原：BCMA、CD4、CD5、CD7、CD8、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD80、CD126、CD138、B7、MUC-1、Ia、HM1.24、HLA-DR、腱生蛋白、血管生成因子、VEGF、PIGF、ED-B纤连蛋白、致癌基因、致癌基因产物、CD66a-d、坏死抗原、Ii、IL-2、T101、TAC、IL-6、DR4、DR5、tEGFR、Her2、L1-CAM、间皮素、CEA、乙型肝炎表面抗原、抗叶酸受体、CD24、CD30、CD44、EGFR、EGP-2、EGP-4、EPHa2、ErbB2、ErbB3、ErbB4、ErbB二聚体、EGFR vIII、FBP、FCRL5、FCRH5、胎儿乙酰胆碱受体、GD2、GD3、GPRC5D、HMW-MAA、IL-22R-α、IL-13R-α2、kdr、κ轻链、Lewis Y、L1-CAM、MAGE-A1、MAGE-A3、MAGE-A6、PRAME、生存素、EGP2、EGP40、TAG72、B7-H6、IL-13Ra2、CA9、CD171、G250/CAIX、HLA-A1、HLA-A2、NY-ESO-1、PSCA、叶酸受体-a、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、NKG2D配体、双抗原、与通用标签相关的抗原、癌症-睾丸抗原、MUC1、MUC16、NY-ESO-1、MART-1、gp100、癌胚胎抗原、VEGF-R2、CEA、前列腺特异性抗原、PSMA、Her2/neu、雌激素受体、孕酮受体、肝配蛋白B2、CD123、c-Met、GD-2、OGD2、CE7、WT-1、细胞周期蛋白A2、CCL-1、hTERT、MDM2、CYP1B、WT1、活素、AFP、p53、细胞周期蛋白(D1)、CS-1、BAFF-R、TACI、CD56、TIM-3、CD123、L1-细胞粘附分子、细胞周期蛋白(如细胞周期蛋白A1(CCNA1))和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子；和/或新表位或新抗原。
核酸、载体、宿主细胞
在另一方面中，本发明涉及编码本发明的抗ROR1抗体或多特异性抗体的核酸分子。本发明的核酸可为RNA、DNA或cDNA。根据本发明的一个实施方案，本发明的核酸是基本上分离的核酸。
在一个实施方案中，编码所述抗ROR1抗体的核酸分子与选自SEQ ID NO：39-43、71、75、78、83、87、91和95的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％序列同一性，并且其编码的抗ROR1抗 体能够特异性结合ROR1(即，几乎不与非靶标抗原结合)。优选地，编码所述抗ROR1抗体的核酸分子选自SEQ ID NO：39-43、71、75、78、83、87、91和95。
本发明的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒或YAC。载体可尤其为表达载体，即可提供ROR1抗体在体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本发明的核酸分子，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。对所述调控元件及其序列进行选择以便在特定宿主中表达是本领域技术人员熟知的。对本发明的ROR1抗体的表达有用或必需的调控元件及其他元件的具体实例包括但不限于启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
在另一方面中，本发明还提供表达本发明的ROR1抗体、多特异性抗体和/或含有本发明的核酸或载体的宿主细胞。本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
适合的细菌细胞包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichiapastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本发明也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
重组受体
在另一方面，本发明还提供包含如上所述的抗ROR1抗体的重组受体，例如T 细胞融合蛋白、T细胞抗原耦合器、重组TCR受体或嵌合抗原受体。优选地，本发明还提供包含如上所述的抗ROR1抗体的嵌合抗原受体。
如本文所用，术语“T细胞融合蛋白”或“TFP”是指由TCR各组分衍生的重组多肽，通常由TCR亚基和与其连接的抗体组成并在细胞表面表达。其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内信号结构域。
如本文所用，术语“T细胞抗原耦合器”或“TAC”包括三个功能结构域：1肿瘤靶向结构域，包括单链抗体(例如本发明的抗ROR1抗体)、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAM)磷酸化作为T细胞活化的初始步骤。
如本文所用，术语“T细胞受体”或“TCR”是T细胞表面的特征性标志，以非共价键与CD3结合形成复合物。抗原呈递细胞通过主要组织相容性复合体分子(MHC)将抗原肽呈递至T细胞并且结合至TCR复合物以诱发一系列胞内信号传导。TCR由分别形成异二聚体的六条肽链组成，其一般分为αβ型和γδ型。每条肽链包括恒定区和可变区，其中可变区负责结合特异性的抗原和MHC分子。
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括配体结合结构域(例如抗体的抗原结合部分)、跨膜结构域、任选的共刺激结构域和细胞内信号传导结构域，各个结构域之间通过接头连接。CAR能够利用抗体的抗原结合特性以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。
在一个实施方案中，本发明提供一种嵌合抗原受体，其包含如上所述的抗ROR1抗体或其抗原结合片段或含有所述抗ROR1抗体的多特异性抗体、跨膜结构域和胞内信号传导结构域。
如本文所用，术语“跨膜结构域”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、 CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154及其功能性片段。或者，跨膜结构域可以是合成的并且可以主要地包含疏水性残基如亮氨酸和缬氨酸。优选地，所述跨膜结构域源自CD8α链或CD28，其与SEQ ID NO:44或46所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:45或47所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。在一个实施方案中，本发明的嵌合抗原受体包含的胞内信号传导结构域可以是T细胞受体和共受体的胞内区序列，其在抗原受体结合以后一同起作用以引发信号传导，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。胞内信号传导结构域可以包含许多免疫受体酪氨酸激活基序(Immunoreceptor Tyrosine-based Activation Motifs,ITAM)。本发明的胞内信号传导结构域的非限制性施例包括但不限于FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d等的胞内区。在优选的实施方式中，本发明CAR的信号传导结构域可以包含CD3ζ胞内区，其与SEQ ID NO:52或54所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:53或55所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的嵌合抗原受体还可以包含位于抗体和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗体的任何寡肽或多肽。具体地，铰链区用来为抗体提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。在优选的实施方式中，所述铰链区包含CD8α、CD28、FcγRIIIα受体、IgG4或IgG1的铰链区部分，更优选CD8α、CD28或IgG4铰链，其与SEQ ID NO:60、62或64所示的氨基酸序列具有至少70％， 优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:61、63或65所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，嵌合抗原受体还可以包含一个或多个共刺激结构域。共刺激结构域可以是来自共刺激分子的细胞内功能性信号传导结构域，其包含所述共刺激分子的整个细胞内部分，或其功能片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。共刺激分子包括但不限于1类MHC分子、BTLA和Toll配体受体。本发明的共刺激结构域的非限制性施例包括但不限于源自以下蛋白质的共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、ROR1、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM以及ZAP70。优选地，本发明CAR的共刺激结构域来自4-1BB、CD28或4-1BB+CD28。在一个实施方案中，4-1BB共刺激结构域与SEQ ID NO:50所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:51所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。在一个实施方案中，CD28共刺激结构域与SEQ ID NO:48所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:49所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的CAR还可以包含信号肽，使得当其在细胞例如T细胞中表达时，新生蛋白质被引导至内质网并随后引导至细胞表面。信号肽的核心可以含有长的疏水性氨基酸区段，其具有形成单个α-螺旋的倾向。在信号肽的末端，通常有被信号肽酶识别和切割的氨基酸区段。信号肽酶可以在移位期间或完成后切割，以产生游离信号肽和成熟蛋白。然后，游离信号肽被特定蛋白酶消化。可用于本发明的信号肽是本领域技术人员熟知的，例如衍生自B2M、CD8α、IgG1、GM-CSFRα等的信号肽。在一个实施方案中，可用于本发明的信号肽来自B2M或CD8α，其 与SEQ ID NO:56或58所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:57或59所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，所述CAR含有如本文所提供的抗ROR1抗体或其抗原结合片段或含有所述抗ROR1抗体的多特异性抗体、CD8α或CD28跨膜区、CD28和/或4-1BB共刺激结构域，和CD3ζ胞内信号传导结构域。在该实施方案中，所述CAR还可以进一步包含来自B2M、CD8α、IgG1或GM-CSFRα的信号肽。
本发明还提供编码如上所定义的靶向ROR1的嵌合抗原受体的核酸分子，以及包含所述核酸分子的载体。
如本文所用，术语“载体”是用作将(外源)遗传材料转移到宿主细胞中的媒介核酸分子，在该宿主细胞中所述核酸分子可以例如复制和/或表达。载体一般包括靶向载体和表达载体。“靶向载体”是通过例如同源重组或使用特异性靶向位点处序列的杂合重组酶将分离的核酸递送至细胞内部的介质。“表达载体”是用于异源核酸序列(例如编码本发明的嵌合抗原受体多肽的那些序列)在合适的宿主细胞中的转录以及它们的mRNA的翻译的载体。可用于本发明的合适载体是本领域已知的，并且许多可商购获得。在一个实施方案中，本发明的载体包括但不限于质粒、病毒(例如逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV、多瘤病毒和腺相关病毒(AAV)等)、噬菌体、噬菌粒、粘粒和人工染色体(包括BAC和YAC)。载体本身通常是核酸分子，通常是包含插入物(转基因)的DNA序列和作为载体“骨架”的较大序列。工程化载体通常还包含在宿主细胞中自主复制的起点(如果需要多核苷酸的稳定表达)、选择标记和限制酶切割位点(如多克隆位点，MCS)。载体可另外包含启动子、多聚腺苷酸尾(polyA)、3’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。
工程化免疫细胞
在一个方面，本发明还提供表达本发明所述重组受体，例如T细胞融合蛋白、T细胞抗原耦合器、重组TCR受体或嵌合抗原受体的工程化免疫细胞。
如本文所用，术语“免疫细胞”是指免疫系统的具有一种或多种效应子功能(例 如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。例如，免疫细胞可以是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞。在一个实施方案中，免疫细胞衍生自干细胞，例如成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞等。优选地，免疫细胞是T细胞。T细胞可以是任何T细胞，如体外培养的T细胞，例如原代T细胞，或者来自体外培养的T细胞系例如Jurkat、SupT1等的T细胞，或获得自受试者的T细胞。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。T细胞也可以被浓缩或纯化。T细胞可以处于任何发育阶段，包括但不限于，CD4+/CD8+T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。在一个优选的实施方案中，免疫细胞是人T细胞。可以使用本领域技术人员已知的多种技术，如Ficoll分离从受试者的血液获得T细胞。
采用本领域已知的常规方法(如通过转导、转染、转化等)可以将编码嵌合抗原受体的核酸序列引入免疫细胞。“转染”是将核酸分子或多核苷酸(包括载体)引入靶细胞的过程。一个例子是RNA转染，即将RNA(比如体外转录的RNA，ivtRNA)引入宿主细胞的过程。该术语主要用于真核细胞中的非病毒方法。术语“转导”通常用于描述病毒介导的核酸分子或多核苷酸的转移。动物细胞的转染通常涉及在细胞膜中打开瞬时的孔或“洞”，以允许摄取材料。可以使用磷酸钙、通过电穿孔、通过细胞挤压或通过将阳离子脂质与材料混合以产生与细胞膜融合并将它们的运载物沉积入内部的脂质体，进行转染。用于转染真核宿主细胞的示例性技术包括脂质囊泡介导的摄取、热休克介导的摄取、磷酸钙介导的转染(磷酸钙/DNA共沉淀)、显微注射和电穿孔。术语“转化”用于描述核酸分子或多核苷酸(包括载体)向细菌中、也向非动物真核细胞(包括植物细胞)中的非病毒转移。因此，转化是细菌或非动物真核细胞的基因改变，其通过细胞膜从其周围直接摄取并随后并入外源遗传材料(核酸分子)而产生。转化可以通过人工手段实现。为了发生转化，细胞或细菌必须处于感受态的状态。对于原核转化，技术可包括热休克介导的摄取、与完整细胞的细菌原生质体融合、显微注射和电穿孔。将核酸或载体引入免疫细胞后，本领域技术人员可以通过常规技术对所得免 疫细胞进行扩增和活化。
在一个实施方案中，为减少移植物抗宿主病的风险，所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：CD52、GR、dCK、TCR/CD3基因(例如TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ)、MHC相关基因(HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA)和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。优选地，所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA、PD1、LAG3、TIM3、CTLA4，更优选TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA。
抑制基因表达或使基因沉默的方法是本领域技术人员熟知的。例如，可以使用反义RNA、RNA诱饵、RNA适体、siRNA、shRNA/miRNA、反式显性阴性蛋白(TNP)、嵌合/抗体偶联物、趋化因子配体、抗感染性细胞蛋白、细胞内抗体(sFv)、核苷类似物(NRTI)、非核苷类似物(NNRTI)、整合酶抑制剂(寡核苷酸、二核苷酸和化学剂)和蛋白酶抑制剂来抑制基因的表达。另外，也可以通过例如大范围核酸酶、锌指核酸酶、TALE核酸酶或CRISPR系统中的Cas酶介导DNA断裂，从而使基因沉默。
在一个实施方案中，所述工程化免疫细胞还包含靶向其他肿瘤抗原的第二嵌合抗原受体。所述第二嵌合抗原受体靶向的其他肿瘤抗原可以选自例如BCMA、CD4、CD5、CD7、CD8、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD80、CD126、CD138、B7、MUC-1、HM1.24、血管生成因子、VEGF、PIGF、ED-B纤连蛋白、CD66a-d、IL-2、T101、TAC、IL-6、DR4、DR5、tEGFR、Her2、L1-CAM、间皮素、CEA、乙型肝炎表面抗原、抗叶酸受体、CD24、CD30、CD44、EGFR、EGP-2、EGP-4、EPHa2、ErbB2、ErbB3、ErbB4、ErbB二聚体、EGFR vIII、FBP、FCRL5、FCRH5、胎儿乙酰胆碱受 体、GD2、GD3、GPRC5D、HMW-MAA、IL-22R-α、IL-13R-α2、κ轻链、Lewis Y、L1-CAM、MAGE-A1、MAGE-A3、MAGE-A6、PRAME、生存素、EGP2、EGP40、TAG72、B7-H6、IL-13Ra2、CA9、CD171、G250/CAIX、HLA-A1、HLA-A2、NY-ESO-1、PSCA、叶酸受体-a、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、MUC1、MUC16、NY-ESO-1、MART-1、gp100、癌胚胎抗原、VEGF-R2、CEA、前列腺特异性抗原、PSMA、Her2/neu、雌激素受体、孕酮受体、肝配蛋白B2、CD123、c-Met、GD-2、O-乙酰化GD2(OGD2)、CE7、WT-1、细胞周期蛋白A2、CCL-1、hTERT、MDM2、CYP1B、WT1、活素、AFP、p53、D1、CS-1、BAFF-R、TACI、CD56、TIM-3、CD123、L1-细胞粘附分子、MAGE-A1、MAGEA3、CCNA1和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子。
在一个实施方案中，提供多种免疫细胞，每种免疫细胞被改造为表达一种或多种嵌合抗原受体。例如，在一些实施方案中，将一种免疫细胞改造为表达结合和/或靶向ROR1的嵌合抗原受体(例如包含本发明所述抗ROR1抗体的CAR)，并且将另一种细胞改造为表达结合和/或靶向其他抗原的嵌合抗原受体。在一个实施方案中，免疫细胞也可以表达多特异性嵌合抗原受体，其靶向包括ROR1在内的一种或多种抗原。例如，这种多特异性嵌合抗原受体可以包含靶向ROR1的多特异性抗体，或者同时包含本发明所述的抗ROR1抗体和靶向其他抗原的抗体。在此类实施方案中，所述多种工程化免疫细胞可以一起或单独施用。在一个实施方案中，所述多种免疫细胞可以在同一组合物中或在不同组合物中。细胞的示例性组合物包括本申请以下章节中所描述的组合物。
抗体偶联物
在一个方面，本发明提供一种抗体偶联物，其包含本发明所定义的抗ROR1抗体和第二功能性结构，其中所述第二功能性结构选自Fc、放射性同位素、延长半衰期的结构部分、可检测标记物和药物。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗ROR1抗体和Fc。如本文所用，术语“Fc”用于定义免疫球蛋白重链的C末端区，其包括天然Fc和变体Fc。“天然Fc”是指包含通过消化完整抗体产生的、无论是单体形式或是多聚体形式的非抗原结合片段的分子或序列。产生天然Fc的免疫球蛋白源优选来源于人类。天然Fc片段由可以通过共价连接(例如二硫键)和非共价连接 而连接为二聚体或多聚体形式的单体多肽构成。根据类别(例如IgG、IgA、IgE、IgD、IgM)或亚型(例如IgG1、IgG2、IgG3、IgA1、IgGA2)的不同，天然Fc分子单体亚基之间具有1-4个分子间二硫键。天然Fc的一个实例是通过用木瓜蛋白酶消化IgG产生的二硫键连接的二聚体(参见Ellison等(1982)，Nucleic Acids Res.10：4071-9)。本文所用的术语“天然Fc”一般是指单体、二聚体和多聚体形式。“变体Fc”是指由于至少一个本文定义的“氨基酸修饰”而与“天然”或“野生型”Fc的氨基酸序列不同的氨基酸序列，也称为“Fc变体”。因此，“Fc”也包括单链Fc(scFc)，即，由多肽接头连接的两个Fc单体组成的单链Fc，其能够自然折叠成功能性二聚体Fc区域。在一个实施方案中，所述Fc优选是人免疫球蛋白的Fc，更优选是人IgG1的Fc。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗ROR1抗体和放射性同位素。可用于本发明的放射性同位素的实例包括但不限于At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32、Pb 212、 99mTc、 123I、 18F和 68Ga。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗ROR1抗体和延长半衰期的结构部分，所述延长半衰期的结构部分选自白蛋白的结合结构、转铁蛋白的结合结构、聚乙二醇分子、重组聚乙二醇分子、人血清白蛋白、人血清白蛋白的片段和结合人血清白蛋白的白多肽(包括抗体)。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗ROR1抗体和可检测标记物。术语“可检测标记物”在本文中意指产生可检测信号的化合物。例如，可检测标记物可以是MRI造影剂、闪烁扫描造影剂、X射线成像造影剂、超声造影剂、光学成像造影剂。可检测标记物的实施例包括荧光团(如荧光素、Alexa或花青)、化学发光化合物(如鲁米诺)、生物发光化合物(如荧光素酶或碱性磷酸酶)、酶(如辣根过氧化物酶、葡萄糖-6-磷酸酶、β-半乳糖苷酶)、抗生素(例如卡那霉素、氨苄霉素、氯霉素、四环素等)抗性基因和造影剂(如纳米颗粒或钆)。本领域技术人员可以根据所用的检测系统选择合适的可检测标记物。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗ROR1抗体和与所述抗ROR1抗体偶联的药物，例如细胞毒素或免疫调节剂(即，抗体药物偶联物)。通常药物通过共价与抗体连接，并且通常依赖于接头。在一个实施方案中，所述药物是细胞毒素。在另一个实施方案中，所述药物是免疫调节剂。细胞 毒素的实例包括但不限于甲氨蝶呤、氨基蝶呤、6-巯基嘌呤、6-硫鸟嘌呤、阿糖胞苷、5-氟尿嘧啶、达卡巴嗪、氮芥、噻替派、苯丁酸氮芥、美法仑、卡莫司汀(BSNU)、洛莫司汀(CCNU)、1-甲基亚硝基脲、环磷酰胺、氮芥、白消安、二溴甘露醇、链佐星、丝裂霉素、顺-二氯二胺铂(II)(DDP)、顺铂、卡铂、佐柔比星、多柔比星、地托比星、卡米诺霉素、伊达比星、表柔比星、米托蒽醌、放线菌素D、博来霉素、刺孢霉素、光辉霉素、安曲霉素(AMC)、长春新碱、长春花碱、紫杉醇、蓖麻毒素、假单胞菌外毒素、吉西他滨、细胞松弛素B、短杆菌肽D、溴乙锭、依米丁、依托泊苷、替尼泊苷、秋水仙素、二羟基蒽二酮、1-脱氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普萘洛尔、嘌呤霉素、丙卡巴肼、羟基脲、天冬酰胺酶、皮质类固醇、米托坦(O,P'-(DDD))、干扰素，以及它们的组合。免疫调节剂的实例包括但不限于更昔洛韦、依那西普、他克莫司、西罗莫司、伏环孢素、环孢灵、雷帕霉素、环磷酰胺、硫唑嘌呤、霉酚酸酯、甲氨蝶呤、糖皮质素及其类似物、细胞因子、干细胞生长因子、淋巴毒素、肿瘤坏死因子(TNF)、造血因子、白介素(例如IL-1、IL-2、IL-3、IL-6、IL-10、IL-12、IL-18及IL-21)、集落刺激因子(例如G-CSF及GM-CSF)、干扰素(例如干扰素-α、干扰素-β及干扰素-γ)、命名为“S1因子”的干细胞生长因子、红细胞生成素和血小板生成素，或其组合。
试剂盒和药物组合物
在另一个方面，本发明还提供一种检测试剂盒，其包含本发明所述的人源化抗体、多特异性抗体、抗体偶联物、工程化免疫细胞或嵌合抗原受体。
在另一个方面，本发明还提供一种药物组合物，其包含本发明所述的人源化抗体、嵌合抗原受体、多特异性抗体、工程化免疫细胞或抗体偶联物，和一种或多种药学上可接受的赋形剂。
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包 覆剂、等渗剂、吸收延迟剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本发明期望的药物组合物。用于本发明的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。
根据本发明的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。
根据本发明的药物组合物也可以制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏、乳膏、透皮贴剂、凝胶、粉末、片剂、溶液、气雾剂、颗粒、丸剂、混悬剂、乳剂、胶囊、糖浆、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的免疫细胞的药物组合物通常以溶液形式提供，并且优选包含药学上可接受的缓冲剂。
根据本发明的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。此外，本发明的药物组合物也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
治疗/预防/诊断用途
在另一个方面，本发明还提供一种治疗和/或预防和/或诊断与ROR1表达相关的疾病的方法，包括向受试者施用如上所述的人源化抗体、嵌合抗原受体、多特异性抗 体、抗体偶联物、工程化免疫细胞或药物组合物。
在一个实施方案中，与ROR1表达相关的疾病包括但不限于B细胞白血病、淋巴瘤、B细胞慢性淋巴细胞性白血病(CLL)、非霍奇金淋巴瘤(NHL)、急性髓细胞样白血病(AML)、急性淋巴细胞性白血病(ALL)、伯基特淋巴瘤、套细胞淋巴瘤(MCL)、非小细胞肺癌(NSCLC)、神经母细胞瘤、肾癌、结肠癌、结直肠癌、乳腺癌、上皮鳞状细胞癌、黑素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、子宫癌、肾上腺癌和头颈癌。
下面将参考附图并结合实例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了例举的目的，并不能对本发明构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
附图说明
图1：示出了表达ROR1鼠源ScFv的CAR-T细胞中的scFv表达水平。
图2：示出了表达ROR1鼠源ScFv的CAR-T细胞在不同效靶比下对靶细胞的杀伤效果。
图3：示出了表达ROR1鼠源ScFv的CAR-T细胞与各种靶细胞和非靶细胞共培养后的IFN-γ的释放水平。
图4：示出了表达ROR1人源化ScFv的CAR-T细胞中的scFv表达水平。
图5：示出了表达ROR1人源化ScFv的CAR-T细胞在不同效靶比下对靶细胞Jeko-1-luci(A)和非靶细胞K562-luci(B)的杀伤效果。
图6：示出了表达ROR1人源化ScFv的CAR-T细胞对靶细胞MDA-MB-231、Jeko-1、A549和非靶细胞K562的脱颗粒作用。
图7：示出了表达ROR1人源化ScFv的CAR-T细胞与各种靶细胞MDA-MB-231、Jeko-1、A549和非靶细胞K562共培养后的IL2(A)和IFN-γ(B)的释放水平。
具体实施方式
实施例1.制备ROR1抗体
将含有ROR1蛋白胞外区序列克隆到带有His标签的pCP载体上，将测序正确的质粒瞬时感染CHO细胞，培养8-10天后收获细胞培养液，采用亲和层析方式进行 蛋白纯化，获得人源LIR1蛋白胞外区，作为免疫原。
采用6-8周雌性Balb/c小鼠进行初次和加强免疫，采用ELISA和FACS检测血清中蛋白免疫原的抗体效价和特异性，综合评定后对小鼠进行最后一次强化免疫。3-4天后处死小鼠，收集脾细胞，将其按照一定比例与小鼠骨髓瘤细胞(SP2/0)混合，并将增殖后的杂交瘤细胞经由FACS和ELISA方法鉴定后筛选到阳性细胞株。经过多轮亚克隆鉴定，最终获得可稳定分泌ROR1抗体的杂交瘤细胞，将杂交瘤细胞上清中的抗体进行纯化后，获得5个抗ROR1抗体。
采用胰蛋白酶进行酶解抗体，通过DNA数据采集获得高质量的一级和二级谱图，将谱图用peaks软件进行de novo解析后获得初步的氨基酸序列结果；采用多种蛋白酶对抗体进行酶解，MSE采集数据后可获得所有肽段峰以及每个肽段各个价态完整和碎裂的信息；将获得的更加全面的质谱数据与初步的氨基酸序列进行匹配，最终获得的5个ROR1抗体的氨基酸序列如下表1所示。
表1.ROR1抗体的序列
  ROR1-1 ROR1-2 ROR1-3 ROR1-4 ROR1-5
CDR-L1 SEQ ID NO:1 SEQ ID NO:1 SEQ ID NO:10 SEQ ID NO:16 SEQ ID NO:1
CDR-L2 SEQ ID NO:2 SEQ ID NO:2 SEQ ID NO:11 SEQ ID NO:17 SEQ ID NO:22
CDR-L3 SEQ ID NO:3 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:7
CDR-H1 SEQ ID NO:4 SEQ ID NO:4 SEQ ID NO:13 SEQ ID NO:19 SEQ ID NO:4
CDR-H2 SEQ ID NO:5 SEQ ID NO:8 SEQ ID NO:14 SEQ ID NO:20 SEQ ID NO:5
CDR-H3 SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:15 SEQ ID NO:21 SEQ ID NO:23
VL SEQ ID NO:24 SEQ ID NO:27 SEQ ID NO:30 SEQ ID NO:33 SEQ ID NO:36
VH SEQ ID NO:25 SEQ ID NO:28 SEQ ID NO:31 SEQ ID NO:34 SEQ ID NO:37
scFv SEQ ID NO:26 SEQ ID NO:29 SEQ ID NO:32 SEQ ID NO:35 SEQ ID NO:38
实施例2.制备包含ROR1鼠源抗体的CAR-T细胞
合成编码以下蛋白的序列，并将其克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号:PPL00157-4a)：CD8α信号肽(SEQ ID NO：58)、抗ROR1抗体、CD8α铰链区(SEQ ID NO：60)、CD8α跨膜区(SEQ ID NO：44)、4-1BB胞内区(SEQ ID NO：50)、CD3ζ胞内信号传导结构域(SEQ ID NO：52)，并通 过测序确认目标序列的正确插入。其中，ROR1-1CAR包含的抗ROR1scFv的氨基酸序列如SEQ ID NO：26所示；ROR1-2CAR包含的抗ROR1scFv的氨基酸序列如SEQ ID NO：29所示；ROR1-3CAR包含的抗ROR1scFv的氨基酸序列如SEQ ID NO：32所示；ROR1-4CAR包含的抗ROR1scFv的氨基酸序列如SEQ ID NO：35所示；ROR1-5CAR包含的抗ROR1scFv的氨基酸序列如SEQ ID NO：38所示。
在无菌管中加入3mL Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒:病毒包装载体：病毒包膜载体＝4:2:1的比例加入包装载体psPAX2(Addgene，货号12260)和包膜载体pMD2.G(Addgene，货号12259)。然后，加入120μL X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28CTS TM(Gibco,货号40203D)激活T细胞，并在37℃和5％CO2下继续培养1天。然后，加入浓缩的慢病毒，持续培养3天后，获得表达不同ROR1鼠源scFv的CAR-T细胞，未经修饰的野生型T细胞用作阴性对照(NT)。
使用Biotin-SP(long spacer)AffiniPure Goat Anti-Mouse IgG,F(ab') 2Fragment Specific(min X Hu,Bov,Hrs Sr Prot)(jackson immunoresearch，货号115-065-072)作为一抗，APC Streptavidin(BD Pharmingen，货号554067)或PE Streptavidin(BD Pharmingen，货号554061)作为二抗)，通过流式细胞仪检测ROR1-1CAR-T细胞、ROR1-2CAR-T细胞、ROR1-3CAR-T细胞、ROR1-4CAR-T细胞和、ROR1-5CAR-T细胞上的scFv的表达水平，结果如图1所示。
可以看出，本发明制备的表达不同ROR1鼠源scFv的CAR-T细胞中的抗ROR1scFv均可以有效表达。
实施例3.检测表达ROR1鼠源ScFv的CAR-T细胞对靶细胞的杀伤效果和细胞因子释放
3.1检测CAR-T细胞对靶细胞的杀伤效果
以1×10 4个细胞/孔的浓度将携带荧光素基因的MDA-MB-231靶细胞铺入96孔板中，然后以10:1或5:1的效靶比(即效应T细胞与靶细胞之比)将NT细胞和表达 ROR1鼠源scFv的CAR-T细胞铺入到96孔板进行共培养，16-18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图2所示。
可以看出，与NT相比，本发明制备的表达ROR1鼠源ScFv的CAR-T细胞在不同效靶比下对靶细胞均有显著的特异性杀伤。
3.2检测CAR-T细胞的细胞因子释放水平
以1×10 5个细胞/孔的浓度将靶细胞(MDA-MB-231细胞、Hs-578T细胞)或非靶细胞(Jurkat细胞、Huh7细胞)铺于96孔板中，然后以1:1的比例加入表达ROR1鼠源ScFv的CAR-T细胞或NT细胞进行共培养，18-24小时后收集细胞共培养上清液。
使用捕获抗体Purified anti-human IFN-γAntibody(Biolegend，货号506502)包被96孔板4℃孵育过夜，然后移除抗体溶液，加入250μL含有2％BSA(sigma，货号V900933-1kg)的PBST(含0.1％吐温的1XPBS)溶液，37℃孵育2小时。然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。每孔加入50μL细胞共培养上清液或标准品，并在37℃孵育1小时，然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。然后向各孔分别加入50μL检测抗体Anti-Interferon gamma抗体[MD-1](Biotin)(abcam，货号ab25017)，在37℃孵育1小时后，用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。再加入HRP Streptavidin(Biolegend，货号405210)，在37℃孵育30分钟后，弃上清液，加入250μL PBST(含0.1％吐温的1XPBS)，清洗5次。向各孔加入50μL TMB底物溶液。使反应在室温下于暗处发生30分钟，之后向各孔中加入50μL 1mol/L H 2SO 4以停止反应。在停止反应的30分钟内，使用酶标仪检测450nm处吸光度，并根据标准曲线(根据标准品的读值和浓度绘制)计算细胞因子的含量，结果如图3所示。
可以看出，本发明制备的表达ROR1鼠源ScFv的细胞与非靶细胞Jurkat和Huh27共培养后均没有释放细胞因子IFN-γ，而与靶细胞MDA-MB-231细胞或Hs-578T细胞共培养后IFN-γ的释放水平显著升高。这表明，本发明制备的表达ROR1鼠源ScFv的CAR-T细胞可以对靶细胞产生特异性杀伤。
实施例4.ROR1抗体的人源化
对鼠源抗体ROR1-4进行人源化，具体方法如下：首先通过IG BLAST数据库(https://www.ncbi.nlm.nih.gov/igblast/)检索相似度较高的人源抗体序列，然后将单链抗体中的FR区替换为相应的人源序列；再根据氨基酸残基的不同理化性质对个别氨基酸残基进行替换，最终获得7个人源化单链抗体，其序列如表2所示。
表2.ROR1人源化抗体的序列。
  VL VH ScFv(aa) ScFv(nt)
6V7 SEQ ID NO：68 SEQ ID NO：69 SEQ ID NO：70 SEQ ID NO：71
6V8 SEQ ID NO：72 SEQ ID NO：73 SEQ ID NO：74 SEQ ID NO：75
6V9 SEQ ID NO：76 SEQ ID NO：77 SEQ ID NO：78 SEQ ID NO：79
6V10 SEQ ID NO：80 SEQ ID NO：81 SEQ ID NO：82 SEQ ID NO：83
6V11 SEQ ID NO：84 SEQ ID NO：85 SEQ ID NO：86 SEQ ID NO：87
6V12 SEQ ID NO：88 SEQ ID NO：89 SEQ ID NO：90 SEQ ID NO：91
6V13 SEQ ID NO：92 SEQ ID NO：93 SEQ ID NO：94 SEQ ID NO：95
实施例5.制备包含ROR1人源化抗体的CAR-T细胞
合成编码以下蛋白的序列，并将其克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号：PPL00157-4a)：CD8α信号肽(SEQ ID NO：58)、抗ROR1人源化抗体(SEQ ID NO：70、74、78、82、86、90、94之一)、IgG4铰链区(SEQ ID NO：64)、CD8α跨膜区(SEQ ID NO：44)、4-1BB胞内区(SEQ ID NO：50)、CD3ζ胞内信号传导结构域(SEQ ID NO：52)，并通过测序确认目标序列的正确插入。
根据实施例2所述的方法将上述载体包装入慢病毒并感染激活的T细胞，获得包含ROR1人源化抗体的CAR-T细胞。
在37℃和5％CO 2下培养11天之后，将上述CAR-T细胞、ROR1-4CAR-T-2细胞(将ROR1-4CAR-T细胞中的CD8α铰链区替换为IgG4铰链区)以及野生型T细胞(NT，用作阴性对照)与ROR1-hFc(Acrobio品牌，货号RO1-H5250)蛋白共孵育45min，清洗后加入PE anti-human IgG Fc Antibody(Biolegend品牌，货号410708)共孵育30min。并通过流式细胞仪检测CAR-T细胞上的抗ROR1单链抗体的表达水平，结果如图4所示。
可以看出，本发明制备的CAR-T细胞中的抗ROR1人源化抗体可以有效表达。
实施例6.检测表达ROR1人源化ScFv的CAR-T细胞对靶细胞的杀伤效果、脱颗粒作用和细胞因子释放水平
6.1检测CAR-T细胞对靶细胞的杀伤效果
以1×10 4个细胞/孔的浓度将靶细胞(Jeko-1-luci细胞)或非靶细胞(K562-luci细胞)铺入96孔板中，然后以16:1、8:1、4:1、2:1、1:1的效靶比(即效应T细胞与靶细胞之比)将NT细胞和各CAR-T细胞铺入到96孔板进行共培养，16-18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图5所示。
可以看出，在不同效靶比下，本发明制备的表达ROR1人源化ScFv的CAR-T细胞显示出对靶细胞Jeko-1-luci细胞的强烈杀伤作用，而对非靶细胞K562-luci细胞的杀伤则较弱，表明表达ROR1人源化ScFv的CAR-T细胞仅对表达ROR1的细胞呈现特异性杀伤。
6.2检测CAR-T细胞的脱颗粒作用
以1×10 5个细胞/孔的浓度将靶细胞(MDA-MB-231细胞、Jeko-1细胞、A549细胞)和非靶细胞(K562细胞)分别铺于96孔板中，按1:1的比例加入表达ROR1人源化ScFv的CAR-T细胞、ROR1-4CAR-T-2细胞和NT细胞(阴性对照)，然后向各孔加入10μL PE Mouse anti-human CD107a antibody(BD,货号555801)，并于37℃、5％CO 2条件下避光孵育。1h后，向各孔加入20μL Golgi Stop(BD,货号51-2092K2)，并于37℃、5％CO 2条件下避光孵育2.5h。然后向各孔加入10μL APC anti-human CD8(BD,货号555369)，并于37℃、5％CO 2条件下避光孵育0.5h。通过流式细胞术检测各孔细胞样品，并分别分析CD107a+CD8+细胞占T细胞的比例，结果如图6所示。
可以看出，与NT细胞相比，本发明制备的表达ROR1人源化ScFv的CAR-T细胞对三种靶细胞MDA-MB-231细胞、Jeko-1细胞、A549细胞均显示出显著升高的特异性的脱颗粒作用，而对非靶细胞则没有观察到显著升高的脱颗粒作用。
6.3检测CAR-T细胞的细胞因子释放水平
以1×10 5个细胞/孔的浓度将靶细胞(MDA-MB-231细胞、Jeko-1细胞、A549 细胞)和非靶细胞(K562细胞)铺于96孔板中，按1:1的比例分别加入表达ROR1人源化ScFv的CAR-T细胞和NT细胞(阴性对照)，共培养18-24小时后收集细胞共培养上清液。
按照制造商的建议，分别使用Human IL-2 DuoSet ELISA Kit(R&D systems,货号DY202)、Human IFN-gamma DuoSet ELISA Kit(R&D systems,货号DY285)检测共培养上清液中IL2和IFN-γ的含量，结果如图7所示。
可以看出，与NT细胞相比，本发明的表达ROR1人源化ScFv的CAR-T细胞与三种靶细胞共培养后，细胞因子IL2和IFN-γ的释放水平均显著升高，并且这种细胞因子释放是特异性的。
需要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (26)

  1. 一种靶向ROR1的抗体，其包含：
    (1)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：3所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：6所示的CDR-H3；
    (2)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：7所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：8所示的CDR-H2和如SEQ ID NO：9所示的CDR-H3；
    (3)如SEQ ID NO：10所示的CDR-L1、如SEQ ID NO：11所示的CDR-L2、如SEQ ID NO：12所示的CDR-L3、如SEQ ID NO：13所示的CDR-H1、如SEQ ID NO：14所示的CDR-H2和如SEQ ID NO：15所示的CDR-H3；
    (4)如SEQ ID NO：16所示的CDR-L1、如SEQ ID NO：17所示的CDR-L2、如SEQ ID NO：18所示的CDR-L3、如SEQ ID NO：19所示的CDR-H1、如SEQ ID NO：20所示的CDR-H2和如SEQ ID NO：21所示的CDR-H3；或
    (5)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：22所示的CDR-L2、如SEQ ID NO：7所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：23所示的CDR-H3。
  2. 权利要求1所述的抗体，所述抗体包含重链可变区和轻链可变区，所述重链可变区与选自SEQ ID NO：25、28、31、34、37、69、73、77、81、85、89和93的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：25、28、31、34、37、69、73、77、81、85、89和93的氨基酸序列相比具有一个或几个氨基酸的保守性修饰；所述轻链可变区与选自SEQ ID NO：24、27、30、33和36的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：24、27、30、33、36、68、72、76、80、84、88和92的氨基酸序列相比具有一个或 几个氨基酸的保守性修饰。
  3. 权利要求1所述的抗体，其中所述抗体的氨基酸序列与选自SEQ ID NO：26、29、32、35、38、70、74、78、82、86、90和94的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：26、29、32、35、38、70、74、78、82、86、90和94的氨基酸序列相比具有一个或几个氨基酸的保守性修饰。
  4. 权利要求1-3任一项所述的抗体，其中所述抗体是鼠源抗体、嵌合抗体、人源化抗体或人抗体。
  5. 权利要求1-4任一项所述的抗体，其中所述抗体选自IgG、Fab、Fab'、F(ab') 2、Fd、Fd′、Fv、scFv、sdFv、线性抗体和双体。
  6. 一种核酸分子，其编码权利要求1-5任一项所述的抗体。
  7. 权利要求6所述的核酸分子，其与选自SEQ ID NO：39-43、71、75、79、83、87、91和95的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％序列同一性，并且其编码的抗体能够特异性结合ROR1。
  8. 一种多特异性抗体，其包含权利要求1-5任一项所述的抗体和一个或多个与其他抗原特异性结合的第二抗体或其抗原结合部分。
  9. 权利要求8所述的多特异性抗体，其中所述第二抗体或其抗原结合部分选自全长抗体、Fab、Fab'、(Fab') 2、Fv、scFv、scFv-scFv、微抗体、双抗体或sdAb。
  10. 一种载体，其包含编码权利要求1-5任一项所述的抗体或权利要求8或9所述的多特异性抗体的核酸分子。
  11. 一种宿主细胞，其表达权利要求1-5任一项所述的抗体或权利要求8或9所述的多特异性抗体。
  12. 一种重组受体，其包含权利要求1-5任一项所述的抗体或权利要求8或9所述的多特异性抗体，所述重组受体是T细胞融合蛋白、T细胞抗原耦合器、重组TCR受体或嵌合抗原受体。
  13. 权利要求12所述的重组受体，其进一步包含跨膜结构域和胞内信号传导结构域。
  14. 权利要求13所述的重组受体，所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、 CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154
  15. 权利要求13所述的重组受体，所述胞内信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
  16. 权利要求13所述的重组受体，所述重组受体还包含一个或多个共刺激结构域，其选自以下蛋白质的共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、ROR1、CD8、CD18、CD27、CD28、CD30、CD40、CD54、CD83、CD134、CD137、CD270、CD272、CD276、CD278、CD357、DAP10、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM以及ZAP70。
  17. 一种工程化免疫细胞，其包含权利要求12-16任一项所述的重组受体。
  18. 权利要求17所述的工程化免疫细胞，其选自T细胞、NK细胞、NKT细胞、巨噬细胞、树突细胞。
  19. 权利要求18所述的工程化免疫细胞，其还包含靶向其他肿瘤抗原的第二嵌合抗原受体。
  20. 权利要求17-19任一项所述的工程化免疫细胞，还包含至少一种选自以下的基因的表达被抑制或沉默：TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA、PD1、LAG3、TIM3和CTLA4。
  21. 一种抗体偶联物，其包含权利要求1-5任一项所述的抗体或权利要求8或9所述的多特异性抗体，和第二功能性结构，其中所述第二功能性结构选自Fc、放射性同位素、延长半衰期的结构部分、可检测标记物和药物。
  22. 权利要求21所述的抗体偶联物，其中所述延长半衰期的结构部分选自：白蛋白的结合结构、转铁蛋白的结合结构、聚乙二醇分子、重组聚乙二醇分子、人血清白蛋白、人血清白蛋白的片段和结合人血清白蛋白的白多肽；所述可检测标记物选自荧光团、化学发光化合物、生物发光化合物、酶、抗生素抗性基因和造影剂；所述药物选自细胞毒素和免疫调节剂。
  23. 一种检测试剂盒，其包含权利要求1-5任一项所述的抗体、权利要求8或9所述的多特异性抗体、权利要求12-16任一项所述的重组受体、权利要求 17-20任一项所述的工程化免疫细胞、或权利要求21或22所述的抗体偶联物。
  24. 一种药物组合物，包含权利要求1-5任一项所述的抗体、权利要求8或9所述的多特异性抗体、权利要求12-16任一项所述的重组受体、权利要求17-20任一项所述的工程化免疫细胞、或权利要求21或22所述的抗体偶联物，和一种或多种药学上可接受的赋形剂。
  25. 权利要求1-5任一项所述的抗体、权利要求8或9所述的多特异性抗体、权利要求12-16任一项所述的重组受体、权利要求17-20任一项所述的工程化免疫细胞、或权利要求21或22所述的抗体偶联物、或权利要求24所述的药物组合物在制备用于治疗和/或预防和/或诊断与ROR1表达相关的疾病的药物中的用途。
  26. 权利要求25所述的用途，其中所述与ROR1表达相关的疾病选自B细胞白血病、淋巴瘤、B细胞慢性淋巴细胞性白血病(CLL)、非霍奇金淋巴瘤(NHL)、急性髓细胞样白血病(AML)、急性淋巴细胞性白血病(ALL)、伯基特淋巴瘤、套细胞淋巴瘤(MCL)、非小细胞肺癌(NSCLC)、神经母细胞瘤、肾癌、结肠癌、结直肠癌、乳腺癌、上皮鳞状细胞癌、黑素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、子宫癌、肾上腺癌和头颈癌。
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