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WO2022111425A1 - 与cldn18.2特异性结合的分子 - Google Patents

与cldn18.2特异性结合的分子 Download PDF

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WO2022111425A1
WO2022111425A1 PCT/CN2021/132218 CN2021132218W WO2022111425A1 WO 2022111425 A1 WO2022111425 A1 WO 2022111425A1 CN 2021132218 W CN2021132218 W CN 2021132218W WO 2022111425 A1 WO2022111425 A1 WO 2022111425A1
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amino acid
seq
acid sequence
sequence shown
antibody
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PCT/CN2021/132218
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French (fr)
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谭永聪
郎国竣
刘婵娟
闫鑫甜
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三优生物医药(上海)有限公司
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Priority to EP21896926.9A priority Critical patent/EP4253411A4/en
Priority to US18/254,206 priority patent/US20240092891A1/en
Priority to CN202180078658.2A priority patent/CN116568809A/zh
Priority to AU2021387558A priority patent/AU2021387558A1/en
Priority to JP2023532547A priority patent/JP2023551027A/ja
Priority to CA3200052A priority patent/CA3200052A1/en
Publication of WO2022111425A1 publication Critical patent/WO2022111425A1/zh

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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • 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

  • This application relates generally to antibodies. More specifically, the present application relates to molecules that specifically bind to CLDN18.2, methods for their preparation and uses thereof.
  • Tight junctions also known as occlusive junctions, are structures formed between endothelial or epithelial cells that prevent interstitial substances from diffusing from the intercellular space and can only enter cells by active transport. Tight junction structures are formed by dozens of Claudin proteins through intracellular and intercellular protein interactions, and the expression of these proteins is tissue-specific. CLDN18 is an important protein in Claudin proteins present in tight junctions.
  • CLDN18 is a membrane protein with a four-transmembrane domain containing two extracellular domains.
  • CLDN18.1 and CLDN18.2 also known as "CLD18A2"
  • the two are distributed in different tissues, the former is mainly expressed in lung epithelial cells, and the latter is specifically expressed in gastric epithelial cells, but not in gastric stem cells.
  • CLD18A2 the sequence similarity between CLDN18.1 and CLDN18.2 is extremely high. The main difference between the two is at the N-terminus. There is only 8 amino acid difference between the two in the first extracellular domain. The C-terminus sequence is completely Consistent.
  • Antibody therapy is emerging as one of the most promising ways to treat cancer patients. Due to its specific expression in tumor cells and normal tissues, CLDN18.2 has become a very potential antibody drug target. However, due to the extremely high sequence similarity between CLDN18.1 and CLDN18.2, it is extremely difficult to develop antibodies against CLDN18.2 but not CLDN18.1. The earlier development of antibody drugs against the CLDN18.2 target was the German company Ganymed, which developed IMAB362, a monoclonal antibody composed of two heavy chains and two light chains. However, up to now, there is no report of a clinical drug under development with a single-domain antibody targeting CLDN18.2.
  • Single domain antibody is referred to as single domain antibody (sdAb), which is an antibody composed of a single monomer variable antibody domain (such as antibody heavy chain variable domain). Like whole antibodies (eg IgG), it is capable of selectively binding to specific antigens. However, the molecular weight of single-domain antibodies is much smaller than that of common antibodies composed of two heavy and two light chains.
  • the first single domain antibodies were engineered from heavy chain antibodies found in camelid (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993). ) Naturally occurring antibodies devoid of light chains. Nature 363(6428):446–448.); heavy chain antibodies found in these camelid species are also known as VHH fragments.
  • sdAb single domain antibody
  • the present disclosure provides novel compounds, methods of making, compositions and articles of manufacture of antibodies.
  • the benefits provided by the present disclosure are broadly applicable to the fields of antibody therapy and diagnostics. More specifically, the present disclosure provides single domain antibodies targeting CLDN18.2, as well as methods of making the antibodies, expression vectors and host cells for expressing the antibodies, and the like.
  • the antibodies of the present disclosure provide methods of treating or preventing disorders associated with Claudin proteins, particularly CLDN18.2, and uses thereof.
  • the present inventors discovered a molecule that specifically binds to CLDN18.2, that is, a CLDN18.2-binding molecule that can specifically bind to the extracellular domain 1 (ECD1) of human CLDN18.2 (eg, a single domain targeting CLDN18.2). Antibody).
  • ECD1 extracellular domain 1
  • the present disclosure includes at least the following embodiments, each ordered and enumerated by "N” (where “N” represents a number).
  • N represents a number
  • a CLDN18.2 binding molecule comprising at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, and wherein
  • amino acid sequence of CDR1 and the sequence shown in SEQ ID NO: 1, 4 or 7 have differences in amino acid addition, deletion or substitution of no more than 2 amino acids;
  • CDR2 has the same amino acid sequence as SEQ ID NO: 2,
  • the sequence shown in 5 or 8 has an amino acid addition, deletion or substitution difference of no more than 2 amino acids; and/or CDR3 in the amino acid sequence and the sequence shown in SEQ ID NO: 3, 6 or 9 exist no more than 2 Differences in amino acid additions, deletions or substitutions of amino acids; or
  • CDR1 differs in amino acid sequence from the sequence shown in SEQ ID NO: 30, 37 or 45 by amino acid addition, deletion or substitution of no more than 2 amino acids
  • CDR2 differs in amino acid sequence from SEQ ID NO: 31
  • the sequence shown in 38 or 46 has an amino acid addition, deletion or substitution difference of no more than 2 amino acids
  • CDR3 in the amino acid sequence and the sequence shown in SEQ ID NO: 32, 39 or 47 exist no more than 2 Differences in amino acid additions, deletions or substitutions of amino acids.
  • the CLDN18.2 binding molecule of any one of embodiments 1-3, wherein the immunoglobulin single variable domain comprises:
  • the CLDN18.2 binding molecule of any one of embodiments 1-3, wherein the immunoglobulin single variable domain comprises:
  • the CLDN18.2 binding molecule of any one of embodiments 1-4, wherein the immunoglobulin single variable domain comprises:
  • (C) has one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) compared to SEQ ID NO: 10, 11, 12, 13, 14, or 15 Amino acid sequence of additions, deletions and/or substitutions of amino acids, preferably, the additions, deletions and/or substitutions do not occur in the CDR regions.
  • the CLDN18.2 binding molecule of any one of embodiments 1-5, wherein the immunoglobulin single variable domain comprises or consists of the following sequence:
  • CLDN18.2 binding molecule of any one of embodiments 1-6, wherein the CLDN18.2 binding molecule is a single domain antibody, eg, a heavy chain single domain antibody; a chimeric antibody; or a humanized antibody.
  • CLDN18.2 binding molecule of embodiment 8 wherein the CLDN18.2 binding molecule is a chimeric antibody comprising a VHH from camelid and the Fc domain of a human IgG (eg, human IgGl or IgG4).
  • a human IgG eg, human IgGl or IgG4
  • CLDN18.2 binding molecule of any one of embodiments 1-10, wherein the CLDN18.2 binding molecule binds the extracellular domain 1 (ECD1) of human CLDN18.2.
  • a CLDN18.2 binding molecule that competes for the same epitope as the CLDN18.2 binding molecule of any one of embodiments 1-11.
  • CLDN18.2 binding molecule of any one of embodiments 1-12, which specifically binds CLDN18.2, but does not bind CLDN18.1.
  • An expression vector comprising the isolated nucleic acid molecule of embodiment 14.
  • a host cell comprising the expression vector of embodiment 15.
  • the host cell of embodiment 16 which is a bacterial cell (eg, E. coli), a fungal cell (eg, yeast), or a mammalian cell.
  • a pharmaceutical composition comprising at least one CLDN18.2 binding molecule according to any one of embodiments 1-13 and a pharmaceutically acceptable carrier.
  • a method of treating a disorder associated with CLDN18.2 in a subject comprising: administering to the subject a therapeutically effective amount of the CLDN18.2 binding molecule of any one of embodiments 1-13.
  • the cancer comprises bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer Cancer, Stomach Cancer, Colon Cancer, Breast Cancer, Prostate Cancer, Uterine Cancer, Sexual and Reproductive Organ Cancer, Hodgkin's Disease, Esophageal Cancer, Small Intestine Cancer, Endocrine System Cancer, Thyroid Cancer, Parathyroid Cancer, Adrenal Cancer, Soft Tissue Sarcoma , bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancers, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas.
  • CNS central nervous system
  • kits for treating or diagnosing a disorder associated with CLDN18.2, comprising a container comprising the CLDN18.2 binding molecule of any one of embodiments 1-13.
  • Figures 1a and 1b show the results of flow cytometry identification of the overexpression cell lines and tumor cell lines in Example 1 using IMAB362, hIgG and anti-CLDN18 antibodies.
  • Figure 1a shows the flow cytometry identification results of human CLDN18.2-HEK293T, human CLDN18.1-HEK293, murine CLDN18.2-HEK293 and murine CLDN18.1-HEK293 overexpressing cell lines;
  • Figure 1b shows human CLDN18.
  • Figures 2a-2f show that candidate antibodies NA4-S, NA7-S and NA8-S were expressed in human CLDN18.2-HEK293T, human CLDN18.1-HEK293, murine CLDN18.2-HEK293 and murine CLDN18.1-HEK293 overexpressing cell lines Results of cross-binding experiments on .
  • Figures 3a, 3b and 3c show the results of experiments comparing the binding of candidate antibodies NA4-S, NA7-S, NA8-S and control antibodies at the cellular level.
  • Figure 3a shows the experimental results of the flow level comparison of antibodies NA4-S and NA7-S on human CLDN18.2-HEK293T overexpressing cells
  • Figure 3b shows antibody NA8-S on human CLDN18.2-HEK293T overexpressing cells
  • Figure 3c shows the results of the flow level comparison experiment of antibodies NA4-S, NA7-S, NA8-S and the control antibody on the human CLDN18.2-KATOIII tumor cell line.
  • Figures 4a-4e show complement-dependent cytotoxicity mediated by candidate antibodies NA4-S, NA7-S and NA8-S on human CLDN18.2-HEK293T overexpressing cell line and human CLDN18.2-KATOIII tumor cell line ( CDC).
  • Figures 4a, 4b and 4c show the CDC cytotoxic effects mediated by antibodies NA4-S, NA7-S and NA8-S on human CLDN18.2-HEK293T, respectively
  • Figure 4d and Figure 4e show antibodies NA7-S and NA8-S, respectively NA8-S-mediated CDC on the human CLDN18.2-KATOIII tumor cell line.
  • Figures 5a, 5b, 5c, and 5d show antibody-dependent cell-mediated cytotoxicity mediated by candidate antibodies NA7-S and NA8-S on human CLDN18.2-HEK293T and human CLDN18.2-KATOIII tumor cell lines ( ADCC).
  • Figures 5a and 5b show the cytotoxic effect of ADCC mediated by antibodies NA7-S and NA8-S on human CLDN18.2-HEK293T cell line
  • Figures 5c and 5d show that antibodies NA7-S and NA8-S in human CLDN18.
  • ADCC mediated on 2-KATOIII tumor cell line.
  • Figures 6a and 6b show the tumor suppressor effect of candidate antibodies NA7-S and NA8-S in a human CLDN18.2-HEK293T xenograft model.
  • Figure 6a shows the tumor suppressor effect of the candidate antibody NA7-S in the human CLDN18.2-HEK293T xenograft model;
  • Figure 6b shows the tumor suppressor effect of the candidate antibody NA8-S in the human CLDN18.2-HEK293T xenograft model.
  • the mice were immunodeficient mice SCID, and the doses of NA7-S, NA8-S and IMAB362 were calculated according to the mass concentration per kilogram of body weight, and the administration method was cross-administration through the tail vein and the abdominal cavity. medicine.
  • Figures 7a-7g show the binding epitopes of candidate antibodies NA7-S, NA8-S and IMAB362 on the CLDN18.2 protein and the key amino acid sites for binding.
  • Figure 7a shows the competitive binding test of candidate antibodies NA7-S and NA8-S on human CLDN18.2-HEK293T with IMAB362
  • Figure 7b shows the antibody IMAB362 and NA8-S on human CLDN18.2-HEK293T with NA7 -Competitive binding assay for S
  • Figure 7c uses anti-CLDN18 antibody to measure the expression levels of mutant CLDN18.2 and wild CLDN18.2 on human CLDN18.2 wild and mutant cell lines
  • Figure 7d shows the expression levels of NA7-S and IMAB362 in human Binding levels to CLDN18.2 mutants on CLDN18.2 mutant cell lines
  • Figure 7e shows the relative percentage binding of NA7-S and IMAB362 on human CLDN18.2 mutant cell lines relative to wild-type cell
  • Figure 7f shows the binding levels of NA8-S and IMAB362 on human CLDN18.2 mutant cell lines to CLDN18.2 mutants;
  • Figure 7g shows NA8-S and IMAB362 on human CLDN18.2 mutant cell lines relative to Relative percent binding of wild-type cell lines.
  • Figures 8a-8b show the cell killing efficiency of NA7-S, NA8-S and IMAB362 against human CLDN18.2-HEK293T in the presence of Fab-ZAP.
  • Figure 8a shows the cell killing efficiency of NA7-S and IMAB362 against human CLDN18.2-HEK293T
  • Figure 8b shows the cell killing efficiency of NA8-S and IMAB362 against human CLDN18.2-HEK293T, wherein hIgG and Fab-ZAP were tested As a control, long-term incubation of Fab-ZAP with cells also had certain toxic effects on cells.
  • Figures 9a-9c show the binding activity of NA4-S in human CLDN18.2-HEK293T and human CLDN18.1-HEK293 cells before and after sequence modification by humanization, wherein NA4-S-H7 is a humanized molecule.
  • Figure 9a shows the comparison of the binding activities of the molecules in human CLDN18.2-HEK293T cells before and after NA4-S humanization; Binding strength on cells;
  • Figure 9c shows the positive rate of binding of NA4-S, NA4-S-H7 and IMAB362 on human CLDN18.1-HEK293 cells at high concentrations (100 ⁇ g/mL), all using irrelevant antibodies as control.
  • Figures 10a-10c show the binding activity of NA7-S in human CLDN18.2-HEK293T and human CLDN18.1-HEK293 cells before and after sequence modification by humanization, wherein NA7-S-H2 is a humanized molecule.
  • Figure 10a shows the comparison of the binding activities of the molecules in human CLDN18.2-HEK293T cells before and after humanization of NA7-S; Binding strength on cells;
  • Figure 10c shows the positive rate of binding of NA7-S, NA7-S-H2 and IMAB362 on human CLDN18.1-HEK293 cells at high concentrations (100 ⁇ g/mL), all using irrelevant antibodies as control.
  • Figures 11a-11c show the binding activity of NA8-S in human CLDN18.2-HEK293T and human CLDN18.1-HEK293 cells before and after sequence modification by humanization, wherein NA8-S-H9 is a humanized molecule.
  • Figure 11a shows the comparison of the binding activities of the molecules in human CLDN18.2-HEK293T cells before and after NA8-S humanization; Binding strength on cells;
  • Figure 11c shows the positive rate of binding of NA8-S, NA8-S-H9 and IMAB362 on human CLDN18.1-HEK293 cells at high concentrations (100 ⁇ g/mL), all using irrelevant antibodies as control.
  • Figure 12 shows the binding activity of NA7-S-H2 in human CLDN18.1-HEK293 cells before and after sequence affinity maturation.
  • Figures 13a-13c show the binding activity of affinity matured molecules on human CLDN18.2-HEK293T cells.
  • Figure 13a shows the binding activity of NA7-S-H2 and its affinity matured molecules on human CLDN18.2-HEK293T cells;
  • Figure 13b shows the binding of NA4-S-H7 and its affinity matured molecules on human CLDN18.2-HEK293T cells Activity;
  • Figure 13c shows the binding activity of NA8-S-H9 and its affinity matured molecules on human CLDN18.2-HEK293T cells.
  • Figure 14 shows the binding activity of NA7-S-H2 and its affinity matured molecules on the human CLDN18.2-KATOIII tumor cell line.
  • Figures 15a-15d show complement-dependent cytotoxicity (CDC) mediated by affinity matured molecules on human CLDN18.2-HEK293T cells.
  • Figure 15a shows the CDC cytotoxic effect mediated by the affinity matured molecules NA7S-H2-418 and NA7S-H2-420 on human CLDN18.2-HEK293T
  • Figure 15b shows the affinity matured molecules NA7S-H2-20 and NA7S-H2 -30-mediated CDC cytotoxic effect on human CLDN18.2-HEK293T
  • Figure 15c shows the CDC cytotoxic effect mediated by affinity matured molecules NA4S-H7-10 and NA4S-H7-3 on human CLDN18.2-HEK293T
  • Figure 15d shows the CDC cytotoxic effect mediated by the affinity matured molecule NA8S-H9-57 on human CLDN18.2-HEK293T.
  • Figures 16a and 16b show the mediated antibody-dependent cell-mediated cytotoxicity (ADCC) of NA7-S-H2 and its affinity matured molecules on human CLDN18.2-HEK293T cells.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Figure 16a shows the ADCC mediated by the affinity matured molecules NA7S-H2-418 and NA7S-H2-420 on human CLDN18.2-HEK293T
  • Figure 16b shows the affinity matured molecules NA7S-H2-20 and NA7S-H2-30 in ADCC mediated on human CLDN18.2-HEK293T.
  • Figures 17a and 17b show antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by affinity matured molecules on the human CLDN18.2-KATOIII tumor cell line.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Figure 17a shows the ADCC mediated by the affinity matured molecules NA4S-H7-10 and NA4S-H7-3 on human CLDN18.2-KATOIII
  • Figure 17b shows the affinity matured molecules NA8S-H9-57 in human CLDN18.2-KATOIII mediated ADCC.
  • antibody generally refers to any form of antibody that exhibits the desired biological or binding activity. It includes, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies and single domain antibodies. Antibodies can contain heavy and light chains. Heavy chains can be classified as mu, delta, gamma, alpha, and epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • VH heavy chain variable region
  • CH heavy chain constant region
  • Heavy chain constant regions typically consist of 3 domains (for gamma, alpha and delta: CH1, CH2 and CH3) or 4 domains (for mu and epsilon: CH1, CH2, CH3 and CH4).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the VH and VL regions can be further divided into relatively conserved regions called framework regions (FRs) and hypervariable regions separated by FRs called complementarity determining regions (CDRs).
  • FRs framework regions
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminal to C-terminal.
  • Antibodies may be of different antibody isotypes, such as IgG (including, for example, IgGl, IgG2, IgG3 or IgG4 subtypes), IgAl, IgA2, IgD, IgE or IgM antibodies.
  • IgG including, for example, IgGl, IgG2, IgG3 or IgG4 subtypes
  • IgAl IgA2, IgD, IgE or IgM antibodies.
  • humanized antibody refers to an antibody in which CDR sequences derived from the germline of another non-human mammalian species are grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.
  • chimeric antibody as used herein broadly refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.
  • a chimeric antibody comprises a variable region derived from a non-human animal antibody and a constant region of another antibody, for example comprising a camel-derived variable region and a human-derived constant region.
  • Chimeric antibodies can also refer to multispecific antibodies that are specific for at least two different antigens.
  • CLDN18.2 binding molecule means a molecule that specifically binds to CLDN18.2.
  • CLDN18.2 antibody antibody against CLDN18.2
  • antibody that specifically binds CLDN18.2 antibody that specifically targets CLDN18.2
  • antibody that specifically recognizes CLDN18.2 as used herein.
  • Antibody of 2 is used interchangeably to mean an antibody capable of specifically binding to the Claudin protein CLDN18.2.
  • an antibody that specifically binds to human CLDN18.2 is meant, in particular an antibody that specifically binds to human CLDN18.2 but not to human CLDN18.1.
  • amino acid sequences of human CLDN18.2 and human CLDN18.1 are shown in SEQ ID NO: 16 and SEQ ID NO: 17 respectively; the amino acid sequences of mouse CLDN18.2 and mouse CLDN18.1 are respectively shown in SEQ ID NO: 18 and SEQ ID NO: 17. shown in SEQ ID NO:19.
  • immunoglobulin single variable domain or "ISV” as used herein is generally defined herein as an amino acid sequence that comprises an immunoglobulin fold or is capable of forming under suitable conditions, such as physiological conditions Immunoglobulin folding (ie, by folding), i.e., thereby forming an immunoglobulin variable domain (eg, a VH, VL or VHH domain); and forming (or capable of forming under suitable conditions) an immunoglobulin variable structure domain, which contains a functional antigen binding site (in the sense that it does not need to interact with another immunoglobulin variable domain (eg, VH-VL interaction) to form a functional antigen binding site).
  • a functional antigen binding site in the sense that it does not need to interact with another immunoglobulin variable domain (eg, VH-VL interaction) to form a functional antigen binding site).
  • Binding molecule, antibody or antibody numbering as used herein eg NA4-S, NA7-S, NA8-S, NA4-S-H7, NA7-S-H2, NA8-S-H9, NA7S-H2-418, NA8S- H9-57, NA7S-H2-420, NA7S-H2-20, NA7S-H2-30, NA4S-H7-10, NA4S-H7-3, NA8S-H9-57, etc.
  • NA4-S, NA7-S, NA8-S, NA4-S-H7, NA7-S-H2, NA8-S-H9, NA7S-H2-418, NA8S- H9-57, NA7S-H2-420, NA7S-H2-20, NA7S-H2-30, NA4S-H7-10, NA4S-H7-3, NA8S-H9-57, etc. are only used to distinguish or identify molecules or products , is not intended to indicate that such identification is characteristic
  • 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%.
  • EC50 also referred to as “50% effective concentration”, as used herein, refers to the concentration of a drug, antibody or toxicant that induces a 50% response between baseline and maximum after a specified exposure time.
  • the unit of EC 50 is “nM” or “ ⁇ g/mL”.
  • 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 steric conformation, which may be “linear” epitope” or “conformational epitope”. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • isolated refers to the state of a substance or component that has been artificially obtained from its natural state. If an "isolated” substance or component occurs in nature, it may be due to a change in its natural environment, the isolation of the substance or component from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same high-purity polynucleotide or polypeptide isolated from this natural state is called an isolated polynucleotide or polypeptide.
  • isolated does not exclude mixed man-made or synthetic substances nor other impure substances that do not affect the activity of the isolated substance.
  • isolated antibody refers to an antibody that is substantially free of other antibodies with different antigenic specificities. Furthermore, the isolated antibody can be substantially free of other cellular material and/or chemicals.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector allows the expression of the protein encoded by the polynucleotide inserted into it, the vector is called an expression vector.
  • the vector can be transformed, transduced or transfected into the host cell so that the carried genetic material elements are expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • bacteriophages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses Viruses (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papovaviruses Viruses (eg SV40).
  • a vector may contain various elements for controlling expression including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain an origin of replication.
  • the term "host cell” refers to any kind of cellular system into which a vector can be introduced, including but not limited to prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • Methods of producing antibodies of the invention using host cells are routine in the art and include expression of the antibody in prokaryotic or eukaryotic cells, followed by isolation of the antibody, and typically purification to pharmaceutically acceptable purity.
  • the nucleic acid encoding the antibody is inserted into an expression vector by standard techniques known in the art and the expression vector is introduced into a suitable prokaryotic or eukaryotic host cell at a concentration sufficient to produce the antibody or functional fragment thereof of the invention.
  • Cultivate host cells such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6(R) cells, yeast or E. coli cells, under the conditions and times of recovery and purification of antibodies from liquid or cells).
  • identity refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by aligning and comparing the sequences.
  • Percent identity refers to the percentage of identical residues between amino acids or nucleotides in compared molecules, and is calculated based on the size of the smallest molecule being compared. For these calculations, the gaps (if any) in the alignment are preferably addressed by a specific mathematical model or computer program (ie, an "algorithm").
  • the term "immunogenicity” refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the nature of antigens to stimulate the activation, proliferation and differentiation of specific immune cells to eventually produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response of antibodies or sensitized T lymphocytes that can stimulate an organism with an antigen. formed in the immune system of the organism. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in the host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunity.
  • transfection refers to the process of introducing nucleic acid into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection, chemical and physical transfection such as electroporation. Many transfection techniques are well known in the art, see e.g. Graham et al, 1973, Virology 52:456; Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual; Davis et al, 1986, Basic Methods in Molecular Biology, Elsevier ; Chu et al, 1981, Gene 13:197.
  • FACS fluorescence activated cell sorting
  • Such instruments include the FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, CA), the Epics C from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).
  • subject includes any human or non-human animal, preferably a human.
  • disorder associated with CLDN18.2 refers to any disorder caused, exacerbated or otherwise associated with increased or decreased expression or activity of CLDN18.2 (eg, human CLDN18.2).
  • cancer refers to any tumor or malignant cell growth, proliferation or metastasis-mediated solid or non-solid tumor such as leukemia that causes a medical condition.
  • treatment as used herein in the context of treating a condition generally relates to treatment and therapy in humans or animals in which some desired therapeutic effect is achieved, eg, inhibition of disease progression, including decreased rate of progression, stalled rate of progression, regression of disease, The condition improves and the condition is cured. Treatment as a preventive measure (ie, prophylaxis) is also included.
  • treating may refer to inhibiting or slowing tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof.
  • treatment includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.
  • a “therapeutically effective amount” relates to an amount of an active compound or a material, composition or dosage form comprising an active compound which, when administered according to the desired therapeutic regimen, is effective to produce a reasonable benefit/risk ratio commensurate with Certain desired therapeutic effects.
  • a “therapeutically effective amount” means an amount or concentration of an antibody or antigen-binding portion thereof effective to treat a disorder associated with CLDN18.2.
  • 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 present disclosure provides CLDN18.2 binding molecules.
  • CLDN18.2 binding molecules can include any molecule that specifically binds CLDN18.2.
  • the CLDN18.2 binding molecule can be a polypeptide or protein, such as an antibody, more specifically an antibody that specifically binds to CLDN18.2 (eg, human CLDN18.2).
  • Antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, or single domain antibodies, and the like.
  • the CLDN18.2 binding molecule is a single domain antibody, which generally refers to an antibody consisting of a single monomeric variable antibody domain. Like whole antibodies, single-domain antibodies are capable of selectively binding to specific antigens.
  • the CLDN18.2 binding molecule is a heavy chain single domain antibody, which can be combined with the terms "VHH”, “VHH antibody”, “VHH domain”, “VHH antibody fragment”, “ VHH “ or “nanobody”. Antibody” etc. are used interchangeably.
  • VHH molecules from camelid antibodies are one of the smallest known intact antigen-binding domains (about 15 KDa, or 1/10 the size of conventional IgG), and are therefore well suited for delivery to dense tissues and into the confined spaces between macromolecules.
  • the single domain antibodies disclosed herein can be prepared by one of skill in the art according to methods known in the art or any future methods.
  • VHHs can be obtained using methods known in the art, such as by immunizing camels and obtaining therefrom VHHs that bind to and neutralize target antigens, or by cloning VHHs of the invention using molecular biology techniques known in the art Libraries are then selected by using phage display.
  • the single domain antibodies of the invention are naturally produced in camelids, ie, produced by immunizing camels with CLDN18.2 or a fragment thereof using the techniques described herein for other antibodies.
  • single domain antibodies are obtained by immunizing llamas or alpacas with the desired antigen and subsequently isolating mRNA encoding the heavy chain antibody.
  • gene libraries containing millions of clones of single domain antibodies are generated. Screening techniques such as phage display and ribosome display aid in the identification of antigen-binding clones.
  • phage display an antibody library is synthesized on phage, the library is screened with an antigen of interest or an antibody-binding portion thereof, and an antigen-binding phage is isolated, from which immunoreactive fragments can be obtained.
  • kits for generating phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and Stratagene SurfZAP. TM Phage Display Kit, Cat. No. 240612). Still other methods and reagents can be used to generate and screen antibody display libraries (see, eg, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).
  • one or more CDRs of an "affinity matured" antibody comprise one or more additions, deletions and/or substitutions of amino acids compared to one or more CDRs of a parent antibody such that the affinity matured antibody is compatible with the parent antibody Compared with the present antibody, the affinity for the antigen is improved.
  • Methods for affinity maturation of antibodies are known in the art, see e.g.
  • the single domain antibodies of the invention can be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding the naturally occurring VHH domain; (3) ) by "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by a naturally occurring VHH domain from any animal species, in particular mammalian species, such as from humans “camelization” of the VH domain of the , Nature 341:544-546), or by expressing a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (7) by using Synthetic techniques produce nucleic acid encoding the VHH, and then express the nucleic acid thus obtained; and/or (8) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing will be apparent to those skilled in the
  • Single domain antibodies are typically produced by PCR cloning of variable domain libraries into phage display vectors from cDNAs of blood, lymph node or spleen lymphocytes obtained from immunized animals.
  • Antigen specificity is typically selected by panning the corresponding library on immobilized antigens (eg, antigens coated on the plastic surface of test tubes, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the cell surface) Sexual single domain antibody.
  • the affinity of sdAbs can be improved by mimicking this strategy in vitro, for example by site-directed mutagenesis of CDR regions and under increased stringency conditions (higher temperature, high or low salt concentration, high or low pH and low antigen concentration) for immobilized antigen Further panning was performed (Wesolowski et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol (2009) 198:157-174).
  • the VHH in the CLDN18.2 binding molecule is fused to the Fc domain of an antibody (eg, the Fc domain of an IgG (eg, IgGl or IgG4)).
  • an antibody eg, the Fc domain of an IgG (eg, IgGl or IgG4)
  • VHHs By fusing VHHs to the Fc domain, effector functions such as ADCC and CDC can be recruited more efficiently.
  • the fusion of VHH to the Fc domain can help CLDN18.2 binding molecules to form dimers, and can also help prolong the in vivo half-life of CLDN18.2 binding molecules.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic effector cells such as natural killer (NK) cells, neutrophils, and macrophages.
  • FcR Fc receptor
  • NK cells the primary cells mediating ADCC, express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • ADCC activity of a molecule of interest can be assessed in vitro, eg, in animal models as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
  • complement-dependent cytotoxicity refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (the appropriate subclass) that bind to its cognate antigen.
  • a CDC assay can be performed, eg, by the method described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
  • CLDN18.2 binding molecule is hereinafter described as a CLDN18.2 antibody.
  • CLDN18.2 antibody capable of specifically binding to a specific epitope of CLDN18.2
  • the present disclosure relates to single domain antibodies that specifically bind CLDN18.2, but do not bind or substantially do not bind CLDN18.1.
  • CLDN18.2-binding molecule such as a single-domain antibody targeting CLDN18.2 capable of specifically binding to the extracellular domain 1 (ECD1) of human CLDN18.2.
  • CLDN18.2 has two extracellular domains (ECDs), wherein the full-length sequence of human CLDN18.2 is shown in SEQ ID NO:16, and the sequence of ECD1 is shown in SEQ ID NO:20.
  • ECDs extracellular domains
  • Mouse CLDN18.2 is shown in SEQ ID NO:18.
  • CLDN18.2 antibody comprising CDRs with sequence identity to specific sequences
  • the CLDN18.2 antibody of the present disclosure comprises at least one immunoglobulin single variable domain (eg, VHH), wherein the VHH comprises CDR1, CDR2, and CDR3, and wherein CDR1 comprises the same sequence as SEQ ID NO: 1 , 4 or 7 are at least 80% identical to the amino acid sequence, CDR2 comprises an amino acid sequence at least 80% identical to SEQ ID NO: 2, 5 or 8, and CDR3 comprises at least 80% identical to SEQ ID NO: 3, 6 or 9 amino acid sequence.
  • VHH immunoglobulin single variable domain
  • each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, including, for example, antibody-based three-dimensional Structure and topology of CDR loops of Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997 )), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S.
  • the CLDN18.2 antibody of the present disclosure uses IMGT or AbM to define the amino acid sequence of the CDR region, but the definition of CDR is not unique and limited, nor does it mean that the present disclosure only protects the CDR region defined by IMGT or AbM. Represented antibody molecules.
  • the CLDN18.2 antibody molecules of the present disclosure can also be characterized in various other CDR region definitions. It should be noted that the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may be different, that is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may be different. The CDR sequences defined under these various assignment systems and the antibody molecules they characterize are also intended to be protected by the present disclosure.
  • Variable regions and CDRs in antibody sequences can be identified according to general rules that have been developed in the art (eg, the Kabat numbering system, as described above) or by aligning the sequences with databases of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described at and available from the "Abysis" website (maintained by A.C.
  • sequences are preferably analyzed using the Abyss database, which integrates sequence data from Kabat, IMGT and the Protein Data Bank (PDB) with structural data from the PDB, see the book Protein Sequence and Structure Analysis of Antibody Variable by Dr. Andrew C.R. Martin Domains.In: Antibody Engineering Lab Manual (Ed.: Duebel, S.
  • the Abysis database website also includes general rules that have been developed for identifying CDRs that can be used in accordance with the teachings herein.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table with a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol.
  • the amino acid sequences of the CDRs may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% identical to the respective sequences given above , 95%, 96%, 97%, 98% or 99% are the same.
  • CLDN18.2 antibody comprising CDRs with amino acid additions, deletions or substitutions
  • a CLDN18.2 antibody of the present disclosure comprises at least one immunoglobulin single variable domain (eg, a VHH), wherein the VHH comprises CDRl, CDR2, and CDR3.
  • the CDR1 has no more than 2 (eg, 0, 1 or 2) amino acid additions, deletions or substitutions in the amino acid sequence from the sequence shown in SEQ ID NO: 1, 4 or 7. Differences; CDR2 differs in amino acid sequence from the sequence shown in SEQ ID NO: 2, 5 or 8 by amino acid additions, deletions or substitutions of no more than 2 (e.g.
  • CDR3 differs from the sequence shown in SEQ ID NO: 3, 6 or 9 by no more than 2 (such as 0, 1 or 2) amino acid additions, deletions or substitutions.
  • CDR1, CDR2 and CDR3 have an amino acid addition of only one amino acid to the amino acid sequences shown in SEQ ID NO: 1, 4 or 7, SEQ ID NO: 2, 5 or 8 and SEQ ID NO: 3, 6 or 9, respectively , deletion or substitution differences.
  • the coding system of the above-mentioned CDR can use IMGT.
  • CDR1 has an amino acid addition, deletion or substitution of no more than 2 (eg, 0, 1 or 2) amino acids in the amino acid sequence with the sequence shown in SEQ ID NO: 30, 37 or 45
  • the difference between CDR2 and the sequence shown in SEQ ID NO:31, 38 or 46 in amino acid sequence is not more than 2 (such as 0, 1 or 2) amino acid addition, deletion or substitution of amino acid difference; and /or CDR3 differs in amino acid sequence from the sequence shown in SEQ ID NO: 32, 39 or 47 by amino acid additions, deletions or substitutions of no more than 2 (such as 0, 1 or 2) amino acids.
  • the coding system of the above-mentioned CDR can adopt AbM.
  • the CDRs of the isolated antibody or antigen-binding portion thereof contain conservative substitutions of no more than 2 amino acids or no more than 1 amino acid.
  • conservative substitution refers to amino acid substitutions that do not adversely affect or alter the essential properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art (eg, site-directed mutagenesis and PCR-mediated mutagenesis).
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced by another amino acid residue with a similar side chain, such as a physically or functionally similar residue (e.g., having similar size, shape, charge, chemical properties including formation of covalent bonds or capacity for hydrogen bonding, etc.) by substitution of the corresponding amino acid residues. Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine. Accordingly, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family.
  • Methods for identifying conservative amino acid substitutions are well known in the art (see, eg, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999) ); and Burks et al, Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which is incorporated herein by reference).
  • the immunoglobulin single variable domain of the CLDN18.2 antibody comprises:
  • the immunoglobulin single variable domain of the CLDN18.2 antibody comprises:
  • the immunoglobulin single variable domain of the CLDN18.2 antibody comprises:
  • the CLDN18.2 antibody of the present disclosure comprises at least one immunoglobulin single variable domain (eg, VHH), wherein the VHH comprises CDR1, CDR2, and CDR3, and wherein CDR1, CDR2, and CDR3 are selected from:
  • VHH immunoglobulin single variable domain
  • the CLDN18.2 antibody of the present disclosure comprises at least one (eg, one) immunoglobulin single variable domain (eg, VHH), wherein the VHH comprises:
  • (C) has one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) compared to SEQ ID NO: 10, 11, 12, 13, 14, or 15 Amino acid sequence of additions, deletions and/or substitutions of amino acids, preferably, the additions, deletions and/or substitutions do not occur in the CDR regions.
  • the CLDN18.2 antibody of the present disclosure comprises at least one (eg, one) immunoglobulin single variable domain (eg, VHH), wherein the VHH comprises:
  • (C) has one or more (eg, 1, 2, 3, 4) compared to SEQ ID NO: 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, or 29 , 5, 6, 7, 8, 9 or 10) amino acid sequence of additions, deletions and/or substitutions of amino acids, preferably, the additions, deletions and/or substitutions do not occur in the CDR regions.
  • the amino acid sequence of the VHH may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% identical to each of the above-mentioned sequences , 96%, 97%, 98% or 99% are the same.
  • an antibody may comprise at least 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of VHHs.
  • the CLDN18.2 antibodies of the present disclosure may comprise conservative substitutions or modifications of amino acids in the variable region of the heavy chain. It is understood in the art that certain conservative sequence modifications can be made that do not eliminate antigen binding properties. See, eg, Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.
  • the present disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding CLDN18.2 antibodies of the present disclosure.
  • Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For antibodies obtained from immunoglobulin gene libraries (eg, using phage display techniques), nucleic acid encoding such antibodies can be recovered from the gene library.
  • Nucleic acid molecules encoding the CLDN18.2 antibodies of the present disclosure can be inserted into vectors for further cloning (DNA amplification) or for expression using recombinant techniques known in the art.
  • Many vectors are available.
  • Vectors or vector components typically include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters (eg, SV40, CMV, EF-1 ⁇ ), and transcription termination sequence.
  • a selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, eg, US Pat. Nos. 4,399,216; 4,634,665 and 5,179,017).
  • a selectable marker gene confers resistance to a drug, such as G418, hygromycin or methotrexate, to a host cell into which the vector has been introduced.
  • the selectable marker genes may include the dihydrofolate reductase (DHFR) gene (for dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • neo gene for G418 selection.
  • antibodies can be produced by homologous recombination as known in the art. DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional methods (eg, by using oligonucleotide probes capable of binding specifically to the gene encoding the antibody heavy chain).
  • vector systems include mammalian, bacterial, yeast systems, etc., and include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., among others and commercially available vectors.
  • plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, p
  • Suitable vectors may include plasmid or viral vectors (eg, replication-defective retroviruses, adenoviruses and adeno-associated viruses).
  • the vector may be pET, such as pETbac containing a hexahistidine tag and a c-Myc-tag gene.
  • Vectors comprising nucleic acid sequences encoding the CLDN18.2 antibodies of the present disclosure can be introduced into host cells for cloning or gene expression.
  • Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotic, yeast or higher eukaryotic cells.
  • Suitable prokaryotes for this purpose include eubacteria, eg Gram-negative or Gram-positive organisms, eg Enterobacteriaceae, eg Escherichia, eg Escherichia coli, Enterobacter, Erwinia, Grams Lebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescens and Shigella, and Bacillus such as Bacillus subtilis and Bacillus licheniformis, Pseudobacterium Monomonas such as Pseudomonas aeruginosa and Streptomyces.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable host cells for expressing the CLDN18.2 antibodies of the present disclosure.
  • Saccharomyces cerevisiae or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms.
  • many other genera, species and strains are generally available and can be used in the present disclosure, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis (K. lactis), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178) ), K.
  • suitable host cells for expressing the CLDN18.2 antibodies of the present disclosure are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • a large number of baculovirus strains and variants and corresponding permissive insect host cells have been identified from the following hosts: Spodoptera Frugiperda (caterpillar), Aedes aegypti (mosquito), E. albopictus Mosquito (Aedes albopictus, mosquito), Drosophila melanogaster (fruit fly) and silkworm moth (Bombyx mori).
  • Various viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and according to the present disclosure, these viruses can be used with The transfection procedure for the expression of CLDN18.2 antibody in suitable host cells, especially for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.
  • Host cells are transformed with the vectors described above for CLDN18.2 antibody production of the present disclosure and cultured in conventional nutrient media modified as necessary for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences.
  • Host cells used to produce the CLDN18.2 antibodies of the present disclosure can be cultured in various media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells.
  • Ham's F10 Sigma
  • Minimal Essential Medium MEM
  • RPMI-1640 Sigma
  • DMEM Dulbecco's Modified Eagle's Medium
  • antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, particulate debris (host cells or lysed fragments) is removed as a first step, eg, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a method for the isolation of antibodies secreted into the periplasmic space of E. coli. Briefly, cells were thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) in about 30 minutes. Cell debris can be removed by centrifugation.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonyl fluoride
  • the supernatant from such an expression system is usually first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units.
  • Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants.
  • Antibodies prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being preferred purification technology.
  • the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer with a pH between about 2.5-4.5, preferably at low salt concentrations (eg, about 0-0.25 M salt).
  • the present disclosure relates to pharmaceutical compositions comprising at least one CLDN18.2 binding molecule as disclosed herein (eg, a CLDN18.2 antibody of the present disclosure) and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug.
  • additional pharmaceutically active ingredients such as another antibody or drug.
  • the pharmaceutical compositions of the present disclosure can also be administered in combination with, eg, another immunostimulatory, anticancer, antiviral, or vaccine, such that anti-CLDN18.2 antibodies enhance the immune response to the vaccine.
  • Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, Chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, combinations of various components or more known in the art.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins Refined.
  • Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylbenzene Methyl ether, butylated hydroxytoluene and/or propylarsenate.
  • the antibody or antigen-binding fragment of the composition of the present disclosure may be oxidized in a solvent containing one or more antioxidants such as methionine that reduces the antibody or antigen-binding fragment thereof. Redox prevents or reduces the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Accordingly, in some embodiments, the present disclosure provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present disclosure further provides methods wherein an antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine. Thus, the antibody or antigen-binding fragment thereof can be protected from oxidation to extend its shelf life and/or increase its activity.
  • one or more antioxidants such as methionine
  • a pharmaceutically acceptable carrier can include, for example, an aqueous carrier such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection or Dextrose and Lactated Ringers injection, non-aqueous carriers such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, bacteriostatic or fungistatic concentrations of antimicrobial agents, isotonic agents such as sodium chloride or dextrose, buffers such as phosphoric acid Salt or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents Agents such as polysorbate 80 (TWEEN-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol
  • Antimicrobial agents used as carriers can be added to compounds containing phenol or cresol, mercury formulations, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. ammonium in pharmaceutical compositions in multi-dose containers.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers or additives such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin. reagents.
  • compositions of the present disclosure can be administered in vivo to a subject in need thereof by various routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, Intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, or by implantation or inhalation.
  • the pharmaceutical compositions of the present disclosure can be formulated into preparations in solid, semi-solid, liquid or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalation and aerosols. Appropriate formulations and routes of administration can be selected depending on the intended application and treatment regimen.
  • Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and controlled release dosage forms thereof.
  • Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free solutions in which the active ingredient is dissolved, suspended, or otherwise provided (eg, in liposomes or other microparticles).
  • sterile liquids eg solutions, suspensions
  • These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickening agents and to render the formulation compatible with the blood (or other relevant body fluids of the intended recipient) ) isotonic solute.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • isotonic carriers suitable for use in such formulations include Sodium Chloride Injection, Ringer's Solution or Lactated Ringer's Injection.
  • the particular dosage regimen ie, dose, timing, and repetition
  • the frequency of administration can be determined and adjusted during treatment and based on reducing the number of proliferating or tumorigenic cells, maintaining such tumor cell reduction, reducing tumor cell proliferation or delaying the development of metastases.
  • the dose administered can be adjusted or reduced to control potential side effects and/or toxicity.
  • sustained continuous release formulations of the pharmaceutical compositions of the present disclosure for use in therapy may be suitable.
  • ⁇ dosages may vary from patient to patient. Determining the optimal dose generally involves balancing the level of therapeutic benefit against any risks or harmful side effects.
  • the dose level selected will depend on a variety of factors including, but not limited to, the activity of the particular compound, administration, time of administration, rate of clearance of the compound, duration of treatment, other concomitant drugs, compounds and/or materials, the severity of the disorder , and species, gender, age, weight, condition, general health, and previous medical history of the patient.
  • the amount and route of administration of the compound is ultimately at the discretion of the physician, veterinarian or clinician, but the dosage is generally selected to achieve local concentrations at the site of action to achieve the desired effect without causing substantial deleterious or adverse side effects.
  • CLDN18.2 binding molecules can be administered in various dosage ranges.
  • the CLDN18.2 binding molecules provided herein can be administered at about 0.01 mg/kg to about 100 mg/kg (eg, about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg) of a therapeutically effective dose.
  • the antibody is administered at a dose of about 50 mg/kg or less, and in certain of these embodiments, the dose is 10 mg/kg or less, 5 mg/kg or less, 1 mg /kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less.
  • the dose administered may vary over the course of treatment.
  • the initially administered dose may be higher than the subsequently administered dose.
  • the administered dose may vary over the course of treatment depending on the subject's response.
  • the antibodies or antigen-binding portions thereof of the present disclosure are preferably administered as needed to a subject in need.
  • the frequency of administration can be determined by one of skill in the art, eg, based on the considerations of the attending physician based on the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like.
  • a course of treatment involving an antibody or antigen-binding portion thereof of the present disclosure will comprise multiple doses of the selected drug product administered over a period of weeks or months. More specifically, the antibodies or antigen-binding portions thereof of the present disclosure can be daily, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two month, every ten weeks or every three months. In this regard, it will be appreciated that the dosage may be varied or the interval adjusted based on patient response and clinical practice.
  • Dosages and regimens of the disclosed pharmaceutical compositions for therapy can also be determined empirically in individuals given one or more administrations. For example, an individual may be administered incremental doses of a pharmaceutical composition as described herein. In selected embodiments, the dosage may be determined empirically, respectively, or may be gradually increased or decreased based on observed side effects or toxicity. To assess the efficacy of a selected composition, markers for a particular disease, disorder or condition can be followed.
  • these include direct measurement of tumor size by palpation or visual inspection, indirect measurement of tumor size by X-ray or other imaging techniques; improvement in assessment by direct tumor biopsy and microscopy of tumor samples; measurement according to the methods described herein Identified indirect tumor markers (eg, PSA for prostate cancer) or tumorigenic antigens, reduction in pain or paralysis; improvement in tumor-related speech, vision, breathing, or other disability; increased appetite; An improvement in quality of life or prolongation of survival as measured by the test.
  • the dosage will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and the treatments used in the past and concurrently.
  • Compatible formulations for parenteral administration may comprise a CLDN18.2 binding molecule as provided herein at a concentration of from about 10 ⁇ g/ml to about 100 mg/ml.
  • the concentration of the CLDN18.2 binding molecule can include 20 ⁇ g/ml, 40 ⁇ g/ml, 60 ⁇ g/ml, 80 ⁇ g/ml, 100 ⁇ g/ml, 200 ⁇ g/ml, 300 ⁇ g/ml, 400 ⁇ g/ml, 500 ⁇ g/ml , 600 ⁇ g/ml, 700 ⁇ g/ml, 800 ⁇ g/ml, 900 ⁇ g/ml or 1mg/ml.
  • the concentration of CLDN18.2 binding molecule will include 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg /ml, 16mg/ml, 18mg/ml, 20mg/ml, 25mg/ml, 30mg/ml, 35mg/ml, 40mg/ml, 45mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml , 90mg/ml or 100mg/ml.
  • the concentration of the CLDN18.2 binding molecule can include, for example, 133 nM, 266 nM, 400 nM, 533 nM, 667 nM, 1.3 ⁇ M, 2 ⁇ M, 2.67 ⁇ M, 3.33 ⁇ M, 4 ⁇ M, 4.67 ⁇ M, 5.33 ⁇ M , 6 ⁇ M or 6.67 ⁇ M.
  • CLDN18.2 binding molecules of the present invention have numerous in vitro and in vivo uses.
  • Conditions and disorders associated with CLDN18.2 may be immune-related diseases or disorders, including but not limited to "diseases involving cells expressing CLDN18.2" or “diseases associated with cells expressing CLDN18.2” or similar expressions , meaning that CLDN18.2 is expressed in cells of a diseased tissue or organ.
  • the expression of CLDN18.2 is increased in cells of a diseased tissue or organ as compared to the state in the corresponding healthy tissue or organ.
  • To increase means to increase by at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.
  • diseases associated with cells expressing CLDN18.2 include cancer diseases.
  • cancer diseases are preferably those in which cancer cells express CLDN18.2.
  • cancer refers to or describe the physiological condition in an individual that is often characterized by unregulated cell growth.
  • cancer include, but are not limited to, epithelial cancer, lymphoma, blastoma, sarcoma, and leukemia.
  • examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer , colon cancer, breast cancer, prostate cancer, uterine cancer, sex and reproductive organ cancer, Hodgkin's Disease, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer , soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancers, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas.
  • CNS central nervous system
  • cancer also includes cancer metastasis.
  • a “cancer disease” is characterized by cells expressing CLDN18.2, and cancer cells expressing CLDN18.2.
  • the cells expressing CLDN18.2 are preferably cancer cells, preferably cancer cells of the cancers described herein.
  • tumor refers to abnormal growth of cells (known as neoplastic cells, tumorigenic cells or tumor cells), preferably forming swellings or lesions.
  • Tumor cell means an abnormal cell that grows by rapid uncontrolled cell proliferation and continues to grow after the stimuli that initiate new growth cease. Tumors exhibit a partial or complete loss of structural organization and functional coordination with normal tissue, and often form distinct tissue masses that may be benign, premalignant, or malignant.
  • cancer disease is preferably “tumor disease”. In general, however, the terms “cancer” and “tumor” are used interchangeably herein.
  • the cancer according to the present disclosure involves cancer cells expressing CLDN18.2.
  • the cancer is CLDN18.2 positive.
  • the expression of CLDN18.2 is on the surface of the cell.
  • at least 50%, preferably 60%, 70%, 80% or 90% of the cancer cells are CLDN18.2 positive, and/or at least 40%, preferably at least 50% of the cancer cells are positive for CLDN18.2 positive for surface expression.
  • at least 95% or at least 98% of the cancer cells are CLDN18.2 positive.
  • at least 60%, at least 70%, at least 80%, or at least 90% of the cancer cells are positive for surface expression of CLDN18.2.
  • the CLDN18.2 expressing cancer, cancer involving CLDN18.2 expressing cancer cells, or CLDN18.2 positive cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (eg, non-small cell lung cancer (NSCLC)) ), ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer, and their metastases, especially gastric cancer metastases (eg Krugenberg tumor), peritoneal metastases and lymph node metastases.
  • the cancer is adenocarcinoma, particularly advanced adenocarcinoma.
  • cancer diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung and ovary.
  • the cancer is selected from gastric cancer, esophageal cancer (especially lower esophageal cancer), cancer of the esophago-gastric junction and gastroesophageal cancer.
  • the cancer is gastroesophageal cancer, such as metastatic, refractory or recurrent advanced gastroesophageal cancer.
  • antibodies or antigen-binding portions thereof of the present disclosure can be used in combination with chemotherapy or radiation therapy.
  • Antibodies or antigen-binding portions thereof can be used in combination with anticancer, cytotoxic, or chemotherapeutic agents.
  • anticancer agent or “antiproliferative agent” means any agent that can be used to treat cell proliferative disorders such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, antiangiogenic agents, radiotherapy, and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and antimetastatic and immunotherapeutic agents.
  • anticancer agents may comprise conjugates and may bind to the disclosed site-specific antibodies prior to administration. More specifically, in some embodiments, a selected anticancer agent is linked to an unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Accordingly, such engineered conjugates are expressly contemplated within the scope of the present disclosure.
  • the disclosed anticancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.
  • cytotoxic agent refers to a substance that is toxic to cells and reduces or inhibits cell function and/or causes cell destruction.
  • the substance is a naturally occurring molecule derived from a living organism.
  • cytotoxic agents include, but are not limited to, bacteria (eg, diphtheria toxins, Pseudomonas endotoxins and exotoxins, staphylococcal enterotoxin A), fungi (eg, alpha-ocassin, limited aspergillus), plants (Acacia soy protein, ricin, capsular root toxin, mistletoe, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii protein, Caryophyllene protein, Phytolacca mericana protein (PAPI, PAPII and PAP-S), balsam pear inhibitor, jatrophin protein, crotontoxin, lycop
  • bacteria eg, diphth
  • chemotherapeutic agents include chemical compounds (eg, cytotoxic or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation, and/or survival of cancer cells. These chemicals typically target intracellular processes that are required for cells to grow or divide, and are therefore particularly effective against cancer cells, which typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cell entry into mitosis.
  • chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become sexual or produce tumorigenic progeny (eg, TIC). These agents are usually used in combination and are usually most effective, eg, in regimens such as CHOP or FOLFIRI.
  • anticancer agents that can be used in combination with the site-specific constructs of the present disclosure (either as a component of a site-specific conjugate or in the unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates , aziridine, ethyleneimine and methyl melamine, acetogenins, camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dora statin, docarmicin, eleutherobin, dynemicin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonic acid Salts, espomycin, chromophore protein enediyne antibiotics, aclacinomysins, actinomycins, amplimycin, azoserine, bleomycin, actinomycin C , carabicin (
  • antihormonal agents used to modulate or inhibit hormonal effects on tumors such as antiestrogens and selective estrogen receptor modulators, aromatase inhibition that inhibits aromatase that regulates estrogen production in the adrenal glands and anti-androgens; and troxacitabine (1,3-dioxolane cytosine analog); antisense oligonucleotides, inhibitors of ribozymes such as VEGF expression and HER2 expression inhibitor dose; vaccine, rIL-2; Topoisomerase 1 inhibitor; rmRH; vinorelbine and espomycin, and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing.
  • antihormonal agents used to modulate or inhibit hormonal effects on tumors such as antiestrogens and selective estrogen receptor modulators, aromatase inhibition that inhibits aromatase that regulates estrogen production in the adrenal glands and anti-androgens; and troxacitabine (1,3-dioxolane cytosine analog
  • the present disclosure provides in vitro and in vivo methods for detecting, diagnosing, or monitoring proliferative disorders and methods of screening cells from patients to identify tumor cells, including tumorigenic cells.
  • Such methods include identifying an individual with cancer for treatment or monitoring the progression of cancer, comprising contacting the patient or a sample (in vivo or in vitro) obtained from the patient with an antibody described herein, and detecting in the sample the presence of bound or The presence or absence or level of binding of free target molecule bound antibody.
  • the antibody will comprise a detectable label or reporter.
  • binding of an antibody to a particular cell in a sample can indicate that the sample may contain tumorigenic cells, thereby indicating that an individual with cancer can be effectively treated with the antibodies described herein.
  • Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (eg, ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays.
  • assays such as radioimmunoassays, enzyme immunoassays (eg, ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays.
  • Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (eg, CAT scan), positron emission tomography (eg, PET scan), known to those skilled in the art, Radiography, Ultrasound, etc.
  • a unit dose comprising one or more containers of one or more doses of the antibody or antigen-binding portion thereof.
  • a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, eg, an antibody or antigen-binding portion thereof, with or without one or more other agents.
  • such unit doses are supplied in single-use prefilled injectable syringes.
  • the compositions contained in a unit dose may contain saline, sucrose, or the like; buffers, such as phosphates, etc.; and/or be formulated within a stable and effective pH range.
  • the composition is a conjugate composition, which can be provided as a lyophilized powder and reconstituted after addition of a suitable liquid (eg, sterile water or saline solution). Any label on or associated with the container indicates that the encapsulated conjugate composition is used to treat the tumor condition of choice.
  • the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine.
  • kits typically contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container and, optionally, one or more anticancer agents in the same or a different container.
  • the kit may also contain other pharmaceutically acceptable formulations for diagnosis or combination therapy.
  • such a kit may contain any one or more anti-cancer agents, eg, chemotherapeutic or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targets To anticancer agents; cytotoxic agents; and/or other anticancer agents.
  • kits can have a single container containing the disclosed antibodies or antigen-binding portions thereof, with or without additional components, or they can have separate containers for each desired reagent.
  • a single solution can be premixed in a molar equivalent combination or more of one component than the other.
  • the conjugate and any optional anticancer agent of the kit can be kept separately in separate containers prior to administration to the patient.
  • the kit may also contain a second/second solution for containing sterile pharmaceutically acceptable buffers or other diluents such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • PBS phosphate buffered saline
  • Ringer's solution phosphate buffered saline
  • the liquid solutions are preferably aqueous solutions, particularly preferably sterile aqueous solutions or saline solutions.
  • the components of the kit may be provided as dry powders.
  • the powder can be reconstituted by adding a suitable solvent. It is envisaged that the solvent may also be provided in another container.
  • the kit may also contain means for administering the antibody, or antigen-binding portion thereof, and any optional components to a patient, such as one or more needles, I.V. bags or syringes, or even eye droppers, pipettes A fluid tube or other similar device through which a formulation can be injected or introduced into an animal or administered to a diseased area of the body.
  • Kits of the present disclosure also typically include means for containing vials or the like and other tightly closed components for commercial sale, such as injection or blow-molded plastic containers, in which the desired vials and other devices are placed and held.
  • human CLDN18.2 is highly expressed in cancer cells, while in normal cells, it is only specifically expressed in gastric epithelial cells, so the toxicity and side effects are low, and the possibility of becoming a drug is higher;
  • CLDN18.2 is expressed in the tight junctions of gastric epithelial cells, which are relatively loose in cancer cells, and antibodies are not easy to act on; even if epithelial cells are killed by antibodies, stem cells under the epithelium do not express CLDN18.2. Differentiate and replenish damaged gastric epithelial cells;
  • antibodies against CLDN18.2 can not only kill cells through ADCC and CDC, but also mediate cell apoptosis by cross-linking CLDN18.2 with antibodies, and can inhibit cell proliferation to a certain extent.
  • Nanobodies are a new type of antibody, and one antibody, caplacizumab, has been approved for marketing, which fully demonstrates the druggability of nanobodies.
  • this antibody has significant advantages, such as its half-life can be adjusted by chemical modification or protein fusion transformation, strong permeability, can recognize hidden epitopes that are not accessible by ordinary antibodies, pepsin resistance, acid resistance, heat resistance, It is easy to produce, and because it is a single chain, it is easy to assemble with other types of antibodies into diabodies and multivalent antibodies.
  • the antibody against CLDN18.2 is obtained by screening the alpaca immune library. On this basis, the C-terminus is fused to the Fc fragment of IgG1. At present, some candidate antibodies are tested for CDC and ADCC at the cellular level. The results all showed better or at least comparable activity than the control antibody. Combined with the characteristics of the CLDN18.2 target, this antibody is used for cancer immunotherapy, with lower toxic side effects and better clinical efficacy, and will provide patients with more drug choices.
  • the nucleic acid sequences of full-length human CLDN18.1 (SEQ ID NO: 17), murine CLDN18.2 (SEQ ID NO: 18), murine CLDN18.1 (SEQ ID NO: 19) were constructed into the pLVX-puro plasmid (Clontech, Cat#632164). Then, the resulting plasmid was electroporated into HEK293 cells ( CRL-1573 TM ). Through screening, an overexpression cell line expressing full-length human CLDN18.1 (human CLDN18.1-HEK293), an overexpression cell line expressing murine CLDN18.2 (murine CLDN18.2-HEK293) and a cell line expressing murine CLDN18.1 were obtained.
  • Overexpressing cell line (murine CLDN18.1-HEK293).
  • IMAB362 antibody expressed and purified according to the sequence information disclosed in the patent US20180127489A1
  • anti-CLDN18 antibody [34H14L15] (Abeam, ab203563) that recognizes the C-terminal CLDN18 intracellular segment (GFKASTGFGSNTKN, SEQ ID NO: 22)
  • Flow cytometry identification was performed to obtain the successfully transformed overexpression cell line, and the specific method was as follows.
  • the HEK293 cells were recovered and cultured, and serially passaged 2-3 times.
  • the cells were seeded into a cell culture dish at a density of 3 ⁇ 10 5 cells/mL one day before transfection, and the next day, when the cell confluence reached about 70%, that is, be usable.
  • Trypsin Gibco, 25200-072
  • EDTA EDTA
  • the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% by volume FBS (Gibco, 15140-141) and antibiotic-free, and then the cells were seeded into 10cm ⁇ 10cm cells
  • the cells were cultured in a petri dish for 48 h, and then the cells were dispensed into 96-well cell culture plates at an average density of 0.5 cells/well, and puromycin (Gibco, A111138-03) with a final concentration of 2 ⁇ g/mL was added as the screening pressure, 2
  • the clone growth of the cell line was observed for about a week, and the cloned cell line was picked for identification.
  • the cells were washed three times with FACS buffer, and 200 ⁇ L of FACS buffer was added to the cells to resuspend the cells, and finally detected by a flow cytometer (Beckman, CytoFLEX AOO-1-1102).
  • the specific method is as follows: take 1 ⁇ 10 5 cells, centrifuge at low speed (300g), and remove the supernatant. The cells at the bottom of the centrifuge tube were rinsed once with FACS buffer, then 200 ⁇ L of IC fixative (eBioscience, 00-8222) was added to the rinsed cells, incubated at 4°C for 1 h, and then washed with membrane breaking buffer (eBioscience, 00-8333) rinsed twice, added the aforementioned anti-CLDN18 antibody, and incubated at 4°C for 1 h.
  • IC fixative eBioscience, 00-8222
  • the IMAB362 antibody was used to determine whether it bound to human CLDN18.1-HEK293 and mouse CLDN18.1-HEK293 cell lines.
  • the anti-CLDN18 antibody can recognize human CLDN18.2-HEK293T, human CLDN18.1-HEK293, murine CLDN18.2-HEK293, murine CLDN18.1-HEK293, HEK293 and HEK293T cells; while the antibody IMAB362 only It recognizes mouse CLDN18.2-HEK293 and human CLDN18.2-HEK293T, but does not recognize mouse CLDN18.1-HEK293 and human CLDN18.1-HEK293. This shows that the four cell lines of human CLDN18.2-HEK293T, human CLDN18.1-HEK293, mouse CLDN18.2-HEK293 and mouse CLDN18.1-HEK293 have successfully expressed the corresponding CLDN18 protein.
  • the gastric cancer cell line KATOIII (human CLDN18.2-KATOIII tumor cell line) overexpressing human CLDN18.2 was constructed by lentiviral transfection, and identified by antibody IMAB362. The specific method is as follows:
  • Good state human gastric cancer cells KATOIII HTB-103 TM 5 ⁇ 10 4 , add the packaged lentivirus containing human CLDN18.2 sequence (SEQ ID NO: 16) at an MOI ratio of 30:1, mix well, and then add poly IMDM complete medium (Gibco, 12440061) of brene (Polybrene, Sigma, 107689), mix well, incubate in a constant temperature incubator at 37°C, 5% CO 2 for 20 h, remove the medium, and replace with fresh IMDM complete medium And continue to incubate for 24h, and then inoculate the transfected KATOIII cells into 96-well plates at an average density of 0.5 cells/well, and add puromycin at a final concentration of 2 ⁇ g/mL for resistance pressure screening, at 37°C, Culture in a constant temperature incubator with 5% CO 2 for 2-3 weeks, and pick clones for identification.
  • poly IMDM complete medium Gibco, 12440061
  • brene Polybren
  • FIG. 1b The results of flow cytometry are shown in Figure 1b.
  • the KATOIII cells that were not transfected with CLDN18.2 were hardly recognized by the antibody IMAB362, indicating that the KATOIII cell line hardly expressed CLDN18.2 or the expression level was extremely low; it was successfully constructed by lentivirus transfection
  • the human CLDN18.2-KATOIII tumor cell line can be recognized by the antibody IMAB362, indicating that this cell line was successfully constructed.
  • alpaca immunization was used.
  • the specific operation is as follows: the immunogen adopts the cell line human CLDN18.2-HEK293T (Kangyuan Borchuang, KC-0986) and hCLDN18.2-pLVX-puro plasmid containing human CLDN18.2ECD1 (SEQ ID NO: 20).
  • Alpacas Nanchang Dajia Biological Breeding
  • Alpacas were alternately immunized weekly with 2 ⁇ 10 7 human CLDN18.2-HEK293T cells (subcutaneous multi-point injection) and 2 mg plasmid (muscular multi-point injection), the alpaca code was NSY002, and the total immunization 8 times.
  • boosting was performed with 2 x 107 human CLDN18.2 -HEK293T cells.
  • the immune titer was determined by ELISA method according to the signal of the immune serum on the antigen recombinant protein CLDN18.2 (GenScript, CP0007).
  • the specific method is as follows.
  • the antigen recombinant protein CLDN18.2 was diluted with PBS to 1 ⁇ g/mL to obtain a dilution. 30 [mu]L of the dilution was added to the ELISA plate and coated overnight at 4[deg.]C. On the day of immunotiter determination, the coated plates were rinsed twice with PBS, then blocked with PBST containing 5% nonfat dry milk at room temperature for two hours at room temperature, and rinsed twice with PBS. In another 96-well dilution plate, the unimmunized negative serum and the immunized serum were diluted with PBS, the first well was diluted 1000 times, and then the subsequent 7 wells were diluted by 2 times.
  • the diluted serum was correspondingly added to the first ELISA plate coated with the antigen recombinant protein CLDN18.2, incubated at 37°C for 1 h, washed twice with PBS, and then added the secondary antibody MonoRab TM rabbit anti-camelid VHH antibody at 1:5000 ( GenScript, A01862-200), and finally read the OD value by a microplate reader (Molecular Devices, SpectraMax 190) at a wavelength of 450 nm.
  • the results are shown in Table 1.
  • the immune titer of alpaca is about 1:8000.
  • the upper plasma mixture was carefully aspirated with a sterile Pasteur pipette, and then PBMCs were aspirated with a new sterile Pasteur pipette to obtain isolated PBMCs. Rinse twice with PBS, then centrifuge horizontally at 1500 rpm for 10 min at 4°C, and finally resuspend with 1.5 mL of PBS and count by a cell counter (CountStar, CountStar Altair).
  • RNA of the isolated PBMCs was extracted and reverse transcribed into cDNA by reverse transcription kit (TaKaRa, 6210A). Since the molecular form of alpaca antibody is different from that of ordinary antibodies, it does not contain light chain and heavy chain does not contain CH1, so by designing primers on the front end of VH germline gene and CH2, two fragments of different sizes were obtained by PCR, and recovered by gel tapping.
  • coli SS320 (Lucigen, MC1061F) by electroporation (Bio-Rad, MicroPulser) and plated on ampicillin-resistant 2 -YT solid plate (prepared by tryptone 1.5%, yeast extract 1%, NaCl 0.5%, agar 1.5%, according to mass volume g/mL).
  • ampicillin-resistant 2 -YT solid plate prepared by tryptone 1.5%, yeast extract 1%, NaCl 0.5%, agar 1.5%, according to mass volume g/mL.
  • the total number of clones formed by all electrotransformations was calculated as the clones formed on the plate after dilution of 1 ⁇ L of bacterial solution, that is, the library capacity.
  • the capacity of this immune pool is 1 ⁇ 10 9 cfu.
  • the supernatant was prepared by adding 20% PEG/NaCl (prepared by volume concentration of 20% PEG6000 and 2.5M NaCl), and the phage corresponding to the alpaca library was obtained by precipitation. After rinsing once with PBS , for phage screening.
  • the human CLDN18.2-HEK293T cell line was used as the screening antigen, and the antibody against human CLDN18.2 was screened from the phage display library.
  • the specific method is as follows. Human CLDN18.2-HEK293T or human CLDN18.1-HEK293 were grown in T25 flasks. When the density reaches about 90%, the growth state is the best.
  • SS320 cells (Lucigen, 60512-1) were allowed to stand for 30 minutes, then cultured at 220 rpm for 1 hour, and then by adding VSCM13 helper phage, allowed to stand for 30 minutes, and continued to culture at 220 rpm for 1 hour, centrifuged and replaced to C + /K + 2 In -YT medium, the resulting phages were continued for the second round of screening. This was repeated, and the sequence analysis was performed on 10 randomly selected clones in each round. It was found that after 3 rounds of screening, the sequence enrichment was obvious after the third round of screening.
  • the specific flow level verification method is as follows:
  • APC-labeled anti-mouse Fc secondary antibody (Jackson, 115136071) was added, incubated at 4°C for 1 h, rinsed twice with FACS buffer, and finally passed through a flow cytometer (Beckman, CytoFLEX). AOO-1-1102) for detection. It was found that NA4-S, NA7-S and NA8-S could specifically bind to human CLDN18.2-HEK293T, but not to human CLDN18.1-HEK293 cells. These antibodies were constructed in full length for further verification.
  • VHHs of clones NA4-S, NA7-S and NA8-S and the amino acid sequences (SEQ ID NOs) of CDR1, CDR2 and CDR3 defined using the IMGT or AbM numbering system are shown in Table 2.
  • CLDN18.2 is highly expressed on cancer cells such as gastric cancer.
  • Antibody drugs targeting such tumor-related targets can kill tumors through complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • chimeric antibodies are designed based on the candidate Nanobodies screened in Example 3, and expressed for subsequent CDC and ADCC experiments.
  • a human IgG1 Fc fragment (SEQ ID NO: 21) was fused to the C-terminal of the candidate Nanobody, This fragment includes the linker region and the constant region of IgG1 to mediate effects such as ADCC and CDC.
  • candidate Nanobodies NA4-S (SEQ ID NO: 10), NA7-S (SEQ ID NO: 11) and NA8-S (SEQ ID NO: 12) were selected, and the human IgG1 Fc fragment was fused on their respective basis.
  • NA4-S also referred to in the present disclosure as “candidate antibody NA4-S”, “antibody NA4-S” or “NA4-S antibody”, which is the candidate Nanobody NA4-S chimeric antibody fused to human IgG1 Fc
  • NA7-S also referred to in this disclosure as “candidate antibody NA7-S”, “antibody NA7-S” or “NA7-S antibody”
  • NA8-S also referred to in the present disclosure as “candidate antibody NA8-S”, “antibody NA8-S” or “NA8-S antibody”
  • NA8-S also referred to in the present disclosure as “candidate antibody NA8-S”, “antibody NA8-S” or “NA8-S antibody” which are candidate Nanobodies Chimeric antibody obtained by fusion of NA8-S and human IgG1 Fc
  • Antibodies NA4-S, NA7-S and NA8-S were expressed using the ExpiCHO transient expression system (Gibco, A29133), and the specific methods were as follows:
  • the target protein was eluted with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1 M Tris-HCl, and finally the obtained proteins (ie antibodies NA4-S, NA7-S and NA8-S) were concentrated in tubes (Millipore, UFC901096) into PBS buffer.
  • trypsin (Gibco, 25200-072) containing 0.25% EDTA was used to digest human CLDN18.2-HEK293T, HEK293T, HEK293, human CLDN18.1-HEK293, and mouse CLDN18 in good growth condition.
  • 2-HEK293 and murine CLDN18.1-HEK293 cells 1 ⁇ 10 5 cells were taken and incubated with 10 ⁇ g/mL candidate antibody NA4-S for 1 h, and hIgG1 isotype antibody was used as a control.
  • NA7-S and NA8-S were determined in the same manner as NA4-S.
  • the CDC cell-killing effect of candidate antibodies was determined by MTS assay.
  • the candidate antibodies NA4-S, NA7-S and NA8-S can kill cells through CDC because they contain IgG1Fc fragments, and MTS reagent can be reduced to a colored compound by NADPH or NADH produced by living cells, so the shade of color represents The killing effect of antibody-mediated CDC cell killing effect.
  • the specific operation method is as follows:
  • OD value at the wavelength of 492 nm was determined by a microplate reader.
  • 10% Triton X-100 plus target cells was used as a complete lysis control, only target cells were added as a blank negative control, and rabbit complement plus target cells was used as a background negative control.
  • the results of CDC killing effect of candidate antibody NA4-S on human CLDN18.2-HEK293T cells are shown in Figure 4a.
  • the candidate antibody NA4-S exhibited comparable CDC cell killing effect at equimolar concentrations relative to the control antibody IMAB362, wherein the CDC cell killing EC50 of NA4-S and IMAB362 were 0.7317 nM and 0.6125, respectively nM.
  • the CDC killing effect of candidate antibodies NA7-S and NA8-S on human CLDN18.2-HEK293T cells was determined by the same method, and the results are shown in Figure 4b and Figure 4c.
  • the candidate antibodies NA7-S and NA8-S also exhibited comparable CDC cell killing effects at equimolar concentrations relative to the control antibody IMAB362, among which, the CDC cell killing effects of NA7-S and IMAB362 compared to EC 50 were 0.5069 nM and 0.6285 nM, respectively; the EC50 for CDC cell killing by NA8-S and IMAB362 comparison were 0.4782 nM and 1.863 nM, respectively.
  • NA7-S exhibited stronger CDC cell killing effect than the control antibody IMAB362 at equimolar concentrations, where the EC50 of Nanobody NA7-S was 6.853 nM, while that of IMAB362 was 69.79 nM.
  • NA8-S exhibited stronger CDC cell killing effect than the control antibody IMAB362 at equimolar concentrations, where the EC50 of Nanobody NA8-S was 11.25 nM, while that of IMAB362 was 1865 nM .
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • the ADCC effect was detected by lactate dehydrogenase (LDH) release assay.
  • LDH lactate dehydrogenase
  • the principle is: the variable region of the antibody binds the target antigen on the target cell, and when the Fc segment of the antibody binds to FcRIIIa (aka CD16a) on the NK effector cells in PBMC, the NK cells will release perforin, granzyme, etc.
  • the target cells were lysed, and then LDH lactate dehydrogenase kit (Takara, MK401) was used to detect the release of lactate dehydrogenase in the cell supernatant, so as to determine the killing degree of NK cells to target cells.
  • the specific operations are as follows:
  • Fig. 5a, 5b showed that the candidate antibody NA7-S or NA8-S had comparable cytotoxic activity to the control antibody IMAB362 at equimolar concentrations.
  • the EC50 of ADCC cell killing compared with NA7-S and IMAB362 in Figure 5a were 0.1097nM and 0.0862nM, respectively; the EC50 of ADCC cell killing compared with NA8-S and IMAB362 in Figure 5b were 0.0997nM and 0.0636nM , respectively.
  • the ADCC killing effect of candidate antibody NA7-S or NA8-S on human CLDN18.2-KATOIII tumor cells was determined by the same method. The results are shown in Figures 5c and 5d, the candidate antibody exhibited stronger ADCC cell killing effect than the control antibody IMAB362 at equimolar concentrations, wherein, in the comparison of the candidate antibody NA7-S and IMAB362, NA7-S mediated
  • the ADCC killing efficiency is nearly 50%, while the ADCC killing efficiency of the IMAB362 control antibody is only about 23%; in the comparison of the candidate antibody NA8-S and IMAB362, the ADCC killing efficiency mediated by NA8-S is nearly 44%, while the IMAB362 control antibody has a killing efficiency of nearly 44%.
  • the killing efficiency of ADCC is only about 24%.
  • mice 6-8 week old, female SCID mice (24-26 g).
  • the experimental mice were kept in an independent ventilation box with constant temperature and humidity, the temperature of the rearing room was 21-24°C, and the humidity was 30-53%.
  • Both the candidate antibody and the control antibody IMAB362 specifically bind to CLDN18.2 but not CLDN18.1, and the extramembrane regions of CLDN18.2 and CLDN18.1 only differ by 8 amino acids in the ECD1 region, so it is speculated that the candidate antibody NA7-S , NA8-S and the antigen-binding epitope of the control antibody IMAB362 are on the ECD1 region, in order to confirm whether the antigen-binding positions of the candidate antibody and IMAB362 are consistent, the present embodiment adopts the method of competition binding, and the specific method is as follows:
  • FACS buffer (1X PBS+2% FBS) and use FACS buffer to serially dilute the competing antibodies NA7-S, NA8-S and control antibody IMAB362, and also use FACS buffer to make biotinylated NA7-S ( Or IMAB362) protein was diluted to 13.4nM, 100 ⁇ L of gradient dilution solution and 100 ⁇ L of biotin-labeled antibody dilution solution were added to the 96-well plate, and the 96-well plate was placed at 4°C and incubated for 1 h.
  • CLDN18.2 is highly expressed in various tumors, and it is specifically expressed in the tight junction structure of gastric epithelial cells in normal tissues, so it may become an ideal ADC drug target.
  • the endocytic activity of antibodies was detected by the cytotoxicity of antibody-mediated endocytosis of Fab-ZAP, in which Fab-ZAP (Atsbio, IT-51-100) is an anti-Fc region linked to saporin (saponin). Fab fragments, while saporin is a ribosome inhibitor that inhibits protein synthesis and causes cell death.
  • Fab-ZAP and CLDN18.2 antibody are incubated, the CLDN18.2 antibody is loaded with Fab-ZAP.
  • CLDN18.2-HEK293T cells in logarithmic growth phase were taken, and the cells were digested with trypsin (0.25% (w/v) Trypsin 0.53 mM EDTA) until there was no intercellular adhesion, resulting in a gap, and the digestion was terminated with complete medium. After the cells were thoroughly mixed, the cells were counted and their viability was determined. The cell density was adjusted to 4 ⁇ 10 4 cells/mL, 50 ⁇ L per well was added to the cell culture plate and placed in a 37° C. cell incubator for 16 hours.
  • alpaca-derived nanobodies and human-derived antibodies Compared with mouse-derived antibodies, alpaca-derived nanobodies and human-derived antibodies have higher homology, but their structures are special, so in the process of humanization design of NA4-S, NA7-S and NA8-S, the AbM numbering system was adopted.
  • the CDR sequence and framework region sequence were defined, and the germline gene closest to the framework region sequence of each Nanobody was selected, and the structure of the antibody remained unchanged during back mutation, and finally a series of humanized antibodies were designed.
  • NA4-S-H7, NA7-S-H2 and NA8-S-H9 are the optimal humanized molecules corresponding to the parental antibodies (NA4-S, NA7-S and NA8-S), respectively.
  • amino acid sequences (SEQ ID NOs) of the VHH and CDR1, CDR2 and CDR3 of the humanized antibodies NA4-S-H7, NA7-S-H2 and NA8-S-H9 are shown in Table 3.
  • the humanized molecules NA4-S-H7, NA7-S-H2 and NA8-S-H9 were subjected to affinity maturation modification to improve affinity and biological activity.
  • Affinity maturation transformation is based on M13 phage display technology, using codon-based primers (in the process of primer synthesis, a single codon consists of NNK) to introduce mutations in the CDR region, a total of 4 phage display libraries are constructed: library 1 is CDR1+CDR2+CDR3 single Point combination mutation; library 2 is CDR1+CDR2 double point combination mutation; library 3 is CDR1+CDR3 double point combination mutation, and library 4 is CDR2+CDR3 double point combination mutation.
  • VHH sequence with the mutation site was finally transferred into Escherichia coli SS320 by electroporation.
  • VHH and amino acid sequences (SEQ ID NOs) of CDR1, CDR2 and CDR3 of the antibodies obtained by affinity maturation are shown in Table 4.
  • the affinity matured molecules NA7S-H2-418, NA7S-H2-420, NA7S-H2-20, NA7S-H2-30, NA4S-H7-10, NA4S-H7-3 and NA8S were detected by the same detection method as in Example 5 - Binding of H9-57 on human CLDN18.1-HEK293 cells.
  • the affinity matured molecules NA7S-H2-418, NA7S-H2-420, NA7S-H2-20, NA7S-H2-30, NA4S-H7-10, NA4S-H7-3 and NA8S were detected by the same detection method as in Example 6 - Binding ability of H9-57 on human CLDN18.2-HEK293T and human CLDN18.2-KATOIII cells.
  • NA7S-H2-418, NA7S-H2-420, NA7S-H2-20, NA7S-H2-30, NA4S-H7-10, NA4S-H7-3 and NA8S were determined by the same MTS method as in Example 7 - CDC cell killing effect of H9-57 on human CLDN18.2-HEK293T cells.
  • the Fc end of the affinity matured molecule binds to CD16a (V158) and the VHH end binds to the target cell, it will activate the expression of NF-AT protein in Jurkat cells, and the binding of NF-AT to its response element will trigger the expression of its downstream luciferase.
  • Antibody stimulation with different concentration gradients will obtain antibody concentration-dependent fluorescence reading curves, so that the ADCC activity of the antibody can be evaluated.
  • Luciferase-CD16a(V158) cells stably transfected with CD16a(V158) sequence (UniProtKB-P08637) and pGL4.30 plasmid containing NF-AT-re nucleic acid sequence (promega, #E8481) into Jurkat cells The obtained stably transfected cell line), add 50 ⁇ L of the sample to be tested in gradient dilution, and incubate at 37 °C for 6 h. Add 50 ⁇ L Bright-Lite (vazyme, product number: DD1204-03) to each well, incubate in the dark for 10 min, and detect the fluorescent signal.
  • the detection results of ADCC effect on human CLDN18.2-HEK293T cells are as follows: As shown in Figure 16a and Figure 16b, the ADCC killing effect of affinity matured molecules NA7S-H2-418, NA7S-H2-420 and NA7S-H2-20 was excellent Compared with the parent molecule NA7-S-H2; the affinity matured molecule NA7S-H2-30 is equivalent to the parent molecule NA7-S-H2.
  • the detection results on human CLDN18.2-KATOIII cells are as follows: As shown in Figure 17a, the ADCC killing effect of the affinity matured molecules NA4S-H7-10 and NA4S-H7-3 is better than that of the parent molecule NA4-S-H7; As shown in Figure 17b, the ADCC killing effect of the affinity matured molecule NA8S-H9-57 was better than that of the parent molecule NA8-S-H9.

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Abstract

提供了与CLDN18.2特异性结合的分子，其包含免疫球蛋白单可变结构域，编码所述与CLDN18.2特异性结合的分子的核酸分子、用于表达与CLDN18.2特异性结合的分子的表达载体和宿主细胞；还提供了产生所述与CLDN18.2特异性结合的分子的方法及其用于治疗或预防与CLDN18.2相关的病症的用途。

Description

与CLDN18.2特异性结合的分子
本申请要求2020年11月24日提交的题为“与CLD18A2特异性结合的分子”的第202011332200.9号中国申请的优先权，上述申请的内容整体援引加入本文。
序列表
本申请包含序列表，并且其全部内容通过引用并入本文。
技术领域
本申请一般而言涉及抗体。更具体地，本申请涉及与CLDN18.2特异性结合的分子、其制备方法及其用途。
背景技术
细胞连接是细胞间的联系结构，是多细胞有机体中相邻细胞之间相互联系、协同作用的重要基础。一般而言，动物细胞有四种类型的连接:紧密连接、粘着连接、间隙连接和桥粒/半桥粒。紧密连接，也称为封闭连接，是在内皮细胞或者上皮细胞间形成的结构，可以防止组织间的物质从细胞间隙中扩散，从而只能通过主动运输进入细胞。紧密连接的结构由几十种Claudin蛋白通过细胞内和细胞间的蛋白质相互作用形成，而这些蛋白质的表达具有一定的组织特异性。CLDN18就是紧密连接中存在的Claudin蛋白中的一种重要的蛋白质。
CLDN18是具有四次跨膜区域的膜蛋白，含有两个胞外结构域。人体内存在两种CLDN18变体，分别是CLDN18.1和CLDN18.2(亦称为“CLD18A2”)。两者分布于不同的组织，前者主要表达在肺上皮细胞中，后者则特异性表达在胃上皮细胞中，且在胃干细胞中不表达。在蛋白序列上，CLDN18.1和CLDN18.2的序列相似度极高，两者的主要差别在N端，在第一个胞外结构域两者仅存在8个氨基酸的差别，C端序列完全一致。
抗体疗法正在成为治疗癌症患者的最有前途的方法之一。CLDN18.2由于其在肿瘤细胞和正常组织中的表达特异性，目前已经成为非常有潜力的一个抗体药物作用靶点。然而，由于CLDN18.1和CLDN18.2的序列相似性极高，使得开发只针对CLDN18.2而不针对CLDN18.1的抗体极其困难。针对CLDN18.2靶点研发抗体药物较早的是德国的Ganymed公司，其研制的IMAB362是一种由两条重链和两条轻链组成的单克隆抗体。但是截至目前，尚未见到有靶向CLDN18.2的单结构域抗体的临床在研药物的报道。
单结构域抗体简称单域抗体(single domain antibody，sdAb)，是由单个单体可变 抗体结构域(如抗体重链可变结构域)组成的抗体。像整个抗体(如IgG)一样，它能够选择性结合特定的抗原。但单结构域抗体的分子量却远小于由两条重链和两条轻链组成的常见抗体。第一个单结构域抗体是由骆驼科动物中发现的重链抗体改造而来的(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R(1993)Naturally occurring antibodies devoid of light chains.Nature 363(6428):446–448.)；这些骆驼科动物中发现的重链抗体也被称为VHH片段。目前，对单结构域抗体的大多数研究基于重链可变结构域。
虽然目前已经有靶向CLDN18.2靶点的临床在研单克隆抗体药物，但作为治疗剂，仍有继续开发靶向CLDN18.2靶点抗体的迫切需要。本领域希望开发新的靶向CLDN18.2的抗体，特别是只特异性识别CLDN18.2而不识别CLDN18.1的单结构域抗体。
发明内容
广义而言，本公开提供抗体的新型化合物、制备方法、组合物和制品。本公开提供的益处广泛地适用于抗体治疗和诊断领域。更具体而言，本公开提供了靶向CLDN18.2的单结构域抗体，以及制备所述抗体的方法、用于表达所述抗体的表达载体和宿主细胞等。本公开的抗体提供了治疗或预防与Claudin蛋白相关、特别是与CLDN18.2相关的病症的方法及其应用。
本发明人发现了与CLDN18.2特异性结合的分子，即能够特异性结合人CLDN18.2的胞外结构域1(ECD1)的CLDN18.2结合分子(如靶向CLDN18.2的单结构域抗体)。
本公开至少包括以下实施方案，其分别以“N”(其中“N”表示数字)的方式进行排序和列举。以下列举非穷尽性的，并且本领域技术人员可对不同的技术方案进行组合。
1.CLDN18.2结合分子，其包含至少一个免疫球蛋白单可变结构域，其中所述免疫球蛋白单可变结构域包含CDR1、CDR2和CDR3，并且其中
(1)CDR1在氨基酸序列上与SEQ ID NO:1、4或7所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；CDR2在氨基酸序列上与SEQ ID NO:2、5或8所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；和/或CDR3在氨基酸序列上与SEQ ID NO:3、6或9所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；或者
(2)CDR1在氨基酸序列上与SEQ ID NO:30、37或45所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；CDR2在氨基酸序列上与SEQ ID NO:31、38或46所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；和/或CDR3在氨基酸序列上与SEQ ID NO:32、39或47所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异。
2.实施方案1的CLDN18.2结合分子，其中所述CLDN18.2结合分子是针对CLDN18.2的抗体或其抗原结合片段。
3.实施方案1或2的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域是VHH；例如，来自骆驼科动物(例如羊驼)的VHH。
4.实施方案1-3中任一项的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含:
i)SEQ ID NO:NO:1、4或7所示氨基酸序列的CDR1；
ii)SEQ ID NO:NO:2、5或8所示氨基酸序列的CDR2；和
iii)SEQ ID NO:NO:3、6或9所示氨基酸序列的CDR3。
4a.实施方案1-3中任一项的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含:
i)SEQ ID NO:1、4、7、30、34、37、40、41、45或48所示氨基酸序列的CDR1；
ii)SEQ ID NO:2、5、8、31、35、38、43或46所示氨基酸序列的CDR2；和
iii)SEQ ID NO:3、6、9、32、33、36、39、42、44或47所示氨基酸序列的CDR3。
5.实施方案1-4中任一项的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含:
(a)SEQ ID NO:1所示氨基酸序列的CDR1，SEQ ID NO:2所示氨基酸序列的CDR2和SEQ ID NO:3所示氨基酸序列的CDR3；
(b)SEQ ID NO:4所示氨基酸序列的CDR1，SEQ ID NO:5所示氨基酸序列的CDR2和SEQ ID NO:6所示的氨基酸序列的CDR3；或
(c)SEQ ID NO:7所示氨基酸序列的CDR1，SEQ ID NO:8所示氨基酸序列的CDR2和SEQ ID NO:9所示氨基酸序列的CDR3。
5a.实施方案1-4中任一项的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含:
(d)SEQ ID NO:30所示氨基酸序列的CDR1，SEQ ID NO:31所示氨基酸序列的CDR2和SEQ ID NO:32所示氨基酸序列的CDR3；
(e)SEQ ID NO:30所示氨基酸序列的CDR1，SEQ ID NO:31所示氨基酸序列的CDR2和SEQ ID NO:33所示氨基酸序列的CDR3；
(f)SEQ ID NO:34所示氨基酸序列的CDR1，SEQ ID NO:35所示氨基酸序列的CDR2和SEQ ID NO:36所示氨基酸序列的CDR3；
(g)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
(h)SEQ ID NO:40所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
(i)SEQ ID NO:41所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
(j)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:42所示氨基酸序列的CDR3；
(k)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:43所示氨基酸序列的CDR2和SEQ ID NO:44所示氨基酸序列的CDR3；
(l)SEQ ID NO:45所示氨基酸序列的CDR1，SEQ ID NO:46所示氨基酸序列的CDR2和SEQ ID NO:47所示氨基酸序列的CDR3；
(m)SEQ ID NO:48所示氨基酸序列的CDR1，SEQ ID NO:46所示氨基酸序列的CDR2和SEQ ID NO:47所示氨基酸序列的CDR3。
6.实施方案1-5中任一项的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含或由以下序列组成:
(A)SEQ ID NO:10、11、12、13、14或15所示的氨基酸序列；
(B)与SEQ ID NO:10、11、12、13、14或15所示的氨基酸序列至少80％、85％、90％、95％、98％或99％相同的氨基酸序列；或
(C)与SEQ ID NO:10、11、12、13、14或15相比具有一个或多个(例如，1、2、3、4、5、6、7、8、9或10个)氨基酸的添加、缺失和/或取代的氨基酸序列，优选地，所述添加、缺失和/或取代不发生在CDR区。
6a.实施方案1-5中任一项的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含或由以下序列组成:
(A)SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29所示的氨基酸序列；
(B)与SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29所示的氨基酸序列至少80％、85％、90％、95％、98％或99％相同的氨基酸序列；或(C)与SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29相比具有一个或多个(例如，1、2、3、4、5、6、7、8、9或10个)氨基酸的添加、缺失和/或取代的氨基酸序列，优选地，所述添加、缺失和/或取代不发生在CDR区。
7.实施方案1-6中任一项的CLDN18.2结合分子，其中所述CLDN18.2结合分子是单结构域抗体，例如重链单结构域抗体；嵌合抗体；或人源化抗体。
8.实施方案1-7中任一项的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域与另一分子融合，所述另一分子是例如免疫球蛋白(例如IgG)的Fc结构域或荧光蛋白。
9.实施方案8的CLDN18.2结合分子，其中所述CLDN18.2结合分子是包含来自骆驼科动物的VHH与人IgG(例如，人IgG1或IgG4)的Fc结构域的嵌合抗体。
10.实施方案9的CLDN18.2结合分子，其中所述CLDN18.2结合分子是包含来自羊驼的VHH与人IgG1的Fc结构域的嵌合抗体。
11.实施方案1-10中任一项的CLDN18.2结合分子，其中所述CLDN18.2结合分子结合人CLDN18.2的胞外结构域1(ECD1)。
12.CLDN18.2结合分子，其与实施方案1-11中任一项的CLDN18.2结合分子竞争相同表位。
13.实施方案1-12中任一项的CLDN18.2结合分子，其特异性结合CLDN18.2，但不结合CLDN18.1。
14.分离的核酸分子，其编码如实施方案1-13中任一项的CLDN18.2结合分子。
15.一种表达载体，其包含实施方案14的分离的核酸分子。
16.一种宿主细胞，其包含实施方案15的表达载体。
17.实施方案16的宿主细胞，所述宿主细胞是细菌细胞(例如大肠杆菌(E.coli))，真菌细胞(例如酵母)或哺乳动物细胞。
18.一种药物组合物，其包含至少一种如实施方案1-13中任一项的CLDN18.2结合分子和药学上可接受的载体。
19.制备如实施方案1-13中任一项的CLDN18.2结合分子的方法，包括以下步骤:
-在实施方案16或17的宿主细胞中表达实施方案1-13中任一项的CLDN18.2结合分子；和
-从宿主细胞分离CLDN18.2结合分子。
20.治疗受试者中与CLDN18.2相关的病症的方法，所述方法包括：向所述受试者施用治疗有效量的实施方案1-13中任一项的CLDN18.2结合分子。
21.实施方案20的方法，其中所述与CLDN18.2相关的病症包括涉及表达CLDN18.2的细胞的疾病或与表达CLDN18.2的细胞相关的疾病。
22.实施方案20或21的方法，其中所述与CLDN18.2相关的病症包括癌症。
23.实施方案22的方法，其中所述癌症包括骨癌、血癌、肺癌、肝癌、胰腺癌、皮肤癌、头颈癌、皮肤或眼内黑素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、结肠癌、乳腺癌、前列腺癌、子宫癌、性器官和生殖器官癌、霍奇金病、食管癌、小肠癌、内分泌系统癌症、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、膀胱癌、肾癌、肾细胞癌、肾盂癌、中枢神经系统(CNS)肿瘤、神经外胚层癌症、脊柱轴肿瘤、胶质瘤、脑脊膜瘤和垂体腺瘤。
24.实施方案23的方法，其中所述癌症为胃癌。
25.实施方案1-13中任一项的CLDN18.2结合分子在制备用于治疗或预防与CLDN18.2相关的病症的药物中的用途。
26.用于治疗或诊断与CLDN18.2相关的病症的试剂盒，其包含容器，所述容器包含实施方案1-13中任一项的CLDN18.2结合分子。
以上内容是概括性的描述，必要时可包含细节的简化、概括和省略。因此，本领域技术人员将认识到，该概括性的描述仅是举例说明性的，并不意图以任何方式进行限制。本文所述的方法、组合物和/或装置和/或其他主题的其它方面、特征和优势将在本文所示的教导中变得明显。提供概括性的描述以简化地介绍一些选择的 概念，这些概括性的描述将在下面的详细描述中进一步描述。以上概括性的描述不旨在确定所要求保护的主题的关键特征或基本特征，也不旨在用作确定所要求保护的主题的范围的辅助手段。此外，贯穿本申请引用的所有参考文献、专利和公开的专利申请的内容通过引用整体并入本文。
附图说明
图1a和1b显示实施例1中过表达细胞株和肿瘤细胞株采用IMAB362、hIgG及抗CLDN18的抗体的流式细胞术鉴定结果。其中，图1a显示人CLDN18.2-HEK293T、人CLDN18.1-HEK293、鼠CLDN18.2-HEK293和鼠CLDN18.1-HEK293过表达细胞株的流式细胞术鉴定结果；图1b显示人CLDN18.2-KATOIII肿瘤细胞株的流式细胞术鉴定结果。
图2a-2f显示候选抗体NA4-S、NA7-S和NA8-S在人CLDN18.2-HEK293T、人CLDN18.1-HEK293、鼠CLDN18.2-HEK293和鼠CLDN18.1-HEK293过表达细胞株上的交叉结合实验结果。
图3a、3b和3c显示候选抗体NA4-S、NA7-S、NA8-S和对照抗体在细胞水平上的结合比较实验结果。其中，图3a显示抗体NA4-S和NA7-S在人CLDN18.2-HEK293T过表达细胞上的流式水平比较实验结果；图3b显示抗体NA8-S在人CLDN18.2-HEK293T过表达细胞上的流式水平比较实验结果；图3c显示抗体NA4-S、NA7-S、NA8-S和对照抗体在人CLDN18.2-KATOIII肿瘤细胞株上的流式水平比较实验结果。
图4a-4e显示候选抗体NA4-S、NA7-S和NA8-S在人CLDN18.2-HEK293T过表达细胞株以及人CLDN18.2-KATOIII肿瘤细胞株上介导的补体依赖的细胞毒性作用(CDC)。其中，图4a、4b和4c分别显示抗体NA4-S、NA7-S和NA8-S在人CLDN18.2-HEK293T上介导的CDC细胞毒性效应；图4d和图4e分别显示抗体NA7-S和NA8-S在人CLDN18.2-KATOIII肿瘤细胞株上介导的CDC。
图5a、5b、5c、5d显示候选抗体NA7-S和NA8-S在人CLDN18.2-HEK293T以及人CLDN18.2-KATOIII肿瘤细胞株上介导的抗体依赖的细胞介导的细胞毒性作用(ADCC)。其中，图5a和5b显示抗体NA7-S和NA8-S在人CLDN18.2-HEK293T细胞株上介导的ADCC细胞毒性效应；图5c和5d显示抗体NA7-S和NA8-S在人CLDN18.2-KATOIII肿瘤细胞株上介导的ADCC。
图6a和图6b显示了候选抗体NA7-S和NA8-S在人CLDN18.2-HEK293T异种移植模型中的肿瘤抑制效果。图6a显示了候选抗体NA7-S在人CLDN18.2-HEK293T异种移植模型中的肿瘤抑制效果；图6b显示了候选抗体NA8-S在人CLDN18.2-HEK293T异种移植模型中的肿瘤抑制效果。其中，小鼠采用的是免疫缺陷小鼠SCID，而NA7-S和NA8-S和IMAB362的给药剂量根据每千克体重的质量浓度计算给药，给药方式采用的是尾静脉和腹腔交叉给药。
图7a-7g显示候选抗体NA7-S、NA8-S和IMAB362的在CLDN18.2蛋白上结合表位以及结合的关键氨基酸位点。其中，图7a显示了候选抗体NA7-S和NA8-S在人CLDN18.2-HEK293T上与IMAB362的竞争结合试验；图7b显示了抗体IMAB362和NA8-S在人CLDN18.2-HEK293T上与NA7-S的竞争结合试验；图7c采用抗CLDN18抗体测定人CLDN18.2野生和突变体细胞株上的突变CLDN18.2和野生CLDN18.2的表达水平；图7d显示了NA7-S和IMAB362在人CLDN18.2突变体细胞株上与CLDN18.2突变体的结合水平；图7e显示了NA7-S和IMAB362在人CLDN18.2突变体细胞株上相对于野生细胞株的相对结合百分比。图7f显示了NA8-S和IMAB362在人CLDN18.2突变体细胞株上与CLDN18.2突变体的结合水平；图7g显示了NA8-S和IMAB362在人CLDN18.2突变体细胞株上相对于野生细胞株的相对结合百分比。
图8a-8b显示了NA7-S、NA8-S和IMAB362在Fab-ZAP存在下对人CLDN18.2-HEK293T的细胞杀伤效率。图8a显示了NA7-S和IMAB362对人CLDN18.2-HEK293T的细胞杀伤效率，图8b显示了NA8-S和IMAB362对人CLDN18.2-HEK293T的细胞杀伤效率，其中试验以hIgG和Fab-ZAP作为对照，而Fab-ZAP与细胞长时间孵育也对细胞具有一定的毒性作用。
图9a-9c显示了NA4-S在序列经过人源化修饰前后在人CLDN18.2-HEK293T和人CLDN18.1-HEK293细胞的结合活性，其中NA4-S-H7为人源化后分子。图9a显示的NA4-S人源化前后分子在人CLDN18.2-HEK293T细胞的结合活性比较；图9b显示了不同浓度的NA4-S、NA4-S-H7和IMAB362在人CLDN18.1-HEK293细胞上的结合强度；图9c显示了高浓度下(100μg/mL)，NA4-S、NA4-S-H7和IMAB362在人CLDN18.1-HEK293细胞上的结合阳性率，试验均采用无关抗体作为对照。
图10a-10c显示了NA7-S在序列经过人源化修饰前后在人CLDN18.2-HEK293T和人CLDN18.1-HEK293细胞的结合活性，其中NA7-S-H2为人源化后分子。图10a显示的NA7-S人源化前后分子在人CLDN18.2-HEK293T细胞的结合活性比较；图10b显示了不同浓度的NA7-S、NA7-S-H2和IMAB362在人CLDN18.1-HEK293细胞上的结合强度；图10c显示了高浓度下(100μg/mL)，NA7-S、NA7-S-H2和IMAB362在人CLDN18.1-HEK293细胞上的结合阳性率，试验均采用无关抗体作为对照。
图11a-11c显示了NA8-S在序列经过人源化修饰前后在人CLDN18.2-HEK293T和人CLDN18.1-HEK293细胞的结合活性，其中NA8-S-H9为人源化后分子。图11a显示的NA8-S人源化前后分子在人CLDN18.2-HEK293T细胞的结合活性比较；图11b显示了不同浓度的NA8-S、NA8-S-H9和IMAB362在人CLDN18.1-HEK293细胞上的结合强度；图11c显示了高浓度下(100μg/mL)，NA8-S、NA8-S-H9和IMAB362在人CLDN18.1-HEK293细胞上的结合阳性率，试验均采用无关抗体作为对照。
图12显示了NA7-S-H2在序列经过亲和力成熟前后在人CLDN18.1-HEK293细胞的结合活性。
图13a-13c显示亲和力成熟分子在人CLDN18.2-HEK293T细胞上的结合活性。 图13a显示NA7-S-H2及其亲和力成熟分子在人CLDN18.2-HEK293T细胞上的结合活性；图13b显示NA4-S-H7及其亲和力成熟分子在人CLDN18.2-HEK293T细胞上的结合活性；图13c显示NA8-S-H9及其亲和力成熟分子在人CLDN18.2-HEK293T细胞上的结合活性。
图14显示NA7-S-H2及其亲和力成熟分子在人CLDN18.2-KATOIII肿瘤细胞株上的结合活性。
图15a-15d显示亲和力成熟分子在人CLDN18.2-HEK293T细胞上的介导的补体依赖的细胞毒性作用(CDC)。其中，图15a显示亲和力成熟分子NA7S-H2-418和NA7S-H2-420在人CLDN18.2-HEK293T上介导的CDC细胞毒性效应；图15b显示亲和力成熟分子NA7S-H2-20和NA7S-H2-30在人CLDN18.2-HEK293T上介导的CDC细胞毒性效应；图15c显示亲和力成熟分子NA4S-H7-10和NA4S-H7-3在人CLDN18.2-HEK293T上介导的CDC细胞毒性效应；图15d显示亲和力成熟分子NA8S-H9-57在人CLDN18.2-HEK293T上介导的CDC细胞毒性效应。
图16a和图16b显示NA7-S-H2及其亲和力成熟分子在人CLDN18.2-HEK293T细胞上的介导的抗体依赖的细胞介导的细胞毒性作用(ADCC)。其中，图16a显示亲和力成熟分子NA7S-H2-418和NA7S-H2-420在人CLDN18.2-HEK293T上介导的ADCC；图16b显示亲和力成熟分子NA7S-H2-20和NA7S-H2-30在人CLDN18.2-HEK293T上介导的ADCC。
图17a和图17b显示亲和力成熟分子在人CLDN18.2-KATOIII肿瘤细胞株上的介导的抗体依赖的细胞介导的细胞毒性作用(ADCC)。其中，图17a显示亲和力成熟分子NA4S-H7-10和NA4S-H7-3在人CLDN18.2-KATOIII上介导的ADCC；图17b显示亲和力成熟分子NA8S-H9-57在人CLDN18.2-KATOIII上介导的ADCC。
发明详述
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案。应该强调的是，本发明不限于所举例说明的具体实施方案。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。
除非在此另外定义，否则与本发明结合使用的科学和技术术语将具有本领域普通技术人员通常理解的含义。此外，除非上下文另有要求，单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非上下文另外明确指出，否则单数形式“一”、“一个”和“该”包括复数指示物。因此，例如，提及“一种蛋白质”包括多种蛋白质；提及“一个细胞”包括细胞的混合物等。在本申请中，除非另有说明，否则使用“或”意指“和/或”。此外，术语“包含”以及其他形式(诸如“包括”和“含有”)的使用不是限制性的。此外，说明书和所附权利要求中提供的范围包括端点和端点之间的所有值。
通常，与本文描述的细胞和组织培养、分子生物学、免疫学、微生物学、遗传学和蛋白质以及核酸化学和杂交有关的术语以及其技术是本领域众所周知和常用的术语。除非另有说明，否则本发明的方法和技术通常根据本领域公知的常规方法进行，并如在本说明书全文中引用和讨论的各种通用和更具体的参考文献中所述进行。参见例如Abbas等人，Cellular and Molecular Immunology，6 th ed.，W.B.Saunders Company(2010)；Sambrook J.&Russell D.Molecular Cloning:A Laboratory Manual，3rd ed.，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(2000)；Ausubel等人，Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology，Wiley，John&Sons，Inc.(2002)；Harlow and Lane Using Antibodies:A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(1998)；和Coligan等人，Short Protocols in Protein Science，Wiley，John&Sons，Inc.(2003)。与本文描述的分析化学，合成有机化学和药物化学有关的术语以及实验室程序和技术是本领域中众所周知和常用的术语。此外，本文使用的任何章节标题仅用于组织目的，并且不被解释为限制所描述的主题。
定义
为了更好地理解本发明，相关术语的定义和解释提供如下。
如本文所用，术语“抗体”或“Ab”通常是指表现出所需生物学或结合活性的任何形式的抗体。它包括但不限于人源化抗体、完全人抗体、嵌合抗体和单结构域抗体。抗体可以包含重链和轻链。重链可分为μ、δ、γ、α和ε，它们分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。每条重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区通常由3个结构域(对于γ、α和δ：CH1，CH2和CH3)或4个结构域(对于μ和ε：CH1、CH2、CH3和CH4)组成。每条轻链由轻链可变区(VL)和轻链恒定区(CL)组成。VH和VL区可以进一步分为相对保守的区域(称为框架区(FR))和由FR间隔开的高变区(称为互补决定区(CDR))。每个VH和VL由以下顺序的3个CDR和4个FR组成:从N端到C端的FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。氨基酸在各种区域或结构域中的分布遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987 and 1991))或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883中的定义。抗体可以具有不同的抗体同种型，例如IgG(包括例如IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文所用的术语“人源化抗体”是指其中来源于另一种非人哺乳动物物种种系的CDR序列移植到人框架序列上的抗体。可以在人框架序列内进行额外的框架区修饰。
如本文所用的术语“嵌合抗体”广义上是指含有来自一种抗体的一个或多个区和来自一种或多种其他抗体的一个或多个区的工程化抗体。具体而言，嵌合抗体包含衍生自非人动物抗体的可变区和另一抗体的恒定区，例如包含驼源的可变区和人来 源的恒定区。嵌合抗体还可指对至少两种不同抗原具有特异性的多特异性抗体。
如本文所用的术语“CLDN18.2结合分子”意指特异性结合CLDN18.2的分子。
如本文所用的术语“CLDN18.2抗体”、“针对CLDN18.2的抗体”、“特异性结合CLDN18.2的抗体”、“特异性靶向CLDN18.2的抗体”、“特异性识别CLDN18.2的抗体”可互换地使用，意指能够与Claudin蛋白CLDN18.2特异性结合的抗体。特别地，在具体实施方案中，意指与人CLDN18.2特异性结合的抗体，特别是与人CLDN18.2特异性结合而不与人CLDN18.1特异性结合的抗体。人CLDN18.2和人CLDN18.1的氨基酸序列分别如SEQ ID NO:16和SEQ ID NO:17所示；小鼠CLDN18.2和小鼠CLDN18.1的氨基酸序列分别如SEQ ID NO:18和SEQ ID NO:19所示。
如本文所用的术语“免疫球蛋白单可变结构域”或“ISV”在本文中通常定义为这样的氨基酸序列:其包含免疫球蛋白折叠或在适合的条件下(如生理条件下)能够形成免疫球蛋白折叠(即通过折叠)，即从而形成免疫球蛋白可变结构域(例如，VH，VL或VHH结构域)；和形成(或在适合的条件下能够形成)免疫球蛋白可变结构域，其包含功能性抗原结合位点(在其不需要与另一个免疫球蛋白可变结构域相互作用(如VH-VL相互作用)以形成功能性抗原结合位点的意义上)。
本文所用的结合分子、抗体或抗体编号(如NA4-S、NA7-S、NA8-S、NA4-S-H7、NA7-S-H2、NA8-S-H9、NA7S-H2-418、NA8S-H9-57、NA7S-H2-420、NA7S-H2-20、NA7S-H2-30、NA4S-H7-10、NA4S-H7-3、NA8S-H9-57等)仅为区分或标识分子或产品所用，并不意图表示这样的标识是本发明的分子或产品的特征。本领域技术人员应当理解，例如出于区分或标识的目的，其他分子、抗体或产品也有可能使用这样的标识，但并非是指相同或等同的分子、抗体或产品。类似地，实施例中所用的类似编号或标识也仅仅是用于示例方便，本发明的结合分子或产品由所附权利要求中所描述的特征限定。
如本文所用的术语“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。在一些实施方案中，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％。在一些实施方案中，该抑制可以大于60％，大于70％，大于80％或大于90％。
如本文所用的术语“EC 50”，也被称为“半数有效浓度”，是指在特定的暴露时间后诱导在基线和最大值之间的50％的应答的药物、抗体或毒剂的浓度。在本申请的上下文中，EC 50的单位为“nM”或“μg/mL”。
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WO03/048731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。
如本文所用，术语“分离的”是指通过人工方式从天然状态获得的物质或组分的状态。如果某种“分离的”物质或组分天然存在，则可能是因为其所处的天然环境发生了变化，或者从天然环境下分离出该物质或组分，或者两者兼而有之。例如，某种未分离的多核苷酸或多肽天然存在于某个活体动物体内，从该天然状态分离的相同的高纯度多核苷酸或多肽被称为分离的多核苷酸或多肽。术语“分离的”既不排除混合的人造或合成物质，也不排除不影响分离的物质的活性的其他不纯物质。
如本文所用，术语“分离的抗体”指基本上不含具有不同抗原特异性的其他抗体的抗体。此外，分离的抗体可以基本上不含其他细胞材料和/或化学物质。
如本文所用，术语“载体”是指可以在其中插入多核苷酸的核酸媒介物。当载体允许插入其中的多核苷酸编码的蛋白质的表达时，该载体称为表达载体。该载体可以通过转化、转导或转染入宿主细胞而使携带的遗传物质元件在宿主细胞中表达。载体是本领域技术人员所熟知的，包括但不限于质粒，噬菌体，粘粒，人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生人工染色体(PAC)，噬菌体如λ噬菌体或M13噬菌体和动物病毒。可用作载体的动物病毒包括但不限于逆转录病毒(包括慢病毒)，腺病毒，腺伴随病毒，疱疹病毒(如单纯疱疹病毒)，痘病毒，杆状病毒，乳头瘤病毒，乳多空病毒(如SV40)。载体可以包含用于控制表达的多个元件，包括但不限于启动子序列，转录起始序列，增强子序列，选择元件和报道基因。另外，载体可以包含复制起点。
如本文所用，术语“宿主细胞”是指可以导入载体的任何种类的细胞系统，包括但不限于原核细胞如大肠杆菌(E.coli)或枯草芽孢杆菌(Bacillus subtilis)，真菌细胞如酵母细胞或曲霉属(Aspergillus)，昆虫细胞如S2果蝇细胞或Sf9，以及动物细胞如成纤维细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞。
利用宿主细胞生产本发明的抗体的方法是本领域常规的，包括在原核或真核细胞中表达抗体，然后分离抗体，并通常纯化至可药用的纯度。如在一些实施方案中，通过本领域已知的标准技术将编码抗体的核酸插入到表达载体并将表达载体导入合适的原核或真核宿主细胞，在足以产生本发明的抗体或其功能性片段的条件和时 间下培养宿主细胞，如CHO细胞、NSO细胞、SP2/0细胞、HEK293细胞、COS细胞、PER.C6(R)细胞、酵母或大肠杆菌细胞，并从细胞(裂解后的上清液或细胞)中回收抗体及对抗体进行纯化。用于生产抗体的常规方法是本领域现有技术已知的，描述在例如Makrides,S.C.，Protein Expr.Purif.17(1999)183-202；Geisse,S.等人，Protein Expr.Purif.8(1996)271-282；Kaufman,R.J.，Mol.Biotechnol.16(2000)151-160；fferner,R.G.，Drug Res.48(1998)870-880的综述文章中。
如本文所用，术语“同一性”是指通过比对和比较序列确定的两个或更多个多肽分子或两个或更多个核酸分子的序列之间的关系。“百分比同一性”是指比较分子中氨基酸或核苷酸之间相同残基的百分比，并基于被比较的最小分子的大小计算。对于这些计算，比对中的间隙(如果有的话)优选通过特定的数学模型或计算机程序(即“算法”)来寻址。可以用于计算比对的核酸或多肽的同一性的方法包括在Computational Molecular Biology,(Lesk，A.M.,ed.)，1988,New York:Oxford University Press；Biocomputing Informatics and Genome Projects,(Smith，D.W.,ed.),1993,New York:Academic Press；Computer Analysis of Sequence Data,Part I,(Griffin，A.M.,and Griffin，H.G.，eds.),1994,New Jersey:Humana Press；von Heinje G.,1987,Sequence Analysis in Molecular Biology,New York:Academic Press；Sequence Analysis Primer,(Gribskov M.and Devereux J.,eds.),1991,New York:M.Stockton Press；和Carillo等人，1988，SIAMJ.Applied Math.48:1073中描述的那些。
如本文所用，术语“免疫原性”是指刺激生物体中特异性抗体或致敏淋巴细胞形成的能力。它不仅指抗原刺激特定免疫细胞活化、增殖和分化以最终产生免疫效应物质如抗体和致敏淋巴细胞的性质，还指抗体或致敏T淋巴细胞的特异性免疫应答可以在用抗原刺激生物体后在生物体的免疫系统中形成。免疫原性是抗原最重要的特性。抗原是否能够成功诱导宿主中免疫应答的产生取决于三个因素:抗原的性质，宿主的反应性和免疫手段。
如本文所用，术语“转染”是指将核酸引入真核细胞特别是哺乳动物细胞的过程。用于转染的方案和技术包括但不限于脂质转染，化学和物理方法转染如电穿孔。许多转染技术在本领域是公知的，参见例如Graham等人，1973，Virology 52:456；Sambrook等人，2001，Molecular Cloning:A Laboratory Manual；Davis等人，1986，Basic Methods in Molecular Biology，Elsevier；Chu et al，1981，Gene 13:197。
如本文所用，术语“荧光激活细胞分选”或“FACS”是指专门类型的流式细胞术。它提供了根据每个细胞的特定光散射和荧光特征，将生物细胞的异质混合物以每次一个细胞分拣到两个或更多个容器中的方法(FlowMetric.“Sorting Out Fluorescence Activated Cell Sorting”.2017-11-09)。用于进行FACS的仪器是本领域技术人员已知的并且对于公众是可商购获得的。这种仪器的实例包括Becton Dickinson(Foster City，CA)的FACS Star Plus、FACScan和FACSort仪器、来自Coulter Epics Division(Hialeah，FL)的Epics C和来自Cytomation(Colorado Springs，Colorado)的MoFlo。
术语“受试者”包括任何人或非人动物，优选人。
如本文所用，术语“与CLDN18.2相关的病症”是指由CLDN18.2(如人CLDN18.2)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病症。
如本文所用，术语“癌症”是指引发医学病症的任何肿瘤或恶性细胞生长、增殖或转移介导的实体瘤或非实体瘤如白血病。
本文在治疗病情的情况中使用的术语“治疗”一般涉及人或动物的治疗和疗法，其中实现了一些期望的治疗效果，例如，抑制病情进展，包括进展速度下降，进展速度停滞，病情消退，病情改善和病情治愈。还包括了作为预防措施(即预防)的治疗。对于癌症，“治疗”可能是指抑制或减缓肿瘤或恶性细胞生长、增殖或转移或其某种组合。对于肿瘤，“治疗”包括去除全部或部分肿瘤、抑制或减缓肿瘤生长和转移、预防或延迟肿瘤的发展或其某种组合。
如本文所用，术语“治疗有效量”涉及活性化合物或包含活性化合物的材料、组合物或剂型的量，其在按照所需的治疗方案施用时有效用于产生与合理的益处/风险比相称的某些所需的治疗效果。具体而言，“治疗有效量”意指抗体或其抗原结合部分有效治疗与CLDN18.2相关的病症的量或浓度。
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。
CLDN18.2结合分子
在一些方面，本公开提供了CLDN18.2结合分子。
一般来说，CLDN18.2结合分子可以包括任何特异性结合CLDN18.2的分子。CLDN18.2结合分子可以是多肽或蛋白质，例如抗体，更具体地说是特异性结合CLDN18.2(如人CLDN18.2)的抗体。
抗体包括但不限于嵌合抗体、人源化抗体或单结构域抗体等。在具体的实施方案中，CLDN18.2结合分子是单结构域抗体，其通常是指由单个单体可变抗体结构域组成的抗体。像整个抗体一样，单结构域抗体能够选择性结合特定抗原。
更具体而言，CLDN18.2结合分子是重链单结构域抗体，其可与术语“VHH”、“VHH抗体”、“VHH结构域”、“VHH抗体片段”、“V HH”或“纳米抗体”等互换使用。来自骆驼科抗体的VHH分子是已知的最小完整抗原结合结构域之一(约15KDa，或是常规IgG的1/10)，因此非常适合递送至致密组织和进入大分子之间的有限空间。
本文公开的单结构域抗体可由本领域技术人员根据本领域已知的方法或任何未来的方法制备。例如，可以使用本领域已知的方法获得VHH，例如通过免疫骆驼并从中获得与靶抗原结合并中和靶抗原的VHH，或者通过使用本领域已知的分子生物学技术克隆本发明的VHH的文库，然后通过使用噬菌体展示进行选择。在一些实施方案中，本发明的单结构域抗体在骆驼科动物中天然产生，即，使用本文对于其它抗体描述的技术，用CLDN18.2或其片段免疫骆驼来生产。
在一些实施方案中，通过用所需抗原免疫美洲驼或羊驼并随后分离编码重链抗体的mRNA来获得单结构域抗体。通过逆转录和聚合酶链反应，产生含有数百万克隆的单结构域抗体的基因文库。筛选技术如噬菌体展示和核糖体展示有助于鉴定结合抗原的克隆。其中噬菌体展示是在噬菌体上合成抗体文库，用感兴趣的抗原或其抗体结合部分筛选文库，并分离结合抗原的噬菌体，从其中可以获得免疫反应性片段。用于制备和筛选这种文库的方法是本领域众所周知的，并且用于产生噬菌体展示文库的试剂盒可商购获得(例如，Pharmacia重组噬菌体抗体系统，目录号27-9400-01；以及Stratagene SurfZAP TM噬菌体展示试剂盒，目录号240612)。还有其他方法和试剂可用于产生和筛选抗体展示文库(参见例如Barbas等人，Proc.Natl.Acad.Sci.USA 88:7978-7982(1991))。
当最有效的克隆被鉴定时，可通过例如亲和力成熟或人源化优化其DNA序列，以防止人体对抗抗体的免疫反应。如本文所用，“亲和力成熟”的抗体的一个或多个CDR相比于母本抗体的一个或多个CDR包含一个或多个氨基酸的添加、缺失和/或取代，使得亲和力成熟的抗体与母本抗体相比，对抗原的亲和力得到改进。对抗体进行亲和力成熟的方法是本领域已知的，参见例如Marks et al.,Bio/Technology 10:779-783(1992)；Barbas et al.,Proc.Nat.Acad.Sci.USA 91:3809-3813(1994)；Scier et al.,Gene 169:147-155(1995)；和Hawkins et al.,J.Mol.Biol.226:889-896(1992)。
因此，可通过以下方式获得本发明的单结构域抗体:(1)分离天然存在的重链抗体的VHH结构域；(2)通过表达编码天然存在的VHH结构域的核苷酸序列；(3)通过天然存在的VHH结构域的“人源化”或通过表达编码这种人源化VHH结构域的核酸；(4)通过来自任何动物物种，特别是哺乳动物物种，例如来自人的天然存在的VH结构域的“骆驼化”，或通过表达编码这种骆驼化VH结构域的核酸；(5)通过“结构域抗体”或“dAb”的“骆驼化”(参见例如Ward等人，1989，Nature 341:544-546)，或通过表达编码这种骆驼化VH结构域的核酸；(6)通过使用合成或半合成技术制备蛋白质、多肽或其他氨基酸序列；(7)通过使用用于核酸合成的技术制备编码VHH的核酸，然后表达由此获得的核酸；和/或(8)通过前述的任何组合。基于本文的公开内容，用于执行前述内容的合适方法和技术对于本领域技术人员将是清楚的，并且包括例如下文更详细描述的方法和技术。
单结构域抗体通常通过从免疫动物获得的血液、淋巴结或脾淋巴细胞的cDNA经PCR克隆可变结构域库至噬菌体展示载体中而产生。通常通过在固定化抗原(例 如涂布在试管塑料表面的抗原，固定在链霉抗生物素蛋白珠上的生物素化抗原或细胞表面上表达的膜蛋白)上淘选相应文库来选择抗原特异性单结构域抗体。通过体外模拟该策略可以提高sdAb的亲和力，例如通过CDR区的定点诱变和在增加的严格条件(更高温度，高或低盐浓度，高或低pH和低抗原浓度)下对固定化抗原进行进一步的淘选(Wesolowski等人.，Single domain antibodies:promising experimental and therapeutic tools in infection and immunity.Med Microbiol Immunol(2009)198:157-174)。
用于制备特异性结合抗原或表位的VHH的方法描述于参考文献中，参见例如:R.van der Linden等人，Journal of Immunological Methods，240(2000)185-195；Li等人，J Biol Chem.，287(2012)13713-13721；Deffar等人，African Journal of Biotechnology Vol.8(12)，pp.2645，17 June，2009和WO94/04678。
在一些实施方案中，CLDN18.2结合分子中的VHH与抗体的Fc结构域(例如IgG(例如IgG1或IgG4)的Fc结构域)融合。通过将VHH融合至Fc结构域，可以更有效地募集效应功能，例如ADCC和CDC。而且，VHH与Fc结构域的融合可以帮助CLDN18.2结合分子形成二聚体，并且还可以帮助延长CLDN18.2结合分子的体内半衰期。
如本文所用，术语“抗体依赖的细胞介导的细胞毒性作用”或“ADCC”是指与某些细胞毒性效应细胞(例如天然杀伤(NK)细胞，嗜中性粒细胞和巨噬细胞)上存在的Fc受体(FcR)结合的分泌的抗体使这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞并随后用细胞毒素杀死靶细胞的细胞毒性形式。抗体“武装”细胞毒性细胞对于这种杀伤是绝对需要的。介导ADCC的主要细胞NK细胞仅表达FcγRIII，而单核细胞表达FcγRI，FcγRII和FcγRIII。造血细胞上的FcR表达总结在Ravetch and Kinet，Annu.Rev.Immunol 9:457-92(1991)的464页的表3中。为了评估感兴趣分子的ADCC活性，可以进行体外ADCC测定，例如美国专利US5500362或US5821337中所述的测定方法。可用于此类测定的效应细胞包括外周血单核细胞(PBMC)和自然杀伤(NK)细胞。可选或另外地，感兴趣分子的ADCC活性可以在体内评估，例如在如Clynes等人PNAS(USA)95:652-656(1998)公开的动物模型中评估。
术语“补体依赖的细胞毒性作用”或“CDC”是指在补体存在下靶细胞的裂解。经典补体途径的激活由补体系统的第一组分(C1q)与结合其同源抗原的抗体(适当的亚类)结合而启动。为了评估补体活化，可以执行CDC测定，通过例如Gazzano-Santoro等人，J.Immunol.Methods 202:163(1996)中所述的方法。
为了便于描述，在下文中将CLDN18.2结合分子描述为CLDN18.2抗体。
能够特异性结合CLDN18.2的特定表位的CLDN18.2抗体
在一个方面，本公开涉及特异性结合CLDN18.2、而不结合或基本不结合CLDN18.1的单结构域抗体。
本发明人发现了能够特异性结合人CLDN18.2的胞外结构域1(ECD1)的 CLDN18.2结合分子(如靶向CLDN18.2的单结构域抗体)。
如前所述，CLDN18.2有两个胞外结构域(ECD)，其中人CLDN18.2的全长序列如SEQ ID NO:16所示，其中ECD1的序列如SEQ ID NO:20所示。小鼠CLDN18.2如SEQ ID NO:18所示。
对于人和小鼠的CLDN18.1而言，其序列分别如SEQ ID NO:17和SEQ ID NO:19所示。
包含与特定序列具有序列同一性的CDR的CLDN18.2抗体
在一些实施方案中，本公开的CLDN18.2抗体包含至少一个免疫球蛋白单可变结构域(例如VHH)，其中所述VHH包含CDR1、CDR2和CDR3，并且其中CDR1包含与SEQ ID NO:1、4或7至少80％相同的氨基酸序列，CDR2包含与SEQ ID NO:2、5或8至少80％相同的氨基酸序列，和CDR3包含与SEQ ID NO:3、6或9至少80％相同的氨基酸序列。
在一个给定的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,“Standardconformations 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 ofImmunological Interest,第4版,U.S.Department of Health and Human Services,NationalInstitutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，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。本公开的CLDN18.2抗体采用的是IMGT或AbM方式来定义CDR区的氨基酸序列，但是CDR的定义方式不是唯一和限定式的，更不表示本公开仅保护采用IMGT或AbM方式定义的CDR区所代表的抗体分子。基于前述各类本领域常规的抗体CDR指派系统，本公开的CLDN18.2抗体分子还能够以其他各种不同CDR区定义方式来表征。应该注意的是，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序列可能会有所不同。这些不同指派系统下定义的CDR序列及其表征的抗体分子也是本公开希望保护的。
抗体序列中的可变区和CDR可以根据本领域已经开发的一般规则(如上所述，例如Kabat编号系统)或通过将序列与已知可变区的数据库比对来鉴定。在Kontermann and Dubel,eds.,Antibody Engineering,Springer,New York,NY,2001和Dinarello等人,Current Protocols in Immunology,John Wiley and Sons Inc.,Hoboken,NJ,2000中描述了鉴定这些区域的方法。抗体序列的示例性数据库描述于并可获自www.bioinf.org.uk/abs上的“Abysis”网站(由Department of Biochemistry&Molecular Biology University College London,London,England的A.C.Martin维护)和VBASE2 网站www.vbase2.org，如Retter等人,Nucl.Acids Res.,33(Database issue):D671-D674(2005)中所述。优选使用Abysis数据库分析序列，其整合了来自Kabat、IMGT和蛋白质数据库(PDB)的序列数据与来自PDB的结构数据，参见Dr.Andrew C.R.Martin所著的书中的Protein Sequence and Structure Analysis of Antibody Variable Domains.In:Antibody Engineering Lab Manual(Ed.:Duebel,S.and Kontermann,R.,Springer-Verlag,Heidelberg,ISBN-13:978-3540413547，也可在网站bioinforg.uk/abs上获得)。Abysis数据库网站还包括已经开发用于识别可以根据本文的教导使用的CDR的一般规则。
两个氨基酸序列之间的百分比同一性可以使用E.Meyers和W.Miller的算法(Comput.Appl.Biosci.,4:11-17(1988))确定，该算法已被并入ALIGN程序(版本2.0)，使用PAM120权重残基表，空位长度罚分为12，空位罚分为4。另外，两个氨基酸序列之间的百分比同一性可以通过Needleman和Wunsch的算法(J.Mol.Biol.48:444-453(1970))确定，其已并入GCG软件包(可从http://www.gcg.com获得)中的GAP程序中，使用Blossum 62矩阵或PAM250矩阵，空隙权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6。
另外地或可选地，本公开的蛋白质序列可以进一步用作“查询序列”来执行针对公共数据库的搜索以例如识别相关序列。这种搜索可以使用Altschul,等人(1990)J.MoI.Biol.215:403-10的XBLAST程序(版本2.0)来执行。可用XBLAST程序进行BLAST蛋白质搜索，得分＝50，字长＝3，以获得与本公开的抗体分子同源的氨基酸序列。为了获得用于比较目的的空位比对，可使用空位BLAST，如Altschul等人,(1997)Nucleic Acids Res.25(17):3389-3402中所述的。当使用BLAST和空位BLAST程序时，可以使用各个程序(例如，XBLAST和NBLAST)的默认参数，参见www.ncbi.nlm.nih.gov。
在另一些实施方案中，CDR的氨基酸序列可以与上文给出的各个序列至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。
包含具有氨基酸添加、缺失或取代的CDR的CLDN18.2抗体
在一些实施方案中，本公开的CLDN18.2抗体包含至少一个免疫球蛋白单可变结构域(例如VHH)，其中所述VHH包含CDR1、CDR2和CDR3。在一些实施方案中，CDR1在氨基酸序列上与SEQ ID NO:1、4或7所示的序列存在不超过2个(如0个，1个或2个)氨基酸的氨基酸添加、缺失或取代的差异；CDR2在氨基酸序列上与SEQ ID NO:2、5或8所示的序列存在不超过2个(如0个，1个或2个)氨基酸的氨基酸添加、缺失或取代的差异；和/或CDR3在氨基酸序列上与SEQ ID NO:3、6或9所示的序列存在不超过2个(如0个，1个或2个)氨基酸的氨基酸添加、缺失或取代的差异。例如，CDR1、CDR2和CDR3分别与SEQ ID NO:1、4或7，SEQ ID NO:2、5或8和SEQ ID NO:3、6或9所示的氨基酸序列存在仅一个氨基酸的氨基 酸添加、缺失或取代的差异。上述CDR的编码系统可以采用IMGT。在另一些实施方案中，CDR1在氨基酸序列上与SEQ ID NO:30、37或45所示的序列存在不超过2个(如0个，1个或2个)氨基酸的氨基酸添加、缺失或取代的差异；CDR2在氨基酸序列上与SEQ ID NO:31、38或46所示的序列存在不超过2个(如0个，1个或2个)氨基酸的氨基酸添加、缺失或取代的差异；和/或CDR3在氨基酸序列上与SEQ ID NO:32、39或47所示的序列存在不超过2个(如0个，1个或2个)氨基酸的氨基酸添加、缺失或取代的差异。上述CDR的编码系统可以采用AbM。
优选地，分离的抗体或其抗原结合部分的CDR含有不多于2个氨基酸或不多于1个氨基酸的保守取代。如本文所用，术语“保守取代”是指不会不利地影响或改变包含氨基酸序列的蛋白质/多肽的基本性质的氨基酸取代。例如，保守取代可以通过本领域已知的标准技术(例如定点诱变和PCR介导的诱变)引入。保守氨基酸取代包括其中氨基酸残基被具有相似侧链的另一氨基酸残基取代的取代，例如物理或功能相似的残基(例如具有相似的大小，形状，电荷，化学性质包括形成共价键或氢键的能力等)被相应的氨基酸残基的取代。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸，精氨酸和组氨酸)，具有酸性侧链的氨基酸(例如天冬氨酸和谷氨酸)，具有不带电荷的极性侧链的氨基酸(例如甘氨酸，天冬酰胺，谷氨酰胺，丝氨酸，苏氨酸，酪氨酸，半胱氨酸，色氨酸)，具有非极性侧链的氨基酸(例如丙氨酸，缬氨酸，亮氨酸，异亮氨酸，脯氨酸，苯丙氨酸，甲硫氨酸)，具有β-分支侧链的氨基酸(例如苏氨酸，缬氨酸，异亮氨酸)和具有芳香族侧链的氨基酸(例如酪氨酸，苯丙氨酸，色氨酸，组氨酸)。因此，相应的氨基酸残基优选被来自相同侧链家族的另一个氨基酸残基取代。用于鉴定氨基酸保守取代的方法在本领域中是公知的(参见例如Brummell等人,Biochem.32:1180-1187(1993)；Kobayashi等人,Protein Eng.12(10):879-884(1999)；和Burks等人,Proc.Natl.Acad.Sci.USA 94:412-417(1997)，其通过引用并入本文)。
在某些实施方案中，CLDN18.2抗体的免疫球蛋白单可变结构域包含:
i)SEQ ID NO:1、4或7所示氨基酸序列的CDR1；
ii)SEQ ID NO:2、5或8所示氨基酸序列的CDR2；和
iii)SEQ ID NO:3、6或9所示氨基酸序列的CDR3。
在某些实施方案中，CLDN18.2抗体的免疫球蛋白单可变结构域包含:
i)SEQ ID NO:30、37、或45所示氨基酸序列的CDR1；
ii)SEQ ID NO:31、38或46所示氨基酸序列的CDR2；和
iii)SEQ ID NO:32、39或47所示氨基酸序列的CDR3。
在另一些实施方案中，CLDN18.2抗体的免疫球蛋白单可变结构域包含:
i)SEQ ID NO:1、4、7、30、34、37、40、41、45或48所示氨基酸序列的CDR1；
ii)SEQ ID NO:2、5、8、31、35、38、43或46所示氨基酸序列的CDR2；和
iii)SEQ ID NO:3、6、9、32、33、36、39、42、44或47所示氨基酸序列的CDR3。
包含CDR的CLDN18.2抗体
在一些实施方案中，本公开的CLDN18.2抗体包含至少一个免疫球蛋白单可变结构域(例如VHH)，其中所述VHH包含CDR1、CDR2和CDR3，并且其中CDR1、CDR2和CDR3选自:
(a)SEQ ID NO:1所示氨基酸序列的CDR1，SEQ ID NO:2所示氨基酸序列的CDR2和SEQ ID NO:3所示氨基酸序列的CDR3；
(b)SEQ ID NO:4所示氨基酸序列的CDR1，SEQ ID NO:5所示氨基酸序列的CDR2和SEQ ID NO:6所示氨基酸序列的CDR3；
(c)SEQ ID NO:7所示氨基酸序列的CDR1，SEQ ID NO:8所示氨基酸序列的CDR2和SEQ ID NO:9所示氨基酸序列的CDR3；
(d)SEQ ID NO:30所示氨基酸序列的CDR1，SEQ ID NO:31所示氨基酸序列的CDR2和SEQ ID NO:32所示氨基酸序列的CDR3；
(e)SEQ ID NO:30所示氨基酸序列的CDR1，SEQ ID NO:31所示氨基酸序列的CDR2和SEQ ID NO:33所示氨基酸序列的CDR3；
(f)SEQ ID NO:34所示氨基酸序列的CDR1，SEQ ID NO:35所示氨基酸序列的CDR2和SEQ ID NO:36所示氨基酸序列的CDR3；
(g)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
(h)SEQ ID NO:40所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
(i)SEQ ID NO:41所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
(j)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:42所示氨基酸序列的CDR3；
(k)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:43所示氨基酸序列的CDR2和SEQ ID NO:44所示氨基酸序列的CDR3；
(l)SEQ ID NO:45所示氨基酸序列的CDR1，SEQ ID NO:46所示氨基酸序列的CDR2和SEQ ID NO:47所示氨基酸序列的CDR3；
(m)SEQ ID NO:48所示氨基酸序列的CDR1，SEQ ID NO:46所示氨基酸序列的CDR2和SEQ ID NO:47所示氨基酸序列的CDR3。
通过VHH序列定义的CLDN18.2抗体
在一些实施方案中，本公开的CLDN18.2抗体包含至少一个(例如，一个)免疫球蛋白单可变结构域(例如VHH)，其中所述VHH包含:
(A)SEQ ID NO:10、11、12、13、14或15所示的氨基酸序列；
(B)与SEQ ID NO:10、11、12、13、14或15至少80％、85％、90％、95％、98％或99％相同的氨基酸序列；或
(C)与SEQ ID NO:10、11、12、13、14或15相比具有一个或多个(例如，1、2、3、4、5、6、7、8、9或10个)氨基酸的添加、缺失和/或取代的氨基酸序列，优选地，所述添加、缺失和/或取代不发生在CDR区。
在另一些实施方案中，本公开的CLDN18.2抗体包含至少一个(例如，一个)免疫球蛋白单可变结构域(例如VHH)，其中所述VHH包含:
(A)SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29所示的氨基酸序列；
(B)与SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29所示的氨基酸序列至少80％、85％、90％、95％、98％或99％相同的氨基酸序列；或
(C)与SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29相比具有一个或多个(例如，1、2、3、4、5、6、7、8、9或10个)氨基酸的添加、缺失和/或取代的氨基酸序列，优选地，所述添加、缺失和/或取代不发生在CDR区。
在其他实施方案中，所述VHH的氨基酸序列可以与上述各个序列至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。作为说明性实例，抗体可以包含与SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的VHH。
在一些进一步的实施方案中，本公开的CLDN18.2抗体可以包含重链可变区中的氨基酸的保守取代或修饰。本领域理解的是，可以进行某些不消除抗原结合性质的保守序列修饰。参见例如Brummell等人(1993)Biochem 32:1180-8；de Wildt等人(1997)Prot.Eng.10:835-41；Komissarov等人(1997)J.Biol.Chem.272:26864-26870；Hall等人(1992)J.Immunol.149:1605-12；Kelley and O’Connell(1993)Biochem.32:6862-35；Adib-Conquy等人(1998)Int.Immunol.10:341-6和Beers等人(2000)Clin.Can.Res.6:2835-43。
编码本公开的抗体的核酸分子和产生CLDN18.2抗体的方法
在一些方面，本公开涉及分离的核酸分子，其包含编码本公开的CLDN18.2抗体的核酸序列。
本公开的核酸可以使用标准分子生物学技术获得。对于从免疫球蛋白基因文库获得的抗体(例如，使用噬菌体展示技术)，编码这种抗体的核酸可以从基因文库中回收。
可以使用本领域已知的重组技术将编码本公开的CLDN18.2抗体的核酸分子插入载体以进一步克隆(扩增DNA)或用于表达。许多载体是可用的。载体或载体组分通常包括但不限于以下一种或多种:信号序列，复制起点，一个或多个标记基因，增强子元件，启动子(例如SV40，CMV，EF-1α)，和转录终止序列。选择标记基因有助于选择导入了载体的宿主细胞(参见例如美国专利号4,399,216；4,634,665和5,179,017)。例如，通常选择标记基因赋予载体已经导入其中的宿主细胞对药物(例 如G418，潮霉素或甲氨蝶呤)的抗性。在一个实施方案中，选择标记基因可以包括二氢叶酸还原酶(DHFR)基因(用于具有甲氨蝶呤选择/扩增的dhfr-宿主细胞)和neo基因(用于G418选择)。在另一个实施方案中，抗体可以通过本领域已知的同源重组产生。编码单克隆抗体的DNA易于使用常规方法分离和测序(例如，通过使用能够与编码抗体重链的基因特异性结合的寡核苷酸探针)。
在一些实施方案中，载体系统包括哺乳动物、细菌、酵母系统等，并包括质粒，例如，但不限于，pALTER、pBAD、pcDNA、pCal、pL、pET、pGEMEX、pGEX、pCI、pCMV、pEGFP、pEGFT、pSV2、pFUSE、pVITRO、pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS420、pLexA、pACT2.2等等，以及其他实验室和商业可用的载体。合适的载体可以包括质粒或病毒载体(例如复制缺陷型逆转录病毒，腺病毒和腺伴随病毒)。在本公开的一个实施方案中，载体可以是pET，例如含有六组氨酸标签和c-Myc-标签基因的pETbac。
包含编码本公开的CLDN18.2抗体的核酸序列的载体可以被引入宿主细胞用于克隆或基因表达。用于在本文的载体中克隆或表达DNA的合适宿主细胞是原核生物、酵母或高等真核生物细胞。用于该目的的合适原核生物包括真细菌，例如革兰氏阴性或革兰氏阳性生物体，例如肠杆菌科如埃希氏菌属，例如大肠杆菌，肠杆菌属，欧文氏菌属，克雷伯氏菌属，变形杆菌属，沙门氏菌属，例如鼠伤寒沙门氏菌，沙雷氏菌属，例如粘质沙雷氏菌和志贺氏菌，以及芽孢杆菌属如枯草芽孢杆菌和地衣芽孢杆菌，假单胞菌属如铜绿假单胞菌和链霉菌。
除了原核生物以外，真核微生物如丝状真菌或酵母是合适的用于表达本公开的CLDN18.2抗体的宿主细胞。酿酒酵母或普通面包酵母是低等真核宿主微生物中最常用的。然而，许多其它属、种和菌株通常是可获得的并且可用于本公开，例如粟酒裂殖酵母(Schizosaccharomyces pombe)；克鲁维酵母属(Kluyveromyces)宿主，例如乳克鲁维酵母(K.lactis)、脆壁克鲁维酵母(K.fragilis)(ATCC 12,424)、保加利亚克鲁维酵母(K.bulgaricus)(ATCC 16,045)、威克曼氏克鲁维酵母(K.wickeramii)(ATCC 24,178)、K.waltii(ATCC 56,500)、果蝇克鲁维酵母(K drosophilarum)(ATCC 36,906)、耐热克鲁维酵母(K.thermotolerans)和马克斯克鲁维氏酵母(K.marxianus)；西洋蓍霉(yarrowia)(EP 402,226)；巴斯德毕赤酵母(pichia pastoris)(EP 183,070)；念珠菌属(Candida)；Trichoderma reesia(EP 244,234)；粗糙链孢霉(Neurospora crassa)；许旺氏酵母属(schwanniomyces)如西方许旺氏酵母(schwanniomyces occidentalis)；和丝状真菌，例如链孢霉属(Neurospora)、青霉属(Penicillium)、Tolypocladium以及曲霉属宿主如构巢曲霉(A.nidulans)和黑曲霉(A.niger)。
用于表达本公开的CLDN18.2抗体的其他合适的宿主细胞来源于多细胞生物体。无脊椎动物细胞的实例包括植物和昆虫细胞。目前已经从下述宿主中鉴定了大量的杆状病毒株和变体以及相应的容许型昆虫宿主细胞:草地夜蛾 (Spodoptera Frugiperda，毛虫)、埃及伊蚊(Aedes aegypti，蚊子)、白纹伊蚊(Aedes albopictus，蚊子)、Drosophila melanogaster(果蝇)和家蚕蛾(Bombyx mori)。用于转染的各种病毒株是公众可获得的，例如苜蓿银纹夜蛾(Autographa californica)NPV的L-1变体和家蚕NPV的Bm-5株，并且根据本公开，这些病毒可以用于CLDN18.2抗体在合适的宿主细胞中表达的转染过程，特别是用于转染草地贪夜蛾(Spodoptera frugiperda)细胞。棉花、玉米、马铃薯、大豆、矮牵牛花、番茄和烟草的植物细胞培养物也可用作宿主。
以上述用于本公开的CLDN18.2抗体产生的载体转化宿主细胞，并在用于诱导启动子、选择转化体或扩增编码所需序列的基因的根据需要修改的常规营养培养基中培养。
用于产生本公开的CLDN18.2抗体的宿主细胞可以在各种培养基中培养。诸如Ham's F10(Sigma)，Minimal Essential Medium(MEM)，(Sigma)，RPMI-1640(Sigma)和Dulbecco's Modified Eagle's Medium(DMEM，Sigma)的商业上可获得的培养基适于培养宿主细胞。此外，Ham等人,Meth.Enz.58:44(1979),Barnes等人,Anal.Biochem.102:255(1980),U.S.Pat.No.4,767,704；4,657,866；4,927,762；4,560,655；或5,122,469；WO 90/03430；WO 87/00195；或U.S.Pat.Re.30,985中描述的任何培养基可用作宿主细胞的培养基。必要时可以用激素和/或其他生长因子(如胰岛素、转铁蛋白或表皮生长因子)，盐(如氯化钠、钙、镁和磷酸盐)，缓冲液(如HEPES)，核苷酸(如腺苷和胸腺嘧啶)，抗生素(如GENTAMYCIN药物)，微量元素(定义为无机化合物，通常以微摩尔范围内的终浓度存在)和葡萄糖或等同能量来源添加至任何这些培养基。任何其他必需的补充剂也可以以本领域技术人员已知的适当浓度包含在内。诸如温度、pH等培养条件是与之前选择用于表达的宿主细胞一起使用的那些，并且对普通技术人员而言将是明显的。
当使用重组技术时，抗体可以在细胞内，在周质空间中产生，或者直接分泌到培养基中。如果抗体在细胞内产生，作为第一步，例如通过离心或超滤除去微粒碎片(宿主细胞或裂解片段)。Carter等人，Bio/Technology 10:163-167(1992)描述了分离分泌到大肠杆菌周质空间的抗体的方法。简而言之，在约30分钟内，在乙酸钠(pH3.5)，EDTA和苯甲基磺酰氟(PMSF)的存在下融化细胞。细胞碎片可以通过离心去除。在抗体分泌到培养基中的情况下，通常首先使用市售的蛋白质浓缩过滤器，例如Amicon或Millipore Pellicon超滤单元浓缩来自这种表达系统的上清液。蛋白酶抑制剂例如PMSF可包括在任何前述步骤中以抑制蛋白水解，并可包含抗生素以防止外来污染物的生长。
可以使用例如羟基磷灰石层析，凝胶电泳，透析，DEAE-纤维素离子交换层析，硫酸铵沉淀，盐析和亲和层析来纯化从细胞制备的抗体，其中亲和层析是优选的纯化技术。
在任何初步纯化步骤之后，包含目标抗体和污染物的混合物可以使用pH介于 约2.5-4.5之间的洗脱缓冲液进行低pH疏水相互作用色谱，优选在低盐浓度(例如约0-0.25M盐)下进行。
药物组合物
在一些方面，本公开涉及药物组合物，其包含至少一种如本文所公开的CLDN18.2结合分子(例如，本公开的CLDN18.2抗体)和药学上可接受的载体。
药物组合物的组分
药物组合物可以任选地含有一种或多种另外的药物活性成分，例如另一种抗体或药物。本公开的药物组合物还可以与例如另一种免疫刺激剂、抗癌剂、抗病毒剂或疫苗组合施用，使得抗CLDN18.2抗体增强对疫苗的免疫反应。药学上可接受的载体可以包括例如药学上可接受的液体，凝胶或固体载体，水性介质，非水性介质，抗微生物剂，等渗剂，缓冲剂，抗氧化剂，麻醉剂，悬浮/分散剂，螯合剂，稀释剂，佐剂，赋形剂或无毒的辅助物质，本领域已知的各种组分的组合或更多。
合适的组分可以包括例如抗氧化剂，填充剂，粘合剂，崩解剂，缓冲剂，防腐剂，润滑剂，调味剂，增稠剂，着色剂，乳化剂或稳定剂如糖和环糊精。合适的抗氧化剂可包括例如甲硫氨酸，抗坏血酸，EDTA，硫代硫酸钠，铂，过氧化氢酶，柠檬酸，半胱氨酸，巯基甘油，巯基乙酸，巯基山梨糖醇，丁基甲基苯甲醚，丁基化羟基甲苯和/或丙基砷酸盐。在本公开中，在包含还原抗体或其抗原结合片段的一种或多种抗氧化剂如甲硫氨酸的含有本公开的组合物的抗体或抗原结合片段的溶剂中，其可被氧化。氧化还原可防止或减少结合亲和力的降低，从而增强抗体稳定性并延长保质期。因此，在一些实施方案中，本公开提供了包含一种或多种抗体或其抗原结合片段和一种或多种抗氧化剂如甲硫氨酸的组合物。本公开进一步提供了多种方法，其中将抗体或其抗原结合片段与一种或多种抗氧化剂如甲硫氨酸混合。从而，抗体或其抗原结合片段可以被防止氧化，以延长其保质期和/或增加活性。
为了进一步说明，药学上可接受的载体可以包括例如水性载体，例如氯化钠注射液，林格氏注射液，等渗右旋糖注射液，无菌水注射液或右旋糖和乳酸林格氏注射液，非水性载体如植物来源的固定油，棉籽油，玉米油，芝麻油或花生油，抑菌剂或抑真菌浓度的抗微生物剂，等渗剂如氯化钠或葡萄糖，缓冲剂如磷酸盐或柠檬酸盐缓冲剂，抗氧化剂如硫酸氢钠，局部麻醉剂如盐酸普鲁卡因，悬浮剂和分散剂如羧甲基纤维素钠，羟丙基甲基纤维素或聚乙烯吡咯烷酮，乳化剂如聚山梨酯80(TWEEN-80)，螯合剂如EDTA(乙二胺四乙酸)或EGTA(乙二醇四乙酸)，乙醇，聚乙二醇，丙二醇，氢氧化钠，盐酸，柠檬酸或乳酸。用作载体的抗微生物剂可以添加到包含酚或甲酚、汞制剂、苯甲醇、氯丁醇、对羟基苯甲酸甲酯和对羟基苯甲酸丙酯、硫柳汞、苯扎氯铵和苄索氯铵的多剂量容器中的药物组合物中。合适的赋形剂可以包括例如水，盐水，右旋糖，甘油或乙醇。合适的无毒辅助物质可以包括例如润湿剂或乳化剂，pH缓冲剂，稳定剂，溶解度增强剂或诸如乙酸钠，脱水山梨糖醇单月桂酸酯，三乙醇胺油酸酯或环糊精的试剂。
施用、制剂和剂量
本公开的药物组合物可以通过各种途径体内施用至有需要的受试者，所述途径包括但不限于口服，静脉内，动脉内，皮下，肠胃外，鼻内，肌内，颅内，心内，心室内，气管内，口腔，直肠，腹膜内，皮内，局部，经皮和鞘内，或者通过植入或吸入。本公开的药物组合物可以配制成固体、半固体、液体或气体形式的制剂；包括但不限于片剂，胶囊剂，粉剂，颗粒剂，软膏剂，溶液剂，栓剂，灌肠剂，注射剂，吸入剂和气雾剂。根据预期的应用和治疗方案可以选择合适的制剂和施用途径。
用于肠内施用的合适制剂包括硬或软的明胶胶囊，丸剂，片剂，包括包衣片剂，酏剂，混悬剂，糖浆剂或吸入剂及其控释剂型。
适用于肠胃外施用(例如通过注射)的制剂包括活性成分溶解、悬浮于其中或以其他方式提供的(例如，在脂质体或其他微粒中)的水性或非水性、等渗、无热原、无菌液体(例如溶液，混悬液)。这些液体可以另外含有其它药学上可接受的成分，例如抗氧化剂，缓冲剂，防腐剂，稳定剂，抑菌剂，悬浮剂，增稠剂和使制剂与预期接受者的血液(或其他相关体液)等渗的溶质。赋形剂的实例包括例如水，醇，多元醇，甘油，植物油等。适用于此类制剂的等渗载体的实例包括氯化钠注射液，林格溶液或乳酸林格氏注射液。类似地，特定剂量方案(即剂量，时间和重复)将取决于具体个体和个体的病史以及诸如药代动力学(例如半衰期，清除率等)的经验考虑。
施用频率可以在治疗过程中确定和调整，并且基于减少增殖或致瘤细胞的数量，维持这种肿瘤细胞的减少，减少肿瘤细胞的增殖或延迟转移的发展。在一些实施方案中，施用的剂量可以被调节或减少以控制潜在的副作用和/或毒性。或者，本公开的用于治疗的药物组合物的持续连续释放制剂可能是合适的。
本领域技术人员将会理解，合适的剂量可因患者而异。确定最佳剂量通常涉及治疗益处水平与任何风险或有害副作用的平衡。所选择的剂量水平将取决于多种因素，包括但不限于特定化合物的活性，施用，施用时间，化合物清除速率，治疗持续时间，其他联合使用的药物、化合物和/或材料，病症的严重程度，以及物种，患者的性别、年龄、体重、病情、一般健康状况和以前的病史。化合物的量和施用途径最终由医生、兽医或临床医师决定，但通常选择剂量以达到实现所需效果的作用部位处的局部浓度，而不会导致实质性的有害或不利副作用。
通常，CLDN18.2结合分子可以以各种剂量范围施用。在一些实施方案中，本文提供的CLDN18.2结合分子可以以约0.01mg/kg至约100mg/kg(例如约0.01mg/kg、约0.5mg/kg、约1mg/kg、约2mg/kg、约5mg/kg、约10mg/kg、约15mg/kg、约20mg/kg、约25mg/kg、约30mg/kg、约35mg/kg、约40mg/kg、约45mg/kg、约50mg/kg、约55mg/kg、约60mg/kg、约65mg/kg、约70mg/kg、约75mg/kg、约80mg/kg、约85mg/kg、约90mg/kg、约95mg/kg或约100mg/kg)的治疗有效剂量施用。在这些实施方案的某些中，抗体以约50mg/kg或更低的剂量施用，并且在 这些实施方案中的某些中，剂量为10mg/kg或更低，5mg/kg或更低，1mg/kg或更低，0.5mg/kg或更低，或者0.1mg/kg或更低。在某些实施方案中，施用剂量可以在治疗过程中改变。例如，在某些实施方案中，初始施用剂量可以高于后续施用剂量。在某些实施方案中，取决于受试者的反应，施用剂量可以在治疗过程中变化。
无论如何，本公开的抗体或其抗原结合部分优选根据需要施用于有需要的受试者。本领域技术人员可以确定施用频率，例如主治医生基于所治疗病症、所治疗受试者的年龄、所治疗病症的严重程度、所治疗受试者的一般健康状况等的考虑。
在某些优选的实施方案中，涉及本公开的抗体或其抗原结合部分的治疗过程将包含在数周或数月的时间内施用的多剂量的所选药物产品。更具体地说，本公开的抗体或其抗原结合部分可以每天，每两天，每四天，每周，每十天，每两周，每三周，每月，每六周，每两个月，每十周或每三个月施用。就此而言，可以理解的是，可以基于患者响应和临床实践来改变剂量或者调整间隔。
在给予一次或多次施用的个体中也可凭经验确定所公开的用于治疗的药物组合物的剂量和方案。例如，可给予个体增量剂量的如本文所述的药物组合物。在选定的实施方案中，剂量可分别根据经验确定，或根据观察到的副作用或毒性逐渐增加或减少。为了评估所选择的组合物的功效，可以跟踪特定疾病、病症或病情的标志物。对于癌症，这些包括通过触诊或视觉观察直接测量肿瘤大小，通过X射线或其他成像技术间接测量肿瘤大小；通过直接肿瘤活组织检查和肿瘤样本的显微镜检查评估的改善；测量根据本文所述方法鉴定的间接肿瘤标志物(例如用于前列腺癌的PSA)或致瘤性抗原，疼痛或麻痹的减轻；与肿瘤相关的言语、视力、呼吸或其他失能的改善；食欲增加；或通过接受的测试测量的生活质量的提高或生存期的延长。本领域技术人员将明白，剂量将根据个体、肿瘤病情的类型、肿瘤病情的阶段、肿瘤病情是否已开始转移至个体中的其他位置以及过去使用的治疗和并行使用的治疗而变化。
用于肠胃外施用(例如静脉内注射)的相容制剂可包含浓度为约10μg/ml至约100mg/ml的如本文提供的CLDN18.2结合分子。在一些实施方案中，CLDN18.2结合分子的浓度可包括20μg/ml，40μg/ml，60μg/ml，80μg/ml，100μg/ml，200μg/ml，300μg/ml，400μg/ml，500μg/ml，600μg/ml，700μg/ml，800μg/ml，900μg/ml或1mg/ml。在其他优选的实施方案中，CLDN18.2结合分子的浓度将包括2mg/ml，3mg/ml，4mg/ml，5mg/ml，6mg/ml，8mg/ml，10mg/ml，12mg/ml，14mg/ml，16mg/ml，18mg/ml，20mg/ml，25mg/ml，30mg/ml，35mg/ml，40mg/ml，45mg/ml，50mg/ml，60mg/ml，70mg/ml，80mg/ml，90mg/ml或100mg/ml。若以摩尔浓度计算，在一些实施方案中，CLDN18.2结合分子的浓度可包括例如133nM，266nM，400nM，533nM，667nM，1.3μM，2μM，2.67μM，3.33μM，4μM，4.67μM，5.33μM，6μM或6.67μM。
本发明的应用
本发明的CLDN18.2结合分子具有许多体外和体内用途。
治疗疾病
与CLDN18.2相关的病症和障碍可以是与免疫相关的疾病或病症，包括但不限于“涉及表达CLDN18.2的细胞的疾病”或“与表达CLDN18.2的细胞相关的疾病”或类似表述，意指CLDN18.2在患病组织或器官的细胞中表达。在一个实施方案中，与对应健康组织或器官中的状态相比，CLDN18.2在患病组织或器官的细胞中的表达提高。提高是指提高至少10％，特别地至少20％、至少50％、至少100％、至少200％、至少500％、至少1000％、至少10000％或甚至更多。在一个实施方案中，表达仅见于患病组织，而相应健康组织中的表达被抑制。根据本公开，与表达CLDN18.2的细胞相关的疾病包括癌症疾病。此外，根据本公开，癌症疾病优选为其中癌细胞表达CLDN18.2的那些。
术语“癌症疾病”或“癌症”是指或描述个体中通常以不受调节的细胞生长为特征的生理状况。癌症的实例包括但不限于上皮癌、淋巴瘤、母细胞瘤、肉瘤和白血病。更特别地，这样的癌症的实例包括骨癌、血癌、肺癌、肝癌、胰腺癌、皮肤癌、头颈癌、皮肤或眼内黑素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、结肠癌、乳腺癌、前列腺癌、子宫癌、性器官和生殖器官癌、霍奇金病(Hodgkin′s Disease)、食管癌、小肠癌、内分泌系统癌症、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、膀胱癌、肾癌、肾细胞癌、肾盂癌、中枢神经系统(CNS)肿瘤、神经外胚层癌症、脊柱轴肿瘤、胶质瘤、脑脊膜瘤和垂体腺瘤。根据本公开的术语“癌症”还包括癌症转移。优选地，“癌症疾病”的特征在于表达CLDN18.2的细胞，以及癌细胞表达CLDN18.2。表达CLDN18.2的细胞优选是癌细胞，优选本文中所述的癌症的癌细胞。
根据本公开，术语“肿瘤”或“肿瘤疾病”是指细胞(被称为赘生性细胞、肿瘤发生性细胞或肿瘤细胞)的异常生长，优选形成肿胀或病变。“肿瘤细胞”意指通过迅速的不受控制的细胞增殖而生长并且在引发新生长的刺激停止之后继续生长的异常细胞。肿瘤显示出结构组织和与正常组织的功能协调的部分或完全缺失，并且通常形成独特的组织团块，其可以是良性的、恶化前的或恶性的。根据本公开，“癌症疾病”优选为“肿瘤疾病”。然而，通常术语“癌症”和“肿瘤”在本文中可互换使用。
在一个实施方案中，根据本公开的癌症涉及表达CLDN18.2的癌细胞。在一个实施方案中，癌症是CLDN18.2阳性的。在一个实施方案中，CLDN18.2的表达是位于细胞的表面。在一个实施方案中，至少50％，优选60％、70％、80％或90％的癌细胞是CLDN18.2阳性的，和/或至少40％，优选至少50％的癌细胞对CLDN18.2的表面表达呈阳性。在一个实施方案中，至少95％或至少98％的癌细胞是CLDN18.2阳性的。在一个实施方案中，至少60％、至少70％、至少80％或至少90％的癌细胞对于CLDN18.2的表面表达是阳性的。
在一个实施方案中，表达CLDN18.2的癌症、涉及表达CLDN18.2的癌细胞的癌症或CLDN18.2阳性癌症选自:胃癌、食管癌、胰腺癌、肺癌(例如非小细胞肺癌 (NSCLC))、卵巢癌、结肠癌、肝癌、头颈癌和胆囊癌，及其转移，特别是胃癌转移(例如克鲁肯贝格瘤)、腹膜转移和淋巴结转移。在一个实施方案中，癌症是腺癌，特别是晚期腺癌。特别优选的癌症疾病是胃、食管、胰管、胆管、肺和卵巢的腺癌。在一个实施方案中，癌症选自胃癌、食管癌(特别是下食管癌)、食管-胃接合部的癌症和胃食管癌。在一个特别优选的实施方案中，癌症是胃食管癌，例如转移性、顽固性或复发性晚期胃食管癌。
此外，本公开的抗体或其抗原结合部分可以与化学疗法或放射疗法组合使用。
与化疗组合使用
抗体或其抗原结合部分可以与抗癌剂、细胞毒性剂或化学治疗剂组合使用。
术语“抗癌剂”或“抗增殖剂”意指可用于治疗细胞增殖性病症例如癌症的任何药剂，并且包括但不限于细胞毒性剂，细胞抑制剂，抗血管生成剂，放射疗法和放射治疗剂，靶向抗癌剂，BRM，治疗性抗体，癌症疫苗，细胞因子，激素疗法，放射疗法和抗转移剂和免疫治疗剂。应该理解的是，在如上所述的选定实施方案中，此类抗癌剂可以包含缀合物并且可以在施用之前与公开的位点特异性抗体结合。更具体而言，在一些实施方案中，将选择的抗癌剂连接至工程化抗体的不配对半胱氨酸以提供如本文所述的工程化偶联物。因此，这样的工程化缀合物被明确地考虑在本公开的范围内。在其他实施方案中，所公开的抗癌剂将与包含如上所述的不同治疗剂的位点特异性缀合物组合施用。
如本文所用，术语“细胞毒性剂”是指对细胞有毒并降低或抑制细胞功能和/或引起细胞破坏的物质。在一些实施方案中，该物质是源自活生物体的天然存在的分子。细胞毒性剂的实例包括但不限于细菌(例如，白喉毒素，假单胞菌内毒素和外毒素，葡萄球菌肠毒素A)，真菌(例如α-八叠球菌素，局限曲霉素)，植物(相思豆毒蛋白，蓖麻毒素，蒴莲根毒素，槲寄生素，美洲商陆抗病毒蛋白，皂草素，白树毒素，momoridin，天花粉蛋白，大麦毒素，油桐(Aleurites fordii)蛋白，石竹素蛋白，Phytolacca mericana蛋白(PAPI，PAPII和PAP-S)，苦瓜抑制剂，麻风树毒蛋白，巴豆毒素，石碱草抑制剂，白树毒素，mitegellin，局限曲霉素，酚霉素，新霉素和单端孢霉烯族化合物)或动物(例如细胞毒性RNA酶，如胞外胰腺RNA酶；DNA酶I，包括其片段和/或变体)的小分子毒素或酶促活性毒素。
为了本公开的目的，“化学治疗剂”包括非特异性降低或抑制癌细胞的生长、增殖和/或存活的化学化合物(例如细胞毒性剂或细胞抑制剂)。这些化学试剂通常针对细胞生长或分裂所需的细胞内过程，因此对于通常快速生长和分裂的癌细胞特别有效。例如，长春新碱使微管解聚，从而抑制细胞进入有丝分裂。通常，化学治疗剂可以包括抑制或被设计用于抑制癌细胞或可能变成性或产生致瘤后代(例如TIC)的细胞的任何化学药剂。这些药剂通常是组合使用的，并且通常是最有效的，例如，在诸如CHOP或FOLFIRI的方案中。
可以与本公开的位点特异性构建体组合使用的抗癌剂(作为位点特异性缀合物 的组分或未缀合状态)的实例包括但不限于烷化剂、烷基磺酸盐、氮丙啶、乙烯亚胺和甲基三聚氰胺、多聚乙酰(acetogenins)、喜树碱、苔藓抑素、卡利士他汀(callystatin)、CC-1065、克瑞托欣(cryptophycins)、多拉司他汀、多卡米星、艾榴素(eleutherobin)、水鬼蕉碱、沙克迪因(sarcodictyin)、海绵素(spongistatin)、氮芥、抗生素、烯二炔类抗生素、dynemicin、双膦酸盐、埃斯波霉素、色素蛋白烯二炔抗生素发色团、阿克拉霉素类(aclacinomysins)、放线菌素、安曲霉素、偶氮丝氨酸、博莱霉素、放线菌素C、卡拉宾辛(carabicin)、洋红霉素、嗜癌霉素、色霉素类(chromomycinis)、更生霉素、柔红霉素、地托比星、6-重氮基-5-氧代-L-正亮氨酸、
Figure PCTCN2021132218-appb-000001
多柔比星、表柔比星、依索比星、伊达比星、麻西罗霉素、丝裂霉素、霉酚酸、诺加霉素、橄榄霉素、培洛霉素、博地霉素(potfiromycin)、嘌呤霉素、三铁阿霉素、罗多比星、链黑菌素、链脲菌素、杀结核菌素、乌苯美司、净司他丁、佐柔比星；抗-代谢物、埃罗替尼、威罗菲尼、克唑替尼、索拉非尼、依鲁替尼、恩杂鲁胺、叶酸类似物、嘌呤类似物、雄激素、抗-肾上腺素、叶酸补充剂如弗林酸(frolinic acid)、醋葡醛内酯、醛磷酰胺糖苷、氨基乙酰丙酸、恩尿嘧啶、安吖啶、贝斯布希(bestrabucil)、比生群、依达曲沙、迪夫法明(defofamine)、秋水仙胺、地吖醌、艾夫尼辛(elfornithine)、依利醋铵、爱波喜龙、依托格鲁、硝酸镓、羟基脲、香菇多糖、氯尼达明、美坦生类化合物(maytansinoids)、米托胍腙、米托蒽醌、莫丹摩尔(mopidanmol)、尼特林(nitraerine)、喷司他丁、蛋氨氮芥、吡柔比星、洛索蒽醌、鬼臼酸、2-乙基肼、丙卡巴肼、
Figure PCTCN2021132218-appb-000002
多糖复合物(JHS Natural Products,Eugene,OR)、雷佐生；根霉素；西佐喃；锗螺胺；替奴佐酸；三亚胺醌；2,2',2”-三氯三乙胺；单端孢霉烯类(尤其是T-2毒素、维拉库林A(verracurin A)、杆孢菌素A和蛇形菌素)；乌拉坦；长春地辛；达卡巴嗪；甘露莫司汀；二溴甘露醇；二溴卫矛醇；哌泊溴烷；卡西托欣(gacytosine)；阿拉伯糖苷(“Ara-C”)；环磷酰胺；噻替派；紫杉烷类；苯丁酸氮芥(chloranbucil)；
Figure PCTCN2021132218-appb-000003
吉西他滨；6-硫代鸟嘌呤；巯嘌呤；氨甲喋呤；铂类似物；长春碱；铂；依托泊苷(VP-16)；异环磷酰胺；米托蒽醌；长春新碱，
Figure PCTCN2021132218-appb-000004
长春瑞滨；诺消灵；替尼泊苷；依达曲沙；柔红霉素；氨基蝶呤；希罗达；伊班膦酸盐；伊立替康(Camptosar，CPT-11)；拓扑异构酶抑制剂RFS 2000；二氟甲基鸟氨酸；类视色素；卡培他滨；考布他汀；甲酰四氢叶酸；奥沙利铂；PKC-α、Raf、H-Ras、EGFR和VEGF-A的抑制剂(其减少细胞增殖)，以及上述任一项的药学上可接受的盐、酸或衍生物。这一定义中还包括用于调节或抑制对肿瘤的激素作用的抗激素剂，诸如抗雌激素和选择性雌激素受体调节剂，抑制调节肾上腺中的雌激素产生的芳香酶的芳香酶抑制剂，和抗-雄激素；以及曲沙他滨(1，3-二氧杂环戊烷核苷胞嘧啶类似物)；反义寡核苷酸、核酶诸如VEGF表达抑制剂和HER2表达抑制剂；疫苗，
Figure PCTCN2021132218-appb-000005
rIL-2；
Figure PCTCN2021132218-appb-000006
拓扑异构酶1抑制剂；
Figure PCTCN2021132218-appb-000007
rmRH；长春瑞滨和埃斯波霉素，以及上述任一项的药学上可接受的盐、酸或衍生物。
与放射疗法组合使用
本公开还提供了抗体或其抗原结合部分与放射疗法(即，用于在肿瘤细胞内局部诱导DNA损伤的任何机制，例如γ-照射，X-射线，UV-照射，微波，电子发射等)的组合。还考虑了使用放射性同位素至肿瘤细胞的定向递送的联合疗法，并且所公开的缀合物可以与靶向的抗癌剂或其他靶向手段结合使用。通常，放射疗法在约1周至约2周的时间段内以脉冲方式施用。放射疗法可以对患有头颈癌的受试者施用约6至7周。任选地，放射疗法可以作为单剂量或作为多个顺序剂量施用。
诊断
本公开提供了用于检测、诊断或监测增殖性病症的体外和体内方法以及筛选来自患者的细胞以鉴定肿瘤细胞包括致瘤细胞的方法。这样的方法包括鉴定用于治疗的患有癌症的个体或监测癌症的进展，包括将患者或从患者获得的样品(体内或体外)与本文所述的抗体接触，并检测样品中与结合的或游离的靶分子的结合的抗体的存在或不存在或结合水平。在一些实施方案中，抗体将包含可检测标记或报道分子。
在一些实施方案中，抗体与样品中特定细胞的结合可表示样品可能含有致瘤细胞，从而表明具有癌症的个体可用本文所述的抗体有效治疗。
可以通过多种测定法分析样品，例如放射免疫测定法，酶免疫测定法(例如ELISA)，竞争结合测定法，荧光免疫测定法，免疫印迹测定法，Western印迹分析和流式细胞术测定法。兼容的体内诊断或诊断测定可以包括本领域公知的成像或监测技术，例如本领域技术人员已知的磁共振成像，计算机化断层摄影(例如CAT扫描)，正电子断层扫描(例如PET扫描)，放射线照相术，超声波等。
药物包装和试剂盒
本公开还提供了包含一个或多个剂量的抗体或其抗原结合部分的一个或多个容器的药物包装和试剂盒。在一些实施方案中，提供单位剂量，其中单位剂量含有预定量的组合物，所述组合物包含例如抗体或其抗原结合部分，具有或不具有一种或多种其他试剂。对于其他实施方案，这种单位剂量以一次性使用的预充式注射用注射器供应。在其他实施方案中，单位剂量中包含的组合物可以包含盐水、蔗糖或类似物；缓冲液，如磷酸盐等；和/或配制在稳定和有效的pH范围内。或者，在一些实施方案中，所述组合物是缀合物组合物，其可以作为冻干粉末提供并在加入合适的液体(例如无菌水或盐溶液)后重建。容器上或与容器相关联的任何标签指示封装的缀合物组合物用于治疗选择的肿瘤疾病状况。在一些优选的实施方案中，组合物包含一种或多种抑制蛋白质聚集的物质，包括但不限于蔗糖和精氨酸。
本公开还提供了用于产生位点特异性缀合物以及任选地一种或多种抗癌剂的单剂量或多剂量施用单元的试剂盒。该试剂盒包括容器以及在容器上或与容器相关联的标签或包装插页。合适的容器包括例如瓶，小瓶，注射器等。容器可以由多种材料形成，例如玻璃或塑料，并且包含药学有效量的所公开的缀合或非缀合形式的缀合物。在其他优选实施例中，容器包括无菌存取口(例如容器可以是静脉内溶液袋或具有可被皮下注射针头刺穿的塞子的小瓶)。这样的试剂盒通常在合适的容器中包 含工程化偶联物的药学上可接受的制剂，并且任选地在相同或不同的容器中包含一种或多种抗癌剂。试剂盒还可以含有其他药学上可接受的制剂，用于诊断或组合治疗。例如，除了本公开的抗体或其抗原结合部分之外，这样的试剂盒可以含有任何一种或多种抗癌剂，例如化学治疗剂或放射治疗剂；抗血管生成剂；抗转移剂；靶向抗癌剂；细胞毒性剂；和/或其他抗癌剂。
更具体地说，试剂盒可以具有含有所公开的抗体或其抗原结合部分的单个容器，其含有或不含另外的组分，或者它们可以具有用于每种所需试剂的不同容器。在提供用于缀合的组合治疗剂的情况下，可以按摩尔当量组合或一种组分多于另一种的方式预混合单一溶液。或者，试剂盒的缀合物和任何任选的抗癌剂可以在施用于患者之前分开保存在不同的容器中。试剂盒还可以包含用于容纳无菌药学上可接受的缓冲液或其他稀释剂例如抑菌注射用水(BWFI)、磷酸盐缓冲盐水(PBS)、林格氏溶液和葡萄糖溶液的第二/第三容器装置。
当试剂盒的组分以一种或多种液体溶液提供时，液体溶液优选为水溶液，特别优选无菌水溶液或盐水溶液。然而，试剂盒的组分可以作为干粉提供。当试剂或组分以干粉形式提供时，可以通过添加合适的溶剂来重构粉末。可以设想溶剂也可以提供于另一个容器中。
如上简要所述，所述试剂盒还可含有向患者施用抗体或其抗原结合部分和任何任选组分的工具，例如一种或多种针，I.V.袋或注射器，或者甚至滴眼器、移液管或其他类似装置，通过其可以将制剂注射或引入动物体内或将其施用于身体的患病区域。本公开的试剂盒通常还包括用于容纳小瓶或类似物的装置以及用于商业销售的其他紧密封闭的部件，例如注射或吹塑塑料容器，其中放置并且保持所需的小瓶和其他装置。
本发明的优势
针对CLDN18.2、特别是人CLDN18.2开发抗体至少具有如下优势：
第一，人CLDN18.2高表达于癌症细胞，而在正常细胞中，仅特异性表达于胃上皮细胞中，因此毒副作用低，成药可能性更高；
第二，CLDN18.2表达于胃上皮细胞的紧密连接中，相对较松散的癌症细胞，抗体不易于作用；即使上皮细胞被抗体杀伤，上皮细胞之下的干细胞因为不表达CLDN18.2，可以通过分化进而补充损伤的胃上皮细胞；
第三，针对CLDN18.2的抗体不仅可以通过ADCC和CDC杀伤细胞，还可以通过抗体交联CLDN18.2从而介导细胞的凋亡，而且可以一定程度抑制细胞的增殖。
纳米抗体作为一种新型的抗体，已经有一个抗体caplacizumab获批上市，充分说明纳米抗体的可成药性。此种抗体相对于其它抗体具有显著的优点，如其半衰期长短可通过化学修饰或者蛋白融合改造进行调节，渗透性强，可识别普通抗体不可及的隐蔽表位，耐胃蛋白酶，耐酸，耐热，易生产，而且因为是单链，易于和其它类型抗体组装为双价抗体和多价抗体。
在本公开的部分实施方案中，针对CLDN18.2的抗体是通过羊驼免疫库筛选得到，在此基础上，C末端和IgG1的Fc片段融合，目前部分候选抗体在细胞水平CDC和ADCC的实验结果都表现出比对照抗体更好或至少相当的活性。结合CLDN18.2靶点的特性，此抗体用于癌症的免疫治疗，具有更低的毒副作用和更佳的临床药效，将给患者提供更多的药物选择。
实施例
通过参考以下实施例将更容易地理解本文一般地描述的本发明，这些实施例是以举例说明的方式提供的，并且不旨在限制本发明。这些实施例并不旨在表示下面的实验是全部或仅进行的实验。
实施例1
过表达细胞株以及肿瘤细胞株的构建与鉴定
在本实施例中，分别构建了不同类型的过表达细胞株以及肿瘤细胞株，并对其进行流式鉴定。
1.过表达细胞株的构建与鉴定
将全长人CLDN18.1(SEQ ID NO:17)、鼠CLDN18.2(SEQ ID NO:18)、鼠CLDN18.1(SEQ ID NO:19)的核酸序列构建至pLVX-puro质粒(Clontech，Cat#632164)上。然后，将所得到的质粒通过电转化至HEK293细胞(
Figure PCTCN2021132218-appb-000008
CRL-1573 TM)中。通过筛选，得到表达全长人CLDN18.1的过表达细胞株(人CLDN18.1-HEK293)、表达鼠CLDN18.2的过表达细胞株(鼠CLDN18.2-HEK293)和表达鼠CLDN18.1的过表达细胞株(鼠CLDN18.1-HEK293)。其后，通过IMAB362抗体(根据专利US20180127489A1所披露的序列信息表达、纯化制得)以及识别C端CLDN18胞内段(GFKASTGFGSNTKN，SEQ ID NO:22)的抗CLDN18抗体[34H14L15](Abcam，ab203563)进行流式细胞术鉴定，获得成功转化的过表达细胞株，具体方法如下。
1.1电转化
首先，复苏及培养HEK293细胞，连续传代2-3次，转染前一天将细胞以3×10 5个/mL的密度接种至细胞培养皿中，第二天待细胞汇合度达到约70％即可使用。用含体积百分比为0.25％的EDTA的Trypsin(Gibco，25200-072)消化细胞2min后收集细胞，于常温、100g离心细胞5min，弃上清后加入1×DPBS(源培，B210)重悬细胞并计数，取5×10 6个细胞离心并收集，用250μL Buffer R(Invitrogen，Neon TM Kit，PK10096)缓冲液重悬细胞，并向其中加入25μg目的质粒，用移液器轻轻混合均匀。后将悬液置入电转仪(Invitrogen，NeonTM Transfection System，MP922947)进行电转化，设置的反应条件为1100V/20ms/2次进行电转。
1.2细胞培养
电转化后，将所得到的细胞分别转移至含有体积百分比为10％FBS(Gibco， 15140-141)且不含抗生素的DMEM培养基(Gibco，11995065)中，然后将细胞接种入10cm×10cm细胞培养皿中培养48h，接着以平均0.5个/孔的密度将细胞分装至96孔细胞培养板中，加入终浓度为2μg/mL的嘌呤霉素(Gibco，A111138-03)作为筛选压力，2周左右观察细胞株克隆生长情况，并挑取形成克隆的细胞株进行鉴定。
1.3过表达细胞株的流式鉴定
1.3.1人CLDN18.2-HEK293T和鼠CLDN18.2-HEK293细胞株的流式鉴定
对于人CLDN18.2-HEK293T(康源博创，KC-0986)和鼠CLDN18.2-HEK293细胞株，直接采用IMAB362抗体进行鉴定。
取1×10 5个细胞，低速离心(300g)去上清。将离心管底部的细胞通过配制好的FACS缓冲液(含体积百分比为2％FBS的1×PBS缓冲液)润洗一次，然后向润洗后的细胞中加入12.5μg/mL抗体IMAB362，在4℃孵育1h，接着再用上述FACS缓冲液润洗三次，加入PE标记的山羊抗人IgG Fc抗体(Abcam，ab98596)0.5μg，在4℃孵育1h。其后，经FACS缓冲液润洗三次，并向细胞中加入200μL FACS缓冲液重悬细胞，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)进行检测。
1.3.2人CLDN18.1-HEK293和鼠CLDN18.1-HEK293细胞株的流式鉴定
由于IMAB362抗体只识别CLDN18.2而不识别CLDN18.1，因此需要结合其它方法对人CLDN18.1-HEK293、鼠CLDN18.1-HEK293细胞株进行鉴定。考虑到人CLDN18.1和鼠CLDN18.1的序列一致性，首先按细胞破膜试剂盒(eBioscience，88-8824-00)的说明书方法对细胞进行固定和破膜，然后根据抗CLDN18抗体[34H14L15]对C端CLDN18胞内段的识别结果及与IMAB362抗体的特异性结合结果进行流式细胞术鉴定。
具体方法如下:取1×10 5个细胞，低速离心(300g)，去上清。将离心管底部的细胞用FACS缓冲液润洗一次，然后向润洗后的细胞中加入200μL IC固定液(eBioscience，00-8222)，在4℃孵育1h，接着用破膜缓冲液(eBioscience，00-8333)润洗两次，加入前述抗CLDN18抗体，在4℃孵育1h。再次用上述破膜缓冲液润洗三遍之后，加入Alexa
Figure PCTCN2021132218-appb-000009
488荧光标记的驴抗兔IgG H&L(abcam，ab150073)0.5μg，在4℃孵育1h，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)进行检测。
同时采用前述1.3.1的方法，用IMAB362抗体测定是否结合人CLDN18.1-HEK293和鼠CLDN18.1-HEK293细胞株。
流式鉴定的结果如图1a所示。从图1a中可以看出:抗CLDN18抗体可以识别人CLDN18.2-HEK293T、人CLDN18.1-HEK293、鼠CLDN18.2-HEK293、鼠CLDN18.1-HEK293、HEK293和HEK293T细胞；而抗体IMAB362仅识别鼠CLDN18.2-HEK293、人CLDN18.2-HEK293T，不识别鼠CLDN18.1-HEK293、人CLDN18.1-HEK293。这说明人CLDN18.2-HEK293T、人CLDN18.1-HEK293、鼠CLDN18.2-HEK293、鼠CLDN18.1-HEK293四个细胞株都成功地表达了对应的 CLDN18蛋白。
2.过表达肿瘤细胞株的构建与鉴定
对于过表达人CLDN18.2的胃癌细胞株KATOIII(人CLDN18.2-KATOIII肿瘤细胞株)的构建则是采用慢病毒转染的方式，并通过抗体IMAB362进行鉴定。具体方法如下:
取状态良好的人胃癌细胞KATOIII(
Figure PCTCN2021132218-appb-000010
HTB-103 TM)5×10 4个，以30:1的MOI比例加入包装好的含有人CLDN18.2序列(SEQ ID NO:16)的慢病毒，充分混匀，然后加入含有8μg/mL聚凝胺(Polybrene，Sigma，107689)的IMDM完全培养基(Gibco，12440061)，混合均匀，于37℃、5％CO 2的恒温培养箱中孵育20h后去除培养基，更换新鲜的IMDM完全培养基并继续孵育24h，然后以平均0.5个细胞/孔密度将转染后的KATOIII细胞接种至96孔板中，并添加终浓度为2μg/mL嘌呤霉素进行抗性加压筛选，于37℃、5％CO 2的恒温培养箱中培养2-3周，并挑取克隆进行鉴定。
以上述1.3.1节同样的方式，通过抗体IMAB362对经抗性筛选的细胞株进行流式鉴定。
流式鉴定的结果如图1b所示。从图1b中可以看出，没有转染CLDN18.2的KATOIII细胞几乎不被抗体IMAB362识别，说明此KATOIII细胞株上几乎不表达CLDN18.2或者表达量极低；通过慢病毒转染而成功构建的人CLDN18.2-KATOIII肿瘤细胞株能够被抗体IMAB362识别，说明此细胞株构建成功。
实施例2
动物免疫和血清免疫效价检测
1.免疫接种
在本实施例中，采用的是羊驼免疫。具体操作如下:免疫原采用细胞株人CLDN18.2-HEK293T(康源博创，KC-0986)和含人CLDN18.2ECD1(SEQ ID NO:20)的hCLDN18.2-pLVX-puro质粒。分别用2×10 7个人CLDN18.2-HEK293T细胞(皮下多点注射)和2mg质粒(肌肉多点注射)按周交替免疫羊驼(南昌大佳生物饲养)，羊驼代号为NSY002，总计免疫8次。最后，用2×10 7个人CLDN18.2-HEK293T细胞进行加免。
2.血清免疫效价测定
免疫效价测定是通过ELISA方法根据免疫血清在抗原重组蛋白CLDN18.2(金斯瑞，CP0007)上的信号进行测定。具体方法如下。
在免疫效价测定的前一天，将抗原重组蛋白CLDN18.2用PBS稀释到1μg/mL，获得稀释液。将30μL稀释液加入到ELISA板中，于4℃包被过夜。在免疫效价测定当日，包被板用PBS润洗两遍，然后用含有体积百分比为5％脱脂奶粉的PBST室温封闭两小时，再用PBS润洗两遍。在另外一块96孔稀释板上将未经免疫的阴 性血清和免疫后血清用PBS进行稀释，首孔1000倍稀释，然后后续7个孔采用2倍梯度稀释。稀释好的血清对应加入到包被了抗原重组蛋白CLDN18.2的第一块ELISA板中，37℃孵育1h，PBS洗两遍后以1:5000加入二抗MonoRab TM兔抗骆驼科VHH抗体(金斯瑞，A01862-200)，最后通过酶标仪(Molecular Devices，SpectraMax 190)在450nm波长下读取OD值。结果如表1所示，羊驼免疫效价大致在1:8000左右。
表1
Figure PCTCN2021132218-appb-000011
实施例3
羊驼免疫库构建和筛选
动物免疫结束之后，将羊驼采血80mL，通过Ficoll-Paque密度梯度分离液(GE，17144003S)分离PBMC用于羊驼免疫库构建。具体方法如下:
取Ficoll-Paque密度梯度分离液15mL缓缓加入至50mL离心管中，然后缓缓加入15mL采集的羊驼血液，使得两种液体保持清晰的分离界面。在15℃左右以下述条件进行离心:400g，20min，加速度为3，减速为0。离心之后，整个液面分为四层，上层为血浆混合物，下层为红细胞和粒细胞，中层为Ficoll-Paque液体，在上、中层交界处有以PBMC为主的白色云雾层狭窄带，即PBMC细胞层。先用无菌巴氏吸管小心地吸去上层的血浆混合物，然后再用新的无菌巴氏吸管吸取PBMC，获得分离的PBMC。用PBS润洗两遍，然后于4℃，1500rpm水平离心10min，最后用1.5mL PBS重悬，通过细胞计数仪(CountStar，CountStar Altair)计数。
将分离的PBMC抽取其RNA，并通过反转录试剂盒(TaKaRa，6210A)反转录成cDNA。由于羊驼抗体的分子形式不同于普通抗体，其不含有轻链而且重链不含有CH1，因此在VH germline基因前端和CH2上通过设计引物，PCR得到两个不同大小的片段，通过割胶回收较小的目的片段；通过比对VHH抗体所有V基因和J基因的所有序列，设计含有NcoI和NotI酶切位点的简并引物，然后以回收的DNA片段产物为模板扩增所有的VHH基因，最后通过双酶切和连接将目的抗体基因片段插入至噬菌体展示用载体上，其中VHH在C端融合了GIII基因。连接产物通过回收试剂盒(Omega，D6492-02)回收，最后通过电转仪(Bio-Rad，MicroPulser)转化至 感受态大肠杆菌SS320(Lucigen，MC1061F)中，并涂布于具有氨苄抗性的2-YT固体平板(由胰蛋白胨1.5％，酵母提取物1％，NaCl 0.5％，琼脂1.5％，按质量体积g/mL配制而成)。为了计算库容，以1μL菌液稀释后在平板上形成的克隆来计算所有电转化形成的总克隆数，即库容容量。此免疫库的库容为1×10 9cfu。
基于库容容量，挑取50个OD(1个OD为5×10 8cfu)的羊驼库菌加入到新鲜的2-YT液体培养基中，使得初始OD值为0.05。置于37℃，220rpm培养至对数生长期，此时以5倍于细菌数的数量加入VSCM13辅助噬菌体，充分混匀，静置30min，然后在220rpm条件下培养1h，通过10000rpm离心5min后，弃上清，置换至C+/K+2-YT培养基中，并于30℃，220rpm培养过夜。次日，13000g离心10min，其上清通过加入20％PEG/NaCl(由体积浓度为20％的PEG6000和2.5M NaCl配制而成)，沉淀得到羊驼库对应的噬菌体，经PBS润洗一次之后，用于噬菌体筛选。
采用细胞筛选的方法，以人CLDN18.2-HEK293T细胞株作为筛选抗原，从噬菌体展示库中筛选针对人CLDN18.2的抗体，具体方法如下所述。在T25培养方瓶中培养人CLDN18.2-HEK293T或者人CLDN18.1-HEK293。当生长至90％左右密度时，此时生长状态最佳，去除培养上清，并用PBS(源培，B310KJ)润洗一次，然后加入5mL 4％多聚甲醛(生工，E672002-0500)进行固定1h，最后用PBS润洗两次，便可作为抗原材料用于噬菌体的细胞筛选。筛选时，羊驼库对应的噬菌体首先和固定的人CLDN18.1-HEK293细胞培养方瓶室温孵育1h，然后吸取经过吸附后的上清噬菌体，和固定的人CLDN18.2-HEK293T细胞培养方瓶孵育2h。PBS润洗两次后，加入3mL甘氨酸-HCl(pH2.0)轻轻混匀10min，以洗脱特异性结合目的膜蛋白CLDN18.2的噬菌体，接着将洗脱上清侵染对数期的SS320菌体(Lucigen，60512-1)静置30min，然后220rpm条件下培养1h，再通过加入VSCM13辅助噬菌体，静置30min，继续在220rpm条件下培养1h，离心并置换至C +/K +2-YT培养基中，最终得到的噬菌体继续用于第二轮的筛选。如此反复，并对每轮随机挑选的10个克隆进行序列分析，结果发现经过3轮的筛选，第三轮筛选后序列富集明显。
挑取第三轮筛选的克隆于96孔板中制备噬菌体上清，通过噬菌体ELISA筛选针对CLDN18.2重组蛋白的阳性克隆，然后挑取所有阳性克隆测序分析，接着将序列唯一的克隆制备噬菌体上清进一步在流式水平上验证，筛选到只结合人CLDN18.2而不结合人CLDN18.1的候选抗体。
具体流式水平验证方法如下:
首先分别取1×10 5个人CLDN18.2-HEK293T与人CLDN18.1-HEK293细胞，500g低速离心去上清，细胞用如1.3.1所述的FACS缓冲液润洗后，加入用10％FBS(Gibco，15140-141)封闭1h的噬菌体制备上清，在4℃孵育1h，然后用FACS缓冲液润洗两次，以1:50加入抗M13鼠源单克隆抗体(Sino Biological,11973-MM05T)，并在4℃孵育1h。然后用FACS缓冲液润洗两次，加入APC标记的抗鼠Fc二抗(Jackson,115136071)，在4℃孵育1h，以FACS缓冲液润洗两次，最后通过流式细 胞仪(Beckman，CytoFLEX AOO-1-1102)进行检测。结果发现NA4-S、NA7-S和NA8-S能够特异性的结合人CLDN18.2-HEK293T，而不是人CLDN18.1-HEK293细胞，将这些抗体进行全长构建，用于进一步验证。
克隆NA4-S、NA7-S和NA8-S的VHH以及采用IMGT或AbM编号系统定义的CDR1、CDR2和CDR3的氨基酸序列(SEQ ID NO)示于表2。
表2
Figure PCTCN2021132218-appb-000012
实施例4
嵌合VHH-Fc(hIgG1)抗体的产生和表达
CLDN18.2在胃癌等癌症细胞上高表达，针对此类肿瘤相关靶点的抗体药物可以通过补体依赖的细胞毒性作用(CDC)和抗体依赖的细胞介导的细胞毒性作用(ADCC)来杀伤肿瘤。本实施例在实施例3筛选到的候选纳米抗体基础上设计嵌合抗体，并进行表达用于后续的CDC和ADCC实验。考虑到靶点的特殊性，在将候选纳米抗体基因构建到瞬转表达质粒pcDNA3.4(Thermofisher，A14697)上时，在候选纳米抗体的C端融合人IgG1Fc片段(SEQ ID NO:21)，这个片段包括连接区和IgG1的恒定区，用以介导ADCC和CDC等效应。本实施例选取了候选纳米抗体NA4-S(SEQ ID NO:10)、NA7-S(SEQ ID NO:11)和NA8-S(SEQ ID NO:12)，在各自基础上融合了人IgG1Fc片段，分别得到Fc融合的嵌合抗体NA4-S(在本公开中也称为“候选抗体NA4-S”、“抗体NA4-S”或“NA4-S抗体”，其为候选纳米抗体NA4-S与人IgG1Fc融合得到的嵌合抗体)、NA7-S(在本公开中也称为“候选抗体NA7-S”、“抗体NA7-S”或“NA7-S抗体，其为候选纳米抗体NA7-S与人IgG1Fc融合得到的嵌合抗体”和NA8-S(在本公开中也称为“候选抗体NA8-S”、“抗体NA8-S”或“NA8-S抗体”，其为候选纳米抗体NA8-S与人IgG1Fc融合得到的嵌合抗体)。
抗体NA4-S、NA7-S和NA8-S的表达采用的是ExpiCHO瞬转表达系统(Gibco，A29133)，具体方法如下:
转染当天，确认细胞密度为7×10 6至1×10 7个活细胞/mL左右，细胞活率>98％，此时用37℃预热的新鲜ExpiCHO表达培养基25mL将细胞调整到终浓度为6×10 6个细胞/mL，用4℃预冷的OptiPRO TMSFM 1mL稀释目的质粒(共25μg)，同时用920μL OptiPRO TMSFM稀释80μL ExpiFectamine TMCHO，再将两者混合并轻轻吹打混匀制备成ExpiFectamine TMCHO/质粒DNA混合液，室温孵育1-5min之后转移到准备好的细胞悬液中，缓慢加入并同时轻轻摇晃细胞悬液，最后置于细胞培养摇床中，在37℃，8％CO 2条件下培养。
在转染后18-22个小时内添加ExpiCHO TMEnhancer和ExpiCHO TMFeed，摇瓶放置于32℃摇床和5％CO 2条件下继续培养，在转染后第五天，添加相同体积的ExpiCHO TMFeed，缓慢加入的同时轻轻混匀细胞混悬液，转染12-15天后，将细胞表达上清高速离心(15000g，10min)，所得上清用Protein A(Millipore，P2545)进行亲和纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和，最后所得蛋白(即抗体NA4-S、NA7-S和NA8-S)用浓缩管(Millipore，UFC901096)的方法置换至PBS缓冲液当中。
实施例5
候选抗体NA4-S、NA7-S和NA8-S的特异性结合和物种交叉特异性结合测定
以候选抗体NA4-S为例，先用含0.25％EDTA的Trypsin(Gibco，25200-072)消化生长状态良好的人CLDN18.2-HEK293T、HEK293T、HEK293、人CLDN18.1-HEK293、鼠CLDN18.2-HEK293和鼠CLDN18.1-HEK293细胞，取1×10 5个细胞和10μg/mL候选抗体NA4-S孵育1h，另外以hIgG1同型抗体作为对照。用FACS缓冲液润洗两次，然后和0.5μg PE标记的山羊抗人IgG-Fc二抗(Abcam，ab98596)在4℃孵育1h。其后，用FACS缓冲液洗三次，通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测候选抗体在细胞上的结合。
采用与NA4-S相同的方法测定候选抗体NA7-S和NA8-S的特异性结合及物种交叉特异性。
流式检测的结果(如图2所示)显示，类似于对照抗体IMAB362，候选抗体NA4-S、NA7-S和NA8-S特异性结合人CLDN18.2-HEK293T和鼠CLDN18.2-HEK293，而在HEK293T、HEK293、人CLDN18.1-HEK293和鼠CLDN18.1-HEK293细胞上不结合。这说明本公开的候选抗体NA4-S、NA7-S和NA8-S能够特异性结合CLDN18.2而不结合CLDN18.1，而且在人和鼠CLDN18.2上交叉识别。
实施例6
候选抗体NA4-S、NA7-S和NA8-S在人CLDN18.2-HEK293T细胞株和人CLDN18.2-KATOIII肿瘤细胞株上结合能力的比较
取培养状态良好的人CLDN18.2-HEK293T细胞，分别与三倍梯度稀释后的候选抗体NA4-S、NA7-S和NA8-S在4℃孵育1h。经FACS缓冲液润洗两遍，然后加入PE标记的山羊抗人IgG-Fc抗体(Abcam，ab98596)0.5μg，在4℃孵育1h，再经FACS缓冲液润洗两遍之后，通过流式细胞仪检测。
流式检测结果如图3a和图3b所示，结果显示：在人CLDN18.2-HEK293T细胞上，候选抗体NA4-S、NA7-S和NA8-S均表现出来和对照抗体相当甚至更优的细胞结合活性。
采用相同的方法测定候选抗体NA4-S、NA7-S和NA8-S在人CLDN18.2-KATOIII肿瘤细胞株上的结合能力。流式检测结果如图3c所示，结果显示:在人CLDN18.2-KATOIII肿瘤细胞株上，候选抗体NA7-S、NA8-S均表现出来和对照抗体相当甚至更优的细胞结合活性；NA4-S几乎不结合人CLDN18.2-KATOIII肿瘤细胞株，这说明了来自CLDN18.2-HEK293T和CLDN18.2-KATOIII两个细胞上的CLDN18.2蛋白存在结构或者糖基化等翻译后修饰的差异。
实施例7
候选抗体NA4-S、NA7-S和NA8-S的补体依赖的细胞毒性作用(CDC)
采用MTS法测定候选抗体的CDC细胞杀伤效应。候选抗体NA4-S、NA7-S和NA8-S由于包含有IgG1Fc片段，可以通过CDC对细胞进行杀伤，而MTS试剂能够被活细胞产生的NADPH或者NADH还原成一种有色化合物，因此颜色的深浅代表了抗体介导CDC细胞杀伤效应的杀伤效果。具体操作方法如下:
以候选抗体NA4-S为例。先经trypsin消化培养状态良好的人CLDN18.2-HEK293T细胞之后，取5×10 4个细胞和稀释5倍的兔血清混合，然后分别加入梯度稀释的候选抗体NA4-S或对照抗体IMAB362各50μL，于37℃孵育3h。然后，向候选抗体NA4-S或对照抗体IMAB362中分别加入MTS试剂(Promega，G3580)30μL，充分混合均匀，置于37℃、5％CO 2的恒温培养箱培养4h，期间观察培养基颜色变化，通过酶标仪测定492nm波长下的OD值。其中以10％Triton X-100加靶细胞作为全裂解的对照，以只加靶细胞作为空白阴性对照，以兔补体加靶细胞作为背景阴性对照。
细胞杀伤率按如下公式计算：细胞杀伤率(％)＝(候选抗体孔OD值-背景孔OD值)/(全裂解孔OD值-空白孔OD值)×100％。
候选抗体NA4-S在人CLDN18.2-HEK293T细胞上的CDC杀伤效应结果如图4a所示。从图中可以看出，相对于对照抗体IMAB362，候选抗体NA4-S在等摩尔浓度下展示出相当的CDC细胞杀伤效果，其中NA4-S和IMAB362的CDC细胞杀伤EC 50分别是0.7317nM和0.6125nM。采用同样的方法测定候选抗体NA7-S和NA8-S在人CLDN18.2-HEK293T细胞上的CDC杀伤效应，其结果如图4b和图4c所示。从图中可以看出，相对于对照抗体IMAB362，候选抗体NA7-S和NA8-S在等摩尔浓度下也展示出相当的CDC细胞杀伤效果，其中，NA7-S和IMAB362比较的CDC细胞杀伤EC 50分别是0.5069nM和0.6285nM；NA8-S和IMAB362比较的CDC细胞杀伤EC 50分别是0.4782nM和1.863nM。
采用同样的方法测定候选抗体NA7-S和NA8-S在人CLDN18.2-KATOIII肿瘤细胞上的CDC杀伤效应，其结果如图4d和4e所示。
从图4d中可以看出，NA7-S在等摩尔浓度下展示出比对照抗体IMAB362更强的CDC细胞杀伤效应，其中纳米抗体NA7-S的EC 50为6.853nM，而IMAB362的 EC 50为69.79nM。
从图4e中可以看出，NA8-S在等摩尔浓度下展示出比对照抗体IMAB362更强的CDC细胞杀伤效应，其中纳米抗体NA8-S的EC 50为11.25nM，而IMAB362的EC 50为1865nM。
综上所述，候选抗体NA4-S、NA7-S和NA8-S在CLDN18.2-HEK293T和CLDN18.2-KATOIII细胞上介导的CDC杀伤效应优于对照抗体IMAB362。
实施例8
抗体依赖的细胞介导的细胞毒作用(ADCC)
利用乳酸脱氢酶(LDH)释放法检测ADCC效应。其原理是:抗体的可变区结合靶细胞上的目标抗原，当抗体的Fc段与PBMC中的NK效应细胞上的FcRIIIa(又名CD16a)结合后，NK细胞会释放穿孔素、颗粒酶等裂解靶细胞，然后通过LDH乳酸脱氢酶试剂盒(Takara，MK401)可以检测细胞上清中乳酸脱氢酶的释放，以此来测定NK细胞对靶细胞的杀伤程度。具体操作如下:
在96孔细胞培养板中每孔加入50μL密度为2×10 5个/mL的人CLDN18.2-HEK293T细胞，置于37℃培养箱中培养过夜(16-20h)。加入梯度稀释的候选抗体NA7-S或NA8-S 50μL，混匀后于37℃培养箱孵育20min，再加入复苏好的5×10 5个/孔的人PBMC细胞(效应细胞/靶细胞比例为50:1)，37℃培养箱孵育4h后，通过300g离心得到上清，然后加入LDH检测试剂，反应60min，最后通过酶标仪(Molecular Devices，SpectraMax190)测定在492nm波长下的OD值并进行检测结果的分析。其中以10％Triton X-100加靶细胞作为全裂解的对照，以只加靶细胞作为空白阴性对照，以PBMC加靶细胞作为背景阴性对照。
细胞杀伤率按如下公式计算：杀伤率(％)＝(候选抗体孔OD值-背景孔OD值)/(全裂解孔OD值-空白孔OD值)×100％。
结果(图5a，5b)显示，候选抗体NA7-S或NA8-S在等摩尔浓度下具有和对照抗体IMAB362相当的细胞杀伤活性。其中，从图5a中NA7-S和IMAB362比较的ADCC细胞杀伤EC 50分别是0.1097nM和0.0862nM；从图5b中NA8-S和IMAB362比较的ADCC细胞杀伤EC 50分别是0.0997nM和0.0636nM。
采用同样的方法测定候选抗体NA7-S或NA8-S在人CLDN18.2-KATOIII肿瘤细胞上的ADCC杀伤效应。结果如图5c和5d所示，在等摩尔浓度下候选抗体展示出比对照抗体IMAB362更强的ADCC细胞杀伤效应，其中，在候选抗体NA7-S和IMAB362的比较中，NA7-S介导的ADCC杀伤效率近50％，而IMAB362对照抗体的ADCC杀伤效率只有23％左右；在候选抗体NA8-S和IMAB362的比较中，NA8-S介导的ADCC杀伤效率近44％，而IMAB362对照抗体的ADCC杀伤效率只有24％左右。
实施例9
体内抑瘤实验(以人CLDN18.2-HEK293T作为成瘤细胞株)
实验使用6-8周龄，雌性SCID小鼠(24-26g)。实验小鼠饲养在恒温恒湿的独立通风盒内，饲养室温度21-24℃，湿度30-53％。
将1×10 7个人CLDN18.2-HEK293T细胞进行右侧腋窝皮下接种注射。待皮下瘤块体积达80-100mm 3时，剔除肿瘤体积差异较大的小鼠样本，依据肿瘤体积进行随机分组(每组8只小鼠):分别是PBS处理组，IMAB362抗体处理组，候选抗体NA7-S或NA8-S处理组。细胞接种8天后即开始用抗体处理(候选抗体NA7-S或NA8-S根据分子量采用与IMAB362抗体等摩尔的剂量和等质量的剂量，分别为候选抗体10mg/kg，而IMAB362抗体20mg/kg和10mg/kg剂量)，每个星期两次给药处理，分别是静脉注射和腹膜内注射两种方式交替给药。随时观察和记录肿瘤长(mm)和宽(mm)，计算其肿瘤生长体积(V)，计算方式为:V＝(长×宽 2)/2。
抗体抑瘤的结果如图6a和6b所示，从中可以看出:在同等质量的剂量下，候选抗体NA7-S或NA8-S在抑制肿瘤生长方面显著优于对照抗体IMAB362。
实施例10
候选抗体在人CLDN18.2上的结合表位测定
候选抗体和对照抗体IMAB362都特异性结合CLDN18.2而不结合CLDN18.1，而CLDN18.2和CLDN18.1的膜外区域只有在ECD1区域有8个氨基酸的差别，因此推测候选抗体NA7-S、NA8-S和对照抗体IMAB362的抗原结合表位在ECD1区域上，为了确认候选抗体和IMAB362的抗原结合位置是否一致，本实施例采用了竞争结合的方法，具体方法如下:
将传代2-4次且生长状态良好的CLDN18.2-HEK293T细胞用于实验。于4℃、300g离心去除上清，随后将细胞用FACS缓冲液重悬，计数后将细胞密度调整为2×10 6个细胞/mL，以每孔100μL加至新的96孔圆底板中，4℃、300g离心并去除上清。
同时配制FACS缓冲液(1X PBS+2％FBS)并使用FACS缓冲液将竞争抗体NA7-S、NA8-S和对照抗体IMAB362进行梯度稀释，同样使用FACS缓冲液将生物素标记的NA7-S(或者IMAB362)蛋白稀释至13.4nM，分别向96孔板中加入100μL的梯度稀释液和100μL生物素标记的抗体稀释液，用排枪轻轻吹打混匀并将96孔板放置于4℃孵育1h。将孵育后的抗体和细胞混合液于4℃、300g离心并去除上清，随后向对应孔中各加入200μL的FACS缓冲液并重悬细胞，4℃、300g离心去上清；重复此步骤2次。使用FACS缓冲液将PE标记的链霉亲和素(eBioscience,12-4317-87)以1:200进行稀释，使用排枪向对应细胞中加入200μL/孔并轻轻吹打重悬细胞，随后将细胞放置于4℃避光孵育30min，孵育结束后将细胞于4℃、300g离心去除上清，加入FACS缓冲液重悬细胞，重复该步骤两次。最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测。
流式结果如图7a显示，随着NA7-S、NA8-S或对照抗体IMAB362浓度的增加，IMAB362-biotin在CLDN18.2-HEK293T细胞上的结合减少。同样，在如图7b中的流式结果显示随着NA7-S、NA8-S或对照抗体IMAB362浓度的增加，NA7-S-biotin在CLDN18.2-HEK293T细胞上的结合减少，这些都说明NA7-S、NA8-S和IMAB362在CLDN18.2上结合的抗原表位类似或者存在表位重叠。
为了进一步了解NA7-S、NA8-S和IMAB362在CLDN18.2上结合的关键氨基酸位点，基于CLDN18.2和CLDN18.1在ECD1存在8个氨基酸差别，本实施例构建了8个突变体HEK293T细胞株，将CLDN18.2上的8个差别氨基酸分别突变成对应CLDN18.1的氨基酸，然后通过测定待测抗体在野生株和突变株上的结合能力差别判断此氨基酸是否为抗体结合的关键氨基酸。具体方法如下:
首先，测定CLDN18.2野生和突变体HEK293T细胞株的表达情况，方法如1.3.2的方法，按细胞破膜试剂盒(eBioscience，88-8824-00)的说明书方法对细胞进行固定和破膜，然后根据抗CLDN18抗体[34H14L15]对C端CLDN18胞内段的结合能力判定表达情况。结果如图7c所示，各个突变细胞株以及野生株的CLDN18.2都有较高的表达。
基于构建的细胞株，参考1.3.1的方法，测定候选抗体NA7-S、NA8-S和IMAB362在CLDN18.2野生和突变体HEK293T细胞株上的结合强度，同时将候选抗体的结合强度除以各个细胞株的表达水平，并以候选抗体在野生株的结合作为100％进行归一化处理并作为最终的相对结合强度。如图7d-7g所示，NA7-S和NA8-S的表位较为接近，CLDN18.2ECD1上的1个位点(E56)对于NA7-S和NA8-S在人CLDN18.2-HEK293T的结合非常关键，而A42、E56和G65是IMAB362结合CLDN18.2的3个关键氨基酸。
实施例11
候选抗体介导的Fab-ZAP细胞杀伤效应
CLDN18.2在多种肿瘤上都高表达，而且在正常组织中其特异性的表达在胃上皮细胞的紧密连接结构中，因此有可能成为理想的ADC药物靶点。本实验通过抗体介导Fab-ZAP内吞的细胞毒性来检测抗体的内吞活性，其中Fab-ZAP(Atsbio,IT-51-100)是一种连接了saporin(皂素)的抗Fc区域的Fab片段，而saporin是一种核糖体抑制剂，能够抑制蛋白质的合成而使细胞死亡。Fab-ZAP和CLDN18.2的抗体孵育后使CLDN18.2的抗体带上Fab-ZAP，当CLDN18.2的抗体内吞时，Fab-ZAP随着抗体进入到细胞内，从而杀死细胞，然后通过MTS(Promega，G3580)检测细胞的活性来检测并比较候选抗体和对照抗体的内吞活性。具体方法如下:
取处于对数生长期的CLDN18.2-HEK293T细胞，用胰酶(0.25％(w/v)Trypsin 0.53mM EDTA)消化细胞至细胞间不黏连，产生间隙，用完全培养基终止消化。充分混匀细胞后，细胞计数并测定其活率。将细胞密度调整为4×10 4个细胞/mL，以每孔50μL加至细胞培养板并置于37℃细胞培养箱孵育16小时。同时用DMEM完全培养基稀释Fab-ZAP至2μg/mL，以此为稀释液进一步将候选抗体NA7-S、NA8-S和对照抗体进 行梯度稀释，并用排枪取50μL稀释液加入至细胞培养板中与CLDN18.2-HEK293T轻轻吹打混匀，将细胞培养板放入37℃细胞培养箱继续孵育72小时。接着使用排枪向每孔中加入20μL MTS并轻轻吹打混匀，然后于37℃孵育2-4小时，最后将细胞板用台式离心机以1000rpm的转速离心5分钟后，于酶标仪(Molecular Devices，SpectraMax190)中读取数据，检测波长为492nm。
结果如图8a-8b，相对于对照抗体IMAB362，候选抗体NA7-S和NA8-S可以更有效的通过Fab-ZAP对目标细胞CLDN18.2-HEK293T进行杀伤，其中在NA7-S和IMAB362的比较中，IC 50分别为0.0487nM和0.1284nM；同时在NA8-S和IMAB362的比较中，IC 50分别为0.0492nM和0.1362nM。
这不仅充分说明了候选抗体可以通过CLDN18.2进入细胞，而且优于对照抗体IMAB362，具有更大的潜力用于开发ADC抗体偶联药物。
实施例12
候选抗体NA4-S、NA7-S和NA8-S的人源化改造
相对鼠源抗体，羊驼来源纳米抗体和人源抗体的同源性较高，但其结构特殊，因此在NA4-S、NA7-S和NA8-S人源化设计过程中，采用AbM编号系统定义CDR序列和框架区序列，选择了与各纳米抗体的框架区序列最接近人的种系基因，而且在做回复突变时兼顾抗体的结构维持不变，最终设计了一系列人源化抗体，其中NA4-S-H7、NA7-S-H2和NA8-S-H9分别为对应母本抗体(NA4-S、NA7-S和NA8-S)的最优人源化分子。
人源化抗体NA4-S-H7、NA7-S-H2和NA8-S-H9的VHH以及CDR1、CDR2和CDR3的氨基酸序列(SEQ ID NO)示于表3。
表3
抗体名称 CDR1 CDR2 CDR3 VHH
NA4-S-H7 30 31 32 13
NA7-S-H2 37 38 39 14
NA8-S-H9 45 46 47 15
为了考察NA4-S、NA7-S和NA8-S人源化后亲和力是否不下降，采用了1.3.1所述方法比较其在人CLDN18.2-HEK293T细胞上的结合能力。
如图9a所示，NA4-S经过人源化改造后NA4-S-H7和母本抗体具备相似的亲和力，人源化前后分子EC 50分别为0.8822μg/mL和0.8771μg/mL，并且在最高浓度下的抗体结合强度MFI远远优于IMAB362对照抗体，分别是129181、127633和82136。
如图10a所示，NA7-S经过人源化改造后NA7-S-H2和母本抗体具备相似的亲和力，人源化前后分子EC 50分别为0.3790μg/mL和0.4824μg/mL，并且在最高浓度下的抗体结合强度MFI远远优于IMAB362对照抗体，分别是163000、184000和106000。
如图11a所示，只有NA8-S经过人源化改造后NA8-S-H9的亲和力劣于母本抗体， 人源化前后分子EC 50分别为0.09844μg/mL和0.1492μg/mL，而NA8-S-H9在最高浓度下的抗体结合强度MFI与IMAB362对照抗体相似，三个抗体MFI分别是101714、65195和76447。
实施例13
候选抗体NA4-S、NA7-S和NA8-S人源化前后在高浓度抗体下的结合特异性
CLDN18.2和CLDN18.1的候选抗体结合区域ECD1上只存在8个氨基酸的差别，而后者表达于肺部上皮细胞中，因此抗体药物如果非特异性结合CLDN18.1将导致严重的肺部损伤或者毒性从而限制临床的应用，因此本实施例在体外采用不同浓度的抗体(包括100μg/mL高浓度)和人CLDN18.1-HEK293孵育，方法如1.3.1所述，将不同浓度的抗体和目标细胞人CLDN18.1-HEK293在4℃孵育1h，接着再用上述FACS缓冲液润洗三次，加入PE标记的山羊抗人IgG Fc抗体(Abcam，ab98596)0.5μg(0.5mg/ml)，在4℃孵育1h。其后，经FACS缓冲液润洗三次，并向细胞中加入200μL FACS缓冲液重悬细胞，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)进行检测，记录结合强度和细胞结合阳性率。
如图9b所示，在结合强度方面，NA4-S、NA4-S-H7和对照抗体IMAB362在人CLDN18.1-HEK293细胞上的结合不随着浓度的增加而增加，与无关对照的结合强度几乎一致，而在图9c的细胞结合阳性率方面，即使在100μg/mL高浓度下，NA4-S、NA4-S-H7的阳性率和对照抗体IMAB362以及无关对照hIgG1非常接近，分别为0.434％、0.247％、0.095％和0.049％。
对于NA7-S人源化前后的特异性比较，如图10b所示，在结合强度方面，NA7-S、NA7-S-H2和对照抗体IMAB362在人CLDN18.1-HEK293细胞上的结合强度非常微弱，与无关对照的结合强度几乎一致；而在图10c的细胞结合阳性率方面，在100μg/mL高浓度下，NA7-S、NA7-S-H2、对照抗体IMAB362以及无关对照hIgG1的阳性率分别为10.801％、5.499％、1.433％和0.851％，其中，NA7-S-H2的阳性率或非特异性甚至优于母本抗体NA7-S。
对于NA8-S人源化前后的特异性比较，如图11b所示，在结合强度方面，NA8-S、NA8-S-H9和对照抗体IMAB362在人CLDN18.1-HEK293的结合强度非常微弱，但NA8-S和NA8-S-H9的结合强度明显高于对照抗体IMAB362；同时在图11c的细胞结合阳性率方面，在100μg/mL高浓度下，NA8-S、NA8-S-H9、对照抗体IMAB362以及无关对照hIgG1的阳性率分别为14.438％、5.459％、0.070％和0.067％，其中，NA8-S-H9的阳性率或非特异性甚至优于母本抗体NA8-S。
实施例14
人源化分子的亲和力成熟改造
本实施例中，对人源化分子NA4-S-H7、NA7-S-H2和NA8-S-H9进行亲和力成熟改造，用于提高亲和力和生物学活性。亲和力成熟改造是基于M13噬菌体展示技术，采用codon-based引物(引物合成过程中，单个密码子由NNK组成)引入CDR区突变，共构建4个噬菌体展示文库：文库1为CDR1+CDR2+CDR3单点组合突变；文库2为CDR1+CDR2双点组合突变；文库3为CDR1+CDR3双点组合突变，文库4为CDR2+CDR3双点组合突变。以人源化分子为模板，通过PCR方式获得单个CDR区突变片段，再通过Overlapping PCR方式获得VHH全长片段，双酶切(HindⅢ和NotⅠ)和双粘端连接将点突变抗体连接到噬菌体展示载体中，最后通过电转将带有突变位点的VHH序列转入大肠杆菌SS320中。
构建好的4个文库包装成噬菌体后，进行固相淘筛。以包被在免疫管上的抗原结合展示VHH全长片段的噬菌体，通过降低包被抗原量压力淘选亲和力高的抗体。经过淘洗，洗脱，侵染大肠杆菌SS320进行下一个循环的淘选，淘选2-3个循环后，挑选单克隆进行FACS检测，根据亲和力和序列分析，选择了7个候选亲和力成熟分子，包括NA7S-H2-418、NA7S-H2-420、NA7S-H2-20、NA7S-H2-30、NA4S-H7-10、NA4S-H7-3和NA8S-H9-57，选取上述亲和力成熟分子进行样品制备，制备方法详见实施例4。
通过亲和力成熟获得的抗体的VHH以及CDR1、CDR2和CDR3的氨基酸序列(SEQ ID NO)示于表4。
表4
抗体名称 CDR1 CDR2 CDR3 VHH
NA4S-H7-10 30 31 33 27
NA4S-H7-3 34 35 36 28
NA7S-H2-20 37 38 42 25
NA7S-H2-30 37 43 44 26
NA7S-H2-418 40 38 39 23
NA7S-H2-420 41 38 39 24
NA8S-H9-57 48 46 47 29
实施例15
亲和力成熟分子的特异性结合测定
采用与实施例5相同的检测方法检测亲和力成熟分子NA7S-H2-418、NA7S-H2-420、NA7S-H2-20、NA7S-H2-30、NA4S-H7-10、NA4S-H7-3和NA8S-H9-57在人CLDN18.1-HEK293细胞上的结合。
对于NA7-S-H2亲和力成熟前后的特异性比较，结果如图12所示，亲和力成熟分子NA7S-H2-418、NA7S-H2-420、NA7S-H2-20和NA7S-H2-30在人CLDN18.1-HEK293细胞上不结合且非特异性优于NA7-S-H2；对于NA4-S-H7和NA8-S-H9亲和力成熟前 后的特异性比较，结果显示与母本分子相当，故此处结果未予显示。
实施例16
亲和力成熟分子在人CLDN18.2-HEK293T细胞株和人CLDN18.2-KATOIII肿瘤细胞株上结合能力的比较
采用与实施例6相同的检测方法检测亲和力成熟分子NA7S-H2-418、NA7S-H2-420、NA7S-H2-20、NA7S-H2-30、NA4S-H7-10、NA4S-H7-3和NA8S-H9-57在人CLDN18.2-HEK293T和人CLDN18.2-KATOIII细胞上的结合能力。
对于NA7-S-H2亲和力成熟前后的结合能力比较，结果如图13a所示，亲和力成熟分子NA7S-H2-418、NA7S-H2-420、NA7S-H2-20、NA7S-H2-30在人CLDN18.2-HEK293T细胞上均表现出来和对照抗体相当甚至更优的细胞结合活性；对于NA4-S-H7亲和力成熟前后的结合能力比较，结果如图13b所示，亲和力成熟分子NA4S-H7-10和NA4S-H7-3在人CLDN18.2-HEK293T细胞上均表现出来和对照抗体相当的细胞结合活性；对于NA8-S-H9亲和力成熟前后的结合能力比较，结果如图13c所示，亲和力成熟分子NA8S-H9-57在人CLDN18.2-HEK293T细胞上表现出更优的细胞结合活性。
对于NA7-S-H2亲和力成熟前后在人CLDN18.2-KATOIII细胞上的结合能力比较，结果如图14所示，亲和力成熟分子表现出来的结合能力显著优于母本分子。
实施例17
亲和力成熟分子的补体依赖的细胞毒性作用(CDC)
采用与实施例7相同的MTS法测定亲和力成熟分子NA7S-H2-418、NA7S-H2-420、NA7S-H2-20、NA7S-H2-30、NA4S-H7-10、NA4S-H7-3和NA8S-H9-57在人CLDN18.2-HEK293T细胞上的CDC细胞杀伤效应。
CDC检测结果如下:如图15a-15d所示，所有亲和力成熟分子在人CLDN18.2-HEK293T细胞上的CDC杀伤效应与母本分子NA4-S-H7、NA7-S-H2或NA8-S-H9相当或更优。
实施例18
亲和力成熟分子的ADCC活性
亲和力成熟分子的Fc端与CD16a(V158)结合、VHH端与靶细胞结合后，会激活Jurkat细胞内部NF-AT蛋白表达，NF-AT与其反应元件结合会触发其下游的荧光素酶表达，用不同浓度梯度的抗体刺激，会得到具有抗体浓度依赖性的荧光读数曲线，从而可评价抗体的ADCC活性。
在96孔细胞培养板中每孔加入总体积为50μL的1×10 4个/孔的人CLDN18.2-HEK293T细胞或人CLDN18.2-KATOIII细胞和1×10 5个/孔的 Jurkat-NFAT-Luciferase-CD16a(V158)细胞(稳定转染CD16a(V158)序列(UniProtKB-P08637)和含有NF-AT-re核酸序列的pGL4.30质粒(promega，#E8481)至Jurkat细胞
Figure PCTCN2021132218-appb-000013
获得的稳转细胞株)，加入50μL梯度稀释的待测样品，37℃培养箱孵育6h。每孔加入50μL Bright-Lite(vazyme，货号：DD1204-03)，避光孵育10min，检测荧光信号。
在人CLDN18.2-HEK293T细胞上的ADCC效应检测结果如下:如图16a和图16b所示，亲和力成熟分子NA7S-H2-418、NA7S-H2-420和NA7S-H2-20的ADCC杀伤效应优于母本分子NA7-S-H2；亲和力成熟分子NA7S-H2-30与母本分子NA7-S-H2相当。
在人CLDN18.2-KATOIII细胞上的检测结果如下:如图17a所示，亲和力成熟分子NA4S-H7-10和NA4S-H7-3的ADCC杀伤效应优于母本分子NA4-S-H7；如图17b所示，亲和力成熟分子NA8S-H9-57的ADCC杀伤效应优于母本分子NA8-S-H9。
序列表概述
Figure PCTCN2021132218-appb-000014
Figure PCTCN2021132218-appb-000015
Figure PCTCN2021132218-appb-000016
Figure PCTCN2021132218-appb-000017
本领域技术人员将进一步认识到，在不脱离其精神或中心特征的情况下，本发明可以以其他具体形式来实施。由于本公开的前述描述仅公开了其示例性实施方案，应该理解的是，其他变化被认为是在本发明的范围内。因此，本发明不限于在此详细描述的特定实施方案。相反，应当参考所附权利要求来指示本发明的范围和内容。

Claims (20)

  1. CLDN18.2结合分子，其包含至少一个免疫球蛋白单可变结构域，其中所述免疫球蛋白单可变结构域包含CDR1、CDR2和CDR3，并且其中
    (1)CDR1在氨基酸序列上与SEQ ID NO:1、4或7所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；CDR2在氨基酸序列上与SEQ ID NO:2、5或8所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；和/或CDR3在氨基酸序列上与SEQ ID NO:3、6或9所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；或者
    (2)CDR1在氨基酸序列上与SEQ ID NO:30、37或45所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；CDR2在氨基酸序列上与SEQ ID NO:31、38或46所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异；和/或CDR3在氨基酸序列上与SEQ ID NO:32、39或47所示的序列存在不超过2个氨基酸的氨基酸添加、缺失或取代的差异。
  2. 权利要求1所述的CLDN18.2结合分子，其中所述CLDN18.2结合分子是针对CLDN18.2的抗体或其抗原结合片段。
  3. 权利要求1或2所述的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域是VHH；例如，来自骆驼科动物(例如羊驼)的VHH。
  4. 权利要求1-3中任一项所述的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含:
    i)SEQ ID NO:1、4、7、30、34、37、40、41、45或48所示氨基酸序列的CDR1；
    ii)SEQ ID NO:2、5、8、31、35、38、43或46所示氨基酸序列的CDR2；和
    iii)SEQ ID NO:3、6、9、32、33、36、39、42、44或47所示氨基酸序列的CDR3。
  5. 权利要求1-4中任一项所述的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含:
    (a)SEQ ID NO:1所示氨基酸序列的CDR1，SEQ ID NO:2所示氨基酸序列的CDR2和SEQ ID NO:3所示氨基酸序列的CDR3；
    (b)SEQ ID NO:4所示氨基酸序列的CDR1，SEQ ID NO:5所示氨基酸序列的CDR2和SEQ ID NO:6所示氨基酸序列的CDR3；
    (c)SEQ ID NO:7所示氨基酸序列的CDR1，SEQ ID NO:8所示氨基酸序列的CDR2和SEQ ID NO:9所示氨基酸序列的CDR3；
    (d)SEQ ID NO:30所示氨基酸序列的CDR1，SEQ ID NO:31所示氨基酸序列的CDR2和SEQ ID NO:32所示氨基酸序列的CDR3；
    (e)SEQ ID NO:30所示氨基酸序列的CDR1，SEQ ID NO:31所示氨基酸序列的CDR2和SEQ ID NO:33所示氨基酸序列的CDR3；
    (f)SEQ ID NO:34所示氨基酸序列的CDR1，SEQ ID NO:35所示氨基酸序列的 CDR2和SEQ ID NO:36所示氨基酸序列的CDR3；
    (g)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
    (h)SEQ ID NO:40所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
    (i)SEQ ID NO:41所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:39所示氨基酸序列的CDR3；
    (j)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:38所示氨基酸序列的CDR2和SEQ ID NO:42所示氨基酸序列的CDR3；
    (k)SEQ ID NO:37所示氨基酸序列的CDR1，SEQ ID NO:43所示氨基酸序列的CDR2和SEQ ID NO:44所示氨基酸序列的CDR3；
    (l)SEQ ID NO:45所示氨基酸序列的CDR1，SEQ ID NO:46所示氨基酸序列的CDR2和SEQ ID NO:47所示氨基酸序列的CDR3；
    (m)SEQ ID NO:48所示氨基酸序列的CDR1，SEQ ID NO:46所示氨基酸序列的CDR2和SEQ ID NO:47所示氨基酸序列的CDR3。
  6. 权利要求1-5中任一项所述的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域包含或由以下序列组成:
    (A)SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29所示的氨基酸序列；
    (B)与SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29所示的氨基酸序列至少80％、85％、90％、95％、98％或99％相同的氨基酸序列；或
    (C)与SEQ ID NO:10、11、12、13、14、15、23、24、25、26、27、28或29相比具有一个或多个(例如，1、2、3、4、5、6、7、8、9或10个)氨基酸的添加、缺失和/或取代的氨基酸序列，优选地，所述添加、缺失和/或取代不发生在CDR区。
  7. 权利要求1-6中任一项所述的CLDN18.2结合分子，其中所述CLDN18.2结合分子是单结构域抗体，例如重链单结构域抗体；嵌合抗体；或人源化抗体。
  8. 权利要求1-7中任一项所述的CLDN18.2结合分子，其中所述免疫球蛋白单可变结构域与另一分子融合，所述另一分子是例如免疫球蛋白(例如IgG)的Fc结构域或荧光蛋白。
  9. 权利要求8所述的CLDN18.2结合分子，其中所述CLDN18.2结合分子是包含来自骆驼科动物的VHH与人IgG(例如，人IgG1或IgG4)的Fc结构域的嵌合抗体。
  10. 权利要求9所述的CLDN18.2结合分子，其中所述CLDN18.2结合分子是包含来自羊驼的VHH与人IgG1的Fc结构域的嵌合抗体。
  11. 权利要求1-10中任一项所述的CLDN18.2结合分子，其中所述CLDN18.2结合分子结合人CLDN18.2的胞外结构域1(ECD1)。
  12. CLDN18.2结合分子，其与权利要求1-11中任一项所述的CLDN18.2结合 分子竞争相同表位。
  13. 权利要求1-12中任一项所述的CLDN18.2结合分子，其特异性结合CLDN18.2，但不结合CLDN18.1。
  14. 分离的核酸分子，其编码权利要求1-13中任一项所述的CLDN18.2结合分子。
  15. 一种表达载体，其包含权利要求14所述的分离的核酸分子。
  16. 一种宿主细胞，其包含权利要求15所述的表达载体。
  17. 一种药物组合物，其包含至少一种权利要求1-13中任一项所述的CLDN18.2结合分子和药学上可接受的载体。
  18. 制备权利要求1-13中任一项所述的CLDN18.2结合分子的方法，包括以下步骤:
    -在权利要求16所述的宿主细胞中表达权利要求1-13中任一项所述的CLDN18.2结合分子；和
    -从宿主细胞分离所述CLDN18.2结合分子。
  19. 权利要求1-13中任一项所述的CLDN18.2结合分子在制备用于治疗或预防与CLDN18.2相关的病症的药物中的用途。
  20. 用于治疗或诊断与CLDN18.2相关的病症的试剂盒，其包含容器，所述容器包含权利要求1-13中任一项所述的CLDN18.2结合分子。
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WO2023025306A1 (zh) * 2021-08-27 2023-03-02 三优生物医药(上海)有限公司 靶向pd-l1和cldn18.2的双特异性抗体及其制备方法和应用
CN115991784A (zh) 2021-10-19 2023-04-21 宝船生物医药科技(上海)有限公司 抗cd47-cldn18.2双特异性抗体及其用途
JP2024540062A (ja) * 2021-11-05 2024-10-31 チア タイ ティエンチン ファーマシューティカル グループ カンパニー リミテッド 抗ｃｌｄｎ１８．２抗体及びその用途
WO2024140670A1 (en) * 2022-12-26 2024-07-04 Full-Life Technologies Hk Limited Conjugate and uses thereof

Citations (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD120403A5 (zh) 1974-05-28 1976-06-12
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
EP0183070A2 (en) 1984-10-30 1986-06-04 Phillips Petroleum Company Transformation of yeasts of the genus pichia
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
WO1987000195A1 (en) 1985-06-28 1987-01-15 Celltech Limited Animal cell culture
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
EP0244234A2 (en) 1986-04-30 1987-11-04 Alko Group Ltd. Transformation of trichoderma
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
WO1990003430A1 (en) 1988-09-23 1990-04-05 Cetus Corporation Cell culture medium for enhanced cell growth, culture longevity and product expression
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
EP0402226A1 (en) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Transformation vectors for yeast yarrowia
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO2003048731A2 (en) 2001-12-03 2003-06-12 Abgenix, Inc. Antibody categorization based on binding characteristics
EP2852408A1 (en) * 2012-05-23 2015-04-01 GANYMED Pharmaceuticals AG Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
CN107667118A (zh) * 2015-04-15 2018-02-06 加尼梅德药物有限公司 包含针对密封蛋白18.2之抗体的药物缀合物
US20180127489A1 (en) 2005-11-24 2018-05-10 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
CN109762067A (zh) * 2019-01-17 2019-05-17 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途
CN111518214A (zh) * 2019-02-03 2020-08-11 上海健信生物医药科技有限公司 靶向cldn18.2的双特异性抗体及其制备方法和应用
CN111836644A (zh) * 2018-03-08 2020-10-27 东莞凡恩世生物医药有限公司 抗密蛋白18.2抗体及其用途
CN111867630A (zh) * 2018-06-17 2020-10-30 上海健信生物医药科技有限公司 靶向cldn18.2的抗体、双特异性抗体、adc和car及其应用
CN111848809A (zh) * 2019-04-08 2020-10-30 上海健信生物医药科技有限公司 靶向Claudin18.2的CAR分子、其修饰的免疫细胞及用途
CN111961135A (zh) * 2020-07-13 2020-11-20 北京亦庄国际蛋白药物技术有限公司 一种用于预防或治疗癌症的抗体
CN112480248A (zh) * 2020-11-24 2021-03-12 三优生物医药(上海)有限公司 与cld18a2特异性结合的分子

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2815888C (en) * 2010-10-25 2020-06-30 National Research Council Of Canada Clostridium difficile-specific antibodies and uses thereof
CN111434692B (zh) * 2019-01-15 2021-12-31 浙江道尔生物科技有限公司 抗cld18a2纳米抗体及其应用
JP7600218B2 (ja) * 2019-05-24 2024-12-16 サンヨウ バイオファーマシューティカルズ カンパニー リミテッド 新規なｃｌｄｎ１８．２結合分子
CN111235113A (zh) * 2020-01-21 2020-06-05 南京北恒生物科技有限公司 包含嵌合抗原受体的免疫细胞及其用途
TW202229335A (zh) * 2020-10-12 2022-08-01 美商健生生物科技公司 用於多方向性生物輸送之生物合成材料及方法

Patent Citations (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD120403A5 (zh) 1974-05-28 1976-06-12
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
EP0183070A2 (en) 1984-10-30 1986-06-04 Phillips Petroleum Company Transformation of yeasts of the genus pichia
WO1987000195A1 (en) 1985-06-28 1987-01-15 Celltech Limited Animal cell culture
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
EP0244234A2 (en) 1986-04-30 1987-11-04 Alko Group Ltd. Transformation of trichoderma
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
WO1990003430A1 (en) 1988-09-23 1990-04-05 Cetus Corporation Cell culture medium for enhanced cell growth, culture longevity and product expression
EP0402226A1 (en) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Transformation vectors for yeast yarrowia
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO2003048731A2 (en) 2001-12-03 2003-06-12 Abgenix, Inc. Antibody categorization based on binding characteristics
US20180127489A1 (en) 2005-11-24 2018-05-10 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
EP2852408A1 (en) * 2012-05-23 2015-04-01 GANYMED Pharmaceuticals AG Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
CN107667118A (zh) * 2015-04-15 2018-02-06 加尼梅德药物有限公司 包含针对密封蛋白18.2之抗体的药物缀合物
CN111836644A (zh) * 2018-03-08 2020-10-27 东莞凡恩世生物医药有限公司 抗密蛋白18.2抗体及其用途
CN111867630A (zh) * 2018-06-17 2020-10-30 上海健信生物医药科技有限公司 靶向cldn18.2的抗体、双特异性抗体、adc和car及其应用
CN109762067A (zh) * 2019-01-17 2019-05-17 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途
CN111518214A (zh) * 2019-02-03 2020-08-11 上海健信生物医药科技有限公司 靶向cldn18.2的双特异性抗体及其制备方法和应用
CN111848809A (zh) * 2019-04-08 2020-10-30 上海健信生物医药科技有限公司 靶向Claudin18.2的CAR分子、其修饰的免疫细胞及用途
CN111961135A (zh) * 2020-07-13 2020-11-20 北京亦庄国际蛋白药物技术有限公司 一种用于预防或治疗癌症的抗体
CN112480248A (zh) * 2020-11-24 2021-03-12 三优生物医药(上海)有限公司 与cld18a2特异性结合的分子

Non-Patent Citations (51)

* Cited by examiner, † Cited by third party
Title
"Antibody Engineering", 2001, SPRINGER
ABBAS ET AL.: "Cellular and Molecular Immunology", 2010, W.B. SAUNDERS COMPANY
ADIB-CONQUY ET AL., INT. IMMUNOL., vol. 10, 1998, pages 341 - 6
AL-LAZIKANI ET AL.: "Standard Conformations for the Canonical Structures of Immunoglobulins", JOURNAL OF MOLECULAR BIOLOGY, vol. 273, 1997, pages 927 - 948, XP004461383, DOI: 10.1006/jmbi.1997.1354
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, no. 17, 1997, pages 3389 - 3402
AUSUBEL ET AL.: "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology", 2002, WILEY, JOHN & SONS, INC.
BARBAS ET AL., PROC. NAT. ACAD. SCI. USA, vol. 91, 1994, pages 3809 - 3813
BARBAS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 7978 - 7982
BARNES ET AL., ANAL. BIOCHEM., vol. 102, 1980, pages 255
BEERS ET AL., CLIN. CAN. RES., vol. 6, 2000, pages 2835 - 43
BRUMMELL ET AL., BIOCHEM, vol. 32, 1993, pages 1180 - 8
BURKS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 412 - 417
CARILLO ET AL., SIAMJ. APPLIED MATH., vol. 48, 1988, pages 1073
CARTER ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 163 - 167
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 877 - 883
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHU ET AL., GENE, vol. 13, 1981, pages 197
CLYNES ET AL., PNAS (USA, vol. 95, 1998, pages 652 - 656
COLIGAN ET AL.: "Short Protocols in Protein Science", 2003, WILEY, JOHN & SONS, INC.
DAVIS ET AL.: "Basic Methods in Molecular Biology", 1986, ELSEVIER
DEFFAR ET AL., AFRICAN JOURNAL OF BIOTECHNOLOGY, vol. 8, no. 12, 17 June 2009 (2009-06-17), pages 2645
DR. ANDREW C. R. MARTIN: "Antibody Engineering Lab Manual", SPRINGER-VERLAG, article "Protein Sequence and Structure Analysis of Antibody Variable Domains"
E. MEYERSW. MILLER, COMPUT. APPL. BIOSCI., vol. 4, 1988, pages 11 - 17
FFERNER, R. G., DRUG RES., vol. 48, 1998, pages 870 - 880
FLOWMETRIC, SORTING OUT FLUORESCENCE ACTIVATED CELL SORTING, 9 November 2017 (2017-11-09)
GAZZANO-SANTORO ET AL., J. IMMUNOL. METHODS, vol. 202, 1996, pages 163
GEISSE, S. ET AL., PROTEIN EXPR. PURIF., vol. 66, 1996, pages 271 - 282
GRAHAM ET AL., VIROLOGY, vol. 52, 1973, pages 456
HALL ET AL., J. IMMUNOL., vol. 149, 1992, pages 1605 - 12
HAM ET AL., METH. ENZ., vol. 58, 1979, pages 44
HAMERS-CASTERMAN CATARHOUCH TMUYLDERMANS SROBINSON GHAMERS CSONGA EBBENDAHMAN NHAMERS R: "Naturally occurring antibodies devoid of light chains", NATURE, vol. 363, no. 6428, 1993, pages 446 - 448, XP002535892, DOI: 10.1038/363446a0
HAWKINS ET AL., J. MOL. BIOL., vol. 226, 1992, pages 889 - 896
J. MOL. BIOL., vol. 48, 1970, pages 444 - 453
KAUFMAN, R.J., MOL. BIOTECHNOL., vol. 16, 2000, pages 151 - 160
KELLEYO' CONNELL, BIOCHEM., vol. 32, 1993, pages 6862 - 1187
KOBAYASHI ET AL., PROTEIN ENG., vol. 12, no. 10, 1999, pages 879 - 884
KOMISSAROV ET AL., J. BIOL. CHEM., vol. 272, 1997, pages 26864 - 26870
LI ET AL., J BIOL CHEM., vol. 287, 2012, pages 13713 - 13721
MAKRIDES, S.C., PROTEIN EXPR. PURIF., vol. 17, 1999, pages 183 - 202
R. VAN DER LINDEN ET AL., JOURNAL OF IMMUNOLOGICAL METHODS, vol. 240, 2000, pages 185 - 195
RAVETCHKINET, ANNU. REV. IMMUNOL, vol. 9, 1991, pages 457 - 92
RETTER ET AL., NUCL. ACIDS RES., vol. 33, 2005, pages D671 - D674
SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL, 2001
SCIER ET AL., GENE, vol. 169, 1995, pages 147 - 155
See also references of EP4253411A4
STEFAN WÖLL; ANNA MELISSA SCHLITTER; KARL DHAENE; MARC ROLLER; IRENE ESPOSITO; UGUR SAHIN; ÖZLEM TÜRECI: "Claudin 18.2 is a target for IMAB362 antibody in pancreatic neoplasms", INTERNATIONAL JOURNAL OF CANCER, vol. 134, no. 3, 20 November 2013 (2013-11-20), US , pages 731 - 739, XP071288537, ISSN: 0020-7136, DOI: 10.1002/ijc.28400 *
VON HEINJE G.: "Sequences of Proteins of Immunological Interest", 1987, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
WESOLOWSKI ET AL.: "Single Domain Antibodies: Promising Experimental and Therapeutic Tools in Infection and Immunity", MED MICROBIOL IMMUNOL, vol. 198, 2009, pages 157 - 174
WILDT ET AL., PROT. ENG., vol. 10, 1997, pages 835 - 41
XU, LIANG'E ET AL.: "Advances of CLDN18.2 Protein in the Therapy of Malignant Tumors", CHINESE JOURNAL OF CLINICAL ONCOLOGY, vol. 46, no. 6, 30 March 2019 (2019-03-30), pages 311 - 315, XP009528623 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024245407A1 (en) * 2023-05-31 2024-12-05 Full-Life Technologies Hk Limited Conjugates and uses thereof

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