WO2023143315A1 - Ror1-targeted antibody or antigen-binding fragment thereof and use thereof - Google Patents

Ror1-targeted antibody or antigen-binding fragment thereof and use thereof Download PDF

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Publication number
WO2023143315A1
WO2023143315A1 PCT/CN2023/072941 CN2023072941W WO2023143315A1 WO 2023143315 A1 WO2023143315 A1 WO 2023143315A1 CN 2023072941 W CN2023072941 W CN 2023072941W WO 2023143315 A1 WO2023143315 A1 WO 2023143315A1
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seq
amino acid
acid sequence
variable region
chain variable
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PCT/CN2023/072941
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French (fr)
Chinese (zh)
Inventor
林晨
束庆玉
刘东舟
徐培芳
刘婵娟
郎国竣
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杭州中美华东制药有限公司
三优生物医药(上海)有限公司
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Publication of WO2023143315A1 publication Critical patent/WO2023143315A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present application relates to an antibody specifically recognizing ROR1, its preparation method and use.
  • the present application also relates to antibody-drug conjugates (ADCs) targeting ROR1 and compositions containing such molecules.
  • ADCs antibody-drug conjugates
  • the present invention also relates to the therapeutic and diagnostic uses of these antibodies, antibody fragments and antibody-drug conjugates.
  • Antibody-Drug Conjugates are derivative drugs of traditional antibody drugs, which are composed of monoclonal antibody drugs targeting specific antigens and small molecule cytotoxic drugs coupled through linkers, and have the functions of antibody drugs.
  • Specific targeting and killing effect of traditional small molecule drugs its main indication is malignant tumors. With its excellent tumor killing effect, ADC has quickly become a hot spot in the research and development of anti-tumor drugs.
  • the toxicity of ADCs is usually high, so the specificity requirements for target antigens are high.
  • Targets suitable for the development of ADCs are generally highly expressed in tumors and low or almost non-expressed in healthy tissues; the antigen is upregulated by tumor cells surface receptors that promote tumor growth or survival, and the target antigen should have internalization properties.
  • Receptor tyrosine kinase-like orphan receptor 1 (ROR1, also known as receptor-associated neurotrophic tyrosine kinase 1, NTRKR1) and receptor tyrosine kinase-like Orphan receptor 2 (receptor tyrosine kinase-like orphan receptor 2, ROR2) is a single transmembrane protein belonging to the receptor tyrosine kinase (RTK) family, and its extracellular structure consists of an immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD domain and KRD domain), intracellularly consists of a tyrosine kinase domain, two serine- or threonine-rich domains and a Domain composition of proline.
  • RTK receptor-associated neurotrophic tyrosine kinase 1
  • ROR2 receptor tyrosine kinase-like orphan receptor 2
  • ROR2 receptor tyros
  • ROR1 and ROR2 participate in the non-canonical Wnt signaling pathway through FZD domain binding ligand Wnt5a (Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, Koshida I, Suzuki K, Yamada G, Schwabe GC et al( 2003). Genes Cells 8:645–654; Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al (2008). Proc Natl Acad Sci USA 105:3047–3052; Paganoni S, Bernstein J, Ferreira A (2010).
  • ROR1 inhibits apoptosis, enhances EGFR signaling, and induces epithelial-mesenchymal transition (EMT) (Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al (2008). Proc Natl Acad Sci USA 105:3047–3052; Yamaguchi T, Yanagisawa K, Sugiyama R, Hosono Y, Shimada Y, Arima C, Kato S, Tomida S, Suzuki M, Osada He et al (2012). Cancer Cell 21:348–361; Cui B, Zhang S, Chen L, Yu J, Widhopf GF, Fecteau J-F, Rassenti LZ, Kipps TJ (2013). Cancer Res 73:3649–3660).
  • EMT epithelial-mesenchymal transition
  • ROR1 is a conserved embryonic protein. Its expression gradually decreases with embryonic development, and it is almost absent or lowly expressed in most tissues of adults. More and more literatures have found that ROR1 is expressed in a variety of cancer cells, such as B cells with chronic Certain other cancer cell lines of lymphocytic leukemia (CLL) and other hematological malignancies, renal cell carcinoma, colon cancer, and breast cancer. Furthermore, ROR1 plays an important role in the progression of many hematological and solid malignancies. Therefore, as a cancer marker, ROR1 becomes an ideal drug target for cancer therapy.
  • CLL lymphocytic leukemia
  • anti-ROR1 antibody drugs have been disclosed in the prior art, as a pan-cancer tumor marker, there is still an urgent need to develop high-quality anti-ROR1 antibodies, which can be used as antibody-based targets for developing ROR1-expressing cancers.
  • ROR1-containing ADCs with effective therapeutic effects, and the present invention meets these needs.
  • the present invention provides an antibody targeting human ROR1, which has the following advantages:
  • the anti-ROR1 antibody provided by the present invention has a complete human sequence and is a human antibody. It is expected that the anti-ROR1 antibodies of the invention will be minimally immunogenic to human subjects, and that the anti-ROR1 antibodies will be well tolerated by human subjects.
  • the present invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising:
  • HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:52 and 3 contained in the light chain variable region as shown in SEQ ID NO:51 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 3 heavy chain CDRs contained in the heavy chain variable region as shown in SEQ ID NO:54 and 3 contained in the light chain variable region as shown in SEQ ID NO:53 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO:56 and 3 included in the light chain variable region as shown in SEQ ID NO:55 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 3 heavy chain CDRs included in the heavy chain variable region as shown in SEQ ID NO:58 and 3 included in the light chain variable region as shown in SEQ ID NO:57 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:60 and 3 contained in the light chain variable region as shown in SEQ ID NO:59 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:62 and 3 contained in the light chain variable region as shown in SEQ ID NO:61 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:64 and 3 contained in the light chain variable region as shown in SEQ ID NO:63 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 3 heavy chain CDRs contained in the heavy chain variable region as shown in SEQ ID NO:66 and 3 contained in the light chain variable region as shown in SEQ ID NO:53 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 3 heavy chain CDRs contained in the heavy chain variable region as shown in SEQ ID NO:68 and 3 contained in the light chain variable region as shown in SEQ ID NO:67 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:70 and 3 contained in the light chain variable region as shown in SEQ ID NO:69 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • HCDR1, HCDR2, HCDR3 3 heavy chain CDRs contained in the heavy chain variable region as shown in SEQ ID NO:62 and 3 contained in the light chain variable region as shown in SEQ ID NO:166 a light chain CDR (LCDR1, LCDR2, LCDR3);
  • the present invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, comprising:
  • sequences as shown in SEQ ID NO: 91, 99 and 100 or sequences containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to said sequences HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 111, 112 and 113, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
  • a sequence comprising a sequence as shown in SEQ ID NO: 121, 122 and 3, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 19, 136 and 21, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
  • a sequence comprising a sequence as shown in SEQ ID NO: 13, 14 and 15, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and the sequences shown in SEQ ID NO: 31, 138 and 33, or the LCDR1 of the sequence containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of no more than 3 amino acids relative to the sequence, LCDR2, LCDR3;
  • the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising a heavy chain variable region, wherein:
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 52 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 52;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 54, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 54 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 54;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 56, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 56 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 56;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 58 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 58;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 60, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 60 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 60;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 62 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 62;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 64, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 64 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 64;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 66, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 66 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 66;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 68, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 68 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 68;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 70 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 70;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 145, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 145 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 145;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 147, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 147 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 147;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 141, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 141 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 141;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 143, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 143 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 143;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 149, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 149 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 149;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 161, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 161 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 161;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 163, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 163 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 163;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 165, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 165 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 165;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 151, or with the amino acid sequence of SEQ ID NO: 151
  • the amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence, or consists of SEQ ID NO: 151;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 153, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 153 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 153;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 155, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 155 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 155;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 157, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 157 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 157;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 168, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 168 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 168; or
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 169, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 169 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO:169.
  • the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising a light chain variable region, wherein:
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 51, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 51 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 51;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 53, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 53 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 53;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 55, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 55 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 55;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 57, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 57 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 57;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 59, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 59 , 96%, 97%, 98% or 99% identical amino acid sequence column, or consists of SEQ ID NO: 59;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 61, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 61 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 61;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 63, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 63 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 63;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 67, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 67 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 67;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 69, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 69 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 69;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 144, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 144 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 144;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 146, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 146 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 146;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 140, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 140 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 140;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 142, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 142 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 142;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 148, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 148 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 148;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 160, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 160 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 160;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 162, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 162 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 162;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 164, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 164 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 164;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 150, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 150 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 150;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 152, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 152 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 152;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 154, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 154 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 154;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 156, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 156 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 156;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 158, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 158 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 158;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 159, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 159 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 159;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 166, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 166 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 166;
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 167, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 167 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO:167.
  • the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 52 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 52
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, or with SEQ ID NO: 52
  • the amino acid sequence of NO: 51 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 51 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 54, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 54 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 54, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53, or with SEQ ID NO: 54
  • the amino acid sequence of NO: 53 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 53 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 56, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 56 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 56, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55, or with SEQ ID NO: 56
  • the amino acid sequence of NO: 55 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 55 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 58 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 58, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57, or with SEQ ID NO: 58
  • the amino acid sequence of NO: 57 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 57 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 60, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 60 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 60, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, or with SEQ ID NO:
  • the amino acid sequence of NO: 59 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 59 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 62 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 62, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or with SEQ ID NO: 62
  • the amino acid sequence of NO: 61 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 61 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 64, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 64 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 64, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 63, or with SEQ ID NO: 64
  • the amino acid sequence of NO: 63 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 63 composition;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 66 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 66
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53, or with SEQ ID NO: 53
  • the amino acid sequence of NO: 53 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or an amino acid sequence with 99% identity, or consisting of SEQ ID NO: 53;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 68, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 68 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 68, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67, or with SEQ ID NO: 68
  • the amino acid sequence of NO: 67 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 67 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 70 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 70, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 69, or with SEQ ID NO: 70
  • the amino acid sequence of NO: 69 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 69 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 145, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 145 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 145, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 144, or with SEQ ID NO: 145
  • the amino acid sequence of NO: 144 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 144 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 147, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 147 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 147, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 146, or with SEQ ID
  • the amino acid sequence of NO: 146 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 146 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 141, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 141 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 141, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 140, or with SEQ ID NO: 140
  • the amino acid sequence of NO: 140 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 140 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 143, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 143 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 143, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 142, or with SEQ ID NO: 143
  • the amino acid sequence of NO: 142 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 142 composition;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 149, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 149 , an amino acid sequence of 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 149, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 148, Or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 148, or from SEQ ID NO: 148 ID NO: 148 composition;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 161, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 161 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 161, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 160, or with SEQ ID NO: 161
  • the amino acid sequence of NO: 160 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 160 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 163, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 163 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 163, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 162, or with SEQ ID NO: 162
  • the amino acid sequence of NO: 162 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 162 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 165, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 165 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 165, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 164, or with SEQ ID NO: 164
  • the amino acid sequence of NO: 164 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 164 composition;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 151, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 151 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 151
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 150, or with SEQ ID NO: 150
  • the amino acid sequence of NO: 150 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 150 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 153, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 153 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 153, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 152, or with SEQ ID NO: 152
  • the amino acid sequence of NO: 152 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 152 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 155, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 155 , 96%, 97%, 98% or 99% identical amino acid sequence, or composed of SEQ ID NO: 155, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 154, or with SEQ ID NO: 154
  • the amino acid sequence of NO: 154 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 154 composition;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 157, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 157 , 96%, 97%, 98% or 99% identical amino groups acid sequence, or consist of SEQ ID NO: 157
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 156, or has at least 90%, 91%, 92% of the amino acid sequence of SEQ ID NO: 156 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of SEQ ID NO: 156;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 52 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 52, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 158, or with SEQ ID NO: 158
  • the amino acid sequence of NO: 158 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 158 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 52 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 52, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 159, or with SEQ ID NO: 52
  • the amino acid sequence of NO: 159 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 159 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 62 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 62, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 166, or with SEQ ID NO: 62
  • the amino acid sequence of NO: 166 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 166 composition;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 168, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 168 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 168, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 167, or with SEQ ID NO: 168
  • the amino acid sequence of NO: 167 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 167 composed; or
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 169, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 169 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 169, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or with SEQ ID NO: 169
  • the amino acid sequence of NO: 61 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 61 composition.
  • the aforementioned antibodies or antigen-binding fragments thereof further comprise heavy and/or light chain constant region sequences derived from human antibody germline consensus sequences.
  • the light chain constant region is preferably a human ⁇ or ⁇ chain constant region.
  • the heavy chain constant region can be ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ chain, and in some embodiments, the heavy chain constant region is preferably derived from the constant region sequence of human IgG1, IgG2, IgG3, IgG4.
  • the light chain constant region comprises or consists of the sequence shown in SEQ ID NO: 49 or 50.
  • the heavy chain constant region comprises the Fc sequence shown in SEQ ID NO:47.
  • the heavy chain constant region comprises or consists of the sequence shown in SEQ ID NO: 48.
  • sequence variants of these constant region domains may also be used, for example comprising one or more amino acid modifications, wherein the amino acid positions are identified according to the EU index system of Kabat et al. (1991).
  • the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising a heavy chain and a light chain, wherein:
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 71, or an amino acid sequence with SEQ ID NO: 71 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 71;
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 74, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 74 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 74
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 73, or an amino acid sequence with SEQ ID NO: 73
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 76, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 76 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 76
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 75, or an amino acid sequence with SEQ ID NO: 75
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 78, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 78 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 78, the light chain comprising an amino acid sequence as shown in SEQ ID NO: 77, or an amino acid sequence with SEQ ID NO: 77 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 77;
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 80, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 80 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 80
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 79, or an amino acid sequence with SEQ ID NO: 79
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 82, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 82 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 82, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 81, or an amino acid sequence identical to SEQ ID NO: 81 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 84, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 84 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 84
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 83, or an amino acid sequence with SEQ ID NO: 83
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 86, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 86 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 86
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 73, or an amino acid sequence with SEQ ID NO: 73
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 88, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 88 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 88, the light chain comprising the amino acid sequence shown in SEQ ID NO: 87, or an amino acid sequence with SEQ ID NO: 87 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 87;
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 90, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 90 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 90
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 89, or an amino acid sequence with SEQ ID NO: 89
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 175, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 175 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 175, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 174, or an amino acid sequence with SEQ ID NO: 174 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 174;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 177, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 177 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 177, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 176, or an amino acid sequence with SEQ ID NO: 176 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 176;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 171, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 171 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 171, said light chain comprising the amino acid sequence shown in SEQ ID NO: 170, or with SEQ ID NO:
  • the amino acid sequence of 170 has an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, or consists of, SEQ ID NO: 170;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 173, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 173 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 173, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 172, or an amino acid sequence with SEQ ID NO: 172 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 172;
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 179, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 179 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 179, the light chain comprising the amino acid sequence shown in SEQ ID NO: 178, or an amino acid sequence with SEQ ID NO: 178 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 178;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 191, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 191 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 191,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 190, or an amino acid sequence with SEQ ID NO: 190
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 193, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 193 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 193,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 192, or an amino acid sequence with SEQ ID NO: 192
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 195, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 195 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 195
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 194, or an amino acid sequence with SEQ ID NO: 194
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 181, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 181 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 181,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 180, or an amino acid sequence with SEQ ID NO: 180
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 183, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 183 , 97%, 98% or 99% identical amino acid sequences, Or consist of SEQ ID NO: 183, said light chain comprises the amino acid sequence shown in SEQ ID NO: 182, or has at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 182 %, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consisting of SEQ ID NO: 182;
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 185, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 185 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 185, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 184, or an amino acid sequence with SEQ ID NO: 184 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 184;
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 187, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 187 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 187, the light chain comprising an amino acid sequence as shown in SEQ ID NO: 186, or an amino acid sequence with SEQ ID NO: 186 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 186;
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 188, or an amino acid sequence with SEQ ID NO: 188 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 188;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 189, or an amino acid sequence with SEQ ID NO: 189
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 82, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 82 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 82, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 196, or an amino acid sequence with SEQ ID NO: 196 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 196;
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 198, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 198 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 198, the light chain comprising the amino acid sequence shown in SEQ ID NO: 197, or an amino acid sequence with SEQ ID NO: 197 An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 197; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 199, or the amino acid sequence with SEQ ID NO: 199 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 199, the light
  • the chain comprises the amino acid sequence shown in SEQ ID NO: 81, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 81 An amino acid sequence that is % or 99% identical, or consists of SEQ ID NO:81.
  • the antibody is monoclonal.
  • the antibody is a full length antibody.
  • the anti-ROR1 antibody of the invention is a whole antibody, such as an IgG1, IgG2, IgG3, IgG4 antibody.
  • the anti-ROR1 antibody of the present invention only encompasses its antigen-binding portion, such as: Fab, Fab'-SH, Fv, scFv or (Fab') 2 fragments.
  • the present invention provides an antibody-conjugated drug targeting human ROR1, which has the following advantages:
  • the ADC of the present invention has high endocytosis efficiency
  • Treating, preventing, or improving a subject's disease related to ROR1 abnormal function or expression for example, cancer, such as blood cancer, solid tumor, or treating, preventing, or improving one or more symptoms of the disease;
  • the present application provides a conjugate comprising the anti-ROR1 antibody of the first aspect.
  • molecules that can be conjugated to an anti-ROR1 antibody are, for example, cytotoxic agents, immunomodulators, imaging agents, fluorescent proteins, molecular markers, therapeutic proteins, biopolymers, and oligonucleotides wait.
  • the present invention provides an antibody-drug conjugate (ADC), which comprises the anti-ROR1 antibody or its antigen-binding fragment described in the first aspect and at least one therapeutically active substance or pharmaceutically active ingredient, having an Ab -The structure of (L-D)n, wherein: Ab is the antibody or antigen-binding fragment thereof that binds to ROR1 according to the first aspect of the present invention; L is a linker; D is a therapeutically active substance or a pharmaceutically active ingredient, and n represents 1-20 Integers, such as 1, 2, 3, 4, 5....
  • ADC antibody-drug conjugate
  • the antibody drug conjugate comprises multiple D components, and the multiple D components may be a combination of different therapeutic active substances or pharmaceutical active ingredients, or a combination of the same therapeutic active substances or pharmaceutical active ingredients.
  • the antibody drug conjugate has a drug/antibody ratio (DAR) of 1-20, for example values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, DAR of 13, 14 or 15.
  • the DAR is an average DAR.
  • the average DAR is 1-15, such as 1-10, 2-8, 2-6 or 3-5. In a specific embodiment, the average DAR is 3.7.
  • said therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a phytotoxin, a Toxins, radioisotopes, maytansinoids, etc.
  • the cytotoxin is dolastatin (dolastatin) and its auristatin derivatives, such as 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy Base-1- ⁇ (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3- ⁇ [(1S)-2-phenyl-1-(1 ,3-Thiazol-2-yl)ethyl]amino ⁇ propyl]pyrrolidin-1-yl ⁇ -5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valline amide), 8261(2-methylalanyl-N-[(3R,4S,5S)-1- ⁇ (
  • the dolastatin and its auristatin derivatives are auristatin, dolastatin, MMAE, MMAF, auristatin F hydroxypropylamide or auristatin F phenylenediamine.
  • the cytotoxin is covalently linked to the anti-ROR1 antibody or antigen-binding fragment thereof in a non-site-specific manner or via a linker in a site-specific manner.
  • the linker is selected from maleimido-caproyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine- Valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propionyl, and maleimide caproyl (mc), maleimide propionyl (MP ), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2- Pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl Imino(4-iodo-acetyl
  • the antibody drug conjugate comprises the anti-ROR1 antibody or antigen-binding fragment thereof of the first aspect and tubulin inhibitor (MMAE).
  • MMAE is conjugated to the sulfhydryl group of the cysteine on the anti-ROR1 antibody through an MC-VC-PAB linker, having the structure of anti-ROR1 antibody-MC-VC-PAB-MMAE.
  • IgG1 antibodies have 16 pairs of cysteine residues, of which there are 12 intrachain and 4 interchain disulfide bonds. Interchain disulfide bonds are solvent-accessible, and can be reduced by reducing agents to form eight sulfhydryl groups, which then become coupling targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J .2015;17:339-51).
  • the antibody-drug conjugate comprises anti-ROR1 monoclonal antibody N99 and MC-VC-PAB-MMAE, or consists of them.
  • MMAE is conjugated to the sulfhydryl group of cysteine on N99 through a MC-VC-PAB linker.
  • the antibody drug conjugate comprises or consists of anti-ROR1 monoclonal antibody N147-135 and MC-VC-PAB-MMAE.
  • MMAE is conjugated to the sulfhydryl group of cysteine on N147-135 through the MC-VC-PAB linker.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (1) the antibody or antigen-binding fragment thereof of the first aspect or the antibody-drug conjugate of the second aspect, and (2) a pharmaceutically acceptable carrier.
  • the present invention provides an isolated polynucleotide molecule encoding any antibody or antigen-binding fragment thereof of the first aspect.
  • the present invention provides a vector comprising the nucleic acid molecule of the fourth aspect.
  • the vector is an expression vector.
  • the present invention provides a host cell comprising the vector of the fifth aspect or the nucleic acid molecule of the fourth invention.
  • the host cell is prokaryotic, such as E. coli.
  • the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells.
  • the present invention provides a method for preventing or treating a disease related to abnormal expression of ROR1 in a subject in need, comprising administering to the subject a prophylactically effective amount or a therapeutically effective amount of the antibody of the present invention or an antigen-binding fragment thereof, or a prophylactically or therapeutically effective amount of the antibody-conjugated drug of the present invention, or a prophylactically or therapeutically effective amount of the pharmaceutical composition of the present invention.
  • the disease associated with the abnormal expression of ROR1 is a cancer with high ROR1 expression, such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer , breast cancer, neuroblastoma, lung cancer, head and neck cancer, and melanoma.
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • mantle cell lymphoma such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer , breast cancer, neuroblastoma, lung cancer, head and neck cancer, and melanoma.
  • said lung cancer is non-small cell lung cancer
  • said breast cancer is triple negative breast cancer.
  • the ADC molecules or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents, for the uses described herein, e.g., for prophylaxis and and/or treat a related disease or condition mentioned herein.
  • therapies e.g., therapeutic modalities and/or other therapeutic agents, for the uses described herein, e.g., for prophylaxis and and/or treat a related disease or condition mentioned herein.
  • the present invention provides a method of killing ROR1-expressing cells or inhibiting the growth of ROR1-expressing cells, comprising contacting said cells with an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, or an effective amount The antibody-conjugated drug of the present invention, or an effective amount of the pharmaceutical composition of the present invention.
  • the present invention provides the use of an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of an antibody-coupled drug for preventing or treating cancer.
  • the present invention also provides the use of an antibody-coupled drug comprising an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of a drug for preventing or treating cancer.
  • Figure 1A-1F shows the binding activity of each antibody clone Fab obtained in the present application to huROR1-HEK293 cells determined based on the FACS method;
  • Figure 1A shows the binding activity of clone NW37 Fab and NW79 Fab to huROR1-HEK293 cells;
  • Figure 1B shows The binding activity of clones N137 Fab, N174 Fab and N147 Fab to huROR1-HEK293 cells;
  • Figure 1C shows the binding activity of clones N218 Fab and N204 Fab to huROR1-HEK293 cells;
  • Figure 1D shows the binding activity of clone N99 Fab to huROR1-HEK293 cells Binding activity;
  • Figure 1E shows the binding activity of clone N31 Fab Binding activity on huROR1-HEK293 cells;
  • Figure 1F shows the binding activity of clones N100 Fab and NW29 Fab
  • Figure 2A-2K shows the SEC-HPLC result of the antibody of the present application;
  • Figure 2A shows the SEC-HPLC result of N137;
  • Figure 2B shows the SEC-HPLC result of N147;
  • Figure 2C shows the SEC-HPLC result of N218;
  • Figure 2D shows the SEC-HPLC results of N99;
  • Figure 2F shows the SEC-HPLC results of NW37;
  • Figure 2G shows the SEC-HPLC results of N100;
  • Figure 2H shows the SEC-HPLC results of N174 HPLC results;
  • Figure 2I shows the SEC-HPLC results of N204;
  • Figure 2J shows the SEC-HPLC results of NW29;
  • Figure 2K shows the SEC-HPLC results of NW79.
  • Figure 3A-3F shows the binding activity of the antibody of the present application to the antigenic protein huROR1-His determined based on the ELISA method;
  • Figure 3A shows the binding activity of N100, N137 and N147 to the antigenic protein huROR1-His;
  • Figure 3B shows the binding activity of N204 and N218 The binding activity with the antigenic protein huROR1-His;
  • Figure 3C shows the binding activity of N31 with the antigenic protein huROR1-His;
  • Figure 3D shows the binding activity of NW29, N99 and NW37 with the antigenic protein huROR1-His;
  • Figure 3E shows the binding activity of N174 with the antigenic protein huROR1-His The binding activity of antigenic protein huROR1-His;
  • Fig. 3F shows the binding activity of NW79 and antigenic protein huROR1-His.
  • Figure 4A-4D shows the binding activity of the antibody of the present application and A549 cells measured based on the FACS method;
  • Figure 4A shows the binding activity of N100, N137, N174 and N147 and A549 cells;
  • Figure 4B shows the binding activity of N204, N218 and N31 and A549 Cell binding activity;
  • Figure 4C shows the binding activity of N99, NW29 and NW37 to A549 cells;
  • Figure 4D shows the binding activity of NW79 to A549 cells.
  • Figures 5A-5C show the binding activity of the antibody of the present application to huROR1-HEK293 cells determined based on the FACS method;
  • Figure 5A shows the binding activity of N100, N174, N204, N137, N147, N218 and N31 to huROR1-HEK293 cells;
  • 5B shows the binding activity of N99, NW29 and NW37 to huROR1-HEK293 cells;
  • Figure 5C shows the binding activity of NW79 to huROR1-HEK293 cells.
  • Figure 6A-6D shows the binding activity of the antibody of the present application and the antigenic protein MusROR1-His determined based on the ELISA method;
  • Figure 6A shows the binding activity of N100, N174, N137 and N147 and the antigenic protein MusROR1-His;
  • Figure 6B shows the binding activity of N204 , N31 and N218 and the binding activity of the antigenic protein MusROR1-His;
  • Figure 6C shows the binding activity of NW29, NW37 and N99 and the antigenic protein MusROR1-His;
  • Figure 6D shows the binding activity of NW79 and the antigenic protein MusROR1-His.
  • Figure 7A-7D shows the binding activity of the antibody of the present application to the antigenic protein huROR2-His determined based on the ELISA method;
  • Figure 7A shows that N100, N174, N137 and N147 are not combined with the antigenic protein huROR2-His;
  • Figure 7B shows that N204, The binding activity of N31 and N218 to the antigenic protein huROR2-His;
  • Figure 7C shows the binding activity of NW29, NW37 and N99 to the antigenic protein huROR2-His;
  • Figure 7D shows the binding activity of NW79 to the antigenic protein huROR2-His.
  • Figure 8A-8B shows the endocytic efficiency of the antibody of the present application on huROR1-HEK293 cells determined based on the FACS method;
  • Figure 8A shows the endocytic efficiency of N204, N147 and NW37 on huROR1-HEK293 cells;
  • Figure 8B shows the endocytic efficiency of N100 , N174, NW29, NW79, N137, N218, N31 and N99 endocytosis efficiencies on huROR1-HEK293 cells.
  • Figure 9A-9F shows the endocytic efficiency of the antibody of the present application on huROR1-HEK293 cells determined based on the Fab-Zap method;
  • Figure 9A shows the endocytic efficiency of N31 and N99 on huROR1-HEK293 cells;
  • Figure 9B shows N174 and N218 Endocytic efficiency on huROR1-HEK293 cells;
  • Figure 9C shows the endocytic efficiency of N147 on huROR1-HEK293 cells;
  • Figure 9D shows the endocytic efficiency of N100 and N137 on huROR1-HEK293 cells;
  • Figure 9E shows N204 and NW37 endocytosis efficiencies on huROR1-HEK293 cells;
  • Figure 9F shows the endocytosis efficiencies of NW29 and NW79 on huROR1-HEK293 cells.
  • Figures 10A-10D show the binding activity of antibodies to A549 cells after affinity maturation based on FACS method;
  • Figure 10A shows the binding activities of N31-6, N31-14, N31-18 and N31-56 to A549 cells;
  • Figure 10B Shows the binding activity of N37-37, N37-99, N37-113 and N37-136 to A549 cells;
  • Figure 10C shows the binding activity of N137-47, N137-53, N137-60 and N137-72 to A549 cells;
  • Figure 10D shows the binding activity of N147-2, N147-86, N148-93 and N147-135 to A549 tumor cells.
  • Figure 11A-11E shows the endocytic efficiency of antibodies on huROR1-HEK293 cells after affinity maturation based on the Fab-Zap method;
  • Figure 11A shows the endocytic efficiency of N31-6 on huROR1-HEK293 cells;
  • Figure 11B shows The endocytic efficiency of NW37-37 on huROR1-HEK293 cells;
  • Figure 11C shows the endocytic efficiency of NW137-72 on huROR1-HEK293 cells;
  • Figure 11D shows the endocytic efficiency of N147-135 on huROR1-HEK293 cells;
  • Figure 1 IE shows the endocytic efficiency of N147-2 on huROR1-HEK293 cells.
  • Figures 12A-12D show the results of the combination of the ADC of the present application with A549 cells and HT-29 cells;
  • Figure 12A shows the combination of N99-MMAE and A549 cells;
  • Figure 12B shows the combination of N147-135-MMAE and A549 cells;
  • Figure 12C shows the binding of N99-MMAE to HT-29 cells;
  • Figure 12D shows the binding of N147-135-MMAE to HT-29 cells.
  • Figure 13A-13B shows the killing effect of the ADC of the present application on two tumor cells A549 and HT-29 detected based on the MTS method;
  • Figure 13A shows the killing effect of N99-MMAE and N147-135-MMAE on A549 cells;
  • Figure 13B shows the killing effect of N99-MMAE and N147-135-MMAE on HT-29 cells.
  • Figure 14A-14F shows the killing effect of the ADC of the present application detected based on CCK8 method on three tumor cells Jeko-1, MDA-MB-468 and NCI-H1944;
  • Figure 14A shows N99-MMAE on Jeko-1 cells
  • Figure 14B shows the killing effect of N99-MMAE on MDA-MB-468 cells
  • Figure 14C shows the killing effect of N99-MMAE on NCI-H1944 cells
  • Figure 14D shows the killing effect of N147-135-MMAE on Killing effect on Jeko-1 cells
  • Figure 14E shows the killing effect of N147-135-MMAE on MDA-MB-468 cells
  • Figure 14F shows the killing effect of N147-135-MMAE on NCI-H1944 cells.
  • Figure 15 shows the antigen-dependent killing effect of the ADC of the present application on huROR1-HEK293 cells determined based on the CCK8 method.
  • Figure 16A-16D shows the endocytic efficiency of the ADC of the present application detected based on FACS method on two tumor cells A549 and HT-29;
  • Figure 16A shows the endocytic efficiency of N99-MMAE on A549 cells
  • Figure 16B shows The endocytic efficiency of N147-135-MMAE on A549 cells
  • Figure 16C shows the endocytic efficiency of N99-MMAE on HT-29 cells
  • Figure 16D shows the endocytic efficiency of N147-135-MMAE on HT-29 cells efficiency.
  • Figure 17A-17C has shown the antitumor effect of the ADC of the present application in mice;
  • Figure 17A has shown different experiments in the experimental cycle The change of tumor volume in the mice of different groups, and the arrow marks the time points of administration;
  • Figure 17B shows the body weight changes of mice in different experimental groups during the experimental period;
  • Figure 17C shows the weight of tumors in mice of different experimental groups after the experiment .
  • Figures 18A-18C show the anti-tumor effect of the ADC of the present application in mice of the A549 tumor model;
  • Figure 18A shows the tumor volume changes in different experimental groups of mice within the experimental period, and the arrows mark the time points of administration;
  • 18B shows the body weight changes of mice in different experimental groups during the experimental period;
  • FIG. 18C shows the tumor weight in mice of different experimental groups after the experiment.
  • Figures 19A-19B show the tumor-suppressing effect of the ADC of the present application in mice of the NCI-N87 tumor model;
  • Figure 19A shows the tumor volume changes in different experimental groups of mice within the experimental period, and the arrows mark the time points of administration ;
  • Figure 19B shows the body weight changes of mice in different experimental groups during the experimental period.
  • Figures 20A-20B show the tumor-suppressive effect of the ADC of the present application in mice of the MDA-MB-231 tumor model;
  • Figure 20A shows the tumor volume changes in mice of different experimental groups within the experimental period, and the arrows are marked as the administration Time points;
  • Figure 20B shows the body weight changes of mice in different experimental groups during the experimental period.
  • Figures 21A-21B show the tumor-suppressive effect of the ADC of the present application in mice of the MDA-MB-468 tumor model;
  • Figure 21A shows the changes in tumor volume in mice of different experimental groups within the experimental period, and the arrows are marked as administration Time points;
  • Figure 21B shows the body weight changes of mice in different experimental groups during the experimental period.
  • Figures 22A-22B show the tumor-suppressing effect of the ADC of the present application in mice of the Jeko-1 tumor model;
  • Figure 22A shows the tumor volume changes in different experimental groups of mice within the experimental period, and the arrows mark the time points of administration ;
  • Figure 22B shows the body weight changes of mice in different experimental groups during the experimental period.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • ROR1 refers to any recombinant or naturally occurring form of receptor tyrosine kinase-like orphan receptor 1 (ROR1), a variant or homolog thereof, which maintains, e.g., at least 50% of, the activity of ROR1 , 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity.
  • Said variant or homologue has at least 90%, 95%, 96%, 97%, 90%, 95%, 96%, 97% or more of the complete or partial sequence (e.g., 50, 100, 150 or 200 contiguous amino acid portions) compared to the naturally occurring ROR1 protein. 98%, 99% or 100% amino acid sequence identity.
  • the ROR1 protein comprises the amino acid sequence of Uniprot ID: Q01973.
  • ROR1 is highly expressed in the embryo, after which its expression level decreases significantly in the adult stage. However, ROR1 expression was found to be significantly elevated in a variety of hematologic cancers and solid tumors.
  • Blood cancers that highly express ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid blood cancers.
  • CLL B-cell chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • NHL non-Hodgkin's lymphoma
  • myeloid blood cancers in solid tumors, cancer types expressing ROR1 include breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer and other cancers.
  • antibody is used herein in the broadest sense and encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they It is sufficient that the desired antigen-binding activity is exhibited.
  • a whole antibody will usually comprise at least two full-length heavy chains and two full-length light chains, but in some cases may comprise fewer chains, for example antibodies naturally occurring in camelids may comprise only heavy chains.
  • anti-ROR1 antibody refers to an antibody molecule that specifically binds to ROR1 and is capable of inhibiting ROR1 activity.
  • the anti-ROR1 antibody is capable of inhibiting ROR1 activity relative to the absence of the ROR1 antibody, for example, by at least partially or completely blocking stimulation of ROR1, reducing, preventing or delaying activation of ROR1, or inactivating, desensitizing or downregulating signaling of ROR1 transduction, activity or amount.
  • the antibody can inhibit ROR1 activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control.
  • antibody fragment refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and is capable of binding an antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single chain antibodies (e.g. scFv); Specific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and multispecific antibodies formed from antibody fragments.
  • variable region refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen.
  • the variable domains of the heavy and light chains of native antibodies typically have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co. p. 91 (2007)).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point”).
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs in a variable domain are generally referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including, for example, based on the three-dimensional structure of the antibody and Chothia of the topology of CDR loops (Chothia et al.
  • CDRs can also be determined based on having the same AbM numbering position as a reference CDR sequence (eg, any of the exemplified CDRs of the invention).
  • the CDRs of an antibody of the invention are positioned according to the AbM numbering scheme.
  • residue positions in antibody variable regions and CDRs including heavy chain variable region residues, this refers to numbered positions according to the AbM numbering system.
  • an "affinity matured” antibody is one that has one or more alterations in one or more CDRs of the antibody that result in an improved affinity of the antibody for antigen compared to a parental antibody without these alterations.
  • Affinity matured antibodies can be generated by methods known in the art.
  • Human antibody or “fully human antibody” or “fully human antibody” are used interchangeably and refer to an antibody having an amino acid sequence corresponding to that of an antibody derived from a human Or human cells are produced or derived from non-human sources that utilize human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • bind or “specifically bind” means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antigen binding site to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD method or surface plasmon resonance (SPR).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • MSD method surface plasmon resonance
  • half effective concentration refers to the concentration of drug, antibody or poison that induces 50% of the response between baseline and maximum after a specified exposure time.
  • therapeutic agent encompasses any substance effective in the prevention or treatment of tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g. immunosuppressants).
  • antibody drug conjugate refers to an antibody or antibody fragment to which a therapeutically active substance or active pharmaceutical ingredient is covalently coupled such that the therapeutically active substance or active pharmaceutical ingredient is targeted to the antibody's binding target for expression its pharmacological function.
  • the therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin capable of killing cells targeted by the ADC, preferably cancer cells.
  • the covalent linking of therapeutically active substances, active pharmaceutical ingredients or cytotoxins can take place in a non-site-specific manner using linkers, or in a site-specific manner.
  • site-specific conjugation refers to an attachment method that specifically attaches a therapeutically active substance or an active pharmaceutical ingredient to a specific site of an antibody.
  • the coupling is accomplished via a linker.
  • cytotoxic agent may be used interchangeably with "cytotoxin", and in the present invention refers to a substance that inhibits or disrupts cell function and/or causes cell death or destruction.
  • cytotoxic agents may include, but are not limited to, bacterial toxins (such as diphtheria toxin), plant toxins (such as ricin), small molecule toxins, radioactive isotopes, etc., specifically, such as anthracycline ), camptothecin, combretastain, dolastatin and its auristatin derivatives, duocarmycin, enediyne, geldanamycin ( geldanamycin), indolino-benzodiazepine dimer, maytansine and its derivatives, puromycin, pyrrolobenzodiazepine dimer ( pyrrolobenzodiazepine dimer, taxane, vinca alkaloid, tubulysin, hemiasterlin, spliceostatin, pladienolide
  • Any antibody-coupled drug of the present invention can be prepared by coupling dolastatin and its auristatin derivatives with antibodies.
  • Dolastatin and its auristatin derivatives are important cytotoxins used in antibody-drug conjugates (ADC), which interfere with microtubule dynamics, cell division, etc., and have anti-tumor and anti-fungal activities.
  • ADC antibody-drug conjugates
  • the modification of its backbone has been widely reported in the literature, mainly the modification of the terminal subunits: P1 (N-terminal) and P5 (C-terminal), and the modification of the central peptide subunit also leads to the effective cytotoxicity of this class of substances in vitro active.
  • dolastatin (dolastatin) and its auristatin derivatives can be, for example, 0101(2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1- ⁇ ( 2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3- ⁇ [(1S)-2-phenyl-1-(1,3-thiazole-2 -yl)ethyl]amino ⁇ propyl]pyrrolidin-1-yl ⁇ -5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), 8261 (2 -Methylalanyl-N-[(3R,4S,5S)-1- ⁇ (2S)-2-[(1R,2R)-3- ⁇ [(1S)-1-carboxy-2-phenylethyl ]amino ⁇ -1-methoxy-2-methyl-3-oxypropyl]pyrrolidin-1
  • MMAE Monomethyl auristatin
  • demethyl-auristatin E is a well-known member of the auristatin compound family, and its structural formula is as follows:
  • MMAE which acts as a potent mitotic inhibitor by inhibiting tubulin polymerization, cannot be used as a drug because of its cytotoxicity, but is widely used to prepare antibody conjugates.
  • MMAE is coupled to a monoclonal antibody (MAB) via a linker to form MMAE-MAB.
  • MAB monoclonal antibody
  • MMAE-MAB targets tumor cells through antibodies, and the linker is cleaved after MMAE-MAB enters tumor cells, thereby releasing MMAE, making it exert cytotoxic effect and kill tumor cells.
  • linker and “linker” are used interchangeably herein to refer to a chemical moiety that covalently links an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC.
  • the linker may comprise amino acid residues linking the antigen to the payload.
  • the amino acid residues may form dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.
  • Amino acid residues include those that occur naturally, as well as non-naturally occurring analogs of amino acids, such as citrulline or ⁇ -amino acids, such as ⁇ -alanine, or ⁇ -amino acids such as 4-amino-butyric acid.
  • the linkers suitable for the present invention can be cathepsin-degradable linkers, such as valine-citrulline (val-cit) linkers, cBu-Cit linkers and CX linkers; non-cleavable linkers such as SMCC linkers or MD Linkers; acid-sensitive linkers, silicone grease-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, UV-sensitive linkers such as PC4AP.
  • valine-citrulline (val-cit) linkers cBu-Cit linkers and CX linkers
  • non-cleavable linkers such as SMCC linkers or MD Linkers
  • acid-sensitive linkers silicone grease-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphat
  • the linker of the present invention can also be a combination of one or more linkers, for example, a linker degraded by cathepsin can be combined with other types of linkers to form a new linker. Therefore, the "linker" of the present invention covers a single type of linker, or a combination of different types of linkers, as long as it can couple the antibody of the present invention to the drug.
  • linkers include, but are not limited to, maleimido-caproyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine- Valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propionyl, and maleimide caproyl (mc), maleimide propionyl (MP ), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2- Pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl Imino(4-iodo
  • load or “drug load” or “payload” refers to the average number of payloads per antibody within an ADC molecule (herein “payload” is used interchangeably with “therapeutically active substance or active pharmaceutical ingredient”) .
  • Drug loading may range from 1-20 therapeutically active substances or active pharmaceutical ingredients per antibody.
  • drug/antibody ratio or “DAR” refers to the ratio of therapeutically active substance or active pharmaceutical ingredient (D) conjugated to an antibody to the antibody.
  • ADCs described herein typically have a DAR of 1-20, and in certain embodiments 1-8, 2-8, 2-6, 2-5, 2-18, 4-16, 5-12, 6 DARs of -10, 3-8, 4-6, 6-10, and 2-4.
  • DAR values are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, usually expressed as a combination of the letter D and a number, where the number indicates the value of DAR,
  • D2 represents a drug/antibody ratio with a DAR value of 2.
  • the DAR is the average DAR, i.e., measured by detection methods (e.g., by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays, electrophoresis, and/or HPLC) in products coupled to those described herein.
  • the overall ratio of the small molecule drug fraction (D) to the Ab fraction of the Ab fraction. DARs may be limited by the number of attachment sites on the antibody.
  • the antibody may have only one or a few cysteine thiol groups or may have only one or a few sufficiently reactive thiol groups ( Alcohol groups can link linking units).
  • the conjugates of the invention have an average DAR value of 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4 -6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 ,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4,4.1,4.2,4.3,4.4,4.5,4.6 , 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,
  • treating refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
  • an effective amount refers to the amount or dosage of an antibody or conjugate or composition of the present invention, which produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as: the species of mammal; body weight, age and general health; the particular disease involved; the extent or severity of the disease; the individual The patient's response; the specific antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
  • therapeutically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition thereof are outweighed by the therapeutically beneficial effects.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%.
  • a measurable parameter e.g., tumor growth rate, tumor volume, etc.
  • Compounds can be evaluated for their ability to inhibit a measurable parameter (eg, cancer) in animal model systems predictive of efficacy in human tumors.
  • prophylactically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • composition refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.
  • the present invention provides a composition, preferably a pharmaceutical composition, comprising any of the anti-ROR1 antibodies described herein, or an ADC molecule thereof.
  • the composition further comprises pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • a composition eg, a pharmaceutical composition
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories.
  • liquid solutions eg, injectable solutions and infusible solutions
  • powders or suspensions e.g., liposomes and suppositories.
  • liposomes e.g., liposomes and suppositories.
  • compositions of the invention are according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or focal Internal route; by sustained release system or by implanted device.
  • the compositions can be administered by bolus injection or by continuous infusion or by implanted device.
  • the subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, an individual suffering from or at risk of having a disease described herein).
  • the subject has or is at risk of having a disease described herein (eg, cancer).
  • the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy.
  • the subject has previously received or is currently receiving immunotherapy.
  • a medicament comprising an antibody described herein can be prepared by mixing an anti-ROR1 antibody of the invention, or an ADC molecule thereof, of the desired purity with one or more optional pharmaceutical excipients, preferably in a lyophilized formulation or in the form of an aqueous solution.
  • compositions or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immune checkpoint inhibitors or agonists), among others.
  • the active ingredients are suitably present in combination in amounts effective for the intended use.
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles such as films or microcapsules.
  • the present invention provides a method for preparing an anti-ROR1 antibody, wherein said method comprises culturing a nucleic acid comprising or comprising said nucleic acid encoding an anti-ROR1 antibody under conditions suitable for expressing said nucleic acid encoding said anti-ROR1 antibody. host cells for the expression vector, and optionally isolate the anti-ROR1 antibody. In a certain embodiment, the method further comprises recovering the anti-ROR1 antibody from the host cell (or host cell culture medium).
  • the nucleic acid encoding the anti-ROR1 antibody of the present invention is first isolated and inserted into a vector for further cloning and/or expression in host cells.
  • Such nucleic acids are readily isolated and sequenced using conventional procedures, for example by using oligonucleotide probes that are capable of specifically binding to nucleic acid encoding an anti-ROR1 antibody of the invention.
  • Anti-ROR1 antibodies of the invention prepared as described herein can be tested by known prior art techniques such as high performance liquid chromatography, ion exchange Purification by exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc.
  • the actual conditions used to purify a particular protein will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art.
  • the purity of the anti-ROR1 antibodies of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the conjugation reaction can be carried out in the antibody through the amino group of the lysine side chain and the amino group at the N-terminus of the antibody, the carboxyl group of aspartic acid, glutamic acid and C-terminus, or the activated cysteine sulfhydryl group. .
  • Embodiment 1 Raw material preparation and identification
  • the eukaryotic expression vector pcDNA3.4 (Invitrogen) was constructed by homologous recombination, and the constructed recombinant protein expression vectors were transformed into Escherichia coli DH5 ⁇ , cultured overnight at 37°C, and then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) for plasmid extraction to obtain the desired expression vector for expressing cetuzumab, the clone number is 99961.1, and the expressed cetuzumab will be referred to as 99961.1 for short below.
  • OEGA endotoxin-free plasmid extraction kit
  • the expression vector was transfected into 293 cells by ExpiFectamine TM CHO transfection kit (Thermo Fisher, A29129) to express cetuzumab. After 7 days of transfection, the cell culture supernatant was collected and centrifuged at 15000 g for 10 min. After the liquid was filtered through a 0.22 ⁇ m filter membrane, the antibody in the supernatant was affinity purified using a Protein A/G affinity chromatography column. The target antibody was eluted with 100 mM glycine salt (pH 3.0), and the eluted antibody was exchanged into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).
  • ROR1 control antibody Use the purchased huROR1-His antigen protein (Kaijia Biology, ROR-HM401) to detect the activity of the prepared positive control antibody 99961.1 (IgG1), the specific method is as follows: coat huROR1-His on a 96-well ELISA plate (2 ⁇ g/mL, 30 ⁇ L/well), coated overnight at 4°C; after washing the plate 3 times, block with 5% skimmed milk in PBS for 1 hour at room temperature; after washing the plate 3 times, add the control antibody 99961.1 serially diluted with PBS Incubate at room temperature for 1 hour; after washing the plate, add secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted in PBS (1:6000) and incubate at room temperature for 1 hour, wash the plate 6 times and add TMB Color development was performed for 5-20 minutes.
  • Antigen protein preparation through genetic manipulation at the coding gene level, human ROR1 protein huROR1 ECD AA30-406 (Uniprot ID: Q01973), mouse ROR1 protein MusROR1 ECD AA30-406 (Uniprot ID: Q9Z139), human ROR2 protein huROR2 A His tag was added to the C-terminus of the sequence of ECD AA34-403 (Uniprot ID: Q01974).
  • the obtained nucleic acid sequences were respectively constructed into pcDNA3.4 vectors, and then transformed into Escherichia coli DH5 ⁇ , cultured overnight at 37°C, and then the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01).
  • OEGA endotoxin-free plasmid extraction kit
  • the resulting plasmid was transiently transfected into HEK293 cells ( CRL-1573 TM ), after 7 days of expression, the cell culture supernatant was collected, and the protein containing the His tag was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then imidazole Gradient elution of the target protein.
  • the eluted proteins were respectively exchanged into PBS buffer solution through ultrafiltration concentrator tubes (Millipore, UFC901096), and finally the antigenic proteins (huROR1-His, MusROR1-His, huROR2-His) were obtained.
  • Antigen identification the antibody 99961.1 (IgG1) qualified for quality inspection obtained in Example 1.1 was used to detect the prepared antigen (huROR1-His).
  • the specific method is as follows: respectively coat the Elisa plate with 2 ⁇ g/mL huROR1-His at 4°C overnight, and use the purchased antigenic protein huROR1-His (Kaijia Biological ROR-HM201) as a positive control; after washing the plate 3 times, use PBS prepared Block with 5% skimmed milk at room temperature for 1 hour; after washing the plate 3 times, add antibody 99961.1 serially diluted with PBS and incubate at room temperature for 1 hour; after washing the plate, add secondary antibody Anti-human-IgG-Kappa diluted in PBS (1:6000) +Lambda-HRP (Millipore, AP502P+AP506P) was incubated at room temperature for 1 hour, the plate was washed 6 times, and TMB was added for 5-20 minutes
  • the data was read by microplate reader OD450, and the processed data was plotted with Graphpad prism.
  • the results show that the antibody 99961.1 can bind the antigen huROR1-His constructed and expressed by the inventors themselves with an affinity comparable to that of the purchased ROR1 antigen protein.
  • HEK293 cell line overexpressing human ROR1 (hereinafter referred to as huROR1-HEK293): The coding nucleic acid sequence of full-length human ROR1 (Uniprot ID: Q01973) was constructed on pLVX-puro plasmid (Clontech, Cat#632164). Then, the resulting plasmid was electrotransformed into HEK293 cells ( CRL-1573 TM ).
  • the obtained cells were respectively transferred to DMEM medium (Gibco, 11995065) containing 10% by volume of FBS (Gibco, 15140-141) without antibiotics, and then the cells were transferred into a 10 ⁇ 10 cm Culture the cells in a culture dish for 48 hours, then divide the cells into 96-well cell culture plates at an average density of 10 4 cells/well, add puromycin at a final concentration of 2 ⁇ g/mL as a selection pressure, and pick for about 2 weeks. The cloned cell lines were taken for identification.
  • Flow cytometric identification of huROR1-HEK293 cells Digest and plate the above cell lines in the logarithmic growth phase into 96-well plates, wash with FACS buffer (1 ⁇ PBS buffer containing 2% FBS by volume) , adding the primary antibody (99961.1) diluted in PBS gradients and incubating for 30min at 4°C; after washing, adding the prepared fluorescent secondary antibody anti human IgG Fc (abcam, 98596), and incubating for 30min at 4°C; finally by flow cytometry (Beckman , CytoFLEXAOO-1-1102) for detection. Test results It shows that the huROR1-HEK293 cell line with high surface expression of human ROR1 has been obtained.
  • an antibody gene phage display library was constructed, and the antigenic protein huROR1-His prepared in Example 1.2 and the huROR1-overexpressing cell huROR1-HEK293 prepared in Example 2 were used as screening antigens to screen the library to obtain Multiple antibody molecules with specific binding to human ROR1.
  • Ficoll-Paque density gradient separation medium (purchased from GE, catalog number: 17144003S) was used to separate peripheral blood mononuclear cells (PBMC) from normal human blood, and total RNA was extracted from the isolated PBMC cells by conventional methods. The extracted total RNA was reverse-transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa Company, catalog number: 6210A). Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers were designed at the front end of the V region and the rear end of the first constant region of the heavy chain and light chain respectively, and the heavy chain variable region gene fragment of the antibody was obtained after PCR and light chain variable region gene fragments.
  • PBMC peripheral blood mononuclear cells
  • the fragments containing the variable region of the light chain and the heavy chain of the antibody were amplified by the fusion PCR method, and the PCR product and the carrier for phage display were digested, recovered and connected, and the connected product was recovered by a recovery kit (Omega, catalog number: D6492-02) recovery (Li Xiaolin, Construction and preliminary screening of a large-capacity non-immune human Fab phage antibody library, "China Peking Union Medical College"master's degree thesis, June 2007).
  • Magnetic bead screening is based on biotin-labeling the antigenic protein huROR1-His, and then binding to magnetic beads coupled with streptavidin, incubating and washing the antigen-binding magnetic beads and antibody gene phage display library and elution panning process. Usually undergoes 3-4 rounds of panning, whereby specific monoclonal antibodies against antigens can be enriched in large quantities.
  • biotin-labeled huROR1-His was used for phage display library screening, and after three rounds of panning, the monoclonal antibody Fab against human ROR1 was screened.
  • Example 2.4.1 in patent CN112250763B .
  • the principle of immunotube screening is to coat the antigenic protein huROR1-His on the surface of the immunotube with high adsorption force, add the phage display antibody library to the immunotube and incubate with the antigenic protein adsorbed on the surface of the immunotube, wash and Eluted pan During the selection process, after 2-4 rounds of panning, the specific monoclonal antibody Fab targeting the antigen is finally enriched. In this example, the monoclonal antibody Fab against human ROR1 was enriched after three rounds of panning. For the specific method, refer to Example 2.4.2 in patent CN112250763B.
  • Antigen protein huROR1-His was used for the primary screening of monoclonal ELISA, and the antibody Fab combined with huROR1-His obtained from the primary screening was prepared into Fab lysate, and then the overexpressed cell huROR1-HEK293 prepared in Example 2.1 was used for flow cytometric analysis ( FACS) for detection and review, a total of 11 antibody Fab molecules specifically binding to human ROR1 were screened out, and the obtained 11 antibody Fabs were named according to the corresponding clone numbers (N99, N218, N31, N137, N147, N100, N204, N174, NW29, NW79 and NW37), the specific FACS results are shown in Figures 1A-1F.
  • the amino acid sequence of the CDR region of the obtained antibody Fab is shown in Table 1, and the CDR sequence was determined by using AbM to define the CDR.
  • the VH coding sequence in the Fab sequence of the monoclonal N99, N218, N31, N137, N147, N100, N204, N174, NW29, NW79 and NW37 obtained by screening was combined with the heavy chain constant region of human IgG1 (SEQ ID NO: 48)
  • the coding sequence of the human antibody is connected to obtain the heavy chain coding sequence of the fully human antibody, and the VL coding sequence in the Fab sequence is combined with the Kappa type (SEQ ID NO: 49) or Lambda type (SEQ ID NO: 49) of the human light chain constant region (CL). 50) to obtain the light chain coding sequence of a fully human antibody.
  • the coding sequences of antibody heavy chain and light chain were respectively inserted into eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5 ⁇ , and cultured overnight at 37°C.
  • the endotoxin-free plasmid extraction kit (OMEGA, D6950-01) was used for plasmid extraction to obtain endotoxin-free antibody plasmids for eukaryotic expression.
  • the full-length sequence of the antibody obtained above was expressed by the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, confirm that the CHO cell density was about 7 ⁇ 10 6 to 1 ⁇ 10 7 viable cells/mL, When the cell survival rate is >98%, adjust the cells to a final concentration of 6 ⁇ 10 6 cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C.
  • OptiPRO TM SFM Dilute the target plasmid with OptiPRO TM SFM pre-cooled at 4°C (add 1 ⁇ g plasmid to 1 mL of the medium), and dilute ExpiFectamine TM CHO reagent with OptiPRO TM SFM at the same time, then mix the two in equal volumes and gently blow and mix to prepare Form ExpiFectamine TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension and shake gently at the same time, and finally place in a cell culture shaker at 37°C, 8% CO 2 conditions under cultivation.
  • the relative molecular weight and purity of the candidate antibodies were detected by SDS-PAGE and SEC-HPLC.
  • Preparation of reducing solution Add 2 ⁇ g of each obtained antibody and quality control IPI to 5 ⁇ SDS sample buffer and 5mM DTT, heat in a dry bath at 100°C for 10 minutes, cool to room temperature, and centrifuge at 12,000 rpm for 5 minutes to take the supernatant. The supernatant was added to Bis-tris4-15% gradient gel (GentScript) for gel electrophoresis, and the protein bands were visualized by Coomassie brilliant blue staining.
  • GenetScript Bis-tris4-15% gradient gel
  • EPSON V550 color scanner was used to scan the protein gel with chromogenic protein bands (the decolorization solution was decolorized until the gel background was transparent), and the purity of reduced and non-reduced bands was calculated by ImageJ according to the peak area normalization method.
  • Material preparation 1. Mobile phase: 150mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: Dilute each antibody and quality control IPI to 0.5mg/mL with mobile phase solution.
  • Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph the chromatographic column is XBridge BEH (SEC 3.5 ⁇ m, 7.8mm I.D. ⁇ 30cm), the Waters flow rate is set to 0.8mL/min, the injection volume is 20 ⁇ L, and the wavelength of the VWD detector is 280nm and 214nm. Inject blank solution, IPI quality control solution and antibody sample solution sequentially. Calculate the percentage of high molecular polymers, antibody monomers and low molecular substances in the sample according to the area normalization method.
  • the binding of the expressed antibodies (N99, N218, N147, N137, N31, NW37, N100, N204, N174, NW29 and NW79) to the human ROR1 antigen protein huROR1-His was detected based on the ELISA method.
  • the binding ability of expressed antibodies (N99, N218, N147, N137, N31, NW37, N100, N204, N174, NW29 and NW79) to human ROR1 overexpression cells huROR1-HEK293 and A549 cells was detected based on FACS method.
  • A549 cells are a human non-small cell lung cancer cell line that overexpresses huROR1.
  • a 96-well ELISA plate (30 ⁇ L/well) was coated with 2 ⁇ g/mL huROR1-His overnight at 4°C. The next day, the plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 h. After the plate was washed 3 times with PBST, a gradient dilution (3.00000, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00046, 0.00005 ⁇ g/mL ) and the positive control antibody 99961.1 and incubated for 1 h. After washing with PBST for 3 times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h.
  • Goat-anti-human Fc-HRP abcam, ab97225
  • human ROR1 overexpression cells huROR1-HEK293 and A549 cells were used to evaluate the binding activity of antibodies.
  • the specific method is as follows: Prepare huROR1-HEK293 cells or A549 cells in the logarithmic growth phase into a single cell suspension, adjust the density to 1 ⁇ 10 6 cells/mL, add 100 ⁇ L per well into a 96-well round bottom plate, and store at 4°C. , 300g centrifugation and remove the supernatant.
  • Each antibody and the positive control antibody 99961.1 were added to the corresponding wells with serial dilutions (3.000000, 0.300000, 0.100000, 0.033333, 0.011111, 0.003704, 0.001235, 0.000123 ⁇ g/mL), mixed well and incubated at 4°C for 30 min.
  • a 96-well ELISA plate (30 ⁇ L/well) was coated with 2 ⁇ g/mL MusROR1-His, overnight at 4°C. The next day, the plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 hours. After washing the plate 3 times with PBST, a gradient dilution (1.00000, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, 0.00015, 0.00002 ⁇ g/mL ) and the positive control antibody 99961.1 and incubated for 1 h. After washing with PBST for 3 times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h.
  • Goat-anti-human Fc-HRP abcam, ab97225
  • a 96-well ELISA plate (30 ⁇ L/well) was coated with 2 ⁇ g/mL huROR2-His overnight at 4°C. On the next day, the plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 hours. After the plate was washed 3 times with PBST, the candidate antibody and positive control antibody 99961.1 in gradient dilution were added and incubated for 1 hour. After washing with PBST for 3 times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h.
  • the endocytosis efficiency of the antibody obtained in this application was identified by using two detection methods of FACS and Fab-Zap respectively.
  • the FACS method for endocytosis detection is to use supersaturated antibodies to bind cells to detect the endocytosis efficiency of antibodies in a short period of time;
  • the Fab-Zap method uses different concentrations of antibodies combined with saporin toxin to bind cells, and the toxin is brought in through endocytosis To kill target cells, this method reflects the effect of long-term accumulation of endocytic efficiency.
  • Antibody dilution Dilute the test antibody with DMEM complete medium to a final concentration of 10.0000 ⁇ g/mL.
  • Cell treatment Digest huROR1-HEK293 cells and add DMEM complete medium. After mixing the cells thoroughly, count the cells and determine their viability. Add 106 cells to 1.5mL centrifuge tube, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend with 1mL pre-cooled DMEM medium, centrifuge at 300g for 5 minutes, discard the supernatant.
  • Primary antibody incubation Take 1000 ⁇ L of the pre-cooled diluted antibody above and add it to a centrifuge tube with existing cells to prepare antibody cell suspension, add the antibody cell suspension to a 96-well plate, and incubate at 4°C.
  • Extracellular secondary antibody incubation Quickly transfer the suspension in the 96-well plate to a second 96-well plate. Add 180 ⁇ L pre-cooled FACS buffer to each well of the second 96-well plate and wash 2 times. After that, 100 ⁇ L of diluted secondary antibody FITC-labeled rabit-anit-huFc or RPE-labeled rabbit-anit-huFc was added to each well (the secondary antibody was diluted 1:150 times with FACS buffer); incubate at 4°C for 30 min.
  • Intracellular secondary antibody incubation After washing the 96-well plate, add 180 ⁇ L of preheated Permeabilization Buffer (Invitrogen TM eBioscience TM , 00-8333-56) to each well to wash twice. Add 100 ⁇ L of diluted secondary antibody RPE-labeled rabbit-anit-huFc (diluted 1:150 times with Permeabilization buffer) to each well. Incubate at room temperature for 60min.
  • Permeabilization Buffer Invitrogen TM eBioscience TM , 00-8333-56
  • Detection of fluorescence After washing the 96-well plate, resuspend it with 100 ⁇ L FACS buffer, and detect it by flow cytometry.
  • the first set was FITC+PE, that is, the FITC secondary antibody was used to identify the extracellular primary antibody first, and the PE secondary antibody was used to identify the intracellular primary antibody after membrane rupture.
  • PE is the intracellular signal
  • FITC is the extracellular signal
  • the second group is set to PE+PE, that is, the PE secondary antibody is used to recognize the extracellular primary antibody first, and the PE secondary antibody is used to recognize the intracellular primary antibody after membrane rupture.
  • a set of PEs is the sum of intracellular and extracellular signals.
  • these two groups of samples were detected by FITC and PE channels, and the calculated value of endocytosis rate was the detection value of PE channel.
  • Fab-ZAP is a Fab fragment linked to saporin (saponin), a ribosome inhibitor that can inhibit protein synthesis and cause cell death.
  • the Fab-ZAP used in this experiment is a Fab fragment that can bind to human antibodies.
  • the human antibodies are loaded with toxins. When the human antibodies are endocytosed, the toxins are The antibody enters into the cell to cause cell death, and then the cell activity is detected by MTS (Promega, G3580) to detect whether the antibody is endocytized.
  • Fab-ZAP was diluted to 0.4nM with DMEM complete medium, and then diluted with 0.4nM Fab-ZAP (0.400000, 0.080000, 0.026667, 0.008889, 0.002963, 0.000988, 0.000329, 0.000066 ⁇ g/mL)
  • Antibody diluents were prepared from each antibody and positive control antibody obtained through application.
  • huROR1-HEK293 cells in the logarithmic growth phase as a single cell suspension, adjust the density to 6 ⁇ 106 cells/mL, inoculate them into 96-well plates at 50 ⁇ L per well, and then take the aforementioned antibody dilution solution at 50 ⁇ L per well
  • the affinity of the antibody obtained in this application and the positive control antibody 99961.1 to the antigenic protein huROR1-His was detected based on Biacore equipment.
  • Protein coupling Dilute the huROR1-His protein produced in Example 1.2 to 5.6 ⁇ g/mL with pH 5.0 NaAc buffer, set the flow rate to 10 ⁇ L/min, 1-ethyl-(3-dimethylaminopropyl ) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) mixed solution activation time of the chip is the default value of 420s, using the coupling mode of the preset coupling amount to immobilize the antigen protein huROR1-His to the level of about 75RU, and ethanolamine is used to block the activation group that is not bound to the test product.
  • EDC 1-ethyl-(3-dimethylaminopropyl ) carbodiimide
  • NHS N-hydroxysuccinimide
  • Sample test conditions use PBS buffer (pH7.4) containing 0.05% Tween-20 as the running buffer, use the running buffer as the control test sample, set a series of antibody concentrations (4nM, 20nM), and set the flow rate during sample analysis. 30 ⁇ L/min, the binding time is 120s, and the dissociation time is 360s. After dissociation, 10mM Gly-HCl (pH2.0) was used to regenerate for 20s to completely remove the antibody bound to the ligand.
  • Parameter fitting The experiment was run in multiple cycles, and the response signal took the analysis time as the abscissa, and the response value as the ordinate. After double-reference subtraction, the obtained data were fitted by BIAcore T200 analysis software. The fitting model adopted was a 1:1 Langmuir binding model, and affinity indicators such as binding and dissociation constants were determined.
  • the affinity of N147 is 2 orders of magnitude better than that of the control antibody 99961.1.
  • the KD of N147 reaches 6.62E-11, while the KD of the control antibody 99961.1 is 1.98E-9.
  • the affinity of the other antibodies is similar to that of the control antibody 99961.1 The affinity is quite.
  • the affinity of the antibody obtained in this application and the positive control antibody 99961.1 to the antigenic protein huROR1-His was detected based on Gator equipment.
  • the specific test method is as follows: first, PBS (10mM pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from Yuanpei) was configured as Q buffer buffer , use the configured Q buffer buffer to dilute the storage solution of the antibody to be tested to a working solution with a final concentration of 30nM; configure the antigenic protein huROR1-His storage solution with Q buffer to make multiple dilutions (480, 240, 120, 60, 30, 15, 7.5nM) working solution, and then use the Gator instrument and its supporting software to select the Advanced Kinetics experimental mode for detection and analysis.
  • the test results are shown in Table 5.
  • the affinity of N100, NW29 and NW79 is higher than that of the control antibody 99961.1, among which the KD of N100 reaches 8.85E-9, the KD of NW29 reaches 6.60E-9, and the KD of NW79 reaches 6.94E-8 , while the KD of the control antibody 99961.1 was 1.35E-8, and the affinities of the other antibodies were comparable to those of the control antibody 99961.1.
  • the specific test method is as follows: first, PBS (10mM pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from Yuanpei) was configured as Q buffer buffer , Dilute the storage solution of the antibody to be tested to a working solution with a final concentration of 100nM with the configured Q buffer buffer; configure the storage solution of the antigenic protein huROR1-His into a 50nM working solution with Q buffer, and then use the Gator instrument and its Supporting software, based on the Tandem setting of the Epitope Binning experiment mode for detection and analysis. The result of each antibody epitope grouping thus obtained is shown in Table 6, wherein each group of antibodies has the same epitope.
  • affinity maturation was performed on antibodies N147, N31, N137 and NW37 to improve antibody affinity and other biological activities.
  • the affinity maturation transformation is based on the M13 phage display technology, using codon-based primers (during the primer synthesis process, a single codon consists of NNK) to introduce mutations in the CDR region, and construct 4 phage display libraries: library 1 and library 2 are single-point combinatorial mutations (that is, each CDR contains only one mutation site), library 1 is CDRL1+CDRL3+CDRH3 combined mutation, library 2 is CDRL2+CDRH1+CDRH2 combined mutation; library 3 and library 4 are double point saturation mutations, library 3 is Double point saturation mutation of CDRL3, library 4 is double point saturation mutation of CDRH3.
  • Specific library construction method first, synthesize primers containing point mutations (Jinweizhi Biotechnology Co., Ltd.); second, use the coding sequences of antibodies to be transformed (also called parental antibodies) N147, N31, N137, and NW37 as PCR amplification templates , to amplify the CDR regions
  • the fragments containing different CDR mutations were combined by bridging PCR, and then the point mutant antibody was connected to the phage display vector by double enzyme digestion (HindIII and NotI) and double sticky end ligation, and finally by The antibody sequence with the mutation site was transformed into Escherichia coli SS320 by electroporation.
  • N147 affinity maturation N147-135 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 53), N147-2 (the amino acid sequence of the heavy chain variable region The sequence is shown in SEQ ID NO:161, the light chain variable region amino acid sequence is shown in SEQ ID NO:160), N147-86 (the heavy chain variable region amino acid sequence is shown in SEQ ID NO:163, the light chain variable region amino acid sequence The sequence is shown in SEQ ID NO:162) and N147-93 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:165, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:164); N31 was obtained after affinity maturation N31-6 (the amino acids of the heavy chain variable region are shown in SEQ ID NO:64, and the amino acids of the light chain variable
  • the corresponding antibody protein obtained was expressed and purified by the methods disclosed in the foregoing examples.
  • the molecular weight and purity of the antibody protein were identified by SDS PAGE.
  • the test results showed that the non-reducing gum band of the antibody after affinity maturation was around 150kD, and the bands of the reducing gum were around 55kD and 25kD, which were in line with the expected size. All affinity matured antibodies were >95% pure by reducing gel assay.
  • the affinity ability of the antibody after affinity maturation to A549 cells was detected based on the FACS method.
  • the test results are shown in Figures 10A-10D, and the results show that the affinity of the antibody to A549 cells has been improved after affinity maturation, among which N31-6, N31-14, N31-18, N31-56, NW37-37, NW37
  • the affinity of -99, NW37-113, NW37-136, N137-47, N137-53, N137-60 and N137-72 were significantly better than their corresponding parent antibody molecules N31, NW37 and N137; N147-135, N147-2 , N147-86 and N147-93 had better affinities than their corresponding parent molecule N147 and better than the control antibody 99961.1.
  • the Fab-ZAP endocytosis detection was performed on the affinity-matured antibodies N147-135, N31-6, N137-72, N147-2 and NW37-37 in Example 11.
  • the specific method refer to Example 8.2.
  • the results As shown in Figures 11A-11E, within the antibody concentration range defined in this example, the endocytosis efficiency of the antibody after affinity maturation is comparable to that of the control antibody.
  • Embodiment 16 ADC preparation
  • the antibodies N99 and N147-135 and the control antibody 99961.1 were coupled with the toxin MMAE (a tubulin inhibitor with anticancer activity) to construct an antibody-drug conjugate, and MMAE was linked with the linker MC-VC-PAB to form MC-VC-PAB-MMAE is covalently linked to the sulfhydryl group on the cysteine of the antibody by means of a linker, so that the antibody is conjugated with MMAE to obtain an antibody-coupled drug.
  • IgG1 antibodies have 16 pairs of cysteine residues, of which there are 12 intrachain and 4 interchain disulfide bonds.
  • Interchain disulfide bonds are solvent-accessible, and can be reduced by reducing agents to form eight sulfhydryl groups, which then become coupling targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J .2015;17:339-51).
  • the specific preparation method is as follows:
  • the addition amount of each component in the reduction reaction system is shown in Table 9, so that the final concentration of the antibody in the reduction reaction system is 5 mg/mL (N99 and 999611) or 4 mg/mL (N147-135), the final concentration of DTPA is 1 mM, TCEP and antibody The molar ratio is 2.2 (N99 and 99961.1) or 2.7 (N147-135).
  • the reduction reaction system was placed in a constant temperature mixer at 25° C., 400 rpm, and the reduction reaction was performed for 2 hours.
  • the ADC-containing solution was centrifuged and filtered to obtain an ADC sample, which was transferred to a 15 mL 30KD ultrafiltration centrifuge tube. Add dialysate to 15 mL, centrifuge at 4500 rpm for 20 min, concentrate to 2-3 mL, add dialysate to 15 mL, and repeat dialysis 8-10 times. SEC-HPLC detection, HIC-HPLC detection, concentration detection, free drug detection, etc. were performed on ADC samples after dialysis. See Table 11 for test results. The test results showed that the ADC with a purity of more than 99% was obtained.
  • the binding ability of the prepared antibody-drug conjugates N99-MMAE and N147-135-MMAE to human ROR1 on tumor cells A549 and HT-29 (human colon cancer cells) was detected based on the FACS method.
  • the specific method is as follows: prepare A549 cells or HT-29 cells in the logarithmic growth phase into a single cell suspension, adjust the density to 1 ⁇ 10 6 cells/mL, add 100 ⁇ L per well into a 96-well round bottom plate, and store at 4°C. , 300g centrifugation and remove the supernatant. Add serially diluted (1.0000, 0.3333, 0.1111, 0.0370, 0.0123, 0.0041, 0.0014, 0.0001 ⁇ g/mL) N99, N99-MMAE, N147-135, N147-135-MMAE, 99961.1, 99961.1-MMA to corresponding wells E and For negative control, mix well and incubate at 4°C for 30min.
  • FIG. 12 A549 cell results show (Figure 12A and Figure 12B), the binding activity of N99-MMAE and control 99961.1-MMA to A549 cells is equivalent to that of the corresponding parental antibody, but N99 and N99-MMAE have the same binding activity as A549 cells
  • the binding activity of N147-135-MMAE, N147-135 and A549 cells was comparable to that of the control antibody 99961.1
  • the results of HT-29 cells showed ( Figure 12C and 12D)
  • N99 -The binding activity of MMAE and control 99961.1-MMA to HT-29 cells is equivalent to that of the corresponding parental antibody
  • the binding activity of N99-MMAE to HT-29 cells is equivalent to that of the control 99961.1-MMAE
  • N147-135-MMAE, N147-135 The binding activity to HT-29 cells was comparable to that of the control antibody 99961.1.
  • A549 and HT-29 cells were used to detect the killing effect of candidate ADC and control ADC on tumor cells.
  • A549 cells or HT-29 cells in the logarithmic growth phase were prepared into a single cell suspension, the density of A549 was adjusted to 1 ⁇ 10 4 cells/mL, and the density of HT-29 was adjusted to 1.5 ⁇ 10 4 cells/mL.
  • mL 100 ⁇ L per well was added to a 96-well cell culture plate, and cultured at 37° C., 5% CO 2 for 12 hours.
  • ADC samples with gradient dilution 2000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 1.953nM
  • culture at 37°C, 5% CO 2 for 72h (A549 cells) or 96h (HT-29 cells) were .
  • 40 ⁇ l of MTS Promega, G3580 was added to each well, and after incubation at 37° C. for 1 h, the data were read at OD492 by a microplate reader.
  • Jeko-1 cells human mantle cell lymphoma cells
  • MDA-MB-468 cells human breast cancer cells
  • NCI-H1944 cells human lung cancer cells
  • the specific method is as follows: the Jeko-1 cells, MDA-MB-468 cells or NCI-H1944 cells in the logarithmic growth phase were prepared into a single cell suspension, the density of Jeko-1 was adjusted to 1 ⁇ 10 5 cells/mL, MDA- Adjust the density of MB-468 to 2 ⁇ 10 5 cells/mL, adjust the density of NCI-H1944 to 1.2 ⁇ 10 5 cells/mL, add 90 ⁇ L per well to a 96-well cell culture plate, 37°C, 5% CO 2 , cultivated for 12h.
  • huROR1-HEK293 cells were used to detect the antigen-dependent killing effect of the ADC obtained in the present application and the control ADC.
  • the specific method is as follows: prepare huROR1-HEK293 cells in the logarithmic growth phase into a single-cell suspension, adjust the cell density to 6 ⁇ 104 cells/mL, add 50 ⁇ L per well to a 96-well cell culture plate, and store at 37°C. After incubating for 24 hours in 5% CO 2 , add 50 ⁇ L of 100 ⁇ g/mL antibodies (N99 and N147-135) and positive control antibody 99961.1 to each well.
  • A549 and HT-29 tumor cells were used to detect the endocytic efficiency of the ADC obtained in the present application based on the FACS method.
  • the tumor inhibitory effect of two candidate ADCs (N99-MMAE and N147-135-MMAE) in animals was tested.
  • the tumor cells used were colon cancer cells HT-29 (BNCC337732), and 99961.1-MMAE was used as a positive control .
  • the specific method is as follows: use female nude mice (Balb/C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks and weighing about 20 g, and inject 1 ⁇ 10 6 HT- 29 cells were randomly divided into cages when the tumor-bearing volume reached about 100 mm 3 .
  • L is the longest tumor diameter
  • W is the shortest tumor diameter
  • the antitumor effect of two candidate ADCs was tested.
  • the tumor cells used were non-small cell lung cancer cells A549 (Shanghai Cell Bank, Chinese Academy of Sciences, C2107019), and 99961.1 -MMAE served as a positive control.
  • the specific method is as follows: use female nude mice (Balb/C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks and weighing 18-20 g, and inject 1 ⁇ 10 Six A549 cells were randomly divided into cages when the tumor-bearing volume reached about 100 mm 3 .
  • mice After the end of the administration, the mice were euthanized after observing for a certain period of time, and the tumors were taken out and the tumor weight was measured. The tumor volume, tumor weight and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 18A-18C and Table 13.
  • the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals was tested.
  • the tumor cells used were gastric cancer cells NCI-N87 (Shanghai Cell Bank, Chinese Academy of Sciences, C2009021, P3), and 99961.1-MMAE was used as positive control.
  • the specific method is as follows: use female nude mice (Balb/C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks and weighing 18-20 g, and subcutaneously inject 1 ⁇ 10 6 NCI in the back of each nude mouse. - N87 cells, when the tumor-bearing volume reaches about 100 mm 3 , they are divided into random groups and divided into cages.
  • tumor-bearing nude mice in each group 5 groups in total, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mpk) and 2 positive control ADCs
  • the administration method was venous injection, the administration was once a week and the tumor volume was measured twice, and the administration was administered 3 times/3 weeks (QW*3).
  • Tumor volume (V) calculation method: V L ⁇ W 2 /2 (wherein L is the longest tumor diameter, W is the shortest tumor diameter).
  • the tumor volume and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 19A-19B and Table 14.
  • the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals was tested.
  • the tumor cells used were triple-negative breast cancer cells MDA-MB-231 (Shanghai Cell Bank, Chinese Academy of Sciences, C2006040), with 99961.1 -MMAE served as a positive control.
  • the specific method is as follows: use 6-8 weeks old female nude mice weighing 20-22 g (NSG Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and subcutaneously inject 1 ⁇ 10 6 MDA-MB- 231 cells were randomly divided into cages when the tumor-bearing volume reached about 100 mm 3 .
  • tumor-bearing nude mice in each group 5 groups in total, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mp k ) and 2 positive controls ADC group (99961.1-MMAE 5mpk and 99961.1-MMAE 10mp k ), the administration method was tail vein injection administration, once a week and measuring the tumor volume twice, a total of 3 administrations/3 weeks (QW*3 ).
  • Tumor volume (V) calculation method: V L ⁇ W 2 /2 (wherein L is the longest tumor diameter, W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 20A-20B and Table 15.
  • the tumor-inhibiting effect of a candidate ADC (N147-135-MMAE) in animals was tested.
  • the tumor cells used were triple-negative breast cancer cells MDA-MB-468 (Shanghai Cell Bank, Chinese Academy of Sciences, TCHu136), and 99961.1 -MMAE served as a positive control.
  • the specific method is as follows: use 6-8 weeks old female nude mice weighing 21-25 g (NOD SCID Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), and subcutaneously inject 1 ⁇ 10 7 MDA-MB on the back of each nude mouse -468 cells, when the tumor-bearing volume reached about 100 mm 3 , they were divided into random groups and divided into cages.
  • the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals was tested.
  • the tumor cells used were mantle cell lymphoma cells Jeko-1 (ATCC, CRL-3006), and 99961.1-MMAE was used as positive control.
  • the specific method is as follows: use female nude mice (BALB/c Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks and weighing 21-25 g, and subcutaneously inject 1 ⁇ 10 7 Jeko- 1 cell, and when the tumor-bearing volume reached about 100mm 3 , they were divided into random groups and divided into cages.
  • L is the longest tumor diameter
  • W is the shortest tumor diameter.
  • the tumor volume and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 22A-22B and Tables 18-19.
  • the test results showed that there was no significant change in the body weight of the mice in each group during the treatment period, indicating that the mice tolerated ADC well (Figure 22B); -The tumor growth inhibition rate TGI of mice in the MMAE (10mpk) dose group was about 96%, the tumor inhibition rate was equivalent, and the tumor did not recur 23 days after administration.
  • the tumor inhibitory effect of N147-135-MMAE (8mpk) was slightly inferior to that of 99961.1-MMAE (10mpk), and the TGIs were 76% and 93%, respectively.
  • the 99961.1-MMAE (10mpk) group was administered The tumor did not recur after 23 days, but the N147-135-MMAE (8mpk) group began to rebound in tumor volume 16 days after administration.

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Abstract

The present application relates to an antibody that specifically recognizes ROR1, a preparation method therefor, and a use thereof. The present application further relates to an ROR1-targeted antibody-drug conjugate (ADC) and a composition containing the molecule. In addition, the present invention further relates to therapeutic and diagnostic uses of the antibody, antibody fragment, and antibody-drug conjugate.

Description

一种靶向ROR1的抗体或其抗原结合片段及其应用Antibody or antigen-binding fragment thereof targeting ROR1 and application thereof 技术领域technical field
本申请涉及特异性识别ROR1的抗体、其制备方法和用途。本申请还涉及靶向ROR1的抗体偶联药物(ADC)以及含有该分子的组合物。此外，本发明还涉及这些抗体、抗体片段以及抗体偶联药物的治疗和诊断用途。The present application relates to an antibody specifically recognizing ROR1, its preparation method and use. The present application also relates to antibody-drug conjugates (ADCs) targeting ROR1 and compositions containing such molecules. In addition, the present invention also relates to the therapeutic and diagnostic uses of these antibodies, antibody fragments and antibody-drug conjugates.
背景技术Background technique
抗体偶联药物(Antibody-Drug Conjugates，ADC)是传统抗体药物的衍生药物，由靶向特异性抗原的单克隆抗体药物和小分子细胞毒药物通过连接子偶联而成，兼具抗体药物的特异靶向性和传统小分子药物的杀伤效应，其主要的适应症为恶性肿瘤。ADC凭借其优异的肿瘤杀伤效应，迅速成为抗肿瘤药物研发的热点。但ADC的毒性通常较高，因此对于靶点抗原的特异性要求较高，适合开发ADC的靶点一般是在肿瘤中高表达，在健康组织中低表达或者几乎不表达；抗原为肿瘤细胞表达上调的表面受体，能够促进肿瘤生长或生存，并且该靶点抗原应具有内化特性。Antibody-Drug Conjugates (ADC) are derivative drugs of traditional antibody drugs, which are composed of monoclonal antibody drugs targeting specific antigens and small molecule cytotoxic drugs coupled through linkers, and have the functions of antibody drugs. Specific targeting and killing effect of traditional small molecule drugs, its main indication is malignant tumors. With its excellent tumor killing effect, ADC has quickly become a hot spot in the research and development of anti-tumor drugs. However, the toxicity of ADCs is usually high, so the specificity requirements for target antigens are high. Targets suitable for the development of ADCs are generally highly expressed in tumors and low or almost non-expressed in healthy tissues; the antigen is upregulated by tumor cells surface receptors that promote tumor growth or survival, and the target antigen should have internalization properties.
受体酪氨酸激酶样孤儿受体1(receptor tyrosine kinase-like orphan receptor 1，ROR1，也被称为受体相关的神经营养性酪氨酸激酶1，NTRKR1)和受体酪氨酸激酶样孤儿受体2(receptor tyrosine kinase-like orphan receptor 2，ROR2)是隶属于受体酪氨酸激酶(RTK)家族的单次跨膜蛋白，其胞外由一个免疫球蛋白样结构域(Ig)和两个富含半胱氨酸的结构域(FZD结构域和KRD结构域)组成，胞内由一个酪氨酸激酶结构域、两个富含丝氨酸或苏氨酸的结构域和一个富含脯氨酸的结构域组成。ROR1和ROR2通过FZD结构域结合配体Wnt5a参与非经典Wnt信号通路(Oishi I,Suzuki H,Onishi N,Takada R,Kani S,Ohkawara B,Koshida I,Suzuki K,Yamada G,Schwabe GC et al(2003).Genes Cells 8:645–654；Fukuda T,Chen L,Endo T,Tang L,Lu D,Castro JE,Widhopf GF II,Rassenti LZ,Cantwell MJ,Prussak CE et al(2008).Proc Natl Acad Sci USA 105:3047–3052；Paganoni S,Bernstein J,Ferreira A(2010).Neuroscience 165:1261–1274)。ROR1能抑制细胞凋亡，增强EGFR信号传导，诱导上皮间质转化(EMT)(Fukuda T,Chen L,Endo T,Tang L,Lu D,Castro JE,Widhopf GF II,Rassenti LZ,Cantwell MJ,Prussak CE et al(2008).Proc Natl Acad Sci USA 105:3047–3052；Yamaguchi T,Yanagisawa K,Sugiyama R,Hosono Y,Shimada Y,ArimaC,KatoS,TomidaS,Suzuki M,OsadaHet al(2012).Cancer Cell 21:348–361；Cui B,Zhang S,Chen L,Yu J,Widhopf GF,Fecteau J-F,Rassenti LZ,Kipps TJ(2013).Cancer Res 73:3649–3660)。Receptor tyrosine kinase-like orphan receptor 1 (ROR1, also known as receptor-associated neurotrophic tyrosine kinase 1, NTRKR1) and receptor tyrosine kinase-like Orphan receptor 2 (receptor tyrosine kinase-like orphan receptor 2, ROR2) is a single transmembrane protein belonging to the receptor tyrosine kinase (RTK) family, and its extracellular structure consists of an immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD domain and KRD domain), intracellularly consists of a tyrosine kinase domain, two serine- or threonine-rich domains and a Domain composition of proline. ROR1 and ROR2 participate in the non-canonical Wnt signaling pathway through FZD domain binding ligand Wnt5a (Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, Koshida I, Suzuki K, Yamada G, Schwabe GC et al( 2003). Genes Cells 8:645–654; Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al (2008). Proc Natl Acad Sci USA 105:3047–3052; Paganoni S, Bernstein J, Ferreira A (2010). Neuroscience 165:1261–1274). ROR1 inhibits apoptosis, enhances EGFR signaling, and induces epithelial-mesenchymal transition (EMT) (Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al (2008). Proc Natl Acad Sci USA 105:3047–3052; Yamaguchi T, Yanagisawa K, Sugiyama R, Hosono Y, Shimada Y, Arima C, Kato S, Tomida S, Suzuki M, Osada He et al (2012). Cancer Cell 21:348–361; Cui B, Zhang S, Chen L, Yu J, Widhopf GF, Fecteau J-F, Rassenti LZ, Kipps TJ (2013). Cancer Res 73:3649–3660).
ROR1是保守的胚胎蛋白，随着胚胎发育其表达逐渐下降，在成人大多数组织中几乎不存在或低表达，而越来越多的文献发现ROR1在多种癌细胞中表达，比如B细胞慢性淋巴细胞白血病(CLL)和其他血液恶性肿瘤、肾细胞癌、结肠癌和乳癌的某些其他癌细胞系。此外，ROR1在许多血液和实体恶性肿瘤的进展中起着重要作用。因此，作为癌症标志物，ROR1成为癌症治疗的理想药物靶点。 ROR1 is a conserved embryonic protein. Its expression gradually decreases with embryonic development, and it is almost absent or lowly expressed in most tissues of adults. More and more literatures have found that ROR1 is expressed in a variety of cancer cells, such as B cells with chronic Certain other cancer cell lines of lymphocytic leukemia (CLL) and other hematological malignancies, renal cell carcinoma, colon cancer, and breast cancer. Furthermore, ROR1 plays an important role in the progression of many hematological and solid malignancies. Therefore, as a cancer marker, ROR1 becomes an ideal drug target for cancer therapy.
虽然现有技术公开了几款针对ROR1的抗体药物，但是作为泛癌种的肿瘤标志物，仍然迫切需要开发高质量的抗ROR1抗体，其可以用作开发表达ROR1的癌症的基于抗体的靶向疗法的基础，还可以用作诊断工具以检测ROR1相关病症中的ROR1表达。此外，基于ADC在肿瘤治疗领域展现出的良好前景，目前依然迫切需要具有有效治疗作用的包含ROR1的ADC，本发明满足了这些需求。Although several anti-ROR1 antibody drugs have been disclosed in the prior art, as a pan-cancer tumor marker, there is still an urgent need to develop high-quality anti-ROR1 antibodies, which can be used as antibody-based targets for developing ROR1-expressing cancers. The basis for therapy and can also be used as a diagnostic tool to detect ROR1 expression in ROR1-associated disorders. In addition, based on the promising prospects of ADCs in the field of tumor therapy, there is still an urgent need for ROR1-containing ADCs with effective therapeutic effects, and the present invention meets these needs.
发明内容Contents of the invention
第一方面，本发明提供了靶向人ROR1的抗体、其具有以下优点：In the first aspect, the present invention provides an antibody targeting human ROR1, which has the following advantages:
(1)高亲和力结合人ROR1、表达人ROR1的靶细胞；(1) Target cells that bind to human ROR1 with high affinity and express human ROR1;
(2)具有良好的鼠交叉活性；(2) have good mouse cross activity;
(3)能够通过内吞作用进入细胞；(3) able to enter cells through endocytosis;
(4)适合构建有效治疗的抗体偶联药物；(4) Antibody-drug conjugates suitable for constructing effective treatments;
本发明提供的抗ROR1抗体具有完全的人序列，是一种人抗体。预期本发明的抗ROR1抗体对人类受试者具有最小的免疫原性，人类受试者对该抗ROR1抗体将会具有良好的耐受性。The anti-ROR1 antibody provided by the present invention has a complete human sequence and is a human antibody. It is expected that the anti-ROR1 antibodies of the invention will be minimally immunogenic to human subjects, and that the anti-ROR1 antibodies will be well tolerated by human subjects.
在一个实施方案中，本发明提供了特异性结合ROR1的抗ROR1抗体及其抗原结合片段，其包含：In one embodiment, the present invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising:
1)如SEQ ID NO:52所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:51所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；1) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:52 and 3 contained in the light chain variable region as shown in SEQ ID NO:51 a light chain CDR (LCDR1, LCDR2, LCDR3);
2)如SEQ ID NO:54所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:53所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；2) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:54 and 3 contained in the light chain variable region as shown in SEQ ID NO:53 a light chain CDR (LCDR1, LCDR2, LCDR3);
3)如SEQ ID NO:56所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:55所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；3) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO:56 and 3 included in the light chain variable region as shown in SEQ ID NO:55 a light chain CDR (LCDR1, LCDR2, LCDR3);
4)如SEQ ID NO:58所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:57所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；4) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO:58 and 3 included in the light chain variable region as shown in SEQ ID NO:57 a light chain CDR (LCDR1, LCDR2, LCDR3);
5)如SEQ ID NO:60所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:59所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；5) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:60 and 3 contained in the light chain variable region as shown in SEQ ID NO:59 a light chain CDR (LCDR1, LCDR2, LCDR3);
6)如SEQ ID NO:62所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:61所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；6) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:62 and 3 contained in the light chain variable region as shown in SEQ ID NO:61 a light chain CDR (LCDR1, LCDR2, LCDR3);
7)如SEQ ID NO:64所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:63所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；7) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:64 and 3 contained in the light chain variable region as shown in SEQ ID NO:63 a light chain CDR (LCDR1, LCDR2, LCDR3);
8)如SEQ ID NO:66所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:53所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；8) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:66 and 3 contained in the light chain variable region as shown in SEQ ID NO:53 a light chain CDR (LCDR1, LCDR2, LCDR3);
9)如SEQ ID NO:68所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:67所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)； 9) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:68 and 3 contained in the light chain variable region as shown in SEQ ID NO:67 a light chain CDR (LCDR1, LCDR2, LCDR3);
10)如SEQ ID NO:70所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:69所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；10) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:70 and 3 contained in the light chain variable region as shown in SEQ ID NO:69 a light chain CDR (LCDR1, LCDR2, LCDR3);
11)如SEQ ID NO:145所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:144所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；11) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:145 and 3 contained in the light chain variable region as shown in SEQ ID NO:144 a light chain CDR (LCDR1, LCDR2, LCDR3);
12)如SEQ ID NO:147所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:146所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；12) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:147 and 3 contained in the light chain variable region as shown in SEQ ID NO:146 a light chain CDR (LCDR1, LCDR2, LCDR3);
13)如SEQ ID NO:141所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:140所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；13) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:141 and 3 contained in the light chain variable region as shown in SEQ ID NO:140 a light chain CDR (LCDR1, LCDR2, LCDR3);
14)如SEQ ID NO:143所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:142所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；14) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:143 and 3 contained in the light chain variable region as shown in SEQ ID NO:142 a light chain CDR (LCDR1, LCDR2, LCDR3);
15)如SEQ ID NO:149所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:148所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；15) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:149 and 3 contained in the light chain variable region as shown in SEQ ID NO:148 a light chain CDR (LCDR1, LCDR2, LCDR3);
16)如SEQ ID NO:161所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:160所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；16) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:161 and 3 contained in the light chain variable region as shown in SEQ ID NO:160 a light chain CDR (LCDR1, LCDR2, LCDR3);
17)如SEQ ID NO:163所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:162所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；17) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:163 and 3 contained in the light chain variable region as shown in SEQ ID NO:162 a light chain CDR (LCDR1, LCDR2, LCDR3);
18)如SEQ ID NO:165所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:164所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；18) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:165 and 3 contained in the light chain variable region as shown in SEQ ID NO:164 a light chain CDR (LCDR1, LCDR2, LCDR3);
19)如SEQ ID NO:151所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:150所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；19) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:151 and 3 contained in the light chain variable region as shown in SEQ ID NO:150 a light chain CDR (LCDR1, LCDR2, LCDR3);
20)如SEQ ID NO:153所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:152所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；20) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:153 and 3 contained in the light chain variable region as shown in SEQ ID NO:152 a light chain CDR (LCDR1, LCDR2, LCDR3);
21)如SEQ ID NO:155所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:154所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；21) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:155 and 3 contained in the light chain variable region as shown in SEQ ID NO:154 a light chain CDR (LCDR1, LCDR2, LCDR3);
22)如SEQ ID NO:157所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:156所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；22) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:157 and 3 contained in the light chain variable region as shown in SEQ ID NO:156 a light chain CDR (LCDR1, LCDR2, LCDR3);
23)如SEQ ID NO:52所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:158所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；23) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:52 and 3 contained in the light chain variable region as shown in SEQ ID NO:158 a light chain CDR (LCDR1, LCDR2, LCDR3);
24)如SEQ ID NO:52所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:159所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；24) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:52 and 3 contained in the light chain variable region as shown in SEQ ID NO:159 a light chain CDR (LCDR1, LCDR2, LCDR3);
25)如SEQ ID NO:62所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:166所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；25) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:62 and 3 contained in the light chain variable region as shown in SEQ ID NO:166 a light chain CDR (LCDR1, LCDR2, LCDR3);
26)如SEQ ID NO:168所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:167所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)； 26) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 168 and 3 contained in the light chain variable region as shown in SEQ ID NO: 167 a light chain CDR (LCDR1, LCDR2, LCDR3);
27)如SEQ ID NO:169所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:61所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)。27) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 169 and 3 contained in the light chain variable region as shown in SEQ ID NO: 61 light chain CDRs (LCDR1, LCDR2, LCDR3).
一个实施方案中，本发明提供了特异性结合ROR1的抗ROR1抗体及其抗原结合片段，其包含：In one embodiment, the present invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, comprising:
1)包含如SEQ ID NO:1、2和3所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、20和21所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；1) Containing the sequence shown in SEQ ID NO: 1, 2 and 3, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 19, 20 and 21, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
2)包含如SEQ ID NO:1、2和6所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:22、23和24所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；2) Containing the sequence shown in SEQ ID NO: 1, 2 and 6, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 22, 23 and 24, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
3)包含如SEQ ID NO:7、8和9所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:25、26和27所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；3) Containing the sequence shown in SEQ ID NO: 7, 8 and 9, or a sequence containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of no more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 25, 26 and 27, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
4)包含如SEQ ID NO:10、11和12所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、29和30所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；4) Containing the sequence shown in SEQ ID NO: 10, 11 and 12, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences as shown in SEQ ID NO: 28, 29 and 30, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
5)包含如SEQ ID NO:13、14和15所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:31、32和33所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；5) Containing the sequence shown in SEQ ID NO: 13, 14 and 15, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 31, 32 and 33, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
6)包含如SEQ ID NO:16、17和18所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:34、35和36所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；6) Containing the sequence shown in SEQ ID NO: 16, 17 and 18, or a sequence containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of no more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 34, 35 and 36, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
7)包含如SEQ ID NO:1、2和37所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、20和21所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；7) Comprising a sequence as shown in SEQ ID NO: 1, 2 and 37, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 19, 20 and 21, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
8)包含如SEQ ID NO:38、39和12所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3； 和如SEQ ID NO:28、44和30所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；8) Comprising a sequence as shown in SEQ ID NO: 38, 39 and 12, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and the sequences shown in SEQ ID NO:28, 44 and 30, or the LCDR1 of the sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence, LCDR2, LCDR3;
9)包含如SEQ ID NO:40、41和15所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:31、45和33所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；9) Containing the sequence shown in SEQ ID NO: 40, 41 and 15, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 31, 45 and 33, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
10)包含如SEQ ID NO:42、43和18所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:34、46和36所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；10) Containing the sequence shown in SEQ ID NO: 42, 43 and 18, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 34, 46 and 36, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
11)包含如SEQ ID NO:91、92和93所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:101、102和103所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；11) Containing the sequence shown in SEQ ID NO: 91, 92 and 93, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 101, 102 and 103, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
12)包含如SEQ ID NO:1、2和94所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:104、32和105所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；12) Comprising a sequence as shown in SEQ ID NO: 1, 2 and 94, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 104, 32 and 105, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
13)包含如SEQ ID NO:95、96和97所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:106、107和108所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；13) Containing the sequence shown in SEQ ID NO: 95, 96 and 97, or a sequence containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of no more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 106, 107 and 108, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
14)包含如SEQ ID NO:1、2和98所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:109、110和105所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；14) Comprising a sequence as shown in SEQ ID NO: 1, 2 and 98, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 109, 110 and 105, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
15)包含如SEQ ID NO:91、99和100所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:111、112和113所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；15) Comprising sequences as shown in SEQ ID NO: 91, 99 and 100, or sequences containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to said sequences HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 111, 112 and 113, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
16)包含如SEQ ID NO:121、122和3所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、136和21所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3； 16) A sequence comprising a sequence as shown in SEQ ID NO: 121, 122 and 3, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 19, 136 and 21, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
17)包含如SEQ ID NO:1、123和3所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、137和21所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；17) Containing the sequence shown in SEQ ID NO: 1, 123 and 3, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 19, 137 and 21, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
18)包含如SEQ ID NO:124、2和3所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、23和21所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；18) Comprising a sequence as shown in SEQ ID NO: 124, 2 and 3, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 19, 23 and 21, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
19)包含如SEQ ID NO:114、115和12所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、128和30所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；19) Containing the sequence shown in SEQ ID NO: 114, 115 and 12, or a sequence containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of no more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 28, 128 and 30, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
20)包含如SEQ ID NO:116、117和12所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、129和30所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；20) Containing the sequence shown in SEQ ID NO: 116, 117 and 12, or a sequence containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of no more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 28, 129 and 30, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
21)包含如SEQ ID NO:118、119和12所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、130和30所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；21) Comprising sequences as shown in SEQ ID NO: 118, 119 and 12, or sequences containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 28, 130 and 30, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
22)包含如SEQ ID NO:16、17和120所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:131、35和132所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；22) Containing the sequence shown in SEQ ID NO: 16, 17 and 120, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 131, 35 and 132, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
23)包含如SEQ ID NO:16、17和18所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:34、35和133所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；23) Containing the sequence shown in SEQ ID NO: 16, 17 and 18, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 34, 35 and 133, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
24)包含如SEQ ID NO:16、17和18所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:134、35和135所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；24) Containing the sequence shown in SEQ ID NO: 16, 17 and 18, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 134, 35 and 135, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence;
25)包含如SEQ ID NO:13、14和15所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3； 和如SEQ ID NO:31、138和33所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；25) A sequence comprising a sequence as shown in SEQ ID NO: 13, 14 and 15, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and the sequences shown in SEQ ID NO: 31, 138 and 33, or the LCDR1 of the sequence containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of no more than 3 amino acids relative to the sequence, LCDR2, LCDR3;
26)包含如SEQ ID NO:125、126和15所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:31、139和33所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；或26) Containing the sequence shown in SEQ ID NO: 125, 126 and 15, or a sequence containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to the sequence HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 31, 139 and 33, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence; or
27)包含如SEQ ID NO:125、127和15所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:31、32和33所示序列，或相对于所述序列含有一个或多个且不超过3个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3。27) Comprising sequences as shown in SEQ ID NO: 125, 127 and 15, or sequences containing one or more and no more than 3 amino acid amino acid substitutions (such as conservative substitutions), deletions or insertions relative to said sequences HCDR1, HCDR2, HCDR3; and sequences shown in SEQ ID NO: 31, 32 and 33, or amino acid substitutions (such as conservative substitutions), deletions or LCDR1, LCDR2, LCDR3 of the inserted sequence.
在一个实施方案中，本发明提供了特异性结合ROR1的抗ROR1抗体及其抗原结合片段，其包含重链可变区，其中：In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising a heavy chain variable region, wherein:
1)所述重链可变区包含如SEQ ID NO：52所示氨基酸序列，或与SEQ ID NO：52的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：52组成；1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 52 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 52;
2)所述重链可变区包含如SEQ ID NO：54所示氨基酸序列，或与SEQ ID NO：54的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：54组成；2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 54, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 54 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 54;
3)所述重链可变区包含如SEQ ID NO：56所示氨基酸序列，或与SEQ ID NO：56的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：56组成；3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 56, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 56 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 56;
4)所述重链可变区包含如SEQ ID NO：58所示氨基酸序列，或与SEQ ID NO：58的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：58组成；4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 58 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 58;
5)所述重链可变区包含如SEQ ID NO：60所示氨基酸序列，或与SEQ ID NO：60的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：60组成；5) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 60, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 60 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 60;
6)所述重链可变区包含如SEQ ID NO：62所示氨基酸序列，或与SEQ ID NO：62的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：62组成；6) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 62 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 62;
7)所述重链可变区包含如SEQ ID NO：64所示氨基酸序列，或与SEQ ID NO：64的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：64组成； 7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 64, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 64 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 64;
8)所述重链可变区包含如SEQ ID NO：66所示氨基酸序列，或与SEQ ID NO：66的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：66组成；8) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 66, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 66 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 66;
9)所述重链可变区包含如SEQ ID NO：68所示氨基酸序列，或与SEQ ID NO：68的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：68组成；9) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 68, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 68 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 68;
10)所述重链可变区包含如SEQ ID NO：70所示氨基酸序列，或与SEQ ID NO：70的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：70组成；10) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 70 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 70;
11)所述重链可变区包含如SEQ ID NO：145所示氨基酸序列，或与SEQ ID NO：145的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：145组成；11) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 145, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 145 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 145;
12)所述重链可变区包含如SEQ ID NO：147所示氨基酸序列，或与SEQ ID NO：147的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：147组成；12) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 147, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 147 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 147;
13)所述重链可变区包含如SEQ ID NO：141所示氨基酸序列，或与SEQ ID NO：141的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：141组成；13) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 141, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 141 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 141;
14)所述重链可变区包含如SEQ ID NO：143所示氨基酸序列，或与SEQ ID NO：143的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：143组成；14) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 143, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 143 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 143;
15)所述重链可变区包含如SEQ ID NO：149所示氨基酸序列，或与SEQ ID NO：149的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：149组成；15) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 149, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 149 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 149;
16)所述重链可变区包含如SEQ ID NO：161所示氨基酸序列，或与SEQ ID NO：161的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：161组成；16) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 161, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 161 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 161;
17)所述重链可变区包含如SEQ ID NO：163所示氨基酸序列，或与SEQ ID NO：163的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：163组成；17) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 163, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 163 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 163;
18)所述重链可变区包含如SEQ ID NO：165所示氨基酸序列，或与SEQ ID NO：165的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：165组成；18) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 165, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 165 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 165;
19)所述重链可变区包含如SEQ ID NO：151所示氨基酸序列，或与SEQ ID NO：151的氨 基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：151组成；19) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 151, or with the amino acid sequence of SEQ ID NO: 151 The amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence, or consists of SEQ ID NO: 151;
20)所述重链可变区包含如SEQ ID NO：153所示氨基酸序列，或与SEQ ID NO：153的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：153组成；20) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 153, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 153 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 153;
21)所述重链可变区包含如SEQ ID NO：155所示氨基酸序列，或与SEQ ID NO：155的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：155组成；21) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 155, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 155 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 155;
22)所述重链可变区包含如SEQ ID NO：157所示氨基酸序列，或与SEQ ID NO：157的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：157组成；22) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 157, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 157 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 157;
23)所述重链可变区包含如SEQ ID NO：168所示氨基酸序列，或与SEQ ID NO：168的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：168组成；或23) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 168, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 168 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 168; or
24)所述重链可变区包含如SEQ ID NO：169所示氨基酸序列，或与SEQ ID NO：169的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：169组成。24) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 169, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 169 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO:169.
在一个实施方案中，本发明提供了特异性结合ROR1的抗ROR1抗体及其抗原结合片段，其包含轻链可变区，其中：In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising a light chain variable region, wherein:
1)所述轻链可变区包含如SEQ ID NO：51所示氨基酸序列，或与SEQ ID NO：51的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：51组成；1) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 51, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 51 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 51;
2)所述轻链可变区包含如SEQ ID NO：53所示氨基酸序列，或与SEQ ID NO：53的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：53组成；2) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 53, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 53 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 53;
3)所述轻链可变区包含如SEQ ID NO：55所示氨基酸序列，或与SEQ ID NO：55的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：55组成；3) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 55, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 55 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 55;
4)所述轻链可变区包含如SEQ ID NO：57所示氨基酸序列，或与SEQ ID NO：57的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：57组成；4) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 57, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 57 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 57;
5)所述轻链可变区包含如SEQ ID NO：59所示氨基酸序列，或与SEQ ID NO：59的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序 列，或由SEQ ID NO：59组成；5) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 59, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 59 , 96%, 97%, 98% or 99% identical amino acid sequence column, or consists of SEQ ID NO: 59;
6)所述轻链可变区包含如SEQ ID NO：61所示氨基酸序列，或与SEQ ID NO：61的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：61组成；6) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 61, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 61 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 61;
7)所述轻链可变区包含如SEQ ID NO：63所示氨基酸序列，或与SEQ ID NO：63的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：63组成；7) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 63, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 63 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 63;
8)所述轻链可变区包含如SEQ ID NO：67所示氨基酸序列，或与SEQ ID NO：67的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：67组成；8) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 67, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 67 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 67;
9)所述轻链可变区包含如SEQ ID NO：69所示氨基酸序列，或与SEQ ID NO：69的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：69组成；9) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 69, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 69 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 69;
10)所述轻链可变区包含如SEQ ID NO：144所示氨基酸序列，或与SEQ ID NO：144的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：144组成；10) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 144, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 144 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 144;
11)所述轻链可变区包含如SEQ ID NO：146所示氨基酸序列，或与SEQ ID NO：146的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：146组成；11) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 146, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 146 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 146;
12)所述轻链可变区包含如SEQ ID NO：140所示氨基酸序列，或与SEQ ID NO：140的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：140组成；12) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 140, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 140 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 140;
13)所述轻链可变区包含如SEQ ID NO：142所示氨基酸序列，或与SEQ ID NO：142的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：142组成；13) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 142, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 142 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 142;
14)所述轻链可变区包含如SEQ ID NO：148所示氨基酸序列，或与SEQ ID NO：148的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：148组成；14) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 148, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 148 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 148;
15)所述轻链可变区包含如SEQ ID NO：160所示氨基酸序列，或与SEQ ID NO：160的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：160组成；15) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 160, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 160 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 160;
16)所述轻链可变区包含如SEQ ID NO：162所示氨基酸序列，或与SEQ ID NO：162的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：162组成； 16) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 162, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 162 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 162;
17)所述轻链可变区包含如SEQ ID NO：164所示氨基酸序列，或与SEQ ID NO：164的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：164组成；17) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 164, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 164 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 164;
18)所述轻链可变区包含如SEQ ID NO：150所示氨基酸序列，或与SEQ ID NO：150的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：150组成；18) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 150, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 150 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 150;
19)所述轻链可变区包含如SEQ ID NO：152所示氨基酸序列，或与SEQ ID NO：152的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：152组成；19) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 152, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 152 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 152;
20)所述轻链可变区包含如SEQ ID NO：154所示氨基酸序列，或与SEQ ID NO：154的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：154组成；20) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 154, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 154 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 154;
21)所述轻链可变区包含如SEQ ID NO：156所示氨基酸序列，或与SEQ ID NO：156的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：156组成；21) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 156, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 156 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 156;
22)所述轻链可变区包含如SEQ ID NO：158所示氨基酸序列，或与SEQ ID NO：158的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：158组成；22) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 158, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 158 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 158;
23)所述轻链可变区包含如SEQ ID NO：159所示氨基酸序列，或与SEQ ID NO：159的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：159组成；23) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 159, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 159 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 159;
24)所述轻链可变区包含如SEQ ID NO：166所示氨基酸序列，或与SEQ ID NO：166的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：166组成；24) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 166, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 166 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 166;
25)所述轻链可变区包含如SEQ ID NO：167所示氨基酸序列，或与SEQ ID NO：167的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：167组成。25) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 167, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 167 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO:167.
在另一个实施方案中，本发明提供了特异性结合ROR1的抗ROR1抗体及其抗原结合片段，其包含重链可变区和轻链可变区，其中：In another embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising a heavy chain variable region and a light chain variable region, wherein:
1)所述重链可变区包含如SEQ ID NO：52所示氨基酸序列，或与SEQ ID NO：52的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：52组成，所述轻链可变区包含如SEQ ID NO：51所示氨基酸序列，或与SEQ ID NO：51的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：51组成； 1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 52 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 52, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, or with SEQ ID NO: 52 The amino acid sequence of NO: 51 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 51 composition;
2)所述重链可变区包含如SEQ ID NO：54所示氨基酸序列，或与SEQ ID NO：54的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：54组成，所述轻链可变区包含如SEQ ID NO：53所示氨基酸序列，或与SEQ ID NO：53的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：53组成；2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 54, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 54 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 54, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53, or with SEQ ID NO: 54 The amino acid sequence of NO: 53 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 53 composition;
3)所述重链可变区包含如SEQ ID NO：56所示氨基酸序列，或与SEQ ID NO：56的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：56组成，所述轻链可变区包含如SEQ ID NO：55所示氨基酸序列，或与SEQ ID NO：55的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：55组成；3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 56, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 56 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 56, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55, or with SEQ ID NO: 56 The amino acid sequence of NO: 55 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 55 composition;
4)所述重链可变区包含如SEQ ID NO：58所示氨基酸序列，或与SEQ ID NO：58的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：58组成，所述轻链可变区包含如SEQ ID NO：57所示氨基酸序列，或与SEQ ID NO：57的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：57组成；4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 58 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 58, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57, or with SEQ ID NO: 58 The amino acid sequence of NO: 57 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 57 composition;
5)所述重链可变区包含如SEQ ID NO：60所示氨基酸序列，或与SEQ ID NO：60的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：60组成，所述轻链可变区包含如SEQ ID NO：59所示氨基酸序列，或与SEQ ID NO：59的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：59组成；5) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 60, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 60 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 60, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, or with SEQ ID NO: The amino acid sequence of NO: 59 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 59 composition;
6)所述重链可变区包含如SEQ ID NO：62所示氨基酸序列，或与SEQ ID NO：62的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：62组成，所述轻链可变区包含如SEQ ID NO：61所示氨基酸序列，或与SEQ ID NO：61的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：61组成；6) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 62 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 62, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or with SEQ ID NO: 62 The amino acid sequence of NO: 61 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 61 composition;
7)所述重链可变区包含如SEQ ID NO：64所示氨基酸序列，或与SEQ ID NO：64的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：64组成，所述轻链可变区包含如SEQ ID NO：63所示氨基酸序列，或与SEQ ID NO：63的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：63组成；7) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 64, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 64 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 64, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 63, or with SEQ ID NO: 64 The amino acid sequence of NO: 63 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 63 composition;
8)所述重链可变区包含如SEQ ID NO：66所示氨基酸序列，或与SEQ ID NO：66的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：66组成，所述轻链可变区包含如SEQ ID NO：53所示氨基酸序列，或与SEQ ID NO：53的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％ 或99％同一性的氨基酸序列，或由SEQ ID NO：53组成；8) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 66 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 66, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53, or with SEQ ID NO: 53 The amino acid sequence of NO: 53 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or an amino acid sequence with 99% identity, or consisting of SEQ ID NO: 53;
9)所述重链可变区包含如SEQ ID NO：68所示氨基酸序列，或与SEQ ID NO：68的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：68组成，所述轻链可变区包含如SEQ ID NO：67所示氨基酸序列，或与SEQ ID NO：67的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：67组成；9) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 68, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 68 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 68, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67, or with SEQ ID NO: 68 The amino acid sequence of NO: 67 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 67 composition;
10)所述重链可变区包含如SEQ ID NO：70所示氨基酸序列，或与SEQ ID NO：70的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：70组成，所述轻链可变区包含如SEQ ID NO：69所示氨基酸序列，或与SEQ ID NO：69的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：69组成；10) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 70 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 70, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 69, or with SEQ ID NO: 70 The amino acid sequence of NO: 69 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 69 composition;
11)所述重链可变区包含如SEQ ID NO：145所示氨基酸序列，或与SEQ ID NO：145的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：145组成，所述轻链可变区包含如SEQ ID NO：144所示氨基酸序列，或与SEQ ID NO：144的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：144组成；11) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 145, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 145 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 145, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 144, or with SEQ ID NO: 145 The amino acid sequence of NO: 144 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 144 composition;
12)所述重链可变区包含如SEQ ID NO：147所示氨基酸序列，或与SEQ ID NO：147的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：147组成，所述轻链可变区包含如SEQ ID NO：146所示氨基酸序列，或与SEQ ID NO：146的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：146组成；12) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 147, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 147 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 147, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 146, or with SEQ ID The amino acid sequence of NO: 146 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 146 composition;
13)所述重链可变区包含如SEQ ID NO：141所示氨基酸序列，或与SEQ ID NO：141的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：141组成，所述轻链可变区包含如SEQ ID NO：140所示氨基酸序列，或与SEQ ID NO：140的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：140组成；13) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 141, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 141 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 141, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 140, or with SEQ ID NO: 140 The amino acid sequence of NO: 140 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 140 composition;
14)所述重链可变区包含如SEQ ID NO：143所示氨基酸序列，或与SEQ ID NO：143的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：143组成，所述轻链可变区包含如SEQ ID NO：142所示氨基酸序列，或与SEQ ID NO：142的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：142组成；14) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 143, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 143 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 143, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 142, or with SEQ ID NO: 143 The amino acid sequence of NO: 142 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 142 composition;
15)所述重链可变区包含如SEQ ID NO：149所示氨基酸序列，或与SEQ ID NO：149的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：149组成，所述轻链可变区包含如SEQ ID NO：148所示氨基酸序列， 或与SEQ ID NO：148的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：148组成；15) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 149, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 149 , an amino acid sequence of 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 149, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 148, Or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 148, or from SEQ ID NO: 148 ID NO: 148 composition;
16)所述重链可变区包含如SEQ ID NO：161所示氨基酸序列，或与SEQ ID NO：161的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：161组成，所述轻链可变区包含如SEQ ID NO：160所示氨基酸序列，或与SEQ ID NO：160的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：160组成；16) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 161, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 161 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 161, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 160, or with SEQ ID NO: 161 The amino acid sequence of NO: 160 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 160 composition;
17)所述重链可变区包含如SEQ ID NO：163所示氨基酸序列，或与SEQ ID NO：163的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：163组成，所述轻链可变区包含如SEQ ID NO：162所示氨基酸序列，或与SEQ ID NO：162的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：162组成；17) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 163, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 163 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 163, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 162, or with SEQ ID NO: 162 The amino acid sequence of NO: 162 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 162 composition;
18)所述重链可变区包含如SEQ ID NO：165所示氨基酸序列，或与SEQ ID NO：165的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：165组成，所述轻链可变区包含如SEQ ID NO：164所示氨基酸序列，或与SEQ ID NO：164的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：164组成；18) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 165, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 165 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 165, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 164, or with SEQ ID NO: 164 The amino acid sequence of NO: 164 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 164 composition;
19)所述重链可变区包含如SEQ ID NO：151所示氨基酸序列，或与SEQ ID NO：151的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：151组成，所述轻链可变区包含如SEQ ID NO：150所示氨基酸序列，或与SEQ ID NO：150的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：150组成；19) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 151, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 151 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 151, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 150, or with SEQ ID NO: 150 The amino acid sequence of NO: 150 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 150 composition;
20)所述重链可变区包含如SEQ ID NO：153所示氨基酸序列，或与SEQ ID NO：153的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：153组成，所述轻链可变区包含如SEQ ID NO：152所示氨基酸序列，或与SEQ ID NO：152的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：152组成；20) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 153, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 153 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 153, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 152, or with SEQ ID NO: 152 The amino acid sequence of NO: 152 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 152 composition;
21)所述重链可变区包含如SEQ ID NO：155所示氨基酸序列，或与SEQ ID NO：155的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：155组成，所述轻链可变区包含如SEQ ID NO：154所示氨基酸序列，或与SEQ ID NO：154的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：154组成；21) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 155, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 155 , 96%, 97%, 98% or 99% identical amino acid sequence, or composed of SEQ ID NO: 155, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 154, or with SEQ ID NO: 154 The amino acid sequence of NO: 154 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 154 composition;
22)所述重链可变区包含如SEQ ID NO：157所示氨基酸序列，或与SEQ ID NO：157的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基 酸序列，或由SEQ ID NO：157组成，所述轻链可变区包含如SEQ ID NO：156所示氨基酸序列，或与SEQ ID NO：156的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：156组成；22) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 157, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 157 , 96%, 97%, 98% or 99% identical amino groups acid sequence, or consist of SEQ ID NO: 157, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 156, or has at least 90%, 91%, 92% of the amino acid sequence of SEQ ID NO: 156 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of SEQ ID NO: 156;
23)所述重链可变区包含如SEQ ID NO：52所示氨基酸序列，或与SEQ ID NO：52的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：52组成，所述轻链可变区包含如SEQ ID NO：158所示氨基酸序列，或与SEQ ID NO：158的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：158组成；23) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 52 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 52, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 158, or with SEQ ID NO: 158 The amino acid sequence of NO: 158 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 158 composition;
24)所述重链可变区包含如SEQ ID NO：52所示氨基酸序列，或与SEQ ID NO：52的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：52组成，所述轻链可变区包含如SEQ ID NO：159所示氨基酸序列，或与SEQ ID NO：159的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：159组成；24) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 52 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 52, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 159, or with SEQ ID NO: 52 The amino acid sequence of NO: 159 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 159 composition;
25)所述重链可变区包含如SEQ ID NO：62所示氨基酸序列，或与SEQ ID NO：62的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：62组成，所述轻链可变区包含如SEQ ID NO：166所示氨基酸序列，或与SEQ ID NO：166的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：166组成；25) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 62 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 62, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 166, or with SEQ ID NO: 62 The amino acid sequence of NO: 166 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 166 composition;
26)所述重链可变区包含如SEQ ID NO：168所示氨基酸序列，或与SEQ ID NO：168的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：168组成，所述轻链可变区包含如SEQ ID NO：167所示氨基酸序列，或与SEQ ID NO：167的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：167组成；或26) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 168, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 168 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 168, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 167, or with SEQ ID NO: 168 The amino acid sequence of NO: 167 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 167 composed; or
27)所述重链可变区包含如SEQ ID NO：169所示氨基酸序列，或与SEQ ID NO：169的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：169组成，所述轻链可变区包含如SEQ ID NO：61所示氨基酸序列，或与SEQ ID NO：61的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：61组成。27) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 169, or has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 169 , 96%, 97%, 98% or 99% identical amino acid sequence, or consist of SEQ ID NO: 169, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or with SEQ ID NO: 169 The amino acid sequence of NO: 61 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO: 61 composition.
在一些实施方案中，上述抗体或其抗原结合片段还包含来自人抗体胚系共有序列的重链和/或轻链恒定区序列。所述的轻链恒定区优选是人源的κ或λ链恒定区。重链恒定区可以是γ、μ、α、δ、或ε链，在一些实施方案中，所述的重链恒定区优选来自人源IgG1、IgG2、IgG3、IgG4的恒定区序列。在一个实施方案中，所述轻链恒定区包含SEQ ID NO：49或50所示的序列，或者由所述序列组成。在另一个实施方案中，所述重链恒定区包含SEQ ID NO：47所示的Fc序列。 再一个实施方案中，所述重链恒定区包含SEQ ID NO：48所示的序列，或者由所述序列组成。In some embodiments, the aforementioned antibodies or antigen-binding fragments thereof further comprise heavy and/or light chain constant region sequences derived from human antibody germline consensus sequences. The light chain constant region is preferably a human κ or λ chain constant region. The heavy chain constant region can be γ, μ, α, δ, or ε chain, and in some embodiments, the heavy chain constant region is preferably derived from the constant region sequence of human IgG1, IgG2, IgG3, IgG4. In one embodiment, the light chain constant region comprises or consists of the sequence shown in SEQ ID NO: 49 or 50. In another embodiment, the heavy chain constant region comprises the Fc sequence shown in SEQ ID NO:47. In yet another embodiment, the heavy chain constant region comprises or consists of the sequence shown in SEQ ID NO: 48.
应当理解，也可以使用这些恒定区结构域的序列变体，例如包含一个或多个氨基酸修饰，其中氨基酸位点是应Kabat等人的(1991)EU索引系统标识。It will be appreciated that sequence variants of these constant region domains may also be used, for example comprising one or more amino acid modifications, wherein the amino acid positions are identified according to the EU index system of Kabat et al. (1991).
在一个具体实施方案中，本发明提供了特异性结合ROR1的抗ROR1抗体及其抗原结合片段，其包含重链和轻链，其中：In a specific embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising a heavy chain and a light chain, wherein:
1)所述重链包含如SEQ ID NO：72所示氨基酸序列，或与SEQ ID NO：72的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：72组成，所述轻链包含如SEQ ID NO：71所示氨基酸序列，或与SEQ ID NO：71的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：71组成；1) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 71, or an amino acid sequence with SEQ ID NO: 71 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 71;
2)所述重链包含如SEQ ID NO：74所示氨基酸序列，或与SEQ ID NO：74的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：74组成，所述轻链包含如SEQ ID NO：73所示氨基酸序列，或与SEQ ID NO：73的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：73组成；2) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 74, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 74 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 74, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 73, or an amino acid sequence with SEQ ID NO: 73 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 73;
3)所述重链包含如SEQ ID NO：76所示氨基酸序列，或与SEQ ID NO：76的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：76组成，所述轻链包含如SEQ ID NO：75所示氨基酸序列，或与SEQ ID NO：75的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：75组成；3) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 76, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 76 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 76, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 75, or an amino acid sequence with SEQ ID NO: 75 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 75;
4)所述重链包含如SEQ ID NO：78所示氨基酸序列，或与SEQ ID NO：78的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：78组成，所述轻链包含如SEQ ID NO：77所示氨基酸序列，或与SEQ ID NO：77的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：77组成；4) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 78, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 78 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 78, the light chain comprising an amino acid sequence as shown in SEQ ID NO: 77, or an amino acid sequence with SEQ ID NO: 77 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 77;
5)所述重链包含如SEQ ID NO：80所示氨基酸序列，或与SEQ ID NO：80的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：80组成，所述轻链包含如SEQ ID NO：79所示氨基酸序列，或与SEQ ID NO：79的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：79组成；5) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 80, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 80 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 80, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 79, or an amino acid sequence with SEQ ID NO: 79 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 79;
6)所述重链包含如SEQ ID NO：82所示氨基酸序列，或与SEQ ID NO：82的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：82组成，所述轻链包含如SEQ ID NO：81所示氨基酸序列，或与SEQ ID NO：81的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性 的氨基酸序列，或由SEQ ID NO：81组成；6) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 82, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 82 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 82, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 81, or an amino acid sequence identical to SEQ ID NO: 81 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity The amino acid sequence of, or consists of SEQ ID NO: 81;
7)所述重链包含如SEQ ID NO：84所示氨基酸序列，或与SEQ ID NO：84的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：84组成，所述轻链包含如SEQ ID NO：83所示氨基酸序列，或与SEQ ID NO：83的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：83组成；7) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 84, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 84 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 84, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 83, or an amino acid sequence with SEQ ID NO: 83 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 83;
8)所述重链包含如SEQ ID NO：86所示氨基酸序列，或与SEQ ID NO：86的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：86组成，所述轻链包含如SEQ ID NO：73所示氨基酸序列，或与SEQ ID NO：73的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：73组成；8) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 86, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 86 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 86, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 73, or an amino acid sequence with SEQ ID NO: 73 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 73;
9)所述重链包含如SEQ ID NO：88所示氨基酸序列，或与SEQ ID NO：88的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：88组成，所述轻链包含如SEQ ID NO：87所示氨基酸序列，或与SEQ ID NO：87的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：87组成；9) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 88, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 88 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 88, the light chain comprising the amino acid sequence shown in SEQ ID NO: 87, or an amino acid sequence with SEQ ID NO: 87 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 87;
10)所述重链包含如SEQ ID NO：90所示氨基酸序列，或与SEQ ID NO：90的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：90组成，所述轻链包含如SEQ ID NO：89所示氨基酸序列，或与SEQ ID NO：89的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：89组成；10) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 90, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 90 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 90, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 89, or an amino acid sequence with SEQ ID NO: 89 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 89;
11)所述重链包含如SEQ ID NO：175所示氨基酸序列，或与SEQ ID NO：175的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：175组成，所述轻链包含如SEQ ID NO：174所示氨基酸序列，或与SEQ ID NO：174的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：174组成；11) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 175, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 175 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 175, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 174, or an amino acid sequence with SEQ ID NO: 174 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 174;
12)所述重链包含如SEQ ID NO：177所示氨基酸序列，或与SEQ ID NO：177的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：177组成，所述轻链包含如SEQ ID NO：176所示氨基酸序列，或与SEQ ID NO：176的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：176组成；12) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 177, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 177 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 177, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 176, or an amino acid sequence with SEQ ID NO: 176 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 176;
13)所述重链包含如SEQ ID NO：171所示氨基酸序列，或与SEQ ID NO：171的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：171组成，所述轻链包含如SEQ ID NO：170所示氨基酸序列，或与SEQ ID NO： 170的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：170组成；13) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 171, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 171 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 171, said light chain comprising the amino acid sequence shown in SEQ ID NO: 170, or with SEQ ID NO: The amino acid sequence of 170 has an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, or consists of, SEQ ID NO: 170;
14)所述重链包含如SEQ ID NO：173所示氨基酸序列，或与SEQ ID NO：173的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：173组成，所述轻链包含如SEQ ID NO：172所示氨基酸序列，或与SEQ ID NO：172的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：172组成；14) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 173, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 173 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 173, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 172, or an amino acid sequence with SEQ ID NO: 172 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 172;
15)所述重链包含如SEQ ID NO：179所示氨基酸序列，或与SEQ ID NO：179的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：179组成，所述轻链包含如SEQ ID NO：178所示氨基酸序列，或与SEQ ID NO：178的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：178组成；15) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 179, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 179 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 179, the light chain comprising the amino acid sequence shown in SEQ ID NO: 178, or an amino acid sequence with SEQ ID NO: 178 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 178;
16)所述重链包含如SEQ ID NO：191所示氨基酸序列，或与SEQ ID NO：191的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：191组成，所述轻链包含如SEQ ID NO：190所示氨基酸序列，或与SEQ ID NO：190的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：190组成；16) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 191, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 191 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 191, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 190, or an amino acid sequence with SEQ ID NO: 190 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 190;
17)所述重链包含如SEQ ID NO：193所示氨基酸序列，或与SEQ ID NO：193的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：193组成，所述轻链包含如SEQ ID NO：192所示氨基酸序列，或与SEQ ID NO：192的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：192组成；17) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 193, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 193 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 193, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 192, or an amino acid sequence with SEQ ID NO: 192 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 192;
18)所述重链包含如SEQ ID NO：195所示氨基酸序列，或与SEQ ID NO：195的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：195组成，所述轻链包含如SEQ ID NO：194所示氨基酸序列，或与SEQ ID NO：194的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：194组成；18) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 195, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 195 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 195, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 194, or an amino acid sequence with SEQ ID NO: 194 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 194;
19)所述重链包含如SEQ ID NO：181所示氨基酸序列，或与SEQ ID NO：181的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：181组成，所述轻链包含如SEQ ID NO：180所示氨基酸序列，或与SEQ ID NO：180的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：180组成；19) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 181, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 181 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 181, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 180, or an amino acid sequence with SEQ ID NO: 180 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 180;
20)所述重链包含如SEQ ID NO：183所示氨基酸序列，或与SEQ ID NO：183的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列， 或由SEQ ID NO：183组成，所述轻链包含如SEQ ID NO：182所示氨基酸序列，或与SEQ ID NO：182的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：182组成；20) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 183, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 183 , 97%, 98% or 99% identical amino acid sequences, Or consist of SEQ ID NO: 183, said light chain comprises the amino acid sequence shown in SEQ ID NO: 182, or has at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 182 %, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consisting of SEQ ID NO: 182;
21)所述重链包含如SEQ ID NO：185所示氨基酸序列，或与SEQ ID NO：185的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：185组成，所述轻链包含如SEQ ID NO：184所示氨基酸序列，或与SEQ ID NO：184的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：184组成；21) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 185, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 185 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 185, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 184, or an amino acid sequence with SEQ ID NO: 184 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 184;
22)所述重链包含如SEQ ID NO：187所示氨基酸序列，或与SEQ ID NO：187的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：187组成，所述轻链包含如SEQ ID NO：186所示氨基酸序列，或与SEQ ID NO：186的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：186组成；22) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 187, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 187 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 187, the light chain comprising an amino acid sequence as shown in SEQ ID NO: 186, or an amino acid sequence with SEQ ID NO: 186 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 186;
23)所述重链包含如SEQ ID NO：72所示氨基酸序列，或与SEQ ID NO：72的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：72组成，所述轻链包含如SEQ ID NO：188所示氨基酸序列，或与SEQ ID NO：188的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：188组成；23) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 188, or an amino acid sequence with SEQ ID NO: 188 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 188;
24)所述重链包含如SEQ ID NO：72所示氨基酸序列，或与SEQ ID NO：72的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：72组成，所述轻链包含如SEQ ID NO：189所示氨基酸序列，或与SEQ ID NO：189的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：189组成；24) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 189, or an amino acid sequence with SEQ ID NO: 189 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 189;
25)所述重链包含如SEQ ID NO：82所示氨基酸序列，或与SEQ ID NO：82的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：82组成，所述轻链包含如SEQ ID NO：196所示氨基酸序列，或与SEQ ID NO：196的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：196组成；25) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 82, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 82 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 82, said light chain comprising an amino acid sequence as shown in SEQ ID NO: 196, or an amino acid sequence with SEQ ID NO: 196 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 196;
26)所述重链包含如SEQ ID NO：198所示氨基酸序列，或与SEQ ID NO：198的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：198组成，所述轻链包含如SEQ ID NO：197所示氨基酸序列，或与SEQ ID NO：197的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：197组成；或26) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 198, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 198 , an amino acid sequence of 97%, 98% or 99% identity, or consisting of SEQ ID NO: 198, the light chain comprising the amino acid sequence shown in SEQ ID NO: 197, or an amino acid sequence with SEQ ID NO: 197 An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 197; or
27)所述重链包含如SEQ ID NO：199所示氨基酸序列，或与SEQ ID NO：199的氨基酸序 列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：199组成，所述轻链包含如SEQ ID NO：81所示氨基酸序列，或与SEQ ID NO：81的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：81组成。27) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 199, or the amino acid sequence with SEQ ID NO: 199 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 199, the light The chain comprises the amino acid sequence shown in SEQ ID NO: 81, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 81 An amino acid sequence that is % or 99% identical, or consists of SEQ ID NO:81.
在前述任一项的抗体的某些实施方案中，该抗体是单克隆的。In certain embodiments of the antibody of any of the preceding, the antibody is monoclonal.
在前述任一项的抗体的某些实施方案中，该抗体是全长抗体。In certain embodiments of the antibody of any of the preceding, the antibody is a full length antibody.
在一个实施方案中，本发明的抗ROR1抗体是完整抗体，例如IgG1、IgG2、IgG3、IgG4抗体。在另一个实施方案中，本发明的抗ROR1抗体仅涵盖其抗原结合部分，例如：Fab、Fab'-SH、Fv、scFv或(Fab')2片段。In one embodiment, the anti-ROR1 antibody of the invention is a whole antibody, such as an IgG1, IgG2, IgG3, IgG4 antibody. In another embodiment, the anti-ROR1 antibody of the present invention only encompasses its antigen-binding portion, such as: Fab, Fab'-SH, Fv, scFv or (Fab') 2 fragments.
第二方面，本发明提供了靶向人ROR1的抗体偶联药物，所述抗体偶联药物具有以下优点：In the second aspect, the present invention provides an antibody-conjugated drug targeting human ROR1, which has the following advantages:
(1)结合表达人ROR1的靶细胞，与其具有高亲和力；(1) Binding to target cells expressing human ROR1 with high affinity;
(2)能够通过内吞作用进入细胞，杀伤靶细胞；在一些实施方案中，本发明的ADC具有高内吞效率；(2) capable of entering cells through endocytosis and killing target cells; in some embodiments, the ADC of the present invention has high endocytosis efficiency;
(3)治疗、预防、改善受试者与ROR1异常功能或表达相关的病症(例如癌症，诸如血液癌症、实体瘤)，或治疗、预防、改善所述疾病的一或多种症状；(3) Treating, preventing, or improving a subject's disease related to ROR1 abnormal function or expression (for example, cancer, such as blood cancer, solid tumor), or treating, preventing, or improving one or more symptoms of the disease;
(4)减少或抑制受试者(其具有表达ROR1的肿瘤)中肿瘤生长或进展；(4) reducing or inhibiting tumor growth or progression in a subject having a ROR1-expressing tumor;
(5)诱导表达ROR1的肿瘤消退(例如长期消退)；(5) Inducing regression (e.g., long-term regression) of tumors expressing ROR1;
(6)在表达ROR1的细胞中发挥细胞毒性活性；(6) Exercising cytotoxic activity in cells expressing ROR1;
在一个实施方案中，本申请提供了包含第一方面所述抗ROR1抗体的缀合物。在一个具体的实施方案中，可以与抗ROR1抗体缀合的分子例如是细胞毒性剂、免疫调节剂、显像剂、荧光蛋白、分子标志物、治疗性蛋白质、生物聚合物及寡核苷酸等。In one embodiment, the present application provides a conjugate comprising the anti-ROR1 antibody of the first aspect. In a specific embodiment, molecules that can be conjugated to an anti-ROR1 antibody are, for example, cytotoxic agents, immunomodulators, imaging agents, fluorescent proteins, molecular markers, therapeutic proteins, biopolymers, and oligonucleotides wait.
在一个实施方案中，本发明提供了一种抗体偶联药物(ADC)，其包含第一方面所述的抗ROR1抗体或其抗原结合片段和至少一种治疗活性物质或药物活性成分，具有Ab-(L-D)n的结构，其中：Ab为如本发明第一方面所述的结合ROR1的抗体或其抗原结合片段；L为接头；D为治疗活性物质或药物活性成分，n表示1-20的整数，例如1，2，3，4，5……。In one embodiment, the present invention provides an antibody-drug conjugate (ADC), which comprises the anti-ROR1 antibody or its antigen-binding fragment described in the first aspect and at least one therapeutically active substance or pharmaceutically active ingredient, having an Ab -The structure of (L-D)n, wherein: Ab is the antibody or antigen-binding fragment thereof that binds to ROR1 according to the first aspect of the present invention; L is a linker; D is a therapeutically active substance or a pharmaceutically active ingredient, and n represents 1-20 Integers, such as 1, 2, 3, 4, 5....
在一个实施方案中，所述抗体偶联药物包含多个D成分，所述多个D成分可以是不同治疗活性物质或药物活性成分的组合，或者是相同治疗活性物质或药物活性成分的组合。在一个实施方案中，所述抗体偶联药物具有1-20的药物/抗体比(DAR)，例如数值为2、3、4、5、6、7、8、9、10、11、12、13、14或15的DAR。在一个实施方案中，所述DAR为平均DAR。在一个实施方案中，平均DAR是1-15，例如1-10、2-8、2-6或3-5。在一个具体实施方案中，平均DAR为3.7。In one embodiment, the antibody drug conjugate comprises multiple D components, and the multiple D components may be a combination of different therapeutic active substances or pharmaceutical active ingredients, or a combination of the same therapeutic active substances or pharmaceutical active ingredients. In one embodiment, the antibody drug conjugate has a drug/antibody ratio (DAR) of 1-20, for example values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, DAR of 13, 14 or 15. In one embodiment, the DAR is an average DAR. In one embodiment, the average DAR is 1-15, such as 1-10, 2-8, 2-6 or 3-5. In a specific embodiment, the average DAR is 3.7.
在一个具体的实施方案中，所述治疗活性物质或药物活性成分是细胞毒素、植物毒素、小分 子毒素、放射性同位素、美登木素生物碱等。在一个具体实施方案中，所述细胞毒素是海兔毒素(dolastatin)及其auristatin类衍生物，例如0101(2-甲基丙胺酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧庚烷-4-基]-N-甲基-L-缬氨酰胺)、8261(2-甲基丙胺酰基-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-羧基-2-苯乙基]氨基}-1-甲氧基-2-甲基-3-氧丙基]吡咯烷-1-基}-3-甲氧基-5-甲基-1-氧庚烷-4-基]-N-甲基-L-缬氨酰胺)Dolastatin 10、Dolastatin 15、auristatin E、auristatin PE、monomethyl auristatin D(MMAD)、monomethyl auristatin E(MMAE)、monomethyl auristatin F(MMAF)、auristatin F苯二胺(AFP)、auristatin EB(AEB)、auristatin EFP(AEFP)、auristatin F羟丙基酰胺(AFHPA)。在另一个实施方案中，所述海兔毒素及其auristatin类衍生物是auristatin、多拉司他汀、MMAE、MMAF、auristatin F羟丙基酰胺或auristatin F苯二胺。In a particular embodiment, said therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a phytotoxin, a Toxins, radioisotopes, maytansinoids, etc. In a specific embodiment, the cytotoxin is dolastatin (dolastatin) and its auristatin derivatives, such as 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy Base-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1 ,3-Thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valline amide), 8261(2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy -2-Phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptyl Alk-4-yl]-N-methyl-L-valinamide) Dolastatin 10, Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D(MMAD), monomethyl auristatin E(MMAE), monomethyl auristatin F(MMAF) , auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AFHPA). In another embodiment, the dolastatin and its auristatin derivatives are auristatin, dolastatin, MMAE, MMAF, auristatin F hydroxypropylamide or auristatin F phenylenediamine.
在一个实施方案中，细胞毒素以非位点特异性方式或者位点特异性方式借助接头与抗ROR1抗体或其抗原结合片段共价连接。In one embodiment, the cytotoxin is covalently linked to the anti-ROR1 antibody or antigen-binding fragment thereof in a non-site-specific manner or via a linker in a site-specific manner.
在一个实施方案中，所述接头选自马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基(mc-vc-PAB)、乙酰基-赖氨酸-缬氨酸-瓜氨酸-对氨基苄氧羰基(AcLys-VC-PABC)、氨基PEG6-丙酰基、及马来酰亚胺己酸基(mc)、马来酰亚胺基丙酰基(MP)、缬氨酸-瓜氨酸(val-cit)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)、N-琥珀酰亚胺基4-(2-吡啶硫基)戊酸酯(SPP)、N-琥珀酰亚胺基4-(N-马来酰亚胺基甲基)-环己烷-1-羧酸酯(SMCC)、N-琥珀酰亚胺基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)、N-琥珀酰亚胺基-4-(2-吡啶基二硫代)丁酸酯(SPDB)、N-琥珀酰亚胺基3-(吡啶-2-基二硫代)-丙酸酯(SPDP)。In one embodiment, the linker is selected from maleimido-caproyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine- Valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propionyl, and maleimide caproyl (mc), maleimide propionyl (MP ), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2- Pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl Imino(4-iodo-acetyl)aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl Imino 3-(pyridin-2-yldithio)-propionate (SPDP).
在一个实施方案中，所述抗体偶联药物包含第一方面所述的抗ROR1抗体或其抗原结合片段和微管蛋白抑制剂(MMAE)。在进一步的实施方案中，MMAE通过MC-VC-PAB接头与抗ROR1抗体上半胱氨酸的巯基缀合，具有抗ROR1抗体-MC-VC-PAB-MMAE的结构。IgG1型抗体具有16对半胱氨酸残基，其中以12个链内和4个链间二硫键的形式存在。链间二硫键具有溶剂可及性，可以被还原剂还原形成八个巯基，进而成为偶联目标(McCombs J,Owen S.Antibody drug conjugates:design and selection of linker,payload and conjugation chemistry.AAPS J.2015；17:339-51)。In one embodiment, the antibody drug conjugate comprises the anti-ROR1 antibody or antigen-binding fragment thereof of the first aspect and tubulin inhibitor (MMAE). In a further embodiment, MMAE is conjugated to the sulfhydryl group of the cysteine on the anti-ROR1 antibody through an MC-VC-PAB linker, having the structure of anti-ROR1 antibody-MC-VC-PAB-MMAE. IgG1 antibodies have 16 pairs of cysteine residues, of which there are 12 intrachain and 4 interchain disulfide bonds. Interchain disulfide bonds are solvent-accessible, and can be reduced by reducing agents to form eight sulfhydryl groups, which then become coupling targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J .2015;17:339-51).
在一个具体实施方案中，所述抗体偶联药物包含抗ROR1单克隆抗体N99和MC-VC-PAB-MMAE，或由其组成。在进一步的实施方案中，MMAE通过MC-VC-PAB接头与N99上半胱氨酸的巯基缀合。In a specific embodiment, the antibody-drug conjugate comprises anti-ROR1 monoclonal antibody N99 and MC-VC-PAB-MMAE, or consists of them. In a further embodiment, MMAE is conjugated to the sulfhydryl group of cysteine on N99 through a MC-VC-PAB linker.
在另一个具体实施方案中，所述抗体偶联药物包含抗ROR1单克隆抗体N147-135和MC-VC-PAB-MMAE，或由其组成。在进一步的实施方案中，MMAE通过MC-VC-PAB接头与N147-135上半胱氨酸的巯基缀合。 In another specific embodiment, the antibody drug conjugate comprises or consists of anti-ROR1 monoclonal antibody N147-135 and MC-VC-PAB-MMAE. In a further embodiment, MMAE is conjugated to the sulfhydryl group of cysteine on N147-135 through the MC-VC-PAB linker.
第三方面，本发明提供了一种药物组合物，其包含(1)第一方面的抗体或其抗原结合片段或第二方面的抗体偶联药物，以及(2)可药用载体。In a third aspect, the present invention provides a pharmaceutical composition comprising (1) the antibody or antigen-binding fragment thereof of the first aspect or the antibody-drug conjugate of the second aspect, and (2) a pharmaceutically acceptable carrier.
第四方面，本发明提供了分离的多核苷酸分子，其编码第一方面所述的任一抗体或其抗原结合片段。In a fourth aspect, the present invention provides an isolated polynucleotide molecule encoding any antibody or antigen-binding fragment thereof of the first aspect.
第五方面，本发明提供了载体，其包含第四方面的核酸分子。在一个实施方案中，所述载体是表达载体。In a fifth aspect, the present invention provides a vector comprising the nucleic acid molecule of the fourth aspect. In one embodiment, the vector is an expression vector.
第六方面，本发明提供了宿主细胞，其包含第五方面的载体或第四发明的核酸分子。在一些实施方案中，该宿主细胞是原核的，例如大肠杆菌。在其它实施方案中，该宿主细胞是真核的，例如293细胞，CHO细胞，酵母细胞，或植物细胞。In a sixth aspect, the present invention provides a host cell comprising the vector of the fifth aspect or the nucleic acid molecule of the fourth invention. In some embodiments, the host cell is prokaryotic, such as E. coli. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells.
第七方面，本发明提供了一种在有需要的受试者中预防或治疗与ROR1异常表达相关的疾病的方法，包括向所述受试者施用预防有效量或治疗有效量的本发明抗体或其抗原结合片段、或预防有效量或治疗有效量的本发明的抗体偶联药物、或预防有效量或治疗有效量的本发明药物组合物。In the seventh aspect, the present invention provides a method for preventing or treating a disease related to abnormal expression of ROR1 in a subject in need, comprising administering to the subject a prophylactically effective amount or a therapeutically effective amount of the antibody of the present invention or an antigen-binding fragment thereof, or a prophylactically or therapeutically effective amount of the antibody-conjugated drug of the present invention, or a prophylactically or therapeutically effective amount of the pharmaceutical composition of the present invention.
在一个实施方案中，所述与ROR1异常表达相关的疾病是高表达ROR1的癌症，例如慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、套细胞淋巴瘤、肾细胞癌、结肠癌、乳腺癌、神经母细胞瘤、肺癌、头颈癌和黑色素瘤。在一个具体实施方案中，其中所述肺癌是非小细胞肺癌，所述乳腺癌是三阴性乳腺癌。In one embodiment, the disease associated with the abnormal expression of ROR1 is a cancer with high ROR1 expression, such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer , breast cancer, neuroblastoma, lung cancer, head and neck cancer, and melanoma. In a specific embodiment, wherein said lung cancer is non-small cell lung cancer, said breast cancer is triple negative breast cancer.
在一些实施方案中，本发明的ADC分子或药物组合物还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用，用于本文所述的用途，例如用于预防和/或治疗本文提及的相关疾病或病症。In some embodiments, the ADC molecules or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents, for the uses described herein, e.g., for prophylaxis and and/or treat a related disease or condition mentioned herein.
在一个具体实施方案中，本发明提供了一种杀死表达ROR1的细胞或者抑制表达ROR1的细胞生长的方法，包括使所述细胞接触有效量的本发明抗体或其抗原结合片段、或有效量的本发明的抗体偶联药物、或有效量的本发明药物组合物。In a specific embodiment, the present invention provides a method of killing ROR1-expressing cells or inhibiting the growth of ROR1-expressing cells, comprising contacting said cells with an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, or an effective amount The antibody-conjugated drug of the present invention, or an effective amount of the pharmaceutical composition of the present invention.
第八方面，本发明提供了抗ROR1抗体或其抗原结合片段在制备用于预防或治疗癌症的抗体偶联药物中的用途。In an eighth aspect, the present invention provides the use of an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of an antibody-coupled drug for preventing or treating cancer.
本发明还提供了包含抗ROR1抗体或其抗原结合片段的抗体偶联药物在制备用于预防或治疗癌症的药物中的用途。The present invention also provides the use of an antibody-coupled drug comprising an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of a drug for preventing or treating cancer.
附图说明Description of drawings
图1A-1F显示了基于FACS法测定的本申请获得的各个抗体克隆Fab结合huROR1-HEK293细胞的结合活性；图1A显示了克隆NW37 Fab和NW79 Fab结合huROR1-HEK293细胞的结合活性；图1B显示了克隆N137 Fab、N174 Fab和N147 Fab结合huROR1-HEK293细胞的结合活性；图1C显示了克隆N218 Fab和N204 Fab结合huROR1-HEK293细胞的结合活性；图1D显示了克隆N99 Fab结合huROR1-HEK293细胞的结合活性；图1E显示了克隆N31 Fab结合 huROR1-HEK293细胞上的结合活性；图1F显示了克隆N100 Fab和NW29 Fab结合huROR1-HEK293细胞的结合活性；NC表示阴性对照，MFI表示中位数荧光强度。Figure 1A-1F shows the binding activity of each antibody clone Fab obtained in the present application to huROR1-HEK293 cells determined based on the FACS method; Figure 1A shows the binding activity of clone NW37 Fab and NW79 Fab to huROR1-HEK293 cells; Figure 1B shows The binding activity of clones N137 Fab, N174 Fab and N147 Fab to huROR1-HEK293 cells; Figure 1C shows the binding activity of clones N218 Fab and N204 Fab to huROR1-HEK293 cells; Figure 1D shows the binding activity of clone N99 Fab to huROR1-HEK293 cells Binding activity; Figure 1E shows the binding activity of clone N31 Fab Binding activity on huROR1-HEK293 cells; Figure 1F shows the binding activity of clones N100 Fab and NW29 Fab to huROR1-HEK293 cells; NC indicates negative control, MFI indicates median fluorescence intensity.
图2A-2K显示了本申请抗体的SEC-HPLC结果；图2A显示了N137的SEC-HPLC结果；图2B显示了N147的SEC-HPLC结果；图2C显示了N218的SEC-HPLC结果；图2D显示了N31的SEC-HPLC结果；图2E显示了N99的SEC-HPLC结果；图2F显示了NW37的SEC-HPLC结果；图2G显示了N100的SEC-HPLC结果；图2H显示了N174的SEC-HPLC结果；图2I显示了N204的SEC-HPLC结果；图2J显示了NW29的SEC-HPLC结果；图2K显示了NW79的SEC-HPLC结果。Figure 2A-2K shows the SEC-HPLC result of the antibody of the present application; Figure 2A shows the SEC-HPLC result of N137; Figure 2B shows the SEC-HPLC result of N147; Figure 2C shows the SEC-HPLC result of N218; Figure 2D Figure 2E shows the SEC-HPLC results of N99; Figure 2F shows the SEC-HPLC results of NW37; Figure 2G shows the SEC-HPLC results of N100; Figure 2H shows the SEC-HPLC results of N174 HPLC results; Figure 2I shows the SEC-HPLC results of N204; Figure 2J shows the SEC-HPLC results of NW29; Figure 2K shows the SEC-HPLC results of NW79.
图3A-3F显示了基于ELISA法测定的本申请抗体与抗原蛋白huROR1-His的结合活性；图3A显示了N100、N137和N147与抗原蛋白huROR1-His的结合活性；图3B显示了N204和N218与抗原蛋白huROR1-His的结合活性；图3C显示了N31与抗原蛋白huROR1-His的结合活性；图3D显示了NW29、N99和NW37与抗原蛋白huROR1-His的结合活性；图3E显示了N174与抗原蛋白huROR1-His的结合活性；图3F显示了NW79与抗原蛋白huROR1-His的结合活性。Figure 3A-3F shows the binding activity of the antibody of the present application to the antigenic protein huROR1-His determined based on the ELISA method; Figure 3A shows the binding activity of N100, N137 and N147 to the antigenic protein huROR1-His; Figure 3B shows the binding activity of N204 and N218 The binding activity with the antigenic protein huROR1-His; Figure 3C shows the binding activity of N31 with the antigenic protein huROR1-His; Figure 3D shows the binding activity of NW29, N99 and NW37 with the antigenic protein huROR1-His; Figure 3E shows the binding activity of N174 with the antigenic protein huROR1-His The binding activity of antigenic protein huROR1-His; Fig. 3F shows the binding activity of NW79 and antigenic protein huROR1-His.
图4A-4D显示了基于FACS法测定的本申请抗体与A549细胞的结合活性；图4A显示了N100、N137、N174和N147与A549细胞的结合活性；图4B显示了N204、N218和N31与A549细胞的结合活性；图4C显示了N99、NW29和NW37与A549细胞的结合活性；图4D显示了NW79与A549细胞的结合活性。Figure 4A-4D shows the binding activity of the antibody of the present application and A549 cells measured based on the FACS method; Figure 4A shows the binding activity of N100, N137, N174 and N147 and A549 cells; Figure 4B shows the binding activity of N204, N218 and N31 and A549 Cell binding activity; Figure 4C shows the binding activity of N99, NW29 and NW37 to A549 cells; Figure 4D shows the binding activity of NW79 to A549 cells.
图5A-5C显示了基于FACS法测定的本申请抗体与huROR1-HEK293细胞的结合活性；图5A显示了N100、N174、N204、N137、N147、N218和N31与huROR1-HEK293细胞的结合活性；图5B显示了N99、NW29和NW37与huROR1-HEK293细胞的结合活性；图5C显示了NW79与huROR1-HEK293细胞的结合活性。Figures 5A-5C show the binding activity of the antibody of the present application to huROR1-HEK293 cells determined based on the FACS method; Figure 5A shows the binding activity of N100, N174, N204, N137, N147, N218 and N31 to huROR1-HEK293 cells; 5B shows the binding activity of N99, NW29 and NW37 to huROR1-HEK293 cells; Figure 5C shows the binding activity of NW79 to huROR1-HEK293 cells.
图6A-6D显示了基于ELISA法测定的本申请抗体与抗原蛋白MusROR1-His的结合活性；图6A显示了N100、N174、N137和N147与抗原蛋白MusROR1-His的结合活性；图6B显示了N204、N31和N218与抗原蛋白MusROR1-His的结合活性；图6C显示了NW29、NW37和N99与抗原蛋白MusROR1-His的结合活性；图6D显示了NW79与抗原蛋白MusROR1-His的结合活性。Figure 6A-6D shows the binding activity of the antibody of the present application and the antigenic protein MusROR1-His determined based on the ELISA method; Figure 6A shows the binding activity of N100, N174, N137 and N147 and the antigenic protein MusROR1-His; Figure 6B shows the binding activity of N204 , N31 and N218 and the binding activity of the antigenic protein MusROR1-His; Figure 6C shows the binding activity of NW29, NW37 and N99 and the antigenic protein MusROR1-His; Figure 6D shows the binding activity of NW79 and the antigenic protein MusROR1-His.
图7A-7D显示了基于ELISA法测定的本申请抗体与抗原蛋白huROR2-His的结合活性；图7A显示了N100、N174、N137和N147不与抗原蛋白huROR2-His结合；图7B显示了N204、N31和N218与抗原蛋白huROR2-His的结合活性；图7C显示了NW29、NW37和N99与抗原蛋白huROR2-His的结合活性；图7D显示了NW79与抗原蛋白huROR2-His的结合活性。Figure 7A-7D shows the binding activity of the antibody of the present application to the antigenic protein huROR2-His determined based on the ELISA method; Figure 7A shows that N100, N174, N137 and N147 are not combined with the antigenic protein huROR2-His; Figure 7B shows that N204, The binding activity of N31 and N218 to the antigenic protein huROR2-His; Figure 7C shows the binding activity of NW29, NW37 and N99 to the antigenic protein huROR2-His; Figure 7D shows the binding activity of NW79 to the antigenic protein huROR2-His.
图8A-8B显示了基于FACS法测定的本申请抗体在huROR1-HEK293细胞上的内吞效率；图8A显示了N204、N147和NW37在huROR1-HEK293细胞上的内吞效率；图8B显示了N100、N174、NW29、NW79、N137、N218、N31和N99在huROR1-HEK293细胞上的内吞效率。Figure 8A-8B shows the endocytic efficiency of the antibody of the present application on huROR1-HEK293 cells determined based on the FACS method; Figure 8A shows the endocytic efficiency of N204, N147 and NW37 on huROR1-HEK293 cells; Figure 8B shows the endocytic efficiency of N100 , N174, NW29, NW79, N137, N218, N31 and N99 endocytosis efficiencies on huROR1-HEK293 cells.
图9A-9F显示了基于Fab-Zap法测定的本申请抗体在huROR1-HEK293细胞上的内吞效率；图9A显示了N31和N99在huROR1-HEK293细胞上的内吞效率；图9B显示了N174和N218 在huROR1-HEK293细胞上的内吞效率；图9C显示了N147在huROR1-HEK293细胞上的内吞效率；图9D显示了N100和N137在huROR1-HEK293细胞上的内吞效率；图9E显示了N204和NW37在huROR1-HEK293细胞上的内吞效率；图9F显示了NW29和NW79在huROR1-HEK293细胞上的内吞效率。Figure 9A-9F shows the endocytic efficiency of the antibody of the present application on huROR1-HEK293 cells determined based on the Fab-Zap method; Figure 9A shows the endocytic efficiency of N31 and N99 on huROR1-HEK293 cells; Figure 9B shows N174 and N218 Endocytic efficiency on huROR1-HEK293 cells; Figure 9C shows the endocytic efficiency of N147 on huROR1-HEK293 cells; Figure 9D shows the endocytic efficiency of N100 and N137 on huROR1-HEK293 cells; Figure 9E shows N204 and NW37 endocytosis efficiencies on huROR1-HEK293 cells; Figure 9F shows the endocytosis efficiencies of NW29 and NW79 on huROR1-HEK293 cells.
图10A-10D显示了基于FACS法测定的亲和力成熟后抗体与A549细胞的结合活性；图10A显示了N31-6、N31-14、N31-18和N31-56与A549细胞的结合活性；图10B显示了N37-37、N37-99、N37-113和N37-136与A549细胞的结合活性；图10C显示了N137-47、N137-53、N137-60和N137-72与A549细胞的结合活性；图10D显示了N147-2、N147-86、N148-93和N147-135与A549肿瘤细胞的结合活性。Figures 10A-10D show the binding activity of antibodies to A549 cells after affinity maturation based on FACS method; Figure 10A shows the binding activities of N31-6, N31-14, N31-18 and N31-56 to A549 cells; Figure 10B Shows the binding activity of N37-37, N37-99, N37-113 and N37-136 to A549 cells; Figure 10C shows the binding activity of N137-47, N137-53, N137-60 and N137-72 to A549 cells; Figure 10D shows the binding activity of N147-2, N147-86, N148-93 and N147-135 to A549 tumor cells.
图11A-11E显示了基于Fab-Zap法测定的亲和力成熟后抗体在huROR1-HEK293细胞上的内吞效率；图11A显示了N31-6在huROR1-HEK293细胞上的内吞效率；图11B显示了NW37-37在huROR1-HEK293细胞上的内吞效率；图11C显示了NW137-72在huROR1-HEK293细胞上的内吞效率；图11D显示了N147-135在huROR1-HEK293细胞上的内吞效率；图11E显示了N147-2在huROR1-HEK293细胞上的内吞效率。Figure 11A-11E shows the endocytic efficiency of antibodies on huROR1-HEK293 cells after affinity maturation based on the Fab-Zap method; Figure 11A shows the endocytic efficiency of N31-6 on huROR1-HEK293 cells; Figure 11B shows The endocytic efficiency of NW37-37 on huROR1-HEK293 cells; Figure 11C shows the endocytic efficiency of NW137-72 on huROR1-HEK293 cells; Figure 11D shows the endocytic efficiency of N147-135 on huROR1-HEK293 cells; Figure 1 IE shows the endocytic efficiency of N147-2 on huROR1-HEK293 cells.
图12A-12D显示了本申请ADC与A549细胞和HT-29细胞结合的结果；图12A显示N99-MMAE与A549细胞的结合情况；图12B显示了N147-135-MMAE与A549细胞的结合情况；图12C显示N99-MMAE与HT-29细胞的结合情况；图12D显示了N147-135-MMAE与HT-29细胞的结合情况。Figures 12A-12D show the results of the combination of the ADC of the present application with A549 cells and HT-29 cells; Figure 12A shows the combination of N99-MMAE and A549 cells; Figure 12B shows the combination of N147-135-MMAE and A549 cells; Figure 12C shows the binding of N99-MMAE to HT-29 cells; Figure 12D shows the binding of N147-135-MMAE to HT-29 cells.
图13A-13B显示了基于MTS法检测的本申请ADC在两个肿瘤细胞A549和HT-29上的杀伤效果；图13A显示了N99-MMAE和N147-135-MMAE在A549细胞上的杀伤效果；图13B显示了N99-MMAE和N147-135-MMAE在HT-29细胞上的杀伤效果。Figure 13A-13B shows the killing effect of the ADC of the present application on two tumor cells A549 and HT-29 detected based on the MTS method; Figure 13A shows the killing effect of N99-MMAE and N147-135-MMAE on A549 cells; Figure 13B shows the killing effect of N99-MMAE and N147-135-MMAE on HT-29 cells.
图14A-14F显示了基于CCK8法检测的本申请ADC在三个肿瘤细胞Jeko-1、MDA-MB-468和NCI-H1944上的杀伤效果；图14A显示了N99-MMAE在Jeko-1细胞上的杀伤效果；图14B显示了N99-MMAE在MDA-MB-468细胞上的杀伤效果；图14C显示了N99-MMAE在NCI-H1944细胞上的杀伤效果；图14D显示了N147-135-MMAE在Jeko-1细胞上的杀伤效果；图14E显示了N147-135-MMAE在MDA-MB-468细胞上的杀伤效果；图14F显示了N147-135-MMAE在NCI-H1944细胞上的杀伤效果。Figure 14A-14F shows the killing effect of the ADC of the present application detected based on CCK8 method on three tumor cells Jeko-1, MDA-MB-468 and NCI-H1944; Figure 14A shows N99-MMAE on Jeko-1 cells Figure 14B shows the killing effect of N99-MMAE on MDA-MB-468 cells; Figure 14C shows the killing effect of N99-MMAE on NCI-H1944 cells; Figure 14D shows the killing effect of N147-135-MMAE on Killing effect on Jeko-1 cells; Figure 14E shows the killing effect of N147-135-MMAE on MDA-MB-468 cells; Figure 14F shows the killing effect of N147-135-MMAE on NCI-H1944 cells.
图15显示了基于CCK8法测定的本申请ADC在huROR1-HEK293细胞上的抗原依赖杀伤效果。Figure 15 shows the antigen-dependent killing effect of the ADC of the present application on huROR1-HEK293 cells determined based on the CCK8 method.
图16A-16D显示了基于FACS法检测的本申请ADC在两个肿瘤细胞A549和HT-29上的内吞效率；图16A显示了N99-MMAE在A549细胞上的内吞效率，图16B显示了N147-135-MMAE在A549细胞上的内吞效率；图16C显示了N99-MMAE在HT-29细胞上的内吞效率；图16D显示了N147-135-MMAE在HT-29细胞上的内吞效率。Figure 16A-16D shows the endocytic efficiency of the ADC of the present application detected based on FACS method on two tumor cells A549 and HT-29; Figure 16A shows the endocytic efficiency of N99-MMAE on A549 cells, Figure 16B shows The endocytic efficiency of N147-135-MMAE on A549 cells; Figure 16C shows the endocytic efficiency of N99-MMAE on HT-29 cells; Figure 16D shows the endocytic efficiency of N147-135-MMAE on HT-29 cells efficiency.
图17A-17C显示了本申请ADC在小鼠体内的抑瘤效果；图17A显示了实验周期内不同实验 组小鼠体内的肿瘤体积变化，箭头标注为给药的时间点；图17B显示了实验周期内不同实验组小鼠的体重变化；图17C显示了实验结束后不同实验组小鼠体内的肿瘤重量。Figure 17A-17C has shown the antitumor effect of the ADC of the present application in mice; Figure 17A has shown different experiments in the experimental cycle The change of tumor volume in the mice of different groups, and the arrow marks the time points of administration; Figure 17B shows the body weight changes of mice in different experimental groups during the experimental period; Figure 17C shows the weight of tumors in mice of different experimental groups after the experiment .
图18A-18C显示了本申请ADC在A549肿瘤模型的小鼠体内的抑瘤效果；图18A显示了实验周期内不同实验组小鼠体内的肿瘤体积变化，箭头标注为给药的时间点；图18B显示了实验周期内不同实验组小鼠的体重变化；图18C显示了实验结束后不同实验组小鼠体内的肿瘤重量。Figures 18A-18C show the anti-tumor effect of the ADC of the present application in mice of the A549 tumor model; Figure 18A shows the tumor volume changes in different experimental groups of mice within the experimental period, and the arrows mark the time points of administration; 18B shows the body weight changes of mice in different experimental groups during the experimental period; FIG. 18C shows the tumor weight in mice of different experimental groups after the experiment.
图19A-19B显示了本申请ADC在NCI-N87肿瘤模型的小鼠体内的抑瘤效果；图19A显示了实验周期内不同实验组小鼠体内的肿瘤体积变化，箭头标注为给药的时间点；图19B显示了实验周期内不同实验组小鼠的体重变化。Figures 19A-19B show the tumor-suppressing effect of the ADC of the present application in mice of the NCI-N87 tumor model; Figure 19A shows the tumor volume changes in different experimental groups of mice within the experimental period, and the arrows mark the time points of administration ; Figure 19B shows the body weight changes of mice in different experimental groups during the experimental period.
图20A-20B显示了本申请ADC在MDA-MB-231肿瘤模型的小鼠体内的抑瘤效果；图20A显示了实验周期内不同实验组小鼠体内的肿瘤体积变化，箭头标注为给药的时间点；图20B显示了实验周期内不同实验组小鼠的体重变化。Figures 20A-20B show the tumor-suppressive effect of the ADC of the present application in mice of the MDA-MB-231 tumor model; Figure 20A shows the tumor volume changes in mice of different experimental groups within the experimental period, and the arrows are marked as the administration Time points; Figure 20B shows the body weight changes of mice in different experimental groups during the experimental period.
图21A-21B显示了本申请ADC在MDA-MB-468肿瘤模型的小鼠体内的抑瘤效果；图21A显示了实验周期内不同实验组小鼠体内的肿瘤体积变化，箭头标注为给药的时间点；图21B显示了实验周期内不同实验组小鼠的体重变化。Figures 21A-21B show the tumor-suppressive effect of the ADC of the present application in mice of the MDA-MB-468 tumor model; Figure 21A shows the changes in tumor volume in mice of different experimental groups within the experimental period, and the arrows are marked as administration Time points; Figure 21B shows the body weight changes of mice in different experimental groups during the experimental period.
图22A-22B显示了本申请ADC在Jeko-1肿瘤模型的小鼠体内的抑瘤效果；图22A显示了实验周期内不同实验组小鼠体内的肿瘤体积变化，箭头标注为给药的时间点；图22B显示了实验周期内不同实验组小鼠的体重变化。Figures 22A-22B show the tumor-suppressing effect of the ADC of the present application in mice of the Jeko-1 tumor model; Figure 22A shows the tumor volume changes in different experimental groups of mice within the experimental period, and the arrows mark the time points of administration ; Figure 22B shows the body weight changes of mice in different experimental groups during the experimental period.
发明详述Detailed description of the invention
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methodology and experimental conditions in this specification, as such methods and conditions may vary. Additionally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
I.定义I. Definition
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit 10% less than the stated numerical value and an upper limit 10% greater than the stated numerical value.
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。When the term "and/or" is used to link two or more optional items, it should be understood as meaning any one of the optional items or any two or more items of the optional items.
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。 As used herein, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Herein, when the term "comprising" or "comprises" is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.
术语“ROR1”指任何重组体或天然存在形式的受体酪氨酸激酶样孤儿受体1(ROR1)、其变体或同系物，所述变体或同系物维持ROR1活性的例如至少50％、80％、90％、95％、96％、97％、98％、99％或100％的活性。所述变体或同系物相比于天然存在的ROR1蛋白在完整序列或部分序列(例如，50、100、150或200个连续氨基酸部分)具有至少90％、95％、96％、97％、98％、99％或100％氨基酸序列同一性。在一个实施方案中，ROR1蛋白包括Uniprot ID：Q01973的氨基酸序列。The term "ROR1" refers to any recombinant or naturally occurring form of receptor tyrosine kinase-like orphan receptor 1 (ROR1), a variant or homolog thereof, which maintains, e.g., at least 50% of, the activity of ROR1 , 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity. Said variant or homologue has at least 90%, 95%, 96%, 97%, 90%, 95%, 96%, 97% or more of the complete or partial sequence (e.g., 50, 100, 150 or 200 contiguous amino acid portions) compared to the naturally occurring ROR1 protein. 98%, 99% or 100% amino acid sequence identity. In one embodiment, the ROR1 protein comprises the amino acid sequence of Uniprot ID: Q01973.
ROR1在胚胎中高度表达，之后其表达水平在成人阶段显著下降。但是发现在多种血液癌症和实体瘤中ROR1的表达显著提高。高表达ROR1的血液癌症包括B细胞慢性淋巴细胞白血病(CLL)，急性淋巴细胞白血病(ALL)，非霍奇金淋巴瘤(NHL)和髓系血液癌症。在实体瘤中，表达ROR1的癌症类型包括结乳腺癌、肠癌、肺癌、胰腺癌、卵巢癌等多种癌症。ROR1 is highly expressed in the embryo, after which its expression level decreases significantly in the adult stage. However, ROR1 expression was found to be significantly elevated in a variety of hematologic cancers and solid tumors. Blood cancers that highly express ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid blood cancers. In solid tumors, cancer types expressing ROR1 include breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer and other cancers.
术语“抗体”在本文中以最广意义使用并且涵盖多种抗体结构物，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要它们显示出所需的抗原结合活性即可。完整抗体通常将包含至少两条全长重链和两条全长轻链，但在某些情况下可包括较少的链，例如骆驼中天然存在的抗体可仅包含重链。The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they It is sufficient that the desired antigen-binding activity is exhibited. A whole antibody will usually comprise at least two full-length heavy chains and two full-length light chains, but in some cases may comprise fewer chains, for example antibodies naturally occurring in camelids may comprise only heavy chains.
术语“抗ROR1抗体”是指特异性与ROR1结合的抗体分子，其能够抑制ROR1活性。相对于不存在ROR1抗体，抗ROR1抗体能够抑制ROR1活性，例如，通过至少部分地或完全地阻断ROR1的刺激，减少、预防或延迟ROR1的活化，或失活、脱敏或下调ROR1的信号转导、活性或量。在一些实施方案中，相比于对照，抗体可以抑制ROR1活性的10％、20％、30％、40％、50％、60％、70％、80％、90％或更多。The term "anti-ROR1 antibody" refers to an antibody molecule that specifically binds to ROR1 and is capable of inhibiting ROR1 activity. The anti-ROR1 antibody is capable of inhibiting ROR1 activity relative to the absence of the ROR1 antibody, for example, by at least partially or completely blocking stimulation of ROR1, reducing, preventing or delaying activation of ROR1, or inactivating, desensitizing or downregulating signaling of ROR1 transduction, activity or amount. In some embodiments, the antibody can inhibit ROR1 activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control.
术语“抗体片段”指与完整抗体不同的分子，其包含完整抗体的一部分且能够结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’)2；双抗体；线性抗体；单链抗体(例如scFv)；单结构域抗体；双价或双特异性抗体或其片段；骆驼科抗体(重链抗体)；和由抗体片段形成的多特异性抗体。The term "antibody fragment" refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and is capable of binding an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single chain antibodies (e.g. scFv); Specific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and multispecific antibodies formed from antibody fragments.
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的构架区(FR)和三个互补决定区(CDR)(参见，例如，Kindt等Kuby Immunology，6th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域足以给予抗原结合特异性。The term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies typically have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co. p. 91 (2007)). A single VH or VL domain is sufficient to confer antigen binding specificity.
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。可变结构域中的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。在一个给定的可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基 于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(http://imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。A "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point"). The CDRs are primarily responsible for binding to antigenic epitopes. The CDRs in a variable domain are generally referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. In a given variable domain amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including, for example, based on the three-dimensional structure of the antibody and Chothia of the topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997) ),base Kabat on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http://imgt.cines.fr/), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the above ways.
CDR也可以基于与参考CDR序列(例如本发明示例的CDR之任一)具有相同的AbM编号位置而确定。在一个实施方案中，本发明的抗体的CDR根据AbM编号方案确定位置。CDRs can also be determined based on having the same AbM numbering position as a reference CDR sequence (eg, any of the exemplified CDRs of the invention). In one embodiment, the CDRs of an antibody of the invention are positioned according to the AbM numbering scheme.
除非另有说明，否则在本发明中，当提及抗体可变区和CDR中的残基位置(包括重链可变区残基)时，是指根据AbM编号系统的编号位置。Unless otherwise stated, in the present invention, when referring to residue positions in antibody variable regions and CDRs, including heavy chain variable region residues, this refers to numbered positions according to the AbM numbering system.
“亲和力成熟的”抗体指在抗体的一个或多个CDR中具有一处或多处改变、导致该抗体对抗原的亲和力与没有这些改变的母本抗体相比有所改进的抗体。亲和力成熟的抗体可通过本领域已知方法来生成。An "affinity matured" antibody is one that has one or more alterations in one or more CDRs of the antibody that result in an improved affinity of the antibody for antigen compared to a parental antibody without these alterations. Affinity matured antibodies can be generated by methods known in the art.
“人抗体”或“全人抗体”或“全人源抗体”可以互换使用，其指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于下述抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。"Human antibody" or "fully human antibody" or "fully human antibody" are used interchangeably and refer to an antibody having an amino acid sequence corresponding to that of an antibody derived from a human Or human cells are produced or derived from non-human sources that utilize human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗原结合位点与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法如通过放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)测定。As used herein, the term "bind" or "specifically bind" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD method or surface plasmon resonance (SPR).
术语“半数有效浓度(EC50)”是指在特定的暴露时间后诱导在基线和最大值之间的50％的应答的药物、抗体或毒剂的浓度。The term "half effective concentration ( EC50 )" refers to the concentration of drug, antibody or poison that induces 50% of the response between baseline and maximum after a specified exposure time.
本文所述的术语“治疗剂”涵盖在预防或治疗肿瘤，例如癌症中有效的任何物质，包括化疗剂、细胞因子、血管生成抑制剂、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫抑制剂)。The term "therapeutic agent" as used herein encompasses any substance effective in the prevention or treatment of tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g. immunosuppressants).
术语“抗体偶联药物”或“ADC”是指以共价键偶联治疗活性物质或活性药物成分的抗体或抗体片段，从而使得治疗活性物质或活性药物成分靶向至抗体的结合靶标以表现出其药理学功能。治疗活性物质或活性药物成分可以是能够杀死ADC靶向的细胞(优选癌细胞)的细胞毒素。治疗活性物质、活性药物成分或细胞毒素的共价连接可以以非位点特异性方式利用接头进行，或者以位点特异性方式进行。The term "antibody drug conjugate" or "ADC" refers to an antibody or antibody fragment to which a therapeutically active substance or active pharmaceutical ingredient is covalently coupled such that the therapeutically active substance or active pharmaceutical ingredient is targeted to the antibody's binding target for expression its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin capable of killing cells targeted by the ADC, preferably cancer cells. The covalent linking of therapeutically active substances, active pharmaceutical ingredients or cytotoxins can take place in a non-site-specific manner using linkers, or in a site-specific manner.
术语“位点特异性偶联”是指将治疗活性物质或活性药物成分特异性连接到抗体的特定位点的连接方式。在一个实施方式中，所述偶联借助连接子完成。 The term "site-specific conjugation" refers to an attachment method that specifically attaches a therapeutically active substance or an active pharmaceutical ingredient to a specific site of an antibody. In one embodiment, the coupling is accomplished via a linker.
术语“细胞毒性剂”可以和“细胞毒素”互换使用，在本发明中指抑制或破坏细胞功能和/或引起细胞死亡或破坏的物质。在一个实施方案中，细胞毒性剂可以包括但不限于细菌毒素(例如白喉毒素)、植物毒素(例如蓖麻毒蛋白)、小分子毒素、放射性同位素等，具体地，例如蒽环霉素(anthracycline)、喜树碱(camptothecin)、考布他汀(combretastain)、海兔毒素(dolastatin)及其auristatin类衍生物、多卡米星(duocarmycin)、烯二炔(enediyne)、格尔德霉素(geldanamycin)、引哚琳并-苯二氮杂卓二聚体(indolino-benzodiazepine dimer)、美登素(maytansine)及其衍生物、嘌呤霉素(puromycin)、吡咯并苯二氮杂卓二聚体(pyrrolobenzodiazepine dimer)、紫杉烷(taxane)、长春花生物碱(vinca alkaloid)、特吡莱辛(tubulysin)、哈米特林(hemiasterlin)、斯考他汀(spliceostatin)、普拉地内酯(pladienolide)及卡奇霉素(calicheamicin)。The term "cytotoxic agent" may be used interchangeably with "cytotoxin", and in the present invention refers to a substance that inhibits or disrupts cell function and/or causes cell death or destruction. In one embodiment, cytotoxic agents may include, but are not limited to, bacterial toxins (such as diphtheria toxin), plant toxins (such as ricin), small molecule toxins, radioactive isotopes, etc., specifically, such as anthracycline ), camptothecin, combretastain, dolastatin and its auristatin derivatives, duocarmycin, enediyne, geldanamycin ( geldanamycin), indolino-benzodiazepine dimer, maytansine and its derivatives, puromycin, pyrrolobenzodiazepine dimer ( pyrrolobenzodiazepine dimer, taxane, vinca alkaloid, tubulysin, hemiasterlin, spliceostatin, pladienolide and calicheamicin.
本发明的任何抗体偶联药物均可用海兔毒素(dolastatin)及其auristatin类衍生物与抗体偶联制备。海兔毒素(dolastatin)及其auristatin类衍生物是抗体偶联药物(ADC)中使用的重要细胞毒素，其干扰微管动力学、细胞分裂等，具有抗肿瘤、抗真菌活性。其骨架的修饰已在文献中广泛报道，主要是对末端亚基：P1(N端)和P5(C端)的修饰，中央肽亚基的修饰也导致该类物质在体外具有有效的细胞毒活性。在一个方面中，海兔毒素(dolastatin)及其auristatin类衍生物例如可为0101(2-甲基丙胺酰基-N-[(3R,4S,5S)-3-甲氧基-1-{(2S)-2-[(1R,2R)-1-甲氧基-2-甲基-3-氧-3-{[(1S)-2-苯基-1-(1,3-噻唑-2-基)乙基]氨基}丙基]吡咯烷-1-基}-5-甲基-1-氧庚烷-4-基]-N-甲基-L-缬氨酰胺)、8261(2-甲基丙胺酰基-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-羧基-2-苯乙基]氨基}-1-甲氧基-2-甲基-3-氧丙基]吡咯烷-1-基}-3-甲氧基-5-甲基-1-氧庚烷-4-基]-N-甲基-L-缬氨酰胺)Dolastatin 10、Dolastatin 15、auristatin E、auristatin PE、monomethyl auristatin D(MMAD)、monomethyl auristatin E(MMAE)、monomethyl auristatin F(MMAF)、auristatin F苯二胺(AFP)、auristatin EB(AEB)、auristatin EFP(AEFP)、auristatin F羟丙基酰胺(AFHPA)、及其他auristatin(例如美国公开案第20130129753号中所述的auristatin)等。Any antibody-coupled drug of the present invention can be prepared by coupling dolastatin and its auristatin derivatives with antibodies. Dolastatin and its auristatin derivatives are important cytotoxins used in antibody-drug conjugates (ADC), which interfere with microtubule dynamics, cell division, etc., and have anti-tumor and anti-fungal activities. The modification of its backbone has been widely reported in the literature, mainly the modification of the terminal subunits: P1 (N-terminal) and P5 (C-terminal), and the modification of the central peptide subunit also leads to the effective cytotoxicity of this class of substances in vitro active. In one aspect, dolastatin (dolastatin) and its auristatin derivatives can be, for example, 0101(2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{( 2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazole-2 -yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), 8261 (2 -Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl ]amino}-1-methoxy-2-methyl-3-oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide) Dolastatin 10, Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AFHPA), and other auristatins (such as auristatin described in US Publication No. 20130129753), etc.
Monomethyl auristatin(MMAE)即去甲基-auristatin E，是auristatin化合物家族成员中著名的一员，其结构式如下：Monomethyl auristatin (MMAE), demethyl-auristatin E, is a well-known member of the auristatin compound family, and its structural formula is as follows:
MMAE通过抑制微管蛋白聚合而起到有效的有丝分裂抑制作用，因其细胞毒性而不能用作药物，但是却被广泛用以制备抗体偶联物。MMAE通过接头与单克隆抗体(MAB)偶联形成MMAE-MAB。一般而言，MMAE-MAB经抗体靶向肿瘤细胞，接头在MMAE-MAB进入肿瘤细胞后被裂解，从而释放MMAE，使其发挥细胞毒作用，杀伤肿瘤细胞。 MMAE, which acts as a potent mitotic inhibitor by inhibiting tubulin polymerization, cannot be used as a drug because of its cytotoxicity, but is widely used to prepare antibody conjugates. MMAE is coupled to a monoclonal antibody (MAB) via a linker to form MMAE-MAB. Generally speaking, MMAE-MAB targets tumor cells through antibodies, and the linker is cleaved after MMAE-MAB enters tumor cells, thereby releasing MMAE, making it exert cytotoxic effect and kill tumor cells.
术语“接头”和“连接子”在本申请中可以互换使用，指使抗体与ADC中的治疗活性物质或活性药物成分共价连接的化学模块。在一个实施方案中，接头可以包含将抗原与有效载荷连接的氨基酸残基。氨基酸残基可以形成二肽、三肽、四肽、五肽、六肽、七肽、八肽、九肽、十肽、十一肽或十二肽单元。氨基酸残基包括天然存在的那些以及非天然存在的氨基酸类似物，例如瓜氨酸或β-氨基酸，例如β-丙氨酸，或ω-氨基酸如4-氨基-丁酸。The terms "linker" and "linker" are used interchangeably herein to refer to a chemical moiety that covalently links an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC. In one embodiment, the linker may comprise amino acid residues linking the antigen to the payload. The amino acid residues may form dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Amino acid residues include those that occur naturally, as well as non-naturally occurring analogs of amino acids, such as citrulline or β-amino acids, such as β-alanine, or ω-amino acids such as 4-amino-butyric acid.
根据性质分类，适用于本发明的接头可以是组织蛋白酶可降解的接头，例如缬氨酸-瓜氨酸(val-cit)接头、cBu-Cit接头和CX接头；不可断裂接头例如SMCC接头或MD接头；酸敏接头、硅脂结构的接头、disulfide-carbamate接头、MC-GGFG接头、TRX接头、含半乳糖苷的接头、焦磷酸酯接头、近红外敏感的接头、紫外敏感的接头例如PC4AP。According to the classification of properties, the linkers suitable for the present invention can be cathepsin-degradable linkers, such as valine-citrulline (val-cit) linkers, cBu-Cit linkers and CX linkers; non-cleavable linkers such as SMCC linkers or MD Linkers; acid-sensitive linkers, silicone grease-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, UV-sensitive linkers such as PC4AP.
本发明的接头还可以是一种或多种接头的组合，例如组织蛋白酶降解的接头可以与其它类型的接头组合，构成新的接头。因此，本发明所述的“接头”涵盖单个类型的接头，或不同类型接头的组合，只要其能够将本发明的抗体与药物偶联起来即可。The linker of the present invention can also be a combination of one or more linkers, for example, a linker degraded by cathepsin can be combined with other types of linkers to form a new linker. Therefore, the "linker" of the present invention covers a single type of linker, or a combination of different types of linkers, as long as it can couple the antibody of the present invention to the drug.
在具体实施方案中，接头包括但不限于马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基(mc-vc-PAB)、乙酰基-赖氨酸-缬氨酸-瓜氨酸-对氨基苄氧羰基(AcLys-VC-PABC)、氨基PEG6-丙酰基、及马来酰亚胺己酸基(mc)、马来酰亚胺基丙酰基(MP)、缬氨酸-瓜氨酸(val-cit)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)、N-琥珀酰亚胺基4-(2-吡啶硫基)戊酸酯(SPP)、N-琥珀酰亚胺基4-(N-马来酰亚胺基甲基)-环己烷-1-羧酸酯(SMCC)、N-琥珀酰亚胺基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)、N-琥珀酰亚胺基-4-(2-吡啶基二硫代)丁酸酯(SPDB)、N-琥珀酰亚胺基3-(吡啶-2-基二硫代)-丙酸酯(SPDP)。In specific embodiments, linkers include, but are not limited to, maleimido-caproyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine- Valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propionyl, and maleimide caproyl (mc), maleimide propionyl (MP ), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2- Pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl Imino(4-iodo-acetyl)aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl Imino 3-(pyridin-2-yldithio)-propionate (SPDP).
术语“负载”或“药物负载”或“有效负载”指在ADC分子内每个抗体的平均有效负载数(在本文中“有效负载”可与“治疗活性物质或活性药物成分”互换使用)。药物负载的范围可以为每个抗体1-20个治疗活性物质或活性药物成分。术语“药物/抗体比”或“DAR”是指偶联于抗体的治疗活性物质或活性药物成分(D)与抗体的比例。本文中所述ADC通常具有1-20的DAR，在某些具体实施方案中具有1-8、2-8、2-6、2-5、2-18、4-16、5-12、6-10、3-8、4-6、6-10，和2-4的DAR。代表性DAR值是2、3、4、5、6、7、8、9、10、11、12、13、14或15，通常表示为字母D和数字的组合，其中数字表示DAR的数值，例如D2表示DAR值为2的药物/抗体比。在一些实施方案中，DAR是平均DAR，即通过检测方法(例如通过常规方法如UV/可见光光谱法、质谱法、ELISA测定、电泳和/或HPLC)测得的产品中偶联于本文所述的Ab部分的小分子药物部分(D)与Ab部分的总体比例。DAR可能受限于抗体上的连结位点数目。例如，在连结位点是半胱氨酸硫醇的情形下，抗体可能只有一或几个半胱氨酸硫醇基或可能只有一或几个具有充分反应性的硫醇基(通过此硫醇基可连结连接单元)。在一些实施方案中，本发明偶联物的平均DAR值是1至20，例如2-18、4-16、5-12、6-10、2-8、3-8、2-6、4-6、6-10，例如1.0-8.0，2.0-6.0，例如0.5、0.6、0.7、0.8、0.9、1、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、 5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8.0、7.9、8、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9或10.0，以这些数值中的两个作为端点的范围。The term "load" or "drug load" or "payload" refers to the average number of payloads per antibody within an ADC molecule (herein "payload" is used interchangeably with "therapeutically active substance or active pharmaceutical ingredient") . Drug loading may range from 1-20 therapeutically active substances or active pharmaceutical ingredients per antibody. The term "drug/antibody ratio" or "DAR" refers to the ratio of therapeutically active substance or active pharmaceutical ingredient (D) conjugated to an antibody to the antibody. ADCs described herein typically have a DAR of 1-20, and in certain embodiments 1-8, 2-8, 2-6, 2-5, 2-18, 4-16, 5-12, 6 DARs of -10, 3-8, 4-6, 6-10, and 2-4. Representative DAR values are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, usually expressed as a combination of the letter D and a number, where the number indicates the value of DAR, For example, D2 represents a drug/antibody ratio with a DAR value of 2. In some embodiments, the DAR is the average DAR, i.e., measured by detection methods (e.g., by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays, electrophoresis, and/or HPLC) in products coupled to those described herein. The overall ratio of the small molecule drug fraction (D) to the Ab fraction of the Ab fraction. DARs may be limited by the number of attachment sites on the antibody. For example, where the attachment site is a cysteine thiol, the antibody may have only one or a few cysteine thiol groups or may have only one or a few sufficiently reactive thiol groups ( Alcohol groups can link linking units). In some embodiments, the conjugates of the invention have an average DAR value of 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4 -6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 ,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4,4.1,4.2,4.3,4.4,4.5,4.6 , 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, with two of these values as endpoints.
术语“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的抗体用来延缓疾病发展或用来减慢疾病的进展。The term "treating" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.
术语“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。The term "prevention" includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
术语“有效量”指本发明的抗体或缀合物或组合物的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动物的物种；体重、年龄和一般健康状况；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。The term "effective amount" refers to the amount or dosage of an antibody or conjugate or composition of the present invention, which produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses. An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as: the species of mammal; body weight, age and general health; the particular disease involved; the extent or severity of the disease; the individual The patient's response; the specific antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
术语“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其缀合物或组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其缀合物或组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率、肿瘤体积等)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％或90％。可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。The term "therapeutically effective amount" refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result. A therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition thereof are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%. Compounds can be evaluated for their ability to inhibit a measurable parameter (eg, cancer) in animal model systems predictive of efficacy in human tumors.
术语“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。The term "prophylactically effective amount" refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。The term "pharmaceutical composition" refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.
II.本发明组合物II. Compositions of the Invention
在一些实施方案中，本发明提供包含本文所述的任何抗ROR1抗体、或其ADC分子的组合物，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。在一个实施方案中，组合物(例如，药物组合物)包含本发明的抗ROR1抗体、或其ADC分子，以及一种或多种其它治疗剂的组合。 In some embodiments, the present invention provides a composition, preferably a pharmaceutical composition, comprising any of the anti-ROR1 antibodies described herein, or an ADC molecule thereof. In one embodiment, the composition further comprises pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art. In one embodiment, a composition (eg, a pharmaceutical composition) comprises an anti-ROR1 antibody of the invention, or an ADC molecule thereof, in combination with one or more other therapeutic agents.
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
对于药用辅料的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第八版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、散剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。The compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.
本发明组合物的施用途径是根据已知方法，例如，经口、通过静脉内注射、腹膜内、脑内(实质内)、脑室内、肌内、眼内、动脉内、门脉内或病灶内途径；通过持续释放系统或通过植入装置。在某些实施方案中，组合物可通过弹丸注射或通过连续输注或通过植入装置施用。The route of administration of the composition of the invention is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or focal Internal route; by sustained release system or by implanted device. In certain embodiments, the compositions can be administered by bolus injection or by continuous infusion or by implanted device.
受试者可以是哺乳动物，例如，灵长类，优选地，高级灵长类，例如，人类(例如，患有本文所述疾病或具有患有本文所述疾病的风险的个体)。在一个实施方案中，受试者患有本文所述疾病(例如，癌症)或具有患有本文所述疾病的风险。在某些实施方案中，受试者接受或已经接受过其它治疗，例如化疗治疗和/或放射疗法。在一些实施方案中，受试者之前已经接受过或正在接受免疫疗法。The subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, an individual suffering from or at risk of having a disease described herein). In one embodiment, the subject has or is at risk of having a disease described herein (eg, cancer). In certain embodiments, the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.
可以通过将具有所需纯度的本发明的抗ROR1抗体、或其ADC分子与一种或多种任选的药用辅料混合来制备包含本文所述的抗体的药物，优选地以冻干制剂或水溶液的形式。A medicament comprising an antibody described herein can be prepared by mixing an anti-ROR1 antibody of the invention, or an ADC molecule thereof, of the desired purity with one or more optional pharmaceutical excipients, preferably in a lyophilized formulation or in the form of an aqueous solution.
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂，包括化疗剂、血管生成抑制剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫检查点抑制剂或激动剂)等。所述活性成分以对于目的用途有效的量合适地组合存在。The pharmaceutical compositions or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immune checkpoint inhibitors or agonists), among others. The active ingredients are suitably present in combination in amounts effective for the intended use.
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles such as films or microcapsules.
III.制备本发明的抗体和抗体偶联药物III. Preparation of Antibodies and Antibody-Drug Conjugates of the Present Invention
在一个实施方案中，本发明提供了制备抗ROR1抗体的方法，其中所述方法包括在适于表达编码所述抗ROR1抗体的核酸的条件下培养包含编码抗ROR1抗体的核酸或包含所述核酸的表达载体的宿主细胞，以及任选地分离所述抗ROR1抗体。在某个实施方案中，所述方法还包括从所述宿主细胞(或宿主细胞培养基)回收抗ROR1抗体。In one embodiment, the present invention provides a method for preparing an anti-ROR1 antibody, wherein said method comprises culturing a nucleic acid comprising or comprising said nucleic acid encoding an anti-ROR1 antibody under conditions suitable for expressing said nucleic acid encoding said anti-ROR1 antibody. host cells for the expression vector, and optionally isolate the anti-ROR1 antibody. In a certain embodiment, the method further comprises recovering the anti-ROR1 antibody from the host cell (or host cell culture medium).
为了重组产生本发明的抗ROR1抗体，首先分离编码本发明抗ROR1抗体的核酸，并将所述核酸插入载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序，例如通过使用能够与编码本发明抗ROR1抗体的核酸特异性结合的寡核苷酸探针进行。In order to recombinantly produce the anti-ROR1 antibody of the present invention, the nucleic acid encoding the anti-ROR1 antibody of the present invention is first isolated and inserted into a vector for further cloning and/or expression in host cells. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example by using oligonucleotide probes that are capable of specifically binding to nucleic acid encoding an anti-ROR1 antibody of the invention.
如本文所述制备的本发明的抗ROR1抗体可以通过已知的现有技术如高效液相色谱、离子交 换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的抗ROR1抗体的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。Anti-ROR1 antibodies of the invention prepared as described herein can be tested by known prior art techniques such as high performance liquid chromatography, ion exchange Purification by exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. The purity of the anti-ROR1 antibodies of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
现有技术描述了用于将细胞毒性剂或其他治疗剂与抗体偶联的多种方法。例如，可以通过赖氨酸侧链的氨基和抗体N末端的氨基，天门冬氨酸、谷氨酸和C末端的羧基或活化的半胱氨酸巯基在抗体中进行，以使缀合反应发生。The prior art describes various methods for conjugating cytotoxic or other therapeutic agents to antibodies. For example, the conjugation reaction can be carried out in the antibody through the amino group of the lysine side chain and the amino group at the N-terminus of the antibody, the carboxyl group of aspartic acid, glutamic acid and C-terminus, or the activated cysteine sulfhydryl group. .
实施例Example
以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。The following examples further illustrate the invention, however, it is to be understood that the examples are presented by way of illustration and not limitation and that various modifications may be made by those skilled in the art.
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。The practice of the present invention will employ, unless expressly indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, within the skill of the art.
实施例1原材料制备及鉴定Embodiment 1 Raw material preparation and identification
1.1 ROR1对照抗体制备及鉴定1.1 Preparation and identification of ROR1 control antibody
ROR1对照抗体制备：本申请使用抗ROR1抗体Cirmtuzumab(西妥珠单抗)作为阳性对照抗体，根据WO/2019/173843公开的序列由通用生物科技股份有限公司进行目的片段的基因合成。然后通过同源重组的方法构建至真核表达载体pcDNA3.4(Invitrogen)，将构建好的重组蛋白表达载体分别转化到大肠杆菌DH5α中，37℃过夜培养，然后利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，获得期望的表达西妥珠单抗的表达载体，克隆号为99961.1，下文将表达的西妥珠单抗简称为99961.1。通过ExpiFectamineTM CHO转染试剂盒(Thermo Fisher，A29129)将表达载体转染293细胞以表达西妥珠单抗，转染7天后，收集细胞培养物上清液，15000g离心10min，将所得上清液经0.22μm滤膜过滤后，采用Protein A/G亲和层析柱对上清液中的抗体进行亲和纯化。用100mM甘氨酸盐(pH 3.0)洗脱目的抗体，并将洗脱的抗体通过超滤浓缩管(Millipore，UFC901096)换液至PBS缓冲液中。Preparation of ROR1 control antibody: In this application, the anti-ROR1 antibody Cirmtuzumab (Cetuzumab) was used as a positive control antibody, and the gene synthesis of the target fragment was carried out by General Biotechnology Co., Ltd. according to the sequence disclosed in WO/2019/173843. Then, the eukaryotic expression vector pcDNA3.4 (Invitrogen) was constructed by homologous recombination, and the constructed recombinant protein expression vectors were transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) for plasmid extraction to obtain the desired expression vector for expressing cetuzumab, the clone number is 99961.1, and the expressed cetuzumab will be referred to as 99961.1 for short below. The expression vector was transfected into 293 cells by ExpiFectamine TM CHO transfection kit (Thermo Fisher, A29129) to express cetuzumab. After 7 days of transfection, the cell culture supernatant was collected and centrifuged at 15000 g for 10 min. After the liquid was filtered through a 0.22 μm filter membrane, the antibody in the supernatant was affinity purified using a Protein A/G affinity chromatography column. The target antibody was eluted with 100 mM glycine salt (pH 3.0), and the eluted antibody was exchanged into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).
ROR1对照抗体鉴定：用购买的huROR1-His抗原蛋白(恺佧生物，ROR-HM401)检测制备的阳性对照抗体99961.1(IgG1)的活性，具体方法如下：在96孔ELISA板上包被huROR1-His(2μg/mL、30μL/孔)，4℃包被过夜；洗板3次后，用PBS配置的5％脱脂牛奶室温封闭1小时；洗板3次后，加入用PBS梯度稀释的对照抗体99961.1室温孵育1小时；洗板后，加入PBS稀释(1：6000)的二抗Anti-human-IgG-Kappa+Lambda-HRP(Millipore，AP502P+AP506P)室温孵育1小时后，洗板6次加入TMB显色5-20min，终止显色后酶标仪OD450读取数据，处理数据Graphpad prism作图。结果显示，表达的对照抗体99961.1可以结合ROR1蛋白，具有正常的抗ROR1活性。 Identification of ROR1 control antibody: Use the purchased huROR1-His antigen protein (Kaijia Biology, ROR-HM401) to detect the activity of the prepared positive control antibody 99961.1 (IgG1), the specific method is as follows: coat huROR1-His on a 96-well ELISA plate (2μg/mL, 30μL/well), coated overnight at 4°C; after washing the plate 3 times, block with 5% skimmed milk in PBS for 1 hour at room temperature; after washing the plate 3 times, add the control antibody 99961.1 serially diluted with PBS Incubate at room temperature for 1 hour; after washing the plate, add secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted in PBS (1:6000) and incubate at room temperature for 1 hour, wash the plate 6 times and add TMB Color development was performed for 5-20 minutes. After the color development was terminated, the OD450 data was read by the microplate reader, and the processed data was plotted by Graphpad prism. The results showed that the expressed control antibody 99961.1 could bind to ROR1 protein and had normal anti-ROR1 activity.
1.2 ROR1抗原蛋白制备及鉴定1.2 Preparation and identification of ROR1 antigen protein
抗原蛋白制备：通过在编码基因水平的遗传操作，分别在人ROR1蛋白huROR1 ECD AA30-406(Uniprot ID：Q01973)、小鼠ROR1蛋白MusROR1 ECD AA30-406(Uniprot ID：Q9Z139)、人ROR2蛋白huROR2 ECD AA34-403(Uniprot ID：Q01974)的序列C端加上His标签。分别将获得的核酸序列构建至pcDNA3.4载体中，然后转化到大肠杆菌DH5α中，37℃过夜培养，之后利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)提取质粒。将所得到的质粒用ExpiFectamineTM 293转染试剂盒(GibcoTM，A14524)瞬转至HEK293细胞(CRL-1573TM)中，表达7天后，收取细胞培养物上清液，对含有His标签的蛋白用Ni Smart Beads 6FF(常州天地人和生物科技有限公司，SA036050)进行亲和纯化，然后用咪唑梯度洗脱目的蛋白。洗脱的各蛋白分别通过超滤浓缩管(Millipore，UFC901096)换液至PBS缓冲液中，最后获得抗原蛋白(huROR1-His，MusROR1-His，huROR2-His)。Antigen protein preparation: through genetic manipulation at the coding gene level, human ROR1 protein huROR1 ECD AA30-406 (Uniprot ID: Q01973), mouse ROR1 protein MusROR1 ECD AA30-406 (Uniprot ID: Q9Z139), human ROR2 protein huROR2 A His tag was added to the C-terminus of the sequence of ECD AA34-403 (Uniprot ID: Q01974). The obtained nucleic acid sequences were respectively constructed into pcDNA3.4 vectors, and then transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The resulting plasmid was transiently transfected into HEK293 cells ( CRL-1573 TM ), after 7 days of expression, the cell culture supernatant was collected, and the protein containing the His tag was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then imidazole Gradient elution of the target protein. The eluted proteins were respectively exchanged into PBS buffer solution through ultrafiltration concentrator tubes (Millipore, UFC901096), and finally the antigenic proteins (huROR1-His, MusROR1-His, huROR2-His) were obtained.
抗原鉴定：用实施例1.1获得的质检合格的抗体99961.1(IgG1)检测制得的抗原(huROR1-His)。具体方法如下：分别用2μg/mL huROR1-His包被Elisa板4℃过夜，以购买的抗原蛋白huROR1-His(恺佧生物ROR-HM201)作为阳性对照；洗板3次后，用PBS配置的5％脱脂牛奶室温封闭1小时；洗板3次后，加入用PBS梯度稀释的抗体99961.1室温孵育1小时；洗板后，加入PBS稀释(1：6000)的二抗Anti-human-IgG-Kappa+Lambda-HRP(Millipore，AP502P+AP506P)室温孵育1小时，洗板6次然后加入TMB显色5-20min，终止显色反应，采用酶标仪OD450读取数据，处理数据Graphpad prism作图。结果显示，抗体99961.1可以以与购买的ROR1抗原蛋白相当的亲和力结合本申请发明人自行构建表达的抗原huROR1-His。Antigen identification: the antibody 99961.1 (IgG1) qualified for quality inspection obtained in Example 1.1 was used to detect the prepared antigen (huROR1-His). The specific method is as follows: respectively coat the Elisa plate with 2 μg/mL huROR1-His at 4°C overnight, and use the purchased antigenic protein huROR1-His (Kaijia Biological ROR-HM201) as a positive control; after washing the plate 3 times, use PBS prepared Block with 5% skimmed milk at room temperature for 1 hour; after washing the plate 3 times, add antibody 99961.1 serially diluted with PBS and incubate at room temperature for 1 hour; after washing the plate, add secondary antibody Anti-human-IgG-Kappa diluted in PBS (1:6000) +Lambda-HRP (Millipore, AP502P+AP506P) was incubated at room temperature for 1 hour, the plate was washed 6 times, and TMB was added for 5-20 minutes to develop color, and the color reaction was terminated. The data was read by microplate reader OD450, and the processed data was plotted with Graphpad prism. The results show that the antibody 99961.1 can bind the antigen huROR1-His constructed and expressed by the inventors themselves with an affinity comparable to that of the purchased ROR1 antigen protein.
实施例2构建及鉴定过表达人ROR1的细胞株Example 2 Construction and identification of cell lines overexpressing human ROR1
过表达人ROR1的HEK293细胞株(以下简称huROR1-HEK293)的构建：将全长人ROR1(Uniprot ID：Q01973)的编码核酸序列构建至pLVX-puro质粒(Clontech，Cat#632164)上。然后，将所得到的质粒通过电转仪(Invitrogen，NeonTM Transfection System，MP922947)电转化至HEK293细胞(CRL-1573TM)中。电转化后，将所得到的细胞分别转移至含有体积百分比为10％的FBS(Gibco，15140-141)且不含抗生素的DMEM培养基(Gibco，11995065)中，然后将细胞转入10×10cm细胞培养皿中培养48小时，接着以平均104个细胞/孔的密度将细胞分装至96孔细胞培养板中，加入终浓度为2μg/mL的嘌呤霉素作为筛选压力，2周左右挑取形成克隆的细胞株进行鉴定。Construction of HEK293 cell line overexpressing human ROR1 (hereinafter referred to as huROR1-HEK293): The coding nucleic acid sequence of full-length human ROR1 (Uniprot ID: Q01973) was constructed on pLVX-puro plasmid (Clontech, Cat#632164). Then, the resulting plasmid was electrotransformed into HEK293 cells ( CRL-1573 ™ ). After electroporation, the obtained cells were respectively transferred to DMEM medium (Gibco, 11995065) containing 10% by volume of FBS (Gibco, 15140-141) without antibiotics, and then the cells were transferred into a 10×10 cm Culture the cells in a culture dish for 48 hours, then divide the cells into 96-well cell culture plates at an average density of 10 4 cells/well, add puromycin at a final concentration of 2 μg/mL as a selection pressure, and pick for about 2 weeks. The cloned cell lines were taken for identification.
huROR1-HEK293细胞的流式鉴定：将对数生长期的上述细胞株细胞消化并铺板到96孔板中，用FACS缓冲液(含体积百分比为2％的FBS的1×PBS缓冲液)清洗后，加入用PBS梯度稀释的一抗(99961.1)4℃孵育30min；清洗后，加入配制好的荧光二抗anti human IgG Fc(abcam，98596)，4℃孵育30min；最后通过流式细胞仪(Beckman，CytoFLEXAOO-1-1102)进行检测。检测结果 显示获得了表面高表达人ROR1的huROR1-HEK293细胞株。Flow cytometric identification of huROR1-HEK293 cells: Digest and plate the above cell lines in the logarithmic growth phase into 96-well plates, wash with FACS buffer (1×PBS buffer containing 2% FBS by volume) , adding the primary antibody (99961.1) diluted in PBS gradients and incubating for 30min at 4°C; after washing, adding the prepared fluorescent secondary antibody anti human IgG Fc (abcam, 98596), and incubating for 30min at 4°C; finally by flow cytometry (Beckman , CytoFLEXAOO-1-1102) for detection. Test results It shows that the huROR1-HEK293 cell line with high surface expression of human ROR1 has been obtained.
实施例3人源噬菌体展示重组抗体文库的构建及筛选Example 3 Construction and screening of human phage display recombinant antibody library
在本实施例中，构建了抗体基因噬菌体展示文库，并用实施例1.2制备的抗原蛋白huROR1-His和实施例2制备的过表达huROR1的细胞huROR1-HEK293作为筛选抗原对该文库进行筛选，获得了多个具有特异性结合人ROR1的抗体分子。In this example, an antibody gene phage display library was constructed, and the antigenic protein huROR1-His prepared in Example 1.2 and the huROR1-overexpressing cell huROR1-HEK293 prepared in Example 2 were used as screening antigens to screen the library to obtain Multiple antibody molecules with specific binding to human ROR1.
3.1构建人抗体的基因文库3.1 Construction of human antibody gene library
取Ficoll-Paque密度梯度分离液(购自GE公司，目录号：17144003S)分离正常人血液的外周血单个核细胞(Peripheral Blood Mononuclear Cell，PBMC)，通过常规方法自分离的PBMC细胞提取总RNA。使用反转录试剂盒(购自TaKaRa公司,目录号：6210A)将提取的总RNA反转录成cDNA。基于重链和轻链种系基因的序列相似度，分别在重链和轻链的V区前端和第一个恒定区后端设计简并引物，PCR后得到抗体的重链可变区基因片段和轻链可变区基因片段。通过融合PCR方法扩增得到含有抗体的轻链和重链可变区的片段，对该PCR产物和噬菌体展示用载体进行酶切、回收和连接，连接产物通过回收试剂盒(Omega，目录号：D6492-02)回收(李晓琳，大容量非免疫人源性Fab噬菌体抗体库的构建及初步筛选，《中国协和医科大学》硕士学位论文，2007年6月)。最后，通过电转仪(Bio-Rad，MicroPulser)转化至感受态大肠杆菌SS320(Lucigen，MC1061 F)中，并将经转化的大肠杆菌SS320菌液涂布于具有氨苄青霉素抗性的2-YT固体平板(固体平板由1.5％的胰蛋白胨，1％的酵母提取物，0.5％的NaCl，1.5％的琼脂，按质量体积g/mL配制而成)。通过梯度稀释铺板，测得此文库库容量为3×1011cfu，即3×1011个抗体基因的抗体基因库(库容计算方法参考专利CN112250763B中的实施例2.2)。采用VSCM13辅助噬菌体(购自Stratagene)对其进行包装，获得了抗体基因噬菌体展示文库(抗体基因噬菌体展示文库的制备参考专利CN112250763B中的实施例2.3)。Ficoll-Paque density gradient separation medium (purchased from GE, catalog number: 17144003S) was used to separate peripheral blood mononuclear cells (PBMC) from normal human blood, and total RNA was extracted from the isolated PBMC cells by conventional methods. The extracted total RNA was reverse-transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa Company, catalog number: 6210A). Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers were designed at the front end of the V region and the rear end of the first constant region of the heavy chain and light chain respectively, and the heavy chain variable region gene fragment of the antibody was obtained after PCR and light chain variable region gene fragments. The fragments containing the variable region of the light chain and the heavy chain of the antibody were amplified by the fusion PCR method, and the PCR product and the carrier for phage display were digested, recovered and connected, and the connected product was recovered by a recovery kit (Omega, catalog number: D6492-02) recovery (Li Xiaolin, Construction and preliminary screening of a large-capacity non-immune human Fab phage antibody library, "China Peking Union Medical College"master's degree thesis, June 2007). Finally, transform into competent Escherichia coli SS320 (Lucigen, MC1061 F) by electroporation (Bio-Rad, MicroPulser), and spread the transformed Escherichia coli SS320 bacterial liquid on 2-YT solid with ampicillin resistance Plate (the solid plate is made of 1.5% tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar, prepared by mass volume g/mL). The library capacity of the library was determined to be 3×10 11 cfu by serial dilution plating, that is, an antibody gene library of 3×10 11 antibody genes (refer to Example 2.2 in patent CN112250763B for the calculation method of the library capacity). The VSCM13 helper phage (purchased from Stratagene) was used to package it to obtain the antibody gene phage display library (for the preparation of the antibody gene phage display library, refer to Example 2.3 in patent CN112250763B).
3.2抗体基因噬菌体展示文库的筛选3.2 Screening of antibody gene phage display library
3.2.1磁珠法筛选抗体基因噬菌体展示文库3.2.1 Magnetic bead method to screen antibody gene phage display library
磁珠法筛选是基于将抗原蛋白huROR1-His进行生物素标记后，再与偶联有链霉亲和素的磁珠结合，通过将结合抗原的磁珠和抗体基因噬菌体展示文库进行孵育、洗涤和洗脱的淘选过程。通常经历3-4轮的淘选，由此针对抗原的特异性单克隆抗体可以大量富集。本实施例中，将生物素标记的huROR1-His用于噬菌体展示文库筛选，经过3轮淘选后进行针对人ROR1的单克隆抗体Fab初筛，具体方法参考专利CN112250763B中的实施例2.4.1。Magnetic bead screening is based on biotin-labeling the antigenic protein huROR1-His, and then binding to magnetic beads coupled with streptavidin, incubating and washing the antigen-binding magnetic beads and antibody gene phage display library and elution panning process. Usually undergoes 3-4 rounds of panning, whereby specific monoclonal antibodies against antigens can be enriched in large quantities. In this example, biotin-labeled huROR1-His was used for phage display library screening, and after three rounds of panning, the monoclonal antibody Fab against human ROR1 was screened. For specific methods, refer to Example 2.4.1 in patent CN112250763B .
3.2.2免疫管法筛选抗体基因噬菌体展示文库3.2.2 Immunotube screening of antibody gene phage display library
免疫管法筛选的原理是将抗原蛋白huROR1-His包被在具有高吸附力的免疫管表面，通过将噬菌体展示抗体文库加入免疫管中并和吸附于免疫管表面的抗原蛋白进行孵育、洗涤和洗脱的淘 选过程，经历2-4轮淘选，最终富集针对抗原的特异性单克隆抗体Fab。本实施例中，经过3轮淘选后富集了针对人ROR1的单克隆抗体Fab，具体方法参考专利CN112250763B中的实施例2.4.2。The principle of immunotube screening is to coat the antigenic protein huROR1-His on the surface of the immunotube with high adsorption force, add the phage display antibody library to the immunotube and incubate with the antigenic protein adsorbed on the surface of the immunotube, wash and Eluted pan During the selection process, after 2-4 rounds of panning, the specific monoclonal antibody Fab targeting the antigen is finally enriched. In this example, the monoclonal antibody Fab against human ROR1 was enriched after three rounds of panning. For the specific method, refer to Example 2.4.2 in patent CN112250763B.
3.3单克隆的挑选3.3 Selection of monoclonal
对每轮洗脱下来的噬菌体池进行ELISA检测来评价富集的效果，并从每轮筛选的噬菌体池中随机挑选10个克隆进行序列分析，结合富集效果和所测序列重复性比例综合分析，选择合适的轮次进行单克隆挑选。Perform ELISA detection on the phage pools eluted in each round to evaluate the effect of enrichment, and randomly select 10 clones from the phage pools screened in each round for sequence analysis, combined with the comprehensive analysis of the enrichment effect and the measured sequence repeatability ratio , select the appropriate round for monoclonal selection.
ELISA单克隆初筛使用抗原蛋白huROR1-His，将初筛获得的结合huROR1-His的抗体Fab制备成Fab裂解液，然后用实施例2.1制备的过表达细胞huROR1-HEK293通过流式细胞分析法(FACS)进行检测复核，共筛选到11个特异结合人ROR1的抗体Fab分子，分别以相应的克隆号对获得的11株抗体Fab进行命名(N99、N218、N31、N137、N147、N100、N204、N174、NW29、NW79和NW37)，具体的FACS结果见图1A-1F。所得抗体Fab的CDR区氨基酸序列见表1，采用AbM定义CDR的方式，确定CDR序列。Antigen protein huROR1-His was used for the primary screening of monoclonal ELISA, and the antibody Fab combined with huROR1-His obtained from the primary screening was prepared into Fab lysate, and then the overexpressed cell huROR1-HEK293 prepared in Example 2.1 was used for flow cytometric analysis ( FACS) for detection and review, a total of 11 antibody Fab molecules specifically binding to human ROR1 were screened out, and the obtained 11 antibody Fabs were named according to the corresponding clone numbers (N99, N218, N31, N137, N147, N100, N204, N174, NW29, NW79 and NW37), the specific FACS results are shown in Figures 1A-1F. The amino acid sequence of the CDR region of the obtained antibody Fab is shown in Table 1, and the CDR sequence was determined by using AbM to define the CDR.
表1. 11株抗体CDR区域的氨基酸序列

Table 1. Amino acid sequences of CDR regions of 11 antibody strains

实施例4抗体构建、表达与纯化Example 4 Antibody Construction, Expression and Purification
4.1质粒构建4.1 Plasmid construction
将筛选获得的单克隆N99、N218、N31、N137、N147、N100、N204、N174、NW29、NW79和NW37的Fab序列中的VH编码序列与人IgG1的重链恒定区(SEQ ID NO:48)的编码序列连接获得全人源抗体的重链编码序列，将Fab序列中的VL编码序列与人轻链恒定区(CL)的Kappa型(SEQ ID NO:49)或Lambda型(SEQ ID NO:50)的编码序列连接获得全人源抗体的轻链编码序列。将抗体重链和轻链的编码序列分别插入真核表达载体质粒pcDNA3.4(Invitrogen)中，转化到大肠杆菌DH5α中，37℃过夜培养。利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，得到无内毒素的抗体质粒以供真核表达使用。The VH coding sequence in the Fab sequence of the monoclonal N99, N218, N31, N137, N147, N100, N204, N174, NW29, NW79 and NW37 obtained by screening was combined with the heavy chain constant region of human IgG1 (SEQ ID NO: 48) The coding sequence of the human antibody is connected to obtain the heavy chain coding sequence of the fully human antibody, and the VL coding sequence in the Fab sequence is combined with the Kappa type (SEQ ID NO: 49) or Lambda type (SEQ ID NO: 49) of the human light chain constant region (CL). 50) to obtain the light chain coding sequence of a fully human antibody. The coding sequences of antibody heavy chain and light chain were respectively inserted into eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5α, and cultured overnight at 37°C. The endotoxin-free plasmid extraction kit (OMEGA, D6950-01) was used for plasmid extraction to obtain endotoxin-free antibody plasmids for eukaryotic expression.
4.2抗体的表达和纯化4.2 Expression and purification of antibody
通过ExpiCHO瞬转表达系统(Thermo Fisher，A29133)表达上述获得的抗体全长序列，具体方法如下：转染当天，确认CHO细胞密度为7×106至1×107个活细胞/mL左右，细胞存活率>98％，此时用37℃预温的新鲜ExpiCHO表达培养基将细胞调整到终浓度为6×106个细胞/mL。用4℃预冷的OptiPROTM SFM稀释目的质粒(向1mL所述培养基中加入1μg质粒)，同时用OptiPROTMSFM稀释ExpiFectamineTMCHO试剂，再将两者等体积混合并轻轻吹打混匀制备成ExpiFectamineTMCHO/质粒DNA混合液，室温孵育1-5min，缓慢加入到准备好的细胞悬液中并同时轻轻摇晃，最后置于细胞培养摇床中，在37℃、8％CO2条件下培养。 The full-length sequence of the antibody obtained above was expressed by the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, confirm that the CHO cell density was about 7×10 6 to 1×10 7 viable cells/mL, When the cell survival rate is >98%, adjust the cells to a final concentration of 6×10 6 cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C. Dilute the target plasmid with OptiPRO ™ SFM pre-cooled at 4°C (add 1 μg plasmid to 1 mL of the medium), and dilute ExpiFectamine ™ CHO reagent with OptiPRO ™ SFM at the same time, then mix the two in equal volumes and gently blow and mix to prepare Form ExpiFectamine TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension and shake gently at the same time, and finally place in a cell culture shaker at 37°C, 8% CO 2 conditions under cultivation.
转染18-22h后，向细胞培养液中添加ExpiCHOTMEnhancer试剂和ExpiCHOTMFeed试剂，摇瓶放置于32℃摇床和5％CO2条件下继续培养。在转染后的第5天，添加相同体积的ExpiCHOTMFeed试剂，缓慢加入的同时轻轻混匀细胞混悬液。转染7天后，收集表达有目的抗体蛋白的细胞培养上清液，15000g离心10min，所得上清用MabSelect SuRe LX(GE，17547403)进行亲和纯化，然后用100mM乙酸钠(pH 3.0)洗脱目的抗体蛋白，接着用1M Tris-HCl中和，最后通过超滤浓缩管(Millipore，UFC901096)将所得抗体蛋白换液至PBS缓冲液中。18-22 hours after transfection, add ExpiCHO TM Enhancer reagent and ExpiCHO TM Feed reagent to the cell culture medium, and place the shaker flask in a shaker at 32°C and 5% CO 2 to continue culturing. On day 5 after transfection, add the same volume of ExpiCHO TM Feed Reagent, and gently mix the cell suspension while adding slowly. Seven days after transfection, collect the cell culture supernatant expressing the target antibody protein, centrifuge at 15,000 g for 10 min, and then use MabSelect SuRe LX (GE, 17547403) for affinity purification, and then elute with 100 mM sodium acetate (pH 3.0) The target antibody protein was then neutralized with 1M Tris-HCl, and finally the resulting antibody protein was exchanged into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096).
实施例5抗体的理化性质检测The physicochemical property detection of embodiment 5 antibody
本实施例中，采用SDS-PAGE和SEC-HPLC检测了候选抗体的相对分子量和纯度。In this example, the relative molecular weight and purity of the candidate antibodies were detected by SDS-PAGE and SEC-HPLC.
5.1抗体SDS-PAGE鉴定5.1 Antibody identification by SDS-PAGE
非还原溶液制备：分别将1μg各个获得的抗体以及质控品IPI(伊匹木单抗，Ipilimumab)加入5×SDS上样缓冲液和40mM碘代乙酰胺中，75℃干浴加热10min，冷却到室温后，12000rpm离心5min取上清。Preparation of non-reducing solution: Add 1 μg of each obtained antibody and quality control product IPI (Ipilimumab, Ipilimumab) to 5×SDS loading buffer and 40 mM iodoacetamide, heat in a dry bath at 75°C for 10 min, and cool After reaching room temperature, centrifuge at 12000rpm for 5min to take the supernatant.
还原溶液制备：分别将2μg各个获得的抗体以及质控品IPI加入5×SDS上样缓冲液和5mM DTT中，100℃干浴加热10min，冷却到室温后，12000rpm离心5min取上清。将上清加入Bis-tris4-15％梯度胶(金斯瑞)进行凝胶电泳并通过考马斯亮蓝染色使蛋白条带显色。Preparation of reducing solution: Add 2 μg of each obtained antibody and quality control IPI to 5×SDS sample buffer and 5mM DTT, heat in a dry bath at 100°C for 10 minutes, cool to room temperature, and centrifuge at 12,000 rpm for 5 minutes to take the supernatant. The supernatant was added to Bis-tris4-15% gradient gel (GentScript) for gel electrophoresis, and the protein bands were visualized by Coomassie brilliant blue staining.
使用EPSON V550彩色扫描仪扫描带有显色蛋白条带的蛋白凝胶(脱色液脱色至凝胶背景透明)，通过ImageJ按照峰面积归一法计算还原和非还原条带纯度。EPSON V550 color scanner was used to scan the protein gel with chromogenic protein bands (the decolorization solution was decolorized until the gel background was transparent), and the purity of reduced and non-reduced bands was calculated by ImageJ according to the peak area normalization method.
试验结果表明各个抗体非还原胶的条带在150kD左右，还原胶的条带在55kD左右和25kD左右，符合预期大小。通过还原胶检测的所有候选抗体纯度均大于95％(表2)。The test results showed that the bands of the non-reducing gum of each antibody were around 150kD, and the bands of the reducing gum were around 55kD and 25kD, which were in line with the expected size. All candidate antibodies were more than 95% pure by reducing gel (Table 2).
5.2 SEC-HPLC鉴定抗体的单体纯度5.2 SEC-HPLC identification of antibody monomer purity
材料准备：1、流动相：150mmol/L磷酸缓冲液，pH 7.4；2、样品制备：分别用流动相溶液稀释各个抗体以及质控品IPI到0.5mg/mL。Agilent HPLC 1100或岛津LC2030C PLUS液相色谱仪，色谱柱为XBridge BEH(SEC 3.5μm，7.8mm I.D.×30cm)，Waters流速设为0.8mL/min，进样体积20μL，VWD检测器波长为280nm和214nm。依次进样空白溶液、IPI质控品溶液和抗体样品溶液。按照面积归一法计算样品中高分子聚合物、抗体单体和低分子物质百分比。Material preparation: 1. Mobile phase: 150mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: Dilute each antibody and quality control IPI to 0.5mg/mL with mobile phase solution. Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph, the chromatographic column is XBridge BEH (SEC 3.5μm, 7.8mm I.D.×30cm), the Waters flow rate is set to 0.8mL/min, the injection volume is 20μL, and the wavelength of the VWD detector is 280nm and 214nm. Inject blank solution, IPI quality control solution and antibody sample solution sequentially. Calculate the percentage of high molecular polymers, antibody monomers and low molecular substances in the sample according to the area normalization method.
结果如图2A-2K和表2所示，除了N204和NW79的单体纯度在95％左右，其他抗体的单体纯度均大于98％。The results are shown in Figures 2A-2K and Table 2, except that the monomer purity of N204 and NW79 is about 95%, the monomer purity of other antibodies is greater than 98%.
表2候选抗体表达量和理化性质

Table 2 Candidate antibody expression and physicochemical properties

实施例6抗体的抗原结合活性检测Antigen-binding activity detection of embodiment 6 antibody
在本实施例中，基于ELISA方法检测了表达的抗体(N99、N218、N147、N137、N31、NW37、N100、N204、N174、NW29和NW79)与人ROR1抗原蛋白huROR1-His的结合情况，还基于FACS方法检测了表达的抗体(N99、N218、N147、N137、N31、NW37、N100、N204、N174、NW29和NW79)与人ROR1过表达细胞huROR1-HEK293和A549细胞的结合能力。A549细胞是人类非小细胞肺癌细胞系，其过表达huROR1。In this example, the binding of the expressed antibodies (N99, N218, N147, N137, N31, NW37, N100, N204, N174, NW29 and NW79) to the human ROR1 antigen protein huROR1-His was detected based on the ELISA method. The binding ability of expressed antibodies (N99, N218, N147, N137, N31, NW37, N100, N204, N174, NW29 and NW79) to human ROR1 overexpression cells huROR1-HEK293 and A549 cells was detected based on FACS method. A549 cells are a human non-small cell lung cancer cell line that overexpresses huROR1.
6.1基于ELISA检测抗体对抗原蛋白huROR1-His的结合能力6.1 Detection of antibody binding ability to antigenic protein huROR1-His based on ELISA
以2μg/mL的huROR1-His包被96孔ELISA板(30μL/孔)，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入梯度稀释(3.00000、0.33333、0.11111、0.03704、0.01235、0.00412、0.00046、0.00005μg/mL)的各个抗体及阳性对照抗体99961.1并孵育1h。之后用PBST清洗3次后加入二抗Goat-anti-human Fc-HRP(abcam，ab97225)并孵育1h。孵育完成后，PBST洗板6次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读取吸光度。A 96-well ELISA plate (30 μL/well) was coated with 2 μg/mL huROR1-His overnight at 4°C. The next day, the plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 h. After the plate was washed 3 times with PBST, a gradient dilution (3.00000, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00046, 0.00005 μg/mL ) and the positive control antibody 99961.1 and incubated for 1 h. After washing with PBST for 3 times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plate was washed 6 times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color. According to the color development results, 2M HCl was added to terminate the reaction, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190).
结果如图3A-3F所示，本申请获得的全部抗体和抗原蛋白huROR1-His都具有较好的结合能力，各个抗体与抗原蛋白的结合能力与阳性对照抗体99961.1相当。The results are shown in Figures 3A-3F, all the antibodies obtained in this application have good binding ability to the antigenic protein huROR1-His, and the binding ability of each antibody to the antigenic protein is equivalent to that of the positive control antibody 99961.1.
6.2基于FACS检测抗体对huROR1-HEK293和A549细胞的结合能力6.2 Detection of antibody binding ability to huROR1-HEK293 and A549 cells based on FACS
本实施例中分别用人ROR1过表达细胞huROR1-HEK293和A549细胞两种细胞对抗体的结合活性进行评价。In this example, human ROR1 overexpression cells huROR1-HEK293 and A549 cells were used to evaluate the binding activity of antibodies.
具体方法如下：将对数生长期的huROR1-HEK293细胞或者A549细胞制备成单细胞悬液，密度调整为1×106个细胞/mL，以每孔100μL加至96孔圆底板中，4℃、300g离心并去除上清。向对应孔中分别加入各个梯度稀释(3.000000、0.300000、0.100000、0.033333、0.011111、0.003704、0.001235、0.000123μg/mL)的各个抗体和阳性对照抗体99961.1，混匀并于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入100μL 1：300稀释的二抗Goat F(ab’)2Anti-Human IgG-Fc (abcam,ab98596)，4℃避光孵育30min，洗涤3次后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测。The specific method is as follows: Prepare huROR1-HEK293 cells or A549 cells in the logarithmic growth phase into a single cell suspension, adjust the density to 1×10 6 cells/mL, add 100 μL per well into a 96-well round bottom plate, and store at 4°C. , 300g centrifugation and remove the supernatant. Each antibody and the positive control antibody 99961.1 were added to the corresponding wells with serial dilutions (3.000000, 0.300000, 0.100000, 0.033333, 0.011111, 0.003704, 0.001235, 0.000123 μg/mL), mixed well and incubated at 4°C for 30 min. After the incubated cell mixture was washed 3 times, 100 μL of 1:300 diluted secondary antibody Goat F(ab')2Anti-Human IgG-Fc was added (abcam, ab98596), incubated at 4°C in the dark for 30 min, washed 3 times and detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).
结果如图4A-4D和图5A-5C所示，在A549细胞上，NW29、N147和N99与人ROR1的亲和力优于阳性对照抗体99961.1，其余抗体与人ROR1亲和力稍微弱于阳性对照抗体99961.1；在huROR1-HEK293细胞上，N137、N31与人ROR1亲和力优于阳性对照抗体99961.1，其余抗体亲和力与阳性对照抗体99961.1相当或弱于阳性对照抗体99961.1。The results are shown in Figure 4A-4D and Figure 5A-5C, on A549 cells, the affinity of NW29, N147 and N99 to human ROR1 is better than the positive control antibody 99961.1, and the affinity of other antibodies to human ROR1 is slightly weaker than the positive control antibody 99961.1; On huROR1-HEK293 cells, the affinity between N137 and N31 and human ROR1 is better than that of the positive control antibody 99961.1, and the affinity of other antibodies is comparable to or weaker than that of the positive control antibody 99961.1.
实施例7抗体种属及同族交叉活性检测Example 7 Antibody species and homologous cross activity detection
本实施例中检测了本申请获得的各个抗体在种属及同族上的交叉反应性。采用实施例1.2制备的鼠ROR1抗原蛋白MusROR1-His和人ROR2抗原蛋白huROR2-His进行测定。In this example, the cross-reactivity of each antibody obtained in this application in terms of species and homology was tested. The murine ROR1 antigenic protein MusROR1-His and the human ROR2 antigenic protein huROR2-His prepared in Example 1.2 were used for determination.
7.1本申请抗体的种属交叉反应性检测7.1 Species cross-reactivity detection of antibodies of this application
用2μg/mL的MusROR1-His包被96孔ELISA板(30μL/孔)，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入梯度稀释(1.00000、0.11111、0.03704、0.01235、0.00412、0.00137、0.00015、0.00002μg/mL)的各个抗体及阳性对照抗体99961.1并孵育1h。之后用PBST清洗3次后加入二抗Goat-anti-human Fc-HRP(abcam，ab97225)并孵育1h。孵育完成后，PBST洗板6次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读取吸光度。A 96-well ELISA plate (30 μL/well) was coated with 2 μg/mL MusROR1-His, overnight at 4°C. The next day, the plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 hours. After washing the plate 3 times with PBST, a gradient dilution (1.00000, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, 0.00015, 0.00002 μg/mL ) and the positive control antibody 99961.1 and incubated for 1 h. After washing with PBST for 3 times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plate was washed 6 times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color. According to the color development results, 2M HCl was added to terminate the reaction, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190).
结果如图6A-6D和表3所示，本申请获得的所有抗体均可以与鼠源抗原蛋白MusROR1-His结合，显示出了良好的鼠交叉活性。而阳性对照抗体99961.1不结合鼠源抗原蛋白。因此本申请获得的抗体在基于小鼠模型的实验及测试中具有广泛用途。The results are shown in Figures 6A-6D and Table 3. All the antibodies obtained in this application can bind to the murine antigenic protein MusROR1-His, showing good murine cross-activity. The positive control antibody 99961.1 does not bind to the mouse antigen protein. Therefore, the antibody obtained in the present application has a wide range of applications in experiments and tests based on mouse models.
7.2本申请抗体同族交叉反应性检测7.2 Antibody homologous cross-reactivity detection of this application
用2μg/mL的huROR2-His包被96孔ELISA板(30μL/孔)，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入梯度稀释的候选抗体及阳性对照抗体99961.1并孵育1h。之后用PBST清洗3次后加入二抗Goat-anti-human Fc-HRP(abcam，ab97225)并孵育1h。孵育完成后，PBST洗板6次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读取吸光度。A 96-well ELISA plate (30 μL/well) was coated with 2 μg/mL huROR2-His overnight at 4°C. On the next day, the plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 hours. After the plate was washed 3 times with PBST, the candidate antibody and positive control antibody 99961.1 in gradient dilution were added and incubated for 1 hour. After washing with PBST for 3 times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plate was washed 6 times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color. According to the color development results, 2M HCl was added to terminate the reaction, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190).
结果如图7A-7D和表3所示，N204、NW29、NW79、N218、N31和N99可以与抗原蛋白huROR2-His结合，而其余抗体与抗原蛋白huROR2-His不结合。The results are shown in Figures 7A-7D and Table 3, N204, NW29, NW79, N218, N31 and N99 could bind to the antigenic protein huROR2-His, while the rest of the antibodies did not bind to the antigenic protein huROR2-His.
表3候选抗体种属交叉和同族交叉活性

Table 3 Candidate Antibody Species Crossover and Homologous Crossover Activity

实施例8本申请抗体内吞效率检测Example 8 Detection of Antibody Endocytosis Efficiency of the Application
在本实施例中，分别运用FACS和Fab-Zap两种检测方法对本申请获得的抗体的内吞效率进行鉴定。FACS法内吞检测是用过饱和的抗体结合细胞，检测短时间内的抗体内吞效率；Fab-Zap法是以不同浓度的结合了saporin毒素的抗体去结合细胞，通过内吞将毒素带进去杀伤靶细胞，此种方法反映的是内吞效率长期累加的效果。In this example, the endocytosis efficiency of the antibody obtained in this application was identified by using two detection methods of FACS and Fab-Zap respectively. The FACS method for endocytosis detection is to use supersaturated antibodies to bind cells to detect the endocytosis efficiency of antibodies in a short period of time; the Fab-Zap method uses different concentrations of antibodies combined with saporin toxin to bind cells, and the toxin is brought in through endocytosis To kill target cells, this method reflects the effect of long-term accumulation of endocytic efficiency.
8.1 FACS法检测抗体内吞效率8.1 FACS method to detect antibody endocytosis efficiency
抗体稀释：将测试抗体用DMEM完全培养基稀释成10.0000μg/mL的终浓度。Antibody dilution: Dilute the test antibody with DMEM complete medium to a final concentration of 10.0000 μg/mL.
处理细胞：将huROR1-HEK293细胞消化后加入DMEM完全培养基。充分混匀细胞后，细胞计数并测定其活率。分别取106的细胞加到1.5mL离心管，300g离心5分钟，弃去上清，用1mL预冷的DMEM培养基重悬，300g离心5分钟，弃去上清。Cell treatment: Digest huROR1-HEK293 cells and add DMEM complete medium. After mixing the cells thoroughly, count the cells and determine their viability. Add 106 cells to 1.5mL centrifuge tube, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend with 1mL pre-cooled DMEM medium, centrifuge at 300g for 5 minutes, discard the supernatant.
一抗孵育：取1000μL预冷的上述稀释的抗体，加到已有细胞的离心管中制备抗体细胞混悬液，将抗体细胞混悬液加入96孔板中，4℃孵育。Primary antibody incubation: Take 1000 μL of the pre-cooled diluted antibody above and add it to a centrifuge tube with existing cells to prepare antibody cell suspension, add the antibody cell suspension to a 96-well plate, and incubate at 4°C.
胞外二抗孵育：快速转移96孔板中的混悬液到第二个96孔板中。向第二个96孔板中每孔加入180μL预冷FACS buffer清洗2遍。之后每孔各加入100μL的稀释二抗FITC-labeled rabit-anit-huFc或RPE-labeled rabit-anit-huFc(二抗采用FACS buffer以1：150倍稀释)；4℃孵育30min。Extracellular secondary antibody incubation: Quickly transfer the suspension in the 96-well plate to a second 96-well plate. Add 180 μL pre-cooled FACS buffer to each well of the second 96-well plate and wash 2 times. After that, 100 μL of diluted secondary antibody FITC-labeled rabit-anit-huFc or RPE-labeled rabbit-anit-huFc was added to each well (the secondary antibody was diluted 1:150 times with FACS buffer); incubate at 4°C for 30 min.
细胞固定：孵育结束后，离心弃上清液，每孔加入180μL预冷FACS buffer清洗细胞2遍。离心去上清，每孔用100μL 4％多聚甲醛室温固定细胞30min。Cell fixation: After the incubation, the supernatant was discarded by centrifugation, and 180 μL of pre-cooled FACS buffer was added to each well to wash the cells twice. The supernatant was removed by centrifugation, and the cells were fixed with 100 μL 4% paraformaldehyde per well for 30 min at room temperature.
破碎细胞膜：固定完后，加入180μL FACS buffer清洗2遍。每孔加入100μL预热的0.5％Triton X-100，室温穿孔5min。Broken cell membrane: After fixation, add 180 μL FACS buffer to wash 2 times. Add 100 μL of preheated 0.5% Triton X-100 to each well, and perforate at room temperature for 5 min.
胞内二抗孵育：清洗96孔板之后，每孔加入180μL预热的Permeabilization Buffer(InvitrogenTM eBioscienceTM，00-8333-56)清洗2遍。每孔各加入100μL的稀释二抗RPE-labeled rabit-anit-huFc(采用Permeabilization buffer以1：150倍稀释)。室温孵育60min。Intracellular secondary antibody incubation: After washing the 96-well plate, add 180 μL of preheated Permeabilization Buffer (Invitrogen ™ eBioscience ™ , 00-8333-56) to each well to wash twice. Add 100 μL of diluted secondary antibody RPE-labeled rabbit-anit-huFc (diluted 1:150 times with Permeabilization buffer) to each well. Incubate at room temperature for 60min.
检测荧光：清洗96孔板之后，用100μL FACS buffer重悬，通过流式细胞仪检测。 Detection of fluorescence: After washing the 96-well plate, resuspend it with 100 μL FACS buffer, and detect it by flow cytometry.
本实验采用两组不同荧光设置对每个样品染色，第一组设置为FITC+PE，即先用FITC二抗识别胞外一抗，破膜后用PE二抗识别胞内一抗，在这一组中PE为胞内信号，FITC为胞外信号；第二组设置为PE+PE，即先用PE二抗识别胞外一抗，破膜后用PE二抗识别胞内一抗，这一组PE为胞内和胞外信号的总和。同时这两组样品均用FITC和PE通道去检测，内吞率计算值都是计算PE通道检测值，具体公式为：内吞率＝一组(FITC+PE)PE通道值/二组(PE+PE)PE通道值×100％。In this experiment, two sets of different fluorescence settings were used to stain each sample. The first set was FITC+PE, that is, the FITC secondary antibody was used to identify the extracellular primary antibody first, and the PE secondary antibody was used to identify the intracellular primary antibody after membrane rupture. In one group, PE is the intracellular signal, and FITC is the extracellular signal; the second group is set to PE+PE, that is, the PE secondary antibody is used to recognize the extracellular primary antibody first, and the PE secondary antibody is used to recognize the intracellular primary antibody after membrane rupture. A set of PEs is the sum of intracellular and extracellular signals. At the same time, these two groups of samples were detected by FITC and PE channels, and the calculated value of endocytosis rate was the detection value of PE channel. The specific formula was: endocytosis rate = one group (FITC+PE) PE channel value/two groups (PE +PE) PE channel value × 100%.
结果如图8A-8B所示，本实施例的结果显示N147、N100、NW37、N99和N31在较短的时间内(3h内)内吞效率优于对照抗体99961.1，N174、N218、N204和NW29与对照抗体99961.1相当。The results are shown in Figure 8A-8B. The results of this example show that the endocytosis efficiency of N147, N100, NW37, N99 and N31 is better than that of the control antibody 99961.1, N174, N218, N204 and NW29 in a short period of time (within 3h). Comparable to control antibody 99961.1.
8.2 Fab-Zap法检测抗体内吞效率8.2 Fab-Zap method to detect antibody endocytosis efficiency
本实验通过抗体介导Fab-ZAP内吞的细胞毒性来检测抗体的内吞活性。Fab-ZAP是连接了saporin(皂素)的Fab片段，saporin是一种核糖体抑制剂，能够抑制蛋白质的合成而使细胞死亡。本实验用的Fab-ZAP是一种能够和人源的抗体结合的Fab片段，Fab-ZAP和人源抗体孵育后使人源抗体带上毒素，当人源抗体被内吞时，毒素随着抗体进入到细胞内，使细胞死亡，然后通过MTS(Promega，G3580)检测细胞的活性来检测抗体是否内吞。In this experiment, the endocytic activity of the antibody was detected by antibody-mediated cytotoxicity of Fab-ZAP endocytosis. Fab-ZAP is a Fab fragment linked to saporin (saponin), a ribosome inhibitor that can inhibit protein synthesis and cause cell death. The Fab-ZAP used in this experiment is a Fab fragment that can bind to human antibodies. After incubation with Fab-ZAP and human antibodies, the human antibodies are loaded with toxins. When the human antibodies are endocytosed, the toxins are The antibody enters into the cell to cause cell death, and then the cell activity is detected by MTS (Promega, G3580) to detect whether the antibody is endocytized.
具体实验方法如下：首先将Fab-ZAP用DMEM完全培养基稀释成0.4nM，然后用0.4nM Fab-ZAP梯度稀释(0.400000、0.080000、0.026667、0.008889、0.002963、0.000988、0.000329、0.000066μg/mL)本申请获得的各个抗体及阳性对照抗体制成抗体稀释液。将对数生长期huROR1-HEK293细胞制成单细胞悬液，调整密度为6×106个细胞/mL,以每孔50μL接种到96孔板中，然后取前述抗体稀释液，以每孔50μL加入细胞培养板中，充分吹打混匀。将细胞培养板放入37℃细胞培养箱孵育48小时。孵育结束后，向每孔中加入7.5μL TritonX-100溶液，轻轻拍打混匀，将细胞培养板放入37℃细胞培养箱孵育0.5小时。接着向每孔中继续加入20μL MTS，37℃孵育1-4h。最后以1000rpm离心细胞培养板5min，酶标仪读取数据，检测波长A492。The specific experimental method is as follows: First, Fab-ZAP was diluted to 0.4nM with DMEM complete medium, and then diluted with 0.4nM Fab-ZAP (0.400000, 0.080000, 0.026667, 0.008889, 0.002963, 0.000988, 0.000329, 0.000066μg/mL) Antibody diluents were prepared from each antibody and positive control antibody obtained through application. Prepare huROR1-HEK293 cells in the logarithmic growth phase as a single cell suspension, adjust the density to 6× 106 cells/mL, inoculate them into 96-well plates at 50 μL per well, and then take the aforementioned antibody dilution solution at 50 μL per well Add to the cell culture plate and mix well by pipetting. Place the cell culture plate in a 37°C cell culture incubator and incubate for 48 hours. After the incubation, add 7.5 μL TritonX-100 solution to each well, pat gently to mix, and place the cell culture plate in a 37°C cell culture incubator for 0.5 hours. Then continue to add 20 μL MTS to each well, and incubate at 37°C for 1-4h. Finally, the cell culture plate was centrifuged at 1000 rpm for 5 min, and the data was read with a microplate reader, and the detection wavelength was A492.
结果如图9A-9F所示，表明在本实施例限定的抗体浓度条件下，本申请制得的抗体的内吞效果与对照抗体相当。考虑到细胞对于毒素敏感是在某一个阈值以上才会出现反应，当累加的毒素差异不大时较难拉开差距。The results are shown in Figures 9A-9F, indicating that under the antibody concentration conditions defined in this example, the endocytosis effect of the antibody prepared in this application is equivalent to that of the control antibody. Considering that cells are sensitive to toxins above a certain threshold, it is difficult to widen the gap when the accumulated toxins are not very different.
实施例9抗体亲和动力学(Biacore)分析Example 9 Antibody Affinity Kinetics (Biacore) Analysis
本实施例中，基于Biacore设备检测本申请获得抗体和阳性对照抗体99961.1与抗原蛋白huROR1-His的亲和力。In this example, the affinity of the antibody obtained in this application and the positive control antibody 99961.1 to the antigenic protein huROR1-His was detected based on Biacore equipment.
蛋白的偶联：将实施例1.2生产的huROR1-His蛋白用pH 5.0的NaAc缓冲液稀释至5.6μg/mL，流速设置为10μL/min，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)和N-羟基丁二酰亚胺(NHS)混合液活化芯片时间为默认值420s，采用预设偶联量的偶联模式固定抗原蛋白 huROR1-His至约75RU水平，以乙醇胺封闭未结合供试品的活化基团。Protein coupling: Dilute the huROR1-His protein produced in Example 1.2 to 5.6 μg/mL with pH 5.0 NaAc buffer, set the flow rate to 10 μL/min, 1-ethyl-(3-dimethylaminopropyl ) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) mixed solution activation time of the chip is the default value of 420s, using the coupling mode of the preset coupling amount to immobilize the antigen protein huROR1-His to the level of about 75RU, and ethanolamine is used to block the activation group that is not bound to the test product.
样品测试条件：以含有0.05％Tween-20的PBS缓冲液(pH7.4)作为运行缓冲液，将运行缓冲液作为对照测试样品，设置系列抗体浓度(4nM，20nM)，样品分析时设置流速为30μL/min，结合时间为120s，解离时间为360s。解离结束之后，采用10mM Gly-HCl(pH2.0)再生20s以完全去除与配体结合的抗体。Sample test conditions: use PBS buffer (pH7.4) containing 0.05% Tween-20 as the running buffer, use the running buffer as the control test sample, set a series of antibody concentrations (4nM, 20nM), and set the flow rate during sample analysis. 30μL/min, the binding time is 120s, and the dissociation time is 360s. After dissociation, 10mM Gly-HCl (pH2.0) was used to regenerate for 20s to completely remove the antibody bound to the ligand.
参数拟合：实验采用多循环运行，其响应信号以分析时间为横坐标，响应值为纵坐标。所得数据进行双参比扣减后，通过BIAcore T200分析软件进行拟合，所采用的拟合模型为1:1 Langmuir结合模型，确定其结合解离常数等亲和力指标。Parameter fitting: The experiment was run in multiple cycles, and the response signal took the analysis time as the abscissa, and the response value as the ordinate. After double-reference subtraction, the obtained data were fitted by BIAcore T200 analysis software. The fitting model adopted was a 1:1 Langmuir binding model, and affinity indicators such as binding and dissociation constants were determined.
结果如表4所示，N147的亲和力优于对照抗体99961.1的亲和力2个数量级，其中N147的KD达到6.62E-11，而对照抗体99961.1的KD为1.98E-9，其余抗体亲和力与对照抗体99961.1的亲和力相当。The results are shown in Table 4. The affinity of N147 is 2 orders of magnitude better than that of the control antibody 99961.1. The KD of N147 reaches 6.62E-11, while the KD of the control antibody 99961.1 is 1.98E-9. The affinity of the other antibodies is similar to that of the control antibody 99961.1 The affinity is quite.
表4抗体的KD值
Table 4 KD value of antibody
实施例10抗体亲和动力学(Gator)检测Example 10 Antibody Affinity Kinetics (Gator) Detection
本实施例中，基于Gator设备检测本申请获得抗体和阳性对照抗体99961.1与抗原蛋白huROR1-His的亲和力。In this example, the affinity of the antibody obtained in this application and the positive control antibody 99961.1 to the antigenic protein huROR1-His was detected based on Gator equipment.
具体试验方法如下：首先将PBS(10mM pH 7.4)(IgG-free，购自Jackson ImmunoResearch Lab)+0.02％Tween 20(购自thermo)+0.2％BSA(购自源培)配置成Q buffer缓冲液，用配置好的Q buffer缓冲液将待测抗体的存储液稀释到终浓度为30nM的工作液；将抗原蛋白huROR1-His存储液用Q buffer配置成倍数稀释(480、240、120、60、30、15、7.5nM)的工作液，然后利用Gator仪器及其配套软件，选择Advanced Kinetics实验模式进行检测和分析。试验结果如表5所示，N100、NW29和NW79的亲和力高于对照抗体99961.1的亲和力，其中N100的KD达到8.85E-9，NW29的KD达到6.60E-9，NW79的KD达到6.94E-8，而对照抗体99961.1的KD为1.35E-8，其余抗体亲和力与对照抗体99961.1的亲和力相当。The specific test method is as follows: first, PBS (10mM pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from Yuanpei) was configured as Q buffer buffer , use the configured Q buffer buffer to dilute the storage solution of the antibody to be tested to a working solution with a final concentration of 30nM; configure the antigenic protein huROR1-His storage solution with Q buffer to make multiple dilutions (480, 240, 120, 60, 30, 15, 7.5nM) working solution, and then use the Gator instrument and its supporting software to select the Advanced Kinetics experimental mode for detection and analysis. The test results are shown in Table 5. The affinity of N100, NW29 and NW79 is higher than that of the control antibody 99961.1, among which the KD of N100 reaches 8.85E-9, the KD of NW29 reaches 6.60E-9, and the KD of NW79 reaches 6.94E-8 , while the KD of the control antibody 99961.1 was 1.35E-8, and the affinities of the other antibodies were comparable to those of the control antibody 99961.1.
表5抗体的KD值

Table 5 KD value of antibody

实施例11亲和动力学表位分组Example 11 Affinity Kinetic Epitope Grouping
本实施例采用亲和动力学的方法，对本申请获得的抗体和阳性对照抗体99961.1的表位进行分组。In this example, the method of affinity kinetics was used to group the epitopes of the antibodies obtained in this application and the positive control antibody 99961.1.
具体试验方法如下：首先将PBS(10mM pH 7.4)(IgG-free，购自Jackson ImmunoResearch Lab)+0.02％Tween 20(购自thermo)+0.2％BSA(购自源培)配置成Q buffer缓冲液，用配置好的Q buffer缓冲液将待测抗体的存储液稀释到终浓度为100nM的工作液；将抗原蛋白huROR1-His存储液用Q buffer配置成50nM的工作液，然后利用Gator仪器及其配套软件，基于Epitope Binning实验模式的Tandem设置进行检测和分析。由此获得的各个抗体表位分组的结果如表6所示，其中每组抗体具有相同的表位。The specific test method is as follows: first, PBS (10mM pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from Yuanpei) was configured as Q buffer buffer , Dilute the storage solution of the antibody to be tested to a working solution with a final concentration of 100nM with the configured Q buffer buffer; configure the storage solution of the antigenic protein huROR1-His into a 50nM working solution with Q buffer, and then use the Gator instrument and its Supporting software, based on the Tandem setting of the Epitope Binning experiment mode for detection and analysis. The result of each antibody epitope grouping thus obtained is shown in Table 6, wherein each group of antibodies has the same epitope.
表6表位分组结果汇总
Table 6 Summary of epitope grouping results
实施例12抗体亲和力成熟Example 12 Antibody affinity maturation
本实施例对抗体N147、N31、N137和NW37进行亲和力成熟改造，用于提高抗体亲和力和其他生物学活性。亲和力成熟改造是基于M13噬菌体展示技术，采用codon-based引物(引物合成过程中，单个密码子由NNK组成)引入CDR区突变，构建4个噬菌体展示文库：文库1和文库2为单点组合突变(即，每个CDR仅含有一个突变位点)，文库1为CDRL1+CDRL3+CDRH3组合突变，文库2为CDRL2+CDRH1+CDRH2组合突变；文库3和文库4为双点饱和突变，文库3为CDRL3的双点饱和突变，文库4为CDRH3的双点饱和突变。In this example, affinity maturation was performed on antibodies N147, N31, N137 and NW37 to improve antibody affinity and other biological activities. The affinity maturation transformation is based on the M13 phage display technology, using codon-based primers (during the primer synthesis process, a single codon consists of NNK) to introduce mutations in the CDR region, and construct 4 phage display libraries: library 1 and library 2 are single-point combinatorial mutations (that is, each CDR contains only one mutation site), library 1 is CDRL1+CDRL3+CDRH3 combined mutation, library 2 is CDRL2+CDRH1+CDRH2 combined mutation; library 3 and library 4 are double point saturation mutations, library 3 is Double point saturation mutation of CDRL3, library 4 is double point saturation mutation of CDRH3.
具体的建库方法：首先合成包含点突变的引物(金唯智生物科技有限公司)；其次分别以待改造抗体(也称母本抗体)N147、N31、N137和NW37的编码序列为PCR扩增模板，扩增CDR区 包含突变的序列，通过桥连PCR的方法，将包含不同CDR突变的片段进行组合，然后通过双酶切(HindⅢ和NotⅠ)和双粘端连接将点突变抗体连接到噬菌体展示载体中，最后通过电转将带有突变位点的抗体序列转入大肠杆菌SS320中。库容计算、噬菌体文库制备和文库筛选操作过程详见前述实施例。N147亲和力成熟后获得N147-135(重链可变区氨基酸序列示于SEQ ID NO:66，轻链可变区氨基酸序列示于SEQ ID NO:53)、N147-2(重链可变区氨基酸序列示于SEQ ID NO:161，轻链可变区氨基酸序列示于SEQ ID NO:160)、N147-86(重链可变区氨基酸序列示于SEQ ID NO:163，轻链可变区氨基酸序列示于SEQ ID NO:162)和N147-93(重链可变区氨基酸序列示于SEQ ID NO:165，轻链可变区氨基酸序列示于SEQ ID NO:164)；N31亲和力成熟后获得N31-6(重链可变区氨基酸示于SEQ ID NO:64，轻链可变区氨基酸示于SEQ ID NO:63)、N31-14(重链可变区氨基酸示于SEQ ID NO:151，轻链可变区氨基酸示于SEQ ID NO:150)、N31-18(重链可变区氨基酸示于SEQ ID NO:153，轻链可变区氨基酸示于SEQ ID NO:152)和N31-56(重链可变区氨基酸示于SEQ ID NO:155，轻链可变区氨基酸示于SEQ ID NO:154)；N137亲和力成熟获得N137-72(重链可变区氨基酸示于SEQ ID NO:68，轻链可变区氨基酸示于SEQ ID NO:67)、N137-47(重链可变区氨基酸示于SEQ ID NO:157，轻链可变区氨基酸示于SEQ ID NO:156)、N137-53(重链可变区氨基酸示于SEQ ID NO:52，轻链可变区氨基酸示于SEQ ID NO:158)、N137-60(重链可变区氨基酸示于SEQ ID NO:52，轻链可变区氨基酸示于SEQ ID NO:159)；NW37亲和力成熟后获得NW37-37(重链可变区氨基酸示于SEQ ID NO:70，轻链可变区氨基酸示于SEQ ID NO:69)、NW37-99(重链可变区氨基酸示于SEQ ID NO:62，轻链可变区氨基酸示于SEQ ID NO:166)、NW37-113(重链可变区氨基酸示于SEQ ID NO:168，轻链可变区氨基酸示于SEQ ID NO:167)和NW37-136(重链可变区氨基酸示于SEQ ID NO:169，轻链可变区氨基酸示于SEQ ID NO:61)；亲和力成熟改造后抗体的CDR区氨基酸序列见表7。通过前述实施例公开的方法表达、纯化获得的相应抗体蛋白。并通过SDS PAGE鉴定抗体蛋白的分子量和纯度，测试结果表明亲和力成熟后抗体的非还原胶的条带在150kD左右，还原胶的条带在55kD左右和25kD左右，符合预期大小。通过还原胶检测的所有亲和力成熟后抗体纯度均大于95％。Specific library construction method: first, synthesize primers containing point mutations (Jinweizhi Biotechnology Co., Ltd.); second, use the coding sequences of antibodies to be transformed (also called parental antibodies) N147, N31, N137, and NW37 as PCR amplification templates , to amplify the CDR regions For the sequence containing the mutation, the fragments containing different CDR mutations were combined by bridging PCR, and then the point mutant antibody was connected to the phage display vector by double enzyme digestion (HindⅢ and NotI) and double sticky end ligation, and finally by The antibody sequence with the mutation site was transformed into Escherichia coli SS320 by electroporation. For the operation process of library capacity calculation, phage library preparation and library screening, please refer to the previous examples for details. After N147 affinity maturation, N147-135 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 53), N147-2 (the amino acid sequence of the heavy chain variable region The sequence is shown in SEQ ID NO:161, the light chain variable region amino acid sequence is shown in SEQ ID NO:160), N147-86 (the heavy chain variable region amino acid sequence is shown in SEQ ID NO:163, the light chain variable region amino acid sequence The sequence is shown in SEQ ID NO:162) and N147-93 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:165, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:164); N31 was obtained after affinity maturation N31-6 (the amino acids of the heavy chain variable region are shown in SEQ ID NO:64, and the amino acids of the light chain variable region are shown in SEQ ID NO:63), N31-14 (the amino acids of the heavy chain variable region are shown in SEQ ID NO:151 , light chain variable region amino acids are shown in SEQ ID NO:150), N31-18 (heavy chain variable region amino acids are shown in SEQ ID NO:153, light chain variable region amino acids are shown in SEQ ID NO:152) and N31 -56 (the amino acid of the heavy chain variable region is shown in SEQ ID NO:155, and the amino acid of the light chain variable region is shown in SEQ ID NO:154); N137 affinity maturation obtains N137-72 (the amino acid of the heavy chain variable region is shown in SEQ ID NO:154); NO:68, the light chain variable region amino acid is shown in SEQ ID NO:67), N137-47 (the heavy chain variable region amino acid is shown in SEQ ID NO:157, the light chain variable region amino acid is shown in SEQ ID NO:156 ), N137-53 (the amino acids of the heavy chain variable region are shown in SEQ ID NO:52, and the amino acids of the light chain variable region are shown in SEQ ID NO:158), N137-60 (the amino acids of the heavy chain variable region are shown in SEQ ID NO: NW37-37 (the amino acid of the heavy chain variable region is shown in SEQ ID NO:70, and the amino acid of the light chain variable region is shown in SEQ ID NO:70) after affinity maturation of NW37; ID NO:69), NW37-99 (amino acids of heavy chain variable region are shown in SEQ ID NO:62, light chain variable region amino acids are shown in SEQ ID NO:166), NW37-113 (amino acids of heavy chain variable region are shown in SEQ ID NO:166), NW37-113 (amino acids of heavy chain variable region are shown in In SEQ ID NO:168, amino acids of the light chain variable region are shown in SEQ ID NO:167) and NW37-136 (amino acids of the heavy chain variable region are shown in SEQ ID NO:169, amino acids of the light chain variable region are shown in SEQ ID NO:169) NO:61); the amino acid sequence of the CDR region of the antibody after affinity maturation transformation is shown in Table 7. The corresponding antibody protein obtained was expressed and purified by the methods disclosed in the foregoing examples. The molecular weight and purity of the antibody protein were identified by SDS PAGE. The test results showed that the non-reducing gum band of the antibody after affinity maturation was around 150kD, and the bands of the reducing gum were around 55kD and 25kD, which were in line with the expected size. All affinity matured antibodies were >95% pure by reducing gel assay.
表7亲和力成熟后抗体的CDR区氨基酸序列(采用AbM定义)


Table 7 The amino acid sequence of the CDR region of the antibody after affinity maturation (defined by AbM)


实施例13亲和力成熟后抗体的抗原亲和力检测Example 13 Antigen affinity detection of antibodies after affinity maturation
本实施例中，基于FACS方法检测了亲和力成熟后抗体与A549细胞的亲和能力。具体检测方法参见实施例6。检测结果如图10A-10D所示，结果表明亲和力成熟后抗体与A549细胞的亲和能力均有所提升，其中N31-6、N31-14、N31-18、N31-56、NW37-37、NW37-99、NW37-113、NW37-136、N137-47、N137-53、N137-60和N137-72亲和力均显著优于其对应母本抗体分子N31、NW37和N137；N147-135、N147-2、N147-86和N147-93亲和力均优于其对应母本分子N147且优于对照抗体99961.1。In this example, the affinity ability of the antibody after affinity maturation to A549 cells was detected based on the FACS method. Refer to Example 6 for the specific detection method. The test results are shown in Figures 10A-10D, and the results show that the affinity of the antibody to A549 cells has been improved after affinity maturation, among which N31-6, N31-14, N31-18, N31-56, NW37-37, NW37 The affinity of -99, NW37-113, NW37-136, N137-47, N137-53, N137-60 and N137-72 were significantly better than their corresponding parent antibody molecules N31, NW37 and N137; N147-135, N147-2 , N147-86 and N147-93 had better affinities than their corresponding parent molecule N147 and better than the control antibody 99961.1.
实施例14亲和力成熟后抗体亲和动力学(Biacore)分析Example 14 Antibody affinity kinetics (Biacore) analysis after affinity maturation
本实施例中，基于Biacore设备检测16个亲和力成熟后抗体和母本分子与人ROR1抗原蛋白huROR1-His的亲和力。具体实验方法参照实施例9，结果如表8所示，NW37-37的KD比母本抗体高1个数量级；其余亲和力成熟后抗体与人ROR1抗原蛋白huROR1-His的亲和力也均优于其母本分子。In this example, the affinity of 16 affinity matured antibodies and parent molecules to human ROR1 antigen protein huROR1-His was detected based on Biacore equipment. Refer to Example 9 for specific experimental methods, and the results are shown in Table 8. The KD of NW37-37 is an order of magnitude higher than that of the parental antibody; the affinity of other antibodies after affinity maturation to human ROR1 antigen protein huROR1-His is also better than that of the parental antibody. This molecule.
表8亲和力成熟后抗体的KD值

Table 8 KD value of antibody after affinity maturation

实施例15亲和力成熟后抗体内吞效率检测Example 15 Detection of antibody endocytosis efficiency after affinity maturation
本实施例中，对实施例11中亲和力成熟的抗体N147-135、N31-6、N137-72、N147-2和NW37-37进行Fab-ZAP法内吞检测，具体方法参照实施例8.2，结果如图11A-11E所示，在本实施例限定的抗体浓度范围内，亲和力成熟后抗体与对照抗体的内吞效率相当。In this example, the Fab-ZAP endocytosis detection was performed on the affinity-matured antibodies N147-135, N31-6, N137-72, N147-2 and NW37-37 in Example 11. For the specific method, refer to Example 8.2. The results As shown in Figures 11A-11E, within the antibody concentration range defined in this example, the endocytosis efficiency of the antibody after affinity maturation is comparable to that of the control antibody.
实施例16 ADC制备Embodiment 16 ADC preparation
本实施例中分别将抗体N99和N147-135和对照抗体99961.1与毒素MMAE(一种tubulin抑制剂，具有抗癌活性)偶联构建了抗体偶联药物，MMAE与接头MC-VC-PAB连接构成MC-VC-PAB-MMAE，并借助接头与抗体的半胱氨酸上的巯基共价连接，从而使得抗体与MMAE缀合制得抗体偶联药物。IgG1型抗体具有16对半胱氨酸残基，其中以12个链内和4个链间二硫键的形式存在。链间二硫键具有溶剂可及性，可以被还原剂还原形成八个巯基，进而成为偶联目标(McCombs J,Owen S.Antibody drug conjugates:design and selection of linker,payload and conjugation chemistry.AAPS J.2015；17:339-51)。In this example, the antibodies N99 and N147-135 and the control antibody 99961.1 were coupled with the toxin MMAE (a tubulin inhibitor with anticancer activity) to construct an antibody-drug conjugate, and MMAE was linked with the linker MC-VC-PAB to form MC-VC-PAB-MMAE is covalently linked to the sulfhydryl group on the cysteine of the antibody by means of a linker, so that the antibody is conjugated with MMAE to obtain an antibody-coupled drug. IgG1 antibodies have 16 pairs of cysteine residues, of which there are 12 intrachain and 4 interchain disulfide bonds. Interchain disulfide bonds are solvent-accessible, and can be reduced by reducing agents to form eight sulfhydryl groups, which then become coupling targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J .2015;17:339-51).
具体制备方法如下：The specific preparation method is as follows:
将抗体N99、N147-135和对照抗体99961.1从-80℃冰箱取出，融化后分别转移到15mL 30KD超滤离心管中，补加偶联缓冲液(每1L含量：Na2HPO4·2H2O 6.86g，NaH2PO4·H2O 1.58g，纯化水定容至1000g，pH7.4)至15mL，4500rpm离心约30min，浓缩至2-3mL，重新补加透析液(每1L含量：组氨酸0.73g，一水盐酸组氨酸1.12g，纯化水定容至1000g，pH 6.0)至15mL，反复透析8-10次，获得抗体原液，检测透析后抗体浓度。Take antibodies N99, N147-135 and control antibody 99961.1 out of the -80°C refrigerator, transfer them to 15mL 30KD ultrafiltration centrifuge tubes after melting, and add coupling buffer (per 1L content: Na 2 HPO 4 2H 2 O 6.86g, NaH 2 PO 4 ·H 2 O 1.58g, dilute purified water to 1000g, pH7.4) to 15mL, centrifuge at 4500rpm for about 30min, concentrate to 2-3mL, add dialysate again (per 1L content: group Amino acid 0.73g, histidine hydrochloride monohydrate 1.12g, purified water to 1000g, pH 6.0) to 15mL, repeated dialysis for 8-10 times to obtain antibody stock solution, and detect the antibody concentration after dialysis.
依次加入抗体原液、10mM二硫键还原剂TCEP母液(三(2-羧乙基)膦盐酸盐母液，每1L含量：Na2HPO4·2H2O 6.86g，NaH2PO4·H2O 1.58g)、10mM DTPA母液(二乙烯三胺五乙酸母液，每1L含量：DTPA 3.90g，NaOH 1.20g)和偶联缓冲液构成还原反应体系，以还原抗体的半胱氨酸上的巯基。还原反应体系中各组分的加入量参见表9，使得还原反应体系中抗体终浓度为5mg/mL(N99和999611)或4mg/mL(N147-135)，DTPA终浓度为1mM，TCEP与抗体摩尔比为2.2(N99和99961.1)或2.7(N147-135)。充分混匀之后，将还原反应体系置于25℃恒温混匀仪中，400rpm，还原反应2h。 Add antibody stock solution, 10mM disulfide bond reducing agent TCEP mother solution (tris(2-carboxyethyl)phosphine hydrochloride mother solution, content per 1L: Na 2 HPO 4 ·2H 2 O 6.86g, NaH 2 PO 4 ·H 2 O 1.58g), 10mM DTPA mother solution (diethylenetriaminepentaacetic acid mother solution, content per 1L: DTPA 3.90g, NaOH 1.20g) and coupling buffer constitute a reduction reaction system to reduce the sulfhydryl group on the cysteine of the antibody . The addition amount of each component in the reduction reaction system is shown in Table 9, so that the final concentration of the antibody in the reduction reaction system is 5 mg/mL (N99 and 999611) or 4 mg/mL (N147-135), the final concentration of DTPA is 1 mM, TCEP and antibody The molar ratio is 2.2 (N99 and 99961.1) or 2.7 (N147-135). After thorough mixing, the reduction reaction system was placed in a constant temperature mixer at 25° C., 400 rpm, and the reduction reaction was performed for 2 hours.
称量MC-VC-PAB-MMAE，使用DMSO溶解，配置成5mM MC-VC-PAB-MMAE母液。还原反应结束后，在冰水浴中依次向还原反应体系中添加MC-VC-PAB-MMAE母液，添加量如表10所示，制成偶联反应体系。充分混匀后，将偶联反应体系置于25℃恒温混匀仪中，400rpm，偶联反应1h获得包含ADC的溶液。Weigh MC-VC-PAB-MMAE, dissolve it in DMSO, and prepare 5mM MC-VC-PAB-MMAE stock solution. After the reduction reaction was completed, MC-VC-PAB-MMAE mother liquor was sequentially added to the reduction reaction system in an ice-water bath, and the addition amount was shown in Table 10 to prepare a coupling reaction system. After thorough mixing, the coupling reaction system was placed in a constant temperature mixer at 25° C. at 400 rpm, and the coupling reaction was performed for 1 hour to obtain a solution containing ADC.
偶联结束后，离心包含ADC的溶液并过滤，获得ADC样品，并将其转移到15mL 30KD超滤离心管中。补加透析液至15mL，4500rpm离心20min，浓缩至2-3mL，重新补加透析液至15mL，反复透析8-10次。对透析后的ADC样品进行SEC-HPLC检测、HIC-HPLC检测、浓度检测、游离药物检测等。测试结果参见表11。测试结果显示获得了纯度99％以上的ADC。After the coupling, the ADC-containing solution was centrifuged and filtered to obtain an ADC sample, which was transferred to a 15 mL 30KD ultrafiltration centrifuge tube. Add dialysate to 15 mL, centrifuge at 4500 rpm for 20 min, concentrate to 2-3 mL, add dialysate to 15 mL, and repeat dialysis 8-10 times. SEC-HPLC detection, HIC-HPLC detection, concentration detection, free drug detection, etc. were performed on ADC samples after dialysis. See Table 11 for test results. The test results showed that the ADC with a purity of more than 99% was obtained.
表9还原反应体系组成
Table 9 Reduction reaction system composition
表10 MC-VC-PAB-MMAE母液添加量
Table 10 MC-VC-PAB-MMAE mother liquor addition amount
表11 ADC样品测试结果
Table 11 ADC sample test results
实施例17 ADC的抗原结合活性检测Antigen-binding activity detection of embodiment 17 ADC
本实施例中，基于FACS方法检测了所述制得的抗体偶联药物N99-MMAE和N147-135-MMAE与肿瘤细胞A549和HT-29(人结肠癌细胞)上的人ROR1的结合能力。In this example, the binding ability of the prepared antibody-drug conjugates N99-MMAE and N147-135-MMAE to human ROR1 on tumor cells A549 and HT-29 (human colon cancer cells) was detected based on the FACS method.
具体方法如下：将对数生长期的A549细胞或者HT-29细胞制备成单细胞悬液，密度调整为1×106个细胞/mL，以每孔100μL加至96孔圆底板中，4℃、300g离心并去除上清。向对应孔中加入梯度稀释(1.0000、0.3333、0.1111、0.0370、0.0123、0.0041、0.0014、0.0001μg/mL)的N99、N99-MMAE、N147-135、N147-135-MMAE、99961.1、99961.1-MMAE和阴性对照，混匀并于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入100μL 1：300稀释的二抗Goat F(ab’)2Anti-Human IgG-Fc(abcam,ab98596)，4℃避光孵育30min，洗涤3次后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测。 The specific method is as follows: prepare A549 cells or HT-29 cells in the logarithmic growth phase into a single cell suspension, adjust the density to 1×10 6 cells/mL, add 100 μL per well into a 96-well round bottom plate, and store at 4°C. , 300g centrifugation and remove the supernatant. Add serially diluted (1.0000, 0.3333, 0.1111, 0.0370, 0.0123, 0.0041, 0.0014, 0.0001 μg/mL) N99, N99-MMAE, N147-135, N147-135-MMAE, 99961.1, 99961.1-MMA to corresponding wells E and For negative control, mix well and incubate at 4°C for 30min. Wash the incubated cell mixture 3 times, add 100 μL of 1:300 diluted secondary antibody Goat F(ab') 2 Anti-Human IgG-Fc (abcam, ab98596), incubate at 4°C for 30 min in the dark, wash 3 times Detection by flow cytometry (Beckman, CytoFLEX AOO-1-1102).
结果如图12所示：A549细胞结果显示(图12A和图12B)，N99-MMAE和对照99961.1-MMA与A549细胞的结合活性与对应母本抗体相当，但是，N99和N99-MMAE与A549细胞的结合活性都要优于相应对照99961.1和99961.1-MMAE；N147-135-MMAE、N147-135与A549细胞的结合活性与对照抗体99961.1相当；HT-29细胞结果显示(图12C和12D)，N99-MMAE和对照99961.1-MMA与HT-29细胞的结合活性与对应母本抗体相当，且N99-MMAE与HT-29细胞的结合活性与对照99961.1-MMAE相当；N147-135-MMAE、N147-135与HT-29细胞的结合活性与对照抗体99961.1相当。The results are shown in Figure 12: A549 cell results show (Figure 12A and Figure 12B), the binding activity of N99-MMAE and control 99961.1-MMA to A549 cells is equivalent to that of the corresponding parental antibody, but N99 and N99-MMAE have the same binding activity as A549 cells The binding activity of N147-135-MMAE, N147-135 and A549 cells was comparable to that of the control antibody 99961.1; the results of HT-29 cells showed (Figure 12C and 12D), N99 -The binding activity of MMAE and control 99961.1-MMA to HT-29 cells is equivalent to that of the corresponding parental antibody, and the binding activity of N99-MMAE to HT-29 cells is equivalent to that of the control 99961.1-MMAE; N147-135-MMAE, N147-135 The binding activity to HT-29 cells was comparable to that of the control antibody 99961.1.
实施例18基于MTS法检测ADC肿瘤细胞杀伤效果Example 18 Detection of ADC Tumor Cell Killing Effect Based on MTS Method
本实施例中分别用A549和HT-29细胞检测候选ADC和对照ADC对肿瘤细胞的杀伤情况。In this example, A549 and HT-29 cells were used to detect the killing effect of candidate ADC and control ADC on tumor cells.
具体方法如下：将对数生长期的A549细胞或者HT-29细胞制备成单细胞悬液，A549密度调整为1×104个细胞/mL，HT-29密度调整为1.5×104个细胞/mL，以每孔100μL加至96孔细胞培养板中，37℃，5％CO2，培养12h。之后加入梯度稀释(2000、500、250、125、62.5、31.25、15.625、7.813、1.953nM)的ADC样品，37℃，5％CO2，培养72h(A549细胞)或者96h(HT-29细胞)。然后每孔加入40μl MTS(Promega，G3580)，37℃孵育1h后，通过酶标仪在OD492下读取数据。The specific method is as follows: A549 cells or HT-29 cells in the logarithmic growth phase were prepared into a single cell suspension, the density of A549 was adjusted to 1×10 4 cells/mL, and the density of HT-29 was adjusted to 1.5×10 4 cells/mL. mL, 100 μL per well was added to a 96-well cell culture plate, and cultured at 37° C., 5% CO 2 for 12 hours. Then add ADC samples with gradient dilution (2000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 1.953nM), culture at 37°C, 5% CO 2 for 72h (A549 cells) or 96h (HT-29 cells) . Then 40 μl of MTS (Promega, G3580) was added to each well, and after incubation at 37° C. for 1 h, the data were read at OD492 by a microplate reader.
结果如图13A-13B所示：N99-MMAE在两个肿瘤细胞A549(图13A)和HT-29(图13B)上的杀伤效果均优于对照99961.1-MMAE，其中N99-MMAE在A549和HT-29上的IC50分别是11.07nM和5.952nM；N147-135-MMAE在HT-29肿瘤细胞上的杀伤效果优于对照99961.1-MMAE，在A549肿瘤细胞上的杀伤效果与对照ADC相当。The results are shown in Figures 13A-13B: the killing effect of N99-MMAE on the two tumor cells A549 (Figure 13A) and HT-29 (Figure 13B) was better than that of the control 99961.1-MMAE, wherein N99-MMAE had a higher effect on A549 and HT-29 (Figure 13B). The IC 50 on -29 were 11.07nM and 5.952nM respectively; the killing effect of N147-135-MMAE on HT-29 tumor cells was better than that of the control 99961.1-MMAE, and the killing effect on A549 tumor cells was comparable to that of the control ADC.
实施例19基于CCK8法检测ADC肿瘤细胞杀伤效果Example 19 Detection of ADC Tumor Cell Killing Effect Based on CCK8 Method
本实施例中分别用Jeko-1细胞(人套细胞淋巴瘤细胞)、MDA-MB-468细胞(人乳腺癌细胞)和NCI-H1944细胞(人肺癌细胞)检测N99-MMAE和对照ADC对肿瘤细胞的杀伤情况。In this embodiment, use Jeko-1 cells (human mantle cell lymphoma cells), MDA-MB-468 cells (human breast cancer cells) and NCI-H1944 cells (human lung cancer cells) to detect the effect of N99-MMAE and contrast ADC on tumor cell killing.
具体方法如下：将对数生长期的Jeko-1细胞、MDA-MB-468细胞或NCI-H1944细胞制备成单细胞悬液，Jeko-1密度调整为1×105个细胞/mL，MDA-MB-468密度调整为2×105个细胞/mL，NCI-H1944密度调整为1.2×105个细胞/mL，以每孔90μL加至96孔细胞培养板中，37℃，5％CO2，培养12h。之后加入ADC样品，将药物浓度梯度稀释至500、158、50、15.8、5、1.58、0.5、0.158、0.05nM，37℃，5％CO2，培养72h。然后每孔加入10μl CCK8(Bimake，B34304)，37℃孵育1h后，通过酶标仪在OD450下读取数据。The specific method is as follows: the Jeko-1 cells, MDA-MB-468 cells or NCI-H1944 cells in the logarithmic growth phase were prepared into a single cell suspension, the density of Jeko-1 was adjusted to 1×10 5 cells/mL, MDA- Adjust the density of MB-468 to 2×10 5 cells/mL, adjust the density of NCI-H1944 to 1.2×10 5 cells/mL, add 90 μL per well to a 96-well cell culture plate, 37°C, 5% CO 2 , cultivated for 12h. Afterwards, ADC samples were added, and the drug concentration was gradually diluted to 500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, 0.05 nM, 37°C, 5% CO 2 , and incubated for 72 hours. Then 10 μl of CCK8 (Bimake, B34304) was added to each well, and after incubation at 37° C. for 1 h, the data were read at OD450 by a microplate reader.
结果如图14所示：N99-MMAE在三个肿瘤细胞Jeko-1(图14A)和MDA-MB-468(图14B)和NCI-H1944(图14C)上的杀伤效果均优于对照ADC 99961.1-MMAE，其中N99-MMAE在Jeko-1、MDA-MB-468和NCI-H1944上的IC50分别是5.188nM、5.126nM和48.62nM；N147-135-MMAE在三个肿瘤细胞Jeko-1(图14D)和MDA-MB-468(图14E)和NCI-H1944(图14F)上的杀伤效果 与对照ADC 99961.1-MMAE相当，其中N147-135-MMAE在Jeko-1、MDA-MB-468和NCI-H1944上的IC50分别是52.98nM、16.17nM和113.4nM。The results are shown in Figure 14: the killing effect of N99-MMAE on the three tumor cells Jeko-1 (Figure 14A), MDA-MB-468 (Figure 14B) and NCI-H1944 (Figure 14C) was better than that of the control ADC 99961.1 -MMAE, wherein the IC 50 of N99-MMAE on Jeko-1, MDA-MB-468 and NCI-H1944 were 5.188nM, 5.126nM and 48.62nM; Figure 14D) and the killing effect on MDA-MB-468 (Figure 14E) and NCI-H1944 (Figure 14F) Comparable to the control ADC 99961.1-MMAE, where the IC 50 of N147-135-MMAE on Jeko-1, MDA-MB-468 and NCI-H1944 were 52.98nM, 16.17nM and 113.4nM, respectively.
实施例20基于CCK8法检测ADC抗原依赖的杀伤效果Example 20 Detection of ADC antigen-dependent killing effect based on CCK8 method
本实施例中用huROR1-HEK293细胞检测本申请获得的ADC和对照ADC抗原依赖的杀伤效果。In this example, huROR1-HEK293 cells were used to detect the antigen-dependent killing effect of the ADC obtained in the present application and the control ADC.
具体方法如下：将对数生长期的huROR1-HEK293细胞制备成单细胞悬液，细胞密度调整成6×104个细胞/mL，以每孔50μL加至96孔细胞培养板中，37℃，5％CO2，培养24h后，每孔加入50μL浓度为100μg/mL的抗体(N99和N147-135)和阳性对照抗体99961.1，37℃孵育2h后，加入浓度梯度稀释(85.3333、42.6667、21.3333、10.6667、5.3333、2.6667nM)的对应的ADC(N99-MMAE和N147-135-MMAE)和阳性对照ADC 99961.1-MMAE，37℃，5％CO2，培养96h后，每孔加入30μL CCK8(absin/爱必信，abs50003)，37℃孵育1-4h后，通过酶标仪在OD450下读板。采用未加抗体仅加入ADC的体系作为对照组。The specific method is as follows: prepare huROR1-HEK293 cells in the logarithmic growth phase into a single-cell suspension, adjust the cell density to 6× 104 cells/mL, add 50 μL per well to a 96-well cell culture plate, and store at 37°C. After incubating for 24 hours in 5% CO 2 , add 50 μL of 100 μg/mL antibodies (N99 and N147-135) and positive control antibody 99961.1 to each well. 10.6667, 5.3333, 2.6667nM) corresponding ADC (N99-MMAE and N147-135-MMAE) and positive control ADC 99961.1-MMAE, 37 ℃, 5% CO 2 , after 96 hours of culture, add 30 μL CCK8 (absin/ Absin, abs50003), after incubation at 37°C for 1-4h, the plate was read at OD450 by a microplate reader. A system in which no antibody was added and only ADC was added was used as the control group.
结果如图15A-15B所示，抗体N99、N147-135和99961.1的竞争能够显著抑制其对应ADC对huROR1-HEK293细胞的杀伤效果，证明本申请制得的ADC对于细胞的杀伤依赖于连接在ADC上的抗体与细胞表面的抗原的结合，因而是抗原依赖性杀伤而非连接毒素的非特异性杀伤。The results are shown in Figures 15A-15B, the competition of antibodies N99, N147-135 and 99961.1 can significantly inhibit the killing effect of their corresponding ADCs on huROR1-HEK293 cells, which proves that the killing of cells by the ADC prepared in this application depends on the linking of the ADC The binding of antibodies on the cell surface to antigens on the cell surface is thus antigen-dependent killing rather than non-specific killing of toxin-linked.
实施例21 ADC内吞效率检测Example 21 Detection of ADC endocytosis efficiency
本实施例中用A549和HT-29肿瘤细胞基于FACS法检测了本申请获得的ADC的内吞效率，具体实验方法参见实施例8。In this example, A549 and HT-29 tumor cells were used to detect the endocytic efficiency of the ADC obtained in the present application based on the FACS method. For specific experimental methods, see Example 8.
实验结果表明，在A549细胞上(图16A-16B)，N99-MMAE的内吞效率显著高于对照9996.1-MMAE，且ADC的内吞效率均高于其对应母本抗体，N147-135-MMAE的内吞效率也显著高于对照99961.1-MMAE；在HT-29细胞上(图16C-16D)，N99-MMAE的内吞效率也显著高于对照9996.1-MMAE，且ADC的内吞效率均高于其对应母本抗体；N147-135-MMAE的内吞效率稍弱于阳性对照。The experimental results showed that on A549 cells (Figure 16A-16B), the endocytosis efficiency of N99-MMAE was significantly higher than that of the control 9996.1-MMAE, and the endocytosis efficiency of ADC was higher than that of its corresponding parental antibody, N147-135-MMAE The endocytic efficiency of N99-MMAE was also significantly higher than that of the control 99961.1-MMAE; on HT-29 cells (Fig. 16C-16D), the endocytic efficiency of N99-MMAE was also significantly higher than that of the control 9996.1-MMAE, and the endocytic efficiency of ADC was high The endocytosis efficiency of N147-135-MMAE was slightly weaker than that of the positive control because of its corresponding parental antibody.
实施例22 ADC抑制HT-29小鼠荷瘤模型药效检测Example 22 Detection of drug efficacy of ADC inhibiting HT-29 mouse tumor-bearing model
本实施例检测了2个候选ADC(N99-MMAE和N147-135-MMAE)在动物体内的抑瘤效果，所使用肿瘤细胞为结肠癌细胞HT-29(BNCC337732)，以99961.1-MMAE作为阳性对照。In this example, the tumor inhibitory effect of two candidate ADCs (N99-MMAE and N147-135-MMAE) in animals was tested. The tumor cells used were colon cancer cells HT-29 (BNCC337732), and 99961.1-MMAE was used as a positive control .
具体方法如下：使用6-8周龄、体重在20g左右的雌性裸鼠(Balb/C北京维通利华实验动物技术有限公司)，每只裸鼠皮下单侧注射1×106个HT-29细胞，待荷瘤体积达100mm3左右时，进行随机分组分笼。每组6只荷瘤裸鼠，共9组，包括一个PBS阴性对照组、5个ADC组(N99-MMAE0.4mg/kg(mpk)、N99-MMAE 2mg/kg、N99-MMAE 10mg/kg、N147-135-MMAE 2mg/kg和 N147-135-MMAE 10mg/kg)和3个阳性对照ADC组(99961.1-MMAE 0.4mg/kg、99961.1-MMAE 2mg/kg和99961.1-MMAE 10mg/kg)，给药方式为尾静脉注射给药，每周给药2次并测量2次瘤体积，共给药8次/4周(BIW*4)。瘤体积(V)计算方式：V＝L×W2/2(其中L是肿瘤直径中最长的，W是肿瘤直径中最短的)。给药结束后1周将小鼠安乐死，取肿瘤并测量瘤重。分析瘤体积、瘤重和小鼠体重变化数据，计算抑瘤率。结果示于图17A-17C和表12。The specific method is as follows: use female nude mice (Balb/C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks and weighing about 20 g, and inject 1×10 6 HT- 29 cells were randomly divided into cages when the tumor-bearing volume reached about 100 mm 3 . 6 tumor-bearing nude mice in each group, 9 groups in total, including a PBS negative control group, 5 ADC groups (N99-MMAE0.4mg/kg (mpk), N99-MMAE 2mg/kg, N99-MMAE 10mg/kg, N147-135-MMAE 2mg/kg and N147-135-MMAE 10mg/kg) and three positive control ADC groups (99961.1-MMAE 0.4mg/kg, 99961.1-MMAE 2mg/kg and 99961.1-MMAE 10mg/kg), the administration method was tail vein injection, The drug was administered twice a week and the tumor volume was measured twice, for a total of 8 times/4 weeks (BIW*4). Tumor volume (V) calculation method: V=L×W 2 /2 (wherein L is the longest tumor diameter, W is the shortest tumor diameter). One week after the administration, the mice were euthanized, and the tumors were taken out and the tumor weight was measured. The tumor volume, tumor weight and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 17A-17C and Table 12.
试验结果表明，治疗期间各组小鼠体重均无明显变化，表明小鼠对ADC的耐受性良好(图17B)；N99-MMAE、M147-135MMAE和对照99961.1-MMAE均在高剂量(10mpk)的条件下显示出了显著的抑瘤效果，在高剂量的条件下，N99-MMAE和99961.1-MMAE抑瘤效果相当，N147-135-MMAE的抑瘤率在第41天的时候就几乎达到了100％，显著优于阳性对照99961.1-MMAE(图17A、图17C和表12)。The test results showed that there was no significant change in the body weight of the mice in each group during the treatment period, indicating that the mice tolerated the ADC well (Figure 17B); Under the condition of high dose, N99-MMAE and 99961.1-MMAE showed the same tumor inhibitory effect, and the tumor inhibitory rate of N147-135-MMAE almost reached the 41st day. 100%, significantly better than the positive control 99961.1-MMAE (Fig. 17A, Fig. 17C and Table 12).
表12 ADC在小鼠体内的抑瘤率
Table 12 Tumor inhibition rate of ADC in mice
实施例23 ADC抑制A549小鼠荷瘤模型药效检测Example 23 Detection of the efficacy of ADC in inhibiting the tumor-bearing model of A549 mice
本实施例检测了2个候选ADC(N99-MMAE和N147-135-MMAE)在动物体内的抑瘤效果，所使用肿瘤细胞为非小细胞肺癌细胞A549(中科院上海细胞库，C2107019)，以99961.1-MMAE作为阳性对照。In this example, the antitumor effect of two candidate ADCs (N99-MMAE and N147-135-MMAE) in animals was tested. The tumor cells used were non-small cell lung cancer cells A549 (Shanghai Cell Bank, Chinese Academy of Sciences, C2107019), and 99961.1 -MMAE served as a positive control.
具体方法如下：使用6-8周龄、体重在18-20g的雌性裸鼠(Balb/C北京维通利华实验动物技术有限公司)，每只裸鼠右侧背部皮下单侧注射1×106个A549细胞，待荷瘤体积达100mm3左右时，进行随机分组分笼。每组6只荷瘤裸鼠，共7组，包括一个PBS阴性对照组、4个ADC组(N99-MMAE 5mpk(mg/kg)、N99-MMAE 10mpk、N147-135-MMAE 5mpk和N147-135-MMAE 10mpk)和2个阳性对照ADC组(99961.1-MMAE 5mpk和99961.1-MMAE 10mpk)，给药方式 为尾静脉注射给药，每周给药2次并测量2次瘤体积，共给药6次/3周(BIW*3)。瘤体积(V)计算方式：V＝L×W2/2(其中L是肿瘤直径中最长的，W是肿瘤直径中最短的)。给药结束后观察一定时间后将小鼠安乐死，取肿瘤并测量瘤重。分析瘤体积、瘤重和小鼠体重变化数据，计算抑瘤率。结果示于图18A-18C和表13。The specific method is as follows: use female nude mice (Balb/C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks and weighing 18-20 g, and inject 1×10 Six A549 cells were randomly divided into cages when the tumor-bearing volume reached about 100 mm 3 . 6 tumor-bearing nude mice in each group, 7 groups in total, including a PBS negative control group, 4 ADC groups (N99-MMAE 5mpk(mg/kg), N99-MMAE 10mpk , N147-135-MMAE 5mpk and N147- 135-MMAE 10mpk) and 2 positive control ADC groups (99961.1-MMAE 5mpk and 99961.1-MMAE 10mpk ), administration method For tail vein injection, the drug was administered twice a week and the tumor volume was measured twice, for a total of 6 times/3 weeks (BIW*3). Tumor volume (V) calculation method: V=L×W 2 /2 (wherein L is the longest tumor diameter, W is the shortest tumor diameter). After the end of the administration, the mice were euthanized after observing for a certain period of time, and the tumors were taken out and the tumor weight was measured. The tumor volume, tumor weight and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 18A-18C and Table 13.
试验结果表明，治疗期间各组小鼠体重均无明显变化，表明小鼠对ADC的耐受性良好(图18B)；N99-MMAE在高剂量(10mpk)和低剂量(5mpk)条件下显示出了接近的抑瘤效果，且在低剂量条件下抑瘤效果优于阳性对照ADC 99961.1-MMAE，M147-135MMAE在高剂量的条件下也显示出了一定的抑瘤效果(图18A和表13)；但高剂量组和低剂量组在第41天后均出现了一些肿瘤组织反弹。The test results showed that there was no significant change in the body weight of mice in each group during treatment, indicating that the mice tolerated ADC well (Figure 18B); N99-MMAE showed high-dose (10mpk) and low-dose (5mpk) conditions The anti-tumor effect was close to that of the positive control ADC 99961.1-MMAE under low-dose conditions, and M147-135MMAE also showed a certain anti-tumor effect under high-dose conditions (Figure 18A and Table 13) ; but both the high-dose group and the low-dose group showed some tumor tissue rebound after the 41st day.
表13 ADC在小鼠体内的抑瘤率
Table 13 Tumor inhibition rate of ADC in mice
实施例24 ADC抑制NCI-N87小鼠荷瘤模型药效检测Example 24 Detection of Drug Effect of ADC Inhibiting NCI-N87 Mouse Tumor-bearing Model
本实施例检测了1个候选ADC(N147-135-MMAE)在动物体内的抑瘤效果，所使用肿瘤细胞为胃癌细胞NCI-N87(中科院上海细胞库，C2009021，P3)，以99961.1-MMAE作为阳性对照。In this example, the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals was tested. The tumor cells used were gastric cancer cells NCI-N87 (Shanghai Cell Bank, Chinese Academy of Sciences, C2009021, P3), and 99961.1-MMAE was used as positive control.
具体方法如下：使用6-8周龄、体重在18-20g的雌性裸鼠(Balb/C北京维通利华实验动物技术有限公司)，每只裸鼠进行背部皮下注射1×106个NCI-N87细胞，待荷瘤体积达100mm3左右时，进行随机分组分笼。每组10只荷瘤裸鼠，共5组，包括一个PBS阴性对照组、2个ADC组(N147-135-MMAE 5mpk(mg/kg)和N147-135-MMAE 10mpk)和2个阳性对照ADC组(99961.1-MMAE 5mpk和99961.1-MMAE 10mpk)，给药方式为微静脉注射给药，每周给药1次并测量2次瘤体积，共给药3次/3周(QW*3)。瘤体积(V)计算方式：V＝L×W2/2(其中L是肿瘤直径中最长的，W是肿瘤直径中最短的)。分析瘤体积和小鼠体重变化数据，计算抑瘤率。结果示于图19A-19B和表14。The specific method is as follows: use female nude mice (Balb/C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks and weighing 18-20 g, and subcutaneously inject 1×10 6 NCI in the back of each nude mouse. - N87 cells, when the tumor-bearing volume reaches about 100 mm 3 , they are divided into random groups and divided into cages. 10 tumor-bearing nude mice in each group, 5 groups in total, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mpk) and 2 positive control ADCs In the group (99961.1-MMAE 5mpk and 99961.1-MMAE 10mpk), the administration method was venous injection, the administration was once a week and the tumor volume was measured twice, and the administration was administered 3 times/3 weeks (QW*3). Tumor volume (V) calculation method: V=L×W 2 /2 (wherein L is the longest tumor diameter, W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 19A-19B and Table 14.
试验结果表明，治疗期间各组小鼠体重均无明显变化，表明小鼠对ADC的耐受性良好(图 19B)；和PBS组比较，各剂量组都具有显著的抑瘤效果，N147-135-MMAE分子与99961.1-MMAE分子在10mpk和5mpk同剂量下抑瘤效果均相当，同剂量下均无显著性差异(图19A和表14)。The test results showed that there was no significant change in the body weight of the mice in each group during the treatment, indicating that the mice had a good tolerance to ADC (Fig. 19B); compared with the PBS group, each dose group has significant tumor-inhibiting effects, and the tumor-inhibiting effects of N147-135-MMAE molecules and 99961.1-MMAE molecules are equivalent at the same doses of 10mpk and 5mpk, and there is no significant effect at the same doses Differences (Figure 19A and Table 14).
表14 ADC在小鼠体内的抑瘤率
Table 14 Tumor inhibition rate of ADC in mice
实施例25 ADC抑制MDA-MB-231小鼠荷瘤模型药效检测Example 25 Detection of drug efficacy of ADC inhibiting MDA-MB-231 mouse tumor-bearing model
本实施例检测了1个候选ADC(N147-135-MMAE)在动物体内的抑瘤效果，所使用肿瘤细胞为三阴性乳腺癌细胞MDA-MB-231(中科院上海细胞库，C2006040)，以99961.1-MMAE作为阳性对照。In this example, the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals was tested. The tumor cells used were triple-negative breast cancer cells MDA-MB-231 (Shanghai Cell Bank, Chinese Academy of Sciences, C2006040), with 99961.1 -MMAE served as a positive control.
具体方法如下：使用6-8周龄、体重在20-22g的雌性裸鼠(NSG北京维通利华实验动物技术有限公司)，每只裸鼠背部皮下注射1×106个MDA-MB-231细胞，待荷瘤体积达100mm3左右时，进行随机分组分笼。每组10只荷瘤裸鼠，共5组，包括一个PBS阴性对照组、2个ADC组(N147-135-MMAE 5mpk(mg/kg)和N147-135-MMAE 10mpk)和2个阳性对照ADC组(99961.1-MMAE 5mpk和99961.1-MMAE 10mpk)，给药方式为尾静脉注射给药，每周给药1次并测量2次瘤体积，共给药3次/3周(QW*3)。瘤体积(V)计算方式：V＝L×W2/2(其中L是肿瘤直径中最长的，W是肿瘤直径中最短的)。分析瘤体积和小鼠体重变化数据，计算抑瘤率。结果示于图20A-20B和表15。The specific method is as follows: use 6-8 weeks old female nude mice weighing 20-22 g (NSG Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and subcutaneously inject 1×10 6 MDA-MB- 231 cells were randomly divided into cages when the tumor-bearing volume reached about 100 mm 3 . 10 tumor-bearing nude mice in each group, 5 groups in total, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mp k ) and 2 positive controls ADC group (99961.1-MMAE 5mpk and 99961.1-MMAE 10mp k ), the administration method was tail vein injection administration, once a week and measuring the tumor volume twice, a total of 3 administrations/3 weeks (QW*3 ). Tumor volume (V) calculation method: V=L×W 2 /2 (wherein L is the longest tumor diameter, W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 20A-20B and Table 15.
试验结果表明，各组小鼠给药前期未见明显差异，PBS组于第35天开始出现体重下降，ADC分子组合阳性对照ADC组小鼠体重无明显变化，表明小鼠对ADC的耐受性良好(图20B)；和 PBS组比较，阳性对照分子及候选ADC分子的高剂量组有相似的显著抑瘤效果，且两个分子均具有明显剂量相关性，低剂量组在停药后有一定组织肿瘤反弹(图20A和表15)。The test results showed that there was no significant difference in the pre-administration period of the mice in each group, and the PBS group began to lose weight on the 35th day, and the body weight of the mice in the positive control ADC group of the ADC molecular combination did not change significantly, indicating that the mice were resistant to ADC Good (Fig. 20B); and Compared with the PBS group, the positive control molecule and the high-dose group of the candidate ADC molecule had similar significant anti-tumor effects, and both molecules had a significant dose-correlation, and the low-dose group had certain tissue tumor rebound after drug withdrawal (Figure 20A and Table 15).
表15 ADC在小鼠体内的抑瘤率
Table 15 Tumor inhibition rate of ADC in mice
实施例26 ADC抑制MDA-MB-468小鼠荷瘤模型药效检测Example 26 Detection of the efficacy of ADC in inhibiting MDA-MB-468 mouse tumor-bearing model
本实施例检测了1个候选ADC(N147-135-MMAE)在动物体内的抑瘤效果，所使用肿瘤细胞为三阴性乳腺癌细胞MDA-MB-468(中科院上海细胞库，TCHu136)，以99961.1-MMAE作为阳性对照。In this example, the tumor-inhibiting effect of a candidate ADC (N147-135-MMAE) in animals was tested. The tumor cells used were triple-negative breast cancer cells MDA-MB-468 (Shanghai Cell Bank, Chinese Academy of Sciences, TCHu136), and 99961.1 -MMAE served as a positive control.
具体方法如下：使用6-8周龄、体重在21-25g的雌性裸鼠(NOD SCID浙江维通利华实验动物技术有限公司)，每只裸鼠背部皮下注射1×107个MDA-MB-468细胞，待荷瘤体积达100mm3左右时，进行随机分组分笼。每组6只荷瘤裸鼠，共5组，包括一个PBS阴性对照组、2个ADC组(N147-135-MMAE 5mpk(mg/kg)和N147-135-MMAE 10mpk)和2个阳性对照ADC组(99961.1-MMAE 5mpk和99961.1-MMAE 10mpk)，给药方式为尾静脉注射给药，每周给药1次并每周测量2次瘤体积，共给药3次/3周(QW*3)。瘤体积(V)计算方式：V＝L×W2/2(其中L是肿瘤直径中最长的，W是肿瘤直径中最短的)。分析瘤体积和小鼠体重变化数据，计算抑瘤率。结果示于图21A-21B和表16-17。The specific method is as follows: use 6-8 weeks old female nude mice weighing 21-25 g (NOD SCID Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), and subcutaneously inject 1×10 7 MDA-MB on the back of each nude mouse -468 cells, when the tumor-bearing volume reached about 100 mm 3 , they were divided into random groups and divided into cages. 6 tumor-bearing nude mice in each group, 5 groups in total, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mp k ) and 2 positive controls In the ADC group (99961.1-MMAE 5mpk and 99961.1-MMAE 10mp k ), the administration method was tail vein injection, once a week and the tumor volume was measured twice a week, a total of 3 times/3 weeks (QW *3). Tumor volume (V) calculation method: V=L×W 2 /2 (wherein L is the longest tumor diameter, W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 21A-21B and Tables 16-17.
试验结果表明，治疗期间各组小鼠体重均无明显变化，表明小鼠对ADC的耐受性良好(图21B)；多次给药组，10mg/kg剂量组N147-135-MMAE(第一次给药后第14天全部完全缓解)，其抑瘤效果优于99961.1-MMAE抑瘤效果(第一次给药后第18天全部完全缓解)；5mg/kg剂量组N147-135-MMAE第一次给药后第20天全部完全缓解，抑瘤效果优于同剂量组99961.1-MMAE (第一次给药后第25天6只小鼠中4只达到完全缓解)(图21A和表16-17)。The test results showed that during the treatment period, the body weight of mice in each group had no significant change, indicating that the mice had good tolerance to ADC (Figure 21B); 14 days after the first administration, all were completely relieved), and its tumor inhibitory effect was better than that of 99961.1-MMAE (all were completely relieved on the 18th day after the first administration); the 5mg/kg dose group N147-135-MMAE All patients were completely relieved on the 20th day after one administration, and the antitumor effect was better than that of the same dose group 99961.1-MMAE (4 out of 6 mice achieved complete remission on day 25 after the first dose) (FIG. 21A and Tables 16-17).
表16 ADC在小鼠体内的抑瘤率
Table 16 Tumor inhibition rate of ADC in mice
表17 ADC在小鼠体内的完全缓解率
Table 17 Complete remission rate of ADC in mice
实施例27 ADC抑制Jeko-1小鼠荷瘤模型药效检测Example 27 Detection of drug efficacy of ADC inhibiting Jeko-1 mouse tumor-bearing model
本实施例检测了1个候选ADC(N147-135-MMAE)在动物体内的抑瘤效果，所使用肿瘤细胞为套细胞淋巴瘤细胞Jeko-1(ATCC，CRL-3006)，以99961.1-MMAE作为阳性对照。In this example, the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals was tested. The tumor cells used were mantle cell lymphoma cells Jeko-1 (ATCC, CRL-3006), and 99961.1-MMAE was used as positive control.
具体方法如下：使用6-8周龄、体重在21-25g的雌性裸鼠(BALB/c浙江维通利华实验动物技术有限公司)，每只裸鼠背部皮下注射1×107个Jeko-1细胞，待荷瘤体积达100mm3左右时，进行随机分组分笼。每组7只荷瘤裸鼠，共9组，包括一个PBS阴性对照组、4个ADC组(N147-135-MMAE 2mpk(mg/kg)(QW*3)、N147-135-MMAE 8mpk(QW*3)、N147-135-MMAE 8mpk(单剂量，)和N147-135-MMAE 2+3mpk(Q2W*2即第15天给药2mpk和第29天给药3mpk，简写为D15 2mpk+D29 3mpk))和4个阳性对照ADC组(99961.1-MMAE 2.5mpk(QW*3)、99961.1-MMAE 10mpk(QW*3)、99961.1-MMAE 10mpk(单剂量)和99961.1-MMAE 2.5+3.5 mpk(Q2W*2，即第15天给药2.5mpk和第29天给药3.5mpk，简写为D15 2.5mpk+D29 3.5mpk))，给药方式为尾静脉注射给药，共给药3次/3周(QW*3)或单次给药(single dose)或给药2次/3周(Q2W*2)。瘤体积(V)计算方式：V＝L×W2/2(其中L是肿瘤直径中最长的，W是肿瘤直径中最短的)。分析瘤体积和小鼠体重变化数据，计算抑瘤率。结果示于图22A-22B和表18-19。The specific method is as follows: use female nude mice (BALB/c Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks and weighing 21-25 g, and subcutaneously inject 1×10 7 Jeko- 1 cell, and when the tumor-bearing volume reached about 100mm 3 , they were divided into random groups and divided into cages. 7 tumor-bearing nude mice in each group, 9 groups in total, including a PBS negative control group, 4 ADC groups (N147-135-MMAE 2mpk (mg/kg) (QW*3), N147-135-MMAE 8mpk (QW *3), N147-135-MMAE 8mp k (single dose,) and N147-135-MMAE 2+3mpk (Q2W*2 means 2mpk on day 15 and 3mpk on day 29, abbreviated as D15 2mpk+D29 3mpk)) and 4 positive control ADC groups (99961.1-MMAE 2.5mpk (QW*3), 99961.1-MMAE 10mpk (QW*3), 99961.1-MMAE 10mpk (single dose) and 99961.1-MMAE 2.5+3.5 mpk (Q2W*2, 2.5mpk on the 15th day and 3.5mpk on the 29th day, abbreviated as D15 2.5mpk+D29 3.5mpk)), the administration method is tail vein injection administration, a total of 3 administrations /3 weeks (QW*3) or single dose (single dose) or 2 times/3 weeks (Q2W*2). Tumor volume (V) calculation method: V=L×W 2 /2 (wherein L is the longest tumor diameter, W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 22A-22B and Tables 18-19.
试验结果表明，治疗期间各组小鼠体重均无明显变化，表明小鼠对ADC的耐受性良好(图22B)；QW*3给药高剂量组，N147-135-MMAE(8mpk)和99961.1-MMAE(10mpk)剂量组的小鼠肿瘤生长抑制率TGI为96％左右，抑瘤率相当，给药后23天肿瘤未复发。单次给药(single dose)组，N147-135-MMAE(8mpk)的抑瘤效果略劣于99961.1-MMAE(10mpk)，TGI分别为76％和93％，99961.1-MMAE(10mpk)组给药后23天肿瘤未复发，但N147-135-MMAE(8mpk)组在给药后16天开始发生肿瘤体积的反弹。Q2W*2给药组，N147-135-MMAE(2+3mpk)比99961.1-MMAE(2.5+3.5mpk)剂量组的抑瘤效果略优，TGI分别为47％和32％(图22A和表18-19)。The test results showed that there was no significant change in the body weight of the mice in each group during the treatment period, indicating that the mice tolerated ADC well (Figure 22B); -The tumor growth inhibition rate TGI of mice in the MMAE (10mpk) dose group was about 96%, the tumor inhibition rate was equivalent, and the tumor did not recur 23 days after administration. In the single dose group, the tumor inhibitory effect of N147-135-MMAE (8mpk) was slightly inferior to that of 99961.1-MMAE (10mpk), and the TGIs were 76% and 93%, respectively. The 99961.1-MMAE (10mpk) group was administered The tumor did not recur after 23 days, but the N147-135-MMAE (8mpk) group began to rebound in tumor volume 16 days after administration. In the Q2W*2 administration group, the tumor inhibitory effect of N147-135-MMAE (2+3mpk) was slightly better than that of the 99961.1-MMAE (2.5+3.5mpk) dosage group, with TGIs of 47% and 32% respectively (Figure 22A and Table 18 -19).
表18 ADC在小鼠体内QW给药的抑瘤率
Table 18 Tumor inhibition rate of ADC administered QW in mice
表19 ADC在小鼠体内single dose给药和Q2W给药的抑瘤率

Table 19 Tumor inhibition rate of ADC in single dose administration and Q2W administration in mice

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Claims (29)

  1. 一种特异性结合ROR1的抗ROR1抗体及其抗原结合片段，其包含：An anti-ROR1 antibody and antigen-binding fragment thereof that specifically binds ROR1, comprising:
    1)如SEQ ID NO:52所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:51所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；1) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:52 and 3 contained in the light chain variable region as shown in SEQ ID NO:51 a light chain CDR (LCDR1, LCDR2, LCDR3);
    2)如SEQ ID NO:54所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:53所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；2) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:54 and 3 contained in the light chain variable region as shown in SEQ ID NO:53 a light chain CDR (LCDR1, LCDR2, LCDR3);
    3)如SEQ ID NO:56所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:55所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；3) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO:56 and 3 included in the light chain variable region as shown in SEQ ID NO:55 a light chain CDR (LCDR1, LCDR2, LCDR3);
    4)如SEQ ID NO:58所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:57所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；4) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO:58 and 3 included in the light chain variable region as shown in SEQ ID NO:57 a light chain CDR (LCDR1, LCDR2, LCDR3);
    5)如SEQ ID NO:60所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:59所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；5) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:60 and 3 contained in the light chain variable region as shown in SEQ ID NO:59 a light chain CDR (LCDR1, LCDR2, LCDR3);
    6)如SEQ ID NO:62所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:61所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；6) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:62 and 3 contained in the light chain variable region as shown in SEQ ID NO:61 a light chain CDR (LCDR1, LCDR2, LCDR3);
    7)如SEQ ID NO:64所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:63所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；7) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:64 and 3 contained in the light chain variable region as shown in SEQ ID NO:63 a light chain CDR (LCDR1, LCDR2, LCDR3);
    8)如SEQ ID NO:66所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:53所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；8) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:66 and 3 contained in the light chain variable region as shown in SEQ ID NO:53 a light chain CDR (LCDR1, LCDR2, LCDR3);
    9)如SEQ ID NO:68所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:67所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；9) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:68 and 3 contained in the light chain variable region as shown in SEQ ID NO:67 a light chain CDR (LCDR1, LCDR2, LCDR3);
    10)如SEQ ID NO:70所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:69所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；10) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:70 and 3 contained in the light chain variable region as shown in SEQ ID NO:69 a light chain CDR (LCDR1, LCDR2, LCDR3);
    11)如SEQ ID NO:145所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:144所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；11) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:145 and 3 contained in the light chain variable region as shown in SEQ ID NO:144 a light chain CDR (LCDR1, LCDR2, LCDR3);
    12)如SEQ ID NO:147所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:146所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；12) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:147 and 3 contained in the light chain variable region as shown in SEQ ID NO:146 a light chain CDR (LCDR1, LCDR2, LCDR3);
    13)如SEQ ID NO:141所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:140所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；13) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:141 and 3 contained in the light chain variable region as shown in SEQ ID NO:140 a light chain CDR (LCDR1, LCDR2, LCDR3);
    14)如SEQ ID NO:143所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:142所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；14) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:143 and 3 contained in the light chain variable region as shown in SEQ ID NO:142 a light chain CDR (LCDR1, LCDR2, LCDR3);
    15)如SEQ ID NO:149所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:148所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；15) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:149 and 3 contained in the light chain variable region as shown in SEQ ID NO:148 a light chain CDR (LCDR1, LCDR2, LCDR3);
    16)如SEQ ID NO:161所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3) 和如SEQ ID NO:160所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；16) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 161 and 3 light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 160;
    17)如SEQ ID NO:163所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:162所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；17) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:163 and 3 contained in the light chain variable region as shown in SEQ ID NO:162 a light chain CDR (LCDR1, LCDR2, LCDR3);
    18)如SEQ ID NO:165所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:164所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；18) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:165 and 3 contained in the light chain variable region as shown in SEQ ID NO:164 a light chain CDR (LCDR1, LCDR2, LCDR3);
    19)如SEQ ID NO:151所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:150所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；19) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:151 and 3 contained in the light chain variable region as shown in SEQ ID NO:150 a light chain CDR (LCDR1, LCDR2, LCDR3);
    20)如SEQ ID NO:153所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:152所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；20) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:153 and 3 contained in the light chain variable region as shown in SEQ ID NO:152 a light chain CDR (LCDR1, LCDR2, LCDR3);
    21)如SEQ ID NO:155所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:154所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；21) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:155 and 3 contained in the light chain variable region as shown in SEQ ID NO:154 a light chain CDR (LCDR1, LCDR2, LCDR3);
    22)如SEQ ID NO:157所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:156所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；22) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:157 and 3 contained in the light chain variable region as shown in SEQ ID NO:156 a light chain CDR (LCDR1, LCDR2, LCDR3);
    23)如SEQ ID NO:52所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:158所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；23) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:52 and 3 contained in the light chain variable region as shown in SEQ ID NO:158 a light chain CDR (LCDR1, LCDR2, LCDR3);
    24)如SEQ ID NO:52所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:159所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；24) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:52 and 3 contained in the light chain variable region as shown in SEQ ID NO:159 a light chain CDR (LCDR1, LCDR2, LCDR3);
    25)如SEQ ID NO:62所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:166所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；25) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:62 and 3 contained in the light chain variable region as shown in SEQ ID NO:166 a light chain CDR (LCDR1, LCDR2, LCDR3);
    26)如SEQ ID NO:168所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:167所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；或26) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 168 and 3 contained in the light chain variable region as shown in SEQ ID NO: 167 a light chain CDR (LCDR1, LCDR2, LCDR3); or
    27)如SEQ ID NO:169所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:61所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)。27) 3 heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 169 and 3 contained in the light chain variable region as shown in SEQ ID NO: 61 light chain CDRs (LCDR1, LCDR2, LCDR3).
  2. 一种特异性结合ROR1的抗ROR1抗体及其抗原结合片段，其包含：An anti-ROR1 antibody and antigen-binding fragment thereof that specifically binds ROR1, comprising:
    1)包含如SEQ ID NO:1、2和3所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、20和21所示序列的LCDR1、LCDR2、LCDR3；1) HCDR1, HCDR2, HCDR3 comprising sequences shown in SEQ ID NO:1, 2 and 3; and LCDR1, LCDR2, LCDR3 of sequences shown in SEQ ID NO:19, 20 and 21;
    2)包含如SEQ ID NO:1、2和6所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:22、23和24所示序列的LCDR1、LCDR2、LCDR3；2) HCDR1, HCDR2, HCDR3 comprising sequences shown in SEQ ID NO:1, 2 and 6; and LCDR1, LCDR2, LCDR3 of sequences shown in SEQ ID NO:22, 23 and 24;
    3)包含如SEQ ID NO:7、8和9所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:25、26和27所示序列的LCDR1、LCDR2、LCDR3；3) HCDR1, HCDR2, HCDR3 comprising sequences shown in SEQ ID NO:7, 8 and 9; and LCDR1, LCDR2, LCDR3 of sequences shown in SEQ ID NO:25, 26 and 27;
    4)包含如SEQ ID NO:10、11和12所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、29和30所示序列的LCDR1、LCDR2、LCDR3；4) HCDR1, HCDR2, HCDR3 comprising sequences shown in SEQ ID NO:10, 11 and 12; and LCDR1, LCDR2, LCDR3 of sequences shown in SEQ ID NO:28, 29 and 30;
    5)包含如SEQ ID NO:13、14和15所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO: 31、32和33所示序列的LCDR1、LCDR2、LCDR3；5) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:13, 14 and 15; and as SEQ ID NO: LCDR1, LCDR2, LCDR3 of the sequence shown in 31, 32 and 33;
    6)包含如SEQ ID NO:16、17和18所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:34、35和36所示序列的LCDR1、LCDR2、LCDR3；6) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:16, 17 and 18; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:34, 35 and 36;
    7)包含如SEQ ID NO:1、2和37所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、20和21所示序列的LCDR1、LCDR2、LCDR3；7) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:1, 2 and 37; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:19, 20 and 21;
    8)包含如SEQ ID NO:38、39和12所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、44和30所示序列的LCDR1、LCDR2、LCDR3；8) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:38, 39 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:28, 44 and 30;
    9)包含如SEQ ID NO:40、41和15所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:31、45和33所示序列的LCDR1、LCDR2、LCDR3；9) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:40, 41 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:31, 45 and 33;
    10)包含如SEQ ID NO:42、43和18所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:34、46和36所示序列的LCDR1、LCDR2、LCDR3；或or
    11)包含如SEQ ID NO:91、92和93所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:101、102和103所示序列的LCDR1、LCDR2、LCDR3；11) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:91, 92 and 93; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:101, 102 and 103;
    12)包含如SEQ ID NO:1、2和94所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:104、32和105所示序列的LCDR1、LCDR2、LCDR3；12) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:1, 2 and 94; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:104, 32 and 105;
    13)包含如SEQ ID NO:95、96和97所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:106、107和108所示序列的LCDR1、LCDR2、LCDR3；13) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:95, 96 and 97; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:106, 107 and 108;
    14)包含如SEQ ID NO:1、2和98所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:109、110和105所示序列的LCDR1、LCDR2、LCDR3；14) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:1, 2 and 98; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:109, 110 and 105;
    15)包含如SEQ ID NO:91、99和100所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:111、112和113所示序列的LCDR1、LCDR2、LCDR3；15) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:91, 99 and 100; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:111, 112 and 113;
    16)包含如SEQ ID NO:121、122和3所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、136和21所示序列的LCDR1、LCDR2、LCDR3；16) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:121, 122 and 3; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:19, 136 and 21;
    17)包含如SEQ ID NO:1、123和3所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、137和21所示序列的LCDR1、LCDR2、LCDR3；17) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:1, 123 and 3; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:19, 137 and 21;
    18)包含如SEQ ID NO:124、2和3所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:19、23和21所示序列的LCDR1、LCDR2、LCDR3；18) HCDR1, HCDR2, HCDR3 comprising sequences shown in SEQ ID NO:124, 2 and 3; and LCDR1, LCDR2, LCDR3 of sequences shown in SEQ ID NO:19, 23 and 21;
    19)包含如SEQ ID NO:114、115和12所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、128和30所示序列的LCDR1、LCDR2、LCDR3；19) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:114, 115 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:28, 128 and 30;
    20)包含如SEQ ID NO:116、117和12所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、129和30所示序列的LCDR1、LCDR2、LCDR3；20) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:116, 117 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:28, 129 and 30;
    21)包含如SEQ ID NO:118、119和12所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:28、130和30所示序列的LCDR1、LCDR2、LCDR3；21) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:118, 119 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:28, 130 and 30;
    22)包含如SEQ ID NO:16、17和120所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID  NO:131、35和132所示序列的LCDR1、LCDR2、LCDR3；22) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:16, 17 and 120; and as SEQ ID LCDR1, LCDR2, LCDR3 of the sequence shown in NO:131, 35 and 132;
    23)包含如SEQ ID NO:16、17和18所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:34、35和133所示序列的LCDR1、LCDR2、LCDR3；23) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:16, 17 and 18; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:34, 35 and 133;
    24)包含如SEQ ID NO:16、17和18所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:134、35和135所示序列的LCDR1、LCDR2、LCDR3；24) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:16, 17 and 18; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:134, 35 and 135;
    25)包含如SEQ ID NO:13、14和15所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:31、138和33所示序列的LCDR1、LCDR2、LCDR3；25) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:13, 14 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:31, 138 and 33;
    26)包含如SEQ ID NO:125、126和15所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:31、139和33所示序列的LCDR1、LCDR2、LCDR3；或26) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:125, 126 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:31, 139 and 33; or
    27)包含如SEQ ID NO:125、127和15所示序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:31、32和33所示序列的LCDR1、LCDR2、LCDR3。27) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:125, 127 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NO:31, 32 and 33.
  3. 权利要求1或2所述的抗ROR1抗体及其抗原结合片段，其包含重链可变区和轻链可变区，其中：The anti-ROR1 antibody and antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein:
    1)所述重链可变区包含如SEQ ID NO：52所示氨基酸序列，或与SEQ ID NO：52的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：52组成，所述轻链可变区包含如SEQ ID NO：51所示氨基酸序列，或与SEQ ID NO：51的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：51组成；1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 52, or consists of SEQ ID NO: 52, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 51, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 51, or consists of SEQ ID NO: 51;
    2)所述重链可变区包含如SEQ ID NO：54所示氨基酸序列，或与SEQ ID NO：54的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：54组成，所述轻链可变区包含如SEQ ID NO：53所示氨基酸序列，或与SEQ ID NO：53的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：53组成；2) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 54, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 54, or consists of SEQ ID NO: 54, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 53, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 53, or consists of SEQ ID NO: 53;
    3)所述重链可变区包含如SEQ ID NO：56所示氨基酸序列，或与SEQ ID NO：56的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：56组成，所述轻链可变区包含如SEQ ID NO：55所示氨基酸序列，或与SEQ ID NO：55的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：55组成；3) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 56, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 56, or consists of SEQ ID NO: 56, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 55, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 55, or consists of SEQ ID NO: 55;
    4)所述重链可变区包含如SEQ ID NO：58所示氨基酸序列，或与SEQ ID NO：58的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：58组成，所述轻链可变区包含如SEQ ID NO：57所示氨基酸序列，或与SEQ ID NO：57的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：57组成；4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 58, or consists of SEQ ID NO: 58, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 57, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 57, or consists of SEQ ID NO: 57;
    5)所述重链可变区包含如SEQ ID NO：60所示氨基酸序列，或与SEQ ID NO：60的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：60组成，所述轻链可变区包含如SEQ ID NO：59所示氨基酸序列，或与SEQ ID NO：59的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：59组成； 5) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 60, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 60, or consists of SEQ ID NO: 60, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 59, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 59, or consists of SEQ ID NO: 59;
    6)所述重链可变区包含如SEQ ID NO：62所示氨基酸序列，或与SEQ ID NO：62的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：62组成，所述轻链可变区包含如SEQ ID NO：61所示氨基酸序列，或与SEQ ID NO：61的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：61组成；6) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 62, or consists of SEQ ID NO: 62, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 61, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 61, or consists of SEQ ID NO: 61;
    7)所述重链可变区包含如SEQ ID NO：64所示氨基酸序列，或与SEQ ID NO：64的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：64组成，所述轻链可变区包含如SEQ ID NO：63所示氨基酸序列，或与SEQ ID NO：63的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：63组成；7) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 64, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 64, or consists of SEQ ID NO: 64, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 63, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 63, or consists of SEQ ID NO: 63;
    8)所述重链可变区包含如SEQ ID NO：66所示氨基酸序列，或与SEQ ID NO：66的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：66组成，所述轻链可变区包含如SEQ ID NO：53所示氨基酸序列，或与SEQ ID NO：53的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：53组成；8) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 66, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 66, or consists of SEQ ID NO: 66, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 53, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 53, or consists of SEQ ID NO: 53;
    9)所述重链可变区包含如SEQ ID NO：68所示氨基酸序列，或与SEQ ID NO：68的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：68组成，所述轻链可变区包含如SEQ ID NO：67所示氨基酸序列，或与SEQ ID NO：67的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：67组成；9) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 68, or consists of SEQ ID NO: 68, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 67, or consists of SEQ ID NO: 67;
    10)所述重链可变区包含如SEQ ID NO：70所示氨基酸序列，或与SEQ ID NO：70的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：70组成，所述轻链可变区包含如SEQ ID NO：69所示氨基酸序列，或与SEQ ID NO：69的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：69组成；或10) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 70, or consists of SEQ ID NO: 70, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 69, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 69, or consists of SEQ ID NO: 69; or
    11)所述重链可变区包含如SEQ ID NO：145所示氨基酸序列，或与SEQ ID NO：145的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：145组成，所述轻链可变区包含如SEQ ID NO：144所示氨基酸序列，或与SEQ ID NO：144的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：144组成；11) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 145, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 145, or consists of SEQ ID NO: 145, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 144, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 144, or consists of SEQ ID NO: 144;
    12)所述重链可变区包含如SEQ ID NO：147所示氨基酸序列，或与SEQ ID NO：147的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：147组成，所述轻链可变区包含如SEQ ID NO：146所示氨基酸序列，或与SEQ ID NO：146的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：146组成；12) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 147, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 147, or consists of SEQ ID NO: 147, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 146, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 146, or consists of SEQ ID NO: 146;
    13)所述重链可变区包含如SEQ ID NO：141所示氨基酸序列，或与SEQ ID NO：141的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：141组成，所述轻链可变区包含如SEQ ID NO：140所示氨基酸序列，或与SEQ ID NO：140的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：140组成；13) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 141, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 141, or consists of SEQ ID NO: 141, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 140, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 140, or consists of SEQ ID NO: 140;
    14)所述重链可变区包含如SEQ ID NO：143所示氨基酸序列，或与SEQ ID NO：143的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：143组成，所述轻链可变区包 含如SEQ ID NO：142所示氨基酸序列，或与SEQ ID NO：142的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：142组成；14) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 143, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 143, or consists of SEQ ID NO: 143, The light chain variable region includes Contains an amino acid sequence as shown in SEQ ID NO: 142, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 142, or consists of SEQ ID NO: 142;
    15)所述重链可变区包含如SEQ ID NO：149所示氨基酸序列，或与SEQ ID NO：149的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：149组成，所述轻链可变区包含如SEQ ID NO：148所示氨基酸序列，或与SEQ ID NO：148的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：148组成；15) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 149, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 149, or consists of SEQ ID NO: 149, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 148, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 148, or consists of SEQ ID NO: 148;
    16)所述重链可变区包含如SEQ ID NO：161所示氨基酸序列，或与SEQ ID NO：161的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：161组成，所述轻链可变区包含如SEQ ID NO：160所示氨基酸序列，或与SEQ ID NO：160的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：160组成；16) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 161, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 161, or consists of SEQ ID NO: 161, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 160, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 160, or consists of SEQ ID NO: 160;
    17)所述重链可变区包含如SEQ ID NO：163所示氨基酸序列，或与SEQ ID NO：163的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：163组成，所述轻链可变区包含如SEQ ID NO：162所示氨基酸序列，或与SEQ ID NO：162的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：162组成；17) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 163, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 163, or consists of SEQ ID NO: 163, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 162, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 162, or consists of SEQ ID NO: 162;
    18)所述重链可变区包含如SEQ ID NO：165所示氨基酸序列，或与SEQ ID NO：165的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：165组成，所述轻链可变区包含如SEQ ID NO：164所示氨基酸序列，或与SEQ ID NO：164的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：164组成；18) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 165, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 165, or consists of SEQ ID NO: 165, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 164, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 164, or consists of SEQ ID NO: 164;
    19)所述重链可变区包含如SEQ ID NO：151所示氨基酸序列，或与SEQ ID NO：151的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：151组成，所述轻链可变区包含如SEQ ID NO：150所示氨基酸序列，或与SEQ ID NO：150的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：150组成；19) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 151, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 151, or consists of SEQ ID NO: 151, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 150, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 150, or consists of SEQ ID NO: 150;
    20)所述重链可变区包含如SEQ ID NO：153所示氨基酸序列，或与SEQ ID NO：153的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：153组成，所述轻链可变区包含如SEQ ID NO：152所示氨基酸序列，或与SEQ ID NO：152的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：152组成；20) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 153, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 153, or consists of SEQ ID NO: 153, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 152, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 152, or consists of SEQ ID NO: 152;
    21)所述重链可变区包含如SEQ ID NO：155所示氨基酸序列，或与SEQ ID NO：155的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：155组成，所述轻链可变区包含如SEQ ID NO：154所示氨基酸序列，或与SEQ ID NO：154的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：154组成；21) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 155, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 155, or consists of SEQ ID NO: 155, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 154, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 154, or consists of SEQ ID NO: 154;
    22)所述重链可变区包含如SEQ ID NO：157所示氨基酸序列，或与SEQ ID NO：157的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：157组成，所述轻链可变区包含如SEQ ID NO：156所示氨基酸序列，或与SEQ ID NO：156的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：156组成； 22) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 157, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 157, or consists of SEQ ID NO: 157, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 156, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 156, or consists of SEQ ID NO: 156;
    23)所述重链可变区包含如SEQ ID NO：52所示氨基酸序列，或与SEQ ID NO：52的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：52组成，所述轻链可变区包含如SEQ ID NO：158所示氨基酸序列，或与SEQ ID NO：158的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：158组成；23) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 52, or consists of SEQ ID NO: 52, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 158, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 158, or consists of SEQ ID NO: 158;
    24)所述重链可变区包含如SEQ ID NO：52所示氨基酸序列，或与SEQ ID NO：52的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：52组成，所述轻链可变区包含如SEQ ID NO：159所示氨基酸序列，或与SEQ ID NO：159的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：159组成；24) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 52, or consists of SEQ ID NO: 52, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 159, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 159, or consists of SEQ ID NO: 159;
    25)所述重链可变区包含如SEQ ID NO：62所示氨基酸序列，或与SEQ ID NO：62的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：62组成，所述轻链可变区包含如SEQ ID NO：166所示氨基酸序列，或与SEQ ID NO：166的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：166组成；25) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 62, or consists of SEQ ID NO: 62, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 166, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 166, or consists of SEQ ID NO: 166;
    26)所述重链可变区包含如SEQ ID NO：168所示氨基酸序列，或与SEQ ID NO：168的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：168组成，所述轻链可变区包含如SEQ ID NO：167所示氨基酸序列，或与SEQ ID NO：167的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：167组成；或26) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 168, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 168, or consists of SEQ ID NO: 168, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 167, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 167, or consists of SEQ ID NO: 167; or
    27)所述重链可变区包含如SEQ ID NO：169所示氨基酸序列，或与SEQ ID NO：169的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：169组成，所述轻链可变区包含如SEQ ID NO：61所示氨基酸序列，或与SEQ ID NO：61的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：61组成。27) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 169, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 169, or consists of SEQ ID NO: 169, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 61, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 61, or consists of SEQ ID NO: 61.
  4. 权利要求1至3中任一项所述的特异性结合ROR1的抗体及其抗原结合片段，其中所述抗体是人单克隆抗体。The antibody specifically binding to ROR1 and the antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a human monoclonal antibody.
  5. 权利要求1至4中任一项的分离的抗ROR1抗体或其抗原结合片段，其中所述抗原结合片段选自Fab、Fab’-SH、Fv(例如scFv)或(Fab’)2片段。The isolated anti-ROR1 antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein said antigen-binding fragment is selected from a Fab, Fab'-SH, Fv (e.g. scFv) or (Fab')2 fragment.
  6. 权利要求1至5中任一项的分离的抗ROR1单克隆抗体或其抗原结合片段，其包含恒定区序列，其中所述恒定区序列的至少一部分是人共有恒定区序列。The isolated anti-ROR1 monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 5, comprising a constant region sequence, wherein at least a portion of said constant region sequence is a human consensus constant region sequence.
  7. 如权利要求1-6中任何一项所述的抗体或其抗原结合片段，其中所述抗体的重链恒定区包含SEQ ID NO:47所示的Fc序列或包含SEQ ID NO:48所示的氨基酸序列，轻链恒定区包含SEQ ID NO:49或SEQ ID NO:50所示的氨基酸序列。The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the heavy chain constant region of the antibody comprises the Fc sequence shown in SEQ ID NO:47 or comprises the Fc sequence shown in SEQ ID NO:48 Amino acid sequence, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:50.
  8. 如权利要求1-7中任何一项所述的抗体或其抗原结合片段，其包含重链和轻链，其中：The antibody or antigen-binding fragment thereof of any one of claims 1-7, comprising a heavy chain and a light chain, wherein:
    1)所述重链包含如SEQ ID NO：72所示氨基酸序列，或与SEQ ID NO：72的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：72组成，所述轻链包含如SEQ ID NO：71所示氨基酸序列，或与SEQ ID NO：71的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：71组成； 1) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 72, or consists of SEQ ID NO: 72, the light chain The chain comprises an amino acid sequence as shown in SEQ ID NO: 71, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 71, or consists of SEQ ID NO: 71;
    2)所述重链包含如SEQ ID NO：74所示氨基酸序列，或与SEQ ID NO：74的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：74组成，所述轻链包含如SEQ ID NO：73所示氨基酸序列，或与SEQ ID NO：73的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：73组成；2) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 74, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 74, or consists of SEQ ID NO: 74, the light chain The chain comprises an amino acid sequence as shown in SEQ ID NO: 73, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 73, or consists of SEQ ID NO: 73;
    3)所述重链包含如SEQ ID NO：76所示氨基酸序列，或与SEQ ID NO：76的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：76组成，所述轻链包含如SEQ ID NO：75所示氨基酸序列，或与SEQ ID NO：75的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：75组成；3) the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 76, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 76, or consists of SEQ ID NO: 76, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 75, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 75, or consists of SEQ ID NO: 75;
    4)所述重链包含如SEQ ID NO：78所示氨基酸序列，或与SEQ ID NO：78的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：78组成，所述轻链包含如SEQ ID NO：77所示氨基酸序列，或与SEQ ID NO：77的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：77组成；4) the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 78, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 78, or consists of SEQ ID NO: 78, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 77, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77;
    5)所述重链包含如SEQ ID NO：80所示氨基酸序列，或与SEQ ID NO：80的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：80组成，所述轻链包含如SEQ ID NO：79所示氨基酸序列，或与SEQ ID NO：79的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：79组成；5) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 80, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 80, or consists of SEQ ID NO: 80, the light chain The chain comprises an amino acid sequence as shown in SEQ ID NO: 79, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 79, or consists of SEQ ID NO: 79;
    6)所述重链包含如SEQ ID NO：82所示氨基酸序列，或与SEQ ID NO：82的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：82组成，所述轻链包含如SEQ ID NO：81所示氨基酸序列，或与SEQ ID NO：81的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：81组成；6) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 82, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 82, or consists of SEQ ID NO: 82, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 81, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 81, or consists of SEQ ID NO: 81;
    7)所述重链包含如SEQ ID NO：84所示氨基酸序列，或与SEQ ID NO：84的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：84组成，所述轻链包含如SEQ ID NO：83所示氨基酸序列，或与SEQ ID NO：83的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：83组成；7) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 84, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 84, or consists of SEQ ID NO: 84, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 83, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 83, or consists of SEQ ID NO: 83;
    8)所述重链包含如SEQ ID NO：86所示氨基酸序列，或与SEQ ID NO：86的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：86组成，所述轻链包含如SEQ ID NO：73所示氨基酸序列，或与SEQ ID NO：73的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：73组成；8) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 86, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 86, or consists of SEQ ID NO: 86, the light chain The chain comprises an amino acid sequence as shown in SEQ ID NO: 73, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 73, or consists of SEQ ID NO: 73;
    9)所述重链包含如SEQ ID NO：88所示氨基酸序列，或与SEQ ID NO：88的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：88组成，所述轻链包含如SEQ ID NO：87所示氨基酸序列，或与SEQ ID NO：87的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：87组成；9) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 88, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 88, or consists of SEQ ID NO: 88, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 87, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 87, or consists of SEQ ID NO: 87;
    10)所述重链包含如SEQ ID NO：90所示氨基酸序列，或与SEQ ID NO：90的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：90组成，所述轻链包含如SEQ ID NO： 89所示氨基酸序列，或与SEQ ID NO：89的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：89组成；10) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 90, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 90, or consists of SEQ ID NO: 90, the light chain The chain contains such as SEQ ID NO: The amino acid sequence shown as 89, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 89, or consisting of SEQ ID NO: 89;
    11)所述重链包含如SEQ ID NO：175所示氨基酸序列，或与SEQ ID NO：175的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：175组成，所述轻链包含如SEQ ID NO：174所示氨基酸序列，或与SEQ ID NO：174的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：174组成；11) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 175, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 175, or consists of SEQ ID NO: 175, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 174, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 174, or consists of SEQ ID NO: 174;
    12)所述重链包含如SEQ ID NO：177所示氨基酸序列，或与SEQ ID NO：177的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：177组成，所述轻链包含如SEQ ID NO：176所示氨基酸序列，或与SEQ ID NO：176的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：176组成；12) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 177, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 177, or consists of SEQ ID NO: 177, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 176, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 176, or consists of SEQ ID NO: 176;
    13)所述重链包含如SEQ ID NO：171所示氨基酸序列，或与SEQ ID NO：171的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：171组成，所述轻链包含如SEQ ID NO：170所示氨基酸序列，或与SEQ ID NO：170的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：170组成；13) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 171, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 171, or consists of SEQ ID NO: 171, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 170, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 170, or consists of SEQ ID NO: 170;
    14)所述重链包含如SEQ ID NO：173所示氨基酸序列，或与SEQ ID NO：173的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：173组成，所述轻链包含如SEQ ID NO：172所示氨基酸序列，或与SEQ ID NO：172的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：172组成；14) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 173, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 173, or consists of SEQ ID NO: 173, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 172, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 172, or consists of SEQ ID NO: 172;
    15)所述重链包含如SEQ ID NO：179所示氨基酸序列，或与SEQ ID NO：179的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：179组成，所述轻链包含如SEQ ID NO：178所示氨基酸序列，或与SEQ ID NO：178的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：178组成；15) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 179, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 179, or consists of SEQ ID NO: 179, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 178, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 178, or consists of SEQ ID NO: 178;
    16)所述重链包含如SEQ ID NO：191所示氨基酸序列，或与SEQ ID NO：191的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：191组成，所述轻链包含如SEQ ID NO：190所示氨基酸序列，或与SEQ ID NO：190的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：190组成；16) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 191, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 191, or consists of SEQ ID NO: 191, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 190, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 190, or consists of SEQ ID NO: 190;
    17)所述重链包含如SEQ ID NO：193所示氨基酸序列，或与SEQ ID NO：193的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：193组成，所述轻链包含如SEQ ID NO：192所示氨基酸序列，或与SEQ ID NO：192的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：192组成；17) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 193, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 193, or consists of SEQ ID NO: 193, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 192, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 192, or consists of SEQ ID NO: 192;
    18)所述重链包含如SEQ ID NO：195所示氨基酸序列，或与SEQ ID NO：195的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：195组成，所述轻链包含如SEQ ID NO：194所示氨基酸序列，或与SEQ ID NO：194的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：194组成； 18) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 195, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 195, or consists of SEQ ID NO: 195, the light chain The chain comprises an amino acid sequence as shown in SEQ ID NO: 194, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 194, or consists of SEQ ID NO: 194;
    19)所述重链包含如SEQ ID NO：181所示氨基酸序列，或与SEQ ID NO：181的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：181组成，所述轻链包含如SEQ ID NO：180所示氨基酸序列，或与SEQ ID NO：180的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：180组成；19) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 181, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 181, or consists of SEQ ID NO: 181, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 180, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 180, or consists of SEQ ID NO: 180;
    20)所述重链包含如SEQ ID NO：183所示氨基酸序列，或与SEQ ID NO：183的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：183组成，所述轻链包含如SEQ ID NO：182所示氨基酸序列，或与SEQ ID NO：182的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：182组成；20) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 183, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 183, or consists of SEQ ID NO: 183, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 182, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 182, or consists of SEQ ID NO: 182;
    21)所述重链包含如SEQ ID NO：185所示氨基酸序列，或与SEQ ID NO：185的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：185组成，所述轻链包含如SEQ ID NO：184所示氨基酸序列，或与SEQ ID NO：184的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：184组成；21) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 185, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 185, or consists of SEQ ID NO: 185, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 184, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 184, or consists of SEQ ID NO: 184;
    22)所述重链包含如SEQ ID NO：187所示氨基酸序列，或与SEQ ID NO：187的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：187组成，所述轻链包含如SEQ ID NO：186所示氨基酸序列，或与SEQ ID NO：186的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：186组成；22) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 187, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 187, or consists of SEQ ID NO: 187, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 186, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 186, or consists of SEQ ID NO: 186;
    23)所述重链包含如SEQ ID NO：72所示氨基酸序列，或与SEQ ID NO：72的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：72组成，所述轻链包含如SEQ ID NO：188所示氨基酸序列，或与SEQ ID NO：188的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：188组成；23) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 72, or consists of SEQ ID NO: 72, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 188, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 188, or consists of SEQ ID NO: 188;
    24)所述重链包含如SEQ ID NO：72所示氨基酸序列，或与SEQ ID NO：72的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：72组成，所述轻链包含如SEQ ID NO：189所示氨基酸序列，或与SEQ ID NO：189的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：189组成；24) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 72, or consists of SEQ ID NO: 72, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 189, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 189, or consists of SEQ ID NO: 189;
    25)所述重链包含如SEQ ID NO：82所示氨基酸序列，或与SEQ ID NO：82的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：82组成，所述轻链包含如SEQ ID NO：196所示氨基酸序列，或与SEQ ID NO：196的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：196组成；25) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 82, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 82, or consists of SEQ ID NO: 82, the light The chain comprises an amino acid sequence as shown in SEQ ID NO: 196, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 196, or consists of SEQ ID NO: 196;
    26)所述重链包含如SEQ ID NO：198所示氨基酸序列，或与SEQ ID NO：198的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：198组成，所述轻链包含如SEQ ID NO：197所示氨基酸序列，或与SEQ ID NO：197的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：197组成；或26) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 198, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 198, or consists of SEQ ID NO: 198, the light The chain comprises the amino acid sequence shown in SEQ ID NO: 197, or has an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 197, or consists of SEQ ID NO: 197; or
    27)所述重链包含如SEQ ID NO：199所示氨基酸序列，或与SEQ ID NO：199的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：199组成，所述轻链包含如SEQ ID NO： 81所示氨基酸序列，或与SEQ ID NO：81的氨基酸序列具有至少90％同一性的氨基酸序列，或由SEQ ID NO：81组成。27) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 199, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 199, or consists of SEQ ID NO: 199, the light chain The chain contains such as SEQ ID NO: The amino acid sequence shown in 81, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 81, or consisting of SEQ ID NO: 81.
  9. 一种具有Ab-(L-D)n结构的抗体偶联药物，其中Ab为如权利要求1-8中任一项所述的ROR1的抗体或其抗原结合片段，L为接头，D为治疗活性物质或药物活性成分，n表示1-20的整数。An antibody-drug conjugate with an Ab-(L-D)n structure, wherein Ab is the antibody of ROR1 or an antigen-binding fragment thereof according to any one of claims 1-8, L is a linker, and D is a therapeutically active substance Or active pharmaceutical ingredients, n represents an integer of 1-20.
  10. 权利要求9所述的抗体偶联药物，其中所述治疗活性物质或药物活性成分是细胞毒素、植物毒素、小分子毒素、放射性同位素。The antibody-drug conjugate according to claim 9, wherein the therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a plant toxin, a small molecule toxin, or a radioactive isotope.
  11. 权利要求9或10所述的抗体偶联药物，其中所述治疗活性物质或药物活性成分是海兔毒素(Dolastatin)及其auristatin类衍生物，例如Dolastatin 10、Dolastatin 15、auristatin E、auristatin PE、monomethyl auristatin D(MMAD)、monomethyl auristatin E(MMAE)、monomethyl auristatin F(MMAF)、auristatin F苯二胺(AFP)、auristatin EB(AEB)、auristatin EFP(AEFP)、auristatin F羟丙基酰胺(AF HPA)。The antibody-drug conjugate according to claim 9 or 10, wherein the therapeutic active substance or pharmaceutical active ingredient is Dolastatin (Dolastatin) and its auristatin derivatives, such as Dolastatin 10, Dolastatin 15, auristatin E, auristatin PE, Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AF HPA).
  12. 权利要求9-11中任一项所述的抗体偶联药物，其中所述海兔毒素(Dolastatin)及其auristatin类衍生物是MMAE、MMAF、auristatin F羟丙基酰胺或auristatin F苯二胺。The antibody-drug conjugate according to any one of claims 9-11, wherein the dolastatin (Dolastatin) and its auristatin derivatives are MMAE, MMAF, auristatin F hydroxypropylamide or auristatin F phenylenediamine.
  13. 权利要求9-12中任一项所述的抗体偶联药物，其中所述接头是组织蛋白酶可降解的接头(例如缬氨酸-瓜氨酸(val-cit)接头、cBu-Cit接头和CX接头)、不可断裂接头(例如SMCC接头或MD接头)、酸敏接头、硅脂结构的接头、disulfide-carbamate接头、MC-GGFG接头、TRX接头、含半乳糖苷的接头、焦磷酸酯接头、近红外敏感的接头、紫外敏感的接头(例如PC4AP)。The antibody drug conjugate of any one of claims 9-12, wherein the linker is a cathepsin-degradable linker (such as valine-citrulline (val-cit) linker, cBu-Cit linker and CX linker), non-breakable linker (such as SMCC linker or MD linker), acid-sensitive linker, silicone grease linker, disulfide-carbamate linker, MC-GGFG linker, TRX linker, galactoside-containing linker, pyrophosphate linker, NIR-sensitive linkers, UV-sensitive linkers (eg PC4AP).
  14. 权利要求9-13中任一项所述的抗体偶联药物，其中所述接头选自马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基(mc-vc-PAB)、乙酰基-赖氨酸-缬氨酸-瓜氨酸-对氨基苄氧羰基(AcLys-VC-PABC)、氨基PEG6-丙酰基、及马来酰亚胺己酸基(mc)、马来酰亚胺基丙酰基(MP)、缬氨酸-瓜氨酸(val-cit)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)、N-琥珀酰亚胺基4-(2-吡啶硫基)戊酸酯(SPP)、N-琥珀酰亚胺基4-(N-马来酰亚胺基甲基)-环己烷-1-羧酸酯(SMCC)、N-琥珀酰亚胺基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)、N-琥珀酰亚胺基-4-(2-吡啶基二硫代)丁酸酯(SPDB)、N-琥珀酰亚胺基3-(吡啶-2-基二硫代)-丙酸酯(SPDP)。The antibody-drug conjugate according to any one of claims 9-13, wherein the linker is selected from the group consisting of maleimide-caproyl-valine-citrulline-p-aminobenzyloxy (mc- vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimide caproyl (mc ), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-Succinimidyl 4-(N-maleimidylmethyl)-cyclohexane-1 -carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio ) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).
  15. 权利要求9-14中任一项所述的抗体偶联药物，其中所述接头是MC-VC-PAB、SMCC或MC-GGFG。The antibody drug conjugate according to any one of claims 9-14, wherein the linker is MC-VC-PAB, SMCC or MC-GGFG.
  16. 权利要求9-15中任一项所述的抗体偶联药物，其中所述Ab包含：The antibody-drug conjugate of any one of claims 9-15, wherein the Ab comprises:
    (1)如SEQ ID NO:7、8和9所示序列的HCDR1、HCDR2、HCDR3和如SEQ ID NO:25、26和27所示序列的LCDR1、LCDR2、LCDR3，或(1) HCDR1, HCDR2, HCDR3 of the sequence shown in SEQ ID NO:7, 8 and 9 and LCDR1, LCDR2, LCDR3 of the sequence shown in SEQ ID NO:25, 26 and 27, or
    (2)如SEQ ID NO:1、2和37所示序列的HCDR1、HCDR2、HCDR3和如SEQ ID NO:19、20和21所示序列的LCDR1、LCDR2、LCDR3。(2) HCDR1, HCDR2, HCDR3 of sequences shown in SEQ ID NO:1, 2 and 37 and LCDR1, LCDR2, LCDR3 of sequences shown in SEQ ID NO:19, 20 and 21.
  17. 权利要求9-16中任一项所述的抗体偶联药物，其中所述Ab包含重链可变区和轻链可变区，其中 The antibody-drug conjugate according to any one of claims 9-16, wherein the Ab comprises a heavy chain variable region and a light chain variable region, wherein
    (1)所述重链可变区包含如SEQ ID NO：60所示氨基酸序列，所述轻链可变区包含如SEQ ID NO：59所示氨基酸序列，或(1) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 60, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 59, or
    (2)所述重链可变区包含如SEQ ID NO：66所示氨基酸序列，所述轻链可变区包含如SEQ ID NO：53所示氨基酸序列。(2) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53.
  18. 权利要求9-17中任一项所述的抗体偶联药物，其中所述Ab包含：The antibody-drug conjugate of any one of claims 9-17, wherein the Ab comprises:
    (1)如SEQ ID NO：80所示氨基酸序列的重链和如SEQ ID NO：79所示氨基酸序列的轻链；或(1) the heavy chain of the amino acid sequence shown in SEQ ID NO: 80 and the light chain of the amino acid sequence shown in SEQ ID NO: 79; or
    (2)如SEQ ID NO：86所示氨基酸序列的重链和如SEQ ID NO：73所示氨基酸序列的轻链。(2) The heavy chain of the amino acid sequence shown in SEQ ID NO: 86 and the light chain of the amino acid sequence shown in SEQ ID NO: 73.
  19. 权利要求16-18中任一项所述的抗体偶联药物，其所述接头是MC-VC-PAB，所述细胞毒素是MMAE.The antibody-drug conjugate according to any one of claims 16-18, wherein the linker is MC-VC-PAB, and the cytotoxin is MMAE.
  20. 一种药物组合物，其包含：A pharmaceutical composition comprising:
    (1)如权利要求1-8中任一项所述的抗ROR1抗体或其抗原结合片段，或如权利要求9-19中任一项所述的的抗体偶联药物，以及；(1) The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the antibody-drug conjugate according to any one of claims 9-19, and;
    (2)可药用载体。(2) Pharmaceutically acceptable carrier.
  21. 一种分离的多核苷酸分子，其编码如权利要求1-8中任一项所述的抗ROR1抗体或其抗原结合片段。An isolated polynucleotide molecule encoding the anti-ROR1 antibody or antigen-binding fragment thereof of any one of claims 1-8.
  22. 一种载体，其包含权利要求21所述的核酸分子，优选地，所述载体是表达载体。A vector comprising the nucleic acid molecule of claim 21, preferably, the vector is an expression vector.
  23. 一种宿主细胞，其包含权利要求22所述的载体或权利要求21所述的核酸分子。A host cell comprising the vector of claim 22 or the nucleic acid molecule of claim 21.
  24. 如权利要求1-8中任一项所述的抗ROR1抗体或其抗原结合片段在制备用于预防或治疗癌症的抗体偶联药物中的用途，或在制备用于预防或治疗与ROR1异常表达相关的疾病的药物中的用途。Use of the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1-8 in the preparation of antibody-conjugated drugs for the prevention or treatment of cancer, or in the preparation of anti-ROR1 antibodies associated with abnormal expression of ROR1 Use in medicine for related diseases.
  25. 如权利要求9-19中任一项所述的的抗体偶联药物在制备用于预防或治疗与ROR1异常表达相关的疾病的药物中的用途。Use of the antibody-conjugated drug according to any one of claims 9-19 in the preparation of a drug for preventing or treating a disease related to abnormal expression of ROR1.
  26. 一种杀死表达ROR1的细胞或者抑制表达ROR1的细胞生长的方法，包括使所述细胞接触有效量的如权利要求1-8任一项所述的抗体或其抗原结合片段、或有效量的如权利要求9-19任一项所述的的抗体偶联药物、或有效量的如权利要求20所述的药物组合物。A method for killing cells expressing ROR1 or inhibiting the growth of cells expressing ROR1, comprising contacting the cells with an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or an effective amount of The antibody-drug conjugate according to any one of claims 9-19, or an effective amount of the pharmaceutical composition according to claim 20.
  27. 一种在有需要的受试者中预防或治疗与ROR1异常表达相关的疾病的方法，包括向所述受试者施用预防有效量或治疗有效量的如权利要求1-8任一项所述的抗体或其抗原结合片段、或预防有效量或治疗有效量的如权利要求9-19任一项所述的的抗体偶联药物、或预防有效量或治疗有效量的如权利要求20所述的药物组合物。A method for preventing or treating a disease associated with abnormal expression of ROR1 in a subject in need thereof, comprising administering to the subject a preventive or therapeutically effective amount of the drug as described in any one of claims 1-8. Antibody or antigen-binding fragment thereof, or a preventive effective amount or a therapeutically effective amount of the antibody-conjugated drug as described in any one of claims 9-19, or a preventive effective amount or a therapeutically effective amount as described in claim 20 pharmaceutical composition.
  28. 权利要求25所述的用途或权利要求27所述的方法，其中与ROR1异常表达相关的疾病是高表达ROR1的癌症。The use according to claim 25 or the method according to claim 27, wherein the disease associated with abnormal expression of ROR1 is a cancer with high expression of ROR1.
  29. 如权利要求28所述的用途或方法，其中所述高表达ROR1的癌症是慢性淋巴细胞白血病 (CLL)、急性淋巴细胞白血病(ALL)、套细胞淋巴瘤、肾细胞癌、结肠癌、乳腺癌、神经母细胞瘤、肺癌、胃癌、头颈癌和黑色素瘤。 The use or method according to claim 28, wherein the cancer that highly expresses ROR1 is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer, breast cancer, neuroblastoma, lung cancer, gastric cancer, head and neck cancer, and melanoma.
PCT/CN2023/072941 2022-01-29 2023-01-18 Ror1-targeted antibody or antigen-binding fragment thereof and use thereof WO2023143315A1 (en)

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WO2011159847A2 (en) * 2010-06-15 2011-12-22 The Regents Of The University Of California Receptor tyrosine kinase-like orphan receptor 1 (ror1) single chain fv antibody fragment conjugates and methods of use thereof
US20160208018A1 (en) * 2015-01-16 2016-07-21 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for ror1
CN108884160A (en) * 2015-10-30 2018-11-23 恩比伊治疗股份公司 Anti- ROR1 antibody
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