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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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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seq
amino acid
acid sequence
antibody
variable region
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PCT/CN2021/089059
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English (en)
French (fr)
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张鹏
李百勇
夏瑜
王忠民
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中山康方生物医药有限公司
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Priority to EP21792313.5A priority Critical patent/EP4141033A4/en
Priority to AU2021261803A priority patent/AU2021261803A1/en
Priority to US17/996,878 priority patent/US20230151111A1/en
Priority to KR1020227040580A priority patent/KR20230004726A/ko
Priority to JP2022564073A priority patent/JP2023522730A/ja
Priority to CA3176321A priority patent/CA3176321A1/en
Priority to IL297432A priority patent/IL297432A/en
Priority to MX2022013311A priority patent/MX2022013311A/es
Priority to BR112022021426A priority patent/BR112022021426A2/pt
Publication of WO2021213475A1 publication Critical patent/WO2021213475A1/zh
Priority to ZA2022/11405A priority patent/ZA202211405B/en

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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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Abstract

提供了抗CD73-抗PD-1双特异性抗体、其药物组合物及用途。

Description

抗CD73-抗PD-1双特异性抗体及其用途 技术领域
本发明属于肿瘤治疗和分子免疫学领域，涉及一种抗CD73-抗PD-1双特异性抗体、其药物组合物及其用途。
背景技术
胞外-5’-核苷酸酶(Ecto-5’-nucleotidase)，即CD73蛋白，是NT5E基因编码的一种蛋白分子量为70KD的多功能糖蛋白，其通过糖基磷脂酰肌醇(glyocsyl phosphatidy linositol，GPI)锚定于细胞膜上(Zimmermann H.5′-Nucleotidase：molecular structure and functional aspects.Biochem J.1992；285：345-365)。
CD73广泛分布在人体组织细胞表面，目前研究已经发现CD73高表达于多种实体肿瘤，如肿瘤微环境的癌细胞、树突细胞、调节型T细胞(Treg)、自然杀伤细胞(NK细胞)、髓系起源的抑制细胞(MDSC)、肿瘤相关巨噬细胞(TAM)等。CD73的表达受到TGF-β、EGFR、AKT、β-catenin等分子的调控，特别是行使转录因子功能的HIF-1最为关键。肿瘤微环境的一个重要特征是低氧，低氧诱导因子-1(HIF-1)等分子的上调，进而导致CD73在肿瘤微环境广泛表达(Synnestvedt K，et al.Ecto-5′-nucleotidase(CD73)regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia.J Clin Invest.2002；110：993-1002.)。对临床肿瘤样本的分析显示，CD73高表达是一种潜在的生物标志物，与多种类型肿瘤的不良预后密切相关，包括乳腺癌、肺癌、卵巢癌、肾癌、胃癌、头颈癌等。
CD73具有水解酶活性，又有非水解酶作用。CD73的酶与非酶功能同时存在于肿瘤的相关过程中，并且互相促进，维持肿瘤的演进。越来越多的研究发现，CD73是体外肿瘤细胞增殖、转移和侵袭，体内肿瘤血管生成和肿瘤免疫逃逸机制的关键调控分子，其中很重要的一个免疫抑制机制是由CD73-腺苷(Adenosine)代谢信号通路介导的，CD73上 游的CD39可以催化ATP产生腺苷单磷酸(AMP)，所产生的AMP被CD73转化为腺苷，而腺苷会结合下游的腺苷受体(A2AR)，A2AR通过激活蛋白激酶A(PKA)和Csk激酶，抑制LCK、MAPK、PKC等一系列与免疫激活相关的信号通路，抑制T细胞的免疫杀伤作用，从而发挥免疫抑制作用，使肿瘤实现免疫逃逸(Antonioli L，et al.Immunity，inflammation and cancer：a leading role for adenosine.Nat Rev Cancer.2013；13：842-857)。临床前动物模型研究显示，免疫细胞和非免疫细胞表达的CD73均可以促进肿瘤的免疫逃逸，发展和转移，其中Treg细胞相关的CD73-腺苷信号对CTL(细胞毒性T细胞)和NK细胞功能的抑制最为明显。
对于实体瘤治疗而言，要克服耐药提高疗效，很重要的一个方面就是解除肿瘤微环境(Tumor micro environment，TME)对免疫效应细胞的抑制作用。TME是非常复杂的系统，由多种细胞、胞间质、酶、细胞因子、代射产物等构成，有显著的低氢、低pH和高压的特点，与正常组织差异巨大。放化疗杀伤肿瘤细胞所造成的低氧或ATP富集会促进CD39-CD73腺苷信号的级联反应，有利于各种促癌类细胞的增殖和功能，而不利于抑癌类细胞。(Regateiro，F.S.，Cobbold，S.P.&Waldmann，H.CD73 and adenosine generation in the creation of regulatory microenvironments.Clin.Exp.Immunol.2013；171：1-7)。
在动物模型中使用靶向CD73的抗体或基因敲除CD73可以有效阻断肿瘤生长和转移。近来，利用CD73单克隆抗体、小干扰RNA技术、特异性抑制剂APCP等在动物实验的抗肿瘤治疗中取得了显著疗效，为抗肿瘤治疗提供了新的途径。体内试验的证据显示，对于肿瘤患者，靶向阻断CD73将成为有效的治疗手段。
CD73过表达与肿瘤亚型、预后及患者的药物反应之间的联系已经表明CD73可作为未来个体肿瘤治疗、检测的一个重要标志物。因此，对CD73靶点的研究是不可或缺的。
跨膜受体PD-1(程序性细胞死亡-1)是CD28家族成员之一，在活化的T细胞，B细胞以及骨髓系细胞都有表达。PD-1的受体PDL1和PDL2均属于B7超家族，其中PDL1多种细胞都有表达，包括T细胞，B细胞以及内皮细胞和上皮细胞，PDL2则仅表达于抗原呈递细胞如树突状细胞 和巨噬细胞。
PD-1在负调节T细胞的活化过程中起着非常重要的作用，PD-1介导的对T细胞负调节作用是肿瘤免疫逃避的重要机制之一，肿瘤表面表达的PDL-1可与免疫细胞表面的PD-1结合，进而通过PD-1/PDL-1信号通路抑制免疫细胞对肿瘤组织的杀伤，高表达PD-L1的肿瘤伴随着很难被检测到的癌症(Hamanishi et al.，Proc.Natl.Acad.Sci.USA 2007；104：3360-5)。拮抗PD-1，从而抑制PD-1/PDL-1信号通路的一种有效方法是体内注射抗PD-1抗体。
PD-1抗体具有广谱抗肿瘤前景和惊人的药效，针对PD-1通路的抗体将带来治疗多种肿瘤治疗的突破性的进展：用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色素瘤(Homet M.B.，Parisi G.，et al.，Anti-PD-1 Therapy in Melanoma.Semin Oncol.2015Jun；42(3)：466-473)，血液肿瘤以及贫血(Held SA，Heine A，et al.，Advances in immunotherapy of chronic myeloid leukemia CML.Curr Cancer Drug Targets.2013Sep；13(7)：768-74)。
双功能抗体亦称为双特异性抗体(Bispecific Antibody)，是同时靶向结合两种不同抗原的特异性抗体药物，其可通过免疫分选纯化生产，也可通过基因工程获得。基因工程在结合位点优化，合成形式的考量以及产量等方面都具有相应的灵活性，所以具有一定的优势。目前，双特异性抗体存在形式已被证明有超过45种(Müller D，Kontermann RE.Bispecific antibodies for cancer immunotherapy：Current perspectives.BioDrugs 2010；24：89-98)。IgG-ScFv形式即Morrison模式(Coloma MJ，Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nat Biotechnol.Nature Biotechnology，1997；15：159-163)由于类似于天然存在的IgG的形式，在抗体工程、表达和纯化上具有优势，已被证明是双功能抗体的一种理想存在形式(Miller BR，Demarest SJ，et al.，Stability engineering of scFvs for the development of bispecific and multivalent antibodies.Protein Eng Des Sel 2010；23：549-57；Fitzgerald J，Lugovskoy A.Rational engineering of antibody therapeutics targeting multiple oncogene pathways.MAbs 2011；3：299-309)。
ADCC(antibody-dependent cell-mediated cytotoxicity)是抗体依赖的 细胞介导的细胞毒性作用，是指抗体的Fab段结合病毒感染的细胞或肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的Fc受体(Fc Receptor，FcR)结合，介导杀伤细胞直接杀伤靶细胞。
CDC(complement dependent cytotoxicity)指补体依赖的细胞毒性。CDC作用是由抗体与细胞膜表面相应抗原结合后，进一步与补体C1q的首先结合引起的，接着C2-C9就被激活形成攻膜复合物对靶细胞发挥裂解效应。
IgG家族包含四个成员，IgG1、IgG2、IgG3和IgG4，它们重链恒定区的可结晶片段(fragment crystallizable，Fc)区域存在氨基酸的差异，导致它们与FcγRs的亲和力各不相同。野生型IgG1能够结合各种FcγRs，能够引发ADCC以及CDC效应。Zhang等人(Zhang T等人.Cancer ImmunolImmunother.2018；67(7)：1079-1090.)及Dahan等人(Dahan R等人。Cancer Cell.2015；28(3)：285-95.)的研究表明，靶向PD-1等免疫检查点的抗体的Fc段与Fc受体结合会对抗抗体介导的抗癌活性产生负面影响，这可能是由于Fc依赖性效应子功能诱导的免疫细胞受损，包括抗体依赖性细胞介导的细胞毒性是导致免疫细胞损伤的重要机制。
白细胞介素8(interleutin-8，IL-8)是一种趋化性细胞因子(Chemotactic cytokines)，主要是由单核细胞等分泌。IL-8在正常细胞和肿瘤细胞的增殖中有重要作用，尤其是对肿瘤的发生和发展具有重要的促进作用。研究表明IL-8可以促进肿瘤的发生；肿瘤细胞本身也可分泌IL-8，促进肿瘤的生长和转移(Lo MC et al.Cancer letters，2013，335(1)：81-92.)。因此，IL-8已成为肿瘤微环境之中不可缺少的一种重要的炎症因子。
IL-8作为一种促炎因子与肿瘤的发生发展密切相关。在甲基胂酸(methylarsonate)诱导非肾癌细胞的恶变过程中，IL-8基因的表达增加，IL-8基因沉默则可显著抑制小鼠体内移植瘤的生长，此外IL-8水平的降低可抑制与肿瘤的生长、转移相关的基质金属蛋白酶-9(Matrix metalloproteinase-9)、细胞周期蛋白D1(Cyclin D1)、促凋亡蛋白Bcl-2、血管内皮生长因子(VEGF)的表达(Escudero-Lourdes C et al.Toxicology and applied pharmacology，2012，258(1)：10-18.)。Inoue等 人的研究发现IL-8可诱导非肿瘤性膀胱细胞系(233JP)的恶变及侵袭性的增加，而在IL-8敲除鼠中233JP细胞发生恶性转化的几率明显减少(Inoue K et al.Cancer Res，2000，60(8)：2290-2299.)。此外，在前列腺癌中，IL-8可以促进患者去势抵抗性前列腺癌(CRPC)的发生(Chen K et al.Cancer research，2015，75(10)：1992-2004.)，并且与肿瘤治疗的抗药性有关(Araki S et al.Cancer Res，2007，67(14)：6854-6862.)；IL-8或其受体的基因沉默可诱导肿瘤细胞细胞周期阻滞，抑制肿瘤的增殖(Singh RK，Lokeshwar BL.Molecul Cancer，2009，8：57.)。上述研究表明IL-8的水平与肿瘤的发生发展密切相关。进一步的研究(Mian BM et al.Clin Cancer Res，2003，9(8)：3167-3175.)表明IL-8可以作为肿瘤治疗新的靶点。在膀胱癌的肿瘤模型中，使用抗IL-8抗体可以明显的抑制抑制瘤的生长。
IL-6主要由巨噬细胞迅速产生，响应与病原体相关的分子模式(PAMP)或损伤相关分子模式(DAMP)，并通过除去感染因子，诱导急性期和免疫应答来治愈受损组织，起保护作用。IL-6虽然在感染和组织损伤的抵抗和修复中发挥重要作用，但是高水平的IL-6可以激活凝血途径和血管内皮细胞，进而抑制心肌功能，甚至可以引起“细胞因子风暴”，产生严重的急性全身炎症反应。细胞因子风暴是病毒感染、肿瘤免疫疗法等中一种致死性的并发症和不良反应。
免疫相关不良反应是免疫检查点抑制剂(immune checkpoint inhibitor，ICI)抗肿瘤治疗中一种常见而又危险的不良反应(Spain L et al.Cancer Treat Rev.2016；44：51-60.)。近年来，免疫检查点抑制剂在肿瘤免疫治疗方面取得了巨大成功，但也因为脱靶(off-target)效应导致了全新的毒性谱。其中尤其是主要器官(包括心脏、肺和脑)的严重免疫相关不良事件(irAE)可能危及生命(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).2016；85(3)：331-9.；Prieux-Klotz C et al.Target Oncol.2017；12(3)：301-308.；Tajiri K et al.Jpn J Clin Oncol.2018；48(1)：7-12.)。已有数据表明ICI可能通过4种机制诱导脱靶效应，包括直接结合正 常细胞表面表达的免疫检查点分子，激活补体超敏反应；正常组织与肿瘤细胞存在同源抗原/表位；产生自身抗体；增加前炎症细胞因子的水平，如IL-6等(Martins F et al.，The Lancet Oncology，20(1)，e54-e64.)。
目前抗IL-6疗法，如托珠单抗，一种重组人源化抗IL-6R单克隆抗体，已被用于治疗急性期严重irAE、严重或难治性关节炎、大血管血管炎、葡萄膜炎、心肌炎、肺炎、重症肌无力等(Martins F et al.，The Lancet Oncology，20(1)，e54-e64.)。
巨噬细胞上的FcγRIa与野生型IgG1或者IgG4抗体的结合可由诱导其分泌IL-8和IL-6(Kinder M et al.，mAbs.2015)，而进行抗体Fc段的突变，消除其与FcγRIa的结合可以有效地抑制IL-8的分泌，进而增加抗体的安全性和有效性。
发明内容
本发明人利用哺乳动物细胞表达系统分别表达出重组的CD73和PD-1分别作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过进行对大量样本的筛选，分别得到了如下的杂交瘤细胞株：
杂交瘤细胞株LT014(又称CD73-19F3)，其于2018年6月19日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018137；以及
杂交瘤细胞株LT003(又称PD-1-14C12)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015105。
本发明人惊奇地发现：
杂交瘤细胞株LT014能够分泌产生与人CD73特异性结合的特异性单克隆抗体(命名为19F3)，并且该单克隆抗体能够十分有效地以非底物竞争的方式抑制CD73的酶活反应，降低腺苷的产生，促进T细胞活性及肿瘤抑制效果。
杂交瘤细胞株LT003能够分泌产生与PD-1特异性结合的特异性单克 隆抗体(命名为14C12)，并且该单克隆抗体能够十分有效地阻断PD-1与PDL1的结合。
进一步地，本发明人创造性地制得了抗CD73的人源化抗体(分别命名为19F3H2L2、19F3H2L3、19F3H2L3(hG1M)、19F3H2L3(hG1TM))和抗PD-1的人源化抗体(命名为14C12H1L1和14C12H1L1(hG1TM))。
进一步地，本发明人创造性地将两类人源化抗体进行蛋白重组融合成新的抗体，获得了能够结合CD73和PD-1，抑制CD73活性、阻断PD-1与PDL1的结合的人源化双功能抗体(分别命名为P1D7V01、P1D7V03、NTPDV1、NTPDV2、NTPDV3、NTPDV4(在本文及在申请号为202110270671.X的中国发明专利中又写为NTPDV1(hG1TM)、NTPDV2(hG1TM)、NTPDV3(hG1TM)、NTPDV4(hG1TM))，具有用于制备预防和治疗实体瘤和血液肿瘤的潜力。
由此提供了下述发明：
本发明的一个方面涉及一种抗CD73-抗PD-1双特异性抗体，其包括：
第一蛋白功能区，所述第一蛋白功能区靶向PD-1，和
第二蛋白功能区，所述第二蛋白功能区靶向CD73。
在本发明的一个实施方案中，所述双特异性抗体中
第一蛋白功能区包含氨基酸序列如SEQ ID NO：44所示的重链可变区所包含的HCDR1，HCDR2和HCDR3，优选HCDR1，HCDR2和HCDR3的氨基酸序列分别如SEQ ID NOs：45-47所示的序列或与SEQ ID NOs：45-47所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，或与SEQ ID NOs：45-47所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，和包含
氨基酸序列如SEQ ID NO：49所示的轻链可变区所包含的LCDR1，LCDR2和LCDR3，优选LCDR1，LCDR2和LCDR3的氨基酸序列分别如SEQ ID NOs：50-52所示的序列或与SEQ ID NOs：50-52所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％ 序列同一性的序列，或与SEQ ID NOs：50-52所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
第二蛋白功能区包含氨基酸序列如SEQ ID NO：2所示的重链可变区所包含的HCDR1，HCDR2和HCDR3，优选HCDR1，HCDR2和HCDR3的氨基酸序列如SEQ ID NOs：3-5所示的序列或与SEQ ID NOs：3-5所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：3-5所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，和包含
氨基酸序列如SEQ ID NO：7所示的轻链可变区所包含的LCDR1，LCDR2和LCDR3，优选LCDR1，LCDR2和LCDR3的氨基酸序列分别如SEQ ID NOs：8-10所示的序列或与SEQ ID NOs：8-10所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：8-10所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中：
所述第一蛋白功能区包含
氨基酸序列如SEQ ID NO：44或SEQ ID NO：62所示的序列或与SEQ ID NOs：44或62所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：44或62所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；和包含
分别对应选自氨基酸序列如SEQ ID NO：49或SEQ ID NO：64所示的序列或与SEQ ID NOs：49或64所示的序列具有至少80％、81％、82％、 83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：49或64所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
和/或，
所述第二蛋白功能区包含氨基酸序列选自SEQ ID NO：2、SEQ ID NO：20所示的序列或与SEQ ID NO：2、SEQ ID NO：20所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：2、SEQ ID NO：20所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；和包含
分别对应选自SEQ ID NO：7，或SEQ ID NO：22或与SEQ ID NO：7或SEQ ID NO：22所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：7或SEQ ID NO：22所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
或
所述第二蛋白功能区包含氨基酸序列如SEQ ID NO20所示的序列或与SEQ ID NO：20所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：20所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，和包含
氨基酸序列如SEQ ID NO：24所示的序列或与SEQ ID NO：24示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：24所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的 氨基酸序列。
本发明的一个方面涉及一种抗CD73-抗PD-1双特异性抗体，其包括：
第一蛋白功能区，所述第一蛋白功能区靶向CD73，和
第二蛋白功能区，所述第二蛋白功能区靶向PD-1。
在本发明的一个实施方案中，在所述双特异性抗体中，
第一蛋白功能区包含氨基酸序列如SEQ ID NO：2所示的重链可变区所包含的HCDR1，HCDR2和HCDR3，优选HCDR1，HCDR2和HCDR3的氨基酸序列分别如SEQ ID NOs：3-5所示的序列或与SEQ ID NOs：3-5所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，或与与SEQ ID NOs：3-5所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，和包含
氨基酸序列如SEQ ID NO：7所示的轻链可变区所包含的LCDR1，LCDR2和LCDR3，优选LCDR1，LCDR2和LCDR3的氨基酸序列分别如SEQ ID NOs：8-10所示的序列或与SEQ ID NOs：8-10所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：8-10所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
第二蛋白功能区包含氨基酸序列如SEQ ID NO：44所示的重链可变区所包含的HCDR1，HCDR2和HCDR3，优选HCDR1，HCDR2和HCDR3的氨基酸序列分别如SEQ ID NOs：45-47所示的序列或与SEQ ID NOs：45-47所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：45-47所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，和包含
氨基酸序列如SEQ ID NO：49所示的轻链可变区所包含的LCDR1， LCDR2和LCDR3，优选LCDR1，LCDR2和LCDR3的氨基酸序列分别如SEQ ID NOs：50-52所示的序列或与SEQ ID NOs：50-52所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：50-52所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中：
所述第一蛋白功能区包含
氨基酸序列如SEQ ID NO：2、SEQ ID NO：20所示的序列或与SEQ ID NOs：2或20所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：2或20所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；和包含
分别对应选自氨基酸序列如SEQ ID NO：7，SEQ ID NO：22，SEQ ID NO：24所示的序列或与SEQ ID NO：7，SEQ ID NO：22，SEQ ID NO：24所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：7，22或24所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
和/或，
所述第二蛋白功能区包含氨基酸序列选自SEQ ID NO：44或SEQ ID NO：62所示的序列或与SEQ ID NO：44或SEQ ID NO：62所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：44或SEQ ID NO：62所示所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换， 插入或缺失)的氨基酸序列；和包含
分别对应选自SEQ ID NOs：49或64或与SEQ ID NOs：49或64所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：49或64所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；优选地，所述连接片段为(GGGGS)n，n为正整数，例如1、2、3、4、5或6。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中的所述第一蛋白功能区和第二蛋白功能区独立地为免疫球蛋白或抗原结合片段，例如半抗体、Fab、F(ab’) 2或单链抗体，优选地，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为抗原结合片段；或者，所述第一蛋白功能区为抗原结合片段，所述第二蛋白功能区为免疫球蛋白。
在本发明的一个实施方案中，所述抗原结合片段的重链可变区N端直接(或通过连接片段)连接于所述免疫球蛋白的CH1的C端和所述抗原结合片段的轻链可变区N端直接(或通过连接片段)连接于所述免疫球蛋白的轻链可变区CL的C端；或者所述抗原结合片段的重链可变区N端直接(或通过连接片段)连接于所述免疫球蛋白的的轻链可变区CL的C端和所述抗原结合片段的轻链可变区N端直接(或通过连接片段)连接于所述免疫球蛋白的重链可变区CH1的C端。
在本发明的一个实施方案中，所述抗原结合片段的重链可变区C端直接(或通过连接片段)连接于所述免疫球蛋白的重链N端和所述抗原结合片段的轻链可变区C端直接(或通过连接片段)连接于所述免疫球蛋白的轻链N端；或者所述抗原结合片段的重链可变区C端直接(或通过连接片段)连接于所述免疫球蛋白的的轻链N端和所述抗原结合片段的轻链可变区的C端直接(或通过连接片段)连接于所述免疫球蛋白的重链N端。
在本发明的一个实施方案中，所述抗原结合片段是单链抗体，优选地， 所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；或者，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白。
在本发明的一个实施方案中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中，所述单链抗体为通过连接片段(Linker)连接抗体重链可变区(V H)和抗体轻链可变区(V L)的分子；优选地，可具有一般结构：NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中，所述单链抗体通过连接片段连接在免疫球蛋白的重链的C末端(C H)(或者重链的N端、重链可变区的CH1的C端)时，可以首先连接所述单链抗体的抗体重链可变区(V H)，或者，首先连接所述单链抗体的抗体轻链可变区(V L)；优选地，单链抗体可具有一般结构：C H-连接片段-V H-连接片段-V L-COOH，或者，C H-连接片段-V L-连接片段-V H-COOH，
优选地，
所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：3-5的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：8-10的CDR；
所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：45-47的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：50-52的CDR，
优选地，所述单链抗体(例如NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH)通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：45-47的CDR的抗体重链可变区(V H)，或者，首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：50-52的CDR的抗体轻链可变区(V L)，
或者优选地，
所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：45-47的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：50-52的CDR；和/或，
所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：3-5的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：8-10的CDR，
其中，所述单链抗体(例如NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH)通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：3-5的CDR的抗体重链可变区(V H)，或者，首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：8-10的CDR的抗体轻链可变区(V L)，
优选地，
一个免疫球蛋白分子连接有两个单链抗体分子，更优选地，两个单链抗体分子相同。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中，所述免疫球蛋白为IgG、IgA、IgD、IgE或IgM；优选为IgG，例如IgG1、IgG2、IgG3或IgG4。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中，所述单链抗体连接在免疫球蛋白的重链的C末端。由于免疫球蛋白由两条重链组成，因此，一个免疫球蛋白分子连接有两个单链抗体分子。优选地，两个单链抗体分子相同。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中：
所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：3-5的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：8-10的CDR；
和/或，
所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：45-47的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：50-52的CDR，
优选地，所述单链抗体(例如NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH)通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：45-47的CDR的抗体重链可变区(V H)，或者，首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：50-52的CDR的抗体轻链可变区(V L)。
在本发明的另一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中：
所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：45 -47的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：50-52的CDR；和/或，
所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：3-5的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：8-10的CDR，
其中，所述单链抗体(例如NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH)通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：3-5的CDR的抗体重链可变区(V H)，或者，首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：8-10的CDR的抗体轻链可变区(V L)。
在本发明的一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中：
所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO：2或SEQ ID NO：20；所述免疫球蛋白的轻链可变区的氨基酸序列分别对应选自SEQ ID NO：7或SEQ ID NO：22，或者所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO：20所示的序列；所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO：24所示的序列；
和/或，
所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：44或SEQ ID NO：62；所述单链抗体的轻链可变区的氨基酸序列分别对应选自SEQ ID NO：49或SEQ ID NO：64；
其中，所述单链抗体通过连接片段(Linker)连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的抗体重链可变区(V H)，或者，首先连接所述单链抗体的抗体轻链可变区(V L)。
在本发明的另一个实施方案中，在所述抗CD73-抗PD-1双特异性抗体中：
所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO：44或SEQ ID NO：62；所述免疫球蛋白的轻链可变区的氨基酸序列对应选自SEQ ID NO：49或SEQ ID NO：64，或所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：2或SEQ ID NO：20，所述单链抗体的轻链可变区的氨基酸序列对应选自SEQ ID NO：7或SEQ ID NO：22，或所述单链抗体的 重链可变区的氨基酸序列为SEQ ID NO：20所示的序列，所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO：24所示的序列。
本发明的另一方面涉及分离的核酸分子，其包含能够编码双特异性抗体重链可变区的核酸序列，其中，
所述抗体的重链可变区包含：
氨基酸序列为SEQ ID NO：3-5的CDR、氨基酸序列为SEQ ID NO：45-47的CDR和氨基酸序列为SEQ ID NO：50-52的CDR；
并且所述双特异性抗体重链可变区作为所述双特异性抗体的一部分，特异性结合CD73和PD-1抗原，所述双特异性抗体还包含轻链可变区，所述轻链可变区包含：
氨基酸序列为SEQ ID NO：8-10的CDR；
优选地，轻链可变区的CDR与重链可变区包含的CDR不相同。
在本发明的一个实施方案中，在所述的双特异性抗体中，
所述的免疫球蛋白包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一个实施方案中，所述免疫球蛋白的恒定区是人源化的，例如，重链恒定区采用Ig gamma-1 chain C region，ACCESSION：P01857；轻链恒定区采用Ig kappa chain C region，ACCESSION：P01834。
在本发明的一个实施方案中，所述免疫球蛋白的恒定区是人源化的，例如，重链恒定区采用Ig gamma-1 chain C region，ACCESSION：P01857；轻链恒定区采用Ig kappa chain C region，ACCESSION：P01834；其中，按照EU编号系统(EU numbering system)，所述免疫球蛋白的重链恒定区在234位点、235位点和237位点中的任意2个位点或3个位点发生突变，并且突变后，双特异性抗体与FcγRIa、FcγRIIIa和/或C1q的亲和力常数相比突变前降低；优选地，所述亲和力常数通过Fortebio Octet分子相互作用仪测得。
在本发明的一个或多个实施方式中，所述的双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区在第234位点、235位点和/或237位点具有如下突变：
L234A和L235A；或者
L234A和G237A；或者
L235A和G237A；
或者
L234A、L235A、G237A。
本发明中，如果没有特别说明，位点之前的字母表示突变前的氨基酸，位点之后的字母表示突变后的氨基酸。
在本发明的一个或多个实施方式中，所述的双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区还具有选自如下的一个或多个突变：
N297A、D265A、D270A、P238D、L328E、E233D、H268D、P271G、A330R、C226S、C229S、E233P、P331S、S267E、L328F、A330L、M252Y、S254T、T256E、N297Q、P238S、P238A、A327Q、A327G、P329A、K322A、T394D、G236R、G236A、L328R、A330S、P331S、H268A、E318A和K320A。
在具体的实施方案中，所述的抗CD73-抗PD-1双特异性抗体结构如重链-轻链-连接片段1-scFv所示，所述scFv选自14C12H1V-连接片段2-14C12L1V，14C12H1V-连接片段1-14C12L1V，14C12H1V-连接片段2-14C12L1V和14C12H1V-连接片段1-14C12L1V，具体选自以下各项组成的组：
(1)NTPDV1，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：28所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段2的氨基酸序列如SEQ ID NO：81所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示，
(2)NTPDV2，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：28所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示，
(3)NTPDV3，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：96所示，连接片段1的氨基酸序列如SEQ ID NO：79 所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段2的氨基酸序列如SEQ ID NO：81所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示，和
(4)NTPDV4，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：96所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示。
在本发明的一个实施方案中，所述的双特异性抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的K D结合CD73蛋白和/或PD-1蛋白。
本发明的再一方面涉及一种载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及制备本发明的双特异性抗体的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述双特异性抗体的步骤。
本发明的再一方面涉及偶联物，其包括双特异性抗体以及偶联部分，其中，所述双特异性抗体为本发明的双特异性抗体，所述偶联部分为可检测的标记；具体地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质或酶。
本发明的再一方面涉及试剂盒，其包括本发明的双特异性抗体，或者包括本发明的偶联物；优选地所述试剂盒还包括第二抗体，其特异性识别所述双特异性抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶。
本发明的再一方面涉及本发明的双特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测CD73和/或PD-1在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明的双特异性抗体或者本发明的偶联物；可选地，其还包括药学上可接受的载体和/ 或赋形剂。
本发明的再一方面涉及使用本发明的双特异性抗体或者本发明的偶联物在预防和/或治疗肿瘤或者贫血病中的用途，或者在诊断肿瘤或者贫血病的用途。
本发明的再一方面涉及本发明的双特异性抗体或者本发明的偶联物在制备预防和/或治疗肿瘤或者贫血病的药物中的用途，或者在制备诊断肿瘤或者贫血病的药物中的用途。
本发明的再一方面涉及本发明的双特异性抗体或者本发明的偶联物在制备如下药物中的用途：
检测样品中CD73水平的药物，
抑制CD73的酶活反应；
和/或
阻断PD-1与PDL1结合的药物，
调节(例如下调)PD-1活性或水平的药物，
解除PD-1对机体免疫抑制的药物，
提高T淋巴细胞中IL-2表达的药物，或者
提高T淋巴细胞中IFN-γ表达的药物。
本发明的再一方面涉及一种在体内或体外方法，包括施加细胞或者给予有需求的受试者以有效量的本发明的双特异性抗体或者本发明的偶联物的步骤，
本发明的所涉及的抗CD73-抗PD-1双特异性抗体均能抑制细胞膜表面CD73酶活性，并且均能诱导IFNγ、IL-2的分泌，激活免疫反应。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Bethesda M.d.，Sequences of Proteins of Immunological Interest，Fifth Edition，NIH Publication 1991；1-3：91-3242。
优选地，CDR也可以由IMGT编号系统定义，请参见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].Nucleic acids research，2009；38(suppl_1)：D301-D307。
通过本领域技术人员所熟知的技术手段，例如根据IMGT定义分析下面的(1)-(11)项中的单克隆抗体序列的CDR的氨基酸序列，结果如下：
(1) 19F3
重链可变区的氨基酸序列如SEQ ID NO：2所示，轻链可变区的氨基酸序列如SEQ ID NO：7所示。
其重链可变区的3个CDR的氨基酸序列如下：
HCDR1：GYSFTGYT(SEQ ID NO：3)，
HCDR2：INPYNAGT(SEQ ID NO：4)，
HCDR3：ARSEYRYGGDYFDY(SEQ ID NO：5)；
其轻链可变区的3个CDR的氨基酸序列如下：
LCDR1：QSLLNSSNQKNY(SEQ ID NO：8)，
LCDR2：FAS(SEQ ID NO：9)，
LCDR3：QQHYDTPYT(SEQ ID NO：10)。
(2) 19F3H2L2
重链可变区的氨基酸序列如SEQ ID NO：20所示，轻链可变区的氨基酸序列如SEQ ID NO：22所示。
其重链可变区的3个CDR的氨基酸序列与19F3相同。
其轻链可变区的3个CDR的氨基酸序列与19F3相同。
(3) 19F3H2L3
重链可变区的氨基酸序列如SEQ ID NO：20所示，轻链可变区的氨基酸序列如SEQ ID NO：24所示。
其重链可变区的3个CDR的氨基酸序列与19F3相同。
其轻链可变区的3个CDR的氨基酸序列与19F3相同。
(4) 14C12
重链可变区的氨基酸序列如SEQ ID NO：44所示，轻链可变区的氨基酸序列如SEQ ID NO：49所示。
其重链可变区的3个CDR的氨基酸序列如下：
HCDR1：GFAFSSYD(SEQ ID NO：45)
HCDR2：ISGGGRYT(SEQ ID NO：46)
HCDR3：ANRYGEAWFAY(SEQ ID NO：47)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：QDINTY(SEQ ID NO：50)
LCDR2：RAN(SEQ ID NO：51)
LCDR3：LQYDEFPLT(SEQ ID NO：52)
(5) 14C12H1L1
重链可变区的氨基酸序列如SEQ ID NO：62所示，轻链可变区的氨基酸序列如SEQ ID NO：64所示。
其重链可变区的3个CDR的氨基酸序列与14C12相同。
其轻链可变区的3个CDR的氨基酸序列与14C12相同。
(6) NTPDV1、NTPDV2、NTPDV3、NTPDV4其重链所包含的9个CDR的氨基酸序列依照N端向C端顺序分别为与13F9重链，14C12重链和14C12轻链区域的CDR氨基酸序列一致，按照前述排列序列如下：
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)
HCDR7：QDINTY(SEQ ID NO：50)
HCDR8：RAN(SEQ ID NO：51)
HCDR9：LQYDEFPLT(SEQ ID NO：52)
其轻链的3个CDR的氨基酸序列与19F3轻链三个CDR氨基酸序列一致，序列如下：
LCDR1：QSLLNSSNQKNY(SEQ ID NO：8)
LCDR2：FAS(SEQ ID NO：9)
LCDR3：QQHYDTPYT(SEQ ID NO：10)。
本发明的再一方面涉及杂交瘤细胞株LT014，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018137。
本发明的再一方面涉及杂交瘤细胞株LT003，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015105。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Bethesda M.d.，Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，(1987 and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196：901-917；Chothia等人Nature  1989；342：878-883，或者IMGT编号系统定义，见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].Nucleic acids research，2009；38(suppl_1)：D301-D307的定义。
特别地，重链还可以包含3个以上CDR，例如6、9、或12个。例如在本发明的双特异性抗体中，重链可以是IgG抗体的重链的C端连接一个ScFv，这种情况下重链含有9个CDR。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2021089059-appb-000001
G，Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature，1975；256(5517)：495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.，Nature 1986；321：522525；Reichmann et al.，Nature，1988；332：323 329；Presta，Curr.Op.Struct.Biol.1992；2：593-596；和Clark， Immunol.Today 2000；21：397 402。在一些情况下，抗体的抗原结合片段是双抗体(Diabodies)，其中V H和V L结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.et al.，Proc.Natl.Acad.Sci.USA 1993；90：6444-6448和Poljak R.J.et al.，Structure 1994；2：1121-1123)。
如本文中所使用的，术语”单链抗体(single chain fragment variable，ScFv)”是指，包含通过连接体连接的抗体重链可变区(V H)和抗体轻链可变区(V L)的分子。其中V L和V H结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如，Bird et al，Science 1988；242：423-426和Huston et al，Proc.Natl.Acad.Sci.USA 1988；85：5879-5883)。此类scFv分子可具有一般结构：NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger et al，Proc.Natl.Acad.Sci.USA 1993；90：6444-6448)。可用于本发明的其他接头由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和Roovers et al，Cancer Immunology，Immunotherapy，2001，50(1)：51-59.描述。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白 获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，GS细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，PD-1蛋白)。可以使用本领域技术人员知悉的方法测定K D，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使 用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明的单克隆抗体(例如13F9H2L3)能够很好地特异性与CD73结合，并且能够十分有效地，以非底物竞争的方式抑制CD73的酶活反应，降低腺苷的产生，促进T细胞活性及肿瘤抑制效果。
本发明涉及的双特异性抗体例如NTPDV1、NTPDV2、NTPDV3、NTPDV4能够很好地特异性与PD-1及CD73结合，并且能够十分有效地阻断PD-1与PDL1的结合，特异地解除PD-1对机体免疫抑制，以及抑制CD73的催化活性，解除腺苷对免疫细胞的抑制，激活T淋巴细胞，并且不引起细胞因子IL-8和IL-6的释放，有效地增加安全性和有效性。
本发明的双功能抗体具有应用于制备抗肿瘤的药物的潜力。
附图说明
图1.ELISA检测P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、14C12H1L1、Nivolumab与PD-1-mFc的结合。
图2.ELISA检测P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、19F3H2L3、MEDI9447与人NT5E-Biotin的结合。
图3.P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、14C12H1L1和Nivolumab与人PD-L1-mFc竞争结合人PD-1-mFc-Biotin的活性检测结果。
图4.P1D7V01与PD-1-mFc亲和力常数测定。
图5. 14C12H1L1与PD-1-mFc亲和力常数测定
图6.Nivolumab与PD-1-mFc亲和力常数测定。
图7.P1D7V01与人NT5E(1-552)-his亲和力常数测定。
图8.MEDI 9447与人NT5E(1-552)-his亲和力常数测定。
图9.FACS检测P1D7V01、P1D7V02R、P1D7V03、P1D7V04R和14C12H1L1与293T-PD1细胞表面PD-1的结合活性。
图10.FACS检测P1D7V01、P1D7V03、MEDI9447和19F3H2L3与MDA-MB-231细胞表面CD73的结合活性
图11.抗CD73-抗PD-1双特异性抗体对MDA-MB-231膜表面CD73酶活性的抑制作用。
图12.抗CD73-抗PD-1双特异性抗体对U87-MG膜表面CD73酶活性的抑制作用。
图13.抗CD73-抗PD-1双特异性抗体促Raji-PDL1混合淋巴反应体系分泌IFN-γ的生物活性检测。
图14.抗CD73-抗PD-1双特异性抗体促Raji-PDL1混合淋巴反应体系分泌IL-2的生物活性检测。
图15.抗CD73-抗PD-1双特异性抗体促DC混合淋巴反应体系分泌IFN-γ的生物活性检测。
图16.抗CD73-抗PD-1双特异性抗体促DC混合淋巴反应体系分泌IL-2的生物活性检测。
图17. 14C12H1L1(hG1TM)与PD-1-mFc亲和力常数测定。
图18.Nivolumab与PD-1-mFc亲和力常数测定
图19.NTPDV1与PD-1-mFc亲和力常数测定。
图20.NTPDV2与PD-1-mFc亲和力常数测定。
图21.NTPDV3与PD-1-mFc亲和力常数测定。
图22.NTPDV4与PD-1-mFc亲和力常数测定。
图23. 19F3H2L3(hG1M)与人NT5E(1-552)-his亲和力常数测定。
图24.NTPDV1与人NT5E(1-552)-his亲和力常数测定。
图25.NTPDV2与人NT5E(1-552)-his亲和力常数测定。
图26.NTPDV3与人NT5E(1-552)-his亲和力常数测定。
图27.NTPDV4与人NT5E(1-552)-his亲和力常数测定。
图28.抗CD73-抗PD-1双特异性抗体抑制U87-MG细胞膜表面CD73酶活性的检测。
图29.混合淋巴细胞反应MLR检测抗CD73-抗PD-1双特异性抗体促IFN-γ和IL-2分泌的生物活性。
图30.同型对照、19F3H2L3(hG1M)、不同剂量NTPDV2对小鼠肿瘤体积的影响。
图31.同型对照、19F3H2L3(hG1M)、不同剂量NTPDV2对小鼠体重的影响。
图32.在CHO-K1-PD1细胞和人巨噬细胞共培养体系中，Fc段氨基酸突变有效消除由PD-1/CD73双特异性抗体介导的人巨噬细胞分泌IL-8。
图33.在CHO-K1-PD1细胞和人巨噬细胞共培养体系中，Fc段氨基酸突变有效消除由PD-1/CD73双特异性抗体介导的人巨噬细胞分泌IL-6。
图34.在U87-MG细胞和人巨噬细胞共培养体系中，Fc段氨基酸突变有效消除由PD-1/CD73双特异性抗体介导的人巨噬细胞分泌IL-8。
图35.在U87-MG细胞和人巨噬细胞共培养体系中，Fc段氨基酸突变有效消除由PD-1/CD73双特异性抗体介导的人巨噬细胞分泌IL-6。
生物材料保持信息：
杂交瘤细胞株LT003(又称PD-1-14C12)，其于2015年6月16日保 藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015105，保藏地址为中国.武汉.武汉大学，邮编：430072。
杂交瘤细胞株LT014(又称CD73-19F3)，其于2018年6月21日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018137，保藏地址为中国.武汉.武汉大学，邮编：430072。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。例如MDA-MB-231细胞和U87-MG细胞可以购自ATCC。
在本发明的下述实施例中，使用的BALB/c小鼠购自广东省医学实验动物中心。
在本发明的下述实施例中，所用的阳性对照抗体MEDI9447(通用名：Oleclumab)，生产自中山康方生物医药有限公司，其序列与Medlmmune Limited公开专利，公开号：US 20160129108A1中所述的抗体SEQ ID NOs：21-24相同。
在本发明的下述实施例中，所用的同靶点已上市药物抗体Nivolumab，商品名Opdivo，购自百时美施贵宝公司。
在本发明的下述实施例中，所用的293T-PD1细胞系由中山康方生物医药有限公司构建。293T-PD1细胞系由HEK293T细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T，Zufferey R，Kelly M，Mandel RJ，Nguyen M，Trono D，and Naldini L.J Virol.1998.72(11)：8463-8471.其中所使用的慢病毒表达载体为pCDH-CMV-PD-1FL-Puro(其中PD1，Genebank ID：NM_005018；载体pCDH-CMV-Puro，购自优宝生物，产品编号：VT1480)。
在本发明的下述实施例中，所用的Raji-PDL1细胞系由中山康方生物医药有限公司构建。Raji-PDL1细胞系由Raji细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T，Zufferey R，Kelly M，Mandel RJ，Nguyen M，Trono D，and Naldini L.J Virol.1998.72(11)：8463-8471.其中所使用的慢病毒表达载体为plenti6.3-PDL1(其中PDL1，Genebank ID：NP_054862.1；载体plenti6.3，购自Invitrogen，货号：K5315-20)。
在本发明的下述实施例中，所用的CHO-K1-PD1细胞系由中山康方生物医药有限公司构建。CHO-K1-PD1细胞系由CHO-K1细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T，Zufferey R，Kelly M，Mandel RJ，Nguyen M，Trono D，and Naldini L.J Virol.1998.72(11)：8463-8471.其中所使用的慢病毒表达载体为pCDH-CMV-PD-1FL-Puro(其中PD1，Genebank ID：NM_005018；载体pCDH-CMV-Puro，购自优宝生物，产品编号：VT1480)。
实验例中采用携带有S228P突变的IgG4亚型抗PD-1抗体Nivolumab(商品名Opdivo)作为对照抗体，均购自百时美施贵宝公司。
在本发明的下述实验例中，所用到同型对照抗体，即hIgG1均为靶向为人抗鸡蛋溶酶体(HEL)的抗体，抗体的可变区序列来自于Acierno等人发表的Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies(Acierno等人.J Mol Biol.2007；374(1)：130-46.)，hIgG1的恒定区片段采用Ig gamma-1 chain C region，ACCESSION：P01857作为重链恒定区，Ig kappa chain C region，ACCESSION：P01834为轻链恒定区；hIgG1为在中山康方生物医药有限公司的实验室制得。
实施例1：抗CD73抗体19F3的制备
1.杂交瘤细胞株LT014的制备
制备抗CD73抗体所用的抗原为人NT5E-his(NT5E为 GenbankID：NP_002517.1，位置：1-552)。取免疫后小鼠的脾细胞与小鼠骨髓瘤细胞融合，制成杂交瘤细胞，以人NT5E(NT5E为GenbankID：NP_002517.1，位置：1-552)-Biotin作为抗原，对杂交瘤细胞进行间接ELISA法筛选，获得能够分泌与CD73特异性结合的抗体的杂交瘤细胞。对ELISA法筛选得到的杂交瘤细胞，经过有限稀释法得到稳定的杂交瘤细胞株。将以上杂交瘤细胞株命名为杂交瘤细胞株LT014，其分泌的单克隆抗体命名为19F3。
杂交瘤细胞株LT014(又称CD73-19F3)，其于2018年6月21日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018137，保藏地址为中国.武汉.武汉大学，邮编：430072。
2.抗CD73抗体19F3的制备
用CD培养基(Chemical Defined Medium)对上面制得的LT014细胞株进行培养(CD培养基，内含1％青链霉素，于5％CO 2，37℃细胞培养箱中进行培养)，7天后收集细胞培养上清，通过高速离心、微孔滤膜抽真空过滤以及HiTrap protein A HP柱进行纯化，制得抗体19F3。
实施例2：抗CD73的抗体19F3的序列分析
按照培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)的方法，分别从实施例1中培养的LT014细胞株中提取mRNA。
按照Invitrogen
Figure PCTCN2021089059-appb-000002
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。
PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1 Cloning Kit(Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，测序结果如下：
19F3重链可变区的核酸序列(363bp)如SEQ ID NO：1所示，其编码的氨基酸序列(121aa)如SEQ ID NO：2所示。
根据IMGT编号系统，其中重链CDR1的序列如SEQ ID NO：3所示，重链CDR2的序列如SEQ ID NO：4所示，重链CDR3的序列如SEQ ID NO：5所示。
19F3轻链可变区的核酸序列(339bp)如SEQ ID NO：6所示，其编码的氨基酸序列(113aa)如SEQ ID NO：7所示。
根据IMGT编号系统，其中轻链CDR1的序列如SEQ ID NO：8所示，轻链CDR2的序列如SEQ ID NO：9所示，轻链CDR3的序列如SEQ ID NO：10所示。
19F3重链4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：11至SEQ ID NO：14所示；19F3轻链4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：15至SEQ ID NO：18所示。
实施例3：抗人CD73的人源化抗体的设计、制备和检测
1.人源化抗体19F3H2L3和19F3H2L2的轻链和重链序列的设计
根据人CD73蛋白的三维晶体结构(Hage T，Reinemer P，Sebald W.Crystals of a 1∶1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain.Eur J Biochem.1998；258(2)：831-6)以及实施例2获得的抗体鼠源抗体19F3的序列，通过计算机模拟抗体模型，根据模型设计突变，得到抗体19F3H1L1、19F3H2L3和19F3H2L3的可变区序列，其相应的重链可变区序列分别为19F3H1、19F3H2(氨基酸序列分别如SEQ ID NO：93、SEQ ID NO：97所示)，轻链可变区序列分别为和19F3L1、19F3L2、19F3L3(氨基酸序列分别如SEQ ID NO：95、SEQ ID NO：98、SEQ ID NO：99所示)，抗体恒定区序列，来自NCBI的数据库，重链恒定区采用Ig gamma-1 chain C region，ACCESSION：P01857；轻链恒定区为Ig kappa chain C region，ACCESSION：P01834。其中，19F3H2L3在申请号为202110270671.X的中国发明专利中又写为19F3H2L3(hG1WT)，其中19F3H1L1，19F3H2L2，19F3H2L3的轻、重链可变区又可记为19F3H1V(或者19F3H1 V)，19F3H2V(或者19F3H2 V)，19F3L1V(或者19F3L1 V)，19F3L2V(或者19F3L2 V)，19F3L3V(或者19F3L3 V)。
(1)人源化单克隆抗体19F3H1L1的重链可变区和轻链可变区序列
重链可变区的核酸序列(363bp)如SEQ ID NO：92所示，其编码的氨基酸序列(121aa)如SEQ ID NO：93所示。
轻链可变区的核酸序列(339bp)如SEQ ID NO：94所示，其编码的氨基酸序列(113aa)如SEQ ID NO：95所示。
(2)人源化单克隆抗体19F3H2L2的重链可变区和轻链可变区序列如下：
重链可变区19F3H2的核酸序列(363bp)如SEQ ID NO：19所示，其编码的氨基酸序列(121aa)如SEQ ID NO：20所示。
轻链可变区19F3L3的核酸序列(339bp)如SEQ ID NO：21所示，其编码的氨基酸序列(113aa)如SEQ ID NO：22所示。
(3)人源化单克隆抗体19F3H2L3的重链可变区和轻链可变区序列如下：
重链可变区19F3H2的核酸序列(363bp)如SEQ ID NO：19所示，其编码的氨基酸序列(121aa)如SEQ ID NO：20所示。
轻链可变区19F3L3的核酸序列(339bp)如SEQ ID NO：23所示，其编码的氨基酸序列(113aa)如SEQ ID NO：24所示。
2.人源化抗体19F3H1L1、19F3H2L2和19F3H2L3的制备
重链恒定区采用Ig gamma-1 chain C region，ACCESSION：P01857；轻链恒定区采用Ig kappa chain C region，ACCESSION：P01834。
分别将19F3H1L1、19F3H2L2和19F3H2L3的重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-19F3H1、pUC57simple-19F3L1，pUC57simple-19F3H2、pUC57simple-19F3L2和pUC57simple-19F3L3。参照《分子克隆实验指南(第二版)》介绍的标准技术，EcoRI&HindIII酶切合成的重、轻链全长基因，通过限制酶(EcoRI&HindIII)的酶切亚克隆到表达载体pcDNA3.1中获得表达质粒pcDNA3.1-19F3H1，pcDNA3.1-19F3L1，pcDNA3.1-19F3H2，pcDNA3.1-19F3L2，和pcDNA3.1-19F3L3，并进一步对重组表达质粒的重轻链基因进行测序分析。随后将含有相应的轻、重链重组质粒设计基因组合(pcDNA3.1-19F3H1/pcDNA3.1-19F3L1，pcDNA3.1-19F3H2/pcDNA3.1-19F3L2，pcDNA3.1-19F3H2/pcDNA3.1-19F3L3)分别共转染293F细胞后收集培养液进行纯化。测序验证正确后，制备去内毒素级别的表达质粒并将质粒瞬时转染 HEK293细胞进行抗体表达，培养7天后收集细胞培养液，采用Protein A柱进行亲和纯化获得人源化抗体。
3.人源化抗体19F3H2L3(hG1M)和19F3H2L3(hG1TM)的轻链和重链序列的设计
本发明人在实施例3的1中所获得的19F3H2L3的基础上，通过在其重链的第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)，得到了19F3H2L3(hG1M)，19F3H2L3(hG1M)重链核苷酸及氨基酸序列分别如SEQ ID NO：25和SEQ ID NO：26所示；19F3H2L3(hG1M)轻链恒定区为Ig kappa chain C region，ACCESSION：P01834，19F3H2L3(hG1M)轻链核苷酸及基酸序列分别如SEQ ID NO：27和SEQ ID NO：28所示。
本发明人在实施例3的1中所获得的19F3H2L3的基础上，通过在其重链的第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)，第237号位点引进了甘氨酸到丙氨酸的点突变(G237A)，得到了19F3H2L3(hG1TM)，其重链氨核苷酸及基酸序列分别如SEQ ID NO：29和SEQ ID NO：30所示；其轻链与19F3H2L3(hG1M)一致。
19F3H2的4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：31至SEQ ID NO：34所示；
19F3L2轻链4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：35至SEQ ID NO：38所示；
19F3L3轻链4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：39至SEQ ID NO：42所示。
4.人源化抗体19F3H2L3(hG1M)的制备
分别将19F3H2L3(hG1M)的重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-19F3H2(hG1M)、pUC57simple-19F3L3。参照《分子克隆实验指南(第二版)》介绍的标准技术，EcoRI&HindIII酶切合成的重、轻链全长基因，通过限制酶(EcoRI&HindIII)的酶切亚克隆到表达载体 pcDNA3.1中获得表达质粒pcDNA3.1-19F3H2(hG1M)和pcDNA3.1-19F3L3，并进一步对重组表达质粒的重轻链基因进行测序分析。随后将含有相应的轻、重链重组质粒设计基因组合pcDNA3.1-19F3H2(hG1M)/pcDNA3.1-19F3L3共转染293F细胞后收集培养液进行纯化。测序验证正确后，制备去内毒素级别的表达质粒并将质粒瞬时转染HEK293细胞进行抗体表达，培养7天后收集细胞培养液，采用Protein A柱进行亲和纯化获得人源化抗体19F3H2L3(hG1M)。
实施例4：抗PD-1的抗体14C12的制备
1.杂交瘤细胞株LT003的制备
用PD-1-mFc融合蛋白(PD-1 GenBank：NM_005018，mFc SEQ ID NO：89)作为抗原，取免疫BALB/c小鼠(购自广东医学实验动物中心)的脾细胞与小鼠骨髓瘤细胞融合成杂交瘤细胞，参考目前已确立的方法(例如，Stewart，S.J.，“Monoclonal Antibody Production”，in Basic Methods in antibody Production and Characterization，Eds.G.C.Howard and D.R.Bethell，Boca Raton：CRC Press，2000)。
用PD-1-hFc(PD-1 Genbank ID：NM_005018，hFc为人IgG Fc纯化标签，具体为Ig gamma-1 chain C region，GenbankID：P01857位置114-330)作为抗原包被酶标板，进行间接ELISA法筛选，得到分泌与PD-1特异性结合的新的抗体的杂交瘤细胞。
通过竞争ELISA筛选出能够分泌与配体PD-L1-hFc(PD-L1 Genbank ID：NP_054862.1)竞争结合PD-1的单克隆抗体的杂交瘤细胞株，经过有限稀释法得到稳定的杂交瘤细胞株，并经过有限稀释法得到LT003稳定细胞株(PD-1-14C12)，其分泌的单克隆抗体命名为14C12。
杂交瘤细胞株LT003(又称PD-1-14C12)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015105，保藏地址为中国.武汉.武汉大学，邮编：430072。
2.抗PD-1的抗体14C12的制备
用含10％的低IgG胎牛血清的IMDM培养基对上面制得的LT003细胞株进行培养(IMDM培养基，内含1％青链霉素，于5％CO 2，37℃细 胞培养箱中进行培养)，7天后收集细胞培养上清进行纯化，制得抗体14C12。
实施例5：抗PD-1抗体14C12的序列分析
按照培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1制得的杂交瘤细胞株LT003中提取mRNA。
按照Invitrogen
Figure PCTCN2021089059-appb-000003
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。
PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1 Cloning Kit(Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，测序结果如下：
重链可变区的核酸序列(354bp)如SEQ ID NO：43所示，其编码的氨基酸序列(118aa)如SEQ ID NO：44所示。
根据IMGT编号系统，其中重链CDR1的序列如SEQ ID NO：45所示，重链CDR2的序列如SEQ ID NO：46所示，重链CDR3的序列如SEQ ID NO：47所示。
轻链可变区的核酸序列(321bp)如SEQ ID NO：48所示，其编码的氨基酸序列(107aa)如SEQ ID NO：49所示。
根据IMGT编号系统，其中轻链CDR1的序列如SEQ ID NO：50所示，轻链CDR2的序列如SEQ ID NO：51所示，轻链CDR3的序列如SEQ ID NO：52所示。
14C12重链4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：53至SEQ ID NO：56所示；14C12轻链4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：57至SEQ ID NO：60所示。
实施例6：抗PD-1的人源化抗体14C12H1L1及的14C12H1L1(hG1TM)设计和制备
1.抗PD-1的人源化抗体14C12H1L1的设计
人源化抗体14C12H1L1的轻链和重链序列的设计，根据PD-1蛋白的 三维晶体结构(Shinohara T，et al.，Structure and chromosomal localization of the human PD-1 gene(PDCD1).Genomics 1995，23(3)：704-6)以及实施例5获得的抗体14C12的序列，通过计算机模拟抗体模型，根据模型设计突变，得到抗体14C12H1L1的可变区序列。
设计的可变区序列如下：
人源化单克隆抗体14C12H1L1的重链可变区14C12H1的核酸序列(354bp)如SEQ ID NO：61所示，其编码的氨基酸序列(118aa)如SEQ ID NO：62所示。
人源化单克隆抗体14C12H1L1的轻链可变区14C12L1的核酸序列(321bp)如SEQ ID NO：63所示，其编码的氨基酸序列(107aa)如SEQ ID NO：64所示。
抗体14C12H1L1的抗体恒定区序列，来自NCBI的数据库，重链恒定区采用Ig gamma-1 chain C region，ACCESSION：P01857；轻链恒定区为Ig kappa chain C region，ACCESSION：P01834)，14C12H1L1重链的核苷酸序列和氨基酸序列分别如SEQ ID NO：65和66所示，14C12H1L1轻链的核苷酸序列和氨基酸序列分别如SEQ ID NO：67和68所示。其中，14C12H1L1在本文及申请号为202110270671.X的中国发明专利中又写为14C12H1L1(hG1WT)，其中，14C12H1L1的重链可变区和轻链可变区在本文在申请号为202110270671.X的中国发明专利中又写为14C12H1V(或者14C12H1 V)，和14C12L1 V(或者14C12L1 V)。
2.人源化抗体14C12H1L1(hG1TM)轻链和重链序列的设计
本发明人在实施例6的1中所获得的14C12H1L1的基础上，通过在其重链的第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)，第237号位点引进了甘氨酸到丙氨酸的点突变(G237A)得到了抗体14C12H1L1(hG1TM)。14C12H1L1(hG1TM)的重链核苷酸及氨基酸序列分别入SEQ ID NO：69和SEQ ID NO：70所示；其轻链可变区氨基酸序列与抗体14C12H1L1的轻链可变区一致。
14C12H1的4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：71至SEQ ID NO：74所示；
14C12L1轻链4个骨架区(Framework region)(FR-H1至FR-H4)氨基酸序列分别如SEQ ID NO：75至SEQ ID NO：78所示；
3.人源化抗体14C12H1L1，14C12H1L1(hG1TM)的制备
分别将14C12H1L1(hG1TM)和14C12H1L1的重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-14C12H1、pUC57simple-14C12L1和pUC57simple-14C12H1(hG1TM)。参照《分子克隆实验指南(第二版)》介绍的标准技术，EcoRI&HindIII酶切合成的重、轻链全长基因，通过限制酶(EcoRI&HindIII)的酶切亚克隆到表达载体pcDNA3.1中获得表达质粒pcDNA3.1-14C12H1，pcDNA3.1-14C12L1，和pcDNA3.1-14C12H1(hG1TM)，并进一步对重组表达质粒的重轻链基因进行测序分析。随后将含有相应的轻、重链重组质粒设计基因组合(pcDNA3.1-14C12H1(hG1TM)/pcDNA3.1-14C12L1，pcDNA3.1-14C12H1/pcDNA3.1-14C12L1)分别共转染293F细胞后收集培养液进行纯化。测序验证正确后，制备去内毒素级别的表达质粒并将质粒瞬时转染HEK293细胞进行抗体表达，培养7天后收集细胞培养液，采用Protein A柱进行亲和纯化获得人源化抗体。
实施例7：抗PD-1/CD73双功能抗体的序列设计、表达
1.序列设计
本发明中的双功能抗体的结构模式属于Morrison模式(IgG-scFv)，即在一个IgG抗体的两条重链的C端均连接另一个抗体的scFv片段，其重链和轻链的主要组成设计如下面的表1。表1表1：P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、P1D7V07、P1D7V08的重链和轻链的组成设计
Figure PCTCN2021089059-appb-000004
Figure PCTCN2021089059-appb-000005
上面的表1中：
(1)右下角标注“V”的，是指相应重链的可变区或者相应轻链的可变区。没有标注“V”的，相应重链或者轻链为包含恒定区的全长。这些可变区或者全长的氨基酸序列及其编码核酸序列均参照上面的实施例中记载的相应序列。
(2)Linker1的氨基酸序列为(GGGGS)4((核苷酸序列SEQ ID NO：80，氨基酸序列SEQ ID NO：79)，Linker2的氨基酸序列为(GGGGS)3((核苷酸序列SEQ ID NO：82，氨基酸序列SEQ ID NO：81)。
2.抗体的表达和纯化
分别将P1D7V01的重链cDNA序列和轻链的cDNA序列克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-VP101H和pUC57simple-VP101L质粒。
分别将质粒pUC57simple-VP101H和pUC57simple VP101L进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
按照上述P1D7V01的表达和纯化方法获得纯化的抗体P1D7V02R、P1D7V03、P1D7V04R、P1D7V07、P1D7V08。
实施例8：ELISA方法测定抗CD73-抗PD-1双特异性抗体与抗原的结合活性
1.ELISA方法分别测定P1D7V01、P1D7V02R、P1D7V03、P1D7V04R与抗原PD-1-mFc的结合活性。具体方法如下：
将PD-1-mFc，0.5μg/ml包被酶标板置于4℃孵育过夜，然后使用PBST洗板包被了抗原的酶标板1次，再使用1％BSA的PBS溶液作为封闭液在 37℃下对酶标板进行封闭2小时。酶标板封闭结束后用PBST洗板3次，加入PBST溶液梯度稀释的抗体(抗体稀释梯度详见表2)，加入待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1∶5000比例稀释的HRP标记羊抗人IgG(H+L)(Jackson，货号：109-035-088)二抗工作液，然后置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如表2和图1所示。由图可知，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R与抗原PD-1-mFc能够有效的结合，并且其结合效率呈剂量依赖关系，各剂量的吸光度强度见表2，通过对结合的抗体进行吸光度定量分析，曲线模拟计算获得抗体P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、14C12H1L1和Nivolumab(作为对照)的结合效率EC50分别为0.078nM、0.078nM、0.075nM和0.089nM、0.033nM、0.051nM。
以上实验结果表明，在相同实验条件下，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R结合PD-1-mFc的活性与同靶点阳性药14C12H1L1、Nivolumab相当，提示P1D7V01、P1D7V02R、P1D7V03、P1D7V04R具有有效结合PD-1-mFc的活性。
表2：ELISA检测P1D7V01、P1D7V02R、P1D7V03、P1D7V04R。14C12H1L1、Nivolumab与PD-1-mFc的结合
Figure PCTCN2021089059-appb-000006
Figure PCTCN2021089059-appb-000007
2.ELISA方法分别测定P1D7V01、P1D7V02R、P1D7V03、P1D7V04R与抗原人NT5E-Biotin的结合活性
将链霉亲和素，2μg/ml包被酶标板后置于4℃孵育过夜。孵育结束后使用PBST对包被了链霉亲和素的酶标板洗板1次，1％BSA的PBS溶液作为酶标板封闭液，对酶标板在37℃进行封闭2小时。酶标板封闭结束后用PBST洗板3次。再加入抗原人NT5E-Biotin 0.5μg/ml并置于37℃条件下孵育30分钟，然后用PBST洗板3次。在酶标板孔内加入PBST溶液梯度稀释的抗体(抗体稀释梯度详见表3)，加入待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1∶5000比例稀释的HRP标记羊抗人IgG(H+L)(Jackson，货号：109-035-088)二抗工作液，置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如表3和图2所示。由图可知，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R与抗原人NT5E-Biotin能够有效的结合，并且其结合效率呈剂量依赖关系(各剂量的吸光度强度见表3)。通过对结合的抗体进行吸光度定量分析，曲线模拟计算获得抗体P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、19F3H2L3和MEDI9447(作为对照抗体)的结合效率EC50分别为0.063nM、0.230nM、0.068nM和0.439nM、0.045nM、0.042nM。
以上实验结果表明，在相同实验条件下，双特异抗体P1D7V01、P1D7V02R、P1D7V03、P1D7V04R结合人NT5E-Biotin的活性与同靶点 阳性药19F3H2L3、MEDI9447相当，提示P1D7V01、P1D7V02R、P1D7V03、P1D7V04R具有有效结合人NT5E-Biotin的活性。
表3：ELISA检测P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、19F3H2L3、MEDI9447与人NT5E-Biotin的结合
Figure PCTCN2021089059-appb-000008
实施例9：竞争ELISA方法分别测定抗体抗CD73-抗PD-1双特异性抗体与人PD-L1-mFc竞争结合人PD-1-mFc-Biotin的活性
将人PD-L1-mFc(PD-L1 Genbank ID：NP_054862.1，mFc SEQ ID NO：143)以2μg/mL包被酶标板后，4℃孵育过夜。孵育结束后用1％BSA的PBS溶液在37℃条件下对酶标板进行封闭2小时，封闭结束后洗板三次并拍干。在稀释板上以10μg/mL作为起始浓度按照1∶3的稀释比例将抗体进行梯度稀释至7个浓度，并设空白对照，然后加入等体积的0.3μg/mL的人PD-1-mFc-Biotin溶液，混匀后在室温孵育20分钟。然后将反应后混合液加入到包被好的酶标板，在37℃条件下孵育30分钟。孵育结束用PBST洗板三次并拍干，加入SA-HRP(KPL，14-30-00)工作液，在37℃条件下孵育30分钟。孵育完成后洗板四次拍干，后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值，用SoftMax Pro  6.2.1软件对数据进行分析处理。
检测结果如图3所示。各剂量的OD值见表4。通过对结合的抗体进行吸光强度定量分析，曲线模拟抗体的结合效率获得结合EC50(表4)。
结果表明，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、14C12H1L1和Nivolumab(作为对照)能有效地阻断抗原人PD-1-mFc-Biotin与其受体人PD-L1-mFc的结合，且阻断效率呈现剂量依赖关系，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、14C12H1L1和Nivolumab阻断人PD-1-mFc-Biotin与其配体人PD-L1-mFc结合的EC50分别为1.115nM、1.329nM、1.154nM、1.339nM、1.459nM、1.698nM。
表4：P1D7V01、P1D7V02R、P1D7V03、P1D7V04R、14C12H1L1和Nivolumab与人PD-L1-mFc竞争结合人PD-1-mFc-Biotin的活性检测结果
Figure PCTCN2021089059-appb-000009
实施例10：使用Fortebio分子相互作用仪测定抗CD73-抗PD-1双特异性抗体与抗原人PD-1-mFc结合的动力学参数
样品稀释缓冲液为PBST，0.1％BSA，pH7.4。将抗体以5μg/mL的浓度固定于AHC传感器上，固定高度约为0.4nm，传感器在缓冲液中平衡60s，固定在传感器上的抗体与抗原PD-1-mFc结合，抗原浓度为0.6-50nM(三倍稀释)，结合时间为120s，蛋白在缓冲液中解离，时间180s。检测温度为37度，检测频率为0.3Hz，样品板震动速率为1000rpm。数据以 1∶1模型拟合分析，得到亲和力常数。
人源化抗体P1D7V01、14C12H1L1和Nivolumab(作为对照抗体)与人PD-1-mFc的亲和力常数测定结果见表5，检测结果如图4、图5和图6所示。人源化抗体P1D7V01、14C12H1L1和Nivolumab与人PD-1-mFc的亲和力常数依次为1.76E-10M，1.64E-10M，2.32E-10M。以上实验结果表明：P1D7V01与14C12H1L1、Nivolumab的结合能力相当，提示人源化抗体P1D7V01与人PD-1-mFc有较强的结合能力。
表5：P1D7V01、14C12H1L1、Nivolumab、与PD-1-mFc亲和力常数测定
待测抗体 KD(M) kon(1/Ms) SE(kon) kdis(1/s) S E(kdis) Rmax(nm)
P1D7V01 1.76E-10 4.19E+05 1.52E+04 7.37E-05 3.12E-05 0.13-0.17
14C12H1L1 1.64E-10 4.55E+05 1.61E+04 7.47E-05 2.98E-05 0.24-0.28
Nivolumab 2.32E-10 5.85E+05 2.03E+04 1.36E-04 3.47E-05 0.02-0.14
K D为亲和力常数；K D＝kdis/kon
实施例11：使用Fortebio分子相互作用仪测定抗CD73-抗PD-1双特异性抗体与抗原人NT5E(1-552)-his结合的动力学参数。
样品稀释缓冲液为PBST，pH7.4。将抗体以5μg/mL的浓度固定在Protein A传感器上，固定时间为15s，传感器在缓冲液中平衡120s，固定在传感器上的抗体与抗原人NT5E(1-552)-his结合，抗原浓度为3.125-200nM(两倍稀释)，结合时间为120s，蛋白在缓冲液中解离，时间600s。传感器采用10mM Gly，pH1.5溶液再生。检测温度为37度，检测频率为0.6Hz，样品板震动速率为1000rpm。数据以1∶1模型拟合分析，得到亲和力常数
人源化抗体P1D7V01、MEDI 9447(作为对照抗体)与人NT5E(1-552)-his的亲和力常数测定结果见表6，检测结果如图7和图8所示。人源化抗体P1D7V01和MEDI 9447与人NT5E(1-552)-his的亲和力常数依次为2.29E-10M，1.04E-10M。
以上实验结果表明：P1D7V01与MEDI 9447的结合能力相当，提示人源化抗体P1D7V01与人NT5E(1-552)-his有较强的结合能力。
表6：P1D7V01、MEDI 9447与人NT5E(1-552)-his亲和力常数测定
待测抗体 KD(M) kon(1/Ms) S E(kon) kdis(1/s) S E(kdis) Rmax(nm)
P1D7V01 2.29E-10 1.81E+05 2.30E+03 4.13E-05 4.54E-06 0.47-0.57
MEDI 9447 1.04E-10 2.34E+05 3.20E+03 2.44E-05 5.02E-06 0.59-0.82
K D为亲和力常数；K D＝kdis/kon
实施例12：FACS检测抗CD73-抗PD-1双特异性抗体的结合活性
1.FACS检测抗CD73-抗PD-1双特异性抗体与293T-PD1膜表面PD-1的结合活性
收集对数生长期293T-PD1细胞，按3x10 5个细胞/管将细胞转移到1.5ml离心管，加入500μL PBSA，5600rpm离心5min，去上清；加入PBSA稀释的抗体100μL(终浓度分别为100，33.33，11.11，3.7，1.23，0.41，0.14，0.05nM)，轻柔混匀后于冰上孵育1h；加入500μL PBSA，5600rpm离心5min，去上清；加入500倍稀释的FITC标记羊抗人IgG二抗(Jackson，货号：109-095-098)重悬混匀，冰上避光孵育0.5h；加入500μLPBSA，5600rpm离心5min，去上清；加入200μLPBSA重悬细胞沉淀，并转移至流式管，FACSCalibur检测。
实验结果如表7和图9所示，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R均可特异性与293T-PD1膜表面PD-1结合，且呈剂量依赖关系，与PD1单靶点对照抗体14C12H1L1相比，均强于14C12H1L1。
在相同实验条件下，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R与293T-PD1结合的EC50分别为1.000nM、1.075nM、1.377nM、1.57nM，14C12H1L1与293T-PD1结合的EC50为2.111nM。
以上实验结果表明，在相同实验条件下P1D7V01、P1D7V02R、P1D7V03、P1D7V04R与293T-PD1结合活性均优于PD1单靶点对照抗体14C12H1L1，提示P1D7V01、P1D7V02R、P1D7V03、P1D7V04R具有有 效结合293T-PD1膜表面PD-1的活性。
表7：FACS检测P1D7V01、P1D7V02R、P1D7V03、P1D7V04R和14C12H1L1与293T-PD1细胞表面PD-1的结合活性
浓度(nM) 0.05 0.14 0.41 1.23 3.70 11.11 33.33 100.00 EC50
14C12H1L1 35.69 57.70 129.42 437.62 974.97 1384.91 1174.75 1500.20 2.111
P1D7V01 26.65 58.65 150.57 307.19 530.77 577.76 479.34 531.15 1.000
P1D7V02R 23.44 42.98 108.44 217.56 404.33 426.06 405.29 301.40 1.075
P1D7V03 24.05 49.21 118.03 289.08 384.62 459.40 808.38 343.48 1.377
P1D7V04R 24.79 52.43 112.56 288.79 466.72 1284.73 400.33 360.79 1.57.0
2.FACS检测抗CD73-抗PD-1双特异性抗体与MDA-MB-231膜表面CD73的结合活性
常规胰酶消化对数期MDA-MB-231细胞，按3x10 5个细胞/管将细胞转移到1.5ml离心管，加入500μL PBSA，5600rpm离心5min，去上清；加入PBSA稀释的抗体100μL(终浓度分别为100，33.33，11.11，3.7，1.23，0.41，0.14，0.05nM)，轻柔混匀后于冰上孵育1h；加入500μLPBSA，5600rpm离心5min，去上清；加入500倍稀释的FITC标记羊抗人IgG二抗(Jackson，货号：109-095-098)重悬混匀，冰上避光孵育0.5h；加入500μLPBSA，5600rpm离心5min，去上清；加入200μLPBSA重悬细胞沉淀，并转移至流式管，FACSCalibur检测。
实验结果如表8和图10所示，与MDA-MB-231膜表面CD73的结合活性，P1D7V01和P1D7V03优于19F3H2L3，其中P1D7V01优于同靶点阳性药MEDI9447。在相同实验条件下，P1D7V01、P1D7V03与MDA-MB-231膜表面CD73结合的EC50分别为1.384nM、2.009nM，MEDI9447和19F3H2L3与MDA-MB-231膜表面CD73结合的EC50分别为1.589nM、2.773nM。
以上实验结果表明，P1D7V01、P1D7V03、19F3H2L3和同靶点阳性药MEDI9447均可呈剂量依赖性与MDA-MB-231膜表面CD73特异性结合，P1D7V01和P1D7V03的结合活性优于19F3H2L3，P1D7V01优于同 靶点阳性药MEDI9447。提示P1D7V01、P1D7V03具有有效结合MDA-MB-231膜表面CD73的活性。
表8：FACS检测P1D7V01、P1D7V03、MEDI9447和19F3H2L3与MDA-MB-231细胞表面CD73的结合活性。
浓度(nM) 0.05 0.14 0.41 1.23 3.70 11.11 33.33 100.00 EC50
MEDI9447 32.59 64.38 157.75 388.51 697.80 829.29 930.26 878.04 1.589
19F3H2L3 25.95 45.13 102.56 189.07 466.87 658.72 843.09 639.57 2.773
P1D7V01 23.37 38.02 76.84 271.72 571.56 560.81 525.91 705.50 1.384
P1D7V03 16.96 44.05 106.39 233.32 457.61 525.37 576.19 677.66 2.009
实施例13：抗CD73-抗PD-1双特异性抗体抑制细胞膜表面CD73酶活性的检测
1.检测抗CD73-抗PD-1双特异性抗体对MDA-MB-231细胞膜表面CD73酶活性的抑制
实验步骤如下：取状态良好的对数期MDA-MB-231细胞，用无血清的RPMI-1640培养液将细胞重悬、计数；将MDA-MB-231细胞接种至96孔板，2x104个细胞/100μL/孔；用无血清RPMI-1640培养液按2.5倍进行梯度稀释抗体；将抗体加入96孔板，每孔50μL，37℃孵育1小时，1小时后，每孔加入50μL 1200μM RPMI-1640稀释的AMP(TCL，货号：A0157)；3小时后取25μL细胞培养上清，转移至新96孔板，每孔加入25μL 100μM的ATP(TCL，货号：A0158)；每孔加入50μL CTG(CellTiterGlo，Promega，货号：G8641)显色液，进行显色，于多标记微孔板检测仪(PerkinElmer 2140-0020)读取相对荧光强度RLU。
实验结果如图11所示，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R与阳性对照药MEDI9447的AMP含量相当，并且呈浓度依赖关系。
以上实验结果说明加入的AMP，在无抗体情况下，可被MDA-MB-231细胞表面CD73的酶活性催化而转化为腺苷A，而加入抗体后，由于P1D7V01、P1D7V02R、P1D7V03、P1D7V04R抗体与CD73结合而降低了其酶催化的活性，使AMP不会转化为腺苷A。提示抗体以非底物竞争的方式有效抑制其酶活反应，降低腺苷的产生。
2.检测抗CD73-抗PD-1双特异性抗体对U87-MG细胞膜表面CD73酶活性的抑制
取状态良好的对数期U87-MG细胞，用无血清的RPMI-1640培养液将细胞重悬、计数；将U87-MG细胞接种至96孔板，2x10 4个细胞/100μL/孔；用无血清RPMI-1640培养液按2.5倍进行梯度稀释抗体，将抗体加入96孔板，每孔50μL，37℃孵育1小时；1小时后，每孔加入50μL 1200uM RPMI-1640稀释的AMP；3小时后取25μL细胞培养上清，转移至新96孔板，每孔加入25μL 100μM的ATP；每孔加入50μl CTG(CellTiterGlo)显色液，进行显色后于多标记微孔板检测仪(PerkinElmer 2140-0020)读取相对荧光强度RLU。
实验结果如图12所示，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R与阳性对照药MEDI9447的AMP含量相当，并且呈浓度依赖关系。
以上实验结果说明加入的AMP，在无抗体情况下，可被U87-MG细胞表面CD73的酶活性催化而转化为腺苷A，而加入抗体后，由于P1D7V01、P1D7V02R、P1D7V03、P1D7V04R抗体与CD73结合而降低了其酶催化的功能，使AMP不会转化为腺苷A。提示抗体以非底物竞争的方式有效抑制其酶活反应，降低腺苷的产生。
实施例14：混合淋巴细胞反应MLR检测抗CD73-抗PD-1双特异性抗体促IFN-γ和IL-2分泌的生物活性
1.抗CD73-抗PD-1双特异性抗体促Raji-PDL1混合淋巴反应体系分泌IFN-γ的生物活性检测
Raji-PDL1细胞正常传代培养；复苏PBMC，用10mL 1640完全培养基培养，0.5μg/mL的SEB(德诺泰克，货号：S010201)刺激两天；Raji-PDL1细胞用25μg/mL的MMC(Sigma，货号：M4287)处理，置于37℃培养箱中1小时；收集SEB刺激2天后PBMC和经MMC处理1小时的Raji-PDL1细胞，PBS洗涤两次，完全培养基重悬计数，分别加入到U型96孔板，100000个细胞/孔；按实验设计加入抗体，于培养箱共培养3天；3天后，收集细胞培养上清，ELISA法进行IFN-γ检测。
如图13所示，人PBMC和Raji-PDL1细胞混合培养后对PBMC的IFN-γ的分泌具有显著促进作用，在混合培养体系中同时加入抗体能显著诱导PBMC进一步分泌IFN-γ，促IFN-γ分泌活性水平方面，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R抗体与其母版PD-1单靶点抗体14C12H1L1活性相当。
2.抗CD73-抗PD-1双特异性抗体促Raji-PDL1混合淋巴反应体系分泌IL-2的生物活性检测
Raji-PDL1细胞正常传代培养；复苏PBMC，用10mL 1640完全培养基培养，SEB(0.5μg/mL)刺激两天；Raji-PDL1细胞用25μg/mL的MMC处理，置于37℃培养箱中1小时；收集SEB刺激2天后的PBMC和经MMC处理1小时的Raji-PDL1细胞，PBS洗两次，完全培养基重悬计数，分别加入到U型96孔板，100000个细胞/孔；按实验设计加入抗体，共培养3天；收集细胞培养上清，ELISA法进行IL-2检测。
如图14所示，人PBMC和Raji-PDL1细胞混合培养后对PBMC的IL-2的分泌有一定的促进作用，在混合培养体系中同时加入抗体能显著诱导PBMC进一步分泌IL-2，具有显著剂量依赖关系，促IL-2分泌活性水平方面，双功能抗体P1D7V01、P1D7V02R、P1D7V03、P1D7V04R在低浓度活性略低于其母版PD-1单靶点抗体14C12H1L1，而中高浓度与其相当，与PD1靶点阳性对照药Nivolumab相比，P1D7V01、P1D7V02R、P1D7V03、P1D7V04R在三个不同抗体浓度水平都具更佳的促IL-2分泌潜能。
3.抗CD73-抗PD-1双特异性抗体促DC混合淋巴反应体系分泌IFN-γ的生物活性检测
分离正常人外周血PBMC，完全培养基重悬，接种于培养皿，置于培养箱过夜培养后，收集去除悬浮的PBMC，皿底贴壁细胞用PBS缓冲液洗涤后进行DC成熟诱导：每皿加入含GM-CSF和IL-4的RPMI1640完全培养基10mL，GM-CSF和IL-4浓度均为1000U/mL，置于37℃，5％二氧化碳培养箱中培养三天；半量换液，补加GM-CSF、IL-4各1000U/mL，置于37℃，5％二氧化碳培养箱中继续培养三天；三天后再次半量换液，加 入GM-CSF、IL-4各1000U/mL，TNF-α 100U/mL，继续培养两天；新鲜分离另外供者的PBMC，细胞计数后接种至96孔板，100000个细胞/孔；收集诱导成熟的DC细胞，用完全培养基洗涤一次，细胞计数，接种至含PBMC的96孔板，10000个细胞/孔；按实验设计加入抗体，混匀后置于37℃，5％二氧化碳培养箱中共培养5天；5天后，收集细胞培养上清，ELISA法进行IFN-γ定量检测。
结果如图15所示，相比单独DC或PBMC培养，DC与PBMC的混合培养可显著促进IFN-γ的分泌；与同型对照相比，在DC与PBMC二者混合培养的基础上加入抗CD73-抗PD-1双特异性抗体，可见IFN-γ分泌水平进一步显著提高。
抗CD73-抗PD-1双特异性抗体促DC混合淋巴反应体系分泌IL-2的生物活性检测分离正常人外周血PBMC，完全培养基重悬，接种于培养皿，置于培养箱过夜培养后，收集去除悬浮的PBMC，皿底贴壁细胞用PBS缓冲液洗涤后进行DC成熟诱导；每皿加入含GM-CSF和IL-4的RPMI1640完全培养基10mL，GM-CSF和IL-4浓度均为2000U/mL，置于37℃，5％二氧化碳培养箱中培养三天；半量换液，加入50ng/mL的IFN-γ和100ng/mL的LPS，继续培养两天；复苏另外供者的PBMC，细胞计数后接种至96孔板，100000个细胞/孔；收集诱导成熟的DC细胞，用完全培养基洗涤一次，细胞计数，接种至含PBMC的96孔板，10000个细胞/孔；按实验设计加入抗体，混匀后置于37℃，5％二氧化碳培养箱中共培养5天；5天后，收集细胞培养上清，ELISA法进行IL-2定量检测。结果如图16所示，相比单独DC或PBMC培养，DC与PBMC的混合培养可显著促进IL-2的分泌；与同型对照相比，在DC与PBMC二者混合培养的基础上加入抗CD73-抗PD-1双特异性抗体，可见IL-2分泌水平进一步显著提高。
实施例15：抗PD-1/CD73双特异性抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4的制备
双特异性抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4的结构模式属于Morrison模式(IgG-scFv)，即在一个IgG抗体的两条重链的C端均通过连接片段连接另一个抗体的scFv片段，其重链和轻链的 设计组成如下面的表9。
由于其免疫球蛋白部分的恒定区区域引入了氨基酸突变以消除其与FcγR的结合活性，因此NTPDV1、NTPDV2、NTPDV3、NTPDV4在本文及在申请号为202110270671.X的中国发明专利中又写为NTPDV1(hG1TM)、NTPDV2(hG1TM)、NTPDV3(hG1TM)、NTPDV4(hG1TM)。
表9：NTPDV1、NTPDV2、NTPDV3、NTPDV4的序列设计
Figure PCTCN2021089059-appb-000010
上面的表9中：
右下角标注“V”的，是指相应重链的可变区或者相应轻链的可变区。没有标注“V”的，相应重链或者轻链为包含恒定区的全长。这些可变区或者全长的氨基酸序列及其编码核酸序列均参照上面的实施例中记载的相应序列。
Linker1的氨基酸序列为(GGGGS)结构重复4次，即(GGGGS)4或者(G4S)4(核苷酸序列SEQ ID NO：80，氨基酸序列SEQ ID NO：79)；
Linker2的氨基酸序列为(GGGGS)结构重复3次，即(GGGGS)4或者(G4S)3(核苷酸序列SEQ ID NO：82，氨基酸序列SEQ ID NO：81)。
NTPDV1、NTPDV2、NTPDV3、NTPDV4中免疫球蛋白部分中的轻链19F3L2和19F3L3的3个CDR序列为和19F3中轻链CDR序列一致。
NTPDV1、NTPDV2、NTPDV3、NTPDV4中免疫球蛋白部分中的19F3H2(hG1TM)的3个CDR序列为和19F3中重链CDR序列一致。
NTPDV1、NTPDV2、NTPDV3、NTPDV4中的scFv部分的 14C12H1V-Linker2-14C12L1V和14C12H1V-Linker1-14C12L1V的CDR序列和14C12中的重链CDR和轻链CDR一致。
NTPDV2和NTPDV4重链的氨基酸序列一致，记为NTPDH2/4(SEQ ID NO：83)，NTPDV2、NTPDV4的重链的核苷酸序列为SEQ ID NO：84。
NTPDV1和NTPDV3的重链的氨基酸序列一致，记为NTPDH1/3(SEQ ID NO：85)，NTPDV1和NTPDV3的重链的核苷酸序列为SEQ ID NO：86。
NTPDV1、NTPDV2、NTPDV3、NTPDV4中免疫球蛋白部分中的19F3L3和19F3L2的3个CDR序列和抗体19F3轻链三个CDR一致。
NTPDV1、NTPDV2中的免疫球蛋白部分的轻链19F3L3的氨基酸序列和抗体19F3H2L3(G1M)的轻链序列一致(SEQ ID NO：28)，NTPDV1、NTPDV2中的免疫球蛋白部分的轻链19F3L3的核苷酸序列为SEQ ID NO：27。NTPDV3、NTPDV4中的免疫球蛋白部分的轻链19F3L2的氨基酸序列为SEQ ID NO：96)，NTPDV3、NTPDV4中的免疫球蛋白部分的轻链19F3L2的核苷酸序列为SEQ ID NO：100。
(1)NTPDV1，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：28所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段2的氨基酸序列如SEQ ID NO：81所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示，
(2)NTPDV2，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：28所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示，
(3)NTPDV3，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：96所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段2的氨基酸序列如SEQ ID NO：81所示，14C12L1V的氨基酸序列如SEQ ID NO：68 所示，和
(4)NTPDV4，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：96所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示。
抗体的表达和纯化
分别将NTPDV1、NTPDV3的重链cDNA和NTPDV2、NTPDV4的重链cDNA序列，以及其轻链的cDNA序列克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-NTPDH2/4，pUC57simple-NTPDH1/3和pUC57simple-19F3L3质粒和pUC57simple-19F3L2质粒。
分别将质粒pUC57simple-NTPDH2/4和pUC57simple-19F3L3质粒、pUC57simple-NTPDH2/4和pUC57simple-19F3L2质粒、pUC57simple-NTPDH1/3和pUC57simple-19F3L3质粒、pUC57simple-NTPDH1/3和pUC57simple-19F3L2质粒进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，得到pcDNA3.1-NTPDH2/4和pcDNA3.1-19F3L3质粒、pcDNA3.1-NTPDH2/4和pcDNA3.1-19F3L2质粒、pcDNA3.1-NTPDH1/3和pcDNA3.1-19F3L3质粒、pcDNA3.1-NTPDH1/3和pcDNA3.1-19F3L2质粒，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
按照前述制备例中提及的表达和纯化方法获得纯化的抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4。
实施例16：ELISA方法测定抗CD73-抗PD-1双特异性抗体与抗原的结合活性
1.ELISA方法分别测定NTPDV1、NTPDV2、NTPDV3、NTPDV4与抗原PD-1-mFc的结合活性。
将PD-1-mFc(0.5μg/ml)包被酶标板置于4℃孵育过夜，然后使用PBST洗板包被了抗原的酶标板1次，再使用1％BSA的PBS溶液作为封闭液在37℃下对酶标板进行封闭2小时。酶标板封闭结束后用PBST洗板3次，加入PBST溶液梯度稀释的抗体(抗体稀释梯度详见表2)，加入待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1∶5000比例稀释的HRP标记羊抗人IgG Fc(Jackson，货号：109-035-098)二抗工作液，然后置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光显色8min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如表10所示。可知，NTPDV1、NTPDV2、NTPDV3、NTPDV4与抗原PD-1-mFc能够有效的结合，并且其结合效率呈剂量依赖关系，各剂量的吸光度强度见表10，通过对结合的抗体进行吸光度定量分析，曲线模拟计算获得抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4、14C12H1L1(hG1TM)(作为对照)的结合效率EC50分别为0.101nM、0.119nM和0.110nM、0.123nM、0.031nM。
以上实验结果表明，在相同实验条件下，NTPDV1、NTPDV2、NTPDV3、NTPDV4结合PD-1-mFc的活性与同靶点阳性药14C12H1L1(hG1TM)相当，提示NTPDV1、NTPDV2、NTPDV3、NTPDV4具有有效结合PD-1-mFc的活性。
表10：ELISA检测NTPDV1、NTPDV2、NTPDV3、NTPDV4、14C12H1L1(hG1TM)与PD-1-mFc的结合
Figure PCTCN2021089059-appb-000011
Figure PCTCN2021089059-appb-000012
2.ELISA方法分别测定NTPDV1、NTPDV2、NTPDV3、NTPDV4与抗原人NT5E-Biotin的结合活性
将链霉亲和素SA(2μg/ml)包被酶标板后置于4℃孵育过夜。孵育结束后使用PBST对包被了链霉亲和素的酶标板洗板1次，1％BSA的PBS溶液作为酶标板封闭液，对酶标板在37℃进行封闭2小时。酶标板封闭结束后用PBST洗板3次。再加入抗原人NT5E-Biotin 0.5μg/ml并置于37℃条件下孵育30分钟，然后用PBST洗板3次。在酶标板孔内加入PBST溶液梯度稀释的抗体(抗体稀释梯度详见表11)，加入待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1∶5000比例稀释的HRP标记羊抗人IgG Fc(Jackson，货号：109-035-098)二抗工作液，置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光显色7min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如表11所示。可知，NTPDV1、NTPDV2、NTPDV3、NTPDV4、19F3H2L3(hG1M)与抗原人NT5E-Biotin能够有效的结合，并且其结合效率呈剂量依赖关系(各剂量的吸光度强度见表11)。通过对结合的抗体进行吸光度定量分析，曲线模拟计算获得抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4、19F3H2L3(hG1M)(作为对照抗体)的结合效率EC50分别为0.079nM、0.082nM和0.084nM、0.077nM、0.029nM。
以上实验结果表明，在相同实验条件下，双特异抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4结合人NT5E-Biotin的活性与同靶点阳性 药19F3H2L3(hG1M)相当，提示NTPDV1、NTPDV2、NTPDV3、NTPDV4具有有效结合人NT5E-Biotin的活性。
表11：ELISA检测NTPDV1、NTPDV2、NTPDV3、NTPDV4、19F3H2L3(hG1M)与人NT5E-Biotin的结合
Figure PCTCN2021089059-appb-000013
实施例17：竞争ELISA方法分别测定抗CD73-抗PD-1双特异性抗体与人PD-L1-mFc竞争结合人PD-1-mFc-Biotin的活性
将人PD-L1-mFc(PD-L1 Genbank ID：NP_054862.1，mFc SEQ ID NO：89)以2μg/mL包被酶标板后，4℃孵育过夜。孵育结束后用1％BSA的PBS溶液在37℃条件下对酶标板进行封闭2小时，封闭结束后洗板一次并拍干。在稀释板上以10μg/mL作为起始浓度按照1∶3的稀释比例将抗体进行梯度稀释至7个浓度，并设空白对照，然后加入等体积的0.3μg/mL的人PD-1-mFc-Biotin溶液，混匀后在室温孵育10分钟。然后将反应后混合液加入到包被好的酶标板，在37℃条件下孵育30分钟。孵育结束用PBST洗板三次并拍干，加入SA-HRP(KPL，14-30-00)工作液，在37℃条件下孵育30分钟。孵育完成后洗板四次拍干，后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值，用SoftMax Pro  6.2.1软件对数据进行分析处理。
各剂量的OD值见表12。通过对结合的抗体进行吸光强度定量分析，曲线模拟抗体的结合效率获得结合EC50(表12)。
结果表明，NTPDV1、NTPDV2、NTPDV3、NTPDV4、14C12H1L1(hG1TM)(作为对照)能有效地阻断抗原人PD-1-mFc-Biotin与其受体人PD-L1-mFc的结合，且阻断效率呈现剂量依赖关系，NTPDV1、NTPDV2、NTPDV3、NTPDV4、14C12H1L1(hG1TM)阻断人PD-1-mFc-Biotin与其配体人PD-L1-mFc结合的EC50分别为2.249nM、2.253nM、2.332nM、2.398nM、2.216nM、2.231nM。
表12：NTPDV1、NTPDV2、NTPDV3、NTPDV4、14C12H1L1(hG1TM)与人PD-L1-mFc竞争结合人PD-1-mFc-Biotin的活性检测结果
Figure PCTCN2021089059-appb-000014
实施例18：使用Fortebio分子相互作用仪测定抗CD73-抗PD-1双特异性抗体与抗原人PD-1-mFc结合的动力学参数
样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。将PD1-mFc以5μg/mL的浓度固定于AMC传感器上，固定高度约为0.1nm(时间60s)，传感器在缓冲液中平衡60s，固定在传感器上的PD1-mFc与抗体结合，浓度为0.62-50nM(三倍稀释)，时间120s，蛋白在缓冲液中解离，时间300s。检测温度为30度，检测频率为0.3Hz，样品板震动速率为1000rpm。数据以1∶1模型拟合分析，得到亲和力常数。
人源化抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4和Nivolumab(作为对照抗体)与人PD-1-mFc的亲和力常数测定结果见表13，检测结果如图19、图20、图21、图22和图18所示。人源化抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4和Nivolumab与人PD-1-mFc的亲和力常数依次为1.40E-10M，7.39E-11M，1.25E-10M、1.13E-11M、2.26E-10M。以上实验结果表明：NTPDV1、NTPDV3、Nivolumab的结合能力相当，NTPDV2、NTPDV4的结合能力优于Nivolumab，提示人源化抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4与人PD-1-mFc有较强的结合能力。
表13：14C12H1L1(hG1TM)、NTPDV1、NTPDV2、NTPDV3、NTPDV4、Nivolumab与PD-1-mFc亲和力常数测定
Figure PCTCN2021089059-appb-000015
KD为亲和力常数；KD＝kdis/kon
实施例19：使用Fortebio分子相互作用仪测定抗CD73-抗PD-1双特异性抗体与抗原人NT5E(1-552)-his结合的动力学参数。
样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。将HNT5E(1-552)-His以5μg/mL的浓度固定于HIS1K传感器上，固定高度约为0.4nm(时间50s)，传感器在缓冲液中平衡60s，固定在传感器上的HNT5E(1-552)-His与抗体结合，浓度为0.31-25nM(三倍稀释)，时间100s，蛋白在缓冲液中解离，时间180s。检测温度为30度，检测频率为0.3Hz，样品板震动速率为1000rpm。数据以1∶1模型拟合分析，得到亲和力常数。
人源化抗体19F3H2L3(hG1M)(作为对照抗体)、NTPDV1、NTPDV2、 NTPDV3、NTPDV4与人NT5E(1-552)-his的亲和力常数测定结果见表14，检测结果如图24-27所示。人源化抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4与人NT5E(1-552)-his的亲和力常数依次为3.29E-11M，2.88E-11M、7.92E-11M、5.77E-11M。
以上实验结果表明：19F3H2L3(hG1M)、NTPDV1、NTPDV2、NTPDV3、NTPDV4的结合能力相当，提示人源化抗体NTPDV1、NTPDV2、NTPDV3、NTPDV4与人NT5E(1-552)-his有较强的结合能力。
表14：19F3H2L3(hG1M)、NTPDV1、NTPDV2、NTPDV3、NTPDV4与人NT5E(1-552)-his亲和力常数测定
Figure PCTCN2021089059-appb-000016
K D为亲和力常数；K D＝kdis/kon
实施例20：抗CD73-抗PD-1双特异性抗体抑制U87-MG细胞膜表面CD73酶活性的检测
取状态良好的对数期U87-MG细胞，用无血清的RPMI-1640培养液将细胞重悬、计数；将U87-MG细胞接种至96孔板，2.5x10 4个细胞/60μL/孔；用无血清RPMI-1640培养液按实验设计浓度进行梯度稀释抗体，将抗体加入96孔板，每孔60μL，37℃孵育1小时；1小时后，每孔加入60μL600uM RPMI-1640稀释的AMP；3小时后取100μL细胞培养上清，转移至新96孔板，每孔加入40μL CTG(CellTiterGlo)显色液，室温避光放置5min；5min后，每孔加入10μL 300μM的ATP，进行显色后于多标记微孔板检测仪(PerkinElmer 2140-0020)读取相对荧光强度RLU。
实验结果如表15和图28所示，NTPDV2与阳性对照药MEDI9447 的AMP含量相当。
以上实验结果说明加入的AMP，在无抗体情况下，可被U87-MG细胞表面CD73的酶活性催化而转化为腺苷A，而加入抗体后，由于NTPDV2抗体与CD73结合而降低了其酶催化的功能，使AMP不会转化为腺苷A。提示抗体以非底物竞争的方式有效抑制其酶活反应，降低腺苷的产生。
表15抗CD73-抗PD-1双特异性抗体抑制U87-MG细胞膜表面CD73酶活性的检测
抗体名称 MEDI9447 19F3H2L3(hG1M) NTPDV2
IC50(nM) 0.1189 0.3647 0.5551
实施例21：混合淋巴细胞反应MLR检测抗CD73-抗PD-1双特异性抗体促IFN-γ和IL-2分泌的生物活性
1.抗CD73-抗PD-1双特异性抗体促Raji-PDL1混合淋巴反应体系分泌IFN-γ的生物活性检测
Raji-PDL1细胞正常传代培养；复苏PBMC，用10mL 1640完全培养基培养，0.5μg/mL的SEB(德诺泰克，货号：S010201)刺激两天；Raji-PDL1细胞用25μg/mL的MMC(丝裂霉素C，Stressmarq，目录：SIH-246，批号：SM286474)处理，置于37℃培养箱中1小时；收集SEB刺激2天后PBMC和经MMC处理1小时的Raji-PDL1细胞，PBS洗涤两次，完全培养基重悬计数，分别加入到U型96孔板，1x10 5个细胞/孔；按实验设计加入抗体，于培养箱共培养3天；3天后，收集细胞培养上清，ELISA法进行IFN-γ检测。
如图29所示，人PBMC和Raji-PDL1细胞混合培养后对PBMC的IFN-γ的分泌具有显著促进作用，在混合培养体系中同时加入抗体能显著诱导PBMC进一步分泌IFN-γ，促IFN-γ分泌活性水平方面，NTPDV2抗体与其母版PD-1单靶点抗体14C12H1L1活性相当。
2.抗CD73-抗PD-1双特异性抗体促Raji-PDL1混合淋巴反应体系分泌IL-2的生物活性检测
Raji-PDL1细胞正常传代培养；复苏PBMC，用10mL 1640完全培养基培养，SEB(0.5μg/mL)刺激两天；Raji-PDL1细胞用25μg/mL的MMC处理，置于37℃培养箱中1小时；收集SEB刺激2天后的PBMC和经MMC处理1小时的Raji-PDL1细胞，PBS洗两次，完全培养基重悬计数，分别加入到U型96孔板，1x10 5个细胞/孔；按实验设计加入抗体，共培养3天；收集细胞培养上清，ELISA法进行IL-2检测。
如图29所示，人PBMC和Raji-PDL1细胞混合培养后对PBMC的IL-2的分泌有一定的促进作用，在混合培养体系中同时加入抗体能显著诱导PBMC进一步分泌IL-2，具有显著剂量依赖关系，促IL-2分泌活性水平方面，双功能抗体NTPDV2在低浓度活性与其母版PD-1单靶点抗体14C12H1L1相当，而中高浓度略低于母版PD-1单靶点抗体14C12H1L1。
实施例22：抗CD73-抗PD-1双特异性抗体体内抑制肿瘤生长实验
为检测抗CD73-抗PD-1双特异性抗体的体内抑瘤活性，首先用MC38-hPDL1/hCD73细胞(来源于江苏集萃药康生物科技有限公司)，皮下接种到5-7周龄的雌性C57BL6-hPD1hPDL1hCD73三转基因小鼠(来源于江苏集萃药康生物科技有限公司)，造模与具体的给药方式见表16。给药后测量各组肿瘤的长宽，计算肿瘤体积。
表16：实验设计。
Figure PCTCN2021089059-appb-000017
实验结果如图30、图31所示，结果表明：相比同型对照抗体hIgG， 19F3H2L3(hG1M)，不同剂量NTPDV2均能有效抑制小鼠肿瘤的生长，且高剂量NTPDV2对肿瘤的抑制优于低剂量NTPDV2。此外，NTPDV2对荷瘤小鼠体重无影响。
实验例23：Fc段突变可有效消除由双特异性免疫检查点抑制剂PD-1/CD73双特异性抗体介导的IL-8和IL-6的分泌
HPMM由PBMC诱导而来。本研究中所使用的PBMC均为在中山康方生物医药有限公司分离制备，且经提供者知情同意。
Ficoll-Paque PLUS淋巴细胞分离液(GE，货号：17-1440-03)；RPMI1640(Gibco，货号：22400-105)；CHO-K1-PD1细胞(中山康方生物医药有限公司构建)；U87-MG细胞(细胞来源于ATCC，购自北京中原领先科技有限公司)；FBS(Fetal Bovine Serum，Excell bio，货号：FSP500)；人IFN-γ蛋白(sinobio，货号：11725-HNAS-100)；LPS(Lipopolysaccharides)，脂多糖(sigma，货号：L4391)；96孔细胞培养板(康宁)。
按照分离液Ficoll-PaqueTM Plus试剂说明书分离健康人PBMC并用含2％FBS的1640培养基重悬，在37℃，5％CO2细胞培养箱中放置。2h后去除上清，贴壁细胞用PBS洗涤2次，加入1640完全培养基(含10％FBS)以及100ng/mL人M-CSF诱导7天。第3天和第5天换液并补充M-CSF以诱导HPMM。第7天HPMM诱导完成后收集，用完全培养基调整浓度为10万/mL并分装至96孔板中，并加入重组人IFN-γ(50ng/mL)，放置培养箱孵育24h。24h后收集对数期的表达人PD-1的CHO-K1-PD1细胞或者组成性表达人CD73的U87-MG细胞，重悬后用完全培养基调整浓度为30万/mL。抗体用完全培养基稀释至工作浓度为25nM、2.5nM、0.25nM。并同时设计同型对照抗体和空白对照。将96孔板中上清去掉，加入CHO-K1-PD1或者U87-MG细胞悬液和抗体(终体积200μL)混匀，放置培养箱孵育24h。500xg离心5min后收集上清并用达科为试剂盒检测IL-8、IL-6分泌量。LPS作为阳性对照药物，实验中由完全培养基调整浓度为100ng/mL。
本实施例中，CHO-K1-PD1及U87-MG细胞细胞作为靶细胞与HPMM 共培养可诱导HPMM活化，活化的HPMM通过抗体Fab链接靶细胞后，抗体Fc段与HPMM上的FcγR作用，引起HPMM分泌细胞因子。
3.实验结果
如图32-图35所示。
结果显示，相较于野生型IgG1亚型的PD-1抗体或者CD73抗体，携带有Fc段突变的抗PD-1/CD73双特异性抗体能够有效地消除免疫细胞的IL-6和/或IL-8的分泌。
以上对本发明的较佳实施方式进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明精神的前提下还可做出种种的等同变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
序列表
19F3的重链可变区核苷酸序列：
Figure PCTCN2021089059-appb-000018
(SEQ ID NO：1，下划线表示CDR序列)
19F3的重链可变区氨基酸序列：
Figure PCTCN2021089059-appb-000019
(SEQ ID NO：2，下划线表示CDR序列)
19F3的HCDR1：GYSFTGYT
(SEQ ID NO：3)
19F3的HCDR2：INPYNAGT
(SEQ ID NO：4)
19F3的HCDR3：ARSEYRYGGDYFDY
(SEQ ID NO：5)
19F3轻链可变区的核苷酸序列：
Figure PCTCN2021089059-appb-000020
(SEQ ID NO：6，下划线表示CDR序列)
19F3轻链可变的氨基酸序列：
Figure PCTCN2021089059-appb-000021
(SEQ ID NO：7，下划线表示CDR序列)
19F3的LCDR1氨基酸序列：QSLLNSSNQKNY
(SEQ ID NO：8)
19F3的LCDR2氨基酸序列：FAS
(SEQ ID NO：9)
19F3的LCDR3氨基酸序列：QQHYDTPYT
(SEQ ID NO：10)
19F3重链的骨架区氨基酸序列：
FR-H1：EVQLQQSGPELVKPGASMRMSCKAS(SEQ ID NO：11)
FR-H2：MNWVKQSHGKNLEWIGL(SEQ ID NO：12)
FR-H3：SYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC(SEQ ID NO：13)
FR-H4：WGQGTTLTVSS(SEQ ID NO：14)
19F3轻链的骨架区氨基酸序列：
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)
19F3H2的核苷酸序列：(SEQ ID NO：19，下划线表示CDR序列)
Figure PCTCN2021089059-appb-000022
19F3H2的氨基酸序列：(SEQ ID NO：20，下划线表示CDR序列)
Figure PCTCN2021089059-appb-000023
19F3L2的核苷酸序列：(SEQ ID NO：2l，下划线表示CDR序列)
Figure PCTCN2021089059-appb-000024
19F3L2的氨基酸序列：(SEQ ID NO：22，下划线表示CDR序列)
Figure PCTCN2021089059-appb-000025
19F3L3的核苷酸序列：(SEQ ID NO：23，下划线表示CDR序列)
Figure PCTCN2021089059-appb-000026
19F3L3的氨基酸序列：(SEQ ID NO：24，下划线表示CDR序列)
Figure PCTCN2021089059-appb-000027
19F3H2L3(G1M)的重链核苷酸(SEQ ID NO：25，下划线区域是非可变区序列)
Figure PCTCN2021089059-appb-000028
19F3H2L3(G1M)的重链氨基酸(SEQ ID NO：26，下划线区域是非可变区序列)
Figure PCTCN2021089059-appb-000029
19F3H2L3(G1M)的轻链核苷酸(SEQ ID NO：27，下划线区域是非可变区序列)
Figure PCTCN2021089059-appb-000030
Figure PCTCN2021089059-appb-000031
19F3H2L3(G1M)的轻链氨基酸(SEQ ID NO：28，下划线区域是非可变区序列)
Figure PCTCN2021089059-appb-000032
19F3H2L3(hG1TM)的重链核苷酸(SEQ ID NO：29，下划线区域是非可变区序列)
Figure PCTCN2021089059-appb-000033
19F3H2L3(hG1TM)的重链氨基酸序列：(SEQ ID NO：30)
Figure PCTCN2021089059-appb-000034
19F3H2的骨架区氨基酸序列：
FR-H1：QVQLVQSGAEVVKPGASVKVSCKAS(SEQ ID NO：31)
FR-H2：MNWVRQAPGQNLEWIGL(SEQ ID NO：32)
FR-H3：SYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC(SEQ ID NO：33)
FR-H4：WGQGTTLTVSS(SEQ ID NO：34)
19F3L2的骨架区氨基酸序列：
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)
19F3L3的骨架区氨基酸序列：
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)
14C12重链可变区核苷酸序列：(SEQ ID NO：43)
Figure PCTCN2021089059-appb-000035
14C12重链可变区氨基酸序列：(SEQ ID NO：44)
Figure PCTCN2021089059-appb-000036
14C12的HCDR1氨基酸序列：GFAFSSYD
(SEQ ID NO：45)
14C12的HCDR2氨基酸序列：ISGGGRYT
(SEQ ID NO：46
14C12的HCDR3氨基酸序列：ANRYGEAWFAY
(SEQ ID NO：47)
14C12轻链可变区核苷酸序列：(SEQ ID NO：48)
Figure PCTCN2021089059-appb-000037
14C12轻链可变区氨基酸序列：(SEQ ID NO：49)
Figure PCTCN2021089059-appb-000038
14C12的LCDR1氨基酸序列：QDINTY(SEQ ID NO：50)
14C12的LCDR2氨基酸序列：RAN(SEQ ID NO：51)
14C12的LCDR3氨基酸序列：LQYDEFPLT(SEQ ID NO：52)
14C12重链骨架区氨基酸序列：
FR-H1：EVKLVESGGGLVKPGGSLKLSCAAS(SEQ ID NO：53)
FR-H2：MSWVRQTPEKRLEWVAT(SEQ ID NO：54)
FR-H3：YYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYC(SEQ ID NO：55)
FR-H4：WGQGTLVTVSA(SEQ ID NO：56)
14C12轻链骨架区氨基酸序列：
HR-L1：DIKMTQSPSSMYASLGERVTFTCKAS(SEQ ID NO：57)
HR-L2：LSWFQQKPGKSPKTLIY(SEQ ID NO：58)
HR-L3：RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC(SEQ ID NO：59)
HR-L4：FGAGTKLELK(SEQ ID NO：60)
14C12H1的核苷酸序列：(SEQ ID NO：61)
Figure PCTCN2021089059-appb-000039
14C12H1的氨基酸序列：(SEQ ID NO：62)
Figure PCTCN2021089059-appb-000040
14C12L1的核苷酸序列：(SEQ ID NO：63)
Figure PCTCN2021089059-appb-000041
14C12L1的氨基酸序列：(SEQ ID NO：64)
Figure PCTCN2021089059-appb-000042
14C12H1L1的重链核苷酸(SEQ ID NO：65)
Figure PCTCN2021089059-appb-000043
Figure PCTCN2021089059-appb-000044
14C12H1L1的重链氨基酸序列：(SEQ ID NO：66)
Figure PCTCN2021089059-appb-000045
14C12H1L1的轻链核苷酸序列：(SEQ ID NO：67)
Figure PCTCN2021089059-appb-000046
14C12H1L1的轻链氨基酸序列：(SEQ ID NO：68)
Figure PCTCN2021089059-appb-000047
14C12H1L1(G1TM)的重链核苷酸序列(SEQ ID NO：69)
Figure PCTCN2021089059-appb-000048
Figure PCTCN2021089059-appb-000049
14C12H1L1(G1TM)的重链氨基酸(SEQ ID NO：70)
Figure PCTCN2021089059-appb-000050
14C12H1的重链骨架区氨基酸序列：
FR-H1：EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO：71)
FR-H2：MSWVRQAPGKGLDWVAT(SEQ ID NO：72)
FR-H3：YYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC(SEQ ID NO：73)
FR-H4：WGQGTLVTVSS(SEQ ID NO：74)
14C12L1的轻链骨架区氨基酸序列：
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的氨基酸序列：GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：79)
Linker1的核苷酸序列：(SEQ ID NO：80)
Figure PCTCN2021089059-appb-000051
Linker2的氨基酸序列：GGGGSGGGGSGGGGS(SEQ ID NO：81)
Linker2的核苷酸序列：(SEQ ID NO：82)
Figure PCTCN2021089059-appb-000052
NTPDV2和NTPDV4的重链的氨基酸序列(SEQ ID NO：83)：其中的免疫球蛋白部分中的19F3H2(hG1TM)的CDR区域用 下划线加粗标识，scFv部分中的14C12H1V-Linker1-14C12L1V的CDR区域用分别用 下划线加粗标识，重链区突变氨基酸用斜体加粗标识，linker区域用加粗标识：
Figure PCTCN2021089059-appb-000053
NTPDV2和NTPDV4的重链的核苷酸序列：(SEQ ID NO：84)
Figure PCTCN2021089059-appb-000054
Figure PCTCN2021089059-appb-000055
NTPDV1和NTPDV3的重链的氨基酸序列(SEQ ID NO：85)：其中的免疫球蛋白部分中的19F3H2(hG1TM)的CDR区域用 下划线加粗标识，scFv部分中的14C12H1V-Linker2-14C12L1V的CDR区域用分别用 下划线加粗标识，重链区突变氨基酸用斜体加粗标识，linker区域用加粗标识：
Figure PCTCN2021089059-appb-000056
NTPDV1和NTPDV3的重链的核苷酸序列：(SEQ ID NO：86)
Figure PCTCN2021089059-appb-000057
Figure PCTCN2021089059-appb-000058
NT5E(1-552)-his的氨基酸序列(SEQ ID NO：87)
Figure PCTCN2021089059-appb-000059
NT5E(1-552)-his的核苷酸序列(SEQ ID NO：88)
Figure PCTCN2021089059-appb-000060
Figure PCTCN2021089059-appb-000061
mFc的氨基酸序列：(SEQ ID NO：89)
Figure PCTCN2021089059-appb-000062
19F3H1V-Linker-19F3L2V的氨基酸序列：(SEQ ID NO：90)
Figure PCTCN2021089059-appb-000063
19F3H1V-Linker2-19F3L2V的核苷酸序列：(SEQ ID NO：91)
Figure PCTCN2021089059-appb-000064
19F3H1的核苷酸序列：(SEQ ID NO：92)
Figure PCTCN2021089059-appb-000065
19F3H1的氨基酸序列：(SEQ ID NO：93)
Figure PCTCN2021089059-appb-000066
19F3L1的核苷酸序列：(SEQ ID NO：94)
Figure PCTCN2021089059-appb-000067
19F3L1的氨基酸序列：(SEQ ID NO：95)
Figure PCTCN2021089059-appb-000068
19F3L2的轻链氨基酸(全长)(SEQ ID NO：96，下划线区域是非可变区序列)
Figure PCTCN2021089059-appb-000069
19F3H2可变区的氨基酸序列，下划线表示CDR序列：(SEQ ID NO：97)
Figure PCTCN2021089059-appb-000070
19F3L2可变区的氨基酸序列，下划线表示CDR序列：(SEQ ID NO：98)
Figure PCTCN2021089059-appb-000071
19F3L3可变区的氨基酸序列，下划线表示CDR序列：(SEQ ID NO：99)
Figure PCTCN2021089059-appb-000072
19F3L2的轻链核苷酸(全长)(SEQ ID NO：100)
Figure PCTCN2021089059-appb-000073

Claims (31)

  1. 抗CD73-抗PD-1双特异性抗体，其包括：
    第一蛋白功能区，所述第一蛋白功能区靶向PD-1，和
    第二蛋白功能区，所述第二蛋白功能区靶向CD73，
    其中
    第一蛋白功能区包含氨基酸序列如SEQ ID NO：44所示的重链可变区所包含的HCDR1，HCDR2和HCDR3，优选HCDR1，HCDR2和HCDR3的氨基酸序列分别如SEQ ID NOs：45-47所示的序列或与SEQ ID NOs：45-47所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：45-47所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，和包含
    氨基酸序列如SEQ ID NO：49所示的轻链可变区所包含的LCDR1，LCDR2和LCDR3，优选LCDR1，LCDR2和LCDR3的氨基酸序列分别如SEQ ID NOs：50-52所示的序列或与SEQ ID NOs：50-52所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：50-52所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
    或第二蛋白功能区包含氨基酸序列如SEQ ID NO：2所示的重链可变区所包含的HCDR1，HCDR2和HCDR3，优选HCDR1，HCDR2和HCDR3的氨基酸序列分别如SEQ ID NOs：3-5所示的序列或与SEQ ID NOs：3-5所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：3-5所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，和包含
    氨基酸序列如SEQ ID NO：7所示的轻链可变区所包含的LCDR1，LCDR2和LCDR3，优选LCDR1，LCDR2和LCDR3的氨基酸序列分别如SEQ ID NOs：8-10所示的序列或与SEQ ID NOs：8-10所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：8-10所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
  2. 权利要求1所述的抗CD73-抗PD-1双特异性抗体，其中：
    所述第一蛋白功能区包含
    氨基酸序列如SEQ ID NO：44或SEQ ID NO：62所示的序列或与SEQ ID NOs：44或62所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：44或62所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；和包含
    分别对应选自氨基酸序列如SEQ ID NO：49或SEQ ID NO：64所示的序列或与SEQ ID NOs：49或64所示的序列具有至少80％、81％、82％、83％、84％、85％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NOs：49或64所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，
    和/或，
    所述第二蛋白功能区包含氨基酸序列选自SEQ ID NO：2、SEQ ID NO：20所示的序列或与SEQ ID NO：2或SEQ ID NO：20所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：2或SEQ ID NO：20所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；和包含
    分别对应选自SEQ ID NO：7，或SEQ ID NO：22或与SEQ ID NO：7或SEQ ID NO：22所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：7或SEQ ID NO：22所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；
    或
    所述第二蛋白功能区包含氨基酸序列如SEQ ID NO：20所示的序列或与SEQ ID NO：20所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：20所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，和包含
    氨基酸序列如SEQ ID NO：24所示的序列或与SEQ ID NO：24示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：24所示的序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
  3. 权利要求1或2所述的抗CD73-抗PD-1双特异性抗体，其中：所述第一蛋白功能区和第二蛋白功能区独立地为1个或者2个以上。
  4. 权利要求1-3任一项所述的抗CD73-抗PD-1双特异性抗体，其中：所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；优选地，所述连接片段为(GGGGS)n，n为正整数，例如1、2、3、4、5或6。
  5. 权利要求1-4任一项所述的抗CD73-抗PD-1双特异性抗体，其中：所述第一蛋白功能区和第二蛋白功能区独立地为免疫球蛋白或抗原结合片段，例如半抗体、Fab、F(ab’) 2或单链抗体，优选地，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为抗原结合片段；或者，所述第一蛋白功能区为抗原结合片段，所述第二蛋白功能区为免疫球蛋白。
  6. 权利要求1-5任一项所述的抗CD73-抗PD-1双特异性抗体，其中：所述抗原结合片段的重链可变区N端直接(或通过连接片段)连接于所述免疫球蛋白的CH1的C端和所述抗原结合片段的轻链可变区N端直接(或通过连接片段)连接于所述免疫球蛋白的轻链可变区CL的C端；或者所述抗原结合片段的重链可变区N端直接(或通过连接片段)连接于所述免疫球蛋白的的轻链可变区CL的C端和所述抗原结合片段的轻链可变区N端直接(或通过连接片段)连接于所述免疫球蛋白的重链可变区CH1的C端。或者
    所述抗原结合片段的重链可变区C端直接(或通过连接片段)连接于所述免疫球蛋白的重链N端和所述抗原结合片段的轻链可变区C端直接(或通过连接片段)连接于所述免疫球蛋白的轻链N端；或者所述抗原结合片段的重链可变区C端直接(或通过连接片段)连接于所述免疫球蛋白的的轻链N端和所述抗原结合片段的轻链可变区的C端直接(或通过连接片段)连接于所述免疫球蛋白的重链N端。
  7. 权利要求1-6任一项所述的抗CD73-抗PD-1双特异性抗体，其中：所述抗原结合片段是单链抗体，优选地，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；或者，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白。
  8. 权利要求7所述的抗CD73-抗PD-1双特异性抗体，其中：所述单链抗体为通过连接片段连接抗体重链可变区(V H)和抗体轻链可变区(V L)的分子；优选地，所述单链抗体具有结构：NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH。
  9. 权利要求7或8所述的抗CD73-抗PD-1双特异性抗体，其中：所述单链抗体通过连接片段连接在免疫球蛋白的重链的C末端(C H)(或者重链的N端、重链可变区的CH1的C端)时，首先连接所述单链抗体的抗体重链可变区(V H)，或者，首先连接所述单链抗体的抗体轻链可变区(V L)；优选地，单链抗体可具有结构：连接片段-V H-连接片段-V L-COOH，或者，连接片段-V L-连接片段-V H-COOH，
    优选地，
    所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：3 -5的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：8-10的CDR；
    所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：45-47的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：50-52的CDR，
    优选地，所述单链抗体(例如NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH)通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：45-47的CDR的抗体重链可变区(V H)，或者，首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：50-52的CDR的抗体轻链可变区(V L)，
    或者优选地，
    所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：45-47的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：50-52的CDR；和/或，
    所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：3-5的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：8-10的CDR，
    其中，所述单链抗体(例如NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH)通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：3-5的CDR的抗体重链可变区(V H)，或者，首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：8-10的CDR的抗体轻链可变区(V L)，
    优选地，
    一个免疫球蛋白分子连接有两个单链抗体分子，更优选地，两个单链抗体分子相同。
  10. 权利要求1或2所述的抗CD73-抗PD-1双特异性抗体，其中：
    所述免疫球蛋白为IgG、IgA、IgD、IgE或IgM；优选为IgG，例如IgG1、IgG2、IgG3或IgG4。
  11. 权利要求1或2所述的抗CD73-抗PD-1双特异性抗体，其中：所述单链抗体连接在免疫球蛋白的重链的C末端，优选地，一个免疫球蛋白分子连接有两个单链抗体分子，更优选地，两个单链抗体分子相同。
  12. 权利要求1或2所述的抗CD73-抗PD-1双特异性抗体，其中：所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：45-47的 CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：50-52的CDR；
    和/或，
    所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：3-5的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：8-10的CDR，
    优选地，所述单链抗体通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：3-5的CDR的抗体重链可变区(V H)，或者，首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：8-10的CDR的抗体轻链可变区(V L)。
  13. 权利要求1或2所述的抗CD73-抗PD-1双特异性抗体，其中：
    所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：3-5的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：8-10的CDR；和/或，
    所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：45-47的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：50-52的CDR，
    其中，所述单链抗体通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：45-47的CDR的抗体重链可变区(V H)，或者，首先连接所述单链抗体的包含氨基酸序列为SEQ ID NO：50-52的CDR的抗体轻链可变区(V L)。
  14. 权利要求1或2所述的抗CD73-抗PD-1双特异性抗体，其中：所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO：44或SEQ ID NO：62；所述免疫球蛋白的轻链可变区的氨基酸序列分别对应选自SEQ ID NO：49或SEQ ID NO：64；
    和/或，
    所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：2、或SEQ ID NO：20；所述单链抗体的轻链可变区的氨基酸序列分别对应选自SEQ ID NO：7，SEQ ID NO：22；或所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO：20，所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO：24；
    其中，所述单链抗体通过连接片段连接在免疫球蛋白的重链的C末端时，可以首先连接所述单链抗体的抗体重链可变区(V H)，或者，首先 连接所述单链抗体的抗体轻链可变区(V L)。
  15. 权利要求1或2所述的抗CD73-抗PD-1双特异性抗体，其中：所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO：2或SEQ ID NO：20；所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO：7或SEQ ID NO：22，或所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO：20，所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO：24；
    和/或，
    所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：44或SEQ ID NO：62；所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO：49或SEQ ID NO：64，
    其中所述单链抗体通过连接片段连接在重链的C末端时，可以首先连接所述单链抗体的抗体重链可变区(V H)，或者，首先连接抗体轻链可变区(V L)。
  16. 权利要求5-14任一项所述的抗CD73-抗PD-1双特异性抗体，其中：所述的免疫球蛋白包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体，更优选地，所述免疫球蛋白的恒定区是人源化的，例如，重链恒定区采用Ig gamma-1 chain C region，ACCESSION：P01857；轻链恒定区采用Ig kappa chain C region，ACCESSION：P01834，或者
    所述免疫球蛋白的重链恒定区在Ig gamma-1 chain C region，ACCESSION：P01857的基础上，在234位点、235位点和237位点中的任意2个位点或3个位点发生突变，并且突变后，所述双特异性抗体与FcγRIa、FcγRIIIa和/或C1q的亲和力常数相比突变前降低；
    更优选地，按照EU编号系统，在Ig gamma-1 chain C region，ACCESSION：P01857的基础上，在第234位点、235位点和/或237位点具有如下突变：
    L234A和L235A；或者
    L234A和G237A；或者
    L235A和G237A；
    或者
    L234A、L235A、G237A，
    进一步优选地，所述免疫球蛋白的重链恒定区还具有选自如下的一个或多个突变：
    N297A、D265A、D270A、P238D、L328E、E233D、H268D、P271G、A330R、C226S、C229S、E233P、P331S、S267E、L328F、A330L、M252Y、S254T、T256E、N297Q、P238S、P238A、A327Q、A327G、P329A、K322A、T394D、G236R、G236A、L328R、A330S、P331S、H268A、E318A和K320A，优选地，所述的抗CD73-抗PD-1双特异性抗体结构如重链-轻链-连接片段1-scFv所示，所述scFv选自14C12H1V-连接片段2-14C12L1V，14C12H1V-连接片段1-14C12L1V，14C12H1V-连接片段2-14C12L1V和14C12H1V-连接片段1-14C12L1V，具体选自以下各项组成的组：
    (1)NTPDV1，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：28所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段2的氨基酸序列如SEQ ID NO：81所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示，
    (2)NTPDV2，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：28所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示，
    (3)NTPDV3，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：96所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段2的氨基酸序列如SEQ ID NO：81所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示，和
    (4)NTPDV4，其重链氨基酸序列如SEQ ID NO：85所示，轻链氨基酸序列如SEQ ID NO：96所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12H1V的氨基酸序列如SEQ ID NO：66所示，连接片段1的氨基酸序列如SEQ ID NO：79所示，14C12L1V的氨基酸序列如SEQ ID NO：68所示。
  17. 权利要求1-16任一项所述的抗CD73-抗PD-1双特异性抗体，其中：所述的抗CD73-抗PD-1双特异性抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的K D结合CD73蛋白和/或PD-1蛋白。
  18. 分离的核酸分子，其包含能够编码双特异性抗体重链可变区的核酸序列，其中，
    所述抗体的重链可变区包含：
    氨基酸序列为SEQ ID NO：3-5的CDR、氨基酸序列为SEQ ID NO：45-47的CDR和氨基酸序列为SEQ ID NO：50-52的CDR；
    或者
    氨基酸序列为SEQ ID NO：45-47的CDR、氨基酸序列为SEQ ID NO：3-5的CDR和氨基酸序列为SEQ ID NO：8-10的CDR；
    并且所述双特异性抗体重链可变区作为所述双特异性抗体的一部分，特异性结合CD73和PD-1抗原，所述双特异性抗体还包含轻链可变区，所述轻链可变区包含：
    氨基酸序列为SEQ ID NO：8-10的CDR；
    或者，氨基酸序列为SEQ ID NO：50-52的CDR。
    优选地，轻链可变区的CDR与重链可变区包含的CDR不相同。
  19. 载体，其包含权利要求18所述分离的核酸分子。
  20. 宿主细胞，其包含权利要求18所述分离的核酸分子，或者权利要求19所述的载体。
  21. 制备权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体的方法，其包括在合适的条件下培养权利要求20的宿主细胞，以及从细胞培养物中回收所述双特异性抗体的步骤。
  22. 偶联物，其包括权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体以及偶联部分，其中，所述偶联部分为可检测的标记；具体地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质或酶。
  23. 试剂盒，其包括权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体，或者权利要求22所述的偶联物；优选地所述试剂盒还 包括第二抗体，其特异性识别所述双特异性抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶。
  24. 权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测CD73和/或PD-1在样品中的存在或其水平。
  25. 药物组合物，其包含权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体或者权利要求22所述的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
  26. 权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体或者权利要求22所述的偶联物在预防和/或治疗肿瘤或者贫血病中的用途，或者在诊断肿瘤或者贫血病的用途。
  27. 权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体或者权利要求22所述的偶联物在制备预防和/或治疗肿瘤或者贫血病的药物中的用途，或者在制备诊断肿瘤或者贫血病的药物中的用途。
  28. 权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体或者权利要求22所述的偶联物在制备如下药物中的用途：
    检测样品中CD73水平的药物，
    抑制CD73的酶活反应；
    和/或
    阻断PD-1与PDL1结合的药物，
    调节(例如下调)PD-1活性或水平的药物，
    解除PD-1对机体免疫抑制的药物，
    提高T淋巴细胞中IL-2表达的药物，或者
    提高T淋巴细胞中IFN-γ表达的药物。
  29. 在体内或体外方法，包括施加细胞或者给予有需求的受试者以有效量的权利要求1-17任一项所述的抗CD73-抗PD-1双特异性抗体或者权利要求22所述偶联物的步骤，
  30. 杂交瘤细胞株，其选自：
    杂交瘤细胞株LT014(又称CD73-19F3)，其于2018年6月19日保 藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018137；或
    杂交瘤细胞株LT003(又称PD-1-14C12)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015105。
  31. 抗CD73单克隆抗体，其为19F3H2L3(hG1TM)，其重链氨基酸序列如SEQ ID NO：30所示，轻链氨基酸序列如SEQ ID NO：28所示。
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WO2023201267A1 (en) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Combination therapy for treating trop-2 expressing cancers

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