WO2021213475A1 - 抗cd73-抗pd-1双特异性抗体及其用途 - Google Patents

抗cd73-抗pd-1双特异性抗体及其用途 Download PDF

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Publication number
WO2021213475A1
WO2021213475A1 PCT/CN2021/089059 CN2021089059W WO2021213475A1 WO 2021213475 A1 WO2021213475 A1 WO 2021213475A1 CN 2021089059 W CN2021089059 W CN 2021089059W WO 2021213475 A1 WO2021213475 A1 WO 2021213475A1
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Prior art keywords
seq
amino acid
acid sequence
antibody
variable region
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Ceased
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PCT/CN2021/089059
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English (en)
French (fr)
Chinese (zh)
Inventor
张鹏
李百勇
夏瑜
王忠民
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Akeso Pharmaceuticals Inc
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Akeso Biopharma Inc
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Priority to KR1020227040580A priority Critical patent/KR20230004726A/ko
Priority to AU2021261803A priority patent/AU2021261803B2/en
Priority to IL297432A priority patent/IL297432A/en
Priority to PH1/2022/552785A priority patent/PH12022552785A1/en
Priority to EP21792313.5A priority patent/EP4141033A4/en
Priority to MX2022013311A priority patent/MX2022013311A/es
Priority to US17/996,878 priority patent/US20230151111A1/en
Priority to BR112022021426A priority patent/BR112022021426A2/pt
Application filed by Akeso Biopharma Inc filed Critical Akeso Biopharma Inc
Priority to CA3176321A priority patent/CA3176321A1/en
Priority to JP2022564073A priority patent/JP7763780B2/ja
Publication of WO2021213475A1 publication Critical patent/WO2021213475A1/zh
Priority to ZA2022/11405A priority patent/ZA202211405B/en
Anticipated expiration legal-status Critical
Priority to JP2024218813A priority patent/JP2025038088A/ja
Ceased legal-status Critical Current

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    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells

Definitions

  • the present invention belongs to the field of tumor therapy and molecular immunology, and relates to an anti-CD73-anti-PD-1 bispecific antibody, a pharmaceutical composition and its use.
  • Ecto-5'-nucleotidase (Ecto-5'-nucleotidase), CD73 protein, is a multifunctional glycoprotein with a molecular weight of 70KD encoded by the NT5E gene.
  • the phosphatidy linositol, GPI) is anchored on the cell membrane (Zimmermann H.5'-Nucleotidase: molecular structure and functional aspects. Biochem J. 1992; 285: 345-365).
  • CD73 is widely distributed on the surface of human tissue cells. Current studies have found that CD73 is highly expressed in a variety of solid tumors, such as cancer cells, dendritic cells, regulatory T cells (Treg), natural killer cells (NK cells), and tumor microenvironment. Suppressor cells of myeloid origin (MDSC), tumor-associated macrophages (TAM), etc. The expression of CD73 is regulated by TGF- ⁇ , EGFR, AKT, ⁇ -catenin and other molecules, especially HIF-1, which functions as a transcription factor, is the most critical.
  • hypoxia-inducible factor-1 HIF-1
  • CD73 hypoxia-inducible factor-1 regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest. 2002; 110: 993-1002.
  • the analysis of clinical tumor samples showed that the high expression of CD73 is a potential biomarker, which is closely related to the poor prognosis of many types of tumors, including breast, lung, ovarian, kidney, stomach, head and neck cancer.
  • CD73 has hydrolytic enzyme activity and non-hydrolytic enzyme activity.
  • the enzymatic and non-enzymatic functions of CD73 exist in the tumor-related processes at the same time, and promote each other to maintain the evolution of the tumor. More and more studies have found that CD73 is a key regulator of tumor cell proliferation, metastasis and invasion in vitro, tumor angiogenesis and tumor immune escape mechanism in vivo.
  • CD73-adenosine (Adenosine). Mediated by the metabolic signaling pathway, CD39 upstream of CD73 can catalyze ATP to produce adenosine monophosphate (AMP).
  • the AMP produced is converted to adenosine by CD73, and adenosine binds to the downstream adenosine receptor (A2AR), A2AR
  • A2AR adenosine receptor
  • PKA protein kinase A
  • Csk kinase Csk kinase
  • CD73 expressed by immune cells and non-immune cells can promote the immune escape, development and metastasis of tumors.
  • the CD73-adenosine signal related to Treg cells can affect the functions of CTL (cytotoxic T cells) and NK cells. The most obvious inhibition.
  • TME tumor microenvironment
  • TME tumor microenvironment
  • the hypoxia or ATP enrichment caused by radiotherapy and chemotherapy killing tumor cells will promote the CD39-CD73 adenosine signal cascade reaction, which is beneficial to the proliferation and function of various cancer-promoting cells, but not to tumor suppressor cells.
  • CD73-targeting antibodies or gene knockout CD73 in animal models can effectively block tumor growth and metastasis.
  • CD73 monoclonal antibody, small interfering RNA technology, specific inhibitor APCP, etc. has achieved significant effects in anti-tumor treatment in animal experiments, providing a new way for anti-tumor treatment.
  • Evidence from in vivo experiments shows that targeted blocking of CD73 will become an effective treatment for cancer patients.
  • CD73 The relationship between CD73 overexpression and tumor subtypes, prognosis and patient drug response has shown that CD73 can be used as an important marker for the treatment and detection of individual tumors in the future. Therefore, research on the CD73 target is indispensable.
  • the transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 family and is expressed on activated T cells, B cells, and bone marrow cells. Both PD-1 receptors PDL1 and PDL2 belong to the B7 superfamily. Among them, PDL1 is expressed in a variety of cells, including T cells, B cells, and endothelial cells and epithelial cells. PDL2 is only expressed in antigen-presenting cells such as dendritic cells. And macrophages.
  • PD-1 plays a very important role in the activation of negative regulatory T cells.
  • the negative regulation of T cells mediated by PD-1 is one of the important mechanisms of tumor immune evasion.
  • PDL-1 expressed on the tumor surface can interact with The PD-1 on the surface of immune cells binds to inhibit the killing of tumor tissues by immune cells through the PD-1/PDL-1 signaling pathway.
  • Tumors with high PD-L1 expression are accompanied by cancers that are difficult to detect (Hamanishi et al. , Proc. Natl. Acad. Sci. USA 2007; 104: 3360-5).
  • An effective way to antagonize PD-1 and thereby inhibit the PD-1/PDL-1 signaling pathway is to inject anti-PD-1 antibodies in vivo.
  • PD-1 antibody has broad-spectrum anti-tumor prospects and amazing efficacy. Antibodies against the PD-1 pathway will bring breakthroughs in the treatment of a variety of tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, Ovarian cancer, melanoma (Homet MB, Parisi G., et al., Anti-PD-1 Therapy in Melanoma. Semin Oncol. 2015Jun; 42(3): 466-473), hematological tumors and anemia (Held SA, Heine A, et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets. 2013 Sep; 13(7): 768-74).
  • yield SA Heine A, et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets. 2013 Sep; 13(7): 768-74.
  • Bifunctional antibodies also known as bispecific antibodies, are specific antibody drugs that simultaneously target and bind two different antigens. They can be produced through immune sorting and purification, or they can be obtained through genetic engineering. Genetic engineering has corresponding flexibility in terms of optimization of binding sites, consideration of synthetic forms, and yield, so it has certain advantages.
  • the IgG-ScFv form is the Morrison model (Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fab fragment of the antibody binds to the antigen epitope of virus-infected cells or tumor cells. Its Fc fragment and killer cells (NK cells) , Macrophages, etc.) binding to the Fc receptor (FcR) on the surface, mediating killer cells to directly kill target cells.
  • NK cells Fc fragment and killer cells
  • FcR Fc receptor
  • CDC complement dependent cytotoxicity refers to complement dependent cytotoxicity.
  • the CDC effect is caused by the first binding of the antibody to the corresponding antigen on the cell membrane surface and further binding to complement C1q, and then C2-C9 is activated to form a membrane attack complex to exert a lytic effect on the target cell.
  • the IgG family contains four members, IgG1, IgG2, IgG3, and IgG4. There are amino acid differences in the fragment crystallizable (Fc) region of their heavy chain constant regions, which leads to their different affinities with Fc ⁇ Rs. Wild-type IgG1 can bind various Fc ⁇ Rs and can trigger ADCC and CDC effects. Zhang et al. (Zhang T et al. Cancer Immunol Immunother. 2018; 67(7): 1079-1090.) and Dahan et al. (Dahan R et al. Cancer Cell.
  • Interleutin-8 is a chemotactic cytokines, mainly secreted by monocytes and the like. IL-8 plays an important role in the proliferation of normal cells and tumor cells, especially for the occurrence and development of tumors. Studies have shown that IL-8 can promote the occurrence of tumors; tumor cells themselves can also secrete IL-8 to promote tumor growth and metastasis (Lo MC et al. Cancer letters, 2013, 335(1): 81-92.). Therefore, IL-8 has become an indispensable important inflammatory factor in the tumor microenvironment.
  • IL-8 As a pro-inflammatory factor, IL-8 is closely related to the occurrence and development of tumors. During the malignant transformation of non-kidney cancer cells induced by methylarsonate, the expression of IL-8 gene increases, and IL-8 gene silencing can significantly inhibit the growth of transplanted tumors in mice. In addition, the level of IL-8 decreases. It can inhibit the expression of matrix metalloproteinase-9 (Matrix metalloproteinase-9), cyclin D1 (Cyclin D1), pro-apoptotic protein Bcl-2, and vascular endothelial growth factor (VEGF) related to tumor growth and metastasis (Escudero -Lourdes C et al.
  • matrix metalloproteinase-9 Matrix metalloproteinase-9
  • Cyclin D1 cyclin D1
  • VEGF vascular endothelial growth factor
  • IL-8 can induce malignant transformation and increased invasiveness of a non-tumor bladder cell line (233JP), while the probability of malignant transformation of 233JP cells in IL-8 knockout mice was significantly reduced (Inoue K et al. al. Cancer Res, 2000, 60(8): 2290-2299.).
  • IL-8 can promote the occurrence of castration-resistant prostate cancer (CRPC) in patients (Chen K et al.
  • IL-6 is mainly produced rapidly by macrophages, responding to pathogen-related molecular patterns (PAMP) or damage-related molecular patterns (DAMP), and by removing infectious agents, inducing acute phase and immune response to heal damaged tissues and protect them effect.
  • PAMP pathogen-related molecular patterns
  • DAMP damage-related molecular patterns
  • IL-6 plays an important role in the resistance and repair of infection and tissue damage, high levels of IL-6 can activate blood coagulation pathways and vascular endothelial cells, thereby inhibiting myocardial function, and can even cause a "cytokine storm", resulting in serious Acute systemic inflammation. Cytokine storm is a fatal complication and adverse reaction in viral infections and tumor immunotherapy.
  • Immune-related adverse reactions are a common and dangerous adverse reaction in immune checkpoint inhibitor (ICI) anti-tumor therapy (Spain Let al. Cancer Treat Rev. 2016; 44: 51-60.).
  • immune checkpoint inhibitors have achieved great success in tumor immunotherapy, but they have also led to a new toxicity spectrum due to off-target effects.
  • serious immune-related adverse events (irAE) of major organs may be life-threatening (Bergqvist V, et al. Cancer Immunol Immunother. 2017; 66(5): 581-592.; Gomatou G et al. Respiration. 2020; 1: 1-11.; Joshi MN et al. Clin Endocrinol(Oxf).
  • ICI may induce off-target effects through four mechanisms, including direct binding to immune checkpoint molecules expressed on the surface of normal cells to activate complement hypersensitivity; normal tissues and tumor cells have homologous antigens/epitopes; autoantibodies are produced; Increase the level of pro-inflammatory cytokines, such as IL-6 (Martins F et al., The Lancet Oncology, 20(1), e54-e64.).
  • the binding of Fc ⁇ RIa on macrophages to wild-type IgG1 or IgG4 antibodies can induce the secretion of IL-8 and IL-6 (Kinder M et al., mAbs. 2015), and mutation of the Fc segment of the antibody can eliminate its interaction with Fc ⁇ RIa.
  • the combination can effectively inhibit the secretion of IL-8, thereby increasing the safety and effectiveness of the antibody.
  • the present inventors used a mammalian cell expression system to express recombinant CD73 and PD-1 respectively as antigens to immunize mice, and obtain hybridoma cells by fusion of mouse spleen cells and myeloma cells.
  • the inventors screened a large number of samples and obtained the following hybridoma cell lines:
  • Hybridoma cell line LT014 also known as CD73-19F3
  • CTCC China Center for Type Culture Collection
  • the hybridoma cell line LT003 (also known as PD-1-14C12) was deposited in the China Center for Type Culture Collection (CCTCC) on June 16, 2015, and the deposit number is CCTCC NO: C2015105.
  • the hybridoma cell line LT014 can secrete and produce a specific monoclonal antibody (named 19F3) that specifically binds to human CD73, and this monoclonal antibody can effectively inhibit the enzymatic reaction of CD73 in a non-substrate competition manner, and reduce The production of adenosine promotes T cell activity and tumor suppressor effect.
  • a specific monoclonal antibody named 19F3
  • This monoclonal antibody can effectively inhibit the enzymatic reaction of CD73 in a non-substrate competition manner, and reduce The production of adenosine promotes T cell activity and tumor suppressor effect.
  • the hybridoma cell line LT003 can secrete and produce a specific monoclonal antibody (named 14C12) that specifically binds to PD-1, and this monoclonal antibody can effectively block the binding of PD-1 to PDL1.
  • the present inventors creatively prepared anti-CD73 humanized antibodies (named 19F3H2L2, 19F3H2L3, 19F3H2L3 (hG1M), 19F3H2L3 (hG1TM)) and anti-PD-1 humanized antibodies (named 14C12H1L1 and 14C12H1L1(hG1TM)).
  • the present inventors creatively fused two types of humanized antibodies into a new antibody by protein recombination, and obtained a human source that can bind to CD73 and PD-1, inhibit the activity of CD73, and block the binding of PD-1 to PDL1.
  • Bifunctional antibodies named P1D7V01, P1D7V03, NTPDV1, NTPDV2, NTPDV3, NTPDV4 (respectively named NTPDV1 (hG1TM), NTPDV2 (hG1TM), NTPDV3 ( hG1TM), NTPDV4(hG1TM)
  • an anti-CD73-anti-PD-1 bispecific antibody which comprises:
  • the first protein functional region which targets PD-1, and
  • the second protein functional region, the second protein functional region targets CD73.
  • the bispecific antibody in one embodiment of the invention, the bispecific antibody
  • the first protein functional region contains the amino acid sequence of HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 44, preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NOs: 45-47.
  • sequence shown or the sequence shown in SEQ ID NOs: 45-47 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , Preferably a sequence with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or compared with the sequence shown in SEQ ID NOs: 45-47
  • amino acid sequence of one or more preferably 1, 2, or 3 conservative amino acid mutations (preferably substitutions, insertions or deletions), and comprising
  • the sequence shown by 50-52 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity sequence, or compared with the sequence shown in SEQ ID NOs: 50-52 with one or more ( Preferably 1, 2 or 3) conservative amino acid mutation (preferably substitution, insertion or deletion) amino acid sequence;
  • the second protein functional region contains the amino acid sequence shown in SEQ ID NO: 2.
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3, preferably the amino acid sequence of HCDR1, HCDR2 and HCDR3 is shown in SEQ ID NOs: 3-5
  • the sequence shown in SEQ ID NOs: 3-5 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, Preferably, a sequence with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or a sequence with the sequence shown in SEQ ID NOs: 3-5
  • the first protein functional region comprises
  • the amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85% with the sequence shown in SEQ ID NO: 44 or SEQ ID NO: 62 or the sequence shown in SEQ ID NOs: 44 or 62 , 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, Or an amino acid sequence with one or more (preferably 1, 2, or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequence shown in SEQ ID NOs: 44 or 62; and
  • amino acid sequence selected from the sequence shown in SEQ ID NO: 49 or SEQ ID NO: 64 or the sequence shown in SEQ ID NOs: 49 or 64 has at least 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity Sexual sequence, or an amino acid sequence with one or more (preferably 1, 2, or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequence shown in SEQ ID NOs: 49 or 64;
  • the second protein functional region includes an amino acid sequence selected from the sequence shown in SEQ ID NO: 2, SEQ ID NO: 20 or at least 80% of the sequence shown in SEQ ID NO: 2, SEQ ID NO: 20, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity, or has one or more (preferably 1, 2, or 3) conserved amino acids compared with the sequence shown in SEQ ID NO: 2, SEQ ID NO: 20
  • the amino acid sequence of the mutation (preferably substitution, insertion or deletion); and comprising
  • the corresponding sequence is selected from SEQ ID NO: 7, or SEQ ID NO: 22, or the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 22 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity Sequence, or an amino acid with one or more (preferably 1, 2, or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 22 sequence;
  • the second protein functional region includes an amino acid sequence as shown in SEQ ID NO20 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86% with the sequence shown in SEQ ID NO: 20. %, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or with Compared with the sequence shown in SEQ ID NO: 20, an amino acid sequence with one or more (preferably 1, 2, or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions), and containing
  • the amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the sequence shown in SEQ ID NO: 24 or the sequence shown in SEQ ID NO: 24 , 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or the sequence shown in SEQ ID NO: 24 Compared with the sequence, the amino acid sequence has one or more (preferably 1, 2, or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions).
  • an anti-CD73-anti-PD-1 bispecific antibody which comprises:
  • the first protein functional region which targets CD73, and
  • the second protein functional region, the second protein functional region targets PD-1.
  • the first protein functional region contains the amino acid sequence of HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 2, preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NOs: 3-5.
  • sequence shown or the sequence shown in SEQ ID NOs: 3-5 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , Preferably a sequence with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or compared with the sequence shown in SEQ ID NOs: 3-5
  • the second protein functional region contains the amino acid sequence of HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 44, preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NOs: 45-47.
  • sequence shown or the sequence shown in SEQ ID NOs: 45-47 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , Preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or compared with the sequence shown in SEQ ID NOs: 45-47
  • the first protein functional region comprises
  • the amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85% of the sequence shown in SEQ ID NO: 2, SEQ ID NO: 20 or the sequence shown in SEQ ID NOs: 2 or 20 , 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, Or an amino acid sequence with one or more (preferably 1, 2, or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequence shown in SEQ ID NOs: 2 or 20; and
  • amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 7, SEQ ID NO: 22, SEQ ID NO: 24 The sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, Sequences with 95%, 96%, 97%, 98% or 99% sequence identity, or with one or more (preferably one, two or 3) Amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions);
  • the second protein functional region includes an amino acid sequence selected from the sequence shown in SEQ ID NO: 44 or SEQ ID NO: 62 or at least 80% of the sequence shown in SEQ ID NO: 44 or SEQ ID NO: 62. %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity, or one or more (preferably 1, 2, or 3) compared with the sequence shown in SEQ ID NO: 44 or SEQ ID NO: 62
  • the amino acid sequence of conservative amino acid mutations preferably substitutions, insertions or deletions
  • SEQ ID NOs: 49 or 64 Corresponding to the sequence selected from SEQ ID NOs: 49 or 64 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, and sequences shown in SEQ ID NOs: 49 or 64, respectively. 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or with SEQ ID NOs: 49 Or an amino acid sequence with one or more (preferably 1, 2, or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in 64.
  • conservative amino acid mutations preferably substitutions, insertions or deletions
  • the first protein functional region and the second protein functional region are directly connected or connected by a linking fragment; preferably, the The connecting fragment is (GGGGS)n, where n is a positive integer, such as 1, 2, 3, 4, 5, or 6.
  • the first protein functional region and the second protein functional region in the anti-CD73-anti-PD-1 bispecific antibody are independently immunoglobulins or antigen-binding fragments, such as Half antibody, Fab, F(ab') 2 or single chain antibody, preferably, the first protein functional region is an immunoglobulin, and the second protein functional region is an antigen-binding fragment; or, the first protein The functional region is an antigen-binding fragment, and the second protein functional region is an immunoglobulin.
  • the N-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment) connected to the C-terminus of the immunoglobulin CH1 and the light chain of the antigen-binding fragment
  • the N-terminus of the variable region is directly (or through a linking fragment) connected to the C-terminus of the light chain variable region CL of the immunoglobulin; or the N-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment).
  • the C-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or via a linking fragment) connected to the N-terminus of the heavy chain of the immunoglobulin and the light chain of the antigen-binding fragment
  • the C-terminus of the variable region is directly (or through a linking fragment) connected to the N-terminus of the light chain of the immunoglobulin; or the C-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment) connected to the The N-terminus of the light chain of the immunoglobulin and the C-terminus of the variable region of the light chain of the antigen-binding fragment are directly (or through a linking fragment) connected to the N-terminus of the heavy chain of the immunoglobulin.
  • the antigen-binding fragment is a single-chain antibody, preferably, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; or, the The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin.
  • the bispecific antibody wherein the first protein functional region and the second protein functional region are independently one, two or more than two.
  • the single-chain antibody is a linker that connects the variable region of the antibody heavy chain (V H ) and the antibody molecular light chain variable region (V L); preferably, may have the general structure: NH2-V L - linker segment -V H -COOH or NH2-V H - linker segment -V L -COOH.
  • the PD 1-bispecific antibody anti-anti-CD73-, the single chain antibody to the heavy chain immunoglobulin fragment is connected via the C-terminus (C H) (or When the N-terminus of the heavy chain and the C-terminus of the CH1 of the heavy chain variable region), the antibody heavy chain variable region ( VH ) of the single-chain antibody may be connected first, or the antibody of the single-chain antibody may be connected first a light chain variable region (V L); preferably, the single-chain antibody may have the general structure: C H - linker segment -V H - linker segment -V L -COOH, or, C H - linker segment -V L - connector Fragment -V H -COOH,
  • variable region of the heavy chain includes the CDR with the amino acid sequence of SEQ ID NO: 3-5
  • variable region of the light chain includes the CDR with the amino acid sequence of SEQ ID NO: 8-10;
  • variable region of the heavy chain of the single-chain antibody includes the CDRs with the amino acid sequence of SEQ ID NO: 45-47, and the variable region of the light chain includes the CDRs with the amino acid sequence of SEQ ID NO: 50-52,
  • the single chain antibodies e.g. NH2-V L - linker segment -V H -COOH or NH2-V H - linker segment -V L -COOH
  • the variable region (V H ) of the antibody heavy chain containing the CDRs of the amino acid sequence of SEQ ID NO: 45-47 can be connected first, or the amino acid sequence containing the single chain antibody can be connected first SEQ ID NO: 50-52 CDR of an antibody light chain variable region (V L),
  • variable region of the heavy chain includes the CDRs with the amino acid sequence of SEQ ID NO: 45-47
  • variable region of the light chain includes the CDRs with the amino acid sequence of SEQ ID NO: 50-52; and/or ,
  • variable region of the heavy chain includes the CDR with the amino acid sequence of SEQ ID NO: 3-5
  • variable region of the light chain includes the CDR with the amino acid sequence of SEQ ID NO: 8-10
  • variable region (VH ) of the antibody heavy chain containing the CDRs of the amino acid sequence of SEQ ID NO: 3-5 can be first connected to the single-chain antibody, or the single-chain antibody containing the amino acid sequence of SEQ ID NO: 3-5 can be connected first.
  • One immunoglobulin molecule is connected to two single-chain antibody molecules, and more preferably, the two single-chain antibody molecules are the same.
  • the immunoglobulin in the anti-CD73-anti-PD-1 bispecific antibody, is IgG, IgA, IgD, IgE or IgM; preferably IgG, such as IgG1, IgG2, IgG3 or IgG4.
  • the single-chain antibody in the anti-CD73-anti-PD-1 bispecific antibody, is attached to the C-terminus of the heavy chain of the immunoglobulin. Since immunoglobulin is composed of two heavy chains, one immunoglobulin molecule is connected to two single-chain antibody molecules. Preferably, the two single chain antibody molecules are the same.
  • variable region of the heavy chain includes the CDR with the amino acid sequence of SEQ ID NO: 3-5
  • variable region of the light chain includes the CDR with the amino acid sequence of SEQ ID NO: 8-10;
  • variable region of the heavy chain of the single-chain antibody includes the CDRs with the amino acid sequence of SEQ ID NO: 45-47, and the variable region of the light chain includes the CDRs with the amino acid sequence of SEQ ID NO: 50-52,
  • the single chain antibodies e.g. NH2-V L - linker segment -V H -COOH or NH2-V H - linker segment -V L -COOH
  • the variable region (V H ) of the antibody heavy chain containing the CDRs of the amino acid sequence of SEQ ID NO: 45-47 can be connected first, or the amino acid sequence containing the single chain antibody can be connected first SEQ ID NO: 50-52 CDR of an antibody light chain variable region (V L).
  • variable region of the heavy chain includes the CDRs with the amino acid sequence of SEQ ID NO: 45-47
  • variable region of the light chain includes the CDRs with the amino acid sequence of SEQ ID NO: 50-52; and/or ,
  • variable region of the heavy chain of the single-chain antibody includes the CDRs with the amino acid sequence of SEQ ID NO: 3-5, and the variable region of the light chain includes the CDRs with the amino acid sequence of SEQ ID NO: 8-10,
  • variable region (VH ) of the antibody heavy chain containing the CDRs of the amino acid sequence of SEQ ID NO: 3-5 can be first connected to the single-chain antibody, or the single-chain antibody containing the amino acid sequence of SEQ ID NO: 3-5 can be connected first.
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2 or SEQ ID NO: 20; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7 or SEQ ID NO: 22, or the amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the light chain variable region of the immunoglobulin is SEQ ID NO: the sequence shown in 24;
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 44 or SEQ ID NO: 62; the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 49 or SEQ ID NO: 64;
  • variable region (V H ) of the antibody heavy chain of the single-chain antibody may be connected first, or firstly connected the single-chain antibody antibody light chain variable region (V L).
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 44 or SEQ ID NO: 62; the amino acid sequence of the light chain variable region of the immunoglobulin is correspondingly selected from SEQ ID NO: 49 Or SEQ ID NO: 64, or the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 2 or SEQ ID NO: 20, the amino acid sequence of the light chain variable region of the single-chain antibody Corresponding to SEQ ID NO: 7 or SEQ ID NO: 22, or the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20, and the light chain of the single-chain antibody may The amino acid sequence of the variable region is the sequence shown in SEQ ID NO: 24.
  • Another aspect of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding a variable region of a bispecific antibody heavy chain, wherein,
  • the heavy chain variable region of the antibody comprises:
  • amino acid sequence is the CDR of SEQ ID NO: 3-5, the amino acid sequence is the CDR of SEQ ID NO: 45-47, and the amino acid sequence is the CDR of SEQ ID NO: 50-52;
  • the bispecific antibody also includes a light chain variable region, the light chain The variable region contains:
  • amino acid sequence is the CDR of SEQ ID NO: 8-10;
  • the CDR of the variable region of the light chain is different from the CDR contained in the variable region of the heavy chain.
  • the immunoglobulin includes a non-CDR region, and the non-CDR region is from a species other than murine, for example, from a human antibody.
  • the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834.
  • the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834; where, according to the EU numbering system, the heavy chain constant region of the immunoglobulin is located at any two positions or 3 among positions 234, 235 and 237 There are mutations at each site, and after the mutation, the affinity constant of the bispecific antibody with Fc ⁇ RIa, Fc ⁇ RIIIa and/or C1q is lower than before the mutation; preferably, the affinity constant is measured by the Fortebio Octet molecular interaction instrument.
  • the bispecific antibody wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin is located at positions 234, 235 and/or 237
  • the site has the following mutations:
  • the letter before the site indicates the amino acid before the mutation
  • the letter after the site indicates the amino acid after the mutation
  • the bispecific antibody wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin further has one or more mutations selected from the following:
  • the structure of the anti-CD73-anti-PD-1 bispecific antibody is shown as heavy chain-light chain-linking fragment 1-scFv, and the scFv is selected from 14C12H1V-linking fragment 2-14C12L1V, 14C12H1V-linked fragment 1-14C12L1V, 14C12H1V-linked fragment 2-14C12L1V and 14C12H1V-linked fragment 1-14C12L1V, specifically selected from the group consisting of:
  • NTPDV1 whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, light chain amino acid sequence is shown in SEQ ID NO: 28, and the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, the amino acid of 14C12H1V The sequence is shown in SEQ ID NO: 66, the amino acid sequence of connection fragment 2 is shown in SEQ ID NO: 81, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 68.
  • NTPDV2 the heavy chain amino acid sequence is shown in SEQ ID NO: 85
  • the light chain amino acid sequence is shown in SEQ ID NO: 28
  • the amino acid sequence of the connecting fragment 1 is shown in SEQ ID NO: 79
  • the amino acid of 14C12H1V The sequence is shown in SEQ ID NO: 66
  • the amino acid sequence of connection fragment 1 is shown in SEQ ID NO: 79
  • the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 68.
  • NTPDV3 whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, light chain amino acid sequence is shown in SEQ ID NO: 96, and the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, the amino acid of 14C12H1V The sequence is shown in SEQ ID NO: 66, the amino acid sequence of connecting fragment 2 is shown in SEQ ID NO: 81, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 68, and
  • NTPDV4 whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, light chain amino acid sequence is shown in SEQ ID NO: 96, and the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, the amino acid of 14C12H1V The sequence is shown in SEQ ID NO: 66, the amino acid sequence of connection fragment 1 is shown in SEQ ID NO: 79, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 68.
  • the bispecific antibody has a molecular weight of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K D of M or less binds to CD73 protein and/or PD-1 protein.
  • Yet another aspect of the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.
  • Another aspect of the present invention relates to a host cell, which contains the isolated nucleic acid molecule of the present invention, or the vector of the present invention.
  • Another aspect of the present invention relates to a method for preparing the bispecific antibody of the present invention, which includes the steps of culturing the host cell of the present invention under suitable conditions, and recovering the bispecific antibody from the cell culture.
  • a conjugate which includes a bispecific antibody and a coupling part, wherein the bispecific antibody is the bispecific antibody of the present invention, and the coupling part is a detectable label;
  • the coupling part is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.
  • kits which includes the bispecific antibody of the present invention, or includes the conjugate of the present invention; preferably, the kit further includes a second antibody, which specifically recognizes the bispecific antibody Antibody;
  • the second antibody also includes a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.
  • Another aspect of the present invention relates to the use of the bispecific antibody of the present invention in the preparation of a kit for detecting the presence or level of CD73 and/or PD-1 in a sample.
  • Another aspect of the present invention relates to a pharmaceutical composition, which comprises the bispecific antibody of the present invention or the conjugate of the present invention; optionally, it also includes a pharmaceutically acceptable carrier and/or excipient.
  • Another aspect of the present invention relates to the use of the bispecific antibody of the present invention or the conjugate of the present invention in the prevention and/or treatment of tumors or anemia, or the use of diagnosing tumors or anemia.
  • Another aspect of the present invention relates to the use of the bispecific antibody of the present invention or the conjugate of the present invention in the preparation of drugs for the prevention and/or treatment of tumors or anemia, or in the preparation of drugs for the diagnosis of tumors or anemia. use.
  • Another aspect of the present invention relates to the use of the bispecific antibody of the present invention or the conjugate of the present invention in the preparation of the following drugs:
  • Drugs that regulate e.g. down-regulate the activity or level of PD-1
  • Another aspect of the present invention relates to an in vivo or in vitro method, including the steps of applying cells or administering to a subject in need an effective amount of the bispecific antibody of the present invention or the conjugate of the present invention,
  • the anti-CD73-anti-PD-1 bispecific antibodies involved in the present invention can all inhibit the CD73 enzyme activity on the cell membrane surface, and can all induce the secretion of IFN ⁇ and IL-2, and activate the immune response.
  • variable regions of the light chain and the heavy chain determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, called the complementarity determining region (CDR) (the CDR of the heavy chain (H) includes HCDR1, HCDR2, HCDR3
  • CDR of the light chain (L) includes LCDR1, LCDR2, LCDR3; they are named by Kabat et al., see Bethesda Md, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3: 91-3242.
  • CDR can also be defined by the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc M P.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J].
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7.
  • amino acid sequences of the 3 CDRs of the variable region of the heavy chain are as follows:
  • HCDR1 GYSFTGYT (SEQ ID NO: 3),
  • HCDR2 INPYNAGT (SEQ ID NO: 4),
  • HCDR3 ARSEYRYGGDYFDY (SEQ ID NO: 5);
  • amino acid sequences of the 3 CDRs of the light chain variable region are as follows:
  • LCDR1 QSLLNSSNQKNY (SEQ ID NO: 8),
  • LCDR2 FAS (SEQ ID NO: 9),
  • LCDR3 QQHYDTPYT (SEQ ID NO: 10).
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22.
  • amino acid sequence of the three CDRs in the variable region of the heavy chain is identical to 19F3.
  • amino acid sequence of the 3 CDRs of the light chain variable region is the same as 19F3.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 24.
  • amino acid sequence of the three CDRs in the variable region of the heavy chain is identical to 19F3.
  • amino acid sequence of the 3 CDRs of the light chain variable region is the same as 19F3.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 49.
  • amino acid sequences of the 3 CDRs of the variable region of the heavy chain are as follows:
  • HCDR1 GFAFSSYD (SEQ ID NO: 45)
  • HCDR2 ISGGGRYT (SEQ ID NO: 46)
  • HCDR3 ANRYGEAWFAY (SEQ ID NO: 47)
  • amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:
  • LCDR1 QDINTY (SEQ ID NO: 50)
  • LCDR2 RAN (SEQ ID NO: 51)
  • LCDR3 LQYDEFPLT (SEQ ID NO: 52)
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 62
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 64.
  • amino acid sequence of the three CDRs in the variable region of the heavy chain is identical to that of 14C12.
  • amino acid sequence of the 3 CDRs of the light chain variable region is the same as 14C12.
  • amino acid sequences of the 9 CDRs contained in the heavy chains of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are the same as those of the 13F9 heavy chain, 14C12 heavy chain and 14C12 light chain regions according to the N-terminal to C-terminal sequence. According to the aforementioned arrangement sequence is as follows:
  • HCDR1 GYSFTGYT (SEQ ID NO: 3)
  • HCDR2 INPYNAGT (SEQ ID NO: 4)
  • HCDR3 ARSEYRYGGDYFDY (SEQ ID NO: 5)
  • HCDR4 GFAFSSYD (SEQ ID NO: 45)
  • HCDR5 ISGGGRYT (SEQ ID NO: 46)
  • HCDR6 ANRYGEAWFAY (SEQ ID NO: 47)
  • HCDR8 RAN (SEQ ID NO: 51)
  • HCDR9 LQYDEFPLT (SEQ ID NO: 52)
  • amino acid sequence of the three CDRs of the light chain is consistent with the amino acid sequence of the three CDRs of the 19F3 light chain, and the sequence is as follows:
  • LCDR1 QSLLNSSNQKNY (SEQ ID NO: 8)
  • LCDR2 FAS (SEQ ID NO: 9)
  • LCDR3 QQHYDTPYT (SEQ ID NO: 10).
  • Another aspect of the present invention relates to the hybridoma cell line LT014, which is deposited in the China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: C2018137.
  • CTCC China Center for Type Culture Collection
  • Another aspect of the present invention relates to the hybridoma cell line LT003, which is deposited in the China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: C2015105.
  • CTCC China Center for Type Culture Collection
  • EC 50 refers to the concentration for 50% of maximal effect (concentration for 50% of maximal effect), which refers to the concentration that can cause 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain).
  • Antibody light chains can be classified into kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antibody binding site.
  • the allocation of amino acids to each region or domain follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): Definition of D301-D307.
  • the heavy chain may also include more than 3 CDRs, such as 6, 9, or 12 CDRs.
  • the heavy chain may be a ScFv connected to the C-terminus of the heavy chain of an IgG antibody. In this case, the heavy chain contains 9 CDRs.
  • antibody is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, in addition to natural mutations that may occur spontaneously, A group of identical antibody molecules.
  • the monoclonal antibody has high specificity for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256(5517): 495), but it can also be obtained by recombinant DNA technology (for example, see US Patent 4,816,567).
  • humanized antibody means that all or part of the CDR region of a human immunoglobulin (acceptor antibody) is replaced by a CDR region of a non-human antibody (donor antibody).
  • the donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the expected specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by corresponding non-human antibody amino acid residues, or by other antibody amino acid residues, to further improve or optimize the performance of the antibody.
  • the antigen-binding antibody fragment is a bis (Diabodies), wherein the V H and V L, domains are expressed on a single polypeptide chain, but using a linker that is too short to not allow the two domains on the same chain Pairing between the domains to force the domain to pair with the complementary domain of the other chain and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90: 6444 -6448 and Poljak RJet al., Structure 1994; 2: 1121-1123).
  • single chain fragment variable refers to an antibody heavy chain variable region (V H ) and an antibody light chain variable region (V L ) Numerator. Wherein V L and V H domains by a linker makes it possible to produce a single polypeptide chain pair to form monovalent molecules (see, e.g., Bird et al, Science 1988; 242: 423-426 and Huston et al, Proc.Natl. Acad. Sci. USA 1988; 85: 5879-5883).
  • Such scFv molecules can have the general structure: NH2-V L - linker segment -V H -COOH or NH2-V H - linker segment -V L -COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448).
  • Other linkers that can be used in the present invention are described by Alfthan et al, Protein Eng. 1995; 8: 725-731, Choi et al, Eur. J. Immunol. 2001; 31: 94-106, Hu et al, Cancer Res. 1996; 56: 3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293: 41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59.
  • isolated refers to those obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolation a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 fruit fly cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed.
  • an antibody that specifically binds to a certain antigen means that the antibody has a concentration of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, The affinity (K D ) of 10 -8 M, 10 -9 M, or 10 -10 M or less binds the antigen.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the antibody has a dissociation equilibrium constant (K D ) less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less Binding antigen (for example, PD-1 protein).
  • K D can be determined using methods known to those skilled in the art, for example, using a Fortebio molecular interaction analyzer.
  • amino acids are usually represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but not limited to: pH regulators, surfactants, adjuvants, ions Strength enhancer.
  • pH adjusting agents include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., tumor
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent a patient who already has a disease. The amount of disease and its complications. It is completely within the abilities of those skilled in the art to determine such an effective amount.
  • the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general conditions such as age, weight and sex, the way the drug is administered, and other treatments that are administered at the same time and many more.
  • the monoclonal antibody of the present invention (such as 13F9H2L3) can specifically bind to CD73, and can effectively inhibit the enzymatic reaction of CD73 in a non-substrate competition manner, reduce the production of adenosine, and promote T cell activity And tumor suppressor effect.
  • the bispecific antibodies involved in the present invention such as NTPDV1, NTPDV2, NTPDV3, NTPDV4 can specifically bind to PD-1 and CD73, and can block the binding of PD-1 and PDL1 very effectively, and specifically release PD- 1Immune suppression of the body and inhibition of the catalytic activity of CD73, relieve the suppression of immune cells by adenosine, activate T lymphocytes, and do not cause the release of cytokines IL-8 and IL-6, effectively increasing safety and effectiveness .
  • the bifunctional antibody of the present invention has the potential to be applied to the preparation of anti-tumor drugs.
  • ELISA detects the binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1, Nivolumab and PD-1-mFc.
  • ELISA detects the binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3, MEDI9447 and human NT5E-Biotin.
  • FACS detects the binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 14C12H1L1 to PD-1 on the surface of 293T-PD1 cells.
  • Figure 14 Detection of the biological activity of the anti-CD73-anti-PD-1 bispecific antibody to promote IL-2 secretion by the Raji-PDL1 mixed lymphatic response system.
  • Figure 15 Detection of the biological activity of the anti-CD73-anti-PD-1 bispecific antibody in promoting the secretion of IFN- ⁇ in the mixed lymphatic response system of DC.
  • Figure 23 19F3H2L3(hG1M) and human NT5E(1-552)-his affinity constant determination.
  • Figure 25 Determination of the affinity constant between NTPDV2 and human NT5E(1-552)-his.
  • Figure 28 Detection of the anti-CD73-anti-PD-1 bispecific antibody inhibiting the CD73 enzymatic activity on the U87-MG cell membrane surface.
  • Figure 29 Mixed lymphocyte reaction MLR to detect the biological activity of anti-CD73-anti-PD-1 bispecific antibody to promote the secretion of IFN- ⁇ and IL-2.
  • FIG. 30 The effect of isotype control, 19F3H2L3 (hG1M), and different doses of NTPDV2 on tumor volume in mice.
  • FIG. 31 The effect of isotype control, 19F3H2L3 (hG1M), and different doses of NTPDV2 on the body weight of mice.
  • FIG. 32 In the co-culture system of CHO-K1-PD1 cells and human macrophages, Fc amino acid mutations effectively eliminated IL-8 secretion by human macrophages mediated by the PD-1/CD73 bispecific antibody.
  • FIG. 33 In the co-culture system of CHO-K1-PD1 cells and human macrophages, Fc amino acid mutations effectively eliminated IL-6 secretion by human macrophages mediated by the PD-1/CD73 bispecific antibody.
  • FIG. 34 In the co-culture system of U87-MG cells and human macrophages, Fc amino acid mutations effectively eliminated IL-8 secretion by human macrophages mediated by the PD-1/CD73 bispecific antibody.
  • FIG. 35 In the co-culture system of U87-MG cells and human macrophages, Fc amino acid mutations effectively eliminated IL-6 secretion by human macrophages mediated by the PD-1/CD73 bispecific antibody.
  • Hybridoma cell line LT003 also known as PD-1-14C12
  • CTCC China Center for Type Culture Collection
  • the deposit number is CCTCC NO: C2015105
  • the deposit address is Wuhan, China. Wuhan University, Zip Code: 430072.
  • Hybridoma cell line LT014 also known as CD73-19F3
  • CTCC China Center for Type Culture Collection
  • the deposit number is CCTCC NO: C2018137
  • the deposit address is Wuhan University, Wuhan, China , Zip Code: 430072.
  • the BALB/c mice used were purchased from Guangdong Medical Experimental Animal Center.
  • the positive control antibody MEDI9447 (generic name: Oleclumab) used is produced by Zhongshan Kangfang Biomedical Co., Ltd., and its sequence is the same as that described in the Medlmmune Limited public patent, publication number: US 20160129108A1
  • the antibody SEQ ID NOs: 21-24 are the same.
  • Nivolumab a marketed drug antibody with the same target, with the trade name Opdivo, was purchased from Bristol-Myers Squibb.
  • the 293T-PD1 cell line used was constructed by Zhongshan Kangfang Biomedicine Co., Ltd.
  • the 293T-PD1 cell line is prepared by virus infection of HEK293T cells.
  • the virus preparation uses 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Manage RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998.72(11): 8463-8471.
  • the lentiviral expression vector used is pCDH-CMV-PD-1FL-Puro (where PD1, Genebank ID: NM_005018; vector pCDH- CMV-Puro, purchased from Ubao Bio, product number: VT1480).
  • the Raji-PDL1 cell line used was constructed by Zhongshan Kangfang Biomedicine Co., Ltd.
  • the Raji-PDL1 cell line is prepared by virus infection from Raji cells.
  • the virus preparation uses 3rd Generation Lentiviral Systems. See, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Manage RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998.72(11): 8463-8471.
  • the lentiviral expression vector used is plenti6.3-PDL1 (where PDL1, Genebank ID: NP_054862.1; vector plenti6.3, Purchased from Invitrogen, article number: K5315-20).
  • the CHO-K1-PD1 cell line used was constructed by Zhongshan Kangfang Biomedicine Co., Ltd.
  • the CHO-K1-PD1 cell line is made from CHO-K1 cells through virus infection.
  • the virus preparation uses 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M , Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998.72(11): 8463-8471.
  • the lentiviral expression vector used is pCDH-CMV-PD-1FL-Puro (where PD1, Genebank ID: NM_005018 ; Vector pCDH-CMV-Puro, purchased from Ubao Bio, product number: VT1480).
  • the IgG4 subtype anti-PD-1 antibody Nivolumab (trade name Opdivo) carrying the S228P mutation was used as the control antibody, all of which were purchased from Bristol-Myers Squibb.
  • the isotype control antibodies used namely hIgG1
  • HEL human anti-egg lysosome
  • the variable region sequence of the antibody comes from Affinity maturation increases published by Acierno et al. the stability and plasticity of the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol.
  • hIgG1 uses Ig gamma-1 chain C region, ACCESSION : P01857 is the heavy chain constant region, Ig kappa chain C region, ACCESSION: P01834 is the light chain constant region; hIgG1 is prepared in the laboratory of Zhongshan Kangfang Biomedical Co., Ltd.
  • the antigen used to prepare the anti-CD73 antibody is human NT5E-his (NT5E is GenbankID: NP_002517.1, position: 1-552).
  • the spleen cells of the immunized mice were fused with mouse myeloma cells to make hybridoma cells.
  • Human NT5E (NT5E is GenbankID: NP_002517.1, position: 1-552)-Biotin was used as the antigen to perform hybridoma cell analysis.
  • Screening by indirect ELISA method to obtain hybridoma cells capable of secreting antibodies that specifically bind to CD73.
  • a stable hybridoma cell line was obtained through the limiting dilution method.
  • the above hybridoma cell line was named hybridoma cell line LT014, and the monoclonal antibody secreted by it was named 19F3.
  • Hybridoma cell line LT014 also known as CD73-19F3
  • CTCC China Center for Type Culture Collection
  • the deposit number is CCTCC NO: C2018137
  • the deposit address is Wuhan University, Wuhan, China , Zip Code: 430072.
  • CD medium Cosmetic Defined Medium
  • penicillin cultured in a 5% CO 2 , 37°C cell incubator
  • HiTrap protein A HP column was prepared by high-speed centrifugation, vacuum filtration with a microporous membrane, and HiTrap protein A HP column to prepare antibody 19F3.
  • mRNA was extracted from the LT014 cell line cultured in Example 1 respectively.
  • the PCR amplification product is directly subjected to TA cloning, and the specific operation refers to the pEASY-T1 Cloning Kit (Transgen CT101) kit manual.
  • the TA cloned product was directly sequenced, and the sequencing results are as follows:
  • the nucleic acid sequence (363bp) of the variable region of the 19F3 heavy chain is shown in SEQ ID NO:1, and the encoded amino acid sequence (121aa) is shown in SEQ ID NO:2.
  • the sequence of the heavy chain CDR1 is shown in SEQ ID NO: 3
  • the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 4
  • the sequence of the heavy chain CDR3 is shown in SEQ ID NO: 5.
  • the nucleic acid sequence (339bp) of the variable region of the 19F3 light chain is shown in SEQ ID NO: 6, and the encoded amino acid sequence (113aa) is shown in SEQ ID NO: 7.
  • the sequence of light chain CDR1 is shown in SEQ ID NO: 8
  • the sequence of light chain CDR2 is shown in SEQ ID NO: 9
  • the sequence of light chain CDR3 is shown in SEQ ID NO: 10.
  • the amino acid sequences of the 4 framework regions (FR-H1 to FR-H4) of the 19F3 heavy chain are shown in SEQ ID NO: 11 to SEQ ID NO: 14, respectively; the 4 framework regions of the 19F3 light chain (Framework region) ( The amino acid sequences of FR-H1 to FR-H4) are shown in SEQ ID NO: 15 to SEQ ID NO: 18, respectively.
  • Example 3 Design, preparation and detection of humanized antibodies against human CD73
  • variable region sequences of the antibodies 19F3H1L1, 19F3H2L3 and 19F3H2L3 were obtained, and their corresponding heavy chain variable
  • the sequence of the region is 19F3H1, 19F3H2 (amino acid sequence is shown in SEQ ID NO: 93, SEQ ID NO: 97), and the light chain variable region sequence is 19F3L1, 19F3L2, 19F3L3 (amino acid sequence is shown in SEQ ID NO: 95, SEQ ID NO: 98, SEQ ID NO: 99), antibody constant region sequence, from NCBI database, heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; light chain constant region is Ig kappa chain C region, ACCESSION: P01834.
  • 19F3H2L3 is written as 19F3H2L3 (hG1WT) in the Chinese invention patent with the application number 202110270671.
  • X of which the light and heavy chain variable regions of 19F3H1L1, 19F3H2L2, 19F3H2L3 can be recorded as 19F3H1V (or 19F3H1 V2V), 19F3H1 V2 (or 19F3H1 V2).
  • 19F3H2 V 19F3L1V (or 19F3L1 V ), 19F3L2V (or 19F3L2 V ), 19F3L3V (or 19F3L3 V ).
  • the nucleic acid sequence (363bp) of the heavy chain variable region is shown in SEQ ID NO: 92, and the encoded amino acid sequence (121aa) is shown in SEQ ID NO: 93.
  • the nucleic acid sequence (339bp) of the light chain variable region is shown in SEQ ID NO: 94, and the encoded amino acid sequence (113aa) is shown in SEQ ID NO: 95.
  • the nucleic acid sequence (363bp) of the heavy chain variable region 19F3H2 is shown in SEQ ID NO: 19, and the encoded amino acid sequence (121aa) is shown in SEQ ID NO: 20.
  • the nucleic acid sequence (339bp) of the light chain variable region 19F3L3 is shown in SEQ ID NO: 21, and the encoded amino acid sequence (113aa) is shown in SEQ ID NO: 22.
  • the nucleic acid sequence (363bp) of the heavy chain variable region 19F3H2 is shown in SEQ ID NO: 19, and the encoded amino acid sequence (121aa) is shown in SEQ ID NO: 20.
  • the nucleic acid sequence (339bp) of the light chain variable region 19F3L3 is shown in SEQ ID NO: 23, and the encoded amino acid sequence (113aa) is shown in SEQ ID NO: 24.
  • the heavy chain constant region uses Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region uses Ig kappa chain C region, ACCESSION: P01834.
  • the heavy chain cDNA and light chain cDNA of 19F3H1L1, 19F3H2L2, and 19F3H2L3 were cloned into the pUC57simple (provided by GenScript) vector to obtain pUC57simple-19F3H1, pUC57simple-19F3L1, pUC57simple-19F3H2, pUC57simple-19F3L2, respectively -19F3L3.
  • the full-length heavy and light chain genes synthesized by EcoRI&HindIII digestion are subcloned into the expression vector pcDNA3.1 by restriction enzymes (EcoRI&HindIII) to obtain expression.
  • the inventor introduced a leucine to alanine point mutation (L234A) at position 235 of its heavy chain at position 234
  • L234A leucine to alanine point mutation
  • the point mutation (L235A) from leucine to alanine was introduced, and the heavy chain nucleotide and amino acid sequences of 19F3H2L3 (hG1M) and 19F3H2L3 (hG1M) were obtained as shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
  • the inventor introduced a leucine to alanine point mutation (L234A) at position 235 of its heavy chain at position 234
  • L235A The point mutation from leucine to alanine
  • G237A the point mutation from glycine to alanine
  • hG1TM 19F3H2L3
  • Its heavy chain amino nucleotides and The base acid sequence is shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively; its light chain is consistent with 19F3H2L3 (hG1M).
  • amino acid sequences of the 4 Framework regions (FR-H1 to FR-H4) of 19F3H2 are shown in SEQ ID NO: 31 to SEQ ID NO: 34, respectively;
  • amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3L2 light chain are shown in SEQ ID NO: 35 to SEQ ID NO: 38, respectively;
  • amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3L3 light chain are shown in SEQ ID NO: 39 to SEQ ID NO: 42, respectively.
  • the heavy chain cDNA and light chain cDNA of 19F3H2L3 were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-19F3H2 (hG1M) and pUC57simple-19F3L3, respectively.
  • pUC57simple provided by GenScript
  • the full-length heavy and light chain genes synthesized by EcoRI&HindIII digestion are subcloned into the expression vector pcDNA3.1 by restriction enzymes (EcoRI&HindIII) to obtain expression.
  • PD-1-mFc fusion protein (PD-1 GenBank: NM_005018, mFc SEQ ID NO: 89) as the antigen
  • BALB/c mice purchased from Guangdong Medical Experimental Animal Center
  • mouse myeloma were collected Cell fusion into hybridoma cells, refer to the established methods (for example, Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. GC Howard and DRBethell, Boca Raton: CRC Press, 2000 ).
  • PD-1-hFc (PD-1 Genbank ID: NM_005018, hFc is human IgG Fc purification tag, specifically Ig gamma-1 chain C region, GenbankID: P01857 position 114-330) as the antigen-coated microtiter plate for indirect Screened by ELISA method to obtain hybridoma cells secreting new antibodies specifically binding to PD-1.
  • the hybridoma cell line that can secrete the monoclonal antibody that competes with the ligand PD-L1-hFc (PD-L1 Genbank ID: NP_054862.1) to bind to PD-1 was screened by competitive ELISA, and a stable hybridoma was obtained by the limiting dilution method.
  • Cell line, and the LT003 stable cell line (PD-1-14C12) was obtained by the limiting dilution method, and the monoclonal antibody secreted by it was named 14C12.
  • Hybridoma cell line LT003 also known as PD-1-14C12
  • CTCC China Center for Type Culture Collection
  • the deposit number is CCTCC NO: C2015105
  • the deposit address is Wuhan, China. Wuhan University, Zip Code: 430072.
  • the LT003 cell line prepared above was cultured with IMDM medium containing 10% low IgG fetal bovine serum (IMDM medium, containing 1% penicillin, in 5% CO 2 , 37°C cell incubator Culture). After 7 days, the cell culture supernatant was collected and purified to prepare antibody 14C12.
  • IMDM medium 10% low IgG fetal bovine serum
  • the mRNA was extracted from the hybridoma cell line LT003 prepared in Example 1 according to the method of the cultured cell bacterial total RNA extraction kit (Tiangen, article number DP430).
  • the PCR amplification product is directly subjected to TA cloning, and the specific operation refers to the pEASY-T1 Cloning Kit (Transgen CT101) kit manual.
  • the TA cloned product was directly sequenced, and the sequencing results are as follows:
  • the nucleic acid sequence (354bp) of the heavy chain variable region is shown in SEQ ID NO: 43, and the encoded amino acid sequence (118aa) is shown in SEQ ID NO: 44.
  • the sequence of the heavy chain CDR1 is shown in SEQ ID NO: 45
  • the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 46
  • the sequence of the heavy chain CDR3 is shown in SEQ ID NO: 47.
  • the nucleic acid sequence (321 bp) of the light chain variable region is shown in SEQ ID NO: 48, and the encoded amino acid sequence (107aa) is shown in SEQ ID NO: 49.
  • the light chain CDR1 sequence is shown in SEQ ID NO: 50
  • the light chain CDR2 sequence is shown in SEQ ID NO: 51
  • the light chain CDR3 sequence is shown in SEQ ID NO: 52.
  • the amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 14C12 heavy chain are shown in SEQ ID NO: 53 to SEQ ID NO: 56; the four framework regions of the 14C12 light chain (Framework region) ( The amino acid sequences of FR-H1 to FR-H4) are shown in SEQ ID NO: 57 to SEQ ID NO: 60, respectively.
  • Example 6 Design and preparation of anti-PD-1 humanized antibodies 14C12H1L1 and 14C12H1L1 (hG1TM)
  • the design of the light chain and heavy chain sequences of the humanized antibody 14C12H1L1 is based on the three-dimensional crystal structure of the PD-1 protein (Shinohara T, et al., Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics 1995, 23(3):704-6) and the sequence of the antibody 14C12 obtained in Example 5, the variable region sequence of the antibody 14C12H1L1 was obtained by simulating the antibody model by computer and designing mutations according to the model.
  • the designed variable region sequence is as follows:
  • the nucleic acid sequence (354 bp) of the heavy chain variable region 14C12H1 of the humanized monoclonal antibody 14C12H1L1 is shown in SEQ ID NO: 61, and the encoded amino acid sequence (118aa) is shown in SEQ ID NO: 62.
  • the nucleic acid sequence (321 bp) of the light chain variable region 14C12L1 of the humanized monoclonal antibody 14C12H1L1 is shown in SEQ ID NO: 63, and the encoded amino acid sequence (107aa) is shown in SEQ ID NO: 64.
  • the antibody constant region sequence of antibody 14C12H1L1 uses Ig gamma-1 chain C region, ACCESSION: P01857; light chain constant region is Ig kappa chain C region, ACCESSION: P01834), 14C12H1L1 heavy chain
  • the nucleotide sequence and amino acid sequence are shown in SEQ ID NOs: 65 and 66, respectively, and the nucleotide sequence and amino acid sequence of the 14C12H1L1 light chain are shown in SEQ ID NOs: 67 and 68, respectively.
  • 14C12H1L1 is written as 14C12H1L1 (hG1WT) in this article and the Chinese invention patent with application number 202110270671.X.
  • the heavy chain variable region and light chain variable region of 14C12H1L1 are in this paper with application number 202110270671.
  • the Chinese invention patents are also written as 14C12H1V (or 14C12H1 V ) and 14C12L1 V (or 14C12L1 V ).
  • the inventor introduced a leucine-to-alanine point mutation (L234A) at position 234 of its heavy chain, position 235 A point mutation from leucine to alanine (L235A) was introduced, and a point mutation from glycine to alanine (G237A) was introduced at position 237 to obtain the antibody 14C12H1L1 (hG1TM).
  • L234A leucine-to-alanine point mutation
  • G237A point mutation from glycine to alanine
  • the nucleotide and amino acid sequences of the heavy chain of 14C12H1L1 (hG1TM) are shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; the amino acid sequence of the light chain variable region is consistent with that of the antibody 14C12H1L1.
  • amino acid sequences of the four framework regions (FR-H1 to FR-H4) of 14C12H1 are shown in SEQ ID NO: 71 to SEQ ID NO: 74, respectively;
  • amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 14C12L1 light chain are shown in SEQ ID NO: 75 to SEQ ID NO: 78, respectively;
  • the heavy chain cDNA and light chain cDNA of 14C12H1L1 (hG1TM) and 14C12H1L1 were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-14C12H1, pUC57simple-14C12L1 and pUC57simple-14C12H1 (hG1TM).
  • pUC57simple provided by GenScript
  • pUC57simple-14C12H1 obtained by GenScript
  • pUC57simple-14C12H1 obtained by GenScript
  • the full-length heavy and light chain genes synthesized by EcoRI&HindIII digestion are subcloned into the expression vector pcDNA3.1 by restriction enzymes (EcoRI&HindIII) to obtain expression.
  • the structural mode of the bifunctional antibody in the present invention belongs to the Morrison mode (IgG-scFv), that is, the C-terminus of the two heavy chains of an IgG antibody are connected to the scFv fragment of the other antibody.
  • the main components of the heavy chain and light chain are The design is shown in Table 1 below.
  • variable region of the corresponding heavy chain or the variable region of the corresponding light chain refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. Without “V”, the corresponding heavy chain or light chain is the full length including the constant region.
  • the amino acid sequence of Linker1 is (GGGGS)4((nucleotide sequence SEQ ID NO: 80, amino acid sequence SEQ ID NO: 79), and the amino acid sequence of Linker2 is (GGGGS) 3((nucleotide sequence SEQ ID NO: 82, amino acid sequence SEQ ID NO: 81).
  • the heavy chain cDNA sequence and the light chain cDNA sequence of P1D7V01 were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-VP101H and pUC57simple-VP101L plasmids, respectively.
  • the plasmids pUC57simple-VP101H and pUC57simple VP101L were digested (HindIII&EcoRI), and the heavy and light chains recovered by electrophoresis were subcloned into pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After the cells were cultured for 7 days, the culture solution was centrifuged at a high speed, the supernatant was concentrated, and then loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer and the target sample was recovered and the medium was changed to PBS.
  • Purified antibodies P1D7V02R, P1D7V03, P1D7V04R, P1D7V07, P1D7V08 were obtained according to the above-mentioned P1D7V01 expression and purification method.
  • Example 8 ELISA method to determine the binding activity of anti-CD73-anti-PD-1 bispecific antibody to antigen
  • ELISA method was used to determine the binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and the antigen PD-1-mFc.
  • the specific method is as follows:
  • test results are shown in Table 2 and Figure 1. It can be seen from the figure that P1D7V01, P1D7V02R, P1D7V03, P1D7V04R can effectively bind to the antigen PD-1-mFc, and the binding efficiency is dose-dependent.
  • the absorbance intensity of each dose is shown in Table 2, and the absorbance of the bound antibody is quantitatively analyzed.
  • Curve simulation calculation obtained the binding efficiency EC50 of antibodies P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab (as a control) as 0.078nM, 0.078nM, 0.075nM and 0.089nM, 0.033nM, 0.051nM, respectively.
  • the streptavidin, 2 ⁇ g/ml was coated on the ELISA plate and incubated overnight at 4°C. After the incubation, the ELISA plate coated with streptavidin was washed once with PBST, 1% BSA in PBS was used as the ELISA plate blocking solution, and the ELISA plate was blocked at 37°C for 2 hours. Wash the plate with PBST three times after the end of the plate. Then add the antigen human NT5E-Biotin 0.5 ⁇ g/ml and incubate at 37°C for 30 minutes, then wash the plate with PBST 3 times.
  • the plate was washed 4 times with PBST, and then TMB (Neogen, 308177) was added for 5 minutes to develop color in the dark, and stop solution was added to terminate the color reaction.
  • TMB Neogen, 308177
  • stop solution was added to terminate the color reaction.
  • the microplate select the 450nm light wavelength to read the OD value of each well of the microplate.
  • SoftMax Pro 6.2.1 software to analyze and process the data.
  • Example 9 Competitive ELISA method was used to determine the activity of the antibody anti-CD73-anti-PD-1 bispecific antibody and human PD-L1-mFc to compete with human PD-1-mFc-Biotin
  • Human PD-L1-mFc (PD-L1 Genbank ID: NP_054862.1, mFc SEQ ID NO: 143) was coated with 2 ⁇ g/mL microtiter plate, and incubated overnight at 4°C. After the incubation, the ELISA plate was blocked with 1% BSA in PBS at 37°C for 2 hours. After the blocking, the plate was washed three times and patted dry.
  • the plate was washed four times and patted dry, and then TMB (Neogen, 308177) was added for 5 minutes to develop color in the dark, and a stop solution was added to terminate the color reaction.
  • TMB Neogen, 308177
  • test results are shown in Figure 3.
  • the OD value of each dose is shown in Table 4.
  • the curve simulates the binding efficiency of the antibody to obtain the binding EC50 (Table 4).
  • Example 10 Determination of the kinetic parameters of the binding of anti-CD73-anti-PD-1 bispecific antibody to antigen human PD-1-mFc using Fortebio molecular interaction instrument
  • the sample dilution buffer is PBST, 0.1% BSA, pH 7.4.
  • the antibody is fixed on the AHC sensor at a concentration of 5 ⁇ g/mL, the fixed height is about 0.4nm, the sensor is equilibrated in the buffer for 60s, the antibody fixed on the sensor is bound to the antigen PD-1-mFc, and the antigen concentration is 0.6-50nM (Three-fold dilution), the binding time is 120s, and the protein dissociates in the buffer, the time is 180s.
  • the detection temperature is 37 degrees, the detection frequency is 0.3 Hz, and the sample plate vibration rate is 1000 rpm.
  • the data is analyzed by 1:1 model fitting, and the affinity constant is obtained.
  • the determination results of the affinity constants of humanized antibodies P1D7V01, 14C12H1L1 and Nivolumab (as control antibodies) with human PD-1-mFc are shown in Table 5, and the detection results are shown in Figs. 4, 5 and 6.
  • the affinity constants of humanized antibodies P1D7V01, 14C12H1L1 and Nivolumab to human PD-1-mFc are 1.76E-10M, 1.64E-10M, and 2.32E-10M, respectively.
  • the above experimental results show that the binding ability of P1D7V01 and 14C12H1L1 and Nivolumab is equivalent, suggesting that the humanized antibody P1D7V01 has a strong binding ability to human PD-1-mFc.
  • K D kdis/kon
  • Example 11 Determination of the kinetic parameters of the binding of the anti-CD73-anti-PD-1 bispecific antibody to the antigen human NT5E(1-552)-his using the Fortebio molecular interaction instrument.
  • the sample dilution buffer is PBST, pH 7.4.
  • the antibody was immobilized on the Protein A sensor at a concentration of 5 ⁇ g/mL. The immobilization time was 15s.
  • the sensor was equilibrated in buffer for 120s.
  • the antibody immobilized on the sensor combined with the antigen human NT5E(1-552)-his, and the antigen concentration was 3. 125-200nM (two-fold dilution), binding time is 120s, protein dissociates in buffer, time 600s.
  • the sensor is regenerated with 10mM Gly, pH1.5 solution.
  • the detection temperature is 37 degrees, the detection frequency is 0.6 Hz, and the sample plate vibration rate is 1000 rpm.
  • the data is analyzed with a 1:1 model to get the affinity constant
  • the determination results of the affinity constants of humanized antibodies P1D7V01, MEDI 9447 (as a control antibody) and human NT5E(1-552)-his are shown in Table 6, and the detection results are shown in Figures 7 and 8.
  • the affinity constants of humanized antibodies P1D7V01 and MEDI 9447 to human NT5E(1-552)-his are 2.29E-10M and 1.04E-10M, respectively.
  • K D kdis/kon
  • Example 12 FACS detection of the binding activity of anti-CD73-anti-PD-1 bispecific antibodies
  • FACS detects the binding activity of anti-CD73-anti-PD-1 bispecific antibody to PD-1 on the surface of 293T-PD1 membrane
  • P1D7V01, P1D7V02R, P1D7V03, P1D7V04R can specifically bind to PD-1 on the surface of 293T-PD1 membrane in a dose-dependent manner. Compared with the single-target PD1 control antibody 14C12H1L1, Both are stronger than 14C12H1L1.
  • the EC50 of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R combined with 293T-PD1 were 1.000nM, 1.075nM, 1.377nM, 1.57nM, and the EC50 of 14C12H1L1 combined with 293T-PD1 was 2.111nM.
  • Table 7 FACS detection of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 14C12H1L1 and 293T-PD1 cell surface PD-1 binding activity
  • FACS detects the binding activity of anti-CD73-anti-PD-1 bispecific antibody to CD73 on the surface of MDA-MB-231 membrane
  • the experimental results are shown in Table 8 and Figure 10.
  • the binding activity of CD73 on the surface of MDA-MB-231 membrane, P1D7V01 and P1D7V03 are better than 19F3H2L3, and P1D7V01 is better than the same target positive drug MEDI9447.
  • the EC50s of P1D7V01, P1D7V03 and CD73 on the surface of MDA-MB-231 membrane were 1.384nM and 2.009nM, respectively, and the EC50s of MEDI9447 and 19F3H2L3 bound to CD73 on the surface of MDA-MB-231 membrane were 1.589nM and 2.773, respectively nM.
  • Table 8 FACS detects the binding activity of P1D7V01, P1D7V03, MEDI9447 and 19F3H2L3 to CD73 on the surface of MDA-MB-231 cells.
  • Example 13 Detection of anti-CD73-anti-PD-1 bispecific antibody inhibiting CD73 enzyme activity on the cell membrane surface
  • the experimental procedure is as follows: Take MDA-MB-231 cells in good logarithmic phase, resuspend and count the cells in serum-free RPMI-1640 medium; inoculate MDA-MB-231 cells into a 96-well plate, 2x104 cells /100 ⁇ L/well; use serum-free RPMI-1640 culture medium to dilute the antibody by 2.5 times; add the antibody to a 96-well plate, 50 ⁇ L per well, incubate at 37°C for 1 hour, after 1 hour, add 50 ⁇ L 1200 ⁇ M RPMI-1640 to each well Diluted AMP (TCL, catalog number: A0157); after 3 hours, take 25 ⁇ L of cell culture supernatant, transfer to a new 96-well plate, add 25 ⁇ L 100 ⁇ M of ATP (TCL, catalog number: A0158) to each well; add 50 ⁇ L CTG (CellTiterGlo) , Promega, item number: G8641) color developing solution, perform color development, and read the relative
  • Example 14 Detection of the biological activity of the anti-CD73-anti-PD-1 bispecific antibody to promote the secretion of IFN- ⁇ and IL-2 by the mixed lymphocyte reaction MLR
  • Raji-PDL1 cells were subcultured normally; resuscitated PBMCs, cultured with 10mL 1640 complete medium, and stimulated with 0.5 ⁇ g/mL SEB (Denotek, catalog number: S010201) for two days; Raji-PDL1 cells were cultured with 25 ⁇ g/mL MMC (Sigma , Item No.: M4287), placed in a 37°C incubator for 1 hour; collected PBMC after SEB stimulation for 2 days and Raji-PDL1 cells treated with MMC for 1 hour, washed twice with PBS, resuspended in complete medium, counted, and added to U-shaped 96-well plate, 100,000 cells/well; add antibodies according to the experimental design, and co-culture for 3 days in the incubator; after 3 days, collect the cell culture supernatant and perform IFN- ⁇ detection by ELISA.
  • SEB Denotek, catalog number: S010201
  • the mixed culture of human PBMC and Raji-PDL1 cells has a significant effect on the secretion of IFN- ⁇ of PBMC.
  • the simultaneous addition of antibodies in the mixed culture system can significantly induce PBMC to further secrete IFN- ⁇ , which promotes IFN- ⁇ secretion.
  • the activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R antibody and its parent PD-1 single-target antibody 14C12H1L1 are comparable.
  • Raji-PDL1 cells were subcultured normally; resuscitated PBMC, cultured with 10mL 1640 complete medium, stimulated with SEB (0.5 ⁇ g/mL) for two days; Raji-PDL1 cells were treated with 25 ⁇ g/mL MMC and placed in a 37°C incubator1 Hours; collect PBMC after 2 days of SEB stimulation and Raji-PDL1 cells treated with MMC for 1 hour, wash twice with PBS, resuspend in complete medium and count, add to U-shaped 96-well plate, 100000 cells/well; according to the experiment Designed to add antibody, co-culture for 3 days; collect cell culture supernatant, ELISA method for IL-2 detection.
  • the mixed culture of human PBMC and Raji-PDL1 cells can promote the secretion of IL-2 of PBMC to a certain extent.
  • the simultaneous addition of antibodies in the mixed culture system can significantly induce PBMC to secrete IL-2 further.
  • the bifunctional antibodies P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R at low concentrations are slightly less active than their parent PD-1 single-target antibody 14C12H1L1, while the medium and high concentrations are equivalent to PD1
  • the target positive control drug Nivolumab P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R have better IL-2 secretion potential at three different antibody concentration levels.
  • Anti-CD73-anti-PD-1 bispecific antibody promotes the biological activity of the DC mixed lymphatic reaction system to secrete IL-2. Isolate normal human peripheral blood PBMC, resuspend in complete medium, inoculate it in a petri dish, and place it in an incubator overnight.
  • Example 15 Preparation of anti-PD-1/CD73 bispecific antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4
  • the structural mode of bispecific antibodies NTPDV1, NTPDV2, NTPDV3, and NTPDV4 belongs to Morrison mode (IgG-scFv), that is, the C-terminus of the two heavy chains of one IgG antibody is connected to the scFv fragment of the other antibody through a linking fragment.
  • the design composition of the chain and light chain is shown in Table 9 below.
  • NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are written as NTPDV1 in this article and in the Chinese invention patent with application number 202110270671.X (hG1TM), NTPDV2 (hG1TM), NTPDV3 (hG1TM), NTPDV4 (hG1TM).
  • variable region of the corresponding heavy chain or the variable region of the corresponding light chain refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. Without “V”, the corresponding heavy chain or light chain is the full length including the constant region.
  • the amino acid sequence of Linker1 is (GGGGS) structure repeated 4 times, namely (GGGGS)4 or (G4S)4 (nucleotide sequence SEQ ID NO: 80, amino acid sequence SEQ ID NO: 79);
  • the amino acid sequence of Linker2 is (GGGGS) structure repeated 3 times, that is, (GGGGS)4 or (G4S)3 (nucleotide sequence SEQ ID NO: 82, amino acid sequence SEQ ID NO: 81).
  • the 3 CDR sequences of the light chain 19F3L2 and 19F3L3 in the immunoglobulin portion of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are consistent with the light chain CDR sequence of 19F3.
  • the 3 CDR sequences of 19F3H2 (hG1TM) in the immunoglobulin part of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are consistent with the heavy chain CDR sequence of 19F3.
  • the CDR sequences of 14C12H1V-Linker2-14C12L1V and 14C12H1V-Linker1-14C12L1V in the scFv part of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are consistent with the heavy chain CDR and light chain CDR of 14C12.
  • NTPDH2/4 amino acid sequences of the heavy chains of NTPDV2 and NTPDV4 are identical and are denoted as NTPDH2/4 (SEQ ID NO: 83), and the nucleotide sequences of the heavy chains of NTPDV2 and NTPDV4 are SEQ ID NO: 84.
  • NTPDH1/3 amino acid sequences of the heavy chains of NTPDV1 and NTPDV3 are identical and are denoted as NTPDH1/3 (SEQ ID NO: 85), and the nucleotide sequences of the heavy chains of NTPDV1 and NTPDV3 are SEQ ID NO: 86.
  • the 3 CDR sequences of 19F3L3 and 19F3L2 in the immunoglobulin part of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are consistent with the three CDRs of the antibody 19F3 light chain.
  • the amino acid sequence of the light chain 19F3L3 of the immunoglobulin portion of NTPDV1 and NTPDV2 is the same as the light chain sequence of the antibody 19F3H2L3 (G1M) (SEQ ID NO: 28), and the light chain of the immunoglobulin portion of NTPDV1 and NTPDV2 is the core of the light chain 19F3L3.
  • the nucleotide sequence is SEQ ID NO: 27.
  • the amino acid sequence of the light chain 19F3L2 of the immunoglobulin portion in NTPDV3 and NTPDV4 is SEQ ID NO: 96
  • the nucleotide sequence of the light chain 19F3L2 of the immunoglobulin portion in NTPDV3 and NTPDV4 is SEQ ID NO: 100.
  • NTPDV1 whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, light chain amino acid sequence is shown in SEQ ID NO: 28, and the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, the amino acid of 14C12H1V The sequence is shown in SEQ ID NO: 66, the amino acid sequence of connection fragment 2 is shown in SEQ ID NO: 81, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 68.
  • NTPDV2 the heavy chain amino acid sequence is shown in SEQ ID NO: 85
  • the light chain amino acid sequence is shown in SEQ ID NO: 28
  • the amino acid sequence of the connecting fragment 1 is shown in SEQ ID NO: 79
  • the amino acid of 14C12H1V The sequence is shown in SEQ ID NO: 66
  • the amino acid sequence of connection fragment 1 is shown in SEQ ID NO: 79
  • the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 68.
  • NTPDV3 whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, light chain amino acid sequence is shown in SEQ ID NO: 96, and the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, and the amino acid sequence of 14C12H1V
  • the sequence is shown in SEQ ID NO: 66, the amino acid sequence of connection fragment 2 is shown in SEQ ID NO: 81, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 68, and
  • NTPDV4 whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, light chain amino acid sequence is shown in SEQ ID NO: 96, and the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, the amino acid of 14C12H1V The sequence is shown in SEQ ID NO: 66, the amino acid sequence of connection fragment 1 is shown in SEQ ID NO: 79, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 68.
  • the heavy chain cDNA sequence of NTPDV1, NTPDV3, the heavy chain cDNA sequence of NTPDV2, NTPDV4, and the cDNA sequence of its light chain were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-NTPDH2/4 and pUC57simple-NTPDH1, respectively.
  • pUC57simple provided by GenScript
  • the plasmids pUC57simple-NTPDH2/4 and pUC57simple-19F3L3 plasmids, pUC57simple-NTPDH2/4 and pUC57simple-19F3L2 plasmids, pUC57simple-NTPDH1/3 and pUC57simple-19F3L3 plasmids, pUC57simple-NTPDH1/3 and pUC57simple-19F3L2 plasmids were digested respectively ( HindIII&EcoRI), the heavy and light chains recovered by electrophoresis were subcloned into the pcDNA3.1 vector to obtain pcDNA3.1-NTPDH2/4 and pcDNA3.1-19F3L3 plasmids, pcDNA3.1-NTPDH2/4 and pcDNA3.1-19F3L2 Plasmids, pcDNA3.1-NTPDH1/3 and pcDNA3.1-19F3L3 plasm
  • the culture solution was centrifuged at a high speed, the supernatant was concentrated, and then loaded onto the HiTrap MabSelect SuRe column.
  • the protein was eluted in one step with Elution Buffer and the target sample was recovered and the medium was changed to PBS.
  • NTPDV1, NTPDV2, NTPDV3, and NTPDV4 were obtained according to the expression and purification methods mentioned in the aforementioned preparation examples.
  • Example 16 ELISA method to measure the binding activity of anti-CD73-anti-PD-1 bispecific antibody to antigen
  • the test results are shown in Table 10. It can be seen that NTPDV1, NTPDV2, NTPDV3, and NTPDV4 can effectively bind to the antigen PD-1-mFc, and the binding efficiency is dose-dependent.
  • the absorbance intensity of each dose is shown in Table 10.
  • the curve Simulation calculations obtained the binding efficiency EC50 of antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1 (hG1TM) (as a control) were 0.101nM, 0.119nM and 0.110nM, 0.123nM, 0.031nM, respectively.
  • the streptavidin SA (2 ⁇ g/ml) was coated on the ELISA plate and incubated overnight at 4°C. After the incubation, the ELISA plate coated with streptavidin was washed once with PBST, 1% BSA in PBS was used as the ELISA plate blocking solution, and the ELISA plate was blocked at 37°C for 2 hours. Wash the plate with PBST three times after the end of the plate. Then add the antigen human NT5E-Biotin 0.5 ⁇ g/ml and incubate at 37°C for 30 minutes, then wash the plate with PBST 3 times.
  • NTPDV1, NTPDV2, NTPDV3, NTPDV4, 19F3H2L3 can effectively bind to the antigen human NT5E-Biotin, and the binding efficiency is dose-dependent (see Table 11 for the absorbance intensity of each dose).
  • the binding efficiency EC50 of antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4, 19F3H2L3 (hG1M) (as a control antibody) were obtained by quantitative analysis of the absorbance of the bound antibody, and curve simulation calculation was 0.079nM, 0.082nM and 0.084nM, 0.077nM, 0.029nM.
  • Example 17 Competitive ELISA method to determine the activity of anti-CD73-anti-PD-1 bispecific antibody and human PD-L1-mFc to compete with human PD-1-mFc-Biotin
  • Human PD-L1-mFc (PD-L1 Genbank ID: NP_054862.1, mFc SEQ ID NO: 89) was coated with 2 ⁇ g/mL microtiter plate, and incubated overnight at 4°C. After the incubation, the ELISA plate was blocked with 1% BSA in PBS at 37°C for 2 hours. After the blocking, the plate was washed once and patted dry.
  • the plate was washed four times and patted dry, and then TMB (Neogen, 308177) was added for 5 minutes to develop color in the dark, and a stop solution was added to terminate the color reaction.
  • TMB Neogen, 308177
  • the OD value of each dose is shown in Table 12.
  • the curve simulates the binding efficiency of the antibody to obtain the binding EC50 (Table 12).
  • NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1 (as a control) can effectively block the binding of the antigen human PD-1-mFc-Biotin to its receptor human PD-L1-mFc, and the blocking efficiency is shown
  • the EC50 of NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1 (hG1TM) blocking the binding of human PD-1-mFc-Biotin to its ligand human PD-L1-mFc are 2.249nM, 2.253nM, 2.332nM, 2.398, respectively nM, 2.216nM, 2.231nM.
  • NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1 (hG1TM) compete with human PD-L1-mFc to bind to human PD-1-mFc-Biotin activity detection results
  • Example 18 Determination of the kinetic parameters of the binding of the anti-CD73-anti-PD-1 bispecific antibody to the antigen human PD-1-mFc using the Fortebio molecular interaction instrument
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. Fix PD1-mFc on the AMC sensor at a concentration of 5 ⁇ g/mL, the fixed height is about 0.1nm (time 60s), the sensor is equilibrated in the buffer for 60s, and the PD1-mFc fixed on the sensor binds to the antibody at a concentration of 0.62 -50nM (three-fold dilution), time 120s, protein dissociation in the buffer, time 300s.
  • the detection temperature is 30 degrees
  • the detection frequency is 0.3 Hz
  • the sample plate vibration rate is 1000 rpm.
  • the data is analyzed by a 1:1 model to get the affinity constant.
  • the determination results of the affinity constants of humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and Nivolumab (as control antibodies) with human PD-1-mFc are shown in Table 13.
  • the detection results are shown in Figure 19, Figure 20, Figure 21, Figure 22 and Figures. 18 shown.
  • the affinity constants of humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and Nivolumab to human PD-1-mFc are 1.40E-10M, 7.39E-11M, 1.25E-10M, 1.13E-11M, and 2.26E-10M.
  • KD is the affinity constant
  • Example 19 Using the Fortebio molecular interaction instrument to determine the kinetic parameters of the binding of anti-CD73-anti-PD-1 bispecific antibodies to the antigen human NT5E(1-552)-his.
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. Fix HNT5E(1-552)-His on the HIS1K sensor at a concentration of 5 ⁇ g/mL, the fixed height is about 0.4nm (time 50s), the sensor is equilibrated in the buffer for 60s, and the HNT5E(1-552) fixed on the sensor )-His binds to the antibody, the concentration is 0.31-25nM (three-fold dilution), the time is 100s, and the protein is dissociated in the buffer, the time is 180s.
  • the detection temperature is 30 degrees, the detection frequency is 0.3 Hz, and the sample plate vibration rate is 1000 rpm.
  • the data is analyzed by a 1:1 model to get the affinity constant.
  • K D kdis/kon
  • Example 20 Detection of anti-CD73-anti-PD-1 bispecific antibody inhibiting the activity of CD73 on the cell membrane of U87-MG
  • the antibody was added to a 96-well plate, 60 ⁇ L per well, incubated at 37°C for 1 hour; after 1 hour, 60 ⁇ L 600uM AMP diluted with 600uM RPMI-1640 was added to each well; 3 hours Then take 100 ⁇ L of cell culture supernatant, transfer to a new 96-well plate, add 40 ⁇ L CTG (CellTiterGlo) color developing solution to each well, and place it in the dark at room temperature for 5 minutes; after 5 minutes, add 10 ⁇ L 300 ⁇ M ATP to each well.
  • the relative fluorescence intensity RLU was read by the labeled microplate detector (PerkinElmer 2140-0020).
  • Anti-CD73-anti-PD-1 bispecific antibody inhibits the detection of CD73 enzyme activity on the surface of U87-MG cell membrane
  • Example 21 Detection of the biological activity of anti-CD73-anti-PD-1 bispecific antibodies in promoting the secretion of IFN- ⁇ and IL-2 by mixed lymphocyte reaction MLR
  • Raji-PDL1 cells were subcultured normally; resuscitated PBMCs, cultured with 10mL 1640 complete medium, and stimulated with 0.5 ⁇ g/mL SEB (Denotec, catalog number: S010201) for two days; Raji-PDL1 cells were cultured with 25 ⁇ g/mL MMC (silk) Schitomycin C, Stressmarq, catalog: SIH-246, batch number: SM286474), placed in a 37°C incubator for 1 hour; collected PBMC 2 days after SEB stimulation and Raji-PDL1 cells treated with MMC for 1 hour, washed twice with PBS Second, resuspend the complete medium for counting, add them to a U-shaped 96-well plate, 1x10 5 cells/well; add antibodies according to the experimental design, and incubate in an incubator for 3 days; after 3 days, collect the cell culture supernatant by ELISA method Perform IFN- ⁇ detection.
  • SEB Denotec, catalog number: S010201
  • the mixed culture of human PBMC and Raji-PDL1 cells has a significant effect on the secretion of IFN- ⁇ of PBMC.
  • the simultaneous addition of antibodies in the mixed culture system can significantly induce PBMC to further secrete IFN- ⁇ and promote IFN- ⁇ .
  • the activity of NTPDV2 antibody and its parent PD-1 single-target antibody 14C12H1L1 are comparable.
  • Raji-PDL1 cells were subcultured normally; resuscitated PBMC, cultured with 10mL 1640 complete medium, and stimulated with SEB (0.5 ⁇ g/mL) for two days; Raji-PDL1 cells were treated with 25 ⁇ g/mL MMC and placed in a 37°C incubator1 Hours; collect PBMC after 2 days of SEB stimulation and Raji-PDL1 cells treated with MMC for 1 hour, wash twice with PBS, resuspend in complete medium and count, and add them to U-shaped 96-well plates, 1x10 5 cells/well; In the experimental design, antibody was added and cultured for 3 days; the cell culture supernatant was collected, and IL-2 was detected by ELISA.
  • SEB 0.5 ⁇ g/mL
  • the mixed culture of human PBMC and Raji-PDL1 cells can promote the secretion of IL-2 of PBMC to a certain extent. Adding antibodies to the mixed culture system can significantly induce PBMC to further secrete IL-2, which has a significant effect.
  • the activity of the bifunctional antibody NTPDV2 at low concentrations is comparable to that of its parent PD-1 single-target antibody 14C12H1L1, while the medium-to-high concentration is slightly lower than that of the parent PD-1 single-target antibody 14C12H1L1 .
  • MC38-hPDL1/hCD73 cells (from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were first used to subcutaneously inoculate females aged 5-7 weeks.
  • C57BL6-hPD1hPDL1hCD73 three transgenic mice (from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), the model and specific administration methods are shown in Table 16. After the administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.
  • the experimental results are shown in Figure 30 and Figure 31.
  • the results show that: compared with the isotype control antibody hIgG, 19F3H2L3 (hG1M), different doses of NTPDV2 can effectively inhibit the growth of tumors in mice, and high-dose NTPDV2 inhibits tumors better than low Dose NTPDV2.
  • NTPDV2 had no effect on the body weight of tumor-bearing mice.
  • HPMM is induced by PBMC.
  • the PBMCs used in this study were all isolated and prepared in Zhongshan Kangfang Biomedicine Co., Ltd., and with the informed consent of the provider.
  • Ficoll-Paque PLUS Lymphocyte Separation Solution (GE, item number: 17-1440-03); RPMI1640 (Gibco, item number: 22400-105); CHO-K1-PD1 cells (built by Zhongshan Kangfang Biomedical Co., Ltd.); U87- MG cells (cells sourced from ATCC, purchased from Beijing Zhongyuan Leading Technology Co., Ltd.); FBS (Fetal Bovine Serum, Excell Bio, catalog number: FSP500); human IFN- ⁇ protein (sinobio, catalog number: 11725-HNAS-100); LPS (Lipopolysaccharides), lipopolysaccharide (sigma, catalog number: L4391); 96-well cell culture plate (Corning).
  • Ficoll-PaqueTM Plus reagent instructions separate healthy human PBMCs and resuspend them in 1640 medium containing 2% FBS, and place them in a 37°C, 5% CO2 cell incubator. After 2 hours, the supernatant was removed, the adherent cells were washed twice with PBS, and 1640 complete medium (containing 10% FBS) and 100 ng/mL human M-CSF were added to induce 7 days. On the 3rd and 5th day, the fluid was changed and supplemented with M-CSF to induce HPMM.
  • HPMM was collected after induction, adjusted to a concentration of 100,000/mL with complete medium, and aliquoted into 96-well plates, and added recombinant human IFN- ⁇ (50ng/mL), and placed in an incubator for 24 hours.
  • log-phase CHO-K1-PD1 cells expressing human PD-1 or U87-MG cells constitutively expressing human CD73 were collected, and the concentration was adjusted to 300,000/mL with complete medium after resuspension.
  • the antibody was diluted with complete medium to working concentration of 25nM, 2.5nM, 0.25nM. And design isotype control antibody and blank control at the same time.
  • CHO-K1-PD1 and U87-MG cells as target cells can be co-cultured with HPMM to induce HPMM activation.
  • the activated HPMM is linked to the target cells through the antibody Fab, the antibody Fc segment interacts with the Fc ⁇ R on HPMM, causing HPMM secretes cytokines.
  • the anti-PD-1/CD73 bispecific antibody with Fc-segment mutation can effectively eliminate IL-6 and/or IL in immune cells -8 secretion.
  • variable amino acid sequence of the 19F3 light chain is variable amino acid sequence of the 19F3 light chain:
  • FR-H1 EVQLQQSGPELVKPGASMRMSCKAS (SEQ ID NO: 11)
  • FR-H3 SYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC (SEQ ID NO: 13)
  • FR-L1 DIVMTQSPSSLAMSVGQKVTMSCKSS (SEQ ID NO: 15)
  • FR-L2 LAWYQQKPGQSPKLLVY (SEQ ID NO: 16)
  • FR-L3 TRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFC (SEQ ID NO: 17)
  • FR-L4 FGGGTKLEIK (SEQ ID NO: 18)
  • amino acid sequence of 19F3H2 (SEQ ID NO: 20, underlined indicates the CDR sequence)
  • amino acid sequence of 19F3L2 (SEQ ID NO: 22, underlined indicates the CDR sequence)
  • the heavy chain nucleotides of 19F3H2L3 (SEQ ID NO: 25, the underlined area is the non-variable region sequence)
  • the heavy chain amino acid of 19F3H2L3 (SEQ ID NO: 26, the underlined region is the non-variable region sequence)
  • the light chain nucleotides of 19F3H2L3 (G1M) (SEQ ID NO: 27, the underlined region is the non-variable region sequence)
  • the light chain amino acids of 19F3H2L3 (SEQ ID NO: 28, the underlined area is the non-variable region sequence)
  • the heavy chain nucleotides of 19F3H2L3 (hG1TM) (SEQ ID NO: 29, the underlined region is the non-variable region sequence)
  • FR-H3 SYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 33)
  • FR-L1 DIVMTQSPSSLAVSVGERVTISCKSS (SEQ ID NO: 35)
  • FR-L2 LAWYQQKPGQAPKLLIY (SEQ ID NO: 36)
  • FR-L3 TRESGVPDRFSGSGSGTDFTLTISSVQAEDVADYYC (SEQ ID NO: 37)
  • FR-L4 FGGGTKLEIK (SEQ ID NO: 38)
  • FR-L1 DIVMTQSPSSLAVSVGERVTISCKSS (SEQ ID NO: 39)
  • FR-L2 LAWYQQKPGQAPKLLIY (SEQ ID NO: 40)
  • FR-L3 TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 41)
  • FR-L4 FGGGTKLEIK (SEQ ID NO: 42)
  • FR-H1 EVKLVESGGGLVKPGGSLKLSCAAS (SEQ ID NO: 53)
  • FR-H3 YYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYC (SEQ ID NO: 55)
  • nucleotide sequence of the light chain of 14C12H1L1 (SEQ ID NO: 67)
  • the heavy chain nucleotide sequence of 14C12H1L1 (G1TM) (SEQ ID NO: 69)
  • FR-H1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 71)
  • FR-H3 YYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC (SEQ ID NO: 73)
  • FR-H4 WGQGTLVTVSS (SEQ ID NO: 74)
  • FR-L1 DIQMTQSPSSMSASVGDRVTFTCRAS (SEQ ID NO: 75)
  • FR-L2 LSWFQQKPGKSPKTLIY (SEQ ID NO: 76)
  • FR-L3 RLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYC (SEQ ID NO: 77)
  • FR-L4 FGAGTKLELK (SEQ ID NO: 78)
  • Linker1 The amino acid sequence of Linker1: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 79)
  • Linker2 GGGGSGGGGSGGGGS (SEQ ID NO: 81)
  • NTPDV2 NTPDV4 and heavy chain (SEQ ID NO: 83): wherein the immunoglobulin moiety in 19F3H2 (hG1TM) of the CDR regions are underlined in bold identity, CDR regions of the scFv portion of 14C12H1V-Linker1-14C12L1V with bold underlined amino acids are mutations identified heavy chain region identified by bold italics, bold Linker region identified by:
  • nucleotide sequence of the heavy chain of NTPDV2 and NTPDV4 (SEQ ID NO: 84)
  • NTPDV1 and amino acid sequence (SEQ ID NO: 85): a heavy chain NTPDV3 19F3H2 (hG1TM) wherein the immunoglobulin moiety in the CDR regions are underlined in bold identity, CDR regions of the scFv portion of 14C12H1V-Linker2-14C12L1V with bold underlined amino acids are mutations identified heavy chain region identified by bold italics, bold Linker region identified by:
  • nucleotide sequence of the heavy chain of NTPDV1 and NTPDV3 (SEQ ID NO: 86)
  • NT5E(1-552)-his nucleotide sequence SEQ ID NO: 88
  • nucleotide sequence of 19F3H1V-Linker2-19F3L2V (SEQ ID NO: 91)
  • amino acid sequence of the variable region of 19F3H2 underlined indicates the CDR sequence: (SEQ ID NO: 97)
  • variable region of 19F3L2 underlined indicates the CDR sequence: (SEQ ID NO: 98)
  • amino acid sequence of the variable region of 19F3L3, underlined indicates the CDR sequence: (SEQ ID NO: 99)

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