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CN-116375875-B - Anti-LAG 3 single domain antibody and application thereof

CN116375875BCN 116375875 BCN116375875 BCN 116375875BCN-116375875-B

Abstract

The invention belongs to the field of immunology, and relates to a single-domain antibody for resisting LAG3 and application thereof. The single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1 shown in any one of SEQ ID NO. 22-SEQ ID NO. 26, a heavy chain CDR2 shown in any one of SEQ ID NO. 27-SEQ ID NO. 29 and a heavy chain CDR3 shown in any one of SEQ ID NO. 30-SEQ ID NO. 34. Compared with the prior art, the method has the beneficial effects that the biological genetic engineering technology is used for screening out the single-domain antibody specific to LAG3, and the affinity of the antibody is good.

Inventors

  • SU ZHIPENG
  • ZHANG YUN
  • MENG JINGUO
  • WANG LEFEI

Assignees

  • 南京融捷康生物科技有限公司

Dates

Publication Date
20260508
Application Date
20230516

Claims (11)

  1. 1. A single domain antibody for resisting LAG3 is characterized in that the single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; The amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are (1) or (2) as follows: (1) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 32; (2) CDR1 shown in SEQ ID NO. 23, CDR2 shown in SEQ ID NO. 27, and CDR3 shown in SEQ ID NO. 33.
  2. 2. The anti-LAG 3 single domain antibody according to claim 1, further comprising a framework region FR comprising the amino acid sequences of FR1, FR2, FR3 and FR4, wherein the amino acid sequences of the framework region FR are: 13, or a variant of FR1 as set forth in SEQ ID No. 13, said variant of FR1 comprising up to 5 amino acid substitutions in said FR 1; an FR2 or a variant of FR2 as shown in SEQ ID No. 15, said variant of FR2 comprising up to 5 amino acid substitutions in said FR 2; 20 or 19, said FR3 variant comprising up to 5 amino acid substitutions in said FR 3; FR4 shown in SEQ ID NO. 21 or a variant of FR4, said variant of FR4 comprising at most 5 amino acid substitutions in said FR 4.
  3. 3. A single domain antibody for resisting LAG3 is characterized in that the amino acid sequence of the single domain antibody is shown as SEQ ID NO.2 or 1 respectively, or at least 1 amino acid residue in FR1, FR2, FR3 or FR4 sequence is substituted by conservative amino acid compared with any one of SEQ ID NO.2 or 1.
  4. 4. The Fc fusion antibody or humanized antibody of the single domain antibody against LAG3 of any one of claims 1-3.
  5. 5. A polynucleotide molecule encoding the anti-LAG 3 single domain antibody according to any one of claims 1 to 3, wherein the nucleotide sequence is shown in SEQ ID NO. 7 or 6, respectively, or the amino acid sequence encoded by said nucleotide sequence is identical to the amino acid sequence encoded by SEQ ID NO. 7 or 6.
  6. 6. An expression vector comprising a polynucleotide molecule encoding the anti-LAG 3 single domain antibody of any one of claims 1 to 3 or the Fc fusion antibody or humanized antibody of claim 4 or the polynucleotide molecule of claim 5.
  7. 7. A host cell capable of expressing the anti-LAG 3 single domain antibody according to any one of claims 1 to 3 or the Fc fusion antibody or humanized antibody according to claim 4, or comprising the expression vector according to claim 6.
  8. 8. A pharmaceutical composition comprising an anti-LAG 3 single domain antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.
  9. 9. Agent for the treatment of diseases, characterized in that it comprises as active ingredient a single domain antibody against LAG3 according to any one of claims 1 to 3.
  10. 10. Use of the anti-LAG 3 single domain antibody of any one of claims 1 to 3 or the pharmaceutical composition of claim 8 for the manufacture of a medicament for the treatment of a disease, wherein the disease is melanoma, colorectal cancer, esophageal cancer, gastric cancer, glioblastoma, lung cancer, renal tumor, liver cancer, hodgkin's lymphoma, endometrial cancer, breast cancer, diffuse large B-cell lymphoma, ovarian cancer, squamous cell carcinoma or biliary tract tumor.
  11. 11. The method according to claim 10, wherein the squamous cell carcinoma is advanced head and neck squamous cell carcinoma.

Description

Anti-LAG 3 single domain antibody and application thereof Technical Field The present invention relates to a single domain antibody capable of specifically binding to LAG3 (hereinafter, abbreviated as "LAG3 single domain antibody"), a pharmaceutical composition containing the single domain antibody as an active ingredient, and a pharmaceutical therapeutic use thereof. Background LAG-3 (lymphocyte activating gene-3, also known as CD 223) is an immune checkpoint receptor protein mainly expressed in activated T cells, NK cells, B cells and plasma cell dendritic cells, whose main function is to negatively regulate the function of T cells, belonging to the immunoglobulin superfamily members. It is located on chromosome 12 and is structurally and genetically related to CD 4. LAG3 can down regulate T cell activity through binding to MHC II molecules. Meanwhile, LAG3 may also enhance the inhibitory activity of regulatory T cells (tregs). The therapeutic antibody can inhibit LAG3, relieve inhibition of T cells, and enhance immune response of organism. Direct binding of LAG-3 to MHC class II molecules has been proposed to play a role in down-regulating antigen dependent stimulation of CD4 + T lymphocytes (Huard et al (1994) Eur. J. Immunol. 24:3216-3221) and LAG-3 blockade has also been shown to rejuvenate CD8 lymphocytes in tumor or autoantigen models (Gross et al (2007) J Clin invest. 117:3383-3392) and viral models (Blackburn et al (2009) Nat. Immunol. 10:29-37). In addition, the cytoplasmic region of LAG-3 can interact with LAP (LAG-3 related protein), a signaling molecule involved in down-regulation of the CD3/TCR activation pathway (Iouzalen et al (2001) Eur. J. Immunol. 31:2885-2891)). In addition, CD4+CD25+ regulatory T cells (Treg) have been shown to express LAG-3 upon activation, which contributes to the suppressive activity of the Treg cells (Huang, C.et al (2004) Immunity 21:503-513). LAG-3 can also negatively regulate T cell homeostasis by Treg cells in both T cell-dependent and independent mechanisms (Workman, c.j. And Vignali, d.a. (2005) j.immunol.174:688-695). Nanobodies are a new proposition in the antibody community, and because of the small molecular weight, bivalent, trivalent or bispecific antibodies can be obtained by simple molecular cloning techniques. Because of the characteristic of small molecules, the nanobody can achieve high yield in both prokaryotic expression systems (escherichia coli) and eukaryotic expression systems (CHO cells, 293 cells and the like). The rapid development of nanobodies has become a potential-unlimited force in antibody drug development, representing an important development direction of antibody drugs from now on. Disclosure of Invention The invention of this patent aims to provide a single domain antibody capable of specifically binding to LAG3 and uses thereof. In a first aspect, the invention provides a single domain antibody against LAG3, said single domain antibody comprising a heavy chain CDR1 as shown in any one of SEQ ID NO. 22-SEQ ID NO. 26 and a heavy chain as shown in any one of SEQ ID NO. 27-SEQ ID NO. 29 CDR2 and SEQ ID NO:30-SEQ ID NO: 34. Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1) to (5): (1) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 32; (2) CDR1 shown in SEQ ID NO. 23, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 33; (3) CDR1 shown in SEQ ID NO. 24, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 31; (4) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 29, CDR3 shown in SEQ ID NO. 34; (5) CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 28, and CDR3 shown in SEQ ID NO. 30. The above 5 CDR combinations (1) - (5) correspond in turn to single domain antibodies 4C10, 2D4, 4F1, 4D9, 4G 12. All of the above sequences may be replaced with sequences having "at least 80% homology" or sequences with only one or a few amino acid substitutions, preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology". In one embodiment, wherein any one to five of the amino acid residues in any one or more of the CDRs of heavy chain CDR1, CDR2 and CDR3 may be substituted with their conserved amino acids, respectively. Specifically, 1 to 5 amino acid residues in the heavy chain CDR1 can be replaced by the conserved amino acid, 1 to 5 amino acid residues in the heavy chain CDR2 can be replaced by the conserved amino acid, and 1 to 5 amino acid residues in the heavy chain CDR3 can be replaced by the conserved amino acid. As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compare