WO2022179580A1 - 一种抗NKp30抗体及其应用 - Google Patents

一种抗NKp30抗体及其应用 Download PDF

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WO2022179580A1
WO2022179580A1 PCT/CN2022/077787 CN2022077787W WO2022179580A1 WO 2022179580 A1 WO2022179580 A1 WO 2022179580A1 CN 2022077787 W CN2022077787 W CN 2022077787W WO 2022179580 A1 WO2022179580 A1 WO 2022179580A1
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seq
cdr2
cdr1
cdr3
antibody
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PCT/CN2022/077787
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French (fr)
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周金花
朱彩林
吴崇兵
姜晓玲
殷刘松
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盛禾(中国)生物制药有限公司
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Priority claimed from CN202110213137.5A external-priority patent/CN114957469B/zh
Application filed by 盛禾(中国)生物制药有限公司 filed Critical 盛禾(中国)生物制药有限公司
Priority to US18/279,014 priority Critical patent/US20240150460A1/en
Publication of WO2022179580A1 publication Critical patent/WO2022179580A1/zh

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Definitions

  • the invention belongs to the field of tumor immunotherapy and molecular immunology, in particular to an anti-NKp30 single domain antibody and its application.
  • T-cell immune checkpoint therapies such as CTLA-4 and PD-1 have significantly improved the prognosis of patients with a variety of metastatic and refractory cancers, however, they are only effective in a small number of patients, with an effective rate of around 20%, and face drug resistance question.
  • NK cells are recognized as the first line of defense in the medical community. Compared with other anti-cancer immune cells, NK cells are stronger and more effective in killing tumors and virus-infected cells. Its activation does not depend on tumor cell surface antigens, and it does not need to go through the antigen recognition reaction of the immune system to determine the "attack" target like T cells. NK cells swim in the blood vessels of the whole body to perform immune surveillance. They can detect and quickly activate immune defense and immune stabilization functions at the first time, and kill diseased and cancerous cells. After NK cells act on target cells, the killing effect can be seen in 1 hour in vitro and 4 hours in vivo.
  • Major human NK cell activating receptors include CD16, NKG2D, and natural cytotoxicity receptors (NCRs), the latter including NKp30, NKp44, and NKp46.
  • NK cells the activation of NK cells is mainly through the binding of CD16 to the Fc region of the antibody, but the affinity between Fc and CD16 is low.
  • CD16 agonists which can more effectively activate NK cells and play an anti-tumor effect, but the lack of CD16 or CD16 polymorphisms limit the application of CD16 agonists.
  • ⁇ T cells are immune cells that can not only kill cancer cells, tumor stem cells, but also recognize cancer antigens. At the same time, ⁇ T cells are mainly distributed in the skin and mucosal tissues, so they have outstanding therapeutic effects on mucosal cancers, such as those in the digestive tract, respiratory tract, and reproductive system. There are no reports on biological agonists of ⁇ T cells.
  • NKp30 Natural cytotoxicity triggering receptor 3
  • NCRs natural cytotoxicity triggering receptors
  • Camelids such as camels or alpacas can produce a heavy chain antibody that lacks the light chain naturally. Antigen-binding function, and does not aggregate as easily as engineered single-chain antibody fragments (scFv). Due to its special structural properties, heavy-chain single-domain antibodies combine the advantages of traditional antibodies and small molecule drugs, and overcome the shortcomings of traditional antibodies such as long development cycles, low stability, and harsh storage conditions, representing a new generation of antibody therapy. development direction.
  • single-domain antibodies are much smaller in size than common monoclonal antibodies with two heavy and two light chains, they can bind antigen with similar affinity and specificity as monoclonal antibodies (mAbs).
  • mAbs monoclonal antibodies
  • single-domain antibodies When single-domain antibodies are used as building blocks, they can be fused to an IgG Fc domain to generate IgG-like antibodies, including bivalent and multivalent antibodies.
  • IgG Fc domain to generate IgG-like antibodies, including bivalent and multivalent antibodies.
  • the present invention takes NKp30 as the target of immunotherapy, and develops a new anti-NKp30 single domain antibody, which is used for the development of bifunctional antibody, multifunctional antibody or multifunctional fusion protein.
  • the present invention provides an anti-NKp30 single domain antibody, which can activate NK cells or ⁇ T cells to release cytokines.
  • the cytokine is a lymphokine, preferably IL2, IL3, IL4, IL5, IL6, IL9, IL10, IFN- ⁇ or TNF- ⁇ , more preferably IFN- ⁇ , TNF- ⁇ or IL2.
  • the anti-NKp30 single domain antibody comprises an immunoglobulin single variable domain comprising the complementarity determining regions CDR1, CDR2 and CDR3, wherein,
  • CDR1 selected from any amino acid sequence of SEQ ID NOs: 47-69, 141, 142, or having at least 80%, 85%, A sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, or to SEQ ID NOs: 47-69,141,142
  • CDR2 selected from any amino acid sequence of SEQ ID NO: 70-92, 140, or having at least 80%, 85%, 90%, 91% with any amino acid sequence of SEQ ID NO: 70-92, 140 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared to any of the amino acid sequences of SEQ ID NOs: 70-92,140
  • CDR3 selected from any amino acid sequence of SEQ ID NO: 93-115, or having at least 80%, 85%, 90%, 91%, 92% with any amino acid sequence of SEQ ID NO: 93-115 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more ( Preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) in the amino acid sequence.
  • the single variable domain of the anti-NKp30 single domain antibody comprises CDR1, CDR2 and CDR3 selected from the group consisting of:
  • the immunoglobulin single variable domain is a VHH.
  • the VHH comprises at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence described in any of SEQ ID NOs: 1-23, 117-139 Sequences of %, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the VHH is selected from the amino acid sequences set forth in any of SEQ ID NOs: 1-23.
  • the VHH is selected from the amino acid sequences set forth in any of SEQ ID NOs: 117-139.
  • the antibody binds NKp30 with a KD of 20.1 nM or less.
  • the antibody further comprises an immunoglobulin Fc region selected from the group consisting of IgGl, IgG2, IgG3 and/or IgG4.
  • amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO:116.
  • the present invention also provides a nucleic acid molecule encoding any of the anti-NKp30 single-domain antibodies described above.
  • the present invention also provides expression vectors comprising the above-described nucleic acid molecules operably linked to expression control elements.
  • the present invention also provides a recombinant cell, which is transformed with the above-mentioned nucleic acid molecule or the above-mentioned expression vector, and is capable of expressing the anti-NKp30 single-domain antibody.
  • the present invention also provides a multifunctional fusion protein comprising any of the anti-NKp30 single-domain antibodies described above.
  • the multifunctional fusion protein thereof further comprises one or more secondary antibodies or antigen-binding portions thereof that specifically bind to other antigens.
  • the antigen that binds the second antibody or antigen-binding portion thereof is selected from tumor-associated antigens (TAAs) or immune checkpoints.
  • TAAs tumor-associated antigens
  • the tumor associated antigen is selected from the group consisting of BCMA, CD38, HER2, PSMA, Claudin18.2, GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, AFP, ALK or B7H3.
  • the second antibody or antigen-binding portion thereof is an NK cell agonist.
  • the antigen that binds the second antibody or antigen-binding portion thereof is selected from NKP30, NKP46, CD16, NKP44, CD244, CD226, NKG2E, NKG2D, NKG2C or KIR.
  • its multifunctional fusion protein further comprises cytokines.
  • the cytokine is selected from IL8, IL10, IL15, IL18, TGF, VEGF, IFN ⁇ , IFN ⁇ or GM-CSF.
  • the present invention also provides the use of any of the above-mentioned anti-NKp30 single-domain antibodies and multifunctional fusion proteins in the preparation of medicaments for treating and/or preventing and/or diagnosing diseases.
  • the use is accomplished by one or more of tumor immunotherapy, cell therapy, and gene therapy.
  • the present invention also provides the use of any of the above-mentioned anti-NKp30 single-domain antibodies and multifunctional fusion proteins in medicines for treating cancer.
  • the cancer is lung cancer, liver cancer, melanoma, glioblastoma, head and neck cancer, colorectal cancer, stomach cancer, prostate cancer, ovarian cancer, bladder cancer, pancreatic cancer, stomach cancer, colon cancer, Cervical cancer or related tumors.
  • the present invention also provides a pharmaceutical composition comprising any of the anti-NKp30 single domain antibodies described above and an acceptable carrier, diluent or excipient.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising any of the multifunctional fusion proteins described above and an acceptable carrier, diluent or excipient.
  • the anti-NKp30 single-domain antibody provided by the present invention can specifically bind to NKp30, activate NK immune response, and promote the release of IFN- ⁇ , TNF- ⁇ and other cytokines by NK cells; the above functions are close to or exceed the level of current NKp30 monoclonal antibodies.
  • IgG Immunoglobulin G
  • FACS Fluorescence Activated Cell Sorting
  • domain refers to a folded protein structure capable of maintaining its tertiary structure independently of the rest of the protein. In general, domains are responsible for individual functional properties of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or the domain.
  • tumor-associated antigen refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed in whole or as fragments on the surface of cancerous cells, and which can be used to preferentially Pharmacological agents target cancerous cells.
  • tumor-associated antigens include, eg, BCMA, CD38, HER2, PSMA, Claudin18.2, GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138 , CDK4, CEA, AFP, ALK or B7H3.
  • antibody or "immunoglobulin”, which are used interchangeably herein, whether referring to heavy chain antibodies or conventional 4-chain antibodies, are used as general terms to include full-length antibodies, individual chains thereof, and all parts thereof , domains or fragments (including but not limited to antigen binding domains or fragments such as VHH domains or VH/VL domains, respectively).
  • sequence as used herein (eg, in terms of “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) should be generally understood To include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding said sequence, unless a more limited interpretation is required herein.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain capable of specifically binding an epitope without pairing with other immunoglobulin variable domains.
  • An example of an immunoglobulin single variable domain within the meaning of the present invention is a "domain antibody”, eg an immunoglobulin single variable domain is a "VHH domain” of Camelidae as defined below (or simply "" VHH").
  • VHH domains also known as heavy chain single domain antibodies, single domain antibodies, VHH, VHH domains, VHH antibody fragments, and VHH antibodies, are known as “heavy chain antibodies” (ie, “light chain-deficient antibodies”)
  • the variable domains of antigen-binding immunoglobulins Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature363, 446-448 (1993)).
  • VHH domain is used to relate the variable domains to the heavy chain variable domains (which are referred to herein as "VH domains”) present in conventional 4-chain antibodies and to those present in conventional 4-chain antibodies
  • VH domains heavy chain variable domains
  • VL domains Light chain variable domains
  • the VHH domain binds specifically to an epitope without the need for additional antigen binding domains (in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain).
  • VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain.
  • single domain antibody In the context of the present invention, the terms “single domain antibody”, “heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH domain”, “VHH antibody fragment”, “VHH antibody”, “ Nanobody” and “Nanobody” are used interchangeably.
  • immunoglobulin variable domain refers to what is essentially referred to in the art and hereinafter as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3” or “FR3”, respectively ", and an antibody domain consisting of four “framework regions” of "framework region 4" or “FR4", wherein the framework regions are referred to in the art and hereinafter as “complementarity determining region 1" or "CDR1", " The three “complementarity determining regions” or “CDRs” of “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer antigen specificity to antibodies by having an antigen-binding site.
  • the term "specificity” refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (eg, an immunoglobulin single variable domain of the invention) can bind.
  • the specificity of an antigen binding protein can be determined based on its affinity and/or avidity.
  • the affinity expressed by the dissociation equilibrium constant (K D ) of the antigen and the antigen-binding protein is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the K D value, the more the epitope binds to the antigen
  • K A association constant
  • affinity can be determined in a known manner depending on the particular antigen of interest.
  • Affinity is a measure of the strength of binding between an antigen-binding protein (eg, an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing the same) and a relevant antigen. Affinity is related to both: the affinity between its antigen-binding site on an antigen-binding protein, and the number of associated binding sites present on the antigen-binding protein.
  • an antigen-binding protein eg, an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing the same
  • polypeptide refers to an amino acid chain of any length, regardless of modification (eg, phosphorylation or glycosylation).
  • polypeptide includes proteins and fragments thereof.
  • Polypeptides may be "foreign”, meaning that they are “heterologous”, ie foreign to the host cell being utilized, eg, human polypeptides produced by bacterial cells.
  • Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written left to right in amino-terminal to carboxy-terminal direction.
  • amino acid residue sequences are named by three-letter or one-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), Partic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F) , proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps where necessary to obtain maximum percent sequence identity percentage of residues. Alignment for purposes of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program Bag.
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest.
  • the term includes progeny of a parent cell, whether or not the progeny is identical in morphology or genetic composition to the original parent cell, so long as the progeny has the selected gene of interest.
  • Commonly used host cells include bacteria, yeast, mammalian cells, and the like.
  • transfection refers to the uptake of foreign or exogenous DNA into a cell, a technique that can be used to introduce one or more exogenous DNA moieties into a suitable host cell.
  • Cells can be induced by physicochemical methods (eg, by calcium chloride treatment) into a physiological state optimal for uptake and accommodation of foreign DNA, ie, "competent.”
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • Figures 1a-1b are FACS results of chimeric anti-NKp30 single domain antibody binding to stable expression cell lines
  • Figures 2a-2c are the experimental results of chimeric anti-NKp30 single domain antibody stimulating NK cells to activate and release cytokines;
  • Figures 3a-3h are the FACS results of the binding of humanized anti-NKp30 antibody and/or CDR-engineered anti-NKp30 antibody to stable expression cell lines;
  • Figure 4 is the experimental result of the humanized anti-NKp30 antibody stimulating NK cells to activate and release cytokines.
  • immunization was performed on the 0th day, the 21st day, the 42nd day, and the 63rd day, respectively.
  • 10 mL of blood was collected from the neck vein of the camel, and 50 mL of blood was collected on the 49th day and the 70th day, each time. A part of the blood was taken out for serum titer detection.
  • Immunization once every 2 weeks, a total of 7 immunizations. Blood was collected at intervals of 5-7 days after the 6th and 7th immunization, and 25-30 mL of blood was taken each time and collected in 3 blood collection tubes. Before the 4th, 5th, and 6th immunization, blood was collected for immune evaluation.
  • the blood was collected from the neck vein of the camel, and 5 mL of blood was taken each time. On the same day, the blood was centrifuged at 400 x g for 30 minutes in a pre-cooled 25 °C centrifuge, and the upper serum was separated and stored. . Then, the lymphocytes were separated, that is, 3 mL of cell separation solution was firstly added to a 15 mL centrifuge tube, and then 3 mL of blood was slowly added. When adding blood, be careful and slow to prevent mixing of blood and separation solution. After that, the centrifuge was pre-cooled to room temperature.
  • RNA in PBMCs was extracted and reverse-transcribed with PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A), and a total of 5 ⁇ g RNA was transcribed.
  • the cDNA stock solution was mixed in equal proportions, diluted 5 times, and 5.0 ⁇ L was added for the first round of amplification.
  • the amplified product was recovered by gel tapping.
  • the recovered product was used as a template for the second round of amplification, and the amplified product was recovered by gel tapping as the target fragment.
  • the vector and the target fragment were digested with SfiI respectively, digested at 50°C overnight, and then the target fragment was recovered.
  • a total of 10 electroconversions were performed.
  • 1 mL of 2YT medium preheated at 37°C was added to the electric shock cup for recovery, and the electric shock products were aspirated and washed with 2YT medium to obtain a total of 100 ml of recovery products.
  • Resuscitate for 45 min under conditions take 100 ⁇ L of gradient dilution to 10 -3 and 10 -4 to determine the number of library transformants, spread on 90mm plates, centrifuge the rest, add 8mL 2YT to resuspend, and spread on 8 200mm plates. The next day, the number of transformants in the library was determined, and the capacity of the library was calculated.
  • the target molecule NKp30his was diluted with a carbonate buffer with a pH value of 9.6 to a final concentration of 5 ⁇ g/mL, added to the enzyme-labeled wells at 100 ⁇ L/well, and each target molecule was coated with 8 wells (the second round of screening was coated with 4 Wells, the third and fourth rounds of screening were coated with 2 wells each), and were coated overnight at 4°C. Discard the coating solution, wash 3 times with PBS, add 300 ⁇ L of 3% BSA-PBS blocking solution to each well, and block at 37°C for 1 h. After washing 3 times with PBS, 100 ⁇ L of phage library was added and incubated at 37°C for 1 h.
  • Unbound phage was aspirated and washed 6 times with PBST and 2 times with PBS.
  • the panning eluate was mixed with 5 mL of the E.coli TG1 culture in the early logarithmic growth stage, left at 37°C for 30 min, and shaken at 220 r/min for 30 min; centrifuged at 1000 g for 15 min, removed the supernatant, and added 500 ⁇ L of 2 ⁇ YT Resuspend and coat on a 200mm 2 ⁇ YT-GA plate.
  • 96 clones (numbered 1-96) were randomly selected from the second round titer plate and 96 randomly selected from the first round titer plate with a sterile toothpick
  • the clones (numbered 97-192) were inoculated into 1 mL of 2 ⁇ YT-A, and cultured with shaking at 230 r/min for 8 h at 37°C.
  • Take 200 ⁇ L of the above culture, add M13K07 phage at the ratio of cell:phage 1:20, stand at 37° C. for 15 min, and culture with shaking at 220 r/min for 45 min.
  • the target molecule NKp30 antigen was diluted with a carbonate buffer with a pH value of 9.6 to a final concentration of 2 ⁇ g/mL, added to the enzyme-labeled well at 100 ⁇ L/well, and coated overnight at 4°C. Discard the coating solution, wash three times with PBST, add 300 ⁇ L of 5% skim milk to each well, and block at 37°C for 1 h. PBST was washed three times, and 50 ⁇ L of phage culture supernatant and 50 ⁇ L of 5% skim milk were added to each well, and incubated at 37°C for 1 h.
  • PBST was washed for 5 times, and anti-M13 antibody labeled with horseradish peroxidase (1:10000 dilution with PBS) was added, 100 ⁇ L/well, and acted at 37° C. for 1 h. Plates were washed 6 times with PBST. Add TMB chromogenic solution for color development, 100 ⁇ L/well, 37° C., 7 min, add stop solution to stop the reaction, 50 ⁇ L/well, and measure the optical density at 450 nm.
  • Antibody gene sequencing was performed on the sequences obtained by screening the phage library. 23 antibody sequences were selected, and their amino acid/nucleotide sequences were:
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 1 are: SEQ ID NO: 47, SEQ ID NO: 70 and SEQ ID NO: 93;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 2 are: SEQ ID NO: 48, SEQ ID NO: 71 and SEQ ID NO: 94;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 3 are: SEQ ID NO: 49, SEQ ID NO: 72 and SEQ ID NO: 95;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 4 are: SEQ ID NO: 50, SEQ ID NO: 73 and SEQ ID NO: 96;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 5 are: SEQ ID NO: 51, SEQ ID NO: 74 and SEQ ID NO: 97;
  • the nucleotide sequence of the variable domain of sequence 5 is SEQ ID NO: 28;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 6 are respectively: SEQ ID NO: 52, SEQ ID NO: 75 and SEQ ID NO: 98;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 7 are: SEQ ID NO: 53, SEQ ID NO: 76 and SEQ ID NO: 99;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 8 are: SEQ ID NO: 54, SEQ ID NO: 77 and SEQ ID NO: 100;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 9 are: SEQ ID NO: 55, SEQ ID NO: 78 and SEQ ID NO: 101;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 10 are respectively: SEQ ID NO: 56, SEQ ID NO: 79 and SEQ ID NO: 102;
  • the nucleotide sequence of the variable domain of sequence 10 is SEQ ID NO: 33;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 11 are respectively: SEQ ID NO: 57, SEQ ID NO: 80 and SEQ ID NO: 103;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 12 are: SEQ ID NO: 58, SEQ ID NO: 81 and SEQ ID NO: 104;
  • the nucleotide sequence of the variable domain of sequence 12 is SEQ ID NO: 35;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 13 are respectively: SEQ ID NO: 59, SEQ ID NO: 82 and SEQ ID NO: 105;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 14 are: SEQ ID NO: 60, SEQ ID NO: 83 and SEQ ID NO: 106;
  • the nucleotide sequence of the variable domain of sequence 14 is SEQ ID NO: 37;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 15 are: SEQ ID NO: 61, SEQ ID NO: 84 and SEQ ID NO: 107;
  • the nucleotide sequence of the variable domain of sequence 15 is SEQ ID NO: 38;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 16 are respectively: SEQ ID NO: 62, SEQ ID NO: 85 and SEQ ID NO: 108;
  • the nucleotide sequence of the variable domain of sequence 16 is SEQ ID NO: 39;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 17 are: SEQ ID NO: 63, SEQ ID NO: 86 and SEQ ID NO: 109;
  • the nucleotide sequence of the variable domain of sequence 17 is SEQ ID NO: 40;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 18 are: SEQ ID NO: 64, SEQ ID NO: 87 and SEQ ID NO: 110;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 19 are: SEQ ID NO: 65, SEQ ID NO: 88 and SEQ ID NO: 111;
  • the nucleotide sequence of the variable domain of sequence 19 is SEQ ID NO: 42;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 20 are respectively: SEQ ID NO: 66, SEQ ID NO: 89 and SEQ ID NO: 112;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 21 are respectively: SEQ ID NO: 67, SEQ ID NO: 90 and SEQ ID NO: 113;
  • the nucleotide sequence of the variable domain of sequence 21 is SEQ ID NO: 44;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 22 are: SEQ ID NO: 68, SEQ ID NO: 91 and SEQ ID NO: 114;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the variable domain of sequence 23 are respectively: SEQ ID NO: 69, SEQ ID NO: 92 and SEQ ID NO: 115;
  • the nucleotide sequence of the variable domain of sequence 23 is SEQ ID NO:46.
  • the sequence obtained by screening the phage library is subjected to antibody gene sequencing, and the antibody fragment obtained by sequencing is subjected to gene synthesis to construct into a human IgG framework, and then the antibody fragment is inserted into the pcDNA3.1 vector using molecular cloning technology to construct mammalian cells.
  • the expression plasmid was introduced into the host cell line CHO cells by lipofection, and the fermentation supernatant was obtained by cell fed-batch, and the fermentation supernatant was taken for a series of purification steps such as affinity chromatography and ion exchange chromatography.
  • the amino acid sequences of CDRs and variable domains of Antibody 1 to Antibody 23 correspond to the amino acid sequences of CDRs and variable domains of Sequences 1 to 23 in Example 4, respectively.
  • amino acid sequences of the constant regions of Antibody 1 to Antibody 23 are all identical, as shown in SEQ ID NO:116.
  • Example 6 Affinity verification between anti-NKp30 antibody and NKp30
  • the AHC sensor was soaked with 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) as a buffer for 600 s before use to remove the sucrose covered on the sensor surface.
  • PBST 0.02% Tween 20, pH 7.4, 1*PBS
  • the experimental temperature is set to 30 °C
  • the shaking speed is set to 1000 rpm.
  • the AHC sensor was equilibrated with 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) as a buffer for 60 s, the NKp30 antibody in the sample plate was solidified for 300 s, and the secondary equilibration buffer was 180 s.
  • 100 nM of human NKp30-his protein (KACTUS; Cat: NKp-HM430) was bound to NKp30 antibody for 300 s and then dissociated for 600 s. After dissociation, 10 mM glycine (pH 2.0) was used as regeneration buffer for regeneration for 30 s.
  • the sensor was regenerated with 10 mM glycine (pH 2.0).
  • the supernatant was discarded, and 100 ⁇ L of primary antibody solution and irrelevant antibody solution were added to the experimental group and the irrelevant antibody group respectively. After resuspending the cells, they were incubated at room temperature for 1 h. Blank and NC groups were incubated with the same amount of PBS. After 1 h, the cells were centrifuged and washed twice with PBS. After discarding the supernatant, 100 ⁇ L of fluorescent secondary antibody diluent (goat anti-human Fc-FITC Abcam cat: ab97224) was added to the other sample groups except the Blank group, which was added with 100 ⁇ L PBS. . After the supernatant was discarded, 120 ⁇ L of PBS buffer was added to resuspend and flow cytometry was performed in sequence to measure the average fluorescence intensity. The results are shown in Figure 1a and Figure 1b.
  • Example 8 Antibody stimulates NK cells to activate and release cytokines
  • the 96-well plate was taken out, and the antibody to be tested, positive control antibody (anti-CD337 (NKp30) antibody selected from Biolegend) and isotype control antibody were initially diluted at 150nM, 3-fold dilution, 7 serial dilutions were performed, and 2 parallel wells were set , dissolved in PBS buffer and placed in a 96-well plate. Incubate overnight in a 4°C refrigerator for about 16 hours and then take out for subsequent operations. Remove the 96-well plate, discard the antibody incubation solution, and wash twice with PBS.
  • positive control antibody anti-CD337 (NKp30) antibody selected from Biolegend
  • isotype control antibody were initially diluted at 150nM, 3-fold dilution, 7 serial dilutions were performed, and 2 parallel wells were set , dissolved in PBS buffer and placed in a 96-well plate. Incubate overnight in a 4°C refrigerator for about 16 hours and then take out for subsequent operations. Remove the 96-well plate
  • NK cells were taken out and counted, and the number of cells was set to 4 ⁇ 10 4 cells/well, resuspended in medium containing IL-2 (STEMCELL, 78036) with a final concentration of 400U, and 200 ⁇ L/well of 96 cells incubated with antibody was added.
  • IL-2 STMCELL, 78036
  • the treated 96-well plate was placed in a 37°C CO 2 constant temperature incubator for about 24 hours, and the supernatant was extracted. The supernatant after centrifugation was measured with a kit (Biolegend Cat: 430104) for its maximum IFN- ⁇ secretion. The results are shown in Figures 2a-2c.
  • variable region framework region 1-3 of antibody 10 contains 15 camel-derived sites (VHH genes), and the variable region of antibody 18 contains 15.
  • the framework regions 1-3 contain 14 camel-derived sites (VHH genes).
  • the design template selects the IGHV3 category, designs the humanized sequence, and mutates the sequence into a humanized sequence.
  • CDR1, CDR2 of the variable domain and the amino acid sequences of CDR3 are: SEQ ID NO: 56, SEQ ID NO: 79 and SEQ ID NO: 102, respectively.
  • Four humanized antibodies with the same three CDRs were obtained from antibody 18: antibody 18-hu1, antibody 18-hu2, antibody 18-hu3 and antibody 18-hu4, the amino acid sequences of CDR1, CDR2 and CDR3 of the variable domains They are: SEQ ID NO: 64, SEQ ID NO: 87 and SEQ ID NO: 110.
  • amino acid sequences of the variable domains of the humanized antibodies are shown in Table 2.
  • Antibody 10-hu4 SEQ ID NO: 120 Antibody 10-hu5 SEQ ID NO: 121
  • the constant regions of the 9 humanized antibodies in Table 2 are the same, and the amino acid sequence of the constant region is SEQ ID NO: 116.
  • the CDR2 of antibody 23 in Example 5 was subjected to post-translational modification, that is, the amino acid sequence of CDR2 was transformed from GITGNGLTDYA DS VKG to GITGNGLTDYA ES VKG to obtain a chimeric antibody: antibody 23-p.
  • the antibody 23 in Example 5 was humanized, and the CDRs were subjected to post-translational modification to obtain 13 humanized antibodies.
  • the constant regions of the 14 antibodies in Table 3 are the same, and the amino acid sequence of the constant region is SEQ ID NO: 116.
  • Human-NKp30-His (purchased from KACTUS, Cat. No. NKP-HM430) was diluted to 0.2 ⁇ g/mL with coating solution (1 ⁇ PBS, pH 7.4), and coated on a 96-well microtiter plate, 100 ⁇ L/well, 4°C overnight. Pour off the coating solution, wash the plate with 300 ⁇ L per well of 1 ⁇ PBST, wash 4 times with a plate washer, and pat dry on flat paper. Blocked with 3% nonfat milk powder, 300 ⁇ L/well, incubated at 37°C for 1 h, poured off the blocking solution, washed 4 times with a plate washer, and patted dry on flat paper.
  • the reference product and the test product were diluted with 3% skimmed milk powder to 10 ⁇ g/mL, and the initial concentration was used as the initial concentration for 3-fold dilution. A total of 11 gradients were diluted, and 1 blank hole was added, and only the diluent was added. 100 ⁇ L/well, incubated at 37°C for 1 h. The liquid in the wells was discarded, washed 4 times in a plate washer, and patted dry on flat paper. Goat anti-human Fc was diluted 1:20,000 with 3% nonfat milk powder, 100 ⁇ L/well, and incubated at 37°C for 1 h. Plate washer 6 times and pat dry on flat paper.
  • Example 13 Humanized antibody stimulates NK cells to activate and release cytokines
  • the humanized antibody 18-hu3 was subjected to an experiment of stimulating NK cells to activate and release cytokines, and the experimental results are shown in FIG. 4 .
  • NK cells stimulated by antibody 18-hu3 could secrete IFN- ⁇ with an EC 50 value of 8.834nM, while NK cells stimulated by isotype control at various concentrations did not produce IFN- ⁇ , indicating that antibody 18-hu3 can specifically sexually activated NK cells.

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Abstract

本发明公开了一种抗NKp30单域抗体及其编码核酸，该抗体能够激活NK细胞或γδT细胞释放细胞因子。本发明还公开了包含抗NKp30单域抗体的多功能融合蛋白和组合物，及其在制备治疗、预防或诊断疾病的药物中的用途。

Description

一种抗NKp30抗体及其应用 技术领域
本发明属于肿瘤免疫疗法和分子免疫学领域，具体涉及一种抗NKp30单域抗体及其应用。
背景技术
肿瘤是目前世界范围内危害人类健康的重大疾病。机体免疫***的功能异常与肿瘤的发生和发展具有十分密切的关系。CTLA-4和PD-1等T细胞免疫检查点疗法显著改善了多种转移性和难治性癌症患者的预后，然而它们仅对少数患者有效，有效率在20％左右，并且面临耐药性问题。
NK细胞是医学界公认的第一道防线，与其他抗癌免疫细胞相比，NK细胞杀灭肿瘤和病毒感染细胞的作用更强、更有效。它的激活不依赖于肿瘤细胞表面抗原，也不需要像T细胞一样，要经过免疫***的抗原辨识反应才确定“攻击”目标。NK细胞游弋在全身血管行使免疫监视作用，它能在第一时间发现并迅速启动免疫防御和免疫稳定功能，杀死病变、癌变的细胞。NK细胞作用于靶细胞后杀伤作用，在体外1小时、体内4小时即可见到杀伤效应。人类主要NK细胞活化性受体包括CD16、NKG2D和自然细胞毒性受体(NCRs)，后者包括NKp30、NKp44和NKp46。
目前NK细胞的激活主要是通过抗体Fc区结合CD16，但Fc与CD16的亲和力低，科学家又开发了CD16激动剂，能更有效的引起NK细胞的活化而发挥抗肿瘤作用，但是CD16的缺失或CD16的多态性限制了CD16激动剂的应用。
γδT细胞是一种既能杀伤癌细胞，肿瘤干细胞，又能识别癌抗原的免疫细胞，它的杀伤性较强，但肿瘤干细胞杀伤不如NK细胞。同时，γδT细胞主要分布于皮肤和黏膜组织上，因此对于黏膜方面的癌症治疗效果突出，比如消化道、呼吸道、生殖***方面的癌症效果显著。目前还未有γδT细胞的生物制品激动剂相关报道。
NKp30(Natural cytotoxicity triggering receptor 3)由NCR3基因编码，属于自然细胞毒性引发受体(NCRs)家族成员，是细胞表面的一种激活型受体。NKp30表达于所有静息与活化的NK细胞、多种效应NKT细胞、γδT细胞、MAIT细胞(黏膜相关恒定的T淋巴细胞)，激活NKp30能激活NK细胞、γδT细胞等肿 瘤杀伤性细胞。重要的是，NKp30可以在没有CD16A结合的情况下激活NK细胞，且激活后杀伤效果强于anti-CD16A，抗NKp30抗体与抗CD16A抗体有协同放大作用。
骆驼或是羊驼等骆驼科动物能够产生一种天然缺失轻链的重链抗体，其分子只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，但却具有完整的抗原结合功能，而且不像人工改造的单链抗体片段(scFv)那样容易聚集。重链单域抗体因其特殊的结构性质，兼具了传统抗体与小分子药物的优势，克服了传统抗体的开发周期长，稳定性较低，保存条件苛刻等缺陷，代表了新一代抗体治疗开发的方向。
尽管单域抗体比具有两条重链和两条轻链的常见单克隆抗体的尺寸小得多，但其可以与单克隆抗体(mAb)相似的亲和力和特异性结合抗原。单域抗体用作结构单元时，其可融合至IgG Fc域以产生IgG样抗体，包括二价和多价抗体。开发靶向肿瘤相关抗原与NKp30的双特异性抗体，桥接肿瘤细胞与NK细胞。仅激活肿瘤微环境中的NK细胞，有助于减少副作用，避免因全身性NK细胞的高度活化，对丢失MHC分子的异常细胞敏感性降低。
本发明将NKp30作为免疫治疗的靶点，开发了新的抗NKp30单域抗体，用于双功能抗体、多功能抗体或多功能融合蛋白的开发。
发明内容
本发明提供了一种抗NKp30单域抗体，其能够激活NK细胞或γδT细胞释放细胞因子。
在可选的实施方案中，所述细胞因子为淋巴因子，优选为IL2、IL3、IL4、IL5、IL6、IL9、IL10、IFN-γ或TNF-α，更优选为IFN-γ、TNF-α或IL2。
在可选的实施方案中，所述抗NKp30单域抗体包含免疫球蛋白单一可变结构域，其免疫球蛋白单一可变结构域包含互补决定区CDR1、CDR2和CDR3，其中，
(a)CDR1，选自SEQ ID NO：47-69，141，142的任一氨基酸序列，或与SEQ ID NO：47-69，141，142的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：47-69，141，142的任一氨基酸序列相比具有一个或多个(优 选2个或3个)保守氨基酸突变(优选置换、***或缺失)的氨基酸序列；
(b)CDR2，选自SEQ ID NO：70-92，140的任一氨基酸序列，或与SEQ ID NO：70-92，140的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：70-92，140的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、***或缺失)的氨基酸序列；
(c)CDR3，选自SEQ ID NO：93-115的任一氨基酸序列，或与SEQ ID NO：93-115的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：93-115的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、***或缺失)的氨基酸序列。
在可选的实施方案中，所述抗NKp30单域抗体的单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
(1)SEQ ID NO：47所示的CDR1，SEQ ID NO：70所示的CDR2，SEQ ID NO：93所示的CDR3；
(2)SEQ ID NO：48所示的CDR1，SEQ ID NO：71所示的CDR2，SEQ ID NO：94所示的CDR3；
(3)SEQ ID NO：49所示的CDR1，SEQ ID NO：72所示的CDR2，SEQ ID NO：95所示的CDR3；
(4)SEQ ID NO：50所示的CDR1，SEQ ID NO：73所示的CDR2，SEQ ID NO：96所示的CDR3；
(5)SEQ ID NO：51所示的CDR1，SEQ ID NO：74所示的CDR2，SEQ ID NO：97所示的CDR3；
(6)SEQ ID NO：52所示的CDR1，SEQ ID NO：75所示的CDR2，SEQ ID NO：98所示的CDR3；
(7)SEQ ID NO：53所示的CDR1，SEQ ID NO：76所示的CDR2，SEQ ID NO：99所示的CDR3；
(8)SEQ ID NO：54所示的CDR1，SEQ ID NO：77所示的CDR2，SEQ ID NO：100所示的CDR3；
(9)SEQ ID NO：55所示的CDR1，SEQ ID NO：78所示的CDR2，SEQ ID  NO：101所示的CDR3；
(10)SEQ ID NO：56所示的CDR1，SEQ ID NO：79所示的CDR2，SEQ ID NO：102所示的CDR3；
(11)SEQ ID NO：57所示的CDR1，SEQ ID NO：80所示的CDR2，SEQ ID NO：103所示的CDR3；
(12)SEQ ID NO：58所示的CDR1，SEQ ID NO：81所示的CDR2，SEQ ID NO：104所示的CDR3；
(13)SEQ ID NO：59所示的CDR1，SEQ ID NO：82所示的CDR2，SEQ ID NO：105所示的CDR3；
(14)SEQ ID NO：60所示的CDR1，SEQ ID NO：83所示的CDR2，SEQ ID NO：106所示的CDR3；
(15)SEQ ID NO：61所示的CDR1，SEQ ID NO：84所示的CDR2，SEQ ID NO：107所示的CDR3；
(16)SEQ ID NO：62所示的CDR1，SEQ ID NO：85所示的CDR2，SEQ ID NO：108所示的CDR3；
(17)SEQ ID NO：63所示的CDR1，SEQ ID NO：86所示的CDR2，SEQ ID NO：109所示的CDR3；
(18)SEQ ID NO：64所示的CDR1，SEQ ID NO：87所示的CDR2，SEQ ID NO：110所示的CDR3；
(19)SEQ ID NO：65所示的CDR1，SEQ ID NO：88所示的CDR2，SEQ ID NO：111所示的CDR3；
(20)SEQ ID NO：66所示的CDR1，SEQ ID NO：89所示的CDR2，SEQ ID NO：112所示的CDR3；
(21)SEQ ID NO：67所示的CDR1，SEQ ID NO：90所示的CDR2，SEQ ID NO：113所示的CDR3；
(22)SEQ ID NO：68所示的CDR1，SEQ ID NO：91所示的CDR2，SEQ ID NO：114所示的CDR3；
(23)SEQ ID NO：69所示的CDR1，SEQ ID NO：92所示的CDR2，SEQ ID NO：115所示的CDR3；和/或
(24)SEQ ID NO：69所示的CDR1，SEQ ID NO：140所示的CDR2，SEQ  ID NO：115所示的CDR3；
(25)SEQ ID NO：141所示的CDR1，SEQ ID NO：140所示的CDR2，SEQ ID NO：115所示的CDR3；和/或
(26)SEQ ID NO：142所示的CDR1，SEQ ID NO：140所示的CDR2，SEQ ID NO：115所示的CDR3。
在可选的实施方案中，所述免疫球蛋白单一可变结构域是VHH。
在可选的实施方案中，所述VHH包含与SEQ ID NO：1-23，117-139中任一所述的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列。
在可选的实施方案中，所述VHH选自SEQ ID NO：1-23中任一所述的氨基酸序列。
在可选的实施方案中，所述VHH选自SEQ ID NO：117-139中任一所述的氨基酸序列。
在可选的实施方案中，所述抗体以20.1nM或更小的K D结合NKp30。
在可选的实施方案中，所述抗体还包含免疫球蛋白Fc区，所述免疫球蛋白Fc区选自IgG1、IgG2、IgG3和/或IgG4。
在可选的实施方案中，所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO：116所示。
本发明还提供了核酸分子，其编码上述任一所述的抗NKp30单域抗体。
本发明还提供了表达载体，其包含与表达调控原件可操作地连接的上述核酸分子。
本发明还提供了重组细胞，其包含上述核酸分子或上述的表达载体转化，并能够表达所述抗NKp30单域抗体。
本发明还提供了一种多功能融合蛋白，其包含上述任一所述的抗NKp30单域抗体。
在可选的实施方案中，其多功能融合蛋白还包含一个或多个与其他抗原特异性结合的第二抗体或其抗原结合部分。
在可选的实施方案中，其中所述结合第二抗体或其抗原结合部分的抗原选自肿瘤相关抗原(TAA)或免疫检查点。
在可选的实施方案中，其中所述肿瘤相关抗原(TAA)选自BCMA、CD38、 HER2、PSMA、Claudin18.2、GPC3、CD19、CD20(MS4A1)、CD22、CD24、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、AFP、ALK或B7H3。
在可选的实施方案中，其中所述第二抗体或其抗原结合部分是NK细胞激动剂。
在可选的实施方案中，其中所述结合第二抗体或其抗原结合部分的抗原选自NKP30、NKP46、CD16、NKP44、CD244、CD226、NKG2E、NKG2D、NKG2C或KIR。
在可选的实施方案中，其多功能融合蛋白还包含细胞因子。
在可选的实施方案中，所述细胞因子选自IL8、IL10、IL15、IL18、TGF、VEGF、IFNγ、IFNα或GM-CSF。
本发明还提供了上述任一所述的抗NKp30单域抗体、多功能融合蛋白在制备用于治疗和/或预防和/或诊断疾病的药物中的用途。
在可选的实施方案中，所述用途通过肿瘤免疫疗法、细胞疗法和基因疗法中的一种或多种来实现。
本发明还提供了上述任一所述的抗NKp30单域抗体、多功能融合蛋白在治疗癌症的药物中的用途。
在可选的实施方案中，所述癌症为肺癌、肝癌、黑色素瘤、恶性胶质瘤、头颈癌、结肠直肠癌、胃癌、***癌、卵巢癌、膀胱癌、胰腺癌、胃癌、结肠癌、***或相关肿瘤。
本发明还提供了一种药物组合物，其包含上述任一所述的抗NKp30单域抗体和可接受的载体、稀释剂或赋形剂。
本发明还提供了一种药物组合物，其包含上述任一所述的多功能融合蛋白和可接受的载体、稀释剂或赋形剂。
本发明提供的抗NKp30单域抗体能够特异性地与NKp30结合，激活NK免疫应答，促进NK细胞释放IFN-γ、TNF-α等细胞因子；上述功能接近或超过目前NKp30单抗的水准。
为了帮助理解本文中阐述的发明，现提供以下缩写解释及术语定义。
在本文中使用以下缩写：
IgG：免疫球蛋白G
ELISA：酶联免疫吸附测定
FACS：荧光激活细胞分选术
术语(多肽或蛋白的)“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白而不损失蛋白的其余部分和/或结构域的功能。
术语“肿瘤相关抗原”或“TAA”是指在癌性细胞的表面上完全或作为片段表达的分子(典型地是蛋白质、碳水化合物、脂质或它们的一些组合)，并且其可用于优先将药理学药剂靶向癌性细胞。“肿瘤相关抗原”的非限定示例包含，例如BCMA、CD38、HER2、PSMA、Claudin18.2、GPC3、CD19、CD20(MS4A1)、CD22、CD24、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、AFP、ALK或B7H3。
可互换使用的术语“抗体”或“免疫球蛋白”在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
术语“免疫球蛋白单一可变结构域”是指能够在不与其他免疫球蛋白可变结构域配对的情况下特异性结合抗原表位的免疫球蛋白可变结构域。本发明含义中的免疫球蛋白单一可变结构域的一个实例为“结构域抗体”，例如免疫球蛋白单一可变结构域为如下文定义的骆驼科的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、单域抗体、VHH、VHH结构域、VHH抗体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,BendahmanN,Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature363,446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文 中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
在本发明的上下文中，术语“单域抗体”、“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”、“纳米抗体”以及“Nanobody”可互换使用。
术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的抗体结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本发明的免疫球蛋白单一可变结构域)可结合的不同类型抗原或表位的数目。可基于抗原结合蛋白的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(K D)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：K D值越小，表位与抗原结合蛋白之间的结合强度越强(或者，亲和力也可表示为缔合常数(K A)，其为1/K D)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合蛋白(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合蛋白上的抗原结合位点之间的亲和力，以及存在于抗原结合蛋白上的相关结合位点的数目。
术语“多肽”是指任何长度的氨基酸链，而与修饰(例如磷酸化或糖基化)无关。术语多肽包括蛋白质及其片段。多肽可以是“外源的”，意指它们是“异源的”，即是所利用的宿主细胞外来的，例如由细菌细胞产生的人多肽。本文将多肽公开为氨基酸残基序列。那些序列按氨基末端到羧基末端的方向从左到右书写。根据标准命名法，氨基酸残基序列以三字母或单字母代码命名，如下所示：丙氨酸(Ala， A)、精氨酸(Arg，R)、天冬酰胺(Asn，N)、天冬氨酸(Asp，D)、半胱氨酸(Cys，C)、谷氨酰胺(Gln，Q)、谷氨酸(Glu，E)、甘氨酸(Gly，G)、组氨酸(His，H)、异亮氨酸(Ile，I)、亮氨酸(Leu，L)、赖氨酸(Lys，K)、甲硫氨酸(Met，M)、苯丙氨酸(Phe，F)、脯氨酸(Pro，P)、丝氨酸(Ser，S)、苏氨酸(Thr，T)、色氨酸(Trp，W)、酪氨酸(Tyr，Y)和缬氨酸(Val，V)。
关于参照多肽序列的“百分比(％)氨基酸序列同一性”定义为比对序列并在必要时引入缺口以获取最大百分比序列同一性后，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST，BLAST-2，Clustal W，Megalign(DNASTAR)软件或FASTA程序包。
术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
术语“转染”指外来或外源DNA被细胞摄入，该技术可用于将一种或多种外源DNA部分导入适宜的宿主细胞。可通过理化方法(例如通过氯化钙处理)诱导细胞，使其处于最适摄取和容纳外来DNA的生理状态，即“感受态”。
术语“载体”指能够增殖与其连接的另一种核酸的核酸分子。该术语包括作为自身复制型核酸结构的载体及并入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达。此类载体在本文中称为“表达载体”。
附图说明
图1a-1b为嵌合抗NKp30单域抗体与稳定表达细胞系结合FACS结果；
图2a-2c为嵌合抗NKp30单域抗体刺激NK细胞激活释放细胞因子实验结果；
图3a-3h为人源化的抗NKp30抗体和/或CDR改造后的抗NKp30抗体与稳定表达细胞系结合FACS结果；
图4为人源化的抗NKp30抗体刺激NK细胞激活释放细胞因子实验结果。
具体实施方式
以下结合附图与具体实施例对本发明做进一步的描述，本发明的保护内容不局限于以下实施例。还应该理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求及其任何等同物为本发明的保护范围。在本发明的说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。实施本发明的过程、条件、试剂、实验方法等，除专门提及的内容之外，均为本领域技术人员的普遍知识和公知常识，本发明没有特别限制内容。
以下通过更具体的实施例来说明本发明。
实施例1 动物免疫
准备重组人NKp30，Fc tag蛋白(ACRO，Cat：NC3-H5259)作为免疫原，每次免疫总的抗原量保持在1-2mg之间，体积在2mL以下，将抗原和佐剂1：1乳化使其形成均匀混合物4℃保存。记录骆驼耳号后开始免疫实验，每次在骆驼颈部***附近分左右两侧注射，每侧分2点注射，每点注射0.4mL混合好的抗原，免疫后观察半小时确认骆驼状态良好，无不适症状，分别在第0天，第21天，第42天，第63天进行免疫，在第28天，从骆驼颈部静脉采血10mL，第49天，第70天采血各50mL，每次采血均取出一部分进行血清效价检测。每2周免疫一次，一共进行7次免疫。在第6、7次免疫后间隔5-7天进行采血，每次取25-30mL血液，分3个采血管收集。在第4、5、6次免疫前进行采血用于免疫评价，采血从骆驼颈部静脉采取，每次取5mL血液，血液当天使用预冷25℃离心机，400xg离心30分钟，分离保存上层血清。而后分离淋巴细胞，即在15mL的离心管中先加入3mL细胞分离液，然后缓慢加入3mL血液。加入血液时小心缓慢以防止血液和分离液混匀，之后离心机预冷至室温，400g离心30分钟后，观察离心管中血液分离情况，用200μL移液器小心吸取出中间棉状上层免疫细胞至新的15mL离心管中，上层血清保存在新的离心管中，-80℃保存。每管加入室温放置的10mL PBS缓冲液，25℃、400g离心20分钟，去除上清液，每管加入室温放置的5mL PBS缓冲液，25℃，400g离心20分钟。使用血球计数板 计算细胞数目。去除上清液，根据细胞数目使用RNAiso Plus溶解分离得到的淋巴细胞得到10 7/mL溶解液，-80℃保存。
实施例2 噬菌体文库构建
第二次和第三次的采血用淋巴细胞分离液分离PBMC。提取PBMC中的总RNA，用反转录试剂盒进行反转录PrimeScript TM II 1st Strand cDNA Synthesis Kit(Takara，货号：6210A)，共转录5μg RNA。cDNA原液等比例混合之后稀释5倍，加5.0μL进行第一轮扩增，扩增产物割胶回收，回收产物作为模板进行第二轮扩增，扩增产物割胶回收，为目的片段。将载体与目的片段分别用SfiI进行酶切，50℃过夜酶切，然后回收目的片段。连接摩尔比例为Vector:VHH＝1:3。共进行10次电转化，电击之后立即向电击杯中加入1mL 2YT培养基(37℃预热)复苏，吸出电击产物并用2YT培养基洗净电击杯，共计获得100ml复苏产物，在37℃、180rpm条件下复苏45min，取100μL梯度稀释至10 -3和10 -4测定库转化子数目，涂布于90mm的平板上，其余离心，加入8mL 2YT重悬，涂布于8块200mm的平板上。第二天在测定库转化子数目，计算库容量。
将细菌文库接入2×300mL 2YT+A+G(Amp:100ug/ml、Glu:1％)培养基中，至其初始OD600＝0.1-0.2,37℃、230rpm培养至OD600＝0.8以上。根据OD600值加入辅助噬菌体M13KO7(辅助噬菌体：细菌＝20:1)。加入M13KO7后，混匀，37℃静置30min。37℃，180rpm缓摇30min。5000rpm离心10min，弃尽上清，用等体积2YT+A+K(Amp:100μg/ml、Kan:50μg/ml)培养基重悬沉淀，30℃，220rpm过夜。过夜培养物在4℃、10000rpm条件下离心20min，收取上清，弃沉淀。更换离心筒，在4℃、10000rpm条件下离心20min，收取上清。按上清体积的1/5加入PEG8000/NaCl，混匀，冰浴沉淀2小时以上。4℃，10000rpm，离心20min，弃上清，空离一次除尽上清。1mL 1×PBS悬起沉淀，加入1/5体积的PEG8000/NaCl二次沉淀1h。4℃，12000rpm，离心10min弃上清，空离一次除尽上清。根据沉淀的量,加入1×PBS重悬沉淀。加100％甘油至终浓度为50％，混匀后分装到1.5mL EP管中，-80℃保存。取10uL库噬菌体用2YT梯度稀释，从10 -8和10 -9管中取10ul加到90μL的TG1菌液中，轻柔混匀。37℃静置15min，分别涂布Amp抗性平板，过夜培养。第二天，计算滴度板上的克隆金属噬菌体文库滴度。
实施例3 噬菌体文库筛选阳性抗体
将靶分子NKp30his用pH值为9.6的碳酸盐缓冲液稀释至终浓度为5μg/mL，按100μL/孔加入酶标孔中，每个靶分子包被8孔(第二轮筛选包被4孔，第三轮和第四轮筛选各包被2孔)，4℃包被过夜。弃包被液，PBS洗涤3次，每孔加入300μL 3％BSA-PBS封闭液，37℃封闭1h。PBS洗涤3次，加入100μL噬菌体文库，37℃孵育1h。吸出未结合的噬菌体，用PBST洗涤6次，PBS洗涤2次。加入100μL Gly-HCl洗脱液，37℃孵育8min，洗脱特异性结合的噬菌体；将该洗脱液转移至1.5mL无菌离心管中，迅速用10μL Tris-HCl中和缓冲液中和。取10μL进行梯度稀释，测定滴度，计算淘选回收率，其余洗脱物混合后进行扩增和纯化，用于下一轮亲和淘选。
将淘选洗脱物与处于对数生长前期的E.coli TG1培养物5mL混匀，37℃，静置30min，220r/min振荡培养30min；1000g离心15min，去上清，用500μL 2×YT重悬涂布于200mm 2×YT-GA平板。用10ml 2×YT液体培养基刮菌，取500μl悬液加入50ml 2×YT液体培养基中，37℃振摇30min；按cell:phage＝1:20的比例加入M13K07辅助噬菌体，37℃静置30min，220r/min振摇培养30min；将培养物分装于离心管中，25℃，5000r/min，离心10min，细胞沉淀以50mL 2×YT-AK液体培养基重悬，30℃，230r/min振荡培养过夜。将过夜培养物4℃，10000r/min离心20min，将上清转移至新离心管，加入1/5体积的PEG/NaCl，混匀后置于4℃2h以上。4℃，10000r/min，离心20min，去除上清，将沉淀重悬于1mL PBS中，加入1/5体积的PEG/NaCl，混匀后置于4℃1h以上。4℃，12000r/min，离心2min，去除上清，将沉淀悬浮于200μL PBS中，即为扩增产物，测定滴度，用于下一轮淘选或者分析。
从淘选洗脱物滴度的平板上，用灭菌牙签从从第二轮滴度测定平板上随机挑选96个克隆(编号为1-96)，第一轮滴度平板上随机挑选96个克隆(编号为97-192)接种于1mL 2×YT-A中，37℃，230r/min振荡培养8h。取200μL上述培养物，按cell：phage＝1：20的比例加入M13K07噬菌体，37℃，静置15min，220r/min振荡培养45min。补加800μL体积的2×YT-AK，30℃，剧烈振荡培养过夜。第二天12000rpm离心2min，取上清，用于单克隆ELISA鉴定。
将靶分子NKp30抗原用pH值为9.6的碳酸盐缓冲液稀释至终浓度为2μg/mL，按100μL/孔加入酶标孔中，4℃包被过夜。弃包被液，PBST洗涤3次，每 孔加入300μL 5％脱脂牛奶，37℃封闭1h。PBST洗涤3次，每孔加入50μL噬菌体培养菌液上清和50μL 5％脱脂牛奶，37℃，孵育1h。PBST洗涤5次，加入辣根过氧化物酶标记的抗M13抗体(用PBS按1:10000稀释)，100μL/孔，37℃作用1h。PBST洗板6次。加入TMB显色液显色，100μL/孔，37℃，7min，加入终止液终止反应，50μL/孔，于450nm下测光密度。
实施例4 抗体基因序列测序
将噬菌体文库筛选得到的序列进行抗体基因测序。选出23条抗体序列，其氨基酸/核苷酸序列分别为：
(1)序列1可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：47、SEQ ID NO：70和SEQ ID NO：93；
序列1可变结构域的氨基酸序列为SEQ ID NO：1；
序列1可变结构域的核苷酸序列为SEQ ID NO：24；
(2)序列2可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：48、SEQ ID NO：71和SEQ ID NO：94；
序列2可变结构域的氨基酸序列为SEQ ID NO：2；
序列2可变结构域的核苷酸序列为SEQ ID NO：25；
(3)序列3可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：49、SEQ ID NO：72和SEQ ID NO：95；
序列3可变结构域的氨基酸序列为SEQ ID NO：3；
序列3可变结构域的核苷酸序列为SEQ ID NO：26；
(4)序列4可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：50、SEQ ID NO：73和SEQ ID NO：96；
序列4可变结构域的氨基酸序列为SEQ ID NO：4；
序列4可变结构域的核苷酸序列为SEQ ID NO：27；
(5)序列5可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：51、SEQ ID NO：74和SEQ ID NO：97；
序列5可变结构域的氨基酸序列为SEQ ID NO：5；
序列5可变结构域的核苷酸序列为SEQ ID NO：28；
(6)序列6可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为： SEQ ID NO：52、SEQ ID NO：75和SEQ ID NO：98；
序列6可变结构域的氨基酸序列为SEQ ID NO：6；
序列6可变结构域的核苷酸序列为SEQ ID NO：29；
(7)序列7可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：53、SEQ ID NO：76和SEQ ID NO：99；
序列7可变结构域的氨基酸序列为SEQ ID NO：7；
序列7可变结构域的核苷酸序列为SEQ ID NO：30；
(8)序列8可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：54、SEQ ID NO：77和SEQ ID NO：100；
序列8可变结构域的氨基酸序列为SEQ ID NO：8；
序列8可变结构域的核苷酸序列为SEQ ID NO：31；
(9)序列9可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：55、SEQ ID NO：78和SEQ ID NO：101；
序列9可变结构域的氨基酸序列为SEQ ID NO：9；
序列9可变结构域的核苷酸序列为SEQ ID NO：32；
(10)序列10可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：56、SEQ ID NO：79和SEQ ID NO：102；
序列10可变结构域的氨基酸序列为SEQ ID NO：10；
序列10可变结构域的核苷酸序列为SEQ ID NO：33；
(11)序列11可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：57、SEQ ID NO：80和SEQ ID NO：103；
序列11可变结构域的氨基酸序列为SEQ ID NO：11；
序列11可变结构域的核苷酸序列为SEQ ID NO：34；
(12)序列12可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：58、SEQ ID NO：81和SEQ ID NO：104；
序列12可变结构域的氨基酸序列为SEQ ID NO：12；
序列12可变结构域的核苷酸序列为SEQ ID NO：35；
(13)序列13可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：59、SEQ ID NO：82和SEQ ID NO：105；
序列13可变结构域的氨基酸序列为SEQ ID NO：13；
序列13可变结构域的核苷酸序列为SEQ ID NO：36；
(14)序列14可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：60、SEQ ID NO：83和SEQ ID NO：106；
序列14可变结构域的氨基酸序列为SEQ ID NO：14；
序列14可变结构域的核苷酸序列为SEQ ID NO：37；
(15)序列15可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：61、SEQ ID NO：84和SEQ ID NO：107；
序列15可变结构域的氨基酸序列为SEQ ID NO：15；
序列15可变结构域的核苷酸序列为SEQ ID NO：38；
(16)序列16可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：62、SEQ ID NO：85和SEQ ID NO：108；
序列16可变结构域的氨基酸序列为SEQ ID NO：16；
序列16可变结构域的核苷酸序列为SEQ ID NO：39；
(17)序列17可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：63、SEQ ID NO：86和SEQ ID NO：109；
序列17可变结构域的氨基酸序列为SEQ ID NO：17；
序列17可变结构域的核苷酸序列为SEQ ID NO：40；
(18)序列18可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：64、SEQ ID NO：87和SEQ ID NO：110；
序列18可变结构域的氨基酸序列为SEQ ID NO：18；
序列18可变结构域的核苷酸序列为SEQ ID NO：41；
(19)序列19可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：65、SEQ ID NO：88和SEQ ID NO：111；
序列19可变结构域的氨基酸序列为SEQ ID NO：19；
序列19可变结构域的核苷酸序列为SEQ ID NO：42；
(20)序列20可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：66、SEQ ID NO：89和SEQ ID NO：112；
序列20可变结构域的氨基酸序列为SEQ ID NO：20；
序列20可变结构域的核苷酸序列为SEQ ID NO：43；
(21)序列21可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为： SEQ ID NO：67、SEQ ID NO：90和SEQ ID NO：113；
序列21可变结构域的氨基酸序列为SEQ ID NO：21；
序列21可变结构域的核苷酸序列为SEQ ID NO：44；
(22)序列22可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：68、SEQ ID NO：91和SEQ ID NO：114；
序列22可变结构域的氨基酸序列为SEQ ID NO：22；
序列22可变结构域的核苷酸序列为SEQ ID NO：45；
(23)序列23可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：69、SEQ ID NO：92和SEQ ID NO：115；
序列23可变结构域的氨基酸序列为SEQ ID NO：23；
序列23可变结构域的核苷酸序列为SEQ ID NO：46。
实施例5 嵌合抗体的构建与表达
将噬菌体文库筛选得到的序列进行抗体基因测序，将测序得到的抗体片段进行基因合成，构建到人IgG框架中，而后利用分子克隆技术，将抗体片段***PCDNA3.1载体中，构建成哺乳动物细胞表达质粒，利用脂质体转染方式，导入宿主细胞株CHO细胞，利用细胞fed-batch获得发酵上清液，取发酵液上清进行亲和层析、离子交换层析等一系列步骤的纯化，最终纯化得到构建的嵌合抗体：抗体1、抗体2、抗体3、抗体4、抗体5、抗体6、抗体7、抗体8、抗体9、抗体10、抗体11、抗体12、抗体13、抗体14、抗体15、抗体16、抗体17、抗体18、抗体19、抗体20、抗体21、抗体22和抗体23。抗体1至抗体23的CDR和可变结构域的氨基酸序列分别对应实施例4中序列1至序列23的CDR和可变结构域的氨基酸序列。
抗体1至抗体23的恒定区的氨基酸序列都相同，如SEQ ID NO：116所示。
实施例6 抗NKp30抗体与NKp30亲和力验证
设备：OCTET Red96e(Fortebio)。
传感器：AHC。
(1)实验设置：
传感器准备：在使用前以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作 为缓冲液浸润AHC传感器600s，除去传感器表面覆盖的蔗糖。
根据样品的实际加样位置设置样品板及传感器位置。
设置需要进行的步骤，并设置时间和转速，实验温度设置为30℃，震荡速度为1000rpm。
(2)固化及捕获：
AHC传感器以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液平衡60s，固化样品板中的NKp30抗体300s，二次平衡缓冲液180s。100nM的人NKp30-his蛋白(KACTUS；Cat：NKp-HM430)与NKp30抗体结合300s，然后解离600s。解离后以10mM甘氨酸(pH2.0)作为再生缓冲液，再生30s。
(3)再生：
用10mM甘氨酸(pH2.0)再生传感器。
(4)数据分析：
从测试结果图中减去参考通道H1的结果图。实验数据符合1：1结合模型。用分子量35kDa计算人NKp30蛋白的摩尔浓度。结果见表1。
由表1可知，除了抗体5的K D为1.04×10 -7M，抗体21的K D为2.01×10 -8M外，其他抗体的K D值都为纳摩尔浓度级甚至更小，说明NKp30单域抗体与NKp30亲和力高。
表1
Figure PCTCN2022077787-appb-000001
Figure PCTCN2022077787-appb-000002
实施例7 抗体与细胞结合活性检测
取待测抗体按照40μg/mL为初始终浓度，3倍稀释，稀释8个梯度。取出培养箱内NK细胞，将细胞悬液转至15mL离心管中，离心，PBS重悬计数。留出空白对照组(Blank)、阴性对照组(NC)、实验组和无关抗体组。按照约3×10 5个细胞/孔，将细胞悬液铺于96孔板中。离心(1000rpm，5min)后用PBS清洗，再离心，重复两次去除培养基残留。弃去上清，实验组、无关抗体组分别加入100μL的一抗溶液、无关抗体溶液，重悬细胞后，室温孵育1h。Blank、NC组使用等量的PBS进行孵育。1h后离心，加入PBS清洗两次。弃上清后，除Blank组加入100μLPBS外，其余样品组分别加入100μL荧光二抗稀释液(羊抗人Fc-FITC Abcam cat：ab97224)，室温避光孵育0.5h后，离心加入PBS清洗两次。弃去上清后，加入120μL的PBS缓冲液重悬并按顺序进行流式细胞检测，测其平均荧光强度，结果见图1a和图1b。
分析结果表明，抗NKp30抗体的结合活性都比较好。
实施例8 抗体刺激NK细胞激活释放细胞因子实验
取出96孔板，将待测抗体，阳性对照抗体(选自Biolegend公司的抗CD337(NKp30)抗体)和同型对照抗体按照150nM起始，3倍稀释，进行7个梯度稀释，设置2个平行孔，用PBS缓冲液溶解，置于96孔板中。放于4度冰箱孵育过夜约16h后取出进行后续操作。取出96孔板，弃抗体孵育液，用PBS清洗2次。取出NK细胞并进行细胞计数，设置细胞数为4×10 4个/孔用含终浓度为400U的IL-2(STEMCELL，78036)的培养基进行重悬，200μL/孔加入抗体孵育完成的96孔板中，设置空白对照孔和阴性对照孔。处理好的96孔板放置在37℃CO 2恒温培养箱孵育约24h后，提取上清，将离心后的上清用试剂盒(Biolegend Cat：430104)测其最大IFN-γ分泌量。结果见图2a-2c。
分析结果表明，抗NKp30抗体刺激的NK细胞释放细胞因子的效果显著优于对照抗体和同型抗体。
实施例9 抗体的人源化
使用IgBLAST工具将实施例5所述得到的驼源序列与人Germline序列比对，结果显示抗体10可变区框架区1-3含15个驼源位点(VHH基因)，抗体18可变区框架区1-3含14个驼源位点(VHH基因)。设计模板选取IGHV3大类，进行人源化序列的设计，将序列突变成人源化序列。由抗体10得到3个CDR都相同的5条人源化抗体：抗体10-hu1、抗体10-hu2、抗体10-hu3、抗体10-hu4和抗体10-hu5，可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：56、SEQ ID NO：79和SEQ ID NO：102。由抗体18得到3个CDR都相同的4条人源化抗体：抗体18-hu1、抗体18-hu2、抗体18-hu3和抗体18-hu4，可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别为：SEQ ID NO：64、SEQ ID NO：87和SEQ ID NO：110。
人源化抗体的可变结构域的氨基酸序列如表2所示。
表2
人源化抗体 可变区氨基酸序列
抗体10-hu1 SEQ ID NO：117
抗体10-hu2 SEQ ID NO：118
抗体10-hu3 SEQ ID NO：119
抗体10-hu4 SEQ ID NO：120
抗体10-hu5 SEQ ID NO：121
抗体18-hu1 SEQ ID NO：122
抗体18-hu2 SEQ ID NO：123
抗体18-hu3 SEQ ID NO：124
抗体18-hu4 SEQ ID NO：125
表2中的9条人源化抗体的恒定区相同，其恒定区的氨基酸序列为SEQ ID NO：116。
实施例10 抗体的人源化和CDR改造
对实施例5中抗体23的CDR2进行翻译后修饰改造，即CDR2的氨基酸序列由GITGNGLTDYA DSVKG改造为GITGNGLTDYA ESVKG，得到1条嵌合抗体：抗体23-p。
按照实施例9的方法，对实施例5中的抗体23进行人源化，并且对CDR进行翻译后修饰的改造，得到13条人源化抗体。
得到的14条抗体序列如表3所示。
表3
抗体名称 可变结构域 CDR1 CDR2 CDR3
抗体23-p SEQ ID NO：126 SEQ ID NO：69 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu41 SEQ ID NO：127 SEQ ID NO：69 SEQ ID NO：92 SEQ ID NO：115
抗体23-hu42 SEQ ID NO：128 SEQ ID NO：69 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu43 SEQ ID NO：129 SEQ ID NO：69 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu44 SEQ ID NO：130 SEQ ID NO：69 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu45 SEQ ID NO：131 SEQ ID NO：69 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu46 SEQ ID NO：132 SEQ ID NO：141 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu47 SEQ ID NO：133 SEQ ID NO：141 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu48 SEQ ID NO：134 SEQ ID NO：141 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu49 SEQ ID NO：135 SEQ ID NO：141 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu50 SEQ ID NO：136 SEQ ID NO：142 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu51 SEQ ID NO：137 SEQ ID NO：142 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu52 SEQ ID NO：138 SEQ ID NO：142 SEQ ID NO：140 SEQ ID NO：115
抗体23-hu53 SEQ ID NO：139 SEQ ID NO：142 SEQ ID NO：140 SEQ ID NO：115
表3中的14条抗体的恒定区相同，其恒定区的氨基酸序列为SEQ ID NO：116。
实施例11 人源化抗体与细胞结合能力检测
用包被液(1×PBS，pH7.4)将人-NKp30-His(购自KACTUS，货号NKP-HM430)稀释为0.2μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤4次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h，倒掉封闭液，洗板机洗涤4次，平板纸上拍干。参比品和供试品用3％脱脂奶粉稀释为10μg/mL，以此为初始浓度进行3倍稀释，共稀释11个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤4次，平板纸上拍干。用3％脱脂奶粉将羊抗人Fc按1：20000稀释，100μL/孔，37℃孵育1h。洗板机洗涤6次，平板纸上拍干。加入TMB显色液，100μL/孔，并用铝箔纸包好，37℃避光显色8分钟。加入终止液1M HCl终止显色反应，100μL/孔。在酶标仪上450nm处读数。结果见图3a-3h。
由图3a-3b可知，人源化的抗体10-hu1、抗体10-hu2、抗体10-hu3、抗体10-hu4和抗体10-hu5与细胞结合能力优于或相当于嵌合抗体10。
由图3c-3d可知，人源化的抗体18-hu1、抗体18-hu2、抗体18-hu3和抗体18-hu4与细胞结合能力与嵌合抗体18相当。
由图3e-3h可知，抗体23-p、抗体23-hu44、抗体23-hu45和抗体23-hu49与细胞结合能力显著优于或相当于嵌合抗体23。
实施例12 人源化抗体的亲和力测定
根据实施例6的方法，对CDR修饰的抗体和人源化抗体进行亲和力测定，结果如表4所示。
表4
抗体名称 亲和力K D(M)
抗体23 8.44×10 -10
抗体23-p 1.27×10 -9
抗体23-hu43 3.77×10 -10
抗体23-hu44 1.30×10 -9
抗体23-hu45 9.91×10 -10
抗体23-hu48 5.10×10 -9
抗体23-hu52 5.93×10 -9
由表4可知，抗体23、抗体23-p、抗体23-hu43、抗体23-hu44、抗体23-hu45、抗体23-hu48和抗体23-hu52的K D值都为纳摩尔浓度级甚至更小，说明NKp30单域抗体与NKp30亲和力高。
实施例13 人源化抗体刺激NK细胞激活释放细胞因子实验
按照实施例8的方法，对人源化抗体18-hu3进行刺激NK细胞激活释放细胞因子实验，实验结果如图4所示。
实验结果显示，经过抗体18-hu3刺激的NK细胞，能够分泌IFN-γ，EC 50值为8.834nM，而各个浓度的同型对照刺激NK细胞均未产生IFN-γ，说明抗体18-hu3能够特异性激活NK细胞。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (27)

  1. 一种抗NKp30单域抗体，其特征在于，其能够激活NK细胞或γδT细胞释放细胞因子。
  2. 根据权利要求1所述的抗NKp30单域抗体，其特征在于，所述细胞因子为淋巴因子，优选为IL2、IL3、IL4、IL5、IL6、IL9、IL10、IFN-γ或TNF-α，更优选为IFN-γ、TNF-α或IL2。
  3. 根据权利要求1或2所述的抗NKp30单域抗体，其包含免疫球蛋白单一可变结构域，其特征在于，其免疫球蛋白单一可变结构域包含互补决定区CDR1、CDR2和CDR3，其中，
    (a)CDR1，选自SEQ ID NO：47-69，141，142的任一氨基酸序列，或与SEQ ID NO：47-69，141，142的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：47-69，141，142的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、***或缺失)的氨基酸序列；
    (b)CDR2，选自SEQ ID NO：70-92，140的任一氨基酸序列，或与SEQ ID NO：70-92，140的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：70-92，140的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、***或缺失)的氨基酸序列；
    (c)CDR3，选自SEQ ID NO：93-115的任一氨基酸序列，或与SEQ ID NO：93-115的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：93-115的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、***或缺失)的氨基酸序列。
  4. 根据权利要求3所述的抗NKp30单域抗体，其中所述单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
    (1)SEQ ID NO：47所示的CDR1，SEQ ID NO：70所示的CDR2，SEQ ID NO：93所示的CDR3；
    (2)SEQ ID NO：48所示的CDR1，SEQ ID NO：71所示的CDR2，SEQ ID NO：94所示的CDR3；
    (3)SEQ ID NO：49所示的CDR1，SEQ ID NO：72所示的CDR2，SEQ ID  NO：95所示的CDR3；
    (4)SEQ ID NO：50所示的CDR1，SEQ ID NO：73所示的CDR2，SEQ ID NO：96所示的CDR3；
    (5)SEQ ID NO：51所示的CDR1，SEQ ID NO：74所示的CDR2，SEQ ID NO：97所示的CDR3；
    (6)SEQ ID NO：52所示的CDR1，SEQ ID NO：75所示的CDR2，SEQ ID NO：98所示的CDR3；
    (7)SEQ ID NO：53所示的CDR1，SEQ ID NO：76所示的CDR2，SEQ ID NO：99所示的CDR3；
    (8)SEQ ID NO：54所示的CDR1，SEQ ID NO：77所示的CDR2，SEQ ID NO：100所示的CDR3；
    (9)SEQ ID NO：55所示的CDR1，SEQ ID NO：78所示的CDR2，SEQ ID NO：101所示的CDR3；
    (10)SEQ ID NO：56所示的CDR1，SEQ ID NO：79所示的CDR2，SEQ ID NO：102所示的CDR3；
    (11)SEQ ID NO：57所示的CDR1，SEQ ID NO：80所示的CDR2，SEQ ID NO：103所示的CDR3；
    (12)SEQ ID NO：58所示的CDR1，SEQ ID NO：81所示的CDR2，SEQ ID NO：104所示的CDR3；
    (13)SEQ ID NO：59所示的CDR1，SEQ ID NO：82所示的CDR2，SEQ ID NO：105所示的CDR3；
    (14)SEQ ID NO：60所示的CDR1，SEQ ID NO：83所示的CDR2，SEQ ID NO：106所示的CDR3；
    (15)SEQ ID NO：61所示的CDR1，SEQ ID NO：84所示的CDR2，SEQ ID NO：107所示的CDR3；
    (16)SEQ ID NO：62所示的CDR1，SEQ ID NO：85所示的CDR2，SEQ ID NO：108所示的CDR3；
    (17)SEQ ID NO：63所示的CDR1，SEQ ID NO：86所示的CDR2，SEQ ID NO：109所示的CDR3；
    (18)SEQ ID NO：64所示的CDR1，SEQ ID NO：87所示的CDR2，SEQ  ID NO：110所示的CDR3；
    (19)SEQ ID NO：65所示的CDR1，SEQ ID NO：88所示的CDR2，SEQ ID NO：111所示的CDR3；
    (20)SEQ ID NO：66所示的CDR1，SEQ ID NO：89所示的CDR2，SEQ ID NO：112所示的CDR3；
    (21)SEQ ID NO：67所示的CDR1，SEQ ID NO：90所示的CDR2，SEQ ID NO：113所示的CDR3；
    (22)SEQ ID NO：68所示的CDR1，SEQ ID NO：91所示的CDR2，SEQ ID NO：114所示的CDR3；
    (23)SEQ ID NO：69所示的CDR1，SEQ ID NO：92所示的CDR2，SEQ ID NO：115所示的CDR3；
    (24)SEQ ID NO：69所示的CDR1，SEQ ID NO：140所示的CDR2，SEQ ID NO：115所示的CDR3；
    (25)SEQ ID NO：141所示的CDR1，SEQ ID NO：140所示的CDR2，SEQ ID NO：115所示的CDR3；和/或
    (26)SEQ ID NO：142所示的CDR1，SEQ ID NO：140所示的CDR2，SEQ ID NO：115所示的CDR3。
  5. 根据权利要求3或4所述的抗NKp30单域抗体，其中所述免疫球蛋白单一可变结构域是VHH。
  6. 根据权利要求5所述的抗NKp30单域抗体，其中所述VHH包含与SEQ ID NO：1-23，117-139中任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列。
  7. 根据权利要求6所述的抗NKp30单域抗体，其中所述VHH选自SEQ ID NO：1-23中任一氨基酸序列。
  8. 根据权利要求6所述的抗NKp30单域抗体，其中所述VHH选自SEQ ID NO：117-139中任一氨基酸序列。
  9. 根据权利要求1-8任一所述的抗NKp30单域抗体，其中所述抗体以20.1nM或更小的K D结合NKp30。
  10. 根据权利要求1-9任一所述的抗NKp30单域抗体，其还包含免疫球蛋白Fc区，所述免疫球蛋白Fc区选自IgG1、IgG2、IgG3和/或IgG4。
  11. 根据权利要求10所述的抗NKp30单域抗体，所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO：116所示。
  12. 核酸分子，其编码权利要求1-11任一所述的抗NKp30单域抗体。
  13. 表达载体，其包含与表达调控原件可操作地连接的权利要求12的核酸分子。
  14. 重组细胞，其包含权利要求12的核酸分子或以权利要求13的表达载体转化，并能够表达所述抗NKp30单域抗体。
  15. 一种多功能融合蛋白，其包含权利要求1-11任一所述的抗NKp30单域抗体。
  16. 根据权利要求15所述的多功能融合蛋白，其还包含一个或多个与其他抗原特异性结合的第二抗体或其抗原结合部分。
  17. 根据权利要求16所述的多功能融合蛋白，其中所述结合第二抗体或其抗原结合部分的抗原选自肿瘤相关抗原(TAA)或免疫检查点。
  18. 根据权利要求17所述的多功能融合蛋白，其中所述肿瘤相关抗原(TAA)选自BCMA、CD38、HER2、PSMA、Claudin18.2、GPC3、CD19、CD20(MS4A1)、CD22、CD24、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、AFP、ALK或B7H3。
  19. 根据权利要求16所述的多功能融合蛋白，其中所述第二抗体或其抗原结合部分是NK细胞激动剂。
  20. 根据权利要求19所述的多功能融合蛋白，其中所述结合第二抗体或其抗原结合部分的抗原选自NKP30、NKP46、CD16、NKP44、CD244、CD226、NKG2E、NKG2D、NKG2C或KIR。21、根据权利要求15-20任一所述的多功能融合蛋白，其还包含细胞因子。
  21. 根据权利要求21所述的多功能融合蛋白，所述细胞因子选自IL8、IL10、IL15、IL18、TGF、VEGF、IFNγ、IFNα或GM-CSF。
  22. 权利要求1-11任一所述的抗NKp30单域抗体、权利要求15-22任一所述的多功能融合蛋白在制备用于治疗和/或预防和/或诊断疾病的药物中的用途。
  23. 根据权利要求23所述的用途，其中所述用途通过肿瘤免疫疗法、细胞疗法和基因疗法中的一种或多种来实现。
  24. 权利要求1-11任一所述的抗NKp30单域抗体、权利要求15-22任一所 述的多功能融合蛋白在制备治疗癌症的药物中的用途。
  25. 根据权利要求25所述的用途，其中所述癌症为肺癌、肝癌、黑色素瘤、恶性胶质瘤、头颈癌、结肠直肠癌、胃癌、***癌、卵巢癌、膀胱癌、胰腺癌、胃癌、结肠癌、***或相关肿瘤。
  26. 一种药物组合物，其包含权利要求1-11任一所述抗NKp30单域抗体和可接受的载体、稀释剂或赋形剂。
  27. 一种药物组合物，其包含权利要求15-22任一所述的多功能融合蛋白和可接受的载体、稀释剂或赋形剂。
PCT/CN2022/077787 2021-02-26 2022-02-25 一种抗NKp30抗体及其应用 WO2022179580A1 (zh)

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