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CN-117384286-B - Single-domain antibody for GPA33 and derived protein and application thereof

CN117384286BCN 117384286 BCN117384286 BCN 117384286BCN-117384286-B

Abstract

The invention belongs to the field of immunology, and relates to a single-domain antibody aiming at GPA33, a derivative protein thereof 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:41-SEQ ID NO:47, a heavy chain CDR2 shown in any one of SEQ ID NO:48-SEQ ID NO:55 and a heavy chain CDR3 shown in any one of SEQ ID NO:56-SEQ ID NO: 68. Compared with the prior art, the invention has the beneficial effects that the biological genetic engineering technology is used for screening out the single-domain antibodies specific to GPA33, the antibodies have obvious initial affinity, can block specific cells from releasing cytokines, and have good binding activity and good drug property through prokaryotic expression.

Inventors

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

Assignees

  • 南京融捷康生物科技有限公司

Dates

Publication Date
20260508
Application Date
20220303

Claims (8)

  1. 1. The single domain antibody for GPA33 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 (a) or (b) as follows: (a) CDR1 as shown in SEQ ID NO. 46, CDR2 as shown in SEQ ID NO. 50, CDR3 as shown in SEQ ID NO. 63; (b) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 49, and CDR3 shown in SEQ ID NO. 63.
  2. 2. The single domain antibody against GPA33 of claim 1, wherein the antibody sequence further comprises 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: a variant of FR1 or FR1 as shown in SEQ ID No. 72, said variant of FR1 comprising up to 3 amino acid substitutions in said FR 1; An FR2 or a variant of FR2 as shown in SEQ ID No. 75, said variant of FR2 comprising up to 3 amino acid substitutions in said FR 2; 78 or 81, said FR3 variant comprising up to 3 amino acid substitutions in said FR 3; FR4 shown in SEQ ID NO. 91 or a variant of FR4, said variant of FR4 comprising at most 3 amino acid substitutions in said FR 4.
  3. 3. The single domain antibody for GPA33 is characterized in that the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 2 or 6 respectively.
  4. 4. The Fc fusion antibody or humanized antibody of any one of claims 1-3 to a single domain antibody of GPA 33.
  5. 5. A polynucleotide molecule encoding a single domain antibody against GPA33 according to any one of claims 1 to 3, wherein the nucleotide sequence is shown in SEQ ID NO. 22 or 26, respectively.
  6. 6. An expression vector comprising a polynucleotide molecule encoding the single domain antibody of any one of claims 1-3 or the Fc fusion antibody of claim 4 or the polynucleotide molecule of claim 5.
  7. 7. A host cell capable of expressing a single domain antibody against GPA33 according to any of claims 1-3, or comprising an expression vector according to claim 6.
  8. 8. Use of a single domain antibody against GPA33 as claimed in any of claims 1-3 in the manufacture of an anti-tumour or anti-arthritic medicament, wherein the tumour is colorectal cancer and the arthritis is osteoarthritis.

Description

Single-domain antibody for GPA33 and derived protein and application thereof Technical Field The invention relates to the technical field of biotechnology or immunology, and relates to a single domain antibody aiming at GPA33, a derivative protein thereof and application thereof. Background Colorectal cancer is one of the most common malignant tumors in western countries and is also the leading cause of cancer death. Statistics data in 2018 show that the number of concurrent cancers in the world is 1810 ten thousand, the number of deaths is 960 ten thousand, wherein the cure rate of intestinal cancer is 6.1%, and the death rate is 9.2%. Because of the high resistance of micro-disseminated colorectal cancer to traditional therapies in recent years, the development of novel therapeutic methods and medicaments is urgent. Creating less immunogenic, constructing humanized or chimeric antibodies to reduce the immune response of the patient, allowing for antibody reuse, more specific antigen recognition is a goal we pursue. GPA33 (Cell surface A33 anti, swiss Prot database accession number: Q99795) is a type I single transmembrane protein with a molecular weight of 35.6kDa, and consists of 319 amino acids, and the glycosylation modification position is between 1 and 235 amino acids and is located outside the Cell membrane. GPA33 is a cell surface differentiation antigen belonging to the immunoglobulin superfamily. The GPA33 allele is located on chromosome 1q24 and comprises 7 exons, and the total length of genomic DNA is 37787bp. GPA33 presents tissue-specific expression. The specific expression of the polypeptide is carried out on gastrointestinal epithelial tissues, is mainly distributed on mucosal epithelial cells, and has lower content in other tissues. Although the function of GPA33 is not fully understood at present, oncology studies have found that GPA33 is highly expressed in more than 95% of colorectal cancers. Since the expression of GPA33 has very high intestinal tissue specificity, the GPA33 not only provides an index for intestinal tumor detection, but also plays an important role in researching the occurrence and progress of intestinal tumor. French researchers as early as 2009 have found that PPARgamma ligands up-regulate GPA33 mRNA and protein levels in a time and concentration dependent manner. Using DNA chip technology, researchers found that GPA33 gene was the target gene of PPARgamma in HT29-Cl.16E cells, and that administration of PPARgamma agonist GW7845 to different human colon cancer cells (including HT29-Cl.16E, caco2, SW1116, etc.) showed a positive correlation between the two. The main mechanism involved is that activation of PPARgamma induces expression of KLF4, and that KLF4 binds to the promoter of GPA33 thereby increasing GPA33 expression. GPA33 also has a certain correlation with osteoarthritis. The current clinical diagnosis of osteoarthritis is based mainly on imaging examinations. However, osteoarthritis has no or few symptoms at an early stage, and only when secondary inflammation, pain, and joint movement are affected, will it seek medical attention. At this time, joint damage occurs for a long time. Developing a method for early diagnosis of osteoarthritis is a highly desirable problem. In 2018, research reports that GPA33 gene expression in normal synovial tissue and osteoarthritis synovial tissue has significant difference, so that GPA33 can be used for developing products for diagnosing osteoarthritis. Experimental staff find that compared with normal synovial tissue, GPA33 gene expression content in the synovial tissue of osteoarthritis is about 10 times higher by using a fluorescent quantitative PCR technology. In addition, the same experimental results were obtained using immunodetection, in situ hybridization, chip or high throughput sequencing. Compared with the traditional means, the gene diagnosis is more timely and sensitive, and the death rate of osteoarthritis is reduced. Immune disorders refer to the damage of the autoimmune response to the tissues and organs of the individual and the appearance of symptoms. 2021 has been reported to determine the expression pattern of GPA33 in human leukocyte subpopulations using mass spectrometry and flow cytometry. The results show that GPA33 is expressed in B cells, dendritic cells, natural killer cells and innate lymphocytes, with significant cd4+ T cell expression. Primary T cells and cxcr5+ regulatory T cells express high levels of GPA33, and primary cd4+ T cells express moderate levels of GPA33. The expression pattern of GPA33 highlights to some extent the functional heterogeneity of the population of CD4+ central memory T cells. GPA33+CD4+ central memory T cells are completely undifferentiated, true central memory T cells lack immediate effector function, while GPA33+ central memory T cells exhibit rapid effector function. GPA33 expression in traditional CD4+ T cells has been shown to play a role in the localization of t