CN115057934A - anti-Trop-2 antibodies and uses thereof - Google Patents
anti-Trop-2 antibodies and uses thereof Download PDFInfo
- Publication number
- CN115057934A CN115057934A CN202111456440.4A CN202111456440A CN115057934A CN 115057934 A CN115057934 A CN 115057934A CN 202111456440 A CN202111456440 A CN 202111456440A CN 115057934 A CN115057934 A CN 115057934A
- Authority
- CN
- China
- Prior art keywords
- seq
- amino acid
- acid sequence
- cdr1
- cdr2
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of biology, and relates to an anti-Trop-2 antibody or an antigen binding fragment thereof, a pharmaceutical composition thereof and application thereof. The anti-Trop-2 antibody and the antigen binding fragment thereof can effectively bind human Trop-2 protein, have excellent specificity and antigen affinity, and have excellent internalization activity and very wide application prospect.
Description
Technical Field
The invention belongs to the field of biology, and relates to an anti-Trop-2 antibody.
Background
Tumor-associated Calcium Signal Transducer2 (Trop-2), a single transmembrane cell surface glycoprotein encoded and expressed by the tactd 2 gene, is known as Tumor-associated Calcium Signal Transducer2 (tactd 2), and belongs to the family of Tumor-associated Calcium Signal transducers along with the epithelial adhesion molecule Ep-CAM (Trop 1). Trop-2 consists of 323 amino acids and includes a hydrophobic leader, an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The cytoplasmic tail has a highly conserved phosphatidylinositol 4, 5-bisphosphate (PIP2) binding sequence that overlaps with the protein kinase C phosphorylation site, indicating that PIP2 plays an important role in signal transduction by Trop-2. The N-terminal of the Trop-2 protein is an extracellular domain (Trop-2 EC), and is connected with an intracellular short tail (Trop-2 IC) through a single-direction transmembrane helix (TM) so as to be fixed on a cell membrane. Phosphorylation of Trop-2 IC results in a significant conformational change in the functional domain, including the C-terminal order, and these structural features may be important in modulating Trop-2 activity.
Trop-2 is not only involved in the regulation of intracellular calcium ion concentration and cyclin D1(cyclin D1) and phosphokinase C (PKC), but also may activate the ERK pathway, thereby regulating the growth, invasion and metastasis of tumor cells. Trop-2 is expressed in 65-90% of tumors in a large amount, such as pancreatic cancer, colon cancer, gastric cancer, lung cancer, prostate cancer, cervical cancer, ovarian cancer, pancreatic cancer and the like, and is not expressed or rarely expressed in normal human tissues. Clinical data show that the high expression of Trop-2 in tumor cells can promote tumor proliferation, invasion, metastasis and diffusion and the like, and is closely related to poor prognosis of tumor patients, shortened survival time of patients and the like, so that the Trop-2 becomes an important target point for research and development of anti-tumor targeted drug therapy. In recent years, antibody drugs and ADC drugs targeting Trop-2 have become new hot spots for treating Trop-2 positive tumors.
Disclosure of Invention
The invention aims to provide a novel anti-Trop-2 antibody, which can be specifically combined with human Trop-2 and has great potential for treating tumors; meanwhile, the antibody has high internalization capacity and is suitable for developing ADC medicines.
In one aspect, the invention provides an anti-Trop-2 antibody or antigen-binding fragment thereof that binds to Trop-2 or a fragment thereof, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising three CDRs, VH CDR1, VH CDR2 and VH CDR3 respectively, and a light chain variable region (VL) comprising three CDRs, VL CDR1, VL CDR2 and VL CDR3 respectively; wherein,
VH CDR1 comprises SEQ ID NO: 2. 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106 or 114, and VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107 or 115, and VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4. 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108 or 116, and a VL CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6. 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110 or 118, and a VL CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7. 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111 or 119, and a VL CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8. 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, or 120, or a pharmaceutically acceptable salt thereof.
In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO. 2;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO. 3;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO. 4;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 6;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 7;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 8;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 10;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO. 11;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 12;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 14;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 15;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 16;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 18;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 19;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 20;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 22;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 23;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 24;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 26;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 27;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 28;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 30;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 31;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 32;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 34;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO. 35;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 36;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 38;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 39;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 40;
in a preferred embodiment, the antibody comprises VH CDRs 1, VH CDRs 2, and VH CDRs 3, and VL CDRs 1, VL CDRs 2, and VL CDRs 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 42;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 43;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO. 44;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 46;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 47;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 48;
in a preferred embodiment, the antibody comprises VH CDRs 1, VH CDRs 2, and VH CDRs 3, and VL CDRs 1, VL CDRs 2, and VL CDRs 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 50;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 51;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 52;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 54;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 55;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 56;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 58;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 59;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 60;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 62;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 63;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 64;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 66;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 67;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 68;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 70;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 71;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 72;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 74;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 75;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 76;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 78;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 79;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 80;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 82;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 83;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 84;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 86;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 87;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 88;
in a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 90;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 91;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 92;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 94;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 95;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 96;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO. 98;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 99;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 100;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 102;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 103;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 104;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 106;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 107;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 108;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 110;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 111;
the amino acid sequence of VL CDR3 is shown as SEQ ID NO. 112;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 114;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 115;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 116;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 118;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 119;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO 120.
In some embodiments, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105 or 113, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the above-described sequences.
In some embodiments, the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 5. 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109 or 117, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the aforementioned sequences.
In certain preferred embodiments, the 3 CDRs contained in the heavy chain variable region, and/or the 3 CDRs contained in the light chain variable region, are defined by the IgBlast system.
In some embodiments, an antibody or antigen-binding fragment thereof of the invention may further comprise one or more of a heavy chain constant region, a light chain constant region, an Fc region. In a further preferred embodiment, the light chain constant region is a lambda chain or a kappa chain constant region. In some preferred embodiments, the antibody or antigen binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 type.
In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody or antigen-binding fragment thereof.
In some embodiments, the heavy chain of the antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO 121 or SEQ ID NO 123 and the light chain of the antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO 122 or SEQ ID NO 124.
In one aspect, the invention provides a biomaterial that is:
(1) a nucleic acid encoding the antibody or antigen-binding fragment thereof of the invention;
(2) a vector, host cell or microorganism comprising (1);
(3) the expression product, suspension or supernatant of the above (2).
The skilled person can easily select and prepare a vector, a host cell or a microorganism comprising the coding sequence of the antibody according to the amino acid sequence of the antibody, and can know how to culture such host cell or microorganism to obtain the corresponding expression product, suspension, supernatant, etc. to obtain the corresponding antibody. This is all a routine technical measure in the art.
In another aspect, the invention provides a conjugate of an antibody or antigen-binding fragment thereof according to the invention, wherein the antibody or antigen-binding fragment thereof is conjugated to a drug, wherein the drug is a cytotoxic agent, such as an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent, and a RNA polymerase II inhibitor or any active pharmaceutical ingredient that interferes with a specific cellular pathway.
In another aspect, the invention provides a composition comprising an antibody or antigen-binding fragment thereof or an antibody or antigen-binding fragment conjugate of the invention; preferably, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
In another aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the invention, comprising: culturing the above host cell or microorganism to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the host cell.
In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the invention or a conjugate of the invention or a composition of the invention or a biomaterial of the invention in the manufacture of a medicament for the treatment of a tumor, for the treatment of a Trop-2-high expressing cancer; preferably, the Trop-2 high-expression cancer is gastric cancer, pancreatic cancer, prostate cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, breast cancer or cervical cancer.
In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the invention or a conjugate of the invention or a composition of the invention or a biomaterial of the invention in the manufacture of a product for binding Trop-2.
In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the invention or a composition of the invention or a conjugate of the invention or a biomaterial of the invention in combination with one or more other cancer therapeutic agents in the manufacture of a medicament for the treatment of a tumour. Other cancer therapeutic agents include, but are not limited to: paclitaxel, mitomycin, vincristine, colchicine, doxorubicin, daunorubicin, actinomycin D, emetine, puromycin, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan, docetaxel, pemetrexed, sorafenib, oxaliplatin, 5-FU, lapatinib, and analogs or homologs thereof. The cancer therapeutic agent also includes radiotherapeutic agent and biomacromolecule drug.
The antibody has good internalization capacity, and the chimeric antibody and the humanized antibody can promote Trop-2 to internalize in human cells. Thus, the antibodies of the invention have potential for the development of ADC drugs.
Drawings
FIG. 1 shows the binding of chimeric antibodies to Trop-2 protein.
FIG. 2 shows the binding of the chimeric antibody to Trop-2 on the cell membrane.
FIG. 3 shows the amount of internalization of Trop-2 on the MDA-MB-468 cell membrane by the chimeric antibody.
FIG. 4 shows the results of antigen binding epitope analysis of Trop-2 antibody.
FIG. 5 shows binding of humanized antibodies to Trop-2 protein, (A) ELISA detects binding of humanized antibodies HuA3-1, HuA3-3 and HuA3-4 to Trop-2 protein, (B) ELISA detects binding of humanized antibody HuA3-2 to Trop-2 protein.
FIG. 6 shows binding of humanized antibodies to Trop-2 protein, (A) ELISA detects binding of humanized antibodies HuA4-1, HuA4-3 and HuA4-6 to Trop-2 protein, (B) ELISA detects binding of humanized antibodies HuA4-2, HuA4-4 and HuA4-5 to Trop-2 protein.
FIG. 7 shows binding of humanized monoclonal antibody to Trop-2 on cell membrane, (A) FACS detection of binding of humanized antibodies HuA3-1 to HuA3-4 to Trop-2 on cell membrane, and (B) FACS detection of binding of humanized antibodies HuA4-1 to HuA4-6 to Trop-2 on cell membrane.
FIG. 8 shows the amount of the humanized antibody that endocytoses Trop-2 on MDA-MB-468 cell membranes, (A) FACS measures the amount of the humanized antibodies HuA3-1 to HuA3-4 that endocytoses Trop-2 on MDA-MB-468 cell membranes, and (B) FACS measures the amount of the humanized antibodies HuA4-1 to HuA4-6 that endocytoses Trop-2 on MDA-MB-468 cell membranes.
FIG. 9 shows the humanized antibody thermostability assay, (A) ELISA detects the binding of humanized antibodies HuA 3-1-HuA 3-4 to Trop-2 protein at different denaturation temperatures, and (B) ELISA detects the binding of humanized antibodies HuA 4-1-HuA 4-6 to Trop-2 protein at different denaturation temperatures.
Detailed Description
The present invention will now be described with reference to specific examples. Unless otherwise specified, the reagents and apparatus used in the following methods are those commonly used in the art and are commercially available; the methods used are conventional in the art and can be carried out unambiguously by the person skilled in the art from the description of the examples and corresponding results are obtained.
Defining:
in the present invention, the term "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally occurring or partially or wholly synthetically (e.g., recombinantly) produced, including any fragment thereof that comprises at least a portion of the variable region of an immunoglobulin molecule that retains the binding specificity capability of a full-length immunoglobulin. The term encompasses intact monoclonal antibodies (including murine, human, humanized and chimeric monoclonal antibodies), multispecific antibodies (such as bispecific, trispecific, tetraspecific, etc.), single chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen binding site with the desired specificity. Antibodies include antibodies of any class or subclass (e.g., IgG1, IgG2, IgG3, and IgG 4; IgM, IgD, IgE, and IgA).
As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to a portion of an immunoglobulin molecule that binds an antigen. Antigen binding fragments may be synthetic, enzymatically obtainable, or genetically engineered polypeptides, including but not limited to Fab, Fab ', F (ab') 2 Single chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, and other fragments. The fragments may comprise multiple chains linked together, for example by disulphide bonds and/or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), results in an antibody that immunospecifically binds an antigen.
As used herein, "conventional antibody" refers to an antibody comprising two heavy chains (which may be designated as H and H ') and two light chains (which may be designated as L and L') and two antigen binding sites. A variable domain or variable region is a domain of an antibody heavy or light chain that recognizes and specifically binds an antigen, and comprises an amino acid sequence that varies between different antibodies. The heavy chain is composed of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. The light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can also be divided into hypervariable regions, called Complementarity Determining Regions (CDRs), which are separated by more conserved Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains comprise binding domains that interact with antigens.
As used herein, "hypervariable region," "complementarity determining region," and "CDR" are used interchangeably to refer to the amino acid sequences of the variable regions of the heavy and light chains of an immunoglobulin, providing the primary contact residues for binding of the antibody to an antigen or epitope.
As used herein, Framework Region (FR) refers to variable domain residues other than hypervariable region residues. The FR of a variable domain typically consists of 4 FR regions: FR1, FR2, FR3 and FR 4. The FR regions are relatively more conserved in amino acid sequence than the hypervariable regions.
As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that comprises an amino acid sequence that is relatively more conserved than the amino acid sequence of the variable region domain.
The term "murine antibody" is used herein to refer to a murine monoclonal antibody to human Trop-2 prepared according to the knowledge and skill in the art. The preparation is carried out by injecting a test subject mouse with Trop-2 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.
The term "chimeric antibody" is an antibody in which the variable region of a murine antibody is fused to the constant region of a human antibody. Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding murine Fc, and inserted into a human vector after substitution with an equivalent portion of the gene encoding human Fc constant region, and finally the chimeric antibody molecule is expressed.
The term "humanized antibody" refers to an antibody produced by grafting mouse CDR sequences into the framework of a human antibody variable region. The constant regions of the antibody molecule are derived from the constant regions of human antibodies, murine antibodies or antibodies from other species may be humanized using techniques well known in the art.
The term "epitope" refers to any protein determinant capable of specifically binding to an immunoglobulin or otherwise interacting with a molecule. Epitopic determinants are typically composed of chemically active surface groupings of molecules, such as amino acids or carbohydrates or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be "linear" or "conformational". If two antibodies exhibit competitive binding to the antigen, it is possible to bind to the same epitope on the antigen.
As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. These terms encompass transcription of genes into RNA. These terms also encompass translation of the RNA into one or more polypeptides, and also encompass all naturally occurring post-transcriptional and post-translational modifications. Expression or production of the antibody or antigen-binding fragment thereof may be within the cytoplasm of the cell, or in an extracellular environment such as the growth medium of a cell culture.
As used herein, "host cell" includes individual cells or cell cultures that may or have received a vector for introduction of a polynucleotide insert. Host cells include progeny of a single host cell, and such progeny may not necessarily be identical (in morphology or genomic DNA complementarity) to the original parent cell due to natural, accidental, or deliberate mutation. The host cell may be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, HeLa cells, HEK cells such as HEK 293 cells.
As used herein, an "expression vector" is a replicon, a vector capable of expressing DNA, wherein a segment may be operably inserted into another nucleic acid segment to cause replication or expression of the segment. An expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, cosmid, virus, or other vector, which when introduced into an appropriate host cell results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors which are replicable in eukaryotic and/or prokaryotic cells, as well as expression vectors which remain episomal or which integrate into the genome of the host cell.
As used herein, "treatment" refers to any sign of success or success in reducing or ameliorating an injury, pathology, or condition, including any objective or subjective parameter, such as a reduction in symptoms, alleviation, weakening, or making a patient more tolerant to the condition, slowing the rate of degeneration or decline, decreasing the extent of degenerative endpoint failure, improving the physical or mental well-being of the subject, or prolonging survival. Treatment can be assessed by objective or subjective parameters; including results of physical examination, neurological examination, or mental assessment.
As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to a dose or concentration of a certain drug effective to treat the relevant disease or condition, i.e., an amount necessary to prevent, cure, ameliorate, block, or partially block the symptoms of the disease or disorder. Also, as used herein, a "prophylactically effective amount" or a "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that will have the intended prophylactic effect when administered to a subject, e.g., to prevent or delay the onset or recurrence of a disease or symptom, to reduce the likelihood of onset or recurrence of a disease or symptom. A complete prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.
As used herein, the term "individual" refers to a mammal, such as a human.
Antibodies of the invention
The present invention provides an antibody against Trop-2, an anti-Trop-2 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically recognizes and binds to Trop-2.
In some embodiments, the antibodies or antigen-binding fragments thereof of the invention specifically bind to Trop-2, are superior in internalization activity into hTrop-2 expressing target tumor cells, and have potential for the development of ADC drugs.
In some embodiments, the tumors targeted include, but are not limited to, those described below with respect to neoplastic diseases. In other embodiments, the antibody or antigen-binding fragment thereof of the invention is capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%.
The anti-Trop-2 antibodies or antigen-binding fragments thereof of the invention comprise substitutions, insertions or deletions. The anti-Trop-2 antibody comprises a light chain variable region, a heavy chain variable region, a light chain or a heavy chain which are modified, and the amino acid sequence of the modified anti-Trop-2 antibody is different from the amino acid sequence from which the antibody is derived. For example, an amino acid sequence derived from the same given protein may be similar to the starting sequence, e.g., have a certain percent identity, e.g., it may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% percent identical to the starting sequence.
In certain embodiments, amino acid modifications, which may be one or more, may be introduced into the Fc region of the antibodies provided herein, thereby generating Fc variants. An Fc variant may comprise a human Fc region sequence comprising amino acid modifications at one or more amino acid positions.
Suitable "antibodies and antigen binding fragments thereof" for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunogenic antibodies, or Fab fragments, Fab 'fragments, F (ab') 2 Fragments, single chain antibodies, nanobodies, and epitope-binding fragments of any of the foregoing.
In some embodiments, the antibodies of the invention may be monospecific, bispecific or multispecific. The anti-Trop-2 antibody may be linked to another antibody or antibody fragment to produce a bispecific or multispecific antibody with a second or more binding specificities.
In certain embodiments, the antibodies may be further modified to add functional components, suitable portions for antibody derivatization include, but are not limited to, the following examples: PEG, dextran, proteins, lipids, therapeutic agents or toxins. Antibodies can be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or linkage to other proteins, and the like.
Neoplastic diseases
The antibodies or antigen binding fragments thereof of the invention can be used to treat neoplastic diseases. The antibodies or antigen binding fragments thereof of the present invention can be used for the prevention and/or treatment of various tumors expressing hTROP-2. Non-limiting examples of cancers that can be treated include, but are not limited to, 1 or more of various tumors such as gastric cancer, pancreatic cancer, prostate cancer, intestinal cancer, breast cancer (e.g., triple negative breast cancer), ovarian cancer, lung cancer (e.g., squamous lung cancer, non-small cell lung cancer, etc.), cholangiocarcinoma, urothelial cancer, cervical cancer, and the like.
Nucleic acid, vector and antibody production method
The invention also provides a nucleic acid encoding an antibody or antigen-binding fragment thereof of the invention. The nucleotide sequence of the nucleic acid can be easily known to those skilled in the art from the amino acid sequence of the antibody or antigen-binding fragment thereof, and appropriate codons can be selected as needed to express the antibody or antigen-binding fragment thereof of the present invention.
The present invention also provides an expression vector which can be easily selected and prepared based on the nucleic acid sequence of the aforementioned antibody of the present invention.
An aspect of the present invention also provides a host cell or microorganism transformed with at least one of the aforementioned expression vectors.
Further, the present invention also provides an expression product, suspension or supernatant of said host cell or microorganism. Such expression products, suspensions or supernatants comprise the antibodies or antigen-binding fragments thereof of the invention, as is known in the art.
Yet another aspect of the present invention also relates to a method of producing an anti-Trop-2 antibody or antigen-binding fragment thereof, which comprises: (i) culturing the host cell or microorganism of the invention under conditions suitable for expression by an expression vector, and (ii) isolating and purifying the antibody or antigen-binding fragment thereof expressed by the expression vector.
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment or antibody drug conjugate of the invention and a pharmaceutically acceptable carrier.
As used herein, "antibody drug conjugates" (abbreviated "conjugates") relate to the coupling of an antibody or antigen-binding fragment, antibody-based binding protein, or antibody fragment thereof as described herein to a therapeutically active substance or active pharmaceutical ingredient. The therapeutically active substance or active pharmaceutical ingredient includes, but is not limited to, anti-tubulin agents, DNA alkylating agents, DNA cross-linking agents, DNA intercalating agents and RNA polymerase II inhibitors or any active pharmaceutical ingredient that interferes with a specific cellular pathway that is essential for the survival of a cell and/or for a specific cellular pathway. The therapeutically active substance or active pharmaceutical ingredient may be non-site specifically conjugated to the antibody or antibody fragment through a lysine or cysteine side chain; conjugation can also be site-specifically by chemical, chemo-enzymatic or enzymatic conjugation as known in the art.
As used herein, a "pharmaceutically acceptable carrier" can be any suitable and pharmaceutically acceptable carrier. May be any compatible solid or liquid filler or fillers, diluents, other excipients or encapsulating substances that are administered to the user. Examples of suitable carriers, diluents, and/or excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and any combination thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols or sodium chloride in the composition. The pharmaceutical compositions of the present invention may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents.
The compositions of the present invention may take a variety of forms, including liquid, semi-solid, and solid dosage forms, but the preferred form depends on the intended mode of administration and therapeutic application. Typically preferred compositions are in the form of injectable or infusible solutions.
The route of administration of the pharmaceutical composition of the present invention may be any of enteral administration, topical administration, and parenteral application.
Combination therapy
The invention also provides combination therapies comprising the anti-Trop-2 antibodies or pharmaceutical compositions of the invention in combination with at least one additional cancer therapeutic.
Such additional cancer therapeutic agents include, but are not limited to: paclitaxel, mitomycin, vincristine, colchicine, doxorubicin, daunorubicin, actinomycin D, emetine, puromycin, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan, docetaxel, pemetrexed, sorafenib, oxaliplatin, 5-FU, lapatinib, and analogs or homologs thereof. The cancer therapeutic agent also includes radiotherapeutic agent and biomacromolecule drug.
The antibody of the present invention and the cancer therapeutic agent may be administered all at once or separately. When administered separately (in the case of mutually different administration regimens), they may be administered continuously without interruption or at predetermined intervals.
anti-Trop-2 antibody sequences exemplified herein
Table 1: amino acid sequence numbering of VH, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3 of an antibody of the invention
Table 2: sequence information of the invention
TABLE 3 cA3 antibody heavy and light chain amino acid sequences
TABLE 4 cA4 antibody heavy and light chain amino acid sequences
Example 1: preparation of anti-Trop-2 antibody mouse monoclonal antibody
This example describes the preparation of mouse anti-human Trop-2 monoclonal antibodies using hybridoma technology. Firstly, expressing an extracellular region Trop-2 protein (Uniprot: P09758) as an immunogen, adding a 6xFc tag to the C-terminal of an amino acid sequence (His27-Thr274) of the extracellular region Trop-2 protein, and then cloning the amino acid sequence to a V152 vector (provided by Huaanmab), thereby obtaining V152-Trop-2 ECD-Fc. The cells were transiently transfected into 293 cells (source: ATCC), and after 5 days, cell culture broth was collected and the supernatant was purified by a nickel column (manufacturer: Solarbio, cat # I8090) to obtain Trop-2 protein used in this experiment. To prepare anti-human Trop-2 mouse monoclonal antibodies, 4-week-old BAL B/c mice (supplied with Huaanmab) were first immunized with 100 μ g of Trop-2 protein. On day 14 and 28 after the first immunization, the composition is administeredThe immunized mice were re-immunized with 50. mu.g of Trop-2 protein. The serum titer of the immunized mice was measured by ELISA, and the Trop-2 protein was diluted to a concentration of 1. mu.g/ml with PBS (manufacturer: Corning, cat # 21-040-CV) and coated on a microplate overnight at 4 ℃. Then blocking for 1h at 37 ℃ by using 1% BSA-PBST blocking solution; after PBST washing of the plates, serum dilutions from immunized mice were added to the plates and incubated for 1 hour at 37 ℃. The plate was washed with HRP-labeled goat anti-mouse IgG (manufacturer: Abcam, cat: Ab205719, 1:10000 dilution) and reacted at 37 ℃ for 0.5 hour. After washing the plate, adding TMB solution (manufacturer: Huzhou Yingchuang, cat # TMB-S-001), reacting for 5 minutes at room temperature in the dark, and then adding 2N H 2 SO 4 The reaction was terminated. Absorbance was measured at a wavelength of 450nm on a microplate reader (Molecular Devices, M5). Mice with sufficient titers of anti-Trop-2 antibodies were boosted with 50 μ g of Trop-2 protein at day 42 post-immunization. The mice were sacrificed 3 to 5 days later, splenocytes were collected, cells were washed by centrifugation 2 to 3 times with a basal medium of IMDM (manufacturer: Shanghai culture, cat # L610KJ), then mixed with mouse myeloma cells SP2/0 (supplied by Huaanmab) at a ratio of 1:1, PEG (manufacturer: Roche, cat # 25771700) was added to the mixed cells and gently stirred, and left to stand at 37 ℃ for 30 s. The fused cells were diluted in IMEM selective medium containing 15% fetal bovine serum (manufacturer: Hangzhou organism in Zhejiang, cat # 11011- 2 And an incubator at 37 ℃. The anti-Trop-2 antibody in the hybridoma supernatant was detected 10-14 days later by ELISA assay. 5 different hybridoma clones were identified, including 17F1-G2, A2-H4, 24H2-D10, 31C-1, D7-1.
Flow cytometry (FACS) is adopted to detect the combination of hybridoma supernatant and endogenously expressed Trop-2 on the membrane of human breast cancer cell strain MDA-MB-468 (source: Shanghai cell bank). MDA-MB-468 cells were reacted with hybridoma supernatants diluted at different concentrations for 30 minutes at 4 ℃. After washing the cells 2 times, FITC-labeled goat anti-mouse IgG (manufacturer: Abcam, cat # ab6785, 1:1000 dilution) was added and reacted at 4 ℃ for 30 minutes in the dark. After washing the cells 2 times, they were examined by flow cytometry BD C6, confirming that all 5 hybridoma supernatants could bind to Trop-2 on the MDA-MB-468 cell membrane (data not shown).
Example 2: murine antibody-mediated Trop-2 endocytosis
Hybridoma supernatant-mediated endocytosis of Trop-2 protein in MDA-MB-468 cells was detected using flow cytometry (FACS). MDA-MB-468 cells were reacted with hybridoma supernatant at 4 ℃ for 30 minutes, and after washing the cells 2 times, the cells were divided into two portions and incubated at 4 ℃ and 37 ℃ for 4 hours, respectively. After washing the cells 2 times, FITC-labeled goat anti-mouse IgG (manufacturer: Abcam, cat # ab6785, 1:1000 dilution) was added and the reaction was carried out at 4 ℃ for 30 minutes in the absence of light. After washing the cells 2 times, endocytosis was calculated from the decrease in cell surface fluorescence at 37 ℃ versus 4 ℃ by flow cytometry analysis. The endocytosis rate is calculated according to the following formula: (4 ℃ fluorescence intensity MFI-37 ℃ fluorescence intensity MFI)/4 ℃ fluorescence intensity MFI 100%). The endocytosis rate of the hybridoma supernatants was determined by the above method, and it was confirmed that all 5 hybridoma supernatants could mediate the endocytosis of Trop-2 (data not shown).
Example 3: sequencing of light and heavy chain variable regions and expression of human and murine chimeric antibodies
Hybridoma cells expressing 17F1-G2, A2-H4, 24H2-D10, 31C-1 and D7-1 were RNA-extracted and reverse-transcribed into cDNA according to a conventional method, and the sequences of antibody genes were determined to obtain the sequences of heavy chain variable regions and light chain variable regions of 17F1-G2, A2-H4, 24H2-D10, 31C-1 and D7-1. Further, the amino acid sequence was analyzed, and the CDR sequence was determined by the Igblast database.
For expression of chimeric antibody, the variable regions of the heavy and light chains of 17F1-G2, A2-H4, 24H2-D10, 31C-1 and D7-1 were ligated with the constant regions of human antibody heavy chain IgG1 and human antibody light chain kappa respectively, cloned into vector V152 (supplied by Huaanmab), and then the above vector was transfected into 293 cells (supplied by ATCC) using PEI (supplied by Polysciences), after culturing for about 5 days, the cell supernatant was centrifuged at 3000rpm for 10min, and the supernatant was purified with protein A (manufactured by Solarbio, cat # I8090) so that the antibody purity was > 95%, 17F1-G2, A2-H4, 24H2-D10, 31C-1 and D7-15 hybridoma supernatants corresponded to the expressed chimeric antibodies 686 8, cA 48-G2, cA 3527 and 4, cA 4. The expression levels of 5 chimeric antibodies were 24.63, 41.79, 135.77, 238.91 and 452.31. mu.g/ml, respectively.
Example 4: binding of chimeric antibodies to Trop-2
And (3) quantitatively detecting the binding capacity of the chimeric antibody and the human Trop-2 protein by adopting an ELISA method. Trop-2-His (manufacturer: Acrobiosystems, cat # TR2-H5223) was diluted at 1. mu.g/ml into PBS and the plates were coated overnight at 4 ℃. Then blocking for 1h at 37 ℃ by using 1% BSA-PBST blocking solution; after PBST washing of the plates, cA1, cA2, cA3, cA4, cA5 were diluted to different concentrations (starting concentration 68.5nM, 3-fold gradient dilution, total 12 concentrations) added to the plates and incubated for 1 hour at 37 ℃. The plate was washed with HRP-labeled goat anti-human IgG (manufacturer: Sigma, cat # A0170,1:10000 dilution) and reacted at 37 ℃ for 1 hour. After washing the plate, adding TMB solution, reacting for 10 minutes at room temperature in the dark, and then adding 2N H 2 SO 4 The reaction was terminated. Placing on a microplate reader, and detecting absorbance at a wavelength of 450 nm. The results are shown in FIG. 1, and the EC50 values of the chimeric antibodies cA1, cA2, cA3, cA4 and cA5 for binding with Trop-2 are 0.136nM, 0.075nM, 0.165nM, 0.141nM and 0.114nM respectively, which shows that the chimeric molecules have higher binding activity with Trop-2.
The binding of the chimeric antibody to Trop-2 on the membrane of a human breast cancer cell line endogenously expressing Trop-2 (MDA-MB-468) was tested by FACS as described in example 1. Where the initial concentration of antibody was 68.5nM, 3-fold dilutions were made for 7 concentration points, with hRS7 as a positive control (hRS 7 was generated according to the sequence disclosed in US 20070212350). The results are shown in FIG. 2, the EC50 values of chimeric antibodies cA1, cA2, cA3, cA4 and cA5 for binding with Trop-2 on MDA-MB-468 cell membrane are 2.234nM, 1.274nM, 1.211nM, 2.470nM and 2.274nM respectively, and the control antibody hRS7 for binding with Trop-2 on membrane EC50 is 3.848nM, which shows that the chimeric molecules can bind with Trop-2 on cell membrane and are stronger than the control antibody.
Example 5: chimeric antibody-mediated Trop-2 endocytosis
The chimeric antibody was tested for endocytosis of Trop-2 on the cell membrane of MDA-MB-468 by FACS as described in example 2. MDA-MB-468 cells were reacted with chimeric antibodies (starting concentration 2.5nM, 3-fold gradient dilution, total 4 concentration points, 2.5, 0.8, 0.3, 0.1nM, respectively) at 4 ℃ for 30 minutes, washed 2 times, divided into two portions, and incubated at 4 ℃ and 37 ℃ for 4 hours, respectively. After washing the cells for 2 times, FITC-labeled goat anti-human IgG was added and the reaction was carried out at 4 ℃ for 30 minutes in the absence of light. Cells were washed and analyzed by flow cytometry. Endocytosis rate-4 ℃ fluorescence intensity MFI-37 ℃ fluorescence intensity MFI. As a result, the chimeric antibodies cA1 to cA5 were endocytosed to various degrees at various concentrations as shown in FIG. 3.
Example 6: trop-2 antibody antigen binding epitope analysis
To determine the epitope of the chimeric antibody, human Trop-2-His protein (manufacturer: Acrobiosystems, cat # TR2-H5223) was diluted at a concentration of 1. mu.g/ml in PBS and the plates were coated overnight at 4 ℃. After washing the plate, adding 1% BSA-PBST blocking solution, and blocking for 1h at 37 ℃; after PBST washing, different concentrations of antibody (starting 137nM, 3-fold dilution, 12 concentrations total) and 0.9nM biotinylated Trop-2 antibody hRS7(ThermoFisher antibody labeling kit, cat. A39257) were added to the plates and incubated at 37 ℃ for 1 hour. After washing the plate, streptavidin labeled with HRP (manufactured by Abcam, cat. ab7403) was added thereto, and the reaction was carried out at 37 ℃ for 30 minutes. After washing the plate, adding TMB solution, reacting for 30 minutes at room temperature in the dark, adding 2NH 2 SO 4 The reaction is stopped, and the absorbance at the wavelength of 450nm is detected by an enzyme-labeling instrument. As shown in fig. 4, chimeric antibodies cA3, cA5, similar to hRS7, competitively bound to Trop-2 protein bound to biotin-labeled hRS7 antibody, indicating that cA3, cA5 share epitopes with hRS7, while cA1, cA2, cA4 were unable to competitively bind to Trop-2 protein bound to biotin-labeled hRS7 antibody, indicating that cA1, cA2, cA4 and hRS7 recognize different epitopes.
Example 7: humanization of chimeric antibodies
According to the results of example 6, cA3, cA5 recognized the same epitope, and cA1, cA2, cA4 recognized the same epitope; according to the results of the example 4 and the example 5, cA3 with higher binding and endocytosis activity is selected from cA3 and cA5 groups which recognize the same epitope for humanized modification; of groups cA1, cA2 and cA4, cA4 (238.91. mu.g/ml) with the highest expression level was selected and humanized. The heavy chain variable regions and light chain variable regions of both antibodies cA3 and cA4 were compared to available human IgG gene sequence databases to identify the best matching human germline IgG gene sequences, and then the CDR regions of the chimeric antibody heavy and light chains were grafted onto the framework sequences of the matching heavy chain variable region gene and light chain variable region gene, respectively. Specifically, the heavy chain and the light chain of the human germline IgG selected by the cA3 are (1) IGHV5-51 × 01 and IGKV1-5 × 01 respectively; (2) IGHV5-51 × 01, IGKV1-12 × 01; (3) IGHV1-46 x 01, IGKV1-5 x 01; (4) IGHV1-46 x 01, IGKV3-20 x 01; the corresponding new humanized molecules are HuA3-1, HuA3-2, HuA3-3 and HuA 3-4. The heavy chain and the light chain of the human germline IgG selected by the cA4 are (1) IGHV1-3 x 01 and IGKV3-20 x 01 respectively; (2) IGHV1-3 x 01, IGKV1-27 x 01; (3) IGHV5-51 × 01, IGKV3-20 × 01; (4) IGHV5-51 × 01, IGKV4-1 × 01; (5) IGHV5-51 × 01, IGKV1-27 × 01; (6) IGHV1-18 x 01, IGKV1-33 x 01; the corresponding new humanized molecules are HuA4-1, HuA4-2, HuA4-3, HuA4-4, HuA4-5 and HuA 4-6. The sequences of the heavy chain variable region, the light chain variable region and the CDR region of each of the humanized antibodies obtained after the construction are shown in table 2. The heavy and light chain sequences of cA3, cA4 antibodies are shown in table 3 and table 4. The heavy and light chain constant regions of the humanized antibodies were identical to those of cA3, cA4 antibody. The complete amino acid sequences of the heavy and light chains of each humanized molecule are well known to those skilled in the art from the information disclosed above.
The antibody of the invention was expressed and purified in 293 cells (source: ATCC) as in example 3 with an antibody purity of > 95%. The expression levels of the HuA3-1 to the HuA3-4 are respectively 38.63, 57.78, 86.58 and 112.57 mu g/ml; the expression levels of HuA4-1 to HuA4-6 are 349.45, 316.93, 325.37, 338.42, 333.89 and 279.83 mu g/ml respectively.
Example 8: binding of humanized antibodies to Trop-2
The binding capacity of the human antibody to the human Trop-2 protein was quantitatively determined by ELISA as described in example 4. The initial concentration of the antibody was 68.5nM, and the results are shown in FIGS. 5 and 6, with 3-fold gradient dilutions for 12 concentration points. FIG. 5A shows the results for antibodies HuA3-1, HuA3-3, and HuA3-4 binding to Trop-2 with EC50 values of 0.175nM, 0.229nM, and 0.223nM, respectively, and stronger binding activity than the control antibody hRS7 (0.440 nM for EC 50); the results in fig. 5B show that antibody HuA3-2 has comparable binding activity to hRS7, with EC50 values of 0.132nM and 0.215nM, respectively; the results in FIG. 6A show that antibodies HuA4-1, HuA4-3, HuA4-6 and hRS7 bind to Trop-2 with EC50 values of 0.197nM, 0.252nM, 0.183nM and 0.312nM, respectively, and FIG. 6B shows that antibodies HuA4-2, HuA4-4, HuA4-5 and hRS7 bind to Trop-2 with EC50 values of 0.314nM, 0.268nM, 0.231nM and 0.355nM, respectively, indicating that humanized molecules HuA4-1 to HuA4-6 have higher binding activity to Trop-2 similarly to hRS 7.
The binding of the humanized antibody to Trop-2 on the membrane of a human breast cancer cell line endogenously expressing Trop-2 (MDA-MB-468) was tested by FACS as described in example 1. The initial concentration of the antibody is 7.6nM, the initial concentration is 3 times diluted, and the results are shown in FIGS. 7A and 7B, and the EC50 values of the antibodies HuA 3-1-HuA 3-4 combined with Trop-2 on MDA-MB-468 cell membranes are 1.05nM, 0.941nM, 2.086nM and 1.646nM respectively; the EC50 values of the antibodies HuA 4-1-HuA 4-6 for binding with Trop-2 on the cell membrane of MDA-MB-468 were 2.413nM, 1.285nM, 1.679nM, 1.259nM, 1.144nM and 1.530nM, respectively, indicating that these humanized molecules all have better binding activity with Trop-2 on the cell membrane than the control antibody hRS7(EC50 is 5.239 nM). And the humanized molecule had no decrease in affinity for Trop-2 compared to the chimeric molecule, as seen by comparison with cA3 and cA 4.
Example 9: humanized antibody-mediated Trop-2 endocytosis
Human antibodies were tested for endocytosis of Trop-2 on the cell membrane of MDA-MB-468 by FACS as described in example 2. MDA-MB-468 cells were reacted with different concentrations of antibody (starting concentration 2.5nM, 3-fold gradient dilution, total 4 concentration points) at 4 ℃ for 30 min, and after washing the cells 2 times, the cells were divided into two portions and incubated at 4 ℃ and 37 ℃ for 4h, respectively. After washing the cells 2 times, FITC-labeled goat anti-human IgG (origin Abcam, cat # ab97224) was added and the reaction was carried out for 30 minutes at 4 ℃ in the absence of light. Cells were washed and analyzed by flow cytometry. The results are shown in fig. 8A and 8B, with each humanized antibody mediating Trop-2 endocytosis better than the control antibody hRS 7.
Example 10: analysis of thermal stability of antibodies
The thermal stability of the humanized antibody was tested by ELISA. Trop-2-His protein was diluted at 1. mu.g/ml in PBS and the plates were coated overnight at 4 ℃. Then blocking for 1h at 37 ℃ by using 1% BSA-PBS blocking solution; the antibody was diluted to 205.5nM with 1% BSA-PBS and 50. mu.l per well was placed in PCR plates. Different temperatures were set on the PCR instrument: the antibody was placed at 58 deg.C, 61.5 deg.C, 64 deg.C, 67.4 deg.C, 71.8 deg.C, 76 deg.C, 78.6 deg.C, 80 deg.C, respectively, reacted for 30 minutes, and centrifuged at 1500rpm for 5min to remove the inactivated aggregated precipitated protein. The treated antibody was diluted to 2.5nM and analyzed by ELISA as in example 4, as shown in FIGS. 9A and 9B, the humanized antibody was more than 60 ℃ thermostable, with better thermostability being the highest thermostability of HuA4-6, reaching 73 ℃.
Claims (11)
1. An anti-Trop-2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three CDRs of VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region comprising three CDRs of VL CDR1, VL CDR2 and VL CDR 3; wherein,
VH CDR1 comprises SEQ ID NO: 2. 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106 or 114, and VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107 or 115, and VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4. 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108 or 116, and a VL CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6. 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110 or 118, and a VL CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7. 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111 or 119, and a VL CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8. 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, or 120, or a pharmaceutically acceptable salt thereof.
2. The anti-Trop-2 antibody or antigen-binding fragment thereof of claim 1, comprising a VH CDR1, a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR 3; wherein,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO. 2;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO. 3;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO. 4;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 6;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 7;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 8;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 10;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 11;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 12;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 14;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 15;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 16;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 18;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 19;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 20;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 22;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 23;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 24;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 26;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 27;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 28;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 30;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 31;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 32;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 34;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 35;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO. 36;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 38;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 39;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 40;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 42;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 43;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 44;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 46;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 47;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 48;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 50;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 51;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 52;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 54;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 55;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 56;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO. 58;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 59;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 60;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 62;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 63;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 64;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 66;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO. 67;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 68;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 70;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 71;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 72;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 74;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 75;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 76;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 78;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 79;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 80;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 82;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 83;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 84;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 86;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 87;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 88;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 90;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 91;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 92;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 94;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 95;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO. 96;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 98;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 99;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 100;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO. 102;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO. 103;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 104;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 106;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 107;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 108;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 110;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 111;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 112;
or,
the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 114;
the amino acid sequence of VH CDR2 is shown in SEQ ID NO. 115;
the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 116;
the amino acid sequence of VL CDR1 is shown in SEQ ID NO: 118;
the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 119;
the amino acid sequence of VL CDR3 is shown in SEQ ID NO 120.
3. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105 or 113, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the above-described sequences; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 5. 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109 or 117, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the above-described sequences.
4. The anti-Trop-2 antibody or antigen-binding fragment thereof as recited in any one of claims 1 to 3, further comprising one or more of a heavy chain constant region, a light chain constant region, an Fc region; preferably, the light chain constant region is a lambda chain or a kappa chain constant region; further preferably, the antibody or antigen binding fragment thereof is of the IgG1, IgG2, IgG3 or IgG4 type; preferably, the antibody heavy chain comprises or consists of the sequence shown as SEQ ID NO 121 or 123 and the antibody light chain comprises or consists of the sequence shown as SEQ ID NO 122 or 124; more preferably, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody or antigen-binding fragment thereof.
5. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is conjugated to a drug, wherein the drug is a cytotoxic agent such as an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent, and a RNA polymerase II inhibitor or any active pharmaceutical ingredient that interferes with a specific cellular pathway.
6. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 4 or the conjugate of claim 5, and a pharmaceutically acceptable carrier.
7. A biomaterial, being:
(1) a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 4;
(2) a vector, host cell or microorganism comprising (1);
(3) the expression product, suspension or supernatant of the above (2).
8. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1 to 4; preferably, the method comprises culturing the host cell or microorganism of claim 7 such that the antibody or antigen-binding fragment thereof is expressed and isolated.
9. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a conjugate according to claim 5 or a pharmaceutical composition according to claim 6 or a biomaterial according to claim 7 in the manufacture of a medicament for the treatment of a tumour; preferably, the tumor is a Trop-2 high expression cancer; further preferably, the cancer is gastric cancer, pancreatic cancer, prostate cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, breast cancer or cervical cancer.
10. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a conjugate according to claim 5 or a pharmaceutical composition according to claim 6 or a biomaterial according to claim 7 in the manufacture of a product for binding Trop-2.
11. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a conjugate according to claim 5 or a pharmaceutical composition according to claim 6 or a biomaterial according to claim 7 in the manufacture of a medicament for the treatment of a tumour in combination with one or more other cancer therapeutic agents; preferably, the other cancer therapeutic agents include, but are not limited to: paclitaxel, mitomycin, vincristine, colchicine, doxorubicin, daunorubicin, actinomycin D, emetine, puromycin, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan, docetaxel, pemetrexed, sorafenib, oxaliplatin, 5-FU, lapatinib, or an analog or homolog thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111456440.4A CN115057934A (en) | 2021-12-02 | 2021-12-02 | anti-Trop-2 antibodies and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111456440.4A CN115057934A (en) | 2021-12-02 | 2021-12-02 | anti-Trop-2 antibodies and uses thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115057934A true CN115057934A (en) | 2022-09-16 |
Family
ID=83197333
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111456440.4A Pending CN115057934A (en) | 2021-12-02 | 2021-12-02 | anti-Trop-2 antibodies and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115057934A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104053672A (en) * | 2011-11-11 | 2014-09-17 | 瑞纳神经科学公司 | Trop-2 specific antibody and use thereof |
| CN105925536A (en) * | 2016-06-24 | 2016-09-07 | 安徽未名细胞治疗有限公司 | T lymphocyte modified by Trop2 chimeric antigen receptor and application thereof |
| US20190048095A1 (en) * | 2017-08-11 | 2019-02-14 | Bio-Thera Solutions, Ltd. | Compounds and methods for the treatment of trop2 positive diseases |
| CN112574310A (en) * | 2020-12-11 | 2021-03-30 | 浙江博锐生物制药有限公司 | anti-SIRP alpha antibodies and uses thereof |
-
2021
- 2021-12-02 CN CN202111456440.4A patent/CN115057934A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104053672A (en) * | 2011-11-11 | 2014-09-17 | 瑞纳神经科学公司 | Trop-2 specific antibody and use thereof |
| CN105925536A (en) * | 2016-06-24 | 2016-09-07 | 安徽未名细胞治疗有限公司 | T lymphocyte modified by Trop2 chimeric antigen receptor and application thereof |
| US20190048095A1 (en) * | 2017-08-11 | 2019-02-14 | Bio-Thera Solutions, Ltd. | Compounds and methods for the treatment of trop2 positive diseases |
| CN112574310A (en) * | 2020-12-11 | 2021-03-30 | 浙江博锐生物制药有限公司 | anti-SIRP alpha antibodies and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| 刘金荣;白璐月;唐奇;王一荃;叶春萍;殷郑娜;朱进;冯振卿;童华;张慧林;: "全人源抗Trop-2 IgG的制备及对卵巢癌细胞生物特性的影响", 南京医科大学学报(自然科学版), no. 03, 15 March 2016 (2016-03-15), pages 30 - 36 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7199494B2 (en) | Humanized and Chimeric Monoclonal Antibodies Against CD47 | |
| JP6124377B2 (en) | New anti-DR5 antibody | |
| TWI564306B (en) | Bispecific antibody | |
| CN115052895B (en) | Anti-SIRPα antibodies and uses thereof | |
| CN107922503A (en) | A kind of 1 antibody of PDL, its medical composition and its use | |
| TW201909926A (en) | B7H3 antibody-drug conjugate and its medical use | |
| JP2018504113A (en) | Bispecific antibody or antibody mixture having a common light chain | |
| CN114181310B (en) | Anti-TIGIT antibody, its pharmaceutical composition and use | |
| WO2020114478A1 (en) | Cd3 antibody and pharmaceutical use thereof | |
| CN112830969B (en) | Monoclonal antibody specifically binding to human Claudin18.2, and medicine and kit containing monoclonal antibody | |
| WO2020114479A1 (en) | Multispecific protein molecule | |
| CN113321734A (en) | anti-CD 47/anti-PD-L1 antibodies and uses thereof | |
| KR20150032075A (en) | Anticancer composition containing an anti-Ang2 antibody inducing binding to Tie2 receptor | |
| WO2023001303A1 (en) | Pharmaceutical composition and use | |
| CN112243443B (en) | anti-TROP-2 antibodies, antigen-binding fragments thereof, and medical uses thereof | |
| WO2022194201A1 (en) | Cldn18.2-targeting antibody or antigen binding fragment thereof and use thereof | |
| WO2022068810A1 (en) | Anti-claudin18.2 and cd3 bispecific antibody and use thereof | |
| CN117279950A (en) | Antibody targeting IL-18BP and application thereof | |
| US20230374132A1 (en) | Anti-cd3 antibody and uses thereof | |
| WO2022095934A1 (en) | Anti-siglec-15 antibody and application thereof in preparing drug | |
| WO2022237647A1 (en) | Binding molecule against dll3 and use thereof | |
| CN115057934A (en) | anti-Trop-2 antibodies and uses thereof | |
| KR20150014077A (en) | Anti-Ang2 antibody inducing binding to Tie2 receptor | |
| CN116925222A (en) | anti-PVRIG antibody, pharmaceutical composition and application thereof | |
| CN114729057A (en) | anti-fucosyl-GM 1 antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40073121 Country of ref document: HK |
|
| CB02 | Change of applicant information |
Country or region after: China Address after: 318000 No. 1 Shugang Avenue, Jiaojiang District, Taizhou City, Zhejiang Province Applicant after: Zhejiang Borui biopharmaceutical Co.,Ltd. Applicant after: Hangzhou Bozhirui Biopharmaceutical Co.,Ltd. Address before: 318000 No. 1 Shugang Avenue, Jiaojiang District, Taizhou City, Zhejiang Province Applicant before: Zhejiang Borui biopharmaceutical Co.,Ltd. Country or region before: China Applicant before: Haizheng biopharmaceutical Co.,Ltd. |
|
| CB02 | Change of applicant information |