WO2022193993A1 - 精液凝固蛋白的中和抗体及其表位和应用 - Google Patents

精液凝固蛋白的中和抗体及其表位和应用 Download PDF

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WO2022193993A1
WO2022193993A1 PCT/CN2022/079756 CN2022079756W WO2022193993A1 WO 2022193993 A1 WO2022193993 A1 WO 2022193993A1 CN 2022079756 W CN2022079756 W CN 2022079756W WO 2022193993 A1 WO2022193993 A1 WO 2022193993A1
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antibody
semg1
semg2
cells
binding
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PCT/CN2022/079756
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English (en)
French (fr)
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许杰
孙玉凡
钟佳孟
赵雪花
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上海柏全生物科技有限公司
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Priority to CN202280022072.9A priority Critical patent/CN117062832A/zh
Priority to CA3212368A priority patent/CA3212368A1/en
Priority to AU2022240549A priority patent/AU2022240549A1/en
Priority to EP22770348.5A priority patent/EP4310098A1/en
Publication of WO2022193993A1 publication Critical patent/WO2022193993A1/zh

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    • 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to the field of biomedicine, in particular to a method for suppressing tumor based on semen coagulation protein and use thereof.
  • SEMG1 and SEMG2 are both semen coagulation proteins (Semenogellins), which are the most abundant proteins in human semen.
  • SEMG1 and SEMG2 are 50kda and 63kda proteins, respectively, with 78% similarity at the gene level. They are both secreted into the seminal vesicles in the seminal vesicles, which are then rapidly degraded into small peptides by prostate-specific antigen (PSA, also known as kallikrein peptidase).
  • PSA prostate-specific antigen
  • SEMG proteins and their protein hydrolysates have many important functions. They regulate sperm motility and fertility and provide sperm with antimicrobial defenses.
  • SEMG1 and SEMG2 belong to the cancer-testis antigens (CTA), which are a group of proteins frequently expressed in various tumors but usually limited to germ cells. Recent studies have shown that SEMGs have an anti-proliferation effect on tumor cells. At the same time, our recent study found that SEMG2 can bind to CD27 molecule, and the antibody molecule against SEMG2 was active in killing tumor cells and mouse tumor models by PBMC in vitro.
  • CTA cancer-testis antigens
  • the CD27 molecule belongs to the tumor necrosis factor receptor (TNFR) superfamily and is a type I membrane protein with a molecular weight of about 55 kDa, and exists as a dimer of two monomers linked by disulfide bonds. CD27 is mainly expressed on lymphocytes, and activation of CD27 signaling pathway can increase the infiltration of suppressor T cells (Treg) in solid tumors and reduce anti-tumor immunity (Claus C, Riether C, Schürch C, Matter MS, Hilmenyuk T, Ochsenbein AF). . Cancer Res. 2012 Jul 15;72(14):3664-76).
  • Treg suppressor T cells
  • Treg cells in skin tissue cannot perform normal immune regulation after losing the expression of CD27 (Remedios KA, Zirak B, Sandoval PM, Lowe MM, Boda D, Henley E et al., Sci Immunol.2018Dec 21;3(30).pii:eaau2042). Furthermore, activation of CD27 increases the number of Tregs and reduces atherosclerosis in hyperlipidemic mice (Winkels H, Meiler S, Lievens D, Engel D, Spitz C,soluble C, et al., Eur Heart J. 2017; 38(48):3590-3599).
  • CD27 plays an important role in the functional activation of specific Treg cells, including tumor-infiltrating Treg cells, and thus avoiding the activation of CD27 expressed by tumor-infiltrating Treg cells is a potential cancer treatment strategy.
  • Binding to ligands activates CD27 downstream signaling, and the currently known CD27 ligand molecule is CD70.
  • CD70 the currently known CD27 ligand molecule is CD70.
  • blocking the binding of CD70 to CD27 is a tumor immunotherapy strategy being studied.
  • SEMG1 or SEMG2 is a ligand of CD27.
  • LILRB2 and LILRB4 belong to the family of leukocyte Ig-like receptors (LILRs), which play a very important role in the function of the immune system.
  • LILRB2 a class of immune system inhibitory receptors, is mainly expressed in myeloid cells and B cells, and is involved in the negative regulation of its function.
  • the endogenous ligands of LILRB2 are MHC molecules, which upon binding generate inhibitory signals as a mechanism for buffering immune responses and maintaining immune tolerance.
  • TAMs tumor-associated macrophages
  • LILRB4 is mainly expressed on APCs as an immune tolerance receptor.
  • LILRB4-expressing APCs play a key role in controlling inflammation.
  • LILRB4 has been implicated in a variety of diseases, such as Kawasaki disease and systemic lupus erythematosus (SLE), inflammatory diseases, and some neurological diseases such as multiple sclerosis.
  • LILRB4 is also an important marker of monocytic leukemia, supporting tumor cell infiltration into tissues and suppressing T cell activity in acute myeloid leukemia (AML) cells.
  • AML acute myeloid leukemia
  • TIGIT (also known as WUCAM, Vstm3, VSIG9) is a receptor of the Ig superfamily consisting of an extracellular immunoglobulin (Ig) variable domain, a type 1 transmembrane domain, and an intracellular domain. Intracellular domain It has two inhibitory motifs that are conserved in mice and humans: the immunoreceptor tyrosine-based inhibitory motif (ITIM) and the Ig tail tyrosine-like (ITT) motif.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITT Ig tail tyrosine-like motif.
  • TIGIT is expressed by activated CD8+ T and CD4+ T cells, natural killer (NK) cells, regulatory T cells (Tregs) and follicular helper T cells. TIGIT functions as a negative regulator of T cells and acts as a brake. TIGIT potentially suppresses innate and adaptive immunity through multiple mechanisms. TIGIT can enhance the immunosuppressive function and stability of Tregs
  • SEMG1 and SEMG2 proteins are expressed in tumor cells, and both can act as ligands to bind LILRB2, LILRB4, CD27 and TIGIT receptors, thereby inhibiting T cell killing of tumor cells and inducing the expansion of Treg and MDSC.
  • SEMG1 and SEMG2 the only two members of the semen coagulation protein family, have functions in promoting tumor immune escape.
  • the present invention innovatively develops a dual-targeting antibody that can bind to SEMG1 and SEMG2, which can bind the above-mentioned target protein with high affinity and neutralize its function, inhibiting its interaction with one or more ligands.
  • the antibody of the present invention can be used as an active ingredient to develop a drug for treating tumors.
  • the present invention also discloses technical solutions closely related to the antibody, such as the linear epitope corresponding to the antibody with strong neutralizing function, the screening method of the antibody, the biomarker for administering the antibody, and the like.
  • not more than includes every value less than the stated value.
  • "not more than 100 nucleotides” includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. Also include any smaller numbers or fractions in between.
  • the terms “plurality,” “at least two,” “two or more,” “at least a second,” etc. should be understood to include, but not be limited to, at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19 20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 , 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
  • any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range and, where appropriate, fractions thereof (eg, tenths of an integer) one and one percent).
  • an antibody includes, but is not limited to, glycoprotein immunoglobulins that specifically bind an antigen.
  • an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region contains three constant domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region contains one constant domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the Ab can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the light and heavy chain variable regions comprise "framework” regions interspersed with three hypervariable regions (also referred to as “complementarity determining regions” or “CDRs”).
  • CDRs complementarity determining regions
  • “Complementarity determining regions” or “CDR regions” or “CDRs” or “hypervariable regions” are the variable domains of antibodies that are hypervariable in sequence and Structurally defined loops ("hypervariable loops") and/or regions containing antigen-contacting residues ("antigen contact points”) are formed.
  • the CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
  • the CDRs located within the variable domains of antibody heavy chains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including Example: Chothia based on the three-dimensional structure of antibodies and topology of CDR loops (Chothia et al.
  • the CDR boundaries of the variable regions of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. For example, the residue ranges defined by different assignment systems for CDR regions using Kabat and Chothia numbering are shown in Table A below.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined by the present invention.
  • CDRs of the antibodies of the invention can be manually evaluated to determine boundaries according to any protocol in the art or a combination thereof.
  • the term "CDR” or "CDR sequence” encompasses CDR sequences determined in any of the ways described above.
  • Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins , synthetic antibody, tetrameric antibody comprising two heavy chain and two light chain molecules, antibody light chain monomer, antibody heavy chain monomer, antibody light chain dimer, antibody heavy chain dimer, antibody light chain - antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates”), heteroconjugated antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), Camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), micro Antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody
  • humanized antibody is intended to refer to an antibody obtained by grafting CDR sequences derived from the germline of another mammalian species, such as mouse, onto human framework sequences. Additional framework region modifications can be made in human framework sequences.
  • an "antigen-binding molecule,” “antigen-binding fragment,” or “antibody fragment” refers to any molecule comprising an antigen-binding fragment (eg, CDRs) of an antibody from which the molecule is derived.
  • Antigen binding molecules can include antigenic complementarity determining regions (CDRs).
  • Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules.
  • the antigen binding molecule binds to SEMG1 protein, and in some embodiments, the antigen binding molecule has neutralizing activity capable of inhibiting binding of SEMG1 or SEMG2 to receptors CD27, LILRB2, LILRB4, or TIGIT.
  • the term "antigen" refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen-binding molecule.
  • the immune response may involve antibody production or activation of specific immunocompetent cells or both.
  • any macromolecule including nearly any protein or peptide, can serve as an antigen.
  • Antigens can be expressed endogenously, ie, from genomic DNA, or can be expressed recombinantly. Antigens may be specific to certain tissues, such as cancer cells, or they may be expressed broadly. In addition, fragments of larger molecules can act as antigens.
  • the antigen is a SEMG1 or SEMG2 protein antigen.
  • the antigen binding molecule, scFv, antibody or fragment thereof directly blocks the binding site on the ligand or alters the binding capacity of the ligand by indirect means (eg, structural or energetic changes of the ligand) .
  • the antigen binding molecule, scFv, antibody or fragment thereof prevents the protein to which it binds from performing a biological function.
  • peptide As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds.
  • the protein or peptide contains at least two amino acids and there is no limit to the maximum number of amino acids that can comprise the sequence of the protein or peptide.
  • Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds.
  • the term refers to short chains (which are also commonly referred to in the art as eg peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, which are of many types) ) both.
  • Polypeptide includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like .
  • Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.
  • the term “specifically binds” or “specifically binds to” refers to a non-random binding reaction between two molecules, eg, between an antibody and an antigen.
  • the ability to "inhibit binding,” “block binding,” or “compete for the same epitope” refers to the ability of an antibody to inhibit binding of two molecules to any detectable degree.
  • an antibody that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%.
  • the inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.
  • Ka is intended to represent the on-rate of a particular antibody-antigen interaction
  • Kd is intended to represent the dissociation rate of a particular antibody-antigen interaction
  • KD or “KD value” is intended to refer to the dissociation constant for a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (ie, Kd/Ka) and expressed as molar concentration (M) .
  • M molar concentration
  • high affinity antibody refers to an antibody having 1 x 10-7 M or less, more preferably 5 x 10-8 M or less, even more preferably 1 x 10-8 M or less for the target antigen Low, even more preferably, an antibody with a KD value of 5 x 10-9 M or lower, and even more preferably 1 x 10-9 M or lower.
  • epitope refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. "Epitopes" are also referred to as "antigenic determinants”. Epitopes or antigenic determinants typically consist of chemically active surface groups of molecules such as amino acids, carbohydrates, or sugar side chains, and typically have a specific three-dimensional structure and specific charge characteristics. For example, epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique stereo conformation, which may be “linear” epitope” or “conformational epitope”. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E.
  • nucleic acid or “nucleic acid sequence” in the present invention refers to any molecule, preferably a polymeric molecule, comprising units of ribonucleic acid, deoxyribonucleic acid or analogs thereof.
  • the nucleic acid may be single-stranded or double-stranded.
  • the single-stranded nucleic acid may be the nucleic acid of one strand of denatured double-stranded DNA.
  • the single-stranded nucleic acid may be a single-stranded nucleic acid not derived from any double-stranded DNA.
  • complementary refers to hydrogen bonding base pairing between the nucleotide bases G, A, T, C and U such that when two given polynucleotides or sequences of polynucleotides anneal to each other In DNA, A pairs with T, G pairs with C, and in RNA, G pairs with C, and A pairs with U.
  • cancer refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer” or “cancer tissue” can include tumors.
  • cancer associated with SEMG1 or SMEG2 refers to any cancer caused, exacerbated, or otherwise associated with increased or decreased expression or activity of SEMG1 or SEMG2, and in some embodiments, disclosed herein
  • the method can be used for a cancer selected from the group consisting of colorectal cancer, lung cancer, breast cancer, melanoma, lymphoma, liver cancer, head and neck cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer, breast cancer, and ovary cancer.
  • an “effective dose,” “effective amount,” or “therapeutically effective dose” is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression, the Evidence of disease regression is a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of impairment or disability due to the affliction of the disease.
  • the ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems for predicting efficacy in humans, or by Activity assessment of the assay reagents in an in vitro assay.
  • an "individual” or “subject” is a mammal. Mammals include primates (eg, humans and non-human primates such as monkeys) and rodents (eg, mice and rats). In certain embodiments, the individual or subject is a human.
  • a "subject” may be a “patient” - a patient is a human subject in need of treatment, may be an individual with a SEMG1 or SEMG2-related cancer, such as breast cancer, who is at risk for developing a SEMG1 or SEMG2-related cancer, such as breast cancer subject.
  • in vitro cell refers to any cell cultured in vitro.
  • in vitro cells can include T cells.
  • the term "pharmaceutically acceptable” means that the carrier, diluent, excipient and/or salt thereof is chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient Allow.
  • 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 active agent, which is are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers .
  • pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
  • the term “modulate” generally includes the meaning of up-regulation or down-regulation in two different directions, and in some cases can be understood as inhibition or enhancement, in some cases can be understood as decrease or increase, in some cases can be understood as The following can be understood as reduction or increase, etc., the specific explanation is not limited, and it is understood and explained according to the actual application context.
  • "regulating" tumor cell growth can be understood as inhibiting or enhancing tumor cell growth.
  • the terms “reduce” and “reduce” are used interchangeably and mean any change from the original. “Reduce” and “reduce” are relative terms and require comparison between before and after measurement. “Reduce” and “reduce” include complete consumption; by the same token the terms “increase” and “increase” have opposite interpretations.
  • Treatment or “treating” of a subject refers to any type of intervention or procedure performed on the subject, or administration of an active agent to the subject, to achieve reversal, alleviation, amelioration, suppression, alleviation, or prevention of symptoms , complication or condition or the onset, progression, progression, severity or recurrence of disease-related biochemical markers.
  • “treating” or “treating” includes partial remission. In another embodiment, “treating” or “treating” includes complete remission.
  • the proteins SEMG1 or SEMG2 are both SEMG proteins.
  • SEMG1 or SEMG2 protein has not been deeply understood, and there is no report of tumor inhibition based on SEMG1 or SEMG2.
  • the inventors have confirmed through gene silencing knockout and immunology experiments that SEMG1 can be blocked by inhibiting the function or activity of SEMG1.
  • SEMG2 with receptors CD27, LILRB2, LILRB4 or TIGIT can promote the killing effect of immune cells on tumor cells, which can be used in the field of cancer treatment.
  • the cancer is a SEMG1 or SEMG2-related cancer.
  • cancer associated with SEMG1 or SEMG2 refers to any cancer caused, exacerbated or otherwise associated with increased or decreased expression or activity of SEMG1 or SEMG2.
  • the methods disclosed herein can be used for a cancer selected from the group consisting of colorectal cancer, lung cancer, breast cancer, melanoma, lymphoma, liver cancer, head and neck cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer Endometrial, breast, and ovarian cancers.
  • a method for regulating tumor cells in vitro or in vivo is disclosed.
  • the method modulates the killing effect of PBMC on tumor cells by changing the binding effect between SEMG1 or SEMG2 and any one of the receptors CD27, LILRB2, LILRB4 or TIGIT, changing the cell ratio of immunosuppressive cells MDSC or Treg.
  • regulation may be to regulate the cell activity of tumor cells, or the growth of tumor cells, or the migration of tumor cells, etc., which is not limited here. It can be reasonably expected in the art that such regulation may be multi-faceted. . While “regulation” includes both enhancement and inhibition, in more embodiments, this regulation is the inhibition aspect, that is, the inhibition of tumor cell activity, growth or migration, etc., to achieve the purpose of disease foci removal or treatment .
  • the method may include the steps of:
  • the above-mentioned purpose is achieved by inhibiting the expression, function or activity of SEMG1 or SEMG2.
  • it may also include detecting the expression of SEMG1 or SEMG2 in tumor cells, so as to better implement the above method.
  • step b
  • step b) contacting tumor cells with immune cells; it will be appreciated that step b) can be done before or after step a) without affecting the performance of the method.
  • the above-mentioned inhibiting method can be any of the following:
  • the gene editing system of the present invention is a CRISPR/Cas9 gene editing system
  • the oligomeric DNA sequences used in the CRISPR/Cas9 gene editing system to encode sgRNAs are selected from SEQ ID NOs: 1-8.
  • RNAi Interfere with SEMG1 or SEMG2 gene expression, such as RNAi, to reduce the expression or activity of the gene
  • Inhibition by immune means such as inhibiting the function or activity of SEMG1 or SEMG2 protein by an antibody.
  • the present invention can also provide a method for regulating other aspects, for example, a method for regulating the activity of CD27, LILRB2, LILRB4 or TIGIT in cells in vitro and in vivo; for example, regulating immunosuppressive cells MDSC or Treg cells in vitro and in vivo The ratio method; another example, the method of regulating the killing effect of PBMC on tumor cells in vitro and in vivo.
  • a method for regulating the activity of CD27, LILRB2, LILRB4 or TIGIT in cells in vitro and in vivo for example, regulating immunosuppressive cells MDSC or Treg cells in vitro and in vivo
  • the ratio method another example, the method of regulating the killing effect of PBMC on tumor cells in vitro and in vivo.
  • the above-mentioned modulating method is a method of inhibiting the activity of CD27, LILRB2, LILRB4 or TIGIT; a method of reducing the ratio of immunosuppressive cells MDSC or Treg cells; a method of enhancing the killing effect of PBMC on tumor cells.
  • These methods include the step of inhibiting the function or activity of SEMG1 or SEMG2, respectively, in tumor cells.
  • SEMG1 or SEMG2 protein can bind to receptors such as CD27, LILRB2, LILRB4 or TIGIT, and the expression of SEMG1 or SEMG2 protein in tumor cells has an effect on myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg ) and immune cells.
  • MDSC myeloid-derived suppressor cells
  • Treg regulatory T cells
  • the present invention provides a SEMG1 or SEMG2 inhibitor
  • the "inhibitor” in the present invention can be a protein-level inhibitor or a gene-level inhibitor, and any inhibitor that can inhibit SEMG1 or SEMG2 gene or protein, including but not limited to gene
  • the production, transcription, activity, function, etc. of the protein, and the expression, activity, function, etc. of the protein belong to the category of "inhibitor”.
  • a protein-level inhibitor in the case of a protein-level inhibitor, it is a substance capable of binding a SEMG1 or SEMG2 protein while inhibiting or blocking the interaction between SEMG1 or SEMG2 and any of CD27, LILRB2, LILRB4, or TIGIT.
  • the SEMG1 or SEMG2 inhibitor at the protein level specifically binds to SEMG1 or SEMG2 protein.
  • the SEMG1 or SEMG2 inhibitor at the protein level binds specifically and with high affinity to SEMG1 or SEMG2 protein.
  • the protein-level inhibitor is a compound that agonizes or antagonizes the interaction of SEMG1 or SEMG2 with CD27, LILRB2, LILRB4, or TIGIT.
  • the protein-level inhibitors include, but are not limited to, small molecule inhibitors, polypeptides, antibodies, or antigen-binding fragments.
  • a gene-level inhibitor it is a substance capable of inhibiting or blocking the interaction between SEMG1 or SEMG2 and any of CD27, LILRB2, LILRB4 or TIGIT by altering the activity or function of the SEMG1 gene.
  • the SEMG1 or SEMG2 inhibitor at the gene level includes, but is not limited to, small molecular substances, DNA-based substances or RNA-based substances, etc., which are specific for SEMG1 gene, such as primers, probes, sgRNA and other substances.
  • the present invention also provides a method for screening or identifying the above-mentioned inhibitor, and the method may include the following steps:
  • SEMG1 inhibitory molecules First obtain SEMG1 inhibitory molecules, and then analyze the effect of candidate molecules on the binding of SEMG1 to receptors or LILRB2; preferably, analyze whether the candidate molecules have antagonistic activity.
  • the method may include the steps of:
  • SEMG1 or SEMG2 inhibitory molecules First obtain SEMG1 or SEMG2 inhibitory molecules, and then analyze the effect of the candidate molecules on myeloid-derived suppressor cells (MDSCs); preferably, analyze whether the candidate molecules can reduce the content or ratio of suppressor cells (MDSCs).
  • MDSCs myeloid-derived suppressor cells
  • the method may include the steps of: first obtaining SEMG1 inhibitory molecules, and then analyzing the effect of the candidate molecules on regulatory T cells (Treg); preferably, analyzing whether the candidate molecules can reduce regulatory T cells (Treg) ) content or ratio.
  • the method may include the steps of: first obtaining SEMG1 inhibitory molecules, and then analyzing the effect of the candidate molecules on the killing of tumor cells by immune cells; preferably, analyzing whether the candidate molecules can promote the killing of tumor cells by immune cells or inhibition.
  • the method may include a combination or all of the above steps.
  • antibody against SEMG1 or SEMG2 refers to an antibody of the present invention that is capable of binding SEMG1 or SEMG2 protein with sufficient affinity such that the antibody can be used as a diagnostic, prophylactic and/or therapeutic agent targeting SEMG1 or SEMG2 protein.
  • any antibody or antigen-binding fragment thereof with the following properties theoretically belongs to the inventive concept or scope of rights of the present invention, specifically: the isolated antibody or antigen-binding fragment thereof has:
  • the antibody additionally comprises at least one of the following properties:
  • the antibody reduces the ratio of immunosuppressive cells to MDSC cells
  • the antibody reduces the cellularity of immunosuppressive Treg cells
  • the antibody enhances the killing effect of immune cells (eg PBMC) on tumor cells.
  • immune cells eg PBMC
  • the antibody may comprise one or more of the other aforementioned characteristics.
  • the isolated antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, and the specific sequences can be as follows:
  • amino acid change is an addition, deletion or substitution of an amino acid, and preferably, the amino acid change is a conservative amino acid substitution.
  • the antibody comprises a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 sequences defined according to the IMGT; and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences defined according to the IMGT, the sequences are as follows:
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.15;
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.16;
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.17;
  • amino acid change is an addition, deletion or substitution of an amino acid, and preferably, the amino acid change is a conservative amino acid substitution.
  • the binding KD of the antibody or its antigen-binding fragment to SEMG1 or SEMG2 is ⁇ 2 ⁇ 10 ⁇ 8 ;
  • the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the sequences being selected from the following:
  • the amino acid sequence of the heavy chain variable region comprises or has at least 70%, 80%, 90%, 95% or 99% of the amino acid sequence selected from the group consisting of SEQ ID NOs: 13 sequence identity;
  • the amino acid sequence of the light chain variable region comprises or has at least 70%, 80%, 90%, 95% or 99% of the amino acid sequence selected from the group consisting of SEQ ID NOs: 14 sequence identity.
  • Conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid replace. Exemplary substitutions are shown in the following table:
  • substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp;Gln Asp
  • substitutions Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu, Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
  • the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes described in the present invention occur in the Fc region.
  • anti-SEMG1 protein antibodies are provided comprising an Fc domain containing one or more mutations that enhance or reduce binding of the antibody to the FcRn receptor, eg, at acidic pH compared to neutral pH.
  • Fc modifications include, for example: positions 250 (eg E or Q), 250 and 428 (eg L or F), 252 (eg L/Y/F/W or T), 254 Modification of position (e.g. S or T) and position 256 (e.g.
  • position 428 and/or 433 e.g. H/L/R/S/P/Q or K
  • position 428 and/or 433 e.g. H/L/R/S/P/Q or K
  • 434 e.g. A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]
  • 250 and/or 428 e.g. A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]
  • 250 and/or 428 e.g. A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]
  • 250 and/or 428 e.g. A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]
  • 307 or 308 For
  • the modifications include 428L (eg, M428L) and 434S (eg, N434S) modifications; 428L, 259I (eg, V259I) and 308F (eg, V308F) modifications; 433K (eg, H433K) and 434 (eg, 434Y) modifications; 252, 254 and 256 (eg 252Y, 254T and 256E) modifications; 250Q and 428L modifications (eg T250Q and M428L); and 307 and/or 308 modifications (eg 308F or 308P).
  • the modification includes 265A (eg, D265A) and/or 297A (eg, N297A) modifications.
  • the invention includes anti-SEMG1 protein antibodies comprising an Fc domain comprising one (set) or more (set) of mutations selected from the group consisting of 252Y, 254T and 256E (eg, M252Y, S254T and T256E) 428L and 434S (such as M428L and N434S); 257I and 311I (such as P257I and Q311I); 257I and 434H (such as P257I and N434H); 376V and 434H (such as D376V and N434H); E380A and N434A); and 433K and 434F (eg H433K and N434F).
  • 252Y, 254T and 256E eg, M252Y, S254T and T256E
  • 428L and 434S such as M428L and N434S
  • 257I and 311I such as P257I and Q311I
  • the present invention includes an anti-SEMG1 protein antibody comprising an Fc domain comprising the S108P mutation in the hinge region of IgG4 to facilitate dimer stabilization. Any possible combination of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are included within the scope of the present invention.
  • the SEMG1 or SEMG2 protein antibodies provided herein can be altered to increase or decrease the degree of glycosylation thereof. Addition or deletion of glycosylation sites to SEMG1 or SEMG2 protein antibodies can be conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites.
  • the SEMG1 protein antibody comprises an Fc region
  • the carbohydrate linked to the Fc region can be altered.
  • modifications to remove unwanted glycosylation sites may be useful, such as removal of fucose moieties to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733 ).
  • galactosylation modifications can be made to modulate complement-dependent cytotoxicity (CDC).
  • one or more amino acid modifications can be introduced into the Fc region of the coronavirus S protein antibodies provided herein, thereby generating Fc region variants, in order to enhance, for example, the prevention of the coronavirus S protein antibodies of the invention and/or effectiveness in the treatment of coronavirus infection.
  • the antibody is an IgGl, IgG2, IgG3 or IgG4 antibody; preferably, it is an IgGl or IgG4 antibody; more preferably, it is a human or murine IgGl or IgG4 antibody.
  • the antibody may further comprise a conjugation moiety attached to the polypeptide selected from the group consisting of radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins, lipids, and biotin One or more of the above, wherein the polypeptide or antibody and the coupling moiety are selectively linked by a linker, preferably the linker is a peptide or a polypeptide.
  • the antibody can be prepared or selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies and human antibodies without altering the core functional regions of the antibody; more preferably The antibody can be prepared as or selected from multispecific antibodies, single chain Fv (scFv), single chain antibodies, anti-idiotype (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies , Fab fragments, F(ab') fragments, disulfide-linked bispecific Fv (sdFv) and intrabodies.
  • scFv single chain Fv
  • anti-Id anti-idiotype antibodies
  • the present invention also provides an isolated polynucleotide encoding the aforementioned antibody or antigen-binding fragment.
  • the present invention also provides a recombinant vector comprising the above-mentioned polynucleotide, and optional regulatory sequences;
  • the recombinant vector is a cloning vector or an expression vector; more preferably, the control sequence is selected from a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or its any combination.
  • the present invention also provides a host cell, which is characterized by comprising the above-mentioned recombinant vector; preferably, the host cell is a prokaryotic cell or a eukaryotic cell.
  • the present invention also provides a pharmaceutical composition, which is characterized by comprising one or more of the above-mentioned antibodies or antigen-binding fragments, polynucleotides, recombinant vectors, host cells and pharmaceutical compositions; preferably, the The composition also includes a pharmaceutically acceptable carrier or adjuvant.
  • the present invention also provides a kit, which is characterized in that it comprises one or more of the above-mentioned antibodies or antigen-binding fragments, polynucleotides, recombinant vectors, host cells and pharmaceutical compositions, and is contained in a suitable container middle.
  • Anti-SEMG1 monoclonal antibodies (mAbs) and human sequence antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodologies, such as the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256:495. Any technique for producing monoclonal antibodies can be employed, such as viral or oncogenic transformation of B lymphocytes.
  • One animal system for making hybridomas is the murine system. The production of hybridomas in mice is a well established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art.
  • the present invention is obtained by immunizing mice multiple times with recombinant SEMG1 protein.
  • the SEMG1 protein antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art.
  • the SEMG1 protein antibody of the present invention is tested for its antigen-binding activity, for example, by known methods such as ELISA, Western blotting, and the like. Binding to SEMG1 or SEMG2 protein can be determined using methods known in the art, exemplary methods are disclosed herein.
  • the binding of SEMG1 protein antibody of the invention to SEMG1 or SEMG2 protein is determined using biofilm interferometry, and the binding of SEMG1 or SEMG2 to CD27, LILRB2, LILRB4 or TIGIT is further analyzed by ELISA experiments.
  • the present invention provides compositions comprising any of the compounds described herein, SEMG1 or SEMG2 protein inhibitors, antagonists, antibodies, antigenic peptides, proteins, etc., preferably the compositions are pharmaceutical compositions.
  • the composition comprises an antibody or antigen-binding fragment thereof, or immunoconjugate, or bispecific molecule of the invention, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises a SEMG1 or SEMG2 protein antibody of the invention in combination with one or more other therapeutic agents.
  • the pharmaceutical compositions or formulations of the present invention comprise suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerin , propylene, glycol, water, ethanol, etc. See also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago, for the use of excipients and their uses.
  • the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.
  • compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • Compounds containing the proteins described herein can be prepared by mixing a SEMG1 protein antibody of the invention of the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980)).
  • the pharmaceutical preparation of the SEMG1 or SEMG2 protein antibody is preferably in the form of a lyophilized preparation or an aqueous solution.
  • compositions or formulations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other.
  • active ingredients including but not limited to CTLA4 and PD-1 inhibitors, among others.
  • the active ingredients are suitably combined in amounts effective for the intended use.
  • the present invention provides methods for preventing SEMG1 or SEMG2-related cancer in a subject comprising administering to the subject an inhibitor, antibody or pharmaceutical composition of the present invention.
  • Administration of prophylactic agents can be administered before symptomatic features are manifested in order to prevent the onset of the disease, or alternatively to delay the progression of the disease.
  • the subject has received or is receiving or will receive additional anti-cancer therapy
  • the additional anticancer therapy includes surgery, radiotherapy, chemotherapy, immunotherapy or hormonal therapy.
  • the present invention provides methods for treating a SEMG1 or SEMG2-related cancer in a subject comprising administering to the subject an inhibitor, antibody or pharmaceutical composition of the present invention. Administration of the therapeutic agent can be administered after symptomatic features have been manifested.
  • the subject has received or is receiving or will receive additional anti-cancer therapy
  • the additional anticancer therapy includes surgery, radiotherapy, chemotherapy, immunotherapy or hormonal therapy.
  • SEMG1 or SEMG2 related cancers include but are not limited to various cancers, such as common colorectal cancer, lung cancer, melanoma, lymphoma, liver cancer, head and neck cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer cancer, breast cancer, and ovarian cancer.
  • the treatment method provided by the present invention can also be that the subject's immune cells and tumor cells are contacted with an effective dose of the antibodies or antigen-binding fragments, polynucleotides, recombinant vectors, host cells and pharmaceutical compositions of the present invention;
  • the subject's immune cells and/or tumor cells are contacted with an effective amount of the compound, detecting the expression of SEMG1 or SEMG2 in tumor cells;
  • the immune cells are lymphocytes
  • the lymphocytes are T lymphocytes.
  • inhibitors can at least include the following preventive and therapeutic uses or the preparation of corresponding drugs:
  • SEMG1 or SEMG2 is expressed in tumor cells, and CD27, LILRB2, LILRB4 or TIGIT is expressed in immune cells;
  • SEMG1 or SEMG2 is expressed in tumor cells, and CD27, LILRB2, LILRB4 or TIGIT is expressed in immune cells;
  • companion diagnostics generally refers to providing information about a patient's therapeutic response to a specific therapeutic drug, helping to identify patient populations who could benefit from a therapeutic product, thereby improving treatment outcomes and reducing health care costs.
  • companion diagnostics can help identify patient populations most likely to respond to therapeutic drugs.
  • the present invention confirms that SEMG1 or SEMG2 protein can be used as a therapeutic target for SEMG1 or SEMG2-related cancer, that is, when SEMG1 or SEMG2 protein is expressed in tumor cells, the tumor can be effectively inhibited by introducing a SEMG1 or SEMG2 protein inhibitor (such as an antibody). Therefore, during the treatment of the corresponding tested tumor patients (including before and after treatment), by detecting the expression of SEMG1 or SEMG2 protein in tumor cells, the patient group most likely to respond to the therapeutic drug can be determined. Therefore, the present invention provides a method for companion diagnosis, which is accomplished by detecting the protein expression or function of SEMG1 or SEMG2 in tumor cells of the subject before/after immunotherapy.
  • a SEMG1 or SEMG2 protein inhibitor such as an antibody
  • Kits comprising an antibody or antibody composition of the invention (eg, a human antibody, bispecific or multispecific molecule, or immunoconjugate) and instructions for use are also within the scope of the invention.
  • the kit may further comprise at least one additional agent, or one or more additional antibodies, and the like.
  • Kits typically include a label that indicates the intended use of the contents of the kit.
  • the term label includes any written or recorded material on or provided with the kit or otherwise accompanying the kit.
  • the kit of the present invention may also include components capable of detecting the expression or activity of SEMG1 or SEMG2 protein in tumor cells. Therefore, in addition to the above-mentioned antibody composition, the components of the kit of the present invention may also include primers, probes, etc., or other materials capable of detecting protein expression or activity, which are not limited herein.
  • FIG. 1 Concentration effects of SEMG1 binding to corresponding receptor proteins.
  • the ability of different concentrations of SEMG1 to bind CD27 (2 ⁇ g/mL), LILRB2 (2 ⁇ g/mL), TIGIT (3 ⁇ g/mL) and LILRB4 (2 ⁇ g/mL) increased with the increase of the concentration of SEMG1, indicating that SEMG1 is closely related to the receptor.
  • the proteins CD27, LILRB2, TIGIT or LILRB4 have the ability to specifically bind.
  • the OD 450 readings after color development of the negative controls that have been blank-coated, blocked, and added with the corresponding ligand protein have been subtracted for each positive group in the example graph.
  • Figure 3 Expression identification of SEMG1 and SMEG2 in tumor tissue. Protein suspension prepared by lysing gastric wall and gastric tumor tissue extracts, gastric wall and gastric tumor tissue extracts, colorectal and colorectal tissue extracts of tumor patients for different pathological tissues
  • FIG. 4 Inhibitory effect of overexpression of SEMG1 or SEMG2 on tumor cell killing by PBMC. After co-incubating PBMC cells with tumor cells or tumor cells overexpressing SEMG1 or SEMG2, respectively, the percentage of apoptotic cells was determined. The vertical axis is the percentage of apoptotic tumor cells; the horizontal axis is the different experimental treatment conditions, that is, the different cells added. The results showed that overexpression of SEMG1 or SEMG2 could significantly inhibit the killing effect of activated PBMC on tumor cells.
  • FIG. 1 Concentration effect of murine hybridoma antibody binding to SEMG1 and SEMG2.
  • the ordinate is the OD450 absorption value as the reading of the ELISA experiment, showing the binding degree of 1 ⁇ g/mL SEMG1 (A) or SEMG2 (B) immobilized on the microplate with the added mouse hybridoma monoclonal antibody; the abscissa shows Incubate with different concentrations of antibody.
  • Different curves represent antibodies produced by different clones, with unrelated murine monoclonal antibody mIgG1 as a negative control.
  • the EC50 values of the corresponding antibodies are also indicated in the figure.
  • the fitted curve is based on a representative result of three independent biological experiments (error bars represent standard deviation).
  • FIG. 7 The positive rate of binding of murine hybridoma monoclonal antibodies to HCT116 cells overexpressing SEMG1 was detected by flow cytometry. The results showed that cells overexpressing SEMG1 bound to different clones of hybridoma monoclonal antibodies (5 ⁇ g/mL) with different positive rates, indicating the diversity of antibodies and the ability of antibodies to bind to antigens expressed on the surface of tumor cells.
  • FIG. 8 Blocking effect of murine hybridoma antibody SEMG1 or SEMG2 binding to the corresponding protein.
  • the picture shows that two monoclonal antibodies 33-B5-C1-F5 and 25-E10-F8-B5 block the corresponding proteins of 2ug/mL SEMG1 protein (A) or 22ug/mL SEMG2 protein (B) at two concentrations:
  • the blocking percentage was calculated by comparing the ELISA OD450 values of the coated microwells with ligand protein and PBS.
  • the blocking percentage represents the degree of blocking, and the higher the blocking percentage, the better the effect of blocking the binding of the target protein to the ligand.
  • Figure 9 Identification heat map of antibody binding to SEMG1 and SEMG2 polypeptides.
  • A After coating a 96-well microplate with polypeptide (table) and SEMG1 protein (positive control), 2.5ug/ml of monoclonal antibodies produced by corresponding 29 hybridoma cell lines were added respectively. After conventional ELISA, The OD value was detected with rabbit anti-mouse HRP secondary antibody. If the corresponding value of the antibody-bound polypeptide is more than 3 times higher than the background value, it is considered to be the polypeptide specifically bound by the antibody. All full-length SEMG1-coated wells achieved the highest machine-readable value.
  • peptide sequence peptides are marked as ordinate, antibody response is marked as ordinate, and the intersection of each ordinate and abscissa is the response value of one polypeptide corresponding to one antibody.
  • the isolated antibodies mainly bind to BSA-S1, S1-13, S1-14, S1-19, S1-26, and S1-40.
  • 10 monoclonal antibodies bind to the S1-26 polypeptide, indicating that the sequence of this segment of the polypeptide is the main antigenic determinant for the production of SEMG1 monoclonal antibody, and is the key segment for SEMG1 to stimulate the body to produce antibodies.
  • SEMG1-26 (S1-26) epitope peptide, SEMG2 corresponding epitope peptide (KDIFITQDELLVYNK), SEMG1-S14 (S1-14), SEMG1-S40 (S1-40) peptides were coated on ELISA plate, and then The different antibody clones shown in the figure were bound and detected.
  • the results showed that the antibody capable of binding SEMG1-26 (S1-26) also had significant binding to the epitope peptide corresponding to SEMG2 (ie, the amino acid sequence and the highly similar region sequence KDIFITQDELLVYNK of SEMG1). This indicates that the dominant epitopes of SEMG1 and SEMG2 bind the same antibody clone.
  • FIG. 10 Antibodies against SEMG1 and SEMG2 promote PBMC killing of tumor cell lines.
  • Activated PBMC human peripheral blood mononuclear cells were co-cultured with HCT116 and HCT116 cells overexpressing SEMG1 and SEMG2, respectively, and different antibodies were added: unrelated mouse IgG, MM02 mouse antibody against SMEG2, and SEMG1 and SEMG2 binding.
  • Murine monoclonal antibodies 33-B5-C1-F5 and 25-E10-F8-B5.
  • the vertical axis is the percentage of apoptotic tumor cells; the horizontal axis is the different experimental treatment conditions, that is, the different cells and antibodies added.
  • FIG. 11 Antibodies inhibit tumorigenesis in an in vivo mouse model. After NPSG mice were inoculated with PBMC, and on the 3rd day after inoculation with human breast cancer cells MCF7 that naturally expressed SEMG1 and SEMG2, the mice were divided into groups and treated with SEMG1 monoclonal antibody 33-B5-C1-F5 and SEMG2 monoclonal antibody MM02 respectively. As well as PBS as a tumor suppressor, the tumor-forming mice were treated.
  • the figure shows that the inhibitory effect of monoclonal antibody 33-B5-C1-F5 on tumors is significantly stronger than that of PBS control group, and the therapeutic effect is better than that of SEMG2 monoclonal antibody MM02, showing the therapeutic effect of SEMG1 monoclonal antibody on tumors expressing SEMG1 and SEMG2 .
  • Example 1 Detection of binding of SEMG1 or SEMG2 to receptor protein LILRB2, TIGIT or LILRB4 protein
  • the binding and binding strength of SEMG1 or SEMG2 to receptor proteins CD27, LILRB2, TIGIT or LILRB4 were analyzed by ELISA. Specifically, an ELISA-specific plate (costar, ME, USA) was used. First, the plates were replicated with ELISA coating solution (Solarbio, Beijing, China) containing SEMG1 or SEMG2 recombinant protein at final concentrations of 10ug/mL, 3.3ug/mL, 1.1ug/mL, respectively. Two groups were set up. The negative control was 100 ⁇ l of protein-free coating solution. Coat overnight at 4°C.
  • the cells were blocked with 100 microliters of 5% skim milk (Sangong, Shanghai, China) dissolved in PBS, and blocked in an incubator at 37° C. for 90 minutes.
  • the receptor protein CD27, LILRB2, TIGIT or LILRB4, and SIRP as a negative control, SEMG2 and the action group as a positive control
  • extracellular protein fragments BPS or sense (Qiao Shenzhou, Beijing, China) for incubation and binding in PBS solution
  • the above-mentioned receptor protein was tagged with hFc tag (P-Fc), and the binding was carried out at 37°C for 60 minutes in an incubator.
  • the protein suspensions (Western) prepared by lysing gastric wall and gastric tumor tissue extracts, gastric wall and gastric tumor tissue extracts, and colorectal and colorectal tissue extracts from tumor patients were used to analyze the distribution of SEMG1 and SEMG2 in tumor tissues.
  • Grid Bio, Shanghai, China to detect the protein expression of SEMG1 and SEMG1, and the lysed protein suspension of the corresponding tissue extracts from normal people was set as a control.
  • a 10% PAGE gel (Yase, Shanghai, China) was prepared in a gel plate (Bole, CA, USA), and the prepared gel was placed in an electrophoresis tank (Bole, CA, USA), followed by Good power (Bole, CA, USA) runs the stacking gel under the condition of constant voltage of 80 volts, and runs the separating gel under the condition of constant voltage of 120 volts.
  • the band ran to the bottom of the separating gel, transfer the membrane by wet method and transfer the membrane with a constant flow of 350 mA in a transfer tank (Bole, CA, USA) for 90 minutes. After the membrane transfer was completed, the membrane was sheared according to the mass of SEMG1 and GAPDH proteins.
  • Example 3 Overexpression of SEMG1 in tumor cells can significantly reduce the killing of tumor cells by human peripheral blood mononuclear cells
  • Count the recovered PBMC and HCT116 or overexpressing cells adjust the cell number to 1 ⁇ 10 6 /ml, add 100 microliters of each to a 96-well plate (Thermo Fisher, MA, USA), and incubate in an incubator for 6 hours .
  • the 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), and the supernatant was discarded after centrifugation at 400 rcf for 5 minutes.
  • the washed cells were resuspended in 500 microliters of cell staining buffer (Invitrogen, CA, USA), and centrifugation and washing were repeated.
  • the CD45-APC antibody (Invitrogen, CA, USA) was diluted 1:20 with cell staining buffer, 200 ⁇ l of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Meilun, Shanghai, China); centrifugation and washing were repeated.
  • Example 4 SEMG1 induces transformation of PBMC myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg)
  • cells were first prepared with 1.5*10 5 PBMCs per well, grouped in different experiments, and cultured for 24h (for detecting Treg ratio) or 3 days (for detecting MDSC ratio).
  • the phenotype CD4+CD25+Foxp3+, HLA-DR-CD11b+CD33+ ratio and count were analyzed by flow cytometry.
  • HCT116 cells expressing SEMG1 co-cultured with human peripheral blood mononuclear cells increased the ratio of MDSC cells (HLA-DR-CD11b+CD33+) and Treg (CD4+CD25+Foxp3+) relative to ordinary HCT116 cells , the results are shown in Figure 5.
  • the SEMG1 protein was expressed and purified by the E. coli system, and the purity was 92% after testing.
  • the ELISA experiment verified that the SEMG1 protein had the activity of binding the receptor and LILRB2.
  • 10 mice were immunized with protein, and multiple immunizations were performed to enhance the effect: (1) primary immunization, antigen 50 ⁇ g/mice, subcutaneously injected with Freund's complete adjuvant at multiple points, with an interval of 3 weeks; (2) second immunization, dose The route is the same as above, plus Freund's incomplete adjuvant, with an interval of 3 weeks; (3) The third immunization, the dosage route is the same as above, and cross-immunized with SEMG2 protein at the same time, without adjuvant, intraperitoneal injection, with an interval of 3 weeks; (4) Boost Immunization, dose 50 ⁇ g, intraperitoneal injection.
  • SEMG1 and SEMG2 were cloned into pCDNA3.1 vector and transfected with DH5 ⁇ to prepare sterile plasmids for transfection.
  • 5-10 ⁇ 106 cells were seeded in a 6-well cell plate, cultured overnight in medium containing 10% calf serum, and used for transfection when about 60% to 80% confluent. Discard the supernatant in the culture plate, wash the cells three times with anti-serum-free medium, add 800 microliters of medium to each well, add rewarmed FuGENE HD transfection reagent, and add to 100 microliters of reaction system.
  • Example 7 ELISA and BLI biofilm interferometry to detect the binding strength of mouse hybridoma monoclonal antibody to SEMG1 and SEMG2
  • Example 8 Detection of the positive rate of binding of SEMG1 mouse hybridoma monoclonal antibody to HCT116-SEMG1 overexpressing cells by flow cytometry
  • the cells were resuspended with eBioscience Flow Cytometry Staining Buffer (Thermo fisher) and evenly distributed into 1.5ml EP tubes, and the prepared mouse monoclonal antibody was added to the final concentration of 5ug/mL and incubated for 1 hour. Then, centrifuge at 400 rpm for 5 min at 4°C to discard the supernatant, add PBS to resuspend the cells, and repeat once.
  • the rabbit anti-mouse FITC secondary antibody (Abcam) was diluted 1:400 with eBioscience Flow Cytometry Staining Buffer (Thermo fisher), and then evenly distributed to each reaction tube, and then placed in a shaker at room temperature for 45 minutes.
  • Example 10 ELISA detection of mouse hybridoma monoclonal antibody blocking SEMG1 or SEMG2 binding to receptors CD27, LILRB2, LILRB4 or TIGIT
  • the effect of the antibodies on the binding of SEMG1 or SEMG2 to its receptors CD27, LILRB2, LILRB4 or TIGIT was analyzed by ELISA.
  • the SEMG1 or SEMG2 recombinant protein was coated on a 96-well microtiter plate, and different concentrations of antibodies (0, 1.1, 3.3, 10.0 ⁇ g per ml) were added, and the corresponding recombinant receptors (human antibody Fc fragment) were added.
  • the antibody showed inhibitory effect on the binding of SEMG1 or SMEG2 to its receptor CD27, LILRB2, LILRB4 or TIGIT, and the degree of inhibition was related to the concentration of the antibody, and the higher the concentration, the more significant the degree of inhibition.
  • Trizol TM reagent Invitrogen
  • RT-PCR reverse transcription polymerase chain reaction
  • VH heavy chain variable region
  • VL light chain variable region
  • the obtained DNA encoding the variable region of the heavy chain of the selected lead antibody was cloned into pcDNA3.4-mIgG1 in frame with the constant region of mouse IgG1, and the light chain variable region encoding the light chain was cloned into pcDNA3.4-mIgG1.
  • the DNA of the variable region was cloned into the in-frame light chain expression plasmid pcDNA3.4-m ⁇ c with the murine kappa constant region. After the plasmid was extracted and transfected into 293E cells to express and verify the activity, the amino acid sequences of VH and VL of the determined monoclonal antibody are shown in Table 4.
  • Example 12 In vitro inhibitory effect of SEMG1 monoclonal antibody on tumor cells
  • HCT116-SEMG1 or HCT116-SEMG2 tumor cells were selected, first incubated with the antibody or control mouse IgG, and then incubated with the antibody or control mouse IgG.
  • PBMCs activated by CD3 and CD28 were co-cultured for 12 hours, and the apoptosis ratio of CD45- tumor cells was analyzed by flow cytometry labeled with Annexin-V. The results showed that overexpression of SEMG1 or SEMG2 could inhibit the killing of HCT116 tumor cells by activated PBMC.
  • SEMG1 monoclonal antibody acts on tumor cells overexpressing SEMG1 or SEMG2 to promote the killing effect of PBMC.
  • the SEMG2-specific antibody MM02 previously isolated by our company has a weaker effect on promoting the killing of PBMC than the antibody in this case.
  • the negative control monoclonal Antibodies did not promote killing.
  • Figure 10 This indicates that the SEMG1 antibody works by specifically inhibiting the function of SEMG1 or SEMG2; the hybridoma monoclonal antibody combining SEMG1 and SEMG2 has a stronger killing effect on PBMC than the previously reported SEMG2 hybridoma monoclonal antibody; SEMG1 or SEMG2
  • the expression of the antibody is of great significance for the killing of tumor cells by the antibody.
  • Example 13 Inhibitory effect of SEMG1 monoclonal antibody on tumor cells in vivo
  • mice were randomized into groups based on tumor volume and the proportion of hCD45+ cells in the blood of the mice, and the administration was started immediately.
  • the dosing start date is considered day 0.
  • Dosing regimen SEMG1 antibody was injected intraperitoneally at 5 mg/kg three times a week. After the start of administration, the tumor growth status of the mice was observed every week. After the tumor growth, the body weight and tumor volume were measured 3 times a week, and the relative count of hCD45+ cells in the blood of the mice was monitored by flow 3 times a week. When the tumor volume reached the end-point standard, blood was collected to detect the same indicators as above, and the experiment was ended. The observation of mice includes: daily observation, after inoculation, observation of animal morbidity and death every working day.
  • Tumor inhibitory effect of MM02 (Figure 11). This indicates that the SEMG1 antibody can be used for the inhibition of inoculated SEMG1 and/or SEMG2-expressing tumors in experimental animals, and shows a good inhibitory effect. This indicates that tumor cells expressing SEMG1 and/or SEMG2 are more responsive to SEMG1 antibody, and the expression of specific target protein SEMG1 or SEMG2 is of great significance for the targeted administration of SEMG1 antibody.

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Abstract

一种可特异性结合精液凝固蛋白-1(SEMG1)与精液凝固蛋白-2(SEMG2)并调节这两种蛋白与其受体(LILRB2,LILRB4,CD27或TIGIT)结合的抗体。证明了SEMG1和SEMG2对于免疫细胞有调节作用，并证明了所述抗体具备的中和作用。提供了位于SEMG1和SEMG2蛋白上特定的线性表位，证明了识别上述表位的抗体具备更强的中和功能，可以更有效地解除SEMG1和SEMG2介导的免疫抑制作用，而促进免疫细胞对肿瘤细胞的抑制和杀伤。还提供了所述抗体的制备与鉴定方法，以及施用这种抗体的生物标志物。在制备抗肿瘤药物的方面具备良好的应用前景。

Description

精液凝固蛋白的中和抗体及其表位和应用
相关申请的交叉引用
本申请要求于2021年3月16日提交中国专利局的申请号为202110280166.3、名称为“精液凝固蛋白的中和抗体及其表位和应用”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
本发明涉及生物医药领域，具体涉及一种基于精液凝固蛋白的肿瘤抑制方法及其用途。
背景技术
SEMG1与SEMG2同属精液凝固蛋白(Semenogelins)，是人类精液中含量最丰富的蛋白质。SEMG1和SEMG2分别是50kda和63kda的蛋白质，在基因水平上有78％的相似性。它们都被分泌到精囊内的精液中，然后被前列腺特异性抗原(PSA，又称激肽释放酶肽酶)迅速降解为小肽。SEMG蛋白及其蛋白水解产物具有许多重要的功能。它们调节精子的活力和生育力，并为精子提供抗菌防御。同时SEMG1与SEMG2又属于癌-睾丸抗原(CTA)，是一组在各种肿瘤中频繁表达但通常仅限于生殖细胞的蛋白质。近来有研究表明，SEMGs具有抗肿瘤细胞增殖的作用。同时，我们最近的研究发现SEMG2可以结合CD27分子，并且针对SEMG2的抗体分子在体外PBMC杀伤肿瘤细胞与小鼠肿瘤模型中表现积极。
CD27分子属于肿瘤坏死因子受体(TNFR)超家族的成员，是分子量约为55kDa的I型膜蛋白，并作为两个单体通过二硫键连接的二聚体形式存在。CD27主要表达在淋巴细胞上，CD27信号途径的激活可增加抑制性T细胞(Treg)在实体瘤的浸润，降低抗肿瘤免疫(Claus C,Riether C,Schürch C,Matter MS,Hilmenyuk T,Ochsenbein AF.Cancer Res.2012Jul 15；72(14):3664-76)。与此相一致，研究还发现皮肤组织内的Treg细胞失去CD27的表达后无法发挥正常的免疫调控功能(Remedios KA,Zirak B,Sandoval PM,Lowe MM,Boda D,Henley E等，Sci Immunol.2018Dec 21；3(30).pii:eaau2042)。此外，CD27的激活可增加Treg的数量并减少高脂血症小鼠的动脉粥样硬化(Winkels H,Meiler S,Lievens D,Engel D,Spitz C,Bürger C,等，Eur Heart J.2017；38(48):3590-3599)。上述的最新研究一致表明，CD27在特定Treg(包括肿瘤浸润的Treg)细胞的功能激活中发挥重要作用，因此避免肿瘤浸润Treg细胞表达的CD27激活是潜在的癌症治疗策略。与配体的结合可激活CD27下游信号转导，而目前已知的CD27配体分子是CD70。目前，阻断CD70与CD27的结合是一种正在被研究的肿瘤免疫治疗策略。在已公开发表的信息中，未见到有关于SEMG1或SEMG2为CD27的配体的报道。
LILRB2与LILRB4同属白细胞Ig样受体(LILRs)家族，在免疫系统的功能中起着非常重要的作用。LILRB2，是一类免疫系统抑制性受体，主要表达于髓样细胞和B细胞中，并参与了其功能的负性调控。LILRB2的内源性配体是MHC分子、与其结合后产生抑制信号，作为一个缓冲免疫应答和维持免疫耐受的一个机制。基因学研究显示多种肿瘤微环境中的肿瘤关联巨噬细胞(TAM)高表达LILRB2，抑制LILRB2降低Treg和MDSC在肿瘤组织的侵入。LILRB4主要作为免疫耐受性受体在APC上表达。通过抑制共刺激分子的表达，LILRB4表达的APCs在控制炎症中起着关键作用。LILRB4与多种疾病有关，如川崎病和系统性红斑狼疮(SLE)、炎症性疾病，以及一些神经系统疾病，如多发性硬化症。LILRB4还是单核细胞白血病重要的标志物，在急性髓系白血病(AML)细胞中支持肿瘤细胞浸润到组织中并抑制T细胞活性。在已公开发表的信息中，未见到有关于SEMG1或SEMG2为LILRB2或LILRB4的配体的报道。
TIGIT(也称为WUCAM、Vstm3、VSIG9)是Ig超家族的一种受体，由一个细胞外免疫球蛋白(Ig)可变结构域、1型跨膜结构域和一个胞内结构域组成，胞内结构域其具有两个在小鼠和人类中保守的抑制基序：免疫受体基于酪氨酸的抑制基序(ITIM)和Ig尾酪氨酸样(ITT)基序。TIGIT由活化的CD8+T和CD4+T细胞、自然杀伤(NK)细胞、调节性T细胞(Tregs)和滤泡辅助性T细胞表达。TIGIT作为T细胞负性调节器的作用功能，起着刹车的功能。TIGIT通过多种机制潜在地抑制固有和适应性免疫。TIGIT作用于Tregs可增强免疫抑制功能和稳定性。在已公开发表的信息中，未见到有关于SEMG1或SEMG2为TIGIT的配体的报道。
我们发现SEMG1和SEMG2蛋白表达在肿瘤细胞，且均可作为配体结合LILRB2、LILRB4、CD27和TIGIT受体，从而抑制T细胞对肿瘤细胞的杀伤、诱导Treg和MDSC的扩增。这些结果表明为精液凝固蛋白家族的仅有两种成员SEMG1和SEMG2具有促进肿瘤免疫逃逸的功能。基于上述原创的科学发现，本发明创新性地开发了可以结合SEMG1和SEMG2的双靶向性抗体，能够以高亲和力结合上述靶蛋白并且中和其功能，抑制其与一个或多个配体的结合，从而达到解除肿瘤细胞免疫逃逸功能的作用，促进免疫细胞在体内外对肿瘤细胞的杀伤。本发明所述的抗体可作为有效成分开发用于治疗肿瘤的药物。同时本发明还揭示了具有较强中和功能的抗体对应的线性表位、抗体的筛选方法、施用抗体的生物标志物等与所属抗体密切相关的技术方案。
发明内容
虽然本发明可以以许多不同的形式来实施，但在此公开的是验证本发明原理的其具体的举例说明性实施方式。应该强调的是，本发明不限于所举例说明的具体实施方式。此外，本文使用的任何章节标题仅用于组织目的，并不被解释为限制所描述的主题。
除非在下文中另有定义，本发明具体实施方式中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。
术语“包括”、“包含”、“具有”、“含有”或“涉及”为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。术语“由…组成”被认为是术语“包含”的优选实施方式。如果在下文中某一组被定义为包含至少一定数目的实施方式，这也应被理解为揭示了一个优选地仅由这些实施方式组成的组。
在提及单数形式名词时使用的不定冠词或定冠词例如“一个”或“一种”，“所述”，包括该名词的复数形式。
此外，说明书和权利要求书中的术语第一、第二、第三、(a)、(b)、(c)以及诸如此类，是用于区分相似的元素，不是描述顺序或时间次序必须的。应理解，如此应用的术语在适当的环境下可互换，并且本发明描述的实施方式能以不同于本发明描述或举例说明的其它顺序实施。
术语“和/或”视为具有或不具有另一个的两个指定特征或组件中的每一个的具体公开。因此，如在本文中的短语例如“A和/或B”中所使用的术语“和/或”旨在包括A和B；A或B；A(单独)；和B(单独)。同样地，如在短语例如“A、B和/或C”中所使用的术语“和/或”旨在涵盖以下方面的每一个：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；和C(单独)。
术语“例如”和“即”仅作为实例使用，而无意于限制，并且不应当诠释为仅涉及在说明书中明确列举的那些项目。
术语“或更多”、“至少”、“超过”等，例如“至少一种”应当理解为包括但不限于至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、 45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100或200、300、400、500、600、700、800、900、1000、2000、3000、4000、5000或超过所述值。还包括其间任何更大的数字或分数。
相反地，术语“不超过”包括小于所述值的每个值。例如，“不超过100个核苷酸”包括100、99、98、97、96、95、94、93、92、91、90、89、88、87、86、85、84、83、82、81、80、79、78、77、76、75、74、73、72、71、70、69、68、67、66、65、64、63、62、61、60、59、58、57、56、55、54、53、52、51、50、49、48、47、46、45、44、43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2、1和0个核苷酸。还包括其间任何更小的数字或分数。
术语“多个”、“至少两个”、“两个或更多个”、“至少第二个”等应当理解为包括但不限于至少2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100或200、300、400、500、600、700、800、900、1000、2000、3000、4000、5000或更多。还包括其间任何更大的数字或分数。
术语“大约”、“大体”表示本领域技术人员能够理解的仍可保证论及特征的技术效果的准确度区间。该术语通常表示偏离指示数值的±10％，优选±5％。
如文本所述，除非另有说明，否则任何浓度范围、百分比范围、比率范围或整数范围应当理解为包括所叙述范围内的任何整数的值，并且在适当时包括其分数(例如整数的十分之一和百分之一)。
如本文所用，术语“抗体”(Ab)包括但不限于，特异性结合抗原的糖蛋白免疫球蛋白。通常，抗体可以包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链，或其抗原结合分子。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域，CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域，CL。VH和VL区域可以进一步细分为高变的区域，称为互补决定区(CDR)，散布着更保守的区域，称为框架区(FR)。每个VH和VL包含三个CDR和四个FR，以下列顺序从氨基末端到羧基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。Ab的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，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)。
轻链可变区和重链可变区分别包含间插有三个高变区(也称为“互补决定区”或“CDR”)的“构架”区。“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883， Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
然而，应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。例如，对使用Kabat和Chothia编号的CDR区域在不同指派系统定义下的残基范围如下表A所示。
表A.不同指派系统定义下的CDR残基范围
Figure PCTCN2022079756-appb-000001
因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
本发明抗体的CDR可以根据本领域的任何方案或其组合人工地评估确定边界。除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
抗体可以包括例如，单克隆抗体、重组产生的抗体、单特异性抗体、多特异性抗体(包括双特异性抗体)、人抗体、工程化抗体、人源化抗体、嵌合抗体、免疫球蛋白、合成抗体、包含两个重链和两个轻链分子的四聚体抗体、抗体轻链单体、抗体重链单体、抗体轻链二聚体、抗体重链二聚体、抗体轻链-抗体重链对、胞内抗体、抗体融合物(本文有时称为“抗体缀合物”)、异缀合抗体、单结构域抗体、单价抗体、单链抗体或单链Fv(scFv)、骆驼源化抗体、亲和体、Fab片段、F(ab’)2片段、二硫键连接的Fv(sdFv)、抗独特型(抗Id)抗体(包括例如，抗-抗Id抗体)、微抗体、结构域抗体、合成抗体(本文有时称为“抗体模拟物”)和以上任何的抗原结合片段。
术语“人源化抗体”意图指将源自另一哺乳动物物种，诸如小鼠种系的CDR序列嫁接到人框架序列上获得的抗体。可以在人框架序列中进行别的框架区修饰。
如本文所用，“抗原结合分子”、“抗原结合片段”或“抗体片段”是指包含从该分子所源于的抗体的抗原结合片段(例如，CDR)的任何分子。抗原结合分子可以包括抗原互补决定区(CDR)。抗体片段的实例包括但不限于，从抗原结合分子形成的Fab、Fab’、F(ab’)2和Fv片段、dAb、线性抗体、scFv抗体和多特异性抗体。在一些实施方式中，抗原结合分子结合SEMG1蛋白，在一些实施方式中，抗原结合分子具有中和活性，能够抑制SEMG1或SEMG2与受体CD27、LILRB2、LILRB4或TIGIT结合。
如本文所用，术语“抗原”是指引发免疫应答或能够被抗体或抗原结合分子结合的任何分子。免疫应答可以牵涉抗体产生或特异性免疫活性细胞的活化或这两者。本领域技术人员将 容易理解任何大分子(包括几乎所有蛋白质或肽)均可以充当抗原。抗原可以内源性表达，即由基因组DNA表达或可以重组表达。抗原可以对某些组织，例如癌细胞具有特异性或者其可以广泛地表达。此外，较大分子的片段可以起抗原作用。在一些实施方式中，抗原为SEMG1或SEMG2蛋白抗原。
如本文所用，在一些实施方式中，抗原结合分子、scFv、抗体或其片段直接阻断配体上的结合位点或者通过间接方式(如配体的结构或能量改变)改变配体的结合能力。在一些实施方式中，抗原结合分子、scFv、抗体或其片段防止与其结合的蛋白质行使生物学功能。
如本文所用，术语“肽”、“多肽”和“蛋白质”可互换使用并且是指包含通过肽键共价连接的氨基酸残基的化合物。蛋白质或肽含有至少两个氨基酸并且可以包含蛋白质或肽的序列的氨基酸的最大数量没有限制。多肽包括任何包含两个或更多个通过肽键彼此连接的氨基酸的肽或蛋白质。如本文所用，该术语是指短链(其在本领域中也通常称为例如肽、寡肽和寡聚体)和较长的链(其在本领域中通常称为蛋白质，其具有许多类型)两者。“多肽”包括例如生物活性片段、基本上同源的多肽、寡肽、同源二聚体、异源二聚体、多肽的变体、经修饰的多肽、衍生物、类似物、融合蛋白等。多肽包括天然的肽、重组的肽、合成的肽或其组合。
如本文所用，术语“特异性结合”或“特异性结合至”是指两个分子之间例如抗体和抗原之间的非随机结合反应。
如本文所用，“抑制结合”、“阻断结合”或“竞争相同表位”的能力是指抗体抑制两个分子的结合至任何可检测的程度的能力。在一些实施方式中，阻断两个分子之间结合的抗体将两个分子之间的结合相互作用抑制至少50％。在一些实施方式中，该抑制可以大于60％，大于70％，大于80％或大于90％。
如本文所用，术语“Ka”旨在表示特定抗体-抗原相互作用的缔合速率，而本文所用的术语“Kd”旨在表示特定抗体-抗原相互作用的解离速率。如本文所用，术语“KD”或“KD值”旨在表示特定抗体-抗原相互作用的解离常数，其从Kd与Ka的比率(即，Kd/Ka)获得并且表示为摩尔浓度(M)。抗体的KD值可以使用本领域良好建立的方法来确定。
如本文所用，术语“高亲和力”的抗体是指针对靶抗原具有1×10 -7M或更低，更优选5×10 -8M或更低，甚至更优选1×10 -8M或更低，甚至更优选5×10 -9M或更低，和甚至更优选1×10 -9M或更低的KD值的抗体。
如本文所用，术语“表位”是指免疫球蛋白或抗体特异性结合的抗原部分。“表位”也被称为“抗原决定簇”。表位或抗原决定簇通常由分子例如氨基酸、碳水化合物或糖侧链的化学活性表面基团组成，并且通常具有特定的三维结构和特定的电荷特征。例如，表位通常包含独特立体构象中的至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或不连续的氨基酸，其可以是“线性表位”或“构象表位”。参见例如Epitope Mapping Protocols in Methods in Molecular Biology，Vol.66，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质和相互作用分子(例如抗体)之间的所有相互作用位点沿蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用位点跨越蛋白质中彼此分离的氨基酸残基。取决于通过本领域技术人员已知的常规技术检测的结合相同表位的竞争性，可以筛选抗体。例如，可以进行竞争或交叉竞争研究以获得彼此竞争或交叉竞争结合抗原(例如CLDN18.2)的抗体。在国际专利申请WO 03/048731中描述了用于获得结合相同表位的抗体的高通量方法，其基于它们的交叉竞争。
本发明中的术语“核酸”或“核酸序列”指包含核糖核酸、脱氧核糖核酸或其类似物单元的任何分子、优选聚合分子。所述核酸可为单链的或双链的。单链核酸可为变性双链DNA的一条链的核酸。或者，单链核酸可为不来源于任何双链DNA的单链核酸。
本文所使用的术语“互补”涉及核苷酸碱基G、A、T、C和U之间的氢键碱基配对，以使得当两种给定的多核苷酸或多核苷酸序列彼此退火时，在DNA中A与T配对、G与C配对，在RNA中G与C配对、A与U配对。
如本文所用，术语“癌症”是指一大组各种疾病，其特征为体内异常细胞不受控制的生长。不受调节的细胞分裂和生长导致形成入侵相邻组织且也可以通过淋巴系统或血流转移到身体远程部分的恶性肿瘤。“癌症”或“癌症组织”可以包括肿瘤。比如：骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃肠、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、宫颈癌、阴道癌、阴户癌、何杰金氏病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统的癌症、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童期实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)的赘生物/肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊髓轴(spinal axis)肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏(Kaposi)肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症(包括由石棉诱发的那些癌症)、及所述癌症的组合。
如本文所用，术语“与SEMG1或SMEG2相关的癌症”是指由SEMG1或SEMG2的增加或减少的表达或活性引起、加重或以其它方式与其相关的任何癌症，在一些实施方式中，本文公开的方法可以用于选自结直肠癌、肺癌、乳腺癌、黑色素瘤、淋巴瘤、肝癌、头颈癌、胃癌、肾癌、膀胱癌、前列腺癌、睾丸癌、子宫内膜癌、乳腺癌、和卵巢癌。
如本文所用，“有效剂量”、“有效量”或“治疗有效剂量”是当单独使用或与另一治疗剂组合使用时保护受试者免于疾病发作或者促进疾病消退的任何量，所述疾病消退的证据为疾病症状的严重性降低、疾病无症状期的频率和持续期间增加或防止由于疾病折磨导致的损伤或残疾。可以使用熟练从业人员已知的各种方法评估治疗剂促进疾病消退的能力，例如在临床试验期间在人受试者中评估、在用于预测在人体中的效力的动物模型系统中评估或通过在体外测定法中测定试剂的活性评估。
如本文所用，“个体”或“受试者”是哺乳动物。哺乳动物包括灵长类(例如，人和非人灵长类如猴)和啮齿类(例如，小鼠和大鼠)。在某些实施方式中，个体或受试者是人。“受试者”可以是“患者”–患者是需要治疗的人类受试者，可以是患有SEMG1或SEMG2相关癌症如乳腺癌的个体，处于发生SEMG1或SEMG2相关癌症如结乳腺癌的风险的受试者。
如本文所用，术语“体外细胞”是指离体培养的任何细胞。特别地，体外细胞可以包括T细胞。
如本文所用，术语“药学上可接受”是指载体、稀释剂、赋形剂和/或其盐在化学和/或物理上与制剂中的其他成分相容，并且与接受者在生理学上相容。
如本文所用，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剂相容的载体和/或赋形剂，其在本领域中是公知的(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，佐剂和离子强度增强剂。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、阴离子或非离子表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文所用，术语“调控”总体上包含了上调或下调两个不同方向的意思，在某些情况下可以理解了抑制或增强，在某些情况下可以理解为降低或提高，在某些情况下可以理解为减少或增多等，具体解释不做限制，根据实际应用语境理解和解释。示例性的，在一些实施方式中，“调控”肿瘤细胞生长可以理解为抑制或增强肿瘤细胞生长。
如本文所用，术语“减少”和“降低”可互换使用并且表示小于原来的任何变化。“减少”和“降低”是相对的术语，需要在测量前和测量后间进行比较。“减少”和“降低”包括完全消耗；同理术语“增多”和“提高”是相反解释。
受试者的“治疗”或“处理”是指在受试者上进行任何类型的干预或过程，或对该受试者施 用活性剂，以达到逆转、减轻、改善、抑制、减缓或预防症状、并发症或状况或与疾病相关的生化指标的发作、进展、发展、严重或复发的目的。在一些实施方式中，“治疗”或“处理”包括部分缓解。在另一个实施方式中，“治疗”或“处理”包括完全缓解。
本公开的各种方面进一步详细描述：
1.SEMG1或SEMG2蛋白及其在肿瘤中的作用
蛋白SEMG1或SEMG2同属SEMG蛋白。
然而，SEMG1或SEMG2蛋白功能尚未被深入认识，亦未见基于SEMG1或SEMG2抑制肿瘤的报道，本发明人通过基因沉默敲除以及免疫学等实验证实了通过抑制SEMG1功能或活性，能够阻断SEMG1或SEMG2与受体CD27、LILRB2、LILRB4或TIGIT的相互作用，促进免疫细胞对肿瘤细胞的杀伤作用，从而可用于癌症的治疗领域。
在一些实施方式中，所述癌症为与SEMG1或SEMG2相关的癌症。
如本文所用，术语“与SEMG1或SEMG2相关的癌症”是指由SEMG1或SEMG2的增加或减少的表达或活性引起、加重或以其它方式与其相关的任何癌症。
在一些实施方式中，本文公开的方法可以用于选自结直肠癌、肺癌、乳腺癌、黑色素瘤、淋巴瘤、肝癌、头颈癌、胃癌、肾癌、膀胱癌、前列腺癌、睾丸癌、子宫内膜癌、乳腺癌、和卵巢癌。
2.基于SEMG1或SEMG2的肿瘤细胞调控的方法
基于本发明的上述发现，公开了一种在体外或体内调控肿瘤细胞的方法。该方法通过改变SEMG1或SEMG2与受体CD27、LILRB2、LILRB4或TIGIT任一之间的结合作用，改变免疫抑制性细胞MDSC或Treg的细胞比率，调控PBMC对肿瘤细胞的杀伤作用。
本领域可以理解，这种调控可以是调控肿瘤细胞的细胞活性，或是肿瘤细胞生长，或是肿瘤细胞迁移等，在此并不做限制，本领域可以合理预期这种调控可以是多方面的。而“调控”包含了增强和抑制两个方面，在更多实施方式中，这种调控是抑制方面，即对于肿瘤细胞活性、生长或迁移等的抑制作用，以实现疾病的病灶去除或治疗目的。
在一些具体的抑制实施方式中，所述方法可以包括以下步骤：
a)对表达SEMG1或SEMG2的肿瘤细胞，通过抑制SEMG1或SEMG2的表达、功能或活性进而实现上述目的。可选的，在步骤a之前还可以包括对于肿瘤细胞的SEMG1或SEMG2表达进行检测，进而更好的实现上述方法。
进一步的，所述方法还包括步骤b：
b)使肿瘤细胞与免疫细胞接触；可以理解的是，步骤b)可以在步骤a)之前或之后完成，这都不影响该方法实施。
本领域可以理解，对于特定蛋白的表达、功能或活性的抑制方法有多种，这些方法可以是蛋白层面或基因层面来开展，其都是本领域公知的；
在一些实施方式中，上述抑制方法可以为如下任一种：
a、对SEMG1或SEMG2基因进行基因编辑，以破坏该基因的完整性，进而降低该基因的表达量和活性等；
在一些优选的实施方式中，本发明的基因编辑系统为CRISPR/Cas9基因编辑系统；
在一些更优选的实施方式中，所述CRISPR/Cas9基因编辑系统用于编码sgRNA的寡聚DNA序列选自SEQ ID NO:1-8。
b、干扰SEMG1或SEMG2基因表达，比如RNAi方式，以降低该基因的表达量或活性；
c、免疫手段抑制，比如通过抗体抑制SEMG1或SEMG2蛋白的功能或活性。
另一方面，本发明还可提供了其他方面的调控的方法，比如，在体内外调控细胞中CD27、LILRB2、LILRB4或TIGIT活性的方法；比如，在体内外调控免疫抑制性细胞MDSC或Treg细胞比率的方法；再比如，在体内外调控PBMC对肿瘤细胞的杀伤作用的方法。这些方法都 包括了改变肿瘤细胞中SEMG1或SEMG2的功能或活性的步骤。
在一些实施方式中，上述调控方法是抑制CD27、LILRB2、LILRB4或TIGIT活性的方法；是降低免疫抑制性细胞MDSC或Treg细胞比率的方法；是增强PBMC对肿瘤细胞的杀伤作用的方法。这些方法分别包括抑制肿瘤细胞中SEMG1或SEMG2的功能或活性的步骤。
3.本发明的抑制剂及其筛选或鉴定方法
基于本发明发现了SEMG1或SEMG2蛋白能够与CD27、LILRB2、LILRB4或TIGIT等受体结合，以及肿瘤细胞中SEMG1或SEMG2蛋白表达对于髓系来源的抑制性细胞(MDSC)、调节性T细胞(Treg)以及免疫细胞的影响。
本发明提供了一种SEMG1或SEMG2抑制剂，本发明中的“抑制剂”，其可以是蛋白层面抑制剂或基因层面抑制剂，但凡能够抑制SEMG1或SEMG2基因或蛋白的，包括但不限于基因的生成、转录、活性、功能等，蛋白的表达、活性、功能等，都属于“抑制剂”范畴。
示例性的，当是蛋白层面抑制剂时，其是这样的物质，其能够结合SEMG1或SEMG2蛋白同时抑制或阻断SEMG1或SEMG2与CD27、LILRB2、LILRB4或TIGIT中任一之间的相互作用。
在一些实施方式中，该蛋白层面的SEMG1或SEMG2抑制剂特异性结合于SEMG1或SEMG2蛋白。
在一些实施方式中，该蛋白层面的SEMG1或SEMG2抑制剂特异性并高亲和力结合SEMG1或SEMG2蛋白。
在一些实施方式中，该蛋白层面抑制剂为激动或拮抗SEMG1或SEMG2与CD27、LILRB2、LILRB4或TIGIT相互作用的化合物。
在一些实施方式中，所述蛋白层面抑制剂包括但不限于小分子抑制剂、多肽、抗体或抗原结合片段。
当是基因层面抑制剂时，其是这样的物质，其能够通过改变SEMG1基因的活性或功能，进而抑制或阻断SEMG1或SEMG2与CD27、LILRB2、LILRB4或TIGIT中任一之间的相互作用。
在一些实施方式中，该基因层面的SEMG1或SEMG2抑制剂包括但不限于特异性针对SEMG1基因的小分子物质、DNA类物质或RNA类物质等，比如引物、探针、sgRNA等物质。
本发明还提供上述抑制剂的筛选或鉴定方法，该方法可以包括以下步骤：
首先获得SEMG1抑制性分子，再分析候选分子对SEMG1与受体或LILRB2结合的影响；优选的，分析候选分子是否具有拮抗活性。
在一些实施方式中，该方法可以包括以下步骤：
首先获得SEMG1或SEMG2抑制性分子，再分析候选分子对髓系来源的抑制性细胞(MDSC)的影响；优选的，分析候选分子是否能够降低抑制性细胞(MDSC)的含量或比率。
在一些实施方式中，该方法可以包括以下步骤：首先获得SEMG1抑制性分子，再分析候选分子对调节性T细胞(Treg)的影响；优选的，分析候选分子是否能够降低调节性T细胞(Treg)的含量或比率。
在一些实施方式中，该方法可以包括以下步骤：首先获得SEMG1抑制性分子，再分析候选分子对免疫细胞杀伤肿瘤细胞的影响；优选的，分析候选分子是否能够促进免疫细胞对肿瘤细胞的杀伤或抑制作用。
在一些实施方式中，该方法可以包括上述步骤中的组合或全部。
通过上述方法，本领域可以预期能够筛选和/或鉴定出有价值的SEMG1抑制性分子。
4.本发明的抗体及其制备方法
本发明的抗体
术语“针对SEMG1或SEMG2的抗体”、“抗SEMG1或SEMG2的抗体”、“抗SEMG1或SEMG2蛋白抗体”、“SEMG1或SEMG2蛋白抗体”、或“结合SEMG1或SEMG2的抗体”在本文中可互换地使用。是指这样的本发明的抗体，所述抗体能够以足够的亲和力结合SEMG1或SEMG2蛋白，由此所述抗体可以用作靶向SEMG1或SEMG2蛋白的诊断剂、预防剂和/或治疗剂。
任何具备如下性质的抗体或其抗原结合片段理论上都属于本发明的发明构思或权利范围，具体的：这种分离的抗体或其抗原结合片段具有：
a)能够特异性结合SEMG1或SEMG2蛋白；且
b)能够抑制或阻断SEMG1或SEMG2与CD27、LILRB2、LILRB4或TIGIT中任一之间的相互作用。
在一些实施方式中，所述抗体另外还包含至少一项下列性质：
所述抗体降低免疫抑制性细胞MDSC细胞比率；
所述抗体降低免疫抑制性细胞Treg的细胞比率；或
所述抗体增强免疫细胞(如PBMC)对肿瘤细胞的杀伤作用。
另外/或者，所述抗体可以包含一种或多种其它的上述特征。
在一些具体的实施方式中，本发明的分离的抗体或其抗原结合片段，包含重链可变区和轻链可变区，具体序列可以是如下各种：
SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR；或者与上述6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体。
其中，所述氨基酸变化是氨基酸的添加、缺失或取代，优选的的，所述氨基酸变化是保守氨基酸取代。
在一些实施方式中，抗体包含了根据IMGT定义的HCDR1、HCDR2和HCDR3序列的重链可变区；以及包含根据IMGT定义的LCDR1、LCDR2和LCDR3序列的轻链可变区，序列如下：
i.所述HCDR1的氨基酸序列如SEQ ID NO.15所示；
ii.所述HCDR2的氨基酸序列如SEQ ID NO.16所示；
iii.所述HCDR3的氨基酸序列如SEQ ID NO.17所示；
iv.所述LCDR1的氨基酸序列如SEQ ID NO.18所示；
v.所述LCDR2的氨基酸序列如SEQ ID NO.19所示；
vi.所述LCDR3的氨基酸序列如SEQ ID NO.20所示。
或者与上述i-vi的6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体；
其中，所述氨基酸变化是氨基酸的添加、缺失或取代，优选的的，所述氨基酸变化是保守氨基酸取代。
优选的，所述抗体或其抗原结合片段与SEMG1或SEMG2的结合KD<2×10 -8；
更优选的，所述KD<2×10 -8、<1×10 -8、<9×10 -9、<8×10 -9、<7×10 -9、<6×10 -9、<5×10 -9、<4×10 -9、<3×10 -9、<2×10 -9、<1×10 -9、<1×10 -10。
在一些实施方式中，所述抗体或抗原结合片段包含重链可变区和轻链可变区，序列选自如下：
a)所述重链可变区的氨基酸序列包含选自由SEQ ID NOs﹕13所组成的组中的氨基酸序列或与组中的序列具有至少70％、80％、90％、95％或99％序列同一性；
b)所述轻链可变区的氨基酸序列包含选自由SEQ ID NOs﹕14所组成的组中的氨基酸序列或与组中的序列具有至少70％、80％、90％、95％或99％序列同一性。
保守取代是指一个氨基酸经相同类别内的另一氨基酸取代，例如一个酸性氨基酸经另一酸性氨基酸取代，一个碱性氨基酸经另一碱性氨基酸取代，或一个中性氨基酸经另一中性氨基酸取代。示例性的取代如下表所示：
示例的氨基酸取代
原始残基 示例性取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp、Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
原始残基 示例性取代 优选的取代
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu，Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
在优选的实施方式中，本发明所述的氨基酸变化发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸变化发生在Fc区。在一些实施方式中，提供了包含含有一个或多个突变的Fc结构域的抗SEMG1蛋白抗体，该突变增强或减弱抗体例如与中性pH相比在酸性pH下与FcRn受体的结合。这类Fc修饰的非限制性实例包括，例如：250位(例如E或Q)、250位和428位(例如L或F)、252位(例如L/Y/F/W或T)、254位(例如S或T)和256位(例如S/R/Q/E/D或T)的修饰；或428位和/或433位(例如H/L/R/S/P/Q或K)和/或434位(例如A、W、H、F或Y[N434A、N434W、N434H、N434F或N434Y])的修饰；或250位和/或 428位的修饰；或307位或308位(例如308F、V308F)和434位的修饰。在一个实施方式中，该修饰包括428L(例如M428L)和434S(例如N434S)修饰；428L、259I(例如V259I)和308F(例如V308F)修饰；433K(例如H433K)和434(例如434Y)修饰；252、254和256(例如252Y、254T和256E)修饰；250Q和428L修饰(例如T250Q和M428L)；及307和/或308修饰(例如308F或308P)。还在另一实施方式中，该修饰包括265A(例如D265A)和/或297A(例如N297A)修饰。
例如，本发明包括含有Fc结构域的抗SEMG1蛋白抗体，该Fc结构域包含选自以下的一对(组)或多对(组)突变：252Y、254T和256E(例如M252Y、S254T和T256E)；428L和434S(例如M428L和N434S)；257I和311I(例如P257I和Q311I)；257I和434H(例如P257I和N434H)；376V和434H(例如D376V和N434H)；307A、380A和434A(例如T307A、E380A和N434A)；和433K和434F(例如H433K和N434F)。在一个实施方案中，本发明包括含有Fc结构域的抗SEMG1蛋白抗体，该Fc结构域包含IgG4铰链区中的S108P突变以促进二聚体稳定化。前述Fc结构域突变和本文公开的抗体可变结构域内的其他突变的任意可能的组合都包含在本发明的范围之内。
在另一些实施方式中，本文中所提供的SEMG1或SEMG2蛋白抗体可经改变以增加或降低其糖基化的程度。对SEMG1或SEMG2蛋白抗体的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一个或多个糖基化位点而方便地实现。当SEMG1蛋白抗体包含Fc区时，可以改变与Fc区连接的糖类。在一些应用中，除去不想要的糖基化位点的修饰可以是有用的，例如除去岩藻糖模块以提高抗体依赖性细胞性细胞毒性(ADCC)功能(参见Shield等(2002)JBC277:26733)。在其它应用中，可以进行半乳糖苷化修饰以调节补体依赖性细胞毒性(CDC)。在某些实施方式中，可在本文中所提供冠状病毒S蛋白抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体，以便增强例如本发明的冠状病毒S蛋白抗体预防和/或治疗冠状病毒感染的有效性。
在一些实施方式中，抗体是IgG1、IgG2、IgG3或IgG4抗体；优选地，其是IgG1或IgG4抗体；更优选地，其是人或鼠IgG1或IgG4抗体。
在一些实施方式中，抗体可进一步包含连接至多肽的偶联部分，所述偶联部分选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白、脂类、和生物素中的一种或多种，其中所述多肽或抗体与所述偶联部分可选择性通过连接子相连，优选所述连接子为肽或多肽。
本领域可以理解，在不改变抗体核心功能区的情况下，所述抗体可制备成或选自单克隆抗体、多克隆抗体、抗血清、嵌合抗体、人源化抗体和人抗体；更优选的，所述抗体可制备成或选自多特异性抗体、单链Fv(scFv)、单链抗体、抗独特型(抗-Id)抗体、双抗体、微型抗体、纳米抗体、单结构域抗体、Fab片段、F(ab’)片段、二硫化物连接的双特异性Fv(sdFv)和胞内抗体。
本发明还提供一种分离的多核苷酸，所述多核苷酸编码上述抗体或抗原结合片段。
本发明还提供一种重组载体，其包含上述的多核苷酸，以及任选的调控序列；
优选的，所述重组载体为克隆载体或表达载体；更优选的，所述调控序列选自前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号序列、转录终止子，或其任何组合。
本发明还提供一种宿主细胞，其特征在于，包含上述的重组载体；优选的，所述宿主细胞为原核细胞或真核细胞。
本发明还提供一种药物组合物，其特征在于，包含上述的抗体或抗原结合片段、多核苷酸、重组载体、宿主细胞和药物组合物中的一种或更多种；优选的，所述组合物还包含药学上可接受的载体或辅料。
本发明还提供一种试剂盒，其特征在于，包含上述的抗体或抗原结合片段、多核苷酸、重组载体、宿主细胞和药物组合物中的一种或更多种，并容纳于合适的容器中。
本发明的抗体制备方法
可以通过多种技术来生产本发明的抗SEMG1的单克隆抗体(mAb)和人序列抗体，包括常规单克隆抗体方法学，例如Kohler and Milstein(1975)Nature 256：495的标准体细胞杂交技术。可以采用生产单克隆抗体的任何技术，例如B淋巴细胞的病毒或致癌转化。一种用于制备杂交瘤的动物系统是鼠系统。在小鼠中生产杂交瘤是已完全建立的方法。用于分离供融合用的经免疫脾细胞的免疫方案和技术是本领域已知的。在一些实施方式中，本发明是利用重组SEMG1蛋白多次免疫小鼠获得。
本发明抗体的活性测定法
可以通过本领域中已知的多种测定法对本文中提供的SEMG1蛋白抗体鉴定、筛选或表征其物理/化学特性和/或生物学活性。一方面，对本发明的SEMG1蛋白抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA，Western印迹等来进行。可使用本领域已知方法来测定对SEMG1或SEMG2蛋白的结合，本文中公开了例示性方法。在一些实施方式中，使用生物膜层干涉测定本发明的SEMG1蛋白抗体对SEMG1或SEMG2蛋白的结合，同时进一步通过ELISA实验分析SEMG1或SEMG2与CD27、LILRB2、LILRB4或TIGIT的结合。
5.本发明的药物组合物
在一些实施方式中，本发明提供包含本文所述的任何化合物、SEMG1或SEMG2蛋白抑制剂、拮抗剂、抗体、抗原肽、蛋白等的组合物，优选地组合物为药物组合物。
在一个实施方式中，所述组合物含有本发明的抗体或其抗原结合片段，或免疫偶联物，或双特异性分子，及药学可接受载体。
在一个实施方式中，药物组合物包含本发明的SEMG1或SEMG2蛋白抗体，以及一种或多种其它治疗剂的组合。
在一些实施方式中，本发明的药物组合物或药物制剂包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。适用于本发明的药用载体可以是无菌液体，如水和油，包括那些石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准药用载体和/或赋形剂，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。
可以通过将具有所需纯度的本发明的SEMG1蛋白抗体与一种或多种任选的药用辅料(Remington’s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合来制备包含本文所述的SEMG1或SEMG2蛋白抗体的药物制剂，优选地以冻干制剂或水溶液的形式。
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它活性成分，包括但不限于CTLA4和PD-1抑制剂等。所述活性成分以对于目的用途有效的量合适地组合存在。
6.本发明的预防或治疗用途
预防或治疗方法
本发明提供了用于预防受试者中SEMG1或SEMG2相关癌症的方法，其包括向受试者 施用本发明的抑制剂、抗体或药物组合物。预防剂的施用可以在表现出症状特征之前施用，以便阻止疾病发生，或可选择地延迟疾病的进展。
优选的，所述受试者已经接受或正在接受或将要接受额外的抗癌疗法；
更优选的，所述额外的抗癌疗法包括手术、放疗、化疗、免疫疗法或激素疗法。
本发明提供了用于治疗受试者中SEMG1或SEMG2相关癌症的方法，其包括向受试者施用本发明的抑制剂、抗体或药物组合物。治疗剂的施用可以在表现出症状特征之后施用。
优选的，所述受试者已经接受或正在接受或将要接受额外的抗癌疗法；
更优选的，所述额外的抗癌疗法包括手术、放疗、化疗、免疫疗法或激素疗法。
SEMG1或者SEMG2相关癌症包括但不限于各类癌症，比如常见的结直肠癌、肺癌、黑色素瘤、淋巴瘤、肝癌、头颈癌、胃癌、肾癌、膀胱癌、前列腺癌、睾丸癌、子宫内膜癌、乳腺癌、和卵巢癌等。
本发明提供的治疗方法也可以是，使受试者的免疫细胞和肿瘤细胞与有效剂量的本发明所述的抗体或抗原结合片段、多核苷酸、重组载体、宿主细胞和药物组合物接触；可选的，在使用有效量的化合物和受试者的免疫细胞和/或肿瘤细胞接触前，检测SEMG1或SEMG2在肿瘤细胞的表达；
优选的，所述免疫细胞为淋巴细胞；
更优选的，所述淋巴细胞为T淋巴细胞。
预防或治疗用途
可以理解，利用本发明所述的抑制剂、抗体或抗原结合片段、多核苷酸、重组载体、宿主细胞和药物组合物至少可包括如下预防治疗用途或相应药物的制备用途：
a)在激动或拮抗SEMG1或SEMG2和CD27、LILRB2、LILRB4或TIGIT相互作用中的用途；优选SEMG1或SEMG2表达于肿瘤细胞，CD27、LILRB2、LILRB4或TIGIT表达于免疫细胞；
b)在制备用于激动或拮抗SEMG1或SEMG2和CD27、LILRB2、LILRB4或TIGIT相互作用的产品中的用途；优选SEMG1或SEMG2和表达于肿瘤细胞，CD27、LILRB2、LILRB4或TIGIT表达于免疫细胞；
c)在预防或治疗肿瘤中的用途；
d)在制备用于预防或治疗肿瘤的药物中的用途；
e)在调节针对肿瘤所引起的免疫反应中的用途；
f)在制备用于调节针对肿瘤所引起的免疫反应的药物中的用途；
g)在体内外抑制肿瘤细胞生长中的用途；
h)在制备用于体内外抑制肿瘤细胞生长的试剂中的用途。
7.本发明的诊断用途
术语“伴随诊断”通常是指提供有关患者针对特定治疗药物的治疗反应的信息，有助于确定能够从某一治疗产品中获益的患者群体，从而改善治疗预后并降低保健开支。此外，伴随诊断还有助于确定最有可能针对治疗药物产生响应的患者群体。
本发明证实了SEMG1或SEMG2蛋白可作为SEMG1或SEMG2相关癌症的治疗靶点，即当肿瘤细胞中表达SEMG1或SEMG2蛋白时，可通过引入SEMG1或SEMG2蛋白抑制剂(比如抗体)有效抑制肿瘤。因此，在相应受试肿瘤患者治疗过程中(包括治疗前和治疗后)，可通过检测肿瘤细胞的SEMG1或SEMG2蛋白表达情况，确定最有可能针对治疗药物产生响应的患者群体。所以本发明提供了一种伴随诊断的方法，该方法通过检测经免疫治疗前/后受试者肿瘤细胞的SEMG1或SEMG2蛋白表达量或功能来完成。
8.本发明的试剂盒
包含本发明的抗体或抗体组合物(例如人抗体、双特异性或多特异性分子、或免疫偶联物) 及使用说明书的试剂盒也在本发明的范围内。试剂盒还可进一步包含至少一种别的药剂，或一种或多种别的抗体等。试剂盒通常包括标签，其指明试剂盒内容物的预期用途。术语标签包括在试剂盒上或与试剂盒一起提供的或者以其它方式随附于试剂盒的、任何书写的或记录的材料。
此外，基于本发明的诊断用途，本发明的试剂盒还可以是包含能够检测肿瘤细胞SEMG1或SEMG2蛋白表达量或活性的组分。因此，除了上述抗体组合物外，本发明试剂盒组分还可以是包含诸如引物、探针等，或者其他能够检测蛋白表达或活性的材料，在此不作限制。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1.SEMG1结合相应受体蛋白的浓度效应。不同浓度的SEMG1结合CD27(2μg/mL)、LILRB2(2μg/mL)、TIGIT(3μg/mL)以及LILRB4(2μg/mL)的能力随着SEMG1的浓度的增高而升高，表明SEMG1与受体蛋白CD27、LILRB2、TIGIT或LILRB4具有特异结合的能力。实例图中各阳性组已扣除空白包被、封闭并加入相应配体蛋白的阴性对照在显色后的OD 450读数。
图2.SEMG2结合相应受体蛋白的浓度效应。不同浓度的SEMG2结合2μg/mL的CD27(2μg/mL)、LILRB2(2μg/mL)、TIGIT(3μg/mL)以及LILRB4(2μg/mL)的能力随着SEMG2的浓度的增高而升高，表明SEMG2与受体蛋白CD27、LILRB2、TIGIT或LILRB4具有特异结合的能力。图中各阳性组已扣除空白包被、封闭并加入相应配体蛋白的阴性对照在显色后的OD 450读数。
图3.SEMG1与SMEG2在肿瘤组织中的表达鉴定。对不同病理组织将肿瘤病人的胃壁和胃肿瘤组织提取物、胃壁和胃肿瘤组织提取物、结直肠和结直肠组织提取物裂解制得的蛋白悬液
图4.过表达SEMG1或SEMG2对PBMC杀伤肿瘤细胞的抑制作用。将PBMC细胞与肿瘤细胞或过表达SEMG1或SEMG2的肿瘤细胞分别共孵育后，测定凋亡细胞的百分比。纵坐标是凋亡肿瘤细胞的百分比；横坐标是不同的实验处理条件，即加入的不同细胞。结果表明，过表达SEMG1或SEMG2能够在显著抑制结合激活的PBMC对肿瘤细胞的杀伤作用，
图5.过表达SEMG1与SEMG2的HCT116细胞系对MDSC与Treg的诱导作用。相对于HCT116细胞，过表达SEMG1与SEMG2的HCT116细胞与人外周血单核细胞PBMC共培养，均可显著增加MDSC细胞(HLA-DR-,CD33+,CD11+)或Treg细胞(CD4+FoxP3+CD25+)的比率，这表明SEMG1与SEMG2通过诱导MDSC与Treg的转化，实现肿瘤细胞的免疫逃逸。
图6.鼠杂交瘤抗体结合SEMG1与SEMG2的浓度效应。纵坐标是OD450吸收值作为ELISA实验的读数，显示固定在微孔板上的的1μg/mL SEMG1(A)或SEMG2(B)与加入的鼠杂交瘤单克隆抗体的结合程度；横坐标显示了孵育抗体的不同浓度。不同的曲线代表不同的克隆产生的抗体，不相关鼠单克隆抗体mIgG1作为阴性对照。在图中也表明了相应抗体的EC50值。拟合的曲线是基于三次独立生物学实验的一次代表性结果(误差线代表标准差)。
图7.流式细胞分析技术检测鼠杂交瘤单克隆抗体结合过表达SEMG1的HCT116细胞的结合阳性率。结果显示过表达SEMG1的细胞结合不同克隆的杂交瘤单克隆抗体(5μg/mL)阳性率不同，表明了抗体的多样性以及抗体可结合表达于肿瘤细胞表面的抗原。
图8.鼠杂交瘤抗体SEMG1或SEMG2与相应蛋白结合的阻断效应。图示为两株单克隆 抗体33-B5-C1-F5与25-E10-F8-B5在两个浓度下阻断2ug/mL SEMG1蛋白(A)或22ug/mL SEMG2蛋白(B)相应蛋白：2μg/mL CD27、2μg/mL LILRB2、3μg/mL TIGIT以及2μg/mL LILRB4ELISA结合的测试，与包被后的微孔加入配体蛋白与PBS的ELISA OD450值相比较计算阻断百分率。阻断百分率代表了阻断程度，阻断百分率越高阻断靶蛋白与配体结合的效果越好。
图9.抗体结合SEMG1和SEMG2多肽的鉴定热图。(A)在用多肽(表)以及SEMG1蛋白(阳性对照)包被96孔微量板后，分别加入2.5ug/ml的相应的29株杂交瘤细胞株产生的单克隆抗体，经过常规ELISA后，用兔抗鼠HRP二抗进行检测OD值。抗体结合多肽的对应值高于背景值3倍以上的，认为是抗体特异结合的多肽。所有全长SEMG1包被的反应孔读值均达到机器可读到的最高值。利用graphad prism绘制热图，多肽序列肽标记为纵坐标，抗体反应为纵坐标标记，每一个纵横坐标交汇处为一种多肽对应一种抗体的反应值。从热图可以看到：分离得到的抗体主要是结合BSA-S1、S1-13、S1-14、S1-19、S1-26、S1-40。其中10个单克隆抗体结合S1-26多肽，表明这一段多肽的序列为产生SEMG1单抗的主抗原决定簇，是SEMG1刺激机体产生抗体的关键区段。(B)SEMG1-26(S1-26)表位肽、SEMG2对应的表位肽(KDIFITQDELLVYNK)、SEMG1-S14(S1-14)、SEMG1-S40(S1-40)多肽包被ELISA板，再以图中所示的不同抗体克隆进行结合与检测。结果表明，能够结合SEMG1-26(S1-26)的抗体与SEMG2对应的表位肽(即氨基酸序列合SEMG1高度相近的区域序列KDIFITQDELLVYNK)亦存在显著的结合。这表明，SEMG1和SEMG2的优势表位结合相同的抗体克隆。
图10.针对SEMG1与SEMG2的抗体促进PBMC对肿瘤细胞系的杀伤作用。经过激活的PBMC人外周血单核细胞分别和HCT116以及过表达SEMG1与SEMG2的HCT116细胞共培养，同时加入不同的抗体：不相关的鼠IgG、针对SMEG2的MM02鼠抗、与SEMG1和SEMG2结合的鼠单克隆抗体33-B5-C1-F5与25-E10-F8-B5。纵坐标是凋亡肿瘤细胞的百分比；横坐标是不同的实验处理条件，即加入的不同细胞与抗体。结果表明，过表达SEMG1或SEMG2能够在一定程度上抑制结合激活的PBMC对肿瘤细胞的杀伤作用，而双结合SEMG1和SEMG2的鼠单克隆抗体33-B5-C1-F5与25-E10-F8-B5显著促进了T细胞对肿瘤的杀伤，且优于SEMG2的单克隆抗体MM02，而对照的不相关mIgG未显示此功能。这表明SEMG1与SEMG2表达对肿瘤细胞的免疫逃逸功能非常重要，针对SEMG1与SEMG2的抗体在免疫系统杀伤肿瘤细胞中具有重要作用。
图11.抗体在小鼠体内模型对成瘤的抑制作用。NPSG小鼠接种PBMC后,第3天接种天然表达SEMG1与SEMG2的人乳腺癌细胞MCF7成瘤后，将小鼠分组，分别用SEMG1单克隆抗体33-B5-C1-F5、SEMG2单克隆抗体MM02以及PBS作为肿瘤抑制剂对成瘤小鼠进行治疗。图示表明单克隆抗体33-B5-C1-F5对肿瘤的抑制作用显著强于PBS对照组，且治疗效果优于SEMG2单克隆抗体MM02，显示SEMG1单克隆抗体对表达SEMG1与SEMG2肿瘤的治疗效果。
实施例
下述实施例中的实验方法，如无特殊说明，均为常规方法。将参照下述非限制性实验实施例进一步理解本发明。这些实施例仅用于说明目的，并不限制本文提供的权利要求的范围。
实施例1：SEMG1或SEMG2与受体蛋白LILRB2、TIGIT或LILRB4蛋白的结合的检测
利用ELISA实验分析SEMG1或SEMG2与受体蛋白CD27、LILRB2、TIGIT或LILRB4的结合与结合强度。具体地，使用ELISA专用板(costar，ME，美国)。首先用含终浓度分别为10ug/mL、3.3ug/mL、1.1ug/mL的SEMG1或SEMG2重组蛋白的ELISA包被液(Solarbio， 北京，中国)复孔包板，设两组，阴性对照采用不含蛋白的100微升包被液。4℃包被过夜。PBST清洗后用100微升5％溶于PBS的脱脂乳(生工，上海，中国)进行封闭，温箱37℃封闭90分钟。PBST清洗后，再以用相应浓度的受体蛋白(CD27、LILRB2、TIGIT或LILRB4，以及作为阴性对照的SIRP，其中SEMG2与作用组作为阳性对照)胞外区蛋白质片段(百普赛斯或义翘神州，北京，中国)PBS溶液中进行孵育结合，上述受体蛋白带有hFc标签(P-Fc)，温箱37℃结合60分钟。PBST清洗后，用PBS稀释的HRP标记的特异性抗人Fc段抗体(Abcam，MA，美国)进行孵育结合，温箱37℃结合30分钟。PBST清洗后，用显色液(生工，上海，中国)进行显色，每孔100微升，置于温箱反应5-30分钟，再予以50微升终止液(生工，上海，中国)，置于酶标仪(赛默飞，MA，美国)下450nm显色读数。
结果表明：SEMG1能够结合LILRB2、TIGIT或LILRB4，证明LILRB2、TIGIT或LILRB4为SEMG1或SEMG2的结合受体。SEMG1结合LILRB2、TIGIT或LILRB4的浓度梯度效应如图1所示。SEMG2结合LILRB2、TIGIT或LILRB4的浓度梯度效应如图2所示。
实施例2：SEMG1与SEMG2在不同来源肿瘤组织中表达的检测
为分析SEMG1与SEMG2在肿瘤组织中的分布情况，将肿瘤病人的胃壁和胃肿瘤组织提取物、胃壁和胃肿瘤组织提取物、结直肠和结直肠组织提取物裂解制得的蛋白悬液(西格生物，上海，中国)进行SEMG1与SEMG1蛋白表达的检测，并设正常人的相应组织提取物的裂解蛋白悬液作为对照。具体地，按照说明书于胶板(伯乐，CA，美国)中配置10％PAGE凝胶(雅酶，上海，中国)，将配置好的胶置于电泳槽(伯乐，CA，美国)中，接好电源(伯乐，CA，美国)以80伏恒压的条件让所条带跑完浓缩胶，以120伏恒压的条件跑完分离胶。待条带跑至分离胶底部时，运用湿法转膜，转膜槽(伯乐，CA，美国)350毫安恒流90分钟转膜。待转膜完成后，以SEMG1与GAPDH蛋白的质量大小进行剪膜处理。用快速封闭液(雅酶，上海，中国)封闭十分钟，用SEMG1与SEMG2共反应抗体(制备方式如下所述)、GAPDH抗体(康臣，上海，中国)分别孵育相应条带，四度过夜。次日经TBST清洗后，用5％溶于TBS的脱脂奶粉(生工，上海，中国)稀释的特异性抗鼠二抗或者抗兔二抗(康臣，上海，中国)室温孵育一小时，经TBST清洗后，置于混合发光液(圣尔，上海，中国)中一分钟，于凝胶成像仪(伯乐，CA，美国)下曝光。对9份胃癌样本、3份结直肠癌样本以及10份肺癌样本的分析结果如图3所示。结果表明，在胃癌组织样本和结直肠癌样本中SEMG1与SEMG2均有不同程度的协同表达，在肺癌样品中，6份样品的SEMG1与SEMG2有协同表达，1份只表达SEMG1,2份只表达SEMG2。在分析的所有样品中SEMG1与SEMG2均表达的比率为85.7％。而在正常人的胃组织、结直肠组织以及肺组织中没有SEMG1与SEMG2的表达。上述结果表明SEMG1与SEMG2的表达可以作为肿瘤治疗的标志物用于肿瘤的诊断与作为特异性的治疗靶点。
实施例3：肿瘤细胞过表达SEMG1可显著降低人外周血单核细胞对肿瘤细胞的杀伤
将复苏的PBMC与HCT116或过表达细胞计数，分别调整细胞数为1x10 6/毫升，各加入100微升至96孔板(赛默飞，MA，美国)中，置于培养箱中孵育6小时。取出96孔板，将各孔细胞置于EP管(Axygen，CA,美国)中，400rcf离心5分钟后弃去上清。用500微升细胞染色缓冲液(Invitrogen，CA，美国)重悬洗涤细胞，重复离心与洗涤。用细胞染色缓冲液按1:20稀释CD45-APC抗体(Invitrogen，CA，美国)，各EP管加入200微升混合液重悬，室温孵育30分钟。400rcf离心5分钟后弃去上清，用1毫升binding buffer(美仑，上海，中国)重悬清洗细胞；重复离心与洗涤。设置各实验和对照组，依据条件分别加入100微升binding buffer，5微升Annexin V-FITC(美仑，上海，中国)与10微升PI(美仑，上海，中国)，室温孵育15分钟，再各自加入400微升binding buffer，转移至流式管(Falcon，NY，美国)中上机(美天旎，科隆，德国)测试，结果如图4所示，表明SEMG1过表达抑制了PBMC对肿瘤细胞的杀伤作用。
实施例4：SEMG1诱导PBMC髓系来源的抑制性细胞(MDSC)以及调节性T细胞(Treg)的转化
为了阐明SEMG1对Treg/MDSC的影响，首先进行细胞的准备1.5*10 5每孔的PBMC，进行不同的实验分组，培养24h(用于检测Treg比例)或3天(用于检测MDSC比例)。通过流式细胞术分析表型CD4+CD25+Foxp3+、HLA-DR-CD11b+CD33+比例、计数。
分析结果表明，相对于普通的HCT116细胞，表达SEMG1的HCT116细胞与人外周血单核细胞共培养，增加了MDSC细胞(HLA-DR-CD11b+CD33+)和Treg(CD4+CD25+Foxp3+)的比率，结果如图5所示。
实施例5：鼠杂交瘤单克隆抗体的制备
将SEMG1蛋白用大肠杆菌系统进行表达和纯化，经过检测达到92％纯度，并利用ELISA实验验证了SEMG1蛋白质具备结合受体与LILRB2的活性。利用蛋白免疫10只小鼠，进行多次免疫以增强效果：(1)初次免疫，抗原50μg/只，加福氏完全佐剂皮下多点注射，间隔3周；(2)第二次免疫，剂量途径同上，加福氏不完全佐剂，间隔3周；(3)第三次免疫，剂量途径同上，并同时利用SEMG2蛋白进行交叉免疫，不加佐剂，腹腔注射，间隔3周；(4)加强免疫，剂量50μg，腹腔注射。最后一次注射3天后采血测其效价，检测免疫效果。对免疫效价滴度达标的小鼠处死，收集脾脏和淋巴结进行融合以产生杂交瘤。杂交瘤在96孔组织培养板中生长，并通过ELISA对各个孔的上清液进行筛选，以鉴定目标抗原SEMG1与SEMG2蛋白的阳性结合物。亚克隆后通过ELISA检测单克隆抗体与SEMG1与SEMG2的结合。
实施例6：过表达SEMG1的HCT116-SEMG1细胞系的构建
将SEMG1与SEMG2克隆到pCDNA3.1载体，转染DH5α后，制备转染用无菌质粒。将5-10×106细胞接种于6孔细胞板中，用含10％小牛血清的培养基培养过夜，约60％～80％铺满时用于转染。将培养板中的上清弃去，用细胞用无抗无血清培养基洗涤三次后，每孔加入800微升培养基，加入复温的FuGENE HD转染试剂，在100微升反应体系中加入培养基90ul，浓度为500ng/ul的质粒4ul，振荡器混匀；随后加入6ul FuGENE HD转染试剂，立即震荡混匀，室温静置7-12分钟后，将混合液均匀滴加在细胞平板孔中，吹打混匀或者震荡混匀。放入37℃培养箱孵育。转染2小时后加入含有血清的培养基，使血清终浓度达到1-4％，上清总量达到1.5毫升。细胞平板放入37℃培养箱培养24-48h后，加入600ug/mL的G418筛选压力进行筛选。并设转染pCDNA3.1质粒的对照以及空转对照。待空转对照细胞死亡90％以上时，观察转染组的细胞状态，并换液，持续用600ug/mL的G418的培养基进行筛选。待转染细胞稳定增殖后，用300ug/mL的G418的培养基进行维持。用4℃预冷的PBS漂洗细胞3次，加入含有PMSF的Western细胞裂解液冰上裂解15分钟。随后刮取细胞转移至1.5ml EP管中，12,000rpm离心10分钟，取上清加入5×SDS上样缓冲液，混匀后置于沸水浴中煮沸10分钟。SDS-PAGE后进行western blotting鉴定。
实施例7：ELISA与BLI生物膜干涉法检测鼠杂交瘤单克隆抗体结合SEMG1与SEMG2的强度
为了鉴定筛选到的具备中和作用的单克隆抗体有能否结合SEMG1与SEMG2蛋白，我们将稀释于碳酸盐缓冲液(Solarbio，北京，中国)至终浓度为1ug/mL的SEMG2蛋白包被在专用的ELISA专用板(costar，ME，美国)，初步测试1ug/mL鼠杂交瘤抗体对SEMG2结合情况。根据测定情况，利用ELISA实验进一步分析了SEMG1或SEMG2与纯化的鼠单克隆抗体的的结合与结合强度，取无关鼠单克隆抗体作为阴性对照。具体地，使用ELISA专用板(costar，ME，美国)。首先用不同浓度的SEMG1或者SEMG2重组蛋白各溶于100微升碳酸盐包被缓冲液(Solarbio，北京，中国)至2微克/毫升包板，阴性对照采用不含蛋白的100微升包被液包板。4℃包被过夜。PBST清洗后用100微升5％溶于PBS的脱脂乳(生工，上海，中国)进 行封闭，温箱37℃封闭90分钟。PBST清洗后，再以在PBS中从10ug/mL开始进行3倍浓度梯度稀释的单克隆抗体37℃孵育60分钟。PBST清洗后，用PBS稀释的HRP标记的特异性羊抗鼠抗体(JacksonIR，PA，美国)进行孵育结合，温箱37℃结合30分钟。PBST清洗后，用显色液(生工，上海，中国)进行显色，每孔100微升，置于温箱反应5-30分钟，加入50微升终止液终止反应(生工，上海，中国)，然后置于酶标仪(赛默飞，MA，美国)下450nm显色读数。利用graphpad prism绘制抗体结合包被蛋白的浓度效应曲线，结果显示对SEMG1结合良好的单克隆抗体对SEMG2均具有良好的的结合能力，如图6所示。
表1.ELISA检测抗原抗体结合SEMG1和SEMG2的EC50数值
Figure PCTCN2022079756-appb-000002
实施例8：流式细胞分析技术检测SEMG1鼠杂交瘤单克隆抗体结合HCT116-SEMG1过表达细胞的结合阳性率
将新鲜培养的生长状态良好的HCT116或过表达细胞的培养基弃掉，加入PBS洗一下，然后加入适量0.25％胰酶，上下轻轻晃动，直至观察细胞有要掉落的状态，立即加入完全培养基中和胰酶，并将细胞放入15ml离心管中，800rpm，离心4min。弃上清，加入PBS重悬细胞，800rpm，离心4min，重复一次。计数，分别调整细胞数适量。用eBioscience Flow Cytometry Staining Buffer(Thermo fisher)将细胞重悬后均匀分装至1.5ml EP管中，加入制备的鼠单克隆抗体以终浓度为5ug/mL孵育1小时。然后400rpm，4℃离心5min弃上清，加入PBS重悬细胞，重复一次。用eBioscience Flow Cytometry Staining Buffer(Thermo fisher)按照1:400稀释兔抗鼠FITC二抗(Abcam)，配制好后均匀分至各反应管，然后放至室温摇床慢摇45min。400rpm，4℃离心5min弃上清，加入PBS重悬细胞，再次离心后用400微升binding buffer，转移至流式管(Falcon，NY，美国)中上机(美天旎，科隆，德国)测试，结果如图7所示。
实施例9：抗体结合SEMG1多肽与关键表位的鉴定
设计1/3重叠的多肽序列，合成由45条多肽组成的多肽库(南京金斯瑞)。合成的多肽序列如表2所示。利用多肽包被96孔板(Costar，康宁)制备肽矩阵，利用选定的单克隆抗体作为一抗进行ELISA结合试验，并设定SEMG1包被作为阳性对照，BSA包被作为对照。ELISA程序按照常规操作，如上述实施例2中所述。对于OD实验组/OD空白组>3的包被多肽孔判定为是阳性反应，为对应的抗体结合的多肽序列。根据抗体可结合的多肽，对应抗体的分类如表3所示。
表2.合成多肽序列与对应SEMG1蛋白的氨基酸位置
Figure PCTCN2022079756-appb-000003
Figure PCTCN2022079756-appb-000004
表3.根据结合的多肽对单克隆抗体分类
Figure PCTCN2022079756-appb-000005
实施例10：鼠杂交瘤单克隆抗体阻断SEMG1或SEMG2与受体CD27、LILRB2、LILRB4或TIGIT结合的ELISA检测
为了筛选鉴定能够阻断SEMG1或SEMG2功能的抗体，利用ELISA实验分析了抗体对于SEMG1或SEMG2与其受体受体CD27、LILRB2、LILRB4或者TIGIT结合的影响。具体地，将SEMG1或SEMG2重组蛋白包被在96孔微量板板上，并加入不同浓度的抗体(0，1.1，3.3，10.0微克每毫升)，进一步加入相应的重组受体(人类抗体Fc段或生物素)，再以抗人二抗偶联辣根过氧化物酶(Jacksonimmuno，美国)或亲和素HRP(thermo Fisher，美国)，最后以TMB底物显色，检测450nm的吸收值以表明SEMG1或SEMG2与受体CD27、LILRB2、LILRB4或者TIGIT的结合程度。根据公式1-(OD 测试组-OD 空白)/(OD 阳性对照-OD 空白)计算阻断百分比。
如图8所示，抗体对SEMG1或SMEG2与其受体受体CD27、LILRB2、LILRB4或者TIGIT的结合显示出抑制作用，且抑制的程度与抗体浓度相关，浓度愈高抑制程度越显著。
实施例11：单克隆抗体V-区测序
使用Trizol TM试剂(Invitrogen)从分泌抗人SEMG1的杂交瘤细胞中提取总RNA,用家族特异性引物进行逆转录多聚酶链反应(RT-PCR),分别扩增出重链可变区(VH)基因及轻链可变区(VL)基因.RT-PCR产物通过琼脂糖凝胶电泳分离，将具有预测大小的DNA凝胶纯化并在正向和反向方向上测序。将PCR产物克隆至pGEM-T载体,然后转化JM109细菌。挑取单个个体菌落的使用通用引物用于测序。测序结果分析后，进一步地，将获得的编码所选定的前导抗体的重链可变区的DNA克隆至具有鼠IgG1恒定区的符合读框的pcDNA3.4-mIgG1中，将编码轻链可变区的DNA克隆至具有鼠κ恒定区的符合读框的轻链表达质粒pcDNA3.4-mκc中。提取质粒转染293E细胞表达验证活性后，确定的单克隆抗体的VH与VL的氨基酸序列如表4所示。
表4单克隆抗体的VH与VL序列
Figure PCTCN2022079756-appb-000006
在abysis网站线上对单克隆抗体的VH与VL序列进行CDR区分析，定义的CDR区氨基酸序列如表5所示。
表5单克隆抗体的CDR氨基酸序列
Figure PCTCN2022079756-appb-000007
实施例12：SEMG1单克隆抗体对肿瘤细胞的体外抑制作用
为了鉴定所述的SEMG1抗体对表达SEMG1或者SEMG2的肿瘤细胞的的抑制作用，进行了下述的体外实验：选取HCT116-SEMG1或HCT116-SEMG2肿瘤细胞，先与抗体或对照鼠IgG孵育，再与预先经过CD3和CD28激活的PBMC共培养12小时，利用Annexin-V标记的流式细胞数分析CD45-的肿瘤细胞凋亡比率。结果表明过表达SEMG1或SEMG2对激活的PBMC杀伤HCT116肿瘤细胞有抑制作用。SEMG1单克隆抗体作用于过表达SEMG1或SEMG2的肿瘤细胞对PBMC杀伤效应具有促进作用，本公司以前分离到的SEMG2特异性抗体MM02对促进PBMC杀伤的作用弱于本案中的抗体，阴性对照单克隆抗体则没有促进杀伤作用。结果如图10所示。这表明，SEMG1抗体是通过特异性抑制SEMG1或者SEMG2的功能而发挥作用；结合SEMG1与SEMG2的杂交瘤单克隆抗体对PBMC杀伤的作用强于以前报道的SEMG2的杂交瘤单克隆抗体；SEMG1或SEMG2的表达对抗体作用杀伤肿瘤细胞具有重要意义。
实施例13：SEMG1单克隆抗体对肿瘤细胞在体内的抑制作用
为了鉴定所述的SEMG1抗体对表达SEMG1与SEMG2的肿瘤细胞的的抑制作用，进行了成瘤的小鼠体内实验。将12只6-8周龄雄性NPSG小鼠模型称重。将表达SEMG1与SEMG2的人乳腺癌细胞MCF7进行体外培养，获得1.8×10 8细胞。小鼠接种PBMC后,第3天接种肿瘤细胞，其后每周一次测定小鼠血液中hCD45+细胞比例及体重。接种后，每周1次测量肿瘤体积，当平均肿瘤体积达到约40-80mm 3时测量小鼠血液中hCD45+细胞比例。依据肿瘤体积和小鼠血液中hCD45+细胞比例，小鼠随机分组，随即开始给药。给药开始日期视为第0天。给药方案：SEMG1抗体腹腔注射5mg/kg每周三次。给药开始后，小鼠每周观测瘤体生 长状况，瘤体生长后，每周3次测量体重和瘤体积，每周3次流式监控小鼠血液中hCD45+细胞相对计数。当肿瘤体积达到终点标准后，取血检测同上指标，结束实验。对小鼠的观察包括：日常观察，接种后，每工作日观察动物发病及死亡情况。肿瘤体积测量：接种后至分组前，当肿瘤可见时，每周一次测量实验动物肿瘤体积，接种分组后，实验中的动物肿瘤体积每周测量2次。肿瘤体积测量采用双向测量法，首先利用游标卡尺测量肿瘤长短径，再使用公式TV＝0.5*a*b2计算肿瘤体积。其中a是肿瘤的长径，b是肿瘤的短径。结果显示：对于表达SEMG1与SEMG2的MCF7人乳腺癌细胞比较于不加抗体组，本案筛选得到的SEMG1抗体对小鼠体内的肿瘤生长产生了显著的抑制作用，且优于针对SEMG2的特异性抗体MM02对肿瘤的抑制作用(图11)。这表明，SEMG1抗体可用于在实验动物体内的接种的表达SEMG1和/或SEMG2肿瘤的抑制，且表现了较好的抑制效果。这表明表达SEMG1和/或SEMG2的肿瘤细胞对SEMG1抗体的反应更显著，特定靶蛋白SEMG1或者SEMG2的表达对于SEMG1抗体有针对性的施用具有重要的意义。
前述对本发明的具体示例性实施方式的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式，并且很显然，根据上述教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方式以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。

Claims (30)

  1. 一种结合精液凝固蛋白(Semenogelin)的特异性抗体，其特征在于，能够以高亲和力结合SEMG1与SEMG2蛋白，并调节SEMG1或SEMG2与其任一受体(LILRB2、LILRB4、TIGIT或CD27)的结合；所述的高亲和力定义为，通过ELISA检测得出的抗体结合SEMG1的半数有效浓度(EC50)值低于3.7纳摩尔(nanomolar)，且结合SEMG2的EC50值低于2.4纳摩尔，或亲和力高于通过其它方法检测得出的与EC50数值相应的亲和力水平。
  2. 如权利要求1所述的结合精液凝固蛋白的特异性抗体，其优选的表位包括来自SEMG1或SEMG2的下列氨基酸：KDIFSTQDELLVYNK(SEMG1第251-265氨基酸，Seq ID NO：26)；KDIFITQDELLVYNK(SEMG2第251-265氨基酸，SEQ ID NO:70)；GHFHRVVIHHKGGKA(SEMG1第121-135氨基酸，Seq ID NO：13)；KGGKAHRGTQNPSQD(SEMG1第131-145氨基酸，Seq ID NO：14)、AQKGRKQGGSQSSYV(SEMG1第181-195氨基酸，Seq ID NO：19)、或PNPKQEPWHGENAKG(SEMG1第391-405氨基酸，Seq ID NO：40)。
  3. 一类抗体，其与具有高变区序列(Seq ID NO：47-54)的抗体竞争结合SEMG1或SEMG2。
  4. 用于鉴定权利要求1、2所述的抗体的方法，其特征在于，至少分析候选抗体的以下一种效能：抗体结合分离的SEMG1和SEMG2蛋白的强度；抗体结合表达在细胞表面的SEMG1和SEMG2蛋白的强度；抗体对于SEMG1结合其受体CD27的影响；抗体对于SEMG1结合其受体LILRB2的影响；抗体对于SEMG1结合其受体TIGIT的影响；抗体对于SEMG1结合其受体LILRB4的影响；抗体对于SEMG2结合其受体LILRB2的影响；抗体对于SEMG2结合其受体TIGIT的影响；抗体对于SEMG2结合其受体LILRB4的影响；抗体对髓系来源的抑制性细胞(MDSC)的影响；抗体对调节性T细胞(Treg)的影响；抗体对免疫细胞杀伤肿瘤细胞的影响。
  5. 一种用于判断是否施用权利要求1-3所述抗体的生物标志物，由SEMG1与SEMG2的表达构成的组合，其具体使用步骤为：检测SEMG1或SEMG2的mRNA或蛋白在肿瘤细胞或个体血液样本中的表达；在SEMG1或SEMG2的表达高于正常细胞或个体的情况下，使用权利要求1-3所述的抗体与肿瘤细胞在体外或体内接触；在使用抗体后检测SEMG1或SEMG2的表达，用于评估抗体的抑制效果。
  6. 一种在体外或体内抑制肿瘤细胞的方案，其特征在于，包括以下步骤：采用权利要求5所述的生物标志物进行分析；在适合的情况下采用权利要求1-3所述的抗体与肿瘤细胞表达的SEMG1或SEMG2接触。
  7. 在体外和体内抑制肿瘤细胞的方法，其特征在于，抑制SEMG1或SEMG2的功能或活性，包括但不限于以下方式：对SEMG1或SEMG2基因进行编辑；干扰SEMG1或SEMG2基因表达；通过抗体抑制SEMG1或SEMG2蛋白的功能或活性。
  8. 一种调节体外或体内免疫细胞状态的方法，其特征在于，促进或抑制SEMG1或SEMG2与受体CD27、LILRB2、LILRB4或TIGIT的结合作用。
  9. 一种在体外或体内调控MDSC或Treg细胞比率的方法，其特征在于，促进或抑制SEMG1或SEMG2与受体CD27、LILRB2、LILRB4或TIGIT的结合作用。
  10. 一种在体外或体内调控细胞中CD27、LILRB2、LILRB4或TIGIT受体活性状态的方法，其特征在于，促进或抑制SEMG1或SEMG2与受体CD27、LILRB2、LILRB4或TIGIT的结合作用。
  11. 一种筛选用于预防或治疗肿瘤的药物或试剂的方法，其特征在于，通过筛选抑制SEMG1或SEMG2和受体CD27、LILRB2、LILRB4或TIGIT相互作用的抑制剂或抗体，获 得候选药物或试剂。
  12. 一种激动或拮抗化合物，其特征在于，所述化合物能够调节SEMG2与受体CD27、LILRB2、LILRB4或TIGIT的相互作用；
    优选的，所述化合物为小分子抑制剂、多肽、抗体或抗原结合片段。
  13. 权利要求1-3所述的抗体如下任一用途：
    在拮抗SEMG1或SEMG2和CD27、LILRB2、LILRB4或TIGIT相互作用中的用途，优选的，SEMG1或SEMG2表达于肿瘤细胞，CD27、LILRB2、LILRB4或TIGIT表达于免疫细胞；
    在制备用于拮抗SEMG1或SEMG2和CD27、LILRB2、LILRB4或TIGIT相互作用的产品中的用途，优选的，SEMG1或SEMG2表达于肿瘤细胞，CD27、LILRB2、LILRB4或TIGIT表达于免疫细胞；
    在预防或治疗肿瘤中的用途；
    在制备用于预防或治疗肿瘤的药物中的用途；
    在调节针对肿瘤所引起的免疫反应中的用途；
    在制备用于调节针对肿瘤所引起的免疫反应的药物中的用途；
    在体内外抑制肿瘤细胞生长中的用途；
    在制备用于体内外抑制肿瘤细胞生长的试剂中的用途；
    优选的，所述抑制剂能够抑制或阻断SEMG1或SEMG2与其受体CD27、LILRB2、LILRB4或TIGIT的结合；
    更优选的，所述抑制剂为小分子抑制剂、多肽、抗体或抗原结合片段。
  14. 一种预防或治疗肿瘤的方法，其包括：
    使受试者的免疫细胞或肿瘤细胞与有效剂量的SEMG1或SEMG2抗体接触；所述抗体能够抑制SEMG1或SEMG2与其受体CD27、LILRB2、LILRB4或TIGIT结合；
    优选的，所述受试者已经接受或正在接受或将要接受额外的抗癌疗法；
    更优选的，所述额外的抗癌疗法包括手术、放疗、化疗、免疫疗法或激素疗法。
  15. 一种肿瘤免疫治疗伴随诊断方法，其包括：检测经免疫治疗前/后受试者肿瘤细胞的SEMG1或SEMG2蛋白表达量或功能。
  16. 一种分离的抗体或其抗原结合片段，其特征在于：
    a)所述抗体或其抗原结合片段以高亲和力特异性结合SEMG1或SEMG2蛋白；且
    b)所述抗体能够抑制或阻断SEMG1或SEMG2与CD27、LILRB2、LILRB4或TIGIT中任一之间的相互作用；
    优选的，所述抗体还包含至少一项下列性质：
    i.所述抗体降低免疫抑制性细胞MDSC细胞比率；
    ii.所述抗体降低免疫抑制性细胞Treg的细胞比率；或
    iii.所述抗体增强免疫细胞(如PBMC)对肿瘤细胞的杀伤作用。
  17. 一种分离的抗体或其抗原结合片段，其特征在于，包含SEQ ID NO:47、49、51、53所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:48、50、52、54所示的轻链可变区氨基酸序列中的3个CDR；或者与上述6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体。
  18. 权利要求17所述抗体或抗原结合片段，其特征在于，包含根据通用方法定义的HCDR1、HCDR2和HCDR3序列的重链可变区；以及包含根据通用方法定义的LCDR1、LCDR2和LCDR3序列的轻链可变区，所述抗体或抗原结合片段包含如下CDR序列：
    所述HCDR1的氨基酸序列如SEQ ID NO：55、61、65任一所示；
    所述HCDR2的氨基酸序列如SEQ ID NO：56、62、66任一所示；
    所述HCDR3的氨基酸序列如SEQ ID NO：57、67任一所示；
    所述LCDR1的氨基酸序列如SEQ ID NO：58、63、68任一所示；
    所述LCDR2的氨基酸序列如SEQ ID NO：59所示；
    所述LCDR3的氨基酸序列如SEQ ID NO：60、64、69任一所示；或者与上述i-vi的6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体；
    优选的，所述抗体或其抗原结合片段与SEMG1或SEMG2的结合KD<2×10 -8；
    更优选的，所述KD<2×10 -8、<1×10 -8、<9×10 -9、<8×10 -9、<7×10 -9、<6×10 -9、<5×10 -9、<4×10 -9、<3×10 -9、<2×10 -9、<1×10 -9、<1×10 -10。
  19. 权利要求17-18所述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可变区和轻链可变区，序列选自如下：
    a)所述重链可变区的氨基酸序列包含选自由SEQ ID NO:47、49、51、53所组成的组中的氨基酸序列或与组中的序列具有至少70％、80％、90％、95％或99％序列同一性；
    b)所述轻链可变区的氨基酸序列包含选自由SEQ ID NO:48、50、52、54所组成的组中的氨基酸序列或与组中的序列具有至少70％、80％、90％、95％或99％序列同一性。
  20. 权利要求16-19所述的抗体或抗原结合片段，其特征在于，所述抗体可进一步包含连接至多肽的偶联部分，所述偶联部分选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白、脂类、和生物素中的一种或多种，其中所述多肽或抗体与所述偶联部分可选择性通过连接子相连，优选的，所述连接子为肽或多肽；
    更优选的，所述抗体选自单克隆抗体、多克隆抗体、抗血清、嵌合抗体、人源化抗体和人抗体；更优选的，所述抗体选自多特异性抗体、单链Fv(scFv)、单链抗体、抗独特型(抗-Id)抗体、双抗体、微型抗体、纳米抗体、单结构域抗体、Fab片段、F(ab’)片段、二硫化物连接的双特异性Fv(sdFv)和胞内抗体。
  21. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求17-20任一所述的抗体或抗原结合片段。
  22. 一种重组载体，其包含权利要求21所述的多核苷酸，以及任选的调控序列；
    优选的，所述重组载体为克隆载体或表达载体；
    更优选的，所述调控序列选自前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号序列、转录终止子，或其任何组合。
  23. 一种宿主细胞，其特征在于，包含权利要求22所述的重组载体；
    优选的，所述宿主细胞为原核细胞或真核细胞。
  24. 一种药物组合物，其特征在于，包含如权利要求17-23任一所述的抗体或抗原结合片段、多核苷酸、重组载体和宿主细胞中的一种或更多种；
    优选的，所述组合物还包含药学上可接受的载体或辅料。
  25. 一种试剂盒，其特征在于，包含如权利要求17-24任一所述的抗体或抗原结合片段、多核苷酸、重组载体、宿主细胞和药物组合物中的一种或更多种，并容纳于合适的容器中。
  26. 权利要求17-24任一所述的抗体或抗原结合片段、多核苷酸、重组载体、宿主细胞和药物组合物的如下任一应用：
    在激动或拮抗SEMG1或SEMG2与其受体CD27、LILRB2、LILRB4或TIGIT相互作用中的用途；优选SEMG1或SEMG2表达于肿瘤细胞，CD27、LILRB2、LILRB4或TIGIT表达于免疫细胞；
    在制备用于激动或拮抗SEMG1或SEMG2和其受体CD27、LILRB2、LILRB4或TIGIT相互作用的产品中的用途；选SEMG1或SEMG2表达于肿瘤细胞，CD27、LILRB2、LILRB4或TIGIT表达于免疫细胞；
    在预防或治疗肿瘤中的用途；
    在制备用于预防或治疗肿瘤的药物中的用途；
    在调节针对肿瘤所引起的免疫反应中的用途；
    在制备用于调节针对肿瘤所引起的免疫反应的药物中的用途；
    在体内外抑制肿瘤细胞生长中的用途；
    在制备用于体内外抑制肿瘤细胞生长的试剂中的用途。
  27. 一种预防或治疗肿瘤的方法，其特征在于，包括：
    使受试者的免疫细胞和肿瘤细胞与有效剂量的权利要求17-24任一所述的抗体或抗原结合片段、多核苷酸、重组载体、宿主细胞和药物组合物接触；
    优选的，在使用有效量的化合物和受试者的免疫细胞和/或肿瘤细胞接触前，检测SEMG1或SEMG2在肿瘤细胞的表达；
    优选的，所述免疫细胞为淋巴细胞，更优选的，为T淋巴细胞；
    进一步优选的，所述受试者已经接受或正在接受或将要接受额外的抗癌疗法；
    更进一步优选的，所述额外的抗癌疗法包括手术、放疗、化疗、免疫疗法或激素疗法。
  28. 一种体内外检测生物样本中SEMG1或者SEMG2存在与否的方法，其特征在于，使生物样本与权利要求17-24任一所述的抗体或抗原结合片段、多核苷酸、重组载体、宿主细胞和药物化合物接触。
  29. 一种抑制肿瘤细胞生长的方法，包括以下步骤：
    A)分析SEMG1或SEMG2在肿瘤细胞的表达；
    B)利用可以识别SEMG1或SEMG2的抗体与肿瘤细胞接触，所述的抗体与SEMG1或SEMG2的结合KD<2×10 -8；优选的，所述KD<2×10 -8、<1×10 -8、<9×10 -9、<8×10 -9、<7×10 -9、<6×10 -9、<5×10 -9、<4×10 -9、<3×10 -9、<2×10 -9、<1×10 -9、<1×10 -10；
    C)使T淋巴细胞、所述抗体和肿瘤细胞相接触。
  30. 前述任一权利要求，其特征在于，所述肿瘤选自结直肠癌、肺癌、乳腺癌、黑色素瘤、淋巴瘤、肝癌、头颈癌、胃癌、肾癌、膀胱癌、前列腺癌、睾丸癌、子宫内膜癌、乳腺癌和卵巢癌。
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