WO2019242505A1 - 靶向cldn18.2的抗体、双特异性抗体、adc和car及其应用 - Google Patents

靶向cldn18.2的抗体、双特异性抗体、adc和car及其应用 Download PDF

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WO2019242505A1
WO2019242505A1 PCT/CN2019/090255 CN2019090255W WO2019242505A1 WO 2019242505 A1 WO2019242505 A1 WO 2019242505A1 CN 2019090255 W CN2019090255 W CN 2019090255W WO 2019242505 A1 WO2019242505 A1 WO 2019242505A1
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antibody
seq
region
amino acid
heavy chain
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PCT/CN2019/090255
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English (en)
French (fr)
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刘佳建
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上海健信生物医药科技有限公司
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Priority claimed from CN201810610790.3A external-priority patent/CN110606891B/zh
Priority claimed from CN201811295845.2A external-priority patent/CN111110862A/zh
Priority claimed from CN201910108951.3A external-priority patent/CN111518214B/zh
Priority claimed from CN201910276473.7A external-priority patent/CN111848809A/zh
Application filed by 上海健信生物医药科技有限公司 filed Critical 上海健信生物医药科技有限公司
Priority to CN201980019477.5A priority Critical patent/CN111867630B/zh
Priority to US17/252,259 priority patent/US11912763B2/en
Priority to JP2020571669A priority patent/JP7468903B2/ja
Priority to EP19823355.3A priority patent/EP3808376A4/en
Publication of WO2019242505A1 publication Critical patent/WO2019242505A1/zh

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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention relates to the field of biomedicine, in particular to an antibody, a bispecific antibody, ADC and CAR targeting CLDN 18.2, and applications thereof.
  • Cancer is a huge threat to human health and one of the important causes of death in the field of disease. Cancer treatment has undergone various stages of development such as surgery, chemotherapy, targeted drugs, tumor immunotherapy, and combination therapy, and has achieved great achievements in recent years. Among many cancer patients, the treatment methods for cancer patients such as lung cancer, stomach cancer, pancreatic cancer, esophageal cancer and ovarian cancer are still highly unsatisfactory. Therapies for these tumors include macromolecular targeted drugs such as new monoclonal antibodies, and these monoclonal antibodies and existing tumor immunotherapy methods, such as combined with immune checkpoint inhibitors PD-1, PD-L1 antibodies Treatment offers new possibilities and options for the huge unmet clinical treatment needs.
  • macromolecular targeted drugs such as new monoclonal antibodies
  • these monoclonal antibodies and existing tumor immunotherapy methods such as combined with immune checkpoint inhibitors PD-1, PD-L1 antibodies
  • Cell junction claudin (Claudin, Claudin or CLDN) is expressed in humans, mice and other species. It is a seal-associated protein in the intercellular layer, which controls ion flow between cells, maintains cell polarity, and transmits signals between cells. Has an important role.
  • CLDN family proteins have been found in 29 species, CLDN18 is one of them. CLDN18 has two homologous molecules called clin 18.1 (CLDN 18.1) and clin 18.2 (CLDN 18.2). Human compactin 18.1 (hCLDN18.1) and human compactin 18.2 (hCLDN18.2) are highly homologous, with amino acid homology of up to 92%.
  • hCLDN18.2 expression is very limited in normal tissues, only found in gastric mucosal differentiated epithelial cells, but has a particularly high expression in gastric cancer, including metastatic gastric cancer tissues (Sahin U.et.al. Claudin-18 Splice variant 2 for therapeutic antibody delveopment. Clin Cancer Res. 2008; 14 (23): 7642-34). It was further found that CLDN 18.2 was expressed in different cancer tissues, including approximately 70% of gastric cancer, 50% of pancreatic cancer, 30% of esophageal cancer, 25% of lung cancer and ovarian cancer. Therefore, CLDN18.2 has long been an ideal target for tumor patient markers and anti-tumor drug development. In particular, antibodies targeting CLDN18.2 were developed for tumor treatment.
  • the human CLDN18.2 protein has a total length of 261 amino acids, see the NCBI published sequence NP_001002026.1, claudin-18 isoform 2 of which 1-23 are signal peptides.
  • the CLDN18.2 protein is a transmembrane protein. There are two extra-membrane regions: an extracellular region 1 (ECL1) of about 55 amino acids behind the signal peptide, and an ECL2 of 23 amino acids. This structure is very similar to human CLDN18.1, and the ECL2 regions of human CLDN18.2 and human CLDN18.1 are exactly the same. Therefore, the development of human CLDN18.2 protein target antibodies needs to find antibodies against the ECL1 region or spatial structure of human CLDN18.2 protein. This makes work in this area more difficult.
  • ECL1 extracellular region 1
  • IMAB362 is an antibody against human CLDN18.2, which is a chimeric antibody derived from human and mouse. There is a risk of immunogenicity and its affinity is not high.
  • CAR Chimeric Antigen Receptor T cells (CART or CAR-T for short) are T cells isolated from patients. CARs are used to modify T cells in vitro to enable them to specifically recognize cancer cells. Cells are expanded and returned to the patient to achieve the effect of treating tumors.
  • CN201410341504.X discloses a T lymphocyte targeting CLD18A2 (ie, Claudin 18.2), a preparation method and application thereof, and there are also clinical data reports, but there is still much room for improvement in CART efficacy and safety.
  • CLD18A2 ie, Claudin 18.2
  • the field lacks effective antibodies targeting human CLDN18.2 protein, especially humanized antibodies, and antibodies, bispecific antibodies, antibody-drug conjugates with better cell activity, PK activity, and animal drug efficacy. (ADC) drugs, and more effective chimeric antigen receptor (CAR) and cells containing it.
  • ADC chimeric antigen receptor
  • bispecific antibodies, antibody drug conjugates and CAR molecules that target CLDN18.2 in the art, an antibody, humanized antibody, and bispecific specific to CLDN18.2 are provided. Antibodies and antibody drug conjugates thereof, CAR molecules, and preparation methods and applications thereof.
  • an antibody targeting CLDN18.2 which comprises a light chain variable region (VL) and / or a heavy chain variable region (VH), wherein VL contains the following complementarity determining region (CDR) sequences:
  • the VH contains the following CDR sequences:
  • the antibodies that target CLDN18.2 have better specific binding activity to human and murine CLDN18.2, and higher Emax; higher affinity (KinExA) (preferably reaching 10 pM); better CDC activity in human blood cells; induction of CLDN18. 2+ has better apoptotic activity, better tumor cell growth inhibitory activity, better animal efficacy, and better drug metabolism (PK) in the body, especially longer T1 / 2. It has better binding activity with murine CLDN18.2.
  • the VL comprises VL as shown in SEQ ID NO: 11, CDR1, VL as shown in SEQ ID NO: 13, and CDR2 and SEQ ID NO
  • the VL includes the amino acid sequences of VL1, CDR1 shown in SEQ ID NO: 12, VL2, CDR2 shown in SEQ ID NO: 13, and CDR3, VL shown in SEQ ID NO: 14; and the VH contains, as shown in SEQ ID IDNO: The amino acid sequences of VH CDR1 shown in 15, VH CDR2 shown in SEQ ID NO: 16, and VH CDR3 shown in SEQ ID NO: 17.
  • an antibody targeting CLDN18.2 as described above, wherein the CDR region of the antibody is a CDR sequence optimized for a deamination-sensitive site; preferably, the CDR region
  • the CDR sequence optimized for the deamination sensitive site is a light chain CDR sequence; preferably, the CDR sequence optimized for the deamination sensitive site of the CDR region is a CDR1 sequence optimized for the L30A and / or L30B position of the light chain CDR1.
  • an antibody that targets CLDN18.2 as described above, wherein the NS of the light chain CDR1 L30A and / or L30B of the antibody is mutated to NT, provided that L30E Bit is not Q and L34 is not T.
  • an antibody that targets CLDN18.2 as described above, wherein the NS of the light chain CDR1 L30A and / or L30B of the antibody is mutated to NT, provided that before the mutation
  • the light chain CDR1 is a sequence shown in SEQ ID NO: 12.
  • an antibody targeting CLDN18.2 as described above, wherein the CDR region of the antibody is a CDR sequence optimized for a deamination-sensitive site; preferably, the CDR region
  • the CDR sequence optimized for the deamination sensitive site is a heavy chain CDR sequence; preferably, the CDR sequence optimized for the deamination sensitive site of the CDR region is a CDR sequence optimized for positions H99 and / or H100 of the heavy chain CDR3.
  • an antibody targeting CLDN18.2 as described above wherein the NS of the H99 and / or H100 position of the heavy chain CDR3 of the antibody is mutated to NT, provided that Chain CDR1 is not Q at L30E and T at L34.
  • the aforementioned light chain CDR1 is a sequence shown in SEQ ID NO: 12.
  • the light chain CDR1 at positions L30A, L30B, L30E, and L34, and the heavy chain CDR3 at positions H99 and H100 are defined by the Kabat numbering rule.
  • the CLDN18.2-targeting antibody as described above is a murine antibody; the murine CLDN18.2 antibody matures with affinity, and The affinity is increased 3 to 10 times or more, preferably 10 times or more.
  • the VL of the murine antibody is the amino acid sequence shown in SEQ ID NO: 7 or a mutation thereof; and / or, the VH of the murine antibody is the amino acid sequence shown in SEQ ID NO: 8 or Its mutation
  • the mutation is the deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of the VL and / or VH, and the amino acid sequence of the mutation has the amino acid sequence of the VL and / or VH At least 85% sequence identity and maintains or improves the binding of the antibody to CLDN 18.2; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; most It is preferably at least 99% sequence identity.
  • the CLDN18.2-targeting antibody as described above comprises the variable region of a murine antibody and a murine or human antibody constant region;
  • the murine antibody is constant Regions include the heavy chain constant regions of murine IgG1, IgG2a, IgG2b3, or IgG3 and kappa or lambda type light chain constant regions
  • the human antibody constant regions include the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4 and kappa or lambda types Light chain constant region.
  • the antibody targeting CLDN18.2 is a chimeric antibody combining a variable region of a murine antibody and a constant region of a human antibody.
  • the chimeric antibody matures with affinity, and its affinity is increased by 3-10 times or more, preferably 10 times or more.
  • the light chain amino acid sequence of the chimeric antibody is an amino acid sequence shown in SEQ ID NO: 9 or a mutation thereof; and / or, the heavy chain amino acid sequence of the chimeric antibody is SEQ ID NO: The amino acid sequence shown in 10 or a mutation thereof.
  • the CLDN18.2-targeting antibody is as described above, and the CLDN18.2-targeting antibody is a humanized antibody.
  • the FR sequence preferably contains 1 to 10 amino acid back mutations.
  • an antibody targeting CLDN18.2 as described above wherein the light chain variable region CDR sequence of the humanized antibody can be in accordance with CCG, Kabat, Chothia, AbM or Contact and other numbering rules define that the CDR sequences include the light chain CDR sequences listed in Tables 4 to 8 or mutations thereof.
  • the VL of the humanized antibody comprises the amino acid sequence shown in any one of SEQ ID NOs: 29-33 or a mutation thereof; the mutation is that an amino acid sequence of the VL and / or VH occurs Or deletion, substitution or addition of one or more amino acid residues, and the amino acid sequence of the mutation has at least 85% sequence identity with the amino acid sequence of the VL and / or VH, and maintains or improves the antibody and CLDN18.
  • a combination of 2; said at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; most preferably at least 99% sequence identity.
  • the light chain CDR1 at positions L30E and L34 optimizes (mutates) the amino acid at the corresponding position of the adult germline CDR1.
  • an antibody that targets CLDN18.2 as described above wherein the heavy chain variable region framework (FR) sequence of the humanized antibody is selected from a human germline heavy chain sequence
  • the FR sequence preferably contains 0-10 amino acid back mutations.
  • an antibody targeting CLDN18.2 as described above wherein the heavy chain variable region CDR sequence of the humanized antibody can be in accordance with CCG, Kabat, Chothia, AbM or Contact and other numbering rules define that the CDR sequences include the heavy chain CDR sequences listed in Tables 4 to 8 or mutant sequences thereof.
  • an antibody targeting CLDN18.2 as described above wherein the humanized antibody comprises an amino acid sequence shown in any one of SEQ ID NOs: 29-33 or Its mutated light chain variable region and sequence is the amino acid sequence shown in any one of SEQ ID NOs: 34-37 or a combination of its mutated heavy chain variable region.
  • the antibody that targets CLDN18.2 as described above whose light chain comprises a constant region selected from a human antibody kappa or lambda light chain or a mutation thereof; and / or, the antibody
  • the heavy chain comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3 and IgG4 or a mutation thereof;
  • the heavy chain constant region or a variant thereof comprises mutations at positions 234, 235, and 243, or positions 239, 330, and 332 of the human IgGl Fc region;
  • the heavy chain constant region or a variant thereof comprises a variant in which the human IgGl Fc region is EEM or DEL at positions 356-358.
  • the antibody targeting CLDN18.2 as described above, the light chain comprises SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 45 Amino acid sequence or mutation thereof; and / or,
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 46 or a mutation thereof.
  • the CLDN 18.2-targeting antibody as described above comprises the following light and heavy chains:
  • the heavy chain is shown in the amino acid sequence of SEQ ID NO: 39, and the light chain is shown in the amino acid sequence of SEQ ID NO: 38; or, the heavy chain is shown in the amino acid sequence of SEQ ID NO: 39,
  • the light chain is shown in the amino acid sequence of SEQ ID NO: 40; or, the heavy chain is shown in the amino acid sequence of SEQ ID NO: 41, and the light chain is shown in the amino acid sequence of SEQ ID NO: 38; Or, the heavy chain is shown in the amino acid sequence of SEQ ID NO: 41, and the light chain is shown in the amino acid sequence of SEQ ID NO: 40; or, the heavy chain is shown in the amino acid sequence of SEQ ID NO: 39
  • the light chain is shown in the amino acid sequence of SEQ ID NO: 42; or the heavy chain is shown in the amino acid sequence of SEQ ID NO: 43, and the light chain is shown in the amino acid sequence of SEQ ID NO: 42
  • the heavy chain is shown in the amino acid sequence of SEQ ID NO: 44, and the light
  • the antibody that targets CLDN18.2 as described above, wherein the antibody that targets CLDN18.2 includes an immunoglobulin, Fab, Fab ', F (ab') 2 , Fv or mono Stranded Fv fragment (scFv).
  • the technical solution of the second aspect of the present invention is to provide a bispecific antibody including a first protein functional region and a second protein functional region, wherein the first protein functional region is as in the first
  • the antibody that targets CLDN18.2; the second protein functional region is an antibody that targets a non-CLDN18.2 antigen.
  • the bispecific antibody can not only retain the binding activity and functional activity of a single CLDN18.2 antibody, but also maintain the binding activity and functional activity of another protein functional region.
  • the structure of the bispecific antibody is similar to that of a normal IgG antibody, and the expression and purification can be completed and stabilized according to a conventional antibody expression and purification method.
  • the bispecific IgG antibody of the invention is similar to the structure of the sequence-specific diabodies (S equence-based IgG like bispecific anti body, SBody).
  • the non-CLDN18.2 antigen is an immune checkpoint antigen or a tumor treatment target
  • the immune checkpoint antigen includes PD-1, PD-L1, and Tim3. , LAG3, CD47
  • the tumor treatment targets include SIRP ⁇ (signal regulatory protein ⁇ ), etc.
  • SIRP ⁇ is a membrane protein that is mainly expressed in myeloid cells, including macrophages and dendritic cells.
  • the second protein functional region includes an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Tim3 antibody, an anti-LAG3 antibody, an anti-CD47 antibody, an anti-CD3 antibody, and an anti-CSF-1R antibody.
  • the anti-PD-1 antibody is Nivolumab (abbreviated as Nivo), Pembrolizumab (abbreviated as Pem) or Ba08 (that is, Ba08-1 described in patent application CN201410369300).
  • the anti-PD-L1 antibody is Atezolumab (Atezo), Avelumab (Avel) or Durvalumab (Durv).
  • the anti-CD3 antibody is an antibody constructed by Blincyto or AMG420 that binds to the light and heavy chain variable region sequences of CD3. .
  • the second protein functional region is a cytokine and a cytokine receptor or a fragment thereof; preferably, the cytokine or a fragment thereof includes TGF ⁇ , IL10, and CSF-1, the cytokine receptor or it
  • the fragments include TGF ⁇ RII, IL10 receptor and macrophage colony-stimulating factor 1 receptor (CSF-1R).
  • the antibody is an immunoglobulin, scFv (single chain Fv, also known as single chain variable fragment), Fab, Fab 'or F (ab') 2 .
  • the constant region of the immunoglobulin is a human antibody constant region
  • the human antibody constant region includes a human antibody light chain constant region and a human antibody heavy chain constant region.
  • the human antibody light chain constant region is preferably a ⁇ chain or Lambda chain
  • the human antibody heavy chain constant region is preferably hIgG1, hIgG2 or hIgG4.
  • the bispecific antibody of the present invention has a structure similar to a normal IgG. Specifically, a light chain capable of targeting two targets and / or Or the protein functional region of the heavy chain variable region, two protein functional regions share the same heavy chain Fc region.
  • one target antibody molecule is linked to another target intact antibody in the form of one or more scFvs, or a complete cytokine or a fragment thereof, or a complete cytokine receptor or a fragment thereof.
  • Light or heavy chain at one end.
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is one or more scFv, a cytokine or a fragment thereof, or A cytokine receptor or a fragment thereof; or the second protein functional region is an immunoglobulin and the first protein functional region is one or more scFvs; wherein the scFv includes a heavy chain variable region and A light chain variable region, wherein the heavy chain variable region and the light chain variable region are connected by a linker, and the linker is preferably (Gly-Gly-Gly-Gly-Ser) w [hereinafter referred to as (G 4 S) w ];
  • the scFv, the cytokine or a fragment thereof, or the cytokine receptor or a fragment thereof is connected to the immunoglobulin through a linker, and the linker is selected from peptides or (G 4 S) w ; said w is preferably an immunoglobulin
  • a light chain-containing sequence means that the sequence may include a scFv linked to the light chain sequence in addition to the light chain sequence
  • a heavy chain-containing sequence means that the sequence may include a heavy chain sequence in addition to the heavy chain sequence.
  • Chain sequence linked scFv For the convenience of expression, the scFvs in the table may be sequences of cytokines and cytokine receptors or fragments thereof in addition to the meanings understood by those skilled in the art unless otherwise specified herein.
  • T1 represents the first protein functional region targeting target 1
  • T2 represents the second protein functional region targeting target 2.
  • T1 (scFv) represents the scFv sequence against the target 1 antibody
  • T2 (scFv) represents the scFv sequence against the target 2 antibody.
  • (scFv) n1 , (scFv) n2 , (scFv) n3 , (scFv) n4 n1, n2, n3, n4 are natural numbers, which can be 0, 1, 2, 3, etc., in specific embodiments of the present invention Among them, at least one of n1, n2, n3, and n4 is 1, and the rest are 0.
  • VL represents the light chain variable region sequence of the antibody against target 1 or 2;
  • VH represents the heavy chain variable region sequence of the antibody against target 1 or 2.
  • Lc which represents the constant region sequence of the light chain ( ⁇ or ⁇ ), preferably a human light chain constant region sequence
  • Hc which represents the heavy chain, includes a constant region sequence of IgG1, IgG2, IgG3, IgG4, etc., preferably a human heavy chain constant region sequence
  • the amino acid K at the C-terminus of the heavy chain may be mutated, preferably A.
  • T1 is immunoglobulin and T2 is scFv; in scheme 2, T2 is immunoglobulin and T1 is scFv; scFv targets the same target; in schemes 3 and 4, the scFv at both ends Targeted at two different targets.
  • the scFv in Table 01 is a conventional scFv, that is, it is not a sequence of a cytokine and a cytokine receptor or a fragment thereof, the scFv is a light chain variable region-linker-heavy chain variable region, which The N-terminus of the light chain variable region or the C-terminus of the heavy chain variable region is correspondingly connected to the C-terminus or the N-terminus of the immunoglobulin light and / or heavy chain through a linker; or the scFv for the heavy chain may be Variable region-linker-light chain variable region, the N-terminus of the heavy chain variable region or the C-terminus of the light chain variable region is correspondingly connected to the C of the immunoglobulin light and / or heavy chain by a linker End or N-terminus.
  • the connection mode is that the C-terminus of the light chain variable region is connected to a linker, and the linker is further connected to the heavy chain.
  • the N-terminus of the variable region is linked, thereby exposing the N-terminus of the scFv light chain variable region and the C-terminus of the heavy chain variable region so that it can be connected to the light and / or heavy chain of the immunoglobulin through a linker.
  • the C-terminus of the heavy chain variable region of scFv when it is linked to the light chain of an immunoglobulin, in some specific embodiments, is preferably connected to the N-terminus of the immunoglobulin light chain through a linker; When it is linked to the heavy chain of an immunoglobulin, the N-terminus of the light chain variable region of the scFv is preferably linked to the C-terminus of the immunoglobulin heavy chain in some specific embodiments.
  • the connection mode is that the N-terminus of the light chain variable region is connected to a linker, and the linker is further connected to the heavy chain variable region.
  • the C-terminus is connected to expose the C-terminus of the scFv light chain variable region and the N-terminus of the heavy chain variable region so that it can be connected to the light and / or heavy chain of the immunoglobulin through a linker.
  • the C-terminus of the light chain variable region of the scFv when it is connected to the light chain of the immunoglobulin, in some specific embodiments, it is preferable to use the C-terminus of the light chain variable region of the scFv to be connected to the N-terminus of the light chain of the immunoglobulin;
  • the N-terminus of the heavy chain variable region of scFv is preferably connected to the C-terminus of the immunoglobulin heavy chain.
  • the linker is (G 4 S) 3 , and / or the number of the scFv is two, and the scFv is symmetrically connected to the immunoglobulin light chain and / or heavy chain.
  • the bispecific antibody is selected from any one of the following:
  • the functional region of the first protein is an immunoglobulin, and the immunoglobulin includes an amino acid sequence of a light chain as shown in SEQ ID NO: 38 and a heavy chain as shown in SEQ ID NO: 39; the second The protein functional region is scFv;
  • the C-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the N-termini of the two heavy chains of the immunoglobulin through a linker; and the light chain variable region of the scFv is the light chain of Atezo.
  • the C-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the N-termini of the two heavy chain variable regions of the immunoglobulin through a linker; and the light chain variable regions of the scFv are Hu5F9 A light chain variable region, wherein the scFv heavy chain variable region is a light chain variable region of Hu5F9; or,
  • the N-termini of the heavy chain variable regions of the two scFvs are symmetrically connected to the C-termini of the two heavy chains of the immunoglobulin through a linker; and the light chain variable region of the scFv is a light chain of AMG420.
  • Variable region, the heavy chain variable region of the scFv is the heavy chain variable region of AMG420.
  • the bispecific antibody may further include the following structure, the first protein functional region is an immunoglobulin, and the immunoglobulin includes a light chain as shown in SEQ ID NO: 38 or SEQ ID NO: 42, The heavy chain has the amino acid sequence shown in SEQ ID NO: 39; the second protein functional region is scFv:
  • the light chain variable region of the scFv is the light chain variable region of the iMab (ie the WO2018075857_4 sequence), and the heavy chain variable region is the heavy chain variable region of the iMab (the WO2018075857_3 sequence); or, the light chain of the scFv
  • the sequence of the variable region is the light chain variable region of Tim3 (shown as SEQ ID NO: 27 in the patent application CN201710348699.4), and the heavy chain variable region of the scFv is the heavy chain variable region of Tim3 (such as SEQ ID: NO: 36 in the patent application CN201710348699.4); or, the sequence of the light chain variable region of the scFv is the light chain variable region of BlincytoCD3, and the heavy chain variable region of the scFv is BlincytoCD3 A heavy chain variable region; or, the sequence of the light chain variable region of the scFv is a light chain variable region of Pem, and the heavy
  • the first protein functional region is scFv
  • the second protein functional region is immunoglobulin
  • the C-termini of the heavy chain variable regions of the two scFv are symmetrically connected to the immunoglobulin via a linker.
  • N-termini of two heavy chains the sequence of the light chain variable region of the scFv is shown in SEQ ID NO: 29, and the sequence of the heavy chain variable region of the scFv is shown in SEQ ID NO: 34;
  • the immunoglobulin includes an amino acid sequence of a light chain variable region of Nivo, a light chain constant region of a kappa chain, a heavy chain variable region of Nivo, and a heavy chain constant region of hIgG4; or,
  • the immunoglobulin includes an amino acid sequence of a light chain variable region of Pem, a light chain constant region of ⁇ chain, a heavy chain variable region of Pem, and a heavy chain constant region of hIgG4; or,
  • the immunoglobulin includes the amino acid sequence of the light chain variable region of Atezo, the light chain constant region is a ⁇ chain, the heavy chain variable region of Atezo, and the heavy chain constant region is hIgG1.
  • the bispecific antibody may further include the following immunoglobulins: the first protein functional region is scFv, the second protein functional region is immunoglobulin, the light chain constant region is a ⁇ chain, and the heavy chain
  • the constant region is the amino acid sequence of hIgG1; wherein the light chain variable region of the immunoglobulin is the light chain variable region of iMab (ie, WO2018075857_4 sequence), and the heavy chain variable region is the heavy chain variable region of iMab (ie WO2018075857_3 sequence); or the light chain variable region of the immunoglobulin is the light chain variable region of Tim3 (as shown in SEQ ID NO: 27 in the patent application CN201710348699.4), the weight of the immunoglobulin
  • the chain variable region is the heavy chain variable region of Tim3 (as shown in SEQ ID NO: 36 in the patent application CN201710348699.4); or the light chain variable region of the immunoglobulin is the light chain variable of Hu5
  • T1 (scFv) and T2 (scFv) in Table 01 is a cytokine or a fragment thereof, or a cytokine receptor or a fragment thereof
  • its structure is a Linker-cytokine receptor and its Variant sequence, or cytokine and its variant sequence-Linker.
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is a cytokine or a fragment thereof, or a cytokine receptor or Its fragment
  • the number of the cytokine or its fragment, or the cytokine receptor or its fragment is preferably two or four; it is symmetrical to the two light chains of the immunoglobulin via a linker and / Or the C-terminus and / or N-terminus of two heavy chains, the linker is preferably (G 4 S) 3 ;
  • the immunoglobulin includes a light chain as shown in SEQ ID NO: 38; a heavy chain as shown in SEQ ID ID: 39; wherein,
  • the cytokine or its fragment, or the cytokine receptor or its fragment is TGF ⁇ RII, its sequence is shown in SEQ ID NO: 1, and the number is two; the TGF ⁇ RII is symmetrically connected to the immunoglobulin C-terminus of two heavy chains of which the amino acid at the C-terminus is mutated from K to A; or,
  • the cytokine or its fragment, or the cytokine receptor or its fragment is IL10, its sequence is shown in SEQ ID NO: 2 and the number is two; the IL10 is symmetrically connected to the immunoglobulin The C-terminus of the two heavy chains, the C-terminal amino acid was mutated from K to A.
  • a bispecific antibody as described above includes the following light chain amino acid sequences and heavy chain-containing amino acid sequences:
  • a light chain amino acid sequence as shown in SEQ ID NO: 53, a heavy chain-containing amino acid sequence as shown in SEQ ID NO: 54; or, a light chain amino acid sequence as shown in SEQ ID NO: 55, as shown in SEQ ID NO : 56 contains the heavy chain amino acid sequence; or, the light chain amino acid sequence shown in SEQ ID NO: 38, as shown in SEQ ID NO: 57 contains the heavy chain amino acid sequence; or, as SEQ ID NO : 38 light chain amino acid sequence, as shown in SEQ ID NO: 58 with heavy chain amino acid sequence; or, light chain amino acid sequence as shown in SEQ ID NO: 38, as shown in SEQ ID NO: 59
  • the bispecific antibody according to the present invention is a DVD-Ig (Dual-variable domain Ig) bispecific antibody, and its structure is that the N-terminus of the light and heavy chains of the normal antibody is connected to the VL and VH, achieves dual function through the binding of two antibody variable regions to two targets.
  • DVD-Ig DVD-variable domain Ig
  • T1 and T2 represent target 1 and target 2 respectively.
  • a light chain-containing sequence means that the sequence includes another light chain variable region sequence in addition to the normally complete light chain sequence.
  • a heavy chain-containing sequence means that the sequence includes another heavy chain variable region sequence in addition to the normally complete heavy chain sequence.
  • the light chain variable region and the complete light chain, and the heavy chain variable region and the complete heavy chain are linked by a linker.
  • the bispecific antibody is a DVD-Ig bispecific antibody.
  • the second protein functional region includes a complete light and heavy chain of a normal antibody, and the first protein functional region includes a light chain variable region and a heavy chain variable region; or, the first protein function
  • the region comprises the intact light and heavy chains of a normal antibody, and the second protein functional region comprises a light chain variable region and a heavy chain variable region.
  • the linker is preferably (G 4 S) w , said w is preferably an integer between 0 and 10, more preferably 1, 2, 3 or 4.
  • the bispecific antibody consists of a light chain-containing sequence and a heavy chain-containing sequence.
  • the bispecific antibody is selected from the following combinations: the light chain-containing sequence is Ab10VL- (G 4 S) 3 -NivoVL-Lc ( ⁇ chain), and the heavy chain-containing sequence is Ab10VH- (G 4 S) 3- NivoVH-Hc (hIgG4); or, the sequence containing the light chain is AtezoVL- (G 4 S) 3 -Ab10VL-Lc ( ⁇ chain), and the sequence containing the heavy chain is AtezoVH- (G 4 S) 3 -Ab10VH-Hc (hIgG1); or, the sequence containing the light chain is Hu5F9VL- (G 4 S) 3 -Ab10VL-Lc ( ⁇ chain), and the sequence containing the heavy chain is Hu5F9VH- (G 4 S) 3 -Ab10VH-Hc (hIgG1) .
  • the bispecific antibody according to the present invention includes a first protein functional region and a second protein functional region.
  • One of the protein functional regions is an immunoglobulin and the other protein functional region is Fab 'or F (ab' ) 2 .
  • the technical solution of the third aspect of the present invention is to provide an antibody drug conjugate (ADC), the structure of which is shown in the following formula I:
  • D is a small molecule drug with cytotoxicity
  • L 1 and L 2 are linkers connecting the drug and the antibody, respectively
  • n is 0 or 1
  • y represents the average number of D coupled to Ab, and 0 ⁇ y ⁇ 10, preferably 2 ⁇ y ⁇ 7; more preferably 3 ⁇ y ⁇ 6; most preferably 4.4 or 4.8;
  • the Ab is an antibody that targets CLDN18.2 according to the first aspect of the present invention, or a bispecific antibody according to the second aspect of the present invention.
  • the amount of drug carried by the antibody is determined, and the antibody drug conjugate can be a single product (not a mixture). If the sites are randomly coupled, the number of drug conjugate molecules carried on different antibodies is actually different. Therefore, the drug conjugates of the antibodies of the present invention are actually a mixture, and y in the general formula reflects The average value of antibody conjugates carried in the mixture is shown. After numerical calculation, it usually appears as a non-integer positive number, such as 4.4 or 4.8.
  • the average number of drug moieties per antibody in the ADC formulation from the coupling reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC.
  • the quantitative distribution of ADCs can also be determined.
  • isolating, purifying, and characterizing a homogeneous ADC with a certain value of y from ADCs with other drug loading can be achieved by methods such as reverse-phase HPLC or electrophoresis.
  • the linker L 2 is present (that is, n is 1), the number of L 2 , L 1 and D is the same.
  • the drug conjugate of the antibody is Ab- [L 2 -L 1 -D ] y ; if L 2 does not exist (that is, n is 0), that is, if only one linker is included, the number of L 1 and D is the same.
  • the drug conjugate of the antibody should actually be Ab- [L 1 – D] y .
  • the drug conjugate of the antibody represented by the general formula of the present invention is an ideal drug conjugate of the antibody, that is, the general formula only considers the number of L 2 (if present) to which the linker L 1 is connected, The number of L 1 to which D is connected.
  • the linker L 1 is connected to the linker L 1 to which D is connected.
  • those skilled in the art know that in the actual drug conjugate of the antibody, there should be a linker that is not connected to D, so the average number of drug molecules actually coupled to the antibody should be ⁇ y, that is, y is coupled to the antibody. The theoretical maximum of the drug.
  • an antibody drug conjugate is provided, and the small molecule drug is a cytotoxic agent selected from the group consisting of a toxin, a chemotherapeutic agent, an antibiotic, a radioisotope, and a nucleolytic enzyme.
  • the small molecule drug is selected from the group consisting of monomethyl auristatin, maytansine alkaloids, camptothecin alkaloids, calicheamicin, doxorubicin Star (adriamycin), duocarmycin, or a combination thereof.
  • the monomethyl auristatin is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)
  • the maytansinoids are N 2 '-deacetyl- N 2 '-(3-mercapto-1-oxopropyl) -maytansin (DM1), N 2 ' -deacetyl-N 2 '-(4-mercapto-1-oxopentyl) -mei maytansine (DM3), and N 2 '- deacetyl -N 2' - (4- mercapto-4-methyl-1-oxopentyl) - maytansine (DM4).
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • an antibody drug conjugate is provided, and the L 1 is selected from a cleavable linker, a non-cleavable linker, a hydrophilic linker, a pre-charged linker, and a dicarboxylic acid-based linker.
  • the linker is selected from N-succinimidyl 4- (2-pyridyldithio) valerate (SPP), N-succinimidyl (4-iodoacetyl) aminobenzyl Acid ester (SIAB), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), 6-maleimidohexanoyl (MC), maleyl Iminopropionyl (MP), valine-citrulline (VC), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and MC-VC-PAB.
  • SPP N-succinimidyl 4- (2-pyridyldithio) valerate
  • SIAB N-succinimidyl (4-iodoacetyl) aminobenzyl Acid ester
  • an antibody drug conjugate is provided, wherein L 2 is a compound represented by the following formula II:
  • X 1 is selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • X 2 is selected from alkyl, cycloalkyl, and heterocyclyl; m is 0-5; S is a sulfur atom;
  • X 1 is a hydrogen atom
  • X 2 is an alkyl group
  • m is 1
  • the compound represented by Formula II is S- (3-carbonylpropyl) thioacetate.
  • an antibody drug conjugate is provided, the small molecule drug is DM1, the linker L 1 is SMCC, and n is 0, thereby forming an antibody drug represented by the following formula III Conjugate:
  • the small molecule drug is MMAF
  • the linker L 1 is MC-VC-PAB
  • L 2 is thioacetic acid S- (3-carbonylpropyl) ester
  • n is 1, thereby forming the following formula IV Shown antibody drug conjugates:
  • a drug conjugate of an antibody as described above is provided.
  • the amount of a cytotoxic agent or small molecule drug conjugated to a single antibody molecule linked to a linker is ( (Or load, or DAR) is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, but due to the specificity of the ligation reaction, the DAR of the small molecule drug conjugated to the antibody connected to the linker is actually Above are average values between 0 to 10, 1 to 8, 2 to 7, 3 to 6, or 4 to 5.
  • the antibody conjugate of the present invention is actually a mixture of different numbers of linker-drug or linker-only antibodies, so the y value is the average value of the number of drug couplings and the value is an integer or a non-integer.
  • the drug loading of the ADC of the invention ranges from 1 to about 8; about 2 to about 7; about 3 to about 6; about 4 to about 5; about 4.1 to about 4.9; about 4.2 to about 4.8 ; About 4.3 to about 4.7; about 4.4 to about 4.6; about 4.4, 4.6, or about 4.8.
  • an antibody drug conjugate is provided, which is an antibody drug conjugate shown by the following formula V:
  • the Ab10 includes a light chain as shown in SEQ ID NO: 38 and a heavy chain as shown in SEQ ID NO: 39;
  • the Ab6 includes a light chain as shown in SEQ ID NO: 42 and as shown in SEQ ID ID NO: 42 : 39 as shown in the heavy chain.
  • the technical solution of the fourth aspect of the present invention is to provide a method for preparing the antibody-drug conjugate
  • the preparation method includes the following steps:
  • X 1 is selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • X 2 is selected from alkyl, cycloalkyl, and heterocyclyl; m is 0-5; S is a sulfur atom;
  • X 1 is a hydrogen atom
  • X 2 is an alkyl group
  • m is 1, that is, L 2 is a thioacetic acid S- (3-carbonylpropyl) ester
  • step (3) mixing the solution containing intermediate 1 obtained in step (1) with the solution containing intermediate 2 obtained in step (2), and purifying after the reaction to obtain a solution containing an antibody drug conjugate;
  • the preparation method includes the following steps:
  • step (2) mixing the solution containing intermediate 3 obtained in step (1) with the solution containing the drug, and purifying after the reaction to obtain a solution containing the antibody drug conjugate;
  • the reaction temperature is 25 ° C; the reaction time is 2 to 4 hours; and / or, the purification is purified by gel filtration, more It is preferably desalted and purified by Sephadex G25 gel column.
  • the technical solution of the fifth aspect of the present invention is to provide a chimeric antigen receptor (CAR) targeting Claudin 18.2.
  • CAR antibody genes directed against specific antigens are a key choice. Given the complexity of gene expression in the body and various uncontrollable factors, it is very difficult to select a suitable gene for CAR. In addition, many tumor-specific antigens are difficult to find specific molecules suitable for the construction of CAR cells. After the establishment of CAR, active extracellular binding regions often cannot be obtained, which is also a difficult point in developing CAR technology. In addition, although CAR cells have attractive prospects in tumor immunotherapy, their higher risks also need to be considered. For example, due to the low expression of specific antigens recognized by CAR in some normal tissues, CAR cells may cause damage to normal tissues that express the corresponding antigens.
  • the specific effect of targeting Claudin 18.2 antibody specifically binding Claudin 18.2 is not good, and the effect of CAR cells targeting Claudin 18.2 is not good enough, and it is well known to develop both efficacy and safety CAR cells targeting Claudin 18.2 are extremely difficult.
  • the inventors unexpectedly found through experiments that the high antibody binding activity / affinity of the CAR of the present invention means that the CAR of the present invention can better bind to target cells under the same process (same viral titer and transfection efficiency) , Better specificity, reducing side effects caused by non-target cell binding. With the CAR of the present invention, even a small amount of the CAR can be effective, and has extremely high application value.
  • the antigen-binding portion (antibody) of CAR cells against CLDN 18.2 in the present invention is a humanized sequence, which can reduce the risk of immunogenicity caused by CAR treatment.
  • the cytokines, cytokines and their receptor-binding complexes that can effectively kill tumors are introduced at the C-terminus of CAR, and these factors and / or complexes can secrete extracellular and tumor cells Area to achieve better tumor suppression.
  • the CAR includes: (a) an extracellular binding domain scFv that specifically recognizes CLDN18.2; (b) a hinge domain; (c) a transmembrane domain; (d) a co-stimulated intracellular domain; (e) Signaling domain
  • the extracellular binding domain comprises the light chain variable region and the heavy chain variable region of the antibody targeted to CLDN18.2 according to the first aspect of the present invention.
  • the hinge domain is selected from the hinge regions of one or more of the following molecules: CD8 ⁇ , CD28, CD152, PD1, and IgG1 heavy chains;
  • the transmembrane domain is selected from the transmembrane regions of one or more of the following molecules: ⁇ , ⁇ , ⁇ chains of TCR, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154 and PD1;
  • the co-stimulatory intracellular domain is selected from the intracellular region of one or more of the following molecules: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD134, 4-1BB , CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, NKD2C, SLP76, TRIM, FcsRI ⁇ , and MyD88, preferably the CD28 intracellular region and / or the 4-1BB intracellular region; and / or,
  • the signaling domain is selected from the intracellular region of one or more of the following molecules: Ig ⁇ , Ig ⁇ , TCR ⁇ , FcR1 ⁇ , FcR1 ⁇ , CD3 ⁇ , CD3 ⁇ , CD2, CD5, CD22, CD28, CD79a, CD79b , CD278, CD66d and CD3 ⁇ ; CD3 ⁇ intracellular region is preferred.
  • the hinge domain is a CD8 ⁇ hinge region
  • the transmembrane domain is a CD8 ⁇ transmembrane region
  • the co-stimulatory intracellular domain is a CD28 intracellular region and / Or 4-1BB intracellular region
  • the signal transduction domain is a CD3 ⁇ intracellular region (referred to as CD3 ⁇ in the CAR formula of the embodiment);
  • the CD8 ⁇ hinge region is a human CD8 ⁇ hinge region; the CD8 ⁇ transmembrane region is a human CD8 ⁇ transmembrane region; the CD28 intracellular region is a human CD28 intracellular region; and the 4-1BB intracellular region is a human 4-1BB cell Inner region; and / or, the CD3 ⁇ intracellular region is a human CD3 ⁇ intracellular region.
  • the technical solution of the sixth aspect of the present invention is to provide a nucleic acid construct containing a CAR targeted to Claudin 18.2, the nucleic acid construct having the formula car-[(IRES) -f] Structure shown by q , wherein IRES is the internal ribosome entry site sequence shown in the nucleotide sequence of SEQ ID NO: 55; f encodes a functional protein F, and q is a 0 or non-zero natural number; the car code includes CAR in the fifth aspect above.
  • the functional protein F includes:
  • a cytokine or an active fragment thereof preferably IL10 or IL15 or an active fragment thereof; more preferably, the amino acid sequence of the IL10 is as shown in SEQ ID NO: 54;
  • cytokine receptor or an active fragment thereof, preferably IL15R ⁇ or a fragment thereof, or IL15R ⁇ fragment (sushi) or IL15R ⁇ fragment (sushi +); or,
  • a fusion protein of a cytokine receptor or an active fragment thereof and a cytokine preferably IL15R ⁇ or a fragment thereof, or a fusion fragment of IL15R ⁇ (sushi) or IL15R ⁇ (sushi +) and IL15.
  • the structure of the nucleic acid construct is:
  • scFv-human CD8 ⁇ hinge region-human CD8 ⁇ transmembrane region-human 4-1BB intracellular region-human CD3 ⁇ intracellular region preferably, the encoded amino acid sequence is as shown in SEQ ID NO: 3;
  • a preferred one-nucleotide construct is named CAR1a below; or,
  • the above-mentioned construct is a nucleic acid.
  • the four structures of the construct are omitted from the expression of "encoding nucleotide sequence".
  • the scFv in the above structure is actually a "nucleotide sequence encoding scFv”
  • the human CD8 ⁇ hinge region is actually "a nucleotide sequence encoding a human CD8 ⁇ hinge region”
  • IL10 and IL15 may be wild-type IL10 and IL15, or may be IL10 and IL15 mutants or active fragments thereof.
  • the aforementioned nucleic acid construct may further include a CD27T cell memory and survival signaling domain, and / or a self-destructing domain such as a nucleotide sequence encoding an inducible caspase (iCasp) or a simple encoding The nucleotide sequence of herpes virus thymidine kinase (HSV-TK).
  • the self-destructive domain can regulate the antigen recognition signal pathway, minimize the damage of CAR cells to normal tissues, and reduce off-target effects.
  • the suicide gene is activated under the stimulation of a non-toxic prodrug to induce CAR cell apoptosis and terminate the treatment.
  • the technical solution of the seventh aspect of the present invention is to provide an isolated nucleic acid that encodes the antibody targeting CLDN18.2 as described above or the bispecific antibody as described above.
  • the technical solution of the eighth aspect of the present invention is to provide an expression vector containing the isolated nucleic acid as described above, or the nucleic acid construct as described above.
  • the expression vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector and an adeno-associated virus vector. More preferred is a lentiviral vector, whose backbone plasmid may be pBABEpuro.
  • the technical solution of the ninth aspect of the present invention is to provide a genetically modified cell transfected with the nucleic acid construct as described above and the expression vector; preferably, the genetic modification
  • the cells are eukaryotic cells, more preferably isolated human cells; still more preferably immune cells such as T cells, or NK cells such as the NK92 cell line.
  • the technical solution of the tenth aspect of the present invention is to provide a method for preparing a genetically modified cell, the method including the following steps: the nucleic acid construct, the expression vector, or the The virus is transferred into the cell to be modified.
  • the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably an immune cell such as a T cell or an NK cell; and even more preferably an NK92 cell line.
  • the technical solution of the eleventh aspect of the present invention is to provide a pharmaceutical composition, the pharmaceutical composition comprising the antibody targeting CLDN 18.2, the bispecific antibody, The genetically modified cell or the antibody-drug conjugate, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further includes an immune checkpoint antibody.
  • the technical solution of the twelfth aspect of the present invention is: providing an antibody targeting CLDN 18.2, the bispecific antibody, the antibody drug conjugate, Application of the nucleic acid construct, the expression vector, the virus, the genetically modified cell, or the pharmaceutical composition in the preparation of a medicament for treating a tumor; preferably, the tumor is CLDN18 .2 Positive tumor, preferably gastric cancer, esophageal cancer, lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, glioma or leukemia.
  • the present invention uses innovative immunization and screening methods to carry out a variety of innovative designs, and provides a brand-new antibody targeting CLDN18.2, preferably a humanized antibody, which has better specific binding activity to human and mouse CLDN18.2, and Emax Higher affinity (KinExA) (preferably 10pM level); better CDC activity in human blood cells; better inducing CLDN18.2 + cell apoptosis activity, better tumor cell growth inhibition activity, and better animal efficacy, And in vivo drug metabolism (PK) is good, especially longer T1 / 2. It also has better binding activity with murine CLDN18.2.
  • KinExA Emax Higher affinity
  • PK in vivo drug metabolism
  • the present invention provides a bispecific antibody, one of which targets CLDN 18.2 and the other targets CLDN 18.2.
  • a bispecific antibody SBody
  • the obtained bispecific antibody (SBody) preferred molecule retains the binding activity and functional activity of the dual target; and the dual specific design is similar to the IgG structure, and the process is simple and stable. This has brought convenience to the process of preparing antibodies in the later development.
  • the designed target includes immune checkpoint antigens such as PD-1 / PD-L1
  • it can be combined with CLDN18.2, which can realize the combined action of tumor immunity and targeting antibodies in one molecule, generating 2 molecules equivalent Combined effects or better synergistic effects bring more convenient drug development options for the combination of tumor immunotherapy and targeted therapy.
  • the invention is the first antibody drug conjugate (ADC) against CLDN 18.2.
  • ADC first antibody drug conjugate
  • the effect of the antibody molecule is as described above.
  • the ADC molecule obtained by coupling the antibody and cytotoxin not only retains its excellent specific binding activity against human and murine CLDN 18.2, but also has the characteristics of endocytosis and high-efficiency killing of tumor cells. It also carries cytotoxic toxins and can be more specifically Targeting killing tumor cells, specifically inhibiting tumor cell proliferation, and producing unexpectedly better tumor treatment effects.
  • These characteristics make the ADC drug of the present invention and its pharmaceutically acceptable salts, solvent compounds, or combination with other drugs provide more specific and effective, better treatment options, means and methods for tumor patients, especially patients with CLDN 18.2 positive cancer.
  • the antibody drug conjugate / antibody provided by the present invention has better pharmacokinetic performance, a large safety window, and lower toxic and side effects.
  • the invention provides a new CAR molecule targeting Claudin 18.2, which has higher activity and affinity and can better target tumor cells; it does not bind Claudin 18.1, has excellent specificity, and reduces non-targets. Side effects caused by cell binding; CAR molecule's antigen-binding sequence is preferably humanized, which reduces the risk of immunogenicity and has better safety; CAR molecules are used in combination with cytokines or cytokine receptors to achieve better therapeutic effects; containing the present invention CAR molecules have a better immune cell treatment effect. In particular, in the preferred embodiment, the CAR molecules of the present invention have a nearly 100% anti-cancer effect.
  • Figure 1 shows the binding activity (ELISA) of the mouse-derived anti-human CLDN18.2 antibody mab5b and human CLDN18.2 ( Figure 1a) and mouse CLDN18.2 ( Figure 1b) according to the present invention
  • FIG. 2 is a binding activity (ELISA) of humanized antibody (FIG. 2b) and human CLDN18.2.
  • Figure 3 is a CDC activity evaluation of the humanized anti-human CLDN18.2 antibody and the humanized preferred antibody of the present invention
  • FIG. 4 is the activity of humanized anti-human CLDN18.2 antibody and humanized preferred antibody to induce tumor cell apoptosis (FIG. 4a, FIG. 4b);
  • FIG. 5 is an in vivo evaluation of an animal model of a humanized anti-human CLDN18.2 antibody and a humanized preferred antibody in the present invention (FIG. 5a, FIG. 5b);
  • Figure 6 is an analysis of endocytosis activity of the humanized antibodies Ab10 and Ab6 of the present invention.
  • FIG. 7A is a diagram of the structure of LB302 and related detection data, which includes a schematic diagram of the structure (c), and a detection result of binding activity with human CLDN 18.2 (a), and a detection result of binding activity with human PD-1 (b).
  • FIG. 7B is a diagram of the structure of LB301 and related detection data, which includes a schematic diagram of the structure (c), and the detection result of binding activity with human CLDN 18.2 (a), and the detection result of binding activity with human PD-1 (b).
  • FIG. 7C is a diagram of the structure of LB309 and related detection data, which includes a schematic diagram of the structure (c), and the detection result of the binding activity with human CLDN 18.2 (a), and the detection result of the binding activity with human PD-1 (b).
  • FIG. 7D is a diagram of the structure of LB308 and related detection data, which includes a schematic diagram of the structure (c), and the detection result of the binding activity with human CLDN 18.2 (a), and the detection result of the binding activity with human PD-1 (b).
  • Figure 8 shows the structure of LB401 and related test data, where a is the sample stored at -80 ° C for 60 days (60d), stored at 4 ° C for 30 days, stored at 37 ° C for 7 days and 14 days, and human CLDN 18.2 Binding activity test results; b is a schematic diagram of LB401 structure.
  • FIG. 9 shows the in vivo pharmacological effects of CART cells in animals designed for CLDN 18.2, showing tumor volume changes in mice injected with CART (empty vector), CART1a, CART3ab, CART3ab10, and CART4a cells.
  • amino acid three-letter codes and one-letter codes used in the present invention are known to those skilled in the art or described in J. Biol. Chem, 243, p3558 (1968).
  • compositions and methods include the recited elements but not the exclusion of other elements.
  • Consisting essentially of when used to define compositions and methods should mean excluding other elements that have any substantial effect on the combination for which it is intended. For example, a composition consisting essentially of this element, as defined herein, will not exclude trace contaminants from the methods of isolation and purification and pharmaceutically acceptable carriers (eg, phosphate buffered saline, preservatives, etc.).
  • Consisting of shall mean excluding other ingredients than trace elements and substantial method steps for administering the composition disclosed herein. Aspects defined by each of these transitional terms are within the scope of the invention.
  • CLDN18.2 includes isoforms, mammalian (eg, human) CLDN18.2, species homologues of human CLDN18.2, and analogs comprising at least one common epitope with CLDN18.2.
  • the amino acid sequence of CLDN18.2 e.g., human CLDN18.2 is known in the art, as shown in the NCBI database.
  • CLDN18.1 includes isoforms, mammalian (eg, human) CLDN18.1, species homologues of human CLDN18.1, and analogs comprising at least one common epitope to CLDN18.1.
  • the amino acid sequence of CLDN18.1 (eg, human CLDN18.1) is known in the art, as shown in the NCBI database.
  • the "CLDN18.2 antibody”, “anti-CLDN18.2 antibody”, “CLDN18.2 antibody molecule” and “anti-CLDN18.2 antibody molecule” described in the present invention can be used interchangeably.
  • the term “epitope” refers to the portion of an antigen (eg, human CLDN 18.2) that specifically interacts with an antibody molecule.
  • the term “competing” refers in the present invention to the ability of an antibody molecule to interfere with the binding of an anti-CLDN18.2 antibody molecule to a target (eg, human CLDN18.2). Interference in binding can be direct or indirect (eg, through allosteric regulation of antibody molecules or targets).
  • a competitive binding assay eg, a FACS assay, an ELISA, or a BIACORE assay
  • antibody includes an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by an interchain disulfide bond.
  • immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , Alpha and epsilon chains.
  • Igs of the same type can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by different constant regions.
  • Each of the five types of Ig may have a ⁇ chain or a ⁇ chain.
  • variable region of the antibody light chain according to the present invention may further include a constant region of a light chain, and the constant region of the light chain includes human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain variable region of the present invention may further include a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, 2, 3, 4 or a variant thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (C region).
  • the variable region includes three hypervariable regions (HVR) and four backbone regions (FR) whose sequences are relatively conserved. Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2, and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with high denaturation [referred to as complementarity determining regions (CDRs)], interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminal to the carboxyl terminal.
  • the variable regions (VH and VL) corresponding to each heavy / light chain form an antibody binding site, respectively.
  • the heavy chain may also contain more than 3 CDRs, such as 6, 9, or 12.
  • the heavy chain may be a ScFv whose N-terminus of the heavy chain of an IgG antibody is connected to another antibody, in which case the heavy chain contains 9 CDRs.
  • the CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment according to the present invention conform to the known Kabat, Contact, CCG, AbM, and Chothia numbering rules in number and position.
  • Kabat numbering rules follow the definition of Kabat EA.Et., Sequences of Proteins of Immunological Interest [National Institute of Health, Bethesda, Md. (1987 and 1991)]
  • Chothia numbering rules follow Chothia & Lesk 1987) Mol. Biol. 196: 901-917; definition of Chothia et al. (1989) Nature 342: 877-883.
  • the boundaries of a given CDR can vary depending on the protocol used for identification.
  • the definition rules of the invention and the CDR sequences defined by the antibodies are shown in Tables 3-8.
  • the Kabat scheme is based on structural alignment
  • the Chothia scheme is based on structural information. Numbering for Kabat and Chothia protocols is based on the most commonly used antibody region sequence lengths, and inserts are adapted by insertion of letters (eg, "30a") and deletions are present in some antibodies. Both schemes place certain inserts and deletions ("inserts”) at different locations resulting in differential numbering.
  • the Contact scheme is based on an analysis of the crystalline structure of the complex and is similar in many respects to the Chothia numbering scheme.
  • CDR complementarity determining region
  • individual CDRs of the antibody or region thereof e.g., “CDR-H1, CDR- H2
  • CDR-H1, CDR- H2 complementary decision regions as defined by any of the aforementioned known schemes as described herein.
  • schemes for identifying one or more specific CDRs are specified, for example, as by Kabat, Chothia Or Contact methods to define a CDR.
  • the specific amino acid sequence of a given CDR are specified, for example, as by Kabat, Chothia Or Contact methods to define a CDR.
  • mouse antibody in the present invention is a monoclonal antibody to human CLDN 18.2 prepared according to the knowledge and skill in the art. Test subjects are injected with CLDN18.2 antigen during preparation, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.
  • the mouse-derived CLDN18.2 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived kappa, lambda chain or a variant thereof, or further comprise a mouse-derived IgG1 , IgG2, IgG3 or IgG4, or variants thereof, heavy chain constant regions.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo).
  • the term “human antibody” does not include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human backbone sequence (ie, a "humanized antibody”) .
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response response induced by the murine antibody.
  • To establish a chimeric antibody select a hybridoma that secretes a mouse monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell. Then, clone the constant region gene of the human antibody obtained as needed to convert the mouse variable region gene.
  • the chimeric gene is linked to the human constant region gene and inserted into the vector. Finally, the chimeric antibody molecule is expressed in eukaryotic cells, industrial systems or prokaryotic industrial systems.
  • the antibody light chain variable region of the CLDN18.2 chimeric antibody further comprises a light chain FR region of murine ⁇ , ⁇ type or a variant thereof.
  • the antibody heavy chain variable region of the CLDN18.2 chimeric antibody further comprises a heavy chain RF region of mouse-derived IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human IgG1 or IgG4 heavy chain constant region, or the ADCC (antibody-dependentcell) may be changed after amino acid mutation -mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity), CDC (complement dependent cytotoxicity, CDC, complement dependent cytotoxicity) activity of IgG1. Modification of the Fc segment on the IgG can reduce or eliminate the ADCC and CDC effect functions of the antibody.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody, such as mutations selected from the group consisting of N297A, L234A, L235A; IgG2 / 4 chimera, F235E, or 234A / E235A, F243L, or S239D / A330L / I332E.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the framework of a human antibody variable region.
  • the CDRs of the CLDN18.2 antibody according to the present invention are antibodies generated by transplanting the CDR sequences defined by CCG, Kabat, AbM, Chothia or Contact into human antibody variable region frameworks.
  • the CDRs of the CLDN18.2 antibody according to the present invention preferably at positions 0-5 in the light chain CDR1, mutate the amino acids at the corresponding positions of the CDRs of the adult antibody. These can overcome the strong antibody-variable antibody response induced by the chimeric antibody because it carries a large amount of mouse protein components.
  • Human FR germline sequences can be obtained from the websites of ImMunoGeneTics (IMGT) www.imgt.org and www.vbase2.org.
  • the term "specific binding" with respect to an antibody means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds an antigen from one species can also bind the antigen from one or more species.
  • this cross-species cross-reactivity does not in itself alter the classification of antibodies based on specificity.
  • an antibody that specifically binds an antigen can also bind to different allelic forms of the antigen.
  • this cross-reactivity itself does not change the classification of antibodies according to specificity.
  • the terms “specifically bind” or “specifically bind” can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that the interaction depends on a particular structure on the chemical substance (e.g., The presence of an antigenic determinant or epitope); for example, antibodies generally recognize and bind to specific protein structures rather than proteins. If the antibody is specific for epitope "A”, the presence of a molecule (or free, unlabeled A) containing epitope A will reduce binding to the antibody in a reaction containing labeled "A” and the antibody The amount of labeled A.
  • the CDR sequence of the CLDN18.2 humanized antibody mouse is selected from SEQ ID NOs: 11-28.
  • the human antibody variable region framework is designed and selected, wherein the light chain FR region sequence on the antibody light chain variable region is derived from the combined sequence of the human germline light chain IGKV4-1 * 01 (F) and hJK2.1 SEQ ID NOs: 29-33, comprising FR1, FR2, FR3 regions of human germline light chain IGKV4-1 * 01 (F) and FR4 region of hJK2.1; wherein the heavy chain on the variable region of the heavy chain of the antibody FR region sequence, derived from the combined sequence of human germline heavy chain IGHV1-69 * 01 (F) and hJH4.1 SEQ ID NO: 34-37, contains FR1 of human germline heavy chain IGHV1-69 * 01 (F) , FR2, FR3 and FR4 of hJH4.1.
  • the human antibody variable region may be subjected to minimal back mutation to maintain the activity.
  • the humanized antibody variable region has a back mutation of 0, that is, a fully humanized antibody.
  • deamidation refers to the removal of an amino group at a site or at a site on a molecule.
  • “Deamidation sensitive sites” refers to molecules and certain sites of molecules that are easier and more prone to deamidation.
  • antigen-binding fragment refers to an antigen-binding fragment and an antibody analogue of an antibody, which typically includes at least a portion of a parental antibody's antigen-binding region or variable region (eg, one or more CDRs).
  • the antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when activity is expressed on a molar basis, antibody fragments retain at least 10% of the maternal binding activity. Preferably, the antibody fragment retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more of the binding affinity of the parent antibody for the target.
  • antigen-binding fragments include, but are not limited to, Fab, Fab ', F (ab') 2 , Fv fragments, linear antibodies, single-chain antibodies, Nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.
  • a "Fab fragment” consists of a light chain and a heavy chain of CH1 and a variable region.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.
  • a "Fab 'fragment” contains a light chain and a portion of a heavy chain containing a region between the VH and CH1 domains and between the CH1 and CH2 domains, so that it can be between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F (ab ') 2 molecules.
  • the "F (ab ') 2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains.
  • the F (ab ') 2 fragment therefore consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the term "Fv” means an antibody fragment consisting of the VL and VH domains of one arm of an antibody, but lacking a constant region.
  • the antigen-binding fragment of an antibody is a single-chain binding fragment (e.g., scFv) in which the VL and VH domains form a monovalent molecule by pairing them to enable the production of a linker for a single polypeptide chain [see, eg, Bird et al Human, Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)].
  • scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linker consists of repeated amino acid sequences of G 4 S, or a variant thereof.
  • a linker having an amino acid sequence (G 4 S) 4 or (G 4 S) 3 may be used, but a variant thereof may also be used.
  • multispecific antibody is used in its broadest sense and encompasses antibodies with multi-epitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; has two or more Antibodies for VL and VH regions, each VH-VL unit binds to a different target or different epitopes of the same target; antibodies with two or more single variable regions, each single variable region and Different targets or different epitopes of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies, covalently or non-covalently linked antibody fragments Wait.
  • Antibody molecules include bispecific antibodies (diabody) and single chain molecules and antigen-binding fragments of the antibody (eg, Fab, F (ab ') 2 , scFv, and Fv).
  • An antibody molecule contains or consists of a heavy chain and a light chain (referred to as a half antibody in the present invention).
  • Antibodies and antibody fragments can be from any antibody class, including but not limited to IgG, IgA, IgM, IgD, and IgE and from any antibody subclass (e.g., IgG1, IgG2, IgG3, and IgG4).
  • Antibody molecules can be made monoclonal or polyclonal.
  • the antibody may also be a human antibody, a humanized antibody, a CDR-grafted antibody, or an antibody produced in vitro.
  • the antibody may have, for example, a heavy chain constant region selected from IgG1, IgG2, IgG3, or IgG4.
  • the antibody may also have, for example, a light chain selected from the kappa or lambda type.
  • the antibodies disclosed in the present invention may also be single-domain antibodies.
  • Single domain antibodies can include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies.
  • the single domain antibody can be any antibody of the prior art, or any single domain antibody in the future.
  • Single domain antibodies can be derived from any species, including but not limited to mouse, human, camel, alpaca, goat, rabbit, and cattle. According to some aspects, a single domain antibody is a naturally occurring single domain antibody called a heavy chain antibody lacking a light chain.
  • variable domain derived from a heavy chain antibody that naturally lacks a light chain is referred to herein as a VHH or Nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin.
  • VHH molecules can be derived from antibodies produced in Camelidae species, such as camels, alpacas, dromedaries, llamas, and primates. Species other than camels can produce heavy chain antibodies that naturally lack a light chain, and such VHHs are also considered.
  • the VH region and the VL region can be further divided into hypervariable regions, called “complementarity determining regions" (CDR), with a more conservative region interposed therebetween, called “framework regions” (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • the antibodies of the invention include monoclonal antibodies.
  • the monoclonal antibody or mAb or Ab in the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic, or phage cloned cell line.
  • the host cell of the vector of the present invention may be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell.
  • Suitable eukaryotic cells include, but are not limited to, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, 293T, 293E, BHK cells, and suitable insect cells include, but are not limited to, Sf9 cells.
  • Monoclonal antibodies or antigen-binding fragments can be recombined using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies. Methods of producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the Cold Spring Harbor's Guide to Antibody Experiment Techniques. Antigen-binding fragments can also be prepared by conventional methods.
  • chimeric antigen receptor or "CAR” as used herein refers to: contains an extracellular domain (extracellular binding domain), a hinge domain, a transmembrane domain (transmembrane region), and a cytoplasm capable of binding an antigen.
  • the signal is transmitted to the polypeptide of the domain (ie, the intracellular signal domain).
  • the hinge domain can be considered as part of providing flexibility to the extracellular antigen-binding region.
  • Intracellular signal domain refers to a protein that transmits information into the cell via a defined signaling pathway to regulate cell activity by generating a second messenger, or a protein that functions as an effector by corresponding to such a messenger, producing a CAR that can promote CAR Signals of immune effector function in cells (eg, CART cells).
  • the intracellular signal domain includes a signaling domain and may also include a co-stimulatory intracellular domain derived from a co-stimulatory molecule.
  • signaling domain refers to the part of the CAR that transduces effector function signals and directs cells to perform their specialized functions.
  • Examples of signaling domains include, but are not limited to, the zeta chain of a T cell receptor complex or any homologue thereof.
  • CD3 ⁇ is defined herein as a protein provided by GenBanK accession number BAG36664.1, or an equivalent residue from a non-human species such as a mouse, rodent, monkey, ape, and the like.
  • CD3 ⁇ intracellular region is defined as an amino acid residue from the cytoplasmic domain of the zeta chain, which is sufficient to functionally transmit the initial signal required for T cell activation.
  • the intracellular region of CD3 ⁇ contains residues 52 to 164 of GenBank accession number BAG36664.1, its functional homologues-equivalent residues from non-human species such as mice, rodents, monkeys, apes, and the like.
  • CD3 ⁇ signaling domain or “CD3 ⁇ intracellular region” refers to a specific protein fragment associated with that name and has at least 80% of the amino acid sequence of the CD3 ⁇ intracellular region shown herein, or Alternatively any other molecule having a similar biological function that is at least 90% identical, preferably at least about 95%, more preferably at least about 97%, more preferably at least about 98%, most preferably at least about 99% identical.
  • co-stimulatory intracellular domain refers to the intracellular region of a co-stimulatory molecule, an associated binding partner on a T cell, which specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of immune cells, such as But not limited to proliferation.
  • a co-stimulatory molecule is a non-antigen receptor cell surface molecule or its ligand required for an effective immune response.
  • Co-stimulatory molecules include, but are not limited to, intracellular regions of molecules such as MHCI-like molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18) And 4-1BB (CD137).
  • molecules such as MHCI-like molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18) And 4-1BB (CD137).
  • 4-1BB herein refers to a member of the TNFR superfamily, which has the amino acid sequence of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc .;
  • the "4-1BB” co-stimulatory intracellular domain is defined as the amino acid sequence 214-255 of GenBank Accession No.
  • AAA62478.2 or an equivalent residue from a non-classified species such as mouse, rodent, monkey, ape, etc., or A sequence having at least 80%, or alternatively at least 90% amino acid sequence identity, preferably 95% sequence identity, more preferably at least 97, 98, or 99% sequence identity to a 4-1BB co-stimulatory domain sequence shown herein Any other molecule with similar biological functions.
  • CD28 costimulatory domain is a human CD28 costimulatory domain or refers to a specific protein fragment associated with the name, and the human CD28 costimulatory domain sequence has at least 80%, or alternatively Any other molecule with similar biological function that has at least 90% amino acid sequence identity, preferably 95% sequence identity, and more preferably at least 97, 98, or 99% sequence identity.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, a costimulatory domain, and a signaling domain.
  • the CAR comprises a chimeric fusion protein comprising an extracellular binding domain, a transmembrane domain, a costimulatory domain, and a signaling domain that recognize extracellular antigens.
  • the CAR comprises a chimeric fusion protein comprising an extracellular binding domain, a costimulatory domain, and a signaling domain, the costimulatory domain comprising at least two derived from one or more costimulatory molecules Functional signaling domain.
  • the CAR includes an optional leader sequence (or signal peptide) at the N-terminus of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the N-terminus of the antigen recognition domain (eg, scFv) during the processing and localization of the CAR to the cell membrane of the CAR. under.
  • Homology refers to sequence similarity between two polynucleotide sequences or between two polypeptides.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100. For example, when the sequences are optimally aligned, if 10 positions in the two sequences have 6 matches or are homologous, then the two sequences are 60% homologous.
  • Optimization refers to a mutation that maintains or improves the binding of the antibody to an antigen, and in the present invention refers to a mutation that maintains, maintains, or improves the binding to CLDN 18.2.
  • polypeptide if single chain
  • protein if single chain
  • nucleic acid nucleic acid sequence
  • nucleotide sequence nucleotide sequence
  • polynucleotide sequence nucleotide sequence
  • polynucleotide sequence nucleotide sequence
  • amino acid modification includes amino acid substitutions, additions and / or deletions.
  • amino acid substitutions and “conservative amino acid substitutions” are those in which the amino acid residue is replaced with another amino acid residue and the amino acid residue has a similar side chain. Replacement.
  • IL10 interleukin 10
  • IL15 interleukin 15
  • IL-15 interleukin-15
  • Appropriate amino acid modifications can be easily carried out and ensure that the biological activity of the resulting molecule is not altered. These techniques have taught those skilled in the art that, in general, changing a single amino acid in a non-essential region of a polypeptide will not substantially alter biological activity. Both the active fragment of IL10 or IL15 can be used in the present invention.
  • the meaning of the biologically active fragment means that as a polypeptide, as a part of the full-length polypeptide, it can still maintain all or part of the function of the full-length polypeptide.
  • the biologically active fragment retains at least 50% of the activity of the full-length polypeptide.
  • the active fragment is capable of retaining 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the activity of the full-length polypeptide.
  • modified or improved polypeptides can also be used in the present invention. For example, they can be modified or promoted to promote their half-life, effectiveness, metabolism, and / or the effectiveness of the polypeptide. Improved peptide. That is, any variation that does not affect the biological activity of the polypeptide can be used in the present invention.
  • IL15 binds to the IL15 receptor to exert its biological function.
  • the IL15 receptor has three subunits, namely the IL15 receptor ⁇ , IL15R ⁇ (CD122) and ⁇ (also known as CD132).
  • the extracellular region of IL15R ⁇ is the part that binds to IL15, and the region of which the sushi domain binds to IL15 can exert the biological function of IL15.
  • “sushi +" means that in addition to the sushi fragment, other polypeptide fragments are also included.
  • IL15R ⁇ IL15R ⁇ (sushi) and IL15R ⁇ (sushi +)
  • IL15 and IL15R ⁇ bind, in addition to being activated by their own cells, because IL15R ⁇ is mediated, it can also transmit signals to another cell to activate cell activity. These activities include the selective expansion of CD8 + T cells, NK cells, etc., and do not activate and regulate T cells like IL2, so they may play different functions in the anti-tumor immune response.
  • lentivirus refers to the genus Retroviridae family. Lentiviruses are unique among retroviruses and are capable of infecting non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most effective methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.
  • vector is a composition comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • the term should also be interpreted to include non-plasmid and non-viral compounds, such as polylysine compounds, liposomes, and the like, that facilitate the transfer of nucleic acids into cells.
  • examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • host cell refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli, fungal cells such as yeast cells, or fibroblast cells, CHO cells, COS cells, NSO cells Animal cells such as HeLa cells, BHK cells, HEK 293 cells or human cells.
  • transfection refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposomal fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.
  • immunode refers to a cell that can elicit an immune response
  • immunoreactive cell and “immune cell” and other grammatical forms can refer to immune cells of any origin.
  • Immunune cells include, for example, white blood cells (leukocytes), lymphocytes (T cells, B cells, natural killer (NK) cells, and bone marrow-derived cells (neutrophils) derived from hematopoietic stem cells (HSC) produced in the bone marrow. , Eosinophils, basophils, monocytes, macrophages, dendritic cells).
  • HSC hematopoietic stem cells
  • T cell refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity, and differ from other lymphocytes (such as B cells) in the presence of T cell receptors on the cell surface.
  • T cells include all types of immune cells that express CD3, including T helper cells (CD4 + cells), cytotoxic T cells (CD8 + cells), natural killer T cells, T regulatory cells (Treg), and ⁇ - ⁇ T cells.
  • Cytotoxic cells include CD8 + T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses.
  • NK cells refers to a type of lymphocytes that originate in the bone marrow and play an important role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells, or other stressed cells, even when antibodies and major histocompatibility complexes are not present on the cell surface.
  • immune cells can be derived from blood, such as autologous T cells, allogeneic T cells, autologous NK cells, and allogeneic NK cells. They can also be derived from cell lines, such as using EBV infection to prepare NK cell lines. iPSC induces differentiated NK cells and NK92 cell lines.
  • Optional, “any”, “any”, or “any” means that the event or environment described later may but need not occur, and the description includes the place where the event or environment occurs or does not occur.
  • “optionally comprising 1 antibody heavy chain variable region” means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.
  • “a” and “an” are used in the present invention to refer to one or more grammatical objects. Unless the content clearly indicates otherwise, the term “or” is used in the present invention to mean the term “and / or” and used interchangeably with it.
  • “About” and “approximately” shall generally mean the degree of acceptable error of the measured quantity in view of the nature or accuracy of the measurement.
  • Exemplary levels of error are generally within its 10% range and more generally within their 5% range.
  • the disclosed methods and compositions encompass polypeptides and nucleic acids that have a designated sequence, a variant sequence, or a sequence that is substantially the same or similar thereto, eg, at least 85%, 90%, 95%, 99% of the sequence designation Or more identical sequences.
  • the term "substantially the same" is used in the present invention to refer to a first amino acid sequence.
  • KD refers to the dissociation equilibrium constant (KD) of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • KD dissociation equilibrium constant
  • the antibody is less than about 10 -5 M, such as less than about 10 - 6 M, 10 -7 M , 10 -8 M, or 10 -10 M or less, a dissociation equilibrium constant antigen binding 10 -9 M, e.g. , As measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • KINEXA 400 instrument detected the affinity of the antibody and cell binding.
  • EC 50 refers to the concentration for 50% of maximal effect, that is, the concentration that can cause a 50% maximum effect.
  • the pharmaceutical composition of the present invention can be prepared into various dosage forms according to needs, and can be administered by a physician according to the patient's type, age, weight, general disease status, administration mode and other factors.
  • the method of administration can be, for example, injection or other treatment methods.
  • drug conjugates of antibodies As used herein, the terms “drug conjugates of antibodies”, “conjugates”, “conjugates” or “ADC” are used interchangeably and refer to structures having the formula I, III, IV, V, VI or VII Drug conjugates of the antibodies shown.
  • Auristatin is a fully synthetic drug, and its chemical structural formula is relatively easy to modify in order to optimize its physical properties and pharmaceutical properties.
  • the auristatin derivatives used for antibody coupling mainly include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).
  • the former is a natural tubulin polymerase inhibitor tail sea hare.
  • a synthetic pentapeptide derived from dolastatin-10 was synthesized by adding a 2-amino-1-phenylpropyl-1-ol to the C-terminus.
  • the inhibitory activity of MMAE on various human tumor cell lines is less than one nanomolar.
  • MMAF In order to reduce the cytotoxicity of MMAE itself, MMAF adds a phenylalanine to the C-terminus of cylinolin 10. Because a carboxyl group is introduced into the structure, the membrane permeability of MMAF is poor, so the biological activity of the MMAF is significant. Decreased, but the inhibitory activity on cells was significantly increased after coupling with antibodies (US7750116).
  • the antibody cytotoxic drug conjugate, or a pharmaceutically acceptable salt or solvent compound thereof comprises an antibody of the invention conjugated to one or more maytansinoid molecules.
  • Maytansinoids are mitotic inhibitors that do not work by inhibiting tubulin multimerization. Maytansine was originally isolated from the East African shrub Maytenusserrata (US Patent No. 3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol (U.S. Patent No. 4,151,042).
  • Maytansinoid alkaloid drug modules are attractive drug modules in antibody-drug conjugates because they are: (i) relatively easy to prepare by fermentation or chemical modification or derivatization of fermentation products; (ii) easy Derivatized with functional groups suitable for coupling to antibodies through non-disulfide linkers; (iii) stable in plasma; and (iv) effective against multiple tumor cell lines.
  • Maytansinoids suitable for use as maytansinoid alkaloid drug modules are well known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see Yu et al. (2002) PNAS99: 7968-7973). Maytansinol and maytansinol analogues can also be prepared synthetically according to known methods.
  • Exemplary embodiments of maytansinoid drug modules include: DM1, DM3, and DM4, as disclosed herein.
  • linker refers to groups suitable for use in the present invention for linking antibodies and small molecule drugs of the present invention.
  • exemplary linkers include MC, MP, val-cit, ala-phe, PAB, SPP, SMCC, SIAB.
  • the linker is MC-vc-PAB.
  • Drug loading is represented by y, that is, the average number of drug modules per antibody in molecules of the general formula I, III, IV, V and VI, also known as drug antibody antibody ratio (DAR).
  • the average number of drug modules per antibody in the ADC preparation from the coupling reaction can be characterized by conventional means, such as mass spectrometry, ELISA assays, and HPLC.
  • the quantitative distribution of ADCs in y can also be determined.
  • isolating, purifying, and characterizing a homogeneous ADC with a value of y from ADCs with other drug loads can be achieved by means such as reverse phase HPLC or electrophoresis.
  • Drug loading can range from 0.8-10 drug modules (D) per antibody.
  • ADC loading drug / antibody ratio DAR
  • drug / antibody ratio DAR can be controlled in different ways, for example by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, and (ii) limiting the time or temperature of the coupling reaction , (Iii) cysteine thiol-modified partial or limiting reducing conditions, (iv) engineering of the amino acid sequence of the antibody by recombinant technology, so that the number and position of cysteine residues are in order to control the linker-drug The number and / or location of attachment (such as thioMab or thioFab prepared as described herein and in WO2006 / 034488 (incorporated herein by reference in its entirety)).
  • the methods, compositions, and combination therapies of the present invention may be combined with other active agents or treatments.
  • the methods include administering the anti-CLDN18 of the present invention to the subject in an amount effective to treat or prevent a disease (eg, cancer).
  • a disease eg, cancer
  • .2 antibody molecules optionally, with PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, Tim-3 antibodies (immunotherapy) or other tumor treatment antibodies, Her-2, EGFR, VEGF, VEGFR antibodies, etc., and ADC (antibody drug conjugate, such as T-DM1), bispecific antibodies, chemotherapeutic drugs, one or more combinations of inhibitors, including the administration of anti-CLDN18.2 antibody molecules, additional
  • the active agent or all may be administered in an amount or dose that is higher, lower, or equal to the amount or dose of each active agent used alone (eg, as a monotherapy).
  • Anti-CLDN18.2 antibodies, additional active agents or all are administered in an amount or dose lower than the amount or dose of each active agent used alone (e.g., as monotherapy) (e.g., at least 20%, at least 30%, at least 40% % Or at least 50%).
  • anti-CLDN18.2 antibodies and drug conjugates of CLDN18.2 antibodies can bind CLDN18.2 to induce target cell (tumor cell) apoptosis, inhibit tumor cell growth, and increase the body Effector cells kill tumor cells ADCC and CDC to achieve the purpose of treating cancer patients. Therefore, in certain embodiments, the anti-CLDN18.2 antibody and the drug conjugate of CLDN18.2 antibody described in the present invention show the antitumor effect of the antibody of the present invention through these mechanisms, and a method for inhibiting the growth of tumor cells, including A therapeutically effective amount of an anti-CLDN18.2 antibody and a drug conjugate of the CLDN18.2 antibody described in the present invention is administered to a subject.
  • anti-CLDN18.2 antibody molecules can be administered with other antibodies.
  • the CLDN18.2 antibody and the drug conjugate of the CLDN18.2 antibody are administered in combination with one or more active agents, the combination can be administered in any order or simultaneously to cancer types, particularly tumor patients with high expression of CLDN18.2.
  • it is provided to treat a subject (eg, reduce or alleviate) a hyperproliferative condition or disease (eg, cancer) in the subject.
  • the method includes administering to the subject one or more anti-CLDN18.2 antibodies or drug conjugates of CLDN18.2 antibodies of the invention, alone or in combination with other active agents or treatments.
  • the combination further includes an inhibitor or activator of an immune checkpoint modulator, for example, an anti-PD-L1 antibody molecule, an anti-PD-1 antibody molecule, or a CTLA-4 inhibitor (for example, an anti-CTLA-4 antibody), or a non-immune Inhibitors or activators of checkpoint regulators (such as chemical drugs, small molecule targeted drugs, macromolecules including antibody targeted drugs, such as antibodies such as anti-Her2, anti-VEG, anti-VEGFR, anti-EGFR, etc.) Combination drug, bispecific antibody, CAR-T cell combination, etc.), or any combination thereof.
  • an inhibitor or activator of an immune checkpoint modulator for example, an anti-PD-L1 antibody molecule, an anti-PD-1 antibody molecule, or a CTLA-4 inhibitor (for example, an anti-CTLA-4 antibody), or a non-immune Inhibitors or activators of checkpoint regulators (such as chemical drugs, small molecule targeted drugs, macromolecules including antibody targeted drugs, such as antibodies such as anti-Her2, anti
  • Immune checkpoint refers to a group of molecules on the cell surface of immune cells, which can serve as a "gate” to down-regulate or suppress immune responses, such as anti-tumor immune responses, and then to treat tumors in combination with the antibodies of the invention.
  • Immune checkpoint molecules include but are not limited to PD-1, PD-L1, cytotoxic T lymphocyte antigen 4 (CTLA-4), B7-H1, B7-H3, OX-40, 4-1BB (CD137), CD40 , And lymphocyte activating gene 3 (LAG-3), and so on.
  • anti-CLDN18.2 antibody molecules Treatment with anti-CLDN18.2 antibody molecules, either alone or in combination with another immunomodulator (e.g. anti-LAG-3, anti-Tim-3, anti-PD-1 or anti-PD-L1, anti-CTLA-4 antibody molecules) Gastric cancer, pancreatic cancer, lung cancer, esophageal cancer, ovarian cancer, etc.
  • another immunomodulator e.g. anti-LAG-3, anti-Tim-3, anti-PD-1 or anti-PD-L1, anti-CTLA-4 antibody molecules
  • Anti-CLDN18.2 antibody molecules can be administered in combination with one or more of the following: immune-based strategies, targeted drugs (for example, VEGF inhibitors such as monoclonal antibodies against VEGF); VEGF tyrosine kinase inhibitors such as sunil Tinib, sorafenib, apatinib; inhibitors of RNAi inhibitors or downstream mediators of VEGF signaling, such as inhibitors of the rapamycin mammalian target (mTOR).
  • targeted drugs for example, VEGF inhibitors such as monoclonal antibodies against VEGF
  • VEGF tyrosine kinase inhibitors such as sunil Tinib, sorafenib, apatinib
  • inhibitors of RNAi inhibitors or downstream mediators of VEGF signaling such as inhibitors of the rapamycin mammalian target (mTOR).
  • cancer As used in the present invention, the terms “cancer”, “cancer”, “cancer patient” are intended to include all types of cancerous growths or tumorigenic processes, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of histopathology What is the type or invasive phase. Examples include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.
  • Non-limiting examples of cancers that can be suitably treated using the antibodies, bispecific antibodies, ADC and CAR cells, or combinations thereof that are disclosed in the present invention that target CLDN 18.2 include lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck Cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer and leukemia, etc., or their metastatic lesions.
  • Example 1 Construction of dense protein 18.1, 18.2 (CLDN18.1, CLDN18.2) overexpressing cell lines
  • the human CLDN18.1, human CLDN18.2, mouse CLDN18.1, and mouse CLDN18.2 high-expressing cell lines used in the present invention are completed by the company's stable cell line construction platform. Specific steps are as follows:
  • 293T cells (Cell Bank Cat # GNHu17 of the Typical Culture Collection Committee of the Chinese Academy of Sciences) were seeded into two 6 cm culture dishes, and the number of cells in each culture dish reached 7.5 ⁇ 10 5 cells.
  • the plasmids BioVector such as pGag-pol, pVSV-G, and pBabe, plasmid vector cell cell gene collection center
  • the plasmid pBabe-CLDN18.2 or pBabe cloned with human or mouse CLDN18.2 or CLDN18.1 gene -CLDN18.1 4 ⁇ g each was added to OPTI-MEM (Thermofisher Scientific Cat # 31985070) to a final volume of 200 ⁇ l, and another 200 ⁇ l OPTI-MEM was added to 36 ⁇ l transfection reagent fectin (Shanghai Yuanpei Biotechnology Co., Ltd.
  • the 293T cell supernatant (virus) was collected, filtered through a 0.45 ⁇ m filter to the cultured CHO-K1 cells, and 10 ⁇ g / ml polybrene (Shanghai Pu Sheng Biotechnology Co., Ltd. Cat # 40804ES76) was added, mixed and placed Incubator, change to DMEM / F12 10% FBS medium (source culture, Cat # L310KJ) after 3-4 hours.
  • the CHO-K1 cells were passaged on the 7th day, and the cells passaged on the 8th day were added with 10 ⁇ g / ml puromycin for screening (source culture, Cat # S250J0). A large number of cells died in 2-3 days. Replace the medium and continue to culture. When the cells no longer die, the cells will expand in large numbers. Monoclonal cell lines will be selected, expanded and frozen for seed preservation.
  • the CLDN18 cell lines stably expressed by the present invention are labeled as: human CLDN18.1 + cells (hCLDN18.1 + cell), human CLDN18.2 + cells (hCLDN18.2 + cell), and mouse CLDN18.1 + cells (mCLDN18.1) + cell), murine CLDN18.2 + cells (mCLDN18.2 + cell).
  • the protein sequences used were derived from published databases. The amino acid sequence of each protein is as follows.
  • Human CLDN18.1 (hCLDN18.1) is derived from NCBI database> NP_057453.1, claudin-18 isoform1precursor [Homo sapiens].
  • Human CLDN18.2 (hCLDN18.2) is derived from the NCBI database> NP_001002026.1 claudin-18 isoform 2 [Homo sapiens].
  • the mouse CLDN18.1 (mCLDN18.1) originated from the NCBI database> NP_062789.1claudin-18 isoform A1.1.
  • Mouse CLDN18.2 (mCLDN18.2) is derived from NP_001181850.1 claudin-18 isoform A2.1 [Musmusculus] in the NCBI database.
  • Example 3 Cloning, expression and purification of recombinant proteins and antibodies, and activity detection (ELISA, blocking assay)
  • the cloning, expression and purification of the recombinant protein / antibody used in the present invention are performed according to molecular cloning methods well known to those skilled in the art.
  • the expression vector used in the present invention was purchased from Changsha Youbao Biotechnology Co., Ltd., and was subsequently introduced into EcoRI digestion site (GAATTC) by Shanghai Jianxin Biomedical Technology Co., Ltd. Or homologous recombination method to clone foreign genes. Gene synthesis is performed by companies such as Bio-Engineering Biotechnology (Shanghai) Co., Ltd. (Bio-Engineering Bio). 293 cells and CHO-K were purchased from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences.
  • the recombinant protein and antibody in the present invention are obtained by transient transfection and purification in 293 cells.
  • the specific step is that 293 cells are expanded in Gibco FreeStyle 293 Expression Medium (Gibco, Cat # 12338018) medium.
  • Gibco FreeStyle 293 Expression Medium Gibco, Cat # 12338018
  • FBS Aus Gene X FBS Excellent supplier: AusGeneX, China, Cat # FBSSA500-S
  • 37 ° C 8% CO 2 shaker After 24 hours of incubation, the survival rate after microscopic examination was> 95%, and the cell concentration was 1.2 ⁇ 10 6 cells / ml.
  • Opti-MEM 300 ml cells of the culture system were prepared, 15 ml of Opti-MEM (Gibco, Cat # 31985070) were dissolved in 150 ⁇ g of each of the heavy chain and light chain plasmids, and 0.22 ⁇ m was filtered to sterilize. Another 15 ml of Opti-MEM was dissolved in 600 ⁇ l of 1 mg / ml PEI (Polysciences, Inc, Cat # 23966-2), and then allowed to stand for 5 min. In a 500 ml culture system, 25 ml of Opti-MEM (Gibco, Cat # 31985070) was dissolved in 250 ⁇ g each of the heavy chain and light chain plasmids, and 0.22 ⁇ m was filtered to sterilize.
  • Opti-MEM Opti-MEM was dissolved in 1000mg of 1mg / ml PEI and then allowed to stand for 5min. PEI was slowly added to the plasmid, incubated at room temperature 10min, side edges shaking flask was slowly added dropwise a mixed solution of plasmid PEI, 37 °C 8% CO 2 for 5 days shaking receives the type, 3300G 10min the supernatant was purified.
  • the sample was centrifuged at high speed to remove impurities, and a gravity column (Biotech, Cat # F506606-0001) containing Protein A (Mabselect, GE Healthcare, Science, Cat # 71-5020-91) was equilibrated with PBS pH 7.4. -5 column volumes rinse. Pass the sample through the column. The column was washed with 5-10 column volumes of PBS (Bio-Bio, Cat # B548117-0500). Then the target protein was eluted with pH 3.5 and 0.1 M acetic acid, and then adjusted to neutrality with Tris-HCl pH 8.0. The concentration was measured by a microplate reader, and then packed and stored for later use.
  • the recombinant human CLDN18.2 (claudin 18.2) extracellular region (D-70 at position 20 is the A fragment) and the Fc fusion protein were purified after transient transfection by the 293 system.
  • the protein can be used to detect serum titers in immunized mice.
  • the antibody human CLDN18.2 (anti-hCLDN18.2) antibody (called a control molecule or a positive molecule) is used as a comparison.
  • the antibody is abbreviated as Ref (Reference) in the present invention, and the sequence is derived from WO2014146672.
  • Antigen plate ELISA human PD-1, PD-L1, CD47, LAG3, Tim3, or TGF ⁇ 1 (Cat # 100-21-10), TGF ⁇ 2 ( Cat # 100-35B), TGF ⁇ 3 (Cat # 100-36E).
  • Antigens such as IL10 (Cat. No. SEKA10947), Fc ⁇ R I / CD64 (Cat. # 1257-FC-050, R & D Systems) purchased from Sino biological are diluted to a concentration of 1-2 ⁇ g / ml according to different assays, and added in a volume of 50 ⁇ l / well Place in a 96-well microtiter plate (Corning, CLS3590-100EA) in a 37 ° C incubator for 2 hours.
  • skimmed milk (Shanghai Shengong Biological Engineering Co., Ltd., A600669-0250) blocking solution diluted in PBS, and incubate at 37 ° C for 3 hours or at 4 ° C overnight (16-18 hours) ) For closure. Discard the blocking solution and wash the plate 5 times with PBST buffer (pH 7.4 PBS with 0.05% tweeen-20). Then add 50 ⁇ l / well of the test antibody diluted 5 times with 1% BSA. Incubate for 1 hour at 37 ° C.
  • skimmed milk Shanghai Shengong Biological Engineering Co., Ltd., A600669-0250
  • PBST The plate was washed 5 times, and 50 ⁇ l / well of a 1: 2500 diluted HRP-labeled secondary antibody (Jackson Immuno Research, 115-035-003) was added and incubated at 37 ° C. for 1 hour. After washing the plate 5 times with PBST, add 50 ⁇ l / well TMB chromogenic substrate (KPL, 52-00-03), incubate at room temperature for 5-10 min, add 50 ⁇ l / well 1M H 2 SO 4 to stop the reaction, and use MULTISKAN Go enzyme labeling The instrument (ThermoFisher, 51119200) reads the absorption value at 450nm, and calculates the EC50 based on the OD value.
  • KPL chromogenic substrate
  • the antigens PD-1, PD-L1, and CD47 expressed by the present invention were diluted to a concentration of 2 ⁇ g / ml with PBS buffer at pH 7.4, and added to a 96-well microtiter plate (Corning, CLS3590-100EA) at a volume of 50 ⁇ l / well. Incubate at 37 ° C for 2 hours. After discarding the liquid, 200 ⁇ l / well of blocking solution of 5% skim milk (Shanghai Shengong Biological Engineering Co., Ltd., A600669-0250) diluted with PBS was added, and incubated at 37 ° C. for 3 hours for blocking.
  • Biotin-labeled ligands (PD-1, PD-L1, SIRP ⁇ , etc., expression and purification of the present invention), incubated at 37 ° C for 1 hour, washed the plate 5 times with PBST, and added 50 ⁇ l / well of a 1: 1000 diluted HRP-labeled secondary antibody ( Kingsley Biotechnology Co., Ltd., M00091), incubate at 37 ° C for 1 hour.
  • the Biotin-labeled kit was Biotin Labeling Kit-NH2, which was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., with the product number LK03.
  • the operation method is performed according to the instructions, and the labeled antibody is used after detecting the concentration with a Multiskan GO (ThermoFisher) microplate reader.
  • the anti-human CLDN18.2 monoclonal antibody of the present invention is a mouse immunized with the human CLDN18.2 high-expressing cell line (hCLDN18.2 + cell) obtained in Example 1.
  • the spleens of the immunized mice were fused with a hybridoma. Screened and optimized from one million hybridoma clones.
  • mice Female, 4 weeks of age (SJL was purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Jing) 2016-0011; Balb / c was purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.). After the mice were purchased, they were kept in a laboratory environment for 1 week, adjusted in the light / night cycle during the day, the temperature was 20-25 ° C, and the humidity was 40-60%. Mice were divided into 3 / groups / cages.
  • the human CLDN18.2 high-expressing cell line (hCLDN18.2 + cell, human CLDN18.2 + cells) constructed in Example 1 was cultured, DMEM medium (source culture, Cat # L310KJ) was washed after trypsin digestion, and then reconstituted. Suspended in DMEM medium. Mice were immunized intraperitoneally with 100 ⁇ l / 1 ⁇ 10 7 cells / head. For the first immunization, use Titermax (Sigma-Aldrich, T2684) to mix the cells with the cells 1: 1.
  • mice with high serum titers and plateau titers were selected for splenocyte fusion.
  • mice Before fusion, 200 ⁇ l / 2 ⁇ 10 7 cells / mouse were used to immunize the mice. After 3 days of immunization, remove Mouse spleen lymphocytes and myeloma cells Sp2 / 0 cells ( CRL-8287 TM ) was fused to obtain hybridoma cells in a 96-well plate.
  • the supernatant of hybridoma cells in a 96-well plate was simultaneously plated with human CLDN18.1 + cell and human CLDN18.2 + cell to detect the binding of antibodies produced by the hybridoma cells.
  • Table 1a shows the detection results of the supernatants of some hybridomas.
  • human CLDN18.2 and CLDN18.1 have 92% homology (240/261), and the protein is a transmembrane protein, only a few peptides are extracellular (such as 51 amino acids ECL1), immune The originality is extremely low, and the possibility of producing specific antibodies is very small. Therefore, in the above screening, not only few hybridomas capable of secreting and recognizing CLDN18 can be obtained, but among the few hybridomas, the antibodies in the majority of hybridoma supernatants are antibodies that can simultaneously bind human CLDN18.2 and CLDN18.1. .
  • the present invention found a hybridoma clone whose secreted supernatant only bound to human CLDN18.2 + cell and not to human CLDN18.1 + cell, see mab5 in Table 1a, clone number C13C1 .
  • Table 1a shows that under the same screening conditions, the clone supernatant only binds to human CLDN18.2 + cell, and the detection value is 1.41, while it does not bind to human CLDN18.1 + cell, and the binding activity reads only 0.09.
  • hybridoma cell line C13C1 unexpectedly discovered by the present invention can secrete a unique anti-human CLDN18.2 antibody.
  • the C13C1 hybridoma cells were subjected to multiple limited dilutions, and the clones after each dilution were carefully and carefully optimized. Finally, a monoclonal cell line capable of secreting a unique anti-human CLDN18.2 antibody was found. The results are shown in Table 1b. .
  • the antibody unexpectedly discovered by the present invention can effectively recognize only human CLDN18.2 protein, and has the potential to treat tumors as a monoclonal antibody, especially to treat cancer patients with human CLDN18.2 protein overexpression, including but not limited to pancreatic cancer , Gastric cancer, esophageal cancer, lung cancer, etc. Because it does not bind human CLDN18.1 protein at all, it is expected that the toxic and side effects caused by the non-specific binding of therapeutic antibodies and human CLDN18.1 proteins can be avoided.
  • Example 5 Screening and identification of murine anti-human CLDN18.2 antibody of the present invention
  • the antibody sequence secreted by the cell strain C13C1F1D3G6 (Table 1b) obtained from the hybridoma monoclonal cell line C13C1F1D3G6 obtained in the above-mentioned example is obtained as the mouse antibody mab5b sequence of the present invention.
  • the process of extracting antibody sequences from hybridoma-preferred monoclonal cell lines is well known and commonly used by those skilled in the art.
  • the hybridoma monoclonal cell C13C1F1D3G6 found in the above-mentioned embodiment is amplified and cultured, 1 ⁇ 10 6 cells are collected, and RNA is extracted using Trizol (Invitrogen, 15596-018) (in accordance with the steps of the kit instruction), and The extracted RNA was reverse transcribed into cDNA.
  • the reverse transcription kit was purchased from Biotech Biotechnology (Shanghai) Co., Ltd., Cat # B532435.
  • the reverse transcription cDNA was used as a template, and after PCR amplification, the amplified product was sequenced to obtain the light and heavy chain variable region sequences of the mab5b antibody.
  • the primers used are described in a manual published by Novagen (TB326 Rev. C0308).
  • anti-human CLDN18.2 (anti-hCLDN18.2) monoclonal antibody light chain nucleotide sequence (SEQ ID NO: 5) and heavy chain nucleotide sequence (from the preferred hybridoma monoclonal cell line C13C1F1D3G6 of the present invention) (SEQ ID NO: 6).
  • amino acid sequence of the variable region of the light chain variable region of the mouse-derived anti-human CLDN18.2 (anti-hCLDN18.2) monoclonal antibody mab5b derived from the hybridoma monoclonal cell strain discovered by the present invention is obtained from the translation of the above light chain base sequence:
  • amino acid sequence of the variable region of the heavy chain of the anti-human CLDN18.2 (anti-hCLDN18.2) monoclonal antibody mab5b of the mouse derived from the hybridoma monoclonal cell strain discovered by the present invention is obtained from the base sequence of the heavy chain as follows:
  • the above-mentioned antibody mab5b extracted from the hybridoma monoclonal cell line discovered by the present invention was cloned (sequenced below) and expressed recombinantly using the method described in Example 3, respectively, for the light and heavy chain variable regions and constant regions of the antibody. After purification, and The control antibody Ref simultaneously detected the binding activity to human hCLDN18.1, hCLDN18.2, murine mCLDN18.1, and mCLDN18.2. The results are shown in Table 2a, Table 2b, and Figure 1 below.
  • the anti-hCLDN18.2 antibody mab5b light chain (LChain) of the present invention is shown in SEQ ID NO: 9; the heavy chain (HChain) is shown in SEQ ID NO: 10.
  • Table 2a and Figure 1a show that the anti-hCLDN18.2 murine antibody mab5b and the control antibody (Ref) found by the present invention do not bind to hCLDN18.1 + cell, and the EC50 is not detectable (ND), even at a high concentration of 200nM
  • the binding value Emax (referring to the binding value when the concentration of the sample increased and reached the plateau, that is, the maximum specific binding value) was still the background, that is, the background value. Both antibodies have good binding activity to hCLDN18.2 + cell.
  • the antibody mab5b binding activity of the present invention is more than 1 times better than Ref (EC50 is 0.115 nM vs 0.249 nM). More surprisingly, the maximum binding value Emax that mab5b can reach is more than 36% higher than Ref [(1.92-1.41) /1.41].
  • the activity of the antibody mab5b discovered by the present invention is better than that of Ref, showing that the antibody can be used for the development of tumor treatment drugs.
  • the present invention performs humanization screening on mab5b. , And sequence optimization work. The specific process is described below.
  • the murine anti-human CLDN18.2 antibody mab5b obtained in the above Example 5 is labeled according to various definition methods in Table 3, and its CDR sequence is marked / annotated as follows.
  • Anti-human CLDN18.2 (anti-hCLDN18.2) antibody mab5b according to the present invention defines the CDR sequences
  • the CDR regions of the light and heavy chains of the antibody are identified (as above). Compare with the human antibody germline database (v-base) to find the light and heavy chain germlines of human antibodies with high homology. Based on this, computer modeling will simulate the sites in the antibody structure that may affect the binding to the antigen. , Back to the key sites and combinations of mutations, and select humanized antibody molecules with optimal activity.
  • the human antibody germline which has better homology with mab5b light chain contains IGKV4-1 * 01 (F), IGKV2-28 * 01 (F), IGKV2D-28 * 01 (F), IGKV1-27 * 01 (F), IGKV1-39 * 01 (F), IGKV1D-39 * 01 (F), IGKV2-40 * 01 (F), IGKV2D-29 * 01 (F), IGKV2D-40 * 01 (F), IGKV3-15 * 01 (F).
  • human antibody germline light chain IGKV4-1 * 01 (F) is preferred.
  • the CDR2 sequence of the selected human germline light chain IGKV4-1 * 01 (F) is WASTRES, which is exactly the same as the CDR2 sequence of the mouse antibody mab5b light chain discovered by the present invention. Sequence alignment revealed that the J gene region of mab5b light chain and human antibody germline hJK1, hJK2.1, hJK2.2, hJK2.3, hJK2.4 have high homology.
  • hJK2.1 is preferably used for mab5b Light chain humanized human antibody germline J region for humanized design, screening and sequence optimization.
  • the human antibody germline with better homology with mab5b heavy chain contains IGHV1-69 * 02 (F), IGHV1-69 * 06 (F), IGHV1-69 * 08 (F ), IGHV1-69 * 09 (F), IGHV1-69 * 10 (F), IGHV1-69 * 04 (F), IGHV1-69 * 14 (F), IGHV1 / OR15-2 * 02 (P), IGHV1 -69 * 01 (F), IGHV1-69 * 11 (F).
  • the human germline heavy chain IGHV1-69 * 01 (F) sequence is preferred for humanizing the antibody of the present invention.
  • hJh4.1 is used for the mab5b heavy chain humanized human antibody germline J region of the present invention for humanized design, screening and sequence optimization.
  • the mab5b CDR region of the antibody of the present invention (see the definition of the above CDR) is transplanted onto the selected humanized light and heavy chain human antibody germline template, and then recombined with the IgG light and heavy chain constant regions. Then, based on the three-dimensional structure of the mouse-derived antibody, back mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that have an important effect on the conformation of VL and VH.
  • L24-L34CDR1 differ by only 5 amino acids, which are L29, L30A, L30C, L30E, and L34, respectively, as shown in Table 9a below.
  • the L50-L56 CDR2 is exactly the same (see Table 9b below).
  • amino acids corresponding to the corresponding positions of the adult germline IGKV4-1 * 01 (F) were mutated in five positions of mab5b and CDR1 (L29, L30A, L30C, L30E, and L34).
  • the combined design is shown in Table 9a below.
  • mub anti-hCLDN18.2 antibody mab5b of the present invention is defined by Kabat's amino acid positions L24-L34 (CDR1, ie, SEQ ID NO: 11) and humanized.
  • Table 9b L50-L56 (CDR2) amino acids of the mab5b sequence of the mouse anti-hCLDN18.2 antibody of the present invention
  • the preferred sequence of the humanized light chain variable region of the mab5b antibody of the present invention is as follows:
  • L11 SEQ ID NO: 30;
  • L13 SEQ ID NO: 32;
  • H52 SEQ ID NO: 35; H53: SEQ ID NO: 36;
  • the above light chain variable region sequence including any of the listed sequences such as L14, L11, L12, L13, L15, and not listed, is combined with a human antibody light chain ⁇ -type or ⁇ -type light chain constant region to obtain the present inventors Sourced antibody light chain sequence.
  • the above heavy chain variable region sequence includes the listed heavy chain variable region sequences such as H51, H52, H53, L54, and unlisted heavy chain variable region sequences, and the combination of constant region sequences of different subtypes such as hIgG1,2,3,4, etc.
  • the heavy chain sequence of the antibody of the invention is obtained.
  • the light chain and the heavy chain are arbitrarily combined to obtain the humanized antibody of the present invention, and a part of the humanized antibody sequence is shown in Table 11 below.
  • Light chain of humanized Ab7 antibody amino acid sequence SEQ ID NO: 40; heavy chain: SEQ ID NO: 39;
  • Light chain of humanized Ab8 antibody amino acid sequence SEQ ID NO: 38; heavy chain: SEQ ID NO: 41;
  • Light chain of humanized Ab9 antibody amino acid sequence SEQ ID NO: 40; heavy chain: SEQ ID NO: 41;
  • Light chain of humanized Ab11 antibody amino acid sequence SEQ ID NO: 42; heavy chain: SEQ ID NO: 43;
  • Light chain of humanized Ab12 antibody amino acid sequence SEQ ID NO: 42; heavy chain: SEQ ID NO: 44;
  • Light chain of humanized Ab14 antibody amino acid sequence SEQ ID NO: 45; heavy chain: SEQ ID NO: 44;
  • Light chain of humanized Ab15 antibody amino acid sequence SEQ ID NO: 45; heavy chain: SEQ ID NO: 39;
  • the recombinant antibody was cloned, expressed and purified by the method of Example 3 of the present invention.
  • the ELISA method of Example 2 was used to detect and screen the binding activity of the humanized antibody to hCLDN18.2 + cell and hCLDN18.1 + cell. The results are shown in the table below. 12, and Figure 2a.
  • the results in Table 12 show that after humanizing the antibody mab5b discovered by the present invention, the mouse-bound antibody has a higher binding activity than the reference antibody Ref, and the EC50 of the humanized antibody Ab10 is 2 times better than Ref (0.117 vs 0.345). Not only that, the maximum binding Emax of these humanized optimized molecules is 39.8% -54.1% higher than the control antibody Ref. It is better than the control antibody against the mouse antibody mab5b.
  • the final sequence and human antibody germline light and heavy chains be as close as possible to reduce the immunogenicity that may be caused by a small number of sequences in the mouse antibody.
  • Humanized optimized light chain CDR1 sequences Var3 (see Table 9) designed a series of humanized antibodies and screened for specific binding activity. The results are shown in Table 13 and Figure 2b below.
  • the Ab13 antibody molecule that was found unexpectedly did not have any back mutations, that is, it was a fully humanized antibody molecule, and the CDR1 sequence was optimized for humanization, and its binding activity (EC50 and Emax) was the same as that of Ab6 and Ab10.
  • the antibody molecule obtained by humanization of the mouse-derived antibody mab5b sequence according to the present invention includes only humanized FR regions, and the light chain CDR1 remains wild (no mutation), such as Ab10; or humans in the FR region.
  • the light chain CDR1 was also subjected to a humanized optimized sequence Var3, and the resulting Ab6, Ab11-15, etc. all maintained their binding activity and were better than the control molecule, and the EC50 was 1 times stronger than the control molecule. Emax is 30% -50% higher than the control molecule, and all hCLDN18.1 + cells bind differently.
  • Example 7 Sequence optimization of the deamidation sensitive site of the antibody sequence of the present invention
  • PTMs post-translational modification
  • Spot analysis including antibody aggregation, deamidation-sensitive (asparagine, deamidation, sites (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) -sensitive sites, N glycosylation (N- ⁇ P ⁇ S / T) analysis of sensitive sites and oxidation-sensitive sites found that the light chain CDR1 (CDR1, LChain) of the antibody of the present invention is L30A, L30B is NS, and heavy chain CDR3 (CDR3, HChain) H99, H100 is NS, and the asparagine (N) of L30A and H99 may be sensitive to deamidation.
  • the sequence of the above optimized deamidation sensitive site was designed to express antibodies with different light and heavy chain combinations, and the binding activity was further screened.
  • the antibody combination section is shown in the table below.
  • the above-mentioned preferred antibody was expressed according to the method of Example 3, and after purification, the binding activity with human CLDN18.2 + cell was detected by the method of Example 2. The results are shown in Tables 16a, 16b and 16c.
  • Table 16a Optimized antibody activity by deamidated sensitive sites of the humanized anti-hCLDN18.2 antibody of the present invention
  • the entire anti-molecule has completely lost its binding activity (Ab30).
  • the CDR1 of the antibody of the present invention is Q at L30E and T at L34 (underlined), S at position H100 of the heavy chain cannot be mutated, for example, to avoid potential deamidation of S100 at position H100. T, otherwise the entire antibody loses binding activity.
  • the light chain CDR1L30A and L30B of the antibody of the present invention are NS; the heavy chain CDR3H99 and H100 of NS can be optimized by NS-> NT to reduce the possible risk of deamidation.
  • L34 that is, the sequence is humanized optimized sequence KSSQSLLNSGN N KNYL A , SEQ ID NO: 12
  • the sequence of the region is KSSQSLLNSGN Q KNYL T (SEQ ID NO: 11)
  • the antibody is completely inactive ( The underlined parts have different sequences.)
  • the light and heavy chain constant regions of the antibody variable regions and human antibodies of the present invention include, but are not limited to, the light chain ( ⁇ , ⁇ -type light chain, etc.) and heavy chain constant regions (kappa, lambda light chain, etc.) of the human antibodies listed in Example 3 that are different.
  • hIgG2, hIgG4, hIgG1) combinations, especially human IgG1 Fc sequence variants, such as different forms of DEL or EEM at positions 356-358, can obtain different antibody variant forms.
  • Table 17 lists some of the variants of the antibodies and Fc sequences of the invention, including positions 356-358 of the Fc region sequence as DEL or EEM.
  • the amino acid sequence of the Ab42 antibody is as follows:
  • Light chain SEQ ID NO: 38; Heavy chain: SEQ ID NO: 46;
  • the above preferred antibody was expressed according to the method of Example 3, and after purification, the binding activity with human CLDN18.2 + cell was detected by the method of Example 2. Representative data are shown in Table 18 below. The results showed that the above-mentioned changes in the constant regions of the light and heavy chains, including DEL 356 or 358 of hIgG1, respectively, did not affect the activity of the antibodies of the present invention.
  • Example 9 Sequence optimization of the antibody Fc region (human IgG1) of the present invention against ADCC, CDC activity
  • the antibody human Fc region (hIgG1 Fc) of the present invention mediates the effects of ADCC and CDC of effector cells, can specifically enhance the targeting of tumor cells, and cause non-specific side effects outside the target.
  • ADCC and CDC human antibody Fc region
  • the present invention confirmed the effect of the Fc region of the discovered antibody molecule on human blood cells (ADCC and CDC).
  • the above-mentioned preferred antibody was expressed according to the method of Example 3, and the antibody was obtained after purification, and then ADCC (antibody-dependent cytotoxicity test) and CDC (complement-dependent cytotoxicity test) were performed to detect the optimized activity of the antibody Fc sequence. specifically,
  • the hCLDN18.2 + cells constructed in Example 1 were cultured normally.
  • the culture medium was DMEM / F12 plus 10% FBS (Shanghai Yuanpei Biotechnology Co., Ltd. Cat # L310KJ), which was used as the target cells for ADCC in this experiment.
  • PBMC cells were prepared (the present invention is isolated from human peripheral blood. Human peripheral blood was donated by our volunteers), and PBMCs were suspended in serum-free RPMI1640 medium (Peiyuan Bio, Cat # L210KJ) at a concentration of 150,000 cells / 50 ⁇ l.
  • the drug to be tested was formulated with serum-free RPMI1640, and the initial concentration of 40 ⁇ g / ml was diluted by 3 times.
  • the LDH kit was Cytotoxicity LDH Assay Kit-WST, which was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., article number CK12.
  • the operation method is according to the instructions. Take out the well plate, add 100 ⁇ l of WorkingSolution to each well, cover with aluminum foil to protect from light, and react at room temperature for 10-40min. MultiskanGO (ThermoFisher) microplate reader reads 490nM, detect every 10min, and take the appropriate reaction time Data were analyzed and processed with Graphpadprism5.
  • hCLDN18.2 + cells constructed in Example 1 were used as target cells for CDC in this experiment.
  • hCLDN18.2 + cells were cultured normally with DMEM / F12 plus 10% FBS (same as ADCC).
  • target cells were collected, counted, and prepared at 1 ⁇ 10 5 / ml cells, and 100 ⁇ l / well was added to a 96-well cell culture plate. Incubate at 37 ° C, 5% CO 2 overnight.
  • the medium in the 96-well plate cells was removed and washed twice with PBS until use.
  • the antibody to be tested was diluted with serum-free medium (RPMI1640). The initial antibody concentration was 20 ⁇ g / ml, and the dilution was 5-fold. 50 ⁇ l / well of diluted antibody was added to the target cell culture plate washed with PBS (100 ⁇ l / well of fresh medium for 0 ⁇ g / ml antibody wells as control wells), and six replicates were set at each concentration point. Incubate at 37 ° C, 5% CO 2 for 15 min.
  • RPMI1640 medium 40%: 60%, that is, 40% was serum and 60% was RPMI1640.
  • the final serum concentration was 20%; the initial concentration of the sample (antibody) was 10 ⁇ g / ml.
  • Serum with complement was added in the first 3 replicates, and serum with inactivated complement was added in the last 3 replicates. Incubate for 2 hours at 37 ° C, 5% CO2 in an incubator, and then take it out for detection with an LDH kit.
  • the LDH kit was Cytotoxicity LDH Assay Kit-WST, which was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., article number CK12.
  • the operation method is according to the instructions. Take out the well plate, add 100 ⁇ l Working Solution to each well, wrap the aluminum foil to protect from light, react at room temperature, detect every 10 minutes, take the appropriate reaction time data, read 490nM of MultiskanGO (ThermoFisher) microplate reader Graphpadprism5 analyzes and processes the data.
  • MultiskanGO ThermoFisher
  • Neg IgG a non-specific antibody that does not bind to the target
  • Neg IgG a non-specific antibody that does not bind to the target
  • Example 10 Evaluation of ADCC and CDC activities of different humanized molecules of the antibodies of the present invention
  • Neg IgG a non-specific antibody that does not bind to the target, the same below
  • the humanized antibody ADCC activity of the present invention is comparable to the control antibody (Ref) (Table 21a).
  • the CDC activities of Ab36 and Ab24 were also better than those of the control antibody Ref.
  • Example 11 Evaluation of Apoptosis Activity of Tumor Cells (hCLDN18.2 + cell) Induced by the Antibodies of the Invention
  • hCLDN18.2 + cell In order to detect the antibody of the present invention, especially the humanized antibody that induces the apoptosis of hCLDN18.2 + cell (tumor cells), we used the hCLDN18.2 + cell constructed in Example 1 of the present invention to detect the antibody of the present invention. Induces tumor cell apoptosis activity.
  • hCLDN18.2 + cell was cultured normally (DMEM / F12 containing 10% FBS in the medium, supplier: Shanghai Yuanpei Biotechnology Co., Ltd., article number: L310), as the cells used in this experiment.
  • hCLDN18.2 + cell was plated in a 96-well plate with a plate density of 2 ⁇ 10 4 / well.
  • antibody samples 0 ⁇ g / ml, 1 ⁇ g / ml, 3 ⁇ g / ml, 10 ⁇ g / ml antibody samples were prepared using serum-free DMEM / F12 medium. Remove hCLDN18.2 + cell adherent culture overnight, discard the medium, and wash cells twice with PBS. Add prepared antibody samples of different concentrations, 100 ⁇ l / well. After 24 hours of incubation, LDH was detected.
  • the LDH kit was Cytotoxicity LDH Assay Kit-WST, which was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., article number CK12.
  • the operation method is according to the instructions. Take out the well plate, add 100 ⁇ l of Working Solution to each well, use aluminum foil and other methods to protect from light, react at room temperature, and at different time points (10min, 20min, 30min, 40min, 50min), Multiskan GO (ThermoFisher) Microplate reader reads 490nM, finds the best reaction time, takes the readings and analyzes the data with Graphpad prism5.
  • Ab6 10 ⁇ g / ml increased tumor cell apoptosis activity (46.7%) more than 2 times better than the same concentration (15.3%) of the positive control; even more than 1 time better than 30 ⁇ g / ml (19.6%) of the positive control; and 30 ⁇ g
  • the activity of inducing tumor cell apoptosis (85.1%) was more than three times better than that of the control antibody (19.6%) at the same concentration.
  • Ab6 increased the apoptosis activity of tumor cells by 10 ⁇ g / ml (46.7%) 41% better than the same concentration of Ab10 (33.2%); at 30 ⁇ g / ml concentration, it induced tumor cell apoptosis (85.1%) than the same concentration Ab10 (34.7%) was more than twice as strong.
  • Example 12 Antibodies of the invention inhibit tumor cell (hCLDN18.2 + cell) proliferation
  • hCLDN18.2 + cells constructed in Example 1 were tested for activity. Specifically, hCLDN18.2 + cells were cultured normally (medium: DMEM / F12 containing 10% FBS, supplier: Shanghai Yuanpei Biotechnology Co., Ltd., article number: L310). At the beginning of the experiment, hCLDN18.2 + cells grown in logarithmic phase were plated in 96-well plates with a density of 3 ⁇ 10 3 / well. 37 ° C, 5% CO2 culture overnight adherent culture.
  • antibody samples 1 ⁇ g / ml, 10 ⁇ g / ml, 30 ⁇ g / ml antibody samples were prepared with DMEM / F12 (source culture) containing 10% FBS. Take out hCLDN18.2 + cells adhered to the overnight culture, discard the medium, and wash the cells with PBS once, and then add prepared antibody samples with different concentrations, 100 ⁇ l / well. After 72 hours of incubation, the cells were detected by CCK-8 kit.
  • the CCK-8 kit was Cell Counting Kit-8, purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., with the article number CK04.
  • the operation method is according to the instructions. Take out the 96-well plate, add 10 ⁇ l CCK-8 solution to each well (be careful not to generate bubbles in the well, otherwise it will affect the reading), incubate the culture plate in the incubator for 1-4h, find out Optimal detection time point, 450nM readings of Multiskan GO (ThermoFisher) microplate reader, the values were analyzed and processed by Graphpad prism5. The results are shown in Table 22 below.
  • Table 22 Preferred humanized antibodies of the present invention inhibit tumor cell (hCLDN18.2 + cell) proliferation activity (inhibition rate%)
  • the results in Table 22 show that the negative antibody inhibited tumor cell activity (inhibition rate) at a concentration of 1 ⁇ g / ml, 10 ⁇ g / ml, and 30 ⁇ g / ml (inhibition rate) below 1%, that is, the background level.
  • Ab10 inhibited tumor cell activity (inhibition rate) from 2.17% to 3.16%, which was close to the inhibition rate of Ref from 2.34% to 3.9%.
  • Ab6 inhibited tumor cell activity much stronger than Ref.
  • the inhibition rate of 6.12% at 10 ⁇ g / ml was more than twice that of Ref (2.94%).
  • Example 13 Detection of binding activity of the humanized antibody of the present invention and murine CLDN18
  • the binding activity of the humanized preferred antibody of the present invention to murine CLDN18.1 + cell and murine CLDN18.2 + cell was detected.
  • a clone antibody L180
  • Example 14 Evaluation of in vivo pharmacological activity of antibodies of the present invention
  • hLBND18.2 + cells constructed in Example 1
  • gastric cancer cell line NUGC4 gastric cancer cell line NUGC4 (Shanghai Suer Biotechnology Co., Ltd.) were used to subcutaneously transplant BALB / c nude mice into animal models To evaluate the in vivo efficacy of the antibodies of the present invention.
  • the hCLDN18.2 + cell culture medium is DMEM / F12 (source culture) plus 10% fetal bovine serum (Shanghai Bosheng Biotechnology Co., Ltd., article number BS-0002-500).
  • NUGC4 cell culture medium is RPMI1640 (source culture), plus 10% fetal bovine serum.
  • the culture conditions were 37 ° C and 5% CO 2 .
  • hCLDN18.2 + cell xenograft model For the hCLDN18.2 + cell xenograft model, hCLDN18.2 + cells were taken and washed twice with PBS, and then resuspended to form cells 1 ⁇ 10 8 / ml. The mice were inoculated subcutaneously with 0.1 ml total 1 ⁇ 10 7 cells / head. Three- size mice with a tumor size of about 120-180mm were selected and randomly divided into groups of 5-6 mice.
  • NUGC4 cell xenograft model For a gastric cancer NUGC4 cell xenograft model, NUGC4 cells were washed twice with RPMI1640, and Matrigel was added so that the ratio of RPMI640 to RPMI640 was 1: 1, and the cells were resuspended in a mixed solution to prepare cells 1 ⁇ 10 8 / ml. The mice were inoculated subcutaneously with 0.1 ml total 1 ⁇ 10 7 cells / head. Three- size mice with a tumor size of about 150-200 mm were selected and randomly divided into groups of 5-6 mice.
  • the sample to be tested was prepared with PBS and sterile. Blank is a PBS without sample control, a target-independent antibody, ie, a negative antibody (Neg IgG) control. Intraperitoneal injection, 200 ⁇ g / 100 ⁇ l / head. 2 times / week for several consecutive weeks. The day of sample injection was day 0. Body weight and tumor volume were measured before each administration and data were recorded.
  • Blank is a PBS without sample control, a target-independent antibody, ie, a negative antibody (Neg IgG) control.
  • Intraperitoneal injection 200 ⁇ g / 100 ⁇ l / head. 2 times / week for several consecutive weeks. The day of sample injection was day 0. Body weight and tumor volume were measured before each administration and data were recorded.
  • Relative tumor growth rate (T / C%) 100% * (T-T0) / (C-C0).
  • Tumor inhibition rate (TGI) (1-T / C) * 100%.
  • T0 and T are tumor volumes at the beginning and end of the experiment in the sample group;
  • C0 and C are tumor volumes at the beginning and end of the experiment in the control group, respectively.
  • FIG. 5a shows that the antibodies Ab10 and Ab6 of the present invention show very good pharmacological activity in vivo as well as the positive antibody molecule (Ref). Inhibits tumor cell growth and / or kills tumor cells (inhibition rate) by more than 90%; unexpectedly, light chain CDR1 and heavy chain CDR3 deamination sensitive sites are optimized, and light chain CDR1 humanized and optimized antibody Ab36 is preferred Completely inhibit tumor growth, its efficacy in vivo is significantly better than Ab6, Ab10 and control positive antibodies (Ref).
  • NA Not adapted, that is, blank control.
  • the antibody of the present invention has good binding activity with mouse CLDN18.2, which provides the antibody of the present invention with a selection of non-primate species for preclinical research.
  • the invention evaluates the pharmacokinetic (PK) properties of the antibodies of the invention in mice.
  • mice were purchased from Shanghai Shiple-Bikai Experimental Animal Co., Ltd. After the mice were purchased, there were 6 animals in each cage, and unlimited amounts of feed and water were obtained.
  • the laboratory environment was raised for 3 days, the temperature was 20-25 ° C, the humidity was 40-60%, and the light / dark cycle was adjusted for 12/12 hours.
  • the mice were measured for body weight. 20-25 g mice were taken and numbered in groups of 3 mice / group.
  • each mouse was injected subcutaneously with the test drug Ab10 and control antibody (Ref), the drug was administered at a dose of 10 mg / kg, and subcutaneously injected at 100 ⁇ l / piece / time.
  • Example 16 Affinity analysis of antibodies of the invention (KinExA)
  • CLD18.2 is a cell transmembrane protein, four transmembrane, and the two extracellular regions ECL1 and 2 are only 20-50 amino acids in length.
  • the antibodies of the invention specifically bind the extracellular region of CLDN18.2.
  • Conventional Biacore detection methods that bind antigens (20-50 amino acids) and antibodies cannot well evaluate the specific affinity of the antibody and extracellular region of the target protein. Therefore, the present invention uses the KinExA method to detect the affinity of antibodies and hCLDN18.2 + cells. The method was performed with reference to the KinExA 4000 instrument manual, that is, the antibodies to be tested Ref, Ab10 were Constant binding partners (CBP), and hCLDN18.2 + cells were Titrant.
  • CBP Constant binding partners
  • CBP antibody
  • a signal test was first calculated based on the estimated affinity to determine a reasonable concentration, and 500ul 120pM Ref antibody and 100pM Ab10 antibody were used as the Signal 100%, and a satisfactory detection net was obtained at this concentration.
  • Signal value PBS blank as negative signal value (NSB).
  • the concentration of 120 pM Ref antibody and 100 pM Ab10 antibody was determined as the CBP concentration.
  • two tubes of hCLDN18.2 + cells were collected by centrifugation at 300g for 10 minutes, and the number of cells in each tube was 5 ⁇ 10 8 (positive rate was 100% by FACS detection).
  • the cells were washed once with PBS, centrifuged at 300g for 10 minutes, and the cells were collected into 15ml centrifuge tubes.
  • Example 17 Detection of the endocytosis activity of the antibody of the present invention
  • hCLDN18.2 + cell reached 90%, trypsinize and resuspend the cells with FACS buffer (PBS + 1% BSA) to a final concentration of 1 ⁇ 10 6 / ml cells.
  • FACS buffer PBS + 1% BSA
  • Ab10, Ab6 The antibody was labeled with a mix-n-stain CF488 antibody labeling kit, Sigma-Aldrich, Cat # MX488S100-1kit.
  • a mix-n-stain CF633 antibody labeling kit Sigma-Aldrich, Cat # MX633S100-1kit.
  • the labeling steps are performed according to the instructions in the kit), the final concentration is 1 ⁇ g / ml or 10 ⁇ g / ml, incubated for 1 hour on ice, and washed three times with pre-chilled FACS buffer. Take out 1/5 and place it on ice, and use it as a binding value sample for flow detection directly. Resuspend the remaining 4/5 cells with 1640 + 10% FBS pre-heated at 37 ° C, 1/4 of the cells are directly placed on ice, as a sample for 0 hours of endocytosis, and the rest are incubated in a 37 ° C incubator.
  • the binding values (fluorescence intensity) of the antibodies Ab10 and Ab6 of the present invention are 27200, 16300 are 15 times and 9 times the background, respectively, and 14 times (27200/1939) and 8.4 times (16300/1939) of the control antibody. This further proves that the binding Emax of the antibody of the invention is stronger than that of the control antibody (Ref).
  • the fluorescence intensity value of the negative antibody (the antibody that did not bind to hCLDN18.2 + cells), that is, the background value is 1284-3485 (underlined numbers) ).
  • the fluorescence intensity (binding value) of the control antibody (Ref) was 17,900, which was 4 times higher than the background (3485), indicating that the specific binding of the fluorescently labeled antibody was detected only when Ref was as high as 10 ⁇ g / ml.
  • the binding intensities (fluorescence values) of the antibodies Ab10 and Ab6 of the present invention are 138000 and 86000, respectively, 39 and 24 times the background (3485), and 7.7 times (138000/17900) and Ref respectively. 4.8 times (86000/17900). This further illustrates that the binding Emax of the antibodies of the invention is much stronger than the control antibody (Ref). This is also consistent with the results of the KinExA test described above.
  • the read value is the background value and there is no endocytosis; NA: Not applicable, there is no endocytosis at this point in time
  • the preferred humanized antibody of the present invention is an endocytosis antibody.
  • the control antibody (Ref) is not an endocytosis antibody, or the endocytosis is very weak.
  • a fluorescent dye CF633 (Sigma-Aldrich, Cat # MX633S100-1kit) different from CF488 was used for labeling antibody and endocytosis activity analysis.
  • the detection instrument was BD FACS Calibur flow cytometry instrument. The results are shown in the table below.
  • the binding values (fluorescence intensity) of the antibodies Ab10 and Ab6 of the present invention are 854, 690, which are 37 times (854/23) and 30 times (690/23) the background, respectively, and 14 times (854 / 62.5) the control antibodies. ) And 11 times (690 / 62.5). This further proves that the binding Emax of the antibody of the present invention is much stronger than the control antibody (Ref). This is also consistent with the results of KinExA mentioned above.
  • the fluorescence intensity value of the negative antibody (the antibody that did not bind to hCLDN18.2 + cells), that is, the background value was 23.2-14.9 (underlined numbers) ).
  • the fluorescence intensity (binding value) of the control antibody (Ref) is 198, which is 8.5 times that of the background (23.2), indicating that Ref can detect the specific binding of the fluorescently labeled antibody when the Ref is as high as 10 ⁇ g / ml.
  • the binding intensities (fluorescence values) of the antibodies Ab10 and Ab6 of the present invention are 3229 and 2237, respectively, 139 and 96 times the background (23.2), and 16 times (3229/198) respectively Ref. And 11 times (2237/198). This further illustrates that the binding Emax of the antibody of the present invention is much stronger than the control antibody (Ref) (the fluorescence reading is more than 10 times stronger).
  • the read value is the background value and there is no endocytosis; NA: Not applicable, there is no endocytosis at this point in time
  • Example 18 Preparation of anti-CLDN18.2 antibody Ab10 conjugated toxin SMCC-DM1 (ADC1)
  • step 1 an intermediate is prepared.
  • 1mg SMCC (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester, Shanghai Hanhong Chemical Technology Co., Ltd., batch number BH-4857-111203
  • SMCC (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester, Shanghai Hanhong Chemical Technology Co., Ltd., batch number BH-4857-111203
  • the imide acetonitrile solution was shaken at 25 ° C for 2 hours.
  • the product was desalted and purified with a Sephadex G25 gel column (eluent: 0.05M PBS solution with pH 6.5) to obtain an intermediate solution, that is, the molecule between the first step and the second step in the above synthetic circuit, and After concentrating to about 8 mg / ml, proceed to the next reaction.
  • a Sephadex G25 gel column eluent: 0.05M PBS solution with pH 6.5
  • the obtained ADC1 sample was analyzed by LC-MS method, and it was confirmed that there was no free toxin small molecule in the obtained sample.
  • the product was purified by desalting using a Sephadex G25 gel column (eluent: 0.05M PBS solution with pH 6.5) to obtain a solution of the product 1a (see the schematic diagram above), and concentrated 1a to about 10 mg / ml for intermediate Preparation of body 1b.
  • x ⁇ y the product 1a
  • Step 2 Antibody-toxin coupling. Take 1.6 mg of the compound MC-VC-PAB-MMAF (prepared by the method disclosed in PCT patent WO2005081711) and dissolve it in 0.3 ml of acetonitrile, add 5 mg of the intermediate solution Ab10-propanethiol prepared above, and shake the reaction at 25 ° C. After 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05M PBS solution with pH 6.5) to obtain the anti-CLDN18.2 antibody Ab10 conjugated toxin MC-VC-PAB of the present invention. -MMAF conjugate ADC2 (the structure is shown below).
  • the resulting ADC2 had a final concentration of 1.21 mg / ml, and was stored at 4 ° C for later use.
  • the obtained ADC2 sample was detected and analyzed by LC-MS method, and it was confirmed that there was no free toxin small molecule in the obtained sample.
  • Example 20 Detection of anti-CLDN18.2 antibody Ab10 cytotoxic conjugate ADC1, ADC2 binding activity
  • the binding activity of the antibody Ab10 of the present invention and the cytotoxin conjugates ADC1, ADC2, and hCLDN18.2 + cel of the Ab10 antibody of the present invention was detected by the ELISA method described in Example 2. The results are shown in the table below.
  • Example 21 Detection of anti-CLDN18.2 antibody Ab10 cytotoxic conjugate ADC1, ADC2 endocytosis activity
  • the endothelium activity of the antibody Ab10 of the present invention and the cytotoxin conjugates ADC1 and ADC2 of the Ab10 antibody were measured in the same manner as in the above-mentioned "Detection of antibody endocytosis activity of the invention" (Example 17). Based on the above examples, optimization of labeling method (selection of CF633 dye), antibody concentration (2.5 ⁇ g / ml) and time (1 hour) and other endocytic detection conditions, the results are shown in the table below.
  • Example 22 Anti-CLDN18.2 antibody Ab10 cytotoxic conjugate ADC1 and ADC2 inhibit target cell proliferation activity
  • the CCK8 method (kit purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., article number CK04. Operate according to instructions) was used to detect cell proliferation. Specifically, hCLDN18.2 + cells (target cells) and hCLDN18.1 + cells (non-CLDN18.2 target cells as control cells) were cultured in DMEM / F12 medium containing 10% FBS 2 before the end of 72 hours of culture 2 Hours, 10ul CCK8 was added to each well, and the culture was continued for 2 hours in the incubator.
  • the cytotoxin conjugate ADC1 and ADC2 molecules of the Ab10 antibody of the present invention can significantly inhibit the proliferation of target-specific cells (hCLDN18.2 + cell) with IC50 of 11.2 nM and 0.71 nM, respectively.
  • ADC1 has no inhibitory effect on non-target cells (hCLDN18.1 + cells that did not bind to Ab10 antibody), and the safety window is as high as 3000.
  • ADC2 sees inhibition of non-target specific cells at extremely high concentrations (IC50 of 18.5 nM). This inhibition is due to the non-targeted toxicity directly caused by high doses (up to 10-100 nM). This is consistent with the highly toxic characteristics of MMAF connected to ADC2.
  • ADC2 has a safety window of 26.
  • Example 23 Detection of anti-CLDN18.2 antibody Ab10 cytotoxic conjugate targeted cytotoxic activity
  • this example takes ADC1 as an example, and evaluates the anti-CLDN18.2 antibody Ab10 cytotoxicity of the present invention by detecting the release of LDH in the cell supernatant. Conjugate target cytotoxic activity.
  • hCLDN18.2 + cells target cells
  • hCLDN18.1 + cells non-CLDN18.2 target cells, as control cells
  • DMEM / F12 medium containing 10% FBS
  • the cytotoxic conjugate of the Ab10 antibody of the present invention specifically targets hCLDN18.2 + positive cytotoxicity with an EC50 of less than 1 nM.
  • the cytotoxicity of non-targeted cells was weak (no cytotoxicity).
  • ADC1 has a toxicity (EC50) ratio of up to 156 between targeted and non-specific cells.
  • This specific window shows that the non-targeted toxicity of the molecule is weak and the safety is reliable.
  • Example 24 In vivo efficacy of anti-CLDN18.2 antibody Ab10 cytotoxic conjugate
  • NA Not adapted, that is, blank control.
  • the tumor cells were inoculated into nude mice with BALB / c cells using the gastric cancer cell line NUGC-4 overexpressing hCLDN 18.2 to evaluate the animal drug efficacy of ADC2 of the present invention.
  • gastric cancer cell line NUGC-4 cells were purchased from the Institute of Cells, Chinese Academy of Sciences.
  • the hCDLN 18.2 overexpression cell line NUGC-4-802 was constructed using the method of Example 1.
  • NUGC-4-802 was cultured in RPMI1640 medium (Shanghai Yuanpei Biotechnology Co., Ltd., article number: L210KJ) containing 10% fetal bovine serum (Shanghai Bosheng Biotechnology Co., Ltd., article number: BS-0002-500), The cells were continuously cultured in a cell incubator at 37 ° C containing 5% CO 2 . When the NUGC-4-802 cells reach the logarithmic growth phase (confluence rate is 80% -90%), digest with 0.25% trypsin, collect the cells, wash them twice with serum-free RPMI1640 medium, resuspend and count, The cell concentration was adjusted to 5 ⁇ 10 7 cells / ml.
  • BALB / c- nude mice were inoculated with 5 ⁇ 10 6 cells / 100 ⁇ l, and the right ribs were subcutaneously selected, and tumor cells were selected to grow to a volume of about 120-150 mm 3 in size and randomly divided into groups of 6 animals.
  • test samples ADC2 and Ab10 were prepared with PBS and sterile.
  • the Blank group was PBS.
  • the dose was 5 mg / kg and was given intravenously. 2 times / week for 2 consecutive weeks.
  • the day of administration of each injection sample was day 0.
  • Body weight and tumor volume were measured before each administration and data were recorded.
  • the data statistical analysis method is the same as the foregoing embodiment 14.
  • the actual dosing cycle in this experiment was 2 weeks. The results are shown in Table 35b.
  • human CLDN 18.2 used in the present invention is described in the above examples; PD-1, PD-L1 extracellular domain-human IgG1 Fc fusion protein, -his tag protein, monoclonal antibodies and bispecific antibodies of different structures designed, etc. or It can be obtained by cloning, expression and purification of the present invention, or purchased from Beijing Biosafes Biotechnology Co., Ltd. and Beijing Sinobiological Co., Ltd.
  • the antigen sequence was obtained by querying the NCBI database.
  • the antibodies used in the present invention include recombinant antibodies and bispecific antibodies. Except for the human CLDN18.2 antibody sequence found in the present invention, other sequences can be obtained from the published literature, including anti-PD-1 antibodies Nivo, Pem, Ba08 (sequence from patent WO2016015685A1); anti-PD-L1 antibody Atezo (Atezolizumab / Tecentriq), Avel (Avelumab / Bavencio), Durv (Durvalumab / imfinzi); anti-CD47 antibodies hu5F9, iMab, Blincyto, AMG420 light and heavy chain variable region sequences of anti-CD3 antibodies.
  • PD-1 antibody Nivo (Nivolumab / Opidivo) is from public literature, such as www.drugbank.ca, or WO2013019906.
  • PD-1 antibody Pem (Pembrolizumab / Keytruda) sequence from www.drugbank.ca (. PD-1 antibody Ba08 sequence from patent WO2016015685A1.
  • Avel sequence (Accession Number, DB11714) are available from www.drugbank. Ca directly found.
  • the light chain sequence of the CD47 antibody hu5F9 is US9382320B2_42 and the heavy chain sequence is US9382320B2_37.
  • the iMab light chain is WO2018075857_4, 1F8; the heavy chain is WO2018075857_3, 1F8.
  • the CD3 antibody is derived from the anti-CD3 antibody of blincyto. Variable region. Blincyto sequence is the sequence disclosed by www.drugbank.ca. Another CD3 antibody sequence is from the light and heavy chain variable region of the anti-CD3 antibody sequence in AMG420. The AMG420 sequence is from WO2014140248_340.
  • the light chain variable region and heavy of Tim3 The amino acid sequence of the variable region of the chain can be found in SEQ ID NO: 27 and SEQ ID: NO: 36 of CN201710348699.4; the patent has been published; the amino acid sequences of the light chain variable region and heavy chain variable region of LAG3 can be found in CN201810917684X SEQ ID NO: 33 and SEQ ID NO: 44 in the present invention.
  • TGF ⁇ RII TGF ⁇ receptor II
  • sequence of TGF ⁇ receptor II is derived from the extracellular region of UniProtKB / Swiss-Prot: P37173.2 (in the sequence listing of the present invention, SEQ ID ID NO: 1) ;
  • IL The 10 sequence is from NCBI number NP_000563.1 (SEQ ID NO: 2 in the sequence listing of the present invention).
  • the CD47 ligand SIRP ⁇ sequence is from NCBI number NP_001035111.1.
  • All antigens and antibodies including individual antibodies, bispecific antibodies, antibody-receptors (Trap), antibody-cytokines, etc., are synthesized by gene synthesis to obtain synthetic fragments.
  • the design, clone expression, and purification are all accomplished by the present invention. Specifically the same as the foregoing embodiment 3.
  • the target protein was eluted with 250 mM imidazole 0.5 M NaCl in PBS (pH 7.4).
  • Buffer replacement Centrifuge the eluted target protein through an ultrafiltration tube at 12000g for 10 minutes (Ultrafiltration tube Merck Millipore Cat # UFC500308), add 1ml PBS, measure the concentration, aliquot and store for later use.
  • TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3 and IL10 were purchased from Peprotech.
  • the heavy chain constant region can also be hIgG2 or hIgG3.
  • Flow cytometry was used to detect the CD3 binding activity of the present invention designed with a CD3 targeted bispecific antibody.
  • healthy human peripheral blood mononuclear cells PBMC
  • Dynabeads Human T-Activator CD3 / CD28 Gibco, 11131D
  • the activated cells were washed once with FACS buffer (PBS, 0.5% FBS) (800 g, 3 minutes centrifugation) and resuspended in FACS buffer.
  • the cell density was 4 ⁇ 10 6 / ml, and 25 ⁇ l of each sample was reserved.
  • the samples to be tested are diluted with FACS buffer gradient, the highest concentration is 250nM, 5 times downward dilution, 8 concentrations, volume 25 ⁇ l; 25 ⁇ l of the diluted sample is added to 25 ⁇ l cell suspension, gently mixed, and incubated at room temperature for 20 minutes; After washing the cells with FACS buffer larger than 5 times the staining volume, a secondary antibody (anti-hFc-PE, Biolegend, 409304) was added, and after staining at room temperature for 20 minutes, the cells were washed, resuspended, and detected by flow cytometry (Beckman, CytoFLEX flow Cytometer). FlowJo software was used to analyze the average fluorescence signal MFI of the measured samples. MFI was plotted against concentration, and EC50 was calculated using Graphpad Prism 5 software.
  • PBMC adjusted the cell density to 4 ⁇ 10 6 / ml in 1640 complete medium, and 50 ⁇ l of each well was added to hCLDN182 + cells or hCLDN18.1 + cells, respectively.
  • a 1640 complete culture medium was used to prepare a gradient dilution of the test sample at a concentration of 4, 0.4, 0.04, 0.004 ⁇ g / ml, 50 ⁇ l per well was added to the cells, and the final concentration was 1, 0.1, 0.01, 0.001 ⁇ g / ml.
  • Three compound holes were used to prepare a gradient dilution of the test sample at a concentration of 4, 0.4, 0.04, 0.004 ⁇ g / ml, 50 ⁇ l per well was added to the cells, and the final concentration was 1, 0.1, 0.01, 0.001 ⁇ g / ml.
  • Cell killing percentage (%) 100 * (OD 490 concentration of a drug-OD 490 control well) / (OD 490 cells lysed-OD 490 control well)
  • a bispecific antibody is designed and uniformly purified by a Protein A gravity column. Purified samples were replaced in PBS buffer, pH 7.4, 1 mg / ml. Store under different conditions, including -80 ° C for more than 60 days, 4 ° C for 14 or 30 days, 37 ° C for 7 days, and 37 ° C for 14 days. Samples stored under different conditions were evaluated for degradation by electrophoresis (PAGE). The detection of binding activity was used to evaluate whether different storage conditions affected the sample activity. Under the same detection conditions, the detected activity value (EC 50 ) is compared with the activity value detected by the sample stored at -80 ° C. If the ratio changes outside the 2-fold range, it can be considered that the storage conditions have an impact on the stability / activity of the sample. .
  • bispecific antibodies with different sequence structures are designed for two targets of CLDN18.2 and PD-1, as shown in the following table.
  • ⁇ chain indicates that the light chain is a kappa type light chain constant region of human IgG.
  • the following designs that introduce scFv at the C-terminus of the heavy chain all mutate the terminal K to A.
  • a "heavy (light) chain-containing sequence" includes one or more scFvs in addition to the normal heavy (light) chain.
  • bispecific antibodies were cloned, expressed, and purified according to the method of Example 25 of the present invention.
  • the binding activity of these designed bispecific molecules and human CLDN18.2 and PD-1 were tested by the method of the previous example, and the results are shown in the following table.
  • the ratio of 2 indicates that the designed bispecific antibody has a weaker target binding activity compared to the corresponding monoclonal antibody. 1 times. The ratio is less than 2 indicating that the binding activity is not affected; the ratio is between 2 and 5 indicating that the binding activity is slightly affected. At this time, the ratio of the other target should be considered. If the ratio of the other target is smaller, for example, within 1 , The bispecific antibody still has certain application value.
  • LB302 (Ab10 scFv is at the N-terminus of NivoVH and Hc), LB301 (Ab10 scFv is at the N-terminus of PemVH and Hc), and LB307 (Ab10 scFv is at the N-terminus of Ba08VH and Hc), and their respective effects on PD-1 binding activity are 0.68 and 2.1 And 1.5-fold changes; the effects on the binding activity of CLDN 18.2 were 1.18, 3.2, and 6.1, respectively.
  • LB309 (Ab10 scFv is at the C-terminus of NivoVH-Hc)
  • LB308 Ab10 scFv at the C-terminus of Pem VH-Hc
  • LB310 Ab10 scFv at the C-terminus of Ba08VH-Hc
  • their respective effects on the binding activity of PD-1 are Changes of 1.33, 0.93, and 0.56 times; the effects on the binding activity of CLDN 18.2 were 4.2, 6.5, and 7.3 times, respectively. That is, in the same design manner, three bispecific antibody molecules with different PD-1 antibody sequences have little effect on PD-1 binding activity. But it has a great effect on the binding activity of CLDN18.2, and Ab10 scFv has better activity in the C-terminal design (LB309) of NivoVH-Hc.
  • the activity data of LB302 / LB301 / LB307 and LB309 / LB308 / LB310 were compared, and the same sequence was found.
  • the position of scFv at the H-terminus of the N-terminus is smaller than that of the C-terminus, which has a smaller effect on the activity.
  • the design of the bispecific antibody, scFv is located at the H-terminal Nc for a more optimized bispecific antibody design.
  • scFvs of the same target such as scFvs targeting CLDN 18.2 have different positions and unexpectedly affect the activity significantly.
  • the binding activity of LB302 on PD-1 was changed by 0.68 times (close to 1, ie no effect), and the binding activity on CLDN 18.2 was changed by 1.18 times (no effect).
  • the binding activity of LB309 on PD-1 was changed by 4.2 times, that is, the activity was reduced by 3.2 times, and the binding activity of CLDN 18.2 was almost not affected (the activity was changed by 1.33 times). That is, scFv is better at the N-terminus of the heavy chain than at the C-terminus.
  • scFvs targeting the same target such as bispecific antibodies designed by Ab10 scFv and PD-1 antibody (Pem)
  • Ab10 scFv are connected to the light chain of PD-1 antibody, but the positions are different.
  • the effects of activity also unexpectedly vary greatly.
  • the Ab10 scFv of LB312 is at the N-terminus of Pem and Lc
  • the Ab10 scFv of LB313 is at the C-terminus of Pem and Lc.
  • the binding activity of LB313 to CLDN18.2 decreased by more than 10 times, and LB312 decreased by 1.6 times. Both have similar effects on PD-1 binding activity. That is, Ab10 scFv is better at the N-terminus of the light chain than at the C-terminus.
  • the affinity of LB302 and LB301 to PD-1 was measured using Biacore.
  • the affinity of LB302 vs. Nivo (L101), LB301 vs. Pem (L105), and human PD-1-his were measured with a Biacore T200, GE Healthcare instrument.
  • HBS-EP + 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% P20
  • Protein A (Thermo Pierce, Cat # 21181) was first coupled to the biosensor chip CM5 (Cat.
  • the chip was activated with freshly prepared 50 mM NHS (N-hydroxysuccinimide) and 200 mM EDC (1-ethyl-3- (3-dimethylamino propyl) carbodiimide hydrochloride), and then injected at pH 4.0 10 mM 10 ⁇ g / ml Protein A formulated with NaAC. Dilute the sample to be tested with running buffer to a concentration of 1 ⁇ g / ml, capture the signal at about 50 RU, the concentration gradient of the antigen PD-1-his starts from 100 nM, 3-fold dilution, the flow rate is 30 ⁇ l / min, the binding time is 180 seconds, and the dissociation time is 300 second.
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3- (3-dimethylamino propyl) carbodiimide hydrochloride
  • the chip was washed with 10 mM Glycine-HCl, pH 1.5, 30 ⁇ l / min, and 30 s.
  • the experimental data was fitted with Biacore T200 evaluation 3.0 (GE) software using a 1: 1 Langmuir model to obtain the affinity value KD.
  • the affinity (KD) measured by the Biacore is consistent with the aforementioned ELISA result, that is, the SBody molecules LB302 and LB301 of the present invention maintain the binding activity of PD-1 which is close to the corresponding antibody.
  • LB3022, LB3012 designed based on Ab6 sequence is similar to LB302 and LB301.
  • IgG-like bispecific antibodies (anti-CLDN18.2 and human PD-1) SBody designed based on the antibodies Ab10, Ab6, etc. of the present invention are sequence-dependent and sequence-position-dependent.
  • bi10 specific antibodies designed based on the Ab10 sequence can achieve unexpected results, that is, the preferred design, such as LB302, retains the binding activity of the dual target.
  • the method of the foregoing embodiment was used to evaluate the preferred molecular-induced apoptosis activity (%) of the design of the present invention to compare the functional activity against CLDN 18.2.
  • Ab10 antibody was used under the same conditions and the same concentration (150 nM in this experiment) as a control.
  • the anti-PD-1 functional activity was evaluated by detecting the PD-1 antigen and ligand PD-L1 binding activity (IC50). The results are shown in the table below.
  • IC50 change factor that is, the ratio of IC50 of the bispecific antibody and the corresponding monoclonal antibody (control antibody). The larger the ratio, the more the functional activity of the designed bispecific antibody against a single target is weakened. For example, the ratio is 2, the functional activity of the designed bispecific antibody on the target is weakened compared to the corresponding monoclonal antibody. 1 times. The ratio is within 2 as the experimental error range, that is, the activity is not affected.
  • NA Ab10 is not suitable for PD-1 functional activity detection because Ab10 has no PD-1 functional activity.
  • LB302, LB308, and LB310 all retain the functional activity of the anti-PD-1 antibody and have not been reduced, but have been enhanced (LB310).
  • the increased activity may reflect the synergistic effects of sequence design relatedness.
  • the functional activity data of Anti-CLDN18.2 showed that Ab10 scFv at the N-terminus of the heavy chain (LB302, LB301, LB307) had an enhancement / synergy (compared with Ab10 antibody) on the activity of Anti-CLDN18.2 cells, and different PD-1 This is true for antibodies (Pem, Nivo, Ba08).
  • Ab10 scFv retained Anti-CLDN 18.2 cell activity at the C-terminus of the heavy chain (LB309, LB308, LB310).
  • the bispecific antibody designed by the present invention has the same ab10 antibody scFv sequence and different PD-1 antibody positions (LB302 vs LB309, LB301 vs LB308, LB307 vs LB310), the same Ab10 sequence scFv sequence position and different PD-1 antibodies (LB302 vs. LB301 vs. LB307, LB309 vs. LB308 vs. LB310), the same Ab10 and PD-1 antibodies scFv or Ab10 scFv sequence and PD-1 antibody (LB156 vs LB301), unexpectedly found that different designs, expression levels and sequence correlation, differ greatly.
  • the highest yield LB309 is 67 times higher than the lowest yield LB156 (28.7 / 0.42).
  • the expression of the same Ab10 scFv at the N-terminus (LB312) and C-terminus (LB313) of the light chain of the same PD-1 antibody (Pem) is also different.
  • the present invention designs bispecific antibodies with different sequence structures for the two targets of CLDN18.2 and PD-L1, as shown in the table below.
  • the sequence of the hIgG1 constant region is the same as that of the corresponding PD-L1 antibody hIgG1.
  • the same Fc as Atezo, with N297A mutation is the same.
  • bispecific antibodies were cloned, expressed, and purified according to the method of Example 25 of the present invention, and the binding activities of these bispecific molecules and human CLDN18.2 and PD-L1 were detected by the methods of the foregoing examples, and the results are shown in the following table.
  • the values in parentheses are the binding activity EC50 of the monoclonal antibody corresponding to the target under the same experimental conditions.
  • * The ratio of the binding activity EC50 of the bispecific antibody and the corresponding monoclonal antibody under the same experimental conditions. The larger the ratio, the more the binding ability of the designed bispecific antibody to a single target is weakened. For example, the ratio of 2 indicates that the designed bispecific antibody has a weaker target binding activity compared to the corresponding monoclonal antibody. 1 times. The ratio is within 2 indicating that the binding activity is not affected.
  • bispecific antibody with similar IgG structure designed by the combination of the antibody Ab10 and Atezo sequences of the present invention can retain the binding activity to two targets, which can be obtained by conventional protein purification method Protein A, the process is simple, and the stability is good.
  • the LB311 data showed that the SBody of Ab10 scFv linked to the C-terminus of the PD-L1 antibody Atezo heavy chain did not affect the binding activity to hCLDN18.2 and PD-L1.
  • LB316 and LB317 show that in the SBody designed by Ab10 scFv and PD-L1 antibody Avel, Ab10 scFv performs better at the N-terminus of Avel heavy chain, while Ab10 scFv performs better at both ends of Atezo heavy chain.
  • the LB319 and LB320 data show that in the SBody designed by Ab10 scFv and PD-L1 antibody Durv, Ab10 scFv at the N-terminus of the Durv heavy chain has no effect on the binding activity of CLDN18.2 and PD-L1. Ab10 scFv at the C-terminus of the Durv heavy chain slightly affects the binding activity of CLDN18.2, but has no effect on the binding activity of PD-L1.
  • the affinity of LB157, LB305, LB185 (Atezo), and PD-L1 was measured using Biacore.
  • the Biacore method is the same as the method described in LB302 above.
  • PD-L1-his available from Sinobiological, Cat #: 10084-H08H was used in place of PD-1-his.
  • mice from Ccnc line purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Zhejiang) 2018-0001
  • SCXK Zhejiang
  • MC38 cells purchased from the Institute of Cell Sciences, Chinese Academy of Sciences
  • MC38-804 required for this experiment (the construction method was the same as in Example 1 above, and CHO-K1 was used instead of MC38).
  • MC38-804 is cultured with 10% fetal bovine serum (Shanghai Bosheng Biotechnology Co., Ltd., article number: BS-0002-500), 1% Hepes (Thermo Fisher Scientific (China) Co., Ltd., article number: 15630080)
  • DMEM / high sugar medium Shanghai Yuanpei Biotechnology Co., Ltd., article number: L110KJ
  • the cells were continuously cultured in a cell incubator at 37 ° C containing 5% CO 2 .
  • C57BL / 6cnc female mice, 6 weeks old, 5 / cages were housed in an SPF environment, temperature 20-25 ° C, humidity 40% -60%, free access to food and water, regular replacement of litter.
  • MC38-804 cells grow to the logarithmic growth phase (confluence rate is 80% -90%), digest with 0.25% trypsin, collect the cells, and wash the cells twice with serum-free DMEM / high sugar medium, and finally Resuspend the serum-free DMEM / high-sugar medium, count the cells, and adjust the cell concentration to 1 ⁇ 10 7 cells / ml with Matrigel (purchased from Biodi Medical Devices Shanghai Co., Ltd., article number: 354234).
  • MC38-804 cell suspension (1 ⁇ 10 6 cells) was inoculated with 100 ⁇ l subcutaneously in the right ribs of mice. Tumor cells were selected to grow to a volume of about 120-150 mm 3 in size and randomly divided into groups of 6 animals.
  • the test sample and the positive control were prepared with PBS and sterile.
  • the Blank group was PBS.
  • PD-L1 antibody LB185 + Ab10 was used as a control group.
  • LB157 and LB305 are the test groups of their respective bispecific antibody drugs.
  • the administration method was intraperitoneal injection.
  • the mice in the LB185 + Ab10 combined control group were administered with 20 ⁇ g of each antibody and 200 ⁇ l per mouse.
  • LB157 mice were administered at a dose of 26 ⁇ g / 200 ⁇ l / head, and LB305 was administered at a dose of 26 ⁇ g / 200 ⁇ l / head (and LB185 and Ab10 in the combined group were equimolar doses).
  • the administration frequency of each group was 2 times / week for 1.5 consecutive weeks.
  • the day of administration of each injection sample was day 0. Body weight and tumor volume were measured before each administration and data were recorded. The actual administration period in this experiment was 1.5 weeks, and the measurement period was 21 days.
  • the present invention designs bispecific antibodies with different sequence structures for the two targets of CLDN18.2 and CD47, as shown in the table below.
  • bispecific antibodies were cloned, expressed, and purified according to the method of Example 25 of the present invention, and the binding activities of these bispecific molecules and human CLDN18.2 and CD47 were detected by the method of the foregoing example, and the results are shown in the following table.
  • the value in parentheses is the binding activity EC50 of the monoclonal antibody corresponding to the target under the same experimental conditions.
  • * The ratio of the binding activity EC50 of the bispecific antibody and the corresponding monoclonal antibody under the same experimental conditions. The larger the ratio, the more the binding ability of the designed bispecific antibody to a single target is weakened. For example, the ratio of 2 indicates that the designed bispecific antibody has a weaker target binding activity compared to the corresponding monoclonal antibody. 1 times. The ratio is within 2 indicating that the binding activity is not affected.
  • the functional activity of CLDN18.2 was evaluated by inducing tumor cell apoptosis. LB158 induced tumor cell apoptosis by 11%. Under the same conditions, Ab10 induced tumor cell apoptosis by 10.3%. That is, the functional activity of LB158 anti-CLDN 18.2 was not reduced.
  • the stability test results showed that LB158 was stored at -80 ° C for 60 days, 4 ° C for 14 days, and 37 ° C for 7 days and 14 days. No degradation was detected by electrophoresis (PAGE) analysis.
  • the activity test results showed that the binding activities of these 4 samples to CLDN 18.2 were 0.68 nM, 0.98 nM, 0.67 nM, and 0.73 nM, respectively.
  • the binding activities to CD47 were 0.08 nM, 0.12 nM, 0.16 nM, and 0.10 nM, respectively. It shows that under these storage conditions, LB158 is not only molecularly stable (electrophoresis results), but also stable in activity.
  • the LB321 results showed that the anti-CLDN18.2 antibody and another CD47 antibody (iMab) designed as a bispecific antibody (iMab scFv at the N-terminus of the heavy chain of Ab10) retained the anti-CLDN18.2 binding activity, and the CD47 Binding activity increased by a factor of 1.5 (EC 50 of 0.484 nM vs 1.38 nM).
  • the functional activity test results showed that LB321 prevented CD47 and ligand SIRP ⁇ activity and IC50 and LS956 (iMab) changed by 1.64 times. This shows that the functional activity of anti-CD47 antibody of LB321 is not affected.
  • the stability test results showed that the binding activities of LB321 and CLDN18.2 and CD47 remained unchanged at -80 ° C for 60 days and 4 ° C for 14 days; the EC50 and CLDN18.2 binding activity at 50 ° C for 14 days remained at 0.99 nM When it became 10.1 nM, the activity was reduced by nearly 10 times; when the binding activity with CD47 was changed from 0.77 nM to 12.3 nM, the activity was decreased by 15 times. It shows that LB321 is stable under 4 °C or below. It also shows that the SBody of the same design is different from the CD47 antibody sequence. For example, in terms of stability, LB158 is more stable than LB321.
  • bispecific antibodies with different sequence structures are designed for two targets of CLDN18.2 and CD3, as shown in the following table.
  • Example 25 of the present invention the above-mentioned bispecific antibodies were cloned, expressed, and purified, and the bispecific molecules and human CLDN18.2 + cell binding (ELISA) and CD3 (T cell) FACS binding activities were detected by the method of the previous embodiment. The results are shown in the table below.
  • the value in parentheses is the binding activity EC50 of the monoclonal antibody (Ab10) corresponding to the target under the same experimental conditions.
  • Ab10 monoclonal antibody
  • ELISA binding activity
  • the larger the ratio the more the binding of the designed bispecific antibody to CLDN18.2 is weakened.
  • the ratio of 2 indicates that the designed bispecific antibody has a doubled CLDN18.2 binding activity compared with Ab10.
  • the ratio is less than 2, indicating that the binding activity is not affected significantly (1 times the experimental error range).
  • the values in parentheses are the combined EC50 (FACS) values of LB155 under the same experimental conditions. **: Ratio of the binding activity of the bispecific antibody and LB155 under the same conditions. This ratio reflects the difference in binding activity between different CD3 antibodies and different structures such as VH-linker-VL and VL-linker-VH.
  • the preferred molecule LB1952 substantially retains the binding activity to CLDN18.2 and CD3.
  • the functional activity analysis showed that LB195 and LB193 activated CDC activity (using the method of the previous example to evaluate the function of anti-CLDN 18.2) relative to Ab10 activity (EC 50 ) and the fold changes were 1.7 and 2.2 times, respectively. These multiples of change are within the experimental error range, that is, LB195 and LB193 retain their functional activity on CLDN 18.2.
  • the activity of killing target cells by activating PBMC (see the previous example for the method). It was found that the activities of LB195 and LB193 are equivalent, and the specific killing effect on target cells is dose-effect relationship. There is 30% -40% of target cells at a concentration of 0.1 ⁇ g / ml. Cracked.
  • the present invention designs bispecific antibodies with different sequences similar to the IgG structure for the two targets of CLDN18.2 and TGF ⁇ .
  • the light or heavy N- or C-terminus of the antibody Ab10, Ab6 of the present invention preferably the C-terminus of the heavy chain Fusion of TGF ⁇ receptor II, the resulting molecule (TRAP) can bind to CLDN18.2, and at the same time can bind TGF ⁇ 1, 2, 3, the design is shown in the table below.
  • LB824 is the control molecule.
  • the sequences are derived from WO2015118175_1 ( ⁇ chain) and WO2015118175_3. It is a Trap molecule composed of PD-L1 antibody Avel and TGF ⁇ receptor II (TGF ⁇ RII). Its TGF ⁇ RII binding activity can be used as a control of the molecule LB401 of the present invention.
  • bispecific antibodies were cloned, expressed, and purified according to the method of Example 25 of the present invention, and the binding activities of these bispecific molecules and human CLDN18.2 and TGF ⁇ were detected by the methods of the foregoing examples, and the results are shown in the following table.
  • LB401 binding activity results indicate that the C-terminus of the antibody Ab10 heavy chain linked to TGF ⁇ RII not only retains its binding activity to human CLDN18.2, but also retains its binding activity to TGF ⁇ I and a high selectivity to TGF ⁇ II and TGF ⁇ III.
  • LB401 and LB824 on TGF ⁇ it was found that the binding activity of LB401 to TGF ⁇ 1, 2, 3 obtained by the antibody Ab10 of the present invention to TGF ⁇ RII is consistent with the Trap (LB824) binding profile (Profile) of TGF ⁇ RII fused by PD-L1 antibody.
  • LB401 was stable at -80 ° C for 60 days, 4 ° C for 30 days, 37 ° C for 7 days, and 37 ° C for 14 days.
  • Electrophoresis (PAGE) analysis revealed that degradation occurred at 14 ° C for 14 days. TGF ⁇ I, II, III binding activity was weakened, and CLDN18.2 binding activity was not affected, as shown in Figure 8. Therefore, LB401 is stable at -80 ° C or 4 ° C.
  • the same animal model (MC38-804) and method described in Example 30 were used to evaluate the in vivo pharmacodynamic effect of the bispecific antibody (TRAP) designed by the present invention against CLDN 18.2 and TGF ⁇ .
  • the test sample and the positive control were prepared with PBS and sterile.
  • the Blank group was PBS.
  • Ab10 is the control group for single medication
  • LB824 is the control group for bispecific antibody.
  • LB401 is the preferred bispecific antibody drug test group of the present invention.
  • the administration method is intraperitoneal injection, the dosage of Ab10 is 120 ⁇ g / 200 ⁇ l / head, and the dosage of LB824 and LB401 is 160 ⁇ g / 200 ⁇ l / head.
  • the administration frequency of each group was 2 times / week for 3 consecutive weeks. The results are shown in the table below.
  • the anti-CLDN18.2 antibody and TGF ⁇ RII of the present invention are optimized for designing a bispecific molecule that retains good activity against dual targets, has significant animal efficacy, and is stable in molecules.
  • the purification process (Protein A binding) is simple and easy to implement.
  • LB401 light chain sequence (SEQ ID NO: 38), which is the Ab10 light chain, see Example 6.
  • the Ab10 antibody and cytokine fusion molecule are designed for two targets of CLDN18.2 and IL10.
  • an IL10 molecule is fused to the light and heavy N and / or C-terminus of the antibody Ab10, Ab6 of the present invention, preferably the C-terminus and N-terminus of the heavy chain, and the design is shown in the table below.
  • # LB4333 is the same as LB433 except that its L234 mutation in the Fc region is A; # LB4334 is the same as LB433 except that its L235 mutation in the Fc region is A; # LB4335 is the same as LB433 except that its Fc region L234 and L235 are mutant A
  • bispecific antibodies were cloned, expressed, and purified according to the method of Example 25 of the present invention, and the binding activities of these bispecific molecules and human CLDN18.2 and IL10 were detected by the methods of the foregoing examples, and the results are shown in the following table.
  • the value in parentheses is the binding activity EC50 of the monoclonal antibody (Ab10) corresponding to the target under the same experimental conditions.
  • binding activity results show that the bispecific molecules LB432 and LB433, LB4332 and CLDN18.2 designed by the antibody Ab10 and cytokine IL10 of the present invention have the same binding activity as Ab10 and Ab6 (the activity changes about 2 times).
  • Binding activity to IL10 was evaluated using antigen-coated ELISA and sandwich ELISA. Specifically, the antigen coating ELISA detection method is described in Example 3 above. Results The EC 50 of LB432, LB433 and LB4332 were 0.34, 0.14 and 0.2nM, respectively. The activity difference is about 2 times, which is close to the experimental error range.
  • Sandwich ELISA method Take Example 2 to construct human CLDN18.2 +, apply 10x10 4 / well to 96-well plate (Corning, Cat # CLS3599-100EA), incubate at 37 ° C overnight, remove the supernatant, and use immunostaining Fixing solution (Shanghai Biyuntian Biotechnology Co., Ltd., Cat # P0098) was fixed at 100 ⁇ l / well for half an hour at room temperature. After washing once in PBS, 230 ⁇ l of 5% milk was blocked at 37 ° C. for 2 hours, and washed 3 times in PBST.
  • LB432 and LB433 were evaluated for serum stability. Specifically, the blood of C57BL / 6 mice (six-week-old female, purchased from Shanghai Xipuer-Bikai Laboratory Animal Co., Ltd.) was centrifuged at 12,000 rpm for 10 minutes, and the serum was collected for use. A 3 ⁇ l sample (1 ⁇ g / ⁇ l, pH 7.4 PBS) was diluted with 27 ⁇ l of the above serum to a final concentration of 0.1 ⁇ g / ml. After being treated at 37 ° C for 0h, 24h and 72h, the binding activity to human CLDN18.2 + cells and IL10 antibody was detected. The results are as follows.
  • the same animal model (MC38-804) and method described in Example 30 were used to evaluate the in vivo efficacy of the bispecific antibodies designed for CLDN 18.2 and IL10 of the present invention.
  • the test sample and the positive control were prepared with PBS and sterile.
  • the Blank group was PBS.
  • Ab10 is a control group used alone.
  • LB433 is a test group of bispecific antibody drugs.
  • the method of administration is intraperitoneal injection, the dosage of Ab10 is 60 ⁇ g / 200 ⁇ l / head, and the dosage of LB433 is 80 ⁇ g / 200 ⁇ l / head.
  • the administration frequency of each group was 2 times / week for 1.5 consecutive weeks. The results are shown in the table below.
  • LB4333 light chain sequence (SEQ ID NO: 38); LB4333 heavy chain sequence (SEQ ID NO: 59);
  • LB4331 light chain sequence (SEQ ID NO: 38); LB4331 heavy chain sequence (SEQ ID NO: 60);
  • LB4335 light chain sequence (SEQ ID NO: 38); LB4335 heavy chain sequence (SEQ ID NO: 61):
  • bispecific antibodies are designed for two targets of CLDN18.2 and LAG3, and two targets of CLDN18.2 and Tim3, as shown in the table below.
  • bispecific antibodies were cloned, expressed, and purified according to the method of Example 25 of the present invention, and the binding activities of these bispecific molecules and human CLDN18.2 and LAG3 and Tim3 were respectively detected by the aforementioned ELISA method.
  • the value in parentheses is the binding activity EC50 of the monoclonal antibody (Ab10) corresponding to the target under the same experimental conditions.
  • the antibodies of the present invention Ab10, LAG3, and Tim3 are designed to be bispecific antibody molecules (scFv at the N-terminus of Ab10 heavy chain) similar to the IgG structure, which can retain the binding activity to CLDN18.2, LAG3, and Tim3, and can be stable Expression purification.
  • Example 36 Structural design and evaluation of DVD with CDDN 18.2 and PD-1, PD-L1, and CD47 dual-target antibodies, respectively
  • the present invention designs bispecific antibodies against CLDN18.2 and PD-1, PD-L1, and CD47 in the form of DVD, respectively, as shown in the table below.
  • Table 56 Design of dual specific antibodies in the form of DVD against the dual targets of CLDN18.2.2 / PD-1, CLDN18.2.2 / PD-L1, CLDN18.2.2 / CD47
  • bispecific antibodies were cloned, expressed, and purified according to the method of Example 25 of the present invention.
  • the results of gel electrophoresis (PAGE) showed that the light and heavy chains of these antibodies were susceptible to interlinker breakage.
  • one target antibody is connected to the N or C terminus of the light or heavy chain of another target antibody, and a design-optimized bispecific antibody can be obtained by screening, which can avoid / reduce interlinker breaks (see the previous implementation) Example), and retain the binding activity and functional activity to the dual target.
  • the bispecific antibody (referred to as SBody in the present invention) is not only stable, but also has a simple purification process because it resembles a conventional IgG structure, which provides great convenience for the process and purification in the later development process.
  • the new CAR molecule design of the present invention directed to CLDN 18.2 refers to the previously published patent CN106755107A.
  • the nucleic acid construct of the CAR molecule designed by the present invention has the general formula CAR-[(IRES) -f] q .
  • CAR stands for chimeric antigen receptor, including, scFv-H-TM-S-CD3 ⁇ , where scFv (single chain Fv) is a single-chain variable fragment that specifically targets the CLDN18.2 antigen, or Single chain antibodies, single chain variable regions. Its sequence consists of the variable region sequences of the anti-CLDN18.2 antibodies (see the previous examples) found in the present invention.
  • H is a hinge domain
  • TM is a transmembrane domain
  • S is a costimulatory signal transduction region.
  • the costimulatory signaling region includes a costimulatory molecule derived from CD28, and / or a costimulatory molecule derived from 4-1BB.
  • CD3 ⁇ is a cytoplasmic signal transduction sequence (intracellular region) derived from CD3 ⁇ .
  • IRES represents the internal ribosome entry site sequence (Internal Ribosome Entry Site, IRES); f represents the functional protein F, and q is a 0 or non-zero natural number.
  • the functional protein includes a cytokine IL10, IL15 or an active fragment thereof, and / or a cytokine receptor such as an IL15 receptor or an active fragment thereof, and / or a cytokine such as IL10, IL15 or an active fragment thereof and IL15. Fusion fragment of the receptor sushi + fragment.
  • the CAR molecule also does not have an IRES sequence.
  • IRES and (IRES) represent the same meaning.
  • IRES contains parentheses "()"
  • the nucleotide sequence before and after the IRES sequence encodes different protein fragments (ie CAR and f), and the different protein fragments are separated from each other.
  • the sequence of the scFv designed based on the variable region of the anti-CLDN18.2 antibody sequence is the novel antibody sequence of the present invention, see the above examples.
  • the scFV can also be a Fab or single domain antibody (sdFv) structure.
  • sequences other than scFv can be searched from the National Medical Library website http://www.pubmed.com, GenBank database, including human CD8 ⁇ signal peptide, human CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, human CD28 intracellular region, human 4-1BB intracellular region, human CD3 ⁇ intracellular region, internal ribosome entry elements (IRES elements), human IL15 (same as SEQ ID NO. 22 in patent CN106755107A), human IL15 receptor alpha ( IL15R ⁇ ) wild type and mutation / sushi portion (US2014 / 01314; WO2007 / 046006), human IL10 (SEQ ID NO: 2) protein sequence and the like. All base sequences constructed into the clone are codon optimized based on the protein sequence to ensure that it is more suitable for human cell expression without changing the coding amino acid sequence.
  • the nucleotide sequence of IL10 is, for example, the sequence shown by GenBank Accession No. NM_000572.
  • the representative CAR molecule construction method of the present invention refers to the method described in Patent Application Publication No. CN106755107A.
  • the plasmid JX1a of the CAR new molecule CAR1a of the present invention is obtained.
  • the amino acid sequence of CAR1a encoded by plasmid JX1a is:
  • positions 1-246 are scFv coding sequences that bind to Ab10; positions 247-293 are human CD8 ⁇ hinge region coding sequences (underlined); positions 294-315 are human CD8 ⁇ transmembrane coding regions; positions 316-357 are 4-1BB coding sequence for the intracellular region; positions 358-469 are coding sequences for the intracellular signal region of CD3zeta ( ⁇ ).
  • the first to 63 nucleotides are signal peptide coding regions.
  • Positions 64-801 are the scFv coding sequences of the Ab10 antibody that binds CLDN18.2; positions 802-942 are the coding sequences of the human CD8 ⁇ hinge region; positions 943-1008 are the coding sequences of the human CD8 ⁇ transmembrane region; positions 1009-1134 are 4-1BB intracellular region coding sequence; 1135-1470th is the CD3zeta ( ⁇ ) intracellular signal region coding sequence.
  • the scFv encoding the c-Met antibody scFv in the JX007 plasmid constructed in Example 5 of the patent application with publication number CN106755107A was replaced with the Ab10 scFv of the present invention (the method is the same as the method of constructing the JX1a of the present invention) to obtain the new CAR3ab molecule CAR3ab of the present invention.
  • Plasmid JX3ab The amino acid sequence encoded by the plasmid JX3ab is the same as the above-mentioned coding sequence of JX1a, and in addition, it encodes the cytokine IL15 active fragment (wild type).
  • the nucleotide sequence of the IL15 active fragment can be any sequence used in the art to encode it, such as the nucleotide sequence of positions 699-1040 of SEQ ID NO: 31 of CN106755107A.
  • the IL15-encoding sequence (for example, the nucleotide sequence of positions 699-1040 of SEQ ID NO: 31 of CN106755107A) in the plasmid JX3ab constructed above is replaced with a sequence encoding IL10 (for example, the sequence shown by GenBank accession number NM_000572)
  • the plasmid JX3ab10 of the new CAR molecule CAR3ab10 of the present invention was obtained.
  • the above plasmid JX3ab10 encodes the following IL10 protein sequence:
  • the scFv encoding the c-Met antibody scFv in the JX008 plasmid constructed in Example 6 of CN106755107A patent is replaced with the Ab10 scFv of the present invention (the method is the same as the method of constructing the JX1a of the present invention), and the plasmid JX4a used for the CAR new molecule CAR4a of the present invention is obtained.
  • the amino acid sequence encoded by JX4a also encodes a fusion protein of the cytokine IL15 active fragment (mutant) and IL15R ⁇ (sushi +); sushi + means that in addition to the sushi fragment, other polypeptide fragments are included.
  • the scFv of the Ab6 antibody sequence was used instead of the scFv of the Ab10 antibody to construct CAR new molecules CAR1a.2, CAR3ab.2, CAR3ab10.2, and CAR4a.2.
  • the virus preparation method refers to the method used in the patent CN106755107A, using a three-plasmid virus packaging system pGag-Pol, pVSVG and each new CAR molecule expression plasmid pBABEpuro (all purchased from Youbao Bio), such as JX1a, JX3ab 293 cells were co-transfected with JX3ab10 or JX4a to obtain the virus supernatant, and the virus was concentrated by ultracentrifugation.
  • the virus supernatant was carefully spread on top of the sucrose solution, 125,000 g, centrifuged for 1.5 hours, the pellet was resuspended in PBS at low temperature, divided into packs, and frozen at -80 ° C.
  • the viruses are denoted as 1a, 3ab, 3ab10, and 4a, respectively.
  • Virus titer detection 293 cells were infected with a gradient dilution of the virus, and 48 hours later, protein L staining was used to determine the positive rate of scFv-expressing cells to determine the virus titer. Specifically, 20 ⁇ l of the virus (1a, 3ab, 3ab10, 4a) were taken, and RPMI 1640 medium (Peiyuan Bio, Cat # L210KJ) contained 10% FBS (Gibco, Cat #: 10099141), 0.8 ⁇ g polybrene (Shanghai ⁇ Sheng Biotechnology Co., Ltd. Cat #: 40804ES76) 5 times gradient dilution, the final system is 250 ⁇ l.
  • the total culture system is 500 ⁇ l / well, 293 5 ⁇ 10 4 cells / well. After 48 hours, cells were collected and labeled with biotin-protein L (Gensray, article number M00097) at 293, 1 ⁇ l / sample. After incubating at room temperature for 20 min, 1 ml of FACS buffer was added and the cells were washed by centrifugation. Resuspend 100 ⁇ l of FACS buffer, add 0.4 ⁇ l / sample of PE-labeled Strepavidin (eBioscience, Cat. No. 12-4317-87), incubate at room temperature for 20 min, add 1 ml of FACS buffer, and centrifuge cells to wash.
  • PE-labeled Strepavidin eBioscience, Cat. No. 12-4317-87
  • the results showed that the titers of the 1a, 3ab, 3ab10 and 4a viruses obtained this time were 9.2, 1.3, 3.2 and 1.5 ⁇ 10 6 IU / ml, respectively.
  • PBMC peripheral blood mononuclear cells
  • the medium was RPMI 1640 containing 10% FBS, 500 IU / mL IL2 (Beijing Sihuan Biopharmaceutical Co., Ltd., Cat #: S20040007). Change the fluid every other day and expand the culture volume 1: 2 until enough cells are obtained for in vivo and in vitro experiments.
  • the T cells (CART cells) of each CAR molecule after infection were recorded as CART1a, CART3ab, CART3ab10, and CART4a cells; uninfected T cells (empty vector) were used as negative cells (control).
  • CART1a.2, CART3ab.2., CART3ab10.2 and CART4a.2. Were prepared in the same manner as described above.
  • the positive rate of Ab6 antibody scFv was similar to that of CART1a, CART3ab, CART3ab10 and CART4a, as shown in Table 57b.
  • Table 57b Positive rate of Ab6 antibody scFv expression in CART cells of the present invention
  • CART3ab The new CART cells designed by the present invention, CART3ab, CART4a express secreted cytokines IL15, IL15 / IL15R; CART3ab10 expresses secreted cytokines IL10.
  • the CART cell culture supernatant was taken and the expressions of IL10 and IL15 were detected using an ELISA kit (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product number SEKA10947, SEK10360).
  • the ELISA method could not detect the expression and secretion of IL15 and IL15 / IL15R ⁇ in the supernatant of CART3ab and CART4a, indicating that the amount of cytokines IL15 and IL15 / IL15R ⁇ secreted by cultured CART3ab and CART4a was low.
  • mice Since the cell culture supernatant could not detect the expression of IL15 and IL15 / IL15R ⁇ , the four CART cells produced were injected into mice. Balb / c nude mice were taken and given 1 ⁇ 10 6 CART cells by intravenous injection. Serum was taken on the 7th and 14th day after injection, and the levels of IL10 and IL15 in the serum were detected by ELISA. The results are shown in the table below.
  • CART3ab and CART4a cells designed by the present invention express and secrete IL15 active fragments.
  • CART3ab10 cells express and secrete IL10.
  • human CLDN18.2 overexpressing cells (hCLDN18.2 + cells) were used as target cells, and human CLDN18.1 overexpressing cells (hCLDN18.1 + cells) were used as control negative target cells.
  • the target cell survival ratio was used to evaluate the specific in vitro killing activity of CART cells.
  • hCLDN18.2 + cells were labeled with CFSE (Biolegend, Cat. No. 423801).
  • a target cell suspension was prepared, and hCLDN18.1 + cells and CFSE-labeled hCLDN18.2 + cells were mixed in equal proportions, each 1.5 ⁇ 10 5 cells / ml.
  • Target cell killing experiments were performed in 24-well plates, with 100 ⁇ l of target cell suspension per well.
  • CART1a, CART3ab, CART3ab10, CART4a cells and negative cells (empty vector) were diluted with the same medium to form different ratios of CART cells and target cells, respectively 20: 1, 10: 1, 3: 1, and 1: 1 .
  • a non-kill group was set up, that is, no CART cells, only the above target cell group.
  • Target cell specific lysis (kill) rate 1- [CART cells 7AAD negative hCLDN18.2 + / hCLN18.1 +] / [negative control (empty vector) cells 7AAD negative hCLDN18.2 + / hCLN18.1 +].
  • the target cell has a high specific lysis (kill) rate, that is, the specific killing effect of CART cells is strong.
  • the following is the calculation result of the CART1a kill rate when CART cells and target cells are 10: 1.
  • the data of the negative control CART cells showed that the live cells CLDN18.1: CLDN18.2 were 50.5% vs. 48.6%, close to 1: 1, indicating that the negative control CART cells had no killing effect on non-target cells and target cells.
  • CAR1a cell results show that CLDN18.1: CLDN18.2 is 57.5% vs. 37.5% in living cells, indicating that CART1a cells have no killing effect on non-target cells (close to 50%), but have killing effect on target cells (CLDN18.2). Effect (reduced from 50% to 37.5%).
  • mice were subcutaneously inoculated with hCLDN18.2 + cells to establish a tumor model.
  • CART cells were injected intravenously, and tumor volume (TV) and body weight (BW) were measured to evaluate the anti-tumor effect and safety of CART cells.
  • TV tumor volume
  • BW body weight
  • hCLDN18.2 + cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, and continuously cultured to a logarithmic growth phase in a cell incubator at 37 ° C containing 5% CO2 (the confluence rate was 80% -90%), trypsinize, collect cells, wash the cells twice with serum-free DMEM / F12, resuspend in PBS, count, and adjust the cell concentration to 1 ⁇ 10 8 / ml.
  • Balb / c nude mice were each inoculated with 100ul of hCLDN18.2 + cell suspension, subcutaneously in the right rib.
  • mice with a tumor volume of 80-130 mm 3 were selected and grouped (2 mice / group) by intravenous injection of CART cells 2 ⁇ 10 6 / mice. The day of dosing was day 0. After that, the tumor volume was measured twice a week, the body weight was weighed, and the data was recorded.
  • Example 40 CARNK cell activity obtained by transfecting CAR with NK cells designed against CLDN 18.2
  • NK92 cells purchased from Mall Bei Na Chuanglian Biotechnology Co., Ltd.
  • virus 1a, 3a, 3ab10, 4a MOI in the range of 0.5-5
  • polybrene final concentration 8 ⁇ g / mL
  • the culture medium was a special medium for NK92 (Mandabei Chuanglian Biological Technology Co., Ltd.). Change the fluid every other day and expand the culture volume 1: 2 until enough cells are obtained for in vivo and in vitro experiments.
  • Infected NK92 cells were recorded as CARNK1a, CARNK3a, CARNK3ab10, and CARNK4a cells; uninfected NK92 cells were recorded as CARNK (empty vector) as a negative control. Seven days after infection, the surface expression of CARNK cells was detected by the same method as in Example 38, and the results are shown in the following table.
  • CARNK cells Positive rate (%) CARNK1a twenty one CARNK3a twenty two CARNK3ab10 30 CARNK4a 41
  • the animal model of the CARNK cells of the present invention was evaluated in the same animal model as in Example 7 above. Take the prepared CARNK (empty vector) control, CARNK1a, CARNK3ab, CARNK3ab10, CARNK4a cells 2 ⁇ 10 5 / each, 2 in each group, Day 0 and Day 3 were injected once. The tumor size was measured twice a week thereafter, and the results are shown in Table 61 below.

Abstract

提供了一种靶向CLDN18.2的抗体，其包含VL和/或VH，所述VL包含以下的CDR序列：如SEQ ID NO：11或SEQ ID NO：12所示的VL CDR1氨基酸序列；如SEQ ID NO：13所示的VL CDR2氨基酸序列；如SEQ ID NO：14所示的VL CDR3氨基酸序列；所述VH包含以下的CDR序列：如SEQ ID NO：15所示的VH CDR1氨基酸序列；如SEQ ID NO：16所示的VH CDR2氨基酸序列；如SEQ ID NO：17所示的VH CDR3氨基酸序列。还公开了一种靶向CLDN18.2的双特异性抗体、上述抗体的偶联体、靶向CLDN18.2的CAR分子及含其的细胞，以及他们的应用。

Description

靶向CLDN18.2的抗体、双特异性抗体、ADC和CAR及其应用
本申请要求申请日为2018年6月17日的中国专利申请CN 201810610790.3、2018年11月01日的中国专利申请CN 201811295845.2、2019年02月03日的中国专利申请CN 201910108951.3、2019年04月08日的中国专利申请CN 201910276473.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药领域，尤其涉及一种靶向CLDN18.2的抗体、双特异性抗体、ADC和CAR及其应用。
背景技术
癌症是人类健康的巨大威胁，也是疾病领域导致死亡的重要原因之一。癌症治疗经历手术、化疗、靶向药物、肿瘤免疫治疗、联合治疗等各阶段的发展后，近年已经取得了巨大成就。在众多的癌症患者中，肺癌、胃癌、胰腺癌、食道癌和卵巢癌等癌症患者的治疗手段仍然高度未满足。针对这些肿瘤的治疗手段，包括大分子靶向药物如新的单克隆抗体，以及这些单克隆抗体和已有的肿瘤免疫治疗手段，如与免疫检查点抑制剂PD-1、PD-L1抗体联合治疗，为巨大的未满足的临床治疗的需求提供了新的可能和选择。
细胞连接密蛋白(密蛋白，Claudin或CLDN)在人、鼠等物种中都有表达，是细胞间层密封关联蛋白，在控制对细胞层间离子流、维持细胞极性和细胞间信号转递具有重要作用。CLDN家族蛋白已经发现的有29种之多，CLDN18就是其中之一。CLDN18有两个同源分子，分别称为密蛋白18.1(CLDN18.1)和密蛋白18.2(CLDN18.2)。人密蛋白18.1(hCLDN18.1)和人密蛋白18.2(hCLDN18.2)高度同源，氨基酸同源性高达92％。hCLDN18.2在正常组织表达非常有限，仅见于胃黏膜分化上皮细胞，但在胃癌包括转移胃癌组织有特别的高表达(Sahin U.et al.Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody delveopment.Clin Cancer Res.2008；14(23):7642-34)。进一步发现，CLDN18.2在不同癌症，包括大约70％的胃癌、50％的胰腺癌、30％的食道癌、25％的肺癌和卵巢癌等的患者组织都有表达。因此，CLDN18.2早已经成为理想的肿瘤患者标记物和抗肿瘤药物开发靶点，特别是靶向CLDN18.2的抗体开发用于肿瘤治疗，但因为靶点的特殊性，开发针对CLDN18.2治疗性抗体非常困难。人CLDN18.2蛋白全长261个氨基酸，见NCBI公开序列NP_001002026.1 claudin-18 isoform 2，其中1-23为信 号肽。CLDN18.2蛋白是一个跨膜蛋白，有两个膜外区域分别为信号肽后面大约55个氨基酸的胞外区1(Extracellular loop 1，ECL1)和23个氨基酸ECL2。这一结构和人CLDN18.1非常相似，而且人CLDN18.2和人CLDN18.1的ECL2区域则完全相同。因而针对人CLDN18.2蛋白靶点抗体的开发，需要寻找针对人CLDN18.2蛋白的ECL1区域或空间结构的抗体。这使得这方面的工作变得更加困难。
此外，针对人CLDN18.2膜蛋白的抗体发挥其功效至少包括诱导肿瘤细胞凋亡、抑制肿瘤细胞生长，通过和病人免疫细胞的效应作用，包括抗体依赖细胞毒性(ADCC)，和补体依赖细胞毒性(CDC)效应细胞介导的杀伤肿瘤细胞作用。而要寻找这样功能的抗体会更加困难。目前针对人CLDN18.2抗体研究只有IMAB362抗体(参见WO 2014/146672)在临床试验阶段。IMAB362是针对人CLDN18.2的抗体，是人、鼠源嵌合抗体，存在免疫原性风险，亲和力不很高。细胞学实验证明其无内吞活性，不适合做ADC开发，且治疗效果极为有限，这在本发明的肿瘤模型药效评价中也得到了验证。此外，针对CLDN18.2靶点还没有进入临床或前期研发的双特异抗体。针对CLDN18.2靶点目前还没有在临床上开发的抗体药物偶联体(antibody drug conjugates，ADC)药物。嵌合抗原受体(CAR)T细胞(简称CART或CAR-T)是从患者体内分离出T细胞，在体外利用CAR改造修饰T细胞，使其能够特异性识别癌细胞，将改造后的CART细胞扩增、回输至患者体内，达到治疗肿瘤的效果。CN201410341504.X公开了一种及靶向CLD18A2(即Claudin18.2)的T淋巴细胞及其制备方法和应用，也有临床数据报道，但是CART药效和安全性还有很大的提升空间。
因此，本领域缺乏有效的靶向人CLDN18.2蛋白的抗体特别是人源化的抗体，以及细胞活性、PK活性和动物药效等更好的抗体、双特异性抗体、抗体药物偶联体(ADC)药物，以及更加有效的嵌合抗原受体(CAR)及含其的细胞。
发明内容
为克服本领域缺乏靶向CLDN18.2的抗体、双特异抗体、抗体药物偶联体和CAR分子的缺陷的技术问题，提供了一种靶向CLDN18.2的抗体、人源化抗体、双特异性抗体及其抗体药物偶联体、CAR分子及其制备方法和应用。
为解决上述技术问题，本发明第一方面的技术方案为：一种靶向CLDN18.2的抗体，其包含轻链可变区(VL)和/或重链可变区(VH)，所述VL包含以下的互补决定区(CDR)序列：
如SEQ ID NO:11或SEQ ID NO:12所示的VL CDR1氨基酸序列；如SEQ ID NO: 13所示的VL CDR2氨基酸序列；如SEQ ID NO:14所示的VL CDR3氨基酸序列；
所述VH包含以下的CDR序列：
如SEQ ID NO:15所示的VH CDR1氨基酸序列；如SEQ ID NO:16所示的VH CDR2氨基酸序列；如SEQ ID NO:17所示的VH CDR3氨基酸序列。
所述靶向CLDN18.2的抗体和人、鼠CLDN18.2特异结合活性更好，Emax更高；亲和力(KinExA)高(优选达到了10pM级)；人血细胞中CDC活性更好；诱导CLDN18.2+细胞凋亡活性更好，抑制肿瘤细胞生长活性更好，动物药效更优，且体内药物代谢(PK)好，特别是更长的T1/2。其和鼠CLDN18.2有更好的结合活性。
在一优选的实施例中，如上所述的靶向CLDN18.2的抗体，所述VL包含如SEQ ID NO:11所示的VL CDR1、SEQ ID NO:13所示的VL CDR2和SEQ ID NO:14所示的VL CDR3的氨基酸序列；所述VH包含如SEQ ID NO:15所示的VH CDR1、SEQ ID NO:16所示的VH CDR2和SEQ ID NO:17所示的VH CDR3的氨基酸序列；或，
所述VL包含如SEQ ID NO:12所示的VL CDR1、SEQ ID NO:13所示的VL CDR2和SEQ ID NO:14所示的VL CDR3的氨基酸序列；所述VH包含如SEQ ID NO:15所示的VH CDR1、SEQ ID NO:16所示的VH CDR2和SEQ ID NO:17所示的VH CDR3的氨基酸序列。
在本发明一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述抗体的CDR区为脱氨基化敏感位点优化的CDR序列；优选地，所述CDR区脱氨基化敏感位点优化的CDR序列为轻链CDR序列；优选地，所述CDR区脱氨基化敏感位点优化的CDR序列为轻链CDR1第L30A和/或L30B位优化的CDR1序列。
在本发明的一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述抗体的轻链CDR1第L30A和/或L30B的NS突变为NT，且前提是第L30E位不为Q和第L34位不为T。
在本发明的一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述抗体的轻链CDR1第L30A和/或L30B的NS突变为NT，且前提是突变前所述轻链CDR1为如SEQ ID NO:12所示的序列。
在本发明一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述抗体的CDR区为脱氨基化敏感位点优化的CDR序列；优选地，所述CDR区脱氨基化敏感位点优化的CDR序列为重链CDR序列；优选地，所述CDR区脱氨基化敏感位点优化的CDR序列为重链CDR3第H99和/或H100位优化的CDR序列。
在本发明的一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其 中所述抗体的重链CDR3第H99和/或H100位的NS突变为NT，且前提是轻链CDR1第L30E位不为Q和第L34位不为T。
在本发明的一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述抗体的重链CDR3第H99和/或H100位的NS突变为NT，且前提是突变前所述轻链CDR1为如SEQ ID NO:12所示的序列。
其中，如上所述的轻链CDR1第L30A、L30B、L30E和L34位，重链CDR3第H99和H100位以Kabat编号规则定义。
在一优选的实施例中，如上所述的靶向CLDN18.2的抗体，所述靶向CLDN18.2的抗体为鼠源抗体；所述鼠源CLDN18.2抗体经亲和力(affinity)成熟，其亲和力提高3-10倍或以上，优选10倍或以上。
优选地，所述鼠源抗体的VL为如SEQ ID NO:7所示的氨基酸序列或其突变；和/或，所述鼠源抗体的VH为如SEQ ID NO:8所示的氨基酸序列或其突变；
所述突变为所述VL和/或VH的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所述VL和/或VH的氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体与CLDN18.2的结合；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％序列同一性；最优选为至少99％序列同一性。
在一优选的实施例中，如上所述的靶向CLDN18.2的抗体，所述靶向CLDN18.2的抗体包含鼠源抗体的可变区和鼠或人抗体恒定区；所述鼠抗体恒定区包括鼠IgG1、IgG2a、IgG2b3或IgG3的重链恒定区和κ或λ型轻链恒定区，所述人抗体恒定区包括人IgG1、IgG2、IgG3或IgG4的重链恒定区和κ或λ型轻链恒定区。
优选地，所述靶向CLDN18.2的抗体为鼠源抗体的可变区和人抗体恒定区组合成的嵌合抗体。所述嵌合抗体经亲和力(affinity)成熟，其亲和力提高3-10倍或以上，优选10倍或以上。
更优选地，所述嵌合抗体的轻链氨基酸序列为如SEQ ID NO:9所示的氨基酸序列或其突变；和/或，所述嵌合抗体的重链氨基酸序列为如SEQ ID NO:10所示的氨基酸序列或其突变。
在一优选的实施例中，如上所述的靶向CLDN18.2的抗体，所述靶向CLDN18.2的抗体为人源化抗体。
在本发明一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述人源化抗体的轻链可变区框架(FR)序列选自人种系轻链序列，FR序列优选包含1- 10个氨基酸回复突变。
在本发明一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述人源化抗体的轻链可变区CDR序列可以分别按照CCG、Kabat、Chothia、AbM或Contact等编号规则定义，所述CDR序列包含表4-表8所列轻链CDR序列或其突变。
优选地，所述人源化抗体的VL包含如SEQ ID NO:29-33中任一个所示的氨基酸序列或其突变；所述突变为所述VL和/或VH的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所述VL和/或VH的氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体与CLDN18.2的结合；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％序列同一性；最优选为至少99％序列同一性。
更优选地，轻链CDR1第L30E和L34位优化(突变)成人种系CDR1相应位点的氨基酸。
在本发明一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述人源化抗体的重链可变区框架(FR)序列选自人种系重链序列，FR序列优选包含0-10个氨基酸回复突变。
在本发明一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述人源化抗体的重链可变区CDR序列可以分别按照CCG、Kabat、Chothia、AbM或Contact等编号规则定义，所述CDR序列包含表4-表8所列重链CDR序列或其突变序列。
在本发明一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述人源化抗体的重链可变区序列包含SEQ ID NO:34-37中任一个所示的氨基酸序列或其突变。
在本发明一个优选的实施方案中，提供一种如上所述靶向CLDN18.2的抗体，其中所述人源化抗体包含序列为SEQ ID NO:29-33中任一个所示的氨基酸序列或其突变的轻链可变区和序列为SEQ ID NO:34-37中任一个所示的氨基酸序列或其突变的重链可变区的组合。
在一优选的实施例中，如上所述的靶向CLDN18.2的抗体，所述抗体的轻链包含选自人抗体κ或λ型轻链恒定区或其突变；和/或，所述抗体的重链包含选自人IgG1、IgG2、IgG3和IgG4的重链恒定区或其突变；
优选地，所述重链恒定区或其变体，包含人IgG1 Fc区第234位、第235位和第243位，或第239、第330和第332位突变；
更优选地，所述重链恒定区或其变体，包含人IgG1 Fc区第356-358位为EEM或DEL的变体。
在一优选的实施例中，如上所述的靶向CLDN18.2的抗体，所述轻链包含SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42或SEQ ID NO:45所示的氨基酸序列或其突变；和/或，
所述重链包含SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:44或SEQ ID NO:46所示的氨基酸序列或其突变。
在一优选的实施例中，如上所述的靶向CLDN18.2的抗体，其包含以下轻链和重链：
所述重链如SEQ ID NO:39的氨基酸序列所示，所述轻链如SEQ ID NO:38的氨基酸序列所示；或，所述重链如SEQ ID NO:39的氨基酸序列所示，所述轻链如SEQ ID NO:40的氨基酸序列所示；或，所述重链如SEQ ID NO:41的氨基酸序列所示，所述轻链如SEQ ID NO:38的氨基酸序列所示；或，所述重链如SEQ ID NO:41的氨基酸序列所示，所述轻链如SEQ ID NO:40的氨基酸序列所示；或，所述重链如SEQ ID NO:39的氨基酸序列所示，所述轻链如SEQ ID NO:42的氨基酸序列所示；或，所述重链如SEQ ID NO:43的氨基酸序列所示，所述轻链如SEQ ID NO:42的氨基酸序列所示；或，所述重链如SEQ ID NO:44的氨基酸序列所示，所述轻链如SEQ ID NO:42的氨基酸序列所示；或，所述重链如SEQ ID NO:43的氨基酸序列所示，所述轻链如SEQ ID NO:45的氨基酸序列所示；或，所述重链如SEQ ID NO:44的氨基酸序列所示，所述轻链如SEQ ID NO:45的氨基酸序列所示；或，所述重链如SEQ ID NO:39的氨基酸序列所示，所述轻链如SEQ ID NO:45的氨基酸序列所示；或，所述重链如SEQ ID NO:46的氨基酸序列所示，所述轻链如SEQ ID NO:38的氨基酸序列所示。
在一优选的实施例中，如上所述的靶向CLDN18.2的抗体，其中所述靶向CLDN18.2的抗体包括免疫球蛋白、Fab、Fab’、F(ab’) 2、Fv或单链Fv片段(scFv)。
为解决上述技术问题，本发明的第二方面的技术方案为：提供一种双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，所述第一蛋白功能区为如第一方面的技术方案所述的靶向CLDN18.2的抗体；所述第二蛋白功能区为靶向非CLDN18.2抗原的抗体。所述的双特异性抗体既能保留单个CLDN18.2抗体所具有的结合活性、功能活性，也能够保持另一个蛋白功能区的结合和功能活性。而且，所述双特异性抗体结构类似正常IgG抗体，可以按常规抗体表达纯化方法，完成表达和纯化，并且稳定。
本发明的双特异性抗体为序列特异的IgG结构类似的双特异抗体( Sequence-based IgG like bispecific anti body,SBody)。
在一具体实施例中，如上所述的双特异性抗体，所述非CLDN18.2抗原为免疫检查点抗原或肿瘤治疗靶点，所述免疫检查点抗原包括PD-1、PD-L1、Tim3、LAG3、CD47；所述肿瘤治疗靶点包括SIRPα(signal regulatory proteinα，信号调节蛋白α)等，SIRPα是一种膜蛋白，主要在髓系细胞，包括巨噬细胞、树突状细胞等表达。更优选地，所述第二蛋白功能区包括抗PD-1抗体、抗PD-L1抗体、抗Tim3抗体、抗LAG3抗体、抗CD47抗体、抗CD3抗体和抗CSF-1R抗体。最优选地，所述抗PD-1抗体为Nivolumab(简称Nivo)、Pembrolizumab(简称Pem)或者Ba08(即专利申请CN201410369300中所述Ba08-1)。所述抗PD-L1抗体为Atezolumab(简称Atezo)、Avelumab(简称Avel)或者Durvalumab(简称Durv)，所述抗CD3抗体为Blincyto或AMG420中结合CD3的轻、重链可变区序列构建的抗体。
或，所述第二蛋白功能区为细胞因子和细胞因子受体或它们的片段；优选地，所述细胞因子或它的片段包括TGFβ、IL10和CSF-1，所述细胞因子受体或它的片段包括TGFβRII、IL10受体和巨噬细胞集落刺激因子1受体(CSF-1R)。
在一些具体实施例中，如上所述的双特异性抗体，所述抗体为免疫球蛋白、scFv(single chain Fv，也称为单链可变片段)、Fab、Fab’或F(ab’) 2。较佳地，所述免疫球蛋白的恒定区为人抗体恒定区，所述人抗体恒定区包括人抗体轻链恒定区和人抗体重链恒定区，所述人抗体轻链恒定区优选κ链或者λ链，所述人抗体重链恒定区优选hIgG1、hIgG2或者hIgG4。
为了设计生产工艺简单且保留有效活性的双特异抗体，本发明的双特异性抗体的形式为类似正常IgG的结构，具体地，在其结构上设计能够靶向两个靶点的轻链和/或者重链可变区的蛋白功能区，两个蛋白功能区共享相同的重链Fc区域。较佳地，将一个靶点的抗体分子以一个或者多个scFv的形式，或者完整的细胞因子或它的片段，或者完整的细胞因子受体或它的片段，连到另一靶点完整抗体的轻链或重链的一端。这样既避免表达不同重链Fc和/或不同轻链带来表达产物的不均一，例如Knob形式的Fc和Hole形式的Fc共表达，其表达过程中会有不均一的Fc-Fc配对形式，给纯化工艺带来很多不便利；也可以避免轻、重链部分区域交换(cross)设计可能对结构活性的影响，以及工艺过程中出现的Fc错配现象。通过一个或者多个scFv设计，还可以调节针对特定靶点的活性。
在一些具体实施例中，如上所述的双特异性抗体，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为一个或多个scFv、细胞因子或它的片段、或细胞因子受体或它的片段；或者，所述第二蛋白功能区为免疫球蛋白，所述的第一蛋白功能区为一个或多个scFv；其中，所述scFv包括重链可变区与轻链可变区，所述重链可变区与轻链可变 区通过连接子连接，所述连接子优选(Gly-Gly-Gly-Gly-Ser) w[以下简称(G 4S) w]；所述scFv、细胞因子或它的片段、或细胞因子受体或它的片段通过连接子与所述免疫球蛋白连接，所述连接子选自可以是本领域常见的肽段或(G 4S) w；所述的w优选为0～10之间的整数，更优选为1、2、3或者4。
所述双特异设计概括(通式1)如下表。
表01 双特异性抗体的设计
Figure PCTCN2019090255-appb-000001
表01中，含轻链的序列指该序列除了包括轻链序列以外，还可以包括与轻链序列连接的scFv；含重链的序列指该序列除了包括重链序列以外，还可以包括与重链序列连接的scFv。为表述方便，表中的scFv在本文中如非特别指明，除本领域技术人员理解的含义外，还可以是细胞因子和细胞因子受体或它们的片段的序列。其中，T1代表针对靶点1的第一蛋白功能区，T2代表针对靶点2的第二蛋白功能区。T1(scFv)代表针对靶点1抗体的scFv序列；T2(scFv)代表针对靶点2抗体的scFv序列。
(scFv) n1，(scFv) n2，(scFv) n3，(scFv) n4中的n1，n2，n3，n4分别为自然数，可以是0、1、2、3等，在本发明的具体实施例中，n1，n2，n3，n4中其中至少1个数值为1，其余为0。VL，代表针对靶点1或者2的抗体轻链可变区序列；VH，代表针对靶点1或者2的抗体重链可变区序列。Lc，代表轻链(κ或者λ)的恒定区序列，优选人轻链恒定区序列；Hc代表重链，包括IgG1、IgG2、IgG3、IgG4等的恒定区序列，优选人的重链恒定区序列。重链恒定区C末端连接scFv或其它蛋白序列的时候，其C末端末位氨基酸K可以突变，优选突变为A。由此，在方案1中，T1为免疫球蛋白，T2为scFv；在方案2中，T2为免疫球蛋白，T1为scFv；scFv针对的靶点相同；在方案3、4中，两端的scFv针对两个不同的靶点。
当表01中的scFv为常规的scFv，即其不为细胞因子和细胞因子受体或它们的片段的序列时，所述scFv为轻链可变区-连接子-重链可变区，其轻链可变区N末端或重链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；或所述scFv为重链可变区-连接子-轻链可变区，其重链可变区N末端或者轻链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端。
需说明的是，当上述scFv为轻链可变区-连接子-重链可变区时，其连接方式为轻链可变区的C末端与连接子连接，所述连接子再与重链可变区的N末端连接，从而将scFv轻链可变区的N末端和重链可变区的C末端暴露出来，使其可以通过连接子与免疫球蛋白的轻链和或重链连接。在本发明中，当其连接免疫球蛋白的轻链时，在一些具体的实施例中优选地使用scFv的重链可变区的C末端通过连接子与免疫球蛋白轻链的N末端连接；当其连接免疫球蛋白的重链时，在一些具体的实施例中优选地使用scFv的轻链可变区的N末端与免疫球蛋白重链的C末端连接。
当所述scFv为重链可变区-连接子-轻链可变区时，其连接方式为轻链可变区的N末端与连接子连接，所述连接子再与重链可变区的C末端连接，从而将scFv轻链可变区的C末端和重链可变区的N末端暴露出来，使其可以通过连接子与免疫球蛋白的轻链和或重链连接。在此情况下，当其连接免疫球蛋白的轻链时，在一些具体的实施例中优选地使用scFv的轻链可变区的C末端与免疫球蛋白轻链的N末端连接；当其连接免疫球蛋白的重链时，在一些具体的实施例中优选地使用scFv的重链可变区的N末端与免疫球蛋白重链的C末端连接。优选地，所述连接子为(G 4S) 3，和/或，所述scFv的数量为两个，对称地连接在所述的免疫球蛋白轻链和/或重链。
更佳地，所述的双特异性抗体选自以下任一种：
(1)所述第一蛋白功能区为免疫球蛋白，所述免疫球蛋白包括轻链如SEQ ID NO:38所示、重链如SEQ ID NO:39所示的氨基酸序列；所述第二蛋白功能区为scFv；其中，
两个scFv的重链可变区的C末端通过连接子对称地连接在所述免疫球蛋白的两条重链的N末端；且，所述scFv的轻链可变区为Atezo的轻链可变区，所述scFv的重链可变区为Atezo的重链可变区；或，
两个scFv的重链可变区的C末端通过连接子对称地连接在所述免疫球蛋白的两条重链可变区的N末端；且，所述scFv的轻链可变区为Hu5F9的轻链可变区，所述scFv的重链可变区为Hu5F9的轻链可变区；或，
两个scFv的重链可变区的N末端通过连接子对称地连接在所述免疫球蛋白的两条重链的C末端；且，所述scFv的轻链可变区为AMG420的轻链可变区，所述scFv的重 链可变区为AMG420的重链可变区。
此外，所述的双特异性抗体还可以包括以下结构，所述第一蛋白功能区为免疫球蛋白，所述免疫球蛋白包括轻链如SEQ ID NO:38或SEQ ID NO:42所示、重链如SEQ ID NO:39所示的氨基酸序列；所述第二蛋白功能区为scFv：
所述scFv的轻链可变区为iMab的轻链可变区(即WO2018075857_4序列)，重链可变区为iMab的重链可变区(即WO2018075857_3序列)；或，所述scFv的轻链可变区的序列为Tim3的轻链可变区(如专利申请CN201710348699.4中的SEQ ID NO:27所示)，所述scFv的重链可变区为Tim3的重链可变区(如专利申请CN201710348699.4中的SEQ ID NO:36所示)；或，所述scFv的轻链可变区的序列为BlincytoCD3的轻链可变区，所述scFv的重链可变区为BlincytoCD3的重链可变区；或，所述scFv的轻链可变区的序列为Pem的轻链可变区，所述scFv的重链可变区为Pem的重链可变区；或，
(2)所述第一蛋白功能区为scFv，所述第二蛋白功能区为免疫球蛋白；两个scFv的重链可变区的C末端通过连接子对称地连接在所述免疫球蛋白的两条重链的N末端；所述scFv的轻链可变区的序列如SEQ ID NO:29所示，所述scFv的重链可变区的序列如SEQ ID NO:34所示；其中，
所述免疫球蛋白包括Nivo的轻链可变区、轻链恒定区为κ链、Nivo的重链可变区以及重链恒定区为hIgG4的氨基酸序列；或，
所述免疫球蛋白包括Pem的轻链可变区、轻链恒定区为κ链、Pem的重链可变区以及重链恒定区为hIgG4的氨基酸序列；或，
所述免疫球蛋白包括Atezo的轻链可变区、轻链恒定区为κ链、Atezo的重链可变区以及重链恒定区为hIgG1的氨基酸序列。
同样地，所述的双特异性抗体还可以包括以下免疫球蛋白：所述第一蛋白功能区为scFv，所述第二蛋白功能区为免疫球蛋白，轻链恒定区为κ链、重链恒定区为hIgG1的氨基酸序列；其中，所述免疫球蛋白的轻链可变区为iMab的轻链可变区(即WO2018075857_4序列)，重链可变区为iMab的重链可变区(即WO2018075857_3序列)；或，所述免疫球蛋白的轻链可变区为Tim3的轻链可变区(如专利申请CN201710348699.4中的SEQ ID NO:27所示)，所述免疫球蛋白的重链可变区为Tim3的重链可变区(如专利申请CN201710348699.4中的SEQ ID NO:36所示)；或，所述免疫球蛋白的轻链可变区为Hu5F9的轻链可变区，重链可变区为Hu5F9的轻链可变区；所述免疫球蛋白的轻链可变区的序列为Avel的轻链可变区，所述免疫球蛋白的重链可变区为Avel的重链可变区；或，所述免疫球蛋白的轻链可变区的序列为BlincytoCD3的轻链可变区，所述免疫球 蛋白的重链可变区为BlincytoCD3的重链可变区。
当表01中T1(scFv)，T2(scFv)中的scFv为细胞因子或它的片段、或细胞因子受体或它的片段时，其结构为连接子(Linker)-细胞因子受体及其变体序列，或者细胞因子及其变体序列-Linker。Linker的另一端连接免疫球蛋白的轻链和或重链的N和或C末端，优选(G 4S) w，w为0、1、2、3、4；优选w＝3或w＝4。
在一具体的实施例中，如上所述的双特异性抗体，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为细胞因子或它的片段、或细胞因子受体或它的片段；所述细胞因子或它的片段、或细胞因子受体或它的片段的数量优选为两个或四个；其通过连接子对称地与所述免疫球蛋白两条轻链和/或两条重链的C末端和/或N末端连接，所述连接子优选为(G 4S) 3；
优选地，所述免疫球蛋白包括轻链如SEQ ID NO:38所示；重链如SEQ ID NO:39所示的氨基酸序列；其中，
所述细胞因子或它的片段、或细胞因子受体或它的片段为TGFβRII，其序列如SEQ ID NO:1所示，且数量为两个；所述TGFβRII对称地连接在所述免疫球蛋白的两条重链的C末端，其C末端氨基酸由K突变为A；或，
所述细胞因子或它的片段、或细胞因子受体或它的片段为IL10，其序列如SEQ ID NO:2所示，且数量为两个；所述IL10对称地连接在所述免疫球蛋白的两条重链的C末端，其C末端氨基酸由K突变为A。
在一些较佳的具体实施例中，如上所述的双特异性抗体，所述的双特异性抗体包括以下轻链氨基酸序列和含重链的氨基酸序列：
如SEQ ID NO:53所示的轻链氨基酸序列，如SEQ ID NO:54所示的含重链的氨基酸序列；或，如SEQ ID NO:55所示的轻链氨基酸序列，如SEQ ID NO:56所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:57所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:58所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:59所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:60所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:61所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:4所示的含重链的氨基酸序列。
或者，本发明所述的双特异性抗体为DVD-Ig(Dual-variable domain Ig)双特异性抗体，其结构是在正常抗体轻、重链的N末端分别再接入另一个抗体的VL和VH，通过两 个抗体可变区结合双靶点实现双功能。
所述双特异性抗体的设计概括(通式2)如下表
表02 双特异性抗体的DVD结构设计
Figure PCTCN2019090255-appb-000002
T1、T2分别代表针对靶点1和靶点2。表02中，含轻链的序列指该序列除了包括正常完整的轻链序列以外，还包括另一个轻链可变区序列。含重链的序列指该序列除了包括正常完整的重链序列以外，还包括另一个重链可变区序列。轻链可变区和完整的轻链之间、重链可变区和完整的重链之间通过连接子(Linker)连接。
在一具体实施例中，所述的双特异性抗体为DVD-Ig双特异性抗体。较佳地，所述第二蛋白功能区包含正常抗体完整的轻链和重链，所述第一蛋白功能区包含轻链可变区和重链可变区；或，所述第一蛋白功能区包含正常抗体完整的轻链和重链，所述第二蛋白功能区包含轻链可变区和重链可变区。更佳地，所述轻链与所述轻链可变区之间，所述的重链与所述的重链可变区之间均通过连接子连接；所述的连接子优选(G 4S) w，所述的w优选为0～10之间的整数，更优选为1、2、3或者4。
在一优选的具体实施例中，所述的双特异性抗体由含轻链的序列和含重链的序列组成。所述的双特异性抗体选自以下组合：含轻链的序列为Ab10VL-(G 4S) 3-NivoVL-Lc(κ链)，含重链的序列为Ab10VH-(G 4S) 3-NivoVH-Hc(hIgG4)；或，含轻链的序列为AtezoVL-(G 4S) 3-Ab10VL-Lc(κ链)，含重链的序列为AtezoVH-(G 4S) 3-Ab10VH-Hc(hIgG1)；或，含轻链的序列为Hu5F9VL-(G 4S) 3-Ab10VL-Lc(κ链)，含重链的序列为Hu5F9VH-(G 4S) 3-Ab10VH-Hc(hIgG1)。
或者，本发明所述的双特异性抗体为包括第一蛋白功能区和第二蛋白功能区，其中之一的蛋白功能区为免疫球蛋白，另一蛋白功能区为Fab’或F(ab’) 2。
为解决上述技术问题，本发明的第三方面技术方案为：提供一种抗体药物偶联体(ADC)，其结构如下式I所示：
Ab-[(L 2) n-L 1–D] y   式I
其中，D为具有细胞毒性的小分子药物，L 1和L 2为分别连接所述药物和所述抗体的接头；n为0或1；y表示偶联于Ab的D的平均数，且0<y≤10，优选2≤y≤7；更优选3≤y≤6；最优选4.4或4.8；
所述Ab为如本发明的第一方面所述的靶向CLDN18.2的抗体，或如本发明的第二 方面所述的双特异性抗体。
在抗体的药物偶联体的制备过程中，依据偶联的方法，如果是定点偶联，则抗体携带药物的数量确定，抗体药物偶联体则可以是单一产物(非混合物)。如果是位点随机偶联，不同抗体上携带的药物偶联体分子数量实际上是不同的，因此本发明所述的抗体的药物偶联体实为一种混合物，且通式中的y反映了混合物中抗体携带药物偶联体的平均值。其在数值计算后通常表现为非整数的正数，例如4.4或4.8。来自偶联反应的ADC制剂中的每个抗体的药物部分的平均数可以通过常规方法例如质谱法、ELISA测定和HPLC来表征。还可以测定ADC的定量分布，在一些情况下，将y为某个值的均匀ADC从具有其它载药量的ADC分离、纯化和表征可以通过诸如逆相HPLC或电泳的方法来实现。
本发明的通式中，接头L 2若存在(即n为1)，则L 2、L 1和D的数量相同，此时抗体的药物偶联体为Ab-[L 2-L 1–D] y；若L 2不存在(即n为0)，即仅包括一种接头的情况下，L 1和D的数量相同，此时抗体的药物偶联体实际应为Ab-[L 1–D] y。此外，本发明通式所示的抗体的药物偶联体实为理想状态下的抗体的药物偶联体，即通式只考虑了连接有连接子L 1的L 2(若存在)的数量、连接有D的L 1的数量。而本领域技术人员皆知，在实际合成的抗体的药物偶联体中，应存在未连接D的接头，因此抗体实际偶联的药物分子的平均数量应≤y，即y为偶联至抗体的药物的理论最大值。
在本发明一个优选的实施方案中，提供一种抗体药物偶联体，所述小分子药物为选自毒素、化疗剂、抗生素、放射性同位素和核溶酶的细胞毒剂。
优选地，所述小分子药物选自以下组：单甲基澳瑞他汀(monomethyl auristatin)、美登素(maytansine)类生物碱、喜树碱类生物碱、加利车霉素、多柔比星(阿霉素)、多卡霉素(duocarmycin)，或其组合。更优选地，所述单甲基澳瑞他汀为单甲基澳瑞他汀E(MMAE)或单甲基澳瑞他汀F(MMAF)，所述美登素类为N 2'-脱乙酰基-N 2'-(3-巯基-1-氧代丙基)-美登素(DM1)，N 2'-脱乙酰基-N 2'-(4-巯基-1-氧代戊基)-美登素(DM3)和N 2'-脱乙酰基-N 2'-(4-巯基-4-甲基-1-氧代戊基)-美登素(DM4)。
在本发明一个优选的实施方案中，提供一种抗体药物偶联体，所述L 1选自可切割接头、不可切割接头、亲水接头、预先带电荷的接头和基于二羧酸的接头。优选地，所述接头选自N-琥珀酰亚胺基4-(2-吡啶基二硫代)戊酸酯(SPP)、N-琥珀酰亚胺基(4-碘乙酰基)氨基苯甲酸酯(SIAB)、N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷羧酸酯(SMCC)、6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、缬氨酸-瓜氨酸(VC)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)和MC-VC-PAB。
在本发明一个优选的实施方案中，提供一种抗体药物偶联体，所述L 2为以下式II所 示的化合物：
Figure PCTCN2019090255-appb-000003
其中，X 1选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
X 2选自烷基、环烷基和杂环基；m为0-5；S为硫原子；
优选地，当X 1为氢原子、X 2为烷基、m为1时，式II所示的化合物为硫代乙酸S-(3-羰基丙基)酯。
在本发明一个优选的实施方案中，提供一种抗体药物偶联体，所述小分子药物为DM1，所述接头L 1为SMCC，n为0，由此形成如下式III所示的抗体药物偶联体：
Figure PCTCN2019090255-appb-000004
或，所述小分子药物为MMAF，所述接头L 1为MC-VC-PAB，L 2为硫代乙酸S-(3-羰基丙基)酯，n为1，由此形成如下式IV所示的抗体药物偶联体：
Figure PCTCN2019090255-appb-000005
在本发明一个优选的实施方案中，提供一种如上所述的抗体的药物偶联体，在一些实施方式中，与接头连接的单个抗体分子缀合的细胞毒剂或小分子药物的数量y(或载荷，或DAR)为1、2、3、4、5、6、7、8、9或10，但由于连接反应的特殊性，连接有接头的抗体上缀合的小分子药物的DAR实际上为介于0至10、1至8、2至7、3至6或4至5的平均值。即本发明的抗体偶联物实为抗体连接了不同数目的接头-药物或仅接头的混合物，因此y值为药物偶联数目的平均值且数值为整数或非整数。在某些实施方案中，本发明ADC的药物载荷的范围为1到约8；约2到约7；约3到约6；约4到约5；约4.1到约4.9；约4.2到约4.8；约4.3到约4.7；约4.4到约4.6；约4.4、4.6或约4.8。
在本发明一个优选的实施方案中，提供一种抗体药物偶联体，其为如下式V所示的抗体药物偶联体：
Figure PCTCN2019090255-appb-000006
或，如下式VI所示的抗体药物偶联体：
Figure PCTCN2019090255-appb-000007
或，如下式VII所示的抗体药物偶联体：
Figure PCTCN2019090255-appb-000008
或，如下式VIII所示的抗体药物偶联体：
Figure PCTCN2019090255-appb-000009
其中，所述Ab10包含如SEQ ID NO:38所示的轻链和如SEQ ID NO:39所示的重链；所述Ab6包含如SEQ ID NO:42所示的轻链和如SEQ ID NO:39所示的重链。
为解决上述技术问题，本发明的第四方面的技术方案为：提供一种所述的抗体药物偶联体的制备方法，
当n为1时，所述制备方法包括以下步骤：
(1)制备中间体1：取所述抗体与所述接头L 2在溶液中混合，反应后纯化即得含中间体1的溶液，所述中间体1如下式IX所示：
Figure PCTCN2019090255-appb-000010
其中，X 1选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
X 2选自烷基、环烷基和杂环基；m为0-5；S为硫原子；
优选地，X 1为氢原子，X 2为烷基，m为1，即L 2为硫代乙酸S-(3-羰基丙基)酯；
(2)制备中间体2：取所述接头L 1与所述药物制备成中间体2：L 1-D；
(3)将步骤(1)获得的含中间体1的溶液，与步骤(2)获得的含中间体2的溶液混合，反应后纯化即得含抗体药物偶联体的溶液；
当n为0时，所述制备方法包括以下步骤：
(1)制备中间体3：取所述抗体与所述接头L 1在溶液中混合，反应后纯化即得含中间体3的溶液；
(2)将步骤(1)获得的含中间体3的溶液，与含所述药物的溶液混合，反应后纯化即得含抗体药物偶联体的溶液；
优选地，所述步骤(1)和/或(2)中，所述反应温度为25℃；所述反应时间为2～4小时；和/或，所述纯化为用凝胶过滤纯化，更优选为Sephadex G25凝胶柱脱盐纯化。
为解决上述技术问题，本发明的第五方面的技术方案为：提供一种靶向Claudin18.2的嵌合抗原受体(CAR)。
设计CAR时，针对特定抗原的抗体基因是一种关键性的选择，鉴于体内基因表达的复杂性以及各种不可控因素，选择到一个合适的用于CAR的基因是非常困难的。并且，很多肿瘤特异性的抗原，很难找到针对其的且适合于构建CAR细胞的特异性分子，在建立CAR后，往往无法获得有活性的胞外结合区，这也是发展CAR技术的难点。此外，尽管CAR细胞在肿瘤免疫治疗中具有诱人的前景，但亦需要考虑其较高的风险。比如，由于某些正常组织低表达CAR所能识别的特异性抗原可能造成CAR细胞对表达相应抗原的正常组织的损伤。现有技术中，靶向Claudin18.2抗体特异性结合Claudin18.2的效果不佳，靶向Claudin18.2的CAR细胞的效果也不够优异，而众所周知的是，开发出兼具有效性和安全性的靶向Claudin18.2的CAR细胞难度极大。发明人通过实验意外地发现，本发明CAR中的抗体结合活性/亲和力高，意味着在同样的工艺(同病毒滴度，转染效率)下，本发明的CAR能更好地结合到靶细胞，特异性更好，减少非靶细胞结合所产生的副作用。使用本发明的CAR，即使很少的剂量就可以发挥作用，有极高的应用价值。本发明中针对CLDN18.2的CAR细胞的抗原结合部分(抗体)是人源化的序列，可以减 少CAR治疗产生免疫原性风险。通过新一代CAR设计，具体地是在CAR的C末端引入能有效杀伤肿瘤的细胞因子、细胞因子和其受体结合复合物等，并使得这些因子和/或复合物能够分泌胞外、肿瘤细胞区域达到更好的抑制肿瘤的效果。
所述CAR包括：(a)特异性识别CLDN18.2的胞外结合结构域scFv；(b)铰链结构域；(c)跨膜结构域；(d)共刺激胞内结构域；(e)信号传导结构域；
其中，所述胞外结合结构域包括如本发明的第一方面所述的靶向CLDN18.2的抗体的轻链可变区和重链可变区。
在一较佳的具体实施例中，如上所述的CAR中，
(1)所述铰链结构域选自一种或多种以下分子的铰链区：CD8α、CD28、CD152、PD1和IgG1重链；
(2)所述跨膜结构域选自一种或多种以下分子的跨膜区：TCR的α、β、ζ链，CD3ε、CD3ζ，CD4，CD5，CD8α，CD9，CD16，CD22，CD27，CD28，CD33，CD37，CD45，CD64，CD80，CD86，CD134，4-1BB，CD152，CD154和PD1；
(3)所述共刺激胞内结构域选自一种或多种以下分子的胞内区：CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54、CD83、OX40、CD134、4-1BB、CD150、CD152、CD223、CD270、PD-L2、PD-L1、CD278、DAP10、NKD2C SLP76、TRIM、FcεRIγ和MyD88，优选CD28胞内区和/或4-1BB胞内区；和/或，
(4)所述信号传导结构域选自一种或多种以下分子的胞内区：Igα、Igβ，TCRξ，FcR1γ、FcR1β，CD3γ、CD3δ、CD3ε，CD2，CD5，CD22，CD28，CD79a、CD79b，CD278，CD66d和CD3ζ；优选CD3ζ胞内区。
在一较佳的实施例中，所述CAR中，所述铰链结构域为CD8α铰链区，所述跨膜结构域为CD8α跨膜区，所述共刺激胞内结构域为CD28胞内区和/或4-1BB胞内区，所述信号传导结构域为CD3ζ胞内区(在实施例的CAR通式中简称CD3ζ)；
优选地，所述CD8α铰链区为人CD8α铰链区；所述CD8α跨膜区为人CD8α跨膜区；所述CD28胞内区为人CD28胞内区；所述4-1BB胞内区为人4-1BB胞内区；和/或，所述CD3ζ胞内区为人CD3ζ胞内区。
为解决上述技术问题，本发明的第六方面的技术方案为：提供一种含有靶向Claudin18.2的CAR的核酸构建体，所述核酸构建体具有如式car-[(IRES)-f] q所示的结构，其中，IRES为如SEQ ID NO:55的核苷酸序列所示的内部核糖体进入位点序列；f编码功能性蛋白F，q为0或非0自然数；car编码包括上述第五方面的CAR。
在一较佳的实施例中，如上所述的核酸构建体中，当q为非0自然数优选为1时， 所述功能性蛋白F包括：
(1)细胞因子或其活性片段，优选IL10或IL15或其活性片段；更优选地，所述IL10的氨基酸序列如SEQ ID NO:54所示；
(2)细胞因子受体或其活性片段，优选IL15Rα或其片段或IL15Rα片段(sushi)或IL15Rα片段(sushi+)；或，
(3)细胞因子受体或其活性片段与细胞因子的融合蛋白，优选IL15Rα或其片段或IL15Rα(sushi)或IL15Rα(sushi+)与IL15的融合片段。
在一较佳的实施例中，所述核酸构建体的结构为：
(1)scFv-人CD8α铰链区-人CD8α跨膜区-人4-1BB胞内区-人CD3ζ胞内区；优选地，其编码的氨基酸序列如SEQ ID NO:3所示；此处较佳的一核苷酸构建体在下文中命名为CAR1a；或，
(2)scFv-人CD8α铰链区-人CD8α跨膜区-人4-1BB胞内区-人CD3ζ胞内区-(IRES)-IL15；此处较佳的一核苷酸构建体在下文中命名为CAR3ab；或，
(3)scFv-人CD8α铰链区-人CD8α跨膜区-人4-1BB胞内区-人CD3ζ胞内区-(IRES)-IL10；此处较佳的一核苷酸构建体在下文中命名为CAR3ab10；或，
(4)scFv-人CD8α铰链区-人CD8α跨膜区-人4-1BB胞内区-人CD3ζ胞内区-(IRES)-IL15Rα(sushi+)-IL15；此处较佳的一核苷酸构建体在下文中命名为CAR4a。
本领域技术人员需知，上述的构建体为核酸，为了简明起见，该构建体的四种结构省略了“编码…的核苷酸序列”的表述。例如上述构建体结构中的scFv实为“编码scFv的核苷酸序列”，人CD8α铰链区实为“编码人CD8α铰链区的核苷酸序列”，以此类推。此外，上述结构中IL10和IL15除了可以为野生型的IL10和IL15以外，还可以是IL10和IL15的突变体或其活性片段。
上述的核酸构建体中，进一步还可以包括CD27T细胞记忆和存活信号传导结构域，和/或，可自毁结构域例如编码诱导型半胱天冬酶(iCasp)的核苷酸序列或编码单纯疱疹病毒胸苷激酶(HSV-TK)的核苷酸序列。可自毁结构域能调控抗原识别信号通路，最大限度降低CAR细胞对正常组织的伤害，减少脱靶效应。当不良反应发生时，在无毒性前体药物刺激下，激活自杀基因，诱导CAR细胞凋亡，终止治疗。
为解决上述技术问题，本发明的第七方面的技术方案为：提供一种分离的核酸，其编码如上所述的靶向CLDN18.2的抗体或如上所述的双特异性抗体。
为解决上述技术问题，本发明的第八方面的技术方案为：提供一种表达载体，其包含如上所述的分离的核酸，或如上所述的核酸构建体。
优选地，所述表达载体选自逆转录病毒载体、慢病毒载体、腺病毒载体和腺相关病毒载体。更优选慢病毒载体，其骨架质粒可以为pBABEpuro。
为解决上述技术问题，本发明的第九方面的技术方案为：提供一种基因修饰的细胞，其转染有如上所述的核酸构建体、所述的表达载体；优选地，所述基因修饰的细胞为真核细胞，更优选分离的人细胞；进一步更优选免疫细胞如T细胞，或NK细胞如NK92细胞系。
为解决上述技术问题，本发明的第十方面的技术方案为：提供一种制备基因修饰的细胞的方法，所述方法包括以下步骤：将所述的核酸构建体、所述的表达载体或所述的病毒转入待修饰的细胞内即得；
较佳地，所述基因修饰的细胞为真核细胞，优选分离的人细胞；更优选免疫细胞如T细胞或NK细胞；进一步更优选为NK92细胞系。
为解决上述技术问题，本发明的第十一方面的技术方案为：提供一种药物组合物，所述药物组合物包含所述的靶向CLDN18.2的抗体、所述的双特异性抗体、所述的基因修饰的细胞或所述的抗体药物偶联体，以及药学上可接受的载体；优选地，所述药物组合物还包括免疫检查点抗体。
为解决上述技术问题，本发明的第十二方面的技术方案为：提供一种所述的靶向CLDN18.2的抗体、所述的双特异性抗体、所述的抗体药物偶联体、所述的核酸构建体、所述的表达载体、所述的病毒、所述的基因修饰的细胞或所述的药物组合物在制备治疗肿瘤的药物中的应用；较佳地，所述肿瘤为CLDN18.2阳性肿瘤，优选胃癌、食管癌、肺癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌、神经胶质瘤或者白血病。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明通过创新免疫和筛选方法，进行多种创新设计，提供了一种全新的靶向CLDN18.2的抗体，优选人源化抗体，其和人、鼠CLDN18.2特异结合活性更好，Emax更高；亲和力(KinExA)高(优选达到了10pM级)；人血细胞中CDC活性更好；诱导CLDN18.2+细胞凋亡活性更好，抑制肿瘤细胞生长活性更好，动物药效更优，且体内药物代谢(PK)好，特别是更长的T1/2。其和鼠CLDN18.2也具有更好的结合活性。
本发明提供了一种双特异抗体，其一个靶点是针对CLDN18.2，另一个则针对CLDN18.2以外的靶点。创新设计意外发现所得到的双特异性抗体(SBody)优选分子保 留了双靶点的结合活性、功能活性；而且双特异设计类似IgG结构，工艺纯化简单，而且稳定。这为后期开发中工艺制备抗体带来了便利。同时，所设计的靶点包括PD-1/PD-L1等免疫检查点抗原时，其和CLDN18.2结合，可以将肿瘤免疫和靶向抗体联合作用在一个分子中实现，产生等同2个分子联用效果或者更好的协同效果，为肿瘤免疫治疗和靶向治疗结合带来更加便利的药物开发选择。
本发明首创针对CLDN18.2的抗体药物偶联体(ADC)。其中抗体分子效果如上所述。由该抗体和细胞毒素偶联得到的ADC分子不仅保留了其抗人、鼠CLDN18.2的优良特异结合活性，内吞活性与高效杀伤肿瘤细胞的特点，同时携带细胞毒性毒素，能更加特异地、靶向杀伤肿瘤细胞，靶向特异抑制肿瘤细胞增殖，产生意想不到的的更加优秀的治疗肿瘤药效。这些特点使得本发明ADC药物以及其药用盐、溶剂化合物或者和其它药物联用的组合，为肿瘤患者特别是CLDN18.2阳性癌症患者提供更加特异有效的、更好治疗选择、手段和方法。此外，本发明提供的抗体药物偶联体/抗体具有更好的药代性能，安全窗口大以及更低的毒副作用。
本发明提供了一种新的靶向Claudin18.2的CAR分子，其活性和亲和力更高，能更好地靶向肿瘤细胞；其不结合Claudin18.1，具有极佳的特异性，减少非靶细胞结合所产生的副作用；CAR分子的抗原结合序列优选人源化，减少免疫原性风险，安全性更佳；CAR分子结合细胞因子或细胞因子受体一起使用，治疗效果更佳；含本发明CAR分子的免疫细胞治疗效果更好，尤其在较佳实施例中，本发明的CAR分子接近100％的抑癌效果。
附图说明
图1为本发明鼠源抗人CLDN18.2抗体mab5b和人CLDN18.2(图1a)以及鼠CLDN18.2(图1b)的结合活性(ELISA)；
图2为本发明抗人CLDN18.2抗体mab5b人源化抗体(图2a)以及人源化优化抗体(图2b)和人CLDN18.2的结合活性(ELISA)；
图3为本发明人源化抗人CLDN18.2抗体、人源化优选抗体CDC活性评价；
图4为本发明人源化抗人CLDN18.2抗体、人源化优选抗体诱导肿瘤细胞凋亡的活性(图4a,图4b)；
图5为本发明人源化抗人CLDN18.2抗体、人源化优选抗体动物模型体内药效评价(图5a,图5b)；
图6为本发明人源化抗体Ab10和Ab6内吞活性分析；
图7A为LB302结构及相关检测数据图，其包括结构示意图(c)，以及和人CLDN18.2的结合活性检测结果(a)，和人PD-1的结合活性检测结果(b)。
图7B为LB301结构及相关检测数据图，其包括结构示意图(c)，以及和人CLDN18.2的结合活性检测结果(a)，和人PD-1的结合活性检测结果(b)。
图7C为LB309结构及相关检测数据图，其包括结构示意图(c)，以及和人CLDN18.2的结合活性检测结果(a)，和人PD-1的结合活性检测结果(b)。
图7D为LB308结构及相关检测数据图，其包括结构示意图(c)，以及和人CLDN18.2的结合活性检测结果(a)，和人PD-1的结合活性检测结果(b)。
图8为LB401结构及相关检测数据图，其中a为样品在-80℃保存60天(60d)，在4℃保存30天、在37℃保存7天以及14天的样品和人CLDN18.2的结合活性检测结果；b为LB401结构示意图。
图9为针对CLDN18.2设计的CART细胞动物体内药效，其显示了注射CART(空载体)、CART1a、CART3ab、CART3ab10和CART4a细胞的小鼠的肿瘤体积变化。
具体实施方式
术语解释：
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
本发明所用氨基酸三字母代码和单字母代码如本领域技术人员知晓，或J.Biol.Chem,243,p3558(1968)中所述。
如本文使用的，术语“包括”或“包含”旨在表示组合物和方法包括所述的元素但不排除其他元素。“基本上由……组成”当被用来定义组合物和方法时，应该表示排除对于预期用途的组合来说具有任何实质性作用的其他元素。例如，如本文定义的基本上由该元素组成的组合物，将不会从分离和纯化方法和药学上可接受的载体(例如磷酸盐缓冲盐水、防腐剂等)中排除微量污染物。“由……组成”应该表示排除多于微量元素的其他成分和用于施用本文公开的组合物的实质性方法步骤。通过这些过渡性术语的每一个进行定义的方面都在本发明的范围之内。
术语“CLDN18.2”包括同种型、哺乳动物(例如人)的CLDN18.2、人CLDN18.2的物种同源物和包含至少一个与CLDN18.2的共同表位的类似物。CLDN18.2(例如人 CLDN18.2)的氨基酸序列是本领域中己知的，如NCBI数据库显示。
术语“CLDN18.1”包括同种型、哺乳动物(例如人)的CLDN18.1、人CLDN18.1的物种同源物和包含至少一个与CLDN18.1的共同表位的类似物。CLDN18.1(例如人CLDN18.1)的氨基酸序列是本领域中己知的，如NCBI数据库显示。
本发明所述的“CLDN18.2抗体”、“抗CLDN18.2抗体”、“CLDN18.2抗体分子”与“抗CLDN18.2抗体分子”可互换使用。术语“表位”指抗原(例如，人CLDN18.2)中与抗体分子特异性相互作用的部分。术语“竞争”在本发明中指抗体分子干扰抗CLDN18.2抗体分子与靶(例如，人CLDN18.2)结合的能力。对结合作用的干扰可以是直接或间接的(例如，通过抗体分子或靶的变构调节作用)。可以使用竞争结合测定法(例如，FACS测定法、ELISA或BIACORE测定法)确定抗体分子是否能够干扰另一种抗体分子与其靶结合的程度。
本发明所述的术语“抗体”包括免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3和IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。在本发明中，本发明所述的抗体重链可变区可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、2、3、4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定 区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域[称为互补决定区(CDR)]，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末排列的3个CDR和4个FR组成。各重链/轻链对应的可变区(VH和VL)分别形成抗体结合部位。特别地，重链还可以包含3个以上CDR，例如6、9或12个。例如在本发明的双特异性抗体中，重链可以是IgG抗体的重链的N端连接另一个抗体的ScFv，这种情况下重链含有9个CDR。
本发明所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat、Contact、CCG、AbM和Chothia编号规则。例如，Kabat编号规则遵循Kabat EA.Et al.,Sequences of Proteins of Immunological Interest[National Institutes of Health,Bethesda,Md.(1987and 1991)]的定义，Chothia编号规则遵循Chothia&Lesk 1987)Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:877-883的定义。给定CDR的边界可取决于用于识别的方案而变化，本发明所述定义规则以及所述抗体定义之CDR序列，见表3-8。例如，Kabat方案基于结构比对，而Chothia方案基于结构信息。用于Kabat及Chothia方案的编号基于最常用的抗体区序列长度，及通过插入字母调适插入物(例如，“30a”)及一些抗体中呈现删除部分。两种方案在不同位置放置某些插入物及删除部分(“插入缺失(indel)”)导致差异性编号。Contact方案基于对复合物结晶结构的分析且在许多方面中类似于Chothia编号方案。因此，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”及该抗体或其区的个别CDR(例如，“CDR-H1、CDR-H2)应了解为涵盖如通过本文描述的上述已知方案中的任何一种界定的互补决定区。在一些情况中，规定用于识别一个或多个特定CDR的方案，例如如通过Kabat、Chothia或Contact方法界定的CDR。在其他情况下，给定CDR的特定氨基酸序列。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人CLDN18.2的单克隆抗体。制备时用CLDN18.2抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源CLDN18.2抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的 人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆所得人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入载体中，最后在真核细胞，工业系统或原核工业系统中表达嵌合抗体分子。在本发明一个优选的实施方案中，所述的CLDN18.2嵌合抗体的抗体轻链可变区进一步包含鼠源κ、λ型或其变体的轻链FR区。所述的CLDN18.2嵌合抗体的抗体重链可变区进一步包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链RF区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1或IgG4重链恒定区，或者使用氨基酸突变后改变ADCC(antibody-dependentcell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)、CDC(complement dependent cytotoxicity、CDC、补体依赖细胞毒作用)活性的IgG1。可通过对IgG上Fc段的修饰，可降低或消除，或增强抗体的ADCC、CDC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A，L234A，L235A；IgG2/4 chimera，IgG4的F235E、或L234A/E235A，F243L、或S239D/A330L/I332E突变。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架中产生的抗体。特别的，本发明所述CLDN18.2抗体的CDR是按CCG、Kabat、AbM、Chothia或Contact等编号规则定义的各CDR序列，移植到人的抗体可变区框架中产生的抗体。优选地，本发明所述CLDN18.2抗体的CDR，优选轻链CDR1中的0-5个位点突变成人抗体CDR对应位点的氨基酸。这些可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站www.imgt.org和www.vbase2.org得到。
如本文所用，关于抗体的术语“特异性结合”意指识别特异性抗原但基本上不识别或结合样品中的其他分子的抗体。例如，特异性结合来自一个物种的抗原的抗体也可以结合来自一个或更多个物种的该抗原。但是，这种种间交叉反应性本身不改变抗体根据特异性的分类。在另一个实例中，特异性结合抗原的抗体也可以结合该抗原的不同等位 基因形式。然而，这种交叉反应性本身不改变抗体根据特异性的分类。在一些情况下，术语“特异性结合”或“特异性地结合”可用于指抗体、蛋白质或肽与第二化学物质的相互作用，意味着该相互作用取决于化学物质上特定结构(例如，抗原决定簇或表位)的存在；例如，抗体一般识别并结合特定的蛋白质结构，而不是蛋白质。如果抗体对表位“A”具有特异性，则在含有经标记的“A”和抗体的反应中，含有表位A的分子(或游离的，未标记的A)的存在将减少结合于抗体的标记的A的量。
在本发明一个优选的实施方案中，所述的CLDN18.2人源化抗体小鼠的CDR序列选自SEQ ID NO:11-28。人的抗体可变区框架经过设计选择，其中所述抗体轻链可变区上的轻链FR区序列，来源于人种系轻链IGKV4-1*01(F)和hJK2.1的组合序列SEQ ID NO:29-33，包含人种系轻链IGKV4-1*01(F)的FR1，FR2，FR3区和hJK2.1的FR4区；其中所述抗体重链可变区上的重链FR区序列，来源于人种系重链IGHV1-69*01(F)和hJH4.1的组合序列SEQ ID NO:34-37，包含人种系重链IGHV1-69*01(F)的FR1，FR2，FR3区和hJH4.1的FR4区。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少回复突变，以保持活性。在本发明一个优选的实施方案中，所述的人源化抗体可变区回复突变为0，即全人源化抗体。
术语“脱氨基化(deamidation)”指移除位点上或分子某个位点上的氨基。“脱氨基化(deamidation)敏感位点，指更容易、更倾向产生脱氨基化作用的分子和分子某个位点。
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少50％、60％、70％、80％、90％、95％、99％或100％或更多的母体抗体对靶标的结合亲和力。抗原结合片段实例包括但不限于：Fab、Fab’、F(ab’) 2、Fv片段、线性抗体(linear antibody)、单链抗体、纳米抗体、结构域抗体和多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson(2005)Nat.Biotechnol.23:1126-1136中。
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fc”区含有包含抗体的CH1和CH2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’) 2分子。“F(ab’) 2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定 区的部分的重链，由此在两条重链间形成链间二硫键。因此F(ab’) 2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。术语“Fv”意指向抗体的单臂的VL和VH结构域组成的抗体片段，但缺少恒定区。
在一些情况下，抗体的抗原结合片段是单链结合片段(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子[参见，例如，Bird等人，Science 242:423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)]。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的G 4S氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(G 4S) 4或(G 4S) 3接头，但也可使用其变体。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双特异性抗体(diabodies)、和三抗体(triabodies)、共价或非共价连接在一起的抗体片段等。
抗体分子包含双特异性抗体(diabody)和单链分子以及抗体的抗原结合片段(例如，Fab、F(ab’) 2，scFv和Fv)。抗体分子包含一条重链和一条轻链(在本发明中称作半抗体)或由其组成。Fab'、F(ab’) 2、Fc、Fd、Fv、单链抗体(例如scFv)、单一可变结构域抗体、双特异性抗体(Dab)(双价和双特异性)和嵌合(例如，人源化)抗体，它们可以通过修饰完整抗体产生，或使用重组DNA技术从头合成的那些抗体分子。这些功能性抗体片段保留选择性地与其相应抗原或受体结合的能力。抗体和抗体片段可以来自任何抗体类别，包括但不限于IgG、IgA、IgM、IgD和IgE并且来自任何抗体亚类(例如，IgG1、IgG2、IgG3和IgG4)。抗体分子的制备可以是单克隆或多克隆的。抗体也可以是人抗体、人源化抗体、CDR移植抗体或体外生成的抗体。抗体可以具有例如选自IgG1、IgG2、IgG3或IgG4的重链恒定区。抗体还可以具有例如选自κ或λ型的轻链。
本发明所公开的抗体也可以是单结构域抗体。单结构域抗体可以包括其互补决定区是单结构域多肽组成部分的抗体。例子包括但不限于重链抗体、天然缺少轻链的抗体、衍生自常规4链抗体的单结构域抗体、工程化抗体和除衍生自抗体的那些支架之外的单结构域支架。单结构域抗体可以是现有技术的任何抗体，或将来的任何单结构域抗体。单结构域抗体可以衍生自任何物种，包括但不限于小鼠、人、骆驼、羊驼、山羊、兔和牛。根据一些方面，单结构域抗体是天然存在的单结构域抗体，称作缺少轻链的重链抗体。 出于清晰原因，从天然缺少轻链的重链抗体衍生的这种可变结构域在本发明中称作VHH或纳米体以将它与四链免疫球蛋白的常规VH区分。这种VHH分子可以衍生自骆驼科(Camelidae)物种(例如骆驼、羊驼、单峰驼、驼羊和原驼)中产生的抗体。除骆驼之外的其他物种可以产生天然缺少轻链的重链抗体，这类VHH也被考虑。VH区和VL区可以再划分为超变区，称作“互补性决定区”(CDR)，其间插有更保守的区域，称作“框架区”(FR)。框架区和CDR的范围己有多种方法定义。
本发明的抗体包括单克隆抗体。本发明所述的单克隆抗体或mAb或Ab，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。本发明所述载体的宿主细胞，可以是但不限于真核细胞、细菌细胞、昆虫细胞或人细胞。合适的真核细胞包括但不限于Vero细胞、Hela细胞、COS细胞、CHO细胞、HEK293细胞、293T、293E、BHK细胞、合适的昆虫细胞包括但不限于Sf9细胞。
单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南。抗原结合片段同样可以用常规方法制备。
本文中使用的术语“嵌合抗原受体”或“CAR”指：包含能够结合抗原的胞外域(胞外结合结构域)、铰链结构域、跨膜结构域(跨膜区)和使胞质信号传到结构域的多肽(即胞内信号域)。铰链结构域可以被认为是用于向细胞外抗原结合区提供柔性的一部分。胞内信号域指经由确定的信号传导途径通过产生第二信使而将信息传递到细胞内以调节细胞活性的蛋白质、或通过相应于此类信使而作为效应子发挥作用的蛋白质，产生可以促进CAR的细胞(例如CART细胞)的免疫效应子功能的信号。胞内信号域包含信号传导结构域，还可以包括源自共刺激分子的共刺激胞内结构域。
术语“信号传导结构域”是指CAR转导效应子功能信号并指导细胞执行其专门功能的部分。信号传导结构域的实例包括但不限于T细胞受体复合物的ζ链或其任何同系物。
本文中术语“CD3ζ”定义为GenBanK登录号BAG36664.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD3ζ胞内区”定义为来自ζ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。一方面，CD3ζ胞内区包含GenBank登录号BAG36664.1的残基52至164、其功能性同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。如本文使用的，术语“CD3ζ信号传导结构域”或“CD3ζ胞内区”是指与该名称相关的特定的蛋白片段，以及与本文所示的CD3ζ胞内区氨基酸序列具有至少80％、或替代地至少90％同一性、优选至少 约95％、更优选至少约97％、更优选至少约98％、最优选至少约99％同一性的具有类似的生物功能的任何其他分子。
术语“共刺激胞内结构域”指共刺激分子的胞内区，为T细胞上的关连结合性配偶体，其特异性结合共刺激配体，由此介导免疫细胞的共刺激反应，例如但不限于增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。共刺激分子包括但不限于以下分子的胞内区，如MHCI类分子、BTLA和Toll配体受体、以及OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)和4-1BB(CD137)。
本文中术语“4-1BB”指TNFR超家族的成员，其具有GenBank登录号AAA62478.2的氨基酸序列、或来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基；“4-1BB”共刺激胞内结构域定义为GenBank登录号AAA62478.2的氨基酸序列214-255，或来自非分类物种例如小鼠、啮齿类动物、猴、猿等的等价残基，或与本申请所示的4-1BB共刺激结构域序列具有至少80％、或替代地至少90％氨基酸序列同一性、优选95％序列同一性、更优选至少97、98或99％序列同一性的具有类似的生物功能的任何其他分子。
如本文使用的，术语“CD28共刺激结构域”是人CD28共刺激结构域或是指与该名称相关的特定的蛋白片段，以及与人CD28共刺激结构域序列具有至少80％、或替代地至少90％氨基酸序列同一性、优选95％序列同一性、更优选至少97、98或99％序列同一性的具有类似的生物功能的任何其他分子。
在本发明中，一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、共刺激结构域和信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含识别胞外抗原的胞外结合结构域、跨膜结构域、共刺激结构域和信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外结合结构域、共刺激结构域和信号传导结构域，所述共刺激结构域包含源自一个或多个共刺激分子的至少两个功能性信号传导结构域。一方面，CAR在CAR融合蛋白的N端包含可选的前导序列(或信号肽)。一方面，CAR在胞外抗原识别结构域的N端还包含前导序列，其中前导序列任选地在CAR的细胞加工和定位至细胞膜的过程中从抗原识别结构域(例如scFv)的N端切下。
“同源性”、“变异序列”、“突变”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那 么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。“优化”指保持或改善了所述抗体与抗原结合的突变，在本发明中，指保持、维持或改善了与CLDN18.2的结合的突变。
术语“多肽”、“肽”和“蛋白质”(如果为单链)在本发明中互换地使用。术语“核酸”、“核酸序列”，“核苷酸序列”或“多核苷酸序列”和“多核苷酸”互换使用。
术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”和“保守性氨基酸取代”分别是其中氨基酸残基以另一种氨基酸残基置换和以具有相似侧链的氨基酸残基置换。
术语“IL10”、“白介素10”及“interleukin-10”可以互换使用，具有相同意思。同样地，术语“IL15”、“白介素15”及“interleukin-15”可以互换使用，具有相同意思。适当氨基酸修饰可以很容易地被实施并且确保不改变所得分子的生物活性。这些技术使本领域人员认识到，一般来说，在一种多肽的非必要区域改变单个氨基酸基本上不会改变生物活性。所述IL10或IL15的活性片段都可以应用到本发明中。在这里，所述的生物活性片段的含义是指作为一种多肽，其作为全长多肽的一部分，仍然能保持全长的多肽的全部或部分功能。通常情况下，所述的生物活性片段至少保持50％的全长多肽的活性。在更优选的条件下，所述活性片段能够保持全长多肽的80％、90％、95％、97％、98％、99％、或100％的活性。在所述IL10或IL10多肽序列的基础上，经修饰或改良的多肽也可以应用到本发明中，比如，可采用为了促进其半衰期、有效性、代谢、和/或多肽的效力而加以修饰或改良的多肽。也就是说，任何不影响多肽的生物活性的变化形式都可用于本发明中。
本领域技术人员皆知，IL15与IL15受体结合发挥其生物学功能。IL15受体有三个亚基，分别为IL15受体α、IL15Rβ(CD122)和γ(也称CD132)。IL15Rα细胞外区域为结合IL15的部分，其中的区域sushi结构域与IL15结合可发挥IL15的生物学功能。在本发明中，“sushi+”意指除了sushi片段以外，还包括了其它多肽片段。上述IL15Rα、IL15Rα(sushi)和IL15Rα(sushi+)的氨基酸序列可参见CN106755107A。IL15和IL15Rα结合，除自身细胞被激活外，因为IL15Rα的介导，还可以将信号传递到另一个细胞激活细胞活性。这些活性包括选择性地扩增CD8+T细胞，NK细胞等，而且不像IL2那样活化调节T细胞，从而在抗肿瘤免疫反应中可能发挥不同的功能。
本文所使用的“慢病毒”是指逆转录病毒科(Retroviridae family)的属。慢病毒在逆转录病毒中是独特的，其能够感染非分裂细胞；它们可以将显著量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体的一种最有效方法。HIV、SIV和FIV都是 慢病毒的实例。来自慢病毒的载体提供了在体内实现显著水平基因转移的手段。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语还应被解释为包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于腺病毒载体、腺相关病毒载体、逆转录病毒载体等。
本发明使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌等原核细胞，如酵母细胞等的真菌细胞，或者如纤维原细胞、CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞、HEK 293细胞或人细胞等的动物细胞。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“免疫细胞”指可以引发免疫应答的细胞，“免疫细胞”及其语法上的其他形式可以指任何来源的免疫细胞。“免疫细胞”包括例如衍生自在骨髓中产生的造血干细胞(HSC)的白血细胞(白细胞)、淋巴细胞(T细胞、B细胞、自然杀伤(NK)细胞和骨髓来源的细胞(嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、单核细胞、巨噬细胞、树突状细胞)。术语“免疫细胞”也可以是人或非人的。
如本文使用的，术语“T细胞”是指在胸腺中成熟的一类淋巴细胞。T细胞在细胞介导的免疫中起重要作用，并且与与其他淋巴细胞(例如B细胞)的不同点在于细胞表面上存在T细胞受体。“T细胞”包括表达CD3的所有类型的免疫细胞，包括T辅助细胞(CD4+细胞)、细胞毒性T细胞(CD8+细胞)、自然杀伤T细胞、T调节细胞(Treg)和γ-δT细胞。“细胞毒性细胞”包括CD8+T细胞、自然杀伤(NK)细胞和嗜中性粒细胞，这些细胞能够介导细胞毒性反应。如本文使用的，术语“NK细胞”是指起源于骨髓并且在先天免疫系统中起重要作用的一类淋巴细胞。NK细胞提供针对病毒感染的细胞、肿瘤细胞或其他应激细胞的快速免疫反应，即使是细胞表面上不存在抗体和主要组织相容性复合体。
例如，免疫细胞可以是来自血液的，如自体的T细胞、异体T细胞、自体NK细胞、异体NK细胞，也可以来源自细胞系，如利用EBV病毒感染来制备NK细胞系，从胚胎 干细胞和iPSC诱导分化来的NK细胞以及NK92细胞系等。
“任选”、“任一”、“任意”或“任一项”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。本发明所用，“一个”和“一种”在本发明中用来指的一个或多于一个的语法对象。除非内容明确提示，否则术语“或”在本发明中用来意指术语“和/或”并且与之互换使用。“约”和“大约”应当通常意指鉴于测量的性质或精度，所测量的量的可接受误差程度。示例性误差程度一般在其10％范围内和更一般在其5％范围内。本发明公开的方法和组合物涵盖这样的多肽和核酸，它们具有指定的序列，变异序列或与其基本上相同或相似的序列，例如，与序列指定至少85％、90％、95％、99％或更多相同的序列。在氨基酸序列的情况下，术语“基本上相同”在本发明中用来指第一氨基酸序列。
如本文中所使用的，术语“KD”是指特定抗体一抗原相互作用的解离平衡常数(KD)，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体一抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 - 6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数结合抗原，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。例如用KINEXA方法KINEXA 400仪器上检测到的抗体和细胞结合的亲和力。如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，即能引起50％最大效应的浓度。
本发明的药物组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它治疗方式。
如本文所用，术语“抗体的药物偶联体”、“偶联物”、“偶联体”或“ADC”可互换使用，指具有式I、III、IV、V、VI或VII等结构所示的抗体的药物偶联体。
澳瑞他汀是全合成药物，化学结构式相对容易改造，以便优化其物理性质和成药特性。用于和抗体偶联的澳瑞他汀衍生物主要包括单甲基澳瑞他汀E(MMAE)和单甲基澳瑞他汀F(MMAF)，前者是由天然微管蛋白聚合酶抑制剂尾海兔素10(dolastatin-10)衍生出的合成五肽，在C-端加上一个2-氨基-1-苯基丙基-1-醇而合成。MMAE对多种人类肿瘤细胞株的抑制活性小于一纳摩尔。为了降低MMAE自身细胞毒活性，MMAF在尾海兔素10的C-端加上一个苯丙氨酸，因为在结构上引入一个羧基，MMAF的细胞膜通过性较差，因此对细胞的生物活性显著降低，但是和抗体偶联后对细胞的抑制活性大幅度提高(US7750116)。
在有些实施方案中，抗体细胞毒性药物偶联物或其可药用盐或溶剂化合物包含偶联有一个或多个美登木素生物碱分子的本发明抗体。美登木素生物碱是通过抑制微管蛋白多聚化未发挥作用的有丝分裂抑制剂。美登素最初从东非灌木齿叶美登木(Maytenusserrata)分离得到(美国专利No.3,896,111)。随后发现某些微生物也生成美登木素生物碱，诸如美登醇和C-3美登醇醋(美国专利No.4,151，042)。美登木素生物碱药物模块在抗体-药物偶联物中是有吸引力的药物模块，因为它们：(i)相对易于通过发酵或发酵产物的化学修饰或衍生化来制备；(ii)易于用适于通过非二硫化物接头偶联至抗体的官能团衍生化；(iii)在血浆中稳定；且(iv)有效针对多种肿瘤细胞系。适于用作美登木素生物碱药物模块的美登素化合物是本领域公知的，而且可以依照己知方法从天然来源分离，或是使用遗传工程技术生产(参见Yu等(2002)PNAS99：7968-7973)。美登醇和美登醇类似物也可以依照己知方法合成制备。美登木素生物碱药物模块的例示性实施方案包括：DM1、DM3和DM4，正如本文中所公开的。
本发明中使用的术语“连接单元”、“连接子”和“接头”指适用于本发明的用于连接本发明的抗体和小分子药物的基团。示例性的接头包括MC、MP、val-cit、ala-phe、PAB、SPP、SMCC、SIAB。在一个实施方案中，所述接头是MC-vc-PAB。
药物载荷(Loading)由y表示，即通式I、III、IV、V和VI分子中每个抗体的平均药物模块数，又称为药物抗体偶联比率(drug antibody ratio,DAR)。来自偶联反应的ADC制备物中每个抗体的平均药物模块数可以通过常规手段来表征，诸如质谱、ELISA测定法、和HPLC。还可以测定ADC在y方面的定量分布。在有些情况中，将y为某数值的同质ADC从具有其它药物载荷的ADC中分离、纯化、和表征可以通过诸如反相HPLC或电泳的手段来实现。药物载荷的范围可以为每个抗体0.8-10个药物模块(D)。
ADC的载荷(药物/抗体比率DAR)可以以不同方式来控制，例如通过：(i)限制药物一接头中间物或接头试剂相对于抗体的摩尔过量，(ii)限制偶联反应的时间或温度，(iii)半胱氨酸硫醇修饰的部分或限制还原性条件，(iv)通过重组技术对抗体的氨基酸序列进行工程改造，使得半胱氨酸残基的数目和位置为了控制接头-药物附着的数目和/或位置而进行改变(诸如如本文中和W02006/034488(完整收入本文作为参考)中所述而制备的thioMab或thioFab)。
本发明所述的方法、组合物、联合治疗可以与其他活性剂或治疗方式，所述的方法包括向对象以有效治疗或预防疾病(例如，癌症)的量，施用本发明所述的抗CLDN18.2抗体分子，任选地，与PD-1、PD-L1、PD-L2、LAG-3、CTLA-4、Tim-3抗体(免疫治疗)或其它肿瘤治疗抗体，Her-2、EGFR、VEGF、VEGFR抗体等，以及ADC(抗体药物偶 联，如T-DM1)，双特异抗体，化疗药物等的一种或多种抑制剂的组合，还包括施用抗CLDN18.2抗体分子、额外的活性剂或全部可以按这样的量或剂量施用，所述量或剂量高于、低于或等于单独(例如，作为单一疗法)使用的每种活性剂的量或剂量。抗CLDN18.2抗体，额外的活性剂或全部的施用量或剂量比单独(例如，作为单一疗法)使用的每种活性剂的量或剂量低(例如，至少20％、至少30％、至少40％或至少50％)。
此外，正如本发明的实施例中描述的那样，抗CLDN18.2抗体以及CLDN18.2抗体的药物偶联体可以结合CLDN18.2诱导靶细胞(肿瘤细胞)凋亡，抑制肿瘤细胞生长，增加体内效应细胞对肿瘤细胞ADCC，CDC杀伤作用来达到治疗癌症患者的目的。因此，在某些实施方案中，本发明所描述的抗CLDN18.2抗体以及CLDN18.2抗体的药物偶联体通过这些机理显示本发明抗体抗肿瘤效应，以及抑制肿瘤细胞生长的方法，包括将治疗有效量的本发明中所述的抗CLDN18.2抗体以及CLDN18.2抗体的药物偶联体施用于受试者。该方法适用于癌症的体内治疗。为了获得靶向特异治疗效果，抗CLDN18.2抗体分子可以与其它抗体一起施用。在将CLDN18.2抗体以及CLDN18.2抗体的药物偶联体组合一种或多种活性剂施用时，该组合可以以任何顺序或同时施用癌症类型，特别是CLDN18.2高表达的肿瘤患者。在某些方面中，提供在对象中治疗对象(例如，减少或缓解)过度增生性病状或疾病(例如，癌症)。该方法包括向对象单独或与其他活性剂或治疗方式组合地施用本发明所述的一种或多种抗CLDN18.2抗体或CLDN18.2抗体的药物偶联体。
组合进一步包括免疫检查点调节物的抑制物或激活物，例如，抗PD-L1抗体分子、抗PD-1抗体分子，或CTLA-4抑制物(例如，抗CTLA-4抗体)，或非免疫检查点调节物的抑制物或激活物(例如化学药物，小分子靶向药物，大分子包括抗体靶向药物，例如anti-Her2、anti-VEG、anti-VEGFR、anti-EGFR等抗体，抗体偶联药物，双特异抗体，CAR-T细胞组合等)，或其任意组合。CLDN18.2抗体治疗也可以与标准癌症治疗组合。
术语“免疫检查点”是指在免疫细胞的细胞表面上的一组分子，其可以充当“闸”以下调或抑制免疫应答，例如抗肿瘤免疫应答，进而和本发明抗体联合治疗肿瘤。免疫检查点分子包括但是不同限于PD-1、PD-L1、细胞毒性的T淋巴细胞抗原4(CTLA-4)、B7-H1、B7-H3、OX-40，4-1BB(CD137)、CD40、和淋巴细胞活化基因3(LAG-3)，等等。
使用单独的或与另一种免疫调节剂(例如抗LAG-3，抗Tim-3,抗PD-l或抗PD-L1、抗CTLA-4抗体分子)组合的抗CLDN18.2抗体分子来治疗胃癌、胰腺癌、肺癌、食管道癌、卵巢癌等。抗CLDN18.2抗体分子可以与以下一者或多者组合施用：基于免疫的 策略、靶向药物(例如，VEGF抑制剂如针对VEGF的单克隆抗体)；VEGF酪氨酸激酶抑制剂如舒尼替尼、索拉非尼、阿帕替尼；RNAi抑制剂或VEGF信号传导的下游介导物的抑制剂，例如，雷帕霉素哺乳动物靶(mTOR)的抑制剂。
如本发明所用，术语“癌”、“癌症”、“癌症病人”意在包括全部类型的癌性生长物或致瘤过程、转移性组织或恶性转化的细胞、组织或器官，无论组织病理学类型或侵袭力阶段是什么。例子包括但不限于实体瘤、血液学癌、软组织肿瘤和转移性病灶。
可以利用本发明中公开的靶向CLDN18.2的抗体、双特异性抗体、ADC及CAR细胞或其组合适合治疗的癌症的非限定性的实例包括，肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌和白血病等，或其转移性病灶。
实施例1：密蛋白18.1、18.2(CLDN18.1、CLDN18.2)高表达细胞株构建
本发明所用的人CLDN18.1、人CLDN18.2、小鼠CLDN18.1、小鼠CLDN18.2高表达细胞株通过公司稳定细胞株构建平台完成。具体步骤如下：
实验开始第1天，将293T细胞(中国科学院典型培养物保藏委员会细胞库Cat#GNHu17)接种于两个6cm培养皿，每个培养皿里的细胞数达到7.5×10 5个。第2天将包裹质粒(pGag-pol、pVSV-G、pBabe等BioVector，质粒载体菌种细胞基因保藏中心)和克隆有人或鼠CLDN18.2或CLDN18.1基因的质粒pBabe-CLDN18.2或pBabe-CLDN18.1各4μg加入OPTI-MEM(Thermofisher Scientific Cat#31985070)，使最终体积为200μl，另准备200μl OPTI-MEM加入36μl转染试剂fectin(上海源培生物科技股份有限公司Cat#F210)，二者混匀，室温放置5min，然后将混合物(每皿各200μl)滴加入培养好的293T细胞。第3天将293T细胞培养液换为4ml DMEM高糖培养基(上海源培生物科技股份有限公司/源培生物：Cat#310KJ)。第4天将CHO-K1细胞(中国科学院典型培养物保藏委员会细胞库Cat#SCSP-507)接种于10cm培养皿，使细胞数达到5×10 5。第5天收集293T细胞上清(病毒)，用0.45μm滤膜过滤至培养好的CHO-K1细胞，同时加入10μg/ml polybrene(上海翊圣生物科技有限公司Cat#40804ES76)，混匀后放置培养箱，3～4h后换成DMEM/F12 10％FBS培养基(源培生物，Cat#L310KJ)。第7天将CHO-K1细胞传代，第8天传代的细胞开始加入10μg/ml puromycin进行筛选(源培生物，Cat#S250J0)。2-3天细胞大量死亡，更换培养基继续培养，直到细胞不再死亡时，细胞大量扩增，筛选单克隆细胞株、扩培并冻存保种。
本发明构建稳定表达CLDN18细胞株分别标记为：人CLDN18.1+细胞(hCLDN18.1+cell)、人CLDN18.2+细胞(hCLDN18.2+cell)、鼠CLDN18.1+细胞 (mCLDN18.1+cell)、鼠CLDN18.2+细胞(mCLDN18.2+cell)。所用的蛋白序列源自公开发表的数据库，各蛋白氨基酸序列如下。
人CLDN18.1(hCLDN18.1)源于NCBI数据库中>NP_057453.1，claudin-18 isoform1precursor[Homo sapiens]。人CLDN18.2(hCLDN18.2)源于NCBI数据库中>NP_001002026.1 claudin-18 isoform 2[Homo sapiens]。鼠CLDN18.1(mCLDN18.1)源于NCBI数据库中>NP_062789.1 claudin-18 isoform A1.1 precursor。鼠CLDN18.2(mCLDN18.2)源于NCBI数据库中NP_001181850.1 claudin-18 isoform A2.1[Mus musculus]。
实施例2：抗CLDN18.2抗体和CLDN18.2+和CLDN18.1+细胞株结合(ELISA)实验
将实施例1得到的细胞人CLDN18.1、人CLDN18.2、鼠CLDN18.1或鼠CLDN18.2高表达的单克隆细胞株扩培后，按5×10 4个/孔铺96-well plate，37℃培养箱过夜孵育后去除上清，用免疫染色固定液(上海碧云天生物技术有限公司Cat#P0098)100μl/孔室温固定半小时。PBS(源培生物，Cat#B320)洗一遍后5％牛奶37℃封闭2小时，PBST洗3遍。加入待测样品(人或鼠源抗体，Jackson Immuno Research)。37℃孵育1小时，后PBST洗3遍。加Anti-human or mouse HRP 1:2500 50μl/孔37℃孵育1小时，后PBST洗3遍，TMB(Surmodic Cat#TTMB-1000-01)显色，加入50μl/孔1M H 2SO 4终止反应。酶标仪(MultiskanGO Thermo型号51119200)读数，Graphpad prism 5进行数据分析。
实施例3：重组蛋白、抗体的克隆、表达和纯化，活性检测(ELISA,blocking assay)
本发明用到的重组蛋白/抗体的克隆，表达和纯化均按本领域技术人员熟知的分子克隆方法进行。
具体地，本发明用到的表达载体购自长沙优宝生物科技有限公司，后由上海健信生物医药科技有限公司(健信生物)引入EcoRI酶切位点(GAATTC)，便于双酶切切或同源重组方法克隆外源基因。基因合成由生工生物工程(上海)股份有限公司(生工生物)等公司完成。293细胞、CHO-K购自中国科学院典型培养物保藏委员会细胞库。
本发明中重组蛋白和抗体是在293细胞瞬时转染表达并纯化得到。具体步骤是，293细胞在Gibco FreeStyle 293 Expression Medium(Gibco，Cat#12338018)培养基扩培。开始瞬转之前，调节细胞浓度至6～8×10 5cell/ml，1％FBS(Aus Gene X FBS Excellent供应商：AusGeneX，China，Cat#FBSSA500-S)，37℃8％CO 2摇床培养24h，再次镜检存活率>95％，细胞浓度在1.2×10 6cell/ml。
准备300ml培养体系细胞，15ml Opti-MEM(Gibco，Cat#31985070)溶入重链、轻链质粒各150μg，0.22μm过滤除菌。再取15ml Opti-MEM溶入1mg/ml PEI(Polysciences，Inc，Cat#23966-2)600μl后静置5min。在500ml培养体系中，25ml Opti-MEM(Gibco，Cat#31985070)溶入重链、轻链质粒各250μg，0.22μm过滤除菌。再取25ml Opti-MEM溶入1mg/ml PEI 1000μl后静置5min。把PEI缓慢加入质粒中，室温孵育10min，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，37℃8％CO 2摇床培养5天收样，3300G 10min取上清进行纯化。
纯化：将样品高速离心去除杂质，用PBS pH 7.4平衡含有Protein A(Mabselect，GE Healthcare Life Science，Cat#71-5020-91 AE)的重力柱(生工生物，Cat#F506606-0001)，2-5倍柱体积冲洗。将样品过柱。用5-10倍柱体积的PBS(生工生物，Cat#B548117-0500)冲洗柱子。再用pH 3.5 0.1M乙酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，酶标仪测定浓度，分装、储存备用。
本发明中的重组人CLDN18.2(claudin18.2)胞外区域(20位D-70为A片段)和Fc融合蛋白是通过293系统瞬时转染后纯化。该蛋白可用于免疫小鼠血清滴度检测。
本发明有的实验中用到抗体人CLDN18.2(anti-hCLDN18.2)抗体(称为对照分子或阳性分子)作为比较。该抗体在本发明中简写为Ref(Reference)，序列源自WO2014146672。
抗原包板ELISA：用pH 7.4的PBS缓冲液将本发明表达的人PD-1、PD-L1、CD47、LAG3、Tim3、或者购自Peprotech的TGFβ1(Cat#100-21-10)、TGFβ2(Cat#100-35B)、TGFβ3(Cat#100-36E)。购自Sino biological的IL10(货号SEKA10947)，FcγR I/CD64(Cat.#1257-FC-050，R&D Systems)等抗原根据不同assay，稀释至1-2μg/ml浓度，以50μl/孔的体积加入96孔酶标板(Corning，CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(上海生工生物工程有限公司，A600669-0250)封闭液200μl/孔，37℃孵育箱孵育3小时或4℃放置过夜(16-18小时)进行封闭。弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用1％BSA 5倍连续稀释的待测抗体，37℃孵育1小时，PBST洗板5次，加入50μl/孔1：2500稀释的HRP标记的二抗(Jackson Immuno Research，115-035-003)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，52-00-03)，室温孵育5-10min，加入50μl/孔1M H 2SO 4终止反应，用MULTISKAN Go酶标仪(ThermoFisher，51119200)在450nm处读取吸收值，根据OD值计算EC50。
抗体阻止抗原和其配体结合实验(Blocking assay)
用pH为7.4的PBS缓冲液将本发明表达的抗原PD-1、PD-L1、CD47稀释至2μg/ml 浓度，以50μl/孔的体积加入96孔酶标板(Corning，CLS3590-100EA)中，于37℃孵育2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(上海生工生物工程有限公司，A600669-0250)封闭液200μl/孔，37℃孵育3小时进行封闭。弃去封闭液，用PBST(pH7.4 PBS含0.05％tweeen-20)洗板5次后，每孔加入25μl用1％BSA 5倍连续稀释的待测样品和25μl终浓度为10μg/ml的biotin标记的配体(PD-1，PD-L1，SIRPα等，本发明表达纯化)，37℃孵育1小时，PBST洗板5次，加入50μl/孔1：1000稀释的HRP标记的二抗(金斯瑞生物科技有限公司，M00091)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，52-00-03)，室温孵育5-10min，加入50μl/孔1M H 2SO 4终止反应，用MULTISKAN Go酶标仪(ThermoFisher，51119200)在450nm处读取吸收值，根据OD值计算IC50。
Biotin标记的试剂盒为Biotin Labeling Kit-NH2，购自东仁化学科技(上海)有限公司，货号LK03。操作方法按说明书进行，标记好的抗体用Multiskan GO(ThermoFisher)酶标仪检测浓度后使用。
实施例4：抗人CLDN18.2抗体的发现
本发明抗人CLDN18.2单克隆抗体是用实施例1中得到的人CLDN18.2高表达细胞株(hCLDN18.2+cell)免疫的小鼠，取免疫小鼠脾进行杂交瘤融合，从数百万株杂交瘤克隆中筛选、优化得到的。
实验用小鼠，雌性，4周龄(SJL购自北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2016-0011；Balb/c购自上海西普尔-必凯实验动物有限公司)。小鼠购进后，实验室环境饲养1周，白天光/夜晚暗周期调节，温度20-25℃；湿度40-60％。小鼠分成3只/组/笼。
培养实施例1中构建的人CLDN18.2高表达细胞株(hCLDN18.2+cell，人CLDN18.2+细胞)，胰酶消化后DMEM培养基(源培生物，Cat#L310KJ)洗涤后，重悬于DMEM培养基中。按100μl/1×10 7细胞/只，腹腔注射免疫小鼠。首次免疫的时候用Titermax(Sigma-Aldrich，T2684)按1:1和细胞混匀免疫。随后免疫1周一次，免疫10次后，用实施例子2之ELISA方法，人CLDN18.1+cell和人CLDN18.2+cell同时铺板，或用上述实施例子3中的重组表达的人CLDN18.2胞外(ECL1)蛋白铺板，检测免疫小鼠血清效价，以人CLDN18.1+cell铺板的ELISA值作为背景，计算小鼠血清免疫效价(滴度)。在12-15次免疫以后，选择血清滴度高并且滴度处于平台期的小鼠进行脾细胞融合，融合前以200μl/2×10 7细胞/只冲免小鼠，冲免3天后，取小鼠脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2019090255-appb-000011
CRL-8287 TM)进行融合得到杂交瘤细胞铺96孔板。
取96孔板中杂交瘤细胞上清同时用人CLDN18.1+cell和人CLDN18.2+cell铺板检测杂交瘤细胞产生的抗体结合情况。表1a是部分杂交瘤上清的检测结果。
表1a 杂交瘤融合克隆和人CLDN18.2+cell、人CLDN18.1+cell结合活性检测
Figure PCTCN2019090255-appb-000012
因为人的CLDN18.2和CLDN18.1有高达92％的同源性(240/261)，且该蛋白是跨膜蛋白，只有少部分的肽段在胞外(比如51氨基酸的ECL1)，免疫原性极低，产生特异抗体的可能性非常小。所以上述筛选中，能得到分泌识别CLDN18的杂交瘤不仅很少，而且在很少的杂交瘤中，绝大部分杂交瘤上清中的抗体是能同时结合人CLDN18.2和CLDN18.1的抗体。
非常意料之外的是，本发明发现一株杂交瘤克隆，其分泌的上清只同人CLDN18.2+cell结合，和人CLDN18.1+cell不结合，见表1a中的mab5，克隆号C13C1。表1a数据显示，同样的筛选条件下，该克隆上清只和人CLDN18.2+cell结合，检测值是1.41，而同人CLDN18.1+cell几乎不结合，结合活性读值只为0.09。
进一步对本发明意外发现的该杂交瘤细胞株C13C1能分泌独特的抗人CLDN18.2抗体进行确认。将C13C1杂交瘤细胞进行了多次有限稀释，精心又精细地优化筛选每次稀释后的单克隆，最终发现了能分泌独特的抗人CLDN18.2的抗体的单克隆细胞株，结果见表1b。
表1b 杂交瘤C13C1优化筛选发现的杂交瘤单克隆细胞株
Figure PCTCN2019090255-appb-000013
Figure PCTCN2019090255-appb-000014
从表1b的结果可以看出，本发明得到初始杂交瘤克隆C13C1经精细、优化筛选得到的的单克隆细胞株C13C1F1D3G6和C13C1F1D3H5所分泌的抗体均保留了和人CLDN18.2细胞结合，读值分别为0.8895和0.8778。而和人CLDN18.1细胞没有结合，读值分别为0.0859和0.0756。该读值接近本次ELISA本底0.081。初始杂交瘤克隆F2A4经同步筛选也得到了单克隆细胞株F2A4F6F3E4和F2A4F6F3H7。这些单克隆细胞株和预期一样，和人CLDN18.2细胞、人CLDN18.1细胞有同样的结合活性，数据见表1b。这些结果表明，本发明发现单克隆细胞株，比如C13C1F1D3G6，能够分泌独特的抗体，意外地，所分泌的抗体只能同人CLDN18.2结合，而不结合人CLDN18.1。
这意味着本发明意外发现的抗体能够有效地只识别人CLDN18.2蛋白，具有作为单克隆抗体治疗肿瘤的潜力，特别是治疗人CLDN18.2蛋白过表达的癌症患者，包括但不限于胰腺癌、胃癌、食道癌、肺癌等。因为完全不结合人CLDN18.1蛋白，从而预期可以避免治疗性抗体和人CLDN18.1等蛋白非特异结合而导致的毒、副作用。
实施例5：本发明鼠源抗人CLDN18.2抗体筛选、鉴定
从上述实施例子中得到的杂交瘤单克隆细胞株C13C1F1D3G6(表1b)中提取该细胞株所分泌的抗体序列，即得到本发明鼠源抗体mab5b序列。从杂交瘤优选的单克隆细胞株中提取抗体序列过程为本领域技术人员熟知并常用的方法。
具体地，本发明通过扩增培养上述实施例子中发现的杂交瘤单克隆细胞C13C1F1D3G6，收取1×10 6个细胞，用Trizol(Invitrogen，15596-018)提取RNA(按照试剂盒说明书步骤)，将提取的RNA反转录成cDNA，反转录试剂盒购自生工生物技术(上海)股份有限公司，Cat#B532435。以反转录得到的cDNA为模板，进行PCR扩增后，扩增产物测序即得到mab5b的抗体轻重链可变区序列。所用引物参阅Novagen发表的手册(TB326 Rev.C0308)。
从本发明优选的杂交瘤单克隆细胞株C13C1F1D3G6中获得抗人CLDN18.2(anti-hCLDN18.2)单克隆抗体轻链核苷酸序列(SEQ ID NO:5)和重链核苷酸序列(SEQ ID NO:6)。
由上述轻链碱基序列翻译即得到本发明发现的杂交瘤单克隆细胞株提取的鼠源抗人CLDN18.2(anti-hCLDN18.2)单克隆抗体mab5b轻链可变区的氨基酸序列为：
Figure PCTCN2019090255-appb-000015
由上述重链碱基序列即得到本发明发现的杂交瘤单克隆细胞株提取的鼠源抗人CLDN18.2(anti-hCLDN18.2)单克隆抗体mab5b重链可变区的氨基酸序列为：
Figure PCTCN2019090255-appb-000016
本发明发现的杂交瘤单克隆细胞株中提取的上述抗体mab5b，用实施例3所述方法将抗体轻、重链可变区和恒定区分别进行克隆(序列如下)重组表达，纯化后，和对照抗体Ref同时检测对人hCLDN18.1、hCLDN18.2、鼠mCLDN18.1和mCLDN18.2的结合活性，结果如下表2a，表2b和图1。
本发明anti-hCLDN18.2抗体mab5b轻链(L Chain)如SEQ ID NO:9所示；重链(H Chain)如SEQ ID NO:10所示。
表2a 本发明anti-hCLDN18.2鼠源抗体mab5b和人CLDN18+cell的结合活性
Figure PCTCN2019090255-appb-000017
表2b 本发明anti-hCLDN18.2鼠源抗体mab5b和鼠CLDN18+cell的结合活性
Figure PCTCN2019090255-appb-000018
表2a，图1a结果表明，本发明发现的anti-hCLDN18.2鼠源抗体mab5b和对照抗体(Ref)都不和hCLDN18.1+cell结合，EC50检测不到(ND)，即使在200nM高浓度的时候仍检测不到结合活性，结合值Emax(指在样品浓度提高，结合达到平台时的结合值，即最大特异结合数值)仍然是本底，即背景值。而两个抗体均和hCLDN18.2+cell有很好的结合活性。意外的是，本发明的抗体mab5b结合活性比Ref好1倍以上(EC50为0.115nM vs 0.249nM)。更加意外的是，mab5b所能达到的最大结合数值Emax比Ref要高36％以上[(1.92-1.41)/1.41]。
表2b、图1b结果表明，本发明发现的anti-hCLDN18.2鼠源抗体mab5b和对照抗体(Ref)都不和鼠CLDN18.1+cell结合，EC50检测不到(ND)，即使在200nM高浓度的 时候仍检测不到结合活性，结合值Emax仍然是本底，即背景值。而两个抗体均和鼠CLDN18.2+cell有很好的结合活性。意外的是，本发明的抗体mab5b结合活性比Ref好4倍以上(EC50为0.182nM vs 1.04nM)。更加意外的是，mab5b所能达到的最大结合数值Emax比Ref要高1倍以上[2.21 vs 1.0]。
上述结果表明本发明所发现的新分子mab5b的结合活性(EC50和Emax)比对照分子都要好。并且和hCLDN18.1和mCLDN18.1都没有任何结合活性，说明mab5b不仅结合活性更好，而且特异性也非常优秀。这表明mab5b用于肿瘤治疗产品开发能提供更好疗效和安全性的优势。并且和鼠CLDN18.2也有更好的结合，为在鼠上进行临床前研究提供了更加便利的非灵长类动物选择。
实施例6：本发明抗体mab5b的人源化
本发明发现的抗体mab5b活性优于Ref，显示该抗体可以用于肿瘤治疗药物开发。为了减少药物开发过程中的免疫原性等方面的风险，比如由鼠源抗体完成人源化，并且优化人源化后分子特性，便于用于药物开发，本发明对mab5b进行了人源化筛选，以及序列优化工作。具体过程描述如下。
抗体的CDR定义本领域还有多种不同的方法，这些标记CDR方法可总结如下表3。
表3 本领域抗体CDR定义不同方法汇总*
区域 CCG定义 Kabat定义 AbM定义 Chothia定义 Contact定义
轻链CDR1 L24-L34 L24-L34 L24-L34 L24-L34 L30-L36
轻链CDR2 L50-L56 L50-L56 L50-L56 L50-L56 L45-L55
轻链CDR3 L89-L97 L89-L97 L89-L97 L89-L97 L89-L96
重链CDR1 H26-H35 H31-H35 H26-H35 H26-H32 H30-H35
重链CDR2 H50-H65 H50-H65 H50-H58 H52-H56 H47-H58
重链CDR3 H95-H102 H95-H102 H95-H102 H95-H102 H93-H101
*更多信息可以参阅网站：http://www.bioinf.org.uk/abs/#cdrdef
上述实施例5中得到的鼠源抗人CLDN18.2抗体mab5b按照表3各种定义方法，其CDR序列标记/注释如下。
表4 本发明抗人CLDN18.2(anti-hCLDN18.2)抗体mab5b按CCG定义CDR序列
Figure PCTCN2019090255-appb-000019
Figure PCTCN2019090255-appb-000020
表5本发明抗人抗体按Kabat定义CDR序列
Figure PCTCN2019090255-appb-000021
表6 本发明抗体按AbM定义CDR序列
Figure PCTCN2019090255-appb-000022
表7 本发明抗体按Chothia定义CDR序列
Figure PCTCN2019090255-appb-000023
Figure PCTCN2019090255-appb-000024
表8 本发明抗体按Contact定义CDR序列
Figure PCTCN2019090255-appb-000025
对本发明anti-hCLDN18.2抗体(mab5b)上述CDR分析，根据抗体标记系统，标记识别抗体轻、重链的CDR区(如上)基础上，将鼠源抗体mab5b轻、重链可变区序列分别和人抗体种系数据库(v-base)比较，找出同源性高的人抗体轻、重链种系，在此基础上，计算机建模，模拟抗体结构中可能影响和抗原结合的位点，回复突变关键位点和组合，筛选出活性优选的人源化抗体分子。
具体地，通过序列同源性比较分析，发现和mab5b轻链同源性比较好的人抗体种系包含有IGKV4-1*01(F)，IGKV2-28*01(F)，IGKV2D-28*01(F)，IGKV1-27*01(F)，IGKV1-39*01(F)，IGKV1D-39*01(F)，IGKV2-40*01(F)，IGKV2D-29*01(F)，IGKV2D-40*01(F)，IGKV3-15*01(F)。进一步比较、分析，优选人抗体种系轻链IGKV4-1*01(F)。特别地，所选定的人种系轻链IGKV4-1*01(F)的CDR2序列为WASTRES，和本发明发现的鼠源抗体mab5b轻链CDR2序列完全相同。序列比对发现mab5b轻链的J基因区和人抗体种系hJK1，hJK2.1，hJK2.2，hJK2.3，hJK2.4同源性高，进一步比较、分析，优选hJK2.1用于mab5b轻链人源化人抗体种系J区，进行人源化设计、筛选和序列优化。
通过序列同源性比较分析，发现和mab5b重链同源性比较好的人抗体种系包含有 IGHV1-69*02(F)，IGHV1-69*06(F)，IGHV1-69*08(F)，IGHV1-69*09(F)，IGHV1-69*10(F)，IGHV1-69*04(F)，IGHV1-69*14(F)，IGHV1/OR15-2*02(P)，IGHV1-69*01(F)，IGHV1-69*11(F)，进一步比较、分析，优选人种系重链IGHV1-69*01(F)序列用于本发明抗体人源化。序列比对发现mab5b的重链J基因区和人抗体种系重链J基因hJh4.1，hJh4.2，hJh4.3，hJh1，hJh2，hJh3.1，hJh3.2同源性高，进一步比较、分析，优选hJh4.1用于本发明mab5b重链人源化人抗体种系J区，进行人源化设计、筛选和序列优化。
将本发明抗体mab5b CDR区(见上述CDR之定义)移植到所选择的人源化轻、重链人抗体种系模板上，再与IgG轻、重链恒定区重组。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，筛选这些突变以及突变组合，看对抗体活性的影响，并对CDR区化学不稳定氨基酸残基优化，得到结构、活性等优化的抗体分子序列，即本发明抗人CLDN18.2鼠源抗体mab5b的人源化系列优化抗体分子。
具体地，在对本发明抗体mab5b轻链分析发现，包含CDR1(L24-L34位)和CDR2(L50-L56位)序列和本发明人源化优选人种系轻链IGKV4-1*01(F)的序列同源性很高。其中，L24-L34CDR1只有5个氨基酸不同，分别是L29、L30A、L30C、L30E和L34位，见下表9a。而L50-L56位CDR2则完全相同(见下表9b)。本发明首先对mab5b CDR1五个位点(L29、L30A、L30C、L30E和L34位)进行了回复突变成人种系IGKV4-1*01(F)对应位点的氨基酸，组合设计如下表9a。
表9a 本发明鼠源anti-hCLDN18.2抗体mab5b序列按Kabat定义的L24-L34位(CDR1，即SEQ ID NO:11)氨基酸以及人源化
Figure PCTCN2019090255-appb-000026
Figure PCTCN2019090255-appb-000027
表9b 本发明鼠源anti-hCLDN18.2抗体mab5b序列L50-L56位(CDR2)氨基酸
氨基酸位置编号 L50 L51 L52 L53 L54 L55 L56
mab5b CDR2 W A S T R E S
IGKV4-1*01(F)CDR2 W A S T R E S
将上述人源化设计分子Var1，Var2，Var3，Var4，Var5，Var6，Var7，Var8和mab5b按前述实施例子3克隆表达纯化，按实施例子2检测和人CLDN18.2+细胞结合活性，结果如下表10。
表10 本发明抗体轻链CDR1序列人源化优化
抗体 EC 50(nM) E max
mab5b 0.215 1.93
Var1 0.461 1.83
Var2 0.221 1.7
Var3 0.143 1.46
Var4 0.434 1.7
Var5 0.656 1.37
Var6 7.67 1.14
Var7 9.51 1.02
Var8 0.20 1.79
上述结果表明，本发明抗体mab5b的轻链CDR1人源化优化序列均保留了和mab5b 同样(接近)的结合活性。
进一步，用上述所述人源方法，将mab5b进行人源化设计，优选得到本发明mab5b抗体人源化轻链可变区优选序列如下：
L14：DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:29)
L11：SEQ ID NO:30；
L12：DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:31)
L13：SEQ ID NO:32；
L15：DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTHFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:33)
由上述所述方法得到本发明人源化重链可变区优选序列如下：
H51：QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS(SEQ ID NO:34)
H52：SEQ ID NO:35；H53：SEQ ID NO:36；
H54：QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS(SEQ ID NO:37)
上述轻链可变区序列，包含列出的如L14、L11、L12、L13、L15和未列出的所示任意序列与人抗体轻链κ型或λ型轻链恒定区组合得到本发明人源化抗体轻链序列。上述重链可变区序列，包含列出的如H51、H52、H53、L54和未列出的所示重链可变区序列和hIgG1，2，3，4等不同亚型的恒定区序列组合得到本发明抗体的重链序列。所述轻链、重链任意组合得到本发明人源化抗体，部分人源化抗体序列如下表11所示。
表11 本发明人源化抗体部分优选序列
Figure PCTCN2019090255-appb-000028
Figure PCTCN2019090255-appb-000029
人源化Ab10抗体氨基酸序列：轻链：
Figure PCTCN2019090255-appb-000030
重链：
Figure PCTCN2019090255-appb-000031
Figure PCTCN2019090255-appb-000032
人源化Ab7抗体氨基酸序列的轻链：SEQ ID NO:40；重链：SEQ ID NO:39；
人源化Ab8抗体氨基酸序列的轻链：SEQ ID NO:38；重链：SEQ ID NO:41；
人源化Ab9抗体氨基酸序列的轻链：SEQ ID NO:40；重链：SEQ ID NO:41；
人源化Ab6抗体氨基酸序列的轻链：
Figure PCTCN2019090255-appb-000033
重链：SEQ ID NO:39；
人源化Ab11抗体氨基酸序列的轻链：SEQ ID NO:42；重链：SEQ ID NO:43；
人源化Ab12抗体氨基酸序列的轻链：SEQ ID NO:42；重链：SEQ ID NO:44；
人源化Ab13抗体氨基酸序列的轻链：SEQ ID NO:45；重链：SEQ ID NO:43；
人源化Ab14抗体氨基酸序列的轻链：SEQ ID NO:45；重链：SEQ ID NO:44；
人源化Ab15抗体氨基酸序列的轻链：SEQ ID NO:45；重链：SEQ ID NO:39；
用本发明所述实施例3方法克隆、表达纯化重组抗体，实施例2所述ELISA方法检测筛选上述人源化抗体和hCLDN18.2+cell，hCLDN18.1+cell的结合活性，结果见下表12，和图2a。
表12 本发明人源化anti-hCLDN18.2抗体和hCLDN18.2+cell，hCLDN18.1+cell的结合活性
Figure PCTCN2019090255-appb-000034
NA：不适用；ND：没有检测到
表12结果表明，本发明发现的抗体mab5b人源化后，保持了鼠源抗体结合活性比对照抗体Ref高的优势，人源化抗体Ab10的EC 50比Ref要好2倍(0.117vs 0.345)。不仅如此，这些人源化优化的分子的结合最高值Emax比对照抗体Ref高出39.8％-54.1％。比鼠源抗体mab5b更加优于对照抗体。
为了进一步优化本发明人源化分子，优选最终序列和人抗体种系轻、重链尽量趋于一致，减少鼠源抗体中少量序列可能造成的免疫原性，优选人源化优化轻链CDR1序列Var3(见表9)设计了系列人源化抗体，并进行了特异结合活性筛选。结果如下表13和图2b。
表13 本发明人源化anti-hCLDN18.2抗体优化抗体和hCLDN18.2+cell，hCLDN18.1+cell的结合活性
Figure PCTCN2019090255-appb-000035
ND：没有检测到
上述结果表明，本发明进一步优化得到的人源化抗体(表13和图2b)中，这些人源化抗体的回复突变的氨基酸数目各有不同，包括Ab1-Ab20(见表11)。其中，有6个回复突变的，如Ab10(轻链1个，重链5个)；有6个回复突变，并且轻链CDR1人源化优化的，如Ab6(轻链1个，重链5个)；有2个回复突变如Ab14(轻链0个，重链2个)；有只有1个回复突变如Ab11(轻链1个，重链0个)，以及完全没有回复突变的，如Ab13。这些优化的人源化分子活性，包括EC50和Emax活性，都保持了和Ab10(表12中已经优化的mab5b人源化分子)相同的水平且都不和hCLDN18.1+细胞结合。
特别意外地发现的Ab13抗体分子没有任何回复突变，即是全人源化的抗体分子，且CDR1序列人源化优化，其结合活性(EC50和Emax)和Ab6，Ab10一样。
上述结果表明，本发明在鼠源抗体mab5b序列通过人源化，优化筛选得到的抗体分 子，包含只人源化FR区，轻链CDR1保持wild type(没有突变)，如Ab10；或FR区人源化外，轻链CDR1也进行了人源化优化的序列Var3，所得到的Ab6，Ab11-15等，均保持了其结合活性，且均优于对照分子，EC50比对照分子强1倍，Emax比对照分子高出30％-50％，而且都不同hCLDN18.1+细胞结合。
实施例7：本发明抗体序列脱酰胺基(deamidation)敏感位点序列优化
通过对本发明mab5b序列以及人源化后的优化序列(上述表9a，9b，11)计算机结构模拟分析，对可能存在的，特别是CDR区的转录后修饰(Potential post-translational modifications，PTMs)位点分析，包括抗体的聚集，脱酰胺基敏感(asparagine deamidation，位点(NG，NS，NH等)、天冬氨酸异构(DG，DP)敏感位点，N糖基化(N-{P}S/T)敏感位点，氧化敏感位点等分析，发现本发明抗体轻链CDR1(CDR1，L Chain)第L30A，L30B位为NS，重链CDR3(CDR3，H Chain)第H99，H100为NS，其中的L30A位、H99位的asparagine(N)可能对deamidation敏感。为了降低本发明抗体用于药物制剂时抗体分子的成药性相关风险，我们对这两个可能的敏感位点进行了序列优化。具体地，对本发明抗体轻链CDR1的L30A，L30B位(NS)；重链CDR3的H99、H100位(NS)点进行突变，优选方案如下：
表14 本发明抗体序列脱酰胺基敏感位点序列优化设计
Figure PCTCN2019090255-appb-000036
*：NA，不适用(没有做改变)
将上述优化的脱酰胺基敏感位点优化设计后的序列以不同轻、重链组合表达抗体后进一步筛选结合活性。所述抗体组合部分见下表。
表15 本发明抗体脱酰胺基敏感位点优化抗体
Figure PCTCN2019090255-appb-000037
*：-代表没有突变
上述优选抗体按实施例子3方法表达、纯化后用实施例2之方法检测其和人CLDN18.2+cell的结合活性，结果见表16a，16b和表16c。
表16a 本发明人源化anti-hCLDN18.2抗体脱酰胺基敏感位点优化抗体活性
Figure PCTCN2019090255-appb-000038
Figure PCTCN2019090255-appb-000039
*：Ab14作为本次assay对照样品(没有NT突变)
表16b 本发明人源化anti-hCLDN18.2抗体脱酰胺基敏感位点优化抗体活性
Figure PCTCN2019090255-appb-000040
*：Ab14作为本次assay对照样品(没有NT突变)
表16c 本发明人源化anti-hCLDN18.2抗体脱酰胺基敏感位点优化抗体活性
Figure PCTCN2019090255-appb-000041
表16a结果表明，Ab21(轻链CDR1 NS->TS和重链CDR3 NS->TS)“几乎没有”结合活性(>16nM)。Ab23(轻链CDR1 NS->NT，重链CDR3 NS->TS)也“几乎没有”结合活性(>40nM)。Ab22(轻链CDR1 NS->TS，重链CDR3 NS->NT)结合活性有所下降，但没有完全消失(EC50＝1.37nM vs Ab14 EC50＝0.35)。也就是说虽然轻链CDR1的L30A位N->T，但因为重链CDR3的H99位N没有突变，抗体的结合活性从“几乎没有”回复到1.37nM，比正常Ab14减少了近3倍。而Ab56(轻链CDR1 NS->NT，重链CDR3 NS->NT)的结合活性则和正常分子Ab14一样，其EC50＝0.295nM。
综合表16a结果说明，本发明抗体轻链CDR1的L30A位N和重链CDR3第H99位N对本发明抗体的结合非常重要，这些点的突变(比如变为T)直接导致活性丢失或者完全没有活性。但是轻链的L30B位S和重链CDR3第H100位S突变为T对结合活性没有影响。
表16b数据进一步证明单独轻链的L30B位S突变为T，或者单独重链CDR3的第H100位S突变为T，对结合活性没有影响。
非常意外地，表16b数据表明，Ab30(重链CDR3，NS->NT)完全失去了结合活性(EC50>11nM，即实际上结合曲线已经和阴性对照一样，和抗原没有特异结合作用了)，但Ab34含有同样的重链(CDR3，NS->NT)却没有失去活性。这说明Ab30活性的丢失不是由于CDR3，NS->NT的变化引起的。鉴于这两个分子唯一的差别是轻链CDR1的不同，即Ab30轻链为L14，其CDR1为KSSQSLLNSGN QKNYL T(SEQ ID NO:11)；Ab34轻链为L12，其CDR1为KSSQSLLNSGN NKNYL A(SEQ ID NO:12)，划线部分为两个CDR1序列的不同。这一意外的发现表明，当重链CDR3第H100位S突变为T(以避免潜在的脱酰胺基)的时候，轻链的CDR1序列必须是KSSQSLLNSGN NKNYL A(SEQ ID NO:12，即CDR1序列优化的Var3，见表9a)。轻链CDR1如果是KSSQSLLNSGN QKNYL T的话(SEQ ID NO:11，划线部分为两个CDR1序列的差异)，则整个抗分子完全失去了结合活性(Ab30)。这一发现表明，本发明抗体CDR1如果第L30E位是Q和L34位是T(划线标出)则重链第H100位S不能进行突变，如以避免潜在的脱酰胺基H100位S突变为T，否则整个抗体失去结合活性。
为了进一步确认本发明抗体轻链CDR1序列KSSQSLLNSGN QKNYL T(SEQ ID NO:11，第L30E位Q和L34位T)和人源化优化的CDR1序列KSSQSLLNSGN NKNYL A(SEQ ID NO:12，第L30E位N和L34位A)的不同对本发明抗体轻链CDR1和重链CDR3中NS突变(以避免潜在的脱酰胺基)的影响，我们比较了Ab10(CDR1：KSSQSLLNSGN QKNYL T，SEQ ID NO:11)和Ab6(CDR1：KSSQSLLNSGN NKNYL A，SEQ ID NO:12)，这两个分子唯一的区别在于其轻链CDR1(划线的氨基酸为两者的不同)。以及这两个分子的轻链CDR1和重链CDR3上同时进行NT->NS突变，得到的Ab35，Ab36两个抗体。表16c结果表明Ab35完全失去了结合活性(EC50>62nM)。
这一数据确认了本发明所发现的轻链CDR1序列KSSQSLLNSGN NKNYL A(SEQ ID NO:12)第L30E位是N和L34位是A(划线标出)即CDR1人源化优化的序列不影响轻链CDR1第L30B位S(斜体)，和或重链第H100位S进行突变以避免潜在的脱酰胺基(比如L30B位S突变为T或/和H100位S突变为T)。
如果CDR1序列KSSQSLLNSGN QKNYL T(SEQ ID NO:11)第L30E位是Q和L34位是T(划线标出)则CDR1第L30B位S(斜体)或重链第H100位S均不能进行突变(比如L30B位S突变为T或H100位S突变为T)，任何这样的突变会让抗体的结合活性消失。
综合上述结果表明，本发明抗体轻链CDR1L30A，L30B位NS；重链CDR3H99，H100位NS可以通过NS->NT之优化突变来降低可能的deamidation风险，但只对CDR1的第L30E位是N，L34位是A(即序列为人源化优化的序列KSSQSLLNSGN NKNYL A，SEQ ID NO:12)时候才可以，如果该区序列为KSSQSLLNSGN QKNYL T(SEQ ID NO:11)则抗体完全失去活性(划线部分为两者的序列不同)。
实施例8：本发明抗体Fc序列(变体)活性分析
本发明抗体可变区和人抗体不同的轻、重链恒定区，包括但不限于实施例子3所列出的人抗体不同的轻链(κ、λ型轻链等)、重链恒定区(hIgG2、hIgG4、hIgG1)组合，特别是人的IgG1Fc序列变体，比如第356-358位为DEL或EEM的不同形式，可以得到不同抗体变体形式。表17列出了部分本发明抗体和Fc序列的变体形式，包括Fc区序列第356-358位为DEL或EEM。
表17 本发明抗体轻重链、重链恒定区不同的抗体
编号 轻链 重链 hIgG1，356-358位
Ab10 L14 H51 DEL
Ab42 L14 H51 EEM
Ab6 L12 H51 DEL
Ab43 L12 H51 EEM
Ab13 L13 H53 DEL
Ab48 L13 H53 EEM
Ab51 L21 H64 DEL
Ab52 L21 H64 EEM
Ab14 L13 H54 DEL
Ab53 L13 H54 EEM
Ab24 L21 H61 DEL
Ab56 L21 H61 EEM
所述Ab42抗体氨基酸序列如下：
轻链：SEQ ID NO:38；重链：SEQ ID NO:46；
上述优选抗体按实施例3方法表达、纯化后用实施例2之方法检测其和人CLDN18.2+cell的结合活性，代表数据如下表18。结果表明，轻、重链恒定区的上述变化，包括hIgG1的356-358位分别是DEL或者EEM不影响本发明抗体的活性。
表18 本发明人源化anti-hCLDN18.2抗体IgG1 Fc 356-358位DEL或EEM的结合活性
Figure PCTCN2019090255-appb-000042
实施例9：本发明抗体Fc区(人IgG1)针对ADCC，CDC活性的序列优化
本发明抗体特异结合人CLDN18.2用于肿瘤治疗作用机理之一是可以通过抗体Fc介导效应细胞(effectorcells)对肿瘤细胞的杀伤达到治疗肿瘤的目的。本发明抗体人Fc区(hIgG1 Fc)介导效应细胞ADCC和CDC作用，能特异增强靶向肿瘤细胞作用，对非特异靶点则导致靶点外的副作用。人抗体Fc区(hIgG1 Fc)介导的ADCC和CDC作用有很多研究。本发明对所发现抗体分子的Fc区域针对人血细胞的效应作用(ADCC和CDC)进行了确认。具体地，在本发明抗体的人抗体Fc区域(hIgG1 Fc)进行不同的突变，评价这些突变体的ADCC和CDC活性，突变设计见表19，活性数据见表20a，20b。
表19 本发明抗体Fc(IgG1)针对ADCC，CDC的活性位点设计
编号 轻链 重链 hIgG1，Fc区域
Ab10 L14 H51 WT
Ab57 L14 H51 F243L
Ab572 L14 H51 L234A
Ab573 L14 H51 L235A
Ab573 L14 H51 L234A/L235A
Ab58 L14 H51 S239D/A330L/I332E
Ab6 L12 H51 WT
Ab59 L12 H51 F243L
Ab60 L12 H51 S239D/A330L/I332E
Ab24 L21 H61 WT
Ab65 L21 H61 F243L
Ab66 L21 H61 S239D/A330L/I332E
上述优选抗体按实施例子3方法表达、纯化后得到抗体后，分别进行ADCC(抗体依赖性细胞毒性实验)和CDC(补体依赖细胞毒性实验)检测抗体Fc序列优化后的活性。具体地，
ADCC：
实施例1中构建好的hCLDN18.2+细胞，正常培养。培养基为DMEM/F12加10％FBS(上海源培生物科技股份有限公司Cat#L310KJ)，作为本实验ADCC用的靶细胞。
实验前一天，取培养好的hCLDN18.2+细胞，计数5000个/孔铺96-孔板。实验当天，准备PBMC细胞(本发明分离自人体外周血。人体外周血由本公司志愿者捐献)，按照150000cell/50μl浓度将PBMC悬浮于无血清RPMI1640培养基(培源生物，Cat#L210KJ)。待测药物用无血清RPMI1640配制，起始浓度40μg/ml按3倍比例稀释。
取出培养好的靶细胞(hCLDN18.2+细胞)，小心吸取移去上清，加入配制好的PBMC，50μl/孔；同时加入50μl/孔配制好的不同浓度的待测样品，后将靶细胞于37℃，5％CO 2培养箱中孵育4小时，检测LDH。
LDH试剂盒为Cytotoxicity LDH Assay Kit-WST，购自东仁化学科技(上海)有限公司，货号CK12。操作方法按说明书进行，取出孔板，在每孔中加入100μlWorkingSolution，包裹铝箔避光，在室温反应10-40min，MultiskanGO(ThermoFisher)酶标仪490nM读数，每隔10min检测一次，取合适的反应时间数据，用Graphpadprism5分析处理数据。
CDC：
实施例1中构建好的hCLDN18.2+细胞，作为本实验CDC用靶细胞。hCLDN18.2+细胞正常培养，培养基为DMEM/F12加10％FBS(同ADCC)。实验前一天，收集靶细胞，计数，配制1×10 5/ml细胞，100μl/孔加入到96孔细胞培养板中。37℃，5％CO 2孵育过夜备用。
实验当天，移走96-孔板细胞中的培养基，用PBS洗2遍待用。待测抗体用无血清培养基(RPMI1640)稀释，抗体起始浓度为20μg/ml，按5倍稀释。将50μl/孔稀释抗体加入PBS洗过的靶细胞培养板(0μg/ml抗体孔用新鲜培养基100μl/孔，作为对照孔)，各个浓度点设6个复孔。37℃，5％CO 2孵育15min。
准备补体：取新鲜血清于灭菌的离心管中。分取一半血清于56℃水浴锅孵育30分钟灭活补体，作阴性对照。将灭活和没有灭活的血清，分别用RPMI1640培养基配成血清：RPMI1640培养基＝40％：60％，即40％为血清，60％为RPMI1640。
向含不同浓度待测抗体的靶细胞培养板中加入50μl/孔稀释好的血清，即血清终浓度为20％；样品(抗体)的起始浓度为10μg/ml。前3复孔加入有补体的血清，后3复孔加入灭活补体的血清。37℃，5％CO2度培养箱孵育2小时，后取出用LDH试剂盒检测。
LDH试剂盒为Cytotoxicity LDH Assay Kit-WST，购自东仁化学科技(上海)有限公司，货号CK12。操作方法按说明书进行，取出孔板，在每孔中加入100μl Working Solution，包裹铝箔避光，在室温反应，每隔10min检测一次，取合适的反应时间数据，MultiskanGO(ThermoFisher)酶标仪490nM读数，Graphpadprism5分析处理数据。
表20a 本发明抗体Fc(IgG1)突变体ADCC活性检测
样品 EC 50(μg/ml)
Ab6 1.40
Ab59 检测不到(无结合)
Ab60 0.431
Neg IgG* 检测不到(无结合)
*Neg IgG：为和靶点没有结合的非特异性抗体
表20b 本发明抗体Fc(IgG1)突变体CDC活性检测
样品 EC 50(μg/ml)
Ab6 0.290
Ab59 检测不到(无结合)
Ab60 0.973
Ab24 0.255
Ab65 检测不到(无结合)
Ab66 0.927
Neg IgG* 检测不到(无结合)
*Neg IgG：为和靶点没有结合的非特异性抗体
结果表明，本发明抗体如果用hIgG1的形式，其Fc区域第243位F单个位点突变， 比如F243L、L234A、L235A、L234A/L235A会完全失去本发明抗体的ADCC和/或CDC活性；如Fc区域第239、330、332三个位点组合突变，比如S239D/A330L/I332E，则本发明抗体CDC活性有所减弱。
实施例10：本发明抗体不同人源化分子ADCC、CDC活性评估
为了评估本发明人源化抗体分子ADCC和CDC活性，用同上述实施例子的方法，和对照分子(Ref)一起，检测了本发明人源化分子的ADCC和CDC活性，结果见下表21a、21b和图3。
表21a 本发明抗体不同人源化分子ADCC活性评估
样品 EC 50(μg/ml)
Ab10 1.55
Ab6 1.40
Ref 1.39
Neg IgG* 检测不到(无结合)
*Neg IgG：为和靶点没有结合的非特异性抗体，下同
表21b 本发明抗体不同人源化分子CDC活性评估
样品 EC 50(μg/ml)
Ab10 0.648
Ab35 检测不到(无结合)
Ab6 0.293
Ab36 1.75
Ab14 0.265
Ab24 1.30
Ab13 0.374
Ab51 2.12
Ref 2.31
Neg IgG* 检测不到(无结合)
上述结果表明，本发明人源化抗体ADCC活性和对照抗体(Ref)相当(表21a)。意外地，本发明人源化分子，包括含不同回复突变的人源化分子之间，Ab6，Ab13，Ab14等的CDC活性接近(EC50分别为0.293μg/ml，0.374μg/ml，0.265μg/ml)且比Ab10 (0.648μg/ml)好1倍以上，尤为意料之外的是，Ab6、Ab13及Ab14等的CDC活性比对照抗体(EC50＝2.31μg/ml)好近10倍(见表21b、图3)。Ab36、Ab24的CDC活性也都比对照抗体Ref要好。
Ab35(CDR1，CDR3 NS突变体，参见实施例子7，表16c)因为失去了结合活性，而检测不到CDC活性。
实施例11：本发明抗体诱导肿瘤细胞(hCLDN18.2+cell)凋亡活性评价
为检测本发明抗体，特别是优选人源化抗体诱导hCLDN18.2+cell(肿瘤细胞)凋亡的作用，我们用本发明实施例1中构建好的hCLDN18.2+cell，检测了本发明抗体诱导肿瘤细胞凋亡活性。hCLDN18.2+cell正常培养(培养基含10％FBS的DMEM/F12，供应商：上海源培生物科技股份有限公司，货号：L310)，作为本实验所用细胞。实验开始，hCLDN18.2+cell铺96-孔板，铺板密度为2×10 4/孔。37℃，5％CO2培养过夜贴壁。抗体样品准备：用无血清DMEM/F12培养基制备0μg/ml，1μg/ml，3μg/ml，10μg/ml抗体样品。取出过夜贴壁培养的hCLDN18.2+cell，弃培养基，并用PBS洗细胞两次。分别加入准备好的不同浓度的抗体样品，100μl/well。继续培养24h后，检测LDH。
LDH试剂盒为Cytotoxicity LDH Assay Kit-WST，购自东仁化学科技(上海)有限公司，货号CK12。操作方法按说明书进行，取出孔板，在每孔中加入100μl Working Solution，采用包裹铝箔等方法避光，在室温反应，在不同的时间点(10min，20min，30min，40min，50min)，Multiskan GO(ThermoFisher)酶标仪490nM读数，找出最佳的反应时间，取读数值用Graphpad prism 5分析处理数据。
结果见图4a、图4b。图4a、4b结果表明，本发明人源化抗体Ab10，Ab6，Ab14，Ab24，Ab36等诱导肿瘤细胞凋亡的活性都比对照分子(Ref)好，活性好/强3-10倍，这些分子10μg/ml浓度已经比对照分子的30μg/ml活性相当或者甚至比阳性分子30μg/ml活性还强。
具体地，Ab6 10μg/ml增加肿瘤细胞凋亡活性(46.7％)比阳性对照同浓度(15.3％)好2倍以上；甚至比阳性对照30μg/ml(19.6％)还好1倍以上；而30μg/ml浓度时诱导肿瘤细胞凋亡活性(85.1％)比同浓度下对照抗体(19.6％)的好3倍以上。
更意外地，Ab6 10μg/ml增加肿瘤细胞凋亡活性(46.7％)比Ab10同浓度(33.2％)好41％；30μg/ml浓度时诱导肿瘤细胞凋亡活性(85.1％)则比同浓度下Ab10(34.7％)的强1倍多。
实施例12：本发明抗体抑制肿瘤细胞(hCLDN18.2+cell)增殖作用
为检测本发明抗体对肿瘤细胞的增殖抑制所用，用实施例子1构建的hCLDN18.2+细胞进行了活性检测。具体地，hCLDN18.2+细胞正常培养(培养基：含10％FBS的DMEM/F12，供应商：上海源培生物科技股份有限公司，货号：L310)。实验开始时，取对数期生长的hCLDN18.2+细胞铺96-孔板，铺板密度为3×10 3/孔。37℃，5％CO2培养过夜贴壁培养。抗体样品准备：用含10％FBS的DMEM/F12(源培生物)配制1μg/ml，10μg/ml，30μg/ml抗体样品。取出过夜贴壁培养的hCLDN18.2+细胞，弃培养基，并用PBS洗细胞一次，后分别加入准备好的不同浓度的抗体样品，100μl/well。继续培养72h后，用CCK-8试剂盒检测。
CCK-8试剂盒为Cell Counting Kit-8，购自东仁化学科技(上海)有限公司，货号CK04。操作方法按说明书进行，取出96孔板，在每孔中加入10μl CCK-8溶液(注意不要在孔中生成气泡，否则会影响读数)，将培养板在培养箱中孵育1-4h，找出最优检测时间点，Multiskan GO(ThermoFisher)酶标仪450nM读数，数值用Graphpad prism 5分析处理数据。结果见下表22。
表22 本发明优选人源化抗体抑制肿瘤细胞(hCLDN18.2+cell)增殖活性(抑制率％)
样品/浓度 1μg/ml 10μg/ml 30μg/ml
Neg IgG 0 0.34 0.1
Ab10 2.17 2.93 3.16
Ab6 3.29 6.12 6.2
Ref 2.34 2.94 3.9
表22结果表明，阴性抗体1μg/ml，10μg/ml，30μg/ml浓度下抑制肿瘤细胞活性(抑制率)在1％以下，即本底水平。1μg/ml，10μg/ml，30μg/ml Ab10抑制肿瘤细胞活性(抑制率)在2.17％-3.16％，这和Ref的抑制率2.34％-3.9％接近。而Ab6抑制肿瘤细胞活性则比Ref强很多，例如10μg/ml抑制率6.12％是Ref(2.94％)的2倍以上。
实施例13：本发明人源化抗体和鼠CLDN18的结合活性检测
按照前述实施例2之方法，检测了本发明人源化优选抗体和鼠CLDN18.1+cell和鼠CLDN18.2+cell的结合活性。在筛选过程中，我们筛选得到了一个克隆(抗体L180)，该抗体和人和鼠的CLDN18.1均有结合。作为本assay的对照，L180和mCLDN18.1+cell结合活性EC50＝0.48nM，表明本发明构建鼠CLDN18.1+cell和抗CLDN18.1抗体有特异结合。而本发明优选人源化抗体和mCLDN18.1+cell均没有结合，且都保留了同鼠源抗体mab5b一样的和mCLDN18.2+cell的结合活性，见下表23。
表23 本发明优选人源化抗体和mCLDN18.2+cell的结合活性
样品 EC 50(nM) E max
mab5b 0.375 2.17
Ab10 0.371 2.27
Ab35 ND# ND
Ab6 0.594 2.47
Ab36 0.518 2.18
Ab14 0.399 2.39
Ab24 0.574 2.22
Ab13 0.422 2.37
Ab51 0.474 1.91
Neg IgG* 无结合 0.19(本底)
#：未检测。
上述结果表明，本发明优选人源化抗体都保留了和mCLDN18.2+cell的结合活性。EC50和人源化前的mab5b(同一assay中EC50＝0.375nM)具有相同的结合活性。Emax在1.91(Ab51)–2.47(Ab6)之间，和mab5b(2.17)接近。
实施例14：本发明抗体体内药效活性评估
为了评价本发明抗体的抗肿瘤活性，用BALB/c裸小鼠皮下移植hCLND18.2+细胞(实施例1构建)或者胃癌细胞系NUGC4(上海素尔生物科技有限公司)建立的动物药效模型，进行本发明抗体体内药效评估。
具体地，hCLDN18.2+细胞培养基为DMEM/F12(源培生物)加10％胎牛血清(上海博升生物科技有限公司，货号BS-0002-500)。NUGC4细胞培养基为RPMI1640(源培生物)，加10％胎牛血清。培养条件为37℃，5％CO 2。BALB/c Nude小鼠，雌性，4周龄，体重18-20g，购自上海西浦尔必凯实验动物有限公司(生产许可证编号：SCXK(京)2012-0001)，室温20-25℃，湿度40-60％，自由进食进水，适应性饲养3-4天。适时更换垫料、洁净笼具。取对数生长期细胞，收集并计数。
对于hCLDN18.2+细胞异体移植模型，取hCLDN18.2+细胞用PBS洗2次后，重悬配成细胞1×10 8/ml。在小鼠左翼皮下接种0.1ml共1×10 7细胞/只。挑选肿瘤长至体积约120-180mm 3大小小鼠，随机分组，每组5-6只。
对于胃癌NUGC4细胞异体移植模型，取NUGC4细胞用RPMI1640洗2次后，加入Matrigel，使其与RPMI640比例为1:1，用混合液重悬配成细胞，1×10 8/ml。在小鼠左翼 皮下接种0.1ml共1×10 7细胞/只。挑选肿瘤长至体积约150-200mm 3大小小鼠，随机分组，每组5-6只。
待测样品用PBS配制，无菌。Blank为PBS无样品对照，和靶点无关的抗体，即阴性抗体(Neg IgG)对照。腹腔注射，200μg/100μl/只。2次/周，连续数周。注射样品当天为第0天。每次给药前测量体重，肿瘤体积，记录数据。
肿瘤大小计算公式：肿瘤体积TV(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。相对肿瘤增长率(T/C％)＝100％*(T-T0)/(C-C0)。抑瘤率(TGI)＝(1-T/C)*100％。其中T0、T分别为样品组实验开始时及实验结束时的肿瘤体积；C0、C分别为对照组实验开始时及实验结束时的肿瘤体积。
动物体内药效结果见图5a，5b(hCLDN18.2+细胞异体移植模型)和下表24(NUGC4肿瘤细胞异体移植模型)。
用人CLDN18.2高表达细胞(hCLDN18.2+cell)建立的肿瘤异体移植模型中，图5a结果显示，本发明抗体Ab10，Ab6和阳性抗体分子(Ref)一样显示非常好的体内药效活性，抑制肿瘤细胞生长和/或杀死肿瘤细胞(抑制率)达到90％以上；意想不到的是，轻链CDR1和重链CDR3脱氨基敏感位点优化，轻链CDR1人源化优化的优选抗体Ab36完全抑制肿瘤生长，其体内药效明显优于Ab6，Ab10以及对照阳性抗体(Ref)。图5b结果显示，本发明完全人源化抗体(没有回复突变)Ab13，及其轻链CDR1和重链CDR3脱氨基敏感位点优化的完全人源化抗体Ab51，以及轻链CDR1和重链CDR3脱氨基敏感位点优化，重链仅有2个回复突变的人源化抗体Ab24均显示了和对照抗体一样的体内药效。
表24 本发明抗体在胃癌细胞系NUGC4肿瘤模型中的药效评价
Figure PCTCN2019090255-appb-000043
Figure PCTCN2019090255-appb-000044
NA:不适应，即空白对照。
在人胃癌细胞系NUGC4建立的肿瘤模型中，上述结果(表24)表明，本发明人源化优选抗体Ab10，Ab6均显示了一定药效，肿瘤抑制率在10％-20％，并且呈现剂量依赖。而同样的模型中，对照抗体(Ref)则和PBS对照，阴性抗体(Neg IgG)一样，没有抑制肿瘤效果。这一结果表明，本发明抗体具有优于阳性对照抗体的体内药效效果。
实施例15：本发明抗体小鼠药物代谢动力学(PK)评价
如上述实施例子13所述，本发明抗体和小鼠CLDN18.2有很好的结合活性，这为本发明抗体提供了临床前研究非灵长类物种选择。本发明评估了本发明抗体在小鼠体内的药物代谢动力学(PK)特性。
具体地，实验用Balb/c小鼠，雌性，6周龄，购自上海西普尔-必凯实验动物有限公司。小鼠购进后，每笼6只，无限量获取饲料和水。实验室环境饲养3天，温度20-25℃；湿度40-60％，12/12小时光/暗周期调节。实验开始前一天，对小鼠进行体重测量，取20-25g小鼠，进行分组编号，3只/组。实验当天，每只小鼠分别皮下注射受试药物Ab10和对照抗体(Ref)，药物给药剂量为10mg/kg，皮下注射100μl/只/次。
注射小鼠后0、1、6、24、26、30、50、55、71、79、98、143、167、191、215、240小时分别眼眶取血。所取血样离心，取上清，-20度保存，待测。等收集完血液样本后，用前述实施例子2之ELISA方法检测血清中的血药浓度。正式检测之前，取一只小鼠血清，进行梯度稀释，确定血清最佳稀释比例。所有样品按照最佳稀释比例进行ELISA检测，检测结果用T1/2计算公式和EXCEL软件对数据进行分析，结果如下表25。
表25 本发明抗体在小鼠体内PK评价
Figure PCTCN2019090255-appb-000045
上述表25结果表明，本发明抗体Ab10的Cmax比对照抗体(Ref)高57％([407.7-259.3]/259.3)。更加意外的是，本发明抗体Ab10的半衰期T1/2比对照抗体(Ref)要长30.7多个小时(170.1-139.4)，小鼠体内长达30小时的T1/2优势，预期人体内优势会更 大。这些结果表明，本发明人源化抗体具有比对照抗体优良的PK性能，特别是半衰期T1/2的显著优势，预期为临床至少可以带来药效(时间长)和成本(给药频率低)等优势。
实施例16：本发明抗体亲和力分析(KinExA)
CLD18.2为细胞跨膜蛋白，四次跨膜，两个胞外区ECL1和2均只有20-50氨基酸长度。本发明抗体特异结合CLDN18.2胞外区。常规表达抗原(20-50氨基酸)和抗体结合的Biacore检测方法不能很好评估本发明抗体和靶蛋白胞外区域特异亲和力。因此，本发明用KinExA方法检测抗体和hCLDN18.2+细胞的亲和力。方法参考KinExA 4000仪器说明书进行，即以待测抗体Ref，Ab10分别为Constant binding Partner(CBP)，以hCLDN18.2+细胞为Titrant。用固定浓度的抗体(CBP)梯度稀释细胞(Titrant)，孵育后通过抗人IgG Fc的柱子捕获未结合细胞的游离抗体(free CBP)，用anti-human Fc Alexa Flour 647获得信号值，通过KinExA自带软件计算获得抗体亲和力。
具体地，为了确定合适的抗体稀释液的浓度，首先根据预估的亲和力推算合理浓度进行信号测试，确定分别以500ul 120pM Ref抗体及100pM Ab10抗体作为Signal 100％，该浓度下获得满意的检测净信号值，PBS空白作为阴性信号值(NSB)。将120pM Ref抗体及100pM Ab10抗体浓度确定为CBP浓度。接下来的平衡实验中，300g离心10分钟分别收集两管hCLDN18.2+细胞，每管细胞数为5×10 8个(阳性率经FACS检测为100％)。用PBS清洗一次细胞，300g离心10分钟，将细胞分别收集到15ml离心管中。分别准备15ml 120pM Ref及100pM Ab10抗体溶液。向5×10 8个细胞中加120pM Ref抗体溶液至2ml，并以120pM Ref抗体溶液作为缓冲液2倍梯度稀释细胞，起始浓度2.5×10 8细胞/ml，18个梯度，每个梯度0.6ml。向5×10 8个细胞中加100pM Ab10抗体溶液至2ml，并以100pM Ab10抗体溶液作为缓冲液2倍梯度稀释细胞，起始浓度2.5×10 8细胞/ml，18个梯度，每个梯度0.6ml。将细胞与抗体的悬液室温震荡孵育2h。孵育结束后，450g，离心10分钟，取上清。准备1μg/ml anti human Fc Alexa Flour 647溶液。将样品放在管架中相应的位置。用KinExA3200仪器检测信号值并获得亲和力数据。结果如下表所示。结果显示：Ab10的亲和力(13.3pM)比Ref抗体亲和力高10倍以上。
表26 本发明抗体亲和力(M)检测
样品 Ab10 Ref
KD 1.33×10 -11 1.44×10 -10
实施例17：本发明抗体内吞活性检测
hCLDN18.2+cell长到90％时，胰酶消化，用FACS buffer(PBS+1％BSA)重悬细胞，终浓度为1x10 6/ml细胞。加细胞悬液500μl到1.5ml离心管，加带荧光标记的抗体对照抗体(和hCLDN18.2+cell没有结合的抗体)，对照抗体Ref，待测样品(本发明优选人源化抗体)Ab10，Ab6(抗体标记用的是mix-n-stain CF488 antibody labeling kit标记，Sigma-Aldrich，Cat#MX488S100-1kit。或mix-n-stain CF633 antibody labeling kit标记，Sigma-Aldrich，Cat#MX633S100-1kit。标记步骤均按照试剂盒中的说明书进行)，终浓度为1μg/ml或10μg/ml，冰上孵育1小时，用预冷的FACS buffer洗涤三次。取出1/5在冰上放置，作为结合值样品直接用于流式检测。余下的4/5细胞，用37℃预热的1640+10％FBS重悬细胞，分1/4直接放于冰上，做为内吞0小时样品，余下的放在37℃培养箱孵育，分别在1hr，2hr，3hr取出，冰上预冷，终止内吞，4℃ 1300rpm离心3分钟，弃上清。将0hr，1hr，2hr，3hr加入strip buffer(0.05M glycine，pH 2.45+0.1M NaCl)250μl，室温7min，4℃ 1300rpm离心3分钟，弃上清，FACS buffer洗涤一次。所有样品加入150μl 4％多聚甲醛(生工生物工程Cat#E672002)，4℃固定半小时后，上机检测(Beckman CytoFLEX流式细胞仪)。结果见下表。
表27 本发明抗体的内吞活性(FACS荧光强度，CF488标记抗体)
Figure PCTCN2019090255-appb-000046
上述结果(用CF488标记待测抗体)表明，在抗体浓度为1μg/ml的时候(上表左半部分)，对照抗体(Ref)的结合荧光强度(1939)和阴性抗体(和hCLDN18.2+细胞没有结合的抗体)的荧光强度值(1747)接近，即本底值。而且，0h，1h，2h，3h荧光强度值(划线数值)也都接近本底(1747)。而本发明抗体Ab10，Ab6的结合值(荧光强度)分别为27200，16300是本底的15倍和9倍，分别是对照抗体的14倍(27200/1939)和8.4倍(16300/1939)。这进一步证明了本发明抗体的结合Emax比对照抗体(Ref)强。
当在抗体浓度增加到10μg/ml的时候(上表右半部分)，阴性抗体(和hCLDN18.2+细胞没有结合的抗体)的荧光强度值，即本底值为1284-3485(划线数字)。对照抗体(Ref)的荧光强度(结合值)为17900，比本底(3485)高出4倍，说明Ref在高达10μg/ml的时候，才检测到荧光标记抗体的特异结合。而同样的浓度下，本发明抗体Ab10，Ab6的 结合强度(荧光值)分别为138000，86000，是本底(3485)的39倍和24倍，分别是Ref的7.7倍(138000/17900)和4.8倍(86000/17900)。这进一步说明本发明抗体的结合Emax比对照抗体(Ref)要强得多。这和上述KinExA检测的结果也是一致的。
将上表中的数据按照公式内吞百分比(％)＝(待测时间点荧光强度-0h荧光强度)/结合值，得到如下表数据。
表28 本发明抗体(CF488标记)的内吞活性(内吞％)
Figure PCTCN2019090255-appb-000047
本底：即读值为本底值，没有内吞；NA：不适用，该时间点没有内吞
上述结果表明，本发明优选抗体Ab10，Ab6在1μg/ml浓度的时候(上表左半部分)，1h，2h，3h均显示了比较好的内吞作用，3h内吞分别为28％和25％。而同等条件下，Ref抗体和阴性对照抗体一样，没有看到内吞。
当抗体的量加大到10μg/ml(上表右半部分)时候，对照抗体仍然只有非常小的内吞(7％-8％)，几乎接近本底，即没有内吞。而本发明抗体Ab10，Ab6内吞仍然随时间延长而增加，到3h，分别为23％和18％。这不及1μg/ml抗体浓度的28％和25％，说明因为本发明抗体结合活性优于Ref很多，10μg/ml对于本发明抗体过饱和而非最优化的抗体内吞浓度。
上述这些结果表明，本发明优选人源化抗体是内吞抗体。而对照抗体(Ref)不是内吞抗体，或者内吞非常微弱。为了进一步证明本发明优选抗体的内吞活性，选用不同于CF488的荧光染料CF633(Sigma-Aldrich，Cat#MX633S100-1kit)进行了标记抗体和内吞活性分析，检测仪器为BD FACS Calibur流式细胞仪。结果如下表。
表29 本发明抗体的内吞活性(FACS荧光强度，CF633标记抗体)
Figure PCTCN2019090255-appb-000048
Figure PCTCN2019090255-appb-000049
上述结果(用CF633标记待测抗体)表明，在抗体浓度为1μg/ml的时候(上表左半部分)，阴性抗体和hCLDN18.2+细胞没有结合的荧光强度值即本底值为23。0h，1h，2h，3h荧光强度值(划线数值)也都接近本底(12.7-13.7，即2倍以内小于100均为本底值)。对照抗体(Ref)的结合荧光强度(62.5)接近本底(阴性抗体，23)，在2倍范围内(62.5/23＝2.7)。而且在0，1，2，3h结合荧光强度值均比较弱(15.5-33.4)，特别是1，2，3h读值没有变化(33.9，33，7，33.4)说明这些数值基本接近本底水平。
而本发明抗体Ab10，Ab6的结合值(荧光强度)分别为854，690是本底的37倍(854/23)和30倍(690/23)，分别是对照抗体的14倍(854/62.5)和11倍(690/62.5)。这进一步证明了本发明抗体的结合Emax比对照抗体(Ref)要强很多。这和上述KinExA的结果也是一致的。
当在抗体浓度增加到10μg/ml的时候(上表右半部分)，阴性抗体(和hCLDN18.2+细胞没有结合的抗体)的荧光强度值，即本底值为23.2-14.9(划线数字)。对照抗体(Ref)的荧光强度(结合值)为198，是本底(23.2)的8.5倍，说明Ref在高达10μg/ml的时候，能检测到荧光标记抗体的特异结合。而同样的浓度下，本发明抗体Ab10，Ab6的结合强度(荧光值)分别为3229和2237，分别是本底(23.2)的139倍和96倍，分别是Ref的16倍(3229/198)和11倍(2237/198)。这进一步说明本发明抗体的结合Emax比对照抗体(Ref)要强很多(荧光读值强10倍以上)。
将上表中的数据按照公式内吞百分比(％)＝(待测时间点荧光强度-0h荧光强度)/结合值，得到如下表数据。
表30 本发明抗体(CF633标记)的内吞活性(内吞％)
Figure PCTCN2019090255-appb-000050
本底：即读值为本底值，没有内吞；NA：不适用，该时间点没有内吞
上述结果表明，本发明优选抗体Ab10，Ab6在1μg/ml浓度的时候，1h，2h，3h均显示了比较好的内吞作用，3h内吞分别为35.1％和30.4％。而同等条件下，Ref抗体(结合强度在本底2-3倍内)和阴性对照抗体一样，没有内吞作用。
当抗体的量加大到10μg/ml(上表右半部分)时候，对照抗体仍然只有非常小的内吞(5％-8％)，几乎接近本底，即没有内吞。而本发明抗体Ab10，Ab6内吞随时间延长而增加，到3h，分别为27.3％和28.9％(见图6)。这不及1μg/ml抗体浓度的35.1％和30.4％，说明因为本发明抗体结合活性优于Ref很多，10μg/ml对于本发明抗体已是过饱和浓度，而非最优化内吞作用的浓度。
综合上述两个不同荧光染料(CF488和CF633)实验结果表明，本发明优选人源化抗体Ab10，Ab6是内吞抗体。这个完全不同于对照抗体(Ref)。对照抗体没有内吞作用的，或者内吞非常微弱的，接近本底水平。
实施例18：抗CLDN18.2抗体Ab10偶联毒素SMCC-DM1(ADC1)制备
本发明抗CLDN18.2抗体Ab10偶联毒素SMCC-DM1(ADC1)的制备方法参考专利CN106188293A和US2009202536A1所公开的方法，具体地步骤如下。
Figure PCTCN2019090255-appb-000051
第1步，制备中间体。将1mg SMCC(4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯，上海瀚鸿化工科技有限公司，批号BH-4857-111203)溶解于0.55mL乙腈溶液，备用；取Ab10抗体(pH＝6.5，PBS缓冲液)50mg(5ml)加入上述备用的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯的乙腈溶液，于25℃下振荡反应2小时。反应结束后用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后得到中间体溶液，即上述合成线路中第一步和第二步之间的分子，并浓缩到约8mg/ml 后进行下一步反应。
第2步，抗体-毒素偶联。取第1步得到的中间体溶液5mg，加入L-DM1乙醇溶液(3.0mg L-DM1/ml乙醇)。L-DM1可采用公知的方法文献“Journal of Medicinal Chemistry.2006，49，4392-4408”制备得到，加入L-DM1的量按照L-DM1：中间体＝3：8mg比例加入。于25℃下振荡反应约4.0小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，即得本发明抗CLDN18.2抗体Ab10偶联毒素SMCC-DM1产物ADC1溶液。所得ADC1终浓度1.3mg/ml，分装于4℃储存备用。
所得ADC1样品用LC-MS方法检测分析，证明所得样品中没有游离的毒素小分子。用分光光度计(UV方法)检测A252，A280吸收峰，测定所得ADC1毒素和抗体的比例DAR＝4.4。
实施例19：抗CLDN18.2抗体Ab10偶联毒素MC-VC-PAB-MMAF(ADC2)制备
本发明抗CLDN18.2抗体Ab10偶联毒素MC-VC-PAB-MMAF(ADC2)的制备方法参考专利CN106188293A和US20140127211A1所公开的方法，具体地步骤如下。
Figure PCTCN2019090255-appb-000052
第1步，制备中间体。取0.7mg硫代乙酸S-(3-羰基丙基)酯溶解于0.9mL乙腈溶液，备用。取Ab10抗体(pH＝4.3的乙酸/乙酸钠缓冲液)50mg(5ml)加入上述备用的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加1.0ml的氰基硼氢化钠(14.1mg)水溶液，于25℃下振荡反应2小时。反应结束后，用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后，得产物1a溶液(见上述线路示意)，将1a浓缩到约10mg/ml进行中间体1b的制备。其中，所述x≤y。
取5ml上述1a溶液，加入0.15ml的2.0M盐酸羟胺溶液，于25℃下振荡反应30分钟后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后，得到上述线路中的中间体1b，即Ab10-丙硫醇溶液。
第2步，抗体-毒素偶联。取1.6mg化合物MC-VC-PAB-MMAF(可采用PCT专利WO2005081711公开的方法制备得到)溶解于0.3ml乙腈中，加入上述制备的中间体Ab10-丙硫醇溶液5mg，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，即得到本发明抗CLDN18.2抗体Ab10偶联毒素MC-VC-PAB-MMAF偶联物ADC2(结构示意如下式)。
Figure PCTCN2019090255-appb-000053
所得ADC2的终浓度为1.21mg/ml，分装于4℃储存备用。所得ADC2样品用LC-MS方法检测分析，证明所得样品中没有游离的毒素小分子。用分光光度计(UV方法)检测A252，A280吸收峰，测定所得ADC2毒素和抗体的比例DAR即y＝4.8。
实施例20：抗CLDN18.2抗体Ab10细胞毒性偶联物ADC1，ADC2结合活性检测
用实施例子2所述ELISA方法检测本发明抗体Ab10，以及Ab10抗体细胞毒素偶联物ADC1，ADC2和hCLDN18.2+cel的结合活性，结果见下表。
表31 本发明Ab10抗体细胞毒性偶联物ADC1，ADC2结合活性(EC50)
样品 ADC1 ADC2 Ab10
EC50(nM) 0.344 0.310 0.164
上述结果表明，Ab10抗体细胞毒素偶联物ADC1，ADC2和靶细胞(hCLDN18.2+cell)的结合活性(EC50)和抗体Ab10比稍微有点减弱，但在2倍(误差)范围内。
实施例21：抗CLDN18.2抗体Ab10细胞毒性偶联物ADC1，ADC2内吞活性检测
用上述“发明抗体内吞活性检测”(实施例17)同样的方法检测本发明抗体Ab10，以及Ab10抗体细胞毒素偶联物ADC1、ADC2的内吞活性。在上述实施例的基础上，优化标记方法(选用CF633染料)，抗体浓度(2.5μg/ml)和时间(1小时)等内吞检测条件，结果如下表。
表32 本发明Ab10抗体细胞毒性偶联物ADC1，ADC2内吞活性检测
样品 结合值 1) 内吞前(0h) 内吞后(1h) 内吞比例(％) 3)
阴性抗体 932 2) 935 944 NA
Ab10 6135 1191 2055 14.1
ADC1 4136 1200 2119 22.2
ADC2 4254 1788 2754 22.7
1)结合值，内吞前，内吞后数值为CF633标记抗体FACS检测读值；
2)划线数字为本FACS结合的本底值；
3)内吞比例(1小时)＝100*(内吞后-内吞前)/结合值；NA：不适用
上述结果表明本发明Ab10抗体细胞毒素偶联物ADC1，ADC2保留了抗体Ab10的 内吞活性。也就是说，连接毒素的抗体Ab10没有影响抗体的内吞活性。
实施例22：抗CLDN18.2抗体Ab10细胞毒性偶联物ADC1、ADC2抑制靶胞增殖活性
为了检测Ab10抗体细胞毒性偶联物ADC1，ADC2抑制靶细胞增殖活性，用CCK8方法(试剂盒购自东仁化学科技(上海)有限公司，货号CK04。按说明书进行操作)检测细胞增殖作用。具体地，用含10％FBS的DMEM/F12培养基培养hCLDN18.2+细胞(靶细胞)和hCLDN18.1+细胞(非CLDN18.2靶细胞，作为对照细胞)，在培养72小时结束前2小时，每孔加入10ul CCK8，培养箱中继续培养2小时，用Multiskan GO酶标仪获得OD450nM读数，用Graphpad prism 5分析处理数据。细胞增殖抑制百分比(％)＝100*(1-(OD450样品/OD450对照孔))，结果见下表。
表33 本发明Ab10抗体细胞毒性偶联物ADC1，ADC2抑制靶细胞增殖活性(IC50，nM)
Figure PCTCN2019090255-appb-000054
上述结果，本发明Ab10抗体细胞毒素偶联物ADC1和ADC2分子可以显著抑制靶点特异性细胞(hCLDN18.2+cell)的增殖，IC50分别为11.2nM和0.71nM。ADC1对于非靶点细胞(本实验用的是和Ab10抗体没有结合的hCLDN18.1+细胞)没有抑制作用，安全窗口高达3000。ADC2在极高的浓度(IC50为18.5nM)看到对非靶点特异细胞的抑制，这种抑制是因为高剂量(高达10-100nM)直接导致的非靶向毒性。这和ADC2所连的MMAF的高毒性特点一致。ADC2的安全窗口达到了26。
实施例23：抗CLDN18.2抗体Ab10细胞毒性偶联物靶向细胞毒性活性检测
为检测本发明Ab10抗体细胞毒素偶联物特异靶向CLDN18.2+细胞毒性，本实施例以ADC1为例子，通过检测细胞上清中LDH的释放评价本发明抗CLDN18.2抗体Ab10细胞毒性偶联物靶细胞毒性活性。
具体地，用含10％FBS的DMEM/F12培养基培养hCLDN18.2+细胞(靶细胞)和hCLDN18.1+细胞(非CLDN18.2靶细胞，作为对照细胞)至对数生长期。实验前一天，取培养好的hCLDN18.2+细胞和hCLDN18.1+细胞，用含10％FBS的DMEM/F12培养基重悬细胞，调整细胞浓度为4×10 4/ml细胞，100μl/孔加入到96孔细胞培养板中；37℃，5％CO 2孵育过夜；实验当天，移走96孔板细胞中的培养基，每孔加入含2％FBS的 DMEM/F12培养基待用；用含2％FBS的DMEM/F12培养基配制梯度稀释的待测药物ADC1，0μg/ml抗体孔用新鲜培养基，作为对照孔；向细胞中加入准备好的不同浓度的抗体样品，每孔50μl，每个浓度三个复孔；37℃，5％CO 2度培养箱孵育72小时后取出上清用LDH试剂盒(购自东仁化学科技(上海)有限公司，货号CK12)检测LDH的释放。检测方法按照说明书进行。用Multiskan GO酶标仪490nM读数，Graphpad prism 5分析处理数据。细胞杀伤百分比(％)＝100*(OD490待测样品-OD490对照孔)/(OD490细胞全部裂解-OD490对照孔)。实验结果如下表所示。
表34 本发明Ab10抗体细胞毒性偶联物ADC1靶向细胞毒性(EC50，nM)评价
Figure PCTCN2019090255-appb-000055
上述结果表明，本发明Ab10抗体细胞毒性偶联物特异靶向hCLDN18.2+阳性细胞毒性强，EC50在1nM以下。而针对非靶向细胞(本实验用的是和Ab10抗体没有结合的hCLDN18.1+细胞)的细胞毒性作用弱(没有细胞毒性)。特别地，ADC1在靶向特异细胞和非特异细胞之间的毒性(EC50)的比率高达156。这一特异性窗口(安全窗口)显示该分子的非靶向毒性很弱，安全性可靠。
实施例24：抗CLDN18.2抗体Ab10细胞毒性偶联物的体内药效
用同实施例子14的方法中的BALB/c裸小鼠皮下移植hCLND18.2+细胞动物模型和试验方法，用ADC1为例子，评价了本发明抗体Ab10抗体细胞毒性偶联物体内药效。结果见表35。
表35 本发明抗体偶联药物ADC1在肿瘤模型中的药效评价
Figure PCTCN2019090255-appb-000056
NA：不适应，即空白对照。
表35结果表明，本发明抗体毒素偶联药物ADC1在低浓度(2mg/kg)，仅4次给药，2周(14天)就看到了非常好的肿瘤抑制效果，46％抑制率，和对照组差异极显著(Pvalue<0.001)。
用同实施例14方法中，将其中的肿瘤细胞，用胃癌细胞系NUGC-4过表达hCLDN18.2 的细胞系接种BALB/c裸小鼠，评估本发明ADC2的动物药效。具体地，胃癌细胞系NUGC-4细胞购自中科院细胞所。用实施例1方法构建hCDLN18.2过表达细胞系NUGC-4-802。NUGC-4-802培养于含10％胎牛血清(上海博升生物科技有限公司，货号：BS-0002-500)的RPMI1640培养基中(上海源培生物科技股份有限公司，货号：L210KJ)，在含5％CO 2的37℃的细胞培养箱中连续培养。待NUGC-4-802细胞长至对数生长期(汇合率在80％-90％)时，用0.25％胰酶消化，收集细胞，并用无血清的RPMI1640培养基洗涤两次，重悬计数，调整细胞浓度为5×10 7细胞/ml。接种5×10 6个/100μl于BALB/c-裸鼠，右肋部皮下，挑选肿瘤细胞长至体积约120-150mm 3大小后随机分组，每组6只。
待测样品ADC2、Ab10用PBS配制，无菌。Blank组为PBS。给药剂量为5mg/kg，静脉注射。2次/周，连续2周。各注射样品给药当天为第0天。每次给药前测量体重，肿瘤体积，记录数据。数据统计分析方法同前述实施例14。本次实验实际给药周期2周。结果见表35b。
表35b 本发明抗体偶联药物ADC2在肿瘤模型中的药效评价
Figure PCTCN2019090255-appb-000057
表35b结果表明，本发明抗体毒素偶联药物ADC2在5mg/kg，仅4次给药，2周(14天)在NUGC-4-802肿瘤模型上就看到了非常好的肿瘤抑制效果，抑制率达62％，和对照组PBS差异显著(P value<0.05)。特别意想不到的是，该模型中，同样浓度的抗体Ab10抑瘤效果很弱(仅10％)。这一意想不到的结果表明本发明抗体毒素偶联药物ADC2有突出优于单独抗体(裸抗)的抑瘤效果。
实施例25 双特异性抗体中抗原、抗体的克隆、表达和纯化
本发明所用的人CLDN18.2设计见上述实施例；PD-1、PD-L1细胞外区-人IgG1 Fc融合蛋白、-his tag蛋白、单克隆抗体及设计的不同结构的双特异抗体等或由本发明克隆、表达纯化得到，或购自北京百普赛斯生物科技有限公司和北京义翘神州科技有限公司(Sino biological)。
抗原序列由NCBI数据库查询得到。本发明所用抗体，包括重组抗体、双特异性抗体。除本发明所发现的抗体人CLDN18.2抗体序列外，其它序列均能从公开的文献中得 到，包括抗PD-1抗体Nivo，Pem，Ba08(序列来自专利WO2016015685A1)；抗PD-L1抗体Atezo(Atezolizumab/Tecentriq)、Avel(Avelumab/Bavencio)、Durv(Durvalumab/imfinzi)；抗CD47抗体hu5F9，iMab，Blincyto，AMG420中抗CD3抗体轻、重链可变区序列。比如，PD-1抗体Nivo(Nivolumab/Opidivo)序列来自公开文献，如www.drugbank.ca，或WO2013019906。PD-1抗体Pem(Pembrolizumab/Keytruda)序列来自www.drugbank.ca(。PD-1抗体Ba08序列来自专利WO2016015685A1。Atezo序列，Avel序列，Durv序列(Accession Number，DB11714)均可从www.drugbank.ca直接查询到。CD47抗体hu5F9的轻链序列为US9382320B2_42，重链序列为US9382320B2_37。iMab轻链为WO2018075857_4，1F8；重链为WO2018075857_3，1F8。CD3抗体来自blincyto的抗CD3抗体的轻链、重链可变区。Blincyto序列为www.drugbank.ca公开之序列。另一CD3抗体序列来自AMG420中的抗CD3抗体序列的轻重链可变区。AMG420序列来自WO2014140248_340。Tim3的轻链可变区和重链可变区的氨基酸序列分别见CN201710348699.4中的SEQ ID NO:27和SEQ ID NO:36；该专利已公开；LAG3的轻链可变区和重链可变区的氨基酸序列分别见CN201810917684X中的SEQ ID NO:33和SEQ ID NO:44。TGFβ受体II(TGFβRII)序列来自UniProtKB/Swiss-Prot:P37173.2的胞外区(在本发明序列表中为SEQ ID NO:1)；IL10序列来自NCBI号NP_000563.1(在本发明序列表中为SEQ ID NO:2)。CD47配体SIRPα序列来自NCBI号NP_001035111.1。
所有抗原、抗体，包括单独抗体，双特异抗体，抗体-受体(Trap)，抗体-细胞因子等的通过基因合成得到合成片段，其设计、克隆表达、纯化均由本发明完成。具体同前述实施例3。
His Tagged蛋白纯化：将样品高速离心去除杂质。平衡镍柱(Ni smart beads 6FF常州天地人和生物科技有限公司Cat#SA036010)：用含有10mM咪唑0.5M NaCl的PBS pH7.4溶液平衡镍柱，2-5倍柱体积冲洗。将样品上清过柱。漂洗杂蛋白：使用含有10mM咪唑0.5M NaCl的PBS pH7.4溶液冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。用含有250mM咪唑0.5M NaCl的PBS(pH为7.4)洗脱目的蛋白。Buffer置换：将洗脱的目的蛋白过超滤管12000g离心10min(超滤管Merck Millipore Cat#UFC500308)，再补加1ml PBS，测定浓度，分装、储存备用。TGFβ1，TGFβ2，TGFβ3和IL10均购自Peprotech。
人抗体轻链恒定区序列Lc(κ链)，Lc(λ链)：现有技术；人抗体重链恒定区序列hIgG4或Hc(hIgG4)；hIgG1或Hc(hIgG1)；hIgG1p或Hc(hIgG1p)，即第356-358位为EEM的hIgG1的另外一种形式，均为现有技术。除上述所列序列外，重链恒定区也可以 是hIgG2或hIgG3。
表36 双特异性抗体技术方案部分克隆表达的单克隆抗体(对照用)编号、轻重链序列及说明
Figure PCTCN2019090255-appb-000058
实施例26 抗体和T细胞(CD3)结合实验(FACS)
用流式细胞术(FACS)检测本发明设计的带有CD3靶向的双特异抗体的CD3结合活性。具体地，取健康人外周血单核细胞(PBMC)经Dynabeads Human T-Activator CD3/CD28(Gibco，11131D)活化三天。将活化细胞用FACS buffer(PBS，0.5％FBS)洗一次(800g，3分钟离心)后重悬到FACS buffer中，细胞密度为4×10 6/ml，每样品25μl备用。待测样品用FACS buffer梯度稀释，最高浓度250nM，向下5倍稀释，8个浓度，体积25μl；将稀释好的样品25μl加入25μl细胞悬液中，轻轻混匀后，室温孵育20分钟；用大于5倍染色体积的FACS buffer清洗细胞后加入二抗(anti-hFc-PE，Biolegend，409304)，室温染色二十分钟后，清洗，重悬，用流式细胞仪检测(Beckman，CytoFLEX流式细胞仪)。用FlowJo软件分析所测样品平均荧光信号MFI。MFI和浓度做曲线，Graphpad Prism 5软件计算EC50。
实施例27 激活PBMC靶细胞杀伤实验
针对本发明靶向CD3的双特异抗体，用本实验评价其功能。用上述实施例1中构建好的hCLDN18.2+细胞作为靶细胞，hCLDN18.1+细胞作为阴性对照细胞(构建hCLDN18.1+细胞方法同实施例1，即同样的构建步骤，将hCLDN18.2换成hCLDN18.1质粒)。取健康人外周血单个核细胞(PBMC)作为效应细胞。具体地，分别收集hCLDN18.2+细胞和hCLDN18.1+细胞，调整细胞密度为2×10 5/ml于1640完全培养基中(RPMI1640，10％FBS)，每孔100μl加入96孔板中备用。PBMC调整细胞密度为4×10 6/ml于1640完全培养基中，每孔50μl分别加入hCLDN18.2+细胞或hCLDN18.1+细胞中。用1640完全培养基配置梯度稀释的待测样品，浓度为4、0.4、0.04、0.004μg/ml、每孔50μl加入细胞中，其终浓度为1、0.1、0.01、0.001μg/ml。三个复孔。于37℃，5％CO 2培养箱中孵育48小时后，取上清用LDH试剂盒(上海同仁生物科技有限公司，目录号：CK12)检测LDH的释放，反应靶细胞的杀伤。操作方法按说明书，用Multiskan GO酶标仪490nM读数，用Graphpad prism 5分析处理数据。
细胞杀伤百分比(％)＝100*(OD 490某药物浓度-OD 490对照孔)/(OD 490细胞全部裂解-OD 490对照孔)
实施例28 本发明双特异抗体稳定性评价
本发明设计双特异抗体，统一用Protein A重力柱纯化。纯化样品置换到pH 7.4的PBS buffer中，1mg/ml。于不同条件保存，包括-80℃保存60天以上，4℃保存14或30天，37℃保存7天，37℃保存14天等。不同条件保存的样品经电泳(PAGE)评价样品的降解程度。通过检测结合活性评价不同保存条件对样品活性是否受到影响。同样检测条件下，检测出的活性值(EC 50)和-80℃保存的样品所检测到的活性值比较，如果比值变化超出2倍范围，可以认为保存条件对样品稳定性/活性产生了影响。
实施例29 针对CLDN18.2和PD-1双靶点的双特异性抗体设计和活性评价
本发明针对CLDN18.2和PD-1两个靶点设计了不同序列结构的双特异抗体，见下表。
表37a 针对CLDN18.2和PD-1双靶点设计的双特异抗体
Figure PCTCN2019090255-appb-000059
Figure PCTCN2019090255-appb-000060
*:κ链表示轻链为人IgG的κ型轻链恒定区。#：IgG的C末端连接连接子的时候，其最末端氨基酸K突变为A。以下重链C末端引入scFv的设计均将最末端K突变为A。本发明中的“含重(轻)链的序列”，其除了包括正常的重(轻)链外连接有一个或多个scFv。
按本发明实施例25的方法分别克隆表达、纯化上述双特异抗体，用前述实施例子方法分别检测这些设计的双特异分子和人CLDN18.2以及PD-1的结合活性，结果如下表。
表37b 针对CLDN18.2和PD-1双靶点设计的双特异抗体的结合活性
Figure PCTCN2019090255-appb-000061
#：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体(对照抗体)的结合活性EC 50。*：同样实验条件下，双特异抗体和对应的单克隆抗体的结合活性EC 50的比值。
比值越大，说明所设计的双特抗体对单靶点的结合力减弱越多，比如比值为2，则说明所设计的双特异抗体对靶点结合活性和对应的单克隆抗体相比减弱了1倍。比值在2以内，说明结合活性没有受到影响；比值在2至5之间，说明结合活性略微有影响，此时应考量另一靶点的比值，若另一靶点比值较小例如在1以内，则该双特异性抗体仍有一定的应用价值。
上表结果分别显示了LB302、LB301、LB308和LB309的人CLDN18.2的结合活性检测结果(图7A-7D，a)、人PD-1的结合活性检测结果(图7A-7D，b)和双特异性抗体的结构示意图(图7A-7D，c)。LB302(Ab10 scFv在NivoVH Hc的N末端)，LB301(Ab10 scFv在PemVH Hc的N末端)，LB307(Ab10 scFv在Ba08VH Hc的N末端)，各自对PD-1的结合活性影响分别是0.68、2.1、1.5倍的改变；对CLDN18.2的结合活性影响分别为1.18、3.2、6.1。LB302保持了其和人CLDN18.2以及PD-1的结合活性，而LB307对人CLDN18.2的结合活性影响最大，减弱了5.1倍(6.1-1＝5.1)。也就是说，在 同样的类似IgG结构的双特异设计分子中，虽然LB302、LB301、LB307同时抗PD-1，但PD-1抗体Nivo、Pem、Ba08的序列不同，因此造成其抗CLDN18.2的效果也不一样。意外的发现，本发明抗体Ab10的scFv和Nivolumab(Nivo)设计的双特异抗体LB302对双靶点CLDN18.2和PD-1的结合活性没有受到影响。
这些数据表明，本发明anti-CLDN18.2抗体序列和不同的PD-1抗体序列设计的序列特异的IgG结构类似的双特异抗体( Sequence-based IgG like bispecific anti body,本发明简称SBody)，其活性和序列以及scFv所在的位置相关。SBody因序列组合、scFv所在的位置产生意想不到的的针对双靶点的活性。
比如，LB309(Ab10 scFv在NivoVH-Hc C末端)，LB308(Ab10 scFv在Pem VH-Hc C末端)，LB310(Ab10 scFv在Ba08VH-Hc C末端)，各自对PD-1的结合活性影响分别是1.33、0.93、0.56倍的改变；对CLDN18.2的结合活性影响分别为4.2、6.5、7.3倍。也就是说，同样的设计方式，三个PD-1抗体序列不同的双特异抗体分子对PD-1结合活性几乎没有影响。但对CLDN18.2结合活性影响很大，Ab10 scFv在和NivoVH-Hc C末端设计(LB309)中活性更优。
比较LB302/LB301/LB307和LB309/LB308/LB310活性数据，发现同样的序列，scFv的位置在Hc N末端比C末端对活性影响更小，针对本发明CLDN18.2抗体序列和PD-1抗体序列的双特异抗体设计，scFv位置在Hc N末端为更优化的双特异抗体设计。
比较LB301和LB156，发现本发明设计的双特异抗体，scFv不同，但位置相同(均在重链N末端)，如果scFv是针对PD-1，其(LB156)对PD-1的结合活性没有减弱(活性改变0.34倍，即活性增强)，但对CLDN18.2活性改变4.8倍，即减弱3.8倍。如果N末端scFv是针对CLDN18.2，其(LB301)对PD-1的结合改变3.2倍，即减弱2.2倍；对CLDN18.2的结合活性改变2.1倍，即减弱1.1倍。这种结构对活性的影响和LB156差别很大。
比较LB302和LB309，发现同样靶点的scFv，比如都是针对CLDN18.2的scFv，其位置不同，对活性影响也意外地差别很大。LB302对PD-1的结合活性改变0.68倍(接近1，即没有影响)，对CLDN18.2的结合活性改变1.18倍(没有影响)。LB309对PD-1的结合活改变4.2倍，即活性减弱3.2倍，对CLDN18.2的结合活性几乎没有影响(活性改变1.33倍)。也就是说，scFv在重链N末端的比在C末端更优。
比较LB312和LB313，发现针对同样靶点的scFv，比如都是Ab10 scFv和PD-1抗体(Pem)设计的双特异抗体，Ab10 scFv均连接PD-1抗体的轻链上，但位置不同，对活性影响也意外地差别很大。LB312的Ab10 scFv在Pem Lc的N末端，LB313的Ab10  scFv在Pem Lc的C末端。LB313对CLDN18.2的结合活减弱了10倍多，LB312则减弱1.6倍。两者对PD-1结合活性影响接近。也就是说，Ab10 scFv在轻链N末端的比在C末端更优。
在PD-1抗体重链C末端增加1个copy的Ab10 scFv(LB314)，意外发现，这样并没有能提高对CLDN18.2的结合活性(和LB302比较)，而且减弱了PD-1的结合活性，改变倍数为5.2倍。也就是说，在Nivo重链的C末端增加一个copy的Ab10 scFv对双靶点的结合活性均有减弱甚至阻碍作用。
此外，为了更加准确评估本发明双特异抗体的结合力，用Biacore检测了LB302、LB301对PD-1的亲和力。具体地，用Biacore T200，GE Healthcare仪器测定LB302 vs Nivo(L101)，LB301 vs Pem(L105)和人PD-1-his(本发明实施例1方法表达)的亲和力。用pH 7.4的运行缓冲液HBS-EP+(10mM HEPES，150mM NaCl，3mM EDTA和0.05％的P20)，先将Protein A(Thermo Pierce,Cat#21181)偶联到生物传感芯片CM5(Cat.#BR-1005-30，GE)上，将芯片用新配制的50mM NHS(N-hydroxysuccinimide)和200mM EDC(1-ethyl-3-(3-dimethylamino propyl)carbodiimide hydrochloride)激活，然后注入pH4.0 10mM NaAC配制的10μg/ml的Protein A。用运行缓冲液稀释待测样品浓度为1μg/ml,捕获信号在50RU左右，抗原PD-1-his浓度梯度从100nM开始，3倍稀释，流速30μl/分钟，结合时间180秒，解离时间300秒。实验后，用10mM Glycine-HCl，pH 1.5，30μl/min，30s清洗芯片。实验数据用Biacore T200 evaluation version 3.0(GE)软件以1:1 Langmuir模型进行拟合，得出亲和力数值KD。结果：LB302，10.4nM vs L101，8.6nM；LB301，6.5nM vs L105，4.4nM。该Biacore测得的亲和力(KD)和前述ELISA结果一致，即本发明SBody分子LB302、LB301保持了和对应抗体接近的和PD-1的结合活性。
基于Ab6序列设计的LB3022、LB3012活性(见上表)和LB302、LB301相似。
这些数据说明，基于本发明抗体Ab10、Ab6等设计的类似IgG结构的双特异抗体(抗CLDN18.2和人PD-1)SBody是序列依赖和序列位置依赖性的。比如说，基于Ab10序列特定的、设计的双特异抗体能得到意想不到的效果，即优选设计，比如LB302很好保留了双靶点的结合活性。
用前述实施例子方法评价本发明设计优选分子诱导细胞凋亡活性(％)来比较抗CLDN18.2功能活性。细胞凋亡活性用Ab10抗体在同样条件、同等浓度(本实验用150nM)作为对照。检测阻止PD-1抗原和配体PD-L1结合活性(IC50)来评价抗PD-1的功能活性，结果如下表。
表38a 本发明优选双特异抗体功能活性评估
Figure PCTCN2019090255-appb-000062
#：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体阻断抗原和配体结合活性IC50。*：IC50改变倍数，即双特异抗体和对应的单克隆抗体(对照抗体)的IC50的比值。比值越大，说明所设计的双特抗体对单靶点的功能活性减弱越多，比如比值为2，则说明所设计的双特异抗体对靶点功能活性和对应的单克隆抗体相比减弱了1倍。比值在2以内为实验误差范围，即活性没有受到影响。NA：Ab10不适用PD-1功能活性检测，因为Ab10无PD-1功能活性。
上述结果表明，LB302、LB308、LB310都保留了抗PD-1抗体的功能活性没有减弱，相反还有增强(LB310)。活性增强可能反应了序列设计相关产生的协同作用。
Anti-CLDN18.2的功能活性数据表明，Ab10 scFv在重链N末端(LB302、LB301、LB307)对Anti-CLDN18.2细胞活性有增强/协同(和Ab10抗体比较)作用，且不同PD-1抗体(Pem、Nivo、Ba08)都是如此。而Ab10 scFv在重链C末端(LB309、LB308、LB310)保留了Anti-CLDN18.2细胞活性。这些数据表明，本发明基于Ab10抗体设计的双特异抗体(SBody)因设计不同，产生意想不到的活性效果。
稳定性评价发现(方法见前述实施例)，双特异抗体(SBody)LB302、LB309、LB156、LB301、LB308、LB307、LB310、LB312、LB313、LB314、LB3022、LB3012在37℃保存14天后，均出现不同程度降解，对PD-1和CLDN18.2的结合活性有所减弱，有些减弱10倍以上。在-80℃保存60天，4℃保存14天活性没有改变，表明这些双特异分子-80℃，4℃能稳定保存。
表38b 针对CLDN18.2和PD-1双靶点设计的双特异抗体的表达水平
Figure PCTCN2019090255-appb-000063
Figure PCTCN2019090255-appb-000064
上述结果表明，本发明设计双特异抗体，同样Ab10抗体scFv序列和PD-1抗体位置的不同(LB302 vs LB309、LB301 vs LB308、LB307 vs LB310)，同样Ab10序列scFv序列位置和不同PD-1抗体(LB302 vs LB301 vs LB307、LB309 vs LB308 vs LB310)，相同的Ab10和PD-1抗体scFv或者Ab10 scFv序列和PD-1抗体(LB156 vs LB301)，意外地发现不同设计，表达量和序列相关，差别很大。产量最高LB309比产量最低LB156高67倍(28.7/0.42)。同样的Ab10 scFv在同样的PD-1抗体(Pem)轻链的N末端(LB312)和C末端(LB313)表达量也有差别。增加一个copy的Ab10 scFv(LB314)其表达量比单copy Ab10 scFv(LB302)表达量降低12倍(2.57/0.2＝12.9)。这些数据表明，基于本发明anti-CLDN18.2抗体所设计的双特异抗体(SBody)表达产量是序列特异的。Ab10抗体在双特异抗体设计中序列位置，结构等都和表达产量相关。
LB302轻链序列：SEQ ID NO:53；含重链的序列：SEQ ID NO:54。
实施例30 针对CLDN18.2和PD-L1双靶点的双特异性抗体设计和活性评价
本发明针对CLDN18.2和PD-L1两个靶点设计了不同序列结构的双特异抗体，见下表。
表39 针对CLDN18.2和PD-L1双靶点设计的双特异抗体
Figure PCTCN2019090255-appb-000065
Figure PCTCN2019090255-appb-000066
*此处的hIgG1恒定区序列和对应的PD-L1抗体hIgG1序列相同。例如和Atezo的Fc相同，带有N297A突变
按本发明实施例25的方法分别克隆表达、纯化上述双特异抗体，用前述实施例方法分别检测这些双特异分子和人CLDN18.2以及PD-L1的结合活性，结果如下表。
表40 针对CLDN18.2和PD-L1双靶点设计的双特异抗体的结合活性
Figure PCTCN2019090255-appb-000067
Figure PCTCN2019090255-appb-000068
#：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体的结合活性EC50。*：同样实验条件下，双特异抗体和对应的单克隆抗体的结合活性EC50的比值。比值越大，说明所设计的双特抗体对单靶点的结合力减弱越多，比如比值为2，则说明所设计的双特异抗体对靶点结合活性和对应的单克隆抗体相比减弱了1倍。比值在2以内，说明结合活性没有受到影响。
比较LB157和LB305发现，本发明抗体Ab10和PD-L1抗体Atezo设计成类似IgG结构的双特异抗体，意想不到的是，重链N末端不管是Ab10 scFv或者Atezo scFv都不影响所得到的双特异抗体对CLDN18.2和PD-L1的结合活性。不仅如此，稳定性实验结果表明，LB157和LB305在37℃保存14天，活性没有变化，而且电泳(PAGE)分析显示抗体没有见到明显的降解。说明本发明抗体Ab10和Atezo序列组合设计成的类似IgG结构的双特异抗体能够保留对两个靶点的结合活性，用常规抗体纯化方法Protein A纯化即可得到，工艺简单，并且稳定性好。
LB311数据表明，Ab10 scFv连在PD-L1抗体Atezo重链C末端得到的SBody不影响对hCLDN18.2和PD-L1的结合活性。
LB316、LB317数据表明，Ab10 scFv和PD-L1抗体Avel设计的SBody中，Ab10 scFv在Avel重链的N末端效果更优，而Ab10 scFv在Atezo重链的两端的效果均优。
LB319、LB320数据表明，Ab10 scFv和PD-L1抗体Durv设计的SBody中，Ab10 scFv在Durv重链的N末端对CLDN18.2，PD-L1的结合活性均没有影响。Ab10 scFv在Durv重链的C末端对CLDN18.2结合活性稍有影响，对PD-L1的结合活性没有影响。
这些数据说明3个PD-L1抗体和Ab10 scFv所设计的SBody中，对双靶点的结合活性序列之间存在差别。即SBody的活性是序列依赖的。Ab10 scFv和Atezo设计的SBody出乎意料之外，表现最优。
此外，为了更加准确评估本发明双特异抗体的结合力，用Biacore检测了LB157、LB305 vs LB185(Atezo)同PD-L1的亲和力。Biacore方法同前述LB302所述方法。用PD-L1-his(购自Sino biological，Cat#:10084-H08H)代替PD-1-his。所得亲和力(KD)结果：LB157为5.22nM，LB305为3.08nM，LB185为1.97nM。该结果说明，本发明anti-CLDN18.2和PD-L1(atezo)设计的SBody中Ab10 scFv对atezo结合活性几乎没有影响(LB305 vs LB185)，atezo scFv在Ab10重链N末端稍微减弱了其对PD-L1的结合力(LB157 vs LB185)。这和上述ELISA检测结果基本一致，即本发明优化设计SBody LB157、 LB305保留其对双靶点的结合活性。
此外，基于Ab6序列，设计的LB1572、LB3052对CLDN18.2和PD-L1活性几乎都没有影响。
对上述双特异抗体进行功能评估，包括针对CLDN18.2的CDC细胞活性，阻断PD-1/PD-L1结合活性，其结果见下表。
表41 本发明针对CLDN18.2和PD-L1设计优选双特异抗体功能活性评估
Figure PCTCN2019090255-appb-000069
#：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体的活性EC 50或IC 50。*：同样实验条件下，双特异抗体和对应的单克隆抗体活性比值。比值在2(实验误差范围)以内表明活性没有差别。##：LB305w重链恒定区没有LB305重链恒定区的突变，以便评估该分子对CLDN18.2+细胞的CDC活性。
用C57BL/6 cnc品系小鼠(购自浙江维通利华实验动物技术有限公司，生产许可证编号：SCXK(浙)2018-0001)建立动物药效模型，对本发明双特异抗体LB157，LB305进行了体内药效评估。
用MC38细胞(购自中科院细胞所)构建本实验所需的稳定表达细胞系MC38-804(构建方法同前述实施例1，将CHO-K1替代MC38)。MC38-804培养于含10％胎牛血清(上海博升生物科技有限公司，货号：BS-0002-500)，1％Hepes(赛默飞世尔科技(中国)有限公司，货号：15630080)的DMEM/高糖培养基中(上海源培生物科技股份有限公司，货号：L110KJ)，在含5％CO 2的37℃的细胞培养箱中连续培养。C57BL/6cnc雌性小鼠，6周龄，5只/笼饲养于SPF级环境，温度20-25℃；湿度40％-60％，自由进食进水，定期更换垫料。
待MC38-804细胞长至对数生长期(汇合率在80％-90％)时，用0.25％胰酶消化，收集细胞，并用无血清的DMEM/高糖培养基洗涤细胞两次，最后用无血清的DMEM/高糖培养基重悬，细胞计数，用基质胶(购自碧迪医疗器械上海有限公司，货号：354234)按1：1的比例调整细胞浓度为1×10 7细胞/ml，用于接种。接种MC38-804细胞悬液(1×10 6个)100μl于小鼠右肋部皮下，挑选肿瘤细胞长至体积约120-150mm 3大小后随机分组，每组6只。
待测样品与阳性对照用PBS配制，无菌。Blank组为PBS。PD-L1抗体LB185+Ab10为联合用药对照组。LB157，LB305为各自双特异抗体药物待测组。给药方式为腹腔注射，LB185+Ab10联合用药对照组小鼠给药剂量为每种抗体各20μg，200μl/只。LB157小鼠给药剂量为26μg/200μl/只，LB305给小鼠药剂量为26μg/200μl/只(和联合组的LB185,Ab10为等摩尔剂量)。各组给药频率均为2次/周，连续1.5周。
各注射样品给药当天为第0天。每次给药前测量体重，肿瘤体积，记录数据。本次实验实际给药周期1.5周，测量周期21天。
肿瘤大小计算公式：肿瘤体积TV(mm 3)＝0.5x(肿瘤长径x肿瘤短径 2)；肿瘤相对体积(RTV)＝T/T0或者C/C0。相对肿瘤增长率(T/C％)＝100％*(T-T0)/(C-C0)；抑瘤率(TGI)＝(1-T/C)*100％；其中T0,T分别为样品组实验开始时及实验结束时的肿瘤体积；C0,C分别为对照组实验开始时及实验结束时的肿瘤体积。
结果见下表。
表41b 本发明针对CLDN18.2和PD-L1设计优选双特异抗体体内药效
Figure PCTCN2019090255-appb-000070
上述结果表明，本发明针对CLDN18.2和PD-L1的双特异抗体LB157和同摩尔剂量的两个单抗联合产生同样的药效，第21天抑瘤率分别为54％和52％。非常意外地，LB305的抑瘤率第21天达到80％,显著好于个单抗联合产生抑瘤率52％。
LB305轻链序列：SEQ ID NO:55；含重链的序列：SEQ ID NO:56；
实施例31 针对CLDN18.2和CD47双靶点的双特异性抗体设计和活性评价
本发明针对CLDN18.2和CD47两个靶点设计了不同序列结构的双特异抗体，见下表。
表42 针对CLDN18.2和CD47双靶点设计的双特异抗体
Figure PCTCN2019090255-appb-000071
Figure PCTCN2019090255-appb-000072
按本发明实施例25的方法分别克隆表达、纯化上述双特异抗体，用前述实施例子方法分别检测这些双特异分子和人CLDN18.2以及CD47的结合活性，结果如下表。
表43 针对CLDN18.2和CD47双靶点设计的双特异抗体的结合活性(ELISA，nM)
Figure PCTCN2019090255-appb-000073
#:括号里面的数值为同实验条件下，同靶点对应的单克隆抗体的结合活性EC50。*:同样实验条件下，双特异抗体和对应的单克隆抗体的结合活性EC50的比值。比值越大，说明所设计的双特抗体对单靶点的结合力减弱越多，比如比值为2，则说明所设计的双特异抗体对靶点结合活性和对应的单克隆抗体相比减弱了1倍。比值在2以内，说明结合活性没有受到影响。
上述结果(LB158和LB304)表明，本发明抗体Ab10和CD47抗体Hu5F9设计成类似IgG结构的形式，所设计的双特异抗体Ab10 scFv在N末端的时候(LB304)，对Ab10的结合活性影响很大，EC50减弱了7.5倍(8.5-1＝7.5，见上表)。同样的序列组合，不同的位置，比如当Hu5F9 scFv在所述双特异抗体N末端的时候(LB158)，对CLDN18.2和CD47的结合活性影响小，EC50减弱在1倍左右(见上表)。
LB158功能活性检测结果表明，其阻止CD47和配体SIRPα活性IC50为1.13nM，比CD47单克隆抗体Hu5F9 LB157的IC50＝2.37nM增强了1.1倍，即功能活性没有减弱。CLDN18.2功能活性用诱导肿瘤细胞凋亡评估，LB158诱导肿瘤细胞凋亡11％，同条件，同浓度的Ab10诱导肿瘤细胞凋亡为10.3％。即LB158 anti-CLDN18.2的功能活性没有减弱。
稳定性检测结果表明，LB158在-80℃保存60天，4℃保存14天，37℃保存7天、14 天，电泳(PAGE)分析没有发现降解。活性检测结果表明这4份样品对CLDN18.2的结合活性为分别为0.68nM、0.98nM、0.67nM、0.73nM。对CD47的结合活性分别为0.08nM、0.12nM、0.16nM、0.10nM。说明LB158在这些保存条件下，不仅分子稳定(电泳结果)，而且活性也稳定。
LB321结果表明，本发明抗CLDN18.2抗体和另一CD47抗体(iMab)设计成的双特异抗体(iMab scFv在Ab10的重链N末端)保留了anti-CLDN18.2的结合活性，且CD47的结合活性增加了1.5倍(EC 50为0.484nM vs 1.38nM)。功能活性检测结果表明，LB321阻止CD47和配体SIRPα活性IC50和LS956(iMab)改变了1.64倍。说明LB321的抗CD47抗体功能活性没有受到影响。
稳定性检测结果表明，LB321在-80℃保存60天，4℃保存14天和CLDN18.2、CD47的结合活性均没有改变；37℃保存14天其和CLDN18.2的结合活性EC50由0.99nM变为10.1nM，活性减弱近10倍；和CD47的结合活性由0.77nM变为12.3nM，活性减弱15倍。说明LB321在4℃或4℃以下保存稳定。同时说明同样设计的SBody因CD47抗体序列的差别，比如在稳定性方面，LB158的稳定性优于LB321。
比较表本发明设计的双特异抗体表达量结果见下表。
表44a 针对CLDN18.2和CD47双靶点设计的双特异抗体的表达水平
Figure PCTCN2019090255-appb-000074
上述结果表明，本发明设计双特异抗体，同样的序列，位置的不同(LB158 vs LB304)，同样条件下表达产量有很大差别。LB158比LB304产量高1倍多。LB158 vs LB321说明不同序列(针对同一个靶点)以同样的方式和Ab10设计的双特异抗体，同样条件下表达产量也有比较大的差别(37.9 vs 13.6)。
这些结果说明本发明抗体Ab10和CD47抗体通过优化设计(包括序列组成，和scFv位置等)得到的优化双特异分子能同时保留对两靶点的结合活性，而且稳定性好。类似IgG结构，可以像常规IgG一样的工艺纯化，让后续开发变得更加简单，易行。
实施例32 针对CLDN18.2和CD3双靶点的双特异性抗体设计和活性评价
本发明针对CLDN18.2和CD3两个靶点设计了不同序列结构的双特异抗体，见下表。
表45 针对CLDN18.2和CD3双靶点设计的双特异抗体
Figure PCTCN2019090255-appb-000075
按本发明实施例25的方法分别克隆表达、纯化上述双特异抗体，用前述实施例子方法分别检测这些双特异分子和人CLDN18.2+细胞结合(ELISA)以及CD3(T细胞)FACS的结合活性，结果如下表。
表46 针对CLDN18.2和CD3双靶点设计的双特异抗体的结合活性
Figure PCTCN2019090255-appb-000076
Figure PCTCN2019090255-appb-000077
#：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体(Ab10)的结合活性EC50。*：同样实验条件下，双特异抗体和对应的单克隆抗体(Ab10)结合活性(ELISA)EC50的比值。比值越大，说明所设计的双特抗体对CLDN18.2的结合力减弱越多，比如比值为2，则说明所设计的双特异抗体对CLDN18.2结合活性和Ab10相比减弱了1倍。比值在2以内，说明结合活性受到影响不大(1倍实验误差范围)。##：括号里面的数值为同实验条件下，LB155的结合EC50(FACS)值。**：同样条件下双特异抗体和LB155结合活性的比值。该比值反应不同的CD3抗体，不同的结构如VH-linker-VL，VL-linker-VH之间对结合活性的差异。
上述结果(LB155，LB195；LB194，LB193)表明，本发明抗体Ab10和CD3抗体设计成类似IgG结构双特异抗体，CD3 scFv(来自Blincyto或AMG420)在N端对Ab10和CLDN18.2的结合活性都有比较大的影响，其中AMG420 scFv和Ab10设计的双特异抗体LB194对CLDN18.2的影响最大，EC50减弱了6.2倍(7.2-1＝6.2)。LB195比LB155，LB194比LB195结果说明，对于CD3抗体和本发明Ab10设计的双特异抗体中，CD3scFv在N末端的情况下，VH-Linker-VL比VL-linker-VH更好地保留了双靶点的结合活性。
此外，比较LB155、LB195、LB194、LB193和LB303结果说明，Ab10 scFv和CD3抗体设计的类似IgG结构的双特异抗体比CD3 scFv和Ab10抗体组成的双特异抗体更好保留了双特异抗体结合活性。
LB196结果说明CD3 scFv在C-末端对CD3的结合活性没有影响(活性改变倍数0.77(小于1)，但对CLDN18.2的结合活性的影响比较大。同条件下，LB196对CLDN18.2的结合活性比Ab10和CLDN18.2的结合活性减弱2.5倍(3.5-1＝2.5)。说明，同样是CD3 scFv和Ab10抗体组成的双特异抗体，CD3 scFv在N端能更好保留双靶点的结合活性。
基于Ab6和CD3抗体设计的双特异抗体中，优选分子LB1952基本上保留了和CLDN18.2和CD3的结合活性。
功能活性分析表明，LB195、LB193激活CDC活性(用前述实施例方法评价anti- CLDN18.2功能)相对Ab10活性(EC 50)改变倍数分别为1.7、2.2倍。这些改变倍数在实验误差范围内，也就是说LB195、LB193保持了其对CLDN18.2的功能活性。激活PBMC杀伤靶细胞的活性(方法见前述实施例子)检测发现，LB195、LB193活性相当，对靶细胞的特异杀伤作用呈量效关系，在0.1μg/ml浓度下有30％-40％靶细胞裂解。
稳定性分析结果表明，LB195和LB193在-80℃保存60天，4℃保存14天，37℃保存7天，37℃保存14天等条件下，仅37℃、14天在电泳(PAGE)上发现少许降解，其它的均稳定未见降解，而且活性都没有明显改变。
这些说明基于anti-CD3 scFv和Ab10的双特异设计优选分子LB195、LB193保留了对双靶点的结合活性和功能活性。能够通过常规IgG纯化方法得到，且能稳定保存。
表47a 针对CLDN18.2和CD3双靶点设计的双特异抗体的表达水平
Figure PCTCN2019090255-appb-000078
上述结果表明，本发明基于Ab10和CD3抗体设计双特异抗体表达产量最优为LB196。
代表序列LB193的轻链序列：SEQ ID NO:38，其为Ab10轻链，参见实施例6；含重链的序列：SEQ ID NO:4。
实施例33 针对CLDN18.2和TGFβ双靶点的双特异性抗体设计和活性评价
本发明针对CLDN18.2和TGFβ两个靶点设计了不同序列类似IgG结构的双特异抗体，具体地，在本发明抗体Ab10，Ab6的轻链、重链N或者C末端，优选重链C末端融合TGFβ受体II，所得分子(TRAP)能和CLDN18.2结合，同时能TGFβ1、2、3结合，设计见下表。
表48 针对CLDN18.2和TGFβ双靶点设计的双特异抗体
Figure PCTCN2019090255-appb-000079
*:hIgG1的重链C末端K突变为A。**:LB824为对照分子。序列源于WO2015118175_1(λ链)和WO2015118175_3。是PD-L1抗体Avel和TGFβ受体II(TGFβRII)组成的Trap分子。其TGFβRII结合活性可作为本发明分子LB401的对照。
按本发明实施例25的方法分别克隆表达、纯化上述双特异抗体，用前述实施例方法分别检测这些双特异分子和人CLDN18.2以及TGFβ的结合活性，结果如下表。
表49 针对CLDN18.2和TGFβ设计的双特异抗体(TRAP)的结合活性(ELISA，nM)
Figure PCTCN2019090255-appb-000080
#:同样实验条件下Ab10的EC50＝0.157nM，即LB401重链C末端加入TGFβRII没有影响其对CLDN18.2结合活性。NA：不适应，该分子不针对CLDN18.2。
上述LB401结合活性结果表明，本发明抗体Ab10重链C末端连接TGFβRII不仅保留了其对人CLDN18.2的结合活性，而且保留了其对TGFβI结合活性，以及对TGFβII和TGFβIII的高度选择性。此外，比较LB401和LB824对TGFβ的结合活性，发现本发明抗体Ab10融合TGFβRII得到LB401对TGFβ1、2、3的结合活性和由PD-L1抗体融合TGFβRII的Trap(LB824)结合特性(Profile)一致。
Ab6代替Ab10得到LB4012同样保留对双靶点的结合活性。
稳定性分析结果表明，LB401在-80℃保存60天，4℃保存30天，37℃保存7天，37℃保存14天等条件下稳定，电泳(PAGE)分析发现37℃保存14天出现降解，TGFβI、II、III结合活性减弱，CLDN18.2的结合活性没有受到影响，见图8。因此，LB401在-80℃或4℃保存稳定。
此外，对LB401和LB824平行进行了血清稳定性评估。具体地，取C57BL/6小鼠(六周龄雌性，购自上海西普尔-必凯实验动物有限公司)血液，12000rpm,离心10分钟，取血清备用。取3μl样品(1μg/μl,pH7.4PBS)用上述血清27μl稀释,终浓度为0.1μg/ml。分别于37℃处理0h，24h和72h后检测其与人CLDN18.2+细胞及TGFβI的结合活性。结果如下。
表50a 针对CLDN18.2和TGFβ设计的双特异抗体(TRAP)的血清稳定性实验(ELISA,EC50,nM)
Figure PCTCN2019090255-appb-000081
#NA：不适应。
上述结果表明，LB401在血清中孵育24小时，对CLDN18.2和TGFβI的结合活性均没有影响。72小时，对CLDN18.2的结合活性减弱了2倍多(0.424 vs 0.11)；对TGFβI的结合活性也有减弱，这和目前的临床试验同类型分子LB824稳定性相似。
用前述实施例30相同的动物模型(MC38-804)和方法对本发明针对CLDN18.2和TGFβ设计的双特异抗体(TRAP)进行了体内药效评估。待测样品与阳性对照用PBS配制，无菌。Blank组为PBS。Ab10为单独用药对照组，LB824为双特异抗体药物对照组。LB401为本发明优选双特异抗体药物待测组。给药方式为腹腔注射，Ab10给药剂量为120μg/200μl/只，LB824、LB401给药剂量为160μg/200μl/只。各组给药频率均为2次/周，连续3周。结果见下表。
表50b 本发明针对CLDN18.2和TGFβ设计的双特异抗体(TRAP)体内药效
Figure PCTCN2019090255-appb-000082
上述结果表明，在这个动物模型中，Ab10单独药效很弱，第24天仅显示12％的肿瘤抑制率。针对PD-L1和TGFβ分子LB824(作为药效模型对照分子)第24天显示56％的抑瘤率。非常意外地，本发明设计针对CLDN18.2和TGFβ的双特异抗体(TRAP)LB401在和Ab10同摩尔剂量下，第24天抑瘤率达到61％，比LB824(56％)好，显著优于单独Ab10(12％)。
上述结果表明，本发明anti-CLDN18.2抗体和TGFβRII优化设计双特异分子保留很好的对双靶点的活性，动物药效显著，而且分子稳定，纯化工艺(Protein A结合)简单易行。
LB401轻链序列(SEQ ID NO:38)，其为Ab10轻链，参见实施例6。
LB401含重链的序列(SEQ ID NO:57)：
Figure PCTCN2019090255-appb-000083
实施例34 针对CLDN18.2和IL10双靶点的双特异性抗体设计和活性评价
本发明针对CLDN18.2和IL10两个靶点设计了Ab10抗体和细胞因子融合分子。具体地，在本发明抗体Ab10、Ab6的轻链、重链N和/或C末端，优选重链C末端和N末端融合了IL10分子，设计见下表。
表51 针对CLDN18.2和IL10双靶点设计的双特异抗体
Figure PCTCN2019090255-appb-000084
*:hIgG1重链的C末端K突变为A。#LB4333同LB433除了其Fc区第L234突变为A；#LB4334同LB433除了其Fc区第L235突变为A；#LB4335同LB433除了其Fc 区第L234,L235均突变为A
按本发明实施例25的方法分别克隆表达、纯化上述双特异抗体，用前述实施例方法分别检测这些双特异分子和人CLDN18.2以及IL10的结合活性，结果如下表。
表52 针对CLDN18.2和IL10双靶点设计的双特异抗体的结合活性(nM)
Figure PCTCN2019090255-appb-000085
#：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体(Ab10)的结合活性EC50。*：同样实验条件下，双特异抗体和对应的单克隆抗体(Ab10)结合活性(ELISA)EC50的比值。比值越大，说明所设计的双特抗体对CLDN18.2的结合力减弱越多，比如比值为2，则说明所设计的双特异抗体对CLDN18.2结合活性和Ab10相比减弱了1倍。比值在2以内，说明结合活性受到影响不大(1倍实验误差范围)。
上述结合活性结果显示，本发明抗体Ab10和细胞因子IL10设计的双特异分子LB432和LB433，LB4332和CLDN18.2的结合活性同Ab10，Ab6(活性改变在2倍左右)。和IL10的结合活性用抗原包被ELISA和夹心法ELISA进行了评估。具体地，抗原包被ELISA检测方法见前述实施例3。结果LB432、LB433和LB4332的EC 50分别为0.34、0.14和0.2nM。活性相差在2倍左右，接近实验误差范围。
同样条件下，ELISA检测到IL10(Peprotech，Cat#:200-10)的结合活性EC50＝4.53nM。也就是说本发明双特异分子LB432、LB433和LB4332的IL10结合活性比重组IL10结合活性强。
夹心法ELISA方法：取实施例2构建人CLDN18.2+，按10x10 4/孔铺96-well plate(Corning，Cat#CLS3599-100EA)，37℃培养箱过夜孵育后去除上清，用免疫染色固定液(上海碧云天生物技术有限公司，Cat#P0098)100μl/孔室温固定半小时。PBS洗一遍后230μl 5％牛奶37℃封闭2小时，PBST洗3遍。加入50μl/孔用1％BSA 5倍连续稀释的待测样品，37℃孵育1小时，PBST洗板5次，加入50μl/孔1：400稀释的HRP标记兔抗人IL10(sino biological，Cat#SEKA10947)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，52-00-03)，室温孵育5-10min，加入50μl/孔1M H 2SO 4终止反应，用MΜLTISKAN Go酶标仪(ThermoFisher，51119200)在450nm处读取吸收 值，根据OD值计算EC50。夹心法ELISA结果表明，LB432的结合活性(11.3nM)显著弱于LB433(0.77nM)。说明同样的IL10序列在Ab10的N末端或者C末端，其分别结合CLDN18.2和IL10活性影响不大，但是LB432结合CLDN18.2后，再结合IL10的活性和LB433比减弱了10倍以上(11.3nM vs 0.77nM)。
也就是说，LB432的N末端连接了IL10存在“位阻效应”，即所得双特异分子单独结合两个靶点活性不受影响，但是结合了其中一个则阻止/影响该分子再结合另外一个靶点。在本发明的其它双特异分子SBody中，包括LB302、LB301、LB157、LB305、LB158、LB195、LB196、LB401等做过夹心法ELISA的检测，都没有发现其中任一靶点的结合影响另一靶点的结合(位阻效应)。
上述数据表明，本发明意外发现，抗体Ab10和IL10设计双特异抗体优化序列为LB433，即IL10融合到Ab10的重链C末端为最优。
稳定性分析结果表明，LB432和LB433在-80℃保存60天，4℃保存30天，37℃保存7天，37℃保存14天等条件下稳定，电泳(PAGE)分析没有发现明显降解，表明LB432和LB433稳定性好。
此外，对LB432和LB433进行了血清稳定性评估。具体地，取C57BL/6小鼠(六周龄雌性，购自上海西普尔-必凯实验动物有限公司)血液，12000rpm,离心10分钟，取血清备用。取3μl样品(1μg/μl,pH7.4PBS)用上述血清27μl稀释,终浓度为0.1μg/ml。分别于37℃处理0h，24h和72h后检测其与人CLDN18.2+细胞及IL10抗体的结合活性。结果如下。
表53a 针对CLDN18.2和IL10双靶点设计的双特异抗体的血清稳定性实验(ELISA,EC50,nM)
Figure PCTCN2019090255-appb-000086
上述血清稳定性结果表明，LB433血清孵育72h后，和CLDN18.2的结合活性改变1.64倍(0.264/0.161)，同条件下LB432为3.4(1.2/0.357)；和IL10的结合活性,LB433改变0.67倍(0.113/0.168)，同条件下LB432为1.4(0.115/0.083)。说明LB433的血清稳定性比LB432要好。
表53b 针对CLDN18.2和IL10双靶点设计的双特异抗体Fc突变体的结合活性(nM)
Figure PCTCN2019090255-appb-000087
Figure PCTCN2019090255-appb-000088
#说明同表52：
上述结果表明，LB4331，LB4333,LB4334,LB4335结合活性(CLDN18.2,IL10)和LB433接近。这些分子和LB433的不同在于其对CD64(FcγR I)的结合活性显著减弱。
用前述实施例30相同的动物模型(MC38-804)和方法对本发明针对CLDN18.2和和IL10设计的双特异抗体进行体内药效评估。待测样品与阳性对照用PBS配制，无菌。Blank组为PBS。Ab10为单独用药对照组。LB433为双特异抗体药物待测组。给药方式为腹腔注射，Ab10给药剂量为60μg/200μl/只，LB433给药剂量为80μg/200μl/只。各组给药频率均为2次/周，连续1.5周。结果见下表。
表53c 本发明针对CLDN18.2和IL10设计的双特异抗体体内药效
Figure PCTCN2019090255-appb-000089
上述结果表明，在这个动物模型中，Ab10单独药效很弱，第21天仅显示4％的肿瘤抑制率，即看不到动物药效。非常意外地，本发明设计针对CLDN18.2和IL10设计的双特异抗体LB433在和Ab10同摩尔剂量下，第21天抑瘤率达到81％，显著优于单独Ab10(4％)，T test分析P value<0.05。
LB433轻链序列(SEQ ID NO:38)，其为Ab10轻链，参见实施例6；
LB433含重链的序列(SEQ ID NO:58)：
Figure PCTCN2019090255-appb-000090
Figure PCTCN2019090255-appb-000091
LB4333轻链序列(SEQ ID NO:38)；LB4333含重链的序列(SEQ ID NO:59)；
LB4331轻链序列(SEQ ID NO:38)；LB4331含重链的序列(SEQ ID NO:60)；
LB4335轻链序列(SEQ ID NO:38)；LB4335含重链的序列(SEQ ID NO:61)：
Figure PCTCN2019090255-appb-000092
实施例35 针对CLDN18.2和LAG3，CLDN18.2和Tim3双靶点的双特异性抗体设计和活性评价
本发明针对CLDN18.2和LAG3两个靶点，以及CLDN18.2和Tim3两个靶点分别设计了双特异抗体，见下表。
表54针对CLDN18.2和LAG3双靶点，CLDN18.2和Tim3双靶点设计的双特异抗体
Figure PCTCN2019090255-appb-000093
Figure PCTCN2019090255-appb-000094
按本发明实施例25的方法分别克隆表达、纯化上述双特异抗体，用前述ELISA方法分别检测这些双特异分子和人CLDN18.2以及LAG3，Tim3的结合活性，结果如下表。
表55 针对CLDN18.2和LAG3，CLDN18.2和Tim3双特异抗体的结合活性(ELISA，nM)
Figure PCTCN2019090255-appb-000095
#:括号里面的数值为同实验条件下，同靶点对应的单克隆抗体(Ab10)的结合活性EC50。*:同样实验条件下，双特异抗体和对应的单克隆抗体(Ab10)结合活性(ELISA)EC50的比值。比值越大，说明所设计的双特抗体对CLDN18.2的结合力减弱越多，比如比值为2，则说明所设计的双特异抗体对CLDN18.2结合活性和Ab10相比减弱了1倍。比值在2以内，说明结合活性受到影响不大(实验误差范围)。**:同样实验条件下，双特异抗体和对应的单克隆抗体结合活性ELISA(LAG3抗体EC50:0.39nM；Tim3抗体EC50:0.06nM)EC50的比值。LAG3抗体序列及其它信息见专利号：201810917684.X。Tim3抗体序列及其它信息见专利申请号：201710348699.4。
上述结果表明，本发明抗体Ab10和LAG3，Tim3抗体设计成的类似IgG结构的双特异抗体分子(scFv在Ab10重链N末端)能保留对CLDN18.2和LAG3、Tim3的结合活性，且能够稳定表达纯化。
实施例36 CLDN18.2分别和PD-1，PD-L1，CD47双靶点抗体DVD结构设计和评价
本发明针对CLDN18.2和PD-1，PD-L1，CD47分别以DVD形式设计了双特异抗体，见下表。
表56 针对CLDN18.2/PD-1，CLDN18.2/PD-L1，CLDN18.2/CD47双靶点的DVD形式的双特异抗体设计
Figure PCTCN2019090255-appb-000096
Figure PCTCN2019090255-appb-000097
按本发明实施例25的方法分别克隆表达、纯化上述双特异抗体，凝胶电泳(PAGE)结果表明，这些抗体轻、重链均易出现linker间断裂现象。而以scFv的形式将一个靶点抗体连接在另一个靶点抗体的轻链或重链的N或C末端，通过筛选得到设计优化的双特异抗体，可以避免/减少linker间断裂(见前述实施例)，并且保留对双靶点的结合活性，功能活性。优选双特异抗体(本发明称之为SBody)不仅稳定，而且因其类似常规IgG结构，纯化工艺简单，这为后期开发过程中的工艺、纯化都提供了极大便捷。
实施例37 针对CLDN18.2的CAR分子设计
本发明针对CLDN18.2的新的CAR分子设计参考之前公开专利CN106755107A。
具体地，本发明设计的CAR分子其核酸构建体的通式为CAR-[(IRES)-f] q。该通式中，CAR表示嵌合抗原受体，包括，scFv-H-TM-S-CD3ζ，其中scFv(single chain Fv)为特异性靶向CLDN18.2抗原的单链可变片段，或称单链抗体、单链可变区。其序列为本发明所发现抗CLDN18.2抗体(见前述实施例)的可变区序列组成。其结构为VL-Linker-VH或VH-Linker-VL，Linker优选(G 4S) w，w为0、1、2、3、4；优选w＝3或w＝4。H为绞链结构域，TM为跨膜结构域，S为共刺激信号传导区域。所述共刺激信号传导区包括源自CD28的共刺激分子，和/或源自4-1BB的共激分子。通式中CD3ζ为源于CD3ζ的胞浆信号传导序列(胞内区)。
IRES表示内部核糖体进入位点序列(Internal ribosome entry site，IRES)；f表示编码功能性蛋白F，q为0或非0自然数。所述功能性蛋白包括细胞因子IL10、IL15或其活性片段，和/或所述细胞因子的受体如IL15受体或其活性片段，和/或细胞因子如IL10、IL15或其活性片段与IL15受体sushi+片段的融合片段。
如果所设计的CAR分子结构中没有f部分，则该CAR分子也没有IRES序列。此外，IRES和(IRES)代表相同的含义，当IRES外含括号“()”时，意指IRES序列仅存在于核酸构建体中，当含IRES序列的核苷酸用于编码蛋白时，该IRES序列不编码对应的蛋白，从而IRES序列之前与其后的核苷酸序列分别编码不同的蛋白质片段(即CAR和f)，且不同的蛋白质片段之间各自是分离的。
上述CAR或者CAR-(IRES)-f分子中，根据抗CLDN18.2抗体序列的可变区设计的scFv的序列为本发明的新抗体序列，见上述实施例。scFV处也可以是Fab或单结构域抗体(sdFv)结构。其它序列(scFv以外的序列)可以从美国国立医学图书馆网站 http://www.pub med.com，GenBank数据库中搜索得到，包括人CD8α信号肽、人CD8α铰链区、CD8α跨膜区、人CD28胞内区、人4-1BB胞内区、人CD3ζ胞内区、内部核糖体进入元件(IRES elements)、人IL15(同专利CN106755107A中的SEQ ID NO.22)，人IL15受体alpha(IL15Rα)野生型和突变/sushi部分(US2014/01314；WO2007/046006)，人IL10(SEQ ID NO:2)蛋白序列等。所有构建到克隆中的碱基序列均根据蛋白质序列进行密码子优化，以保证在编码氨基酸序列不变的情况下更适合人类细胞表达。
IL10的核苷酸序列例如GenBank登录号NM_000572所示的序列。
具体地，本发明代表性CAR分子构建参考公开号为CN106755107A的专利申请中所述的方法。将该专利实施例3，JX005质粒(来源于pBABEpuro)中编码c-Met抗体scFv替换为本发明Ab10 scFv，即得到本发明CAR新分子CAR1a的质粒JX1a。质粒JX1a所编码的CAR1a的氨基酸序列为：
Figure PCTCN2019090255-appb-000098
其中，第1-246位为结合Ab10的scFv编码序列；第247-293位为人CD8α铰链区编码序列(下划线部分)；第294-315位为人CD8α跨膜区编码序列；第316-357位为4-1BB胞内区编码序列；第358-469位为CD3zeta(ζ)胞内信号区编码序列。
其中，第1到63个核苷酸(下划线部分)为信号肽编码区。第64-801位为结合CLDN18.2的Ab10抗体的的scFv编码序列；第802-942位为人CD8α铰链区编码序列；第943-1008位为人CD8α跨膜区编码序列；第1009-1134位为4-1BB胞内区编码序列；第1135-1470位为CD3zeta(ζ)胞内信号区编码序列。
将公开号为CN106755107A的专利申请中实施例5构建的JX007质粒中编码c-Met抗体scFv替换为本发明Ab10 scFv(方法同本发明JX1a构建方法)，即得到本发明CAR新分子CAR3ab的所用的质粒JX3ab。质粒JX3ab编码的氨基酸序列同上述JX1a的编码序列，此外，还编码细胞因子IL15活性片段(野生型)。IL15活性片段的核苷酸序列可 为任何现有技术中用于编码其的序列，例如CN106755107A的SEQ ID NO:31的第699-1040位核苷酸序列。
将上述构建的质粒JX3ab中的编码IL15的序列(例如CN106755107A的SEQ ID NO:31的第699-1040位核苷酸序列)用编码IL10的序列(例如GenBank登录号NM_000572所示的序列)替代，得到本发明新的CAR分子CAR3ab10的质粒JX3ab10。
上述质粒JX3ab10编码如下IL10蛋白序列：
Figure PCTCN2019090255-appb-000099
将专利CN106755107A实施例6构建的JX008质粒中编码c-Met抗体scFv替换为本发明Ab10 scFv(方法同本发明JX1a构建方法)，即得到本发明CAR新分子CAR4a所用质粒JX4a。JX4a编码的氨基酸序列除上述CAR1a的编码序列外，还编码细胞因子IL15活性片段(突变型)与IL15Rα(sushi+)的融合蛋白；sushi+则意指除了sushi片段以外，还包括了其它多肽片段。
用上述同样的方法，将Ab6抗体序列的scFv代替Ab10抗体的scFv构建得到CAR新分子CAR1a.2、CAR3ab.2、CAR3ab10.2和CAR4a.2对应的质粒JX1a.2、JX3ab.2、JX3ab10.2和JX4a.2。
实施例38 针对CLDN18.2设计的CAR分子鉴定
病毒包装，制备和浓缩：病毒制备方法参考专利CN106755107A所用方法，用三质粒病毒包装系统pGag-Pol、pVSVG及本发明各个新CAR分子表达质粒pBABEpuro(均购自优宝生物)，例如JX1a、JX3ab、JX3ab10或者JX4a共转染293细胞获得病毒上清，超速离心浓缩获得浓缩的病毒。
具体地，取包装质粒pGag-Pol、pVSVG及表达质粒JX1a、JX3ab、JX3ab10或JX4a各6μg，PEI(Polysciences，Inc，Cat#:23966-2)36μg，混匀，室温静置5min，加入293(中国科学院典型培养物保藏委员会细胞库)。培养48h，收第1次上清；次日，即培养72h，收第2次上清。合并两次上清，加入3毫升20％的蔗糖溶液，将病毒上清小心铺到蔗糖溶液的上面，125000g,离心1.5小时，PBS低温重悬沉淀，分装，-80℃冻存。病毒分别记为1a、3ab、3ab10、4a。超速离心机型号Beckman Coulter Optima XPN-100；转子型号SW32i，超离管Beckman 344058。
病毒滴度检测：采用梯度稀释的病毒感染293细胞，48小时后通过Protein L染色确定表达scFv的细胞阳性率的方法来确定病毒滴度。具体地，分别取20μl病毒(1a、 3ab、3ab10、4a)，用RPMI 1640培养基(培源生物，Cat#L210KJ)含10％FBS(Gibco，Cat#:10099141)、0.8μg polybrene(上海翊盛生物科技有限公司Cat#:40804ES76)5倍梯度稀释，终体系250μl。加入预先铺板的293中，总培养体系500μl/孔，293 5×10 4个/孔。48h后收集细胞，biotin-protein L(金斯瑞，货号M00097)标记293，1μl/样本。室温孵育20min后，加入FACS buffer 1ml，离心清洗细胞。FACS buffer 100μl重悬，加入PE标记的Strepavidin(eBioscience，货号12-4317-87)0.4μl/样本，室温孵育20min后，加入FACS buffer 1ml，离心清洗细胞。FACS检测阳性细胞比例，选取阳性比例在10％时的样品计算病毒滴度，滴度(IU/ml)＝阳性率*293细胞数*稀释倍数/病毒液体积。结果表明，本次所得1a、3ab、3ab10和4a病毒的滴度分别为9.2、1.3、3.2和1.5×10 6IU/ml。
CART细胞的制备:取健康志愿者新鲜外周血，用专利CN106755107A中相同的方法分离外周血单核细胞(peripheral blood mononuclear cell，PBMC)。PBMC用偶联抗人CD3、CD28抗体的磁珠(Gibco，货号11131D)活化40-48h后分别加入病毒1a、3a、3ab10、4a(MOI在0.05-5范围)、polybrene(终浓度8μg/ml)。感染3h，补液至1ml，过夜换液，培养基为RPMI 1640含10％FBS、500IU/mL IL2(北京四环生物制药有限公司，Cat#:S20040007)。每隔一天换液并1:2扩大培养体积，直到获得足够的细胞进行体内外实验。感染后各个CAR分子的T细胞(CART细胞)分别记为CART1a、CART3ab、CART3ab10和CART4a细胞；未感染的T细胞(空载体)作为阴性细胞(对照)。
本发明CART分子Ab10抗体scFv表达鉴定：上述各个CART细胞通过Protein L染色以确定CART分子scFv的表达和感染细胞的阳性率。具体地，收集感染后的T细胞(CART细胞)2×10 5个，biotin-protein L(南京金斯瑞生物科技有限公司，货号M00097)标记细胞(标记方法同前述病毒滴度检测)。用FACS检测CART阳性细胞比例，用阴性细胞作为对照。阳性率＝CART细胞FACS(％)-阴性细胞(对照)FACS(％)，结果见下表57a。
以上述同样的方法制备CART1a.2、CART3ab.2、CART3ab10.2和CART4a.2。检测其Ab6抗体scFv的阳性率与CART1a、CART3ab、CART3ab10和CART4a接近，见表57b。
表57a 本发明CART细胞表达Ab10抗体scFv的阳性率
CART细胞 阳性率(％)
CART1a 47.1
CART3ab 24.1
CART3ab10 28.6
CART4a 25.7
表57b 本发明CART细胞表达Ab6抗体scFv的阳性率
CART细胞 阳性率(％)
CART1a.2 34.2
CART3ab.2 23.3
CART3ab10.2 27.5
CART4a.2 20.9
上述结果表明，本次实验4种CART细胞(包括Ab10和Ab6 scFv)的阳性率为20％-47％，确认了所得的CART细胞都正常表达了Ab10或Ab6抗体的scFv。以下用CART1a、CART3ab、CART3ab10和CART4a为例评估本发明CAR细胞的活性和功能。
本发明设计CART细胞表达分泌细胞因子鉴定：本发明设计的新CART细胞CART3ab，CART4a表达分泌细胞因子IL15、IL15/IL15R；CART3ab10表达分泌细胞因子IL10。为鉴定所述CART细胞表达并分泌所述因子，取CART细胞培养上清，用ELISA试剂盒(北京义翘神州生物技术有限公司，货号SEKA10947、SEK10360)分别检测IL10，IL15表达。结果发现仅CART3ab10上清中可检测出2256pg/ml和844pg/ml(重复制备不同供体PBMC来源T细胞感染得到的CART细胞)，同时制备的CART1a上清只检测到背景值为13.7pg/ml和5.3pg/ml(重复制备不同供体PBMC来源T细胞感染得到的CART细胞)。该数据表明，本发明设计CART3ab10特异表达并分泌了细胞因子IL10。ELISA方法没有能检测到CART3ab、CART4a上清中IL15、IL15/IL15Rα表达和分泌，说明培养的CART3ab、CART4a所分泌的细胞因子IL15和IL15/IL15Rα量偏低。
鉴于细胞培养上清没有能检测到IL15和IL15/IL15Rα的表达，将所生产的4个CART细胞分别注射小鼠体内。取Balb/c裸鼠，静脉注射给予1×10 6CART细胞。注射后的第7天和第14天分别取血清，ELISA检测血清中IL10和IL15的水平。结果见下表。
表58 本发明CART细胞小鼠血清中细胞因子表达量评估
Figure PCTCN2019090255-appb-000100
*：不适应
上述结果表明，CART3ab10在小鼠体内第7天的时候，IL10表达量达到5154.7pg/ml。第14天，IL10表达量下降到350.7pg/ml。CART1a，CART(空)作为对照样品均没有检测到IL10表达，检测值14、152.7和128.7pg/ml均为背景值。CART3ab在第14天检测到IL15活性片段表达，表达量为118.8pg/ml。CART4a则在第7天检测到IL15/IL15Rα表达，表达量206.4pg/ml。CART1a和CART(空)作为对照样品均没有检测到IL15表达，读数值为0(背景)。
上述结果表明本发明所设计的CAR转染的T细胞CART3ab、CART4a细胞表达分泌了IL15活性片段。CART3ab10细胞表达分泌了IL10。
实施例39 针对CLDN18.2设计的CAR细胞活性
为了评价本发明CART细胞的体外活性，用人CLDN18.2高表达细胞(hCLDN18.2+细胞)作为靶细胞，用人CLDN18.1高表达细胞(hCLDN18.1+细胞)作为对照阴性靶细胞，比较CART细胞对两种细胞杀伤后，靶细胞存活比例来评价CART细胞的特异性体外杀伤活性。具体地，用CFSE(Biolegend，货号423801)标记hCLDN18.2+细胞。配制靶细胞悬液，hCLDN18.1+细胞与CFSE标记的hCLDN18.2+细胞等比例混合，各1.5×10 5个/ml。于24孔板进行靶细胞杀伤实验，靶细胞悬液每孔100μl。CART1a、CART3ab、CART3ab10、CART4a细胞及阴性细胞(空载体)分别用同样培养基进行稀释，形成不同的CART细胞和靶细胞比例，分别为20:1，10:1，3:1和1:1。另设未杀伤组，即无CART细胞，仅上述靶细胞组。CART细胞与靶细胞共培养16小时后，弃上清，用PBS轻轻洗掉残余的CART细胞和被杀死的靶细胞，胰酶消化并收集贴壁的靶细胞，用7AAD(Biolegend:420404)染色后，FACS检测7AAD阴性CFSE标记hCLDN18.2+/hCLDN18.1+细胞的比例。
靶细胞特异裂解(杀伤)率＝1-[CART细胞7AAD阴性hCLDN18.2+/hCLN18.1+]/[阴性对照(空载体)细胞7AAD阴性hCLDN18.2+/hCLN18.1+]。靶细胞特异裂解(杀伤)率高，即CART细胞的特异杀伤作用强，以下是CART细胞和靶细胞为10:1时CART1a杀伤率的计算结果。阴性对照CART细胞结果的数据显示，活细胞CLDN18.1:CLDN18.2为50.5％vs 48.6％，接近1:1，表明阴性对照CART细胞对非靶细胞和靶细胞均没有杀伤作用。CAR1a细胞结果的数据显示，活细胞CLDN18.1:CLDN18.2为57.5％vs 37.5％，表明CART1a细胞对非靶细胞没有杀伤作用(接近50％)，但对靶细胞(CLDN18.2)有杀伤作用(由50％减少至37.5％)。杀伤率(％)计算方法为：1-[(37.5％/57.5％)/(48.6％/50.5％)]＝32.2％。各结果见下表。
表59 本发明CART细胞的体外细胞活性(靶细胞特异杀伤率，％)
Figure PCTCN2019090255-appb-000101
上述结果(三次以上重复实验，且每次实验阴性对照CART细胞对非靶细胞和靶细胞均没有杀伤作用)表明，本发明4种CART细胞对靶细胞在20:1的时候，都显示杀伤作用，杀伤率为19.8％-34.8％。随着CART细胞:靶细胞比例下降，对靶细胞的杀伤效应减弱，减弱最快的是CART4a、CART3ab10和CART3a。这3个CART细胞在3:1，1:1的比例时候，基本看不到对靶细胞的杀伤作用了。这说明本发明的CART设计和细胞功能相关，产生的效果随设计的不同显示意想不到的效果。
实施例40 针对CLDN18.2设计的CART细胞动物体内药效
取Balb/c裸鼠，皮下接种hCLDN18.2+细胞建立肿瘤模型，静脉注射CART细胞，测量肿瘤体积(TV)、体重(BW)来评价CART细胞的抗肿瘤作用及其安全性。具体地，hCLDN18.2+细胞培养于含10％胎牛血清的DMEM/F12培养基中，在含5％CO2的37℃的细胞培养箱中连续培养至对数生长期(汇合率在80％-90％)时，胰酶消化，收集细胞，并用无血清的DMEM/F12洗涤细胞二次，PBS重悬，计数，调整细胞浓度为1×10 8/ml。Balb/c裸小鼠,每只接种hCLDN18.2+细胞悬液100ul,右肋部皮下。三周后，挑选肿瘤体积80-130mm 3的小鼠，分组(2只/组)静脉注射CART细胞2×10 6/只。给药当天为第0天。之后每周测2次瘤体积，称量体重，记录数据。
肿瘤大小计算公式：肿瘤体积TV(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)；相对肿瘤体积(RTV)＝TV/TV0,其中TV0为起始时(Day 0)的肿瘤体积，设置为1。TV为检测时的肿瘤体积。相对肿瘤体积(RTV)即为各检测时间点肿瘤体积增长倍数。相对肿瘤增长率(T/C％)＝100％*(T-T0)/(C-C0)；抑瘤率(TGI)＝(1-T/C)*100％。其中T0、T分别为样品组实验开始时及实验结束时的肿瘤体积；C0、C分别为对照组实验开始时及实验结束时的肿瘤体积。
结果表明，注射CART两周后，各小鼠体重没有明显变化，表明CART细胞没有显著的安全性问题。药效方面，CART(空载体)注射小鼠第13天到第19天，肿瘤体积持续增大，增加倍数(相对肿瘤体积)从14持续到39倍(图9)。而注射CART1a、CART3ab、 CART3ab10和CART4a细胞的小鼠肿瘤体积增加均在10倍以下(抑瘤率为40％-90％)。特别地，注射CART3ab10和CART4a细胞的小鼠肿瘤体积在第13天到第17天时间段维持几乎没有增长，达到持续抑制(接近100％抑制)效果。这一结果表明，本发明抗CLND18.2抗体设计不同CAR分子产生的CART细胞显示了意想不到的动物药效效果。
实施例40 针对CLDN18.2设计的CAR转染NK细胞得到的CARNK细胞活性
取处于对数生长期状态良好的NK92(购自商城北纳创联生物科技有限公司)细胞中分别加入病毒1a、3a、3ab10、4a(MOI在0.5-5范围)、polybrene(终浓度8μg/mL)。感染3h，补液至1mL，过夜换液，培养基为NK92专用培养基(商城北纳创联生物科技有限公司)。每隔一天换液并1：2扩大培养体积，直到获得足够的细胞进行体内外实验。感染后的NK92细胞分别记为CARNK1a、CARNK3a、CARNK3ab10、CARNK4a细胞；未感染的NK92细胞记为CARNK(空载体)，作为阴性对照。感染7天后，用同上实施例38的方法检测CARNK细胞表面的表达，结果如下表所示。
表60 本发明抗CLDN18.2抗体构建CARNK细胞表达抗体scFv的阳性率
CARNK细胞 阳性率(％)
CARNK1a 21
CARNK3a 22
CARNK3ab10 30
CARNK4a 41
上述结果表明，本发明新设计的CAR在NK细胞里面也能很好地表达抗体Ab10的scFv并识别人hCLDN18.2。
实施例41 针对CLDN18.2设计的CARNK细胞动物体内药效
同上述实施例7的同样的动物模型评价本发明CARNK细胞的动物药效。取制备好的CARNK(空载体)对照、CARNK1a、CARNK3ab、CARNK3ab10、CARNK4a细胞2×10 5/只，2只每组，Day 0和Day 3各注射一次。后每周2两次检测肿瘤大小，结果见下表61。
表61 本发明抗CLDN18.2抗体构建CARNK细胞体内药效
Figure PCTCN2019090255-appb-000102
Figure PCTCN2019090255-appb-000103
表61结果表明，本发明抗CLDN18.2抗体构建CARNK细胞体内第11、第13天持续显示抑制肿瘤效果。抑瘤率在16.5％-60.7％范围。其中，CARNK3ab10显示动物体内药效最优。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (34)

  1. 一种靶向CLDN18.2的抗体，其特征在于，其包含轻链可变区(VL)和/或重链可变区(VH)，所述VL包含以下的互补决定区(CDR)序列：
    如SEQ ID NO:11或SEQ ID NO:12所示的VL CDR1氨基酸序列；如SEQ ID NO:13所示的VL CDR2氨基酸序列；如SEQ ID NO:14所示的VL CDR3氨基酸序列；
    所述VH包含以下的CDR序列：
    如SEQ ID NO:15所示的VH CDR1氨基酸序列；如SEQ ID NO:16所示的VH CDR2氨基酸序列；如SEQ ID NO:17所示的VH CDR3氨基酸序列。
  2. 如权利要求1所述的靶向CLDN18.2的抗体，其特征在于，所述VL包含如SEQ ID NO:11所示的VL CDR1、SEQ ID NO:13所示的VL CDR2和SEQ ID NO:14所示的VL CDR3的氨基酸序列；所述VH包含如SEQ ID NO:15所示的VH CDR1、SEQ ID NO:16所示的VH CDR2和SEQ ID NO:17所示的VH CDR3的氨基酸序列；或，
    所述VL包含如SEQ ID NO:12所示的VL CDR1、SEQ ID NO:13所示的VL CDR2和SEQ ID NO:14所示的VL CDR3的氨基酸序列；所述VH包含如SEQ ID NO:15所示的VH CDR1、SEQ ID NO:16所示的VH CDR2和SEQ ID NO:17所示的VH CDR3的氨基酸序列。
  3. 如权利要求1或2所述的靶向CLDN18.2的抗体，其特征在于，所述靶向CLDN18.2的抗体的CDR突变序列为CDR区发生脱氨基化敏感位点突变的序列；优选地，所述CDR区脱氨基化敏感位点为轻链CDR1第L30A和/或L30B；和/或，重链CDR3第H99和/或H100位；
    更优选地，所述轻链CDR1第L30A和L30B的氨基酸残基由NS突变为TS或NT，且前提是第L30E位不为Q和第L34位不为T；所述重链CDR3第H99和H100位的氨基酸残基由NS突变为TS或NT，且前提是轻链CDR1第L30E位不为Q和第L34位不为T。
  4. 如权利要求1-3中任一项所述的靶向CLDN18.2的抗体，其特征在于，所述靶向CLDN18.2的抗体为鼠源抗体；
    优选地，所述鼠源抗体的VL为如SEQ ID NO:7所示的氨基酸序列或其突变；和/ 或，所述鼠源抗体的VH为如SEQ ID NO:8所示的氨基酸序列或其突变；所述突变为所述VL和/或VH的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所述VL和/或VH的氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体与CLDN18.2的结合；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％序列同一性；最优选为至少99％序列同一性。
  5. 如权利要求4所述的靶向CLDN18.2的抗体，其特征在于，所述靶向CLDN18.2的抗体包含鼠源抗体的可变区和鼠或人抗体恒定区；所述鼠抗体恒定区包括鼠IgG1、IgG2a、IgG2b3或IgG3的重链恒定区和κ或λ型轻链恒定区，所述人抗体恒定区包括人IgG1、IgG2、IgG3或IgG4的重链恒定区和κ或λ型轻链恒定区；
    优选地，所述靶向CLDN18.2的抗体为鼠源抗体的可变区和人抗体恒定区组合成的嵌合抗体；
    更优选地，所述嵌合抗体的轻链氨基酸序列为如SEQ ID NO:9所示的氨基酸序列或其突变；和/或，所述嵌合抗体的重链氨基酸序列为如SEQ ID NO:10所示的氨基酸序列或其突变。
  6. 如权利要求1-3中任一项所述的靶向CLDN18.2的抗体，其特征在于，所述靶向CLDN18.2的抗体为人源化抗体；
    优选地，所述人源化抗体的VL包含如SEQ ID NO:29-33中任一个所示的氨基酸序列或其突变；和/或，所述人源化抗体的VH序列如包含如SEQ ID NO:34-37中任一个所示的氨基酸序列或其突变；所述突变为所述VL和/或VH的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所述VL和/或VH的氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体与CLDN18.2的结合；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％序列同一性；最优选为至少99％序列同一性；
    更优选地，所述VL包含如SEQ ID NO:29所示的氨基酸序列；所述VH如包含如SEQ ID NO:34所示的氨基酸序列；或，所述VL包含如SEQ ID NO:31所示的氨基酸序列；所述VH包含如SEQ ID NO:34所示的氨基酸序列。
  7. 如权利要求6所述的靶向CLDN18.2的抗体，其特征在于，所述抗体的轻链包含选自人抗体κ或λ型轻链恒定区或其突变；和/或，所述抗体的重链包含选自人IgG1、IgG2、IgG3和IgG4的重链恒定区或其突变；
    优选地，所述重链恒定区或其突变，包含人IgG1 Fc区第234位、第235和第243 位，或第239、第330和第332位突变；
    更优选地，所述重链恒定区或其突变，包含人IgG1 Fc区第356-358位为EEM或DEL的突变。
  8. 如权利要求6或7所述的靶向CLDN18.2的抗体，其特征在于，所述抗体的轻链包含SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42或SEQ ID NO:45所示的氨基酸序列或其突变；和/或，所述抗体的重链包含SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:44或SEQ ID NO:46所示的氨基酸序列或其突变。
  9. 如权利要求1-8中任一项所述的靶向CLDN18.2的抗体，其包含以下轻链和重链：所述重链如SEQ ID NO:39的氨基酸序列所示，所述轻链如SEQ ID NO:38的氨基酸序列所示；或，所述重链如SEQ ID NO:39的氨基酸序列所示，所述轻链如SEQ ID NO:42的氨基酸序列所示。
  10. 如权利要求1-9中任一项所述的靶向CLDN18.2的抗体，其中所述靶向CLDN18.2的抗体包括免疫球蛋白、Fab、Fab’、F(ab’) 2、Fv或单链Fv片段(scFv)。
  11. 一种双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其特征在于，所述第一蛋白功能区为如权利要求1-10任一项所述的靶向CLDN18.2的抗体；所述第二蛋白功能区为靶向非CLDN18.2抗原的抗体。
  12. 如权利要求11所述的双特异性抗体，其特征在于，所述非CLDN18.2抗原为免疫检查点抗原或肿瘤治疗靶点，所述免疫检查点抗原优选包括PD-1、PD-L1、Tim3、LAG3、CD47，所述肿瘤治疗靶点优选包括SIRPα；优选地，所述第二蛋白功能区抗PD-1抗体、抗PD-L1抗体、抗Tim3抗体、抗LAG3抗体、抗CD47抗体、抗CD3抗体和抗CSF-1R抗体；最优选地，所述抗PD-1抗体为Nivolumab、Pembrolizumab或者Ba08，所述抗PD-L1抗体为Atezolumab、Avelumab或者Durvalumab，所述抗CD47抗体为hu5F9或iMab，所述抗CD3抗体为Blincyto或AMG420中由结合CD3的轻、重链可变区序列构建的抗体；
    或，所述非CLDN18.2抗原为细胞因子和细胞因子受体或它们的片段；优选地，所述细胞因子包括TGFβ、IL10和CSF-1，所述细胞因子受体包括TGFβRII、IL10受体和CSF-1R。
  13. 如权利要求12所述的双特异性抗体，其特征在于，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为一个或多个scFv、细胞因子或它的片段、或细胞因子受 体或它的片段；或者，所述第二蛋白功能区为免疫球蛋白，所述的第一蛋白功能区为一个或多个scFv；其中，所述scFv包括重链可变区与轻链可变区，所述重链可变区与轻链可变区通过连接子连接；所述scFv、细胞因子或它的片段、或细胞因子受体或它的片段通过连接子与所述免疫球蛋白连接，所述连接子优选(G 4S) w；所述的w优选为0～10之间的整数，更优选为1、2、3或者4。
  14. 根据权利要求13所述的双特异性抗体，其特征在于，所述scFv的结构为轻链可变区-连接子-重链可变区，其轻链可变区N末端或重链可变区C末端分别通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；或所述scFv的结构为重链可变区-连接子-轻链可变区，其重链可变区N末端或者轻链可变区C末端分别通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；优选地，所述连接子为(G 4S) 3，和/或，所述scFv的数量为两个且对称地连接在所述的免疫球蛋白轻链和/或重链；
    更佳地，所述的双特异性抗体选自以下任一种：
    (1)所述第一蛋白功能区为免疫球蛋白，所述免疫球蛋白包括轻链如SEQ ID NO:38所示、重链如SEQ ID NO:39所示的氨基酸序列；所述第二蛋白功能区为scFv；其中，
    两个scFv的重链可变区的C末端通过连接子对称地连接在所述免疫球蛋白的两条重链的N末端；且，所述scFv的轻链可变区为Atezolumab的轻链可变区，所述scFv的重链可变区为Atezolumab的重链可变区；或，
    两个scFv的重链可变区的C末端通过连接子对称地连接在所述免疫球蛋白的两条重链可变区的N末端；且，所述scFv的轻链可变区为Hu5F9的轻链可变区，所述scFv的重链可变区为Hu5F9的轻链可变区；或，
    两个scFv的重链可变区的N末端通过连接子对称地连接在所述免疫球蛋白的两条重链的C末端；且，所述scFv的轻链可变区为AMG420的轻链可变区，所述scFv的重链可变区为AMG420的重链可变区；
    (2)所述第一蛋白功能区为scFv，所述第二蛋白功能区为免疫球蛋白；两个scFv的重链可变区的C末端通过连接子对称地连接在所述免疫球蛋白的两条重链的N末端；所述scFv的轻链可变区的序列如SEQ ID NO:29所示，所述scFv的重链可变区的序列如SEQ ID NO:34所示；其中，
    所述免疫球蛋白包括Nivolumab的轻链可变区、轻链恒定区为κ链、Nivolumab的重链可变区以及重链恒定区为hIgG4的氨基酸序列；或，
    所述免疫球蛋白包括Pembrolizumab的轻链可变区、轻链恒定区为κ链、 Pembrolizumab的重链可变区以及重链恒定区为hIgG4的氨基酸序列；或，
    所述免疫球蛋白包括Atezolumab的轻链可变区、轻链恒定区为κ链、Atezolumab的重链可变区以及重链恒定区为hIgG1的氨基酸序列。
  15. 根据权利要求13所述的双特异性抗体，其特征在于，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为细胞因子或它的片段、或细胞因子受体或它的片段；所述细胞因子或它的片段、或细胞因子受体或它的片段的数量优选为两个或四个；其通过连接子对称地与所述免疫球蛋白两条轻链和/或两条重链的C末端和/或N末端连接，所述连接子优选为(G 4S) 3；
    优选地，所述免疫球蛋白包括轻链如SEQ ID NO:38所示；重链如SEQ ID NO:39所示的氨基酸序列；其中，
    所述细胞因子或它的片段、或细胞因子受体或它的片段为TGFβRII，其序列如SEQ ID NO:1所示，且数量为两个；所述TGFβRII对称地连接在所述免疫球蛋白的两条重链的C末端，其C末端氨基酸由K突变为A；或，
    所述细胞因子或它的片段、或细胞因子受体或它的片段为IL10，其序列如SEQ ID NO:2所示，且数量为两个；所述IL10对称地连接在所述免疫球蛋白的两条重链的C末端，其C末端氨基酸由K突变为A。
  16. 如权利要求14或15所述的双特异性抗体，其特征在于，所述的双特异性抗体包括以下轻链氨基酸序列和含重链的氨基酸序列：
    如SEQ ID NO:53所示的轻链氨基酸序列，如SEQ ID NO:54所示的含重链的氨基酸序列；或，如SEQ ID NO:55所示的轻链氨基酸序列，如SEQ ID NO:56所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:57所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:58所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:59所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:60所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:61所示的含重链的氨基酸序列；或，如SEQ ID NO:38所示的轻链氨基酸序列，如SEQ ID NO:4所示的含重链的氨基酸序列。
  17. 一种分离的核酸，其编码如权利要求1-10中任一项所述的靶向CLDN18.2的抗体或如权利要求11-16中任一项所述的双特异性抗体。
  18. 一种抗体药物偶联体(ADC)，其结构如下式I所示：
    Ab-[(L 2) n-L 1–D] y  式I
    其中，D为具有细胞毒性的小分子药物，L 1和L 2为分别连接所述药物和所述抗体的接头；n为0或1；y表示偶联于Ab的D的平均数，且0<y≤10，优选2≤y≤7；更优选3≤y≤6；最优选4.4或4.8；
    所述Ab为如权利要求1-10中任一项所述的靶向CLDN18.2的抗体，或如权利要求11–16中任一项所述的双特异性抗体。
  19. 如权利要求18所述的抗体药物偶联体，其特征在于，所述小分子药物为MMAE、MMAF、DM1、DM3和DM4中的一种或多种；所述接头为SPP、SIAB、SMCC、MP、VC、ala-phe、PAB和MC-VC-PAB中的一种或多种。
  20. 如权利要求18或19所述的抗体药物偶联体，其特征在于，所述L 2为下式II所示的化合物：
    Figure PCTCN2019090255-appb-100001
    其中，X 1选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；X 2选自烷基、环烷基和杂环基；m为0-5；S为硫原子；
    优选地，当X 1为氢原子、X 2为烷基、m为1时，式II所示的化合物为硫代乙酸S-(3-羰基丙基)酯。
  21. 如权利要求18-20中任一项所述的抗体药物偶联体，其特征在于，所述小分子药物为DM1，所述接头L 1为SMCC，n为0，由此形成如下式III所示的抗体药物偶联体：
    Figure PCTCN2019090255-appb-100002
    或，所述小分子药物为MMAF，所述接头L 1为MC-VC-PAB，L 2为硫代乙酸S-(3-羰基丙基)酯，n为1，由此形成如下式IV所示的抗体药物偶联体：
    Figure PCTCN2019090255-appb-100003
  22. 如权利要求18-21中任一项所述的抗体药物偶联体，其为如下式V所示的抗体药物偶联体：
    Figure PCTCN2019090255-appb-100004
    或，如下式VI所示的抗体药物偶联体：
    Figure PCTCN2019090255-appb-100005
    或，如下式VII所示的抗体药物偶联体：
    Figure PCTCN2019090255-appb-100006
    或，如下式VIII所示的抗体药物偶联体：
    Figure PCTCN2019090255-appb-100007
    其中，所述Ab10包含如SEQ ID NO:38所示的轻链和如SEQ ID NO:39所示的重链；所述Ab6包含如SEQ ID NO:42所示的轻链和如SEQ ID NO:39所示的重链。
  23. 一种如权利要求18-22中任一项所述的抗体药物偶联体的制备方法，其特征在于，
    当n为1时，所述制备方法包括以下步骤：
    (1)制备中间体1：取所述抗体与所述接头L 2在溶液中混合，反应后纯化即得含中间体1的溶液，所述中间体1如下式IX所示：
    Figure PCTCN2019090255-appb-100008
    其中，X 1选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；X 2选自烷基、环烷基和杂环基；m为0-5；S为硫原子；
    优选地，X 1为氢原子，X 2为烷基，m为1，即L 2为硫代乙酸S-(3-羰基丙基)酯；
    (2)制备中间体2：取所述接头L 1与所述药物制备成中间体2：L 1-D；
    (3)将步骤(1)获得的含中间体1的溶液，与步骤(2)获得的含中间体2的溶液混合，反应后纯化即得含抗体药物偶联体的溶液；
    当n为0时，所述制备方法包括以下步骤：
    (1)制备中间体3：取所述抗体与所述接头L 1在溶液中混合，反应后纯化即得含中间体3的溶液；
    (2)将步骤(1)获得的含中间体3的溶液，与含所述药物的溶液混合，反应后纯化即得含抗体药物偶联体的溶液；
    优选地，所述步骤(1)和/或(2)中，所述反应温度为25℃；所述反应时间为2～4小时；和/或，所述纯化为用凝胶过滤纯化，更优选为Sephadex G25凝胶柱脱盐纯化。
  24. 一种嵌合抗原受体(CAR)，其特征在于，所述CAR包括：(a)特异性识别CLDN18.2的胞外结合结构域scFv；(b)铰链结构域；(c)跨膜结构域；(d)共刺激胞内结构域；(e)信号传导结构域；所述胞外结合结构域包括如权利要求1-10中任一项所述的靶向CLDN18.2的抗体的轻链可变区和重链可变区。
  25. 如权利要求22所述的CAR，其特征在于，
    (1)所述铰链结构域选自一种或多种以下分子的铰链区：CD8α、CD28、CD152、PD1 和IgG1重链；
    (2)所述跨膜结构域选自一种或多种以下分子的跨膜区：TCR的α、β、ζ链，CD3ε，CD3ζ，CD8α，CD16，CD28，CD33，CD64，CD80，CD86，CD134，4-1BB，CD152，和PD1；
    (3)所述共刺激胞内结构域选自一种或多种以下分子的胞内区：CD27、CD28、CD30、CD40、OX40、CD134、4-1BB、CD150、CD223、PD-L2、PD-L1、NKD2C、FcεRIγ和，优选CD28胞内区和/或4-1BB胞内区；和/或，
    (4)所述信号传导结构域选自一种或多种以下分子的胞内区：Igα、Igβ，TCRξ，FcR1γ、FcR1β，CD3γ、CD3δ、CD3ε，CD22，CD28，CD79a、CD79b，和CD3ζ；优选CD3ζ胞内区。
  26. 如权利要求24或25所述的CAR，其特征在于，所述铰链结构域为CD8α铰链区，所述跨膜结构域为CD8α跨膜区，所述共刺激胞内结构域为CD28胞内区和/或4-1BB胞内区，所述信号传导结构域为CD3ζ胞内区；
    优选地，所述CD8α铰链区为人CD8α铰链区；所述CD8α跨膜区为人CD8α跨膜区；所述CD28胞内区为人CD28胞内区；所述4-1BB胞内区为人4-1BB胞内区；和/或，所述CD3ζ胞内区为人CD3ζ胞内区；
    更优选地，所述CAR的氨基酸序列如SEQ ID NO:3所示。
  27. 一种核酸构建体，其特征在于，所述核酸构建体具有如式car-[(IRES)-f] q所示的结构，其中，IRES为内部核糖体进入位点序列；f编码功能性蛋白F，q为0或非0自然数；car编码如权利要求24-26中任一项所述的CAR。
  28. 如权利要求27所述的核酸构建体，其特征在于，所述核酸构建体中，当q为非0自然数优选为1时，所述功能性蛋白F包括：
    (1)细胞因子或其活性片段，优选IL10或IL15或其活性片段；更优选地，所述IL10的氨基酸序列如SEQ ID NO:2所示；
    (2)细胞因子受体或其活性片段，优选IL15Rα或其片段或IL15Rα片段(sushi)或IL15Rα片段(sushi+)；或，
    (3)细胞因子受体或其活性片段与细胞因子的融合蛋白，优选IL15Rα或其片段或IL15Rα(sushi)或IL15Rα(sushi+)与IL15的融合片段。
  29. 如权利要求27或28所述的核酸构建体，其特征在于，所述核酸构建体的结构 为：
    (1)scFv-人CD8α铰链区-人CD8α跨膜区-人4-1BB胞内区-人CD3ζ胞内区；
    (2)scFv-人CD8α铰链区-人CD8α跨膜区-人4-1BB胞内区-人CD3ζ胞内区-(IRES)-IL15；；
    (3)scFv-人CD8α铰链区-人CD8α跨膜区-人4-1BB胞内区-人CD3ζ胞内区-(IRES)-IL10；或，
    (4)scFv-人CD8α铰链区-人CD8α跨膜区-人4-1BB胞内区-人CD3ζ胞内区-(IRES)-IL15Rα(sushi+)-IL15。
  30. 一种表达载体，其包含如权利要求17所述的分离的核酸，或权利要求27-29中任一项所述的核酸构建体；优选地，所述表达载体选自逆转录病毒载体、慢病毒载体、腺病毒载体和腺相关病毒载体。
  31. 一种基因修饰的细胞，其特征在于，其转染有如权利要求30所述的表达载体；优选地，所述基因修饰的细胞为真核细胞，更优选分离的人细胞；进一步更优选免疫细胞如T细胞，或NK细胞如NK92细胞系。
  32. 一种制备基因修饰的细胞的方法，其特征在于，所述方法包括以下步骤：将权利要求30所述的表达载体转入待修饰的细胞内即得；较佳地，所述基因修饰的细胞为真核细胞，优选分离的人细胞；更优选免疫细胞如T细胞或NK细胞；进一步更优选为NK92细胞系。
  33. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-10中任一项所述的靶向CLDN18.2的抗体、权利要求11-16中任一项所述的双特异性抗体、权利要求17-22中任一项所述的抗体药物偶联体、权利要求31所述的基因修饰的细胞以及药学上可接受的载体；优选地，所述药物组合物还包括免疫检查点抗体。
  34. 一种如权利要求1-10中任一项所述的靶向CLDN18.2的抗体、权利要求11-16中任一项所述的双特异性抗体、权利要求17-22中任一项所述的抗体药物偶联体、权利要求24-26中任一所述的CAR、权利要求27-29中任一所述的核酸构建体、权利要求30所述的表达载体、权利要求31所述的基因修饰的细胞或权利要求33所述的药物组合物在制备治疗肿瘤的药物中的应用；较佳地，所述肿瘤为CLDN18.2阳性肿瘤，优选胃癌、食管癌、肺癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌、神经胶质瘤或者白血病。
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