WO2021115404A1 - 一种抗cd38的抗体及其用途 - Google Patents

一种抗cd38的抗体及其用途 Download PDF

Info

Publication number
WO2021115404A1
WO2021115404A1 PCT/CN2020/135547 CN2020135547W WO2021115404A1 WO 2021115404 A1 WO2021115404 A1 WO 2021115404A1 CN 2020135547 W CN2020135547 W CN 2020135547W WO 2021115404 A1 WO2021115404 A1 WO 2021115404A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
seq
variable region
cells
Prior art date
Application number
PCT/CN2020/135547
Other languages
English (en)
French (fr)
Inventor
赵新燕
邓婧
李鑫鑫
卢世强
赵晓峰
任晋生
Original Assignee
江苏先声药业有限公司
先声生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏先声药业有限公司, 先声生物医药科技有限公司 filed Critical 江苏先声药业有限公司
Priority to CN202080066375.1A priority Critical patent/CN114616245B/zh
Publication of WO2021115404A1 publication Critical patent/WO2021115404A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to an anti-human CD38 antibody or an antigen-binding fragment thereof, a nucleic acid encoding the same, a pharmaceutical composition containing the antibody or antigen-binding fragment, and its use for treating diseases related to abnormal CD38 expression, such as tumors.
  • CD38 is a type II transmembrane glycoprotein.
  • the binding of CD38 and its ligand CD31 can affect cell migration, and the receptor-mediated adhesion that interacts with hyaluronic acid plays a role.
  • the intensity of CD38 expression increases after lymphocyte activation, and its expression is mainly concentrated in hematopoietic cells; it is widely present in lymphoid and bone marrow cells, but there is almost no expression in most mature resting lymphocytes.
  • CD38 has a variety of biological functions and belongs to ribose cyclase. It can use nicotinamide adenine dinucleotide NAD+ as a substrate to generate ADP ribose and cyclic ADP ribose. This has important regulatory effects on extracellular metabolism, intracellular Ca 2+ , cell adhesion and signal transduction.
  • the natural ligand of CD38 is CD31/PECAM. The combination of CD38 and CD31 induces tyrosine phosphorylation and downstream signal transduction to regulate lymphocyte proliferation and cytokine release.
  • CD38 expression levels are relatively low in bone marrow and lymphocytes and some non-hematopoietic tissue cells.
  • normal plasma cells and multiple myeloma (MM) cells have high levels of CD38 expression, which makes CD38 becomes a good target for therapeutic antibodies to target the surface molecules of myeloma cells.
  • CD38 is highly expressed in a variety of cancers, such as prostate cancer, non-small cell lung cancer, multiple myeloma, melanoma, lymphoma, ovarian cancer, breast cancer, and other tumors.
  • the biological function of CD38 is related to the regulation of calcium homeostasis of lymphocytes expressing CD38.
  • CD38 also has extracellular enzyme activity, participates in the production of nucleotide metabolites, and regulates intracellular calcium storage.
  • CD38 monoclonal antibody in the treatment of multiple myeloma MM
  • the anti-CD38 monoclonal antibody is a targeted antibody drug for the CD38 protein molecule highly expressed on the surface of multiple myeloma cells.
  • Daratumumab the first anti-CD38 monoclonal antibody, can bind to CD38 expressed by tumor cells and has antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity ( CDC), and can induce programmed cell death of tumor cells through cross-linking.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • the second CD38 monoclonal antibody Sanofi's Isatuximab
  • Sanofi's Isatuximab has achieved a breakthrough in clinical phase 3 and pomalidomide combined with dexamethasone in patients with relapsed/refractory MM, and is currently applying for listing by the FDA and EMA.
  • it can help antagonize the inhibitory effect of suppressor cells and suppressor factors on T cells in the tumor microenvironment of solid tumors. Therefore, it is currently developing liver cancer, head and neck cancer, ovarian cancer, and glioblastoma in the United States.
  • Clinical trials of solid tumors such as tumors.
  • the CD38 monoclonal antibody licensed by Morphosys to China Tianjing has dropped from 4-6 hours to about 2 hours during clinical intravenous infusion, which may be related to its weakened CDC activity.
  • the Phase 3 clinical trial of MM is currently being conducted in China.
  • the CD38 antibody is the same or higher CD38 protein binding affinity and ADCC, ADCP and other active functions compared with Daratumumab and Isatuximab, and at the same time has only weak or no CDC activity, but can inhibit the NDA hydrolase activity.
  • Adenosinergic pathway is one of the important mechanisms of drug resistance such as PD-L1 antibody.
  • the administration of PD-L1 antibody will cause high expression of CD38 in tumor cells and Treg in the tumor microenvironment.
  • CD38 promotes the formation of adenosine by catalyzing NAD+, thereby inhibiting T, NK, and macrophages. It can inhibit the formation of adenosine through the enzyme activity inhibition of CD38 antibody, and can resist the drug resistance of PD-L1 antibody.
  • CD38 is an important sign of resistance to immune checkpoint inhibitors such as PD-L1 antibody.
  • these immunosuppressive cells in the tumor microenvironment are reduced, thereby promoting the tumor killing effect of immune cells.
  • inhibiting the NAD+hydrolase action of CD38 protein can promote the differentiation of Th0 cells into anti-tumor T cells, inhibit the transformation of Th0 into Treg cells, and increase the anti-tumor effect.
  • the anti-CD38*X bispecific antibody or dual-target drug of the antigen-binding fragment of CD38 monoclonal antibody is used to improve the efficacy of the treatment of MM, or to treat solid tumors and other refractory malignant tumors, especially solid tumors. Significant social and economic significance.
  • the present invention provides antibodies or antigen-binding fragments that specifically bind to human CD38, nucleic acids encoding these antibodies, including the antibodies, antigen-binding fragments, and pharmaceutical compositions, as well as their functions for killing tumor cells, and their use in the treatment of tumors .
  • the antibody or antigen-binding fragment of the present invention can not only bind to human CD38, but also bind to cynomolgus CD38.
  • the isolated antibody or antigen-binding fragment that specifically binds to human CD38 comprises heavy chain CDRs and light chain CDRs:
  • the heavy chain CDRs include: CDR1-VH, CDR2-VH, and CDR3-VH; the CDR1-VH, CDR2-VH, and CDR3-VH have any combination of sequences selected from the following or are combined with the sequence Than sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:
  • the light chain CDRs include: CDR1-VL, CDR2-VL, and CDR3-VL; the CDR1-VL, CDR2-VL, and CDR3-VL have any combination of sequences selected from the following or are combined with the sequence Than sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:
  • Each of CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is coded according to the common analysis method of KABAT, Chothia or IMGT.
  • an isolated antibody or antigen-binding fragment that specifically binds to human CD38 comprises a combination of heavy chain CDRs selected from: VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10 , VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH24, VH25, VH26, VH27, VH28, VH29, VH30, VH31, VH32, VVH34, VH35, Or VH36, and a combination of CDRs with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared with the sequence of the heavy chain CDRs combination.
  • an isolated antibody or antigen-binding fragment that specifically binds to human CD38 comprises a combination of light chain CDRs selected from the group consisting of VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9, VL10 , VL11, VL12, VL13, VL14, VL15, VL16, VL17, VL18, VL19, VL20, VL21, VL22, VL23, VL24, VL25, VL26, VL27, VL28, VL29, VL30, VL31, VL32, VL33, VL34, VL35 Or VL36, and a combination of CDRs with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared with the sequence of the light chain CDRs combination.
  • an isolated antibody or antigen-binding fragment that specifically binds to human CD38 comprises a combination of heavy chain CDRs and light chain CDRs selected from: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4 , VH5+VL5, VH6+VL6, VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17 +VL17, VH18+VL18, VH19+VL19, VH20+VL20, VH21+VL21, VH22+VL22, VH23+VL23, VH24+VL24, VH25+VL25, VH26+VL26, VH27+VL27, V
  • the isolated antibody or antigen-binding fragment that specifically binds to human CD38 wherein: (1) the heavy chain CDRs comprise:
  • CDR1-VH which includes SEQ ID NO: any of 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 CDR1 of VH;
  • CDR2-VH which includes SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 CDR2 of VH; and,
  • CDR3-VH which includes SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 CDR3 of VH; and/or,
  • the light chain CDRs include:
  • CDR1-VL which includes SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 CDR1 of VL;
  • CDR2-VL which includes SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 CDR2 of VL; and,
  • CDR3-VL which includes SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 VL's CDR3.
  • an isolated antibody or antigen-binding fragment that specifically binds human CD38 wherein:
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26 respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28, respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively;
  • the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 35 and SEQ ID NO: 36 respectively; or,
  • variable region of the heavy chain and the variable region of the light chain have at least 70%, 75%, 80%, 85%, 90%, 95%, 96% of the sequence shown in (1) to (18) above, respectively. , 97%, 98%, 99% or higher identity sequence.
  • the antibody or antigen-binding fragment thereof of the present invention is:
  • the antibody or antigen-binding fragment thereof of the present invention has a dissociation constant (KD) of not greater than 5 nM for binding to human CD38, and a dissociation constant (KD) of not greater than 25 nM for binding to cynomolgus CD38 .
  • the antibody or antigen-binding fragment thereof of the present invention comprises the sequence of the constant region of any one of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably comprising human or murine antibodies
  • the antigen-binding fragment of the present invention is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody, and the smallest recognition unit of antibody.
  • the antibody or antigen-binding fragment thereof of the present invention can be combined with numbers 10, 11, 12, 13, 26, 31, 38, 42, 44, 48, 51, 52, 69, 102, 215.
  • the antibody or antigen-binding fragment of, 245, 286, or 292 competitively binds to CD38 or its epitope, and has the following characteristics:
  • ADCC antibody-dependent cytotoxicity killing
  • ADCP antibody-dependent cell-mediated cell phagocytosis
  • the antibody or antigen-binding fragment is also coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a cytotoxic agent, or an immunomodulator, and the therapeutic agent is selected from the group consisting of radioisotopes, cytotoxic agents or immunomodulators.
  • the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from alkaloids, methotrexate Methotrexate, doxorubicin or taxanes; the toxin compound is preferably DM1, DM4, SN-38, MMAE, MMAF, Duocarmycin, Calicheamicin, DX8951.
  • the antibody or antigen-binding fragment is also connected to another functional molecule, and the functional molecule can be selected from one or more of the following: signal peptide, protein tag, or cytokine;
  • the cytokine may be selected from IL-2, IL-6, IL-12, IL-15, IL-21, IFN or TNF-alpha.
  • the present invention provides an isolated nucleic acid molecule that encodes the above-mentioned antibody, antigen-binding fragment, or any combination thereof of the present invention.
  • the present invention provides an expression vector comprising the isolated nucleic acid molecule described above in the present invention.
  • the present invention provides a host cell, which comprises the aforementioned isolated nucleic acid molecule or expression vector of the present invention.
  • the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the host cells are derived from mammalian cells, yeast cells, insect cells, E. coli and/or Bacillus subtilis; The host cell is selected from Chinese hamster ovary cells (CHO).
  • the present invention provides a method for preparing antibodies or antigen-binding fragments, culturing the above-mentioned host cells of the present invention under appropriate conditions, and isolating the antibodies or antigen-binding fragments.
  • the present invention provides a pharmaceutical composition, the composition comprising the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, and the above-mentioned expression vector of the present invention.
  • the pharmaceutical composition further comprises an additional anti-tumor agent.
  • the present invention provides a method for preventing and/or treating diseases related to abnormal CD38 expression, comprising administering the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned present invention to a patient in need thereof
  • the disease is preferably a tumor.
  • the present invention provides the above-mentioned antibody or antigen-binding fragment, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, the above-mentioned cell of the present invention, and the above-mentioned cell of the present invention.
  • the use of the products (such as antibodies and antigen-binding fragments) prepared by the method, or the above-mentioned pharmaceutical composition of the present invention, in the preparation of drugs for preventing and/or treating diseases related to abnormal CD38 expression, and the diseases are preferably tumors.
  • the present invention provides a kit comprising the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, and the above-mentioned expression vector of the present invention.
  • the above-mentioned cells, or products prepared by the above-mentioned methods of the present invention (such as antibodies and antigen-binding fragments), and instructions for use.
  • the present invention provides a multispecific antibody comprising the antibody or antigen-binding fragment of the first aspect; preferably, the multispecific antibody further comprises specifically binding CD38 Other antigens or antibodies or antigen-binding fragments that bind to a CD38 epitope different from the antibody or antigen-binding fragment described in the first aspect.
  • the antigen other than CD38 may be selected from: CD3, preferably CD3 ⁇ ; CD16, preferably CD16A; CD32B; PD-1; PD-2; PD-L1; VEGF; NKG2D; CD19; CD20 ; CD40; CD47; 4-1BB; CD137; EGFR; EGFRvIII; TNF-alpha; CD33; MSLN; HER2; HER3; HAS; CD5; CD27; EphA2; EpCAM; MUC1; MUC16; CEA; Claudin 18.2; Folate receptor ; Claudin6; WT1; NY-ESO-1; MAGE3; ASGPR1 or CDH16.
  • the multispecific antibody may be bispecific, trispecific or tetraspecific, and the multispecific antibody may be bivalent, tetravalent or hexavalent.
  • the present invention provides a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and the cell
  • the external antigen-binding domain comprises an antibody or antigen-binding fragment optionally described in the present invention.
  • the present invention provides an immune effector cell that expresses the above-mentioned chimeric antigen receptor, or comprises a nucleic acid fragment encoding the above-mentioned chimeric antigen receptor; preferably, the Immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells,
  • the T cells are preferably cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
  • CD38 is a glycoprotein that has the action of cyclic ADP ribose hydrolase and exists on the surface of many immune cells (leukocytes), including T, B lymphocytes and natural killer cells. CD38 also plays a role in cell adhesion, signal transduction, and calcium signal transduction.
  • antibody refers to immunoglobulin molecules that specifically bind to a target antigen or have immunoreactivity, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not limited to chimeric antibodies). , Humanized antibodies, fully human antibodies, heteroconjugate antibodies (such as bispecific, trispecific and tetraspecific antibodies, diabodies, tribodies and tetrabodies), antibody conjugates) and antibody antigens Binding fragments (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • antigen-binding fragment refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • Antibody fragments can be Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer or domain antibody.
  • binding fragments that encompass the "antigen-binding fragment" of the term antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region; (iii) Fd fragment composed of VH and CH1 domains; (iv) VL and VH domains of one arm of an antibody (V) a dAb consisting of VH and VL domains; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); (vii) a VH or VL structure A dAb composed of domains; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker.
  • Fab fragment
  • the two domains VL and VH of the Fv fragment are encoded by independent genes, these two domains can be joined through a linker using a recombination method, which can be made into which the VL and VH regions are paired to form A single protein chain of a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • bispecific antibody refers to antibodies with monoclonal binding specificities for at least two different antigens, which are usually human or humanized antibodies.
  • one of the binding specificities can be detected against an epitope of CD38, and the other can be detected against another epitope of CD38 or any other antigen, for example, against cell surface proteins, receptors, receptor subunits , Tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins or bacterial surface proteins, etc. are detected.
  • chimeric antibody refers to antibodies that have variable sequences derived from immunoglobulins derived from one source organism (such as rat or mouse) and immunoglobulins derived from different organisms (such as human) Constant region. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985J Immunol Methods 125:191-202; the above is incorporated by reference This article.
  • humanized antibody refers to a murine monoclonal antibody modified by gene cloning and DNA recombination technology and re-expressed. Most of its amino acid sequence is replaced by human sequence, which basically retains the affinity and specificity of the parent murine monoclonal antibody. , And reduce its heterogeneity, which is beneficial to be applied to the human body.
  • Humanized antibody means that the constant region of the antibody (ie, CH and CL regions) or all of the antibody is encoded by human antibody genes. Humanized antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body.
  • CDR complementarity determining region
  • FR framework regions
  • variable domains of the natural heavy chain and light chain each contain four framework regions that mainly adopt a sheet-layer configuration, which are connected by three CDRs (CDR1, CDR2, and CDR3), and these three CDRs form a loop that connects the sheet-layer structure. , And in some cases form part of the lamellar structure.
  • CDRs in each chain are closely held together in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 by the FR region, and together with the CDRs from other antibody chains, they contribute to the formation of the antigen-binding site of the antibody (see Kabat Et al., Sequences of Protein sof Immunological Interest, National Institute of Health, Bethesda, Md. 1987; it is incorporated herein by reference).
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the variable region of the heavy chain (VH) respectively. These three CDRs constitute the heavy chain variable region (VH).
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and the first CDR of the light chain variable region (VL), respectively Three CDRs, which constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).
  • VH refers to the variable region of the immunoglobulin heavy chain (including the heavy chain of Fv, scFv, or Fab) of an antibody.
  • VL refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv, or Fab).
  • antibody conjugate refers to a conjugate/conjugate formed by chemically bonding an antibody molecule to another molecule directly or through a linker.
  • ADC antibody-drug conjugates
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), and is not limited to the method of producing the antibody.
  • percent (%) sequence identity refers to the alignment of sequences and the introduction of gaps (if necessary) in order to achieve the maximum percent sequence identity (for example, for optimal alignment, one of the candidate and reference sequences can be Or after introducing a gap between the two, and for the purpose of comparison, non-homologous sequences can be ignored), the amino acid (or nucleotide) residues of the candidate sequence are the same as the amino acid (or nucleotide) residues of the reference sequence Percentage.
  • percent sequence identity the alignment can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software, such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence used for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% of the total length of the candidate sequence or a selected portion of consecutive amino acid (or nucleotide) residues of the candidate sequence.
  • the length of the candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. .
  • a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are the same at that position.
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are, for example, specific for antibodies or antigen-binding fragments thereof. Recognition.
  • the antibody or antigen-binding fragment thereof that specifically binds to the antigen will bind to the antigen with a KD of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD as high as 100 nM (for example, between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not show specific binding to a specific antigen or epitope thereof will show a KD of greater than 100 nM (for example, greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for the specific antigen or epitope.
  • a variety of immunoassay methods can be used to select antibodies that specifically immunoreact with specific proteins or carbohydrates.
  • solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates.
  • chimeric antigen receptor refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and specifically bind to an antigen, which contains at least (1) an extracellular antigen binding domain, such as an antibody Variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CAR can use extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fab segment of the antibody specifically recognizes the epitope of tumor cells, and its Fc segment and killer cells (NK cells, macrophages, etc.) FcR binding on the surface mediates killer cells to directly kill target cells.
  • the strength of antibody-mediated ADCC is related to many factors, such as the affinity between the antibody and the antigen, the affinity between the antibody and the Fc segment receptor, and the characteristics of immune effector cells. In general, the ADCC mediated by antibodies with high affinity for antigens or Fc receptors is stronger.
  • the glycosylation and amino acid sequence modification of the Fc segment of the antibody can improve ADCC activity. In the glycosylation modification of antibodies, fucose is considered to be the most important sugar affecting ADCC activity. Defucosylation can significantly increase the affinity of antibodies to Fc ⁇ RIIIa and ADCC activity.
  • antibody induces programmed cell death through cross-linking refers to the Fc segment of the antibody and the Fc receptor (FcR) or the second cross-linked antibody to induce cell program The process of sex cell death.
  • ADCC and ADCP activate the Fc ⁇ Rs on immune effector cells (such as natural killer cells, macrophages and polymorphonuclear cells) through monoclonal antibody binding, and induce aggregated IgG constant domains (Fc domains).
  • Monoclonal antibodies can interact with the Fc receptor through the Fc segment of the antibody to enhance the agonistic activity of the antibody or induce programmed cell death (PCD).
  • Monoclonal antibodies for agonistic therapy targeting members of the death receptor family induce PCD through the exogenous apoptotic pathway.
  • PCD induced by agonistic monoclonal antibodies targeting these death receptors is enhanced by cross-linking, such as by a second cross-linked antibody or more physiologically binding to Fc ⁇ Rs.
  • Antibody-mediated cross-linking of antigens that has nothing to do with the death receptor family can also induce PCD, but not through the classic apoptosis pathway. This pathway is characterized by homotypic aggregation of cells, including cytoskeletal reorganization, lysosome activation, and the production of reactive oxygen species.
  • This non-inducible PCD pathway can be enhanced by Fc cross-linked secondary antibodies or Fc ⁇ R expressing cells.
  • vector includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors).
  • DNA vectors such as plasmids
  • RNA vectors such as RNA vectors
  • viruses or other suitable replicons such as viral vectors.
  • Various vectors have been developed for delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
  • the expression vector of the present invention contains polynucleotide sequences and, for example, additional sequence elements for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells.
  • Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoter and enhancer regions) that direct gene transcription.
  • kits for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear output of mRNA produced by gene transcription. These sequence elements include, for example, 5'and 3'untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector.
  • the expression vector of the present invention may also contain the following polynucleotide, which encodes a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • subject refers to an organism that receives treatment for a particular disease or condition (such as cancer or infectious disease) as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, receiving treatment for diseases or disorders (e.g., cell proliferative disorders, such as cancer or infectious diseases).
  • diseases or disorders e.g., cell proliferative disorders, such as cancer or infectious diseases.
  • Guinea pigs members of the bovid family (such as cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathologies in the subject to be treated, such as cell proliferative disorders (such as cancer or infectious diseases). )Progress.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduced disease severity, stable disease state (i.e. not worsening), delay or slowing of disease progression, improvement or alleviation of disease state, and alleviation (whether partial Remission or complete remission), whether detectable or undetectable.
  • the subjects in need of treatment include those who already have a condition or disease, as well as those who are susceptible to or who intend to prevent the condition or disease.
  • terms such as mitigation, mitigation, weakening, mitigation, mitigation, etc. their meaning also includes elimination, disappearance, and non-occurrence.
  • an effective amount refers to the amount of the therapeutic agent that can effectively prevent or alleviate the disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject.
  • Effective amount also refers to the amount of the compound that is sufficient to relieve symptoms, for example, to treat, cure, prevent, or alleviate related medical conditions, or to treat, cure, prevent, or alleviate these conditions with increasing speed.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers solely to the ingredient.
  • the therapeutically effective dose refers to the combined amount of the active ingredients that produce a therapeutic effect, regardless of whether it is administered in combination, continuous or simultaneous.
  • cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant cancers are included in this definition.
  • tumor or “tumor” refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • Figure 1A Detection of binding titer to human CD38-His in the serum of CD38-His immunized mice;
  • Figure 1B Detection of binding titer of serum from CD38-His immunized mice to human EGFR-His;
  • Figure 2B Results of CREG-His staining of spleen B cells in mice immunized with CD38-His;
  • FIG. 3A FACS detection of anti-CD38 candidate antibody and Daudi cell direct binding EC50 value
  • FIG. 3B FACS detects the direct binding EC50 value of candidate antibody and CHO-CD38 cells
  • FIG. 4A-4B Anti-CD38 antibody-dependent cell-mediated cell phagocytosis (ADCP) assay
  • Figure 5A Romas cell translocation of phosphatidylserine induced by cross-linking of anti-CD38 antibody
  • Figure 5B Cell death induced by cross-linking of anti-CD38 antibody
  • Figure 6A The dose-dependent relationship of Annexin V-positive cells induced by cross-linking of anti-CD38 antibody is higher than that of PI-positive cells;
  • Figure 6B The dose-dependent relationship of cell death induced by cross-linking of anti-CD38 antibody
  • Figure 7A-7B anti-CD38 antibody-mediated ADCC activity test of NK cells on Daudi cells
  • FIG. 8A-8C ADCC activity test of PBMC mediated by anti-CD38 antibody on Daudi cells
  • Figures 10A-10B complement-dependent cytotoxicity mediated by anti-CD38 antibody.
  • the foot pad and back after the first and second immunization injections, and the third and fourth immunization injections under the skin and back of the tail to obtain high-titer, high-affinity, high-specific antiserum and specific immunity cell.
  • the mice were euthanized and the spleen was aseptically removed, and the lymphocytes of the mouse spleen were aseptically separated and extracted, aliquoted into cryovials, and stored in liquid nitrogen. .
  • Mouse spleen cells immunized with CD38 protein are treated with the antigen CD38-His protein (CD38-His, Novoprotein, Cat: CU65) and the indirect labeled antibody anti-His-APC (R&D Systems, Cat.IC050A) and specific for mouse B cells Anti-mouse B220-Pacfic Blue, BD Biosciences, Cat.558108; anti-mouse IgD-PE, BD Biosciences, Cat.558597; anti-mouse IgM-PE cy7, BD Biosciences, Cat.552867) staining, And before sorting, add the dye 7-AAD (BD Biosciences, Cat.51-68981E) to distinguish between dead and live cells, and use AriaIII (BD company) flow cytometer to sort CD38-specific single B cells ( 7AAD - B220 + IgD - IgM - CD38 + ) into the PCR wells containing cell lysate and RNase inhibitor
  • Example 1 Using the method of Example 1 in the patent "A combination primer for nested amplification and its application” patent application number: 201811618134.4, the mRNA of a single cell was reverse transcribed into cDNA. Then, nested PCR was performed using cDNA as a template to perform antibody heavy chain and light chain amplification respectively. The antibody heavy chain variable region and light chain variable region are amplified and cloned into the heavy chain expression vector and the light chain expression vector by homologous recombination. The constant regions of both the heavy chain expression vector and the light chain expression vector are derived from human IgG1.
  • the complete heavy chain expression sequence is the signal peptide-VH-CH1-hinge region-CH2-CH3, and the complete light chain expression sequence is the signal peptide-V ⁇ -C ⁇ .
  • the cloning and expression of the above-mentioned single B cell antibodies are all in a 96-well plate to achieve rapid identification and discovery of antibodies in a high-throughput manner. After a series of physical and chemical and functional screening of 324 pairs of cloned antibody heavy and light chains, a total of 18 positive candidate antibody molecules were obtained. The CDRs of their sequences were analyzed with IMGT and KABAT software respectively. The corresponding sequence information is shown in Table 1. Table 1 shows the VH and VL sequences of the candidate antibody molecule, and Table 2 shows the IMGT and KABAT analysis results of the candidate antibody molecule).
  • the candidate anti-CD38 antibody is sequenced, and the specific sequence information of the heavy chain variable region and the light chain variable region is as follows:
  • the antibody was diluted with PBST to 1 ⁇ g/ml, and the antigen CD38-His (Novoprotein, Cat: CU65) was diluted with PBST to 100nM as the starting concentration.
  • the 2-fold dilution was performed in 7 gradients and added to 96 wells in sequence.
  • Octet plate (Greiner, Cat.655209), 300 ⁇ l/well; set the cycle program, a total of 8 protein A probes, each cycle first load the antibody to the probe to the height of 1nm, about 3 minutes, and then use the probe binding gradient dilution After the CD38-His antigen, about 10 minutes to the saturation plateau, and finally regenerated with Glycine pH 1.5. Analyze the test data, fit the equilibrium dissociation constant KD of the antibody antigen, and determine the binding rate constant ka and the dissociation rate constant kd.
  • the affinities of antibody candidates for human and cyno CD38 proteins are summarized in Table 3.
  • Binding rate Kon dissociation rate Kdis, dissociation constant KD. It can be seen from Table 3 that compared with the drug control antibody Daratumumab (Janssen, Cat.GIS0503) purchased from commercial sources, the candidate CD38 antibody has a greatly improved binding affinity to humans, but the binding affinity to cynomolgus monkeys is better than that of Daratumumab.
  • the dose-dependent binding ability of the candidate anti-CD38 antibody to the CD38 target on the cell surface was confirmed by flow cytometry (FACS).
  • the reference antibody Daratumumab (positive control) on the surface of Daudi cells ( Figure 3A) has an EC50 of 57.94ng/ml, and on CHO-38 cells.
  • the EC50 on the surface ( Figure 3B) is 60.43ng/ml, while the EC50 of the candidate antibody bound to CD38 on the cell surface is not much different from the reference antibody; when the reference antibody is Mor202-kaps (positive control), the two The EC50 of the candidate antibody on the cells is lower than that of the reference antibody, indicating that the binding force of the candidate antibody to these two cell strains is stronger than Mor202-kaps. See Table 4 for details.
  • Daudi cells purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu140
  • human macrophages derived from PBMC isolated monocytes
  • PBMC normal human peripheral blood mononuclear cells
  • mononuclear cells from PBMC, specifically refer to the instructions of the mononuclear cell isolation kit (EasySep Cat: 19058), and use mononuclear cells supplemented with 100 ⁇ g/ml GM-CSF (Peprotech, Cat: 300-03-20Ug) Differentiation was induced in RPMI1640 containing 10% FBS (Gibco, Cat: 10099-141), cultured in 5% CO 2 , 37°C for one week, resuspended and counted and used as effector cells.
  • target cells The target cells Daudi (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu140) were collected and labeled with CelltraceTM violet (Invitrogen, Cat: C34556). The labeled cells were resuspended with RPMI1640 supplemented with 10% FBS. Used as a target cell.
  • Daudi purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu140
  • CelltraceTM violet Invitrogen, Cat: C34556
  • the labeled cells were resuspended with RPMI1640 supplemented with 10% FBS. Used as a target cell.
  • Phagocytosis rate percentage of phagocytic cells (CD19 + CD11b - )/ percentage of target cells (Celltrace + )*100%
  • the candidate anti-CD38 antibody can improve ADCP effect, and the ADCP effect of candidate antibody 69 is compared with the reference antibody Daratumumab (positive control) at the two doses of 1 ⁇ g/ml and 0.1 ⁇ g/ml Stronger.
  • the programmed cell death induced by CD38 antibody cross-linking was measured on Romas cells (Shanghai Enzyme Research Biotechnology Co., Ltd., catalog number CC-Y1430). In a U-shaped round-bottom 96-well plate, 4 ⁇ 10 4 Romas cells and serially diluted anti-CD38 antibodies, the final concentration is 10 ⁇ g/ml, 1 ⁇ g/ml, 0.1 ⁇ g/ml and 0.01 ⁇ g/ml, pre-incubated for half an hour, Then 5 ⁇ g/ml anti hIgG Fc (Invitrogen, Cat: 31125) was added, and cultured in 5% CO 2 at 37°C for 20 hours.
  • the cells were stained with Annexin V and PI in the programmed cell death detection kit (eBioscience, Cat: BMS500FI/300) and analyzed by flow cytometry.
  • the Annexin V positive percentage and PI positive percentage were calculated and analyzed by Flowjo software.
  • PBMC normal human peripheral blood mononuclear cells
  • NK cells separate and purify NK cells from PBMC, specifically refer to the NK cell isolation kit instructions to separate NK cells (Stemcell Cat: 17955), and resuspend the NK cells with RPMI1640 supplemented with 10% FBS (Gibco, Cat: 10099-141) Count and use as effector cells.
  • PBMC can be directly used as effector cells in ADCC experiments.
  • target cells The target cells Daudi (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu140) were collected and labeled with CelltraceTM violet (Invitrogen, Cat: C34556). The labeled cells were resuspended with RPMI1640 supplemented with 10% FBS. Used as a target cell.
  • Daudi purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu140
  • CelltraceTM violet Invitrogen, Cat: C34556
  • the labeled cells were resuspended with RPMI1640 supplemented with 10% FBS. Used as a target cell.
  • the anti-CD38 antibody was diluted with RPMI1640 supplemented with 10% FBS, and the diluted antibody was divided into a U-shaped bottom 96-well plate at 50 ⁇ l/well, and then the labeled target cells were added to the well.
  • the plate was pre-incubated for half an hour in a 37°C, 5% CO 2 incubator. Subsequently, add effector cells to the wells (effective target ratio NK:Daudi 2:1-4:1; PBMC:Daudi 20:1-40:1), and incubate 3-4 in a 37°C, 5% CO 2 incubator hour.
  • the plate was taken out, and the dead cell labeling dye PI (1 ⁇ l/well) was added to each well, and then flow cytometry was performed to measure the proportion of dead cells that are PI positive in the CelltraceTMviolet-positive target cells. Calculate the cell lysis rate caused by ADCC activity according to the following formula:
  • Cell lysis rate (%) (sample well PI%-only target cell well PI%)/(1-only target cell well PI%).
  • the CD38 hydrolase activity inhibitor screening kit (BPS Bicoscience, Cat. 79287) was used for detection, and the operation was performed according to the instructions.
  • hydrolysis buffer 1 ⁇ g/ml antibody to be detected, 0.5 ⁇ g/ml CD38 protein to a 96-well plate, put the system in a 37°C incubator and incubate for 30 minutes, then add ⁇ -NAD substrate, and proceed with the microplate reader.
  • the excitation wavelength is 300nm
  • the detection wavelength is 410nm.
  • the fluorescence value of each sample hole on the plate is read every 3 minutes for a total of 60 minutes. According to the time-fluorescence value curve, select the time point before the plateau phase, and calculate the inhibition rate:
  • Inhibition rate (%) (Sample fluorescence value-Blank fluorescence value)/(Positive Control fluorescence value-Blank fluorescence value) ⁇ 100%.
  • the results are shown in Figure 9.
  • the reaction curve of the control drug Daratumumab (Dara) coincides with the reaction curve of the negative control antibody group IgG1 ⁇ and the untreated group Drug0, none of which inhibits the activity of CD38 hydrolase; while it has the activity of inhibiting the activity of CD38 hydrolase.
  • the reaction curve of the control drug Isatuximab (Isa) is similar to the reaction curves of anti-CD38 antibodies 102 and 215, and the reaction rate of CD38 hydrolase activity is significantly reduced, indicating that the tested anti-CD38 antibody can effectively inhibit the CD38 hydrolase activity.
  • the results are shown in Figures 10A-10B.
  • Daratumumab which is known to have strong CDC activity, showed dose-dependent CDC activity as expected, while the tested CD38 candidate antibodies except for antibodies 10, 31, 38, 102, and 215 were among the top three. Except for the weak CDC activity at the concentration, the others have no obvious CDC activity. It shows that these antibodies are expected to avoid the infusion related reaction (IRR) caused by CDC activity.
  • IRR infusion related reaction

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

一种特异性结合人CD38的抗体或抗原结合片段，所述抗体或抗原结合片段能介导肿瘤细胞凋亡，可用于肿瘤疾病治疗的用途。

Description

一种抗CD38的抗体及其用途
本申请要求于2019年12月13日提交中国专利局、申请号为201911286272.1、发明名称为“一种抗CD38的抗体及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及抗人CD38抗体或其抗原结合片段，编码其的核酸，包含所述抗体或抗原结合片段的药物组合物，以及其用于治疗CD38表达异常相关的疾病，如肿瘤的用途。
背景技术
CD38生物学活性
CD38是一种II型跨膜糖蛋白，CD38和其配体CD31结合可影响细胞迁移，和透明质酸相互作用的受体介导的黏附发挥作用。淋巴细胞活化后CD38表达强度增加，其表达主要集中在造血细胞中；广泛存在于淋巴和骨髓细胞，但大多数成熟静息的淋巴细胞中几乎无表达。
CD38具有多种生物学作用，属于核糖环化酶，可以利用烟酰胺腺嘌呤二核苷酸NAD+为底物，生成ADP核糖和环ADP核糖。这对细胞外代谢、细胞内Ca 2+、细胞粘附和信号传导等具有重要调控作用。CD38的天然配体为CD31/PECAM，CD38和CD31结合后诱导酪氨酸磷酸化和下游信号传导调节淋巴细胞增殖和细胞因子释放。
在正常人体内，骨髓和淋巴细胞以及一些非造血组织的细胞中CD38表达水平相对较低，相比之下，正常血浆细胞和多发性骨髓瘤(MM)细胞有高水平的CD38表达，这使得CD38成为治疗性抗体靶向骨髓瘤细胞表面分子的一个很好的靶点。此外，CD38在多种癌症，如前列腺癌，非小细胞肺癌，多发性骨髓瘤、黑色素瘤、淋巴癌、卵巢癌、乳腺癌、等肿瘤中高表达。CD38的生物学功能与表达CD38的淋巴细胞钙稳态的调节有关。同时，CD38也具有胞外酶活性，参与核苷酸代谢产物的生成，调节细胞内钙的储存。
CD38单克隆抗体治疗多发性骨髓瘤(MM)
抗CD38单克隆抗体是针对多发性骨髓瘤细胞表面高表达的CD38蛋白分子的靶向抗体药物。第一个抗CD38单克隆抗体Daratumumab可与肿瘤细胞表达的CD38结合，具有抗体依赖的细胞介导的细胞毒性作用(ADCC)、抗体依赖性细胞吞噬作用(ADCP)、补体依赖的细胞毒性作用(CDC)、并可通过交联(cross-linking)诱导肿瘤细胞程序性死亡(programmedcell death)。基于其良好的耐受性和已被证实的确切疗效，Dratumumab在2015年被FDA批准用于复发/难治性MM患者的治疗，在中国则于2019年7月被批上市。第二个CD38单抗，Sanofi的Isatuximab，在临床3期和泊马度胺联合地塞米松在复发/难治性MM病人中取得突破性进展，目前正在FDA和EMA申请上市。鉴于其额外具有抑制NDA水解酶活性的功能可帮助拮抗实体瘤肿瘤微环境中抑制细胞和抑制因子对T细胞得抑制作用，因此目前也在美国开展肝癌，头颈癌，卵巢癌，胶质母细胞瘤等实体瘤的临床试验。Morphosys公司许可给中国天境公司的CD38单抗，在临床静脉输注时与Daratumumab和Isatuximab相比，从4-6小时，降到了2小时左右，可能与其减弱的CDC活性相关。目前在中国进行MM的临床3期 试验。我们旨在研发一类与Daratumumab和Isatuximab相比，具有同等或更高CD38蛋白结合亲和力和ADCC、ADCP等活性功能，同时只具有很弱甚至没有CDC活性，但又能抑制NDA水解酶活性的功能的的CD38抗体。
CD38和PD-L1单抗等免疫检查点抑制药物的耐药性的关系
临床数据分析标明，CD38和PD-L1抗体等免疫检查点抑制药物的耐药性密切相关，CD38高表达的病人对PD-L1抗体类药物的响应率有很强的相关性。相关机理如下：1)adenosinergic pathway是PD-L1抗体等药物耐药的重要机理之一。PD-L1抗体给药后会引起在肿瘤微环境中，肿瘤细胞和Treg高表达CD38，CD38通过催化NAD+，促进腺苷的形成，进而抑制T，NK,巨噬细胞。可通过CD38抗体的酶活抑制作用，抑制腺苷形成，可抵抗PD-L1抗体的耐药性。2)对PD-L1类单抗耐药的病人，肿瘤细胞，Treg，MDSC等免疫抑制细胞上的CD38表达显著上调，CD38是PD-L1抗体等免疫检查点抑制药物耐药的重要标志，可通过CD38单抗的杀伤作用，减少肿瘤微环境中的这些免疫抑制细胞，从而促进免疫细胞的肿瘤杀伤作用。当然与此同时，也需要考虑如何设计药物以避免CD38抗体对激活的T细胞的杀伤作用。3)此外，抑制CD38蛋白的NAD+水解酶作用可以促进Th0细胞分化为anti-tumor T cell，抑制Th0转化为Treg细胞，增加抗肿瘤作用。
因此，开发具有肿瘤杀伤及具有NAD水解酶活性抑制作用，可特异性的杀伤高表达CD38的血液瘤细胞和实体瘤中如Treg,MDSC等抑制细胞的CD38单抗,或者在此基础上开发含CD38单抗的抗原结合片段的抗CD38*X双特异抗体或双靶点药物，用于提高治疗MM的药效，或者用于治疗实体瘤等其它难治的恶性肿瘤，尤其是实体瘤，具有重大的社会和经济意义。
发明内容
本发明提供特异性结合人CD38的抗体或抗原结合片段，编码这些抗体的核酸，包含所述抗体及抗原结合片段和药物组合物，以及他们用于杀伤肿瘤细胞的功能，用于治疗肿瘤的用途。本发明所述抗体或抗原结合片段不仅可以结合人CD38，还可以结合食蟹猴CD38。
在一些实施方案中，特异性结合人CD38的分离的抗体或抗原结合片段，包含重链CDRs和轻链CDRs：
(1)所述重链CDRs包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
Figure PCTCN2020135547-appb-000001
Figure PCTCN2020135547-appb-000002
Figure PCTCN2020135547-appb-000003
和；
(2)所述轻链CDRs包含：CDR1-VL、CDR2-VL和CDR3-VL；所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
Figure PCTCN2020135547-appb-000004
Figure PCTCN2020135547-appb-000005
Figure PCTCN2020135547-appb-000006
各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为根据KABAT、Chothia或IMGT的通行分析方法编码。
在一些实施方案中，特异性结合人CD38的分离的抗体或抗原结合片段，其包含选自以下的重链CDRs组合：VH1、VH2、VH3、VH4、VH5、VH6、VH7、VH8、VH9、VH10、VH11、VH12、VH13、VH14、VH15、VH16、VH17、VH18、VH19、VH20、VH21、VH22、VH23、VH24、VH25、VH26、VH27、VH28、VH29、VH30、VH31、VH32、VH33、VH34、VH35或VH36，以及与所述重链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
在一些实施方案中，特异性结合人CD38的分离的抗体或抗原结合片段，其包含选自以下的轻链CDRs组合：VL1、VL2、VL3、VL4、VL5、VL6、VL7、VL8、VL9、VL10、VL11、VL12、VL13、VL14、VL15、VL16、VL17、VL18、VL19、VL20、VL21、VL22、VL23、VL24、VL25、VL26、VL27、VL28、VL29、VL30、VL31、VL32、VL33、VL34、VL35或VL36，以及与所述轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
在一些实施方案中，特异性结合人CD38的分离的抗体或抗原结合片段，其包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、VH14+VL14、VH15+VL15、VH16+VL16、VH17+VL17、VH18+VL18、VH19+VL19、VH20+VL20、VH21+VL21、VH22+VL22、VH23+VL23、VH24+VL24、VH25+VL25、VH26+VL26、VH27+VL27、VH28+VL28、VH29+VL29、VH30+VL30、VH31+VL31、VH32+VL32、VH33+VL33、VH34+VL34、VH35+VL35或VH36+VL36，以及与所述重链CDRs和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
在一些具体的实施方案中，特异性结合人CD38的分离的抗体或抗原结合片段， 其中：(1)所述重链CDRs包含：
CDR1-VH，其包含SEQ ID NO：1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35任一项所示VH的CDR1；
CDR2-VH，其包含SEQ ID NO：1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35任一项所示VH的CDR2；和，
CDR3-VH，其包含SEQ ID NO：1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35任一项所示VH的CDR3；和/或，
(2)所述轻链CDRs包含：
CDR1-VL，其包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36任一项所示VL的CDR1；
CDR2-VL，其包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36任一项所示VL的CDR2；和，
CDR3-VL，其包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36任一项所示VL的CDR3。
在一些具体的实施方案中，特异性结合人CD38的分离的抗体或抗原结合片段，其中：
(1)重链可变区和轻链可变区分别具有SEQ ID NO:1和SEQ ID NO:2所示序列；
(2)重链可变区和轻链可变区分别具有SEQ ID NO:3和SEQ ID NO:4所示序列；
(3)重链可变区和轻链可变区分别具有SEQ ID NO:5和SEQ ID NO:6所示序列；
(4)重链可变区和轻链可变区分别具有SEQ ID NO:7和SEQ ID NO:8所示序列；
(5)重链可变区和轻链可变区分别具有SEQ ID NO:9和SEQ ID NO:10所示序列；
(6)重链可变区和轻链可变区分别具有SEQ ID NO:11和SEQ ID NO:12所示序列；
(7)重链可变区和轻链可变区分别具有SEQ ID NO:13和SEQ ID NO:14所示序列；
(8)重链可变区和轻链可变区分别具有SEQ ID NO:15和SEQ ID NO:16所示序列；
(9)重链可变区和轻链可变区分别具有SEQ ID NO:17和SEQ ID NO:18所示序列；
(10)重链可变区和轻链可变区分别具有SEQ ID NO:19和SEQ ID NO:20所示序列；
(11)重链可变区和轻链可变区分别具有SEQ ID NO:21和SEQ ID NO:22所示序列；
(12)重链可变区和轻链可变区分别具有SEQ ID NO:23和SEQ ID NO:24所示序列；
(13)重链可变区和轻链可变区分别具有SEQ ID NO:25和SEQ ID NO:26所示序列；
(14)重链可变区和轻链可变区分别具有SEQ ID NO:27和SEQ ID NO:28所示序列；
(15)重链可变区和轻链可变区分别具有SEQ ID NO:29和SEQ ID NO:30所示序列；
(16)重链可变区和轻链可变区分别具有SEQ ID NO:31和SEQ ID NO:32所示序列；
(17)重链可变区和轻链可变区分别具有SEQ ID NO:33和SEQ ID NO:34所示序列；
(18)重链可变区和轻链可变区分别具有SEQ ID NO:35和SEQ ID NO:36所示序列；或，
(19)重链可变区和轻链可变区分别具有与上述(1)至(18)所示序列具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
在一个优选实施方案中，本发明的抗体或其抗原结合片段为：
(1)嵌合抗体或其片段；
(2)全人抗体或其片段；或，
(3)人源化抗体或其片段。
在一个优选实施方案中，本发明的抗体或其抗原结合片段为，其与人CD38结合的解离常数(KD)不大于5nM，与食蟹猴CD38结合的解离常数(KD)不大于25nM。
在一个优选实施方案中，本发明的抗体或其抗原结合片段包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
在一个优选实施方案中，本发明所述抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体、纳米抗体和抗体最小识别单位中的一种或多种。
在一个优选实施方案中，本发明的抗体或其抗原结合片段可与选自编号10、11、12、13、26、31、38、42、44、48、51、52、69、102、215、245、286、或292的抗体或抗原结合片段竞争性地结合CD38或其抗原表位，并且具备以下特性：
1)特异性结合人CD38重组蛋白及表达人CD38的细胞；
2)介导抗体依赖的细胞毒性杀伤(ADCC)活性；
3)介导抗体依赖的细胞介导的细胞吞噬作用(ADCP)；
4)介导交联诱导的细胞死亡；
5)不具备或只具备微弱的抗体介导的补体依赖的细胞毒性(CDC)活性；
6)抑制NAD水解酶活性；或/和
7)抑制肿瘤生长。
进一步的，在一些实施例中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、细胞毒性剂或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂；更优选地，所述细胞毒性剂选自生物碱类(alkaloids)、甲氨蝶呤(methotrexate)、蒽环类抗生素(doxorubicin)或紫杉烷类(taxanes)；所述毒素化合物优选DM1、DM4、SN-38、MMAE、MMAF、Duocarmycin、Calicheamicin、DX8951。
进一步的，在一些实施例中，所述抗体或抗原结合片段还连接有另一功能性分子，所述功能性分子可选自以下一种或多种：信号肽、蛋白标签、或细胞因子；优选地，所述细胞因子可选自IL-2、IL-6、IL-12、IL-15、IL-21、IFN或TNF-alpha。
在一些实施方案中，本发明提供一种分离的核酸分子，所述核酸分子编码本发明上述所述的抗体、抗原结合片段、或其任意组合。
在一些实施方案中，本发明提供一种表达载体，其包含本发明上述所述分离的核酸分子。
在一些实施方案中，本发明提供一种宿主细胞，其包含本发明上述所述分离的核酸分子或表达载体。
在一个优选实施方案中，所述宿主细胞是真核细胞或原核细胞；更优选，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选，所述宿主细胞选自中国仓鼠卵巢细胞(CHO)。
在一些实施方案中，本发明提供一种抗体或抗原结合片段的制备方法，在适当的条件下培养本发明上述所述的宿主细胞，并分离抗体或抗原结合片段。
在一些实施方案中，本发明提供一种药物组合物，组合物包含本发明上述所述的抗体或抗原结合片段、本发明上述所述分离的核酸分子、本发明上述所述表达载体、本发明上述所述细胞，或本发明上述所述方法制备的产品(例如抗体和抗原结合片段)，以及药学上可接受的载体。
在一个优选实施方案中，所述药物组合物还包含额外的抗肿瘤剂。
在一些实施方案中，本发明提供一种预防和/或治疗CD38表达异常相关的疾病的方法，包含向有此需要的患者施用本发明上述所述的抗体或抗原结合片段、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、本 发明上述所述的方法制备的产品(例如抗体和抗原结合片段)、或本发明上述所述药物组合物；所述疾病优选肿瘤。
在一些实施方案中，本发明提供上述所述的抗体或抗原结合片段、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、本发明上述所述的方法制备的产品(例如抗体和抗原结合片段)、或本发明上述所述药物组合物在制备预防和/或治疗CD38表达异常相关的疾病的药物中的用途，所述疾病优选肿瘤。
在一些实施方案中，本发明提供一种试剂盒，其包含本发明上述所述的抗体或抗原结合片段、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、或本发明上述所述的方法制备的产品(例如抗体和抗原结合片段)，以及使用说明。
在另一方面，本发明提供了一种多特异性抗体，所述多特异性抗体包含第一方面所述的抗体或抗原结合片段；优选地，所述多特异性抗体进一步包含特异性结合CD38以外的抗原或结合与第一方面所述抗体或抗原结合片段不同的CD38表位的抗体或抗原结合片段。
在一些实施例中，优选地，所述CD38以外的抗原可选自：CD3，优选CD3ε；CD16，优选CD16A；CD32B；PD-1；PD-2；PD-L1；VEGF；NKG2D；CD19；CD20；CD40；CD47；4-1BB；CD137；EGFR；EGFRvIII；TNF-alpha；CD33；MSLN；HER2；HER3；HAS；CD5；CD27；EphA2；EpCAM；MUC1；MUC16；CEA；Claudin18.2；叶酸受体；Claudin6；WT1；NY-ESO-1；MAGE3；ASGPR1或CDH16。
在一些实施例中，优选地，所述多特异性抗体可为双特异性、三特异性或四特异性，所述多特异性抗体可为二价、四价或六价。
在另一方面，本发明提供一种嵌合抗原受体(CAR)，所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含任选自本发明所述抗体或抗原结合片段。
在另一方面，本发明提供一种免疫效应细胞，所述免疫效应细胞表达上述所述的嵌合抗原受体，或包含编码上述所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
术语和定义：
除非另外说明，本文所用术语具有所属技术领域普通技术人员通常理解的含义。对于本文中明确定义的术语，则该术语的含义以所述定义为准。
术语“CD38”，是一种糖蛋白，具有环ADP核糖水解酶作用，存在于许多免疫细胞(白细胞)的表面，包括T、B淋巴细胞和自然杀伤细胞。CD38也在细胞粘附、信号转导和钙信号传导中起作用。
术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体)，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完 整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容援引加入本文)。
术语“抗原结合片段”是指保留特异性结合靶抗原的能力的一个或更多个抗体片段。抗体的抗原结合功能可以由全长抗体的片段执行。抗体片段可以是Fab、F(ab’)2、scFv、SMIP、双抗体、三抗体、亲和体(affibody)、纳米抗体、适体或结构域抗体。涵盖术语抗体的“抗原结合片段”的结合片段的实例包括但不限于：(i)Fab片段，一种由VL、VH、CL和CHl结构域组成的单价片段；(ii)F(ab)2片段，一种包含由二硫键在铰链区连接的两个Fab片段的双价片段；(iii)由VH和CHl结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)包含VH和VL结构域的dAb；(vi)由VH结构域组成的dAb片段(Ward等人，Nature341:544-546,1989)；(vii)由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)；以及(ix)两个或更多个分离的CDR的组合，所述CDR可以任选地由合成接头连接。此外，虽然Fv片段的两个结构域VL和VH是通过独立的基因编码的，但是这两个结构域可以使用重组方法通过接头接合，该接头能够使其制成其中VL和VH区配对以形成单价分子的单蛋白质链(称为单链Fv(scFv)；参见例如，Bird等人，Science 242:423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA85:5879-5883,1988)。这些抗体片段可以使用本领域技术人员已知的常规技术获得，并且这些片段被筛选用于与完整抗体相同的方式使用。可以通过重组DNA技术、完整免疫球蛋白的酶促或化学裂解、或在一些实施方式中通过本领域已知的化学肽合成程序来产生抗原结合片段。
术语“双特异性抗体”是指对至少两种不同的抗原具有单克隆结合特异性的抗体，其通常是人或人源化的抗体。在本发明中，结合特异性之一可以针对CD38的抗原表位而被检测，另一个可以针对CD38的另一个抗原表位或任何其他抗原，例如针对细胞表面蛋白、受体、受体亚基、组织特异性抗原、病毒来源蛋白、病毒编码的包膜蛋白、细菌来源蛋白或细菌表面蛋白等而被检测。
术语“嵌合”抗体是指以下抗体，其具有源自一种来源生物(如大鼠或小鼠)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985J Immunol Methods 125:191-202；以上通过援引加入并入本文。
术语“人源化抗体”是指鼠源单克隆抗体以基因克隆及DNA重组技术改造，重新表达的抗体，其大部分氨基酸序列为人源序列取代，基本保留亲本鼠单克隆抗体的亲和力和特异性，又降低了其异源性，有利应用于人体。人源化抗体就是指抗体的恒定区部分(即CH和CL区)或抗体所有全部由人类抗体基因所编码。人源化抗体可以大大减少异源抗体对人类机体造成的免疫副反应。
术语“互补决定区”(CDR)指在轻链和重链可变结构域中均发现的高变区。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的高变区之外。这些位置中的一个或更多个也可以在延伸的高变区中找到。本发明包括在这些杂合高变的位置中包含修饰的抗体。天然重链和轻链的可变结构域各自包含主 要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分别是指重链可变区(VH)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了重链(或其可变区)的CDR组合(VHCDR组合)；CDR1-VL、CDR2-VL和CDR3-VL分别是指轻链可变区(VL)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了轻链(或其可变区)的CDR组合(VLCDR组合)。
术语“VH”是指抗体的免疫球蛋白重链(包括Fv、scFv或Fab的重链)的可变区。术语“VL”是指免疫球蛋白轻链(包括Fv、scFv、dsFv或Fab的轻链)的可变区。
术语“抗体缀合物”是指抗体分子直接或者通过连接接头与另一个分子化学键合而形成的偶联体/缀合物。例如抗体-药物缀合物(ADC)，其中药物分子就是所述的另一个分子。
术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
术语“百分比(％)序列一致性”是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM(例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow&Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow&Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件。
术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
术语”ADCC”(antibody-dependent cell-mediated cytotoxicity，ADCC)，即抗体依赖的细胞介导的细胞毒性作用，是指抗体的Fab段特异性识别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FcR结合，介导杀伤细胞直接杀伤靶细胞。抗体介导的ADCC强弱和许多因素有关，如抗体与抗原的亲和力、抗体与Fc段受体的亲和力、免疫效应细胞的特性等。一般情况下，对抗原或Fc受体亲和力高的抗体介导的ADCC作用越强。对抗体Fc段的糖基化及氨基酸序列改造可以提高ADCC活性。在抗体的糖基化修饰中，岩藻糖被认为是影响ADCC活性最重要的糖，去岩藻糖化可以显著提高抗体与FcγRIIIa的亲和力及ADCC活性。
术语“抗体通过交联诱导程序性细胞死亡”(antibody induces programmed cell death via cross-linking)是指抗体的Fc段与Fc受体(FcR)或在第二交联抗体作用下后，诱导细胞程序性细胞死亡过程。ADCC和ADCP是通过单克隆抗体结合激活免疫效应细胞(如自然杀伤细胞、巨噬细胞和多形核细胞)上的FcγRs，诱导成聚集的IgG恒定结构域(Fc结构域)。单克隆抗体可通过抗体的Fc段与Fc受体相互作用进而增强抗体的激动活性或诱导程序性细胞死亡(programmed cell death,PCD)。以死亡受体家族成员为靶点的激动治疗单克隆抗体通过外源性凋亡途径诱导PCD。以这些死亡受体为靶点的激动性单克隆抗体诱导的PCD通过交联增强，比如通过第二交联抗体或更生理学上与FcγRs结合而增强。与死亡受体家族无关的抗体介导的抗原交联也可诱导PCD，但不能通过经典的凋亡途径。这种途径的特点是细胞的同型聚集，包括细胞骨架重组、溶酶体活化和活性氧的产生。这种非诱导PCD通路可以通过Fc交联的二抗或FcγR表达细胞来增强。
术语“载体”包括核酸载体，例如DNA载体(如质粒)，RNA载体，病毒或其他适合的复制子(例如病毒载体)。已经开发了多种载体用于将编码外源蛋白质的多核苷酸递送到原核或真核细胞中。本发明的表达载体含有多核苷酸序列以及例如用于表达蛋白质和/或将这些多核苷酸序列整合到哺乳动物细胞基因组中的附加序列元件。可以用于表达本发明的抗体和抗体片段的某些载体包括含有指导基因转录的调控序列(如启动子和增强子区域)的质粒。用于表达抗体和抗体片段的其他有用的载体含有多核苷酸序列，其增强这些基因的翻译速率或改善由基因转录产生的mRNA的稳定性或核输出。这些序列元件包括例如5’和3’非翻译区、内部核糖体进入位点(IRES)和聚腺苷酸化信号位点，以便指导表达载体上携带的基因的有效转录。本发明的表达载体还可以含有以下多核苷酸，该多核苷酸编码用于选择含有这种载体的细胞的标记。适合的标记的实例包括编码抗生素(如氨苄青霉素、氯霉素、卡那霉素或诺尔丝菌素)抗性的基因。
术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、牛、绵羊、马和野牛等。
术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目 的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。如本文所用，术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
附图说明
下面通过对本发明的详细描述以及附图来清楚地说明本发明前面叙述的方面以及其他方面。本文中附图是为了举例说明本发明的一些优选的实施方案，然而，可以理解，本发明并不限于所公开的特定实施方案。
图1A、CD38-His免疫小鼠血清中与人CD38-His结合滴度检测；
图1B、CD38-His免疫小鼠血清与人EGFR-His结合滴度检测；
图2A、未免疫小鼠脾脏B细胞CD38-His染色结果；
图2B、CD38-His免疫的小鼠脾脏B细胞CREG-His染色结果；
图2C、CD38-His免疫的小鼠脾脏B细胞CD38-His染色结果；
图3A、FACS检测抗CD38候选抗体和Daudi细胞直接结合EC50值；
图3B、FACS检测候选抗体和CHO-CD38细胞直接结合EC50值；
图4A-4B、抗CD38抗体的抗体依赖的细胞介导的细胞吞噬作用(ADCP)测定；
图5A、抗CD38抗体通过交联诱导的Romas细胞发生了磷脂酰丝氨酸易位；
图5B、抗CD38抗体通过交联诱导的细胞死亡；
图6A、抗CD38抗体通过交联诱导的Annexin V阳性细胞的剂量依赖关系，比例高于PI阳性细胞比例；
图6B、抗CD38抗体通过交联诱导的细胞死亡的剂量依赖关系；
图7A-7B、抗CD38抗体介导的NK细胞对Daudi细胞的ADCC活性测试；
图8A-8C、抗CD38抗体介导的PBMC对Daudi细胞的ADCC活性测试；
图9、抗CD38抗体抑制CD38蛋白的NAD水解酶活性的检测；
图10A-10B、抗CD38抗体介导的补体依赖的细胞毒性反应。
具体实施方式
下面结合实施例和附图对本发明进行详细描述，本文中附图是为了举例说明本发明的一些优选的实施方案，然而，可以理解，本发明并不限于所公开的特定实施方案或看作对本发明范围的限制。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1小鼠免疫产生特异性结合CD38的单克隆抗体
对6-8周龄的雌性SJL小鼠(购自北京维通利华实验动物技术有限公司)或者Balb/c小鼠(购自上海斯莱克实验动物有限公司)，使用人CD38-His(Novoprotein,Cat：CU65)与弗氏完全佐剂(complete freund's adjuvant,Sigma，Cat：F5881)进行第一次免疫；使用上述人CD38-His与弗氏不完全佐剂(incomplete freund's adjuvant，IFA,Sigma，Cat：F5506)加未甲基化的胞嘧啶鸟嘌呤二核苷酸(CpGODN1826，合成自上海生工生物)进行后三次免疫；免疫时注射50μg/只/次通过乳化操作形成的均一稳定的乳剂。特别地，第一次和第二次免疫注射后足垫和背部，第三次和第四次免疫注射尾部皮下及背部，以获得高滴度高亲和力高特异性的抗血清及特异性的免疫细胞。在末次免疫(第四次免疫)后的第5-7天，安乐死小鼠并无菌取出脾脏，无菌分离提取小鼠脾脏淋巴细胞，分装至冻存管中，冻存于液氮中。分别在二次免疫，三次免疫后10天及安乐死小鼠当天进行小鼠的采血操作，分离血清，使用酶联免疫吸附(ELISA)方法测定血清中抗CD38特异性抗体的滴度。
实验结果如图1A、1B，显示经过四次免疫后，所免疫小鼠的血清与人CD38-His结合的滴度都能很很高。且与His无关蛋白(人EGFR-His)结合滴度都很低，说明了使用此方法进行小鼠的免疫，可以使小鼠产生高滴度高特异性的抗CD38抗体。
实施例2 CD38特异性单个B细胞的流式细胞荧光分选(FACS)
CD38蛋白免疫的小鼠脾脏细胞，经抗原CD38-His蛋白(CD38-His,Novoprotein，Cat：CU65)及间接标记抗体anti-His-APC(R&D Systems,Cat.IC050A)和针对小鼠B细胞特有标志的抗体(anti-mouse B220-Pacfic Blue，BD Biosciences,Cat.558108；anti-mouse IgD-PE，BD Biosciences,Cat.558597；anti-mouse IgM-PE cy7,BD Biosciences,Cat.552867)染色，并在分选前加入区分死细胞和活细胞的染料7-AAD(BD Biosciences,Cat.51-68981E)，用AriaIII(BD公司)流式细胞分选仪分选CD38特异性的单个B细胞(7AAD -B220 +IgD -IgM -CD38 +)至含有细胞裂解液、RNA酶抑制剂的PCR孔中，每个孔收集一个细胞。结果显示未免疫对照小鼠脾脏用CD38-His蛋白染色(图2A)或不相关蛋白CREG-His染色的CD38蛋白免疫的小鼠脾脏(图2B)未检测到CD38 +B细胞，而CD38-His免疫的小鼠脾脏(图2C)检测到明显的一群CD38 +B细胞，每10 6个脾脏细胞中约有65个CD38 +B细胞。
实施例3单克隆抗体的扩增和高通量表达
采用专利“一种用于巢式扩增的组合引物及其应用”专利申请号：201811618134.4中实施例1的方法，将单细胞的mRNA反转录成cDNA。然后以cDNA为模板进行巢式PCR，分别进行抗体重链和轻链扩增。扩增得到抗体重链可变区和轻链可变区，分别通过同源重组方法克隆到重链表达载体和轻链表达载体。重链表达载体和轻链表达载体的恒定区都来自于人IgG1。完整重链表达序列是信号肽-VH-CH1-铰链区 -CH2-CH3，完整轻链表达序列是信号肽-Vκ-Cκ。以上所述单B细胞抗体克隆和表达皆在96孔板内以高通量方式达到抗体的快速鉴定和发现。经过一系列理化和功能筛选324对克隆表达的抗体重链和轻链后共获得18个阳性候选抗体分子，其序列的CDRs分别用IMGT和KABAT软件分析，对应的序列信息如下表1所示，其中表1示出候选抗体分子的VH和VL序列，表2示出候选抗体分子的IMGT和KABAT分析结果)。
将候选抗CD38抗体测序，重链可变区和轻链可变区具体的序列信息如下：
表1.抗CD38抗体重链可变区和轻链可变区的具体序列信息
Figure PCTCN2020135547-appb-000007
Figure PCTCN2020135547-appb-000008
分别使用IMGT和KABAT软件分析各抗体的CDRs，具体的序列信息如下：
表2.IMGT和KABAT软件分析各抗体的CDRs具体序列信息
Figure PCTCN2020135547-appb-000009
Figure PCTCN2020135547-appb-000010
Figure PCTCN2020135547-appb-000011
Figure PCTCN2020135547-appb-000012
实施例4 Octet检测抗体结合亲和力和特异性
采用Octet HTX检测系统，抗体用PBST稀释至1μg/ml，抗原CD38-His(Novoprotein，Cat：CU65)用PBST稀释至100nM作为起始浓度，2倍梯度稀释，共7个梯度，依次加入96孔Octet板(Greiner,Cat.655209)，300μl/孔；设置循环程序，一共8个protein A探针，每一个循环先loading抗体到探针至1nm高度，约3分钟，再用探针结合梯度稀释后的CD38-His抗原，约10分钟至饱和平台，最后用Glycine pH1.5再生。分析试验数据，拟合抗体抗原的平衡解离常数KD，确定结合速率常数ka和解离速率常数kd。
抗体候选物对于人和食蟹猴CD38蛋白的亲和力总结于表3。结合速率Kon，解离速率Kdis,解离常数KD。通过表3可以看出候选CD38抗体与商业途径购买的药物对照抗体Daratumumab(Janssen,Cat.GIS0503)相比，与人的结合亲合力有很大提高，但与食蟹猴的结合亲和力要比Daratumumab弱,与根据Morphosys专利发表序列制备的CD38单抗阳性对照Mor202-kaps(Biointron,Cat.B4163)相比，与人及食蟹猴的结合亲合力大致相当或有所提高。
表3.Octet检测CD38抗体结合亲和力和特异性
Figure PCTCN2020135547-appb-000013
Figure PCTCN2020135547-appb-000014
实施例5通过流式细胞仪(FACS)测定候选抗体的EC50
候选抗CD38抗体对细胞表面上的CD38靶标的剂量依赖性结合能力通过流式细胞术(FACS)证实。
本实验选用2株高表达CD38的细胞株：Daudi(购自中国科学院细胞库，Cat：TCHu140)及CHO细胞表面高表达CD38蛋白的CHO-38细胞(定购自勇山生物科技有限公司)。在U型圆底96孔板中，1×10 5 Daudi或CHO-38细胞和系列稀释的抗CD38抗体，起始浓度为10μg/ml，10倍梯度，每个抗体6个梯度，4℃共孵育半小时后，加入5μg/ml二抗anti hIgG Fc(Invitrogen，Cat:31125)，4℃共孵育半小时后上机FACS检测。
结果证实，候选抗体有效地结合在表达CD38的细胞表面上，如图3所示，参比抗体Daratumumab(阳性对照)在Daudi细胞表面(图3A)EC50为57.94ng/ml，在CHO-38细胞表面上(图3B)的EC50为60.43ng/ml，而候选抗体与这两种细胞表面CD38结合的EC50与参比抗体差异不大；参比抗体为Mor202-kaps(阳性对照)时，两种细胞上候选抗体的EC50都小于参比抗体，说明候选抗体与这两株细胞的结合力强于Mor202-kaps，详见表4。
表4.EC50结果图
抗体编号 Daudi-EC50(ng/ml) CHO-38-EC50(ng/ml)
11 58.36 111.9
13 57.97 123.1
42 128 89.55
44 57.47 116.5
48 86.47 99.59
69 131 67.41
102 58.23 86.21
215 82.71 106.8
286 149.8 109.8
292 94.59 61.08
Daratumumab 57.94 60.43
Mor202-Kaps 322.1 178.2
实施例6抗CD38抗体的抗体依赖的细胞介导的细胞吞噬作用(ADCP)测定
利用Daudi细胞(购自中国科学院细胞库，目录号：TCHu140)作为靶细胞和人的巨噬细胞(PBMC分离单核细胞诱导而来)作为效应细胞来测量ADCP。
效应细胞的制备：冻存的正常人的外周血单核细胞PBMC(Stemexpress,Cat:PB0004C)，复苏PBMC细胞后在补充有100IU/ml rhIL-2(Peprotech,Cat:-200-02)的 RPMI1640(Gibco,Cat:22400-089)中孵育过夜。其后，收集细胞后进行活细胞计数。然后从PBMC中分离单核细胞，具体参照单核细胞分离试剂盒说明书(EasySep Cat:19058)，将单核细胞用补充有100μg/ml GM-CSF(Peprotech,Cat:300-03-20Ug)的含10％FBS(Gibco,Cat:10099-141)的RPMI1640中诱导分化，在5％CO 2,37℃培养中培养培养一周后，重悬后进行计数，用作效应细胞。
靶细胞的制备：将靶细胞Daudi(购自中国科学院细胞库，目录号：TCHu140)收集后用CelltraceTMviolet(Invitrogen，Cat:C34556)进行标记，标记的细胞用补充有10％FBS的RPMI1640重悬，用作靶细胞。
在U型圆底96孔板中，5×10 4Daudi细胞和稀释的抗CD38抗体,终浓度为1μg/ml，0.1μg/ml，4℃预孵育半小时，然后加入的2×10 5效应细胞(效应细胞：靶细胞＝4：1)，4℃共孵育3小时。孵育完成后，进行染色操作。
染色过程：Human TruStain FcX(Biolegend，Cat:422302)封闭半小时后，加入染色抗体APC Mouse anti human CD19(BD,Cat:555415),AF488mouseanti human CD11b(BD,Cat:557701)，4℃孵育半小时。用含有2％FBS(Gibco,Cat:10099-141)的DPBS(Hyclone，Cat:SH30256.01)洗两遍后重悬，上机前加入PI(Invitrogen,Cat:P3566)用流式细胞仪进行分析。
根据下面计算公式计算由ADCP活性引起的细胞裂解率：
细胞吞噬率＝被吞噬的细胞所占比(CD19 +CD11b -)/靶细胞所占百分比(Celltrace +)*100％
参见图4A及图4B，可看出候选抗CD38抗体均能提高ADCP作用，候选抗体69的ADCP作用在1μg/ml和0.1μg/ml这两个剂量下都比参比抗体Daratumumab(阳性对照)更强。
实施例7抗CD38抗体通过交联诱导的程序性细胞死亡分析
在Romas细胞(上海酶研生物科技有限公司，货号CC-Y1430)上测定CD38抗体交联诱导的细胞程序性细胞死亡。在U型圆底96孔板中，4×10 4Romas细胞和系列稀释的抗CD38抗体,终浓度为10μg/ml，1μg/ml，0.1μg/ml和0.01μg/ml，预孵育半小时，然后加入5μg/ml anti hIgG Fc(Invitrogen，Cat:31125)，在5％CO 2，37℃培养中培养20小时。分别使用程序性细胞死亡检测试剂盒(eBioscience,Cat:BMS500FI/300)中的Annexin V和PI对细胞进行染色后用流式细胞仪进行分析。Annexin V阳性百分比和PI阳性百分比用Flowjo软件进行计算分析。
结果显示所有候选CD38抗体(0.1μg/ml)在Fc交联二抗anti hIgGFc作用下，都可诱导了Romas细胞发生了磷脂酰丝氨酸易位(图5A)，因此Romas细胞的Annexin V阳性比例都明显提高。PI细胞阳性比例也有明显提高，即细胞发生死亡(图5B)。Annexin V阳性细胞的比例高于PI阳性细胞比例，进一步观察不同浓度CD38抗体对细胞的影响(图6A-6B)，发现在0.01-10μg/ml浓度下，在Fc交联试剂存在时，本发明候选抗CD38抗体均可有效诱导细胞发生凋亡。
实施例8抗CD38抗体的ADCC活性测试
效应细胞的制备：冻存的正常人的外周血单核细胞PBMC(Stemexpress,Cat:PB0004C)，复苏PBMC细胞后在补充有100IU/ml rhIL-2(Peprotech,Cat:-200-02)的RPMI1640(Gibco,Cat:22400-089)中孵育过夜。其后，收集细胞后进行活细胞计数。然后从PBMC中分离纯化NK细胞，具体参照NK细胞分离试剂盒说明书分离NK细 胞(Stemcell Cat:17955)，将NK细胞用补充有10％FBS(Gibco,Cat:10099-141)的RPMI1640重悬后进行计数，用作效应细胞。PBMC可以直接用于ADCC实验的效应细胞。
靶细胞的制备：将靶细胞Daudi(购自中国科学院细胞库，目录号：TCHu140)收集后用CelltraceTMviolet(Invitrogen，Cat:C34556)进行标记，标记的细胞用补充有10％FBS的RPMI1640重悬，用作靶细胞。
用补充有10％FBS的RPMI1640稀释抗CD38抗体，稀释好的抗体以50μl/孔分到U型底96孔板中，然后将标记的靶细胞加入孔中。板子在37℃，5％CO 2培养箱中预孵育半小时。随后，将效应细胞加入孔中(效靶比NK:Daudi 2:1-4:1；PBMC:Daudi 20:1-40:1)，37℃，5％CO 2培养箱中孵育3-4个小时。将板取出，每孔加入死细胞标记染料PI(1μl/孔)后进行流式分析，测量CelltraceTMviolet阳性的靶细胞中死细胞即PI阳性的比例。根据下面计算公式计算由ADCC活性引起的细胞裂解率：
细胞裂解率(％)＝(样品孔PI％-仅有靶细胞孔PI％)/(1-仅有靶细胞孔PI％)。
结果显示于图7A-7B和图8A-8C中，所有抗CD38抗体均显示出以抗体浓度依赖性方式对Daudi细胞的裂解杀伤活性。用NK作为效应细胞时CD38抗体ADCC活性的EC50在0.008-0.029ng/ml之间(图7A-7B)，用PBMC作为效应细胞时CD38抗体ADCC活性的EC50在0.084-0.200ng/ml之间(图8A-8C)。阳性对照Daratumumab(Dara)的ADCC活性比Mor202-kaps强，阴性对照抗体anti-Hel(先声生物自行制备)(和Daudi细胞没有结合)和预期一致，没有ADCC活性。
实施例9 CD38抗体抑制CD38蛋白水解酶活性的检测
本实施例采用CD38水解酶活性抑制剂筛选试剂盒(BPS Bicoscience,Cat.79287)进行检测，按照说明书进行操作。在96孔板中加入水解缓冲液，1μg/ml待检测抗体，0.5μg/ml CD38蛋白后，将体系放入37℃培养箱中孵育30分钟，随后加入ε-NAD底物，酶标仪进行荧光检测，激发波长300nm，检测波长为410nm，每隔3分钟读取板上各样品孔的荧光值，共读取60分钟。根据时间-荧光值曲线，选取进入平台期的前一时间点，计算抑制率：
抑制率(％)＝(Sample荧光值-Blank荧光值)/(Positive Control荧光值-Blank荧光值)×100％。
结果如图9所示，对照药品Daratumumab(Dara)的反应曲线与阴性对照抗体组IgG1κ、未加药组Drug0的反应曲线重合，都不具有抑制CD38水解酶活性；而具有抑制CD38水解酶活性的对照药品Isatuximab(Isa)的反应曲线与抗CD38抗体102和215的反应曲线相近，CD38水解酶活性的反应速率均有明显下降，说明被测试的抗CD38抗体可以有效抑制CD38的水解酶活性。
实施例10 CD38抗体CDC活性检测
在96孔板中加入以下细胞和抗体：Daudi细胞1×10 5cell/孔，25μl/孔，CD38抗体：25μl/孔，4μg/ml起始浓度，1:10稀释，37℃孵育30分钟。加入人血清(先声药业体检用正常人血清)，使终浓度为5％。37℃孵育2.5小时。用RPMI 1640按照1:20稀释PI染料(invitrogen，Lot:1887160)，加入20μl/well，使得最终PI用量为1μl/well，室温孵育5分钟后，上流式细胞仪检测，记录死细胞比例。CDC activity(％)＝PI阳性细胞比例。结果见图10A-10B，已知具有很强的CDC活性的Daratumumab如预期显示了剂量依赖的CDC活性，而被测试的CD38候选抗体除了抗体10，31，38，102， 215在最高的三个浓度有微弱的CDC活性外，其余均无明显CDC活性。表明这些抗体有望避免因CDC活性导致的静脉输液相关的反应(infusion related reaction,IRR)。

Claims (24)

  1. 特异性结合人CD38的分离的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链CDRs和轻链CDRs：
    (1)所述重链CDRs包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    Figure PCTCN2020135547-appb-100001
    Figure PCTCN2020135547-appb-100002
    Figure PCTCN2020135547-appb-100003
    和；
    (2)所述轻链CDRs包含：CDR1-VL、CDR2-VL和CDR3-VL；所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    Figure PCTCN2020135547-appb-100004
    Figure PCTCN2020135547-appb-100005
    Figure PCTCN2020135547-appb-100006
  2. 权利要求1的抗体或其抗原结合片段，其包含选自以下的重链CDRs组合：VH1、VH2、VH3、VH4、VH5、VH6、VH7、VH8、VH9、VH10、VH11、VH12、VH13、VH14、VH15、VH16、VH17、VH18、VH19、VH20、VH21、VH22、VH23、VH24、VH25、VH26、VH27、VH28、VH29、VH30、VH31、VH32、VH33、VH34、VH35或VH36，以及与所述重链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
  3. 权利要求1的抗体或其抗原结合片段，其包含选自以下的轻链CDRs组合：VL1、VL2、VL3、VL4、VL5、VL6、VL7、VL8、VL9、VL10、VL11、VL12、VL13、VL14、VL15、VL16、VL17、VL18、VL19、VL20、VL21、VL22、VL23、VL24、VL25、VL26、VL27、VL28、VL29、VL30、VL31、VL32、VL33、VL34、VL35或VL36，以及与所述轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、VH14+VL14、VH15+VL15、VH16+VL16、VH17+VL17、VH18+VL18、VH19+VL19、VH20+VL20、VH21+VL21、VH22+VL22、VH23+VL23、VH24+VL24、VH25+VL25、VH26+VL26、VH27+VL27、VH28+VL28、VH29+VL29、VH30+VL30、VH31+VL31、VH32+VL32、VH33+VL33、VH34+VL34、VH35+VL35或VH36+VL36，以及与所述重链CDRs和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
  5. 权利要求1-4任一项的抗体或抗原结合片段，其特征在于，
    (1)所述重链CDRs包含：
    CDR1-VH，其包含SEQ ID NO：1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35任一项所示VH的CDR1；
    CDR2-VH，其包含SEQ ID NO：1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35任一项所示VH的CDR2；和，
    CDR3-VH，其包含SEQ ID NO：1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35任一项所示VH的CDR3；和/或，
    (2)所述轻链CDRs包含：
    CDR1-VL，其包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36任一项所示VL的CDR1；
    CDR2-VL，其包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36任一项所示VL的CDR2；和，
    CDR3-VL，其包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36任一项所示VL的CDR3。
  6. 权利要求1的抗体或抗原结合片段，其特征在于，
    (1)重链可变区和轻链可变区分别具有SEQ ID NO:1和SEQ ID NO:2所示序列；
    (2)重链可变区和轻链可变区分别具有SEQ ID NO:3和SEQ ID NO:4所示序列；
    (3)重链可变区和轻链可变区分别具有SEQ ID NO:5和SEQ ID NO:6所示序列；
    (4)重链可变区和轻链可变区分别具有SEQ ID NO:7和SEQ ID NO:8所示序列；
    (5)重链可变区和轻链可变区分别具有SEQ ID NO:9和SEQ ID NO:10所示序列；
    (6)重链可变区和轻链可变区分别具有SEQ ID NO:11和SEQ ID NO:12所示序列；
    (7)重链可变区和轻链可变区分别具有SEQ ID NO:13和SEQ ID NO:14所示序列；
    (8)重链可变区和轻链可变区分别具有SEQ ID NO:15和SEQ ID NO:16所示序列；
    (9)重链可变区和轻链可变区分别具有SEQ ID NO:17和SEQ ID NO:18所示序列；
    (10)重链可变区和轻链可变区分别具有SEQ ID NO:19和SEQ ID NO:20所示序列；
    (11)重链可变区和轻链可变区分别具有SEQ ID NO:21和SEQ ID NO:22所示序列；
    (12)重链可变区和轻链可变区分别具有SEQ ID NO:23和SEQ ID NO:24所示序列；
    (13)重链可变区和轻链可变区分别具有SEQ ID NO:25和SEQ ID NO:26所示序列；
    (14)重链可变区和轻链可变区分别具有SEQ ID NO:27和SEQ ID NO:28所示序列；
    (15)重链可变区和轻链可变区分别具有SEQ ID NO:29和SEQ ID NO:30所示序列；
    (16)重链可变区和轻链可变区分别具有SEQ ID NO:31和SEQ ID NO:32所示序列；
    (17)重链可变区和轻链可变区分别具有SEQ ID NO:33和SEQ ID NO:34所示序列；
    (18)重链可变区和轻链可变区分别具有SEQ ID NO:35和SEQ ID NO:36所示序列；或，
    (19)重链可变区和轻链可变区分别具有与上述(1)至(18)所示序列具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
  7. 权利要求1-6任一项的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段为：(1)嵌合抗体或其片段；(2)全人抗体或其片段；或，(3)人源化抗体或其片段。
  8. 权利要求1-6任一项的抗体或抗原结合片段，其特征在于，其与人CD38结合的解离常数(KD)不大于5nM，与食蟹猴CD38结合的解离常数(KD)不大于25nM。
  9. 权利要求1-6任一项的抗体或抗原结合片段，其特征在于，所述抗体包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
  10. 权利要求1-6任一项的抗体或抗原结合片段，其特征在于，所述抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体、纳米抗体和抗体最小识别单位中的一种或多种。
  11. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段与权利要求1-10任一项所述的抗体或抗原结合片段竞争性地结合CD38或其抗原表位，并且具备以下特性：
    (1)特异性结合CD38重组蛋白及表达CD38的细胞；
    (2)介导抗体依赖的细胞毒性杀伤(ADCC)活性；
    (3)介导抗体依赖的细胞介导的细胞吞噬作用(ADCP)；
    (4)介导交联诱导的细胞死亡；
    (5)不具备或只具备微弱的抗体介导的补体依赖的细胞毒性(CDC)活性；
    (6)抑制NAD水解酶活性；或/和
    (7)抑制肿瘤生长。
  12. 一种分离的核酸分子，其特征在于，所述核酸分子编码权利要求1-11任一项所述的抗体、抗原结合片段、或其任意组合。
  13. 包含权利要求12所述分离的核酸分子的表达载体。
  14. 包含权利要求12所述的分离的核酸分子、或权利要求13所述的表达载体的分离的宿主细胞；优选，所述宿主细胞是真核细胞或原核细胞；更优选，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选，所述宿主细胞选自中国仓鼠卵巢细胞(CHO)。
  15. 一种抗体或抗原结合片段的制备方法，其特征在于，在适当的条件下培养权利要求14所述的宿主细胞，并分离抗体或抗原结合片段。
  16. 一种药物组合物，其特征在于，所述组合物包含权利要求1-11任一项所述的抗体或抗原结合片段、权利要求12的分离的核酸分子、权利要求13的表达载体、权利要求14的细胞，或权利要求15方法制备的产品，以及药学上可接受的载体；优选，所述药物组合物还包含额外的抗肿瘤剂。
  17. 一种试剂盒，其特征在于，所述试剂盒包含权利要求1-11任一项所述的抗体或抗原结合片段、权利要求12的分离的核酸分子、权利要求13的表达载体、权利要求14的细胞，或权利要求15方法制备的产品，以及使用说明。
  18. 权利要求1-11任一项所述的抗体或抗原结合片段、权利要求12的分离的核酸分子、权利要求13的表达载体、权利要求14的细胞、权利要求15方法制备的产品、或权利要求16所述的药物组合物在制备预防和/或治疗CD38表达异常相关的疾病的药物中的用途，所述疾病优选肿瘤。
  19. 一种预防和/或治疗CD38表达异常相关的疾病的方法，包含向有此需要的患者施用权利要求1-11任一项所述的抗体或抗原结合片段、权利要求12的分离的核酸分子、权利要求13的表达载体、权利要求14的细胞、权利要求15方法制备的产品、或权利要求16所述的药物组合物；所述疾病优选肿瘤。
  20. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求1-11任一项所述抗体或抗原结合片段。
  21. 一种免疫效应细胞，其特征在于，所述免疫效应细胞表达权利要求20所述的嵌合抗原受体，或包含编码权利要求20所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
  22. 一种多特异性抗体，其特征在于，所述多特异性抗体包含权利要求1-11任一项所述的抗体或抗原结合片段；优选地，所述多特异性抗体进一步包含特异性结合CD38以外的抗原或结合与权利要求1-11任一项所述抗体或抗原结合片段不同的CD38表位的抗体或抗原结合片段；优选地，所述CD38以外的抗原可选自：CD3，优选CD3ε；CD16，优选CD16A；CD32B；PD-1；PD-2；PD-L1；VEGF；NKG2D；CD19；CD20；CD40；CD47；4-1BB；CD137；EGFR；EGFRvIII；TNF-alpha；CD33； MSLN；HER2；HER3；HAS；CD5；CD27；EphA2；EpCAM；MUC1；MUC16；CEA；Claudin18.2；叶酸受体；Claudin6；WT1；NY-ESO-1；MAGE3；ASGPR1或CDH16；更优选地，所述多特异性抗体可为双特异性、三特异性或四特异性，所述多特异性抗体可为二价、四价或六价。
  23. 权利要求1-11任一项的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、细胞毒性剂或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂；更优选地，所述细胞毒性剂选自生物碱类(alkaloids)、甲氨蝶呤(methotrexate)、蒽环类抗生素(doxorubicin)或紫杉烷类(taxanes)；所述毒素化合物优选DM1、DM4、SN-38、MMAE、MMAF、Duocarmycin、Calicheamicin、DX8951。
  24. 权利要求1-11任一项的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段还连接有另一功能性分子，所述功能性分子可选自以下一种或多种：信号肽、蛋白标签、或细胞因子；优选地，所述细胞因子可选自IL-2、IL-6、IL-12、IL-15、IL-21、IFN或TNF-alpha。
PCT/CN2020/135547 2019-12-13 2020-12-11 一种抗cd38的抗体及其用途 WO2021115404A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202080066375.1A CN114616245B (zh) 2019-12-13 2020-12-11 一种抗cd38的抗体及其用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911286272 2019-12-13
CN201911286272.1 2019-12-13

Publications (1)

Publication Number Publication Date
WO2021115404A1 true WO2021115404A1 (zh) 2021-06-17

Family

ID=76329601

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/135547 WO2021115404A1 (zh) 2019-12-13 2020-12-11 一种抗cd38的抗体及其用途

Country Status (2)

Country Link
CN (1) CN114616245B (zh)
WO (1) WO2021115404A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116120455A (zh) * 2021-08-23 2023-05-16 东莞市朋志生物科技有限公司 一种抗ca15-3蛋白的重组抗体

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1976950A (zh) * 2004-02-06 2007-06-06 莫佛塞斯公司 抗cd38人抗体及其用途
CN106456731A (zh) * 2014-02-28 2017-02-22 詹森生物科技公司 用于治疗急性成淋巴细胞性白血病的抗cd38抗体
WO2018224682A1 (en) * 2017-06-08 2018-12-13 Tusk Therapeutics Ltd Cd38 modulating antibody

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1976950A (zh) * 2004-02-06 2007-06-06 莫佛塞斯公司 抗cd38人抗体及其用途
CN106456731A (zh) * 2014-02-28 2017-02-22 詹森生物科技公司 用于治疗急性成淋巴细胞性白血病的抗cd38抗体
WO2018224682A1 (en) * 2017-06-08 2018-12-13 Tusk Therapeutics Ltd Cd38 modulating antibody

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
XIN GAO, RONGHUI YANG, AIJUN LIAO: "Advances in Anti-CD38 Monoclonal Antibody in the Treatment of Multiple Myeloma", JOURNAL OF MODERN ONCOLOGY, vol. 27, no. 9, 1 May 2019 (2019-05-01), pages 1617 - 1620, XP055792766, ISSN: 1672-4992, DOI: 10.3969/j.issn.1672-4992.2019.09.036 *
YU TAO, QIAO CHUNXIA, LV MING, TANG LUQUN: "Novel anti-CD38 humanized mAb SG003 possessed enhanced cytotoxicity in lymphoma than Daratumumab via antibody-dependent cell-mediated cytotoxicity", BMC BIOTECHNOLOGY, vol. 19, no. 1, 1 December 2019 (2019-12-01), XP055820638, DOI: 10.1186/s12896-019-0524-8 *
ZHOU YUANQING: "Study on the Preparation and Biological Function of Humanized Anti-CD38 Antibody", MEDICINE AND HEALTH SCIENCES, CHINESE MASTER’S THESES FULL-TEXT DATABASE, 1 December 2017 (2017-12-01), XP055820800 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116120455A (zh) * 2021-08-23 2023-05-16 东莞市朋志生物科技有限公司 一种抗ca15-3蛋白的重组抗体
CN116120455B (zh) * 2021-08-23 2023-11-07 东莞市朋志生物科技有限公司 一种抗ca15-3蛋白的重组抗体

Also Published As

Publication number Publication date
CN114616245B (zh) 2024-02-23
CN114616245A (zh) 2022-06-10

Similar Documents

Publication Publication Date Title
RU2744959C2 (ru) Новые анти-pd-l1 антитела
EP3904386A1 (en) Antibody and use thereof
KR102422411B1 (ko) Pd-l1-특이적 항체 및 이를 사용하는 방법
US8652470B2 (en) Monoclonal antibodies directed to CD52
CN110869388A (zh) 用于肿瘤特异性细胞消耗的Fc优化的抗CD25
KR20150002621A (ko) 항 ｓｅｚ6 항체들 및 사용 방법
JP2021522847A (ja) Ｏｘ４０に対する完全ヒト抗体、それを調製する方法、およびその使用
WO2022161425A1 (zh) 抗tnfr2人源化抗体及其用途
WO2021115303A1 (zh) 一种抗Claudin18.2单克隆抗体、其制备方法及用途
JP2022529350A (ja) アンタゴニスト抗ｃｄ７抗体
KR20220042258A (ko) 항 tigit 항체 및 그의 응용
JP2021524251A (ja) Ｃｄ３に特異的な抗体及びその使用
JP2023179450A (ja) 抗－ヒトプログラム細胞死リガンド－１（ｐｄ－ｌ１）の抗体及びその用途
EP4292611A1 (en) Anti-cd112r antibody and use thereof
JP2022502417A (ja) 抗ｏｘ４０抗体、その抗原結合フラグメント、および医薬用途
WO2021115404A1 (zh) 一种抗cd38的抗体及其用途
JP7430137B2 (ja) 抗体および使用方法
EP4378954A1 (en) Anti-pvrig/anti-tigit bispecific antibody and application
TWI816616B (zh) 抗人程序性死亡配體-1 (pd-l1) 的抗體及其用途
WO2023040940A1 (zh) Pvrig/tigit结合蛋白联合免疫检查点抑制剂用于治疗癌症
WO2022152282A1 (zh) 抗人cd22的单克隆抗体及其用途
TW202227488A (zh) 抗人程序性死亡配體－１（ｐｄ－ｌ１）的抗體及其用途
JP2024509946A (ja) 抗ヒトｃｘｃｒ５抗体及びその使用
JP2024517985A (ja) 抗－ＣＤ３００ｃモノクローナル抗体及びその癌の予防または治療用バイオマーカー
CA3206413A1 (en) Antibodies against cd112r and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20900442

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20900442

Country of ref document: EP

Kind code of ref document: A1