WO2021139777A1 - 抗tigit抗体和使用方法 - Google Patents

抗tigit抗体和使用方法 Download PDF

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Publication number
WO2021139777A1
WO2021139777A1 PCT/CN2021/070903 CN2021070903W WO2021139777A1 WO 2021139777 A1 WO2021139777 A1 WO 2021139777A1 CN 2021070903 W CN2021070903 W CN 2021070903W WO 2021139777 A1 WO2021139777 A1 WO 2021139777A1
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heavy chain
variable region
chain variable
seq
amino acid
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PCT/CN2021/070903
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English (en)
French (fr)
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杨明
许文峰
姜伟东
薛杰
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上海复宏汉霖生物技术股份有限公司
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Priority to JP2022542008A priority Critical patent/JP2023509196A/ja
Priority to CA3164281A priority patent/CA3164281A1/en
Priority to EP21738755.4A priority patent/EP4089115A4/en
Priority to BR112022013545A priority patent/BR112022013545A2/pt
Priority to CN202180008448.6A priority patent/CN115052893A/zh
Priority to AU2021206420A priority patent/AU2021206420A1/en
Priority to KR1020227027301A priority patent/KR20220131258A/ko
Publication of WO2021139777A1 publication Critical patent/WO2021139777A1/zh
Priority to US17/811,455 priority patent/US20230134580A1/en
Priority to ZA2022/08807A priority patent/ZA202208807B/en

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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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Definitions

  • the present invention relates to antibodies that bind to T cell immune receptors (TIGIT) with Ig and ITIM domains, and these antibodies include multispecific anti-TIGIT antibodies with binding specificities for TIGIT and one or more additional antigens; and How to use it.
  • TIGIT T cell immune receptors
  • TIGIT T cell immune receptor
  • Ig and ITIM domains are immune checkpoint receptor expressed by immune cells, such as activated T cells and natural killer cells (NK cells), and mediates immune suppression.
  • TIGIT ligands include PVR (CD155), which has been identified in dendritic cells (DC), macrophages, and many human cancer cells, and has been shown to be combined with TIGIT to down-regulate T cell activation and cytokine secretion.
  • the inhibition of TIGIT/PVR interaction can mediate the effective anti-tumor activity of immune cells.
  • the present disclosure provides isolated monoclonal antibodies that specifically bind TIGIT with high affinity, including multispecific antibodies that bind TIGIT and one or more additional targets.
  • the anti-TIGIT antibody comprises a single domain antibody that binds to TIGIT.
  • the present disclosure further provides methods for preparing antibodies, immunoconjugates, and pharmaceutical compositions containing these antibodies, and the use of antibodies, immunoconjugates, and pharmaceutical compositions containing these antibodies, for example, for the treatment of diseases and disorders (e.g., cancer) Methods.
  • the present invention is based in part on the discovery of single domain anti-TIGIT antibodies that bind to TIGIT, which can increase the immune response in immune cells and provide improved anti-tumor efficacy.
  • the anti-TIGIT antibody comprises a single domain antibody that binds to TIGIT.
  • single domain antibodies bind to TIGIT with a KD of about 1 ⁇ 10 -7 M or less.
  • single domain antibodies bind to TIGIT with a KD of about 1 ⁇ 10 -8 M or less.
  • the single domain antibody binds to TIGIT with a KD of about 5 ⁇ 10 ⁇ 9 M or less.
  • single domain antibodies bind TIGIT with a KD of about 2 ⁇ 10 ⁇ 9 M or less.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 94 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 95; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 96.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 98 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 99; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 100.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 102 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 103; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 104.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 106 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 107; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 108.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 110 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 111; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 112.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 114 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 115; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 116.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 118 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 119; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 120.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 122 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 123; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 124.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 126 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 127; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 128.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 130 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 131; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 132.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 134 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 135; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 136.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 138 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 139; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 140.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 142 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 143; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 144.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 146 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 147; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 148.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 150 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 151; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 152.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 154 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 155; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 156.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 158 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 159; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 160.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 162 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 163; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 164.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 166 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 167; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 168.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 170 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 171; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 172.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 174 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 175; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 176.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 178 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 179; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 180.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 182 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 183; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 184.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 186 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 187; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 188.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 190 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 191; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 192.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a) a heavy chain variable region CDR1, the heavy chain variable region CDR1 comprising SEQ ID NO: 94, 98 , 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186 and 190 Amino acid sequence, or a variant with up to about 3 amino acid substitutions; b) heavy chain variable region CDR2, the heavy chain variable region CDR2 comprises SEQ ID NO: 95, 99, 103, 107, 111, 115, The amino acid sequence of any one of 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, and 191, or up to about A variant with 3 amino acid substitutions
  • the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain and a CDR3 domain, wherein the CDR1 domain, CDR2 domain and CDR3 domain They respectively contain the CDR1 domain, CDR2 domain and CDR3 domain contained in the reference heavy chain variable region, the reference heavy chain variable region comprising SEQ ID NO: 97, 101, 105, 109, 113, 117, The amino acid sequence of the group consisting of 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189 and 193.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 94; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 95 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 96.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 98; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 99 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which contains the amino acid having the sequence shown in SEQ ID NO: 100.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid having the sequence shown in SEQ ID NO: 102; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 103 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 104.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 106; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 108.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 110; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 111 The amino acid sequence shown; and the heavy chain variable region CDR3, which includes the amino acid sequence shown in SEQ ID NO: 112.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 114; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 115 The amino acids shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acids having the sequence shown in SEQ ID NO: 116.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 118; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 119 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 120.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 122; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 124.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 126; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 128.
  • the single domain antibody includes: heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 130; heavy chain variable region CDR2, which includes the heavy chain variable region CDR1 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 132.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 134; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 136.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 138; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 140.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 142; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 144.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid having the sequence shown in SEQ ID NO: 146; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 148.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 150; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 151 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 152.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 154; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 155 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 156.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 158; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 160.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 162; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 164.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 166; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 168.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 170; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid whose sequence is shown; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 172.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 174; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 175 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 176.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 178; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid whose sequence is shown; and the heavy chain variable region CDR3, which contains the amino acid whose sequence is shown in SEQ ID NO: 180.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 182; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 183 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 184.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 186; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 187 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 188.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 190; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 191 The amino acid whose sequence is shown; and the heavy chain variable region CDR3, which contains the amino acid whose sequence is shown in SEQ ID NO: 192.
  • the single domain antibody comprises a heavy chain variable region, which comprises and is selected from SEQ ID NO: 97, 101, 105, 109, 113, 117, 121, 125, 129,
  • the amino acid sequence of the group consisting of 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, and 193 has an amino acid sequence with at least about 90% sequence identity.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 97. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 101. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 105. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:109. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 113. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:117.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 121. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 125. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 129. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 133. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:137. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:141.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 145. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:149. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 153. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 157. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 161. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 165.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 169. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 173. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 177. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:181. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 185. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:189. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 193.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO:1 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 2; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 3.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 5 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 6; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 7.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 9 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 10; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 11.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 13 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 14; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 15.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 17 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 18; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 19.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 21 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 22; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 23.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 25 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 26; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 27.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 29 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 30; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 31.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 33 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 34; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 35.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 37 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 38; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 39.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 41 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 42; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 43.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 45 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 46; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 47.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 49 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 50; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 51.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 53 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 54; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 55.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 57 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 58; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 59.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 61 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 62; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 63.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 65 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 66; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 67.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 69 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 70; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 71.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 73 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 74; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 75.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 77 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 78; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 79.
  • the single domain antibody cross-competes with the reference anti-TIGIT single domain antibody for binding to TIGIT
  • the reference anti-TIGIT single domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 81 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 82; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 83.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 1;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 2; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 3.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 5; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 6; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 7.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 9; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 10; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 11.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 13; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 14; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 15.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 17; the heavy chain may The variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 18; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 19.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 21; The variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 22; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 23.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 25;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 26; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 27.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 29;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 30; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 31.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 33; the heavy chain may The variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 34; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 35.
  • the single domain antibody includes a heavy chain variable region, which includes: the heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 37; the heavy chain may The variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 38; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 39.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 41;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 42; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 43.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 45; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 46; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 47.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 49;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 50; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 51.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 53;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 54; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 55.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 57;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 58; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 59.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: the heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 61; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 62; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 63.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 65;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 66; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 67.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 69;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 70; and
  • the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 71.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 73;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 74; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 75.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 77;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 78; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 79.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 81;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 82; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 83.
  • the single domain antibody comprises a heavy chain variable region which comprises and is selected from SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36,
  • the amino acid sequences of the group consisting of 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, and 84 have amino acid sequences with at least about 90% sequence identity.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 , 44, 48, 52, 56, 60, 64, 68, 72, 76, 80 and 84 amino acid sequences.
  • single domain antibodies comprise a humanized framework.
  • the anti-TIGIT antibody comprises an Fc region.
  • the Fc region comprises a human Fc region.
  • the Fc region comprises an Fc region selected from the group consisting of Fc regions of IgG, IgA, IgD, IgE, and IgM.
  • the Fc region comprises an Fc region selected from the group consisting of Fc regions of IgG1, IgG2, IgG3, and IgG4.
  • the Fc region comprises an IgG1 Fc region.
  • the IgG1 Fc region contains one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the IgG1 Fc region contains mutations of L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the IgG1 Fc region contains mutations of S239D, A330L, and I332E. In certain embodiments, the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO: 194.
  • the heavy chain variable region is connected to the Fc region through a linker.
  • the linker is a peptide linker.
  • the peptide linker comprises about 4 to about 30 amino acids.
  • the peptide linker comprises about 4 to about 15 amino acids.
  • the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 195-220.
  • the anti-TIGIT antibody comprises a multispecific antibody, such as a bispecific antibody, full-length immunoglobulin, single chain Fv (scFv) fragment, Fab fragment, Fab' fragment, F(ab')2 , Fv fragment, disulfide bond stabilized Fv fragment (dsFv), (dsFv)2, VHH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody, tetrabody or any Their combination.
  • a bispecific antibody such as a bispecific antibody, full-length immunoglobulin, single chain Fv (scFv) fragment, Fab fragment, Fab' fragment, F(ab')2 , Fv fragment, disulfide bond stabilized Fv fragment (dsFv), (dsFv)2, VHH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody
  • the anti-TIGIT antibody comprises a multispecific antibody (e.g., a bispecific antibody) that includes a second antibody portion that specifically binds to a second antigen.
  • the second antigen is a tumor-associated antigen.
  • the tumor-associated antigen is selected from the group consisting of Her-2, EGFR, PD-L1, c-Met, B cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X) , Carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial Glycoprotein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epiglin-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2, 3 , 4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglio
  • the second antigen is an immune checkpoint modulator.
  • the immune checkpoint modulator is selected from the group consisting of PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof.
  • the anti-TIGIT antibody is conjugated to a therapeutic agent or label.
  • the label is selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes.
  • the present disclosure further provides immunoconjugates that are linked to therapeutic agents and include any of the antibodies disclosed herein.
  • the therapeutic agent is a cytotoxin.
  • the therapeutic agent is a radioisotope.
  • the present disclosure further provides pharmaceutical compositions.
  • the pharmaceutical composition comprises a) an antibody or immunoconjugate disclosed herein, and b) a pharmaceutically acceptable carrier.
  • the present disclosure further provides nucleic acids encoding any of the antibodies disclosed herein.
  • the present disclosure further provides a vector comprising any nucleic acid disclosed herein.
  • the present disclosure further provides a host cell comprising the nucleic acid or vector disclosed herein.
  • the present disclosure further provides methods for preparing the antibodies disclosed herein.
  • the method includes expressing the antibody in the host cell disclosed herein, and isolating the antibody from the host cell.
  • the present disclosure further provides methods for reducing the tumor burden of a subject.
  • the method includes administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.
  • the method reduces the number of tumor cells. In certain embodiments, the method reduces tumor size. In certain embodiments, the method eradicates tumors in the subject.
  • the tumor is selected from the group consisting of: mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma , Thymus cancer, endometrial cancer, stomach tumor, cholangiocarcinoma, head and neck cancer, blood cancer and combinations thereof.
  • the present disclosure further provides methods of treating and/or preventing neoplasms in a subject.
  • the method includes administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.
  • the present disclosure further provides methods for prolonging the survival period of subjects suffering from neoplasms.
  • the method includes administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.
  • the neoplasm is selected from the group consisting of: mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovium Sarcoma, thymus cancer, endometrial cancer, stomach tumor, cholangiocarcinoma, head and neck cancer, blood cancer, and combinations thereof.
  • the present disclosure further provides any antibody disclosed herein for use as a drug.
  • the present disclosure further provides any of the antibodies disclosed herein for use in the treatment of cancer.
  • the present disclosure further provides the pharmaceutical composition disclosed herein for use as a medicament.
  • the present disclosure further provides the pharmaceutical composition disclosed herein for treating cancer.
  • the cancer is selected from the group consisting of: mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma , Thymus cancer, endometrial cancer, stomach tumor, cholangiocarcinoma, head and neck cancer, blood cancer and combinations thereof.
  • kits comprising the antibody, immunoconjugate, pharmaceutical composition, nucleic acid, vector or host cell disclosed herein.
  • the kit includes written instructions for treating and/or preventing neoplasms.
  • FIGS 1A-1C depict the whole cell binding of representative VHH bivalent antibodies to human TIGIT as determined by flow cytometry.
  • Figure 1A shows an assay compared to a reference anti-human TIGIT antibody (reference Ab1).
  • Figure 1B shows a different assay using other VHH antibodies.
  • the Y axis represents the average fluorescence intensity of AlexaFlour 488.
  • the X axis represents the antibody concentration in nanomolar.
  • 2B7, 1G1, 1C12, 3G6, 2B10, 3G7, 3G10, 13H11 and 15A5 are VHH clones against human TIGIT. Prism's nonlinear regression method was used to obtain EC50 values, and these values are expressed in nanomoles in the table.
  • Figure 1C is a schematic diagram of the structure of the TIGIT VHH bivalent antibody.
  • Figures 2A-2B depict the efficacy of representative bivalent antibodies in blocking TIGIT activity as determined by the luciferase reporter gene assay.
  • a representative anti-TIGIT bivalent antibody and a low concentration of staphylococcal enterotoxin Jurkat cells stably transfected with human TIGIT and NFAT reporter genes were co-cultured with Raji cells stably transfected with PVR (CD155).
  • the Y axis represents NFAT luciferase activity in relative luminescence units.
  • the X axis represents the antibody concentration in nanomolar.
  • Reference Ab1 is the reference anti-h-TIGIT antibody.
  • 2B7, 1G1, 1C12, 3G6, 2B10, 3G7 and 3F10 are representative clones against h-TIGIT. Prism's nonlinear regression method was used to obtain EC50 values, and these values are expressed in nanomoles in the table.
  • Figures 3A and 3B depict the whole cell binding of humanized 1C12 and 1G1 clones to human TIGIT as determined by flow cytometry. Representative results of the humanized form of 1C12 are shown in Figure 3A.
  • 1C12 chimeric antibody is an antibody with the Llama VHH sequence of the 1C12 clone and the CH2 and CH3 domains of human IgG1.
  • 1C12 (F-EREF), 1C12 (F-EREW) and 1C12 (F-GLEW) are the human origin of the 1C12 clone The difference is the mutation in Framework 2. Representative results of the humanized form of 1G1 are shown in Figure 3B.
  • the 1G1 chimeric antibody is an antibody with the Llama VHH sequence of the 1G1 clone and the CH2 and CH3 domains of human IgG1.
  • 1G1 (F-G-ERES), 1G1 (F-A-ERES), 1G1 (F-A-EREW) and 1G1 (F-A-GLEW) are four different forms of humanized 1G1 clones with different mutations in framework 2.
  • the Y axis is the average fluorescence intensity value of AlexaFlour 488.
  • the X axis is the value of the antibody concentration in nanomolar.
  • Figures 4A and 4B depict the efficacy of humanized 1C12 and 1G1 clones in blocking TIGIT activity as determined by the luciferase reporter gene assay. Representative results of the humanized form of 1C12 in the TIGIT blocking luciferase reporter gene assay are shown in Figure 4A. Compared with the reference anti-h-TIGIT antibody reference Ab1, all clones are more effective. Representative results of the humanized form of 1G1 are shown in Figure 4B.
  • the Y axis represents NFAT luciferase activity (RLU, relative luminescence unit).
  • the X axis represents the antibody concentration in nanomolar.
  • Figure 5 depicts the correlation between IC50 values (in nanomoles) from blocking ELISA and EC50 values (in nanomoles) from whole cell binding for representative clones.
  • the clone name is marked in the figure.
  • Figures 6A and 6B depict the thermal stability tested against representative clones.
  • the VHH antibody sample was heated from 25 to 70°C for 60 minutes.
  • Figure 6A shows the binding of a heated sample of a representative clone to h-TIGIT ECD in an ELISA assay, where the clone name is marked in the legend.
  • the Y axis represents the OD450 obtained by the ELISA assay.
  • the X axis represents the processing temperature.
  • Figure 6B shows the binding of heated samples of representative clones to h-TIGIT stably expressed in Jurkat cells as determined by flow cytometry.
  • the Y axis represents the percentage of binding to h-TIGIT.
  • the X axis represents the processing temperature.
  • Figures 7A-7C depict the epitope that 2A3-Fc binds to human TIGIT as determined by using ForteBio's Octet binding assay. Load the recombinant protein of human TIGIT ECD (200 nM) with his tag on the sensor. The binding was detected by injecting 3 different concentrations of 2A3-Fc. No additional binding was detected by the second injection of 2A3-Fc shown in Figure 7A, but by injection of three different concentrations of reference Ab2, a reference anti-TIGIT antibody (as shown in Figure 7B), or reference Ab1 (As shown in Figure 7C), strong binding was detected. The results showed that the 2A3 clone has a different binding epitope compared with the reference Ab2 and the reference Ab1.
  • Figures 8A-8C depict the cross-binding activity of 2A3-Fc to human-, cyno- and mouse-TIGIT as determined by an ELISA assay.
  • Figure 8A shows the binding of 2A3-Fc to recombinant human TIGIT ECD.
  • Reference Ab2 is an anti-human TIGIT reference antibody. Both 2A3-Fc and reference Ab2 bind to h-TIGIT with similar affinity.
  • Anti-PDL1 does not bind to h-TIGIT.
  • Figure 8B shows the binding of 2A3-Fc to recombinant cyno-TIGIT. Both 2A3-Fc and reference Ab2 bind to cyno-TIGIT with similar affinity.
  • FIG. 8C shows the binding of 2A3-Fc to recombinant mouse TIGIT.
  • Neither 2A3-Fc nor reference Ab2 binds to mouse TIGIT, but the anti-mouse TIGIT reference antibody (Biolegend #142101) binds to mouse TIGIT with high affinity.
  • the Y axis represents OD450, and the X axis represents antibody concentration in ⁇ g/ml.
  • Figure 9 depicts a comparison of the potency of a representative clone 2A3-Fc and a hotspot corrected version of 2A3-LT-Fc in the whole cell binding assay and the human TIGIT blocking reporter gene assay.
  • the Y axis represents the average fluorescence intensity in the upper graph, and the NFAT luciferase reporter gene activity in relative luminescence units in the lower graph.
  • the X axis represents the antibody concentration in nanomolar.
  • the hotspot corrected form 2A3-LT-Fc has similar potency to the parental clone 2A3-Fc.
  • Figures 10A and 10B depict the affinity comparison of the hotspot corrected version 2A3-LT-Fc and the reference anti-h-TIGIT antibody reference Ab2.
  • Figure 10A shows a comparison of the potency of the hot spot corrected version 2A3-LT-Fc and the reference Ab2 in whole cell binding.
  • the Y axis represents the average fluorescence intensity of AlexaFlour 488 determined by the flow cytometry assay using CytoFlex.
  • the X axis represents the antibody concentration in nanomolar. The data shown are representative results from three different experiments.
  • the affinity of 2A3-LT-Fc to h-TIGIT is slightly higher than that of reference Ab2.
  • Figure 10B shows the comparison of the potency of the hot spot correction form 2A3-LT-Fc and the reference Ab2 in the TIGIT blocking NFAT reporter gene assay.
  • the Y axis represents NFAT luciferase reporter gene activity in relative luminescence units.
  • the X axis represents the antibody concentration in nanomolar. 2A3-LT-Fc has similar potency to reference Ab2.
  • Figure 11 depicts the anti-tumor efficacy of 2A3-LT-Fc in vitro.
  • TIGIT+T cells and PVR+dendritic cells were used. After 48 hours of mixed culture, the secretion of IL-2 of T cells in the culture supernatant was detected.
  • Anti-PD1 antibody was used as a positive control.
  • Anti-HER2 antibody was used as a negative control.
  • Figure 12 depicts the binding of 2A3-LT-Fc wild-type (wt) and Fc mutations (DLE and VLPLL) to human Fc ⁇ RIIIA, human Fc ⁇ RIIB, and mouse Fc ⁇ RIV determined using the Octet binding assay.
  • the sensor was loaded with ECD recombinant proteins of Fc ⁇ RIIIA, human Fc ⁇ RIIB and mouse Fc ⁇ RIV, and the association and dissociation of 5 different concentrations of 2A3-LT-Fc wt, DLE or VLPLL mutants were detected using ForteBio.
  • the Y axis represents association, dissociation and Rmax.
  • the X axis represents time in seconds.
  • the DLE and VLPLL mutants both have enhanced binding affinity to human Fc ⁇ RIIIA, and the DLE mutant also has enhanced binding affinity to human Fc ⁇ RIIB, but the VLPLL mutant reduces the binding affinity to human Fc ⁇ RIIB. All forms have similar affinity to mouse Fc ⁇ RIV.
  • Figures 13A and 13B depict the effects of TIGIT mAb 2A3-LT-Fc wt and DLE mutants on blocking TIGIT activity and human Fc ⁇ RIIIA-mediated activity, respectively.
  • Figure 13A depicts the effect of TIGIT mAb 2A3-LT-Fc wt and DLE mutants on blocking TIGIT activity determined using the NFAT luciferase reporter gene assay.
  • Jurkat cells stably transfected with human TIGIT and NFAT reporter genes were co-cultured with Raji cells stably transfected with PVR. Add 2A3-LT-Fc wt and the mutant and culture for 5 hours. The TCR-mediated activity is measured by luciferase activity.
  • the Y axis represents NFAT luciferase activity in relative luminescence units.
  • the X axis represents the antibody concentration in nanomolar.
  • Figure 13B depicts the effect of TIGIT mAb 2A3-LT-Fc wt and DLE mutants on human Fc ⁇ RIIIA-mediated activity, which is determined by Fc ⁇ RIIIA-mediated NFAT luciferase reporter gene activity.
  • 2A3-LT-Fc wt and DLE mutants at different concentrations, Jurkat cells stably transfected with human Fc ⁇ RIIIA and NFAT were co-cultured with 293T cells stably transfected with human TIGIT for 5 hours.
  • the luciferase activity is measured and expressed in relative luminescence units on the Y axis.
  • the X axis represents the antibody concentration in nanomolar.
  • Figures 14A-14C depict the in vivo effectiveness analysis of anti-TIGIT antibodies.
  • Human TIGIT knock-in C57BL/6 mice and MC38 murine colon cancer models were used. Mice were inoculated with MC38 tumor cells one week before treatment. The treatment started when the average tumor size reached about 51mm 3 and was administered intraperitoneally twice a week for 2.5 weeks.
  • the 2A3-LT-Fc antibody is 6 mg/kg each time or the reference Ab2 is 11 mg/kg each time (equivalent to the 2A3-LT-Fc antibody in mole/kg).
  • Figure 14A depicts the tumor growth curve of tumor-bearing mice administered with blank control, 2A3-Fc-wt, 2A3-Fc with DLE mutation and reference Ab 2. The results of a single tumor volume are shown in Figure 14B.
  • Figure 14C shows that the weight change of each experimental group in the study was not significant.
  • the present disclosure provides isolated monoclonal antibodies that specifically bind TIGIT with high affinity, including multispecific antibodies that bind TIGIT and one or more additional targets.
  • the anti-TIGIT antibody comprises a single domain antibody that binds to TIGIT.
  • the present disclosure further provides methods for preparing antibodies, immunoconjugates, and pharmaceutical compositions containing these antibodies, and the use of antibodies, immunoconjugates, and pharmaceutical compositions containing these antibodies, for example, for the treatment of diseases and disorders (e.g., cancer) Methods.
  • the present invention is based in part on the discovery of single domain anti-TIGIT antibodies that bind to TIGIT, which can increase the immune response in immune cells and provide improved anti-tumor efficacy.
  • antibody herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), single structures Domain antibodies and antibody fragments, as long as they exhibit the desired antigen-binding activity.
  • Antibody fragment refers to a molecule that contains the antigen-binding portion of a complete full-length antibody that binds to the antigen that is bound to the complete antibody.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibody fragments.
  • VHH refers to a single domain antibody isolated from camelid animals. In certain embodiments, the VHH comprises the heavy chain variable region of a camel heavy chain antibody. In certain embodiments, the size of VHH does not exceed 25 kDa. In certain embodiments, the size of VHH does not exceed 20 kDa. In certain embodiments, the size of VHH does not exceed 15 kDa.
  • “Full-length antibody” refers to an antibody comprising two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable regions of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively.
  • the variable regions in the two chains usually contain three highly variable loops, called complementarity determining regions (CDR) (including LC-CDR1, LC-CDR2 and LC-CDR3 light chain (LC) CDR, including HC- CDR1, HC-CDR2 and HC-CDR3 heavy chain (HC) CDR).
  • CDR complementarity determining regions
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by well-known conventions, for example, the conventions of Kabat, Chothia or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDRs of the heavy or light chain are inserted between flanking sections called framework regions (FR), which are more conservative than the CDRs and form a scaffold that supports the hypervariable loop.
  • FR flanking sections
  • the constant regions of the heavy and light chains do not participate in antigen binding, but exhibit multiple effector functions.
  • Antibodies are classified based on the amino acid sequence of the constant region of the antibody heavy chain.
  • the five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
  • Several major antibody categories are divided into subclasses, such as lgG1 ( ⁇ 1 heavy chain), lgG2 ( ⁇ 2 heavy chain), lgG3 ( ⁇ 3 heavy chain), lgG4 ( ⁇ 4 heavy chain), lgA1 ( ⁇ 1 heavy chain) or lgA2 ( ⁇ 2 heavy chain) chain).
  • An antibody that "cross-competes for binding" with a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by more than 50% in a competition assay. On the contrary, in a competition assay, the reference antibody blocks the binding of the antibody to its antigen by more than 50% .
  • Exemplary competitive assays are described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).
  • Fv is the smallest antibody fragment, which contains a complete antigen recognition site and an antigen binding site.
  • the fragment consists of a dimer of a heavy chain variable region and a light chain variable region that are tightly non-covalently associated. From the folding of these two domains, six hypervariable loops (3 loops in each of the heavy chain and light chain) are emitted, which contribute amino acid residues for antigen binding and endow the antibody with antigen binding specificity .
  • six hypervariable loops (3 loops in each of the heavy chain and light chain) are emitted, which contribute amino acid residues for antigen binding and endow the antibody with antigen binding specificity .
  • a single variable domain or half an Fv containing only three CDRs against the original specificity
  • Single-chain an Fv (also abbreviated as “sFv” or “the scFv”) is an antibody fragment and V L, V H antibody domains connected into a single polypeptide chain comprising.
  • the scFv polypeptide further comprises a polypeptide linker between the V H and V L, domain, the polypeptide linker which enables the scFv to form the desired structure for antigen binding.
  • acceptor human framework or “human framework” is a light chain variable domain (VL) framework or heavy chain variable domain (VH) that is derived from a human immunoglobulin framework or a human consensus framework.
  • the framework of the amino acid sequence of the framework may include the same amino acid sequence thereof, or may include amino acid sequence changes. In certain embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 Or less, 3 or less, or 2 or less.
  • the VL acceptor human framework and the VL human immunoglobulin framework sequence or the human consensus framework sequence are identical in sequence.
  • Binding affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity” refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X to its partner Y can usually be represented by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
  • an “affinity mature” antibody refers to an antibody that has one or more changes in one or more CDRs or hypervariable regions (HVR) compared to a parent antibody that does not have such changes, and the changes provide the antibody to the antigen The improved affinity.
  • HVR hypervariable regions
  • T cell immune receptor with Ig and ITIM domains refers to any derived from any vertebrate source (including mammals (e.g., primates (e.g., humans and cynomolgus monkeys)))
  • the native TIGIT polypeptide, or any fragment thereof may optionally contain at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine or Up to ten amino acid substitutions, additions and/or deletions.
  • the term encompasses full-length untreated TIGIT as well as any form of TIGIT produced as a result of processing in the cell.
  • the term also encompasses naturally occurring variants of TIGIT, such as splice variants or allelic variants.
  • Non-limiting examples of the amino acid sequence of human TIGIT targeted by the anti-TIGIT antibodies of the present disclosure are as follows:
  • ECD of TIGIT refers to the extracellular domain of TIGIT.
  • ECD of the exemplary TIGIT protein shown in SEQ ID NO: 221 includes the following amino acid sequence:
  • anti-TIGIT antibody and "antibody that binds TIGIT” refer to an antibody capable of binding to TIGIT with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting TIGIT.
  • the degree of binding of an anti-TIGIT antibody to an unrelated, non-TIGIT protein is less than about 10% of the binding of the antibody to TIGIT, for example by Measured by surface plasmon resonance measurement.
  • the antibody that binds to TIGIT has the following dissociation constant (KD) ⁇ about 1 ⁇ M, ⁇ about 100 nM, ⁇ about 10 nM, ⁇ about 1 nM, ⁇ about 0.1 nM, ⁇ about 0.01 nM, or ⁇ about 0.001 nM (E.g. 10 -8 M or less, e.g. 10 -8 M to 10 -12 M, e.g. 10 -9 M to 10 -10 M).
  • KD dissociation constant
  • anti-TIGIT antibodies bind to TIGIT epitopes that are conserved among TIGITs from different species.
  • the anti-TIGIT antibody binds to an epitope on TIGIT in the ECD of the protein.
  • chimeric antibody refers to an antibody in which a part of the heavy chain and/or light chain is derived from a specific source or species, and the remainder of the heavy chain and/or light chain is derived from a different source or species.
  • the chimeric antibodies disclosed herein comprise a camelid heavy chain variable region and a human Fc region.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • variable domain residues as in Kabat or “number of amino acid positions as in Kabat” and variants thereof refer to the heavy chain variable domain or light chain used in the antibody compilation of Kabat et al. above
  • the numbering system for variable domains the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain.
  • the heavy chain variable domain may comprise a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (for example, residue according to Kabat 82a, 82b, 82c, etc.).
  • the Kabat numbering of residues in a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbering sequence in the region of homology.
  • the amino acid residues encompassing the CDRs of single domain antibodies are defined according to the IMGT nomenclature of Lefranc et al. above.
  • the amino acid residues encompassing the CDRs of the full-length antibody are defined according to the Kabat nomenclature of Kabat et al. above.
  • the numbering of residues in an immunoglobulin heavy chain, such as the Fc region is the numbering of the EU index as described by Kabat et al., supra. "EU index as described by Kabat” refers to the residue numbering of the human IgG1 EU antibody.
  • Framework or "FR” refers to those variable domain residues other than the CDR residues defined herein.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDR/HVR and amino acid residues derived from human FR.
  • a humanized antibody will include substantially all of at least one, and typically two variable domains, wherein all or substantially all of the HVR/CDR correspond to those of a non-human antibody, and FR All or substantially all of the correspond to those of human antibodies.
  • a humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.
  • a "human antibody” is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by humans and/or has been prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. [Journal of Molecular Biology], 227:381 (1991); Marks et al., J. Mol. Biol. [Journal of Molecular Biology], 222:581 (1991). It can also be used to prepare human monoclonal antibodies in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering an antigen to a transgenic animal (e.g., immunized xenomice) that has been modified to produce such an antibody in response to an antigen challenge, but whose endogenous locus has failed (see, e.g., the XENOMOUSE TM technology U.S. Patent Nos. 6,075,181 and 6,150,584).
  • a transgenic animal e.g., immunized xenomice
  • the “percentage (%) of amino acid sequence identity with respect to the polypeptide and antibody sequences identified herein” or “homology” is defined as after the sequence is aligned (considering any conservative substitutions as part of the sequence identity), the candidate sequence is identical to all Compare the percentage of the same amino acid residues in the polypeptide.
  • the alignment can be achieved in a variety of ways in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithm that needs to achieve the maximum alignment over the full length of the sequence being compared.
  • sequence comparison computer program MUSCLE is used to generate the% value of amino acid sequence identity (Edgar, RC, Nucleic Acids Research [Nucleic Acid Research] 32(5): 1792-1797, 2004; Edgar, RC, BMC Bioinformatics [BMC Bioinformatics] 5(1): 113, 2004).
  • “Homologous” refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When two positions in two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecule is in that position Are homologous.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, comparisons are made when two sequences are aligned to give maximum homology.
  • constant domain refers to a part of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another part of an immunoglobulin, that is, a variable domain, which contains an antigen binding site.
  • C H constant domain of the heavy chain comprises 1, C L domain, and C H C H 2 domain. 3 (collectively referred to as C H) and light chains.
  • the "light chain” of antibodies of any mammalian species can be assigned to one of two distinct types based on the amino acid sequence of its constant domain, called kappa (" ⁇ ") and lambda ( " ⁇ ").
  • CH1 domain (also referred to as “C1" of the “H1” domain) generally ranges from about amino acid 118 to about amino acid 215 (EU numbering system).
  • the "hinge region” is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. [Molecular Immunology] 22:161-206 (1985)).
  • the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues forming the S-S bond between the heavy chains in the same position.
  • the "CH2 domain" of the human IgG Fc region (also referred to as the "C2" domain) generally ranges from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique because it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the complete natural IgG molecule. It is speculated that carbohydrates can provide a substitute for domain-domain pairing and help stabilize the CH2 domain.
  • the "CH3 domain” (also known as the "C2" domain) contains the residues between the CH2 domain and the C-terminus of the Fc region (ie, from about amino acid residue 341 to the C-terminus of the antibody sequence), usually in the IgG Amino acid residues 446 or 447).
  • Fc region or "fragment crystallizable region” as used herein is used to define the C-terminal region of immunoglobulin heavy chains, including native sequence Fc regions and variant Fc regions.
  • Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is generally defined as an amino acid residue at position Cys226 or extending from Pro230 to its carboxyl terminus.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain.
  • composition of intact antibodies may include an antibody population with all K447 residues removed, an antibody population without K447 residues removed, and an antibody population with a mixture of antibodies with and without K447 residues.
  • Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • a preferred FcR is a natural human FcR.
  • a preferred FcR is an FcR that binds IgG antibodies ( ⁇ receptors) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (" Activating receptor") and Fc ⁇ RIIB ("inhibiting receptor”), they have similar amino acid sequences, the main difference lies in their cytoplasmic domains.
  • the activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • epitope refers to a specific atom or amino acid group on the antigen to which the antibody or antigen-binding portion binds. If two antibodies or antigen-binding portions have competitive binding to the antigen, they can bind to the same epitope within the antigen.
  • the terms “specific binding”, “specific recognition” and “specific to” refer to a measurable and reproducible interaction, such as the binding between a target and an antibody or antibody portion, which determines The existence of the target in the presence of a population of heterogeneous molecules (including biomolecules).
  • an antibody or antibody portion that specifically recognizes a target is an antibody or antibody portion that binds to the target, and its affinity, avidity, readiness, and/or duration are longer than binding to other targets.
  • the degree of binding of the antibody to the unrelated target is less than about 10% of the degree of binding of the antibody to the target as measured by radioimmunoassay (RIA), for example.
  • the dissociation constant (K D ) of the antibody that specifically binds to the target is ⁇ 10 -5 M, ⁇ 10 -6 M, ⁇ 10 -7 M, ⁇ 10 -8 M, ⁇ 10 -9 M, ⁇ 10 -10 M, ⁇ 10 -11 M, or ⁇ 10 -12 M.
  • the antibody specifically binds to an epitope of a protein that is conserved from proteins of different species.
  • specific binding may include but does not require exclusive binding.
  • the binding specificity of an antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM -test, and peptide scanning.
  • an “isolated” antibody is an antibody that has been identified, separated, and/or recovered from a component (eg, natural or recombinant) of its production environment.
  • the isolated polypeptide has no or substantially no association with all other components in its production environment.
  • an "isolated" nucleic acid molecule encoding a construct, antibody or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule that is normally associated with it in its production environment.
  • the isolated nucleic acid has no or substantially no association with all components related to the production environment.
  • the form of the isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is different from the naturally occurring form or background. Therefore, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that are naturally present in the cell.
  • An isolated nucleic acid includes the nucleic acid molecule contained in a cell that usually contains the nucleic acid molecule, but the nucleic acid molecule exists outside the chromosome or at a chromosomal location different from its natural chromosomal location.
  • control sequence refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a specific host organism.
  • suitable control sequences for prokaryotes include promoters, optional operator sequences, and ribosome binding sites. It is known that eukaryotic cells utilize promoters, polyadenylation signals and enhancers.
  • a nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence.
  • the DNA of the pre-sequence or the secretory leader sequence is expressed as a pre-protein involved in the secretion of the polypeptide, the DNA of the pre-sequence or the secretory leader sequence is operably linked to the DNA of the polypeptide; if the promoter or enhancer affects For transcription of the coding sequence, the promoter or enhancer is operably linked to the sequence; or if the ribosome binding site is positioned so as to facilitate translation, the ribosome binding side is operably linked to the coding sequence.
  • operably linked means that the linked DNA sequences are continuous and, in the case of a secreted leader sequence, are continuous and in reading frame.
  • the enhancer need not be continuous.
  • the connection is achieved by connecting at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid linked to it.
  • the term includes vectors that are self-replicating nucleic acid structures, as well as vectors that have been incorporated into the genome of a host cell into which they have been introduced. Certain vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • transfected or “transformed” or “transduced” refers to the process of transferring or introducing exogenous nucleic acid into a host cell.
  • a “transfected” or “transformed” or “transduced” cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid, and the cell includes the primary target cell and its progeny.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the nucleic acid content of the offspring may not be exactly the same as the parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as the function or biological activity screened or selected in the original transformed cell are included herein.
  • subject refers to mammals, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the subject is a human.
  • the "effective amount” of an agent refers to an amount effective to achieve the desired therapeutic or preventive result within the necessary dose and time period.
  • the specific dose can be changed according to one or more of the following: the specific agent selected, the subsequent dosing regimen (regardless of whether it is combined with other compounds), the time of administration, the tissue that is imaged, and where it is carried Physical delivery system.
  • the "therapeutically effective amount" of the substance/molecule, agonist or antagonist of the present application can be based on, for example, the disease state, age, sex, and individual weight, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual And other factors.
  • the therapeutically effective amount is also the amount at which any toxic or harmful effects of the substance/molecule, agonist or antagonist are offset by the beneficial effects of the treatment.
  • the therapeutically effective amount can be delivered by one or more administrations.
  • prophylactically effective amount refers to an effective amount in a dose meter and for a required period of time to achieve the desired preventive result. Typically, but not necessarily, because the preventive dose is used in the subject before or early in the disease, the preventive effective amount will be less than the therapeutically effective amount.
  • treatment or treating is a method used to obtain beneficial or desired results (including clinical results).
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, reduction of the degree of the disease, stabilization of the disease (for example, Prevent or delay the deterioration of the disease), prevent or delay the spread of the disease (for example, metastasis), prevent or delay the recurrence of the disease, delay or slow the progression of the disease, improve the disease state, provide relief (partial or full), and reduce The dosage of one or more other drugs required to treat the disease, delay the progression of the disease, increase or improve the quality of life, increase weight gain and/or prolong survival.
  • Treatment also encompasses reducing the pathological consequences of cancer (like, for example, tumor volume). The method of the application considers any one or more of these therapeutic aspects. "Treatment” does not necessarily mean that the disease being treated will be cured.
  • the term “about” or “approximately” means that a specific value determined by a person of ordinary skill in the art is within an acceptable error range, which will depend in part on how the value is determined or Deterministic, that is, limited by the measurement system. In certain embodiments, “about” may mean within 3 or more standard deviations according to practice in the art. In certain embodiments, “about” may mean a range of at most 20% (e.g., at most 10%, at most 5%, or at most 1%) of a given value. In certain embodiments, particularly for biological systems and methods, the term may mean within the order of a certain value, such as within 5 times or within 2 times.
  • modulation refers to a positive or negative change.
  • exemplary adjustments include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.
  • the term "increase” refers to a positive change of at least about 5%.
  • the change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.
  • the term "decrease” refers to a negative change of at least about 5%.
  • the change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.
  • Antibody effector functions refer to those biological activities attributed to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, cell surface receptors (such as B cell receptors) Body) down-regulation and B cell activation.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • phagocytosis phagocytosis
  • cell surface receptors such as B cell receptors
  • Immunoconjugate refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.
  • pharmaceutical formulation refers to a formulation that is in a form that allows the biological activity of the active ingredients contained therein to be effective and does not contain other components that have unacceptable toxicity to the subject to which the formulation is administered.
  • pharmaceutically acceptable carrier refers to ingredients in a pharmaceutical formulation that are not toxic to the subject except for the active ingredients.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • variable region refers to the domain of the heavy or light chain of an antibody, which participates in the binding of the antibody to the antigen.
  • variable domains VH and VL, respectively
  • FR conserved framework regions
  • a single VH or VL domain may be sufficient to confer Antigen binding specificity.
  • VH or VL domains can be used to isolate antibodies that bind to specific antigens from antibodies that bind to antigens to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. [Journal of Immunology] 150:880-887 (1993); Clarkson et al., Nature [Nature] 352:624-628 (1991).
  • the present invention is based in part on the discovery of single domain antibodies that bind to TIGIT, which can be used in anti-tumor therapy, wherein the antibodies selectively inhibit TIGIT receptors and induce immune cell (eg, T cells) Beneficial immune response. Therefore, the present disclosure provides anti-TIGIT antibodies.
  • the anti-TIGIT antibodies disclosed herein are antagonist antibodies that inhibit the function of the TIGIT receptor.
  • the anti-TIGIT antibody blocks the interaction between the TIGIT receptor and the ligand.
  • the anti-TIGIT antibody blocks the immunosuppressive signal from the TIGIT receptor.
  • anti-TIGIT antibodies comprise single domain antibodies, such as camelid antibodies or VHH antibodies. In certain embodiments, since the anti-TIGIT antibody has a smaller size compared to traditional antibodies in the form of IgG, Fab, and/or scFv, it has improved tissue infiltration ability.
  • the antibodies of the present disclosure may be or comprise monoclonal antibodies (including chimeric antibodies, humanized antibodies, or human antibodies).
  • the antibodies disclosed herein comprise humanized antibodies.
  • the antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.
  • the antibodies of the present disclosure may be antibody fragments, such as Fv, Fab, Fab', scFv, diabodies, or F(ab')2 fragments.
  • the antibody is a full-length antibody, such as a complete IgG 1 antibody, or other antibody types or isotypes as defined herein.
  • the antibodies of the present disclosure may incorporate any (as described in this application (e.g., Sections 2.1-2.11 detailed herein)) features, alone or in combination.
  • neoplasias and cancers whose growth can be inhibited using the antibodies of the present disclosure include neoplasias and cancers that generally respond to immunotherapy.
  • neoplasia and cancer include breast cancer (e.g., breast cell carcinoma), ovarian cancer (e.g., ovarian cell carcinoma), and renal cell carcinoma (RCC).
  • melanoma e.g., metastatic malignant melanoma
  • prostate cancer colon cancer
  • lung cancer bone cancer
  • pancreatic cancer skin cancer
  • brain tumor chronic or acute Leukemia (including acute myeloid leukemia, chronic granulocytic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), lymphoma (such as Hodgkin’s lymphoma and non-Hodgkin’s lymphoma, lymphocytic lymphoma)
  • CNS Primary central nervous system
  • lymphoma T-cell lymphoma
  • nasopharyngeal cancer head or neck cancer
  • nasopharyngeal cancer head or neck cancer
  • nasopharyngeal cancer head or neck cancer
  • nasopharyngeal cancer head or neck cancer
  • nasopharyngeal cancer head or neck cancer
  • nasopharyngeal cancer head or neck cancer
  • the present disclosure provides isolated antibodies that bind to TIGIT protein.
  • the anti-TIGIT antibodies of the present disclosure bind to the ECD of TIGIT.
  • the anti-TIGIT antibody binds to the ECD of TIGIT comprising the amino acid sequence shown in SEQ ID NO: 222.
  • the anti-TIGIT antibody binds to the same epitope as the anti-TIGIT antibody described herein (e.g., 2A3).
  • the anti-TIGIT antibodies disclosed herein can be used as antagonists of the TIGIT receptor.
  • the anti-TIGIT antibody can reduce the activity of the TIGIT receptor by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%. , About 90%, about 99%, or about 99.9%.
  • anti-TIGIT antibodies can block downstream immunosuppressive signaling of TIGIT receptors.
  • anti-TIGIT antibodies increase the immune response and/or anti-tumor effects of immune cells (eg, T cells and/or NK cells).
  • treatment with anti-TIGIT antibodies exhibits anti-tumor efficacy in the subject, thereby reducing tumor growth and/or prolonging the survival of the subject.
  • an anti-TIGIT antibody comprising a single domain antibody e.g., VHH
  • VHH has a smaller molecular size compared to a full-length antibody, because compared with the Fab domain of a full-length antibody, the single structure The size of domain antibodies is small, which can lead to superior tissue infiltration (e.g., at tumor sites) compared to full-length antibodies.
  • the treatment using the anti-TIGIT antibody exhibits superior anti-tumor efficacy compared with the treatment using the full-length anti-TIGIT antibody (for example, reference Ab1 and reference Ab 2).
  • Reference Ab1 has the same amino acid sequence as BMS22G2 disclosed in U.S. 2016/0176963 A1
  • reference Ab2 has the same amino acid sequence as Tiragolumab, and its sequence is disclosed in U.S. 2017/0088613 A1.
  • the anti-TIGIT antibody comprises a single domain antibody that binds to TIGIT.
  • single domain antibodies comprise VHH.
  • a single domain antibody comprises a variable region of a heavy chain (VH).
  • single domain antibodies are linked to the Fc region. In certain embodiments, single domain antibodies are not linked to the Fc region.
  • single domain antibodies bind to TIGIT with a KD of about 1 ⁇ 10 -7 M or less. In certain embodiments, single domain antibodies bind to TIGIT with a KD of about 1 ⁇ 10 -8 M or less. In certain embodiments, the single domain antibody binds to TIGIT with a KD of about 5 ⁇ 10 ⁇ 9 M or less. In certain embodiments, single domain antibodies bind to TIGIT with a KD of about 1 ⁇ 10 ⁇ 9 M or less. In certain embodiments, the single domain antibody binds to TIGIT with a KD between about 1 ⁇ 10 -9 M to about 1 ⁇ 10 -7 M.
  • the single domain antibody binds to TIGIT with a KD between about 1 ⁇ 10 -9 M to about 1 ⁇ 10 -8 M. In certain embodiments, the single domain antibody binds to TIGIT with a KD between about 2 ⁇ 10 -9 M to about 1 ⁇ 10 -8 M. In certain embodiments, the single domain antibody binds to TIGIT with a KD between about 2 ⁇ 10 -9 M to about 5 ⁇ 10 -8 M. In certain embodiments, a single domain antibody from about 1x10 -9 KD between about 5x10 -9 M M binding to TIGIT.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 94 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 95; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 96.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 98 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 99; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 100.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 102 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 103; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 104.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 106 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 107; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 108.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 110 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 111; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 112.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 114 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 115; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 116.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 118 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 119; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 120.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 122 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 123; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 124.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 126 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 127; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 128.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 130 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 131; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 132.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 134 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 135; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 136.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 138 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 139; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 140.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 142 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 143; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 144.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 146 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 147; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 148.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 150 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 151; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 152.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 154 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 155; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 156.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 158 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 159; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 160.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 162 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 163; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 164.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 166 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 167; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 168.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 170 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 171; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 172.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 174 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 175; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 176.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 178 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 179; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 180.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 182 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 183; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 184.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 186 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 187; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 188.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 190 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 191; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 192.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a) a heavy chain variable region CDR1, the heavy chain variable region CDR1 comprising SEQ ID NO: 94, 98 , 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186 and 190 Amino acid sequence, or a variant with up to about 3 amino acid substitutions; b) heavy chain variable region CDR2, the heavy chain variable region CDR2 comprises SEQ ID NO: 95, 99, 103, 107, 111, 115, The amino acid sequence of any one of 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, and 191, or up to about A variant with 3 amino acid substitutions
  • the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain and a CDR3 domain, wherein the CDR1 domain, CDR2 domain and CDR3 domain They respectively contain the CDR1 domain, CDR2 domain and CDR3 domain contained in the reference heavy chain variable region, the reference heavy chain variable region comprising SEQ ID NO: 97, 101, 105, 109, 113, 117, The amino acid sequence of the group consisting of 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189 and 193.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 94; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 95 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 96.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 98; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 99 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which contains the amino acid having the sequence shown in SEQ ID NO: 100.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid having the sequence shown in SEQ ID NO: 102; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 103 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 104.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 106; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 107 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 108.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 110; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 111 The amino acid sequence shown; and the heavy chain variable region CDR3, which includes the amino acid sequence shown in SEQ ID NO: 112.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 114; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 115 The amino acids shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acids having the sequence shown in SEQ ID NO: 116.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 118; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 119 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 120.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 122; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 124.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 126; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 128.
  • the single domain antibody includes: heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 130; heavy chain variable region CDR2, which includes the heavy chain variable region CDR1 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 132.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 134; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 136.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 138; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 140.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 142; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 144.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid having the sequence shown in SEQ ID NO: 146; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 148.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 150; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 151 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 152.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 154; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 155 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 156.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 158; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 159 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 160.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 162; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 164.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 166; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 168.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 170; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid whose sequence is shown; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 172.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 174; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR1, which includes the sequence shown in SEQ ID NO: 175 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 176.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 178; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the The amino acid whose sequence is shown; and the heavy chain variable region CDR3, which contains the amino acid whose sequence is shown in SEQ ID NO: 180.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 182; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 183 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 184.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 186; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 187 The amino acid shown in the sequence; and the heavy chain variable region CDR3, which includes the amino acid having the sequence shown in SEQ ID NO: 188.
  • the single domain antibody includes: the heavy chain variable region CDR1, which includes the amino acid sequence shown in SEQ ID NO: 190; the heavy chain variable region CDR2, which includes the heavy chain variable region CDR2, which includes the sequence shown in SEQ ID NO: 191 The amino acid whose sequence is shown; and the heavy chain variable region CDR3, which contains the amino acid whose sequence is shown in SEQ ID NO: 192.
  • the single domain antibody comprises a heavy chain variable region, which comprises and is selected from SEQ ID NO: 97, 101, 105, 109, 113, 117, 121, 125, 129,
  • the amino acid sequence of the group consisting of 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189 and 193 has at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.
  • the single domain antibody comprises a heavy chain variable region comprising a variable region selected from SEQ ID NO: 97, 101, 105, 109, 113, 117, 121, 125, 129, 133 , 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189 and 193 consisting of amino acid sequences.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 97. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 101. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 105. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:109. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 113. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:117.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 121. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 125. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 129. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 133. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:137. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:141.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 145. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:149. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 153. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 157. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 161. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 165.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 169. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 173. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 177. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:181. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 185. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:189. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 193.
  • any amino acid sequence contained in the variable region of the heavy chain may comprise up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. Amino acid substitutions, deletions and/or additions. In certain embodiments, the amino acid substitutions are conservative substitutions.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO:1 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 2; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 3.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 5 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 6; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 7.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 9 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 10; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 11.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 13 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 14; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 15.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 17 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 18; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 19.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 21 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 22; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 23.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 25 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 26; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 27.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 29 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 30; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 31.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 33 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 34; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 35.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 37 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 38; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 39.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 41 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 42; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 43.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 45 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 46; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 47.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 49 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 50; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 51.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 53 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 54; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 55.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 57 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 58; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 59.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 61 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 62; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 63.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 65 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 66; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 67.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 69 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 70; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 71.
  • the single-domain antibody cross-competes with a reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 73 The amino acids of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 74; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 75.
  • the single-domain antibody cross-competes with the reference anti-TIGIT single-domain antibody for binding to TIGIT
  • the reference anti-TIGIT single-domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 77 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 78; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 79.
  • the single domain antibody cross-competes with the reference anti-TIGIT single domain antibody for binding to TIGIT
  • the reference anti-TIGIT single domain antibody includes: heavy chain variable region CDR1, which includes SEQ ID NO: 81 The amino acid of the sequence shown; the heavy chain variable region CDR2, which includes the amino acid having the sequence shown in SEQ ID NO: 82; and the heavy chain variable region CDR3, which includes the amino acid having the sequence of SEQ ID NO: 83.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 1;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 2; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 3.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 5; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 6; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 7.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 9; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 10; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 11.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 13; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 14; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 15.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 17; the heavy chain may The variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 18; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 19.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 21; The variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 22; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 23.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 25;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 26; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 27.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 29;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 30; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 31.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 33; the heavy chain may The variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 34; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 35.
  • the single domain antibody includes a heavy chain variable region, which includes: the heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 37; the heavy chain may The variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 38; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 39.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 41;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 42; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 43.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 45; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 46; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 47.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 49;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 50; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 51.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 53;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 54; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 55.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 57;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 58; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 59.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: the heavy chain variable region CDR1, which comprises an amino acid having the sequence shown in SEQ ID NO: 61; the heavy chain may
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 62; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 63.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 65;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 66; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 67.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 69;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 70; and
  • the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 71.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 73;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 74; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 75.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 77;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 78; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 79.
  • the single domain antibody includes a heavy chain variable region, which includes: a heavy chain variable region CDR1, which includes an amino acid having the sequence shown in SEQ ID NO: 81;
  • the variable region CDR2 includes the amino acid sequence shown in SEQ ID NO: 82; and the heavy chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO: 83.
  • the single domain antibody comprises a heavy chain variable region which comprises and is selected from SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36,
  • the amino acid sequence of the group consisting of 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80 and 84 has at least about 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences with 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.
  • the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 , 44, 48, 52, 56, 60, 64, 68, 72, 76, 80 and 84 amino acid sequences.
  • any amino acid sequence contained in the variable region of the heavy chain may comprise up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. Amino acid substitutions, deletions and/or additions. In certain embodiments, the amino acid substitutions are conservative substitutions.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:4. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:8. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:20. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:24.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:28. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:32. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:40. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:44. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 48.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:52. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:56. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:60. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 64. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 72.
  • the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 76. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:80. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 84.
  • single domain antibodies comprise a humanized framework.
  • the humanized framework comprises a framework sequence selected from the heavy chain variable region sequence of the group consisting of SEQ ID NO: 85-93.
  • the humanized framework comprises an FR2 sequence selected from the heavy chain variable region sequence of the group consisting of SEQ ID NO: 85-93.
  • the anti-TIGIT antibody does not comprise an Fc region.
  • the anti-TIGIT antibody comprises an Fc region.
  • the Fc region comprises a human Fc region.
  • the Fc region comprises an Fc region selected from the group consisting of Fc regions of IgG, IgA, IgD, IgE, and IgM.
  • the Fc region comprises an Fc region selected from the group consisting of Fc regions of IgG1, IgG2, IgG3, and IgG4.
  • the Fc region comprises an IgG1 Fc region.
  • the IgG1 Fc region contains one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region contains mutations of L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the IgG1 Fc region contains mutations of S239D, A330L, and I332E. In certain embodiments, the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO: 194.
  • the heavy chain variable region is connected to the Fc region through a linker.
  • the linker is a peptide linker.
  • the peptide linker comprises about 4 to about 30 amino acids.
  • the peptide linker comprises about 4 to about 15 amino acids.
  • the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 195-220.
  • the anti-TIGIT antibody comprises a multispecific antibody, such as a bispecific antibody, full-length immunoglobulin, single chain Fv (scFv) fragment, Fab fragment, Fab' fragment, F(ab')2 , Fv fragment, disulfide bond stabilized Fv fragment (dsFv), (dsFv)2, VHH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody, tetrabody or any Their combination.
  • the antibody comprises a multispecific antibody (e.g., a bispecific antibody) that includes a second antibody portion that specifically binds to a second antigen.
  • the second antigen is a tumor-associated antigen.
  • the tumor-associated antigen is selected from the group consisting of Her-2, EGFR, PD-L1, c-Met, B cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X) , Carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial Glycoprotein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epiglin-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2, 3 , 4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor
  • FBP fetal
  • the second antigen is an immune checkpoint modulator.
  • the immune checkpoint modulator is selected from the group consisting of PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof.
  • the anti-TIGIT antibody is conjugated to a therapeutic agent or label.
  • the label is selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes.
  • the antigen binding portion of the antibody or multispecific antibody disclosed herein has a high binding affinity for its target antigen.
  • the antibody or antigen-binding portion binds to the target with a KD of about 1 ⁇ 10 ⁇ 7 M or less.
  • the antibody or antigen-binding portion binds to the target with a KD of about 1 ⁇ 10 ⁇ 8 M or less.
  • the antibody or antigen-binding portion binds to the target with a KD of about 5 ⁇ 10 ⁇ 9 M or less.
  • the antibody or antigen-binding portion binds to the target with a KD of about 1 ⁇ 10 ⁇ 9 M or less.
  • the antibody or antigen binding portion binds to the target with a KD between about 1 ⁇ 10 -9 M to about 1 ⁇ 10 -7 M. In certain embodiments, the antibody or antigen-binding portion binds to an antibody with a target between about 1 ⁇ 10 ⁇ 9 M and about 1 ⁇ 10 ⁇ 8 M with a KD. In certain embodiments, the antibody or antigen binding portion binds to the target with a KD between about 2 ⁇ 10 ⁇ 9 M and about 1 ⁇ 10 ⁇ 8 M. In certain embodiments, the antibody or antigen binding portion binds to the target with a KD between about 2x10 -9 M to about 5x10 -8 M. In certain embodiments, the antibody or antigen-binding portion of about 1x10 -9 KD of target binding between about 5x10 -9 M M.
  • the KD of the antibody or antigen-binding portion can be determined by methods known in the art. Such methods include but are not limited to Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, -Test and peptide scan.
  • KD is measured by surface plasmon resonance measurement. For example, but not limited to, about 10 response units (RU) used at 25°C on a fixed antigen CMS chip or 3000 (Biacore, Piscataway, New Jersey (NJ)) for measurement.
  • RU response units
  • the carboxymethylated dextran biosensor chip (CMS, Biacore) is used with N-ethyl-N′-(3-dimethylaminopropyl)-carbon according to the supplier’s instructions.
  • Diimine hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are activated.
  • the antigen was diluted to 5 ⁇ g/ml (approximately 0.2 ⁇ M) with 10 mM sodium acetate at pH 4.8, and then injected at a flow rate of 5 ⁇ l/min to achieve approximately 10 response units (RU) of the coupled protein. After the antigen was injected, 1M ethanolamine was injected to block unreacted groups.
  • double serial dilutions (0.78 nM to 500 nM) of Fab in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25° C. The flow rate of 25 ⁇ l/min was injected.
  • the association rate (k on ) and the dissociation rate (k off ) are based on a simple one-to-one Langmuir association model ( Evaluation software version 3.2) is calculated by simultaneously fitting association and dissociation sensorgrams.
  • the equilibrium dissociation constant (KD) can be calculated as the ratio k0fffkon. See, for example, Chen et al., J. Mol. Biol. [Journal of Molecular Biology] 293:865-881 (1999).
  • a spectrometer such as a spectrophotometer (Aviv instrument) with a shut-off configuration or a 8000 series SLM-AMINCO TM spectrophotometer with a stirred absorption cell (measured by ThermoSpectronic), measure PBS (pH 7.2) at 25°C
  • the fluorescence emission intensity (excitation 295nm
  • the antibodies of the present disclosure comprise antigen-binding fragments or antibody fragments.
  • Antibody fragments include but are not limited to Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments, and other fragments described below.
  • Fab fragment antigen-binding fragments or antibody fragments.
  • Fab' fragment antigen-binding fragments
  • Fab'-SH fragment antigen-binding fragments
  • F(ab')2 fragment antigen-binding fragments or antibody fragments.
  • scFv fragments see, for example, Pluckthtin, The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), Pages 269-315 (1994); see also WO 93/16185; and US Patent Nos. 5,571,894 and 5,587,458.
  • the antibodies of the present disclosure may be diabodies.
  • Diabodies are antibody fragments that have two antigen binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. [Natural Medicine] 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA [National Academy of Sciences Journal] 90: 6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. [Natural Medicine] 9:129-134 (2003).
  • the antibodies of the present disclosure may comprise single domain antibodies.
  • Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody.
  • the single domain antibody is a human single domain antibody (Domantis, Waltham, Massachusetts (MA); see, for example, US Patent No. 6,248,516Bl).
  • the single domain antibody is a camelid single domain antibody.
  • the single domain antibody is VHH.
  • single domain antibodies are humanized.
  • Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of whole antibodies and the production of recombinant host cells (e.g., E. coli or phage), as described herein.
  • recombinant host cells e.g., E. coli or phage
  • the antibodies of the present disclosure that include the antigen-binding portion of a multispecific antibody are chimeric antibodies.
  • Certain chimeric antibodies are described in, for example, US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 81:6851-6855 (1984)).
  • the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region.
  • chimeric antibodies are "class-switched" antibodies in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include their antigen-binding fragments.
  • the antibodies of the present disclosure that include the antigen-binding portion of a multispecific antibody may be humanized antibodies.
  • non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibodies.
  • a humanized antibody contains one or more variable domains, where the HVR, such as CDR, (or a portion thereof) is derived from a non-human antibody, and the FR (or any portion thereof) is derived from a human antibody sequence.
  • the humanized antibody may also optionally comprise at least a part of a human constant region.
  • certain FR residues in the humanized antibody are replaced by corresponding residues from non-human antibodies (eg, antibodies derived from HVR residues), for example, to restore or improve antibody specificity or affinity .
  • Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, for example, Sims et al. J. Immunol. [Journal of Immunology] 151:2296 (1993)) ; A framework region derived from the consensus sequence of a human antibody of a specific subgroup of light chain or heavy chain variable regions (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 89 : 4285 (1992); and Presta et al. J. Immunol.
  • the antibodies of the present disclosure may be human antibodies (e.g., human domain antibodies or human DAb).
  • Human antibodies can be produced using different techniques known in the art. Human antibodies are generally found in van Dijk and van de Winkel, Curr.Opin.Pharmacol. [pharmacological review] 5:368-74 (2001), Lonberg, Curr.Opin.Immunol. [Immunology review] 20:450- 459 (2008), and Chen, Mol. Immunol. [Molecular Immunology] 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single domain antibodies (or DAb) are known in the art. See, for example, US20090307787A1, US Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.
  • Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to respond to an antigen challenge to produce a complete human antibody or a complete antibody with human variable regions.
  • Such animals usually contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or exists outside the chromosomes or is randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated.
  • Human antibodies can also be prepared by hybridoma-based methods.
  • Human myeloma and mouse human heterologous myeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol. [Journal of Immunology], 133:3001 (1984); Brodeur et al. , Monoclonal Antibody Production Techniques and Applications, pp. 51-63 Marcel Dekker, Inc., New York, 1987; and Boerner et al., J. Immunol . [Journal of Immunology], 147:86 (1991)). Li et al., Proc. Natl. Acad. Sci.
  • Trioma technology Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histopathology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology [Methods and Discoveries of Experimental and Clinical Pharmacology], 27(3): 185-91 (2005).
  • Human antibodies (such as human DAb) can also be produced by isolating Fv clone variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. The technique for selecting human antibodies from the antibody library is described below.
  • Antibody fractions can be isolated by screening combinatorial libraries for antibodies having the desired activity or multiple activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are described in, for example, Hoogenboom et al. Methods in Molecular Biology [Molecular Biology] 178:1-37 (O'Brien et al. Edited, Human Press, Totowa, New Jersey) (NJ), 2001) and further described in the following documents: McCafferty et al., Nature [Nature] 348:552-554; Clackson et al., Nature [Nature] 352:624-628 (1991); Marks et al. Human, J. Mol. Biol.
  • phage display methods by polymerase chain reaction (PCR) and cloned V H and V L, gene libraries, and recombined randomly in phage libraries, which can then be in accordance with Winter et al., Ann.Rev.Immunol. [Immunology Annual Review], 12:433-455 (1994) describes screening for antigen-binding phages. Phages usually display antibody fragments as scFv fragments or Fab fragments. Libraries from immunogenic sources can provide high-affinity antibodies against immunogens without the need to construct hybridomas.
  • PCR polymerase chain reaction
  • a natural library (for example, obtained from humans) can be cloned without any immunization as described in Griffiths et al., EMBO J [Journal of the European Society of Molecular Biology], 12:725-734 (1993) to provide A single source of antibodies against a wide range of non-self and self-antigens.
  • a natural library for example, obtained from humans
  • EMBO J Journal of the European Society of Molecular Biology]
  • 12:725-734 (1993) to provide A single source of antibodies against a wide range of non-self and self-antigens.
  • Patent publications describing human antibody phage libraries include: US Patent No. 5,750,373 and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/ 0002360.
  • An antibody or antibody fragment isolated from a human antibody library is considered herein to be a human antibody or human antibody fragment.
  • amino acid sequence variants of the disclosed antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, but are not limited to, deletion and/or insertion and/or substitution of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final (i.e., modified) antibody has the desired properties (eg, antigen binding).
  • antibody variants with one or more amino acid substitutions are provided.
  • Target sites for substitution mutagenesis include HVR (or CDR) and FR.
  • Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions”. More substantial changes are provided in Table 2 under the heading of "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes.
  • Amino acid substitutions can be introduced into the antibody of interest, and the product screened for the desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val
  • Amino acids can be grouped according to common side chain characteristics: (1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe. In certain embodiments, non-conservative substitutions will require the exchange of members of one of these categories for another category.
  • one type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody.
  • the resulting variants selected for further research will have modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity, decreased immunogenicity) and/or will substantially retain Some biological characteristics of the parental antibody are described.
  • Exemplary substitution variants are affinity matured antibodies, which can be conveniently produced, for example, using phage display-based affinity maturation techniques (such as those disclosed herein).
  • one or more HVR (or CDR) residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (such as binding affinity).
  • Changes can be made in the HVR (or CDR), for example to improve antibody affinity.
  • HVR or CDR
  • Such changes can be made in HVR (or CDR) "hot spots” (ie residues encoded by codons that undergo mutations at a high frequency during the somatic maturation process) (see, for example, Chowdhury, Methods Mol. Biol. [Molecular Biological methods] 207:179-196 (2008)) and/or SDR (a-CDR), and test the binding affinity of the obtained variant VH or VL.
  • Affinity maturation through construction and reselection from secondary libraries has been described in, for example, Hoogenboom et al.
  • affinity maturation Diversity is introduced by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis) to select the variable for maturation In the genes. Then a secondary library is generated. The library is then screened to identify any antibody variants with the desired affinity.
  • methods e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis
  • HVR HVR
  • CDR CDR
  • Alanine scanning mutagenesis or modeling can be used, for example, to specifically identify HVR (or CDR) residues involved in antigen binding.
  • CDR-H3 and CDR-L3 are often targets.
  • substitutions, insertions, or deletions can occur within one or more HVRs (or CDRs), as long as such changes do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes e.g., conservative substitutions as provided herein
  • HVRs or CDRs
  • Such changes may be outside of HVR (or CDR) "hot spots" or CDRs.
  • each HVR (or CDR) is unaltered or contains no more than one, two or three amino acid substitutions.
  • alanine scanning mutagenesis a useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis.
  • the residue or residue group of the target residue is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu), and neutral or negatively charged amino acids (e.g., Alanine or polyalanine) substitution to determine whether the interaction between the antibody and the antigen is affected. Additional substitutions can be introduced at amino acid positions, demonstrating functional sensitivity to the initial substitution.
  • the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. You can filter the variants to determine whether they contain the required attributes.
  • Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions, ranging in length from one residue to polypeptides containing one hundred or more residues, and insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with N-terminal methionyl residues.
  • Other insertional variants of antibody molecules include fusions to the N- or C-terminus of the antibody with enzymes (for example, for ADEPT) or polypeptides that increase the serum half-life of the antibody.
  • portions of the antibody are modified to increase or decrease the degree of glycosylation of the construct.
  • the addition or deletion of glycosylation sites to an antibody can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites.
  • the carbohydrates attached to it can be changed.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally connected by a bond to an Fc region N- C H 2 domain of Asn297. See, for example, Wright et al. TIBTECH 15:26-32 (1997).
  • Oligosaccharides can include a variety of carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure .
  • the oligosaccharides in the antibody portion can be modified to produce antibody variants with certain improved properties.
  • the antibody portion has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region.
  • the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose in the sugar chain of Asn297, relative to all sugar structures attached to Asn297 measured by MALDI-TOF mass spectrometry (e.g., complex, hybrid, and high mannose The sum of sugar structure) is as described in WO 2008/077546.
  • Asn297 refers to the asparagine residue at position 297 in the Fc region (EU numbering of residues in the Fc region); however, due to minor sequence changes in antibodies, Asn297 can also be located about ⁇ 3 amino acids upstream or downstream of position 297 , That is, between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of cell lines capable of producing defucosylated antibodies include protein-fucosylation-deficient Lec13CHO cells (Ripka et al. Arch.Biochem.Biophys. [Biochemistry and Biophysics Collection] 249:533- 545 (1986); US Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.), and knock-out cell lines, such as ⁇ -1,6-fucosyltransferase Gene FUT8, knock out CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng.
  • the antibody portion has bisected oligosaccharides, for example, where the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc.
  • Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
  • Also provided are antibody variants having at least one galactose residue linked to the Fc region on the oligosaccharide. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • one or more amino acid modifications may be introduced into the Fc region (e.g., scFv-Fc) of the antibody portion to generate Fc region variants.
  • the Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.
  • Fc fragments with some (but not all) effector functions make the fragments an ideal candidate for applications in which the half-life of the antibody part in vivo is important. But some effector functions (for example, complement and ADCC) are unnecessary or harmful.
  • In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity.
  • an Fc receptor (FcR) binding assay can be performed to ensure that the antibody has no Fc ⁇ R binding ability (and therefore may lack ADCC activity), but can retain FcRn binding ability.
  • NK cells The primary cells used to mediate ADCC, NK cells, only express FcyRIII, while monocytes express FcyRI, FcyRII, and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, in Table 2 on page 464 of Annu. Rev. Immunol. [Annual Review of Immunology] 9:457-492 (1991).
  • a non-limiting example of an in vitro assay for assessing the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (for example, see Hellstrom, I. et al., Proc. Nat'l Acad. Sci.
  • a non-radioactive assay method can be used (for example, see ACTI TM Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc.) Mountain View, California; and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells.
  • the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models such as Clynes et al., Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences] 95:652-656 (1998).
  • a C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity.
  • C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods [Journal of Immunological Methods] 202:163 (1996); Cragg, MS et al., Blood [blood] 101: 1045 -1052 (2003); and Cragg, MS and MJ Glennie, Blood [Blood] 103: 2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life determination can also be performed using methods known in the art (for example, see: Petkova, SB et al., Int'l. Immunol. [International Immunology] 18(12): 1759-1769 (2006) )).
  • Antibodies with reduced effector function include (US Patent No. 6,737,056), antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329.
  • Fc mutants include Fc mutants having substitutions of two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" having residues 265 and 297 substituted by alanine Fc mutant (US Patent No. 7,332,581).
  • Described herein are certain antibody variants that have increased or decreased binding to FcR. (See, for example, U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. [Journal of Biological Chemistry] 9(2): 6591-6604 (2001).)
  • the Fc fragment is an IgG1 Fc fragment. In certain embodiments, the IgG1 Fc fragment contains the L234A mutation and/or the L235A mutation. In certain embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In certain embodiments, the Fc fragment is an IgG4 Fc fragment containing S228P, F234A and/or L235A mutations.
  • the antibody portion comprises an Fc region with one or more amino acid substitutions, these substitutions (e.g., substitutions at positions 298, 333, and/or 334 in the Fc region (EU numbering of residues)) improve ADCC.
  • changes in the Fc region result in changes in C1q binding and/or complement dependent cytotoxicity (CDC) (ie, increase or decrease), for example, as in US Patent No. 6,194,551, WO 99/51642 and Idusogie et al., J. Immunol. [Journal of Immunology], 164: 4178-4184 (2000).
  • CDC complement dependent cytotoxicity
  • the antibody portion (e.g., scFv-Fc) variant comprises a variant Fc region comprising one or more altered half-life and/or alteration with neonatal Fc receptor (FcRn) Combined amino acid substitution.
  • FcRn neonatal Fc receptor
  • An antibody with an extended half-life and improved binding to the neonatal Fc receptor (FcRn) which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. [Journal of Immunology] 117:587 (1976) and Kim et al., J. Immunol. [Journal of Immunology] 24:249 (1994)), as described in US2005/0014934A1 (Hinton et al.).
  • Those antibodies comprise an Fc region with one or more amino acid substitutions, where these substitutions change the binding of the Fc region to FcRn.
  • Such Fc variants include those with substitutions on one or more Fc region residues (e.g., substitution of Fc region residue 434) (US Patent No. 7,371,826).
  • cysteine engineered antibody portion such as "thioMAb", in which one or more residues of the antibody are substituted with cysteine residues.
  • the substituted residues appear at the accessible site of the antibody.
  • the reactive thiol group is thus located at the accessible site of the antibody and can be used to couple the antibody to other parts, such as drug moieties or linker-drug moieties, to generate immunity Conjugates, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the Fc region of the heavy chain.
  • the cysteine engineered antibody portion can be produced as described in, for example, U.S. Patent No. 7,521,541.
  • the antibody portions described herein may be further modified to include other non-protein portions known in the art and readily available.
  • Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3 -Dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly(n-vinylpyrrolidone) )
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer can have any molecular weight and can be branched or unbranched.
  • the number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules.
  • the amount and/or type of polymer used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the antibody to be improved, and whether antibody derivatives are used for definite diagnosis Conditions etc.
  • the antibody portion may be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof.
  • biologically active or “biologically active” refers to the biological activity shown to perform a specific function in the body. For example, it may mean binding to a specific biomolecule (eg protein, DNA, etc.) and then promoting or inhibiting the activity of this biomolecule.
  • biologically active proteins or fragments thereof include: proteins and polypeptides administered to patients as active drug substances; proteins and polypeptides used for the prevention or treatment of diseases or disorders, and for diagnostic purposes (such as diagnostic tests or Enzymes used in in vitro assays); and proteins and polypeptides (e.g., vaccines) administered to patients for disease prevention.
  • the subject matter of the present disclosure also provides isolated nucleic acids encoding the antibodies disclosed herein.
  • the isolated nucleic acid may encode the amino acid sequence of the VL of the antibody and/or the amino acid sequence of the VH of the antibody, such as the light chain and/or heavy chain of the antibody.
  • the nucleic acid may be present in one or more vectors (e.g., expression vectors).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vector expression vectors can direct the expression of genes to which they are operably linked.
  • expression vectors used in recombinant DNA technology are often in the form of plasmids (vectors).
  • the disclosed subject matter is intended to include other forms of expression vectors with equivalent functions, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).
  • the different portions of the antibodies disclosed herein can be constructed in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors.
  • elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, for example, FGF-1 IRES, FGF-2IRES, VEGF IRES, IGF-II IRES, NF -kB IRES, RUNX1IRES, p53IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornavirus IRES, polio virus IRES and encephalomyocarditis virus IRES) and cleavable linkers (such as 2A peptides, such as P2A, T2A, E2A and F2A peptides).
  • Combinations of retroviral vectors and suitable packaging lines are also suitable, where the capsid protein will have the function of infecting human cells.
  • a variety of cell lines producing amphoteric viruses are known, including but not limited to PA12 (Miller et al. (1985) Mol. Cell. Biol. [Molecular Cell Biology] 5:431-437); PA317 (Miller et al. (1986) Mol Cell. Biol. [Molecular Cell Biology] 6: 2895-2902); and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 85: 6460-6464).
  • Non-amphiphilic particles are also suitable, such as coating with VSVG, RD114 or GALV and any other pseudotyped particles known in the art.
  • the nucleic acid encoding the antibody of the present disclosure and/or one or more vectors including the nucleic acid may be introduced into the host cell.
  • nucleic acids can be introduced into cells by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with viruses or phage vectors containing nucleic acid sequences, and cells Fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • the host cell may include, for example, a host cell that has been transformed with a vector comprising a nucleic acid encoding an amino acid sequence comprising a single domain antibody and/or a VH of a single domain antibody.
  • the host cell may include, for example, a host cell that has been transformed with: (1) a vector comprising a nucleic acid encoding the amino acid sequence of the VL of the antibody and the amino acid sequence of the VH of the antibody, Or (2) a first vector comprising a nucleic acid encoding the amino acid sequence of the VL of the antibody, and a second vector comprising a nucleic acid encoding the amino acid sequence of the VH of the antibody.
  • the host cell is eukaryotic, such as Chinese Hamster Ovary (CHO) cells or lymphoid cells (eg, YO, NSO, Sp20 cells).
  • the method of preparing the antibodies disclosed herein may include culturing the host cell into which the nucleic acid encoding the antibody has been introduced under conditions suitable for antibody expression, and optionally culturing from the host cell and/or the host cell
  • the antibody is recovered from the base.
  • the antibody is recovered from the host cell by chromatographic techniques.
  • the nucleic acid encoding the antibody as described above can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • Such nucleic acids can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding genes encoding the heavy and light chains of antibodies).
  • Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein.
  • antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required.
  • For expression of antibody fragments and polypeptides in bacteria see, for example, U.S. Patent Nos.
  • eukaryotic microorganisms such as filamentous fungi or yeasts
  • fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in Partially or fully human glycosylation pattern antibody.
  • Suitable host cells for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells.
  • plant cell cultures can be used as host cells. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describes PLANTIBODIES TM technology for producing antibodies in transgenic plants).
  • vertebrate cells can also be used as hosts.
  • mammalian cell lines suitable for growth in suspension may be useful.
  • a non-limiting example of a useful mammalian host cell line is the monkey kidney CV1 line transformed by SY40 (COS-7); the human embryonic kidney line (293 or 293 cells, as for example in Graham et al., J Gen Viral. Journal of Virology] 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, for example in Mather, Biol. Reprod.
  • techniques for preparing bispecific and/or multispecific antibodies include, but are not limited to, recombinant expression of two immunoglobulin heavy chain light chain pairs with the same specificity, one or two of which The chain or light chain is fused to an antigen-binding portion with different specificities (for example, single domain antibodies, such as VHH), and the recombinant co-expression of two immunoglobulin heavy chain light chain pairs with different specificities (see Milstei'n and Cuello, Nature [Nature] 305:537 (1983)), PCT Patent Application No.
  • Bispecific antibodies can also be prepared by engineering the electrostatic steering effect to prepare antibody Fc-heterodimer molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, for example, U.S. Patent No.
  • the bispecific and multispecific molecules of the present disclosure can also use chemical techniques (see, for example, Kranz (1981) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 78: 5807), "polyoma” Technology (see, for example, U.S. Patent 4,474,893) or recombinant DNA technology. It is also possible to use methods known in the art and described herein to prepare bispecific and multi-specificity of the currently disclosed subject matter by conjugating constitutive binding specificities, such as the first epitope and second epitope binding specificities. Specific molecules.
  • each binding specificity of bispecific and multispecific molecules can be produced together by recombinant fusion protein technology, or can be produced separately and then conjugated to each other.
  • the binding specificity is protein or peptide
  • a variety of coupling agents or cross-linking agents can be used for covalent binding.
  • Non-limiting examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-( 2-pyridyldithio)propionate (SPDP) and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) )
  • SATA N-succinimidyl-S-acetyl-thioacetate
  • SPDP N-succinimidyl-3-( 2-pyridyldithio)propionate
  • sulfo-SMCC sulfo-SMCC
  • binding specificities are antibodies (for example, two humanized antibodies), they can be conjugated by sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains.
  • the hinge region may be modified to include an odd number (e.g., one) sulfhydryl residue.
  • the two binding specificities of the bispecific antibody can be encoded in the same vector, and expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecules are MAb x MAb, MAb x Fab, Fab x F(ab')2 or ligand x Fab fusion proteins.
  • the bispecific antibody of the present disclosure may be a single-chain molecule, such as a single-chain bispecific antibody, a single-chain bispecific molecule comprising a single-chain antibody and a binding determinant, or a single-chain bispecific molecule comprising two binding determinants. Clusters of single-stranded bispecific molecules.
  • Bispecific and multispecific molecules may also be single-stranded molecules or may comprise at least two single-stranded molecules.
  • Methods of preparing bispecific and multispecific molecules are described in, for example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; US Patent No. 5,258,498; and US Patent No. 5,482,858.
  • engineered antibodies with three or more functional antigen binding sites e.g., epitope binding sites
  • octopus antibodies see, e.g., US 2006/0025576A1.
  • animal systems can be used to produce the antibodies of the present disclosure.
  • One animal system used to prepare hybridomas is the murine system.
  • hybridomas in mice is a very well established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g. murine myeloma cells) and fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual], Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ( Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York)).
  • Fusion partners e.g. murine myeloma cells
  • fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual], Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ( Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York)).
  • the antibodies of the present disclosure provided herein can be identified, screened or characterized by various assays known in the art and provided herein for their physical/chemical properties and/or biological activities.
  • the antigen binding activity of the antibodies of the present disclosure can be tested by known methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or western blot assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • western blot assay Each of these assays typically detects the presence of protein-antibody complexes of particular interest by using labeled reagents (e.g., antibodies) specific for the complex of interest.
  • labeled reagents e.g., antibodies
  • an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody can be used to detect the antibody.
  • any of a variety of other immunoassays can be used to detect antibodies.
  • the antibody can be radiolabeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays [principles of radioimmunoassays], Seventh Training Course on Radiolig and Assay Techniques [About Radioimmunoassays] The seventh training course on body measurement technology], The Endocrine Society [Endocrine Society], March 1986, which is incorporated herein by reference).
  • the radioisotope can be detected by such means as the use of a Geiger counter or a scintillation counter or by autoradiography.
  • competition assays can be used to identify antibodies that compete with antibodies of the present disclosure (e.g., 1C12, 2A3, or 1G1) for binding to TIGIT.
  • such competitive antibodies bind to the same epitope (e.g., linear or conformational epitope) bound by 1C12, 2A3, or 1G1.
  • Morris 1996 "Epitope Mapping Protocols [Epitope Mapping Protocol]," Methods in Molecular Biology [Molecular Biology], Volume 66 (Human Press), Totowa, New Jersey (NJ) provides a detailed exemplary method for locating the epitope bound by the antibody.
  • the immobilized TIGIT can be incubated in a solution containing a first labeled antibody (for example, 1C12, 2A3, or 1G1) and a second unlabeled antibody that binds to TIGIT, and the second unlabeled antibody can be tested.
  • a first labeled antibody for example, 1C12, 2A3, or 1G1
  • a second unlabeled antibody that binds to TIGIT
  • the second antibody may be present in the hybridoma supernatant.
  • the immobilized TIGIT was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody.
  • the present disclosure provides an assay for identifying its anti-TIGIT antibodies with biological activity.
  • the biological activity may include, for example, activation of immune cells or immune activation of reporter genes (such as NFAT reporter genes).
  • reporter genes such as NFAT reporter genes.
  • the subject of the present disclosure also provides immunoconjugates, which comprise a combination with one or more detection probes and/or cytotoxic agents (e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, bacteria, Enzymatically active toxins of fungal, plant or animal origin, or fragments thereof)) or radioisotope-conjugated antibodies disclosed herein.
  • cytotoxic agents e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, bacteria, Enzymatically active toxins of fungal, plant or animal origin, or fragments thereof)
  • toxins e.g., protein toxins, bacteria, Enzymatically active toxins of fungal, plant or animal origin, or fragments thereof
  • radioisotope-conjugated antibodies disclosed herein e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, bacteria, Enzymatically active
  • the immunoconjugate is an antibody drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoid (see U.S. Patent No. 5,208,020, 5,416,064 and European Patent EP 0 425235); auristatin, such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); (dolastatin); Calicheamicin or its derivatives (see U.S. Patent Nos.
  • ADC antibody drug conjugate
  • drugs including but not limited to maytansinoid (see U.S. Patent No. 5,208,020, 5,416,064 and European Patent EP 0 425235); auristatin, such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,
  • the immunoconjugate comprises the antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, non-binding activity of diphtheria toxin Fragment, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, acacia soytoxin A chain, capsula root toxin A chain, ⁇ -crytoxin, tung protein , Carnation toxin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, jatropha toxin protein, crotontoxin protein, sapaonaria officinalis inhibition Drugs, leucocephala toxin, mitogellin, restrictocin, phenomycin, economycin and trichothecins.
  • an enzymatically active toxin or fragment thereof including but not limited to diphtheria A chain, non
  • the immunoconjugate comprises an antibody described herein that is conjugated to a radioactive atom to form a radioconjugate.
  • a radioactive atom to form a radioconjugate.
  • a variety of radioisotopes can be used to produce radioconjugates. Non-limiting examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu.
  • radioconjugate When used for detection, it can include radioactive atoms used in scintillation imaging studies, such as tc99m or 1123, or autonomy used in nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI) Rotation labels, such as iodine 123, iodine 131, indium 11, fluorine 19, carbon 13, nitrogen 15, oxygen 17, gadolinium, manganese, or iron.
  • NMR nuclear magnetic resonance
  • MRI magnetic resonance imaging
  • Rotation labels such as iodine 123, iodine 131, indium 11, fluorine 19, carbon 13, nitrogen 15, oxygen 17, gadolinium, manganese, or iron.
  • bifunctional protein coupling agents can be used (e.g., N-succinimidyl-3-(2-pyridinedimercapto)propionate (SPDP), succinimidyl-4-(N-maleic acid) Imidomethyl) cyclohexane-l-carboxylate (SMCC), iminosulfane (IT), bifunctional derivatives of imidate (such as dimethyl adipimidate HCl), Active esters (such as suberic acid disuccinimidyl ester), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl) hexamethylene diamine), double nitrogen derivatives (such as Bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bi-reactive fluorine compounds (such as 1,5-difluoro-2,4 -Dinitrobenzen
  • the ricin immunotoxin can be prepared as described in Vitetta et al., Science [Science], 238:1098 (1987).
  • Carbon 4-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine-pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies .
  • the linker may be a "cleavable linker" that promotes the release of cytotoxic drugs from the cell.
  • acid-labile linkers, peptidase-sensitive linkers, light-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. [Cancer Research] 52:127-131 (1992); US Patent No. 5,208,020).
  • the immunoconjugates or ADCs herein explicitly cover but are not limited to such conjugates prepared with cross-linking agents, including but not limited to commercially available BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP , SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, and Sulfo-SMPB, and SVSB ( Succinimidyl-(4-vinylsulfone) benzoate) (for example, from Pierce Biotechnology, Inc., Rockford, IL., USA).
  • the subject matter of the present disclosure further provides methods of using the disclosed antibodies (e.g., anti-TIGIT antibodies). In certain embodiments, these methods involve the therapeutic use of the currently disclosed antibodies. In certain embodiments, these methods involve diagnostic uses of the currently disclosed antibodies.
  • antibodies and/or pharmaceutical compositions comprising the antibodies disclosed herein can be administered to a subject (e.g., a mammal (e.g., human)) to treat diseases and disorders or to increase an immune response.
  • these diseases and conditions involve immune checkpoint suppression and/or abnormal TIGIT activity.
  • diseases and conditions that can be treated by the antibodies disclosed herein include, but are not limited to, neoplasia (e.g., cancer).
  • the present disclosure provides the anti-TIGIT antibodies (or fragments thereof) described herein for use in the manufacture of drugs. In certain embodiments, the present disclosure provides the anti-TIGIT antibodies (or fragments thereof) described herein for use in the manufacture of drugs for the treatment of cancer. In certain embodiments, the present disclosure provides the anti-TIGIT antibodies (or fragments thereof) described herein for use in the treatment of cancer in a subject. In certain embodiments, the present disclosure provides pharmaceutical compositions comprising the anti-TIGIT antibodies (or fragments thereof) provided herein for the treatment of cancer in a subject.
  • the cancer may be blood cancer (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer , Skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma and a variety of cancers (including prostate cancer and small cell lung cancer).
  • blood cancer e.g., leukemia, lymphoma, and myeloma
  • ovarian cancer breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer , Skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue s
  • Suitable cancers also include any known cancers in the field of oncology, including but not limited to astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblasts Tumor, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchoalveolar carcinoma , Epithelial adenocarcinoma, and its liver metastases, lymphangiosarcoma, lymphatic endothelial sarcoma, liver cancer, cholangiocarcinoma, synovial tumor, mesothelioma, Ewing’s tumor, rhab
  • the cancer may be melanoma, NSCLC, head and neck cancer, urothelial cancer, breast cancer (e.g., triple-negative breast cancer, TNBC), gastric cancer, cholangiocarcinoma, classic Hodgkin’s lymphoma ( cHL), non-Hodgkin’s lymphoma, primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (for example, small cell lung cancer), esophageal cancer, nasopharyngeal cancer (NPC), biliary tract Cancer, colorectal cancer, cervical cancer or thyroid cancer.
  • breast cancer e.g., triple-negative breast cancer, TNBC
  • gastric cancer cholangiocarcinoma
  • cHL classic Hodgkin’s lymphoma
  • NHL PMBCL primary mediastinal B-cell lymphoma
  • mesothelioma ovarian cancer
  • lung cancer for
  • the subject to be treated is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In certain embodiments, the subject is a human. In certain embodiments, the subject is suspected of having cancer or is at risk of cancer, or has been diagnosed with cancer with abnormal TIGIT expression or activity, or any other disease.
  • a mammal e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.
  • the subject is a human.
  • the subject is suspected of having cancer or is at risk of cancer, or has been diagnosed with cancer with abnormal TIGIT expression or activity, or any other disease.
  • the diagnostic methods for many cancers or any other diseases that exhibit abnormal TIGIT activity and the clinical description of these diseases are known in the art. Such methods include, but are not limited to, for example, immunohistochemistry, PCR, fluorescence in situ hybridization (FISH). Other details on the diagnostic method of abnormal TIGIT activity or expression are described in, for example, Gupta et al. (2009) Mod Pathol. [Modern Pathology] 22(1): 128-133; Lopez-Rios et al. (2013) J Clin Pathol .[Journal of Clinical Pathology]66(5):381-385; Ellison et al. (2013) J Clin Pathol[Journal of Clinical Pathology] 66(2):79-89; And and Guha et al. (2013) PLoS ONE [Public Science Library ⁇ General] 8(6):e67782.
  • the anti-TIGIT antibodies (or fragments thereof) and/or compositions provided herein are combined with a second, third, or fourth agent (including, for example, antineoplastic agents, growth inhibitors, cytotoxic agents, or chemotherapeutic agents). ) Administered in combination to treat diseases or disorders involving abnormal TIGIT activity.
  • Such agents include, for example, docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil and folinic acid), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (anti-VEGF antibody) , FOLFOX-4, infusion of fluorouracil, leucovorin and oxaliplatin, afatinib, gemcitabine, capecitabine, pemetrexed, tevantinib, everolimus, CpG-ODN, rapa Mycin, lenalidomide, verofenib, endostatin, lapatinib, PX-866, Imprime PGG and ilotinib.
  • the anti-TIGIT antibody (or fragment thereof) is conjugated to another agent.
  • the anti-TIGIT antibodies (or fragments thereof) and/or compositions provided herein are administered in combination with one or more other therapies (e.g., radiation therapy, surgery, chemotherapy, and/or targeted therapy) .
  • the anti-TIGIT antibodies (or fragments thereof) and/or compositions provided herein are administered in combination with radiation therapy.
  • the combination of anti-TIGIT antibodies (or fragments thereof) and/or compositions provided herein with radiation therapy is used to treat neoplasms or cancers disclosed herein.
  • the anti-TIGIT antibodies or fragments thereof provided herein will be administered at a dose effective to treat the indication while minimizing toxicity and side effects.
  • a typical dosage may be, for example, in the range of 0.001 to 1000 ⁇ g; however, dosages lower or higher than this exemplary range are within the scope of the present invention.
  • the daily dose may be about 0.1 ⁇ g/kg to about 100 mg/kg of total body weight, about 0.1 ⁇ g/kg to about 100 ⁇ g/kg of total body weight, or about 1 ⁇ g/kg to about 100 ⁇ g/kg of total body weight.
  • the efficacy of treatment or prevention can be monitored by regularly evaluating treated patients. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs.
  • the desired dose can be delivered by administering the composition by a single bolus injection, by administering the composition by multiple bolus injections, or by administering the composition by continuous infusion.
  • compositions containing anti-TIGIT antibodies or fragments thereof can be administered once, twice, three or four times a day.
  • the composition may also be administered less frequently than daily administration, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, Once every three weeks, once a month, once every two months, once every three months, or once every six months.
  • the composition can also be administered, for example, in a sustained-release formulation in an implant that gradually releases the composition for use over a period of time, and allows the composition to be administered at a lower frequency, such as once a month, every time 2-6 months once, once a year, or even a single application.
  • Sustained release devices e.g. pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.
  • the antibody may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three, or four times daily.
  • the composition may also be administered less frequently than daily administration, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, Once every three weeks, once a month, once every two months, once every three months, or once every six months.
  • the antibody (or fragment thereof) can also be administered in a sustained-release formulation, for example, in an implant that gradually releases the composition for use over a period of time, and allows the composition to be administered at a lower frequency, such as every Once a month, once every 2-6 months, once a year, or even a single administration.
  • Sustained release devices (such as pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) can be administered by injection or surgical implantation in various locations.
  • Cancer treatment can be evaluated by, for example, but not limited to, tumor regression, tumor weight or size reduction, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression, and/or activity.
  • Methods to determine the efficacy of the therapy can be used, including, for example, measuring the response by radiography.
  • the efficacy of treatment is measured as the percentage of tumor growth inhibition (%TGI), calculated using the equation 100-(T/C x 100), where T is the average relative tumor volume of the treated tumor, and C Is the average relative tumor volume of untreated tumors.
  • %TGI is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, About 92%, about 93%), about 94%), about 95%, or more than 95%.
  • the labeled anti-TIGIT antibodies, fragments thereof, and derivatives and analogs thereof can be used for diagnostic purposes to detect, diagnose or monitor diseases and/or disorders related to the expression, abnormal expression and/or activity of TIGIT.
  • the anti-TIGIT antibodies (or fragments thereof) provided herein can be used in in situ, in vivo, ex vivo, and in vitro diagnostic assays or imaging assays.
  • the method for detecting TIGIT polypeptide expression includes (a) using one or more antibodies of the present invention to determine the expression of the polypeptide in the cells (such as tissue) or body fluid of an individual, and (b) comparing the gene expression level with a standard Gene expression level, where an increase or decrease in the measured gene expression level compared to the standard expression level is indicative of abnormal expression.
  • Additional embodiments provided herein include methods for diagnosing diseases or disorders related to the expression or abnormal expression of TIGIT in animals (e.g., mammals, such as humans). These methods involve the detection of TIGIT molecules in mammals.
  • the diagnosis includes: (a) administering an effective amount of labeled anti-PD-1 antibody (or a fragment thereof) to the mammal; (b) waiting for a period of time after administration to allow the labeled Anti-TIGIT antibodies (or fragments thereof) are preferentially concentrated in the subject's TIGIT molecule-expressing site (and the unbound labeled molecules are eliminated to background levels); (c) determine the background level; and (d) detect the subject A labeled molecule in the human body such that the detection of a labeled molecule above the background level indicates that the subject has a specific disease or disorder related to the expression or abnormal expression of TIGIT.
  • the background level can be determined by different methods, which include comparing the amount of labeled molecules detected with a standard value previously determined for a specific system.
  • the anti-TIGIT antibodies (or fragments thereof) provided herein can be used to determine protein levels in biological samples using classical immunohistological methods known to those skilled in the art (for example, see Jalkanen et al., J. Cell. Biol. The Journal of Biology] 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. [Journal of Cell Biology] 105:3087-3096 (1987)).
  • Other antibody-based methods that are useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels include enzyme labels (e.g., glucose oxidase); radioisotopes, such as iodine (131 I, 125 I, 123 I, 121 I), carbon (14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In, 112 In, 111 In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), Palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc , 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru; Luminol; and fluorescent labels (such as fluorescein and rhodamine and biotin).
  • enzyme labels e.
  • the level of nucleic acid or mRNA encoding a TIGIT polypeptide in a cell can be measured, for example, via fluorescence in situ hybridization using a nucleic acid-based probe corresponding to a nucleic acid encoding EGFR or its complementary sequence; (FISH; See WO 98/45479 published in October 1998), Southern blotting, Northern blotting or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR).
  • TIGIT overexpression can also be studied by measuring shedding antigens in biological fluids (e.g., serum), for example, using antibody-based assays (see also, e.g., U.S. Patent No.
  • cells in a mammal can be exposed to an antibody, which is optionally labeled with a detectable label (e.g., a radioisotope), and can be taken from a mammal previously exposed to the antibody, for example, by external scanning radioactivity or by analysis To evaluate the binding of antibodies to cells (e.g., biopsy or other biological samples).
  • a detectable label e.g., a radioisotope
  • the subject matter of the present disclosure also provides pharmaceutical formulations comprising one or more of the antibodies disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may comprise a combination of multiple (e.g., two or more) antibodies and/or antigen binding portions thereof of the currently disclosed subject matter.
  • the pharmaceutical composition of the present disclosure may include one or more anti-TIGIT antibodies.
  • the disclosed pharmaceutical formulations can be prepared by combining an antibody of the required purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences [Remington's Pharmaceutical Sciences], 16th edition, Osol, A. editor (1980))
  • the combination is prepared in the form of a lyophilized formulation or an aqueous solution.
  • lyophilized antibody formulations are described in U.S. Patent No. 6,267,958.
  • aqueous antibody formulations may include those described in US Patent No. 6,171,586 and WO2006/044908, the latter formulations including histidine-acetate buffer.
  • the antibody may have greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than 95%, greater than about 96%, greater than About 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than A purity of about 99.8% or greater than about 99.9%.
  • the pharmaceutically acceptable carrier is generally non-toxic to the recipient at the dose and concentration used, and includes, but is not limited to: buffers (such as phosphate, citrate, and other organic acids); including ascorbic acid and methyl sulfide Antioxidants of acid; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride, phenethylammonium chloride; phenol, butanol or benzyl alcohol; alkyl Parabens (such as methyl or propyl parabens); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less (About 10 residues) polypeptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acid, such as glycine,
  • Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International).
  • sHASEGP soluble neutral active hyaluronidase glycoprotein
  • rHuPH20 Baxter International
  • Certain exemplary sHASEGP and methods of use are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968, including rHuPH20.
  • sHASEGP is combined with one or more additional glycosaminoglycanase (eg, chondroitinase).
  • the carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound ie, anti-TIGIT antibody
  • the active compound can be coated in a material to protect the compound from acids and other natural conditions that can inactivate the compound.
  • the pharmaceutical composition of the present disclosure can also be combined therapy, that is, administered in combination with other agents.
  • the pharmaceutical composition disclosed herein may also contain one or more active ingredients, which are necessary for the specific indication being treated, for example, those with complementary activities that do not adversely affect each other. Those ones.
  • the pharmaceutical formulation may include a second active ingredient for treating the same disease treated by the first therapeutic agent.
  • Such active ingredients are suitably present in combination in amounts effective for the intended purpose.
  • the formulations of the present disclosure may also contain one or more active ingredients, which are necessary for the specific indication being treated, preferably those that have complementary activities and do not adversely affect each other .
  • Such active ingredients are suitably present in combination in amounts effective for the intended purpose.
  • composition of the present disclosure can be applied by a variety of methods known in the art.
  • the route and/or mode of administration depend on the desired result.
  • These active compounds can be prepared with carriers that protect the compound to avoid rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are described by, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, editor, Marcel Decker, New York, 1978.
  • the pharmaceutical composition is produced under Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration (U.S. Food and Drug Administration).
  • GMP Good Manufacturing Practice
  • Sustained-release preparations containing the disclosed antibodies can also be prepared.
  • suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles (e.g., films or microcapsules).
  • the active ingredient may be embedded in microcapsules prepared by, for example, coacervation technology or by interfacial polymerization, such as in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions), respectively.
  • Nanoparticles and nanocapsules or hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in a coarse emulsion.
  • Such technology is disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Editor (1980).
  • the compound can be administered to the subject in a suitable carrier (e.g., liposome) or diluent.
  • suitable carrier e.g., liposome
  • diluents include saline and buffered aqueous solutions.
  • Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J Neuroimmunol. [Journal of Neuroimmunology] 7:27).
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the use of such media and reagents for pharmaceutical active substances is known in the art.
  • any conventional medium or agent is incompatible with the active compound, it may be considered for use in the pharmaceutical composition of the present disclosure.
  • Supplementary active compounds can also be incorporated into the composition.
  • the therapeutic composition must generally be sterile, substantially isotonic, and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.
  • the carrier may be a solvent or dispersion medium containing the following substances: for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersions by the maintenance of the required particle size, and by the use of surfactants.
  • isotonic agents such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride.
  • Prolonged absorption of these injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the required amount of one or more of the disclosed antibodies in combination with a suitable solvent and one or more of the ingredients listed above as required, and then performing sterilization and microfiltration (for example, by sterile microfiltration). Bacteria membrane filtration) to prepare.
  • the dispersion is prepared in the following way:
  • the active compound is incorporated into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those enumerated above.
  • a sterile vehicle that contains a basic dispersion medium and other required ingredients from those enumerated above.
  • the preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produce the active ingredient and any other from its previous sterile filtered solution. Powder of the desired ingredient.
  • Medical devices known in the art can also be used to administer the therapeutic composition.
  • the therapeutic composition of the present invention can be administered with a needle-free hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • a needle-free hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • implants and modules that can be used in the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a A therapeutic device for administering drugs through the skin; US Patent No.
  • the formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and/or parenteral administration. These formulations can conveniently be presented in unit dosage form and can be prepared by any method well known in the pharmaceutical arts.
  • the amount of antibody that can be combined with carrier materials to produce a single dosage form varies depending on the subject being treated and the particular mode of administration.
  • the amount of antibody that can be combined with a carrier material to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally, in percentage terms, this amount ranges from about 0.01% to about 99% of the active ingredient, from about 0.1% to about 70%, or from about 1% to about 30%.
  • Dosage forms for topical or transdermal administration of the composition of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.
  • the active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.
  • parenteral administration refers to modes of administration generally by injection other than enteral administration and topical administration, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, saccular Intra-orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by the sterilization procedure described above, as well as by the inclusion of different antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in these compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be caused by including agents that delay absorption, such as aluminum monostearate and gelatin.
  • the antibodies of the present disclosure when administered to humans and animals as drugs, they can be administered alone or in combination with a pharmaceutically acceptable carrier as a pharmaceutical composition comprising, for example, about 0.01% to about 99.5% (or about 0.1% to 90%) of the antibodies described herein.
  • the currently disclosed subject matter also provides an article that contains materials that can be used to treat, prevent, and/or diagnose the above-mentioned diseases.
  • the article includes a container and a label or package insert on or associated with the container.
  • suitable containers include bottles, vials, syringes, IV solution bags, and the like.
  • the container can be formed of a variety of materials, such as glass or plastic.
  • the container may contain (by itself or in combination with another composition) a composition that is effective in treating, preventing, and/or diagnosing disorders, and may have a sterile access port (for example, the container may be an intravenous solution bag or have a hypodermic injection needle Vial with pierced stopper).
  • At least one active agent in the composition is an antibody of the currently disclosed subject matter.
  • the label or package insert can indicate that the composition is used to treat the condition of choice.
  • the product may include (a) a first container containing a composition, wherein the composition contains the antibody of the present invention; and (b) a second container containing a composition Where the composition contains other cytotoxic or therapeutic agents.
  • the article of manufacture may further comprise a package insert, which indicates that the composition can be used to treat a particular condition.
  • the product may further include another container, such as a second or third container, which includes a pharmaceutically acceptable buffer, such as but not limited to bacteriostatic water for injection (BWFI), phosphate buffered saline , Ringer's solution and dextrose solution.
  • a pharmaceutically acceptable buffer such as but not limited to bacteriostatic water for injection (BWFI), phosphate buffered saline , Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate buffered saline such as but not limited to bacteriostatic water for injection (BWFI), phosphate buffered saline , Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate buffered saline such as but not limited to bacteriostatic water for injection (BWFI), phosphate buffered saline , Ringer's solution and dextrose solution.
  • Example 1 Immunization, the production of anti-human TIGIT VHH antibodies, and the discovery of sprout antibodies
  • the antigen of recombinant human TIGIT extracellular domain (ECD) protein was purchased from Arco Bio. According to protocols known in the art, llamas are used for TIGIT immunization. The titer of serum antibody was determined by ELISA assay. After 3 rounds of immunization, high titer (1:100,000) was observed. Then whole blood is collected, and PBMCs are separated. Then RNA is isolated from PBMC.
  • the VHH antibody gene was amplified by PCR, purified by DNA agarose gel, constructed into the phage vector pADL-23c (Antibody Design Labs), and transformed into TG1 electroporation Competent cells (from Lucigen).
  • the transformed TG1 cells were cultured in Y2T medium. By adding helper phage and co-cultivating overnight, phage with target VHH can be produced.
  • h-TIGIT human-TIGIT
  • cynomolgus monkey -TIGIT cyno-TIGIT
  • the conjugate of h-TIGIT or cyno-TIGIT was eluted and used to infect SS320 cells. The colonies of SS320 cells were selected and cultured in Y2T medium, and IPTG was added to secrete VHH antibodies.
  • the h-TIGIT ECD coated plate was used to screen the supernatant with VHH antibody by ELISA analysis. Select the positive h-TIGIT binding agent for sequencing. 29 clones with different sequences were selected. The binding ability of VHH antibody to cyno-TIGIT was also checked by ELISA.
  • the sequence listing (SEQ ID NO: 1-84) shows the first 21 binding agents and their CDRs and VHs.
  • the blocking ELISA assay was also used to determine the effect of the VHH antibody clone on blocking the binding of the poliovirus receptor (PVR, also known as CD155) to TIGIT.
  • PVR poliovirus receptor
  • Nine clones (clone names: 2B7, 1G1, 1C12, 3G6, 2B10, 3G7, 3F10, 13H11, and 15A5) that inhibited PVR binding to h-TIGIT by more than 90% were selected.
  • the antibody clone identified in Example 1 was constructed by adding the human constant heavy chain 2 (CH2) and constant heavy chain 3 (CH3) domains shown in FIG. 1C to prepare a bivalent antibody.
  • the constructed bivalent VHH antibody was expressed in ExpiCHO cells, and the protein in the supernatant was collected and purified by protein A.
  • the binding affinity of the bivalent clone to Jurkat cells transfected with human TIGIT was confirmed by flow cytometry.
  • electroporation was used to transfect Jurkat cells stably expressing human TIGIT with the NFAT reporter gene.
  • the cells expressing the NFAT reporter gene were selected by 300ug/ml hygromycin in the culture medium.
  • Raji cells were stably transfected with human PVR, and the transfected cells were selected with hygromycin in the culture medium at 125ug/ml.
  • 1G1 and 1C12 Two representative clones, 1G1 and 1C12, were selected to humanize their frameworks.
  • two cloned sequences were used for Igblast to search the database of human germline genes.
  • the ideal germline sequence is selected and the framework sequence is mutated to change the framework sequence from llama to human.
  • human germline IGHV-3-30*10 was used, and three forms of humanized 1C12 (1C12-EREF, 1C12-EREW and 1C12-GLEW) were made.
  • the human germline IGHV-3-30*01 was used, and six forms of humanized 1G1 (1G1-FG-ERES, 1G1-FA-ERES, 1G1-FA-EREW, 1G1-FA- GLEW, 1G1-FA-GREL and 1G1-FA-GRES).
  • the construct was cloned into an expression vector, and the antibody protein was produced by transient transfection of ExpiCHO, and purified by protein A.
  • the binding affinity of the humanized bivalent antibody to human TIGIT was determined by binding to whole cells of h-TIGIT stably expressed on Jurkat cells.
  • the antibody was incubated with h-TIGIT-transfected Jurkat cells (100 ⁇ l of 0.2 ⁇ 10 6 /well) in FACS buffer for 30 minutes. The cells were washed once and then incubated with anti-human IgG Fc AlexaFluor488 (1:500). CytoFlex was used to determine the average fluorescence intensity, and GraphPad Prism 8.0 was used for nonlinear regression to calculate the binding affinity of the antibody to h-TIGIT, as shown in Figure 3A.
  • the humanized form of the 1C12 clone has similar efficacy in blocking TIGIT. Except that the two forms, 1G1-(F-A-EREW) and 1G1-(F-A-GLEW), lost their blocking effect, the all humanized form of 1G1 has similar efficacy to the chimeric parental clone in blocking TIGIT.
  • the 1G1-F-A-ERES clone was subjected to affinity maturation. Design primers for single mutation of amino acids in each CDR region.
  • the mutation library was prepared using assembly PCR and cloned into a phage vector. The quality of the library was measured by DNA sequencing of transformed TG1 cells and clones.
  • the supernatant was checked for VHH antibodies by ELISA assay.
  • the positive clones against h- and cyno-TIGIT were selected for whole-cell combination with cells stably expressing h- and cyno-TIGIT.
  • the selected clones were also tested by PVR blocking ELISA and human TIGIT blocking NFAT reporter gene.
  • the sequence listing shows the top 25 binding agents and their CDRs and VHHs.
  • a bivalent 2A3 antibody (2A3-Fc) was constructed using human IgG1 CH2 and CH3 domains.
  • the antibody is expressed in ExpiCHO cells and purified by protein A column.
  • the Octet binding test was used to study the combination with human TIGIT Bound 2A3-Fc epitope.
  • the 2A3 clone binds to a different epitope compared to the reference Ab1 and the reference Ab2.
  • the analysis of the CDR region of 2A3 identified two hot spots, namely methionine in CDR2 and aspartic acid in CDR3, and then serine. Mutation of methionine to leucine and isoleucine, and mutation of aspartic acid to threonine and glutamic acid.
  • the CDR and VHH of the modified antibodies (2A3 ML, 2A3 MI, 2A3 ML_DT (also known as 2A3LT) and 2A3 ML_DE) are shown in the sequence listing (SEQ ID NO: 178-193).
  • the modified form was tested in whole-cell binding and NFAT luciferase reporter gene detection. Compared with the parental 2A3 clone, all these modified antibodies showed similar characteristics.
  • Figure 9 shows the representative data of 2A3-LT-Fc (2A3 ML DT), where M becomes L and D becomes T (2A3 ML DT), where compared with the parental 2A3-Fc, in the whole cell binding assay Similar human TIGIT binding affinities were observed in, and similar efficacy was observed in the NFAT reporter gene assay.
  • anti-tumor ability of anti-TIGIT antibodies continues to be explored in in vitro experiments.
  • T cells expressing TIGIT are in contact with dendritic cells (DC) expressing PVR
  • DC dendritic cells
  • the PVR expressed on the DC will bind to the TIGIT on the T cell, thereby inhibiting the anti-tumor activity of the T cell, such as anti-tumor cells Secretion of factors.
  • Effective anti-TIGIT antibody treatment can block the combination of TIGIT and PVR, thereby enhancing the ability of T cells to kill tumors.
  • Dendritic cell-T cell mixed lymphatic response is used to simulate this phenomenon that occurs in the tumor microenvironment.
  • CD14-expressing monocytes isolated from PBMCs of healthy donors were cultured in RPMI1640 medium containing 50ng/ml GM-CSF and 50ng/ml IL-4 for 7 days (change the medium on the 4th day) ) To differentiate into DC. Afterwards, the DCs were transferred to RPMI medium containing 100ng/ml LPS and cultured for 1 day to obtain mature DCs.
  • the dendritic cells obtained by this method stably express CD11c (biomarker of mature DC), MHC class II (major histocompatibility complex), CD80 (ligand of CD28), and PVR (ligand of TIGIT) ).
  • CD11c biomarker of mature DC
  • MHC class II major histocompatibility complex
  • CD80 ligand of CD28
  • PVR ligand of TIGIT
  • PerkinElmer's IL-2AlphaLisa detection kit (AlphaLisa human IL-2kit: Cat#AL221C) was used to detect the level of IL-2 in the supernatant.
  • Anti-PD1 antibody was used as a positive control, and anti-HER2 antibody was used as a negative control.
  • the 2A3-LT-Fc antibody increased the level of IL-2 secreted by T cells in a dose-dependent manner, which was comparable to the positive control anti-PD1 antibody.
  • Tiragolumab analogs did not increase the level of IL-2 secreted by T cells. Because IL-2 is an important anti-tumor cytokine, this result indicates that the anti-TIGIT antibody 2A3-LT-Fc can significantly enhance the anti-tumor ability of T cells in the tumor microenvironment, and it exhibits better anti-tumor properties than Tiragolumab analogs ability.
  • the mechanism of anti-TIGIT antibody enhancing immune function and anti-tumor activity involves not only the blocking effect of TIGIT, but also the binding effect between antigen-presenting cells (APC) and effective T cells (CD8+T cells).
  • the Fc ⁇ RIIIA binds to the Fc region of antibodies, and effectively combines the VHH domain on T cells with TIGIT.
  • two different Fc-enhanced forms were prepared, one with DLE mutations (S239D, A330L, and I332E), and one with VPVLL mutations (L235V, F243L, R292P, Y300L, and P396L).
  • the binding of the Fc mutant to human Fc ⁇ RIIIA and Fc ⁇ RIIB was checked by the Octet binding assay using the recombinant protein of the ECD of Fc ⁇ RIIIA and Fc ⁇ RIIB, and the results are shown in FIG. 12.
  • Both DLE and VPVLL mutants have enhanced binding affinity to human Fc ⁇ RIIIA.
  • the DLE mutant also has an enhanced binding affinity to human Fc ⁇ RIIB, while the VPVLL mutant has a reduced binding affinity to human Fc ⁇ RIIB.
  • TIGIT blocking activity of the Fc mutant was also examined, where the DLE mutant showed a reduced TIGIT blocking function, as shown in Figure 13A.
  • variable region of an antibody when the variable region of an antibody binds to its specific antigen, its Fc region can cross-link Fc ⁇ RIIIA and trigger downstream signal transduction.
  • Jurkat cells transfected with human Fc ⁇ RIIIA and NFAT reporter genes and 293T cells overexpressing TIGIT were used to examine the effect of Fc mutants on Fc ⁇ RIIIA-mediated activity.
  • the DLE mutant significantly increased the activity of the human Fc ⁇ RIIIA-mediated NFAT reporter gene compared with the wild-type Fc form.
  • Table 3 corresponds to the detection data of Figure 13A
  • Table 4 corresponds to the detection data of Figure 13B
  • mice were inoculated with MC38 tumor cells one week before treatment. The treatment started when the average tumor size reached about 51mm 3 and was administered intraperitoneally twice a week for 2.5 weeks.
  • the 2A3-LT-Fc antibody is 6 mg/kg each time or the reference Ab2 is 11 mg/kg each time (equivalent to the 2A3-LT-Fc antibody in mole/kg). 16 days after treatment, the tumor size in the mice in the control group reached the upper limit (2000 mm 3 ).
  • the average tumor volume was 1548.76 ⁇ 191 mm 3 (mean ⁇ standard error) at 16 days after treatment.
  • the treatment with 2A3-LT-Fc wt, 2A3-LT-Fc-DLE significantly reduced tumor growth, resulting in 38% and 50% TGI (tumor suppression) followed by 985.05 ⁇ 123mm 3 and 802.20 ⁇ 126mm 3 tumor volume (compared with the blank control, the P value by Mann-Whitney test is 0.037 and 0.007 respectively).

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Abstract

本发明提供了与"具有Ig和ITIM结构域的T细胞免疫受体（TIGIT）"结合的抗TIGIT抗体，这些抗体包括对TIGIT和一种或多种另外的抗原具有结合特异性的多特异性抗TIGIT抗体；以及其使用方法。这些抗TIGIT抗体包含与TIGIT结合的单结构域抗体。

Description

抗TIGIT抗体和使用方法
相关申请的交叉引用
本申请要求2020年1月10日提交的申请号为CN202010024565.9的中国专利申请的优先权，其中全部内容引入本文以供参考，并要求其优先权。
技术领域
本发明涉及与具有Ig和ITIM结构域的T细胞免疫受体(TIGIT)结合的抗体，这些抗体包括对TIGIT和一种或多种另外的抗原具有结合特异性的多特异性抗TIGIT抗体；以及其使用方法。
背景技术
具有Ig和ITIM结构域的T细胞免疫受体(TIGIT)是由免疫细胞(例如活化的T细胞和自然杀伤细胞(NK细胞))表达的免疫检查点受体，并介导免疫抑制。TIGIT的配体包括PVR(CD155)，其已在树突状细胞(DC)、巨噬细胞以及许多人类癌细胞中鉴定出，并已显示与TIGIT结合后可下调T细胞活化和细胞因子分泌。TIGIT/PVR相互作用的抑制可以介导免疫细胞有效的抗肿瘤活性。考虑到TIGIT在免疫检查点调节中的重要作用，在本领域中仍然需要开发用于调节TIGIT介导的免疫细胞调节以用于免疫疗法和癌症治疗的治疗性分子和方法。
发明内容
本公开提供了以高亲和力特异性结合TIGIT的分离的单克隆抗体，其包括结合TIGIT和一种或多种另外的靶标的多特异性抗体。在某些实施例中，抗TIGIT抗体包含与TIGIT结合的单结构域抗体。本公开进一步提供了制备抗体、免疫缀合物和包含这些抗体的药物组合物的方法，以及使用抗体、免疫缀合物和包含这些抗体的药物组合物例如用于治疗疾病和病症(例如癌症)的方法。本发明部分地基于发现与TIGIT结合的单结构域抗TIGIT抗体，这些抗体可以增加免疫细胞中的免疫应答并提供改善的抗肿瘤功效。
在某些实施例中，抗TIGIT抗体包含与TIGIT结合的单结构域抗体。在某些实施例中，单结构域抗体以约1x10 -7M或更小的KD结合TIGIT。在某些实施例中，单结构域抗体以约1x10 -8M或更小的KD结合TIGIT。在某些实施例中，单结构域抗体以约5x10 -9M或更小的KD结合TIGIT。在某些实施例中，单结构域抗体以约2x10 -9M或更小的KD结合TIGIT。
在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:94所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:95所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:96所示序列的氨基酸。在某些实施例中，单结构域抗 体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:98所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:99所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:100所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:102所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:103所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:104所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:106所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:107所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:108所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:110所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:111所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:112所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:114所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:115所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:116所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:118所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:119所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:120所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:122所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:123所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:124所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:126所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:127所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:128所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:130所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:131所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:132所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:134所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:135所示序列的氨基酸；和重链可 变区CDR3，其包含具有SEQ ID NO:136所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:138所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:139所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:140所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:142所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:143所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:144所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:146所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:147所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:148所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:150所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:151所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:152所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:154所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:155所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:156所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:158所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:159所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:160所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:162所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:163所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:164所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:166所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:167所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:168所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:170所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:171所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:172所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:174所示序 列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:175所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:176所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:178所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:179所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:180所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:182所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:183所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:184所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:186所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:187所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:188所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:190所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:191所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:192所示序列的氨基酸。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：a)重链可变区CDR1，该重链可变区CDR1包含SEQ ID NO:94、98、102、106、110、114、118、122、126、130、134、138、142、146、150、154、158、162、166、170、174、178、182、186和190中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体；b)重链可变区CDR2，该重链可变区CDR2包含SEQ ID NO:95、99、103、107、111、115、119、123、127、131、135、139、143、147、151、155、159、163、167、171、175、179、183、187和191中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体；以及c)重链可变区CDR3，该重链可变区CDR3包含SEQ ID NO:96、100、104、108、112、116、120、124、128、132、136、140、144、148、152、156、160、164、168、172、176、180、184、188和192中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含CDR1结构域、CDR2结构域和CDR3结构域，其中该CDR1结构域、CDR2结构域和CDR3结构域分别含有包含在参考重链可变区中的CDR1结构域、CDR2结构域和CDR3结构域，该参考重链可变区包含选自由SEQ ID NO:97、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181、185、189和193组成的组的氨基酸序列。
在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:94所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:95所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:96所示序列的氨基酸。在某些实施例中，单结 构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:98所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:99所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:100所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:102所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:103所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:104所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:106所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:107所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:108所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:110所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:111所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:112所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:114所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:115所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:116所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:118所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:119所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:120所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:122所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:123所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:124所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:126所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:127所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:128所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:130所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:131所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:132所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:134所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:135所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:136所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:138所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:139所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:140所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:142所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:143所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:144所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:146所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:147所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:148所示序列的氨基酸。在某些实施例中，单结构域抗体 包含：重链可变区CDR1，其包含具有SEQ ID NO:150所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:151所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:152所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:154所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:155所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:156所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:158所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:159所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:160所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:162所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:163所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:164所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:166所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:167所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:168所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:170所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:171所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:172所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:174所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:175所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:176所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:178所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:179所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:180所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:182所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:183所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:184所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:186所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:187所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:188所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:190所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:191所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:192所示序列的氨基酸。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含与选自由SEQ ID NO:97、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181、185、189和193组成的组的氨基酸序列具有至少约90％序列同一性的氨基酸序列。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:97所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID  NO:101所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:105所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:109所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:113所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:117所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:121所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:125所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:129所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:133所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:137所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:141所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:145所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:149所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:153所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:157所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:161所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:165所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:169所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:173所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:177所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:181所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:185所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:189所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:193所示的氨基酸序列。
在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:1所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:2所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:3所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:5所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:6所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:7所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT 结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:9所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:10所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:11所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:13所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:14所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:15所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:17所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:18所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:19所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:21所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:22所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:23所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:25所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:26所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:27所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:29所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:30所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:31所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:33所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:34所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:35所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:37所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:38所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:39所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:41所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:42所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:43所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:45所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:46所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:47所示序列的氨基酸。在某些 实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:49所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:50所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:51所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:53所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:54所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:55所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:57所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:58所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:59所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:61所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:62所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:63所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:65所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:66所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:67所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:69所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:70所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:71所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:73所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:74所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:75所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:77所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:78所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:79所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:81所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:82所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:83所示序列的氨基酸。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:1所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:2所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:3所示序列的氨基酸。在 某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:5所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:6所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:7所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:9所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:10所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:11所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:13所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:14所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:15所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:17所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:18所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:19所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:21所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:22所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:23所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:25所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:26所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:27所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:29所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:30所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:31所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:33所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:34所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:35所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:37所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:38所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:39所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:41所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:42所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:43所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:45所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:46所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:47所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:49所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:50 所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:51所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:53所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:54所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:55所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:57所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:58所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:59所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:61所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:62所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:63所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:65所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:66所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:67所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:69所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:70所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:71所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:73所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:74所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:75所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:77所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:78所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:79所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:81所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:82所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:83所示序列的氨基酸。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含与选自由SEQ ID NO:4、8、12、16、20、24、28、32、36、40、44、48、52、56、60、64、68、72、76、80和84组成的组的氨基酸序列具有至少约90％序列同一性的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含选自由SEQ ID NO:4、8、12、16、20、24、28、32、36、40、44、48、52、56、60、64、68、72、76、80和84组成的组的氨基酸序列。
在某些实施例中，单结构域抗体包含人源化框架。
在某些实施例中，抗TIGIT抗体包含Fc区。在某些实施例中，Fc区包含人Fc区。在某些实施例中，Fc区包含选自下组的Fc区，该组由以下组成：IgG、IgA、IgD、IgE和IgM的Fc区。在某些实施例中，Fc区包含选自下组的Fc区，该组由以下组成：IgG1、IgG2、IgG3和IgG4的Fc区。在某些实施例中，Fc区包含IgG1Fc区。在某些实施例中，IgG1Fc区包 含一种或多种增强抗体依赖性细胞介导的细胞毒性(ADCC)的突变。在某些实施例中，IgG1Fc区包含L235V、F243L、R292P、Y300L和P396L的突变。在某些实施例中，IgG1Fc区包含S239D、A330L和I332E的突变。在某些实施例中，抗TIGIT抗体包含SEQ ID NO:194所示的氨基酸序列。
在某些实施例中，重链可变区通过接头连接至Fc区。在某些实施例中，接头是肽接头。在某些实施例中，肽接头包含约4至约30个氨基酸。在某些实施例中，肽接头包含约4至约15个氨基酸。在某些实施例中，肽接头包含选自由SEQ ID NO:195-220组成的组的氨基酸序列。
在某些实施例中，该抗TIGIT抗体包含多特异性抗体，例如双特异性抗体，全长免疫球蛋白，单链Fv(scFv)片段，Fab片段，Fab'片段，F(ab’)2，Fv片段，二硫键稳定的Fv片段(dsFv)，(dsFv)2，VHH，Fv-Fc融合物，scFv-Fc融合物，scFv-Fv融合物，双抗体，三抗体，四抗体或任何它们的组合。
在某些实施例中，抗TIGIT抗体包含多特异性抗体(例如双特异性抗体)，其包含特异性结合第二抗原的第二抗体部分。在某些实施例中，第二抗原是肿瘤相关抗原。在某些实施例中，肿瘤相关抗原选自下组，该组由以下组成：Her-2，EGFR，PD-L1，c-Met，B细胞成熟抗原(BCMA)，碳酸酐酶IX(CA1X)，癌胚抗原(CEA)，CD5，CD7，CD10，CD19，CD20，CD22，CD30，CD33，CD34，CD38，CD41，CD44，CD49f，CD56，CD74，CD123，CD133，CD138，CD276(B7H3)，上皮糖蛋白(EGP2)，滋养层细胞表面抗原2(TROP-2)，上皮糖蛋白-40(EGP-40)，上皮细胞粘附分子(EpCAM)，受体酪氨酸蛋白激酶erb-B2、3、4，叶酸结合蛋白(FBP)，胎儿乙酰胆碱受体(AChR)，叶酸受体-a，神经节苷脂G2(GD2)，神经节苷脂G3(GD3)，人端粒酶逆转录酶(hTERT)，激酶插入结构域受体(KDR)，Lewis A(CA 1.9.9)，Lewis Y(LeY)，磷脂酰肌醇蛋白聚糖-3(GPC3)，L1细胞粘附分子(L1CAM)，粘蛋白16(Muc-16)，粘蛋白1(Muc-1)，NG2D配体，癌胚抗原(h5T4)，前列腺干细胞抗原(PSCA)，前列腺特异性膜抗原(PSMA)，肿瘤相关糖蛋白72(TAG-72)，密封蛋白18.2(CLDN18.2)，血管内皮生长因子R2(VEGF-R2)，肾母细胞瘤蛋白(WT-1)，1型酪氨酸蛋白激酶跨膜受体(ROR1)及其任何组合。
在某些实施例中，第二抗原是免疫检查点调节剂。在某些实施例中，免疫检查点调节剂选自由以下组成的组：PD1、CTLA4、LAG-3、2B4、BTLA及其任何组合。
在某些实施例中，抗TIGIT抗体缀合至治疗剂或标记。在某些实施例中，标记选自下组，该组由以下组成：放射性同位素、荧光染料和酶。
本公开进一步提供了与治疗剂连接的、包含本文公开的任何抗体的免疫缀合物。在某些实施例中，治疗剂是细胞毒素。在某些实施例中，治疗剂是放射性同位素。
本公开进一步提供了药物组合物。在某些实施例中，药物组合物包含a)本文公开的抗体或免疫缀合物，和b)药学上可接受的载剂。
本公开进一步提供了编码本文公开的任何抗体的核酸。本公开进一步提供了包含本文公开的任何核酸的载体。本公开进一步提供了包含本文公开的核酸或载体的宿主细胞。
本公开进一步提供了用于制备本文公开的抗体的方法。在某些实施例中，该方法包括在本文公开的宿主细胞中表达抗体，并从宿主细胞分离抗体。
本公开进一步提供了减轻受试者的肿瘤负荷的方法。在某些实施例中，该方法包括向受试者施用有效量的本文公开的抗体、免疫缀合物或药物组合物。
在某些实施例中，该方法减少肿瘤细胞的数量。在某些实施例中，该方法减小肿瘤大小。在某些实施例中，该方法根除受试者的肿瘤。在某些实施例中，肿瘤选自由以下组成的组：间皮瘤、肺癌、胰腺癌、卵巢癌、乳腺癌、结肠癌、胸膜肿瘤、成胶质细胞瘤、食道癌、胃癌、滑膜肉瘤、胸腺癌、子宫内膜癌、胃肿瘤、胆管癌、头颈癌、血液癌及其组合。
本公开进一步提供了治疗和/或预防受试者的赘生物的方法。在某些实施例中，该方法包括向受试者施用有效量的本文公开的抗体、免疫缀合物或药物组合物。
本公开进一步提供了延长患有赘生物的受试者的存活期的方法。在某些实施例中，该方法包括向受试者施用有效量的本文公开的抗体、免疫缀合物或药物组合物。
在某些实施例中，赘生物选自由以下组成的组：间皮瘤、肺癌、胰腺癌、卵巢癌、乳腺癌、结肠癌、胸膜肿瘤、成胶质细胞瘤、食道癌、胃癌、滑膜肉瘤、胸腺癌、子宫内膜癌、胃肿瘤、胆管癌、头颈癌、血液癌及其组合。
本公开进一步提供了本文公开的用作药物的任何抗体。本公开进一步提供了本文公开的用于治疗癌症的任何抗体。本公开进一步提供了本文公开的用作药物的药物组合物。本公开进一步提供了本文公开的用于治疗癌症的药物组合物。在某些实施例中，癌症选自由以下组成的组：间皮瘤、肺癌、胰腺癌、卵巢癌、乳腺癌、结肠癌、胸膜肿瘤、成胶质细胞瘤、食道癌、胃癌、滑膜肉瘤、胸腺癌、子宫内膜癌、胃肿瘤、胆管癌、头颈癌、血液癌及其组合。
本公开进一步提供了试剂盒，其包含本文公开的抗体、免疫缀合物、药物组合物、核酸、载体或宿主细胞。在某些实施例中，试剂盒包含用于治疗和/或预防赘生物的书面说明书。
附图说明
图1A-1C描绘了通过流式细胞术测定确定的代表性VHH二价抗体与人TIGIT的全细胞结合。图1A显示了与一种参考抗人TIGIT抗体(参考Ab1)相比的一种测定。图1B显示了使用其他VHH抗体的不同测定。Y轴表示AlexaFlour 488的平均荧光强度。X轴表示以纳摩尔计的抗体浓度。2B7、1G1、1C12、3G6、2B10、3G7、3G10、13H11和15A5是抗人TIGIT的VHH克隆。使用Prism的非线性回归方法获得EC50值，并且在表中以纳摩尔表示这些值。图1C是TIGIT VHH二价抗体的结构示意图。
图2A-2B描绘了代表性二价抗体在阻断TIGIT活性中的功效，该活性由萤光素酶报告基因测定确定。在代表性抗TIGIT二价抗体和低浓度葡萄球菌肠毒素存在下，将人TIGIT和NFAT报告基因稳定转染的Jurkat细胞与PVR(CD155)稳定转染的Raji细胞共培养。Y轴 以相对发光单位表示NFAT萤光素酶活性。X轴表示以纳摩尔计的抗体浓度。参考Ab1是参考抗h-TIGIT抗体。2B7、1G1、1C12、3G6、2B10、3G7和3F10是抗h-TIGIT的代表性克隆。使用Prism的非线性回归方法获得EC50值，并且在表中以纳摩尔表示这些值。
图3A和3B描绘了通过流式细胞术测定确定的人源化1C12和1G1克隆与人TIGIT的全细胞结合。1C12人源化形式的代表性结果如图3A所示。1C12嵌合抗体是具有1C12克隆的Llama VHH序列以及人IgG1的CH2和CH3结构域的抗体，1C12(F-EREF)、1C12(F-EREW)和1C12(F-GLEW)是1C12克隆的人源化形式，差异为框架2中的突变。1G1人源化形式的代表性结果如图3B所示。1G1嵌合抗体是具有1G1克隆的Llama VHH序列以及人IgG1的CH2和CH3结构域的抗体。1G1(F-G-ERES)、1G1(F-A-ERES)、1G1(F-A-EREW)和1G1(F-A-GLEW)是框架2中具有不同突变的人源化1G1克隆的四种不同形式。Y轴是AlexaFlour 488的平均荧光强度值。X轴是以纳摩尔计的抗体浓度的值。
图4A和4B描述了人源化的1C12和1G1克隆在阻断TIGIT活性中的功效，该活性由萤光素酶报告基因测定确定。TIGIT阻断萤光素酶报告基因测定中1C12人源化形式的代表性结果如图4A所示。与参考抗h-TIGIT抗体参考Ab1相比，所有克隆更有效。1G1人源化形式的代表性结果如图4B所示。Y轴代表NFAT萤光素酶活性(RLU，相对发光单位)。X轴表示以纳摩尔计的抗体浓度。
图5描绘了针对代表性克隆的来自阻断ELISA的IC50值(以纳摩尔计)和来自全细胞结合的EC50值(以纳摩尔计)之间的相关性。克隆名称在图中进行了标记。使用GraphPad Prism分析相关性。
图6A和6B描绘了针对代表性克隆测试的热稳定性。将VHH抗体样品从25加热到70℃持续60分钟。使用ELISA或全细胞结合检查加热的样品与人TIGIT的结合。图6A显示了在ELISA测定中代表性克隆的加热样品与h-TIGIT ECD的结合，其中在图例中标记了克隆名称。Y轴代表由ELISA测定法获得的OD450。X轴代表处理温度。图6B显示了代表性克隆的加热样品与通过流式细胞术确定的在Jurkat细胞中稳定表达的h-TIGIT的结合。Y轴代表与h-TIGIT结合的百分比。X轴代表处理温度。
图7A-7C描绘了通过使用ForteBio的Octet结合测定法确定的2A3-Fc与人TIGIT结合的表位。将带有his标签的人TIGIT ECD(200nM)的重组蛋白加载到传感器上。通过注射三种不同浓度的2A3-Fc检测结合。通过第二次注射图7A中所示的2A3-Fc未检测到额外的结合，但是通过注射三种不同浓度的参考Ab2，一种参考抗TIGIT抗体(如图7B中所示)，或参考Ab1(如图7C中所示)，检测到强结合。结果表明，与参考Ab2和参考Ab1相比，2A3克隆具有不同的结合表位。
图8A-8C描绘了通过ELISA测定法确定的2A3-Fc对人-、食蟹猴-和小鼠-TIGIT的交叉结合活性。图8A显示了2A3-Fc与重组人TIGIT ECD的结合。参考Ab2是一种抗人TIGIT参考抗体。2A3-Fc和参考Ab2都以相似的亲和力与h-TIGIT结合。抗PDL1不与h-TIGIT结合。图8B显示了2A3-Fc与重组cyno-TIGIT的结合。2A3-Fc和参考Ab2都以相似的亲和力 与cyno-TIGIT结合。抗PDL1不与cyno-TIGIT结合。图8C显示了2A3-Fc与重组小鼠TIGIT的结合。2A3-Fc和参考Ab2均未与小鼠TIGIT结合，但是抗小鼠TIGIT参考抗体(博奇公司(Biolegend)#142101)与小鼠TIGIT以高亲和力结合。Y轴代表OD450，X轴代表以μg/ml计的抗体浓度。
图9描绘了在全细胞结合测定和人TIGIT阻断报告基因测定中代表性克隆2A3-Fc和热点校正形式的2A3-LT-Fc的效力的比较。Y轴在上图中代表平均荧光强度，在下图中代表以相对发光单位计的NFAT萤光素酶报告基因活性。X轴表示以纳摩尔计的抗体浓度。热点校正形式2A3-LT-Fc具有与亲本克隆2A3-Fc相似的效力。
图10A和10B描绘了热点校正形式2A3-LT-Fc与参考抗-h-TIGIT抗体参考Ab2的亲和力的比较。图10A显示了在全细胞结合中热点校正形式2A3-LT-Fc与参考Ab2的效力的比较。Y轴代表通过使用CytoFlex的流式细胞术测定法确定的AlexaFlour 488的平均荧光强度。X轴表示以纳摩尔计的抗体浓度。显示的数据是来自三个不同实验的代表性结果。2A3-LT-Fc与h-TIGIT的亲和力比与参考Ab2的亲和力略高。图10B显示了在TIGIT阻断NFAT报告基因测定中，热点校正形式2A3-LT-Fc与参考Ab2的效力的比较。Y轴代表以相对发光单位计的NFAT萤光素酶报告基因活性。X轴表示以纳摩尔计的抗体浓度。2A3-LT-Fc具有与参考Ab2相似的效力。
图11描绘了2A3-LT-Fc体外抗肿瘤效力。在混合淋巴细胞反应试验使用了TIGIT+T细胞和PVR+树突细胞(DCs).48小时混合培养后检测了培养上清中T细胞IL-2的分泌。抗PD1抗体被用作正对照。抗HER2抗体被用作负对照。
图12描绘了使用Octet结合测定法确定的2A3-LT-Fc野生型(wt)和Fc突变(DLE和VLPLL)与人FcγRIIIA、人FcγRIIB和小鼠FcγRIV的结合。传感器加载有FcγRIIIA、人FcγRIIB和小鼠FcγRIV的ECD重组蛋白，使用ForteBio检测到5种不同浓度的2A3-LT-Fc wt、DLE或VLPLL突变体的缔合和解离。Y轴表示缔合、解离和Rmax。X轴表示以秒计的时间。与野生型相比，DLE和VLPLL突变体均具有增强的与人FcγRIIIA的结合亲和力，DLE突变体也具有增强的与人FcγRIIB的结合亲和力，但是VLPLL突变体降低了与人FcγRIIB的结合亲和力。所有形式都具有与小鼠FcγRIV相似的亲和力。
图13A和13B分别描绘了TIGIT mAb 2A3-LT-Fc wt、DLE突变体对阻断TIGIT活性和人FcγRIIIA介导的活性的影响。图13A描绘了使用NFAT萤光素酶报告基因测定法确定的TIGIT mAb 2A3-LT-Fc wt、DLE突变体对阻断TIGIT活性的影响。将经人TIGIT和NFAT报告基因稳定转染的Jurkat细胞与经PVR稳定转染的Raji细胞共培养。加入2A3-LT-Fc wt和突变体并培养5小时。通过萤光素酶活性来测量TCR介导的活性。Y轴以相对发光单位表示NFAT萤光素酶活性。X轴表示以纳摩尔计的抗体浓度。图13B描述了TIGIT mAb 2A3-LT-Fc wt、DLE突变体对人FcγRIIIA介导的活性的影响，该活性由FcγRIIIA介导的NFAT萤光素酶报告基因活性确定。在不同浓度的2A3-LT-Fc wt、DLE突变体存在下，将人FcγRIIIA和NFAT稳定转染的Jurkat细胞与人TIGIT稳定转染的293T细胞共培养5小时。测量萤光素酶 活性，并在Y轴上以相对发光单位表示。X轴表示以纳摩尔计的抗体浓度。
图14A-14C描绘了对抗TIGIT抗体的体内有效性分析。使用了人TIGIT敲入C57BL/6小鼠和MC38鼠结肠癌模型。在治疗前一周给小鼠接种了MC38肿瘤细胞。当平均肿瘤大小达到约51mm 3时治疗开始，在腹膜内一周两次给药，持续2.5周。2A3-LT-Fc抗体每次6mg/kg或参考Ab2每次11mg/kg(与2A3-LT-Fc抗体在mole/kg上等同)。图14A描绘了用空白对照，2A3-Fc-wt，有DLE突变的2A3-Fc和参考Ab 2给药的长有肿瘤的小鼠的肿瘤生长曲线。单个肿瘤体积的结果显示在图14B中。图14C显示了在研究中各实验组的体重变化并不显著。
具体实施方式
本公开提供了以高亲和力特异性结合TIGIT的分离的单克隆抗体，其包括结合TIGIT和一种或多种另外的靶标的多特异性抗体。在某些实施例中，抗TIGIT抗体包含与TIGIT结合的单结构域抗体。本公开进一步提供了制备抗体、免疫缀合物和包含这些抗体的药物组合物的方法，以及使用抗体、免疫缀合物和包含这些抗体的药物组合物例如用于治疗疾病和病症(例如癌症)的方法。本发明部分地基于发现与TIGIT结合的单结构域抗TIGIT抗体，这些抗体可以增加免疫细胞中的免疫应答并提供改善的抗肿瘤功效。
为了清楚起见而不是作为限制，目前公开的主题的具体实施方式分为以下小节：
1.定义；
2.抗体；
3.使用方法；
4.药物配制品；以及
5.制品。
1.定义
本文中术语“抗体”以最广泛的含义使用，并且涵盖多种抗体结构，这些抗体结构包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、单结构域抗体和抗体片段，只要它们表现出所需的抗原结合活性即可。
“抗体片段”是指包含完整的全长抗体的抗原结合部分的分子，该完整的全长抗体结合与完整的抗体结合的抗原。抗体片段的实例包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2、双抗体、线性抗体、单链抗体分子(例如，scFv)、由抗体片段形成的多特异性抗体、单结构域抗体、VHH纳米抗体、结构域抗体、二价结构域抗体或与抗原结合的抗体的任何其他片段。“VHH”是指从骆驼科动物中分离的单结构域抗体。在某些实施例中，VHH包含骆驼重链抗体的重链可变区。在某些实施例中，VHH的大小不超过25kDa。在某些实施例中，VHH的大小不超过20kDa。在某些实施例中，VHH的大小不超过15kDa。
“全长抗体”是指包含两条重链和两条轻链的抗体。轻链和重链的可变区对抗原结合负责。重链和轻链的可变区可以分别称为“VH”和“VL”。两条链中的可变区通常包含三个高度可变的环，称为互补决定区(CDR)(包括LC-CDR1、LC-CDR2和LC-CDR3的轻链(LC) CDR，包括HC-CDR1、HC-CDR2和HC-CDR3的重链(HC)CDR)。本文公开的抗体和抗原结合片段的CDR边界可以通过公知的惯例来定义或鉴定，例如，Kabat、Chothia或Al-Lazikani的惯例(Al-Lazikani 1997；Chothia 1985；Chothia 1987；Chothia 1989；Kabat 1987；Kabat 1991)。重链或轻链的三个CDR被插入称为框架区(FR)的侧翼区段之间，它们比CDR更为保守，并形成了支持高变环的支架。重链和轻链的恒定区不参与抗原结合，但是表现出多种效应子功能。根据抗体重链恒定区的氨基酸序列将抗体分类。抗体的五种主要类别或同种型是IgA、IgD、IgE、IgG和IgM，分别以存在α、δ、ε、γ和μ重链为特征。几种主要抗体类别分为亚类，例如lgG1(γ1重链)、lgG2(γ2重链)、lgG3(γ3重链)、lgG4(γ4重链)、lgA1(α1重链)或lgA2(α2重链)。
与参考抗体“交叉竞争结合的抗体”是指在竞争测定中阻断参考抗体与其抗原的结合达50％以上的抗体，相反，在竞争测定中参考抗体阻断抗体与其抗原的结合达50％以上。在Antibodies[抗体],Harlow和Lane(冷泉港出版社，冷泉港，纽约(Cold Spring Harbor Press,Cold Spring Harbor,NY))中描述了示例性竞争测定法。
“Fv”是最小抗体片段，其含有完整抗原识别位点和抗原结合位点。该片段由紧密非共价缔合的一个重链可变区和一个轻链可变区的二聚体组成。从这两个结构域的折叠中发出六个高变环(重链和轻链各自中的3个环)，该高变环贡献用于抗原结合的氨基酸残基并赋予抗体与抗原结合特异性。然而，甚至单个可变结构域(或仅包含三个对抗原有特异性的CDR的半个Fv)可以识别并结合抗原，虽然有时以比完整结合位点更低的亲和力进行。
“单链Fv”(也缩写为“sFv”或“scFv”)是包含连接成单个多肽链的V H和V L抗体结构域的抗体片段。在一些实施例中，scFv多肽进一步包含在V H和V L结构域之间的一种多肽接头，该多肽接头使得scFv形成所希望的用于抗原结合的结构。关于scFv的综述，参见Plückthun的The Pharmacology of Monoclonal Antibodies[单克隆抗体的药理学],第113卷,Rosenburg和Moore编辑Springer-Verlag[施普林格出版社],纽约,第269-315页(1994)。
出于本文的目的，“受体人框架”或“人框架”是包含源自人免疫球蛋白框架或人共有框架的轻链可变结构域(VL)框架或重链可变结构域(VH)框架的氨基酸序列的框架。“源自”人免疫球蛋白框架或人共有框架的受体人框架可以包含其相同的氨基酸序列，或者可以包含氨基酸序列变化。在某些实施例中，氨基酸变化的数目是10个或更少、9个或更少、8个或更少、7个或更少、6个或更少、5个或更少、4个或更少、3个或更少、或2个或更少。在某些实施例中，VL受体人框架与VL人免疫球蛋白框架序列或人共有框架序列在序列方面是相同的。
“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配偶体(例如，抗原)之间非共价相互作用的总和的强度。除非另外指明，如本文所用，“结合亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间1:1相互作用。分子X对其配偶体Y的亲和力通常可以由解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些)测量。下面描述了用于测量结合亲和力的具体说明性和示例性实施例。
“亲和力成熟的”抗体是指与不具有这种改变的亲本抗体相比，在一个或多个CDR或高变区(HVR)中具有一个或多个改变的抗体，该改变提供了抗体对抗原的改善的亲和力。
如本文所用，“具有Ig和ITIM结构域的T细胞免疫受体”或“TIGIT”是指来自任何脊椎动物来源(包括哺乳动物(例如灵长类动物(例如人和食蟹猴)))的任何天然TIGIT多肽、或其任何片段，并且可以任选地包含至多一个、至多两个、至多三个、至多四个、至多五个、至多六个、至多七个、至多八个、至多九个或至多十个氨基酸取代、添加和/或缺失。该术语涵盖全长未处理的TIGIT以及由于在细胞中进行处理而产生的任何形式的TIGIT。该术语还涵盖TIGIT的天然存在的变体，例如剪接变体或等位基因变体。本公开的抗TIGIT抗体靶向的人TIGIT氨基酸序列的非限制性实例如下：
1 MRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST TAQVTQVNWE
61 QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV NDTGEYFCIY HTYPDGTYTG
121 RIFLEVLESS VAEHGARFQI PLLGAMAATL VVICTAVIVV VALTRKKKAL RIHSVEGDLR
181 RKSAGQEEWS PSAPSPPGSC VQAEAAPAGL CGEQRGEDCA ELHDYFNVLS YRSLGNCSFF
241 TETG[SEQ ID NO:221]。
术语“TIGIT的ECD”是指TIGIT的细胞外结构域。例如，SEQ ID NO:221所示的示例性TIGIT蛋白的ECD包含以下氨基酸序列：
MRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST TAQVTQVNWE
QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV NDTGEYFCIY HTYPDGTYTG
RIFLEVLESS VAEHGARF[SEQ ID NO:222]。
术语“抗TIGIT抗体”和“结合TIGIT的抗体”是指能够以足够的亲和力结合TIGIT的抗体，使得该抗体可用作靶向TIGIT的诊断剂和/或治疗剂。在一个实施例中，与无关的、非TIGIT蛋白的抗TIGIT抗体的结合程度小于该抗体与TIGIT结合的约10％，例如通过
Figure PCTCN2021070903-appb-000001
表面等离子体共振测定法所测量的。在某些实施例中，与TIGIT结合的抗体具有以下解离常数(KD)<约1μM，<约100nM，<约10nM，<约1nM，<约0.1nM，<约0.01nM或<约0.001nM(例如10 -8M或更小，例如10 -8M至10 -12M，例如10 -9M至10 -10M)。在某些实施例中，抗TIGIT抗体结合来自不同物种的TIGIT中保守的TIGIT表位。在某些实施例中，抗TIGIT抗体结合在蛋白质的ECD中的TIGIT上的表位。
术语“嵌合”抗体是指这样的抗体，其中重链和/或轻链的一部分源自特定来源或物种，而重链和/或轻链的其余部分源自不同来源或物种。在某些实施例中，本文公开的嵌合抗体包 含骆驼科动物重链可变区和人Fc区。
如本文所用，术语“CDR”或“互补决定区”是指重链和/或轻链的可变区内的非连续抗原结合位点。这些特定区已经描述于：Kabat等人,J.Biol.Chem.[生物化学杂志],252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest[具有免疫学意义的蛋白质序列]”(1991)；Chothia等人,J.Mol.Biol.[分子生物学杂志]196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.[分子生物学杂志],273:927-948(1997)；MacCallum等人,J.Mol.Biol.[分子生物学杂志]262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.[分子免疫学],45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.[发展与比较免疫学],27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.[分子生物学杂志],309:657-670(2001)，其中定义包括当彼此比较时氨基酸残基的重叠或子集。然而，应用任一定义来指代抗体或移植的抗体或其变体的CDR旨在落入如本文定义和使用的术语的范围内。涵盖如上述每篇参考文献定义的CDR的氨基酸残基列于下表1中作为比较。CDR预测算法和接口是本领域已知的，包括例如Abhinandan和Martin，Mol.Immunol.[分子免疫学],45:3832-3839(2008)；Ehrenmann F.等人,Nucleic Acids Res.[核酸研究],38:D301-D307(2010)；和Adolf-Bryfogle J.等人,Nucleic Acids Res.[核酸研究],43:D432-D438(2015)。在本段中引用的参考文献的内容通过引用以其整体并入本文，以用于本申请中并且可能包含在本文的一项或多项权利要求中。
表1：CDR定义
  Kabat 1 Chothia 2 MacCallum 3 IMGT 4 AHo 5
V H CDR1 31-35 26-32 30-35 27-38 25-40
V HCDR2 50-65 53-55 47-58 56-65 58-77
V H CDR3 95-102 96-101 93-101 105-117 109-137
V L CDR1 24-34 26-32 30-36 27-38 25-40
V L CDR2 50-56 50-52 46-55 56-65 58-77
V L CDR3 89-97 91-96 89-96 105-117 109-137
1残基编号遵循Kabat等人的命名法(同上)。
2残基编号遵循Chothia等人的命名法(同上)。
3残基编号遵循MacCallum等人的命名法(同上)。
4残基编号遵循Lefranc等人的命名法(同上)。
5残基编号遵循Honegger和Plückthun的命名法(同上)。
表述“如Kabat中的可变结构域残基编号”或“如Kabat中的氨基酸位置编号”及其变体是指用于上文Kabat等人的抗体汇编的重链可变结构域或轻链可变结构域的编号系统。使用这个编号系统，实际直链氨基酸序列可以含有对应于可变结构域的FR或CDR的缩短或插入的更少的或另外的氨基酸。例如，重链可变结构域可以包含在H2的残基52之后的单个氨基酸插入(根据Kabat的残基52a)以及在重链FR残基82之后的插入残基(例如，根据Kabat 的残基82a、82b和82c等)。可以通过在抗体序列与“标准”Kabat编号序列的同源性区域进行比对来确定给定抗体的残基的Kabat编号。
在某些实施例中，涵盖单结构域抗体(例如，本文公开的单结构域抗TIGIT抗体)的CDR的氨基酸残基是根据上文Lefranc等人的IMGT命名法定义的。在某些实施例中，涵盖全长抗体的CDR的氨基酸残基是根据上文Kabat等人的Kabat命名法定义的。在某些实施例中，免疫球蛋白重链例如Fc区中的残基编号是如上文Kabat等人所述的EU索引的编号。“如Kabat所述的EU索引”是指人IgG1EU抗体的残基编号。
“框架”或“FR”是指除了本文定义的CDR残基以外的那些可变结构域残基。
“人源化”抗体是指包含来自非人CDR/HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在某些实施例中，人源化抗体将包括基本上至少一个、并且典型地两个可变结构域的全部，其中HVR/CDR的全部或基本上全部对应于非人类抗体的那些，并且FR的全部或基本上全部对应于人类抗体的那些。人源化抗体可任选地包含源自人抗体的抗体恒定区的至少一部分。抗体(例如非人抗体)的“人源化形式”是指已经人源化的抗体。
“人抗体”是具有氨基酸序列的抗体，该氨基酸序列对应于由人产生的抗体的氨基酸序列和/或已经使用本文公开的任何用于制备人抗体的技术制备。人抗体的此定义特别排除了包含非人抗原结合残基的人源化抗体。可以使用本领域已知的各种技术(包括噬菌体展示文库)产生人抗体。Hoogenboom和Winter,J.Mol.Biol.[分子生物学杂志],227:381(1991)；Marks等人,J.Mol.Biol.[分子生物学杂志],222:581(1991)。也可用于制备人单克隆抗体的是在Cole等人，Monoclonal Antibodies and Cancer Therapy[单克隆抗体和癌症疗法],Alan R.Liss,第77页(1985)；Boerner等人,J.Immunol.[免疫学杂志],147(1):86-95(1991)中描述的方法。另请参见van Dijk和van de Winkel,Curr.Opin.Pharmacol.[药理学新见],5:368-74(2001)。人抗体可以通过将抗原施用至已被修饰以响应抗原攻击而产生这种抗体但其内源基因座已失效的转基因动物(例如已免疫的xenomice)来制备(参见，例如，有关XENOMOUSE TM技术的美国专利号6,075,181和6,150,584)。关于通过人B细胞杂交瘤技术产生的人抗体，还参见例如Li等人，Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]103:3557-3562(2006)。
将“关于本文鉴定的多肽和抗体序列的氨基酸序列同一性百分比(％)”或“同源性”定义为在对齐序列(考虑任何保守取代作为序列同一性的一部分)后，候选序列中与所比较多肽中的氨基酸残基相同的氨基酸残基的百分比。出于确定氨基酸序列同一性百分比的目的，可以用本领域技术中的多种方式来实现比对，例如使用公众可得的计算机软件，例如BLAST、BLAST-2、ALIGN、Megalign(DNASTAR)或MUSCLE软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。然而，出于本文的目的，使用序列比较计算机程序MUSCLE生成氨基酸序列同一性％值(Edgar,R.C.,Nucleic Acids Research[核酸研究]32(5):1792-1797,2004；Edgar,R.C.,BMC Bioinformatics[BMC生物信息学]5(1):113,2004)。
“同源的”是指两个多肽之间或两个核酸分子之间的序列相似性或序列同一性。当两个 比较序列中两个的一个位置被相同的碱基或氨基酸单体亚基占据时，例如，如果两个DNA分子中的每一个的一个位置被腺嘌呤占据，则该分子在该位置是同源的。两个序列之间的同源性百分比是两个序列共享的匹配或同源位置数除以比较的位置数乘以100的函数。例如，如果两个序列中10个位置中的6个是匹配或同源的，那么两个序列是60％同源的。举例来说，DNA序列ATTGCC和TATGGC具有50％的同源性。通常，当两个序列比对以给出最大同源性时进行比较。
术语“恒定结构域”是指免疫球蛋白分子的一部分，其相对于免疫球蛋白的另一部分，即可变结构域，具有更保守的氨基酸序列，其包含抗原结合位点。恒定结构域包含重链的C H1、C H2和C H3结构域(统称为C H)和轻链的C L结构域。
任何哺乳动物物种的抗体(例如免疫球蛋白)的“轻链”都可以根据其恒定结构域的氨基酸序列指定为两种明显不同的类型之一，分别称为kappa(“κ”)和lambda(“λ”)。
“CH1结构域”(也称为“H1”结构域的“C1”)通常从约氨基酸118至约氨基酸215(EU编号系统)。
“铰链区”通常定义为IgG中对应于人IgG1的Glu216至Pro230的区域(Burton,Molec.Immunol.[分子免疫学]22:161-206(1985))。其他IgG同种型的铰链区可以通过将第一个和最后一个形成重链间S-S键的半胱氨酸残基置于相同位置而与IgG1序列比对。
人IgG Fc区(也称为“C2”结构域)的“CH2结构域”通常从约氨基酸231至约氨基酸340。CH2结构域是独特的，因为其不与另一个结构域紧密配对。而是，两个N连接的支链碳水化合物链插入完整天然IgG分子的两个CH2结构域之间。据推测，碳水化合物可以提供结构域-结构域配对的替代物并有助于稳定CH2结构域。Burton,Molec Immunol.[分子免疫学],22:161-206(1985)。
“CH3结构域”(也称为“C2”结构域)包含CH2结构域与Fc区的C末端之间的残基(即，从约氨基酸残基341到抗体序列的C末端)，通常在IgG的氨基酸残基446或447处)。
本文中的术语“Fc区”或“片段可结晶区”用于定义免疫球蛋白重链的C末端区域，包括天然序列Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以变化，但是通常将人IgG重链的Fc区定义为从Cys226位置处的氨基酸残基或从Pro230延伸至其羧基末端。可以例如抗体的生产或纯化期间或通过对编码抗体重链的核酸进行重组工程化来去除Fc区的C末端赖氨酸(根据EU编号系统的残基447)。因此，完整抗体的组合物可以包括去除了所有K447残基的抗体群体，没有去除K447残基的抗体群体以及具有带有和不带有K447残基的抗体混合物的抗体群体。用于本文所述抗体的合适天然序列Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4。
“Fc受体”或“FcR”描述结合抗体的Fc区的受体。优选的FcR是天然人FcR。此外，优选的FcR是结合IgG抗体(γ受体)并包括FcγRI、FcγRII和FcγRIII亚类的受体的FcR，包括等位基因变体和这些受体的剪接形式，FcγRII受体包括FcγRIIA(“活化受体”)和FcγRIIB(“抑制受体”)，它们具有相似的氨基酸序列，主要区别在于其胞质结构域。活化受体FcγRIIA 在其细胞质结构域中含有基于免疫受体酪氨酸的活化基序(ITAM)。抑制性受体FcγRIIB在其胞质结构域中包含基于免疫受体酪氨酸的抑制基序(ITIM)。(参见M.
Figure PCTCN2021070903-appb-000002
Annu.Rev.Immunol.[免疫学年度评论]15:203-234(1997)。FcR综述于Ravetch和Kinet，Annu.Rev.Immunol.[免疫学年度评论],9:457-92(1991)；Capel等人,Immunomethods[免疫方法]4:25-34(1994)；和de Haas等人,J.Lab.Clin.Med.[实验与临床医学杂志]126:330-41(1995)。本文的术语“FcR”涵盖其他FcR，包括将来鉴定的FcR。
如本文所用，术语“表位”是指抗体或抗原结合部分所结合的抗原上的特定原子或氨基酸基团。如果两个抗体或抗原结合部分对抗原具有竞争性结合，则它们可以结合抗原内的相同表位。
如本文所用，术语“特异性结合”、“特异性识别”和“对……有特异性”是指可测量和可再现的相互作用，例如靶标与抗体或抗体部分之间的结合，其决定了在异质分子(包括生物分子)群体存在时靶标的存在。例如，特异性识别靶标(可以是表位)的抗体或抗体部分是与该靶标结合的抗体或抗体部分，其亲和力、亲合力、就绪性和/或持续时间长于与其他靶标的结合。在一些实施例中，抗体与不相关靶标的结合程度小于例如通过放射免疫测定法(RIA)测量的抗体与靶标的结合程度的约10％。在一些实施例中，特异性结合靶标的抗体的解离常数(K D)≤10 -5M、≤10 -6M、≤10 -7M、≤10 -8M、≤10 -9M、≤10 -10M、≤10 -11M、或≤10 -12M。在一些实施例中，抗体特异性结合对从不同物种的蛋白中为保守的蛋白的表位。在一些实施例中，特异性结合可以包括但不要求排他结合。抗体或抗原结合结构域的结合特异性可以通过本领域已知的方法通过实验确定。这类方法包括但不限于蛋白质印迹、ELISA-、RIA-、ECL-、IRMA-、EIA-、BIACORE TM-检验和肽扫描。
“分离的”抗体(或构建体)是已经从其生产环境的组分(例如天然或重组)中鉴定、分离和/或回收的抗体。在某些实施例中，分离的多肽在其生产环境中没有或基本上没有与所有其他组分缔合。
编码本文所述的构建体、抗体或其抗原结合片段的“分离的”核酸分子是与在其生产环境中从通常与其相关联的至少一种污染物核酸分子中鉴定并分离的核酸分子。在某些实施例中，分离的核酸没有或基本没有与生产环境有关的所有组分缔合。编码本文所述的多肽和抗体的分离的核酸分子的形式不同于天然存在的形式或背景。因此，分离的核酸分子不同于编码天然存在于细胞中的本文所述的多肽和抗体的核酸。分离的核酸包括通常包含核酸分子的细胞中所含的该核酸分子，但是该核酸分子存在于染色体外或与其天然染色体位置不同的染色体位置。
术语“控制序列”是指在特定宿主生物体中表达可操作地连接的编码序列所必需的DNA序列。例如，适用于原核生物的控制序列包括启动子、任选的操纵子序列和核糖体结合位点。已知真核细胞利用启动子、聚腺苷酸化信号和增强子。
当核酸与另一核酸序列处于功能关系时，该核酸是“可操作地连接的”。例如，如果将前序列或分泌性前导序列的DNA表达为参与多肽分泌的前蛋白，则该前序列或分泌性前导序 列的DNA可操作地连接至该多肽的DNA；如果启动子或增强子影响编码序列的转录，则该启动子或增强子可操作地连接至该序列；或者如果核糖体结合位点被定位成使得有助于翻译，则该核糖体结合侧可操作地连接至编码序列。通常，“可操作地连接”意指所连接的DNA序列是连续的，并且在分泌性前导序列的情形下是连续的并处于阅读框中。然而，增强子不必需是连续的。通过在方便的限制位点处连接来实现连接。如果不存在此类位点，则根据常规实践使用合成的寡核苷酸衔接子或连接子。
如本文所用，术语“载体”是指能够繁殖与其连接的另一核酸的核酸分子。该术语包括作为自我复制核酸结构的载体，以及掺入已引入其的宿主细胞基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达。此类载体在本文中称为“表达载体”。
如本文所用，术语“转染的”或“转化的”或“转导的”是指将外源核酸转移或引到宿主细胞中的过程。“转染的”或“转化的”或“转导的”细胞是使用外源核酸转染、转化或转导的细胞，该细胞包括原代目标细胞及其子代。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，是指已引入外源核酸的细胞，包括此类细胞的子代。宿主细胞包括“转化体”和“转化细胞”，其包括原代转化细胞和从其衍生的后代，而与传代次数无关。后代的核酸含量可能与亲代细胞不完全相同，并且可能含有突变。具有与在原始转化细胞中筛选或选择的功能或生物学活性相同的功能或生物学活性的突变后代包括在本文中。
术语“受试者”、“个体”和“患者”在本文可互换使用，是指哺乳动物，包括但不限于人、牛、马、猫、犬、啮齿动物或灵长类动物。在一些实施例中，受试者是人。
药剂的“有效量”是指在必要的剂量和时间段内有效达到所需治疗或预防结果的量。该特定剂量可以根据以下各项中的一种或多种来改变：所选择的具体药剂、随后的给药方案(无论它是否与其他化合物组合)、施用时间、成像的组织、以及其中携带它的物理递送系统。
本申请的物质/分子、激动剂或拮抗剂的“治疗有效量”可以根据例如疾病状态、年龄、性别和个体体重以及该物质/分子、激动剂或拮抗剂在个体中引发希望的应答的能力等因素而变化。治疗有效量也是该物质/分子、激动剂或拮抗剂的任何毒性或有害作用均被治疗有益作用所抵消的量。治疗有效量可以一次或多次施用来递送。
“预防有效量”是指以剂量计并且持续所需的时间段以实现所希望的预防结果的有效的量。典型地，但非必需的，因为预防的剂量是在疾病之前或早期在受试者体内使用的，所以这种预防有效量将小于治疗有效量。
如本文所用，“治疗(treatment或treating)”是用于获得有益的或所希望的结果(包括临床结果)的方法。出于本申请的目的，有益的或所希望的临床结果包括但不限于以下中的一种或多种：缓解由疾病引起的一种或多种症状、减少疾病的程度、稳定疾病(例如，预防或延迟疾病的恶化)、预防或延迟疾病的传播(例如，转移)、预防或延迟疾病的重现、延迟或减缓疾病的进展，改善疾病状态、提供疾病的缓解(部分或全部)、减少治疗疾病所需的一种或多种其他药物的剂量、延迟疾病的进展、增加或改善生活质量、增加体重增长和/或延长存 活。“治疗”还涵盖减少癌症的病理后果(像例如，肿瘤体积)。本申请的方法考虑了这些治疗方面中的任何一个或多个。“治疗”并不一定意味着所治疗的疾病将得到治愈。
应当理解，本文所述的申请的实施例包括“由……组成”和/或“基本上由……组成”。
如本文所用，术语“约(about)”或“大约(approximately)”意指由本领域普通技术人员确定的特定值在可接受的误差范围之内，这将部分地取决于该值是怎样测定或确定的，即，受到测量系统的限制。在某些实施例中，“约”可以意指根据本领域的实践在3个或大于3个标准差之内。在某些实施例中，“约”可以表示给定值的至多20％(例如，至多10％、至多5％或至多1％)的范围。在某些实施例中，特别是对于生物学系统和方法，该术语可以意指在某一值的数量级内，例如在5倍内或在2倍内。
如本文所用，术语“调节”是指正向或负向变化。示例性调节包括约1％、约2％、约5％、约10％、约25％、约50％、约75％或约100％的变化。
如本文所用，术语“增加”是指正向地改变至少约5％。改变可以为约5％、约10％、约25％、约30％、约50％、约75％、约100％或更多。
如本文所用，术语“减少”是指负向地变化至少约5％。改变可以为约5％、约10％、约25％、约30％、约50％、约75％或甚至约100％。
本文使用的术语“约X-Y”具有与“约X至约Y”相同的含义。
当在本文和附带的权利要求中使用时，单数形式“一个/种(a)”、“或(or)”和“这些/该(the)”包括复数指代物，除非上下文明确地指示其他的情况。
“效应子功能”是指归因于抗体的Fc区的那些生物学活性，其随抗体同种型不同而变化。抗体效应子功能的实例包括：C1q结合和补体依赖性细胞毒性(CDC)、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、吞噬作用、细胞表面受体(例如B细胞受体)的下调和B细胞活化。
“免疫缀合物”是指缀合至一个或多个异源分子(包括但不限于细胞毒性剂)的抗体。
术语“药物配制品”是指这样的制剂，其处于允许包含其中的活性成分的生物学活性有效的形式，并且不含对配制品所施用的受试者具有不可接受的毒性的其他组分。
如本文所用，“药学上可接受的载剂”是指药物配制品中除活性成分以外的对受试者无毒的成分。药学上可接受的载剂包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
术语“可变区”或“可变结构域”是指抗体重链或轻链的结构域，其参与抗体与抗原的结合。在某些实施例中，天然抗体的重链和轻链的可变结构域(分别为VH和VL)通常具有相似的结构，每个结构域包含四个保守框架区(FR)和三个CDR。(参见，例如Kindt等人Kuby Immunology[库比免疫学],第61版,W.H.弗里曼公司(W.H.Freeman and Co.),第91页(2007)。)单个VH或VL结构域可足以赋予抗原结合特异性。此外，可以使用VH或VL结构域从结合抗原的抗体中分离结合特定抗原的抗体，以分别筛选互补的VL或VH结构域的文库。参见，例如Portolano等人,J.Immunol.[免疫学杂志]150:880-887(1993)；Clarkson等人,Nature[自然]352:624-628(1991)。
2.抗体
在某些实施例中，本发明部分基于发现与TIGIT结合的单结构域抗体，其可用于抗肿瘤治疗中，其中所述抗体选择性抑制TIGIT受体并诱导免疫细胞(例如，T细胞)的有益免疫应答。因此，本公开提供了抗TIGIT抗体。在某些实施例中，本文公开的抗TIGIT抗体是抑制TIGIT受体功能的拮抗剂抗体。在某些实施例中，抗TIGIT抗体阻断TIGIT受体和配体之间的相互作用。在某些实施例中，抗TIGIT抗体阻断来自TIGIT受体的免疫抑制信号。在某些实施例中，抗TIGIT抗体包含单结构域抗体，例如骆驼科动物抗体或VHH抗体。在某些实施例中，由于抗TIGIT抗体与IgG、Fab和/或scFv形式的传统抗体相比尺寸较小，因此其具有改善的组织浸润能力。
在某些实施例中，本公开的抗体可以是或包含单克隆抗体(包括嵌合抗体、人源化抗体或人抗体)。在某些实施例中，本文公开的抗体包含人源化抗体。在某些实施例中，抗体包含受体人框架，例如人免疫球蛋白框架或人共有框架。
在某些实施例中，本公开的抗体可以是抗体片段，例如Fv、Fab、Fab'、scFv、双抗体或F(ab')2片段。在某些实施例中，抗体是全长抗体，例如完整的IgG 1抗体、或本文定义的其他抗体类型或同种型。在某些实施例中，本公开的抗体可以单独或组合地掺入任何(如本申请中所述的(例如，本文详述的第2.1-2.11节))特征。
本公开的抗体可用于例如诊断或治疗赘生物或癌症。在某些实施例中，使用本公开的抗体可以抑制其生长的瘤形成和癌症包括通常对免疫疗法有响应的瘤形成和癌症。在某些实施例中，瘤形成和癌症包括乳腺癌(例如，乳腺细胞癌)、卵巢癌(例如，卵巢细胞癌)和肾细胞癌(RCC)。可以使用本公开的方法治疗的其他癌症的实例包括黑素瘤(例如，转移性恶性黑素瘤)，前列腺癌，结肠癌，肺癌，骨癌，胰腺癌，皮肤癌，脑瘤，慢性或急性白血病(包括急性髓细胞性白血病，慢性粒细胞性白血病，急性淋巴细胞性白血病，慢性淋巴细胞性白血病)，淋巴瘤(例如霍奇金淋巴瘤和非霍奇金淋巴瘤，淋巴细胞性淋巴瘤，原发性中枢神经系统(CNS)淋巴瘤，T细胞淋巴瘤)，鼻咽癌，头或颈癌，皮肤癌或眼内恶性黑色素瘤，子宫癌，直肠癌，肛门区域癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，子宫颈癌，阴道癌，外阴，食道癌，小肠癌，内分泌系统癌，甲状腺癌，甲状旁腺癌，乳腺旁癌(cancer of the adbreast gland)，软组织肉瘤，尿道癌，阴茎癌，儿童实体瘤，膀胱癌，肾癌或输尿管癌，乳腺骨盆癌，中枢神经系统(CNS)赘生物，肿瘤血管生成，脊柱肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西氏肉瘤，表皮样癌，鳞状细胞癌，环境诱导的癌症，包括由石棉(例如间皮瘤)诱导的癌症以及上述癌症的组合。
2.1示例性抗TIGIT抗体
本公开提供了结合TIGIT蛋白的分离的抗体。在某些实施例中，本公开的抗TIGIT抗体结合TIGIT的ECD。在某些实施例中，抗TIGIT抗体与包含SEQ ID NO:222所示氨基酸序列的TIGIT的ECD结合。在某些实施例中，抗TIGIT抗体结合与本文所述的抗TIGIT抗体(例如2A3)相同的表位。
在某些实施例中，本文公开的抗TIGIT抗体可以用作TIGIT受体的拮抗剂。在某些实施例中，抗TIGIT抗体可以使TIGIT受体的活性降低至少约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约99％或约99.9％。在某些实施例中，抗TIGIT抗体可以阻断TIGIT受体的下游免疫抑制信号传导。在某些实施例中，抗TIGIT抗体增加免疫细胞(例如T细胞和/或NK细胞)的免疫应答和/或抗肿瘤作用。在某些实施例中，使用抗TIGIT抗体的治疗在受试者中表现出抗肿瘤功效，从而减少肿瘤生长和/或延长受试者的存活期。在某些实施例中，包含单结构域抗体(例如，VHH)的抗TIGIT抗体与全长抗体相比具有较小的分子尺寸，这是因为与全长抗体的Fab结构域相比，单结构域抗体的尺寸较小，这样与全长抗体相比，可以导致(例如在肿瘤部位)优异的组织浸润。在某些实施例中，与使用全长抗TIGIT抗体(例如参考Ab1和参考Ab 2)的治疗相比，使用抗TIGIT抗体的治疗表现出更优异的抗肿瘤功效。参考Ab1具有与在U.S.2016/0176963 A1中公开的BMS22G2相同的氨基酸序列，参考Ab2具有与Tiragolumab相同的氨基酸序列，其序列在U.S.2017/0088613 A1中公开。
在某些实施例中，抗TIGIT抗体包含与TIGIT结合的单结构域抗体。在某些实施例中，单结构域抗体包含VHH。在某些实施例中，单结构域抗体包含重链可变区(VH)。在某些实施例中，单结构域抗体连接至Fc区。在某些实施例中，单结构域抗体不连接至Fc区。
在某些实施例中，单结构域抗体以约1x10 -7M或更小的KD结合TIGIT。在某些实施例中，单结构域抗体以约1x10 -8M或更小的KD结合TIGIT。在某些实施例中，单结构域抗体以约5x10 -9M或更小的KD结合TIGIT。在某些实施例中，单结构域抗体以约1x10 -9M或更小的KD结合TIGIT。在某些实施例中，单结构域抗体以约1x10 -9M至约1x10 -7M之间的KD与TIGIT结合。在某些实施例中，单结构域抗体以约1x10 -9M至约1x10 -8M之间的KD与TIGIT结合。在某些实施例中，单结构域抗体以约2x10 -9M至约1x10 -8M之间的KD与TIGIT结合。在某些实施例中，单结构域抗体以约2x10 -9M至约5x10 -8M之间的KD与TIGIT结合。在某些实施例中，单结构域抗体以约1x10 -9M至约5x10 -9M之间的KD与TIGIT结合。
在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:94所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:95所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:96所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:98所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:99所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:100所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:102所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:103所示序列的氨 基酸；和重链可变区CDR3，其包含具有SEQ ID NO:104所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:106所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:107所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:108所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:110所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:111所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:112所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:114所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:115所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:116所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:118所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:119所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:120所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:122所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:123所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:124所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:126所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:127所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:128所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:130所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:131所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:132所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:134所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:135所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:136所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:138所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:139所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:140所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ  ID NO:142所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:143所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:144所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:146所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:147所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:148所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:150所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:151所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:152所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:154所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:155所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:156所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:158所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:159所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:160所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:162所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:163所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:164所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:166所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:167所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:168所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:170所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:171所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:172所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:174所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:175所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:176所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:178所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:179所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:180所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉 竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:182所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:183所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:184所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:186所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:187所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:188所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:190所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:191所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:192所示序列的氨基酸。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：a)重链可变区CDR1，该重链可变区CDR1包含SEQ ID NO:94、98、102、106、110、114、118、122、126、130、134、138、142、146、150、154、158、162、166、170、174、178、182、186和190中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体；b)重链可变区CDR2，该重链可变区CDR2包含SEQ ID NO:95、99、103、107、111、115、119、123、127、131、135、139、143、147、151、155、159、163、167、171、175、179、183、187和191中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体；以及c)重链可变区CDR3，该重链可变区CDR3包含SEQ ID NO:96、100、104、108、112、116、120、124、128、132、136、140、144、148、152、156、160、164、168、172、176、180、184、188和192中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含CDR1结构域、CDR2结构域和CDR3结构域，其中该CDR1结构域、CDR2结构域和CDR3结构域分别含有包含在参考重链可变区中的CDR1结构域、CDR2结构域和CDR3结构域，该参考重链可变区包含选自由SEQ ID NO:97、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181、185、189和193组成的组的氨基酸序列。
在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:94所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:95所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:96所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:98所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:99所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:100所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:102所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:103所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:104所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO: 106所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:107所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:108所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:110所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:111所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:112所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:114所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:115所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:116所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:118所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:119所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:120所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:122所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:123所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:124所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:126所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:127所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:128所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:130所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:131所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:132所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:134所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:135所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:136所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:138所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:139所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:140所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:142所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:143所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:144所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:146所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:147所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:148所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:150所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:151所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:152所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:154所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:155所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:156所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:158所示序列 的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:159所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:160所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:162所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:163所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:164所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:166所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:167所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:168所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:170所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:171所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:172所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:174所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:175所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:176所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:178所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:179所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:180所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:182所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:183所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:184所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:186所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:187所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:188所示序列的氨基酸。在某些实施例中，单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:190所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:191所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:192所示序列的氨基酸。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含与选自由SEQ ID NO:97、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181、185、189和193组成的组的氨基酸序列具有至少约80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％序列同一性的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含选自由SEQ ID NO:97、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181、185、189和193组成的组的氨基酸序列。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:97所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:101所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:105所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:109所示的氨基酸序列。在某些实施例中，单结构域抗体包含 重链可变区，该重链可变区包含SEQ ID NO:113所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:117所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:121所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:125所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:129所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:133所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:137所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:141所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:145所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:149所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:153所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:157所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:161所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:165所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:169所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:173所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:177所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:181所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:185所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:189所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:193所示的氨基酸序列。
在某些实施例中，重链可变区中包含的任何氨基酸序列可包含至多约1、约2、约3、约4、约5、约6、约7、约8、约9或约10个氨基酸取代、缺失和/或添加。在某些实施例中，氨基酸取代是保守取代。
在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:1所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:2所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:3所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:5所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:6所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:7所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT 结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:9所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:10所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:11所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:13所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:14所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:15所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:17所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:18所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:19所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:21所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:22所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:23所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:25所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:26所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:27所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:29所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:30所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:31所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:33所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:34所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:35所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:37所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:38所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:39所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:41所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:42所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:43所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:45所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:46所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:47所示序列的氨基酸。在某些 实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:49所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:50所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:51所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:53所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:54所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:55所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:57所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:58所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:59所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:61所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:62所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:63所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:65所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:66所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:67所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:69所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:70所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:71所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:73所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:74所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:75所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:77所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:78所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:79所示序列的氨基酸。在某些实施例中，单结构域抗体与参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该参考抗TIGIT单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:81所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:82所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:83所示序列的氨基酸。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:1所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:2所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:3所示序列的氨基酸。在 某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:5所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:6所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:7所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:9所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:10所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:11所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:13所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:14所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:15所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:17所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:18所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:19所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:21所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:22所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:23所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:25所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:26所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:27所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:29所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:30所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:31所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:33所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:34所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:35所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:37所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:38所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:39所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:41所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:42所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:43所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:45所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:46所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:47所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:49所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:50 所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:51所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:53所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:54所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:55所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:57所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:58所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:59所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:61所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:62所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:63所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:65所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:66所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:67所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:69所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:70所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:71所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:73所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:74所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:75所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:77所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:78所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:79所示序列的氨基酸。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含：重链可变区CDR1，其包含具有SEQ ID NO:81所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:82所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:83所示序列的氨基酸。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含与选自由SEQ ID NO:4、8、12、16、20、24、28、32、36、40、44、48、52、56、60、64、68、72、76、80和84组成的组的氨基酸序列具有至少约80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％序列同一性的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含选自由SEQ ID NO:4、8、12、16、20、24、28、32、36、40、44、48、52、56、60、64、68、72、76、80和84组成的组的氨基酸序列。
在某些实施例中，重链可变区中包含的任何氨基酸序列可包含至多约1、约2、约3、约4、约5、约6、约7、约8、约9或约10个氨基酸取代、缺失和/或添加。在某些实施例中，氨基酸取代是保守取代。
在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:4所示 的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:8所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:12所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:16所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:20所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:24所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:28所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:32所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:36所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:40所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:44所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:48所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:52所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:56所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:60所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:64所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:68所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:72所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:76所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:80所示的氨基酸序列。在某些实施例中，单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:84所示的氨基酸序列。
在某些实施例中，单结构域抗体包含人源化框架。在某些实施例中，人源化框架包含选自由SEQ ID NO:85-93组成的组的重链可变区序列的框架序列。在某些实施例中，人源化框架包含选自由SEQ ID NO:85-93组成的组的重链可变区序列的FR2序列。
在某些实施例中，抗TIGIT抗体不包含Fc区。在某些实施例中，抗TIGIT抗体包含Fc区。在某些实施例中，Fc区包含人Fc区。在某些实施例中，Fc区包含选自下组的Fc区，该组由以下组成：IgG、IgA、IgD、IgE和IgM的Fc区。在某些实施例中，Fc区包含选自下组的Fc区，该组由以下组成：IgG1、IgG2、IgG3和IgG4的Fc区。在某些实施例中，Fc区包含IgG1Fc区。在某些实施例中，IgG1Fc区包含一种或多种增强抗体依赖性细胞介导的细胞毒性(ADCC)的突变。在某些实施例中，IgG1Fc区包含L235V、F243L、R292P、Y300L和P396L的突变。在某些实施例中，IgG1Fc区包含S239D、A330L和I332E的突变。在某些实施例中，抗TIGIT抗体包含SEQ ID NO:194所示的氨基酸序列。
在某些实施例中，重链可变区通过接头连接至Fc区。在某些实施例中，接头是肽接头。在某些实施例中，肽接头包含约4至约30个氨基酸。在某些实施例中，肽接头包含约4至约15个氨基酸。在某些实施例中，肽接头包含选自由SEQ ID NO:195-220组成的组的氨基酸序列。
在某些实施例中，该抗TIGIT抗体包含多特异性抗体，例如双特异性抗体，全长免疫球蛋白，单链Fv(scFv)片段，Fab片段，Fab'片段，F(ab’)2，Fv片段，二硫键稳定的Fv片段(dsFv)，(dsFv)2，VHH，Fv-Fc融合物，scFv-Fc融合物，scFv-Fv融合物，双抗体，三抗体，四抗体或任何它们的组合。在某些实施例中，抗体包含多特异性抗体(例如双特异性抗体)，其包含特异性结合第二抗原的第二抗体部分。
在某些实施例中，第二抗原是肿瘤相关抗原。在某些实施例中，肿瘤相关抗原选自下组，该组由以下组成：Her-2，EGFR，PD-L1，c-Met，B细胞成熟抗原(BCMA)，碳酸酐酶IX(CA1X)，癌胚抗原(CEA)，CD5，CD7，CD10，CD19，CD20，CD22，CD30，CD33，CD34，CD38，CD41，CD44，CD49f，CD56，CD74，CD123，CD133，CD138，CD276(B7H3)，上皮糖蛋白(EGP2)，滋养层细胞表面抗原2(TROP-2)，上皮糖蛋白-40(EGP-40)，上皮细胞粘附分子(EpCAM)，受体酪氨酸蛋白激酶erb-B2、3、4，叶酸结合蛋白(FBP)，胎儿乙酰胆碱受体(AChR)，叶酸受体-a，神经节苷脂G2(GD2)，神经节苷脂G3(GD3)，人端粒酶逆转录酶(hTERT)，激酶插入结构域受体(KDR)，Lewis A(CA 1.9.9)，Lewis Y(LeY)，磷脂酰肌醇蛋白聚糖-3(GPC3)，L1细胞粘附分子(L1CAM)，粘蛋白16(Muc-16)，粘蛋白1(Muc-1)，NG2D配体，癌胚抗原(h5T4)，前列腺干细胞抗原(PSCA)，前列腺特异性膜抗原(PSMA)，肿瘤相关糖蛋白72(TAG-72)，密封蛋白18.2(CLDN18.2)，血管内皮生长因子R2(VEGF-R2)，肾母细胞瘤蛋白(WT-1)，1型酪氨酸蛋白激酶跨膜受体(ROR1)及其任何组合。
在某些实施例中，第二抗原是免疫检查点调节剂。在某些实施例中，免疫检查点调节剂选自由以下组成的组：PD1、CTLA4、LAG-3、2B4、BTLA及其任何组合。
在某些实施例中，抗TIGIT抗体缀合至治疗剂或标记。在某些实施例中，标记选自下组，该组由以下组成：放射性同位素、荧光染料和酶。
2.2抗体亲和力
在某些实施例中，本文公开的抗体或多特异性抗体的抗原结合部分对其靶抗原具有高结合亲和力。在某些实施例中，抗体或抗原结合部分以约1x10 -7M或更小的KD结合靶标。在某些实施例中，抗体或抗原结合部分以约1x10 -8M或更小的KD结合靶标。在某些实施例中，抗体或抗原结合部分以约5x10 -9M或更小的KD结合靶标。在某些实施例中，抗体或抗原结合部分以约1x10 -9M或更小的KD结合靶标。在某些实施例中，抗体或抗原结合部分以约1x10 -9M至约1x10 -7M之间的KD结合靶标。在某些实施例中，抗体或抗原结合部分以KD结合至靶标在约1x10 -9M和约1x10 -8M之间的抗体。在某些实施例中，抗体或抗原结合部分以约2x10 -9M和约1x10 -8M之间的KD结合靶标。在某些实施例中，抗体或抗原结合部分以 约2x10 -9M至约5x10 -8M之间的KD结合靶标。在某些实施例中，抗体或抗原结合部分以约1x10 -9M至约5x10 -9M之间的KD结合靶标。
抗体或抗原结合部分的KD可以通过本领域已知的方法确定。这类方法包括但不限于蛋白质印迹、ELISA-、RIA-、ECL-、IRMA-、EIA-、
Figure PCTCN2021070903-appb-000003
-检验和肽扫描。
在某些实施例中，可以使用
Figure PCTCN2021070903-appb-000004
表面等离子体共振测定法测量KD。例如但不限于，在固定的抗原CMS芯片上以约10个响应单位(RU)在25℃下使用
Figure PCTCN2021070903-appb-000005
或
Figure PCTCN2021070903-appb-000006
3000(Biacore公司，皮斯卡塔韦(Piscataway)，新泽西州(NJ))进行测定。在某些实施例中，根据供应商说明书，将羧甲基化葡聚糖生物传感器芯片(CMS，Biacore公司)用N-乙基-N′-(3-二甲基氨丙基)-碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)进行活化。将抗原用pH 4.8的10mM乙酸钠稀释至5μg/ml(大约0.2μM)，之后以5μl/分钟的流速注入，以实现偶联蛋白质的大约10个响应单位(RU)。在注入抗原之后，将1M乙醇胺注入以封闭未反应的基团。对于动力学测量，在25℃，将在具有0.05％聚山梨醇酯20(TWEEN-20TM)表面活性剂(PBST)的PBS中的Fab的双倍的连续稀释物(0.78nM至500nM)以大约25μl/min的流速注入。缔合速率(k on)和解离速率(k off)是使用简单的一对一朗缪尔结合模型(
Figure PCTCN2021070903-appb-000007
评估软件版本3.2)通过同时拟合缔合与解离传感图来计算。可以将平衡解离常数(KD)计算为比率k0fffkon。参见例如,Chen等人,J.Mol.Biol.[分子生物学杂志]293:865-881(1999)。如果通过以上表面等离子体共振测定法的缔合速率(on-rate)超过10 6M -l s -1，则缔合速率可以通过使用荧光淬灭技术来确定，该技术在增加的抗原浓度(如光谱仪例如截流配置的分光光度计(阿维夫仪器)或具有搅拌吸收池的8000系列SLM-AMINCO TM分光光度计(ThermoSpectronic公司)测量的)的存在下，在25℃测量PBS(pH 7.2)中的20nM抗-抗原抗体(Fab形式)的荧光发射强度(激发＝295nm；发射＝340nm，16nm带通)的增加或减少。
2.3抗体片段
在某些实施例中，本公开的抗体包含抗原结合片段或抗体片段。抗体片段包括但不限于Fab、Fab'、Fab'-SH、F(ab')2、Fv和scFv片段，以及以下描述的其他片段。有关某些抗体片段的综述，请参见Hudson等人，Nat.Med.[自然医学]9:129-134(2003)。关于scFv片段的综述，参见例如，Pluckthtin,The Pharmacology of Monoclonal Antibodies[单克隆抗体的药理学]，第113卷，Rosenburg和Moore编辑，(施普林格出版社(Springer-Verlag),纽约)，第269-315页(1994)；还参见WO 93/16185；以及美国专利号5,571,894和5,587,458。关于包含补救受体结合表位残基并具有增加的体内半衰期的Fab和F(ab) 2片段的讨论，请参见美国专利号5,869,046。
在某些实施例中，本公开的抗体可以是双抗体。双抗体是具有两个可以是二价或双特异性的抗原结合位点的抗体片段。参见例如，EP 404,097；WO 1993/01161；Hudson等人,Nat.Med.[自然医学]9:129-134(2003)；和Hollinger等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]90:6444-6448(1993)。三抗体和四抗体还被描述于Hudson等人，Nat.Med.[自然医学] 9:129-134(2003)中。
在某些实施例中，本公开的抗体可包含单结构域抗体。单结构域抗体是包含抗体的全部或部分重链可变结构域或全部或部分轻链可变结构域的抗体片段。在某些实施例中，单结构域抗体是人单结构域抗体(Domantis公司,沃尔瑟姆(Waltham),马萨诸塞州(MA)；参见例如，美国专利号6,248,516Bl)。在某些实施例中，单结构域抗体是骆驼科动物单结构域抗体。在某些实施例中，单结构域抗体是VHH。在某些实施例中，单结构域抗体是人源化的。
抗体片段可以通过多种技术制备，这些技术包括但不限于完整抗体的蛋白水解消化以及重组宿主细胞(例如大肠杆菌或噬菌体)的生产，如本文所述。
2.4嵌合抗体和人源化抗体
在某些实施例中，本公开的包括多特异性抗体的抗原结合部分的抗体是嵌合抗体。某些嵌合抗体描述于，例如，美国专利号4,816,567；和Morrison等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],81:6851-6855(1984))中。在某些实施例中，嵌合抗体包含非人可变区(例如，源自小鼠的可变区)和人恒定区。在某些实施例中，嵌合抗体是其中类别或亚类已经从亲本抗体的类别或亚类改变的“类别转换”抗体。嵌合抗体包括其抗原结合片段。
在某些实施例中，本公开的包括多特异性抗体的抗原结合部分的抗体可以是人源化抗体。通常，将非人抗体进行人源化以减少对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。通常，人源化抗体包含一个或多个可变结构域，其中HVR，例如CDR，(或其部分)衍生自非人抗体，而FR(或其任何部分)源自人抗体序列。人源化抗体还可以任选地包含人恒定区的至少一部分。在某些实施例中，人源化抗体中的某些FR残基被来自非人抗体(例如，源自HVR残基的抗体)的相应残基取代，例如以恢复或改善抗体特异性或亲和力。
人源化抗体及其制备方法描述于例如Almagro和Fransson,Front.Biosci.[生物科学前沿]13:1619-1633(2008)中,并在例如Riechmann等人,Nature[自然]332:323-329(1988)中进一步描述；Queen等人,Proc.Nat’l Acad.Sci.USA[美国国家科学院院刊]86:10029-10033(1989)；美国专利号5,821,337、7,527,791、6,982,321和7,087,409；Kashmiri等人,Methods[方法]36:25-34(2005)(描述了SDR(a-CDR)移植)；Padlan,Mol.Immunol.[分子免疫学]28:489-498(1991)(描述“重修表面”)；Dall’Acqua等人,Methods[方法]36:43-60(2005)(描述了“FR改组”)；和Osbourn等人,Methods[方法]36:61-68(2005)和Klimka等人,Br.J.Cancer[英国癌症杂志],83:252-260(2000)(描述了FR改组的“指导选择”方法)。
可用于人源化的人类框架区包括但不限于：使用“最佳拟合”方法选择的框架区(参见，例如，Sims等人J.Immunol.[免疫学杂志]151:2296(1993))；源自轻链或重链可变区的特定亚组的人抗体的共有序列的框架区(参见，例如，Carter等人Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],89:4285(1992)；和Presta等人J.Immunol.[免疫学杂志],151:2623(1993))；人类成熟的(体细胞突变的)构架区或人种系框架区(参见，例如Almagro和Fransson,Front.Biosci.[生物科学前沿]13:1619-1633(2008))；以及筛选FR文库得到的框架区(参见，例如Baca等人,J.Biol.Chem.[生物化学杂志]272:10678-10684(1997)和Rosok等人,J.Biol. Chem.[生物化学杂志]271:22611-22618(1996))。
2.5人抗体
在某些实施例中，本公开的抗体可以是人抗体(例如，人结构域抗体或人DAb)。人抗体可以使用本领域中已知的不同技术产生。人抗体一般在van Dijk和van de Winkel,Curr.Opin.Pharmacol.[药理学新见]5:368-74(2001),Lonberg,Curr.Opin.Immunol.[免疫学新见]20:450-459(2008),和Chen,Mol.Immunol.[分子免疫学]47(4):912-21(2010)中描述。能够产生完全人单结构域抗体(或DAb)的转基因小鼠或大鼠是本领域已知的。参见例如US20090307787A1、美国专利号8,754,287、US20150289489A1、US20100122358A1、和WO2004049794。
可以通过向转基因动物施用免疫原来制备人抗体(例如人DAb)，该转基因动物已被修饰以响应抗原攻击而产生完整的人抗体或具有人可变区的完整抗体。这样的动物通常含有全部或部分的人免疫球蛋白基因座，它们取代了内源性免疫球蛋白基因座，或者存在于染色体外或随机整合到动物的染色体中。在这种转基因小鼠中，内源性免疫球蛋白基因座通常已被灭活。对于从转基因动物中获得人抗体的方法的综述，参见Lonberg,Nat.Biotech.[自然生物技术]23:1117-1125(2005)。还参见例如描述XENOMOUSE TM技术的美国专利号6,075,181和6,150,584；描述
Figure PCTCN2021070903-appb-000008
技术的美国专利号5,770,429；描述K-M
Figure PCTCN2021070903-appb-000009
技术的美国专利号7,041,870，和描述
Figure PCTCN2021070903-appb-000010
技术的美国专利申请公开号US 2007/0061900)。来自这类动物产生的完整抗体的人可变区可以(例如通过与不同的人恒定区结合)被进一步修饰。
人抗体(例如人DAb)也可以通过基于杂交瘤的方法来制备。已经描述了用于产生人单克隆抗体的人骨髓瘤和小鼠人异源骨髓瘤细胞系(参见，例如，Kozbor J.Immunol.[免疫学杂志],133:3001(1984)；Brodeur等人,Monoclonal Antibody Production Techniques and Applications[单克隆抗体生产技术和应用],第51-63页马塞尔德克尔公司(Marcel Dekker,Inc.),纽约,1987)；和Boerner等人,J.Immunol.[免疫学杂志],147:86(1991))。Li等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],103:3557-3562(2006)中还描述了通过人B细胞杂交瘤技术产生的人抗体。另外的方法包括例如在美国专利号7,189,826(描述从杂交瘤细胞系生产单克隆人IgM抗体)和Ni,Xiandai Mianyixue[现代免疫学],26(4):265-268(2006)(描述人-人杂交瘤)中描述的那些方法。人杂交瘤技术(Trioma技术)也描述于Vollmers和Brandlein,Histology and Histopathology[组织学和组织病理学],20(3):927-937(2005)和Vollmers和Brandlein,Methods and Findings in Experimental and Clinical Pharmacology[实验和临床药理学的方法和发现],27(3):185-91(2005)中。
人抗体(例如人DAb)也可以通过分离选自人源噬菌体展示文库的Fv克隆可变结构域序列来产生。然后可以将此类可变结构域序列与所需的人恒定域结合。从抗体库中选择人抗体的技术描述如下。
2.6文库衍生的抗体
可以通过在组合文库中筛选具有所需活性或多种活性的抗体来分离抗体部分。例如，本 领域已知多种用于产生噬菌体展示文库并筛选此类文库中具有所需结合特性的抗体的方法。这类方法在例如Hoogenboom等人Methods in Molecular Biology[分子生物学方法]178:1-37(O’Brien等人编辑,哈门那出版社(Human Press),托托瓦(Totowa),新泽西州(NJ),2001)中描述，并且在以下文献中进一步描述：McCafferty等人,Nature[自然]348:552-554；Clackson等人,Nature[自然]352:624-628(1991)；Marks等人,J.Mol.Biol.[分子生物学杂志]222:581-597(1992)；Marks和Bradbury,Methods in Molecular Biology[分子生物学方法]248:161-175(Lo编辑,哈门那出版社(Human Press),托托瓦(Totowa),新泽西州(NJ),2003)；Sidhu等人,J.Mol.Biol.[分子生物学杂志]338(2):299-310(2004)；Lee等人,J.Mol.Biol.[分子生物学杂志]340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]101(34):12467-12472(2004)；和Lee等人,J.Immunol.Methods[免疫学方法杂志]284(1-2):119-132(2004)。已经描述了用于构建单结构域抗体文库的方法，例如，参见美国专利号7371849。
在某些噬菌体展示方法中，通过聚合酶链反应(PCR)分别克隆V H和V L基因库，并在噬菌体文库中随机重组，然后可以按照Winter等人,Ann.Rev.Immunol.[免疫学年度评论],12:433-455(1994)中的描述筛选抗原结合噬菌体。噬菌体通常将抗体片段展示为scFv片段或Fab片段。来自免疫源的文库无需构建杂交瘤即可提供针对免疫原的高亲和力抗体。可替代地，可以如Griffiths等人,EMBO J[欧洲分子生物学学会杂志],12:725-734(1993)所述在无需任何免疫的情况下克隆天然文库(例如从人中获得)以提供针对广泛范围的非自身以及自身抗原的抗体的单一来源。最后，如Hoogenboom和Winter,J.Mol.Biol.[分子生物学杂志],227:381-388(1992)所述，还可以通过从干细胞克隆未重排的V基因片段，并使用包含随机序列的PCR引物来编码高度可变的CDR3区并在体外完成重排，来合成天然文库。描述人抗体噬菌体文库的专利出版物包括：美国专利号5,750,373和美国专利公开号2005/0079574、2005/0119455、2005/0266000、2007/0117126、2007/0160598、2007/0237764、2007/0292936和2009/0002360。
从人抗体文库分离的抗体或抗体片段在本文中被认为是人抗体或人抗体片段。
2.7抗体变体
当前公开的主题还提供了公开的抗体的氨基酸序列变体。例如，可能需要改善该抗体的结合亲和力和/或其他生物特性。可以通过将适当的修饰引入编码抗体的核苷酸序列中或通过肽合成来制备抗体的氨基酸序列变体。这样的修饰包括但不限于抗体的氨基酸序列内的残基的缺失和/或插入和/或取代。可以进行缺失、插入和取代的任何组合以得到最终的构建体，条件是最终的(即经过修饰的)抗体具有所需的特性(例如抗原结合)。
2.7.1取代、插入和缺失变体
在某些实施例中，提供了具有一个或多个氨基酸取代的抗体变体。取代诱变的目标位点包括HVR(或CDR)和FR。保守取代示于表2中“优选取代”标题之下。表2中在“示例性取代”的标题下提供了更实质的变化，并且如下文参考氨基酸侧链类别进一步描述的。氨 基酸取代可以引入目的抗体中，并针对所希望的活性(例如保留/改善的抗原结合、降低的免疫原性、或改善的ADCC或CDC)筛选产物。
表2.氨基酸取代
原始残基 示例性取代 优选取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp、Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
氨基酸可以根据常见的侧链特性进行分组：(1)疏水性：正亮氨酸、Met、Ala、Val、Leu、Ile；(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；(3)酸性：Asp、Glu；(4)碱性：His、Lys、Arg；(5)影响链取向的残基：Gly、Pro；和(6)芳香族的：Trp、Tyr、Phe。在某些实施例中，非保守取代将需要将这些类别中的一个的成员交换为另一个类别。
在某些实施例中，取代变体的一种类型涉及取代亲本抗体(例如，人源化或人抗体)的一个或多个高变区残基。通常，选择用于进一步研究的所得变体将相对于亲本抗体在某些生物学特性(例如，增加的亲和力、降低的免疫原性)上具有修饰(例如，改善)和/或将基本上保留了亲本抗体的某些生物学特性。示例性取代变体是亲和力成熟的抗体，该抗体可以方 便地生成，例如使用基于噬菌体展示的亲和力成熟技术(例如本文披露的那些)。简而言之，对一个或多个HVR(或CDR)残基进行突变，并且将变体抗体在噬菌体上展示并针对特定生物学活性(例如结合亲和力)进行筛选。
可以在HVR(或CDR)中进行改变(例如，取代)，例如以改善抗体亲和力。此类改变可以在HVR(或CDR)“热点”(即由在体细胞成熟过程期间以高频率经历突变的密码子编码的残基)中进行(参见例如，Chowdhury,Methods Mol.Biol.[分子生物学方法]207:179-196(2008))和/或SDR(a-CDR)中进行，测试所得变体VH或VL的结合亲和力。通过构建和从二级文库中重新选择而进行的亲和力成熟已经描述于例如，Hoogenboom等人Methods in Molecular Biology[分子生物学方法]178:1-37(O’Brien等人编辑,哈门那出版社(Human Press),托托瓦(Totowa),新泽西州(NJ),(2001))中。在亲和力成熟的某些实施例中，通过多种方法(例如，易错PCR，链改组，或寡核苷酸定向诱变)中的任一种，将多样性引入选择用于成熟的可变基因中。然后产生二级文库。然后筛选文库以鉴别具有所希望的亲和力的任何抗体变体。引入多样性的另一种方法涉及HVR(或CDR)定向方法，其中使若干HVR(或CDR)残基(例如一次4-6个残基)随机化。可例如使用丙氨酸扫描诱变或模型化来特异性地鉴别抗原结合中涉及的HVR(或CDR)残基。特别是CDR-H3和CDR-L3经常成为靶标。
在某些实施例中，取代、插入或缺失可在一个或多个HVR(或CDR)内发生，只要这样的改变基本上不降低抗体结合抗原的能力。例如，可以在HVR(或CDR)中进行基本上不降低结合亲和力的保守改变(例如，如本文提供的保守取代)。此类改变可能在HVR(或CDR)“热点”或CDR之外。在以上提供的变体VHH序列的某些实施例中，每个HVR(或CDR)是未改变的，或包含不超过一个、两个或三个氨基酸取代。
如Cunningham和Wells(1989)Science[科学],244:1081-1085所述，用于鉴定可以靶向诱变的抗体的残基或区域的有用方法称为“丙氨酸扫描诱变”。在这种方法中，鉴定目标残基的残基或残基组(例如，带电荷的残基，例如Arg、Asp、His、Lys和Glu)，并用中性或带负电荷的氨基酸(例如，丙氨酸或聚丙氨酸)取代，以确定抗体与抗原的相互作用是否受到影响。可以在氨基酸位置引入另外的取代，证明对初始取代的功能敏感性。可替代地或另外地，抗原-抗体复合物的晶体结构用于鉴定抗体和抗原之间的接触点。这样的接触残基和邻近残基可以被靶向或消除作为取代候选。可以筛选变体以确定它们是否包含所需的属性。
氨基酸序列插入包括氨基末端和/或羧基末端融合，长度在一个残基至含有一百个或更多残基的多肽的范围内，以及单一或多个氨基酸残基的序列内插入。末端插入的实例包括具有N末端甲硫氨酰基残基的抗体。抗体分子的其他插入变体包括与抗体的N-或C-末端与增加抗体的血清半衰期的酶(例如，对于ADEPT)或多肽融合。
2.7.2糖基化变体
在某些实施例中，改变抗体部分以增加或减少构建体糖基化的程度。向抗体中添加或缺失糖基化位点可通过改变氨基酸序列以产生或去除一个或多个糖基化位点而方便地实现。
当抗体部分包含Fc区(例如，scFv-Fc)时，与其相连的碳水化合物可以发生改变。由哺乳动物细胞产生的天然抗体通常包含分支的双触角寡糖，其通常通过N-键连接至Fc区C H2结构域的Asn297。参见，例如Wright等人TIBTECH 15:26-32(1997)。寡糖可以包括多种碳水化合物，例如，甘露糖、N-乙酰基葡糖胺(GlcNAc)、半乳糖和唾液酸，以及在双触角寡糖结构的“茎”中附着于GlcNAc的岩藻糖。在某些实施例中，可以对抗体部分中的寡糖进行修饰，以产生具有某些改善的特性的抗体变体。
在某些实施例中，抗体部分具有碳水化合物结构，该碳水化合物结构缺少(直接或间接)附接至Fc区的岩藻糖。例如，此类抗体中的岩藻糖含量可以为1％至80％、1％至65％、5％至65％或20％至40％。岩藻糖的量是通过计算Asn297糖链中岩藻糖的平均量来确定的，相对于通过MALDI-TOF质谱测量的与Asn 297附接的所有糖结构(例如，复合、杂合和高甘露糖结构)的总和，如WO 2008/077546中所述。Asn297是指位于Fc区中约297位的天冬酰胺残基(Fc区残基的EU编号)；然而，由于抗体中的微小序列变化，Asn297也可位于位置297上游或下游约±3个氨基酸，即在位置294和300之间。这类岩藻糖基化变体可以具有改善的ADCC功能。参见例如，美国专利公开号US 2003/0157108(Presta,L.)；US 2004/0093621(协和发酵工业株式会社(Kyowa Hakko Kogyo Co.,Ltd))。与“去岩藻糖基化”或“岩藻糖缺陷型”抗体变体有关的出版物实例包括：US 2003/0157108；WO 2000/61739；WO 2001/29246；US 2003/0115614；US 2002/0164328；US 2004/0093621；US 2004/0132140；US 2004/0110704；US 2004/0110282；US 2004/0109865；WO 2003/085119；WO 2003/084570；WO 2005/035586；WO 2005/035778；WO2005/053742；WO2002/031140；Okazaki等人J.Mol.Biol.[分子生物学杂志]336:1239-1249(2004)；Yamane-Ohnuki等人Biotech.Bioeng.[生物技术和生物工程]87:614(2004)。能够产生去岩藻糖基化抗体的细胞系的实例包括蛋白质岩藻糖基化作用缺陷型的Lec13CHO细胞(Ripka等人Arch.Biochem.Biophys.[生物化学与生物物理学集刊]249:533-545(1986)；美国专利申请号US 2003/0157108 A1,Presta,L；和WO 2004/056312 A1，Adams等人)，以及敲除细胞系，例如α-1,6-岩藻糖基转移酶基因FUT8，敲除CHO细胞(参见例如，Yamane-Ohnuki等人Biotech.Bioeng.[生物技术和生物工程]87:614(2004)；Kanda,Y.等人,Biotechnol.Bioeng.[生物技术和生物工程],94(4):680-688(2006)；和WO2003/085107)。
在某些实施例中，抗体部分具有二等分的寡糖，例如其中附着于抗体的Fc区的双触角寡糖被GlcNAc二等分。此类抗体变体可以具有减少的岩藻糖基化和/或改善的ADCC功能。此类抗体变体的实例描述于例如WO 2003/011878(Jean-Mairet等人)；美国专利号6,602,684(Umana等人)；和US 2005/0123546(Umana等人)中。还提供了在寡糖上具有至少一个连接至Fc区的半乳糖残基的抗体变体。此类抗体变体可以具有改善的CDC功能。此类抗体变体描述于例如WO 1997/30087(Patel等人)；WO 1998/58964(Raju,S.)；和WO 1999/22764(Raju,S.)中。
2.7.3 Fc区变体
在某些实施例中，可将一个或多个氨基酸修饰引入抗体部分的Fc区(例如scFv-Fc)内，从而生成Fc区变体。Fc区变体可包含一个人Fc区序列(例如，人IgG1、IgG2、IgG3或IgG4Fc区)，该序列包含在一个或多个氨基酸位置处的氨基酸修饰(例如，取代)。
在某些实施例中，具有一些(但非全部)效应子功能的Fc片段，此类功能使该片段成为适合应用的理想候选物，在所述应用中，抗体部分在体内的半衰期很重要，但某些效应子功能(例如，补体和ADCC)是非必要或有害的。可以进行体外和/或体内细胞毒性测定确认CDC和/或ADCC活性的降低/消耗。例如，可以进行Fc受体(FcR)结合测定确保抗体没有FcγR结合能力(因此可能缺乏ADCC活性)，但可以保留FcRn结合能力。用于介导ADCC的原代细胞NK细胞仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。造血细胞上的FcR表达总结在Ravetch和Kinet，在Annu.Rev.Immunol.[免疫学年度评论]9:457-492(1991)的第464页的表2中。在美国专利号5,500,362中描述了用于评估感兴趣的分子的ADCC活性的体外测定的非限制性实例(例如，参见Hellstrom,I.等人，Proc.Nat’l Acad.Sci.USA[美国国家科学院院刊]，83:7059-7063(1986))以及Hellstrom,I等人，Proc.Nat'l Acad.Sci.USA[美国国家科学院院刊]，82:1499-1502(1985)；5,821,337(参见Bruggemann,M.等人,J.Exp.Med.[实验医学杂志]，166:1351-1361(1987))。可替代地，可以采用非放射性测定方法(例如，参见用于流式细胞术的ACTI TM非放射性细胞毒性测定(细胞技术公司(CellTechnology,Inc.)山景城(Mountain View),加利福尼亚州；以及CytoTox
Figure PCTCN2021070903-appb-000011
非放射性细胞毒性测定(普洛麦格公司(Promega)，麦迪逊，威斯康星州)。用于此类测定的有用的效应细胞包括外周血单核细胞(PBMC)和自然杀伤(NK)细胞。可替代地或另外地，可以在体内评估目的分子的ADCC活性，例如在动物模型中，如Clynes等人,Proc.Nat’l Acad.Sci.USA[美国国家科学院院刊]95:652-656(1998)中披露的。还可以进行C1q结合测定确认抗体不能结合C1q，并且因此缺乏CDC活性。例如，参见在WO 2006/029879和WO 2005/100402中的C1q和C3c结合ELISA。为了评估补体活化，可以进行CDC测定(参见例如,Gazzano-Santoro等人,J.Immunol.Methods[免疫学方法杂志]202:163(1996)；Cragg,M.S.等人,Blood[血液]101:1045-1052(2003)；以及Cragg,M.S.和M.J.Glennie,Blood[血液]103:2738-2743(2004))。也可以使用本领域已知的方法进行FcRn结合和体内清除/半衰期测定(例如，参见：Petkova,S.B.等人,Int’l.Immunol.[国际免疫学]18(12):1759-1769(2006))。
具有降低的效应子功能的抗体包括(美国专利号6,737,056)，具有Fc区残基238、265、269、270、297、327和329中的一个或多个的取代的抗体。此类Fc突变体包括具有氨基酸位置265、269、270、297和327中的两个或更多个的取代的Fc突变体，包括具有残基265和297被丙氨酸取代的所谓“DANA”Fc突变体(美国专利号7,332,581)。
本文描述了与FcR结合提高或降低的某些抗体变体。(参见，例如美国专利号6,737,056；WO 2004/056312，以及Shields等人,J.Biol.Chem.[生物化学杂志]9(2):6591-6604(2001)。)
在某些实施例中，Fc片段是IgG1Fc片段。在某些实施例中，IgG1Fc片段包含L234A突变和/或L235A突变。在某些实施例中，Fc片段是IgG2或IgG4Fc片段。在某些实施例中， Fc片段是包含S228P、F234A和/或L235A突变的IgG4Fc片段。
在某些实施例中，抗体部分包含具有一个或多个氨基酸取代的Fc区，这些取代(例如，Fc区内的位置298、333和/或334处的取代(残基的EU编号))改善ADCC。
在某些实施例中，Fc区内发生改变，导致C1q结合和/或补体依赖性细胞毒性(CDC)发生改变(即，提高或降低)，例如，如美国专利号6,194,551、WO 99/51642和Idusogie等人,J.Immunol.[免疫学杂志],164:4178-4184(2000)中所描述。
在某些实施例中，抗体部分(例如，scFv-Fc)变体包含变体Fc区，该变体Fc区包含一个或多个改变半衰期和/或改变与新生儿Fc受体(FcRn)的结合的氨基酸取代。具有延长的半衰期和与新生儿Fc受体(FcRn)的改善的结合的抗体，其负责将母体IgG转移至胎儿(Guyer等人,J.Immunol.[免疫学杂志]117:587(1976)和Kim等人,J.Immunol.[免疫学杂志]24:249(1994))，如US2005/0014934A1中所描述(Hinton等人)。那些抗体包含具有一个或多个氨基酸取代的Fc区，其中这些取代改变了Fc区与FcRn的结合。此类Fc变体包括在一个或多个Fc区残基上具有取代(例如Fc区残基434的取代)的那些变体(美国专利号7,371,826)。
还参见Duncan和Winter，Nature[自然]322:738-40(1988)；美国专利号5,648,260；美国专利号5,624,821；以及关于Fc区变体的其他实例的WO 94/29351。
2.7.4半胱氨酸工程化抗体变体
在某些实施例中，可能需要产生半胱氨酸工程化抗体部分，例如“thioMAb”，其中抗体的一个或多个残基被半胱氨酸残基取代。在某些实施例中，取代的残基出现在抗体的可及位点处。通过用半胱氨酸取代那些残基，反应性硫醇基团由此位于抗体的可及位点，并可用于将抗体与其他部分，例如药物部分或接头-药物部分偶联，以产生免疫偶联物，如本文进一步所述。在某些实施例中，以下任何一个或多个残基可以被半胱氨酸取代：重链的A118(EU编号)；以及重链Fc区的S400(EU编号)。半胱氨酸工程化抗体部分可以如例如美国专利号7,521,541中所述产生。
2.8抗体衍生物
在某些实施例中，本文所述的抗体部分可以被进一步修饰以包含本领域已知且容易获得的其他非蛋白质部分。适用于抗体衍生的部分包括但不限于水溶性聚合物。水溶性聚合物的非限制性实例包括但不限于聚乙二醇(PEG)，乙二醇/丙二醇，羧甲基纤维素，葡聚糖，聚乙烯醇，聚乙烯吡咯烷酮，聚-1,3-二氧戊环，聚-1,3,6-三氧杂环己烷，乙烯/马来酸酐共聚物，聚氨基酸(均聚物或无规共聚物)和右旋糖酐或聚(正乙烯基吡咯烷酮)聚乙二醇，丙二醇均聚物，环氧丙烷/环氧乙烷共聚物，聚氧乙烯化多元醇(例如甘油)，聚乙烯醇及其混合物。聚乙二醇丙醛由于在水中的稳定性而在制造中可能具有优势。该聚合物可以具有任何分子量，并且可以是支链或非支链的。连接至抗体的聚合物的数量可以变化，并且如果连接的聚合物多于一种，则它们可以是相同或不同的分子。一般而言，用于衍生化的聚合物的数量和/或类型可以基于以下考虑因素来确定，这些因素包括但不限于待改善抗体的特定性质或功能，是否将抗体衍生物用于确定的诊断条件等。
在某些实施例中，抗体部分可以被进一步修饰以包含一种或多种生物活性蛋白、多肽或其片段。如本文可互换使用的，“生物活性的”或“具有生物学活性的”是指在体内显示出执行特定功能的生物学活性。例如，它可能意味着与特定生物分子(例如蛋白质、DNA等)结合，然后促进或抑制这种生物分子的活性。在某些实施例中，生物活性蛋白或其片段包括：作为活性药物物质施用给患者的蛋白和多肽；用于预防或治疗疾病或病症、以及用于诊断目的的蛋白和多肽(例如诊断测试或体外测定中使用的酶)；以及为预防疾病而施用给患者的蛋白质和多肽(例如疫苗)。
2.9抗体的生产方法
可以使用本领域中任何可用的或已知的技术来产生本文公开的抗体。例如但不限于，可以使用重组方法和组合物产生抗体，例如，如美国专利号4,816,567中所述。产生抗体的详细程序在以下实例中描述。
本公开的主题还提供了编码本文公开的抗体的分离的核酸。例如，分离的核酸可以编码包含抗体的VL的氨基酸序列和/或包含抗体的VH的氨基酸序列，例如抗体的轻链和/或重链。
在某些实施例中，核酸可以存在于一种或多种载体(例如表达载体)中。如本文所用，术语“载体”是指能够转运与其连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，其是指可以将另外的DNA区段连接到其中的环状双链DNA环。另一种类型的载体是病毒载体，其中可以将另外的DNA区段连接到病毒基因组中。某些载体能够在它们被引入至其中的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体以及附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)在引入到宿主细胞后被整合到宿主细胞的基因组中，并且从而随着宿主基因组一起复制。此外，某些载体表达载体能够指导它们可操作地连接的基因的表达。一般而言，用于重组DNA技术中的表达载体常常为质粒(载体)形式。但是，所公开的主题旨在包括具有等效功能的其他形式的表达载体，例如病毒载体(例如复制缺陷型逆转录病毒、腺病毒和腺相关病毒)。
可以在单个多顺反子表达盒、单个载体的多个表达盒或多个载体中构建本文公开的抗体的不同部分。产生多顺反子表达盒的元件的实例包括但不限于多种病毒和非病毒内部核糖体进入位点(IRES，例如，FGF-l IRES，FGF-2IRES，VEGF IRES，IGF-II IRES，NF-kB IRES，RUNX1IRES，p53IRES，甲型肝炎IRES，丙型肝炎IRES，瘟病毒IRES，口蹄疫病毒IRES，小核糖核酸病毒IRES，脊髓灰质炎病毒IRES和脑心肌炎病毒IRES)和可裂解的接头(例如2A肽，例如P2A、T2A、E2A和F2A肽)。逆转录病毒载体和合适的包装线的组合也是合适的，其中衣壳蛋白将具有感染人细胞的功能。已知多种产生两性病毒的细胞系，包括但不限于PA12(Miller等人(1985)Mol.Cell.Biol.[分子细胞生物学]5:431-437)；PA317(Miller等人(1986)Mol.Cell.Biol.[分子细胞生物学]6:2895-2902)；和CRIP(Danos等人(1988)Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]85:6460-6464)。非两亲性颗粒也是合适的，例如用VSVG、RD114或GALV包膜和本领域中任何其他已知的假型颗粒。
在某些实施例中，可以将编码本公开的抗体的核酸和/或包括该核酸的一种或多种载体引入宿主细胞。在某些实施例中，可以通过本领域已知的任何方法将核酸引入细胞中，这些方法包括但不限于转染，电穿孔，显微注射，用含有核酸序列的病毒或噬菌体载体感染，细胞融合，染色体介导的基因转移，微细胞介导的基因转移，原生质球融合等。在某些实施例中，宿主细胞可以包括，例如，已经用以下载体转化的宿主细胞：该载体包含编码包含单结构域抗体和/或单结构域抗体的VH的氨基酸序列的核酸。在某些实施例中，宿主细胞可以包括例如已经用以下转化的宿主细胞：(1)一种载体，其包含编码包含该抗体的VL的氨基酸序列和包含该抗体的VH的氨基酸序列的核酸，或(2)第一载体，其包含编码该抗体的VL的氨基酸序列的核酸，和第二载体，其包含编码该抗体的VH的氨基酸序列的核酸。在某些实施例中，宿主细胞是真核的，例如中国仓鼠卵巢(CHO)细胞或淋巴样细胞(例如，YO、NSO、Sp20细胞)。
在某些实施例中，制备本文公开的抗体的方法可以包括在适合于抗体表达的条件下培养其中已经引入了编码抗体的核酸的宿主细胞，以及任选地从宿主细胞和/或宿主细胞培养基中回收抗体。在某些实施例中，通过色谱技术从宿主细胞回收抗体。
为了重组产生本公开的抗体，可以分离编码如上所述的抗体的核酸，并将其插入一种或多种载体中，以在宿主细胞中进一步克隆和/或表达。可以使用常规程序(例如，通过使用能够特异性结合编码抗体的重链和轻链的基因的寡核苷酸探针)容易地分离和测序此类核酸。用于克隆或表达编码抗体的载体的合适宿主细胞包括本文所述的原核或真核细胞。例如，可以在细菌中产生抗体，特别是在不需要糖基化和Fc效应子功能时。对于在细菌中表达抗体片段和多肽，参见例如，美国专利号5,648,237、5,789,199和5,840,523。(还参见Charlton,Methods in Molecular Biology[分子生物学方法],第248卷(B.K.C.Lo,编辑,哈门那出版社(Human Press),托托瓦(Totowa),新泽西州(NJ),2003),第245-254页，描述了抗体片段在大肠杆菌中的表达)表达后，可以从细菌细胞糊中分离出可溶级分的抗体，并可以进一步纯化。
除原核生物外，真核微生物(例如丝状真菌或酵母菌)也是适合抗体编码载体的克隆或表达宿主，包括其糖基化途径已被“人源化”的真菌和酵母菌株，从而产生具有部分或完全人糖基化模式的抗体。参见Gemgross,Nat.Biotech.[自然生物技术]22:1409-1414(2004),和Li等人,Nat.Biotech.[自然生物技术]24:210-215(2006)。用于表达糖基化抗体的合适宿主细胞也可以源自多细胞生物(无脊椎动物和脊椎动物)。无脊椎动物细胞的实例包括植物和昆虫细胞。已经鉴定出许多杆状病毒株，它们可以与昆虫细胞结合使用，特别是用于转染草地贪夜蛾(Spodoptera frugiperda)细胞。在某些实施例中，植物细胞培养物可以用作宿主细胞。参见例如美国专利号5,959,177、6,040,498、6,420,548、7,125,978和6,417,429(描述了在转基因植物中生产抗体的PLANTIBODIES TM技术)。
在某些实施例中，脊椎动物细胞也可以用作宿主。例如但不限于，适于悬浮生长的哺乳动物细胞系可能是有用的。有用的哺乳动物宿主细胞系的非限制性实例是由SY40(COS-7)转化的猴肾CV1系；人胚胎肾系(293或293细胞，如例如在Graham等人,J Gen Viral.[普通 病毒学杂志]36:59(1977)中描述)；幼仓鼠肾细胞(BHK)；小鼠塞托利(sertoli)细胞(TM4细胞，例如在Mather,Biol.Reprod.[生殖生物学]23:243-251(1980)中描述)；猴肾细胞(CV 1)；非洲绿猴肾细胞(VERO-76)；人宫颈癌细胞(HELA)；犬肾细胞(MDCK；水牛鼠(buffalo rat)肝细胞(BRL 3A)；人肺细胞(W138)；人肝细胞(Hep 02)；小鼠乳房肿瘤(MMT 060562)；TRI细胞，例如在Mather等人,Annals N.Y.Acad.Sci.[纽约科学院年刊]383:44-68(1982)中描述；MRC 5细胞；和FS4细胞。其他有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFK CHO细胞(Urlaub等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]77:42I6(1980))；和骨髓瘤细胞系(例如YO、NSO和Sp2/0)。对于某些适合抗体产生的哺乳动物宿主细胞系的综述，参见例如，Yazaki和Wu,Methods in Molecular Biology[分子生物学方法],第248卷(B.K.C.Lo编辑,哈门那出版社(Human Press),托托瓦(Totowa),新泽西州(NJ)),第255-268页(2003)。
在某些实施例中，用于制备双特异性和/或多特异性抗体的技术包括但不限于重组表达具有相同特异性的两个免疫球蛋白重链轻链对，其中一个或两个重链或轻链融合至具有不同特异性的抗原结合部分(例如，单结构域抗体，例如VHH)，具有不同特异性的两个免疫球蛋白重链轻链对的重组共表达(参见Milstei n和Cuello,Nature[自然]305:537(1983))，PCT专利申请号WO 93/08829和Traunecker等人，EMBO J[欧洲分子生物学学会杂志]10:3655(l991))和“杵臼结构”工程化(参见例如，美国专利号5,731,168)。双特异性抗体也可以通过工程化静电转向效应来制备，以制备抗体Fc-异二聚体分子(WO 2009/089004A1)；交联两个或更多个抗体或片段(参见例如，美国专利号4,676,980和Brennan等人,Science[科学],229:81(1985))；使用亮氨酸拉链产生双特异性抗体(参见例如，Kostelny等人,J Immunol.[免疫学杂志],148(5):1547-1553(1992))；使用“双抗体”技术制备双特异性抗体片段(参见例如，Hollinger等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],90:6444-6448(1993))；和使用单链Fv(sFv)二聚体(参见例如，Gruber等人,J.Immunol.[免疫学杂志],152:5368(1994))；并如例如在Tutt等人J Immunol.[免疫学杂志]147:60(1991)中所述制备三特异性抗体。
本公开的双特异性和多特异性分子也可以使用化学技术(参见，例如，Kranz(1981)Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]78:5807)、“多瘤”技术(参见，例如，美国专利4,474,893)或重组DNA技术制备。还可以使用本领域已知的和本文所述的方法，通过缀合组成型结合特异性，例如第一表位和第二表位结合特异性，来制备当前公开的主题的双特异性和多特异性分子。例如，但不限于，双特异性和多特异性分子的每种结合特异性可以通过重组融合蛋白技术一起产生，或者可以分开产生，然后彼此缀合。当结合特异性是蛋白质或肽时，可以使用多种偶联剂或交联剂进行共价结合。交联剂的非限制性实例包括蛋白A，碳二亚胺，N-琥珀酰亚胺基-S-乙酰基-硫代乙酸酯(SATA)，N-琥珀酰亚胺基-3-(2-吡啶基二硫代)丙酸酯(SPDP)和磺基琥珀酰亚胺基4-(N-马来酰亚胺基甲基)环己烷-1-羧酸酯(磺基-SMCC)(参见例如Karpovsky(1984)J.Exp.Med.[实验医学杂志]160:1686；Liu(1985)Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]82:8648)。其他方法包括由Paulus(Behring  Ins.Mitt.[贝林研究所通报](1985)第78期,118-132；Brennan(1985)Science[科学]229:81-83),Glennie(1987)J Immunol.[免疫学杂志]139:2367-2375)描述的那些方法。当结合特异性是抗体(例如，两种人源化抗体)时，它们可以通过两条重链的C末端铰链区的巯基键合来缀合。在某些实施例中，在缀合之前，铰链区可以被修饰为包含奇数个(例如一个)巯基残基。
在某些实施例中，双特异性抗体的两种结合特异性可以在同一载体中编码，并在同一宿主细胞中表达和组装。当双特异性和多特异性分子是MAb x MAb，MAb x Fab，Fab x F(ab’)2或配体x Fab融合蛋白时，此方法特别有用。在某些实施例中，本公开的双特异性抗体可以是单链分子，例如单链双特异性抗体，包含一个单链抗体和结合决定簇的单链双特异性分子或包含两个结合决定簇的单链双特异性分子。双特异性和多特异性分子也可以是单链分子或可以包含至少两个单链分子。制备双特异性分子和多特异性分子的方法描述于例如美国专利号5,260,203；美国专利号5,455,030；美国专利号4,881,175；美国专利号5,132,405；美国专利号5,091,513；美国专利号5,476,786；美国专利号5,013,653；美国专利号5,258,498；以及美国专利号5,482,858中。本文还包括具有三个或更多个功能性抗原结合位点(例如，表位结合位点)的工程化抗体，包括“章鱼抗体”(参见例如US 2006/0025576A1)。
在某些实施例中，动物系统可以用于产生本公开的抗体。用于制备杂交瘤的一种动物系统是鼠系统。
在小鼠中杂交瘤的产生是非常完善建立的程序。用于分离用于融合的经免疫的脾细胞的免疫方案和技术在本领域中是已知的。融合配偶体(例如鼠骨髓瘤细胞)和融合程序也是已知的(参见例如Harlow和Lane(1988),Antibodies,A Laboratory Manual[抗体实验室手册],纽约州冷泉港冷泉港实验室出版社(Cold Spring Harbor Laboratory Press,Cold Spring Harbor New York))。
2.10测定
本文提供的本公开的抗体可以通过本领域已知的和本文提供的多种测定法对其物理/化学性质和/或生物学活性进行鉴定、筛选或表征。
在某些实施例中，可以通过已知方法(例如酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)或蛋白质印迹测定)来测试本公开的抗体的抗原结合活性。这些测定中的每一种通常通过采用特异性针对感兴趣的复合物的经标记的试剂(例如，抗体)来检测特别感兴趣的蛋白-抗体复合物的存在。例如，可以使用例如识别并特异性结合抗体的酶联抗体或抗体片段来检测抗体。可替代地，可以使用多种其他免疫测定中的任何一种来检测抗体。例如，可以将该抗体进行放射性标记并且在放射性免疫测定(RIA)中使用(参见例如，Weintraub,B.，Principles of Radioimmunoassays[放射性免疫测定的原则]，Seventh Training Course on Radioligand Assay Techniques[关于放射性配体测定技术的第七次培训课程]，The Endocrine Society[内分泌学会]，1986年3月，将其通过引用并入本文)。可以通过这样的手段，如使用盖革(Geiger)计数器或闪烁计数器或通过放射自显影来对放射性同位素进行检测。
在某些实施例中，竞争测定法可用于鉴定与本公开的抗体(例如1C12、2A3或1G1)竞争与TIGIT结合的抗体。在某些实施例中，这样的竞争性抗体结合由1C12、2A3或1G1结合的相同表位(例如，线性或构象表位)。Morris(1996)“Epitope Mapping Protocols[表位定位方案],”Methods in Molecular Biology[分子生物学方法],第66卷(哈门那出版社(Human Press),托托瓦(Totowa),新泽西州(NJ))中提供了定位抗体所结合的表位的详细示例性方法。
在竞争测定的非限制性实例中，可以在包含与TIGIT结合的第一标记抗体(例如1C12、2A3或1G1)和第二未标记抗体的溶液中孵育固定的TIGIT，测试所述第二未标记抗体与第一抗体竞争与TIGIT结合的能力。第二抗体可以存在于杂交瘤上清液中。作为对照，将固定的TIGIT在包含第一标记抗体但不包含第二未标记抗体的溶液中孵育。在允许第一抗体与TIGIT结合的条件下孵育后，去除过量的未结合抗体，并测量与固定的TIGIT相关的标记物的量。如果在测试样品中相比于对照样品的与固定的TIGIT相关的标记的量大大减少，则表明第二抗体正在与第一抗体竞争与TIGIT的结合。参见Harlow和Lane(1988)Antibodies:A Laboratory Manual[抗体实验室手册]第14章纽约州冷泉港冷泉港实验室(Cold Spring Harbor Laboratory,Cold Spring Harbor,NY)。
本公开提供了用于鉴定具有生物学活性的其抗TIGIT抗体的测定法。生物学活性可以包括例如激活免疫细胞或免疫激活报告基因(例如NFAT报告基因)。还提供了在体内和/或体外具有这种生物学活性的抗体。
2.11免疫缀合物
本公开的主题还提供了免疫缀合物，其包含与一种或多种检测探针和/或细胞毒性剂(例如化学治疗剂或药物、生长抑制剂、毒素(例如，蛋白毒素，细菌、真菌、植物或动物来源的酶活性毒素、或其片段))或放射性同位素缀合的本文公开的抗体。例如，所公开的主题的抗体或抗原结合部分可以功能性地连接(例如，通过化学偶联、遗传融合、非共价缔合或其他方式)至一个或多个其他结合分子(例如另一种抗体、抗体片段、肽或结合模拟物)。
在某些实施例中，免疫缀合物是抗体药物缀合物(ADC)，其中抗体缀合至一种或多种药物，包括但不限于美登木素生物碱(参见美国专利号5,208,020、5,416,064和欧洲专利EP 0 425235)；奥瑞他汀(auristatin)，例如单甲基奥瑞他汀药物部分DE和DF(MMAE和MMAF)(参见美国专利号5,635,483和5,780,588，以及7,498,298)；尾海兔素(dolastatin)；卡里奇霉素(calicheamicin)或其衍生物(参见美国专利号5,712,374、5,714,586、5,739,116、5,767,285、5,770,701、5,770,710、5,773,001和5,877,296；Hinman等人,Cancer Res.[癌症研究]53:3336-3342(1993)；和Lode等人,Cancer Res.[癌症研究]58:2925-2928(1998))；蒽环霉素，如道诺霉素(daunomycin)或阿霉素(doxorubicin)(参见Kratz等人,Current Med Chem.[现代药物化学]13:477-523(2006)；Jeffrey等人,Bioorganic&Med.Chem.Letters[生物有机化学与医药化学通讯]16:358-362(2006)；Torgov等人,Bioconj.Chem.[生物共轭化学]16:717-721(2005)；Nagy等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]97:829-834(2000)；Dubowchik等人, Bioorg.&Med.Chem.Letters[生物有机化学与医药化学通讯]12:1529-1532(2002)；King等人,J Med.Chem.[医药化学杂志]45:4336-4343(2002)；和美国专利号6,630,579)；甲氨蝶呤；长春地辛；紫杉烷，例如多西他赛、紫杉醇、拉罗他赛、替塞他赛和奥他赛；单端孢霉烯(trichothecenes)；和CC1065。
在某些实施例中，免疫缀合物包含与酶活性毒素或其片段缀合的本文所述的抗体，所述酶活性毒素或其片段包括但不限于白喉A链、白喉毒素的非结合活性片段、外毒素A链(来自铜绿假单胞菌(Pseudomonas aeruginosa)、蓖麻毒蛋白A链、相思豆毒蛋白A链、蒴莲根毒蛋白A链、α-帚曲毒蛋白、油桐蛋白、香石竹毒蛋白、美洲商陆(Phytolaca americana)蛋白(PAPI、PAPII和PAP-S)、苦瓜(momordica charantia)抑制剂、麻疯树毒蛋白、巴豆毒蛋白、肥阜草(sapaonaria officinalis)抑制剂、多花白树毒蛋白、丝林霉素(mitogellin)、局限曲菌素(restrictocin)、酿霉素(phenomycin)、依诺霉素(enomycin)和单端抱菌素(tricothecenes)。
在某些实施例中，免疫缀合物包含与放射性原子缀合以形成放射性缀合物的本文所述的抗体。多种放射性同位素可用于生产放射性缀合物。非限制性实例包括At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32、Pb 212和Lu的放射性同位素。当将放射性缀合物用于检测时，它可以包括用于闪烁显像研究的放射性原子，例如tc99m或1123，或用于核磁共振(NMR)成像(也称为磁共振成像，MRI)的自旋标记，例如碘123、碘131、铟11、氟19、碳13、氮15、氧17、钆、锰或铁。
可以使用多种双功能蛋白偶联剂(例如，N-琥珀酰亚胺基-3-(2-吡啶二巯基)丙酸酯(SPDP)、琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-l-羧酸酯(SMCC)、亚氨基硫烷(IT)、亚氨酸酯的双功能衍生物(如二甲基己二亚氨酸酯HCl)、活性酯(如辛二酸二琥珀酰亚胺酯)、醛类(如戊二醛)、双叠氮化合物(如双(对叠氮苯甲酰基)己二胺)、双重氮衍生物(如双-(对重氮苯甲酰基)-乙二胺)、二异氰酸酯(如甲代亚苯基2,6-二异氰酸酯)、和双活性氟化合物(如1,5-二氟-2,4-二硝基苯))制备抗体和细胞毒性剂的缀合物。例如，可以如Vitetta等人,Science[科学],238:1098(1987)描述的制备蓖麻毒蛋白免疫毒素。碳4-标记的1-异硫氰酸基苄基-3-甲基二亚乙基三胺-五乙酸(MX-DTPA)是用于将放射性核苷酸与抗体缀合的示例性螯合剂。参见WO94/11026。接头可以是促进细胞中细胞毒性药物释放的“可切割接头”。例如，可以使用酸不稳定性接头、肽酶敏感性接头、光不稳定性接头、二甲基接头或含二硫化物的接头(Chari等人,Cancer Res.[癌症研究]52:127-131(1992)；美国专利号5,208,020)。
本文的免疫缀合物或ADC明确涵盖但不限于用交联剂制备的此类缀合物，包括但不限于可商购的BMPS、EMCS、GMBS、HBVS、LC-SMCC、MBS、MPBH、SBAP、SIA、SIAB、SMCC、SMPB、SMPH、磺基-EMCS、磺基-GMBS、磺基-KMUS、磺基-MBS、磺基-SIAB、磺基-SMCC、和磺基-SMPB、和SVSB(琥珀酰亚胺基-(4-乙烯基砜)苯甲酸酯)(例如，来自美国伊利诺伊州罗克福德的皮尔斯生物技术公司(Pierce Biotechnology,Inc.,Rockford,IL.,U.S.A))。
3.使用方法
本公开的主题进一步提供了使用公开的抗体(例如抗TIGIT抗体)的方法。在某些实施例中，这些方法涉及当前公开的抗体的治疗用途。在某些实施例中，这些方法涉及当前公开的抗体的诊断用途。
3.1治疗方法
本公开提供了本文公开的任何抗体(例如，抗TIGIT抗体)用于治疗疾病和病症或用于增强免疫应答的方法和用途。在某些实施例中，可以将抗体和/或包含本文公开的抗体的药物组合物施用于受试者(例如哺乳动物(例如人))以治疗疾病和病症或增加免疫应答。在某些实施例中，这些疾病和病症涉及免疫检查点抑制和/或异常的TIGIT活性。在某些实施例中，可以通过本文公开的抗体治疗的疾病和病症包括但不限于瘤形成(例如癌症)。
在某些实施例中，本公开提供了本文所述的抗TIGIT抗体(或其片段)，用于制造药物。在某些实施例中，本公开提供了本文所述的抗TIGIT抗体(或其片段)，用于制造用于治疗癌症的药物。在某些实施例中，本公开提供了本文所述的抗TIGIT抗体(或其片段)，用于治疗受试者的癌症。在某些实施例中，本公开提供了包含本文提供的抗TIGIT抗体(或其片段)的药物组合物，用于治疗受试者的癌症。在某些实施例中，癌症可以是血液癌(例如白血病、淋巴瘤和骨髓瘤)、卵巢癌、乳腺癌、膀胱癌、脑癌、结肠癌、肠癌、肝癌、肺癌、胰腺癌、前列腺癌、皮肤癌、胃癌、胶质母细胞瘤、喉癌、黑素瘤、神经母细胞瘤、腺癌、神经胶质瘤、软组织肉瘤和多种癌(包括前列腺癌和小细胞肺癌)。合适的癌还包括肿瘤学领域中的任何已知癌，包括但不限于星形细胞瘤、纤维肉瘤、粘液肉瘤、脂肪肉瘤、少突神经胶质瘤、室管膜细胞瘤、成神经管细胞瘤、原始神经外胚层肿瘤(PNET)、软骨肉瘤、骨原性肉瘤、胰腺导管腺癌、小细胞和大细胞肺腺癌、脊索瘤、血管肉瘤、内皮肉瘤、鳞状细胞癌、支气管肺泡癌、上皮腺癌、和其肝转移瘤、淋巴管肉瘤、淋巴管内皮肉瘤、肝癌、胆管癌、滑膜瘤、间皮瘤、尤文氏瘤、横纹肌肉瘤、结肠癌、基底细胞癌、汗腺瘤、乳头状癌、皮脂腺癌、乳头状腺癌、囊腺癌、髓样癌、支气管癌、肾细胞癌、胆管癌、绒毛膜癌、精原细胞瘤、胚胎性癌、肾母细胞瘤、睾丸瘤、成神经管细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、成血管细胞瘤、听神经瘤、少突神经胶质瘤、脑膜瘤、成神经细胞瘤、视网膜母细胞瘤、白血病、多发性骨髓瘤、华氏巨球蛋白血症(Waldenstrom’s macroglobulinemia)、乳腺肿瘤(如导管腺癌和小叶腺癌)、子宫颈鳞状上皮和腺癌、子宫上皮癌和卵巢上皮性癌、前列腺腺癌、膀胱移行性鳞状细胞癌、B和T细胞淋巴瘤(结节性和弥散性)、浆细胞瘤、急性和慢性白血病、恶性黑色素瘤、软组织肉瘤和平滑肌肉瘤。
在某些实施例中，癌症可以是黑素瘤、NSCLC、头颈癌、尿路上皮癌、乳腺癌(例如，三阴性乳腺癌、TNBC)、胃癌、胆管癌、经典型霍奇金淋巴瘤(cHL)、非霍奇金淋巴瘤原发性纵隔B-细胞淋巴瘤(NHL PMBCL)、间皮瘤、卵巢癌、肺癌(例如，小细胞肺癌)、食管癌、鼻咽癌(NPC)、胆道癌、结肠直肠癌、宫颈癌或甲状腺癌。
在某些实施例中，待治疗的受试者是哺乳动物(例如，人，非人灵长类、大鼠、小鼠、牛、马、猪、绵羊、山羊、狗、猫等)。在某些实施例中，受试者是人。在某些实施例中，受 试者疑似患有癌症或有患癌症的风险、或被诊断患有具有异常TIGIT表达或活性的癌症或任何其他疾病。
表现出异常的TIGIT活性的许多癌症或任何其他疾病的诊断方法以及这些疾病的临床描述是本领域已知的。此类方法包括但不限于例如免疫组织化学、PCR、荧光原位杂交(FISH)。关于异常TIGIT活性或表达的诊断方法的其他细节描述于例如Gupta等人，(2009)Mod Pathol.[现代病理学]22(1):128-133；Lopez-Rios等人(2013)J Clin Pathol.[临床病理学杂志]66(5):381-385；Ellison等人(2013)J Clin Pathol[临床病理学杂志]66(2):79-89；And以及Guha等人(2013)PLoS ONE[公共科学图书馆·综合]8(6):e67782中。
可以通过任何合适的途径施用，包括例如静脉内、肌内或皮下。在一些实施例中，本文提供的抗TIGIT抗体(或其片段)和/或组合物与第二、第三或第四药剂(包括例如抗肿瘤药、生长抑制剂，细胞毒性剂或化学治疗剂)组合施用，以治疗涉及异常TIGIT活性的疾病或失调。这类药剂包括例如多西他赛、吉非替尼、FOLFIRI(伊立替康、5-氟尿嘧啶和亚叶酸)、伊立替康、顺铂、卡铂、紫杉醇、贝伐单抗(抗VEGF抗体)、FOLFOX-4、输注氟尿嘧啶、亚叶酸和奥沙利铂、阿法替尼、吉西他滨、卡培他滨、培美曲塞、替万替尼、依维莫司、CpG-ODN、雷帕霉素、来那度胺、维罗非尼、内皮抑素、拉帕替尼、PX-866、Imprime PGG和伊洛替尼。在一些实施例中，将抗TIGIT抗体(或其片段)缀合至另外的药剂。
在某些实施例中，本文提供的抗TIGIT抗体(或其片段)和/或组合物与一种或多种其他疗法(例如放射疗法、手术、化学疗法、和/或靶向疗法)组合施用。在某些实施例中，本文提供的抗TIGIT抗体(或其片段)和/或组合物与放射疗法组合施用。在某些实施例中，本文提供的抗TIGIT抗体(或其片段)和/或组合物与放射疗法的组合用于治疗本文公开的赘生物或癌症。
取决于待治疗的适应症和本领域技术人员熟悉的与给药相关的因素，本文提供的抗TIGIT抗体或其片段将以在最小化毒性和副作用的同时有效治疗该适应症的剂量施用。对于癌症的治疗，典型的剂量可以是例如在0.001至1000μg的范围内；然而，低于或高于该示例性范围的剂量在本发明的范围内。日剂量可以是总体重的约0.1μg/kg至约100mg/kg，总体重的约0.1μg/kg至约100μg/kg或总体重的约1μg/kg至约100μg/kg。如上提到的，可以通过定期评估治疗的患者来监测治疗或预防功效。对于经若干天或更长时间的重复施用，取决于病症，重复进行治疗直至发生所希望的疾病症状抑制。然而，其他剂量方案可能是有用的并且在本发明的范围内。所希望的剂量可通过单次推注施用组合物、通过多次推注施用组合物、或通过连续输注施用组合物来递送。
包含抗TIGIT抗体或其片段的药物组合物可以每天一次、两次、三次或四次施用。组合物也可以以低于每日施用的频率进行施用，例如，每周六次、每周五次、每周四次、每周三次、每周两次、每周一次、每两周一次、每三周一次、每月一次、每两个月一次、每三个月一次或每六个月一次。组合物也可以例如在植入物中以缓释配制品施用，该植入物逐渐释放该组合物以在一段时间内使用，并且允许该组合物以较低频率施用，例如每月一次、每2-6 个月一次、每年一次、或甚至单次施用。缓释装置(例如圆粒剂型(pellet)、纳米颗粒、微粒、纳米球、微球等)可以通过注射进行施用。
抗体(或其片段)可以以单次日剂量施用，或者总日剂量可以以每日两次、三次或四次的分剂量施用。组合物也可以以低于每日施用的频率进行施用，例如，每周六次、每周五次、每周四次、每周三次、每周两次、每周一次、每两周一次、每三周一次、每月一次、每两个月一次、每三个月一次或每六个月一次。抗体(或其片段)也可以例如在植入物中以缓释配制品施用，该植入物逐渐释放该组合物以在一段时间内使用，并且允许该组合物以较低频率施用，例如每月一次、每2-6个月一次、每年一次、或甚至单次施用。缓释装置(例如圆粒剂型(pellet)、纳米颗粒、微粒、纳米球、微球等)可以通过注射或手术植入各种位置进行施用。
可以通过例如但不限于肿瘤消退、肿瘤重量或尺寸缩小、进展时间、存活持续时间、无进展存活、总体反应率、应答持续时间、生活质量、蛋白质表达和/或活性来评估癌症治疗。可以采用确定疗法功效的方法，包括例如通过放射成像测量反应。
在某些实施例中，将治疗功效测量为肿瘤生长抑制百分比(％TGI)，使用等式100-(T/C x 100)进行计算，其中T是治疗的肿瘤的平均相对肿瘤体积，并且C是未治疗的肿瘤的平均相对肿瘤体积。在某些实施例中，％TGI是约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％)、约94％)、约95％、或多于95％。
3.2诊断和成像方法
经标记的抗TIGIT抗体、其片段，以及其衍生物和类似物可用于诊断目的，以检测、诊断或监测与TIGIT的表达、异常表达和/或活性相关的疾病和/或障碍。例如，本文提供的抗TIGIT抗体(或其片段)可以用于原位、体内、离体和体外诊断测定或成像测定。用于检测TIGIT多肽表达的方法，包括(a)使用本发明的一种或多种抗体测定多肽在个体的细胞(例如组织)或体液中的表达，和(b)比较该基因表达水平和标准基因表达水平，其中与标准表达水平相比，测定的基因表达水平的增加或减少指示异常表达。
本文提供的另外的实施例包括在动物(例如哺乳动物，如人)中诊断与TIGIT的表达或异常表达相关的疾病或障碍的方法。这些方法包含在哺乳动物中检测TIGIT分子。在某些实施例中，诊断包括：(a)向哺乳动物施用有效量的经标记的抗PD-1抗体(或其片段)；(b)在施用后等待一段时间间隔，以允许经标记的抗TIGIT抗体(或其片段)优先浓缩在受试者的表达TIGIT分子的部位(并且将未结合的经标记的分子清除至背景水平)；(c)确定背景水平；和(d)检测受试者中经标记的分子，使得检测到高于背景水平的经标记的分子指示受试者患有与TIGIT表达或异常表达相关的特定疾病或障碍。背景水平可以通过不同方法确定，这些方法包括将所检测到的标记的分子的量与先前针对一个具体系统确定的标准值进行对比。
本文提供的抗TIGIT抗体(或其片段)可以用于使用本领域技术人员已知的经典免疫组织学方法来测定生物样品中蛋白质水平(例如，参见Jalkanen等人,J.Cell.Biol.[细胞生物学杂志]101:976-985(1985)；Jalkanen等人,J.Cell.Biol.[细胞生物学杂志]105:3087-3096(1987))。 有用于检测蛋白质基因表达的其他基于抗体的方法包括免疫测定，例如酶联免疫吸附测定(ELISA)和放射免疫测定(RIA)。合适的抗体测定标记是本领域中已知的并且包括酶标记(例如葡萄糖氧化酶)；放射性同位素，例如碘( 131I、 125I、 123I、 121I)、碳( 14C)、硫( 35S)、氚( 3H)、铟( 115mIn、 113mIn、 112In、 111In)、以及锝( 99Tc、 99mTc)、铊( 201Ti)、镓( 68Ga、 67Ga)、钯( 103Pd)、钼( 99Mo)、氙( 133Xe)、氟( 18F)、 153Sm、 177Lu、 159Gd、 149Pm、 140La、 175Yb、 166Ho、 90Y、 47Sc、 186Re、 188Re、 142Pr、 105Rh、 97Ru；鲁米诺；以及荧光标记(例如荧光素及罗丹明及生物素)。
本领域已知的技术可以应用于本文提供的经标记的抗体(或其片段)。此类技术包括但不限于使用双功能缀合剂(参见例如，美国专利号5,756,065；5,714,631；5,696,239；5,652,361；5,505,931；5,489,425；5,435,990；5,428,139；5,342,604；5,274,119；4,994,560；和5,808,003)。
可替代地，或另外地，可以测量细胞中编码TIGIT多肽的核酸或mRNA的水平，例如经由使用对应于编码EGFR的核酸或其互补序列的基于核酸的探针的荧光原位杂交；(FISH；参见1998年10月公布的WO 98/45479)，DNA印迹法、RNA印迹法或聚合酶链反应(PCR)技术，例如实时定量PCR(RT-PCR)。还可以通过测量生物流体(例如血清)中的脱落抗原来研究TIGIT过表达，例如使用基于抗体的测定(还参见，例如，1990年6月12日发布的美国专利号4,933,294；1991年4月18日发布的WO 91/05264；1995年3月28日发布的美国专利号5,401,638；以及Sias等人,J.Immunol.Methods[免疫方法杂志]132:73-80(1990))。除了上述测定之外，本领域技术人员可获得各种体内和离体测定。例如，可以将哺乳动物体内的细胞暴露于抗体，该抗体任选地用可检测标记(例如放射性同位素)进行标记，并且可以例如通过外部扫描放射性或者通过分析取自先前暴露于该抗体的哺乳动物的样品(例如，活体标本检查或其他生物样品)来评估抗体与细胞的结合。
4.药物配制品
本公开的主题还提供了包含一种或多种本文公开的抗体以及药学上可接受的载剂的药物配制品。在某些实施例中，药物组合物可以包含当前公开的主题的多种(例如，两种或更多种)抗体和/或其抗原结合部分的组合。在某些实施例中，本公开的药物组合物可以包含一种或多种抗TIGIT抗体。
在某些实施例中，所公开的药物配制品可以通过将具有所需纯度的抗体与一种或多种任选的药学上可接受的载剂(Remington's Pharmaceutical Sciences[雷明顿药物科学],第16版,Osol,A.编辑(1980))组合以冻干配制品或水溶液的形式来制备。例如但不限于，冻干抗体配制品在美国专利号6,267,958中描述。在某些实施例中，水性抗体配制品可以包括在美国专利号6,171,586和WO2006/044908中描述的那些，后者的配制品包括组氨酸-乙酸盐缓冲液。在某些实施例中，抗体可以具有大于约80％，大于约90％，大于约91％，大于约92％，大于约93％，大于约94％，大于95％，大于约96％，大于约97％，大于约98％，大于约99％，大于约99.1％，大于约99.2％，大于约99.3％，大于约99.4％，大于约99.5％，大于约99.6％，大于约99.7％，大于约99.8％或大于约99.9％的纯度。
药学上可接受的载剂通常在所采用的剂量和浓度下对接受者无毒，并且包括但不限于：缓冲液(例如磷酸盐、柠檬酸盐、以及其他有机酸)；包括抗坏血酸和甲硫氨酸的抗氧化剂；防腐剂(例如氯化十八烷基二甲基苄基铵；氯化六甲铵；氯化苄烷铵、氯化苯乙铵；苯酚、丁醇或苯甲醇；烷基对羟苯甲酸酯(例如甲基或丙基对羟苯甲酸酯)；邻苯二酚；间苯二酚；环己醇；3-戊醇；以及间甲酚)；低分子量(少于约10个残基)多肽；蛋白，例如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，例如聚乙烯吡咯酮；氨基酸，例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸、或赖氨酸；单糖、二糖、以及其他碳水化合物，包括葡萄糖、甘露糖、或糊精；螯合剂，例如EDTA；糖，例如蔗糖、甘露醇、海藻糖或山梨醇；成盐的计数离子，例如钠；金属络合物(例如Zn-蛋白络合物)；和/或非离子型表面活性剂，例如聚乙二醇(PEG)。本文中的示例性药学上可接受的载剂还包括间质药物分散剂，例如可溶性中性活性透明质酸酶糖蛋白(sHASEGP)，例如人可溶性PH-20透明质酸酶糖蛋白，例如rHuPH20(
Figure PCTCN2021070903-appb-000012
Baxter International公司)。在美国专利公开号2005/0260186和2006/0104968中描述了某些示例性的sHASEGP和使用方法，包括rHuPH20。在某些实施例中，sHASEGP与一种或多种另外的糖胺聚糖酶(例如软骨素酶)组合。
载剂可以适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(例如，通过注射或输注)。根据施用途径，可以将活性化合物(即抗TIGIT抗体)包被在一种材料中，以保护该化合物免受酸和其他可使化合物失活的自然条件的影响。
本公开的药物组合物也可以组合治疗，即与其他药剂组合施用。在某些实施例中，本文所公开的药物组合物还可以包含一种或多种活性成分，这对于所治疗的特定适应症是必需的，例如，具有互补活性且不会产生互相不利影响的那些。在某些实施例中，药物配制品可包含用于治疗由第一治疗剂治疗的相同疾病的第二活性成分。此类活性成分合适地以对于预期目的有效的量组合存在。例如但不限于，本公开的配制品还可以包含一种或多种活性成分，这对于所治疗的特定适应症是必要的，优选地是那些具有互补活性且不会产生互相不利影响的活性成分。例如，可能期望进一步提供可用于治疗相同疾病的第二治疗剂。此类活性成分合适地以对于预期目的有效的量组合存在。
本公开的组合物可以通过本领域已知的多种方法施用。施用途径和/或方式取决于所需结果。这些活性化合物可以用保护该化合物以便避免快速释放的载剂来制备，例如一种受控释放配制品，包括植入物、经皮贴片以及微囊化递送系统。可以使用生物可降解、生物相容的聚合物，如乙烯醋酸乙烯酯、聚酸酐、聚乙醇酸、胶原、聚原酸酯、以及聚乳酸。制备这类配制品的许多方法由例如Sustained and Controlled Release Drug Delivery Systems[缓释和控释药物递送系统],J.R.Robinson,编辑,马塞尔德克尔公司,纽约,1978描述。在某些实施例中，药物组合物是在美国食品和药物管理局(U.S.Food and Drug Administration)的优质生产规范(Good Manufacturing Practice(GMP))条件下生产的。
也可以制备含有所公开的抗体的缓释制剂。持续释放制剂的适合的实例包括含有抗体的固体疏水聚合物的半透性基质，该基质处于成型制品的形式(例如薄膜或微胶囊)。在某些实 施例中，可以将活性成分包埋在例如通过凝聚技术或通过界面聚合制备的微胶囊中，例如分别在胶体药物递送系统中(例如，脂质体、白蛋白微球、微乳液、纳米颗粒和纳米胶囊)或在粗乳液中的羟甲基纤维素或明胶-微胶囊和聚(甲基丙烯酸甲酯)微胶囊。此类技术披露于Remington's Pharmaceutical Sciences[雷明顿药物科学],第16版,Osol,A.编辑(1980)中。
为了通过某些施用途径施用本公开的抗体，可能有必要用防止其失活的材料包被该化合物或与该化合物共同施用。例如，可以在合适的载剂(例如脂质体)或稀释剂中将化合物施用给受试者。药学上可接受的稀释剂包括盐水和缓冲水溶液。脂质体包括水包油包水型CGF乳剂以及常规脂质体(Strejan等人(1984)J Neuroimmunol.[神经免疫学杂志]7:27)。
药学上可接受的载剂包括无菌水溶液或分散液以及用于临时制备无菌可注射溶液或分散液的无菌粉末。使用此类介质和试剂用于药物活性的物质是本领域中已知的。
除非任何常规介质或药剂与活性化合物不相容，否则可考虑将其用于本公开的药物组合物中。还可以将补充性活性化合物掺入组合物中。
治疗组合物通常必须是无菌的、基本等渗的，并且在生产和储存条件下是稳定的。该组合物可以配制为一种溶液、微乳液、脂质体，或适合于高药物浓度的其他有序结构。该载剂可以是含有以下物质的溶剂或分散介质：例如，水、乙醇、多元醇(例如，甘油、丙二醇、和液体聚乙二醇等)，以及其适合的混合物。恰当的流动性可以(例如)通过使用包衣(如卵磷脂)来维持，在分散体的情况下通过维持所需的颗粒大小来维持，以及通过使用表面活性剂来维持。在许多情况下，在组合物中包括等张剂，例如糖、多元醇(如甘露糖醇、山梨糖醇)或氯化钠将为优选的。可以通过在该组合物中包括例如单硬脂酸盐以及明胶的一种延迟吸收的药剂来实现这些可注射组合物的延长的吸收。
无菌注射溶液可通过将所需量的一种或多种公开的抗体根据需要与合适的溶剂与上面列举的一种或多种成分的组合掺入，然后进行灭菌微滤(例如通过无菌滤膜过滤)来制备。通常，分散液通过以下方式制备：
将活性化合物掺入无菌媒介物中，该无菌媒介物含有基础分散介质和来自以上列举的那些的其他所需成分。在用于制备无菌可注射溶液的无菌粉末的情况下，优选的制备方法是真空干燥和冷冻干燥(冻干)，所述方法从其先前的无菌过滤溶液中产生活性成分和任何其他所希望的成分的粉末。
还可以用本领域已知的医学装置来施用治疗性组合物。
例如，本发明的治疗组合物可以与无针皮下注射装置一起施用，例如在美国专利号5,399,163、5,383,851、5,312,335、5,064,413、4,941,880、4,790,824或4,596,556中公开的装置。可用于本公开的植入物和模块的实例包括：美国专利号4,487,603，其公开了一种用于以受控的速率分配药物的植入式微输液泵；美国专利号4,486,194，其公开了一种用于通过皮肤施用药物的治疗装置；美国专利号4,447,233，其公开了一种用于以精确的输注速率输送药物的药物输注泵；美国专利号4,447,224，其公开了一种用于连续药物输送的可变流量可植入输注设备；美国专利号4,439,196，其公开了一种具有多室隔室的渗透药物递送系统；以及美国 专利号4,475,196，其公开了一种渗透药物输送系统。已知许多其他此类植入物、递送系统和模块。
对于治疗组合物，本公开的配制品包括适于口服、经鼻、局部(包括经颊和经舌下)、直肠、阴道和/或肠胃外施用的那些配制品。这些配制品可以方便地以单位剂量形式存在并且可以通过药学领域众所周知的任何方法制备。可以与载剂材料组合以产生单一剂型的抗体的量根据所治疗的受试者和特定的施用方式而变化。可以与载剂材料结合以产生单一剂型的抗体的量通常是产生治疗效果的组合物的量。通常，以百分数计，这个量的范围是从约0.01％至约99％的活性成分，从约0.1％至约70％，或从约1％至约30％。
用于局部或经皮施用本公开的组合物的剂型包括散剂、喷雾剂、软膏剂、糊剂、乳膏剂、洗剂、凝胶剂、溶液、贴剂和吸入剂。可以将活性化合物在无菌条件下与药学上可接受的载剂和可能需要的任何防腐剂、缓冲剂或推进剂混合。
短语“肠胃外施用”和“经肠胃外施用”是指除了肠施用和局部施用之外的、通常通过注射的施用模式，并且包括但不限于静脉内、肌肉内、动脉内、鞘内、囊内、眼眶内、心内、真皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外以及胸骨内注射和输注。
这些药物组合物还可以含有辅助剂，例如防腐剂、润湿剂、乳化剂、和分散剂。预防存在微生物可以通过上述灭菌程序，以及通过包含不同抗细菌和抗真菌剂，例如对羟苯甲酸酯(paraben)、氯代丁醇、苯酚山梨酸等来确保。可能还希望将等渗剂，例如糖、氯化钠等包括于这些组合物中。此外，通过包括延迟吸收的试剂，例如单硬脂酸铝和明胶可以引起可注射药物形式的延长吸收。
在某些实施例中，当将本公开的抗体作为药物施用给人和动物时，它们可以单独或与药学上可接受的载剂组合的作为药物组合物给予，所述药物组合物包含例如约0.01％至约99.5％(或约0.1％至90％)的本文所述的抗体。
5.制品
当前公开的主题还提供一种制品，该制品包含可用于治疗、预防和/或诊断上述疾病的材料。
在某些实施例中，制品包括容器以及在容器上或与容器相关联的标签或包装插入物。合适的容器的非限制性实例包括瓶、小瓶、注射器、IV溶液袋等。容器可以由多种材料(例如玻璃或塑料)形成。容器可以容纳(本身或与另一种组合物组合)有效治疗、预防和/或诊断病症的组合物，并且可具有无菌进入口(例如，容器可以是静脉内溶液袋或具有可由皮下注射针刺穿的塞子的小瓶)。
在某些实施例中，组合物中的至少一种活性剂是当前公开的主题的抗体。标签或包装插入物可指示该组合物用于治疗所选病症。
在某些实施例中，该制品可以包括(a)第一容器，该第一容器中装有组合物，其中该组合物包含本发明的抗体；以及(b)第二容器，其中装有组合物，其中该组合物包含其他细胞 毒性或治疗剂。在某些实施例中，所述制品可以进一步包含包装插入物，其指示所述组合物可以用于治疗特定病症。
可替代地或另外地，制品可以进一步包括另外的容器，例如第二或第三容器，其包括药学上可接受的缓冲剂，例如但不限于注射用抑菌水(BWFI)，磷酸盐缓冲盐水，林格氏溶液和右旋糖溶液。该制品可以包括从商业和用户角度所希望的其他材料，包括其他缓冲液、稀释剂、过滤器、针头和注射器。
序列表
Figure PCTCN2021070903-appb-000013
Figure PCTCN2021070903-appb-000014
Figure PCTCN2021070903-appb-000015
Figure PCTCN2021070903-appb-000016
Figure PCTCN2021070903-appb-000017
Figure PCTCN2021070903-appb-000018
Figure PCTCN2021070903-appb-000019
以下实例仅是对当前公开的主题的说明，不应以任何方式视为限制。
实例
实例1.免疫、抗人TIGIT VHH抗体的产生和苗头抗体的发现
重组人TIGIT细胞外结构域(ECD)蛋白的抗原购自Arco Bio公司。根据本领域已知的方案，使用美洲驼进行TIGIT的免疫。通过ELISA测定法测定血清抗体的效价。在3轮免疫后，观察到高滴度(1:100,000)。然后收集全血，并分离PBMC。然后从PBMC中分离RNA。
在本领域已知的方案下，通过PCR扩增VHH抗体基因，通过DNA琼脂糖凝胶纯化，构建到噬菌体载体pADL-23c(抗体设计实验室(Antibody Design Labs))中，并转化至TG1电感受态细胞(来自Lucigen)。转化的TG1细胞在Y2T培养基中培养。通过添加辅助噬菌体并共培养过夜，可生产出具有目标VHH的噬菌体。通过离心收集培养上清液中的噬菌体，并使用包被生物素化的h-TIGIT或cyno-TIGIT ECD的链霉亲和素偶联的Dynabeads进行人-TIGIT(h-TIGIT)或食蟹猴-TIGIT(cyno-TIGIT)抗原的结合剂淘选。经过三轮淘选后，洗脱了h-TIGIT或cyno-TIGIT的结合物，将其用于感染SS320细胞。挑选SS320细胞的集落并在Y2T培养基中培养，并添加IPTG以分泌VHH抗体。使用h-TIGIT ECD包被的板通过ELISA分析筛选具有VHH抗体的上清液。选择阳性h-TIGIT结合剂进行测序。选择了具有不同序列 的29个克隆。还通过ELISA检查了VHH抗体对cyno-TIGIT的结合能力。序列表(SEQ ID NO:1-84)中显示了前21种结合剂及其CDR和VH。
还使用阻断ELISA测定法确定了VHH抗体克隆对阻断脊髓灰质炎病毒受体(PVR，又名CD155)与TIGIT结合的影响。进一步选择了对PVR结合h-TIGIT有90％以上抑制作用的9个克隆(克隆名称：2B7、1G1、1C12、3G6、2B10、3G7、3F10、13H11和15A5)。
实例2-TIGIT VHH抗体的表征和选择
通过添加图1C中所示的人恒定重链2(CH2)和恒定重链3(CH3)结构域，构建从实例1中鉴定出的抗体克隆，以制备二价抗体。所构建的二价VHH抗体在ExpiCHO细胞中表达，并收集上清液中的蛋白质并通过蛋白质A纯化。
通过流式细胞术测定证实了二价克隆与人TIGIT转染的Jurkat细胞的结合亲和力。建立稳定表达人TIGIT和NFAT报告基因的Jurkat细胞。具体地，通过电穿孔用人TIGIT表达载体转染Jurkat细胞，并在细胞培养期间用1ug/ml嘌呤霉素来选择稳定表达人TIGIT的细胞。将具有代表性的抗体克隆在不同浓度下与h-TIGIT稳定表达Jurkat细胞(0.2x10 6/ml)在96孔板中以100ul/孔的FACS缓冲液(含1.5％FBS的PBS)中孵育30分钟。洗涤后，加入Alexaflour 488偶联的抗人IgG Fc二抗(Alexa
Figure PCTCN2021070903-appb-000020
488AffiniPure山羊抗人IgG，Fcγ片段特异性，杰克逊实验室(Jackson labs)，1:500稀释)，并孵育30分钟。洗涤后，使用CytoFlex(贝克曼库尔特公司(Beckman Coulter))通过门控活细胞群体来测量平均荧光强度。使用GraphPad Prism计算结合亲和力。代表性的结果示于图1A和1B中。与参考Ab1(US2016/0176963 A1中公开的参考抗人TIGIT抗体，根据所公开序列合成)相比，所有测试的抗体均对表达人TIGIT的细胞表现出高结合亲和力。
此外，使用电穿孔用NFAT报告基因转染稳定表达人TIGIT的Jurkat细胞。通过在培养基中的300ug/ml潮霉素选择表达NFAT报告基因的细胞。此外，用人PVR稳定转染Raji细胞，并在培养基中用潮霉素以125ug/ml选择转染的细胞。然后通过在葡萄球菌肠毒素(SEE，0.01ng/ml)的存在下共孵育表达人PVR的Raji细胞和表达人TIGIT和NFAT报告基因的Jurkat细胞持续5小时，测定抗体对PVR介导的抑制TCR诱导的NFAT报告基因活性的影响。加入具有底物(普洛麦格公司(Promega))的Bright-Glo萤光素酶测定缓冲液，并使用读板器通过化学发光活性来测定萤光素酶活性。使用GraphPad Prims的非线性回归方法计算抗体阻断TIGIT活性的能力。代表性结果如图2所示。与参考Ab1相比，除3F10外的所有测试克隆均显示出相似的TIGIT阻断作用。
实例3-抗TIGIT抗体的人源化
选择两个代表性克隆1G1和1C12对其框架进行人源化。简而言之，使用两个克隆的序列进行Igblast来搜索人种系基因的数据库。选择理想的种系序列，并对框架序列进行突变以将框架序列从美洲驼的改变为人的。对于1C12克隆，使用人种系IGHV-3-30*10，并制成了三种形式的人源化1C12(1C12-EREF、1C12-EREW和1C12-GLEW)。对于1G1，使用了人种系IGHV-3-30*01，并制作了六种形式的人源化1G1(1G1-F-G-ERES、1G1-F-A-ERES、 1G1-F-A-EREW、1G1-F-A-GLEW、1G1-F-A-GREL和1G1-F-A-GRES)。将构建体克隆到表达载体中，通过ExpiCHO的瞬时转染产生抗体蛋白，并通过蛋白A进行纯化。
人源化二价抗体与人TIGIT的结合亲和力通过与在Jurkat细胞上稳定表达的h-TIGIT的全细胞结合来确定。简而言之，将抗体与h-TIGIT转染的Jurkat细胞(100μl的0.2x10 6/孔)在FACS缓冲液中孵育30分钟。将细胞洗涤一次，然后与抗人IgG Fc AlexaFluor488(1:500)一起孵育。通过CytoFlex确定平均荧光强度，并使用GraphPad Prism 8.0进行非线性回归，计算抗体与h-TIGIT的结合亲和力，如图3A所示。与嵌合亲本克隆相比，对于所有三个形式的1C12克隆都观察到了相似的结合亲和力。对于1G1克隆，与嵌合克隆相比，两个人源化形式F-G-ERES和F-A-ERES对h-TIGIT表现出相似的亲和力，而两个形式(F-A-EREW和F-A-GLEW)对h-TIGIT失去结合亲和力，如图3B所示。
使用先前描述的方法确定人源化抗体在阻断PVR介导的对TCR介导的NFAT报告基因活性的抑制作用中的活性。简而言之，在PVR转染的Raji细胞和低浓度的葡萄球菌肠毒素(SEE，0.01ng/ml)存在下，将人TIGIT和NFAT报告基因转染的Jurkat细胞与不同浓度的抗体孵育5小时。添加具有底物(普洛麦格公司(Promega))的Bright-Glo萤光素酶测定缓冲液，并测量萤光素酶活性。代表性结果显示在图4A和4B。与嵌合亲本克隆和参考抗h-TIGIT抗体参考Ab1相比，1C12克隆的所有人源化形式在阻断TIGIT方面具有相似的效力。除了两个形式1G1-(F-A-EREW)和1G1-(F-A-GLEW)失去了阻断作用以外，1G1的所有人源化形式在阻断TIGIT方面均具有与嵌合亲本克隆相似的效力。
实例4-亲和力成熟、选择和修改
对1G1-F-A-ERES克隆进行亲和力成熟。设计用于使每个CDR区的氨基酸单突变的引物。使用组装PCR制备突变文库，并将其克隆到噬菌体载体中。通过转化TG1细胞和克隆的DNA测序来测量文库质量。使用辅助噬菌体进行噬菌体生产，并使用涂有生物素化的h-TIGIT ECD或cyno-TIGIT ECD的链霉亲和素偶联的Dynabeads进行噬菌体淘选。经过两轮淘选后，洗脱淘选产物用于感染SS320细胞，挑取菌落并在具有IPTG的Y2T培养基中培养。通过ELISA测定法检查上清液中的VHH抗体。选择针对h-和cyno-TIGIT的阳性克隆与h-和cyno-TIGIT稳定表达的细胞进行全细胞结合。还对选定的克隆进行了PVR阻断ELISA和人TIGIT阻断NFAT报告基因检测。使用GraphPad Prism计算EC50或IC50值。序列表(SEQ ID NO:94-177)中显示了前25种结合剂及其CDR和VHH。
使用GraphPad Prism将EC50/IC50在全细胞结合和阻断ELISA分析中的相关性作图，代表性数据如图5A-5C所示。鉴定出2A3具有最高的亲和力和效力。
通过在25至70℃的温度下热处理60分钟测试12个代表性克隆的热稳定性，然后使用ELISA和全细胞结合流式细胞术检测处理后的样品与人TIGIT的结合，其结果如图6A和6B所示。与其他克隆相比，克隆2A3具有更高的热稳定性。
使用人IgG1CH2和CH3结构域构建了二价2A3抗体(2A3-Fc)。抗体在ExpiCHO细胞中表达，并通过蛋白A柱进行纯化。与抗TIGIT参考抗体参考Ab1和参考Ab2(与Tiragolumab 具有相同氨基酸序列的参考抗人TIGIT抗体，根据在U.S.2017/0088613 A1中公开的Tiragolumab序列合成)相比，使用Octet结合试验研究了与人TIGIT结合的2A3-Fc表位。如图7A-7C所示，与参考Ab1和参考Ab2相比，2A3克隆结合至不同的表位。
2A3-Fc对人、食蟹猴和小鼠TIGIT的物种间结合活性通过ELISA测定法测定，其结果示于图8A-8C中。2A3-Fc与人、食蟹猴结合，但不与小鼠TIGIT结合。
对2A3的CDR区域的分析确定了两个热点，即CDR2中的蛋氨酸和CDR3中的天冬氨酸，然后是丝氨酸。将蛋氨酸突变为亮氨酸和异亮氨酸，将天冬氨酸突变为苏氨酸和谷氨酸。修饰的抗体(2A3 ML，2A3 MI，2A3 ML_DT(又名2A3LT)和2A3 ML_DE)的CDR和VHH在序列表中显示(SEQ ID NO:178-193)。在全细胞结合和NFAT萤光素酶报告基因检测中测试了修饰的形式，与亲本2A3克隆相比，所有这些修饰的抗体都表现出相似的特性。图9显示了2A3-LT-Fc(2A3 ML DT)的代表性数据，其中M变为L，并且D变为T(2A3 ML DT)，其中与亲本2A3-Fc相比，在全细胞结合测定中观察到相似的人TIGIT结合亲和力，在NFAT报告基因测定中观察到了相似的效力。
将2A3-Fc的亲和力和效力与参考抗TIGIT抗体参考Ab2进行了比较。2A3-Fc表现出比参考Ab2显著得高的亲和力。在全细胞结合测定中，2A3-Fc的EC50值为0.32±0.06nM，而参考Ab2(n＝3)为0.61±10.13nM(图10A)。与参考Ab2相比，2A3-Fc在NFAT萤光素酶报告基因检测中也显示出类似的阻断TIGIT的功效。2A3-Fc的EC50值为0.48±10.18nM，而参考Ab2(n＝3)为0.72±0.42nM(图10B)。
实例5-体外有效性研究
抗TIGIT抗体的抗肿瘤能力在体外实验中继续被探究。在肿瘤微环境中，当表达TIGIT的T细胞与表达PVR的树突细胞(DC)接触时，DC上表达的PVR会结合T细胞上的TIGIT，进而抑制T细胞抗肿瘤活性，如抗肿瘤细胞因子的分泌。有效的抗TIGIT抗体的治疗可以阻断TIGIT与PVR的结合，从而增强T细胞对肿瘤的杀伤能力。
树突细胞-T细胞混合淋巴反应(MLR assay)被用来模拟这一发生在肿瘤微环境中的现象。从健康捐献者PBMC中分离出的表达CD14的单核白细胞(monocyte)被培养在含50ng/ml GM-CSF和50ng/ml IL-4的RPMI1640培养基中达7天(第4天更换培养基)以分化成DC。之后DC再转移至含有100ng/ml LPS的RPMI培养基中培养1天，从而获得成熟的DC。由此方法得到的树突细胞稳定表达CD11c(成熟DC的生物标志物)，MHC class II(二类主要组织相容性复合体)，CD80(CD28的配体)，和PVR(TIGIT的配体)。利用ThermoFisher T细胞分离试剂盒按照说明方法从另一个健康捐献者PBMC中分离出CD3+T细胞。此后将10,000个成熟DC和200,000个CD3+T细胞混合培养在含有10％FBS-HI的RPMI1640培养基中，每孔加入不同浓度的抗TIGIT抗体和对照抗体。在37℃，5.0％CO2的环境中混合培养48小时后，利用PerkinElmer的IL-2AlphaLisa检测试剂盒(AlphaLisa human IL-2kit:Cat#AL221C)检测上清中IL-2的水平。抗PD1抗体作为正向对照，抗HER2抗体作为负向对照。
如图11所示，2A3-LT-Fc抗体以剂量依赖的方式提升了T细胞分泌IL-2的水平，其与正对照抗PD1抗体相当。相反，Tiragolumab类似物没有提升T细胞分泌IL-2的水平。因为IL-2是重要的抗肿瘤细胞因子，此结果说明抗TIGIT抗体2A3-LT-Fc可以显著提升肿瘤微环境中T细胞的抗肿瘤能力，且其展现了比Tiragolumab类似物更好的抗肿瘤能力。
实例6-Fc工程化
抗TIGIT抗体增强免疫功能和抗肿瘤活性的作用机制不仅涉及对TIGIT的阻断作用，而且还涉及抗原呈递细胞(APC)与有效T细胞(CD8+T细胞)之间的结合作用，通过APC上的FcγRIIIA结合抗体Fc区，以及有效T细胞上VHH结构域与TIGIT的结合。为了利用这种机制，制得了两种不同的Fc增强形式，一种具有DLE突变(S239D、A330L和I332E)，一种具有VPVLL突变(L235V、F243L、R292P、Y300L和P396L)。Fc突变体与人FcγRIIIA和FcγRIIB的结合通过使用FcγRIIIA和FcγRIIB的ECD的重组蛋白的Octet结合测定来检查，其结果如图12所示。DLE和VPVLL突变体均具有增强的对人FcγRIIIA的结合亲和力。DLE突变体还具有增强的与人FcγRIIB的结合亲和力，而VPVLL突变体具有降低的对人FcγRIIB的结合亲和力。
还检查了Fc突变体的TIGIT阻断活性，其中DLE突变体显示出降低的TIGIT阻断功能，如图13A所示。
此外，当抗体可变区与其特异性抗原结合时，其Fc区可以交联FcγRIIIA并触发下游信号传导。使用这种作用方式，使用人FcγRIIIA和NFAT报告基因转染的Jurkat细胞和过表达TIGIT的293T细胞检查了Fc突变体对FcγRIIIA介导的活性的影响。在该测定系统中，如图13B所示，与野生型Fc形式相比，DLE突变体显著增加了人FcγRIIIA介导的NFAT报告基因活性。
表3对应图13A的检测数据
EC50(nM) 2A3 LT-Fc DLE
Exp 1 0.58 0.67
Exp 2 0.687 0.92
Exp 3 0.45 1.32
平均数 0.57 0.97
SD 0.12 0.3279
表4对应图13B的检测数据
EC50(nM) 2A3 LT-Fc DLE
Exp 1 0.226 0.027
Exp 2 0.175 0.0295
Exp 3 0.144 0.018
Exp 4 0.18 0.02
平均数 0.18 0.024
SD 0.03 0.005
实例7–体内有效性研究
利用人TIGIT敲入的C57BL/6小鼠和MC38鼠结肠癌模型，2A3-LT-Fc wt，2A3-LT-Fc-DLE的有效性得到了评估并与参考Ab2比较。在治疗前一周给小鼠接种了MC38肿瘤细胞。当平均肿瘤大小达到约51mm 3时治疗开始，在腹膜内一周两次给药，持续2.5周。2A3-LT-Fc抗体每次6mg/kg或参考Ab2每次11mg/kg(与2A3-LT-Fc抗体在mole/kg上等同)。治疗后16天，多只对照组的小鼠中肿瘤大小达到上限(2000mm 3)。因此治疗后16天成为分析的数据终点。在对照组中，平均肿瘤体积在治疗后16天为1548.76±191mm 3(平均值±标准误差)。与对照组相比，使用2A3-LT-Fc wt，2A3-LT-Fc-DLE的治疗显著减小了肿瘤生长，造成依次为38％和50％的TGI(肿瘤抑制)及依次为985.05±123mm 3和802.20±126mm 3的肿瘤体积(与空白对照相比通过曼-惠特尼(Mann-Whitney)检验P值依次为0.037和0.007)。使用参考抗体的治疗也减小了肿瘤生长，但由此抗体造成的减小与对照组相比在统计上并不显著，其TGI为26％，肿瘤体积为1156.16±195mm 3(图14A,表5)。单个肿瘤体积的结果显示在图14B中。这些结果表明2A3-LT-Fc和2A3-LT-Fc-DLE都比参考Ab2更有效。在研究中各实验组的体重变化并不显著(图14C)，表明治疗得到了良好的耐受。
表5.MC38同基因人TIGIT小鼠模型第16天的平均肿瘤体积
组 处理 N 肿瘤体积(mm 3) a P b ％TGI(16天)
1 媒介物 8 1548.76±191    
2 2A3-LT-Fc-wt 6mg/kg 8 985.05±123 0.037 38
3 2A3-LT-Fc-DLE 6mg/kg 8 802.20±126 0.007 50
4 参考Ab 2,11mg/kg 8 1156.16±195 0.234 26
a平均值±标准误差
b在治疗后16天通过曼-惠特尼(Mann-Whitney)检验与空白对照相比
除了所描绘和要求保护的各种实施例之外，所公开的主题还针对具有本文所公开和要求保护的特征的其他组合的其他实施例。这样，本文所呈现的特定特征可以在所公开的主题的范围内以其他方式彼此组合，使得所公开的主题包括本文所公开的特征的任何合适的组合。出于展示和说明的目的已经提出了所公开主题的具体实施例的以上说明。以上说明并不旨在是穷尽的或将所公开主题限制于所公开的那些实施例。
对于本领域技术人员将显而易见的是，在不脱离所公开主题的精神或范围的情况下，可以对所公开主题的组成和方法进行多种修改和变化。因此，预期的是所公开主题包括在所附权利要求书及其等同物范围之内的修改和变化。
本文引用了多种出版物、专利和专利申请，其内容通过引用以其整体并入本文。

Claims (107)

  1. 一种与TIGIT结合的抗体，该抗体包含单结构域抗体，该单结构域抗体以1x10 -7M或更小的KD结合TIGIT。
  2. 如权利要求1所述的抗体，其中该单结构域抗体以1x10 -8M或更小的KD结合TIGIT。
  3. 如权利要求1或2所述的抗体，其中该单结构域抗体以5x10 -9M或更小的KD结合TIGIT。
  4. 如权利要求1至3中任一项所述的抗体，其中该单结构域抗体以2x10 -9M或更小的KD结合TIGIT。
  5. 如权利要求1至4中任一项所述的抗体，其中该单结构域抗体包含VHH。
  6. 如权利要求1至5中任一项所述的抗体，其中该单结构域抗体或VHH包含重链可变区(VH)。
  7. 如权利要求1至6中任一项所述的抗体，其中该单结构域抗体与包含重链可变区的参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该重链可变区包含：
    a)重链可变区CDR1，其包含具有SEQ ID NO:94所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:95所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:96所示序列的氨基酸，
    b)重链可变区CDR1，其包含具有SEQ ID NO:98所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:99所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:100所示序列的氨基酸，
    c)重链可变区CDR1，其包含具有SEQ ID NO:102所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:103所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:104所示序列的氨基酸，
    d)重链可变区CDR1，其包含具有SEQ ID NO:106所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:107所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:108所示序列的氨基酸，
    e)重链可变区CDR1，其包含具有SEQ ID NO:110所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:111所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:112所示序列的氨基酸，
    f)重链可变区CDR1，其包含具有SEQ ID NO:114所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:115所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:116所示序列的氨基酸，
    g)重链可变区CDR1，其包含具有SEQ ID NO:118所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:119所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:120所示序列的氨基酸，
    h)重链可变区CDR1，其包含具有SEQ ID NO:122所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:123所示序列的氨基酸；和重链可变区CDR3，其包含具有 SEQ ID NO:124所示序列的氨基酸，
    i)重链可变区CDR1，其包含具有SEQ ID NO:126所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:127所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:128所示序列的氨基酸，
    j)重链可变区CDR1，其包含具有SEQ ID NO:130所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:131所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:132所示序列的氨基酸，
    k)重链可变区CDR1，其包含具有SEQ ID NO:134所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:135所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:136所示序列的氨基酸，
    l)重链可变区CDR1，其包含具有SEQ ID NO:138所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:139所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:140所示序列的氨基酸，
    m)重链可变区CDR1，其包含具有SEQ ID NO:142所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:143所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:144所示序列的氨基酸，
    n)重链可变区CDR1，其包含具有SEQ ID NO:146所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:147所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:148所示序列的氨基酸，
    o)重链可变区CDR1，其包含具有SEQ ID NO:150所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:151所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:152所示序列的氨基酸，
    p)重链可变区CDR1，其包含具有SEQ ID NO:154所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:155所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:156所示序列的氨基酸，
    q)重链可变区CDR1，其包含具有SEQ ID NO:158所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:159所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:160所示序列的氨基酸，
    r)重链可变区CDR1，其包含具有SEQ ID NO:162所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:163所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:164所示序列的氨基酸，
    s)重链可变区CDR1，其包含具有SEQ ID NO:166所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:167所示序列的氨基酸；和重链可变区CDR3，
    其包含具有SEQ ID NO:168所示序列的氨基酸，
    t)重链可变区CDR1，其包含具有SEQ ID NO:170所示序列的氨基酸；重链可变区 CDR2，其包含具有SEQ ID NO:171所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:172所示序列的氨基酸，
    u)重链可变区CDR1，其包含具有SEQ ID NO:174所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:175所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:176所示序列的氨基酸，
    v)重链可变区CDR1，其包含具有SEQ ID NO:178所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:179所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:180所示序列的氨基酸，
    w)重链可变区CDR1，其包含具有SEQ ID NO:182所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:183所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:184所示序列的氨基酸，
    x)重链可变区CDR1，其包含具有SEQ ID NO:186所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:187所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:188所示序列的氨基酸，或
    y)重链可变区CDR1，其包含具有SEQ ID NO:190所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:191所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:192所示序列的氨基酸。
  8. 如权利要求1至7中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含：
    a)重链可变区CDR1，该重链可变区CDR1包含SEQ ID NO:94、98、102、106、110、114、118、122、126、130、134、138、142、146、150、154、158、162、166、170、174、178、182、186和190中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体；
    b)重链可变区CDR2，该重链可变区CDR2包含SEQ ID NO:95、99、103、107、111、115、119、123、127、131、135、139、143、147、151、155、159、163、167、171、175、179、183、187和191中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体；以及
    c)重链可变区CDR3，该重链可变区CDR3包含SEQ ID NO:96、100、104、108、112、116、120、124、128、132、136、140、144、148、152、156、160、164、168、172、176、180、184、188和192中任何一个的氨基酸序列，或其具有多达约3个氨基酸取代的变体。
  9. 如权利要求1至8中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含CDR1结构域、CDR2结构域和CDR3结构域，其中该CDR1结构域、CDR2结构域和CDR3结构域分别含有包含在参考重链可变区中的CDR1结构域、CDR2结构域和CDR3结构域，该参考重链可变区包含选自由SEQ ID NO:97、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181、185、189和193组成的组的氨基酸序列。
  10. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:94所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:95所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:96所示序列的氨基酸。
  11. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:98所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:99所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:100所示序列的氨基酸。
  12. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:102所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:103所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:104所示序列的氨基酸。
  13. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:106所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:107所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:108所示序列的氨基酸。
  14. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:110所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:111所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:112所示序列的氨基酸。
  15. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:114所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:115所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:116所示序列的氨基酸。
  16. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:118所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:119所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:120所示序列的氨基酸。
  17. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:122所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:123所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:124所示序列的氨基酸。
  18. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:126所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:127所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:128所示序列的氨基酸。
  19. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区 CDR1，其包含具有SEQ ID NO:130所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:131所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:132所示序列的氨基酸。
  20. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:134所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:135所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:136所示序列的氨基酸。
  21. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:138所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:139所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:140所示序列的氨基酸。
  22. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:142所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:143所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:144所示序列的氨基酸。
  23. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:146所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:147所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:148所示序列的氨基酸。
  24. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:150所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:151所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:152所示序列的氨基酸。
  25. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:154所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:155所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:156所示序列的氨基酸。
  26. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可
    变区CDR1，其包含具有SEQ ID NO:158所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:159所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:160所示序列的氨基酸。
  27. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:162所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:163所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:164所示序列的氨基酸。
  28. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:166所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:167所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:168所示序列的氨基酸。
  29. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:170所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:171所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:172所示序列的氨基酸。
  30. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:174所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:175所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:176所示序列的氨基酸。
  31. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:178所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:179所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:180所示序列的氨基酸。
  32. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:182所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:183所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:184所示序列的氨基酸。
  33. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:186所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:187所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:188所示序列的氨基酸。
  34. 如权利要求1至9中任一项所述的抗体，其中该单结构域抗体包含：重链可变区CDR1，其包含具有SEQ ID NO:190所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:191所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:192所示序列的氨基酸。
  35. 如权利要求1至34中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含与选自由SEQ ID NO:97、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181、185、189和193组成的组的氨基酸序列具有至少约90％序列同一性的氨基酸序列。
  36. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:97所示的氨基酸序列。
  37. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区， 该重链可变区包含SEQ ID NO:101所示的氨基酸序列。
  38. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:105所示的氨基酸序列。
  39. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:109所示的氨基酸序列。
  40. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:113所示的氨基酸序列。
  41. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:117所示的氨基酸序列。
  42. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:121所示的氨基酸序列。
  43. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:125所示的氨基酸序列。
  44. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:129所示的氨基酸序列。
  45. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:133所示的氨基酸序列。
  46. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:137所示的氨基酸序列。
  47. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:141所示的氨基酸序列。
  48. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:145所示的氨基酸序列。
  49. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:149所示的氨基酸序列。
  50. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:153所示的氨基酸序列。
  51. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可
    变区，该重链可变区包含SEQ ID NO:157所示的氨基酸序列。
  52. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:161所示的氨基酸序列。
  53. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:165所示的氨基酸序列。
  54. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:169所示的氨基酸序列。
  55. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:173所示的氨基酸序列。
  56. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:177所示的氨基酸序列。
  57. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:181所示的氨基酸序列。
  58. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:185所示的氨基酸序列。
  59. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:189所示的氨基酸序列。
  60. 如权利要求1至35中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含SEQ ID NO:193所示的氨基酸序列。
  61. 如权利要求1至60中任一项所述的抗体，其中该单结构域抗体与包含重链可变区的参考抗TIGIT单结构域抗体交叉竞争与TIGIT结合，该重链可变区包含：
    (a)重链可变区CDR1，其包含具有SEQ ID NO:1所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:2所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:3所示序列的氨基酸，
    (b)重链可变区CDR1，其包含具有SEQ ID NO:5所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:6所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:7所示序列的氨基酸，
    (c)重链可变区CDR1，其包含具有SEQ ID NO:9所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:10所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:11所示序列的氨基酸，
    (d)重链可变区CDR1，其包含具有SEQ ID NO:13所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:14所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:15所示序列的氨基酸，
    (e)重链可变区CDR1，其包含具有SEQ ID NO:17所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:18所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:19所示序列的氨基酸，
    (f)重链可变区CDR1，其包含具有SEQ ID NO:21所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:22所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:23所示序列的氨基酸，
    (g)重链可变区CDR1，其包含具有SEQ ID NO:25所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:26所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:27所示序列的氨基酸，
    (h)重链可变区CDR1，其包含具有SEQ ID NO:29所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:30所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:31所示序列的氨基酸，
    (i)重链可变区CDR1，其包含具有SEQ ID NO:33所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:34所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:35所示序列的氨基酸，
    (j)重链可变区CDR1，其包含具有SEQ ID NO:37所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:38所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:39所示序列的氨基酸，
    (k)重链可变区CDR1，其包含具有SEQ ID NO:41所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:42所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:43所示序列的氨基酸，
    (l)重链可变区CDR1，其包含具有SEQ ID NO:45所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:46所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:47所示序列的氨基酸，
    (m)重链可变区CDR1，其包含具有SEQ ID NO:49所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:50所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:51所示序列的氨基酸，
    (n)重链可变区CDR1，其包含具有SEQ ID NO:53所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:54所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:55所示序列的氨基酸，
    (o)重链可变区CDR1，其包含具有SEQ ID NO:57所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:58所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:59所示序列的氨基酸，
    (p)重链可变区CDR1，其包含具有SEQ ID NO:61所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:62所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:63所示序列的氨基酸，
    (q)重链可变区CDR1，其包含具有SEQ ID NO:65所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:66所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:67所示序列的氨基酸，
    (r)重链可变区CDR1，其包含具有SEQ ID NO:69所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:70所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:71所示序列的氨基酸，
    (s)重链可变区CDR1，其包含具有SEQ ID NO:73所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:74所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ  ID NO:75所示序列的氨基酸，
    (t)重链可变区CDR1，其包含具有SEQ ID NO:77所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:78所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:79所示序列的氨基酸，或
    (u)重链可变区CDR1，其包含具有SEQ ID NO:81所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:82所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:83所示序列的氨基酸。
  62. 如权利要求1至61中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含：
    (a)重链可变区CDR1，其包含具有SEQ ID NO:1所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:2所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:3所示序列的氨基酸，
    (b)重链可变区CDR1，其包含具有SEQ ID NO:5所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:6所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:7所示序列的氨基酸，
    (c)重链可变区CDR1，其包含具有SEQ ID NO:9所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:10所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:11所示序列的氨基酸，
    (d)重链可变区CDR1，其包含具有SEQ ID NO:13所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:14所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:15所示序列的氨基酸，
    (e)重链可变区CDR1，其包含具有SEQ ID NO:17所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:18所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:19所示序列的氨基酸，
    (f)重链可变区CDR1，其包含具有SEQ ID NO:21所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:22所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:23所示序列的氨基酸，
    (g)重链可变区CDR1，其包含具有SEQ ID NO:25所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:26所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:27所示序列的氨基酸，
    (h)重链可变区CDR1，其包含具有SEQ ID NO:29所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:30所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:31所示序列的氨基酸，
    (i)重链可变区CDR1，其包含具有SEQ ID NO:33所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:34所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ  ID NO:35所示序列的氨基酸，
    (j)重链可变区CDR1，其包含具有SEQ ID NO:37所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:38所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:39所示序列的氨基酸，
    (k)重链可变区CDR1，其包含具有SEQ ID NO:41所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:42所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:43所示序列的氨基酸，
    (l)重链可变区CDR1，其包含具有SEQ ID NO:45所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:46所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:47所示序列的氨基酸，
    (m)重链可变区CDR1，其包含具有SEQ ID NO:49所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:50所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:51所示序列的氨基酸，
    (n)重链可变区CDR1，其包含具有SEQ ID NO:53所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:54所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:55所示序列的氨基酸，
    (o)重链可变区CDR1，其包含具有SEQ ID NO:57所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:58所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:59所示序列的氨基酸，
    (p)重链可变区CDR1，其包含具有SEQ ID NO:61所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:62所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:63所示序列的氨基酸，
    (q)重链可变区CDR1，其包含具有SEQ ID NO:65所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:66所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:67所示序列的氨基酸，
    (r)重链可变区CDR1，其包含具有SEQ ID NO:69所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:70所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:71所示序列的氨基酸，
    (s)重链可变区CDR1，其包含具有SEQ ID NO:73所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:74所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:75所示序列的氨基酸，
    (t)重链可变区CDR1，其包含具有SEQ ID NO:77所示序列的氨基酸；重链可变区CDR2，其包含具有SEQ ID NO:78所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:79所示序列的氨基酸，或
    (u)重链可变区CDR1，其包含具有SEQ ID NO:81所示序列的氨基酸；重链可变区 CDR2，其包含具有SEQ ID NO:82所示序列的氨基酸；和重链可变区CDR3，其包含具有SEQ ID NO:83所示序列的氨基酸。
  63. 如权利要求1至62中任一项所述的抗体，其中该单结构域抗体包含重链可变区，该重链可变区包含与选自由SEQ ID NO:4、8、12、16、20、24、28、32、36、40、44、48、52、56、60、64、68、72、76、80和84组成的组的氨基酸序列具有至少约90％序列同一性的氨基酸序列。
  64. 如权利要求1至63中任一项所述的抗体，其中该单结构域抗体包含人源化框架。
  65. 如权利要求1至64中任一项所述的抗体，其中该抗体包含Fc区。
  66. 如权利要求1至65中任一项所述的抗体，其中该Fc区包含人Fc区。
  67. 如权利要求1至66中任一项所述的抗体，其中该Fc区包含选自下组的Fc区，该组由以下组成：IgG、IgA、IgD、IgE和IgM的Fc区。
  68. 如权利要求1至67中任一项所述的抗体，其中该Fc区包含选自下组的Fc区，该组由以下组成：IgG1、IgG2、IgG3和IgG4的Fc区。
  69. 如权利要求1至68中任一项所述的抗体，其中该Fc区包含IgG1 Fc区。
  70. 如权利要求69所述的抗体，其中该IgG1 Fc区包含一种或多种增强抗体依赖性细胞介导的细胞毒性(ADCC)的突变。
  71. 如权利要求70所述的抗体，其中该IgG1 Fc区包含L235V、F243L、R292P、Y300L和P396L的突变。
  72. 如权利要求70所述的抗体，其中该IgG1 Fc区包含S239D、A330L和I332E的突变。
  73. 如权利要求1至72中任一项所述的抗体，其中该重链可变区通过接头连接至Fc区。
  74. 如权利要求73所述的抗体，其中该接头是肽接头。
  75. 如权利要求74所述的抗体，其中该肽接头包含约4至约30个氨基酸。
  76. 如权利要求74或75所述的抗体，其中该肽接头包含选自由SEQ ID NO:195-220组成的组的氨基酸序列。
  77. 如权利要求1至76中任一项所述的抗体，其中该抗体包含多特异性抗体，例如双特异性抗体，全长免疫球蛋白，单链Fv(scFv)片段，Fab片段，Fab'片段，F(ab’)2，Fv片段，二硫键稳定的Fv片段(dsFv)，(dsFv)2，VHH，Fv-Fc融合物，scFv-Fc融合物，scFv-Fv融合物，双抗体，三抗体，四抗体或任何它们的组合。
  78. 如权利要求1至77中任一项所述的抗体，其中该抗体包含多特异性抗体，例如双特异性抗体，其包含特异性结合第二抗原的第二抗体部分。
  79. 如权利要求78所述的抗体，其中该第二抗原是肿瘤相关抗原。
  80. 如权利要求79所述的抗体，其中该肿瘤相关抗原选自由以下组成的组：Her-2，EGFR，PDL1，c-Met，B细胞成熟抗原(BCMA)，碳酸酐酶IX(CA1X)，癌胚抗原(CEA)，CD5，CD7，CD10，CD19，CD20，CD22，CD30，CD33，CD34，CD38，CD41，CD44，CD49f，CD56，CD74，CD123，CD133，CD138，CD276(B7H3)，上皮糖蛋白(EGP2)，滋养层细 胞表面抗原2(TROP-2)，上皮糖蛋白-40(EGP-40)，上皮细胞粘附分子(EpCAM)，受体酪氨酸蛋白激酶erb-B2、3、4，叶酸结合蛋白(FBP)，胎儿乙酰胆碱受体(AChR)，叶酸受体-a，神经节苷脂G2(GD2)，神经节苷脂G3(GD3)，人端粒酶逆转录酶(hTERT)，激酶插入结构域受体(KDR)，Lewis A(CA 1.9.9)，Lewis Y(LeY)，磷脂酰肌醇蛋白聚糖-3(GPC3)，L1细胞粘附分子(L1CAM)，粘蛋白16(Muc-16)，粘蛋白1(Muc-1)，NG2D配体，癌胚抗原(h5T4)，前列腺干细胞抗原(PSCA)，前列腺特异性膜抗原(PSMA)，肿瘤相关糖蛋白72(TAG-72)，密封蛋白18.2(CLDN18.2)，血管内皮生长因子R2(VEGF-R2)，肾母细胞瘤蛋白(WT-1)，1型酪氨酸蛋白激酶跨膜受体(ROR1)及其任何组合。
  81. 如权利要求80所述的抗体，其中该第二抗原是免疫检查点调节剂。
  82. 如权利要求81所述的抗体，其中该免疫检查点调节剂选自由以下组成的组：PD1、CTLA4、LAG-3、2B4、BTLA及其任何组合。
  83. 如权利要求1至82中任一项所述的抗体，其中该抗体缀合至治疗剂或标记。
  84. 如权利要求83所述的抗体，其中该标记选自由以下组成的组：放射性同位素、荧光染料和酶。
  85. 一种免疫缀合物，该免疫缀合物包含与治疗剂连接的如权利要求1至84中任一项所述的抗体。
  86. 如权利要求85所述的免疫缀合物，其中该治疗剂是细胞毒素。
  87. 如权利要求85所述的免疫缀合物，其中该治疗剂是放射性同位素。
  88. 一种药物组合物，该药物组合物包含a)如权利要求1至84中任一项所述的抗体或如权利要求85至87中任一项所述的免疫缀合物，和b)药学上可接受的载剂。
  89. 一种核酸，该核酸编码如权利要求1至84中任一项所述的抗体。
  90. 一种载体，该载体包含如权利要求89所述的核酸。
  91. 一种宿主细胞，该宿主细胞包含如权利要求89所述的核酸或如权利要求90所述的载体。
  92. 一种制备如权利要求1至84中任一项所述的抗体的方法，该方法包括在如权利要求91所述的宿主细胞中表达该抗体并从该宿主细胞中分离该抗体。
  93. 一种减轻受试者的肿瘤负荷的方法，该方法包括向该受试者施用有效量的如权利要求1至84中任一项所述的抗体、如权利要求85至87中任一项所述的免疫缀合物或如权利要求88所述的药物组合物。
  94. 如权利要求93所述的方法，其中该方法减少肿瘤细胞的数量。
  95. 如权利要求93或94所述的方法，其中该方法减小肿瘤大小。
  96. 如权利要求93至95中任一项所述的方法，其中该方法根除该受试者的肿瘤。
  97. 如权利要求93至95中任一项所述的方法，其中该肿瘤选自由以下组成的组：间皮瘤、肺癌、胰腺癌、卵巢癌、乳腺癌、结肠癌、胸膜肿瘤、成胶质细胞瘤、食道癌、胃癌、滑膜肉瘤、胸腺癌、子宫内膜癌、胃肿瘤、胆管癌、头颈癌、血液癌及其组合。
  98. 一种治疗和/或预防赘生物的方法，该方法包括向受试者施用有效量的如权利要求1至84中任一项所述的抗体、如权利要求85至87中任一项所述的免疫缀合物或如权利要求88所述的药物组合物。
  99. 一种延长患有赘生物的受试者的存活期的方法，该方法包括向该受试者施用有效量的如权利要求1至84中任一项所述的抗体、如权利要求85至87中任一项所述的免疫缀合物或如权利要求88所述的药物组合物。
  100. 如权利要求98或99所述的方法，其中该赘生物选自由以下组成的组：间皮瘤、肺癌、胰腺癌、卵巢癌、乳腺癌、结肠癌、胸膜肿瘤、成胶质细胞瘤、食道癌、胃癌、滑膜肉瘤、胸腺癌、子宫内膜癌、胃肿瘤、胆管癌、头颈癌、血液癌及其组合。
  101. 如权利要求1至84中任一项所述的抗体，其用作药物。
  102. 如权利要求1至84中任一项所述的抗体，其用于治疗癌症。
  103. 如权利要求88所述的药物组合物，其用作药物。
  104. 如权利要求88所述的药物组合物，其用于治疗癌症。
  105. 如权利要求102所述的抗体或如权利要求104所述的药物组合物，其中该癌症选自由以下组成的组：间皮瘤、肺癌、胰腺癌、卵巢癌、乳腺癌、结肠癌、胸膜肿瘤、成胶质细胞瘤、食道癌、胃癌、滑膜肉瘤、胸腺癌、子宫内膜癌、胃肿瘤、胆管癌、头颈癌、血液癌及其组合。
  106. 一种试剂盒，该试剂盒包含如权利要求1至84中任一项所述的抗体、如权利要求85至87中任一项所述的免疫缀合物、如权利要求88所述的药物组合物、如权利要求89所述的核酸、如权利要求90所述的载体、或如权利要求91所述的宿主细胞。
  107. 如权利要求106所述的试剂盒，该试剂盒进一步包含用于治疗和/或预防赘生物的书面说明书。
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