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CN-116082504-B - Nanometer antibody targeting human LILRB2 and application thereof

CN116082504BCN 116082504 BCN116082504 BCN 116082504BCN-116082504-B

Abstract

The invention discloses an anti-LILRB 2 nanobody, nucleic acid for encoding the nanobody, an expression vector containing the nucleic acid, a pharmaceutical composition containing the nanobody and application of the nanobody in preparation of medicines.

Inventors

  • JIAO SHASHA
  • WANG RONGJUAN
  • MAO GUANFAN
  • ZHANG CHANG
  • WANG SHUANG
  • ZHANG JINCHAO

Assignees

  • 北京科诺信诚科技有限公司

Dates

Publication Date
20260505
Application Date
20220628

Claims (9)

  1. 1. A nanobody against LILRB2, wherein the nanobody is capable of specifically binding to LILRB2 and the complementarity determining region CDRs of the VHH strand of the nanobody comprise: CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, and CDR3 shown in SEQ ID NO. 27.
  2. 2. The nanobody of claim 1, wherein the nanobody is a humanized VHH or a camelized VHH.
  3. 3. Nanobody according to claim 1 or 2, characterized in that it has the amino acid sequence shown in SEQ ID No. 7.
  4. 4. A pharmaceutical composition comprising the nanobody against LILRB2 as claimed in any one of claims 1 to 3, and a pharmaceutically acceptable adjuvant.
  5. 5. An isolated nucleic acid molecule encoding the nanobody of anti-LILRB 2 of any one of claims 1-3.
  6. 6. An expression vector comprising the nucleic acid molecule of claim 5.
  7. 7. A recombinant cell comprising the expression vector of claim 6, wherein the recombinant cell is a prokaryotic or eukaryotic cell, and wherein the eukaryotic cell is selected from the group consisting of a protozoan cell, an animal cell and a fungal cell.
  8. 8. A method for producing the nanobody according to any one of claims 1 to 3, which comprises culturing the recombinant cell according to claim 7, and isolating and purifying the cultured product to obtain the nanobody.
  9. 9. Use of the nanobody against LILRB2 of any one of claims 1-3, the composition of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6, or the recombinant cell of claim 7 in the preparation of a medicament for the treatment and/or amelioration of colorectal, pancreatic, endometrial, lung, breast, ovarian or gastric cancer.

Description

Nanometer antibody targeting human LILRB2 and application thereof Technical Field The invention belongs to the field of antibody engineering, and particularly relates to a therapeutic single domain antibody for diagnosing or treating tumors, in particular to an anti-LILRB 2 nano antibody, a derivative protein thereof and application of the anti-LILRB 2 nano antibody in preparation of medicines. Background Nanobodies are the smallest antibody molecule at present, originally found in camel blood by belgium scientists Hamers, a class of interest in engineering antibody products. The nano antibody has the main advantages that the volume is 1/10 of that of the common antibody, the penetration of the common antibody in animal tissues is strong because of small volume, for example, the common antibody can reach the inside of high-density tumors through brain tissues of human bodies, but the common antibody can not treat certain tumors or brain diseases through the nano antibody, the antigen specificity is good, the gene modification is easy, the artificial modification is convenient to obtain antibodies against different pathogens, the stability is high, for example, the time of the nano antibody which is not naturally decomposed in the human body is longer than that of the common antibody (meaning that the drug effect time is longer), and the nano antibody can even pass through human stomach to keep effectiveness. LILRB2, also known as ILT4, is expressed mainly on myeloid cells, including monocytes, dendritic cells, macrophages and neutrophils. Genetic studies have shown that Tumor Associated Macrophages (TAMs) in a variety of tumor microenvironments highly express LILRB2, inhibiting LILRB2 (the corresponding protein in mice Pirb) reduces invasion of tregs and MDSCs in tumor tissues. Animal experiments show that Pirb antibody inhibits tumor growth and has synergistic effect with PD-1 antibody. LILRB2 is mainly expressed in bone marrow cells, with limited expression in other tissues, which makes on-target, off-tissue less toxic. Human LILRB2 is an important steady-state surface regulator in the process of myelocyte maturation, is a promising myeloimmune checkpoint target which is specially determined for the myelocyte function, and has important therapeutic value. Disclosure of Invention The ScFv, fab or whole IgG type anti-LILRB 2 antibody molecules in the prior art have complex structure and larger molecules, can connect active molecules to LILRB2, but have complex functions and methods affecting the active molecules, lower loading efficiency, and have smaller nano antibody molecules and easy operation, but have low humanization degree, low affinity and further improve the half-life prolonging performance. Aiming at the defects of the prior art, the invention provides a series of anti-human LILRB2 nanometer antibody sequences and a preparation scheme. In a first aspect, the invention provides a nanobody against LILRB2, according to an embodiment of the invention, which nanobody is capable of specifically binding to LILRB2, and the complementarity determining regions CDRs of the VHH strand in the nanobody are one or more selected from the group consisting of: (1) CDR1 as shown in SEQ ID NO. 14, CDR2 as shown in SEQ ID NO.15, and CDR3 as shown in SEQ ID NO. 16; (2) CDR1 as shown in SEQ ID NO. 17, CDR2 as shown in SEQ ID NO. 18, and CDR3 as shown in SEQ ID NO. 19; (3) CDR1 shown in SEQ ID NO. 20, CDR2 shown in SEQ ID NO. 21, and CDR3 shown in SEQ ID NO. 22; (4) CDR1 as shown in SEQ ID NO. 23, CDR2 as shown in SEQ ID NO. 21, and CDR3 as shown in SEQ ID NO. 24; (5) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, and CDR3 shown in SEQ ID NO. 27. (6) CDR1 as shown in SEQ ID NO. 28, CDR2 as shown in SEQ ID NO. 29, and CDR3 as shown in SEQ ID NO. 30. (7) CDR1 as shown in SEQ ID NO. 31, CDR2 as shown in SEQ ID NO. 32, and CDR3 as shown in SEQ ID NO. 33. (8) CDR1 shown in SEQ ID NO. 34, CDR2 shown in SEQ ID NO. 35, and CDR3 shown in SEQ ID NO. 36. (9) CDR1 as shown in SEQ ID NO. 25, CDR2 as shown in SEQ ID NO. 37, and CDR3 as shown in SEQ ID NO. 27. Further, in some embodiments of the invention, the nanobody described above is a humanized VHH or a camelized VHH. Further, in some embodiments of the invention, the nanobody described above has an amino acid sequence as set forth in any one of SEQ ID NO. 3 through SEQ ID NO. 12, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in any one of SEQ ID NO. 3 through SEQ ID NO. 12. "At least 80% identity" as used herein is, for example, any percent identity of ≡80% such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity. In a second aspect, the present invention provides a fusion protein comprising a functional domain capable of specifically binding to LILRB2, said functional domain consisting of an anti-LILRB 2 nan