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US-12617871-B2 - Anti-CD47/anti-TIGIT bispecific antibody, preparation method therefor and application thereof

US12617871B2US 12617871 B2US12617871 B2US 12617871B2US-12617871-B2

Abstract

Anti-CD47/anti-TIGIT bispecific antibody, a preparation method thereof and application thereof. The bispecific antibody comprises: (a) a first antigen binding part, comprising heavy chain variable region (V H ) and light chain variable region (V L ), V H and V L forming an antigen binding site that specifically binds to CD47; and (b) a second antigen binding part, comprising a single domain antibody (sdAb) that specifically binds to TIGIT, wherein the first antigen binding part and the second antigen binding part are fused with each other. The bispecific antibody can block two modes of tumor immune escape at the same time, thus having a good effect in tumor immunotherapy.

Inventors

  • Liusong Yin
  • Zhongdao LI
  • Tielin Zhou
  • Zhuo FANG

Assignees

  • Nanjing GenScript Biotech Co., Ltd.

Dates

Publication Date
20260505
Application Date
20200624
Priority Date
20190625

Claims (6)

  1. 1 . An isolated anti-CD47/anti-TIGIT bispecific antigen binding protein comprising (a) a first antigen binding portion comprising an anti-CD47 antibody, comprising two heavy chains each comprising a heavy chain variable region (V H ), and two light chains each comprising a light chain variable region (V L ), wherein the V H and V L form an antigen binding site that specifically binds to CD47; and (b) a second antigen binding portion comprising two anti-TIGIT single domain antibodies, wherein the first antigen binding portion and the second antigen binding portion are fused to each other; wherein the V H comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively; and the V L comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively; wherein the two anti-TIGIT single domain antibodies each comprise complementarity determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of the CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively; and wherein: the N-termini of the two anti-TIGIT single domain antibodies are fused, respectively, to the C-termini of the two heavy chains of the anti-CD47 antibody to form two heavy chain fusion polypeptides, each comprising a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:12, and the two light chains of the anti-CD47 antibody each comprises a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:6; the C-termini of the two anti-TIGIT single domain antibodies are fused, respectively, to the N-termini of the two heavy chains of the anti-CD47 antibody to form two heavy chain fusion polypeptides, each comprising a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:14, and the two light chains of the anti-CD47 antibody each comprises a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:6; the N-termini of the two anti-TIGIT single domain antibodies are fused, respectively, to the C-termini of the two light chains of the anti-CD47 antibody to form two light chain fusion polypeptides, each comprising a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:16 or SEQ ID NO:20, and the two heavy chains of the anti-CD47 antibody each comprises a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:4; or the C-termini of the two anti-TIGIT single domain antibodies are fused, respectively, to the N-termini of the two light chains of the anti-CD47 antibody to form two light chain fusion polypeptides, each comprising a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:22, and the two heavy chains of the anti-CD47 antibody each comprises a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:4.
  2. 2 . An isolated polynucleotide encoding the bispecific antigen binding protein according to claim 1 .
  3. 3 . A vector comprising the isolated polynucleotide according to claim 2 .
  4. 4 . A host cell comprising the isolated polynucleotide according to claim 2 .
  5. 5 . A method for producing isolated anti-CD47/anti-TIGIT bispecific antigen binding protein, comprising culturing the host cell according to claim 4 under suitable conditions, and recovering antibody or fragments thereof from cells or cell culture fluid.
  6. 6 . A pharmaceutical composition comprising the bispecific antigen binding protein according to claim 1 and a pharmaceutically acceptable carrier.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This is the U.S. National Stage of International Application No. PCT/CN2020/097924, filed Jun. 24, 2020, which was published in Chinese under PCT Article 21(2), which in turn claims the benefit of Chinese Patent Application No. 201910554742.1, filed Jun. 25, 2019. INCORPORATION OF ELECTRONIC SEQUENCE LISTING The present application contains a Sequence Listing, which is submitted as an ASCII text file, named “P10448-PI-US.250618. Amended Sequence Listing.txt,” 143,512 bytes, and created on Jun. 9, 2025, which is incorporated herein by reference. TECHNICAL FIELD The present invention belongs to the field of antibodies, and specifically relates to bispecific antibodies and a preparation method and an application thereof. The bispecific antibodies include a first antigen binding portion that specifically binds to CD47, and a second antigen binding portion that specifically binds to TIGIT. BACKGROUND The mammalian immune system is a host defense system that protects against microbial infections and prevents carcinogenesis (Chen et al., Frontiers Immunol. 9:320 (2018)). The immune system is spread all over the body, which is an extremely complex network system composed of different immune cells, and specific tissues and organs exerting a synergistic effect. When the immune system is functioning normally, the diseased cells in the host body will be recognized and eliminated from the healthy cells, thus ensuring the stability of the environment in the body. Therefore, maintaining the integrity of the immune system is essential to maintaining our own health. Conversely, losing control of the immune system can lead to autoimmune diseases, inflammation, cancer and the like (Ribas et al., Cancer Discovery 5:915-9 (2015); Yao and Chen, Eur. J. Immunol. 43:576-9 (2013)). The immune system can be divided into two categories, namely humoral immunity and cell-mediated immunity. Antibodies and other biological macromolecules regulate the humoral immunity. In contrast, the regulation of cellular immunity is achieved at the cellular level, involving the activation of macrophages, natural killer cells and antigen-specific killer T cells. Activation and suppression of immune response are mainly regulated by two independent signaling pathways (Gorentla and Zhong, J. Clin. Cell. Immunol. (2012); Huse, J. Cell Sci. 122:1269-73 (2009); Mizota et al., J. Anesthesia 27:80-7 (2013)). The first signal is mediated by an antigen. When the T cell receptor specifically recognizes and binds to the antigen peptide presented by the MHC on the surface of the antigen presenting cells (APC), the first signal is generated. The second signal is provided by the interaction between antigen presenting cells and co-stimulatory molecules expressed on the surface of T cells. When the first and second signals are activated in sequence, the tumors can be killed by T cells. If the second signal is in lack, T cells will enter an unresponsive state or immune tolerance, and even cause programmed cell death. As mentioned above, the second signaling pathway is very important for activating immune cells. Specifically, co-stimulatory and co-inhibitory receptors participate in the second signaling pathway, induce immune response and regulation of antigen-receptor presentation, balance positive and negative signals while maintaining immune tolerance of autoantigens, and maximize the immune response to invaders (Chen and Flies, Nat. Rev. Immunol. 13:227-42 (2013); Ewing et al., Int. J. Cardiol. 168:1965-74 (2013); Liu et al., Immunol. Invest. 45:813-31 (2016); Shen et al., Frontiers in Biosci. 24:96-132 (2019); Zhang and Vignali, Immunity 44:1034-51(2016)). TIGIT, known as T cell immunoglobulin and ITIM domain protein, is an inhibitory receptor containing Ig and ITIM domains shared by T cells and NK cells. TIGIT is highly expressed in T cells and natural killer (NK) cells. TIGIT, CD96, CD226 and related ligands together form an immunomodulatory signaling pathway. Similar to the CD28/CTLA-4 signaling pathway, the CD226/TIGIT/CD96 signaling pathway also contains co-stimulatory receptors and co-inhibitory receptors sharing some or all of the ligands, in which CD226 is a co-stimulatory receptor and delivers stimulus signals upon combining with ligands, and TIGIT and CD96 are co-inhibitory receptors and deliver inhibitory signals upon combining with related ligands. TIGIT has two ligands, i.e. CD155 and CD122, which are also ligands of CD226. These two ligands are expressed in APC cells, T cells and tumor cells. The ligands of CD96 include CD155 and CD111. The affinity of TIGIT with the ligand CD155 is significantly higher than that of TIGIT with the ligand CD122, and also significantly higher than the affinity of CD226 or CD96 with the ligand CD155. Similar to PD-1 and CTLA-4 receptors, TIGIT is also an important inhibitory immune receptor. Inhibition of TIGIT can promote the proliferation and function of T cells; blockade of TIGIT