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CN-121975008-A - Double-epitope nano antibody targeting SLAMF7 and application thereof

CN121975008ACN 121975008 ACN121975008 ACN 121975008ACN-121975008-A

Abstract

The invention discloses a nanometer antibody S3E5 targeting SLAMF 7. The invention also discloses a double epitope nano antibody CE7, which comprises the nano antibody S3E5. The invention also discloses a fusion protein. The invention also discloses a polynucleotide, an expression vector, a host cell and an antibody conjugate. The invention also discloses application of the nano-antibody S3E5, the double-epitope nano-antibody CE7, the fusion protein and the antibody conjugate. The invention also discloses a pharmaceutical composition. The nano antibody S3E5, the double epitope nano antibody CE7 and the fusion protein have specific recognition and binding performance on extracellular regions of human SLAMF7 proteins, can target the human SLAMF7 proteins, and can be used for preparing reagents, kits or medicines for diagnosing, treating and/or preventing diseases related to the human SLAMF7 proteins.

Inventors

  • JIN TENGCHUAN
  • ZHENG PEIYI

Assignees

  • 合肥综合性国家科学中心大健康研究院

Dates

Publication Date
20260505
Application Date
20260208

Claims (10)

  1. 1. A nanobody S3E5 targeting SLAMF7, wherein the CDR regions of nanobody S3E5 comprise any one of: 1) In the CDR region, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 in sequence; 2) An amino acid sequence having 80% or more homology to at least one amino acid sequence of 1) and still having a function of targeting SLAMF7; 3) An amino acid sequence which has a function of targeting SLAMF7 after adding, deleting, modifying and/or replacing one or more amino acids to at least one amino acid sequence of 1); preferably, the nanobody S3E5 is a camelid nanobody or a humanized nanobody; preferably, when the nanobody S3E5 is a camelid nanobody, the amino acid sequence thereof includes any one of the following: 1) As shown in SEQ ID NO. 4; 2) An amino acid sequence which has 80% or more homology with the amino acid sequence shown in SEQ ID NO. 4 and still has the function of targeting SLAMF 7; 3) An amino acid sequence which has the function of targeting SLAMF7 after one or more amino acids are added, deleted, modified and/or replaced to the amino acid sequence shown in SEQ ID NO. 4; Preferably, when the nanobody S3E5 is a humanized nanobody, the amino acid sequence thereof includes any one of the following: 1) As shown in SEQ ID NO. 5; 2) An amino acid sequence which has 80% or more homology with the amino acid sequence shown in SEQ ID NO. 5 and still has the function of targeting SLAMF 7; 3) An amino acid sequence which has the function of targeting SLAMF7 after one or more amino acids are added, deleted, modified and/or replaced to the amino acid sequence shown in SEQ ID NO. 5.
  2. 2. A diabody nanobody CE7, wherein the diabody nanobody CE7 comprises nanobody S3E5 according to claim 1; Preferably, the diabody CE7 is a camel-derived diabody or a humanized diabody; preferably, the double epitope nano antibody CE7 is formed by connecting a nano antibody 32C and a nano antibody S3E5 in series; preferably, nanobody 32C and nanobody S3E5 target different epitopes of SLAMF7, respectively; preferably, nanobody 32C is a camelid nanobody or a humanized nanobody; Preferably, nanobody S3E5 is a camelid nanobody or a humanized nanobody; Preferably, in the CDR region of the nanobody 32C, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 in sequence; Preferably, when the nano-antibody 32C is a camel-derived nano-antibody, the amino acid sequence of the nano-antibody is shown as SEQ ID NO. 9; Preferably, when the nanobody 32C is a humanized nanobody, the amino acid sequence of the nanobody is shown as SEQ ID NO. 10; Preferably, nanobody 32C is in tandem with nanobody S3E5 via flexible linker peptide 1; preferably, the amino acid sequence of the flexible connecting peptide 1 is GGGGSGGGGSGGGGSGGGGS or an amino acid sequence with 80% or more of homology thereto; preferably, when the bi-epitope nanobody CE7 is a camel bi-epitope nanobody, the amino acid sequence thereof includes any one of the following: 1) As shown in SEQ ID NO. 11; 2) An amino acid sequence which has 80% or more homology with the amino acid sequence shown in SEQ ID NO. 11 and still has the function of dual epitope targeting SLAMF 7; 3) An amino acid sequence with bi-epitope targeting SLAMF7 function after one or more amino acids are added, deleted, modified and/or replaced to the amino acid sequence shown in SEQ ID NO. 11; preferably, when the bi-epitope nanobody CE7 is a humanized bi-epitope nanobody, the amino acid sequence thereof includes any one of the following: 1) As shown in SEQ ID NO. 12; 2) An amino acid sequence which has 80% or more homology with the amino acid sequence shown in SEQ ID NO. 12 and still has the function of dual epitope targeting SLAMF 7; 3) The amino acid sequence shown in SEQ ID NO. 12 still has the function of bi-epitope targeting SLAMF7 after one or more amino acids are added, deleted, modified and/or replaced.
  3. 3. A fusion protein comprising the nanobody S3E5 targeting SLAMF7 according to claim 1 or the diabody nanobody CE7 according to claim 2; preferably, the fusion protein further comprises a flexible connecting peptide 2 and an Fc domain, preferably the Fc domain is a human IgG1 Fc domain, preferably the Fc domain is connected to the C-terminal of nanobody S3E5 or diabody nanobody CE7 through the flexible connecting peptide 2; preferably, the amino acid sequence of the Fc domain is shown as SEQ ID NO. 13, or an amino acid sequence with 80% or more homology with the amino acid sequence shown as SEQ ID NO. 13; preferably, the amino acid sequence of flexible linker peptide 2 is SRGS or SRGSGS.
  4. 4. A fusion protein according to claim 3 wherein the amino acid sequence of the fusion protein comprises any one of the following: 1) As shown in SEQ ID NO. 14-17; 2) An amino acid sequence which has 80% or more homology with the amino acid sequence shown in SEQ ID NO. 14-17 and still has the function of targeting SLAMF 7; 3) The amino acid sequence shown in SEQ ID NO. 14-17 still has the function of targeting SLAMF7 after one or more amino acids are added, deleted, modified and/or replaced.
  5. 5. A polynucleotide encoding the SLAMF 7-targeting nanobody S3E5 of claim 1, or encoding the diabody CE7 of claim 2, or encoding the fusion protein of claim 3 or 4.
  6. 6. An expression vector comprising the polynucleotide of claim 5.
  7. 7. A host cell comprising the expression vector of claim 6 or the polynucleotide of claim 5 integrated into the genome of the host cell, preferably the host cell is a cell capable of expressing a foreign protein, preferably the host cell is a bacterial, yeast, insect or mammalian cell.
  8. 8. An antibody conjugate, characterized in that, the antibody conjugate comprises: 1) The SLAMF 7-targeting nanobody S3E5 of claim 1, the bi-epitope nanobody CE7 of claim 2, or the fusion protein of claim 3 or 4; 2) A coupling moiety selected from any one or combination of a detectable label, an enzyme, a drug, a therapeutic radioisotope, a toxin, a cytokine, a solid support.
  9. 9. Use of the SLAMF 7-targeting nanobody S3E5 of claim 1, the bi-epitope nanobody CE7 of claim 2, the fusion protein of claim 3 or 4, the antibody conjugate of claim 8, comprising: 1) The application of the reagent and/or the kit for preparing and detecting SLAMF7 and the reagent and/or the kit for diagnosing SLAMF7 related diseases; 2) Application in preparing targeted SLAMF7 drugs; 3) Use in the manufacture of a medicament for the treatment and/or prophylaxis of a disease associated with SLAMF 7; preferably, the SLAMF7 associated disease is a hematological malignancy, and preferably, the SLAMF7 associated disease is multiple myeloma.
  10. 10. A pharmaceutical composition comprising one of the SLAMF 7-targeting nanobody S3E5 of claim 1, the bi-epitope nanobody CE7 of claim 2, the fusion protein of claim 3 or 4, and the antibody conjugate of claim 8, preferably further comprising a pharmaceutically acceptable adjuvant.

Description

Double-epitope nano antibody targeting SLAMF7 and application thereof Technical Field The invention relates to the technical field of biological medicine, in particular to a double-epitope nano antibody targeting SLAMF7 and application thereof. Background Multiple myeloma (Multiple Myeloma, MM) is a malignant proliferative disease of plasma cells. The main characteristic is that plasma cells in bone marrow proliferate without limit, and are mostly accompanied by monoclonal immunoglobulin secretion, resulting in organ or tissue injury. Typical clinical manifestations are hypercalcemia, anemia, bone lesions, renal failure, etc., which present significant challenges to the quality of life and survival of the patient. Clinical studies have shown that the combined treatment regimen based on monoclonal antibody drugs (up to Lei Tuoyou mab, erlotinib, ai Satuo mab, etc.), both in the New Diagnosis of Multiple Myeloma (NDMM) and in the treatment of Relapsed Refractory Multiple Myeloma (RRMM), shows a better effect, and the overall survival of patients can even reach more than 10 years. Although many patients have a certain effect at the beginning of treatment, most patients face the dilemma of relapse and difficulty, MM is still considered an incurable disease. SLAMF7 (SIGNALING LYMPHOCYTE ACTIVATING MOLECULE FAMILY-7) is a member of the family of signaling cell activating molecules, expressed in NK cells, CD8+ T cells and B lymphocytes, and overexpressed in MM cells. SLAMF7 plays an important role in the interaction of MM cells with Bone Marrow Stromal Cells (BMSCs), which is considered to be another promising immunotherapeutic target for MM. The U.S. FDA approved anti-SLAMF 7 erlotinib (Elotuzumab) for the treatment of MM patients who had previously received 1-3 therapies. However, objective relief was not seen with a single drug treatment with Elotuzumab, and the condition of most patients progressed during the treatment. Furthermore, the mechanism of drug resistance in Elotuzumab single drug therapies is not clear. Elotuzumab's low binding capacity to SLAMF7 may be an important reason for the drug resistance mechanism. Based on this, development of new antibodies to SLAMF7 with higher affinity remains necessary for the treatment of MM. The annual incidence rate of MM is 4.5-6.0/10 ten thousand cases, accounting for about 1.3% of all malignant tumors. With the continuous progress of medical level, the treatment of MM has made a long-term progress, but the current treatment effect is still not satisfactory, and MM is still considered as incurable disease. Currently, the field of MM treatment is developing toward precise treatment, and in order to obtain better curative effects and avoid recurrence when possible, the development of novel MM therapeutic drugs is urgent. In view of this, development of an MM therapeutic high-affinity bi-epitope antibody targeting SLAMF7 has significant use value and broad application prospects. Disclosure of Invention Based on the technical problems existing in the background technology, the invention provides a double-epitope nano antibody targeting SLAMF7 and application thereof, and the nano antibody S3E5, the double-epitope nano antibody CE7 and fusion protein have specific recognition and binding performance on extracellular regions of human SLAMF7 proteins, can target human SLAMF7 proteins, and can be used for preparing reagents, kits or medicines for diagnosing, treating and/or preventing diseases related to the human SLAMF7 proteins. The invention provides a nanometer antibody S3E5 targeting SLAMF7, wherein a CDR region of the nanometer antibody S3E5 comprises any one of the following components: 1) In the CDR region, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 in sequence; 2) An amino acid sequence having 80% or more homology to at least one amino acid sequence of 1) and still having a function of targeting SLAMF7; 3) An amino acid sequence which has a function of targeting SLAMF7 after addition, deletion, modification and/or substitution of one or more amino acids to at least one amino acid sequence of 1). The heavy chain-only homodimer, called heavy chain antibody (HCAb), was isolated from camelid serum, and for the purpose of distinguishing from the heavy chain variable region (VH) of conventional antibodies, the single variable domain of HCAb with functional antigen binding properties at the N-terminus was called heavy chain antibody variable region fragment (VHH), also called nanobody (Nbs), and immunoglobulin neoantigen receptor (IgNAR) similar to HCAb structure was also found in shark serum in addition to camelids. The isolated HCAb in camelid serum contains one variable region (VHH) with 4 conserved Framework Regions (FR) and 3 Complementarity Determining Regions (CDRs) and two constant regions (CH 2 and CH 3). The CDR regions recognize a specific antigen (e.g., human SLAMF7 protein) and have good affinity