Anti-b7-h3 antibody and application thereof

Anti-B7-H3 antibody and its application

technical field

0001The invention belongs to the field of biomedicine, and in particular relates to an anti-B7-H3 antibody and its application.

Background technique

0002B7-H3 (B7 homolog 3 protein), also known as CD276, is a type I transmembrane glycoprotein belonging to the B7 ligand family.

0003B7-H3 has been found to exist in two forms: 2Ig-B7-H3 and 4Ig-B7-H3. 2Ig-B7-H3 is expressed in mouse and human cells, and it has an extracellular IgV-IgC structure; 4Ig-B7 -H3 is only expressed in human cells and consists of a tandem repeat IgV-IgC-IgV-IgC structure. The main form of human B7-H3 is 4IgB7-H3.

0004The expression level of B7-H3 in normal tissues is limited, but abnormally high expression in a variety of advanced human solid tumors, including but not limited to head and neck cancer, renal cancer, prostate cancer, lung cancer, breast cancer, gastric cancer and liver cancer. Its overexpression is often associated with poor patient prognosis and poorer clinical outcome. Therefore, B7-H3 is considered to be a diagnostic marker for certain tumors and can be used as an effective target for the development of anticancer drugs.

0005<u style="single">Contents of the invention</u>

0006The present invention provides anti-B7-H3 antibodies or antigen-binding fragments, and these antibodies or antigen-binding fragments can specifically bind to B7-H3. In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment has high affinity and strong internalization ability.

0007Some embodiments provide antibodies or antigen-binding fragments that specifically bind B7-H3 and that comprise one or more of the amino acid sequences in (a)-(f):

0008(a) VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence with one or more site substitutions, deletions or insertions compared with the amino acid sequence shown in SEQ ID NO: 6, or consisting of;

0009(b) VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence with one or more site substitutions, deletions or insertions compared to the amino acid sequence shown in SEQ ID NO: 7, or consisting of;

0010(c) VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence with one or more site substitutions, deletions or insertions compared with the amino acid sequence shown in SEQ ID NO: 8, or consisting of;

0011(d) VL CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NO: 9-13, or has one or more amino acid sequences compared with the amino acid sequence shown in any one of SEQ ID NO: 9-13 An amino acid sequence that is substituted, deleted or inserted, or consists of it;

0012(e) VL CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NO:14-18, or has one or more than the amino acid sequence shown in any one of SEQ ID NO:14-18 An amino acid sequence that is substituted, deleted or inserted, or consists of it;

0013(f) VL CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO: 19-23, or has one or more than the amino acid sequence shown in any one of SEQ ID NO: 19-23 An amino acid sequence that is substituted, deleted or inserted, or consists of it.

0014In some embodiments, the antibody or antigen-binding fragment specifically binds B7-H3 and comprises:

0015(a) VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence with one or more site substitutions, deletions or insertions compared with the amino acid sequence shown in SEQ ID NO: 6, or consisting of it; and

0016(b) VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence with one or more site substitutions, deletions or insertions compared to the amino acid sequence shown in SEQ ID NO: 7, or consisting of it; and

0017(c) VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence with one or more site substitutions, deletions or insertions compared with the amino acid sequence shown in SEQ ID NO: 8, or consisting of it.

0018In some embodiments, the antibody or antigen-binding fragment comprises VH CDR1 set forth in SEQ ID NO:6, VH CDR2 set forth in SEQ ID NO:7, and VH CDR3 set forth in SEQ ID NO:8.

0019In some embodiments, the antibody or antigen-binding fragment thereof specifically binds B7-H3 and comprises:

0020(d) VL CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NO: 9-13, or has one or more amino acid sequences compared with the amino acid sequence shown in any one of SEQ ID NO: 9-13 An amino acid sequence that is substituted, deleted or inserted, or consists of it; and

0021(e) VL CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NO:14-18, or has one or more than the amino acid sequence shown in any one of SEQ ID NO:14-18 An amino acid sequence that is substituted, deleted or inserted, or consists of it; and

0022(f) VL CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO: 19-23, or has one or more than the amino acid sequence shown in any one of SEQ ID NO: 19-23 An amino acid sequence that is substituted, deleted or inserted, or consists of it.

0023In some embodiments, the antibody or antigen-binding fragment comprises a VL as set forth in SEQ ID NO:9

0024CDR1, VL CDR2 as shown in SEQ ID NO:14 and VL CDR3 as shown in SEQ ID NO:19.

0025In some embodiments, the antibody or antigen-binding fragment comprises VL CDR1 set forth in SEQ ID NO:10, VL CDR2 set forth in SEQ ID NO:15, and VL CDR3 set forth in SEQ ID NO:20.

0026In some embodiments, the antibody or antigen-binding fragment comprises VL CDR1 set forth in SEQ ID NO:11, VL CDR2 set forth in SEQ ID NO:16, and VL CDR3 set forth in SEQ ID NO:21.

0027In some embodiments, the antibody or antigen-binding fragment comprises VL CDR1 set forth in SEQ ID NO:12, VL CDR2 set forth in SEQ ID NO:17, and VL CDR3 set forth in SEQ ID NO:22.

0028In some embodiments, the antibody or antigen-binding fragment comprises VL CDR1 set forth in SEQ ID NO:13, VL CDR2 set forth in SEQ ID NO:18, and VL CDR3 set forth in SEQ ID NO:23.

0029In some embodiments, the antibody or antigen-binding fragment specifically binds B7-H3 and comprises:

0030(a) VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence with one or more site substitutions, deletions or insertions compared with the amino acid sequence shown in SEQ ID NO: 6, or consisting of it; and

0031(b) VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence with one or more site substitutions, deletions or insertions compared to the amino acid sequence shown in SEQ ID NO: 7, or consisting of it; and

0032(c) VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence with one or more site substitutions, deletions or insertions compared with the amino acid sequence shown in SEQ ID NO: 8, or consisting of it; and

0033(d) VL CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NO: 9-13, or has one or more amino acid sequences compared with the amino acid sequence shown in any one of SEQ ID NO: 9-13 An amino acid sequence that is substituted, deleted or inserted, or consists of it; and

0034(e) VL CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NO:14-18, or has one or more than the amino acid sequence shown in any one of SEQ ID NO:14-18 An amino acid sequence that is substituted, deleted or inserted, or consists of it; and

0035(f) VL CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO: 19-23, or has one or more than the amino acid sequence shown in any one of SEQ ID NO: 19-23 An amino acid sequence that is substituted, deleted or inserted, or consists of it.

0036In some embodiments, the substitutions are conservative amino acid substitutions.

0037In some embodiments, the antibody or antigen-binding fragment specifically binds B7-H3 and comprises:

0038(a) VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 6; and

0039(b) VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 7; and

0040(c) VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 8; and

0041(d) VL CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 9-13; and

0042(e) VL CDR2 comprising the amino acid sequence shown in any one of SEQ ID NO: 14-18; and

0043(f) VL CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO: 19-23.

0044In some embodiments, the antibody or antigen-binding fragment comprises VH CDR1 as set forth in SEQ ID NO:6, VH CDR2 as set forth in SEQ ID NO:7, VH CDR3 as set forth in SEQ ID NO:8, VL CDR1 as shown in SEQ ID NO:9, VL CDR2 as shown in SEQ ID NO:14 and VL CDR3 as shown in SEQ ID NO:19.

0045In some embodiments, the antibody or antigen-binding fragment comprises VH CDR1 as set forth in SEQ ID NO:6, VH CDR2 as set forth in SEQ ID NO:7, VH CDR3 as set forth in SEQ ID NO:8, VL CDR1 as shown in SEQ ID NO:10, VL CDR2 as shown in SEQ ID NO:15 and VL CDR3 as shown in SEQ ID NO:20.

0046In some embodiments, the antibody or antigen-binding fragment comprises VH CDR1 as set forth in SEQ ID NO:6, VH CDR2 as set forth in SEQ ID NO:7, VH CDR3 as set forth in SEQ ID NO:8, VL CDR1 as shown in SEQ ID NO:11, VL CDR2 as shown in SEQ ID NO:16, and VL CDR3 as shown in SEQ ID NO:21.

0047In some embodiments, the antibody or antigen-binding fragment comprises VH CDR1 as set forth in SEQ ID NO:6, VH CDR2 as set forth in SEQ ID NO:7, VH CDR3 as set forth in SEQ ID NO:8, VL CDR1 as shown in SEQ ID NO:12, VL CDR2 as shown in SEQ ID NO:17, and VL CDR3 as shown in SEQ ID NO:22.

0048In some embodiments, the antibody or antigen-binding fragment comprises VH CDR1 as set forth in SEQ ID NO:6, VH CDR2 as set forth in SEQ ID NO:7, VH CDR3 as set forth in SEQ ID NO:8, VL CDR1 as shown in SEQ ID NO:13, VL CDR2 as shown in SEQ ID NO:18, and VL CDR3 as shown in SEQ ID NO:23.

0049Amino acid sequence of VH CDR (Kabat numbering)

0050<tables num="0001"><table><tgroup cols="3"><colspec colname="c001" colwidth="31%" /><colspec colname="c002" colwidth="41%" /><colspec colname="c003" colwidth="28%" /><thead><row><entry morerows="0">type</entry><entry morerows="0">amino acid sequence</entry><entry morerows="0">serial number</entry></row></thead><tbody><row><entry morerows="0">VH CDR1</entry><entry morerows="0">DYDIN</entry><entry morerows="0">6</entry></row><row><entry morerows="0">VH CDR2</entry><entry morerows="0">WIFPGDDTTKYNEKFKG</entry><entry morerows="0">7</entry></row><row><entry morerows="0">VH CDR3</entry><entry morerows="0">SPSFDY</entry><entry morerows="0">8</entry></row></tbody></tgroup></table></tables>

0051Amino acid sequence of VL CDR (Kabat numbering)

0052<img file="WO2023051663A1_D0001.tif" />

0053<img file="WO2023051663A1_D0002.tif" />

0054In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

0055In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions, relative to the parental Fc region. In some embodiments, the amino acid modification of the Fc region alters effector function activity relative to the activity of the parental Fc region. In some embodiments, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding . In some embodiments, amino acid modifications of the Fc region can alter the affinity of the variant Fc region for FcγR (Fcγ receptor) relative to the parent Fc region. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region mutation is N297A, L234A, or L235A (Eu numbering). In some embodiments, the Fc region mutation is E345R or S440Y (Eu numbering).

0056In some embodiments, the antibody or antigen-binding fragment is scFv, Fab, Fab' or F(ab)₂. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

0057In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 24 and 35-38, or the amino acid sequence shown in any one of SEQ ID NOs: 24 and 35-38. An amino acid sequence having at least 80% or 90% identity compared to the amino acid sequence shown in any one of SEQ ID NO: 24 and 35-38, or having one or Amino acid sequences with multiple conservative amino acid substitutions; and/or

0058The light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 25-29 and 39-41, or the amino acid sequence as shown in any one of SEQ ID NOs: 25-29 and 39-41. An amino acid sequence having at least 80% or 90% identity compared to any of the amino acid sequences shown in any of SEQ ID NO:25-29 and 39-41, or having one or Amino acid sequence with multiple conservative amino acid substitutions.

0059In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24, or has at least 80% of the amino acid sequence set forth in SEQ ID NO:24. Or an amino acid sequence with 90% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 24; and/or

0060The light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NO:25-29, or the amino acid sequence shown in any one of SEQ ID NO:25-29 Compared to an amino acid sequence having at least 80% or 90% identity, or having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 25-29.

0061In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : The amino acid sequence shown in 25.

0062In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : the amino acid sequence shown in 26.

0063In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : the amino acid sequence shown in 27.

0064In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : the amino acid sequence shown in 28.

0065In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : the amino acid sequence shown in 29.

0066In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NOs: 35-38, or any of SEQ ID NOs: 35-38 An amino acid sequence having at least 80% or 90% identity compared to the amino acid sequence shown in Item 1, or having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO:35-38 amino acid sequence; and/or

0067The light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NO:39-41, or the amino acid sequence shown in any one of SEQ ID NO:39-41 Compared to an amino acid sequence having at least 80% or 90% identity, or having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 39-41.

0068In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : Amino acid sequence shown in 39.

0069In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 36, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : Amino acid sequence shown in 39.

0070In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 36, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : The amino acid sequence shown in 40.

0071In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : the amino acid sequence shown in 41.

0072In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 38, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: : the amino acid sequence shown in 41.

0073Amino acid sequence of the variable region (Kabat numbering)

0074<img file="WO2023051663A1_D0003.tif" />

0075<img file="WO2023051663A1_D0004.tif" />

0076In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 32 or 33, or has an amino acid sequence compared to the amino acid sequence shown in SEQ ID NO: 32 or 33 An amino acid sequence that is at least 80% or 90% identical, or has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 32 or 33; and/or

0077The light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 34, or has at least 80% or at least 90% identity compared to the amino acid sequence shown in SEQ ID NO: 34 An amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:34.

0078In some embodiments, an antibody specifically binding to B7-H3 is provided, the heavy chain of the antibody comprising the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and the amino acid shown in SEQ ID NO: 32 The heavy chain constant region of the sequence; the light chain of the antibody comprises the light chain variable region of the amino acid sequence shown in SEQ ID NO:25 and the light chain constant region of the amino acid sequence shown in SEQ ID NO:34.

0079In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:26 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0080In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:27 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0081In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:28 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0082In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:29 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0083In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 35 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:39 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0084In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 36 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:39 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0085In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 36 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:40 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0086In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 37 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:41 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0087In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 38 and a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 32; The light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:41 and a light chain constant region having the amino acid sequence shown in SEQ ID NO:34.

0088Amino acid sequence of constant region

0089<img file="WO2023051663A1_D0005.tif" />

0090<img file="WO2023051663A1_D0006.tif" />

0091In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:50; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:52.

0092In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody (including a full-length monoclonal antibody), a multispecific antibody, or an antigen-binding fragment (eg, a bispecific antibody or antigen-binding fragment).

0093In some embodiments, the antibodies have two heavy chains of identical sequence and two light chains of identical sequence. In some embodiments, the Fc regions pair to form disulfide bonds.

0094In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.

0095The present invention also provides polynucleotides encoding said antibodies or antigen-binding fragments. In some embodiments, the polynucleotide is an isolated polynucleotide. In some embodiments, the polynucleotide sequence is selected from the nucleic acid sequences shown in SEQ ID NO:49 and 51.

0096The present invention also provides an expression vector comprising the polynucleotide. In some embodiments, the expression vector comprising the polynucleotide is a nucleic acid fragment, plasmid, phage or virus. In some embodiments, the expression vector is an isolated expression vector.

0097The present invention also provides host cells comprising said polynucleotide or expression vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cells are CHO cells, HEK293 cells (such as HEK293F cells), BHK cells, Cos1 cells, Cos7 cells, CV1 cells or murine L cells.

0098The present invention also provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment or biological material, and a pharmaceutically acceptable carrier.

0099The present invention also provides the application of the antibody or antigen-binding fragment, the biological material or the pharmaceutical composition in the preparation of medicaments for diagnosing and/or treating diseases. In some embodiments, the present invention provides the use of the antibody or antigen-binding fragment, the biological material or the pharmaceutical composition in diagnosing and/or treating a disease. In some embodiments, the present invention provides a method for diagnosing and/or treating a disease, the method comprising administering an effective dose of the antibody or antigen-binding fragment, the biological material or the pharmaceutical composition to a patient in need.

0100In some embodiments, the drug is a drug that inhibits the activity of B7-H3. In some embodiments, the disease is selected from tumors, respiratory diseases, skin and musculoskeletal diseases, urogenital diseases, nervous system diseases, and digestive system diseases. In some embodiments, the tumor is benign or cancerous. In some embodiments, the tumor is a hematological tumor or a solid tumor. In some embodiments, the disease comprises a tumor positive for B7-H3 expression. In some embodiments, the tumor is selected from the group consisting of melanoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), breast cancer (e.g., triple negative breast cancer), ovarian cancer (e.g., epithelial ovarian cancer), gum Gliomas (e.g., glioblastoma and pediatric brainstem glioma), prostate cancer (e.g., castration-resistant prostate cancer), pancreatic cancer (e.g., pancreatic ductal carcinoma), head and neck cancer, leukemia (e.g., acute Myeloid leukemia (AML)), cervical cancer, kidney cancer, squamous cell neoplasm, squamous cell carcinoma (such as squamous lung cancer or squamous head and neck cancer), colorectal cancer, stomach cancer, liver cancer, mesothelioma, anal cancer, Skin cancer, vulvar cancer, neuroblastoma, desmoplastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignancy, sarcoma, brain cancer, central nervous system tumor, brain metastases.

0101The invention also provides diagnostic methods and uses. In some embodiments, a method for detecting expression of B7-H3 in a sample is provided, the sample is contacted with the antibody or antigen-binding fragment, such that the antibody or antigen-binding fragment binds to B7-H3, and the binding is detected, i.e. The content of B7-H3 in the sample. In some embodiments, the use of the antibody or antigen-binding fragment in the preparation of a kit for diagnosing or prognosing tumors (including benign tumors and cancers) is provided. In some embodiments, a diagnostic or prognostic kit comprising the antibody or antigen-binding fragment is provided.

0102The present invention provides anti-B7-H3 antibody or antigen-binding fragment and application thereof. The anti-B7-H3 antibody or antigen-binding fragment of the present invention can specifically bind to B7-H3, and has high specificity, high affinity and strong internalization ability , can be used in the development of ADC drugs to obtain better anti-tumor activity and efficacy, and can also be used in the diagnosis and prognosis of tumors (including benign tumors and cancers).

Description of drawings

0103Figure 1 shows the binding ability of the chimeric antibody to MDA-MB-468 cells; among them, H1L4 is the control antibody M30-H1-L4.

0104Figure 2 shows the binding ability of chimeric antibodies to Lag3-CHO cells, VISTA-CHO cells, Tim3-CHO cells and Raji cells.

0105Figure 3 shows the binding ability of the humanized antibody to hB7-H3-Fc; among them, H1L4 represents the control antibody M30-H1-L4.

0106Figure 4 shows the binding ability of the humanized antibody to MDA-MB-468 cells; among them, H1L4 represents the control antibody M30-H1-L4.

0107Figure 5 shows the binding ability of the humanized antibody to VISTA-CHO cells, Tim3-CHO cells and Lag3-CHO cells; wherein, H1L4 represents the control antibody M30-H1-L4.

Detailed ways

0108Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

0109definition

0110It should be noted that the term "an" entity refers to one or more of that entity, for example "an antibody" should be understood as one or more antibodies, therefore, the term "a" (or "an" ), "one or more" and "at least one" may be used interchangeably herein.

0111As used herein, the term "comprising" or "comprising" means that the antibody, composition or method, etc. includes the listed elements, such as components or steps, but does not exclude others. "Consisting essentially of" means that the antibody, composition or method, etc. excludes other elements that have a fundamental effect on the characteristics of the combination, but does not exclude elements that do not substantially affect the antibody, composition or method, etc. . "Consisting of" means excluding elements not specifically recited.

0112The term "polypeptide" is intended to encompass the singular as well as the plural "polypeptides" and refers to a molecule formed from amino acid monomers linked linearly by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain" or any other term used to refer to a chain of two or more amino acids is included in the definition of "polypeptide" and the term "polypeptide" may Used in place of any of the above terms, or used interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur. A polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, it may be produced by any means including chemical synthesis.

0113"Amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an α-amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids.

0114As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Standing of twenty conventional amino acids Isomers (e.g., D-amino acids), unnatural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for use in the polypeptides of the present disclosure. . Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine , N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine and other similar amino acids and imino acids (such as 4 - hydroxyproline). In the notation of polypeptides used herein, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxy-terminal direction, consistent with standard usage and convention. Conventional (or natural) amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D ), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

0115Minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present disclosure, provided that the amino acid sequence identity remains at least 75%, such as at least 80%, 90%, 95%, and such as 99%. In some embodiments, the changes are conservative amino acid substitutions. Conservative amino acid substitutions are substitutions that occur within a family of amino acids that are related in their side chains. Amino acids encoded by genes are roughly divided into the following categories: (1) acidic amino acids are aspartate and glutamate; (2) basic amino acids are lysine, arginine, and histidine; (3) non-polar amino acids alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar amino acids are Glycine, Asparagine, Glutamine, Cysteine, Serine, Threonine, Tyrosine. Other families of amino acids include (i) the aliphatic-hydroxyl family of serine and threonine; (ii) the amide-containing family of asparagine and glutamine; (iii) the aliphatic family of alanine, valine, leucine and isoleucine; and (iv) phenylalanine, tryptophan and tyrosine of the aromatic family. In some embodiments, conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, Glutamic acid-aspartic acid, and asparagine-glutamine. For example, it is reasonable to predict that a single substitution of isoleucine or valine for leucine, glutamate for aspartate, serine for threonine, or a structurally related amino acid similar Substitution of an amino acid that does not have a significant effect on the binding or properties of the resulting molecule, especially if the substitution does not involve amino acids within the binding site. Whether an amino acid change results in a functional peptide can readily be determined by measuring the specific activity of the polypeptide derivative. Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art.

0116In some embodiments, amino acid substitutions have the effect of (1) reducing susceptibility to proteolysis, (2) reducing susceptibility to oxidation, (3) altering binding affinity for protein complex formation, ( 4) altering binding affinity, and (5) conferring or improving other physicochemical or functional properties of such analogs. Analogs may include various muteins that differ in sequence from the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in naturally occurring sequences (preferably in portions of the polypeptide other than domains that form intermolecular contacts). Conservative amino acid substitutions should not significantly alter the structural properties of the parental sequence (eg, the substituted amino acid should not tend to disrupt the helical structure present in the parental sequence, or disrupt other types of secondary structure that characterize the parental sequence).

0117The number of conservative amino acid substitutions of VL and VH is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11 , about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values (inclusive), or any value therein. The number of amino acids of the heavy chain constant region, the light chain constant region, the conservative amino acid substitutions of the heavy chain or the light chain is about 1, about 2, about 3, about 4, about 5, about 6, about 8 amino acids , about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (inclusive) or any value therein.

0118The term "isolated" used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., for example, "isolated" DNA, RNA, polypeptides, antibodies refers to the DNA, RNA, polypeptide, antibody, etc. One or more of the isolated molecules. The term "isolated" as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include fragments of nucleic acid that do not occur in their natural state and do not exist in their natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptide is intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. will usually be prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or some of these values The range between any two values of , inclusive, or any value therein.

0119The term "encoding" when applied to a polynucleotide refers to a polynucleotide which is said to "encode" a polypeptide which, in its native state or when manipulated by methods well known to those skilled in the art, is transcribed and/or Or translation may result in the polypeptide and/or fragments thereof.

0120The term "recombinant" refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not occur in nature, non-limiting examples may be produced by combination of polynucleotides or polynucleotides that do not normally exist peptide.

0121"Homology", "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology or identity can be determined by comparing the alignable positions in each sequence. When a position in the sequences being compared is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "At least 80% identity" is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, About 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or these A range (inclusive) between any two values in a value, or any value therein. "At least 90% identity" is about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 95% identity, about 96% identity, about 97% identity, About 98% identity, about 99% identity, or a range between any two of these values, inclusive, or any value therein.

0122A polynucleotide is composed of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when the polynucleotide is RNA Thymine was replaced with uracil (U). A "polynucleotide sequence" may be denoted by the letters of the polynucleotide molecule. This letter designation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications, such as for functional genomics and homology searches.

0123The terms "polynucleotide", "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or its analogues. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribose Somatic RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Structural modifications to the nucleotides, if present, can be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example by conjugation with labeling components. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the present disclosure includes the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

0124"Identity or sequence identity" of a nucleic acid or polynucleotide sequence (or polypeptide or antibody sequence) to another sequence by a certain percentage (e.g. 90%, 95%, 98% or 99%) means when the sequence When aligned, this percentage of bases (or amino acids) is identical in the two sequences being compared. The alignment and percent identity or sequence identity can be determined visually or using software programs known in the art, such as Ausubel et al.eds. (2007) in Current Protocols in Molecular Software program as described in Biology. It is preferred to use the default parameters for the alignment. One such alignment program is BLAST with default parameters, such as BLASTN and BLASTP, both of which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions =50sequences;sortby=HIGHSCORE;Databases=non-redundant;GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that share the above indicated percentages of identity and encode a polypeptide having the same or similar biological activity.

0125"Antibody" and "antigen-binding fragment" refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof. The term "antibody" thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity to bind an antigen. Antibodies and antigen-binding fragments include, but are not limited to, complementarity determining regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions of heavy or light chains or ligand-binding portions thereof (CH), light chain constant region (CL), framework region (FR) or any part thereof, or at least a part of a binding protein. The CDR regions include the CDR regions of the light chain variable region (VL CDR1-3) and the CDR regions of the heavy chain variable region (VH CDR1-3). An antibody or antigen-binding fragment can specifically recognize and bind to a polypeptide or polypeptide complex of one or more (eg, two) antigens. Antibodies or antigen-binding fragments that specifically recognize and bind multiple (eg, two) antigens may be referred to as multispecific (eg, bispecific) antibodies or antigen-binding fragments.

0126The term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, and the composition of the antibody fragment of the present invention may be similar to that of F(ab') in a monospecific antibody fragment.₂,F(ab)₂, Fab', Fab, Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, Spiegelmers and diabodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

0127"Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an immunoglobulin. In some aspects, these regions are linked to short linker peptides of about 10 to about 25 amino acids. Linkers can be rich in glycine for flexibility, and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Although the protein has had its constant regions removed and a linker introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example as described in US Pat. No. 5,892,019.

0128The term "antibody" includes a wide variety of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that classes of heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with some subclasses (eg γ1-γ4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class. The two heavy chains and the two light chains are linked by disulfide bonds in a "Y" configuration, where the light chains begin at the mouth of the "Y" and continue through the variable region surrounding the heavy chains.

0129Antibodies, antigen-binding fragments or derivatives disclosed in the present invention include but are not limited to polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope binding Fragments (e.g. class Fab, class Fab' and class F(ab')₂), similar single-chain Fvs (scFv).

0130Light chains can be classified as kappa (κ) or lambda (λ). Each heavy chain can be associated with a kappa or lambda light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are joined by a covalent bond, and the "tail" portions of the two heavy chains are linked by a covalent disulfide bond or non-covalent bonding. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. Immunoglobulin kappa light chain variable region is V_kappa; The variable region of the immunoglobulin lambda light chain is V_lambda。

0131The terms "constant" and "variable" are used according to function. The variable region of the light chain (VL) and the variable region of the heavy chain (VH) determine antigen recognition and specificity. The light chain constant region (CL) and the heavy chain constant region (CH) confer important biological properties, such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant regions increases as they become farther away from the antibody's antigen-binding site or amino terminus. The N-terminal part is the variable region, and the C-terminal part is the constant region; the CH3 and CL domains actually contain the carboxy-terminals of the heavy and light chains, respectively.

0132In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, A non-contiguous sequence of amino acids that specifically binds to an antigen. The remaining other amino acids in the antigen-binding domain, referred to as the "framework" regions, show less intermolecular variability. Most of the framework regions adopt a β-sheet conformation, and the CDRs form a ring structure connected to it, or in some cases form a part of the β-sheet structure. Therefore, the framework regions position the CDRs in the correct orientation by forming scaffolds through non-covalent interchain interactions. The antigen-binding domain with CDRs in specific positions forms a surface complementary to the epitope on the antigen, which facilitates the non-covalent binding of the antibody to its antigenic epitope. For a given heavy chain or light chain variable region, those of ordinary skill in the art can identify amino acids comprising CDRs and framework regions by known methods.

0133As used herein, the term "CDR" refers to the complementarity determining regions within the variable region of an antibody. There are three CDRs in each of the variable regions of the heavy and light chains, designated for each variable region as CDR1, CDR2, and CDR3 (or specifically, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3). The boundaries of the CDRs of the variable regions of the same antibody may vary according to different assignment systems. Thus, where reference is made to defining antibodies with specific CDR sequences as defined in the present invention, the scope of said antibodies also covers antibodies whose variable region sequences comprise the CDR sequences of the present invention, but which result from the application of a different protocol. The claimed CDR boundaries are not the same as the specific CDR boundaries defined by the present invention. CDRs as defined by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the invention to use either definition to refer to the CDRs of an antibody or variant thereof. The exact residue numbers comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues are included in the CDRs based on the amino acid sequence of the variable region of the antibody.

0134As used herein, the term "framework" or "framework sequence" refers to the sequence remaining after subtracting the CDRs from the variable region. Because the precise definition of the CDR sequences can be determined by different systems, the meaning of the framework sequences correspondingly requires different interpretations. The six CDRs (CDR1, CDR2, and CDR3 of the light chain and CDR1, CDR2, and CDR3 of the heavy chain) divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, Among them, CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Without designating a particular subregion as FR1, FR2, FR3 or FR4, framework regions as by other references refer to the combined FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR means one of the four subregions, and FRs means two or more of the four subregions constituting the framework region.

0135The framework region and CDR region of the humanized antibody do not have to correspond exactly to the parental sequence, for example, the CDR or consensus framework of the donor antibody can be mutated by substitution, insertion and/or deletion of at least one amino acid residue, making the CDR at this site Or the framework residues do not correspond to the donor antibody or the consensus framework. Typically, at least 80%, at least 85%, more at least 90%, or at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework regions in the consensus immunoglobulin sequences. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences. In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that most frequently occurs at that position in that family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

0136Where there are two or more definitions for a term used and/or accepted in the art, the definition of the term used herein includes all such meanings unless clearly indicated to the contrary. A specific example is the use of the term "complementarity determining regions" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides.

0137Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independently of other experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU or Chothia numbering system.

0138Antibodies disclosed in the present invention can be derived from any animal, including but not limited to fish, birds and mammals. Preferably, the antibody is of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin. In another embodiment, the variable region may be of condricthoid origin (eg, from a shark).

0139A "heavy chain constant region" includes at least one of a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant regions of antibodies can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region may comprise a hinge region derived partly from an IgG1 molecule and partly from an IgG3 molecule. In another embodiment, part of the heavy chain may comprise a chimeric hinge region derived partly from an IgG1 molecule and partly from an IgG4 molecule.

0140A "light chain constant region" includes a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

0141A "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain. A "CH1 domain" comprises the first constant region of an immunoglobulin heavy chain. The CH2 domain is not tightly paired with other domains, but rather two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule, and contains approximately 108 residues. A "hinge region" includes part of the heavy chain region connecting the CH1 domain and the CH2 domain. The hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains.

0142"Disulfide bond" refers to a covalent bond formed between two sulfur atoms. A thiol group of cysteine can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond.

0143A "chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial or modified) are derived from a second species. In certain embodiments, the variable regions are from a non-human source (eg, mouse or primate), and the constant regions are from a human source.

0144"Specific binding" generally refers to the complementary binding of an antibody or antigen-binding fragment to a specific antigen through its antigen-binding domain and epitope to form a relatively stable complex. "Specificity" can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a particular antigen or epitope. For example, if antibody "A" has a greater relative affinity for the same antigen than antibody "B", antibody "A" can be said to have a higher specificity for that antigen than antibody "B". Specific binding can be described by the equilibrium dissociation constant (KD), a smaller KD means a tighter binding. Methods of determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, optical interferometry of biofilm layers, and the like. An antibody that "specifically binds" antigen a includes an equilibrium dissociation constant KD of less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM with antigen a.

0145"Treatment" means therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, slow down, ameliorate or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable Relief of symptoms, reduction of disease extent, stabilization of disease state (i.e. not worsening), delay or slowing of disease progression, improvement, remission, alleviation or disappearance of disease state (whether partial or total), prolongation and Expected survival without treatment, etc. Patients in need of treatment include those already suffering from a condition or disorder, those susceptible to having a condition or disorder, or those in need of prevention of the condition or disorder, who can or are expected to benefit from the administration of an antibody or pharmaceutical composition disclosed herein for detection , patients who benefit from the diagnostic process and/or treatment.

0146The term "cancer" means or is intended to describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include glioblastoma, acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), non-small cell lung cancer, lung cancer, colon cancer, colorectal cancer, head and neck cancer, breast cancer Carcinoma (such as triple negative breast cancer), pancreatic cancer, squamous cell tumors, squamous cell carcinoma (such as squamous cell lung cancer or squamous cell head and neck cancer), anal cancer, skin cancer, and vulvar cancer.

0147The terms "overexpressed" or "overexpressed" interchangeably refer to a gene that is usually transcribed or translated at a detectably higher level in certain cells, such as cancer cells, compared to normal cells. Overexpression can be of protein or RNA (due to increased transcription, posttranscriptional processing, translation, posttranslational processing, altered stability, and altered protein degradation), as well as localized overexpression due to altered protein trafficking patterns (nuclear localization increased) and enhanced functional activity, such as, for example, increased enzymatic hydrolysis of substrates. Overexpression can be 50%, 60%, 70%, 80%, 90% or more compared to normal cells or comparison cells. In certain embodiments, the anti-B7-H3 antibodies of the invention are used to treat solid tumors that may overexpress B7-H3.

0148As used herein, the term "administering" means delivering a substance (eg, an anti-B7-H3 antibody) for a therapeutic purpose (eg, treating a B7-H3-related disorder). Modes of administration can be parenteral, enteral and topical. Parenteral administration is usually by injection and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, Subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

0149As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a drug, such as an antibody, sufficient to reduce or ameliorate the severity and/or persistence of a disorder (such as cancer) or one or more symptoms thereof prevent the progression of a condition; cause regression of a condition; prevent the recurrence, development, onset, or progression of one or more symptoms associated with a condition; detect a condition; or enhance or improve the prophylaxis or The amount of therapeutic effect. For example, an effective amount of an antibody can inhibit tumor growth (e.g., inhibit an increase in tumor volume); reduce tumor growth (e.g., reduce tumor volume); reduce the number of cancer cells; one or more symptoms. For example, an effective amount can improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of relapse.

0150The terms "patient" and "subject" are used interchangeably and refer to any mammal in need of diagnosis, prognosis or treatment, including but not limited to humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle etc. In some embodiments, the patient is a human patient.

0151As used herein, the term "in need thereof" means that a patient has been identified as being in need of a particular method or treatment. In some embodiments, identification can be by any diagnostic means. A patient may require it in any of the methods and treatments described herein.

0152The term "administering" as used herein refers to administering a substance for a therapeutic purpose (eg, treating a tumor).

0153The term "agent" as used herein means a chemical compound, a mixture of chemical compounds, a biological macromolecule or an extract made from a biological material.

0154The term "medicament" or "drug" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

0155"About" refers to the usual error range for the corresponding value readily known to those skilled in the relevant art. In some embodiments, "about" mentioned herein refers to the described numerical value and the range of ±10%, ±5% or ±1%.

0156"EC₅₀"That is half maximum effect concentration (concentration for 50% of maximal effect, EC₅₀) refers to the concentration that can cause 50% of the maximum effect.

0157As used herein, the term "label" or "labeled" refers to the incorporation of a detectable label, for example, by incorporation of a radioactively labeled amino acid, or attachment to an avidin that can be labeled (for example, containing a fluorescent label or optically detectable). Enzymatic activity detected by the method or calorimetric method (streptavidin) detected by the biotin-based moiety of the polypeptide. In certain instances, markers or markers can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,³H.¹⁴C.¹⁵N.³⁵S,⁹⁰Y,⁹⁹Tc,¹¹¹In, 1²⁵I.¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemical A luminescent label, biotinyl, is a predetermined polypeptide epitope recognized by the secondary reporter gene (e.g., leucine zipper pair sequence, secondary antibody binding site, metal binding domain, epitope tag). In some embodiments, labels are attached via spacer arms of various lengths to reduce possible steric hindrance.

0158The term "antibody drug conjugate" or "ADC" refers to a binding protein (such as an antibody or antigen-binding fragment) linked to one or more chemical drugs, which may optionally be therapeutic or cytotoxic agents. In preferred embodiments, the ADC comprises an antibody, a drug (eg, a cytotoxic drug) and a linker enabling the attachment or conjugation of the drug to the antibody. The number of binding of an antibody to a small molecule drug in an ADC, that is, the number of drug binding of an antibody, is called the drug-antibody conjugation ratio (DAR). In some embodiments, its value is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. When considering the average binding number of the small molecule drug, that is, the average drug binding number of the antibody, or called the average drug-antibody conjugation ratio, its value is selected from about 0 to 10, or 2 to 8. In some embodiments, the drug-antibody conjugation ratio is about 3 to about 6, and in other embodiments, the drug-antibody conjugation ratio is about 6 to about 8, or about 7 to about 8. Non-limiting examples of drugs that may be included in the ADC are mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemical Protective agents, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors (e.g., TEC-family kinase inhibitors and serine/threonine amino acid kinase inhibitors) and radiosensitizers.

0159The terms "antibody drug conjugate" and "ADC" are used interchangeably. The terms "anti-B7-H3 antibody drug conjugate" and "anti-B7-H3 ADC" are used interchangeably and refer to an ADC comprising an antibody that specifically binds B7-H3, wherein the antibody is conjugated to one or more chemical drugs. couplet. In preferred embodiments, the anti-B7-H3 ADC binds to human B7-H3 (hB7-H3).

0160Other chemical terms herein are used according to conventional usage in the art.

0161All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

0162anti-B7-H3 antibody

0163The present invention provides anti-B7-H3 antibodies or antigen-binding fragments. In some embodiments, the invention provides anti-B7-H3 murine antibodies or antigen-binding fragments. In some embodiments, the invention provides anti-B7-H3 chimeric antibodies or antigen-binding fragments. In some embodiments, the invention provides humanized anti-B7-H3 antibodies or antigen-binding fragments.

0164The anti-B7-H3 antibody or antigen-binding fragment of the present invention includes, but is not limited to, binding to B7-H3 in vitro, binding to cells expressing B7-H3, and strong internalization ability.

0165In some embodiments, the antigen-binding fragment of an anti-B7-H3 antibody is Fab, Fab', F(ab')₂, Fv, disulfide-linked Fv, scFv, single domain antibody or diabody. In some embodiments, the anti-B7-H3 antibody is a multispecific antibody (eg, a bispecific antibody).

0166In some embodiments, antigen-binding fragments such as Fab, F(ab')₂and Fv can be prepared by cleavage of intact proteins, for example by protease or chemical cleavage. Including but not limited to: (i) digestion of antibody molecules with pepsin to obtain F(ab')₂Fragment; (ii) by reducing F(ab')₂Disulfide bonds of the fragments yield Fab fragments; (iii) treatment of antibody molecules with papain and a reducing agent to generate Fab fragments, and (iv) Fv fragments.

0167In some embodiments, the antibody comprises a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment comprises an immunoglobulin selected from a human IgG constant domain, a human IgA constant domain, a human IgE constant domain, a human IgM constant domain, and a human IgD constant domain. Protein heavy chain constant domain. In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment comprises an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region, or an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a kappa light chain constant region.

0168The Fc portion of an antibody mediates several important effector functions (e.g., cytokine induction, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), and antibody and antigen-antibody complexation. drug half-life/clearance). In some cases, these effector functions are desirable for therapeutic antibodies, but in other cases, depending on the therapeutic goal, may be unnecessary or even deleterious. Certain human IgG isotypes, especially IgG1 and IgG3, mediate ADCC and CDC via binding to FcyRs and complement CIq, respectively. The nascent Fc receptor (FcRn) is a key component determining the circulating half-life of antibodies. In other embodiments, at least one amino acid residue in the constant region of the antibody (eg, the Fc region of the antibody) is substituted such that the effector function of the antibody is altered. Substitution of amino acid residues in the Fc region of an antibody to alter antibody effector function is described in US Patent Nos. 5,648,260 and 5,624,821, which are incorporated herein by reference.

0169In some embodiments, the invention includes labeled anti-B7-H3 antibodies or antigen-binding fragments, wherein the antibody is derivatized or linked to one or more functional molecules (eg, another peptide or protein). For example, a labeled antibody can be derived by functionally linking (by chemical conjugation, gene fusion, non-covalent association, or otherwise) an antibody or antigen-binding fragment of the invention to one or more other molecular entities that One or more other molecular entities such as another antibody (e.g., a bispecific or diabody), a detectable reagent, a pharmaceutical agent, can mediate the interaction of an antibody or antigen-binding fragment with another molecule (such as a streptavidin protein core region or polyhistidine tag) and/or a cytotoxic or therapeutic agent selected from the group consisting of mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy , alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerases Inhibitors, kinase inhibitors, radiosensitizers, and combinations thereof.

0170In some embodiments, an antibody or antigen-binding fragment of the invention is linked to a detectable reagent, for example, by incorporation of a radioactively labeled amino acid, or attached to an avidin that can be labeled (for example, contains a fluorescent label or can be detected optically or optically). Enzyme activity detected by calorimetry (streptavidin) detected by the biotinyl moiety of the polypeptide. In certain instances, markers or markers can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,³H.¹⁴C.¹⁵N.³⁵S,⁹⁰Y,⁹⁹Tc,¹¹¹In,¹²⁵I.¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemical A luminescent label, biotinyl, is a predetermined polypeptide epitope recognized by the secondary reporter gene (e.g., leucine zipper pair sequence, secondary antibody binding site, metal binding domain, epitope tag). In some embodiments, labels are attached via spacer arms of various lengths to reduce possible steric hindrance.

0171In other embodiments, the antibodies or antigen-binding fragments of the invention can be used to detect the presence of B7-H3 (eg, human B7-H3) or fragments thereof in a sample. In some embodiments, an antibody comprises a detectable reagent. The antibody is a polyclonal antibody, or more preferably a monoclonal antibody. Whole antibodies or antigen-binding fragments (eg, Fab, scFv or F(ab')2) can be used. The detection method can be used for in vitro and in vivo detection of analyte mRNA, protein or genomic DNA in biological samples. For example, the in vitro detection techniques of analyte mRNA include Northern hybridization and in situ hybridization; the in vitro detection techniques of analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence; the in vitro detection techniques of analyte genomic DNA include Southern hybridization. In addition, in vivo detection techniques for analyte proteins include introducing labeled anti-analyte protein antibodies into the patient. For example, antibodies can be labeled with a radiolabel, the presence and location of which radiolabel can then be detected in a patient by standard imaging techniques.

0172The anti-B7-H3 antibodies disclosed herein may be monoclonal antibodies.

0173The binding specificity of the antibodies or antigen-binding fragments disclosed in the present invention can be determined by in vitro experiments, such as co-immunoprecipitation, radioimmunoassay (RIA), surface plasmon resonance, flow cytometry (Facs) or enzyme-linked immunosorbent assay (ELISA). to test.

0174The invention also includes antibodies that bind to the same epitope as the anti-B7-H3 antibodies described herein. For example, an antibody of the invention specifically binds an epitope comprising one or more amino acid residues on human B7-H3.

0175The amino acid sequence of an exemplary human B7-H3 is provided below as SEQ ID NO: 44 (GenBank Accession No. NP_001019907.1, which is incorporated herein by reference). The signal sequence (amino acids 1-28) is underlined + italics.

SEQ ID NO: 44:

0177<img file="WO2023051663A1_D0007.tif" />

01781) Anti-B7-H3 mouse antibody

0179The heavy chain variable region of an exemplary anti-B7-H3 murine antibody of the invention comprises the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having suitable sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 24. Amino acid sequence, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity; light chain variable region comprising such as SEQ ID NO:25-29 An amino acid sequence as shown in any one of SEQ ID NO: 25-29, or an amino acid sequence having suitable sequence identity compared with the amino acid sequence shown in any one of SEQ ID NO: 25-29, such as having at least 80% sequence identity, or At least 90% sequence identity, alternatively at least 95% sequence identity, alternatively at least 99% sequence identity. In some embodiments, at least the CDRs of these amino acid sequences having suitable sequence identity are unchanged.

01802) Anti-B7-H3 chimeric antibody

0181In some embodiments, the invention provides an anti-B7-H3 chimeric antibody in which the variable region of an anti-B7-H3 murine antibody is linked to a human immunoglobulin constant region.

0182In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24, or with SEQ ID NO: 24 The amino acid sequence shown in ID NO: 24 has an amino acid sequence with suitable sequence identity, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity sex. In some embodiments, at least the CDRs of these amino acid sequences with sequence identity are unchanged.

0183In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises a light chain variable region comprising any one of SEQ ID NOs: 25-29. Amino acid sequence, or an amino acid sequence having suitable sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 25-29, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity. In some embodiments, at least the CDRs of these amino acid sequences with sequence identity are unchanged.

0184In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and the amino acid sequence shown in SEQ ID NO: 25 light chain variable region.

0185In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises: a heavy chain variable region comprising (a) VH CDR1 of the amino acid sequence shown in SEQ ID NO: 6; (b) VH CDR2 of the amino acid sequence shown in SEQ ID NO:7; and (c) VH CDR3 of the amino acid sequence shown in SEQ ID NO:8; and a light chain variable region comprising (a) as SEQ ID NO: VL CDR1 of the amino acid sequence shown in 9; (b) VL CDR2 of the amino acid sequence shown in SEQ ID NO:14; and (c) VL CDR3 of the amino acid sequence shown in SEQ ID NO:19.

0186In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and the amino acid sequence shown in SEQ ID NO: 26 light chain variable region.

0187In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises: a heavy chain variable region comprising (a) VH CDR1 of the amino acid sequence shown in SEQ ID NO: 6; (b) VH CDR2 of the amino acid sequence shown in SEQ ID NO:7; and (c) VH CDR3 of the amino acid sequence shown in SEQ ID NO:8; and a light chain variable region comprising (a) as SEQ ID NO: VL CDR1 of the amino acid sequence shown in 10; (b) VL CDR2 of the amino acid sequence shown in SEQ ID NO:15; and (c) VL CDR3 of the amino acid sequence shown in SEQ ID NO:20.

0188In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and the amino acid sequence shown in SEQ ID NO: 27 light chain variable region.

0189In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises: a heavy chain variable region comprising (a) VH CDR1 of the amino acid sequence shown in SEQ ID NO: 6; (b) VH CDR2 of the amino acid sequence shown in SEQ ID NO:7; and (c) VH CDR3 of the amino acid sequence shown in SEQ ID NO:8; and a light chain variable region comprising (a) as SEQ ID NO: VL CDR1 of the amino acid sequence shown in 11; (b) VL CDR2 of the amino acid sequence shown in SEQ ID NO:16; and (c) VL CDR3 of the amino acid sequence shown in SEQ ID NO:21.

0190In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and the amino acid sequence shown in SEQ ID NO: 28 light chain variable region.

0191In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises: a heavy chain variable region comprising (a) VH CDR1 of the amino acid sequence shown in SEQ ID NO: 6; (b) VH CDR2 of the amino acid sequence shown in SEQ ID NO:7; and (c) VH CDR3 of the amino acid sequence shown in SEQ ID NO:8; and a light chain variable region comprising (a) as SEQ ID NO: VL CDR1 of the amino acid sequence shown in 12; (b) VL CDR2 of the amino acid sequence shown in SEQ ID NO:17; and (c) VL CDR3 of the amino acid sequence shown in SEQ ID NO:22.

0192In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 24 and the amino acid sequence shown in SEQ ID NO: 29 light chain variable region.

0193In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention comprises: a heavy chain variable region comprising (a) VH CDR1 of the amino acid sequence shown in SEQ ID NO: 6; (b) VH CDR2 of the amino acid sequence shown in SEQ ID NO:7; and (c) VH CDR3 of the amino acid sequence shown in SEQ ID NO:8; and a light chain variable region comprising (a) as SEQ ID NO: VL CDR1 of the amino acid sequence shown in 13; (b) VL CDR2 of the amino acid sequence shown in SEQ ID NO:18; and (c) VL CDR3 of the amino acid sequence shown in SEQ ID NO:23.

0194In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention further comprises a heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 32 or 33, or with An amino acid sequence having suitable sequence identity compared to the amino acid sequence shown in SEQ ID NO: 32 or 33, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or At least 99% sequence identity.

0195In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention further comprises a light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 34, or the same as SEQ ID NO: 34 The amino acid sequence shown in NO:34 has suitable sequence identity compared with the amino acid sequence, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity identity.

0196In some embodiments, the heavy chain of an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention further comprises a signal peptide, such as MEFGLSWVFLVAILKGVQC (SEQ ID NO: 45) or MKHLWFFFLLVAAPRWVLS (SEQ ID NO: 46). In some embodiments, the light chain of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention further comprises a signal peptide, such as MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 47) or MVLQTQVFISLLLWISGAYG (SEQ ID NO: 48).

0197In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention binds to B7-H3 with a binding-dissociation equilibrium constant (KD) of about 1 μΜ or less. In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention binds to B7-H3 with a KD of between about 1 μM to about 1 pM. In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention binds to B7-H3 with a KD between about 100 nM and about 1 pM. In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention binds B7-H3 with a KD between about 10 nM and about 1 pM. In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention binds to B7-H3 with a KD between about 1 nM and about 0.1 nM.

0198In some embodiments, the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention binds to hB7-H3-His (as shown in SEQ ID NO: 4) with a KD of about 1 μM or lower. In some embodiments, the KD of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the present invention binding to hB7-H3-His (as shown in SEQ ID NO: 4) is between about 1 μM to about 1 pM; or, Between about 100 nM to about 1 pM; or, between about 10 nM to about 1 pM; or, between about 1 nM to about 1 pM; or, between about 1 nM to about 0.1 nM; or, between about 0.5 nM to Between about 0.3nM.

0199The murine antibodies or chimeric antibodies disclosed herein can be used to generate humanized anti-B7-H3 antibodies. For example, chimeric antibody M1 was chosen for humanization.

02003) Humanized anti-B7-H3 antibody

0201Antibodies described herein include humanized antibodies. These antibodies are suitable for administration to animals (eg, humans) without eliciting a deleterious immune response in the animal (eg, human) to be treated.

0202In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises: a heavy chain variable region comprising (a) VH CDR1 of the amino acid sequence shown in SEQ ID NO: 6; (b) VH CDR2 of the amino acid sequence shown in SEQ ID NO: 7; and (c) VH CDR3 of the amino acid sequence shown in SEQ ID NO: 8; and a light chain variable region comprising (a) the amino acid sequence shown in SEQ ID NO: 8; VL CDR1 of the amino acid sequence shown in ID NO:9; (b) VL CDR2 of the amino acid sequence shown in SEQ ID NO:14; and (c) VL CDR3 of the amino acid sequence shown in SEQ ID NO:19.

0203In some embodiments, the heavy chain variable region of the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises an amino acid sequence as shown in any one of SEQ ID NO: 35-38, or with SEQ ID NO: 35-38 The amino acid sequence shown in any one of ID NOs: 35-38 has suitable sequence identity compared to the amino acid sequence, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity or at least 99% sequence identity; and/or its light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO:39-41, or any one of SEQ ID NO:39-41 An amino acid sequence having suitable sequence identity compared to the amino acid sequence shown in item, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity . In some embodiments, at least the CDRs of these amino acid sequences with sequence identity are unchanged.

0204In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 35 and the amino acid sequence shown in SEQ ID NO: 39 sequence of the light chain variable region.

0205In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 36 and the amino acid sequence shown in SEQ ID NO: 39 sequence of the light chain variable region.

0206In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:36 and the amino acid sequence shown in SEQ ID NO:40 sequence of the light chain variable region.

0207In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:37 and the amino acid sequence shown in SEQ ID NO:41 sequence of the light chain variable region.

0208In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 38 and the amino acid sequence shown in SEQ ID NO: 41 sequence of the light chain variable region.

0209In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention further comprises a heavy chain constant region comprising an amino acid sequence as shown in SEQ ID NO: 32 or 33, or An amino acid sequence having suitable sequence identity compared to the amino acid sequence shown in SEQ ID NO: 32 or 33, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, Or at least 99% sequence identity.

0210In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention further comprises a light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 34, or the same as SEQ ID NO: 34. The amino acid sequence shown in ID NO: 34 has suitable sequence identity compared to the amino acid sequence, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity.

0211In some embodiments, the heavy chain of the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention further comprises a signal peptide, such as MEFGLSWVFLVAILKGVQC (SEQ ID NO: 45) or MKHLWFFFLLVAAPRWVLS (SEQ ID NO: 46). In some embodiments, the light chain of the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention further comprises a signal peptide, such as MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 47) or MVLQTQVFISLLLWISGAYG (SEQ ID NO: 48).

0212In some embodiments, a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention binds to B7-H3 with a binding-dissociation equilibrium constant (KD) of about 1 μM or less. In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention binds to B7-H3 with a KD of between about 1 μM to about 1 pM. In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention binds to B7-H3 with a KD between about 100 nM and about 1 pM. In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention binds to B7-H3 with a KD between about 10 nM and about 1 pM. In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention binds to B7-H3 with a KD between about 1 nM and about 0.1 nM.

0213In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention binds to hB7-H3-His (as shown in SEQ ID NO: 4) with a KD of about 1 μM or lower. In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention binds hB7-H3-His (as shown in SEQ ID NO: 4) with a KD between about 1 μM and about 1 pM; or , between about 100 nM to about 1 pM; or, between about 10 nM to about 1 pM; or, between about 1 nM to about 1 pM; or, between about 1 nM to about 0.1 nM; or, at about 0.5 nM to about 0.3nM.

0214treatment method

0215Antibodies or antigen-binding fragments of the invention are capable of neutralizing B7-H3 activity in vivo and in vitro. Accordingly, the antibodies or antigen-binding fragments of the invention can be used to inhibit the activity of B7-H3, for example, in cell cultures containing B7-H3, in human patients or other mammals with B7-H3 with which the antibodies of the invention cross-react. among animals. In one embodiment, the present invention provides a method for inhibiting the activity of B7-H3, which comprises contacting B7-H3 with the antibody of the present invention, thereby inhibiting the activity of B7-H3. For example, in cell cultures containing or suspected of containing B7-H3, an antibody or antigen-binding fragment of the invention can be added to the culture medium to inhibit B7-H3 activity in the culture. In some embodiments, the B7-H3 is human B7-H3 and is a human patient.

0216In some embodiments, methods are provided for reducing B7-H3 activity in a patient from a patient with a disease or condition in which B7-H3 activity is deleterious. The invention provides methods for reducing B7-H3 activity in a patient suffering from such a disease or disorder, the method comprising administering to the patient an effective dose of an antibody or antigen-binding fragment of the invention such that B7-H3 activity in the patient is reduced . In some embodiments, the B7-H3 is human B7-H3 and is a human patient. Alternatively, the patient may be a mammal expressing B7-H3 to which an antibody of the invention is capable of binding. Furthermore, the patient can be a mammal into which B7-H3 has been introduced (eg, by administering B7-H3 or by expressing a B7-H3 transgene). Antibodies or antigen-binding fragments of the invention can be administered to human patients for therapeutic purposes. In addition, an antibody or antigen-binding fragment of the invention can be administered to a non-human mammal expressing a B7-H3 to which the antibody is capable (for veterinary purposes or as an animal model of human disease). Animal models of such human diseases can be used to evaluate the therapeutic efficacy of the antibodies or antigen-binding fragments of the invention (eg, dose testing and dosing time course).

0217In some embodiments, a method for preventing, treating or improving various types of tumors (including benign tumors and cancers) and other related diseases is provided, the method comprising administering an effective amount of the antibody or antigen-binding fragment to a patient . In some embodiments, the use of the antibody or antigen-binding fragment in preventing, treating or improving tumors (including benign tumors and cancers) and other related diseases is provided. In some embodiments, the use of the antibody or antigen-binding fragment in the preparation of drugs for preventing, treating or improving tumors (including benign tumors and cancers) and other related diseases is provided. In some embodiments, the tumor (including benign tumors and cancers) is a B7-H3 expressing tumor (including benign tumors and cancers). In some embodiments, the tumor (including benign tumors and cancers) is a tumor (including benign tumors and cancers) that overexpresses B7-H3. Methods for identifying B7-H3 expressing tumors (eg, B7-H3 overexpressing tumors) are known in the art. For example, the expression of B7-H3 in normal and tumor tissues was detected by immunohistochemistry.

0218In some embodiments, the present invention relates to a method for treating a disease related to B7-H3 as a therapeutic target, thereby improving, slowing down, inhibiting or preventing any disease or condition related to overexpression of B7-H3; relating to providing treatment for a patient's tumor ( Methods including benign tumors and cancers), methods for alleviating symptoms of tumors (including benign tumors and cancers) in patients, methods for avoiding recurrence of tumors (including benign tumors and cancers) in patients, the methods comprising administering to patients an effective amount of any antibody or antigen-binding fragment.

0219Antibodies or antigen-binding fragments provided by the present invention and pharmaceutical compositions comprising them can be used as therapeutic agents for diagnosis, prognosis, monitoring, treatment, alleviation and/or prevention of abnormal B7-H3 expression, activity and/or Signaling-related diseases and conditions. The antibodies or antigen-binding fragments disclosed herein and pharmaceutical compositions comprising the same can be administered when diseases and conditions associated with aberrant B7-H3 expression, activity and/or signaling are identified in patients by using standard methods.

0220In some embodiments, cancers treated and/or prevented with the antibodies or antigen-binding fragments of the invention include, but are not limited to, solid tumors, hematological tumors, and metastatic lesions. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, or leukemia. More specific examples of such cancers include, but are not limited to, melanoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), ovarian cancer (e.g., ovarian epithelial cancer), glioma (eg, glioblastoma, pediatric brainstem glioma), prostate cancer (eg, castration-resistant prostate cancer), pancreatic cancer (eg, pancreatic ductal carcinoma), head and neck cancer, leukemia (eg, acute myeloid leukemia (AML)), cervical cancer, kidney cancer, squamous cell neoplasm, squamous cell carcinoma (eg, squamous lung cancer or squamous head and neck cancer), colorectal cancer, gastric cancer, liver cancer, mesothelioma , anal cancer, skin cancer, vulvar cancer, neuroblastoma, desmoplastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignancy, sarcoma, brain cancer, central nervous system tumor, brain metastases. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with one or more tumors, including benign tumors or cancer, that overexpress B7-H3. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with a solid tumor that may overexpress B7-H3. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with squamous cell non-small cell lung cancer (NSCLC). In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with a solid tumor, including an advanced solid tumor. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with prostate cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with non-small cell lung cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with glioblastoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with colon cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with head and neck cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with kidney cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with clear cell renal cell carcinoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with glioma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with melanoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with pancreatic cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with gastric cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with ovarian cancer. In some embodiments,An antibody or antigen-binding fragment of the invention is administered to a patient with colorectal cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with small cell lung cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with squamous cell carcinoma of the hypopharynx. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with neuroblastoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with breast cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with endometrial cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with urothelial carcinoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with acute myeloid leukemia (AML).

0221The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody or derivative used, the patient's age and weight, general health, sex, and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are in the judgment of the medical caregiver, who is within the purview of those of ordinary skill in the art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound employed, the severity of the disease and the effect desired. The dosage used can be determined by principles of pharmacology and pharmacokinetics well known in the art. In some embodiments, an effective dosage ranges from about 0.01 mg/kg to about 100 mg/kg, and can be, for example, once a day or up to once a month. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. In addition, it should be understood that for any particular patient, the specific dosing regimen can be adjusted at any time according to the needs of the patient and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges set forth herein are exemplary only. It is not intended to limit the scope or practice of the claimed compositions.

0222Methods of administration of the antibody or antigen-binding fragment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral. The pharmaceutical composition can be administered by any convenient route, such as by infusion or bolus injection, absorbed through the epithelium or mucous membranes (such as oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other biologically active agents. Accordingly, pharmaceutical compositions containing antibodies of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops or transdermal patch), oral administration or nasal spray administration.

0223The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

0224The mode of administration can be systemic administration or local administration. In addition, it may be desirable to introduce an antibody or antigen-binding fragment of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be via an intraventricular catheter connected to, for example, a reservoir (which may be an Ommaya reservoir). fluid sac) to aid in injection. Pulmonary administration is also possible, for example by use of an inhaler or nebulizer, and by use of nebulized formulations.

0225The antibodies or antigen-binding fragments of the invention may be administered topically to the area in need of treatment; this may be by, but not limited to, local infusion during surgery, for example topical application in conjunction with a post-operative wound dressing, by injection, through a catheter, by means of a suppository or This is achieved by means of implants, which are porous, non-porous or gel-like materials comprising membranes (for example silicone rubber membranes) or fibers. Preferably, when administering an antibody or antigen-binding fragment of the invention, care must be taken to use materials that do not absorb proteins.

0226The methods for treating diseases are usually tested in vitro, including administering the antibody or derivative of the present invention, then testing the desired therapeutic or prophylactic activity in an acceptable animal model in vivo, and finally administering to humans. Suitable animal models, including transgenic animals, are known to those of ordinary skill in the art. For example, in vitro assays used to demonstrate therapeutic use of the antibodies, antigen-binding fragments of the invention include the effect of the antibodies on cell lines or patient tissue samples. The effect of antibodies on cell lines and/or tissue samples can be detected using techniques known to those skilled in the art, such as those disclosed elsewhere herein. In vitro assays useful in determining whether to administer an antibody or antigen-binding fragment in accordance with the present invention include in vitro cell culture assays in which patient tissue samples are grown in culture and exposed to or otherwise administered an antibody or antigen-binding fragment, And observe the effect of this antibody or antigen-binding fragment on the tissue sample.

0227Various known delivery systems can be used to administer the antibodies or antigen-binding fragments of the invention, or polynucleotides encoding them, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis, construction of nucleic acids as part of retroviruses or other vectors, etc.

0228combination therapy

0229In some embodiments, an antibody or antigen-binding fragment of the invention may be used or combined in conjunction with other therapeutic or prophylactic regimens, including administration of one or more antibodies or antigen-binding fragments of the invention and one or more other therapeutic agents or methods use. For combination therapy, the antibody or antigen-binding fragment can be administered simultaneously or separately from the other therapeutic agent. When administered separately, the antibody or antigen-binding fragment of the invention can be administered before or after another other therapeutic agent is administered.

0230In some embodiments, when an antibody or antigen-binding fragment of the invention is administered to a patient, the antibody molecule or pharmaceutical composition disclosed herein may also be administered to the patient in combination with one or more other therapies, such as treatment modalities and/or or other agents (eg, therapeutic agents).

0231Such combination therapy encompasses simultaneous administration (where two or more formulations are contained in the same formulation or in separate formulations), and separate administration, in which case it may be followed by administration of other therapies, such as treatment modalities and/or Administration of the antibody or antigen-binding fragment of the invention occurs before, simultaneously with, and/or after the therapeutic agent. Antibody molecules and/or other therapies, such as therapeutic agents or treatment modalities, can be administered during active disease or during remission or less active disease. Antibody molecules can be administered before, concurrently with, after, or during remission of other treatments.

0232In some embodiments, an antibody or antigen-binding fragment of the invention is administered in combination with a therapeutic agent. Therapeutic agents include, but are not limited to: cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents (e.g. systemic anti-inflammatory agents), anti-fibrotic agents, metabolic inhibitors, enzyme inhibitors and/or cytotoxic or cytostatic agents, mitotic inhibitors, antitumor antibiotics, immunomodulators, gene therapy carriers, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormonal agents, corticosteroids, photo Active therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors or radiosensitizers.

0233In some embodiments, an antibody or antigen-binding fragment of the invention is administered in combination with an anticancer or antineoplastic agent. The terms "anticancer agent" and "antineoplastic agent" refer to drugs used in the treatment of malignancies, eg, to inhibit cancerous growth. For example, breast cancer is often stimulated by estrogen and can be treated with drugs that inactivate sex hormones. Similarly, prostate cancer can be treated with drugs that inactivate androgens. Examples of anti-cancer agents include, but are not limited to: anti-PD1 antibodies (e.g., paclizumab), anti-PD-L1 antibodies (e.g., atezolizumab), anti-CTLA-4 antibodies (e.g., ipilimumab) , MEK inhibitors (eg, trametinib), ERK inhibitors, BRAF inhibitors (eg, dabrafenib), ostinib, erlotinib, gefitinib, sorafenib, CDK9 Inhibitors (e.g., denazelib), MCL-1 inhibitors, temozolomide, Bcl-xL inhibitors, Bcl-2 inhibitors (e.g., venetoclax), Ibrutinib, mTOR inhibitors (e.g., everolimus), PI3K inhibitors (e.g., bupaliximab), dulvelix, idelaris, AKT inhibitors, HER2 inhibitors (e.g., Patinib), Herceptin, taxanes (e.g., docetaxel, paclitaxel, nab-paclitaxel), ADCs containing auristatin, ADCs containing PBDs (e.g., rovupituzumab-tecillin) , ADCs containing maytansinoids (e.g., TDM1), TRAIL agonists, proteasome inhibitors (e.g., bortezomib), nicotinamide phosphoribosyltransferase inhibitors, Panorex, rituximab, Gemtuzumab, alemtuzumab, ibritumomab, tositumomab, cetuximab, Avastin, and Herceptin.

0234Examples of cytokines that may be administered in conjunction with the antibodies or antigen-binding fragments of the invention include, but are not limited to, one or more of the following: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 , IL-7, IL-8, IL-15, IL-16, IL-18, IL-21 and IL-31, etc.

0235Examples of other therapeutic agents that may be administered in conjunction with the antibodies or antigen-binding fragments of the invention include, but are not limited to, one or more of the following: inhaled steroids; beta-agonists, e.g., short-acting or long-acting beta-agonists; Antagonists of triene or leukotriene receptors; combination drugs, such as ADVAIR; IgE inhibitors, e.g., anti-IgE antibodies (e.g., omalizumab); phosphodiesterase inhibitors (e.g., PDE4 inhibitors); Xanthines; anticholinergic drugs; mast cell stabilizers such as cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaxin/CCR3 inhibitors; histamine or its receptors (including Antagonists of H1, H2, H3, and H4), and antagonists of prostaglandin D or its receptors (DP1 and CRTH2); anti-PD1 antibodies (e.g., paclizumab), anti-PD-L1 antibodies (e.g., terzolimab), anti-CTLA-4 antibodies (e.g., ipilimumab), MEK inhibitors (e.g., trametinib), ERK inhibitors, BRAF inhibitors (e.g., dabrafenib), Os Irtinib, Erlotinib, Gefitinib, Sorafenib, CDK9 Inhibitors (eg, Denaceri), MCL-1 Inhibitors, Temozolomide, Bcl-xL Inhibitors, Bcl-2 Inhibitors (eg, venetoclax), ibrutinib, mTOR inhibitors (e.g., everolimus), PI3K inhibitors (e.g., buparixi), dulvilised, idelaris, AKT inhibitors, HER2 Inhibitors (e.g., lapatinib), Herceptins, taxanes (e.g., docetaxel, paclitaxel, nab-paclitaxel), ADCs containing auristatin, PBDs containing ADCs containing maytansinoids (e.g., TDM1), TRAIL agonists, proteasome inhibitors (e.g., bortezomib), and nicotinamide phosphoribosyltransferase (NAMPT) Inhibitors; TNF antagonists (e.g., soluble fragments of TNF receptors, such as p55 or p75 human TNF receptors or derivatives thereof, such as 75kD TNFR-IgG (75kD TNF receptor-IgG fusion protein, ENBREL)); TNF enzymes Antagonists such as TNF-converting enzyme inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; pirfenidone; chemotherapeutic agents such as leflunomide or sirolimus or similar Drugs, such as CCI-779; COX2 and cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors,TPL-2, MK-2 and NFkB inhibitors, etc.

0236In some embodiments, antibodies or antigen-binding fragments of the invention may be used with immune checkpoint inhibitors. In some embodiments, antibodies or antigen-binding fragments of the invention are administered in conjunction with other therapeutic or prophylactic regimens, such as radiation therapy.

0237pharmaceutical composition

0238Antibodies described herein, or derivatives, fragments, analogs, homologues thereof, may be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in the preparation of such compositions, as well as guidance for selecting components, are well known in the art.

0239Such compositions generally comprise an antibody or antigen-binding fragment and a pharmaceutically acceptable carrier. In some embodiments, the antigen-binding fragment used is a minimal inhibitory fragment that specifically binds to the binding domain of the target protein. For example, peptides based on the variable region sequences of antibodies and retaining the ability to bind the target protein sequence. In some embodiments, the pharmaceutical composition further comprises an anticancer agent (eg, an immune checkpoint inhibitor).

0240In some embodiments, the term "pharmaceutically acceptable" refers to a substance approved by a regulatory agency of the government or listed in a recognized pharmacopoeia for use in animals, especially in humans. In addition, "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary, etc.

0241The term "carrier" refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, Propylene, ethylene glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are listed in Remington's, EW Martin. Described in Pharmaceutical Sciences, incorporated herein by reference. Such compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in a purified form, together with an appropriate amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the mode of administration. The parent formulation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

0242In some embodiments, the composition is formulated into a pharmaceutical composition (eg, a pharmaceutical composition suitable for intravenous injection into a human body) according to conventional procedures. Compositions for intravenous administration are generally solutions in sterile isotonic aqueous buffer. The composition may also contain a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. The pharmaceutical composition is prepared in the form of dosage unit for easy administration and uniform dosage. Dosage unit form as used herein refers to physically separable units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of a drug calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. or a plurality of said antibodies. Generally, the active ingredients are supplied alone or in combination in unit dosage form, such as a dry lyophilized powder or anhydrous concentrate, in a hermetically sealed container (such as an ampoule or sachet) indicating the quantity of active agent. Where the composition is administered by infusion, the composition may be dispensed with an infusion bottle containing pharmaceutical grade sterile water or saline. In the case of administering the composition by injection, an ampoule of sterile water or saline for injection can be used so that the active ingredient can be mixed before administration.

0243In some embodiments, pharmaceutical compositions to be used for in vivo administration are sterile. This is readily accomplished by filtration through sterile membrane filters.

0244A pharmaceutical composition is generally compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. The pharmaceutical composition may include one or more of the following components: sterile diluents for injection, such as water, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; bacteriostatic agents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as histidine hydrochloride, acetate, Citrates or phosphates; osmotic regulators such as sodium chloride or dextrose; stabilizers such as arginine, methionine, trehalose, sucrose, sorbitol; surfactants such as Tween 20 ,Tween 80. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The pharmaceutical composition can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For use, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent delaying absorption, for example, aluminum monostearate and gelatin.

0245For transmucosal or transdermal administration, penetrants appropriate to the barrier to permeation are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, one or more of the antibodies may be formulated into ointments, ointments, gels, or creams as generally known in the art.

0246The pharmaceutical composition can be placed in a container or dispenser and packaged together with instructions for administration.

0247The pharmaceutical compositions described herein may also contain other active ingredients, preferably those with complementary activities that do not adversely affect each other, depending on the particular condition to be treated. In some embodiments, a composition may include agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutics, or growth inhibitory agents. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

0248The compositions of the present invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from anions such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamine, Salts of cations such as aminoethanol, histidine, and procaine.

0249Preparation

0250Anti-B7-H3 antibodies can be produced by immunizing an animal with, for example, membrane-bound and/or soluble B7-H3 (eg, human B7-H3 or an immunogenic fragment, derivative or variant thereof). Alternatively, monoclonal antibodies can be prepared using, for example, the hybridoma method. In the hybridoma approach, a mouse, hamster or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind the immunizing agent. Alternatively, lymphocytes can be immunized in vitro to produce antibodies.

0251The immunizing agent used in the hybridoma method usually includes a protein antigen, a fragment thereof, or a fusion protein thereof (eg hB7-H3-Fc, the amino acid sequence of which is shown in SEQ ID NO: 1). Typically, peripheral blood lymphocytes are used if cells of human origin are desired, or splenocytes or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are usually rat or mouse myeloma cell lines. Hybridoma cells can be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma typically includes hypoxanthine, aminopterin, and thymine ("HAT medium"), the The substance prevents the growth of HGPRT-deficient cells.

0252Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent No. 4,816,567. DNA encoding the monoclonal antibodies described herein can be isolated and sequenced using conventional methods (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as Chinese hamster ovary (CHO) cells, human embryonic kidney 293 cells (HEK293 cells), simian COS cells that do not otherwise produce immunoglobulins , PER.NS0 cells, SP2/0 cells, YB2/0 cells, or myeloma cells, so as to obtain synthetic monoclonal antibodies in recombinant host cells. DNA can also be obtained by, for example, substituting the coding sequences for the human heavy and light chain constant domains for the homologous murine sequences (see U.S. Patent No. 4,816,567) or by combining immunoglobulin coding sequences with all or part of the coding sequences for non-immunoglobulin polypeptides. Covalently linked for modification. Such non-immunoglobulin polypeptides can replace the constant domains of the antibodies described herein, or can replace the variable domains of one of the antigen binding sites of the antibodies described herein to generate chimeric bivalent antibodies.

0253Single-chain Fv (scFv) can be found in techniques for producing single-chain units (US Patent 4,694,778). A single-chain fusion peptide is generated by amino acid bridging of the heavy and light chain fragments of the Fv region to form a single-chain unit. Techniques for assembling functional Fv fragments in E. coli can also be used.

0254Examples of techniques that can be used to produce scFvs and antibodies include those described in US Pat. Nos. 4,946,778 and 5,258,498.

0255In addition, another highly efficient method for producing recombinant antibodies is disclosed in U.S. Patent Nos. 5,658,570, 5,693,780, and 5,756,096. In particular, this technique enables the production of primate antibodies containing monkey variable region and human constant region sequences. The contents are incorporated herein by reference.

0256In some embodiments, the antibodies do not elicit a deleterious immune response in a treated animal (eg, a human). In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, primatized, deimmunized or chimeric antibodies can be made. These types of antibodies are derived from nonhuman antibodies, usually murine or primate antibodies, that retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans. This can be achieved by a variety of methods, including (a) grafting the entire non-human variable region to the human constant region to generate chimeric antibodies; methods for producing chimeric antibodies are known in the art, see US Patents 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference; (b) grafting at least a portion of one or more non-human complementarity determining regions (CDRs) into framework and constant regions of human origin, with or without retention key framework residues; or (c) grafting entire non-human variable domains, but "hiding" them by replacing surface residues with human-like parts. Typically framework residues in the human framework regions will be replaced by corresponding residues from the CDR donor antibody, such as residues that improve antigen binding. These framework substitutions can be identified by methods known in the art, such as by simulating the interaction of CDRs and framework residues to identify framework residues important for antigen binding and by sequence alignment to identify abnormal framework residues at specific positions ( See US Patent 5,585,089; the entire contents of which are incorporated herein by reference). Antibodies can be humanized using various techniques known in the art, such as CDR grafting (EP 239,400; WO 91/09967; US Patents 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596) , and chain rearrangements (US Patent 5,565,332), the entire contents of which are incorporated herein by reference.

0257Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes altering antibodies to modify T cell epitopes (see eg WO/9852976A1 and WO/0034317A2). For example, the heavy and light chain variable region sequences from a starting antibody are analyzed and a human T cell epitope "map" from each variable region is generated, showing the epitopes relative to the complementarity determining regions (CDRs) and the positions of other key residues within the sequence. Analyze individual T-cell epitopes from T-cell epitope maps to identify alternative amino acid substitutions with low risk of altering antibody activity. A series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed and these sequences are subsequently incorporated into a series of binding polypeptides. Genes for the complete heavy and light chains containing the modified variable and human constant regions are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce complete antibodies. Then use appropriate biochemical and biological experiments to compare antibodies and identify the best antibody.

0258Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. See also U.S. Patents 4,444,887 and 4,716,111, and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each The entire content of the patent is incorporated herein by reference.

0259Fully human antibodies that recognize selective epitopes can be produced using a technique known as "guided selection." In this approach, selected non-human monoclonal antibodies (eg, mouse antibodies) are used to guide the selection of fully human antibodies that recognize the same epitope (see US Pat. No. 5,565,332, the entire contents of which are incorporated herein by reference).

0260In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). sequencing. Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or bone marrow that do not produce other immunoglobulins tumor cells). Isolated or synthetic DNA can also be used to prepare the sequences of the constant and variable regions of antibodies as described in US Pat. No. 5,658,570, the entire contents of which are incorporated herein by reference. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR using Ig-specific primers. Suitable probes for this purpose are also mentioned in US Patent No. 5,658,570.

0261In addition, using conventional recombinant DNA techniques, one or more CDRs of an antibody of the invention can be inserted into a framework region, for example into a human framework region, to construct a humanized non-fully human antibody. The framework regions may be naturally occurring or consensus framework regions, preferably human framework regions. Some polynucleotides may encode an antibody that specifically binds at least one epitope of an antigen of interest produced by a combination of framework regions and CDRs. One or more amino acid substitutions may be made within the framework regions, and amino acid substitutions may be selected to improve binding of the antibody to its antigen. In addition, substitution or deletion of cysteine residues in one or more variable regions involved in interchain disulfide bond formation can be performed in this way, thereby producing antibody molecules lacking one or more interchain disulfide bonds. Other modifications to polynucleotides within the skill of the art are also encompassed in the present invention.

0262Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, and the like can be selected, constructed, and cultured using techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications.

0263In some embodiments, the anti-B7-H3 antibodies or antigen-binding fragments of the invention are glycosylated. For example, an aglycosylated antibody can be prepared (ie, the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for antigen. Such modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region glycosylation sites, thereby eliminating glycosylation at that site. Such deglycosylation increases the affinity of the antibody for antigen. Such methods are described in further detail in PCT Publication WO 2003016466 A2 and US Patents 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.

0264Additionally, modified anti-B7-H3 antibodies or antigen-binding fragments with altered glycosylation patterns, such as hypofucosylated antibodies or antibodies with increased bisected GlcNAc structures, can be prepared. These changes have been shown to increase the ADCC ability of the antibody. Such modifications can be accomplished, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing recombinant antibodies of the invention to thereby produce antibodies with altered glycosylation. See, eg, European Patent Nos.: EP 1,176,195; PCT Publication WO 03/035835; WO 99/5434280, each of which is incorporated herein by reference in its entirety.

0265Antibodies of the present invention (eg, anti-B7-H3 antibodies) can be produced by recombinantly expressing heavy and light chain genes of the antibody in host cells. For example, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding heavy and light chains of antibodies, so that the heavy and light chains are expressed in the host cells, and the expressed antibodies can be secreted into the cultured host cells. within the culture medium from which the antibody can be recovered. "Transfection" refers to a wide variety of techniques commonly used to introduce exogenous DNA into eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Standard recombinant DNA methods for obtaining antibody heavy and light chain genes, incorporating these genes into expression vectors, and introducing the vectors into host cells are well known in the art, such as those described in US Pat. No. 4,816,397. The DNA expressing the antibody heavy chain and light chain can be placed in the same vector or placed in different vectors; if placed in different vectors, the vector expressing the antibody heavy chain and the vector expressing the antibody light chain can be transfected into host cells in an appropriate ratio . In some embodiments, an antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements may include enhancers, Kozak sequences (GCCACC, such as SEQ ID NO:53) and the donor and acceptor sites of RNA splicing on both sides of the inserted sequence. High-efficiency transcription can be obtained through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and early promoters of cytomegalovirus, and other cellular promoters such as muscle Kinetin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), pSVL , pMSG, pRSVcat, pSV2dhfr, pBC12MI, pCS2 or pCHO1.0, etc. Commonly used mammalian cells (host cells) include HEK293 cells (such as HEK293F cells), Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.

0266To express an antibody of the invention (eg, an anti-B7-H3 antibody), DNA encoding the full-length light and heavy chains can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. "Operably linked" means that the antibody gene is linked into the vector so that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

0267Antibody (eg, anti-B7-H3 antibody) genes can be inserted into expression vectors by standard methods (eg, ligation of antibody gene fragments and complementary restriction sites on the vector, or blunt ligation if no restriction sites exist). Before inserting the antibody-related light chain or heavy chain gene sequence, the expression vector may already carry the antibody constant region sequence. For example, one way to convert the VH and VL sequences associated with an anti-B7-H3 antibody into full-length antibody genes is to insert them into expression vectors already encoding the heavy and light chain constant regions, respectively, so that the VH segments can be is operably linked to a CH segment within the vector, and the VL segment is operably linked to a CL segment within the vector. Alternatively, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody heavy and light chains from the host cell. Alternatively, the antibody heavy and light chain genes can be cloned into a vector encoding a signal peptide that promotes secretion of the antibody heavy and light chains from the host cell such that the signal peptide is linked in frame to the amino termini of the antibody heavy and light chain genes. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein), for example, MEFGLSWVFLVAILKGVQC (SEQ ID NO: 45), MKHLWFFFLLLVAAPRWVLS (SEQ ID NO: 46), MDMRVLAQLLGLLLLCFPGARC ( SEQ ID NO:47) or MVLQTQVFISLLLWISGAYG (SEQ ID NO:48).

0268In addition to antibody heavy chain and light chain genes, recombinant expression vectors can also carry regulatory sequences that control the expression of antibody chain genes in host cells. Regulatory sequences include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of the antibody chain genes. Those skilled in the art understand that the design of expression vectors, including the choice of regulatory sequences, may depend on factors such as the choice of host cells to be transformed, the expression level of the desired protein, and the like. Suitable regulatory sequences for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, such as those derived from cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), Simian virus 40 (SV40 ) (eg SV40 promoter/enhancer), adenovirus (eg adenovirus major late promoter (AdMLP)) and polyoma promoters and/or enhancers. For further descriptions of viral regulatory elements and their sequences, see, eg, US Patents 5,168,062, 4,510,245 and 4,968,615.

0269In addition to the antibody chain genes and regulatory sequences, a recombinant expression vector can carry additional sequences, such as sequences that regulate replication of the vector in a host cell (eg, an origin of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, eg, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, marker genes that confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced are often selected. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells with methotrexate selection/amplification), the neo gene (for G418 selection) and the GS gene . For expression of the heavy and light chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques.

0270Antibodies (eg, anti-B7-H3 antibodies) of the invention can be expressed in eukaryotic host cells. In certain embodiments, expression of antibodies is performed in eukaryotic cells, such as mammalian host cells. Exemplary host cells for expressing antibodies of the invention include Chinese hamster ovary cells (CHO cells) or CHO-S, CHO-dhfr-, CHO/DG44 or ExpiCHO modified from CHO cells, NSO myeloma cells, COS cells, SP2 cells, CV1 cells, mouse L cells, HEK293 human embryonic kidney cells, or HEK293T, HEK293F or HEK293E cells modified from HEK293 cells. After the recombinant expression vector encoding the antibody chain gene is introduced into the host cell, the host cell is cultured in the medium to allow the expression of the antibody in the host cell or the secretion of the antibody into the medium for a period of time to produce the antibody. Antibodies can be recovered from the culture medium using standard protein purification methods.

0271For the recombinant expression of the antibody of the present invention (such as anti-B7-H3 antibody), host cells can be co-transfected with two recombinant expression vectors, the first recombinant expression vector encodes the antibody heavy chain, and the second recombinant expression vector encodes the antibody light chain. The two recombinant expression vectors can contain the same selectable marker, or they can each contain separate selectable markers. Alternatively, host cells can be transfected with recombinant expression vectors encoding antibody heavy and light chains.

0272Antibodies of the invention (eg, anti-B7-H3 antibodies) can also be produced by chemical synthesis. Variant antibodies can also be generated using cell-free platforms.

0273Antibodies of the invention (e.g., anti-B7-H3 antibodies) produced by recombinant expression can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity and chromatography and fractional column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. For example, protein A or protein G is used for affinity chromatography, which mainly provides the IgG fraction in immune serum. In addition, the specific antigen or epitope targeted by the immunoglobulin can be immobilized on a column to purify the immune-specific antibody by immunoaffinity chromatography. Antibodies of the invention (eg, anti-B7-H3 antibodies) can be fused to heterologous polypeptide sequences known in the art to facilitate purification. The purification of immunoglobulin can refer to the article of D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Volume 14, No. 8 (April 17, 2000), pages 25-28) .

0274In addition, mutations may be introduced in the nucleotide sequences encoding the antibodies of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutations resulting in amino acid substitutions. Variants (including derivatives) encode substitutions of less than 50 amino acids, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions A substitution of amino acids, a substitution of less than 4 amino acids, a substitution of less than 3 amino acids, or a substitution of less than 2 amino acids. Alternatively mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

0275In some embodiments, the substitutions described herein are conservative amino acid substitutions.

0276In some embodiments, the gene sequence encoding the heavy chain of antibody V3 is shown in SEQ ID NO:49, wherein the underlined part encodes the VH CDR; the amino acid sequence of the heavy chain of antibody V3 is shown in SEQ ID NO:50, wherein the underlined part Some are VH CDRs.

0277SEQ ID NO:49 is as follows:

0278<img file="WO2023051663A1_D0008.tif" />

0279<img file="WO2023051663A1_D0009.tif" />

0280SEQ ID NO:50 is as follows:

0281<img file="WO2023051663A1_D0010.tif" />

0282In some embodiments, the gene sequence encoding the light chain of antibody V3 is shown in SEQ ID NO:51, wherein the underlined part encodes the VL CDR; the amino acid sequence of the light chain of antibody V3 is shown in SEQ ID NO:52, wherein the underlined part Part of the VL CDR.

0283SEQ ID NO:51 is as follows:

0284<img file="WO2023051663A1_D0011.tif" />

0285SEQ ID NO:52 is as follows:

0286<img file="WO2023051663A1_D0012.tif" />

0287Example

0288The technical solutions of the present invention are further described below through specific examples, which do not represent limitations on the protection scope of the present invention. Some non-essential modifications or adjustments made by others according to the concept of the present invention still belong to the protection scope of the present invention.

0289The materials and reagents used in the following examples can be obtained from commercial sources or known methods unless otherwise specified.

0290VISTA-CHO cell construction process: the gene sequence encoding VISTA (NCBI Reference Sequence: NP_071436.1) was inserted into the expression vector, and then stably transfected into CHO cells to obtain VISTA-CHO cells.

0291Tim3-CHO cell construction process: Insert the gene sequence encoding Tim3 (NCBI Reference Sequence: NP_116171.3) into the expression vector, and then stably transfect CHO cells to obtain Tim3-CHO cells.

0292Lag3-CHO cell construction process: Insert the gene sequence encoding Lag3 (NCBI Reference Sequence: NP_002277.4) into the expression vector, and then stably transfect CHO cells to obtain Lag3-CHO cells.

0293The preparation of embodiment 1 antigen

0294Preparation of antigen hB7-H3-Fc: encoding antigen hB7-H3-Fc (the amino acid sequence of antigen hB7-H3-Fc is shown in SEQ ID NO: 1, and the extracellular domain of human B7-H3 (such as SEQ ID NO: 2 Shown) The gene sequence of adding IgG1 Fc (as shown in SEQ ID NO:3) to the C-terminus) was cloned into the expression vector, then transiently transfected into HEK293F cells, and purified by Protein A affinity chromatography to obtain the antigen hB7-H3-Fc.

0295Preparation of antigen hB7-H3-His: encoding antigen hB7-H3-His (the amino acid sequence of antigen hB7-H3-His is shown in SEQ ID NO: 4, and human B7-H3 ectodomain (as shown in SEQ ID NO: 2) shown) the C-terminus was added with 10×HIS tag (constructed by HHHHHHHHHH, as shown in SEQ ID NO: 5), the gene sequence was cloned into the expression vector, and then transiently transfected into HEK293F cells, and purified by nickel column to obtain the antigen hB7- H3-His.

0296Amino acid sequence of antigen hB7-H3-Fc:

0297(as shown in SEQ ID NO: 1).

0298Amino acid sequence of human B7-H3 extracellular domain:

0299MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDA HGSVTITGQPMT(Such asSEQ ID NO:2Shown).

0300Amino acid sequence of Fc of IgG1:

0301DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Such asSEQ ID NO:3Shown).

0302Amino acid sequence of antigen hB7-H3-His:

0303MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTHHHHHHHHHH(Such asSEQ ID NO:4Shown).

0304Example 2 Chimeric Antibody

0305The amino acid sequences of VH CDR, VL CDR, VH and VL of the mouse antibody are shown in Table 1-3.

0306Table 1 Amino acid sequence of VH CDR (Kabat numbering)

0307<img file="WO2023051663A1_D0013.tif" />

0308Table 2 Amino acid sequence of VL CDR (Kabat numbering)

0309<img file="WO2023051663A1_D0014.tif" />

0310<img file="WO2023051663A1_D0015.tif" />

0311Amino acid sequence (Kabat numbering) of table 3 mouse anti-variable region

0312<img file="WO2023051663A1_D0016.tif" />

0313The assembly scheme of the chimeric antibody is shown in Table 4. VH and CH form the heavy chain of the antibody, and VL and CL form the light chain of the antibody; it is represented by "antibody" + antibody number, for example, the heavy chain of antibody M1 is composed of SED ID NO: 24 VH as shown and CH as shown in SED ID NO:32 are composed, and the light chain of antibody M1 is composed of VL as shown in SED ID NO:25 and CL as shown in SED ID NO:34. The heavy chain of the control antibody M30-H1-L4 is shown in SEQ ID NO: 30, and the light chain is shown in SEQ ID NO: 31 (see Table 5). The CDR of the chimeric antibody is shown in Table 6, and the amino acid sequence of the IgG constant region is shown in Table 7.

0314The gene sequences encoding the heavy chain and light chain of the antibody were respectively cloned into expression vectors to obtain recombinant expression vectors, and then HEK293F cells were transiently transfected. After culturing, the antibody was purified by Protein A affinity chromatography, and the sequence was consistent with the expected sequence.

0315Table 4 Chimeric antibody numbering, assembly scheme

0316<img file="WO2023051663A1_D0017.tif" />

0317Table 5 Amino acid sequence of control antibody M30-H1-L4

0318<img file="WO2023051663A1_D0018.tif" />

0319<img file="WO2023051663A1_D0019.tif" />

0320Table 6 CDRs of chimeric antibodies

0321<img file="WO2023051663A1_D0020.tif" />

0322Affinity and specificity of embodiment 3 chimeric antibody

03231. Chimeric antibody affinity verification-1Biacore

0324Use BiaCore T200 (GE Healthcare) for detection: use Protein A chip (GE Healthcare, Cas#29127556) for detection, and use 1×HBS-EP+ for the antibody (preparation of 1×HBS-EP+: take HBS-EP+(10×)( GE Healthcare, cat#BR-1006-69) 50mL, add ultrapure water 450mL, mix well) to 5μg/mL, pass through the experimental flow path (Fc2, Fc4) at a flow rate of 10μL/min, and capture for 20 seconds (s) Make the capture amount about 235RU; then adjust the flow rate to 30μL/min, and then add different concentrations of hB7-H3-His dilutions (0nM, 3.125nM, 6.25nM, 12.5nM, 25nM, 50nM, dilute with 1×HBS-EP+ ), through the surface of the experimental flow path (Fc2, Fc4) and the reference flow path (Fc1, Fc3) at the same time, the binding time was 180s, the dissociation time was 600s, and finally the chip was treated with Glycine pH1.5 (GE Healthcare, cat#BR100354). Regenerate for 60s, and then proceed to the next cycle. Use data analysis software Evaluation Software 3.1 Analyze the test results, subtract the sensing signals obtained from the sample test flow path from the reference flow path and the sample blank, and use the kinetic "1:1" model for fitting, and obtain that each sample is the same as hB7- Kinetic parameters of H3-His binding (ka: association rate; kd: dissociation rate; kD: binding-dissociation equilibrium constant).

0325Table 8 The association rate, dissociation rate and binding dissociation equilibrium constant of chimeric antibody binding to hB7-H3-His

0326<tables num="0002"><table><tgroup cols="4"><colspec colname="c001" colwidth="26%" /><colspec colname="c002" colwidth="24%" /><colspec colname="c003" colwidth="25%" /><colspec colname="c004" colwidth="25%" /><thead><row><entry morerows="0">Antibody</entry><entry morerows="0">ka(1/Ms)</entry><entry morerows="0">kd(1/s)</entry><entry morerows="0">KD(M)</entry></row></thead><tbody><row><entry morerows="0">M1</entry><entry morerows="0">7.504E+5</entry><entry morerows="0">2.794E-4</entry><entry morerows="0">3.723E-10</entry></row><row><entry morerows="0">M2</entry><entry morerows="0">1.282E+5</entry><entry morerows="0">0.04906</entry><entry morerows="0">3.828E-7</entry></row><row><entry morerows="0">M30-H1-L4</entry><entry morerows="0">2.437E+5</entry><entry morerows="0">2.403E-4</entry><entry morerows="0">9.860E-10</entry></row></tbody></tgroup></table></tables>

0327The results are shown in Table 8, both antibodies M1 and M2 can bind hB7-H3-His, and antibody M1 has a higher binding affinity to hB7-H3-His.

03282. Chimeric antibody affinity verification-2 flow cytometry

0329Take MDA-MB-468 cells in the logarithmic growth phase, centrifuge at 1500rpm for 5 minutes (min), remove the supernatant, resuspend with 1×PBS, centrifuge at 1500rpm for 5 minutes, remove the supernatant, and repeat once. Add MDA-MB-468 cells to V-type 96-well plate, 50 μL/well (200,000 cells), centrifuge at 1500 rpm for 5 minutes and discard PBS; add 100 μL/well of serially diluted chimeric antibody (initial concentration is 100 nM, The medium is 1×PBS), resuspend the cells; incubate on ice for 1 hour (h); after the incubation, centrifuge at 2000 rpm for 5 minutes, remove the supernatant, resuspend the cells with 200 μL/well of 1×PBS, and then resuspend the cells Perform centrifugation, remove the supernatant, and repeat twice; add PE-labeled goat anti-human IgG Fc secondary antibody (Invitrogen, Cat. No. 12-4998-82; diluted to 1:500 with 1×PBS) at 100 μL/well, and resuspend the cells; Light, incubate on ice for 30 minutes; after the incubation, centrifuge at 2000rpm for 5 minutes, remove the supernatant, resuspend the cells with 200 μL/well of 1×PBS, and then centrifuge again, remove the supernatant, repeat twice; add 200 μL/well of 1 ×PBS resuspended cells; detected on cytoflex flow cytometer (Beckman).

0330The test results are shown in Figure 1.

03313. Chimeric antibody specificity verification - flow cytometry

0332Take the cells in the logarithmic growth phase (Lag3-CHO cells, VISTA-CHO cells, Tim3-CHO cells or Raji cells), centrifuge at 1500rpm for 5 minutes, remove the supernatant, resuspend with 1×PBS, centrifuge at 1500rpm for 5 minutes, remove the supernatant Clear and repeat. Add the cells to a V-shaped 96-well plate, 50 μL/well (500,000 cells), centrifuge at 1500 rpm for 5 minutes and discard the PBS; add 100 μL/well of antibody M1 (concentration is 100 nM), resuspend the cells; incubate on ice for 1 hour; After the incubation, centrifuge at 2000rpm for 5 minutes, remove the supernatant, resuspend the cells with 200 μL/well of 1×PBS, and centrifuge again, remove the supernatant, repeat twice; add PE-labeled goat anti-human IgG Fc secondary antibody to 100 μL/well (Invitrogen, Cat. No. 12-4998-82; diluted to 1:500 with 1×PBS), resuspended cells; protected from light, incubated on ice for 30 minutes; after the incubation, centrifuged at 2000rpm for 5 minutes, removed the supernatant, and washed After the cells were resuspended in 1×PBS, the cells were centrifuged again, the supernatant was removed, and repeated twice; 200 μL/well was added to resuspend the cells in 1×PBS; the cells were detected on a cytoflex flow cytometer (Beckman).

0333The results were shown in Figure 2, antibody M1 did not bind to Lag3-CHO cells, VISTA-CHO cells, Tim3-CHO cells and Raji cells.

0334Example 4 Humanized Antibody

0335The amino acid sequences of humanized antibody VH and VL are shown in Table 9. The assembly scheme of the humanized antibody is shown in Table 10. VH and CH form the heavy chain of the antibody, and VL and CL form the light chain of the antibody; it is represented by "antibody" + antibody number. For example, the heavy chain of antibody V3 is composed of SED ID NO: VH as shown in 36 and CH as shown in SED ID NO:32, the light chain of antibody V3 is composed of VL as shown in SED ID NO:40 and CL as shown in SED ID NO:34. The heavy chain of the control antibody M30-H1-L4 is shown in SEQ ID NO:30, and the light chain is shown in SEQ ID NO:31. The heavy chain of the control antibody Trop2 is shown in SEQ ID NO:42, and the light chain is shown in SEQ ID NO:43 (see Table 11).

0336Amino acid sequences of table 9 humanized VH and VL

0337<img file="WO2023051663A1_D0021.tif" />

0338Note: CDR (Kabat number) is marked with bold + underline

0339Table 10 Humanized antibody number, assembly scheme

0340<tables num="0003"><table><tgroup cols="5"><colspec colname="c001" colwidth="22%" /><colspec colname="c002" colwidth="19%" /><colspec colname="c003" colwidth="21%" /><colspec colname="c004" colwidth="18%" /><colspec colname="c005" colwidth="20%" /><tbody><row><entry morerows="0">Antibody number</entry><entry morerows="0">VH SEQ ID</entry><entry morerows="0">CH SEQ ID</entry><entry morerows="0">VL SEQ ID</entry><entry morerows="0">CL SEQ ID</entry></row></tbody></tgroup></table></tables>

0341<tables num="0004"><table><tgroup cols="5"><colspec colname="c001" colwidth="22%" /><colspec colname="c002" colwidth="19%" /><colspec colname="c003" colwidth="21%" /><colspec colname="c004" colwidth="18%" /><colspec colname="c005" colwidth="20%" /><thead><row><entry morerows="0"></entry><entry morerows="0">NO:</entry><entry morerows="0">NO:</entry><entry morerows="0">NO:</entry><entry morerows="0">NO:</entry></row></thead><tbody><row><entry morerows="0">V1</entry><entry morerows="0">35</entry><entry morerows="0">32</entry><entry morerows="0">39</entry><entry morerows="0">34</entry></row><row><entry morerows="0">V2</entry><entry morerows="0">36</entry><entry morerows="0">32</entry><entry morerows="0">39</entry><entry morerows="0">34</entry></row><row><entry morerows="0">V3</entry><entry morerows="0">36</entry><entry morerows="0">32</entry><entry morerows="0">40</entry><entry morerows="0">34</entry></row><row><entry morerows="0">V4</entry><entry morerows="0">37</entry><entry morerows="0">32</entry><entry morerows="0">41</entry><entry morerows="0">34</entry></row><row><entry morerows="0">V5</entry><entry morerows="0">38</entry><entry morerows="0">32</entry><entry morerows="0">41</entry><entry morerows="0">34</entry></row></tbody></tgroup></table></tables>

0342Table 11 Amino acid sequence of control antibody Trop2

0343<img file="WO2023051663A1_D0022.tif" />

0344The gene sequences of the heavy chain and light chain of the antibody were cloned into expression vectors respectively to obtain recombinant expression vectors, then HEK293F cells were transiently transfected, cultured and purified by Protein A affinity chromatography to obtain humanized antibodies, and sequenced with the expected sequence unanimous.

0345Example 5 Affinity, internalization ability and specificity of humanized antibody

03461. Humanized antibody affinity verification-1 ELISA

0347The day before the experiment, dilute the target antigen hB7-H3-Fc with 1×PBS to 2 μg/mL, then add 100 μL/well to the ELISA plate (96-well plate), and coat at 4°C overnight; the next day, dry the coating Wash twice with washing solution PBST, pat dry, add blocking solution (PBST containing 1% BSA) at 200 μL/well, and block at 37°C for 2 hours; dry the blocking solution, wash twice with washing solution PBST, and pat dry. After drying, add 100 μL/well of serially diluted humanized antibody (initial concentration is 300 nM, 3-fold serial dilution, solvent is 1×PBS), and react at 37°C for 1 hour; shake dry humanized antibody, rinse with washing solution PBST After washing 5 times and patting dry, add HRP-labeled goat anti-human Kappa light chain secondary antibody (Invitrogen, Cat. No. A18853; diluted to 1:2000 with 1×PBS) at 100 μL/well, and react at 37°C for 1 hour; shake dry the secondary antibody , washed 8 times with washing solution PBST, after patting dry, add TMB chromogenic solution, 100 μL/well, and react at 37°C for 5-10 minutes; Read the absorbance value at the wavelength.

0348The result is shown in Figure 3.

03492. Humanized antibody affinity verification-2 flow cytometry

0350Take MDA-MB-468 cells in the logarithmic growth phase, centrifuge at 1500rpm for 5 minutes, remove the supernatant, resuspend with 1×PBS, centrifuge at 1500rpm for 5 minutes, remove the supernatant, and repeat once. Add MDA-MB-468 cells to V-type 96-well plate, 50 μL/well (200,000 cells), centrifuge at 1500 rpm for 5 minutes and discard PBS; add 100 μL/well of serially diluted humanized antibody (initial concentration is 100 nM, 3-fold serial dilution, the medium is 1×PBS), resuspend the cells; incubate on ice for 1 hour; after the incubation, centrifuge at 2000 rpm for 5 minutes, remove the supernatant, resuspend the cells with 200 μL/well of 1×PBS and centrifuge again , remove the supernatant, and repeat twice; add 100 μL/well of PE-labeled goat anti-human IgG Fc secondary antibody (Invitrogen, Cat. No. 12-4998-82; dilute to 1:500 with 1×PBS), resuspend the cells; protect from light, Incubate on ice for 30 minutes; after the incubation, centrifuge at 2000rpm for 5 minutes, remove the supernatant, resuspend the cells with 200 μL/well of 1×PBS, and then centrifuge again, remove the supernatant, repeat twice; add 200 μL/well of 1×PBS Cells were resuspended; assayed on a cytoflex flow cytometer (Beckman).

0351The result is shown in Figure 4.

03523. Detection of internalization of humanized antibody by flow cytometry

0353Prepare 30 μg/mL humanized antibody with ice-cold cell washing solution (PBS containing 2% FBS), then mix with cells (MDA-MB-468 cells or N87 cells, 500,000 cells) at a volume ratio of 1:1 Cells) were mixed (final volume 100 μL), mixed evenly and ice-bathed for 1 h; centrifuged at 1200 r/min at 4 °C for 5 min, discarded the supernatant; Add 200 μL ice-cold cell washing solution to resuspend the cells, incubate at 37°C for 2 hours to internalize the antibody bound to the cell surface, then transfer to ice bath, and add ice-cold cell washing solution Terminate internalization, centrifuge at 1200 r/min at 4°C for 5 min, discard the supernatant, add 100 μL of PE-labeled goat anti-human IgG Fc secondary antibody dilution (Invitrogen, Cat. No. 12-4998-82; ice-cold cell washing solution: PE-labeled goat Anti-human IgG Fc secondary antibody=500:1 Prepare PE-labeled goat anti-human IgG Fc secondary antibody dilution), resuspend the cells, incubate at 4°C in the dark for 30min, centrifuge at 1200r/min at 4°C for 5min, discard the supernatant; pre-cooled cell wash Cells were resuspended in liquid solution, centrifuged at 1200r/min at 4°C for 5min, the supernatant was discarded, and cells were resuspended with ice-cold 1×PBS, centrifuged at 1200r/min at 4°C for 5min, supernatant was discarded, and 200 μL of ice-cold 1×PBS was added The cells were resuspended, and the average fluorescence intensity was measured by flow cytometry.

0354Table 12 Humanized antibody internalization ability

0355<img file="WO2023051663A1_D0023.tif" />

03564. Humanized antibody specificity verification - flow cytometry

0357Take cells in the logarithmic growth phase (VISTA-CHO cells, Tim3-CHO cells or Lag3-CHO cells), centrifuge at 1500rpm for 5 minutes, remove the supernatant, resuspend with 1×PBS, centrifuge at 1500rpm for 5 minutes, remove the supernatant, repeat once. Add the cells into a V-shaped 96-well plate, 50 μL/well (500,000 cells), centrifuge at 1500 rpm for 5 minutes and discard the PBS; add 100 μL/well of humanized antibody (concentration: 100 nM, vehicle: 1×PBS), resuspend Cells; incubate on ice for 1 hour; after the incubation, centrifuge at 2000rpm for 5 minutes, remove the supernatant, resuspend the cells with 200 μL/well of 1×PBS, and then centrifuge again, remove the supernatant, repeat twice; add 100 μL/well of PE Labeled goat anti-human IgG Fc secondary antibody (Invitrogen, Cat. No. 12-4998-82; diluted to 1:500 with 1×PBS), resuspended cells; protected from light, incubated on ice for 30 minutes; after incubation, centrifuged at 2000rpm for 5 minutes , remove the supernatant, resuspend the cells with 200 μL/well of 1×PBS, centrifuge again, remove the supernatant, and repeat twice; add 200 μL/well of 1×PBS to resuspend the cells; to test.

0358The test results are shown in Figure 5, and none of the antibodies V1-V5 bound to VISTA-CHO cells, Tim3-CHO cells or Lag3-CHO cells.

03595. Species specificity of humanized antibodies

0360The day before the experiment, the target antigens cynomolgus monkey B7-H3 (novoprotein CA61), mouse B7-H3 (SinoBiological, 50973-M08H) and hB7-H3-his were diluted to 2 μg/mL with 1×PBS, and then 100 μL/well Add ELISA plates (96-well plates), two rows each, and coat overnight at 4°C. The next day, dry the coating solution, wash twice with washing solution PBST, pat dry, add blocking solution at 200 μL/well, and block at 37°C for 2 hours; dry the blocking solution, wash twice with washing solution PBST, and pat dry. After drying, add 100 μL/well of serially diluted antibody V3 (initial concentration is 2 μg/mL, 2-fold serial dilution), and react at 37°C for 2 hours; spin dry antibody V3, wash 5 times with washing solution PBST, and pat dry, Add anti-human kappa light Chains HRP (Sigma, A7164; diluted to 1:10000 with 1×PBS) at 100 μL/well, react at 37°C for 1 h; dry the secondary antibody, wash 8 times with washing solution PBST, and pat dry, Add TMB chromogenic solution, 100 μL/well, react at 37°C for 10 minutes; add 0.1MH₂SO₄Stop solution, 100μL/well, read the absorbance value at 450nm wavelength with a microplate reader within 15 minutes.

0361Table 13 The binding affinity of antibody V3 to B7-H3

0362<tables num="0005"><table><tgroup cols="2"><colspec colname="c001" colwidth="51%" /><colspec colname="c002" colwidth="49%" /><thead><row><entry morerows="0">antigen</entry><entry morerows="0">EC₅₀(μg/mL)</entry></row></thead><tbody><row><entry morerows="0">mouse B7-H3</entry><entry morerows="0">NA</entry></row><row><entry morerows="0">Cynomolgus monkey B7-H3</entry><entry morerows="0">0.042</entry></row><row><entry morerows="0">hB7-H3-his</entry><entry morerows="0">0.042</entry></row></tbody></tgroup></table></tables>

0363It can be seen from Table 13 that antibody V3 can bind to human and cynomolgus monkey B7-H3, but not to mouse B7-H3.