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WO2023134766A1 - 靶向cd25的抗体及其制备方法和应用 - Google Patents

靶向cd25的抗体及其制备方法和应用 Download PDF

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Publication number
WO2023134766A1
WO2023134766A1 PCT/CN2023/072439 CN2023072439W WO2023134766A1 WO 2023134766 A1 WO2023134766 A1 WO 2023134766A1 CN 2023072439 W CN2023072439 W CN 2023072439W WO 2023134766 A1 WO2023134766 A1 WO 2023134766A1
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Prior art keywords
amino acid
seq
variable region
chain variable
acid sequence
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PCT/CN2023/072439
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English (en)
French (fr)
Inventor
潘秋明
王承恩
何进秋
李艳
王远东
王永强
赵楚楚
潘宏杰
戎一平
罗海山
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诺纳生物（苏州）有限公司
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Priority to CN202380017645.3A priority Critical patent/CN118541391A/zh
Publication of WO2023134766A1 publication Critical patent/WO2023134766A1/zh

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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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present application relates to the field of biomedicine, in particular to an antibody targeting CD25 or its variant, as well as its preparation method and application.
  • Treg Regulatory T cells
  • CD25 is the alpha chain of interleukin-2 receptor (IL2R ⁇ ), which is constitutively expressed in Treg cells, while the expression of CD25 in Teff cells is lower. This property makes CD25 a potential target that can specifically target Treg cells.
  • IL2R ⁇ interleukin-2 receptor
  • Tff cells Teff cells
  • CD25 is expressed in Hodgkin's lymphoma and various non-Hodgkin's lymphomas, and the expression rate of CD25 is close to 100% in adult T-cell leukemia/lymphoma and hairy cell leukemia.
  • ADC antibody-drug conjugates
  • ADCT-301 has shown good efficacy in clinical research on Hodgkin's lymphoma.
  • the development of antibody drugs for this target can achieve anti-tumor effects through Fc optimization and enhancement of ADCC function. On the other hand, it can also be developed through ADC methods.
  • anti-CD25 antibody drugs focused on blocking antibodies against the ligand interleukin-2 in the early stage, including monoclonal antibodies and ADCs. Recent studies have found that anti-CD25 antibodies that do not block interleukin-2 have shown better anti-tumor activity in mouse experiments, which may be due to the expression of CD25 on effector T cells (Teff). Blocking antibodies Inhibits the signaling pathway of interleukin-2, thereby inhibiting the activity of Teff. Teff is the main function of cells in the process of tumor immunity, and this feature of blocking antibodies weakens its anti-tumor effect. A non-blocking anti-CD25 antibody would avoid this problem and have more pronounced antitumor activity.
  • the invention provides an anti-CD25 interleukin-2 non-blocking antibody or variant thereof with high affinity, high selectivity and high biological activity, capable of being endocytized by cells, and suitable for the development of ADC drugs.
  • the first aspect of the present invention provides an antibody targeting CD25 or a variant thereof, wherein the antibody includes a light chain variable region and a heavy chain variable region, wherein the antibody is selected from the following groups:
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 96, 111 and 133; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 20, 44 and 74; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:104, 111, and 127, respectively; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 as indicated in 22, 46, 76; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:94, 110, and 134 respectively; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 as indicated in 23, 48, 78; or,
  • the light chain variable region comprises amino acid sequences LCDR1, LCDR2 and LCDR3 respectively shown in SEQ ID NO:97, 113 and 129; and/or, the heavy chain variable region comprises amino acid sequences respectively such as SEQ ID NO: 16, 40 and HCDR1, HCDR2 and HCDR3 shown in 70; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:94, 116 and 135; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 as indicated in 23, 47 and 77; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:95, 111, and 127, respectively; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 14, 38, 68; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:98, 114, and 130; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 17, 41, 71; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:99, 115, and 131 respectively; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 18, 42, 72; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:100, 113, and 132, respectively; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 14, 43, 73; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:103, 110, and 134; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 as indicated in 21, 45, 75; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:105, 111, and 136; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 18, 42, 80.
  • CDR of an antibody can be defined by various methods in the art, such as Chothia (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)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, vol.
  • Laa-Lbb can refer to the amino acid sequence from the N-terminal of the antibody light chain, from the aa to the bb position;
  • Haa-Hbb can refer to the amino acid sequence from the aa to the bb position from the N-terminal of the antibody heavy chain sequence.
  • L24-L34 can refer to the amino acid sequence starting from the N-terminus of the antibody light chain and from the 24th to 34th positions according to the Chothia coding rules;
  • H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain according to the Chothia coding rules. Amino acid sequence from position 26 to position 32.
  • CDR complementarity determining region
  • variable region e.g., variable region
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different CDR definition rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • a different protocol e.g. Different CDR definition rules or combinations
  • the variant is an addition, deletion or substitution of 1 to 3 amino acid residues in the amino acid sequence of the CD25-targeting antibody.
  • the variant is a substitution of amino acid residues at position 1 of HCDR1 and position 3 of HCDR2 in the heavy chain variable region of the antibody, and/or, in the variable region of the light chain of the antibody Substitution of amino acid residues occurs at positions 10-11 of LCDR1 and at positions 1 or 4 of LCDR3;
  • the amino acid sequence of the variant has at least 85% sequence identity with the original amino acid sequence, and the binding of the antibody to the target antigen is maintained or improved; the at least 85% sequence identity is preferably at least 90% sequence identity Identity; more preferably at least 95%, 96%, 97% or 98% sequence identity; most preferably at least 99% sequence identity.
  • said variant is selected from the group consisting of:
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:101, 113, and 129, respectively; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 19, 40, 70; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:101, 113, and 139 respectively; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 19, 40, 70; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:102, 113, and 129, respectively; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 19, 40, 70; or,
  • the light chain variable region comprises an amino acid sequence such as SEQ ID NO:94, 116, 137, LCDR1, LCDR2 and LCDR3 shown respectively; and/or, the heavy chain variable region comprises an amino acid sequence such as SEQ ID NO : HCDR1, HCDR2 and HCDR3 shown in 23, 50, 77; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:94, 116, and 138; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 as indicated in 23, 51, 77; or,
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:94, 116, and 138; and/or, the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 23, 50, 77.
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 171 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 171
  • Said heavy chain variable region comprises aminoacid sequence as shown in SEQ ID NO:153 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:153 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 173 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 173
  • the VL of the sex comprises an amino acid sequence as shown in SEQ ID NO: 155 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 155 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 175 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 175 VL of sex;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 157 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 157 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 165 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 165
  • Said heavy chain variable region comprises aminoacid sequence as shown in SEQ ID NO:148 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:148 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 174 or is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identical to SEQ ID NO: 174 sexual VL; the heavy chain variable region VH comprising an amino acid sequence as set forth in SEQ ID NO: 156 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 156; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 163 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 163
  • the VL of the sex; said heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 146 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 146 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 166 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 166
  • Said heavy chain variable region comprises aminoacid sequence as shown in SEQ ID NO:149 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:149 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 167 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 167
  • the VL of sex comprises an amino acid sequence as shown in SEQ ID NO: 150 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 150 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 168 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 168 VL of sex;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 151 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO: 151 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 172 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 172
  • the VL of the sex; said heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 154 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 154 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 177 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 177 VL of sex;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 159 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO: 159 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 169 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 169
  • the VL of the sex comprises an amino acid sequence as shown in SEQ ID NO: 152 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 152 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 180 or has the same sequence as SEQ ID NO: 180 A VL of at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity; said heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 152 or identical to SEQ ID NO: 152 NO: 152 has a VH of at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 170 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 170
  • the VL of the sex comprises an amino acid sequence as shown in SEQ ID NO: 152 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 152 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 178 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 178
  • Said heavy chain variable region comprises aminoacid sequence as shown in SEQ ID NO:160 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:160 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 179 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 179
  • Said heavy chain variable region comprises aminoacid sequence as shown in SEQ ID NO:161 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:161 % amino acid identity of VH; or,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 179 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 179
  • Said heavy chain variable region comprises aminoacid sequence as shown in SEQ ID NO:160 or has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:160 % amino acid identity of VH.
  • the antibody or variant thereof comprises a chimeric antibody, a humanized antibody or a fully human antibody.
  • the structure of said antibody or variant thereof is Fab, Fab', F(ab')2, Fv, IgG, Fd, dAb or a bispecific antibody or multispecific antibody comprising said antibody or variant thereof Antibody, or a monoclonal antibody or a polyclonal antibody prepared from the above-mentioned antibody;
  • the Fv is preferably scFv;
  • the antibody with the IgG structure or its variant includes a light chain and a heavy chain:
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 207 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 207;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 189 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 189; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 209 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 209;
  • the heavy chain comprises at least 85%, 90%, 95%, 96%, 97%, 98% or 99% ammonia as shown in or with SEQ ID NO: 191 amino acid sequence with amino acid identity; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 211 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 211;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 193 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 193; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 201 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 201;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 184 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 184; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 210 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 210;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 192 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 192; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 199 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 199;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 182 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 182; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 202 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 202;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 185 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 185; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 203 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 203;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 186 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 186; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 204 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 204;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 187 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 187; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 208 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 208;
  • the heavy chain includes as SEQ ID NO: 190 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 190; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 213 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 213;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 195 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 195; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 205 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 205;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 188 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 188; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 216 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 216;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 188 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 188; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 206 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 206;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 188 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 188; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 214 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 214;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 196 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 196; or,
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 215 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 215;
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 197 or having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 197; or,
  • the light chain includes the amino acid sequence shown in SEQ ID NO:215; the heavy chain includes the amino acid sequence shown in SEQ ID NO:196.
  • Fab fragment consists of CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc” region contains the two heavy chain fragments comprising the CH2 and CH3 domains of the antibody.
  • the two heavy chain fragments are held by two or more disulfide bonds and are held by hydrophobic interactions of the CH3 domains together.
  • a "Fab'fragment” contains one light chain and part of one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby the two heavy chains of the two Fab' fragments can Interchain disulfide bonds are formed to form F(ab')2 molecules.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains.
  • the F(ab')2 fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the term "Fv” means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacking the constant region.
  • the scFv single chain antibody fragment, single-chain antibody
  • the scFv can be a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids .
  • the VL and VH domains are paired to form a monovalent molecule via a linker that enables them to be produced as a single polypeptide chain [see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad . Sci. USA 85:5879-5883 (1988)].
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated G4S amino acid sequence or variants thereof.
  • linkers having the amino acid sequence (G4S)4 or (G4S)3 may be used, although variants thereof may also be used.
  • the Fd can be a conventional antibody fragment in the field, which includes about 225 amino acid residues in Fab except VL and CL, including VH, CH1 and part of the hinge region.
  • the dAb can be conventional in the field: it is composed of VH or VL domains, is some minimal functional antibody fragments, and retains complete antigen-binding specificity. Its molecular weight is about one tenth of that of normal antibodies.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitopic specificities.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; Antibodies with VL and VH domains, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies with two or more single variable domains, each single variable domain binds to Binding to different targets or different epitopes of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently wait.
  • VH heavy chain variable region
  • VL light chain variable region
  • Antibodies or variants thereof of the invention include monoclonal antibodies.
  • the monoclonal antibody or mAb or Ab in the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain.
  • the second aspect of the present invention provides a chimeric antigen receptor comprising the antibody or variant thereof described in the first aspect of the present invention.
  • the third aspect of the present invention provides an isolated nucleic acid encoding the antibody or variant thereof as described in the first aspect of the present invention, or the chimeric antigen receptor as described in the second aspect of the present invention.
  • the fourth aspect of the present invention provides a recombinant expression vector comprising the isolated Nucleic acid; preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenoviral vector or adeno-associated viral vector.
  • the fifth aspect of the present invention provides a transformant, which comprises the recombinant expression vector as described in the fourth aspect of the present invention in a host cell; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the The host cell is selected from yeast cells, mammalian cells or other cells suitable for producing antibodies or antigen-binding fragments thereof; wherein the mammalian cells are, for example, 293 cells or CHO cells.
  • the sixth aspect of the present invention provides an immune cell comprising the chimeric antigen receptor described in the second aspect of the present invention; preferably, the immune cell is a T cell or an NK cell; more preferably, the NK cell is Peripheral blood NK cells, umbilical cord blood-derived NK cells, stem cell differentiated NK cells or NK cell lines such as NK-92 cell line.
  • the seventh aspect of the present invention provides a method for preparing a CD25-targeting antibody or a variant thereof, which comprises culturing the transformant as described in the fifth aspect of the present invention, and obtaining a CD25-targeting antibody or a variant thereof from the culture .
  • the eighth aspect of the present invention provides an antibody-drug conjugate, which comprises a cytotoxic agent, and the CD25-targeting antibody or variant thereof as described in the first aspect of the present invention.
  • the ninth aspect of the present invention provides a pharmaceutical composition, which comprises the CD25-targeting antibody or its variant according to the first aspect of the present invention, the nucleic acid isolated according to the third aspect of the present invention, and the nucleic acid according to the fourth aspect of the present invention.
  • the pharmaceutical composition also contains hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, and co-stimulatory molecules.
  • hormone preparations targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, and co-stimulatory molecules.
  • the pharmaceutical composition or pharmaceutical preparation of the present invention comprises suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, including buffers known in the art.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, dextrose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin , propylene, glycol, water, ethanol, etc.
  • excipients and their uses see also "Handbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Seskey and SCOwen, Pharmaceutical Press, London, Chicago.
  • compositions can also contain small amounts of wetting or emulsifying agents, or pH buffer.
  • These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations can contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharine. It can be prepared by mixing an antibody or antigen-binding fragment thereof of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed.
  • the pharmaceutical preparation or composition according to the present invention is preferably in the form of a freeze-dried preparation or an aqueous solution.
  • 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.
  • 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 antibodies or antigen-binding fragments thereof of the invention in the form of shaped articles, eg films or microcapsules.
  • the CD25-targeting antibody or its variant according to any one of the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, the isolated nucleic acid as described in the third aspect of the present invention, The recombinant expression vector according to the fourth aspect of the present invention, the transformant according to the fifth aspect of the present invention, the immune cell according to the sixth aspect of the present invention, the antibody drug conjugate according to the eighth aspect of the present invention And/or the application of the pharmaceutical composition described in the ninth aspect of the present invention in the preparation of drugs for diagnosis, prevention and/or treatment of tumors.
  • the tenth aspect of the present invention provides a kit, which includes the CD25-targeting antibody or its variant as described in the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, the The isolated nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the immune cell as described in the sixth aspect of the present invention, or the recombinant expression vector as described in the fourth aspect of the present invention.
  • the kit further comprises (i) a device for administering the antibody or its variant or chimeric antigen receptor or immune cell or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
  • the eleventh aspect of the present invention provides a kit of medicines, which includes a medicine box A and a medicine box B, wherein:
  • the kit A contains the CD25-targeting antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, and the chimeric antigen receptor as described in the third aspect of the present invention.
  • Isolated nucleic acid, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the immune cell as described in the sixth aspect of the present invention, the recombinant expression vector as described in the eighth aspect of the present invention Antibody drug conjugates and/or pharmaceutical compositions according to the ninth aspect of the present invention;
  • the kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, One or more of the group consisting of cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  • the twelfth aspect of the present invention provides a method for detecting CD25, which includes using the CD25-targeting antibody or its variant as described in the first aspect of the present invention, and the chimeric antigen receptor as described in the second aspect of the present invention .
  • the immune cell according to the sixth aspect of the present invention, the antibody drug conjugate as described in the eighth aspect of the present invention and/or the pharmaceutical composition according to the ninth aspect of the present invention includes using the CD25-targeting antibody or its variant as described in the first aspect of the present invention, and the chimeric antigen receptor as described in the second aspect of the present invention .
  • said detection is for non-diagnostic and/or therapeutic purposes.
  • the thirteenth aspect of the present invention provides a method for preventing, treating or alleviating a condition in a subject, comprising administering to the subject a therapeutically effective amount of the CD25-targeting antibody according to any one of the first aspect of the present invention Or its variant, the isolated nucleic acid as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the transformant as described in the sixth aspect of the present invention.
  • the condition in the subject is a proliferative disease, such as a tumor or cancer.
  • the subject has an established tumor, such as a solid tumor.
  • a method for reducing the number of Treg cells in a tumor or in a subject in some embodiments, there is provided a method for eliminating or inhibiting the cell activity of Treg in a tumor or in a tumor in a subject, All include administering to the subject an effective amount of the CD25-targeting antibody or variant thereof as described in any one of the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, such as The isolated nucleic acid as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the immune cell as described in the sixth aspect of the present invention, the The antibody-drug conjugate described in the eighth aspect of the invention and/or the pharmaceutical composition described in the ninth aspect of the invention.
  • a method for increasing the ratio of Teff/Treg in a tumor of a subject comprising administering to the subject an antibody targeting CD25 or a variant thereof according to any one of the first aspect of the present invention,
  • the chimeric antigen receptor as described in the second aspect of the present invention, the isolated nucleic acid as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the recombinant expression vector as described in the fifth aspect of the present invention The transformant, the immune cell according to the sixth aspect of the present invention, the antibody drug conjugate according to the eighth aspect of the present invention and/or the pharmaceutical composition according to the ninth aspect of the present invention.
  • a method for enhancing ADCC against tumor cells in a subject comprising administering to the subject the CD25-targeting antibody or variant thereof according to any one of the first aspect of the present invention, such as The chimeric antigen receptor as described in the second aspect of the present invention, the isolated nucleic acid as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformation as described in the fifth aspect of the present invention body, the immune cell according to the sixth aspect of the present invention, the antibody drug conjugate according to the eighth aspect of the present invention and/or the pharmaceutical composition according to the ninth aspect of the present invention.
  • the first aspect of the present invention such as The chimeric antigen receptor as described in the second aspect of the present invention, the isolated nucleic acid as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformation as described in the fifth aspect of the present invention body, the immune cell according to the sixth aspect of the present invention, the antibody drug conjug
  • ADCC effect against tumor cells in the subject is enhanced.
  • the CD25-targeting antibody or variant thereof as described in any one of the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention
  • the isolated nucleic acid described above, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the immune cell as described in the sixth aspect of the present invention, the immune cell as described in the eighth aspect of the present invention Use of the above-mentioned antibody-drug conjugate and/or the pharmaceutical composition according to the ninth aspect of the present invention for the preparation of a drug for preventing, treating or alleviating a subject’s condition, for preparing a drug that reduces the inside of a subject’s tumor or tumor
  • the disorder of the above-mentioned subject is or has a proliferative disorder (eg, cancer or tumor).
  • a proliferative disorder eg, cancer or tumor.
  • tumors include, but are not limited to, carcinomas, lymphomas, leukemias, blastomas, and sarcomas.
  • cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), Hodgkin's lymphoma, non-Hodgkin's lymphoma , acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer , prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer cancer.
  • the cancer or tumor may be a solid tumor, including but not limited to a sarcoma (including mesenchymal tumors formed from tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue). Cancers arising from transformed cells of stromal origin), carcinomas (including tumors arising from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, and the like.
  • a sarcoma including mesenchymal tumors formed from tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue.
  • Cancers involving solid tumors include, but are not limited to, brain, lung, stomach, duodenum, esophagus, breast, colon and rectum, kidney, bladder, kidney, pancreas, prostate, ovary Carcinoma, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, neck cancer, lymphoma, etc.
  • the cancer involves CD25-expressing tumors, including, but not limited to, lymphomas, such as Hodgkin's lymphoma, and lymphocytic leukemias, such as chronic lymphocytic leukemia (CLL).
  • lymphomas such as Hodgkin's lymphoma
  • lymphocytic leukemias such as chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • the reagents and raw materials used in the present invention are all commercially available.
  • the monoclonal antibody targeting CD25 of the present invention is a naturally occurring antibody with the Monkey CD25 binding activity. After being modified by Fc, it has a stronger ADCC effect, and in vitro experiments show a good effect on eliminating Treg and lymphoma cells; at the same time, it does not block the IL-2 signaling pathway, and has no effect on the activity of Teff cells; and has good internalization Activity is an antibody molecule that is potentially suitable for ADC drug development.
  • Figure 1 shows the binding of anti-CD25 H2L2 antibody to CHO-K1 cells overexpressing human CD25.
  • Figure 2 shows the binding of anti-CD25 H2L2 antibody to HEK293 cells overexpressing monkey CD25.
  • Figure 3 shows the binding of anti-CD25 H2L2 antibody to the tumor cell line SU-DHL-1 endogenously expressing human CD25.
  • Figure 4 shows the binding of anti-CD25 H2L2 antibody to the tumor cell line Karpas299 endogenously expressing human CD25.
  • Figure 5 shows the binding of anti-CD25 H2L2 antibody to human Treg cells and Teff cells.
  • Figure 6 shows that anti-CD25 H2L2 antibody does not block the binding of human interleukin-2 to CHOK1-huCD25 cells.
  • Figure 7 shows that anti-CD25 H2L2 antibody does not block the binding of human interleukin-2 to SU-DHL-1 cells.
  • Figure 8 shows that the anti-CD25 H2L2 antibody does not inhibit the phosphorylation level of human interleukin-2 signaling pathway downstream protein STAT5 detected by AlphsLisa.
  • Figure 9 is the FACS detection of the phosphorylation level of the human interleukin-2 signaling pathway downstream protein STAT5 detected by the anti-CD25 H2L2 antibody.
  • Figure 10 is the detection of ADCC effect of anti-CD25 H2L2 antibody on SU-DHL-1 cell line by reporter gene cell line.
  • Figure 11 is the detection of the ADCC effect of anti-CD25 H2L2 antibody on human Treg cells using a reporter gene cell line.
  • Figure 12 is the detection of the ADCC effect of anti-CD25 H2L2 antibody on human Teff cells using a reporter gene cell line.
  • Figure 13 shows the endocytosis of anti-CD25 H2L2 antibody in Karpas 299 cells.
  • Figure 14 shows the endocytosis of anti-CD25 H2L2 antibody by SU-DHL-1 cells.
  • the CD25 recombinant protein is used to immunize experimental animals to obtain antibody molecules specifically binding to CD25, and the experimental animals can be mice, rats, rabbits, sheep, camels, etc.
  • the resulting antibody molecules are non-human.
  • these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability.
  • the humanization process of antibodies has its own technical complexity, and molecules after humanization often reduce the affinity for antigens.
  • transgenic technology have made it possible to create genetically engineered mice that carry the human immunoglobulin immune repertoire and have their endogenous murine immune repertoire deleted.
  • Harbour Antibodies BV is a transgenic mouse carrying a human immunoglobulin immune library.
  • the antibody produced by this transgenic mouse has a fully human sequence, so no further humanization is required, greatly improving Improve the efficiency of therapeutic antibody development.
  • mice were immunized for multiple rounds with soluble recombinant human CD25-his fusion protein (Sino Biological, #10165-H08H) as antigen.
  • the antigenic protein is mixed with an immune adjuvant to form an immunogenic reagent, which is then injected subcutaneously through the groin or intraperitoneally.
  • each mouse received a total injection dose of 100 microliters.
  • each mouse was immunized with an immunogenic reagent prepared by mixing 50 micrograms of antigenic protein with complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1.
  • each mouse was immunized with 25 micrograms of antigenic protein mixed with Sigma Adjuvant System adjuvant (Sigma, #S6322).
  • the interval between each round of booster immunization is at least two weeks, usually 6 to 7 rounds of booster immunization.
  • the immunization time was the 0th, 14th, 28th, 42nd, 56th, 70th, 84th, and 98th day; and on the 49th and 77th day, the serum antibody titer of the mice was detected.
  • the mouse serum was collected, and the titer of the antibody binding to CD25 in the serum was detected by FACS method.
  • the cDNA encoding human CD25 (its Genebank number is NM000417) was obtained by gene synthesis and cloned into the expression vector pcDNA3.1, and then the expression vector was transfected into CHO cells to construct CHOK1-huCD25 cells that highly express human CD25 .
  • the serially diluted mouse serum was incubated with CHOK1-huCD25 cells at 4°C for 1 hour; after the cells were washed twice, the secondary antibody anti-rat IgG (H+L) (Life technologies, A11006) was added and incubated at 4°C for 1 hour, and then washed twice , resuspended cells were detected by flow cytometry (BD, CantoII). CHOK1 cells were used as background control.
  • the cDNA encoding cynomolgus monkey CD25 (its Genebank number is NM001283704) was obtained by gene synthesis and cloned into the expression vector pcDNA3.1, and then the expression vector was transfected into HEK293 cells to construct a high-expression cynomolgus monkey CD25 HEK293-cynoCD25 cells.
  • the FACS method was used to detect the blocking effect of positive clones on the combination of interleukin-2 and CHOK1-huCD25. Positive wells without blocking effect were further subcloned by limiting dilution method, and further screened by ELISA and FACS methods. Clones with better binding to human and monkey CD25 were selected for sequencing.
  • the sequences of the variable domains of anti-CD25 monoclonal antibody molecules obtained from immunized Harbor H2L2 mice are human antibody sequences.
  • VH antibody heavy chain variable domain sequence
  • the antibody light chain variable domain sequence (VL) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Ig kappa light chain constant domain sequence to encode the full-length light chain that produces the antibody.
  • VL antibody light chain variable domain sequence
  • HEK293 cells were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, the cell concentration was adjusted to 6-8 ⁇ 10 5 cells/ml, and cultured in a 8% CO2 shaker at 37°C for 24 hours, and the cell concentration was 1.2 ⁇ 10 6 cells/ml.
  • the antibody heavy chain variable domain sequence (VH) was synthesized through genes and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence, and S239D was introduced into the CH2 region of the IgG1 heavy chain constant region and I332E mutations (substitution of serine at position 239 to aspartic acid, and isoleucine at position 332 to glutamic acid according to EU numbering) to increase ADCC effector function. Then, the plasmids encoding the antibody heavy chain and antibody light chain were transfected into mammalian host cells using the method described in Example 1.4 to prepare recombinant antibody proteins.
  • the Fc conformation is changed by removing the fucose component on the antibody sugar chain (N297) to enhance the ADCC effector function.
  • the plasmid encoding the heavy chain of the IgG1 antibody and the plasmid encoding the light chain of the antibody were simultaneously transfected into mammalian host cells, and 2-fluoro peracetylated fucose (2FF) (Sigma-Aldrich, #344827 ), and then perform steps such as cell culture, collection, and purification according to the method described in Example 1.4.
  • 2FF 2-fluoro peracetylated fucose
  • the addition of compound 2FF can inhibit the activity of glycosidase and inhibit the production of fucose components on sugar chains.
  • the recombinant antibody produced in this example has the same amino acid sequence as the recombinant antibody produced in Example 1.4, but different sugar chain modifications.
  • this example uses "AF" as the suffix of the antibody sample name to indicate that the sample is a "low-fucose” or "fucose-free” sample.
  • PR004639 is a recombinant antibody containing a normal IgG1 constant region, and the corresponding antibody protein sample with a normal sugar chain structure was prepared using the method described in Example 1.4.
  • PR004639AF is an antibody protein sample with the same amino acid sequence as PR004639, which is obtained by using the method of this example to remove fucose modification.
  • the amino acid sequence of the same antibody can be prepared by the method described in Example 1.4 (number is PRxxxxxx, where xxxxxx is a number), or can be prepared by the method of this example (number is PRxxxxxxAF , where xxxxxx is a number), these samples have differences in sugar chain structure.
  • Table 2-1 lists the amino acid sequences of the light and heavy chain variable domains of the CD25 antibody in the application of the present invention, and the entire light chain Long amino acid sequence, heavy chain full-length amino acid sequence and amino acid sequence of CDRs defined according to Chothia definition rules.
  • control antibody used in various embodiments of the present application is RG6292, whose sequence is from the patent application US20190284287A1, and the corresponding recombinant IgG1 antibody number is PR004639.
  • the method described in Example 1.6 was used to prepare the fucose-deleted antibody protein sample PR004639AF, which is an analog of RG6292.
  • the sequence of PR004639 (PR004639AF, RG6292) is shown in Table 2-5.
  • control antibody used in the examples of the application of the present invention is daclizumab (trade name Zenapax, Roche), the number of the corresponding recombinant IgG1 antibody is PR003689, its heavy chain sequence is as shown in SEQ ID NO.217, and the light chain sequence is as shown in SEQ ID Shown in NO.218.
  • control antibody used in the examples of the present application is HuMax-Tac (Genmab Company), its sequence is from the patent application WO2014057119A1, and the number of the corresponding recombinant IgG1 antibody is PR007722.
  • the heavy chain variable domain sequence of the antibody is derived from the gene rearrangement and somatic high-frequency mutation of the germline gene V, D, and J gene fragments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement and somatic hypermutation of germline gene V and J gene fragments of the light chain gene group. Gene rearrangement and somatic hypermutation are the main factors that increase antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but the overall similarity is high.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • PTM post-translational modifications
  • some PTM positions are very conserved.
  • EU numbering the conserved amino acid asparagine Asn at position 297 (EU numbering) of the constant domain of human IgG1 antibodies usually undergoes glycosylation modification to form sugar chains, and the structure of the sugar chains is crucial for antibody structure and related effector functions.
  • PTMs in the variable domain of the antibody, especially in the antigen-binding region (such as CDR), the presence of these PTMs may have a greater impact on the binding of the antigen, and may also affect the physicochemical properties of the antibody. bring about change.
  • glycosylation, deamidation, isomerization, oxidation, etc. may increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development.
  • PTMs are very important for the development of therapeutic antibodies.
  • some PTMs are highly correlated with the composition of amino acid sequences, especially the "pattern" of adjacent amino acid compositions, so that potential PTMs can be predicted from the primary amino acid sequence of proteins.
  • the sequence pattern of NxS/T (the first position is asparagine, the second position is any amino acid except proline, and the third position is serine or threonine) predicts N-linked glycosylation sites.
  • the amino acid sequence pattern that causes PTM may be derived from the germline gene sequence, for example, the human germline gene fragment IGHV3-33 naturally has a glycosylation pattern NST in the FR3 region; it may also be derived from somatic hypermutation.
  • Table 2-2 lists the predicted PTMs of the variable domains VH and VL of the antibody of Example 1.
  • NSS or NLT may be a glycosylation site
  • NS or NT or NN may be a deamidation site.
  • Amino acid mutations can be used to disrupt the amino acid sequence pattern of PTMs, thereby reducing or eliminating the formation of specific PTMs.
  • mutation design methods One method is to replace the "hotspot" amino acid (such as N or S in the NS pattern) with an amino acid with similar physicochemical properties (such as mutating N to Q), or replace it with any other amino acid by saturation mutation .
  • the PTM sequence pattern is derived from somatic hypermutation and does not exist in the germline gene sequence, another method may be to replace the sequence pattern with the corresponding germline gene sequence.
  • multiple mutation design methods may be used for the same PTM sequence pattern.
  • Tables 2-3 list novel antibody molecules (called PTM variants) obtained by amino acid mutation of the sequence of the antibody with potential PTM sites from Example 1.
  • Table 2-4 lists the light and heavy chain variable domain amino acid sequences of these PTM variants in this embodiment, the full-length amino acid sequence of the light chain, the full-length amino acid sequence of the heavy chain (human IgG1), and according to Chothia definition rule Amino acid sequences of defined CDRs.
  • Purified recombinant antibodies were obtained from all designed PTM variants according to the method described in Example 1.4, and further verified in subsequent functional experiments.
  • Pre-expanded human Treg cells were prepared by isolating Treg cells from fresh human PBMCs (EasySep TM Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit, #18063), adding Treg expansion beads (Miltenyi Biotec Treg Expansion Kit, human#130-095-345) and IL-2 (peprotech, 200-02), placed in an incubator containing 5% CO 2 at 37° C. for 10 days, that is, pre-amplified human Treg cells.
  • T cells were isolated from fresh human PBMC (Miltenyi, #130096535), and added to a 6-well plate coated with OKT3 (Invitrogen, #16-0037-85, 2 ⁇ g/ml) at a density of 1 ⁇ 10 6 cells/ml, and anti-CD28 antibody (Invitrogen, #16-0289-85, 1 ⁇ g/ml) was added. After being placed in a cell culture incubator and cultured for 72 hours, after adding IL-2 (peprotech 200-02, 10ng/ml) and culturing for 72 hours, they were pre-activated Teff cells.
  • OKT3 Invitrogen, #16-0037-85, 2 ⁇ g/ml
  • anti-CD28 antibody Invitrogen, #16-0289-85, 1 ⁇ g/ml
  • This example is to study the activity of anti-human CD25 H2L2 monoclonal antibody binding to human/cynomolgus monkey CD25 in vitro.
  • the CHOK1 cell line overexpressing human CD25 (CHOK1-huCD25, Hebo Medicine)
  • the HEK293 cell line overexpressing cynomolgus CD25 (HEK293-cynoCD25, Hebo Medicine)
  • the cell line SU-DHL- 1 Karpas299 Najing Kebai CBP60271
  • pre-expanded human Treg cells pre-activated human Teff cells for antibody binding experiments at the cellular level.
  • CHOK1-huCD25 cells, HEK293-cynoCD25 cells were digested, SU-DHL-1 cells, Karpas299 cells, Treg and Teff cells were collected and resuspended in PBS containing 2% BSA. Adjust the cell density to 1 x 106 cells/mL, respectively. Inoculated with 100 ⁇ L cells/well Seed in a 96-well V-bottom plate (Corning, #3894), then add 100 ⁇ L/well, 5-fold concentration gradient dilution of 2 times the final concentration of the antibody to be tested and the positive control antibody RG6292. Place the cells at 4°C and incubate for 1 hour in the dark.
  • Anti-CD25 antibodies bind to human CD25 on the cell surface, cynomolgus monkey CD25, and CD25 expressed by tumor cells SU-DHL-1 and Karpas299
  • Anti-CD25 antibodies bind to human CD25 on the cell surface, cynomolgus monkey CD25, and CD25 expressed by tumor cells SU-DHL-1 and Karpas299
  • Table 3 The results of antibody binding to the tumor cell line SU-DHL-1 endogenously expressing human CD25 are shown in Figure 3 (A in Figure 3 - I in Figure 3), combined with Table 4-1, Table 4-2, and Table 4- 3.
  • Table 4-4 and Table 4-9 show that the initial antibodies PR005433, PR005436, PR005497, PR006110, PR006122, PR006927, PR006928, PR006930, PR006931, PR006932, and PR007181 have strong binding activity to SU-DHL-1;
  • Table 4-1 and Table 4-4 show that the binding activity of PR005435 and PR006984 is relatively weak.
  • This example is to study the blocking effect of anti-CD25 antibody on the binding between human interleukin-2 and CD25.
  • CHOK1 cell line overexpressing human CD25 CHOK1-huCD25, Hebo Medicine
  • cell line SU-DHL-1 overexpressing human CD25 with biotin-labeled human interleukin-2 Acro, IL2-H82F3
  • the blocking effect of the antibody was detected by FACS method. Briefly, digest CHOK1-huCD25 cells or collect SU-DHL-1 cells and resuspended in PBS containing 2% BSA. Adjust the cell density to 1 x 106 cells/mL, respectively.
  • FACS method was used to detect the blocking effect of antibodies on the combination of human interleukin-2 and CHOK1-huCD25 cells, as shown in Figure 6 (A- Figure 6C). Interleukin-2 binding to CD25 on the surface of CHOK1-huCD25 cells.
  • the initial antibodies PR006927, PR006930, PR006931 and PR006932 could not block the interaction between interleukin-2 and CD25 on the surface of SU-DHL-1 cells
  • the mutants PR006769, PR006771 and PR008197 obtained by PTM mutation of PR005436 could not block the binding of interleukin-2 to CD25 on the surface of SU-DHL-1 cells
  • the mutants PR008014 and PR008016 obtained by PTM mutation of PR006931 could not block interleukin-2 Binding to CD25 on the surface of SU-DHL-1 cells.
  • Teff cells were cultured with IL-2-free medium for 4 hours, the cells were collected to adjust the cell density to 2.5 ⁇ 10 7 cells/ml, and seeded in 384-well plates at 4 ⁇ L/well. Then add 2 ⁇ L/well of 4 times the final concentration of antibody and control antibody PR003689, and incubate in the incubator for 1 hour. Then add 2 ⁇ L/well of IL-2 which is 5 times the final concentration, and incubate in the incubator for 10 minutes. Immediately add 2 ⁇ L/well of 5 ⁇ Lysis Solution and incubate at room temperature for 15 minutes. Then add 5 ⁇ L/well of Acceptor mixture and incubate at room temperature for 1 hour, then add 5 ⁇ L/well of Donor mixture and incubate at room temperature for 1 hour. Signal values were read using PerkinElimer Envision.
  • the AlphaLisa method was used to detect the effect of the antibody on the phosphorylation level of the human interleukin-2 signaling pathway downstream protein STAT5. As shown in Figure 8, PR006122 had no inhibitory effect on the phosphorylation level of the human interleukin-2 signaling pathway downstream protein STAT5 in Teff.
  • Example 7 FACS detection of the effect of anti-CD25 antibody on the phosphorylation level of downstream signaling molecule STAT5 induced by human interleukin-2 in Teff
  • Teff cells were cultured with IL-2-free medium for 4 hours, the cells were collected and the cell density was adjusted to 2 ⁇ 10 6 cells/mL. Inoculate 96-well V-bottom plate (Corning, #3894) with 50 ⁇ L cells/well, then add 50 ⁇ L/well, 5-fold dilutions of the antibody to be tested and control antibody PR003689 or RG6292 at 2 times the final concentration, in the incubator Incubate for 1 hour. Then add 1 ⁇ L/well of 1 ⁇ g/ml IL-2 and incubate in the incubator for 10 minutes.
  • Example 8 Detection of ADCC effect of anti-CD25 antibody on tumor cell line SU-DHL-1 by reporter gene cell line
  • Collect SU-DHL-1 The cells were adjusted to 1.2 ⁇ 10 6 cells/mL, and seeded in 96-well plates (PerkinElmer, 6005225) at 25 ⁇ L/well.
  • the cDNAs encoding human CD16a (its Genebank number is NM000569.8) and human NFAT (its Genebank number is NM_145912.8) were obtained by gene synthesis, and were cloned into the expression vector pcDNA3.1, and then the expression vectors were transfected To Jurkat cells, construct a Jurkat/FC ⁇ RIII-NFAT stable cell line.
  • Example 9 Using a reporter gene cell line to detect the ADCC effect of anti-CD25 antibody on pre-amplified human Treg cells and pre-activated human Teff cells
  • the pre-amplified human Treg cells and pre-activated human Teff cells were collected, adjusted to 1.2 ⁇ 10 6 cells/mL, and seeded in a 96-well plate (PerkinElmer, 6005225) at 25 ⁇ L/well.
  • Collect Jurkat/FC ⁇ RIII-NFAT cells Hebo Medicine
  • 50 ⁇ L/well of antibody at 2 times the final concentration, and place in a 5% CO 2 incubator at 37°C for 6 hours to incubate.
  • PR005433, PR005436, PR005497 and PR006122 have a strong ADCC effect on human Treg cells, But it has no obvious effect on human Teff.
  • PR005435 has weak ADCC effect on human Treg.
  • Tumor cell line SU-DHL-1 highly expressing human CD25 and Karpas299 (Nanjing Kebai CBP60271), using FACS method to detect the internalization of antibodies.
  • the experimental method is as follows. SU-DHL-1 cells or Karpas299 cells were collected and resuspended with PBS containing 2% BSA. Adjust the cell density to 1 x 106 cells/mL, respectively. 100 ⁇ L cells/well were inoculated in a 96-well V-bottom plate (Corning, #3894), and then 100 ⁇ L/well, 2 times the final concentration of 5-fold dilutions of the antibody to be tested and the control antibody PR007722 were added.

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Abstract

本发明公开了一种靶向CD25的抗体或其变体,所述靶向CD25的单克隆抗体是一种天然产生的抗体，具有与人CD25和食蟹猴CD25结合的活性，经Fc改造后具有更强的ADCC效应，体外实验具有清除Treg和淋巴癌细胞作用，同时不阻断IL-2的信号通路。

Description

靶向CD25的抗体及其制备方法和应用
本申请要求申请日为2022/1/17的中国专利申请2022100512339的优先权。本申请引用上述中国专利申请的全文。
技术领域
本申请涉及生物医药领域，具体涉及一种靶向CD25的抗体或其变体，以及其制备方法和应用。
背景技术
调节性T细胞(Treg)是一类控制体内自身免疫反应性的T细胞亚群，在维持机体免疫耐受，自体免疫和抗肿瘤免疫中起到关键的作用。已有的研究发现，肿瘤浸润的Treg细胞增加与多种肿瘤的预后呈现负相关。另外，肿瘤微环境中效应T细胞(Teff)与Treg的比例的高低将影响肿瘤的进程和治疗效果。尽管这些研究提示针对Treg的抗肿瘤治疗方案具有潜在的价值，但在临床上靶向Treg的抗肿瘤疗法却鲜有成功的例子，主要原因是，可用于设计特异性靶向Treg的标志分子尚不明确。在小鼠实验中，抗CTLA-4的抗体表现出了清除肿瘤浸润Treg细胞的效果，这一效应是通过激活Fc受体(FcRs)实现的。然而，在临床的实验中关于抗CTLA-4抗体对Treg的清除作用尚不明确。一些研究发现增强抗CTLA-4抗体与FcRs的亲和力可以改善抗体临床实验的效果，但是并非所有CTLA-4抗体治疗都会导致Treg细胞的减少。因此，仍然需要开发靶向Treg的特异性标记分子抗体，成为实现靶向Treg抗肿瘤治疗。
CD25是白介素-2受体的α链(IL2Rα)，在Treg细胞中组成性的高表达，而在Teff细胞中CD25的表达量更低。这一特性使CD25成为能够特异性靶向Treg细胞的潜在靶点。然而由于缺乏对抗CD25抗体在小鼠及临床实验中的研究，使其发展受限。近期研究发现，在小鼠实验中，通过增加抗CD25抗体与FcRs的亲和力，从而增强抗体依赖的细胞介导的细胞毒性作用(ADCC)能够显著提高抗体的体内的抗肿瘤活性。另外，研究发现经过Fc优化增强ADCC功能的抗CD25抗体能够显著提高抗PD-1抗体在体内的抗肿瘤活性。另一方面，CD25在霍奇金淋巴癌和多种非霍奇金淋巴癌中均有表达，在成人T-细胞白血病/淋巴瘤和毛细胞白血病中CD25的表达率接近了100％。基于CD25在血液肿瘤中的高表达，以CD25为靶点的抗体偶联药物(ADC)的研究成为热点。已有的抗CD25 的ADC药物ADCT-301在霍奇金淋巴癌的临床研究中体现了较好的疗效。基于CD25在Treg和淋巴癌细胞中的高表达，对这一靶点抗体药物的开发可以通过Fc优化增强ADCC功能实现抗肿瘤效果，另一方面也可以通过ADC的方法进行开发。
抗CD25抗体药物的研发，早期都集中于对配体白介素-2的阻断性抗体，包括单抗和ADC。近期的研究发现，不阻断白介素-2的抗CD25抗体在小鼠实验中体现出了更好的抗肿瘤活性，这可能是由于CD25在效应T细胞(Teff)上也有表达，阻断性抗体抑制了白介素-2的信号通路，从而抑制了Teff的活性。而Teff是肿瘤免疫过程中发挥主要功能的细胞，阻断性抗体的这一特性减弱了其抗肿瘤的作用。而非阻断性的抗CD25抗体将避免这一问题，具有更显著的抗肿瘤活性。
在以抗CD25单抗为开发策略的领域，目前已有多家公司正在致力于开发抗CD25的白介素-2非阻断性抗体，进展最快的是罗氏的RG6292，处于临床1期的研究，适应症为实体瘤。在以ADC为开发策略的领域，同样也有多家公司正在研究，进展最快的是ADCT-301，目前处于临床2期，适应症为霍奇金淋巴癌。然而，临床上尚未有一种高选择性的抗CD25白介素-2非阻断性抗体。
发明内容
本发明提供一种高亲和力、高选择性，高生物活性的抗CD25白介素-2非阻断性抗体或其变体，且具备被细胞内吞的能力，适用于ADC药物开发的抗体分子。
本发明第一方面提供一种靶向CD25的抗体或其变体，其特征在于，所述抗体包括轻链可变区和重链可变区，其中，所述抗体选自以下组：
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:96、111和133所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:20、44和74所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:104、111、127所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:22、46、76所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、110、134所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、48、78所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:97、113和129所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:16、40 和70所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、116和135所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、47和77所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:95、111、127所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:14、38、68所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:98、114、130所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:17、41、71所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:99、115、131所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:18、42、72所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:100、113、132所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:14、43、73所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:103、110、134所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:21、45、75所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:105、111、136所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:18、42、80所示的HCDR1、HCDR2和HCDR3。
本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，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)(万维网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。在本发明中，所列CDR的氨基酸序列均是按照Chothia定义规则所示出的(本发 明的权利要求中也是按照Chothia定义规则所示出的序列)。
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位至第bb位的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位至第bb位的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。
本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应理解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明的权利要求中请求保护的范围是基于Chothia定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的CDR定义规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
在某一具体实施例中，所述变体为在靶向CD25的抗体的氨基酸序列上发生1～3个氨基酸残基的增加、缺失或替换。
优选地，所述变体为在所述抗体的重链可变区的HCDR1的第1位、HCDR2的第3位发生氨基酸残基的替换，和/或，在所述抗体的轻链可变区的LCDR1的第10-11位、LCDR3的第1或4位发生氨基酸残基的替换；
优选地，所述变体的氨基酸序列与原氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体与目标抗原的结合；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％、96％、97％或98％序列同一性；最优选为至少99％序列同一性。
更优选地，所述变体选自以下组：
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:101、113、129所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:19、40、70所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:101、113、139所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:19、40、70所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:102、113、129所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:19、40、70所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、116、137、所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、50、77所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、116、138所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、51、77所示的HCDR1、HCDR2和HCDR3；或，
所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、116、138所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、50、77所示的HCDR1、HCDR2和HCDR3。
在某一具体实施例中，
所述轻链可变区包含氨基酸序列如SEQ ID NO:171所示或与SEQ ID NO:171具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:153所示或与SEQ ID NO:153具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:173所示或与SEQ ID NO:173具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:155所示或与SEQ ID NO:155具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:175所示或与SEQ ID NO:175具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:157所示或与SEQ ID NO:157具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:165所示或与SEQ ID NO:165具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:148所示或与SEQ ID NO:148具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:174所示或与SEQ ID NO:174具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区 包含氨基酸序列如SEQ ID NO:156所示或与SEQ ID NO:156具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:163所示或与SEQ ID NO:163具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:146所示或与SEQ ID NO:146具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:166所示或与SEQ ID NO:166具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:149所示或与SEQ ID NO:149具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:167所示或与SEQ ID NO:167具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:150所示或与SEQ ID NO:150具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:168所示或与SEQ ID NO:168具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:151所示或与SEQ ID NO:151具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:172所示或与SEQ ID NO:172具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:154所示或与SEQ ID NO:154具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:177所示或与SEQ ID NO:177具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:159所示或与SEQ ID NO:159具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:169所示或与SEQ ID NO:169具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:152所示或与SEQ ID NO:152具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:180所示或与SEQ ID NO:180具有 至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:152所示或与SEQ ID NO:152具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:170所示或与SEQ ID NO:170具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:152所示或与SEQ ID NO:152具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:178所示或与SEQ ID NO:178具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:160所示或与SEQ ID NO:160具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:179所示或与SEQ ID NO:179具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:161所示或与SEQ ID NO:161具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
所述轻链可变区包含氨基酸序列如SEQ ID NO:179所示或与SEQ ID NO:179具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:160所示或与SEQ ID NO:160具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH。
在某一具体实施例中，所述抗体或其变体包括嵌合抗体、人源化抗体或全人源抗体。
优选地，所述抗体或其变体的结构为Fab、Fab’、F(ab’)2、Fv、IgG、Fd、dAb或包括所述抗体或其变体的双特异性抗体或多特异性抗体，或由上述抗体制得的单克隆抗体或多克隆抗体；所述Fv优选scFv；较佳地，具有所述IgG结构的抗体或其变体包括轻链和重链：
所述轻链包括如SEQ ID NO:207所示或与SEQ ID NO:207具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:189所示或与SEQ ID NO:189具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:209所示或与SEQ ID NO:209具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:191所示或与SEQ ID NO:191具有至少85％、90％、95％、96％、97％、98％或99％的氨 基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:211所示或与SEQ ID NO:211具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:193所示或与SEQ ID NO:193具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:201所示或与SEQ ID NO:201具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:184所示或与SEQ ID NO:184具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:210所示或与SEQ ID NO:210具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:192所示或与SEQ ID NO:192具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:199所示或与SEQ ID NO:199具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:182所示或与SEQ ID NO:182具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:202所示或与SEQ ID NO:202具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:185所示或与SEQ ID NO:185具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:203所示或与SEQ ID NO:203具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:186所示或与SEQ ID NO:186具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:204所示或与SEQ ID NO:204具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:187所示或与SEQ ID NO:187具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:208所示或与SEQ ID NO:208具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO: 190所示或与SEQ ID NO:190具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:213所示或与SEQ ID NO:213具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:195所示或与SEQ ID NO:195具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:205所示或与SEQ ID NO:205具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:188所示或与SEQ ID NO:188具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:216所示或与SEQ ID NO:216具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:188所示或与SEQ ID NO:188具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:206所示或与SEQ ID NO:206具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:188所示或与SEQ ID NO:188具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:214所示或与SEQ ID NO:214具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:196所示或与SEQ ID NO:196具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:215所示或与SEQ ID NO:215具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:197所示或与SEQ ID NO:197具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
所述轻链包括如SEQ ID NO:215所示的氨基酸序列；重链包括如SEQ ID NO:196所示的氨基酸序列。
本发明中，“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fc”区含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持 在一起。“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。“F(ab’)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。术语“Fv”意指向抗体的单臂的VL和VH结构域组成的抗体片段，但缺少恒定区。
本发明中，所述的scFv(single chain antibody fragment，单链抗体)可为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子[参见，例如，Bird等人，Science 242:423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)]。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的G4S氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(G4S)4或(G4S)3接头，但也可使用其变体。
本发明中，所述的Fd可为本领域常规的抗体片段，其包括Fab中除VL、CL部分，约含225个氨基酸残基，包括VH、CH1和部分绞链区。
本发明中，所述的dAb可为本领域常规：由VH或VL结构域组成，是一些最小功能性抗体片段，保留完整的抗原结合特异性。其分子量约为正常抗体分子量的十分之一。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双特异性抗体(diabodies)、和三抗体(triabodies)、共价或非共价连接在一起的抗体片段等。
本发明的抗体或其变体包括单克隆抗体。本发明所述的单克隆抗体或mAb或Ab，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。
本发明第二方面提供一种嵌合抗原受体，其包含本发明第一方面所述的抗体或其变体。
本发明第三方面提供一种分离的核酸，其编码如本发明第一方面所述的抗体或其变体、或如本发明第二方面所述的嵌合抗原受体。
本发明第四方面提供一种重组表达载体，其包含根据本发明第三方面所述的分离的 核酸；优选地，所述重组表达载体为质粒、粘粒、噬菌体或病毒载体，所述病毒载体优选逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
本发明第五方面提供一种转化体，其在宿主细胞中包含如本发明第四方面所述的重组表达载体；优选地，所述宿主细胞为原核细胞或真核细胞；更优选地，所述宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞；其中，所述哺乳动物细胞为，例如293细胞或CHO细胞。
本发明第六方面提供一种免疫细胞，其包含本发明第二方面所述的嵌合抗原受体；优选地，所述免疫细胞为T细胞或NK细胞；更优选地，所述NK细胞为外周血NK细胞、脐带血来源NK细胞、干细胞分化NK细胞或NK细胞系例如NK-92细胞系。
本发明第七方面提供一种靶向CD25的抗体或其变体的制备方法，其包含培养如本发明第五方面所述的转化体，从培养物中获得靶向CD25的抗体或其变体。
本发明第八方面提供一种抗体药物偶联物，其包含细胞毒性剂，以及如本发明第一方面所述的靶向CD25的抗体或其变体。
本发明第九方面提供一种药物组合物，其包含如本发明第一方面所述的靶向CD25的抗体或其变体、如本发明第三方面分离的核酸、如本发明第四方面的重组表达载体、如本发明第五方面的转化体、如本发明第六方面的免疫细胞和/或如本发明第八方面所述的抗体药物偶联物，以及药学上可接受的载体；
较佳地，所述药物组合物还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
在一些实施方案中，本发明的药物组合物或药物制剂包含合适的药学上可接受的载体例如药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。如本发明所用，“药学上可接受的载体”或“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。适用于本发明的药用载体可以是无菌液体，如水和油，包括那些石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或 pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准药用载体和/或赋形剂，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。可以通过将具有所需纯度的本发明的抗体或其抗原结合片段与一种或多种任选的药用辅料(Remington’s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合来制备包含本发明所述的药物制剂或药物组合物，优选地以冻干制剂或水溶液的形式。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症所需的，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抗感染活性成分，例如其它抗体、抗感染活性剂、小分子药物或免疫调节剂等。所述活性成分以对于目的用途有效的量合适地组合存在。可制备持续释放制剂。持续释放制剂的合适实例包括含有本发明的抗体或其抗原结合片段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。
如本发明第一方面任一项所述的靶向CD25的抗体或其变体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第六方面所述的免疫细胞、本发明第八方面所述的抗体药物偶联物和/或本发明第九方面所述的药物组合物在制备诊断、预防和/或治疗肿瘤的药物中的应用。
本发明第十方面提供一种试剂盒，其包括如本发明第一方面所述的靶向CD25的抗体或其变体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第六方面所述的免疫细胞、或如本发明第八方面中所述的抗体药物偶联物或如本发明第九方面所述的药物组合物；
较佳地，所述试剂盒还包括(i)施用抗体或其变体或嵌合抗原受体或免疫细胞或抗体药物偶联物或药物组合物的装置；和/或(ii)使用说明。
本发明第十一方面提供一种套装药盒，其包含药盒A和药盒B，其中：
所述药盒A含有如本发明第一方面所述的靶向CD25的抗体或其抗原结合片段、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第六方面所述的免疫细胞、如本发明第八方面所述的抗体药物偶联物和/或如本发明第九方面所述的药物组合物；
所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、 细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
本发明第十二方面提供一种检测CD25的方法，其包括使用如本发明第一方面所述的靶向CD25的抗体或其变体、如本发明第二方面所述的嵌合抗原受体、如本发明第六方面所述的免疫细胞、如本发明第八方面中所述的抗体药物偶联物和/或如本发明第九方面所述的药物组合物。
优选地，所述检测为非诊断和/或治疗目的。
本发明第十三方面提供一种预防、治疗或缓解受试者病症的方法，包含向所述受试者施用治疗有效量的如本发明第一方面任一项所述的靶向CD25的抗体或其变体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第六方面所述的免疫细胞、如本发明第八方面所述的抗体药物偶联物和/或如本发明第九方面所述的药物组合物。
一些具体实施方案中，所述受试者病症是增殖性疾病，例如肿瘤或癌症。一些实施方案中，上述受试者患有已形成的肿瘤，例如实体瘤。
一些实施方案中，提供减少受试者肿瘤内部或肿瘤侵润性Treg的细胞数量方法；一些实施方案中，提供消除或抑制受试者肿瘤内部或肿瘤侵润性的Treg的细胞活性的方法，均包括向所述受试者施用有效量的如本发明第一方面任一项所述的靶向CD25的抗体或其变体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第六方面所述的免疫细胞、本发明第八方面所述的抗体药物偶联物和/或如本发明第九方面所述的药物组合物。
一些实施方案中，提供增加受试者肿瘤内部Teff/Treg的比例的方法，包括向所述受试者施用如本发明第一方面任一项所述的靶向CD25的抗体或其变体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第六方面所述的免疫细胞、如本发明第八方面所述的抗体药物偶联物和/或如本发明第九方面所述的药物组合物。
一些实施方案中，提供增强受试者体内针对肿瘤细胞的ADCC的方法，包括向所述受试者施用如本发明第一方面任一项所述的靶向CD25的抗体或其变体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第六方面所述的免疫细胞、如本发明第八方面所述的抗体药物偶联物和/或如本发明第九方面所述的药物组合物。
一些具体实施方案中，受试者体内针对肿瘤细胞的ADCC效应增强。
一些实施方案中，提供如本发明第一方面任一项所述的靶向CD25的抗体或其变体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第六方面所述的免疫细胞、如本发明第八方面所述的抗体药物偶联物和/或如本发明第九方面所述的药物组合物用于制备预防、治疗或缓解受试者病症的药物的用途，用于制备减少受试者肿瘤内部或肿瘤侵润性Treg的细胞数量的药物的用途，用于制备消除或抑制受试者肿瘤内部或肿瘤侵润性的Treg的细胞活性的药物的用途，用于制备增加受试者肿瘤内部Teff/Treg比例的药物的用途，用于制备增强受试者体内针对肿瘤细胞的ADCC的药物的用途。
一些具体实施方案中，上述受试者的病症为增殖性病症(例如癌症或肿瘤)或患有增殖性病症(例如癌症或肿瘤)。所述肿瘤包括但不限于癌、淋巴瘤、白血病、胚细胞瘤和肉瘤。这类癌症的更具体的实例包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、神经胶质瘤、肝细胞癌(HCC)、霍奇金淋巴瘤、非霍奇金淋巴瘤、急性髓性白血病(AML)、多种骨髓瘤、胃肠(道)癌、肾癌、卵巢癌、肝脏癌、淋巴母细胞白血病、淋巴细胞白血病、结直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑色素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性胶质母细胞瘤、宫颈癌、脑癌、胃癌、膀胱癌、肝癌、乳腺癌、结肠癌和头颈癌。
一些具体实施方案中，所述癌症或肿瘤可以是实体瘤，包括但不限于肉瘤(包括由组织(例如松质骨、软骨、脂肪、肌肉、血管、造血细胞或纤维结缔组织)中的间充质来源的转化细胞产生的癌症)、癌(包括由上皮细胞产生的肿瘤)、间皮瘤、神经母细胞瘤、视网膜母细胞瘤等。涉及实体瘤的癌症包括但不限于，脑癌、肺癌、胃癌、十二指肠癌、食道癌、乳腺癌、结肠和直肠癌、肾癌、膀胱癌、肾脏癌、胰腺癌、前列腺癌、卵巢癌、黑色素瘤、口腔癌、肉瘤、眼癌、甲状腺癌、尿道癌、阴道癌、颈癌、淋巴瘤等。
一些具体实施方案中，所述癌症涉及表达CD25的肿瘤，包括但不限于淋巴瘤，例如霍奇金淋巴瘤和淋巴细胞性白血病，例如慢性淋巴细胞白血病(CLL)。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的靶向CD25的单克隆抗体是一种天然产生的抗体，具有与人CD25和食蟹 猴CD25结合的活性。经Fc改造后具有更强的ADCC效应，体外实验呈现很好的清除Treg和淋巴癌细胞作用；同时不阻断IL-2的信号通路，对Teff细胞的活性没有影响；而且具有良好的内化活性，是潜在的适用于ADC药物开发的抗体分子。
附图说明
图1为抗CD25 H2L2抗体结合到过表达人CD25的CHO-K1细胞。
图2为抗CD25 H2L2抗体结合到过表达猴CD25的HEK293细胞。
图3为抗CD25 H2L2抗体结合到内源性表达人CD25的肿瘤细胞系SU-DHL-1。
图4为抗CD25 H2L2抗体结合到内源性表达人CD25的肿瘤细胞系Karpas299。
图5为抗CD25 H2L2抗体结合人Treg细胞和Teff细胞。
图6为抗CD25 H2L2抗体不阻断人白介素-2与CHOK1-huCD25细胞的结合。
图7为抗CD25 H2L2抗体不阻断人白介素-2与SU-DHL-1细胞的结合。
图8为AlphsLisa检测抗CD25 H2L2抗体不抑制人白介素-2信号通路下游蛋白STAT5的磷酸化水平。
图9为FACS检测抗CD25 H2L2抗体不抑制人白介素-2信号通路下游蛋白STAT5的磷酸化水平。
图10为利用报告基因细胞系检测抗CD25 H2L2抗体对SU-DHL-1细胞系的ADCC作用。
图11为利用报告基因细胞系检测抗CD25 H2L2抗体对人Treg细胞的ADCC作用。
图12为利用报告基因细胞系检测抗CD25 H2L2抗体对人Teff细胞的ADCC作用。
图13为Karpas 299细胞对抗CD25 H2L2抗体的内吞作用。
图14为SU-DHL-1细胞对抗CD25 H2L2抗体的内吞作用。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1.抗CD25抗体分子的获得
利用CD25重组蛋白对实验动物进行免疫以获得针对CD25特异性结合的抗体分子，该实验动物可以是小鼠、大鼠、兔、羊、骆驼等。通常，其得到的抗体分子是非人源的。 在获得非人源抗体后，需要对这些分子利用抗体工程技术进行人源化改造，以降低免疫原性并提高成药性。然而，抗体的人源化过程有其技术复杂性，经过人源化改造的分子往往会降低对抗原的亲和力。另一方面，转基因技术的进步使得可以培育出基因工程化小鼠，其携带人免疫球蛋白免疫库并使其内源的鼠的免疫库缺失。Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，这种转基因小鼠产生的抗体具有全人源的序列，因而无需再进一步做人源化改造，大大提高了治疗性抗体开发的效率。
1.1.小鼠免疫
用可溶的重组人CD25-his融合蛋白(Sino Biological，#10165-H08H)作为抗原对Harbour H2L2小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂，然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中，每只小鼠接受的总注射剂量是100微升。在首轮免疫中，每只小鼠接受用50微克抗原蛋白与完全弗氏佐剂(Sigma,#F5881)以体积比1:1混合配制的免疫原试剂的免疫。在随后的每轮增强免疫中，每只小鼠接受用25微克抗原蛋白与Sigma Adjuvant System佐剂(Sigma,#S6322)混合配制的免疫原试剂的免疫。每轮增强免疫的间隔时间至少为两周，通常为6到7轮增强免疫。免疫时间为第0、14、28、42、56、70、84、98天；并且在第49、77天，检测小鼠血清抗体滴度。在进行H2L2小鼠脾B细胞分离前5天，以每只小鼠25微克抗原蛋白的剂量进行最后一次增强免疫。
1.2.血清滴度检测
在特定的时间点，收取小鼠血清，用FACS方法检测血清中抗体结合CD25的滴度。
将编码人CD25(其Genebank号为NM000417)的cDNA通过基因合成获得，并被克隆到表达载体pcDNA3.1中，然后将表达载体转染至CHO细胞中，构建高表达人CD25的CHOK1-huCD25细胞。
梯度稀释的鼠血清与CHOK1-huCD25细胞4℃孵育1小时；细胞洗2次后，加入二抗anti-rat IgG(H+L)(Life technologies,A11006)4℃孵育1小时，洗2次后，重悬细胞用流式细胞仪检测(BD,CantoII)。CHOK1细胞作为本底对照。
1.3.通过杂交瘤技术筛选抗CD25的抗体
将编码食蟹猴CD25(其Genebank号为NM001283704)的cDNA通过基因合成获得，并被克隆到表达载体pcDNA3.1中，然后将表达载体转染至HEK293细胞中，构建高表达食蟹猴CD25的HEK293-cynoCD25细胞。
选择血清效价高的免疫小鼠进行一次终免疫后处死小鼠，取脾细胞和SP2/0骨髓瘤 细胞(ATCC,CRL-1581)进行电融合，细胞比例为4:1，电融合参数为V1:50V,t1:15s,V2:600V,t2:20μs,t3:0.5s,n:1,t4:7s,V+/-:+,fade:on。细胞重悬于含20％FBS和HT的DMEM的培液中铺板1×105/100μL/孔，24小时后再加100μL/孔含20％FBS和2×HT的DMEM继续培养。后续取上清检测抗体滴度。一般在融合后9-15天，重组人CD25-his蛋白免疫的小鼠取上清用ELISA进行初筛，检测与重组人CD25-his蛋白的结合；阳性的克隆然后用FACS进一步确认，检测与过表达人CD25的CHOK1细胞株(CHOK1-huCD25)、过表达食蟹猴CD25的HEK293细胞株(HEK293-cynoCD25)的结合能力。另外用FACS方法检测阳性克隆对白介素-2与CHOK1-huCD25结合的阻断作用。没有阻断作用且阳性的孔用有限稀释法进一步亚克隆，再进一步用ELISA与FACS方法筛选。对人和猴CD25结合较好的克隆被挑选出来进行测序。在本实施例中，从免疫的Harbour H2L2小鼠得到的抗CD25单克隆抗体分子可变结构域的序列是人源抗体序列。
1.4.制备全人重组抗体
在得到编码抗体分子的轻、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻、重链可变结构域序列和相应的人的抗体轻、重链恒定结构域序列进行融合表达，得到重组抗体分子。在本实施例中，抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG1抗体重链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生IgG1抗体的全长重链。抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体Igκ轻链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。在本实施例中，由于从免疫的Harbour H2L2小鼠得到的抗CD25单克隆抗体分子可变结构域的序列是人源抗体序列，因而本实施例也得到全人源的抗CD25重组IgG1抗体。
将编码抗体重链的质粒和编码抗体轻链的质粒同时转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到具有轻重链正确配对组装的纯化的CD25重组抗体。具体说来，将HEK293细胞在FreeStyleTM F17 Expression Medium培养基(Thermo,#A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6～8×105细胞/ml，于37℃ 8％CO2摇床中培养24小时，细胞浓度在1.2×106细胞/ml。准备30ml培养的细胞。将上述编码抗体重链的质粒和编码抗体轻链的质粒以2:3的比例混合共计30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo,31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,Inc#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃ 8％CO2摇床中培养5天。5天后观测细胞活率。收集 培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS(pH7.4)平衡含有MabSelectTM(GE Healthcare Life Science,#71-5020-91AE)的重力柱(Bio-Rad,#7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,UFC901024)浓缩换液至PBS缓冲液，得到纯化的CD25抗体溶液。最后用NanoDrop(Thermo ScientificTM NanoDropTM One)测定浓度，分装、存储备用。
1.5通过Fc区突变来增强ADCC效应功能
将抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG1抗体重链恒定结构域序列的哺乳动物细胞表达质粒载体中，并且在该IgG1重链恒定区的CH2区引入S239D和I332E突变(根据EU编号，第239位丝氨酸取代成天冬氨酸，和第332位异亮氨酸取代成谷氨酸)以增加ADCC效应功能。然后利用实施例1.4中所述方法将编码抗体重链和抗体轻链的质粒转染哺乳动物宿主细胞来制备重组抗体蛋白。
1.6通过去除岩藻糖来增强ADCC效应功能
本实施例通过去除抗体糖链(N297)上的岩藻糖组分来改变Fc构象进而增强ADCC效应功能。然后利用实施例1.4中所述方法，将编码IgG1抗体重链的质粒和编码抗体轻链的质粒同时转染哺乳动物宿主细胞，并且加入2-fluoro peracetylated fucose(2FF)(Sigma-Aldrich，#344827)，然后按照实施例1.4中所述方法进行细胞培养、收集、纯化等步骤。化合物2FF的加入会抑制糖苷酶活性并抑制糖链上的岩藻糖组分的产生。
本实施例产生的重组抗体与实施例1.4中产生的重组抗体具有相同的氨基酸序列，但是不同的糖链修饰。为了区分两种方法产生的具有相同氨基酸序列的不同抗体蛋白样品，本实施例使用“AF”作为抗体样品名称后缀来说明该样品是“低岩藻糖”或“无岩藻糖”的样品。
例如，PR004639是含有正常IgG1恒定区的重组抗体，并用实施例1.4中所述方法制备相应的具有正常糖链结构的抗体蛋白样品。PR004639AF是具有和PR004639相同的氨基酸序列，利用本实施例方法得到的去除岩藻糖修饰的抗体蛋白样品。
在一些实施例中，同一个抗体的氨基酸序列既可以用实施例1.4中所述方法制备抗体样品(编号为PRxxxxxx，其中xxxxxx为数字)，也可以用本实施例方法制备抗体样品(编号为PRxxxxxxAF，其中xxxxxx为数字)，这些样品在糖链结构上有差异。
1.7抗CD25全人重组抗体的序列
表2-1列出了本发明申请中CD25抗体的轻、重链可变结构域氨基酸序列，轻链全 长氨基酸序列，重链全长氨基酸序列和根据Chothia定义规则定义的CDR的氨基酸序列。
本发明申请的多个实施例中使用的对照抗体为RG6292，其序列来自专利申请US20190284287A1，其相应的重组IgG1抗体的编号为PR004639。利用实施例1.6中所述方法制备去除岩藻糖的抗体蛋白样品PR004639AF，即RG6292的类似物。PR004639(PR004639AF，RG6292)的序列见表2-5。
本发明申请的实施例中使用的对照抗体为daclizumab(商品名Zenapax，Roche)，对应的重组IgG1抗体的编号为PR003689，其重链序列如SEQ ID NO.217所示，轻链序列如SEQ ID NO.218所示。
本发明申请的实施例中使用的对照抗体为HuMax-Tac(Genmab公司)，其序列来自专利申请WO2014057119A1，对应的重组IgG1抗体的编号为PR007722。
实施例2抗体的序列分析和序列优化
抗体的重链可变结构域序列来源于染色体上重链基因群的胚系基因V、D、J基因片段的基因重排和体细胞高频突变等事件；轻链可变结构域序列来源于轻链基因群的胚系基因V、J基因片段的基因重排和体细胞高频突变等事件。基因重排和体细胞高频突变是增加抗体多样性的主要因素。来源于相同胚系V基因片段的抗体也可能产生不同的序列，但总体上相似性较高。利用一些算法，例如IMGT/DomainGapAlign(http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)或者NCBI/IgBLAST(https://www.ncbi.nlm.nih.gov/igblast/)可以从抗体的可变结构域序列推测出其发生基因重排时可能的胚系基因片段。将实施例1和表2-1中的抗体序列进行分析，其重链可变结构域(VH)和轻链可变结构域(VL)的胚系基因V基因片段列于表2-2。
蛋白质或多肽氨基酸链在细胞中翻译合成后有时会引入化学修饰，称为翻译后修饰(PTM)。对于抗体而言，一些PTM的位点是非常保守的，例如，在人的IgG1抗体的恒定结构域的第297位(EU编号)的保守的氨基酸天冬酰胺Asn通常会发生糖基化修饰形成糖链，而该糖链结构对于抗体结构和相关的效应子功能是至关重要的。但是，如果在抗体的可变结构域，尤其是抗原结合区域(如CDR)中存在PTM，那么这些PTM的存在有可能会对抗原的结合有较大的影响，也可能对抗体的物理化学性质带来变化。例如，糖基化、脱酰胺、异构化、氧化等都可能增加抗体分子的不稳定性或异质性，从而增加抗体开发的难度和风险。因而避免一些潜在的PTM对于治疗性抗体的开发是非常重要的。随着经验的积累，人们发现一些PTM是和氨基酸序列的组成尤其是相邻氨基酸组成的“模式”是高度相关的，这样使得可以从蛋白质的一级氨基酸序列预测出潜在的PTM。 例如，N-x-S/T(第一位是天冬酰胺，第二位是非脯氨酸以外的任意氨基酸，第三位是丝氨酸或者苏氨酸)的序列模式预测出N-连接糖基化位点。引起PTM的氨基酸序列模式有可能来源于胚系基因序列，例如人胚系基因片段IGHV3-33天然地在FR3区域存在糖基化模式NST；也可能来源于体细胞高频突变。表2-2列出了实施例1的抗体的可变结构域VH和VL的预测的PTM。具体说来，NSS或NLT可能是糖基化位点，NS或NT或NN可能是脱酰胺位点。
可以通过氨基酸突变来破坏PTM的氨基酸序列模式，从而降低或者去除特定PTM的形成。根据抗体序列和PTM序列模式的不同，有不同的突变设计方法。一种方法是将“热点”氨基酸(如NS模式中的N或S)替换成物理化学性质相似的氨基酸(如把N突变为Q)，或者利用饱和突变的方法将其替换成其他任一氨基酸。如果PTM序列模式来源于体细胞高频突变，而并不存在于胚系基因序列中，那么另一种方法可以是把该序列模式替换成对应的胚系基因序列。实际操作中，对同一个PTM序列模式可能采用多种突变设计方法。
表2-3列出了对来自实施例1的具有潜在PTM位点的抗体的序列进行氨基酸突变得到的新的抗体分子(称为PTM变体)。表2-4列出了本实施例中这些PTM变体的轻、重链可变结构域氨基酸序列，轻链全长氨基酸序列，重链(人IgG1)全长氨基酸序列，和根据Chothia定义规则定义的CDR的氨基酸序列。所有设计出来的PTM变体按照实施例1.4中描述的方法得到纯化的重组抗体，并在后续的功能实验中进一步验证。
表2-1筛选得到的抗CD25杂交瘤单克隆及其重组抗体

表2-2 CD25抗体序列的胚系基因分析和翻译后修饰位点(PTM)分析
表2-3 CD25抗体序列的突变位点设计
表2-4突变PTM后得到的抗体
表2-5对照抗体
表2-6抗体的具体序列


实施例3.预扩增的人Treg细胞和预激活的人Teff细胞的制备
预扩增的人Treg细胞制备，从新鲜的人PBMC中分离得到Treg细胞(EasySepTM Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit,#18063)，加入Treg扩增微珠(Miltenyi Biotec Treg Expansion Kit,human#130-095-345)和IL-2(peprotech，200-02)，放置于37℃含5％CO2培养箱中孵育10天，即为预扩增的人Treg细胞。
预激活Teff的制备方法，从新鲜人PBMC中分离T细胞(Miltenyi,#130096535)，加入OKT3(Invitrogen,#16-0037-85，2μg/ml)包被的6孔板中，密度为1×106细胞/ml，再加入anti-CD28抗体(Invitrogen,#16-0289-85，1μg/ml)。放置于细胞培养箱中培养72小时后，加入IL-2(peprotech 200-02，10ng/ml)培养72小时后，即为预激活的Teff细胞。
实施例4.FACS检测抗CD25抗体结合CD25的能力
本实施例是为了研究抗人CD25的H2L2单抗体外结合人/食蟹猴CD25的活性。采用过表达人CD25的CHOK1细胞株(CHOK1-huCD25，和铂医药)、过表达食蟹猴CD25的HEK293细胞株(HEK293-cynoCD25，和铂医药)和高表达人CD25的细胞系SU-DHL-1Karpas299(南京科佰CBP60271)，预扩增的人Treg细胞，预激活的人Teff细胞进行细胞水平上的抗体结合实验。简言之，消化CHOK1-huCD25细胞、HEK293-cynoCD25细胞、收集SU-DHL-1细胞，Karpas299细胞，Treg和Teff细胞，并用含2％BSA的PBS重悬。将细胞密度分别调整为1×106细胞/mL。以100μL细胞/孔接 种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗体以及阳性对照抗体RG6292。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷含2％BSA的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson,#109-545-098,1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷含2％BSA的PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷含2％BSA的PBS重悬细胞，使用BD CantoII流式细胞仪读取荧光发光信号值。
抗体结合细胞表面的人CD25、食蟹猴CD25，以及结合肿瘤细胞SU-DHL-1和Karpas表面的CD25的汇总如下(表4-1、表4-2、表4-3、表4-4、表4-5、表4-6、表4-7、表4-8、表4-9)。
表4-1抗CD25抗体结合细胞表面的人CD25、食蟹猴CD25以及结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
表4-2抗CD25抗体结合细胞表面的人CD25、食蟹猴CD25以及结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
表4-3抗CD25抗体结合细胞表面食蟹猴CD25以及结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
表4-4抗CD25抗体结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
表4-5抗CD25抗体结合细胞表面食蟹猴CD25以及结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
表4-6抗CD25抗体结合细胞表面食蟹猴CD25以及结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
表4-7抗CD25抗体结合细胞表面食蟹猴CD25以及结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
表4-8抗CD25抗体结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
表4-9抗CD25抗体结合肿瘤细胞SU-DHL-1和Karpas299表达的CD25
抗体结合到过表达人CD25的CHO-K1细胞的结果如图1(图1的A和图1的B)、表4-1和表4-2所示，PR005433、PR005435、PR005436及PR005497与对照抗体相比有更强的结合活性。抗体结合到过表达猴CD25的HEK293细胞的结果如图2(图2的A- 图2的F)所示，结合表4-1、表4-2和表4-3，可知：初始抗体PR005433、PR005435、PR005436、PR005497、PR006122、PR006927、PR006930、PR006931及PR006932有较好的食蟹猴CD25交叉结合活性；结合表4-5和表4-7可知：PR005436经过PTM突变得到的突变体PR006769、PR006771及PR008197有较好的食蟹猴CD25交叉结合活性；结合表4-6可知：PR006931经过PTM突变得到的突变体PR008014、及PR008016有较好的食蟹猴CD25交叉结合活性。
抗体结合到内源性表达人CD25的肿瘤细胞系SU-DHL-1结果如图3(图3的A-图3的I)所示，结合表4-1、表4-2、表4-3、表4-4和表4-9可知：初始抗体PR005433、PR005436、PR005497、PR006110、PR006122、PR006927、PR006928、PR006930、PR006931、PR006932、PR007181对SU-DHL-1有较强的结合活性；结合表4-1和表4-4可知：PR005435和PR006984结合活性较弱。结合表4-5和表4-7可知：PR005436经过PTM突变得到的突变体PR006769、PR006771及PR008197对SU-DHL-1有较强的结合活性；结合表4-6和表4-8可知：PR006931经过PTM突变得到的突变体PR008014、PR008015及PR008016对SU-DHL-1有较强的结合活性。
抗体结合内源性表达人CD25的肿瘤细胞系Karpas299结果如图4(图4的A-图4的I)所示，结合表4-1、表4-2、表4-3、表4-4和表4-9可知：初始抗体PR005433、PR005497、PR006110、PR006122、PR006927、PR006928、PR006930、PR006932、PR007181对Karpas299有较强的结合活性；结合表4-1、表4-3和表4-4可知：PR005435、PR005436、PR006931和PR006984结合活性较弱。结合表4-5可知：PR005436经过PTM突变得到的突变体PR006769、PR006771对Karpas299的结合活性与对照抗体RG6292的结合活性相当；而根据表4-7可知：PR008197结合活性相较之下稍弱。结合表4-6和4-8可知：PR006931经过PTM突变得到的突变体PR008015对Karpas299有较强的结合活性；而PR008014和PR008016结合活性稍弱。
抗体结合人Treg细胞和Teff细胞的结果如图5(图5的A-图5的F)所示，PR005436、PR008014、PR008016、PR008197、PR006927能与人Treg结合，对人Teff的结合强度较弱。
实施例5.FACS检测抗CD25抗体对人白介素-2和CD25之间结合的阻断作用
本实例是为了研究抗CD25抗体对人白介素-2和CD25之间结合的阻断作用。采用过表达人CD25的CHOK1细胞株(CHOK1-huCD25，和铂医药)或高表达人CD25的细胞系SU-DHL-1与生物素标记的人白介素-2(Acro，IL2-H82F3)通 过FACS的方法检测抗体的阻断作用。简言之，消化CHOK1-huCD25细胞或收集SU-DHL-1细胞，并用含2％BSA的PBS重悬。将细胞密度分别调整为1×106细胞/mL。以90μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗体以及对照抗体RG6292或PR003689，再加入10μL/孔含生物素标记的人白介素-2浓度为10μg/ml的PBS。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷含2％BSA的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(eBioscience#12-4317-87，1:200稀释)，4℃，避光孵育1小时。用100μL/孔预冷含2％BSA的PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷含2％BSA的PBS重悬细胞，使用BD CantoII流式细胞仪读取荧光发光信号值
利用FACS方法检测抗体对人白介素-2与CHOK1-huCD25细胞的结合的阻断作用结果如图6(图6的A-图6的C)所示，PR005433、PR005435、PR005436、PR005497和PR006122不能阻断白介素-2与CHOK1-huCD25细胞表面CD25的结合。
利用FACS方法检测抗体对人白介素-2与SU-DHL-1细胞的结合的阻断作用结果如图7(图7的A-图7的D)所示，初始抗体PR006927、PR006930、PR006931和PR006932不能阻断白介素-2与SU-DHL-1细胞表面CD25的结合，PR005436经过PTM突变得到的突变体PR006769、PR006771及PR008197不能阻断白介素-2与SU-DHL-1细胞表面CD25的结合，PR006931经过PTM突变得到的突变体PR008014及PR008016不能阻断白介素-2与SU-DHL-1细胞表面CD25的结合。
实施例6.AlphaLisa(PerkinElimer，#ALSU-PST5-B500)检测抗CD25抗体对Teff中人白介素-2诱导的下游信号分子STAT5的磷酸化水平的影响
预激活的Teff细胞用不含IL-2的培养基培养4小时后，收集细胞将细胞密度调整为2.5×107细胞/ml，以4μL/孔接种于384孔板中。再加入2μL/孔4倍于终浓度的抗体以及对照抗体PR003689，在培养箱中孵育1小时。然后加入2μL/孔5倍于终浓度的IL-2，在培养箱中孵育10分钟。立即加入2μL/孔的5×裂解液，室温孵育15分钟。然后加入5μL/孔Acceptor混合液室温孵育1小时，再加入5μL/孔Donor混合液室温孵育1小时。使用PerkinElimer Envision读取信号值。
利用AlphaLisa的方法检测抗体对人白介素-2信号通路下游蛋白STAT5的磷酸化水平的影响结果如图8所示，PR006122对Teff中人白介素-2信号通路下游蛋白STAT5的磷酸化水平没有抑制作用。
实施例7.FACS检测抗CD25抗体对Teff中人白介素-2诱导的下游信号分子STAT5的磷酸化水平的影响
预激活的Teff细胞用不含IL-2的培养基培养4小时后，收集细胞将细胞密度调整为2×106细胞/mL。以50μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入50μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗体以及对照抗体PR003689或RG6292，在培养箱中孵育1小时。再加入1μL/孔1μg/ml的IL-2，在培养箱中孵育10分钟。立即加入100μL/孔固定液(Biolegend，#420801)，37℃孵育15分钟后，于500g、4℃下离心5分钟，弃上清。加入100μL/孔预冷含2％BSA的PBS漂洗细胞，再于500g、4℃下离心5分钟，弃上清。然后加入100μL/孔穿膜缓冲液重悬细胞(Biolegend，#425401)，放置于-20℃冰箱孵育过夜。第二天取出于1000g、4℃下离心5分钟，弃上清，加入100μL/孔预冷含2％BSA的PBS漂洗细胞两次，再于500g、4℃下离心5分钟，弃上清。加入100μL/孔抗磷酸化STAT5的抗体(Biolegend，#936904)，于4℃冰箱孵育1小时。加入100μL/孔预冷含2％BSA的PBS漂洗细胞两次，再于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷含2％BSA的PBS重悬细胞，使用BD CantoII流式细胞仪读取荧光发光信号值。
结果如图9所示，PR008014、PR008016、PR008197和PR006927对Teff中人白介素-2信号通路下游蛋白STAT5的磷酸化水平没有抑制作用。
实施例8.利用报告基因细胞系检测抗CD25抗体对肿瘤细胞系SU-DHL-1的ADCC作用
收集SU-DHL-1细胞并调整到1.2×106细胞/mL，以25μL/孔接种于96孔板中(PerkinElmer，6005225)。将编码人CD16a(其Genebank号为NM000569.8)和编码人NFAT(其Genebank号为NM_145912.8)的cDNA通过基因合成获得，并被克隆到表达载体pcDNA3.1中，然后将表达载体转染至Jurkat细胞中，构建Jurkat/FCγRIII-NFAT稳定细胞株。
收集Jurkat/FCγRIII-NFAT细胞(和铂医药)并调整到1.2×106细胞/mL，以25μL/孔接种于同一块96孔板中。然后加入50μL/孔2倍于终浓度的抗体以及对照抗体RG6292，放置于37℃含5％CO2培养箱中孵育6小时。加入60μL/孔One-Glo(Promega，#E6120)，于室温避光处放置10分钟。使用PerkinElmer Envision读取信号值。
抗体对SU-DHL-1的ADCC结果汇总如下(表8-1、表8-2)。
表8-1利用报告基因细胞系检测抗CD25 H2L2抗体对SU-DHL-1细胞系的ADCC作用
表8-2利用报告基因细胞系检测抗CD25 H2L2抗体对SU-DHL-1细胞系的ADCC作用
结果如图10(图10的A和图10的B)所示，PR006927AF、PR006931AF、PR006769AF、PR006771AF对SU-DHL-1有强ADCC作用。
实施例9.利用报告基因细胞系检测抗CD25抗体对预扩增人Treg细胞和预激活的人Teff细胞的ADCC作用
收集预扩增的人Treg细胞和预激活的人Teff细胞，并调整到1.2×106细胞/mL，以25μL/孔接种于96孔板中(PerkinElmer，6005225)。收集Jurkat/FCγRIII-NFAT细胞(和铂医药)并调整到1.2×106细胞/mL，以25μL/孔接种于同一块96孔板中。然后加入50μL/孔2倍于终浓度的抗体，放置于37℃含5％CO2培养箱中孵育6小时。加入60μL/孔One-Glo(Promega，#E6120)，于室温避光处放置10分钟。使用PerkinElmer Envision读取信号值。
抗体对Treg和Teff的ADCC结果汇总如下(表9-1、表9-2、表9-3)。
表9-1利用报告基因细胞系检测抗CD25 H2L2抗体对Treg细胞和Teff细胞的ADCC作用
表9-2利用报告基因细胞系检测抗CD25 H2L2抗体对Treg细胞和Teff细胞的ADCC作用
表9-3利用报告基因细胞系检测抗CD25 H2L2抗体对Treg细胞和Teff细胞的ADCC作用
结果如图11(图11的A-图11的C)和图12(图12的A-图12的C)所示，PR005433、PR005436、PR005497和PR006122对人Treg细胞有较强的ADCC作用，而对人Teff没有明显作用。PR005435对人Treg的ADCC作用较弱。
实施例10.抗体内化实验
本实例是为了研究抗体的内化作用。采用高表达人CD25的肿瘤细胞株SU-DHL-1和Karpas299(南京科佰CBP60271)，利用FACS方法检测抗体的内化作用。实验方法如下，收集SU-DHL-1细胞或Karpas299细胞，并用含2％BSA的PBS重悬。将细胞密度分别调整为1×106细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗体以及对照抗体PR007722。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷含2％BSA的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。用100μL2％BSA的PBS重悬细胞并放置于37℃含5％CO2的培养箱中孵育4小时。之后，加入100μL/孔预冷含2％BSA的PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG，FcγFragment Specific，Jackson，#109-545-098，1:1000稀释)，4℃，避光孵育1小时。用100μL/孔预冷含2％BSA的PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷含2％BSA的PBS重悬细胞，使用BD CantoII流式细胞仪读取荧光发光信号值。
结果如图13和图14所示，人肿瘤细胞系Karpas299对PR005433、PR005497、PR006122、PR006930、PR006932有明显内吞作用。

Claims (18)

  1. 一种靶向CD25的抗体或其变体，其特征在于，所述抗体包括轻链可变区和重链可变区，其中，所述抗体选自以下组：
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:96、111和133所示的LCDR1、LCDR2和LCDR3；和/或，所述重链可变区包含氨基酸序列分别如SEQ ID NO:20、44和74所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:104、111、127所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:22、46、76所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、110、134所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、48、78所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:97、113和129所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:16、40和70所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、116和135所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、47和77所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:95、111、127所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:14、38、68所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:98、114、130所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:17、41、71所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:99、115、131所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:18、42、72所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:100、113、132所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:14、43、73所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:103、110、134所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:21、45、75所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:105、111、136所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:18、42、80所示的HCDR1、HCDR2和HCDR3。
  2. 如权利要求1所述的靶向CD25的抗体或其变体，其特征在于，所述变体为在靶向CD25的抗体的氨基酸序列上发生1～3个氨基酸残基的增加、缺失或替换；优选地，所述变体为在所述抗体的重链可变区的HCDR1的第1位、HCDR2的第3位发生氨基酸残基的替换，和/或，在所述抗体的轻链可变区的LCDR1的第10-11位、LCDR3的第1或4位发生氨基酸残基的替换；更优选地，所述变体选自以下组：
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:101、113、129所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:19、40、70所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:101、113、139所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:19、40、70所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:102、113、129所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:19、40、70所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、116、137、所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、50、77所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、116、138所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、51、77所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO:94、116、138所示的LCDR1、LCDR2和LCDR3；和/或所述重链可变区包含氨基酸序列分别如SEQ ID NO:23、50、77所示的HCDR1、HCDR2和HCDR3。
  3. 如权利要求1或2所述的抗体或其变体，其特征在于，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:171所示或与SEQ ID NO:171具有 至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:153所示或与SEQ ID NO:153具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:173所示或与SEQ ID NO:173具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:155所示或与SEQ ID NO:155具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:175所示或与SEQ ID NO:175具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:157所示或与SEQ ID NO:157具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:165所示或与SEQ ID NO:165具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:148所示或与SEQ ID NO:148具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:174所示或与SEQ ID NO:174具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:156所示或与SEQ ID NO:156具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:163所示或与SEQ ID NO:163具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:146所示或与SEQ ID NO:146具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:166所示或与SEQ ID NO:166具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:149所示或与SEQ ID NO:149具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:167所示或与SEQ ID NO:167具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:150所示或与SEQ ID NO:150具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:168所示或与SEQ ID NO:168具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:151所示或与SEQ ID NO:151具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:172所示或与SEQ ID NO:172具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:154所示或与SEQ ID NO:154具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:177所示或与SEQ ID NO:177具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:159所示或与SEQ ID NO:159具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:169所示或与SEQ ID NO:169具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:152所示或与SEQ ID NO:152具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:180所示或与SEQ ID NO:180具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:152所示或与SEQ ID NO:152具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:170所示或与SEQ ID NO:170具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:152所示或与SEQ ID NO:152具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:178所示或与SEQ ID NO:178具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:160所示或与SEQ ID NO:160具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:179所示或与SEQ ID NO:179具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:161所示或与SEQ ID NO:161具有至少85％、90％、95％、 96％、97％、98％或99％的氨基酸同一性的VH；或，
    所述轻链可变区包含氨基酸序列如SEQ ID NO:179所示或与SEQ ID NO:179具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VL；所述重链可变区包含氨基酸序列如SEQ ID NO:160所示或与SEQ ID NO:160具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的VH。
  4. 如权利要求1-3任一所述的抗体或其变体，其特征在于，所述抗体选自下组：嵌合抗体、人源化抗体和全人源抗体；优选地，
    所述抗体或其变体的结构为Fab、Fab’、F(ab’)2、Fv、IgG、Fd、dAb或包括所述抗体或其变体的双特异性抗体或多特异性抗体，或由上述抗体制得的单克隆抗体或多克隆抗体；所述Fv优选scFv；
    较佳地，具有所述IgG结构的抗体或其变体包括轻链和重链：
    所述轻链包括如SEQ ID NO:207所示或与SEQ ID NO:207具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:189所示或与SEQ ID NO:189具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:209所示或与SEQ ID NO:209具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:191所示或与SEQ ID NO:191具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:211所示或与SEQ ID NO:211具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:193所示或与SEQ ID NO:193具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:201所示或与SEQ ID NO:201具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:184所示或与SEQ ID NO:184具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:210所示或与SEQ ID NO:210具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:192所示或与SEQ ID NO:192具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:199所示或与SEQ ID NO:199具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:182所示或与SEQ ID NO:182具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:202所示或与SEQ ID NO:202具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:185所示或与SEQ ID NO:185具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:203所示或与SEQ ID NO:203具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:186所示或与SEQ ID NO:186具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:204所示或与SEQ ID NO:204具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:187所示或与SEQ ID NO:187具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:208所示或与SEQ ID NO:208具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:190所示或与SEQ ID NO:190具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:213所示或与SEQ ID NO:213具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:195所示或与SEQ ID NO:195具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:205所示或与SEQ ID NO:205具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:188所示或与SEQ ID NO:188具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:216所示或与SEQ ID NO:216具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:188所示或与SEQ ID NO:188具有至少85％、90％、95％、96％、97％、98％或99％的氨 基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:206所示或与SEQ ID NO:206具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:188所示或与SEQ ID NO:188具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:214所示或与SEQ ID NO:214具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:196所示或与SEQ ID NO:196具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:215所示或与SEQ ID NO:215具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:197所示或与SEQ ID NO:197具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；或，
    所述轻链包括如SEQ ID NO:215所示或与SEQ ID NO:215具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列；重链包括如SEQ ID NO:196所示或与SEQ ID NO:196具有至少85％、90％、95％、96％、97％、98％或99％的氨基酸同一性的氨基酸序列。
  5. 一种嵌合抗原受体，其包含权利要求1-4中任一项所述的抗体或其变体。
  6. 一种分离的核酸，其编码如权利要求1-4中任一项所述的抗体或其变体、或如权利要求5所述的嵌合抗原受体。
  7. 一种重组表达载体，其包含根据权利要求6所述的分离的核酸；优选地，所述重组表达载体为质粒、粘粒、噬菌体或病毒载体，所述病毒载体优选逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
  8. 一种转化体，其在宿主细胞中包含如权利要求7所述的重组表达载体；优选地，所述宿主细胞为原核细胞或真核细胞；更优选地，所述宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞；其中，所述哺乳动物细胞为，例如293细胞或CHO细胞。
  9. 一种免疫细胞，其包含权利要求5所述的嵌合抗原受体；优选地，所述免疫细胞为T细胞或NK细胞；更优选地，所述NK细胞为外周血NK细胞、脐带血来源NK细胞、干细胞分化NK细胞或NK细胞系例如NK-92细胞系。
  10. 一种靶向CD25的抗体或其变体的制备方法，其包含培养如权利要求8所述的转 化体，从培养物中获得靶向CD25的抗体或其变体。
  11. 一种抗体药物偶联物，其包含细胞毒性剂，以及如权利要求1～4任一项所述的靶向CD25的抗体或其变体。
  12. 一种药物组合物，其包含如权利要求1～4任一项所述的靶向CD25的抗体或其变体、如权利要求6所述的分离的核酸、如权利要求7所述的重组表达载体、如权利要求8所述的转化体、如权利要求9所述的免疫细胞和/或如权利要求11所述的抗体药物偶联物，以及药学上可接受的载体；
    较佳地，所述药物组合物还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  13. 如权利要求1～4任一项所述的靶向CD25的抗体或其变体、如权利要求5所述的嵌合抗原受体、如权利要求6所述的分离的核酸、如权利要求7所述的重组表达载体、如权利要求8所述的转化体、如权利要求9所述的免疫细胞、如权利要求11所述的抗体药物偶联物和/或如权利要求12所述的药物组合物在制备诊断、预防和/或治疗肿瘤的药物中的应用。
  14. 一种试剂盒，其包括如权利要求1～4任一项所述的靶向CD25的抗体或其变体、如权利要求5所述的嵌合抗原受体、如权利要求6所述的分离的核酸、如权利要求7所述的重组表达载体、如权利要求8所述的转化体、如权利要求9所述的免疫细胞、或如权利要求11中所述的抗体药物偶联物或如权利要求12所述的药物组合物；
    较佳地，所述试剂盒还包括(i)施用抗体或其变体或嵌合抗原受体或免疫细胞或抗体药物偶联物或药物组合物的装置；和/或(ii)使用说明。
  15. 一种套装药盒，其包含药盒A和药盒B，其中：
    所述药盒A含有如权利要求1～4任一项所述的靶向CD25的抗体或其抗原结合片段、如权利要求5所述的嵌合抗原受体、如权利要求6所述的分离的核酸、如权利要求7所述的重组表达载体、如权利要求8所述的转化体、如权利要求9所述的免疫细胞、如权利要求11所述的抗体药物偶联物和/或如权利要求12所述的药物组合物；
    所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  16. 一种检测CD25的方法，其包括使用如权利要求1-4中任一项所述的抗体或其变 体、如权利要求5所述的嵌合抗原受体、如权利要求9所述的免疫细胞、如权利要求11所述的抗体药物偶联物和/或如权利要求12所述的药物组合物；优选地，所述检测为非诊断和/或治疗目的。
  17. 一种预防、治疗或缓解患有癌症的受试者的方法，其包括向受试者施用治疗有效量的如权利要求1～4任一项所述的靶向CD25的抗体或其变体、如权利要求5所述的嵌合抗原受体、如权利要求6所述的分离的核酸、如权利要求7所述的重组表达载体、如权利要求8所述的转化体、如权利要求9所述的免疫细胞、如权利要求11所述的抗体药物偶联物和/或如权利要求12所述的药物组合物。
  18. 一种减少受试者肿瘤内部或肿瘤侵润性Treg的细胞数量方法，其包括向所述受试者施用有效量的如权利要求1～4任一项所述的靶向CD25的抗体或其变体、如权利要求5所述的嵌合抗原受体、如权利要求6所述的分离的核酸、如权利要求7所述的重组表达载体、如权利要求8所述的转化体、如权利要求9所述的免疫细胞、如权利要求11所述的抗体药物偶联物和/或如权利要求12所述的药物组合物。
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