WO2023025147A1 - 抗原表位修饰 - Google Patents

抗原表位修饰 Download PDF

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WO2023025147A1
WO2023025147A1 PCT/CN2022/114250 CN2022114250W WO2023025147A1 WO 2023025147 A1 WO2023025147 A1 WO 2023025147A1 CN 2022114250 W CN2022114250 W CN 2022114250W WO 2023025147 A1 WO2023025147 A1 WO 2023025147A1
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antigen
antibody
hil
mutant
antibodies
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PCT/CN2022/114250
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English (en)
French (fr)
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王峰
朱朝阳
徐良
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南通壹宸生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides

Definitions

  • the invention belongs to the technical field of biomedicine.
  • Antibodies are an important component of vertebrate adaptive immunity and play a crucial role in preventing bacterial and viral infections and neutralizing most foreign harmful substances (Tomita and Tsumoto, 2010).
  • Mouse hybridoma technology is one of the most common methods for producing high-affinity monoclonal antibodies (Schwaber, 1982; Zhang, 2012).
  • mice immunization and screening of downstream monoclonal antibodies have become the main methods for identifying and developing therapeutic antibodies (Brüggemann, 2001; Brüggemann et al., 2015; Taylor et al., 1992).
  • this approach is not always effective in generating functional antibodies (Kellermann and Green, 2002).
  • a therapeutic antibody must bind a specific epitope of the target antigen to perform its intended function, such as blocking ligand-receptor interactions as an antagonist, or inducing receptor-mediated downstream signaling as an agonist (Weiner et al., 2010 ).
  • Viktoriya Dubrovskaya et al. induced broadly neutralizing antibodies by modifying the glycosylated epitopes of the HIV envelope protein (Dubrovskaya et al., 2019).
  • Fabian Sesterhenn et al. fused respiratory syncytial virus (RSV) epitopes with immunogen antigens to obtain neutralizing antibodies that specifically bind RSV epitopes (Sesterhenn et al, 2020).
  • RSV respiratory syncytial virus
  • the engineered tRNA/aaRS orthogonal pair obtained through screening and selection can specifically recognize unnatural amino acids (UAA) and incorporate them into the coding site of the target in living host cells (Wang et al., 2001).
  • UAA unnatural amino acids
  • Various chemical groups on UAA side chains can endow proteins with new functions, so they have shown powerful applications in the study of protein structure and function, cell imaging, therapeutic protein conjugation, and many other fields (Chin, 2014 Year).
  • pBpa p-benzoyl-1-phenylalanine
  • Antibodies (Chen et al., 2020), suggesting that this may be the first antibody screening method available for target epitopes of all types of antigens. However, this method relies on panning of phage libraries in vitro and affinity maturation of the screened antibodies.
  • none of the current methods can selectively elicit antibody responses to antigen-specific conformational epitopes in vivo.
  • the present invention has developed a A new method based on chemical cross-linking groups to modify the target epitope of the antigen to change its immunogenicity, thereby changing the antibody spectrum elicited by the antigen after immunization of animals, so as to enrich the antibodies against the target antigen.
  • the present invention further provides a screening and preparation method for enriching the immune response of the animal against the target epitope of the antigen and the antibody against the target epitope of the antigen.
  • Antibodies that bind to specific epitopes of antigens can be easily and quickly elicited and identified by the present invention: the chemical cross-linking active group (such as N ⁇ -acryloyl or N ⁇ -crotonyl) in a certain way (such as through the target epitope).
  • the chemical cross-linking active group such as N ⁇ -acryloyl or N ⁇ -crotonyl
  • target antigens such as IL-1 ⁇
  • Epitopes such as E64
  • this effect is so prominent that almost all clones randomly selected from the IL-1 ⁇ E64AK and IL-1 ⁇ E64CK immunized phage libraries were able to bind the target epitope.
  • clones obtained by immunization with the wild-type antigen WT IL-1 ⁇ and the IL-1 ⁇ mutant incorporating an unnatural amino acid without cross-linking activity rarely bind the target epitope.
  • the difference in cross-linking activity between IL-1 ⁇ E64AK and IL-1 ⁇ E64CK resulted in different differences in the final screened clone sequences after immunizing mice, suggesting that it can be adjusted by adjusting the reactivity and structure of chemical cross-linking groups. Antibody affinity and sequence diversity.
  • the present invention further found that the directional antibody response of AK or CK incorporating epitopes is not limited by sequence: immunizing mice after incorporating CK into other sites of IL-1 ⁇ can effectively produce high-titer antibodies against specific epitopes.
  • immunizing mice after incorporating CK into other sites of IL-1 ⁇ can effectively produce high-titer antibodies against specific epitopes.
  • CK-induced epitope-specific antibodies can effectively block IL-1 ⁇ from activating its receptor. Therefore, this epitope-directed antibody response can be used in vaccine development to enhance effective antibody responses to functional epitopes.
  • a chemical cross-linking active group such as N ⁇ -crotonyl
  • One aspect of the present invention provides a reagent for immunizing animals, said reagent comprising:
  • a mutant antigen which is an antigen incorporating a group with chemical cross-linking activity or a derivative thereof on one or more target epitopes in the wild-type antigen
  • the agent stimulates or enhances the production of antibodies against the one or more target epitopes in the animal after being administered to the animal.
  • the group with chemical crosslinking activity is N ⁇ -crotonyl or N ⁇ -acryloyl.
  • the group with chemical crosslinking activity is a natural amino acid or an unnatural amino acid.
  • the unnatural amino acid with a chemically active group is N ⁇ -crotonyl-L-lysine (CK) or N ⁇ -acryloyl-L-lysine (AK).
  • the group with chemical crosslinking activity or derivative thereof is incorporated by extending gene codons or by chemical synthesis.
  • the animal is a rodent, a non-human mammal, or a mammal.
  • the wild-type antigen is a soluble protein, a soluble polypeptide, a transmembrane protein expressed on a phospholipid membrane structure, or a polypeptide expressed on a phospholipid membrane structure.
  • the reagent is a reagent for antibody preparation.
  • the agent is a prophylactic or therapeutic agent.
  • the agent is a vaccine composition.
  • the reagent comprises an immunologically effective amount of an IL-1 ⁇ mutant antigen; in some other embodiments, the E64 position of the IL-1 ⁇ mutant antigen incorporates AK; in some other embodiments, The E64 position of the IL-1 ⁇ mutant antigen incorporates CK.
  • the reagent comprises an immunologically effective amount of the new coronavirus S protein RBD region mutant antigen; in some embodiments, the K417 epitope, L452 epitope, L452 epitope, In some embodiments, the K417 epitope, L452 epitope, Y453 epitope, E484 epitope or N501 epitope of the new coronavirus S protein RBD region mutant antigen
  • the epitope is spiked with CK.
  • the reagent comprises an immunologically effective amount of PTN-CK; in some embodiments, the reagent comprises an effective amount of PTN-CK coupled to a carrier protein; in some embodiments, the carrier The protein is hemocyanin KLH; in some embodiments, the reagent comprises an effective amount of KLH-PTN-CK.
  • Another aspect of the present invention provides a reagent for enhancing the immune response of animals, said reagent comprising:
  • mutant antigen which is an antigen incorporating a chemical cross-linking active group or a derivative thereof on one or more target epitopes in the wild-type antigen
  • Another aspect of the present invention provides a method for regulating the immunogenicity of an antigen, the method comprising incorporating a group with chemical cross-linking activity or a derivative thereof on one or more target epitopes of the antigen.
  • the modulation results in a change in the profile of antigen-elicited antibodies.
  • Another aspect of the present invention provides a method for increasing an animal's immune response against a target epitope of an antigen, the method comprising: administering a certain amount of a mutant antigen to the animal, and the mutant antigen is a wild-type Antigens with chemically cross-linked active groups or their derivatives are incorporated on the target epitope of the antigen.
  • Another aspect of the present invention provides a method of increasing an animal's immune response against an antigen target epitope, the method comprising: administering to the animal a certain amount of a mutant antigen, the mutant antigen being the target epitope of the wild-type antigen
  • the epitope is an antigen formed by incorporating a chemical cross-linking active group or its derivatives.
  • Another aspect of the present invention provides the use of the method for increasing the animal immune response to the antigen target epitope in the preparation of a vaccine for preventing or treating diseases.
  • Another aspect of the present invention provides an application of a mutant antigen in the preparation of a vaccine, the mutant antigen is formed by incorporating a chemical cross-linking active group or a derivative thereof on the target epitope of the wild-type antigen antigen.
  • Another aspect of the present invention provides a method for screening antibodies against a target epitope of a wild-type antigen, the method comprising the steps of: (a) providing a mutant antigen, the mutant antigen being on the target epitope of the wild-type antigen (b) administering the mutant antigen described in step (a) to the animal; (c) isolating serum from the animal; (d) using the Antibodies that specifically bind to the target epitope were screened using the wild-type antigen.
  • Another aspect of the present invention provides a method for screening antibodies against a target epitope of a wild-type antigen, the method comprising the steps of: (a) providing a mutant antigen, the mutant antigen being on the target epitope of the wild-type antigen (b) administering the mutant antigen described in step (a) to the animal; (c) isolating B cells from the animal, and using the B cell The cells are fused with myeloma cells to generate hybridoma cells; (d) using the wild-type antigen to screen the culture supernatant of the hybridoma cells for antibodies that specifically bind to the target epitope.
  • Another aspect of the present invention provides a method for screening antibodies against a target epitope of a wild-type antigen, the method comprising the steps of: (a) providing a mutant antigen, the mutant antigen being on the target epitope of the wild-type antigen (b) administering the mutant antigen described in step (a) to the animal; (c) isolating B cells from the animal, using the B cell The cells construct an antibody library; (d) using the wild-type antigen to screen an antibody that specifically binds to the target epitope from the antibody library.
  • Another aspect of the present invention provides a method for screening antibodies against a target epitope of a wild-type antigen, the method comprising the steps of: (a) providing a mutant antigen, the mutant antigen being on the target epitope of the wild-type antigen (b) administering the mutant antigen described in step (a) to the animal; (c) isolating serum from the animal; (d) applying the mutant antigen described in step (a) to the animal; The mutant antigen is incubated with the serum under certain conditions so that the mutant antigen and antibody are covalently cross-linked to form a mutant antigen-antibody complex; (e) eluting under certain conditions to remove the Mutant antigen covalently cross-linked antibody, and release the antibody covalently cross-linked with the mutant antigen; (f) using the wild-type antigen, further screening for specificity from the covalently cross-linked antibody with the mutant antigen Antibodies that bind to target epitopes.
  • Another aspect of the present invention provides a method for screening antibodies against a target epitope of a wild-type antigen, the method comprising the steps of: (a) providing a mutant antigen, the mutant antigen being on the target epitope of the wild-type antigen (b) administering the mutant antigen described in step (a) to the animal; (c) isolating B cells from the animal, and using the B cell Fusing the cells with myeloma cells to produce hybridoma cells; (d) incubating the mutant antigen with the culture supernatant of the hybridoma cells under certain conditions so that the mutant antigen is covalently cross-linked with the antibody Forming a mutant antigen-antibody complex; (e) removing antibodies not covalently cross-linked with the mutant antigen by eluting under certain conditions, and releasing antibodies covalently cross-linked with the mutant antigen; (f ) using the wild-type antigen to further screen the antibody specifically binding to the target epitope from the antibody covalently cross-linked with
  • Another aspect of the present invention provides a method for screening antibodies against a target epitope of a wild-type antigen, the method comprising the steps of: (a) providing a mutant antigen, the mutant antigen being on the target epitope of the wild-type antigen (b) administering the mutant antigen described in step (a) to the animal; (c) isolating B cells from the animal, using the B cell The cells construct an antibody library; (d) incubating the mutant antigen with the antibody library under certain conditions, so that the mutant antigen and the antibody are covalently cross-linked to form a mutant antigen-antibody complex; (e) Eluting under certain conditions removes antibodies that are not covalently cross-linked with the mutant antigen, and releases antibodies that are covalently cross-linked with the mutant antigen; (f) using the wild-type antigen, from the mutant antigen Antibodies covalently cross-linked to the antigen are further screened for antibodies that specifically bind to the target epitope.
  • the chemically active group or derivative thereof is incorporated by extension of genetic codons or chemical synthesis.
  • the group with chemical crosslinking activity is N ⁇ -crotonyl or N ⁇ -acryloyl.
  • the group with chemical crosslinking activity is a natural amino acid or an unnatural amino acid.
  • the unnatural amino acid with a chemical cross-linking active group is N ⁇ -crotonyl-L-lysine (CK) or N ⁇ -acryloyl-L-lysine (AK).
  • the animal is a rodent, a non-human mammal, or a mammal.
  • the rodent is a mouse or a rat; in a specific embodiment, the non-human mammal is a rabbit, an alpaca or a sheep, etc.; in a specific embodiment, the Mammals are humans.
  • the antigen is a soluble protein, a soluble polypeptide, a transmembrane protein expressed on a phospholipid membrane structure, or a polypeptide expressed on a phospholipid membrane structure.
  • the conditions for incubating the mutant antigens with the serum are alkaline conditions. In some embodiments, the conditions for incubating the mutant antigens with the hybridoma cells are alkaline conditions. In some embodiments, the conditions for incubating the mutant antigens with the antibody library are alkaline conditions.
  • the alkaline condition is a solution of pH8.8; in some embodiments, the alkaline condition is a DPBS solution of pH8.8; in some embodiments, the incubation condition is incubation for 24h , 48h, 3d, 4d, 5d, 6d or 7d; In some embodiments, the incubation condition is in the DPBS solution of pH8.8, incubate 24h, 48h, 3d, 4d, 5d, 6d or 7d; In some implementations In the scheme, the incubation condition is incubation in a DPBS solution with pH 8.8 for 24 hours; in some embodiments, the incubation condition is incubation in a DPBS solution with pH 8.8 for 48 hours.
  • the elution conditions for removing antibodies that are not covalently cross-linked to the mutant antigen are: i) alkaline elution with a high pH elution buffer; ii) low pH elution buffer for acidic elution.
  • the releasing of the antibody covalently cross-linked to the antigen is by enzymatic digestion.
  • Another aspect of the present invention provides the use of the method for screening antibodies against target epitopes of wild-type antigens in preparing vaccines for preventing or treating diseases.
  • Another aspect of the present invention provides a method for preparing an antibody, comprising obtaining an antibody by the method for screening antibodies against a target epitope of a wild-type antigen as provided herein; and providing the antibody thus obtained.
  • the antibody obtained by screening against the target epitope of the wild-type antigen specifically binds to IL-1 ⁇ .
  • the antibody specifically binding to IL-1 ⁇ has: the VH shown in SEQ ID NO.6 and the VL shown in SEQ ID NO.4; the VH shown in SEQ ID NO.10 and VL shown in SEQ ID NO.8; VH shown in SEQ ID NO.14 and VL shown in SEQ ID NO.12; VH shown in SEQ ID NO.18 and SEQ ID NO.16 VL; VL shown in VH shown in SEQ ID NO.22 and SEQ ID NO.20; VL shown in VH shown in SEQ ID NO.26 and SEQ ID NO.24; shown in SEQ ID NO.30 The VH shown and the VL shown in SEQ ID NO.28.
  • IL-1-mediated diseases include adult sti Dyer's disease, systemic juvenile idiopathic arthritis, osteoarthritis, rheumatoid arthritis, gouty arthritis, acute gout, multisystem inflammatory disease of the newborn, Behcet's Disease, cryopyrin Associated periodic syndrome, Familial Mediterranean fever, Hereditary periodic fever, Periodic fever syndrome, TNFR-associated periodic fever syndrome, Atherosclerosis, Atrial fibrillation, Acute myocardial infarction, Peripheral arterial disease, Chronic idiopathic Urticaria, abdominal aortic aneurysm, colorectal cancer, triple negative breast cancer, non-small cell lung cancer, type 1 diabetes, type 2 diabetes, mevalonate kinase deficiency, Schnitzler Syndrome; Urticaria and macroglobulinemia, sickle cell anemia,
  • the mutant antigen is the new coronavirus S protein RBD region incorporating AK on the K417 epitope, Y453 epitope, E484 epitope or N501 epitope; in some embodiments, the mutant The antigen is the RBD region of the new coronavirus S protein that incorporates CK on the K417 epitope, L452 epitope, Y453 epitope, E484 epitope or N501 epitope.
  • Figure 1 is the serum antibody titer of immunized mice
  • Figure 2A is the WB detection results of CL-E2 phage and WT IL1 ⁇ , hIL-1 ⁇ E64AK
  • Figure 2B and C are the WB detection results of CL-E2-mFc fusion protein and WT IL1 ⁇ or its mutants
  • Figure 2D is the results of canakinumab and hIL-1 ⁇ E64AK, WB detection results of hIL-1 ⁇ E64CK under chemical cross-linking conditions.
  • Figure 3A is the ELISA results of CL-E2-mFc and hIL-1 ⁇ , hIL-1 ⁇ E64AK, where the K d of CL-E2-mFc binding to hIL-1 ⁇ and hIL-1 ⁇ E64AK are 3.7 ⁇ 0.2nM and 4.2 ⁇ 0.2nM respectively;
  • 3B is the ELISA detection result of CL-E2 phage binding to hIL-1 ⁇ and hIL-1 ⁇ E64AK, where the abscissa is the pfu value of the phage.
  • Figure 4A is the ELISA result of the combination of panned phage clones with WT hIL-1 ⁇ and hIL-1 ⁇ E64AK
  • Figure 4B is the ELISA result of the combination of E64AK-A9-mFc antibody fusion protein with WT hIL-1 ⁇ and hIL-1 ⁇ E64AK (Kd value 1.6 ⁇ 0.2nM and 1.2 ⁇ 0.2nM respectively)
  • Figure 4C and D are the WB detection results of E64AK-A9-mFc antibody fusion protein and hIL-1 ⁇ E64AK, wherein the antibody used for 4C detection is mouse anti-His-tag antibody , the antibody used for 4D detection is anti-mouse Fc.
  • Figure 5 shows the results of ELISA detection of different phage clones and hIL-1 ⁇ E64AK, hIL-1 ⁇ 63-66A, where * means p ⁇ 0.01, ** means p ⁇ 0.01, *** means p ⁇ 0.001, **** means p ⁇ 0.0001 .
  • Figure 6A is the ELISA detection results of Gevokizumab and hIL-1 ⁇ , hIL-1 ⁇ 63-66A, and Figure 6B is the ELISA detection results of E64AK-F4-mFc and hIL-1 ⁇ or its mutants; Figure 6C and D are E64AK-F4 phage and The ELISA results of the binding of E64AK-F4-mFc to hIL-1 ⁇ competitively inhibited by canakinumab.
  • Figure 7A is the ELISA detection results of different phage clones and hIL-1 ⁇ , hIL-1 ⁇ 64CK
  • Figure 7B and 7C are the ELISA detection results of different phage clones and hIL-1 ⁇ E64CK, hIL-1 ⁇ 63-66A
  • Figure 7D is the ELISA detection results of E64CK-H11 and The ELISA detection results of the binding inhibition of hIL-1 ⁇ by E64AK-A9-mFc competition
  • Figure 7E is the ELISA detection results of the binding inhibition of E64AK-G6 and E64AK-A2 and hIL-1 ⁇ competition inhibition by E64CK-B9-mFc, where, * means p ⁇ 0.01, ** means p ⁇ 0.01, *** means p ⁇ 0.001, **** means p ⁇ 0.0001.
  • Figures 8A and 8B are the ELISA detection results of antibody fusion proteins E64CK-A5-mFc, E64CK-G9-mFc and hIL-1 ⁇ or its mutant proteins;
  • Figure 8C and D are antibody fusion proteins E64CK-A5-mFc and E64CK, respectively - The ELISA results of the binding of G9-mFc and hIL-1 ⁇ being inhibited by canakinumab;
  • Figure 8E and 8F are the ELISA results of the binding of phage clones E64CK-A59 and E64CK-G9 to hIL-1 ⁇ being inhibited by canakinumab;
  • Figure 8G is The WB detection results of antibody fusion proteins E64CK-A5-mFc, E64CK-G9-mFc and hIL-1 ⁇ or its mutant proteins incubated at pH 8.8, 37°C for 48 hours;
  • Figure 8H shows the antibody fusion proteins E64CK-A5-mFc, E64CK
  • Figure 9 shows the results of the hIL-1 ⁇ or hIL-1 ⁇ 63-66A ELISA detection of the clones screened from the phage library constructed by hIL-1 ⁇ immunized mice.
  • Figure 10 shows the ELISA results of the clones selected from the phage library screened after immunizing mice with hIL-1 ⁇ E64BK binding to hIL-1 ⁇ E64BK and hIL-1 ⁇ 63-66A, where **** indicates p ⁇ 0.0001.
  • Figure 11A is the result of SDS-PAGE
  • Figure 11B and Figure 11C are the ELISA detection results of the combination of canakinumab and gevokizumab with WT hIL-1 ⁇ and hIL-1 ⁇ Q15G, respectively.
  • Figure 12 is the ELISA result of the clones screened from the phage library constructed after hIL-1 ⁇ Q15CK immunized mice with hIL-1 ⁇ or its mutants, where ** means p ⁇ 0.01, *** means p ⁇ 0.001, **** indicates p ⁇ 0.0001
  • Figure 13 shows the difference in the affinity of Q15CK-G8-mFc binding to hIL-1 ⁇ and its mutants detected by ELISA.
  • Fig. 14A is the serum titer of mice immunized with hIL-1 ⁇ and its mutants (incorporating CK or pNO2F at different sites); 14B is the result of serum IgG neutralization experiment from hIL-1 ⁇ and its mutants immunized mice; 14C HEK-Blue IL-1R inhibition results for serum IgG from hIL-1 ⁇ Q15CK immunized mice.
  • Figure 15 is the titer of different immune sera against different proteins, wherein 15A is the titer of two groups of (PTN-WT, PTN-CK) immunized mouse serum against respective immune antigens; 15B is the titer of two groups of (KLH-PTN-WT, PTN-CK) KLH-PTN-CK) immune serum titers to their respective immune antigens; 15C is the titer of two groups (KLH-PTN-WT, KLH-PTN-CK) immune serum to KLH protein; 15D is the titer of two groups (KLH-PTN -WT, HLH-PTN-CK) immune serum to the titers of respective immune polypeptides (respectively being PTN-WT, PTN-CK); 15E is KLH-PTN-CK immune serum to PTN-WT, and KLH-PTN-WT The titer of immune serum to PTN-CK; the
  • Figure 16 is the analysis of different subtypes of IgG against KLH (16A) and PTN polypeptide (16B) in the immune sera of two groups (KLH-PTN-WT, KLH-PTN-CK).
  • chemically cross-linking active group refers to a chemical group that can covalently cross-link with amino acid residues adjacent to a protein under suitable conditions.
  • Chemical cross-linking active groups may include natural amino acids, derivatives of natural amino acids, and non-natural amino acids.
  • Non-limiting examples of “chemically cross-linking reactive groups” include N ⁇ -crotonyl, N ⁇ -acryloyl or p-acrylamide groups.
  • Non-limiting examples of "non-natural amino acids with chemical cross-linking active groups” include N ⁇ -crotonyl-L-lysine (N ⁇ -crotonyl-L-lysine, CK), N ⁇ -acryloyl-L-lysine acid (N ⁇ -acryloyl-L-lysine, AK), p-acrylamido-(S)-phenylalanine (p-acrylamido-(S)-phenylalanine), natural amino acids with or with nucleophilic groups ( For example, lysine with ⁇ -amino group) and the like.
  • chemical cross-linking means that a group having chemical cross-linking activity can covalently cross-link with a group of amino acid residues adjacent to a protein under suitable conditions to form a complex.
  • unnatural amino acid refers to an amino acid that is not one of the 20 classical amino acids or selenocysteine or pyrrolysine.
  • Other terms that may be used synonymously with the term “unnatural amino acid” are “non-naturally encoded amino acid", “unnatural amino acid”, “non-naturally occurring amino acid”.
  • the term “unnatural amino acid” also includes, but is not limited to, amino acids that have been modified (e.g., post-translationally) by naturally encoded amino acids, including but not limited to the 20 common amino acids or pyrrolysine and selenocysteine occur, but are not themselves naturally incorporated into the growing polypeptide chain by the translation complex.
  • "Unnatural amino acids” can include various functional groups or reactive groups, which can provide additional functions and/or activities.
  • mutant antigen refers to an antigen formed by incorporating a chemical cross-linking active group or a derivative thereof into the target epitope of the wild-type antigen.
  • wild-type antigen includes not only soluble proteins and soluble polypeptides, but also transmembrane proteins or polypeptides expressed on phospholipid membrane structures; "wild-type antigens" can be derived from animals, plants or microorganisms (such as bacteria, fungi, viruses) .
  • the term "vaccine” refers to an antigen that induces an organism to produce antibodies against an epitope of interest. Antigens that enhance an organism's immune response to an antigenic target epitope are also included in the present invention.
  • a non-limiting example of the vaccine of the present invention includes a mutant antigen formed by incorporating a group with chemical cross-linking activity or a derivative thereof into the target epitope of the wild-type antigen.
  • IL-1 ⁇ single alanine mutant hIL-1 ⁇ E64A
  • IL-1 ⁇ mutant containing four alanine mutations hIL-1 ⁇ 63-66A
  • WT hIL-1 ⁇ or IL-1 ⁇ mutants incorporating unnatural amino acids were injected subcutaneously into 6-8 week-old female Balb/C mice (per group of 3).
  • 50 ⁇ g of antigen was mixed with Freund’s complete adjuvant (sigma, F5881) before injection
  • 30 ⁇ g of antigen was mixed with Freund’s incomplete adjuvant (sigma, F5506) before injection .
  • the interval between two immunizations was 2 weeks.
  • mouse serum was collected and diluted with an equal volume of DPBS (pH 8.0).
  • the sample was incubated with protein A filler (GenScript, L00210) for 3 hours, and after washing with DPBS of 10 times the column volume, the protein bound to protein A was eluted with elution buffer (0.2M glycine, 0.1M NaCl, pH 2.5).
  • elution buffer 0.2M glycine, 0.1M NaCl, pH 2.5
  • Tris-HCl final concentration 100 mM
  • Amicon Ultra spin column Merck Millipore, UFC903096
  • Phage display libraries were constructed using published methods (Barbas et al., 1991). To construct the mouse immune library, Balb/c mice were immunized three times with wild-type hIL-1 ⁇ , hIL-1 ⁇ E64AK, hIL-1 ⁇ E64CK, hIL-1 ⁇ E64BK or hIL-1 ⁇ Q15CK, respectively, with an interval of 2 weeks between the two immunizations. Two weeks after the third immunization, the total RNA of mouse spleen was extracted and used as a template for reverse transcription to construct a cDNA library.
  • the scFv phage display library was constructed using the phagemid vector pSEXRTL2, and the M13KO7 ( ⁇ pIII) helper phage ( PROGEN, catalog number: PRHYPE) to package the library into scFv-pIII phage.
  • Escherichia coli XL1-Blue cells carrying phagemids (displaying scFv-pIII) were inoculated into 20 ml of 2 X YT medium, added ampicillin (100 ⁇ g/ml) and tetracycline (15 ⁇ g/ml), and incubated at 37 ° C, 220rpm cultivation.
  • the culture was centrifuged at 4000g for 10 minutes and the supernatant was transferred to a new tube and centrifuged at 10,000g for 20 minutes to remove cell debris.
  • 5X phage precipitation buffer [100g PEG 8000, 73.3g NaCl dissolved in 500ml ddHO] and incubate on ice for 4 hours. Collect phage by centrifugation at 10,000g for 20 minutes at 4°C, dissolve in 1ml DPBS, and incubate at room temperature for 15 minutes. Filter the phage with a 0.22 ⁇ m filter membrane and store at 4°C.
  • hIL-1 ⁇ E64AK (1 ⁇ g) was coated in a 96-well plate at 4°C overnight, blocked with 200 ⁇ l DPBS containing 3% BSA for 2 hours at room temperature, and 10 10 pfu from hIL-1 ⁇ E64AK immune phage library was added Phage (containing 1% BSA, 1 mM EDTA, pH 8.8) was incubated at 37°C for 48 hours.
  • E64AK-A9, E64AK-F4, E64CK-B9, E64CK-A5, E64CK-G9 or Q15CK-G8 obtained by panning in step 6 was connected to the Fc of mouse IgG2a by a linker ( Named respectively as CL-E2-mFc, E64AK-A9-mFc, E64AK-F4-mFc, E64CK-B9-mFc, E64CK-A5-mFc, E64CK-G9-mFc, Q15CK-G8-mFc), cloned into pFuse for expression in the carrier.
  • a linker Named respectively as CL-E2-mFc, E64AK-A9-mFc, E64AK-F4-mFc, E64CK-B9-mFc, E64CK-A5-mFc, E64CK-G9-mFc, Q15CK-G8-mFc
  • HEK 293F cells (Thermo Scientific, R79007) were cultured, and the scFv-mFc expression plasmid constructed above and PEI were transfected into cells (2.5 ⁇ 10 6 cells/ml) at a ratio of 1:2.5 (mass ratio).
  • the cell culture supernatant was collected, passed through protein A filler (GenScript, L00210) pre-equilibrated in DPBS twice, washed with 10 times column volume of DPBS, and eluted with Buffer (0.2M Glycine, 0.1M NaCl, pH 2.5) eluted the protein bound to the medium.
  • Buffer 0.2M Glycine, 0.1M NaCl, pH 2.5
  • Tris-HCl final concentration 100 mM was added to adjust the pH to 7.5. Then use Amicon Ultra spin column (Merck Millipore, UFC903096) to concentrate and change medium (DPBS, pH 7.5), SEC purification (chromatographic column: Superdex 200 increase 10/300 GL, GE Healthcare, 10263259).
  • Antigen 100 ng was coated on 96-well ELISA plate (Corning Costar, 2592) overnight at 4°C and blocked with 200 ⁇ l of DPBS containing 3% skim milk powder for 2 hours at 37°C. Add antibody or phage in DPBST solution containing 3% skimmed milk powder and incubate at 37°C for 2 hours. After washing four times with 200 ⁇ l DPBST, horseradish peroxidase (HRP)-conjugated detection antibody was added and incubated at room temperature for 1 hour.
  • HRP horseradish peroxidase
  • TMB Biolegend, 002023
  • chromogenic reagent After washing with 200 ⁇ l DPBST five times, add 100 ⁇ l TMB (Biolegend, 002023) chromogenic reagent, incubate at room temperature for 10-30 minutes, and use a microplate reader (BMG LABTECH, ) to read the value.
  • 100ng WT hIL-1 ⁇ was coated on the ELSIA plate overnight at 4°C, and after blocking for 2 hours in DPBS containing 3% BSA, add serially diluted phage (starting from 10 8 pfu, 10-fold dilution) in DPBST containing 3% BSA Incubate at room temperature for 2 hours, add 300 nM canakimab and incubate for another 1 hour. After washing, add HRP-conjugated mouse anti-M13 (antibody) (1:2000) and incubate at room temperature for 1 hour.
  • TMB chromogenic reagent TMB chromogenic reagent
  • BMG LABTECH a microplate reader
  • All P values were calculated using GraphPad Prism 6.0 and have the following meanings: nsp>0.05;*p ⁇ 0.05;**p ⁇ 0.01;***p ⁇ 0.001;****p ⁇ 0.0001. Details of the statistical analysis for each experiment can be found in the figures and legends.
  • HEK-Blue TM IL-1R cells (Invivogen, hkb-il1r) with a density of 70% were washed twice with pre-warmed PBS, and the bottom of the flask was tapped to dislodge the cells from the flask.
  • Cells were resuspended in pre-warmed DMEM medium (containing 10% heat-inactivated FBS) (cell density 330,000 cells/ml medium).
  • pre-warmed DMEM medium containing 10% heat-inactivated FBS
  • 25 ⁇ l of recombinant human IL-1 ⁇ (0.8 ng/ml) was incubated with 25 ⁇ l of purified serum IgG diluted 1:5 (initial concentration 4 ⁇ M) for 30 minutes at room temperature.
  • HEK-Blue IL-1R cell suspension (approximately 50,000 cells) was added to each well.
  • the 96-well cell culture plate was incubated overnight in a 5% CO2, 37°C cell culture incubator. Take 20 ⁇ l of cell culture supernatant and incubate with 180 ⁇ l of QUANTI-Blue TM (Invivogen) at 37° C. for 30 minutes to 3 hours.
  • SEAP Secreted embryonic alkaline phosphatase
  • hIL-1 ⁇ incorporated by AK can induce the production of antibodies with cross-linking activity
  • AK is a lysine-derived unnatural amino acid whose acrylamide group on the side chain can form a covalent bond with an adjacent nucleophilic group (Furman et al., 2014). We speculate that after immunization of mice with AK-incorporated antigens, the mice evolve antibodies that can covalently cross-link with AK-incorporated antigens during B cell hypermutation.
  • mice The pEvol vector encoding MmAKRS/tRNA CUA orthogonal pair and pET28a-hIL-1 ⁇ E64TAG were co-transformed into E.coli BL21(DE3), the hIL-1 ⁇ E64AK mutant protein was purified by nickel column and SEC, and after identification by ESI-MS mass spectrometry, the Balb/ c mice, after the three immunizations, the serum titer was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that mouse serum bound WT hIL-1 ⁇ and hIL-1 ⁇ E64AK ( FIG. 1A ) with comparable potency (approximately 1:10 5 ). Isolate mouse spleen, extract its total RNA, reverse transcribe into cDNA library, and construct scfv phage library displaying antibody (Barbas et al., 1991).
  • CL-E2 One of the clones (named CL-E2) was selected for packaging monoclonal phage antibodies. After incubating CL-E2 phage and hIL-1 ⁇ E64AK in DPBS (pH8.8) for 48 hours, WB detection revealed a specific band whose size matched the size of the hIL-1 ⁇ E64AK+pIII-scfv complex (Fig. 2A).
  • CL-E2 and CL-E2-mFc have similar affinity to WT IL-1 ⁇ or hIL-1 ⁇ E64AK ( Figure 3A and 3B), probably because the body binds to the antibody that binds to the hIL-1 ⁇ E64 epitope during the selection of B cell clones
  • the library evolves B cells that can be chemically cross-linked with hIL-1 ⁇ E64AK, and the covalent combination leads to a large number of proliferation of the B cells, thereby enriching a large number of specific antibodies CL-E2 that can covalently bind to the hIL-1 ⁇ E64 epitope, and then through the above-mentioned specific A phage chemical panning method was identified by us.
  • Antigens incorporated by AK can induce epitope-directed antibody responses
  • AK-incorporated antigens can induce the production of antibodies with chemical cross-linking activity, and we speculate that this unique mechanism can be used for epitope-directed enrichment of antibodies that bind to AK-incorporated epitopes.
  • the output clones obtained in the second round of panning were identified for binding epitopes, and the output clones were divided into 7 clusters according to the scfv amino acid sequence (identity ⁇ 98%).
  • the E64AK-A9 clone appeared 46 times with similar affinity to WT IL-1 ⁇ and hIL-1 ⁇ E64AK (Fig. 4A).
  • the E64AK-A9-mFc fusion antibody which has similar affinity to WT IL-1 ⁇ (1.6 ⁇ 0.2nM) and hIL-1 ⁇ E64AK (1.2 ⁇ 0.2nM) (Fig.
  • scfv antibodies and three scfv antibodies that appeared only once were selected from the remaining antibody clusters and packaged into monoclonal phages.
  • hIL-1 ⁇ E64A an IL-1 ⁇ single alanine mutant
  • hIL-1 ⁇ 63-66A an IL-1 ⁇ mutant containing four alanine mutations
  • E64AK-F4 with the largest affinity difference between WT hIL-1 ⁇ and hIL-1 ⁇ 63-66A mutants to construct E64AK-F4-mFc fusion antibody.
  • Antigens incorporated by CK may also induce epitope-specific antibodies
  • N ⁇ -crotonyl-L-lysine is also a lysine-derived unnatural amino acid, which has weaker chemical cross-linking activity than AK.
  • immunization of mice with CK-inserted antigens could also induce antibodies targeting specific epitopes.
  • hIL-1 ⁇ E64CK by genetic coding technology, and constructed antibody phage library after immunizing mice. After 2 rounds of conventional panning, 96 output clones were randomly selected for sequencing, 84 of which had complete mouse scfv antibody sequences.
  • the phage antibody sequences obtained from hIL1 ⁇ E64AK immunization were evenly distributed in five antibody clusters, which may be due to the chemical cross-linking activity of CK. AK is weak. A representative sequence was selected from each cluster to package monoclonal phages, and the results showed that all of these monoclonal phages could bind to WT hIL-1 ⁇ (Fig. 7A).
  • E64AK-A9 binds to the E64 epitope ( Figure 4C), and the binding of E64CK-H11 to hIL-1 ⁇ can be competitively inhibited by E64AK-A9-mFc ( Figure 7D), it is suggested that E64CK-H11 (sequence homologous to E64AK-A9) also binds the E64 epitope, although its affinity is not (at least not entirely) dependent on binding to this region, which explains why there is little difference in its affinity for binding hIL-1 ⁇ E64CK and hIL-1 ⁇ 63-66A. Likewise, since E64AK-G6 specifically binds the E64 epitope (Fig.
  • E64CK-B9 should also bind the E64 epitope given the sequence homology between E64CK-B9 and E64AK-G6.
  • E64CK-B9-mFc could competitively inhibit the binding of E64AK-G6 and E64AK-A2 to hIL-1 ⁇ (Fig. 7E), suggesting that these clones (E64CK-B9, E64AK-G6, E64AK-A2) all bind to Similar regions on hIL-1 ⁇ .
  • mice immunized with hIL-1 ⁇ E64CK or hIL-1 ⁇ E64AK can directly target the target epitope to generate antibody responses and enrich the immune response during the process of clone selection and overfrequency mutation of B cells through the antigenic properties with site-specific cross-linking activity. set.
  • Epitope-specific antibody responses are due to the chemical cross-linking activity of AK or CK incorporated by IL-1 ⁇
  • CK-induced epitope-specific antibody responses are independent of epitope sequence
  • these phage clones that bind hIL-1 ⁇ Q15CK or hIL-1 ⁇ Q15G with no significant difference in affinity may also bind the hIL-1 ⁇ Q15 epitope, just like the E64 epitope antibody screen described above. However, through a single epitope identification method, it was found that these clones can all bind to the hIL-1 ⁇ Q15 epitope. If the frequency of each cluster of antibodies is weighted, the proportion of antibodies binding to the hIL-1 ⁇ Q15 epitope among the 96 analyzed gram antibodies 60%.
  • IL-1 ⁇ incorporated in CK is expected to be used in the development of subunit vaccines
  • IL-1 ⁇ is a pro-inflammatory cytokine that binds IL-1RI and IL1RII (Afonina et al., 2015). Blocking IL-1 ⁇ and IL-1RI signaling pathways can be used to treat a series of autoimmune diseases, such as type II diabetes, rheumatoid arthritis, gout, etc. (Dinarello et al., 2012). Some vaccine frameworks based on IL-1 ⁇ have been used to evaluate their possibility as vaccines, but the specific effects need to be clinically verified (Cavelti-Weder et al., 2016; Spohn et al., 2008; Spohn et al., 2014) .
  • mice immunized with WT hIL-1B and CK-incorporated IL-1 ⁇ mutant had comparable IgG antibody titers ( ⁇ 1:10 6 , FIG. 14A ).
  • mice immunized with WT IL-1 ⁇ and DBPS did not have any inhibitory effect, while IgG from mice immunized with hIL-1 ⁇ Q15CK, hIL-1 ⁇ G33CK, hIL-1 ⁇ N53CK or hIL-1 ⁇ I106CK could significantly inhibit IL-1 ⁇ -induced The activation of HEK-Blue IL-1R ( Figure 14B), among them, the IgG from hIL-1 ⁇ Q15CK immunized mice had the strongest inhibitory effect, with an IC 50 of about 137.5 ⁇ 0.1nM. Under the same experimental conditions, it had a high The affinity of canikizumab with IC50 was 4.7 ⁇ 0.1 nM ( FIG. 14C ).
  • PTN-WT and PTN-CK were coupled to hemocyanin KLH via N-terminal Cys using SMCC (a bifunctional coupling agent of N-hydroxysuccinimide (NHS) active ester and maleimide) , KLH-PTN-WT and KLH-PTN-CK were prepared.
  • SMCC a bifunctional coupling agent of N-hydroxysuccinimide (NHS) active ester and maleimide
  • the synthetic polypeptides PTN-WT, PTN-CK, KLH-PTN-WT, and KLH-PTN-CK were divided into four groups and used as immunogens to immunize mice BAlb/C.
  • the adjuvant used for the initial immunization was complete Freund's adjuvant.
  • the immune dose is 30ug.
  • the adjuvant used for reimmunization was incomplete Freund's adjuvant.
  • Serum was collected 35 days after immunization, and different proteins (such as PTN-WT, PTN-CK, KLH-PTN-WT or KLH-PTN-CK) were used as the coating protein to measure the titer according to the requirements of the experiment.
  • different proteins such as PTN-WT, PTN-CK, KLH-PTN-WT or KLH-PTN-CK
  • the titers of the immunized sera of the two groups (KLH-PTN-WT, KLH-PTN-CK) against the KLH and PTN polypeptide fractions were compared (Fig. 15F), and the KLH-PTN-WT group produced anti-KLH and PTN polypeptide Antibodies, mostly against KLH.
  • the KLH-PTN-Kcr group generated antibodies against the KLH and PTN polypeptide portions, mostly against the PTN polypeptide portion.

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Abstract

涉及基于化学交联基团的表位修饰及其在改变抗原免疫原性和提高针对抗原的目标表位的动物免疫应答方面的应用。涉及包括将在野生型目标表位掺入具有化学交联活性的基团或其衍生物形成的突变体抗原施用动物，所述突变体抗原将动物体内的抗体反应定向并富集到目标表位；提供一种筛选针对抗原目标表位的抗体的方法及由此获得的抗体；进一步提供了所述方法在用于制备预防和治疗疾病的疫苗中的用途。

Description

抗原表位修饰 技术领域：
本发明属于生物医药技术领域。
背景：
抗体是脊椎动物适应性免疫的重要组成部分，在预防细菌和病毒感染以及中和大多数外来有害物质方面发挥着至关重要的作用(Tomita和Tsumoto，2010)。单克隆抗体具有高亲和力，高特异性，良好的生物相容性和细胞效应等优势，使其成为治疗多种疾病(例如自身免疫性疾病和癌症)的重要疗法(Sharma和Allison，2015年；Singh等，2018年；Sliwkowski和Mellman，2013年)。小鼠杂交瘤技术是产生高亲和力单克隆抗体的最常用方法之一(Schwaber，1982；Zhang，2012)。近年来，随着具有人抗体基因片段插入的转基因小鼠在解决抗体人源化问题中的应用，小鼠免疫与下游单克隆抗体的筛选已成为鉴定和开发治疗性抗体的主要方法(Brüggemann,2001；Brüggemann等,2015；Taylor等,1992)。但是，这种方法并不总能有效地产生功能性抗体(Kellermann和Green，2002)。治疗性抗体必须结合目标抗原的特定表位才能发挥其预期的功能，例如作为拮抗剂阻断配体-受体相互作用，或者作为激动剂诱导受体介导的下游信号传导(Weiner等，2010)。不幸的是，对于治疗有用的有效表位可能仅占整个抗原分子表面的一小部分(Sercarz等，1993)。通过当前的方法，如果将整个抗原蛋白用作免疫原免疫小鼠，则引发针对所需表位的抗体应答的可能性很小。另外，目标抗原上无功能性但具有免疫优势的B细胞表位进一步降低这种可能性。人们必须采用高通量筛选以期从众多的单克隆中年筛选到功能性抗体(Pasqualini和Arap，2004)。更糟糕的是，对于与小鼠蛋白物序列高度同源且结构相似的人类靶标，由于宿主的免疫耐受性，可能很难引发针对功能性表位的抗体应答(Goodnow等，1988；Hartley等，1993年；Nemazee，2017年)，这种情况在很多重要的药物靶标如GPCR中非常常见。如果将具有表位序列的多肽用作免疫原，筛选到的针对线性表位的抗体对靶标中的构象表位可能没有结合活性(Xu等，2018)
为克服这一挑战，科学家们已经尝试了几种增加功能性抗体筛选几率的方法。Viktoriya Dubrovskaya等通过修饰HIV包膜蛋白的糖基化表位来诱导产生广泛的中和性抗体(Dubrovskaya等，2019)。Fabian Sesterhenn等人将呼吸道合胞病毒(RSV)表位与免疫原抗原融合后获得了可特异性结合RSV表位的中和性抗体(Sesterhenn et al，2020)。然而，这些方法仅在特定情况下是可行的，难以将其用于针对其它抗原特定表位的抗体筛选。
通过筛选和选择获得的经工程改造的tRNA/aaRS正交对可特异性识别非天然氨基酸(UAA)并将其掺入活宿主细胞中靶标的编码位点(Wang等，2001)。UAA侧链上的各种化学基团可赋予蛋白质新的功能，因此它们在蛋白质结构和功能、细胞成像、治疗性蛋白质偶联以及许多其他领域的研究中显示出了强大的应用(Chin，2014年)。在前期的工作中，我们利用掺入对苯甲酰基-1-苯丙氨酸(pBpa)的抗原，通过光交联淘选从抗体噬菌体展示库中成功筛选到与抗原的目标表位结合的抗体(Chen等，2020)，提示这可能是第一种可用于所有类型抗原目标表位的抗体筛选方法。但是，此方法依赖于体外噬菌体文库的淘选以及筛选到的抗体亲和力成熟。
综上所述，目前的方法均不能在体内选择性地引发对抗原特定构象表位的抗体应答。
发明内容
鉴于小鼠免疫全抗原后产生的抗体通常会富集到具有免疫优势的B细胞表位，而这通常会降低筛选到针对低免疫应答产生的目标表位抗体的可能性，本发明开发了一种新的方法，基于化学交联基团对抗原的目标表位进行修饰以改变其免疫原性，从而改变该抗原在免疫动物后引发的抗体谱，以富集针对目标抗原的抗体。本发明进一步提供了针对抗原目标表位富集动物免疫应答以及针对抗原目标表位的抗体的筛选和制备方法。
通过本发明可简单快速引发和鉴定与抗原的特定表位结合的抗体：将化学交联活性基团(例如Nε-丙烯酰基或Nε-巴豆酰基)以一定的方式(例如通过在目标表位的氨基酸序列中导入包含化学交联活性基团的天然或非天然氨基酸，或者通过化学修饰目标表位中的特定氨基酸以引入化学交联活性基团)掺入目标抗原(如IL-1β)的目标表位(例如E64)，可以创建“超级”的具有免疫优势的B细胞表位，并在小鼠免疫后将抗体反应定向并富集到目标表位。在具体实施例中，这种效果如此突出，以至于几乎所有从IL-1βE64AK和IL-1βE64CK免疫噬菌体库中随机挑选的克隆都能与目标表位结合。反之， 将野生型抗原WT IL-1β和掺入无交联活性的非天然氨基酸的IL-1β突变体进行免疫得到的克隆很少与目标表位结合。另外，IL-1βE64AK和IL-1βE64CK之间的交联活性的差异导致其免疫小鼠后最终筛选到的克隆序列存在不同的差异，提示可以通过调节化学交联基团的反应性和结构来调节抗体亲和力和序列多样性。
本发明进一步发现，AK或CK掺入表位的定向抗体应答不受序列的限制：将CK掺入IL-1β的其它位点后免疫小鼠均可以有效产生高滴度的针对特定表位特异性的抗体；当目标表位位于IL-1β和IL1RI的结合界面时，CK诱导的表位特异性抗体可有效阻断IL-1β激活其受体。因此，该表位定向的抗体应答可以用于疫苗开发，以增强对功能性表位的有效抗体应答。
本发明进一步发现，在多肽(如三角褐指藻蛋白的其中一段多肽PTN)的特定位置引入具有化学交联活性基团(如Nε-巴豆酰基)的氨基酸CK得到的突变体多肽(PTN-CK)，能够显著增强PTN本身(PTN-WT)的免疫源性，PTN-CK免疫的小鼠血清中针对PTN-CK的抗体效价明显高于PTN-WT免疫小鼠血清。将PTN-CK或PTN-WT偶联到载体蛋白(KLH)后免疫小鼠，KLH-PTN-CK免疫小鼠血清中针对KLH的抗体效价明显变少，从而使抗体更多的针对PTN-CK(与KLH-PTN-WT相比)。
本发明的一个方面提供了一种用于免疫动物的试剂，所述试剂包含：
-免疫有效量的突变体抗原，其是在野生型抗原中的一个或多个目标表位上掺有具有化学交联活性的基团或其衍生物的抗原，和
-生理上可接受的载体，
其中该试剂施用于动物后可刺激或增强该动物中针对所述一个或多个目标表位的抗体的生成。
在一些实施方案中，所述具有化学交联活性的基团为Nε-巴豆酰基或Nε-丙烯酰基。
在一些实施方案中，所述具有化学交联活性的基团为天然氨基酸或非天然氨基酸。
在一些实施方案中，所述具有化学活性基团的非天然氨基酸为Nε-巴豆酰基-L-赖氨酸(CK)或Nε-丙烯酰基-L-赖氨酸(AK)。
在一些实施方案中，所述具有化学交联活性的基团或其衍生物是通过扩展基因密码子或化学合成掺入的。
在一些实施方案中，所述动物为啮齿类动物、非人哺乳动物或哺乳动物。
在一些实施方案中，所述野生型抗原为可溶性蛋白、可溶性多肽、表达在磷脂膜结构上的跨膜蛋白、或表达在磷脂膜结构上的多肽。
在一些实施方案中，所述试剂是抗体制备用试剂。
在一些实施方案中，所述试剂是预防或治疗用试剂。
在一些实施方案中，所述试剂是疫苗组合物。
在一些实施方案中，所述试剂包含免疫有效量的IL-1β突变体抗原；在另外一些实施方案中，所述IL-1β突变体抗原的E64位掺有AK；在另外一些实施方案中，所述IL-1β突变体抗原的E64位掺有CK。
在一些实施方案中，所述试剂包含免疫有效量的新冠病毒S蛋白RBD区域突变体抗原；在一些实施方案中，所述新冠病毒S蛋白RBD区域突变体抗原的K417表位、L452表位、Y453表位、E484表位或N501表位掺有AK；在一些实施方案中，所述新冠病毒S蛋白RBD区域突变体抗原的K417表位、L452表位、Y453表位、E484表位或N501表位掺有CK。
在一些实施方案中，所述试剂包含免疫有效量的PTN-CK；在一些实施方案中，所述试剂包含有效量的偶联至载体蛋白的PTN-CK；在一些实施方案中，所述载体蛋白为血蓝蛋白KLH；在一些实施方案中，所述试剂包含有效量的KLH-PTN-CK。
本发明的另一个方面，提供了一种增强动物免疫应答的试剂，所述试剂包含：
-免疫有效量的突变体抗原，其是在野生型抗原中的一个或多个目标表位上掺有具有化学交联活性基团或其衍生物的抗原，和
本发明的另一个方面，提供了一种调节抗原的免疫原性的方法，所述方法包括在抗原的一个或多个目标表位上掺入具有化学交联活性的基团或其衍生物。
在一些实施方案中，所述调节导致抗原激发抗体的分布(profile)改变。
本发明的另一个方面，提供了一种提高动物针对抗原的目标表位的免疫应答的方法，所述方法包括：对所述动物施用一定量的突变体抗原，所述突变体抗原为野生型抗原的目标表位上掺有具有化学交联活性基团或其衍生物的抗原。
本发明的另一个方面提供了一种提高针对抗原目标表位动物免疫应答的方法，所述方法包括：对所述动物施用一定量的突变体抗原，所述突变体抗原为在野生型抗原目标表位上掺入具有化学交联活性基团或其衍生物后形成的抗原。
本发明的另一个方面提供了所述提高针对抗原目标表位动物免疫应答的方法在用于制备预防或治疗疾病的疫苗中的用途。
本发明的另一个方面提供了一种突变体抗原在制备疫苗中的应用，所述突变体抗原为在野生型抗原的目标表位上掺入具有化学交联活性基团或其衍生物后形成的抗原。
本发明的另一个方面提供了一种筛选针对野生型抗原目标表位的抗体的方法，所述方法包括如下步骤：(a)提供突变体抗原，所述突变体抗原在野生型抗原的目标表位上掺有具有化学交联活性的基团或其衍生物；(b)对动物施用步骤(a)所述的突变体抗原；(c)从所述动物中分离血清；(d)利用所述野生型抗原筛选出特异性结合目标表位的抗体。
本发明的另一个方面提供了一种筛选针对野生型抗原目标表位的抗体的方法，所述方法包括如下步骤：(a)提供突变体抗原，所述突变体抗原在野生型抗原的目标表位上掺有具有化学交联活性的基团或其衍生物；(b)对动物施用步骤(a)所述的突变体抗原；(c)从所述动物中分离B细胞，将所述B细胞与骨髓瘤细胞融合以产生杂交瘤细胞；(d)利用所述野生型抗原从所述杂交瘤细胞的培养上清筛选出特异性结合目标表位的抗体。
本发明的另一个方面提供了一种筛选针对野生型抗原目标表位的抗体的方法，所述方法包括如下步骤：(a)提供突变体抗原，所述突变体抗原在野生型抗原的目标表位上掺有具有化学交联活性的基团或其衍生物；(b)对动物施用步骤(a)所述的突变体抗原；(c)从所述动物中分离B细胞，利用所述B细胞构建抗体文库；(d)利用所述野生型抗原从所述抗体文库中筛选出特异性结合目标表位的抗体。
本发明的另一个方面提供了一种筛选针对野生型抗原目标表位的抗体的方法，所述方法包括如下步骤：(a)提供突变体抗原，所述突变体抗原在野生型抗原的目标表位上掺有具有化学交联活性的基团或其衍生物；(b)对动物施用步骤(a)所述的突变体抗原；(c)从所述动物中分离血清；(d)将所述突变体抗原与所述血清在一定的条件下孵育，以便所述突变体抗原与抗体共价交联形成突变体抗原-抗体复合物；(e)以一定的条件洗脱除去未与所述突变体抗原共价交联的抗体，并释放与所述突变体抗原共价交联的抗体；(f)利用野生型抗原，从所述与突变体抗原共价交联的抗体中进一步筛选特异性结合目标表位的抗体。
本发明的另一个方面提供了一种筛选针对野生型抗原目标表位的抗体的方法，所述方法包括如下步骤：(a)提供突变体抗原，所述突变体抗原在野生型抗原的目标表位上掺有具有化学交联活性的基团或其衍生物；(b)对动物施用步骤(a)所述的突 变体抗原；(c)从所述动物中分离B细胞，将所述B细胞与骨髓瘤细胞融合以产生杂交瘤细胞；(d)将所述突变体抗原与所述杂交瘤细胞的培养上清在一定的条件下孵育，以便所述突变体抗原与抗体共价交联形成突变体抗原-抗体复合物；(e)以一定的条件洗脱除去未与所述突变体抗原共价交联的抗体，并释放与所述突变体抗原共价交联的抗体；(f)利用野生型抗原，从所述与突变体抗原共价交联的抗体中进一步筛选特异性结合目标表位的抗体。
本发明的另一个方面提供了一种筛选针对野生型抗原目标表位的抗体的方法，所述方法包括如下步骤：(a)提供突变体抗原，所述突变体抗原在野生型抗原的目标表位上掺有具有化学交联活性的基团或其衍生物；(b)对动物施用步骤(a)所述的突变体抗原；(c)从所述动物中分离B细胞，利用所述B细胞构建抗体文库；(d)将所述突变体抗原与所述抗体文库在一定的条件下孵育，以便所述突变体抗原与抗体共价交联形成突变体抗原-抗体复合物；(e)以一定的条件洗脱除去未与所述突变体抗原共价交联的抗体，并释放与所述突变体抗原共价交联的抗体；(f)利用野生型抗原，从所述与突变体抗原共价交联的抗体中进一步筛选特异性结合目标表位的抗体。
在一些实施方案中，所述具有化学活性的基团或其衍生物是通过扩展基因密码子或化学合成掺入的。
在一些实施方案中，所述具有化学交联活性的基团为Nε-巴豆酰基或Nε-丙烯酰基。
在一些实施方案中，所述具有化学交联活性的基团为天然氨基酸或非天然氨基酸。
在一些实施方案中，所述具有化学交联活性基团的非天然氨基酸为Nε-巴豆酰基-L-赖氨酸(CK)或Nε-丙烯酰基-L-赖氨酸(AK)。
在一些实施方案中，所述动物为啮齿类动物、非人哺乳动物或哺乳动物。在特定的实施方案中，所述啮齿类动物为小鼠或大鼠；在特定的实施方案中，所述非人哺乳动物为兔子、羊驼或羊等；在特定的实施方案中，所述哺乳动物为人。
在一些实施方案，所述抗原为可溶性蛋白、可溶性多肽、表达在磷脂膜结构上的跨膜蛋白、或表达在磷脂膜结构上的多肽。
在一些实施方案中，所述突变体抗原与所述血清的孵育条件为碱性条件。在一些实施方案中，所述突变体抗原与所述杂交瘤细胞的孵育条件为碱性条件。在一些实施方案中，所述突变体抗原与所述抗体文库的孵育条件为碱性条件。
在一些实施方案中，所述碱性条件为pH8.8的溶液；在一些实施方案中，所述碱性条件为pH8.8的DPBS溶液；在一些实施方案中，所述孵育条件为孵育24h、48h、3d、4d、5d、6d或7d；在一些实施方案中，所述孵育条件为pH8.8的DPBS溶液中，孵育24h、48h、3d、4d、5d、6d或7d；在一些实施方案中，所述孵育条件为pH8.8的DPBS溶液中孵育24h；在一些实施方案中，所述孵育条件为pH8.8的DPBS溶液中孵育48h。
在一些实施方案中，所述除去未与所述突变体抗原共价交联的抗体的洗脱条件为：i)用高pH洗脱缓冲液进行碱性洗脱；ii)用低pH洗脱缓冲液进行酸性洗脱。
在一些实施方案中，所述释放与所述抗原共价交联的抗体为通过酶消化的方式释放。
本发明的另一个方面提供了所述筛选针对野生型抗原目标表位的抗体的方法在用于制备预防或治疗疾病的疫苗中的用途。
本发明的另一个方面提供了制备抗体的方法，包括通过此处提供的所述筛选针对野生型抗原目标表位的抗体的方法获得抗体；且提供如此获得的抗体。
在一些实施方案中，所述针对野生型抗原目标表位筛选获得的抗体特异性结合IL-1β。在一些实施方案中，所述特异性结合IL-1β的抗体具有：如SEQ ID NO.6所示的VH和SEQ ID NO.4所示的VL；如SEQ ID NO.10所示的VH和SEQ ID NO.8所示的VL；如SEQ ID NO.14所示的VH和SEQ ID NO.12所示的VL；如SEQ ID NO.18所示的VH和SEQ ID NO.16所示的VL；如SEQ ID NO.22所示的VH和SEQ ID NO.20所示的VL；如SEQ ID NO.26所示的VH和SEQ ID NO.24所示的VL；SEQ ID NO.30所示的VH和SEQ ID NO.28所示的VL。
本发明的另一个方面提供了所述特异性结合IL-1β的抗体在制备治疗和预防IL-1介导的疾病中的药物中的用途；所述IL-1介导的疾病包括成人斯蒂尔病、全身型幼年特发性关节炎、骨关节炎、类风湿性关节炎、痛风性关节炎、急性痛风、新生儿多系统炎症疾病、贝赫切特病(Behcet's Disease)、冷吡啉相关周期性综合征、家族性地中海热、遗传性周期发热、周期性发热综合征、TNFR相关性周期性发热综合征、动脉粥样硬化、房颤、急性心肌梗死、外周动脉疾病、慢性特发性荨麻疹、腹主动脉瘤、结直肠癌、三阴性乳腺癌、非小细胞肺癌、I型糖尿病、II型糖尿病、甲羟戊酸激酶缺乏症、施尼茨勒综合征(Schnitzler Syndrome)；荨麻疹和巨球蛋白血症、镰刀型细胞贫血病、坏疽性脓皮症、慢性阻塞性肺病、干眼症、肺结节病、川崎氏病、湿性年龄相 关性黄斑变性、巩膜炎、葡萄膜炎、穆-韦二氏综合征(Muckle-Wells Syndrome)、寻常痤疮、坏疽性脓皮症等。
本发明的另一个方面提供了一种所述突变体抗原在用于制备用于预防或治疗疾病的疫苗中的用途。在一些实施方案中，所述突变体抗原为在K417表位、Y453表位、E484表位或N501表位上掺有AK的新冠病毒S蛋白RBD区域；在一些实施方案中，所述突变体抗原为在K417表位、L452表位、Y453表位、E484表位或N501表位上掺有CK的新冠病毒S蛋白RBD区域。
附图说明：
图1为免疫小鼠血清抗体滴度
图2A为CL-E2噬菌体与WT IL1β、hIL-1βE64AK WB检测结果；图2B、C为CL-E2-mFc融合蛋白与WT IL1β或其突变体WB检测结果；图2D为Canakinumab与hIL-1βE64AK、hIL-1βE64CK在化学交联条件下的WB检测结果。
图3A为CL-E2-mFc与hIL-1β、hIL-1βE64AK ELISA结果，其中CL-E2-mFc与hIL-1β、hIL-1βE64AK结合的K d分别为3.7±0.2nM和4.2±0.2nM；图3B为CL-E2噬菌体与hIL-1β、hIL-1βE64AK结合的ELISA检测结果，其中横坐标为噬菌体的pfu值。
图4A为淘选出的噬菌体克隆与WT hIL-1β和hIL-1βE64AK结合的ELISA结果；图4B为E64AK-A9-mFc抗体融合蛋白与WT hIL-1β和hIL-1βE64AK结合的ELISA结果(Kd值分别为1.6±0.2nM、1.2±0.2nM)；图4C、D为E64AK-A9-mFc抗体融合蛋白与hIL-1βE64AK的WB检测结果，其中4C检测用的抗体为小鼠抗-His-tag抗体，4D检测用的抗体为抗小鼠Fc。
图5为不同噬菌体克隆与hIL-1βE64AK、hIL-1β63-66A ELISA检测结果，其中*表示p<0.01,**表示p<0.01,***表示p<0.001,****表示p<0.0001。
图6A为Gevokizumab与hIL-1β、hIL-1β63-66A ELISA检测结果，图6B为E64AK-F4-mFc与hIL-1β或其突变体的ELISA检测结果；图6C、D分别为E64AK-F4噬菌体和E64AK-F4-mFc与hIL-1β的结合被canakinumab竞争抑制的ELISA结果。
图7A为不同的噬菌体克隆与与hIL-1β、hIL-1β64CK ELISA检测结果；图7B、7C为不同的噬菌体克隆与hIL-1βE64CK、hIL-1β63-66A ELISA检测结果，图7D为E64CK-H11与hIL-1β的结合被E64AK-A9-mFc竞争抑制的ELISA检测结果；图7E为E64AK-G6和 E64AK-A2与hIL-1β的结合被E64CK-B9-mFc竞争抑制的ELISA检测结果，其中，*表示p<0.01,**表示p<0.01,***表示p<0.001,****表示p<0.0001。
图8A、8B分别为抗体融合蛋白E64CK-A5-mFc、E64CK-G9-mFc与hIL-1β或其突变体蛋白的ELISA检测结果；图8C、D分别为抗体融合蛋白E64CK-A5-mFc、E64CK-G9-mFc与hIL-1β的结合被canakinumab竞争抑制的ELISA结果；图8E、8F分别为噬菌体克隆E64CK-A59、E64CK-G9与hIL-1β的结合被canakinumab竞争抑制的ELISA结果；图8G为抗体融合蛋白E64CK-A5-mFc、E64CK-G9-mFc与hIL-1β或其突变体蛋白在pH8.8、37℃孵育48h的WB检测结果；图8H为抗体融合蛋白E64CK-A5-mFc、E64CK-G9-mFc与hIL-1β或其突变体蛋白在pH8.8、37℃孵育不同时间的WB检测结果；*表示p<0.01,**表示p<0.01,***表示p<0.001,****表示p<0.0001。
图9为hIL-1β免疫小鼠构建的噬菌体文库中筛选出的克隆与hIL-1β或hIL-1β63-66A ELISA检测的结果。
图10为hIL-1βE64BK免疫小鼠后构建的噬菌体文库筛选出的克隆与hIL-1βE64BK、hIL-1β63-66A结合的ELISA结果，其中****表示p<0.0001。
图11A为SDS-PAGE结果；图11B、C分别为canakinumab和Gevokizumab与WT hIL-1β和hIL-1βQ15G结合的ELISA检测结果。
图12为A为hIL-1βQ15CK免疫小鼠后构建的噬菌体文库筛选出的克隆与hIL-1β或其突变体结合的ELISA结果，其中，**表示p<0.01,***表示p<0.001,****表示p<0.0001
图13为ELISA检测Q15CK-G8-mFc结合hIL-1β及其突变体的亲和力差异。
图14A为hIL-1β及其突变体(不同位点掺入CK或pNO2F)免疫小鼠的血清滴度；14B为来自hIL-1β及其突变体免疫小鼠的血清IgG中和实验结果；14C为来自hIL-1βQ15CK免疫小鼠的血清IgG抑制HEK-Blue IL-1R结果。
图15为不同免疫血清针对不同蛋白的效价，其中15A为两组(PTN-WT、PTN-CK)免疫小鼠血清针对各自免疫抗原的效价；15B为两组(KLH-PTN-WT、KLH-PTN-CK)免疫血清对各自免疫抗原的效价；15C为两组(KLH-PTN-WT、KLH-PTN-CK)免疫血清对KLH蛋白的效价；15D为两组(KLH-PTN-WT、HLH-PTN-CK)免疫血清对各自免疫多肽(分别为PTN-WT、PTN-CK)的效价；15E为KLH-PTN-CK免疫血清对PTN-WT、以及KLH-PTN-WT免疫血清对PTN-CK的效价；15F两组(KLH-PTN-WT、KLH-PTN-CK)免疫血清对KLH/PTN的效价比例。
图16为两组(KLH-PTN-WT、KLH-PTN-CK)免疫血清中针对KLH(16A)、PTN多肽(16B)不同亚型IgG分析。
发明详述：
本发明在此通过对使用下述定义和实施例的引用进行详细描述。所有在本文中提及的专利和公开文献的内容，包括在这些专利和公开中披露的所有序列，明确地通过提述并入本文。
如本文所用，术语“化学交联活性的基团”是指在合适的条件下，可与临近蛋白质的氨基酸残基发生共价键交联的化学基团。“化学交联活性的基团”可以包括天然氨基酸、天然氨基酸的衍生物，也可以包括非天然氨基酸。“化学交联活性的基团”的非限制性实例包括Nε-巴豆酰基、Nε-丙烯酰基或对丙烯酰胺基。“具有化学交联活性基团的非天然氨基酸”的非限制性实例包括Nε-巴豆酰基-L-赖氨酸(Nε-crotonyl-L-lysine，CK)，Nε-丙烯酰基-L-赖氨酸(Nε-acryloyl-L-lysine，AK)、对丙烯酰胺-(S)-苯丙氨酸(p-acrylamido-(S)-phenylalanine)、带有或掺有亲核基团的天然氨基酸(例如具有ε-氨基的赖氨酸)等。
如本文所用，术语“化学交联”是指具有化学交联活性的基团在合适的条件下，其与临近蛋白质的氨基酸残基的基团发生共价键交联，形成复合物。
如本文所用，术语“非天然氨基酸”是指不是20种经典氨基酸或硒代半胱氨酸或吡咯赖氨酸之一的氨基酸。可与术语“非天然氨基酸”同义使用的其他术语是“非天然编码氨基酸”、“非自然氨基酸”、“非天然存在的氨基酸”。术语“非天然氨基酸”还包括但不限于这样的氨基酸，它们通过天然编码氨基酸(包括但不限于20种常见氨基酸或吡咯赖氨酸和硒代半胱氨酸)的修饰(例如翻译后修饰)而发生，但它们本身并不天然地通过翻译复合物掺入到增长的多肽链中。“非天然氨基酸”可以包括多种官能基团或者活性基团，这能提供额外的功能和/或者活性。
如本文所用，术语“突变体抗原”是指在野生型抗原的目标表位掺有化学交联活性的基团或其衍生物形成的抗原。术语“野生型抗原”不仅包括可溶性蛋白、可溶性多肽，也包括表达在磷脂膜结构上的跨膜蛋白或多肽；“野生型抗原”可来源于动物、植物或微生物(如细菌、真菌、病毒)。
如本文所用，术语“疫苗”是指可诱导生物体产生针对目标表位的抗体的抗原。可增强生物体对抗原目标表位免疫应答的抗原也包括在本发明中。本发明疫苗的非限制实例包括在野生型抗原目标表位上掺入具有化学交联活性的基团或其衍生物形成的突变体抗原。
实施例
实验方法和步骤：
1，WT IL-1β及其突变体的表达和纯化
为了过表达WT hIL-1β、IL-1β单丙氨酸突变体(hIL-1βE64A)和包含4个丙氨酸突变的IL-1β突变体(hIL-1β63-66A)，将含有上述基因序列的pET28a表达载体转化大肠杆菌BL21(DE3)感受态细胞。挑克隆将其接种至500ml含有卡那霉素(50μg/ml)的2 x YT培养基中，37℃培养至OD600达到0.6时，加入0.5mM异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG)于30℃下诱导过夜；为过表达AK或CK掺入的hIL-1β突变体，将pEVOL-MmAKRS或pEVOL-MmCKRS分别与含有琥珀密码子(TAG)的相应hIL-1β表达质粒共转化BL21(DE3)感受态细胞，挑取单克隆接种至含有卡那霉素(50μg/ml)和氯霉素(25μg/ml)的2 x YT培养基中，培养约3-5h后，当OD600达到0.8时，加入1mM IPTG，5mM CK或10mM AK，并加入终浓度为0.2％的L-阿拉伯糖(m/v)以诱导表达UAA掺入的蛋白质；至于掺入BK或pNO2F的突变体的表达，相应的正交质粒为pUltra-pNO2RS(Tsao等，2006)或pDule-pylRS(Lang和Chin，2014)。其余步骤与掺入CK的突变体相同。随后将培养物在30℃下继续生长15小时，6000g离心10分钟收获菌体，超声裂解菌体，并在4℃条件下，13,000g离心30分钟，收集细胞裂解物上清。按照制造商的说明，在Ni-NTA树脂(GE Healthcare，17-0575-01)上纯化WThIL-1β和突变体。通过Superdex 200increase10/300GL色谱柱(GE Healthcare，10263259)在DPBS缓冲液中进一步纯化蛋白质。纯化的蛋白在-80℃下保存。
2.小鼠免疫
将WT hIL-1β或掺入非天然氨基酸的IL-1β突变体(hIL-1βE64AK、hIL-1βE64CK、hIL-1βE64BK、hIL-1βQ15CK)皮下注射6-8周大的雌性Balb/C小鼠(每组3只)。首次免疫时，将50μg抗原与弗氏完全佐剂(sigma，F5881)混合后注射，第二次和第三次免疫时，将30ug抗原和弗氏不完全佐剂(sigma，F5506)混合后注射。两次免疫间隔的时间为2周。
3.小鼠总IgG纯化
进行三次免疫后，收集小鼠血清，用等体积DPBS(pH8.0)稀释。将样品与protein A填料(GenScript，L00210)孵育3小时，10倍柱体积的DPBS洗涤后，用洗脱缓冲液(0.2M甘氨酸，0.1M NaCl，pH 2.5)洗脱与protein A结合的蛋白。洗脱后，立即添加Tris-HCl(终浓度100mM)，将pH值调节至7.5。然后使用Amicon Ultra离心柱(Merck Millipore，UFC903096)浓缩并换液(DPBS，pH 7.5)。
4.噬菌体文库构建
使用公开的方法构建噬菌体展示文库(Barbas等，1991)。为了构建小鼠免疫文库，分别用野生型hIL-1β、hIL-1βE64AK，hIL-1βE64CK，hIL-1βE64BK或hIL-1βQ15CK免疫Balb/c小鼠3次，两次免疫之间间隔2周。第三次免疫后2周，提取小鼠脾脏总RNA，将其用作反转录的模板以构建cDNA文库，使用噬菌粒载体pSEXRTL2构建产生scFv噬菌体展示文库，使用M13KO7(ΔpIII)辅助噬菌体(PROGEN，货号：PRHYPE)将文库包装成scFv-pIII噬菌体。
5.噬菌体的产生
将携带噬菌粒(展示scFv-pIII)的大肠杆菌XL1-Blue细胞接种到20ml的2 X YT培养基中，加入氨苄青霉素(100μg/ml)和四环素(15μg/ml)，并在37℃，220rpm培养。当OD600达到0.5时，加入感染复数(MOI)＝20的M13KO7(ΔpIII)辅助噬菌体，于37℃，120rpm孵育1小时后，离心，将沉淀物重悬在40ml的2 X YT培养基(100μg/ml氨苄青霉素、15μg/ml四环素和50μg/ml卡那霉素)于30℃，250rpm培养13小时。将培养物4000g离心10分钟，将上清液转移至新管中，10,000g离心20分钟去除细胞碎片。加入5X噬菌体沉淀缓冲液[100g PEG 8000，73.3g NaCl溶解在500ml ddH2O中]，冰上孵育4小时。4℃下10,000g离心20分钟收集噬菌体，并用1ml DPBS溶解，室温下孵育15分钟。0.22μm滤膜过滤噬菌体，4℃保存。
6.噬菌体淘选
常规噬菌体淘选：将WT IL-1β抗原(1μg)在DPBS中的板孔中于4℃包被过夜，然后用200μl含3％脱脂奶粉的DBPS室温封闭2小时。用DPBST清洗两次后，加入10 10pfu从野生型hIL-1β，hIL-1βE64AK，hIL-1βE64CK，hIL-1βE64BK或hIL-1βQ15CK免疫小鼠构建的文库中获得的噬菌体，室温孵育2小时，用DPBST洗涤10次(每次间隔3分钟)，加入1mg/ml胰蛋白酶(Gibco)消化回收与抗原结合的噬菌体。
化学交联噬菌体淘选：将hIL-1βE64AK(1μg)于96孔板中4℃包被过夜后，用200μl含3％BSA的DPBS室温封闭2小时，加入10 10pfu来自hIL-1βE64AK免疫噬菌体文库 的噬菌体(含1％的BSA，1mM EDTA，pH8.8)37℃孵育48小时。在严格条件下洗涤孔，包括：含有10mM DTT的DPBS洗涤2次(共5分钟)；DPBST洗涤10次(共20分钟)；0.15％SDS溶液洗涤2次(共3分钟)；DPBS洗涤10次(总共20分钟)；酸性缓冲液(0.2M甘氨酸，pH 2.2)洗一次(总共3分钟)；DPBST洗涤10次(总共20分钟)；DPBS两次(总共5分钟)。洗涤后，加入1mg/ml胰蛋白酶(Gibco)孵育20分钟释放与抗原结合的噬菌体。将收集的噬菌体感染大肠杆菌XL1-Blue以产生噬菌体。
随机挑选淘选后的阳性克隆测序，并进行同源性分析。使用ClustalW(MEGA-X；DNA Weight Matrix：IUB；Gap opening penalty:15.00；Gap Extension penalty:6.66)对所有scFv进行序列比对，并计算出最大似然系统树。
7.步骤6淘选获得的阳性克隆的Scfv片段与mFc融合的表达质粒的构建和表达
将步骤6淘选获得的阳性克隆CL-E2、E64AK-A9、E64AK-F4、E64CK-B9、E64CK-A5、E64CK-G9或Q15CK-G8的scfv片段通过linker与小鼠IgG2a的Fc连接后(分别命名为CL-E2-mFc、E64AK-A9-mFc、E64AK-F4-mFc、E64CK-B9-mFc、E64CK-A5-mFc、E64CK-G9-mFc、Q15CK-G8-mFc)，克隆进pFuse表达载体中。培养HEK 293F细胞(Thermo Scientific，R79007)，将上述构建的scFv-mFc表达质粒和PEI以1：2.5(质量比)的比例转染细胞(2.5×10 6细胞/ml)。当细胞活力降至75％以下时，收集细胞培养上清，，过在DPBS中进行预平衡后的protein A填料(GenScript，L00210)二次，用10倍柱体积的DPBS洗涤后，用洗脱缓冲液(0.2M甘氨酸，0.1M NaCl，pH 2.5)洗脱与填料结合的蛋白。洗脱后，立即添加Tris-HCl(终浓度100mM)，将pH值调至7.5。然后使用Amicon Ultra离心柱(Merck Millipore，UFC903096)浓缩并换液(DPBS，pH 7.5)，SEC纯化(色谱柱：Superdex 200 increase 10/300 GL，GE Healthcare，10263259)。
8.ELISA
将抗原(100ng)在96孔ELISA板(Corning Costar，2592)上4℃包被过夜，用200μl含3％脱脂奶粉的DPBS于37℃封闭2小时。加入抗体或噬菌体在含3％脱脂奶粉的DPBST溶液中于37℃孵育2小时。200μl DPBST洗涤四次后，加入辣根过氧化物酶(HRP)偶联的检测抗体，室温下孵育1小时。200μl DPBST洗涤五次后，加入100μl TMB(Biolegend，002023)显色试剂，于室温孵育10-30分钟，酶标仪(BMG LABTECH，
Figure PCTCN2022114250-appb-000001
)读取数值。
竞争ELISA：100ng WT hIL-1β于4℃在ELSIA板上包被过夜，含3％BSA的DPBS于37℃封闭2小时后，加入梯度稀释的canakinumab(含3％BSA的DPBST)室温孵育1小 时。孵育后加入100nM步骤7获得的E64AK-F4-mFc、0.2nM E64CK-A5-mFc或10nM E64CK-G9-mFc，室温孵育1小时。洗涤后，加入HRP-conjugated goat anti-mouse IgG Fc(1：5000)室温下孵育1小时，加入TMB显色试剂(Biolegend，002023)室温下孵育10-30分钟，然后使用酶标仪(BMG LABTECH，
Figure PCTCN2022114250-appb-000002
)读取数值。
通过双向ANOVA分析比较ELISA数据，然后使用Prism 6.0(GraphPad软件)进行多次比较。所有的P值都是使用GraphPad Prism 6.0计算的，具有以下含义：n.s.p>0.05；*p<0.05；**p<0.01；***p<0.001；****p<0.0001。每个实验的统计分析细节可在图中和图例中找到。
9.竞争噬菌体ELISA
100ng WT hIL-1β于4℃在ELSIA平板上包被过夜，含3％BSA的DPBS封闭2小时后，加入梯度稀释的噬菌体(从10 8pfu开始，10倍稀释)于3％BSA的DPBST中室温孵育2小时，加入300nM canakiumab再孵育1小时。洗涤后，加入HRP-conjugated mouse anti-M13(抗体)(1：2000)室温下孵育1小时。加入100μl TMB显色试剂(TMB；Biolegend，002023)室温孵育10-30分钟后，酶标仪(BMG LABTECH，
Figure PCTCN2022114250-appb-000003
)读取数值。通过双向ANOVA分析比较ELISA数据，然后使用Prism 6.0(GraphPad软件)进行多次比较。所有的P值都是使用GraphPad Prism 6.0计算的，具有以下含义：n.s.p>0.05；*p<0.05；**p<0.01；***p<0.001；****p<0.0001。每个实验的统计分析细节可在图中和图例中找到。
10.Western blot
将样品与含有20mM DTT和2％SDS的上样缓冲液混合，95℃加热10分钟后，跑SDS-PAGE胶，再电转PVDF膜(Bio-Rad，1620177)，将电转后的PVDF膜在含5％脱脂奶粉的DPBS中封闭2小时后，加入抗体孵育2小时，DPBST洗涤四次后，加入HRP标记的二抗(1：5000)室温下孵育1小时，DPBST洗涤4次后，加入ECL试剂(Thermo Fisher Scientific，35055)显色，并于Tanon 5200读取图像。
11.化学交联反应
将8μM hIL-1βE64AK，hIL-1βQ15AK，hIL-1βE64CK或hIL-1βQ15CK与4μM相应抗体(CL-E2-mFc，E64AK-A9-mFc，E64CK-A5-mFc和E64CK-G9-mFc)在碱性DPBS(含1mM EDTA，pH8.8)条件下，于37℃孵育2天(48h)或5天。为了进行噬菌体化学交联反应，将10 8pfu噬菌体(CL-E2)与8μM hIL-1βE64AK在相同的碱性条件下孵育2天。所有反应均在无菌条件下进行。
12.hIL-1β中和实验
将70％密度的HEK-Blue TM IL-1R细胞(Invivogen，hkb-il1r)用预热的PBS洗涤两次，轻敲瓶底使细胞从瓶上脱落。将细胞重悬于预热的DMEM培养基(含10％热灭活的FBS)(细胞密度330,000个细胞/ml培养基)。在单独的96孔中，将25μl重组人IL-1β(0.8ng/ml)与25μl以1：5梯度稀释的纯化血清IgG(起始浓度4μM)室温下孵育30分钟。每孔加入150μl HEK-Blue IL-1R细胞悬浮液(约50,000个细胞)。将96孔细胞培养板于5％CO2，37℃细胞培养箱中孵育过夜。取20μl细胞培养上清与180μl QUANTI-Blue TM(Invivogen)于37℃孵育30分钟至3小时不等。使用酶标仪
Figure PCTCN2022114250-appb-000004
于655nm处检测分泌的胚胎碱性磷酸酶(SEAP)。
实验结果
1，AK掺入的hIL-1β可诱导产生具有交联活性的抗体
AK是一种赖氨酸衍生的非天然氨基酸，其侧链上的丙烯酰胺基可与邻近亲核基团形成共价键(Furman et al.,2014)。我们推测掺入AK的抗原免疫小鼠后，在B细胞超频突变过程中，小鼠进化出可与AK掺入的抗原共价交联的抗体。
我们选用人IL-1β验证我们的推测。依据hIL-1β-canakinumab(Fab)复合物晶体结构(PDB：4G6J)，选择hIL-1β与canakinumab的关键结合位点E64进行AK插入。将编码MmAKRS/tRNA CUA正交对的pEvol载体与pET28a-hIL-1βE64TAG共转化E.coli BL21(DE3)，镍柱及SEC纯化hIL-1βE64AK突变体蛋白，ESI-MS质谱鉴定后，免疫Balb/c小鼠，三次免疫结束后，酶联免疫吸附法(ELISA)检测血清效价。结果显示，小鼠血清结合WT hIL-1β和hIL-1βE64AK(图1A)的效价相当(大约1:10 5)。分离小鼠脾脏，提取其总RNA，反转录成cDNA文库，构建展示抗体scfv噬菌体库(Barbas et al.,1991)。
接着，我们对hIL-1βE64AK免疫噬菌体文库进行了化学交联淘选：包被hIL-1βE64AK(1ug/孔)于96孔板，4℃孵育过夜，含3％BSA的DPBST(0.5％Tween-20，DPBS)室温封闭3小时。DPBS(pH7.5)洗一次，加入噬菌体文库(10 10pfu)(DPBS,pH8.8)于37℃孵育48h后，进行如下洗脱：1)含10mM DTT的DPBS洗2次；2)DPBST洗10次；3)0.15％SDS洗2次；4)DBPS洗10次；5)甘氨酸溶液(pH2.2)洗1次；6)DPBST洗10次，DPBS洗2次。最后胰蛋白酶消化。经过2轮的化学交联淘选后，随机挑选其中28个output阳性克隆测序，序列同源性分析显示17个克隆具有相同的氨基酸序列。挑选其中一个克隆(命名为CL-E2)用于包装单克隆噬菌体抗体。将CL- E2噬菌体与hIL-1βE64AK于DPBS(pH8.8)孵育48小时后，WB检测，发现了大小符合hIL-1βE64AK+pIII-scfv复合物大小的特异性条带(图2A)。
为进一步证明scfv与hIL-1βE64AK之间的结合是否为共价结合，我们将CL-E2与鼠IgG2a的Fc融合构建了CL-E2-mFc抗体，将其与hIL-1βE64AK在前述的化学交联条件下孵育，用anti-his-tag(图2B)或anti-Fc(图2C)进行WB检测，结果发现均检测到了交联条带。而在相同的条件下，未见到CL-E2-mFc与WT IL-1β交联。此外，我们将AK掺入到hIL-1βQ15表位后也未检测到其与CL-E2-mFc交联(图2B)。结合E64表位的Canakinumab与hIL-1βE64AK在前述的化学交联条件下也未发生交联(图2D)。综合上述结果可知，抗原抗体的交联反应是抗体序列特异性和表位位点特异性的。CL-E2、CL-E2-mFc与WT IL-1β或hIL-1βE64AK具有相似的亲和力(图3A和3B)，可能是因为机体在B细胞克隆选择的过程中，结合hIL-1βE64表位的抗体库进化出来能够与hIL-1βE64AK化学交联的B细胞，共价结合导致该B细胞大量增值从而富集大量能够共价结合hIL-1βE64表位的特异性抗体CL-E2，随后通过上述特异的噬菌体化学淘选方法被我们所鉴定。
表1 序列表
  核酸序列(SEQ ID NO.) 氨基酸序列(SEQ ID NO.)
hIL-1β 1 2
CL-E2VL 3 4
CL-E2VH 5 6
E64AK-A9VL 7 8
E64AK-A9VH 9 10
E64AK-F4VL 11 12
E64AK-F4VH 13 14
E64CK-B9VL 15 16
E64CK-B9VH 17 18
E64CK-G9VL 19 20
E64CK-G9VH 21 22
E64CK-A5VL 23 24
E64CK-A5VH 25 26
Q15CK-G8VL 27 28
Q15CK-G8VH 29 30
mFc 31 32
2.AK掺入的抗原可以诱导表位定向的抗体反应
AK掺入的抗原可以诱发具有化学交联活性抗体的产生，我们推测这种独特的机制可以用于表位定向的结合AK掺入表位的抗体富集。为了验证这种可能性，我们评估了hIL-1βE64AK免疫噬菌体文库中与hIL-1βE64表位结合的抗体的丰度。经过3轮的常规淘选(基于亲和力的筛选以及常规洗脱条件)，从获得的output克隆中随机挑选96个测序并进行scfv序列同源性分析。与第一轮和第三轮淘选获得的克隆数相比，经过2轮淘选后的output克隆兼具序列多样性和较高亲和力。
将第二轮淘选得到的output克隆进行结合表位的鉴定，根据scfv氨基酸序列(一致性<98％)，将output克隆分为7簇。在80个序列中，E64AK-A9克隆出现了46次，其与WT IL-1β和hIL-1βE64AK的亲和力相似(图4A)。接着，我们构建了E64AK-A9-mFc融合抗体，其与WT IL-1β(1.6±0.2nM)和hIL-1βE64AK(1.2±0.2nM)具有类似的亲和力(图4B)，并且，在前述的化学交联条件下，E64AK-A9-mFc与hIL-1βE64AK形成共价交联复合物(图4C、4D)，可能因为E64AK-A9-mFc是通过基于非共价结合的常规淘选方法筛选获得的，其与hIL-1βE64AK的交联反应看起来不是特别明显。然而，这些结果足以说明hIL-1β上E64AK的化学活性诱导了结合该表位的抗体在其附近富集。
从剩余的抗体簇中选择具有代表性scfv抗体与3个仅出现一次的scfv抗体分别包装成单克隆噬菌体。为便于表位结合鉴定，我们分别构建了IL-1β单丙氨酸突变体(hIL-1βE64A)和包含4个丙氨酸突变的IL-1β突变体(hIL-1β63-66A)。如果这些噬菌体结合在E64位表位附近，则其结合hIL-1βE64AK和丙氨酸突变体的亲和力应该有显著的差异。ELISA检测这些单克隆噬菌体对WT hIL-1β、hIL-1βE64AK和丙氨酸突变体的亲和力，结果显示，所有11个噬菌体均能与WT IL-1β结合(图4A)，除7个单克隆噬菌体(E64AK-A4,E64AK-B2,E64AK-D11,E64AK-F4,E64AK-G6,E64AK-H3and E64AK-H4)对hIL-1β63-66A的亲和力显著下降(与对hIL-1βE64AK相比)外(图5A)，其余的噬菌体结合WT-IL-1βE64AK、hIL-1βE63-66A的亲和力类似(图5B)。不与hIL-1β63-66表位结合的IL-1β高亲和力抗体Gevokizumab(Blech et al.,2013)与WT hIL-1β和hIL-1β63-66A的亲和力类似(图6A)，提示63-66A突变对其构象没有太大影响。上述结果说明，与hIL-1β63-66A亲和力降低(相比于hIL-1βE64AK)的7个噬菌体抗体结合hIL-1βE64表位。我们选择了对WT hIL-1β和hIL-1β63-66A突变体亲和力差异最大的E64AK-F4构建了E64AK-F4-mFc融合抗体，ELISA结果显示E64AK-F4-mFc结合WT hIL-1β和hIL-1βE64AK的亲和力类似(分别为K d＝7.8±0.5nM和6.8±0.6nM)，但是不结合 hIL-1β63-66A，并且与hIL-1βE64A的亲和力显著降低(图6B)。另外，E64AK-F4噬菌体、E64AK-F4-mFc融合抗体与hIL-1β的结合均可被Canakinumab竞争抑制(IC 50＝2.1±0.9nM)，进一步说明E64AK-F4结合在hIL-1βE64表位(图6C、D)。这些实验结果显示hIL-1βE64AK可以诱发针对目标表位的抗体反应。
3.CK掺入的抗原也可能诱发表位特异性的抗体
Nε-crotonyl-L-lysine(CK)也是一种lysine衍生的非天然氨基酸，和AK相比有较弱的化学交联活性。受上述结果启发，我们推测CK插入的抗原免疫小鼠也可以诱导抗体靶向特定表位。我们用遗传编码技术表达了hIL-1βE64CK，将其免疫小鼠后构建了抗体噬菌体文库。经过2轮的常规淘选，随机挑选96个output克隆测序，其中84个克隆具有完整的鼠scfv抗体序列。与hIL1βE64AK免疫获得的噬菌体抗体序列(其中一簇抗体含有接近一半的克隆)不同，hIL1βE64CK免疫获得的噬菌体抗体序列平均分布在5个抗体簇中，造成如上差异可能是由于CK的化学交联活性比AK弱。从每一簇各挑一个代表性序列包装单克隆噬菌体，结果显示，这些单克隆噬菌体均能与WT hIL-1β结合(图7A)。与hIL-1βE64CK相比，E64CK-A5/E10(cluster 1)和E64CK-G9/C9(cluster 2)与hIL-1β63-66A的亲和力显著降低(图7B)，提示这两簇抗体可能结合在hIL-1β的E64表位。有意思的是，E64CK-A4结合hIL-1β63-66A的亲和力显著高于与hIL-1βE64CK的亲和力(图7B)，推测E64CK-A4在该表位的结合可能不是通过与63-66氨基酸的直接相互作用。然而，两者之间的亲和力差异也可以说明该簇抗体结合hIL-1β的E64表位。与E64CK-A5/E10,E64CK-G9/C9,E64CK-A4序列同源性较低的E64CK-H11和E64CK-B9结合hIL-1βE64CK和hIL-1β63-66A之间的亲和力没有显著的差异(图7C)。由于E64AK-A9结合E64表位(图4C)，且E64CK-H11与hIL-1β的结合可被E64AK-A9-mFc竞争抑制(图7D)，提示E64CK-H11(序列与E64AK-A9同源)也结合E64表位，虽然其亲和力不依赖(至少不是完全依赖)于该区域的结合，这也解释了为什么其结合hIL-1βE64CK和hIL-1β63-66A的亲和力几乎没有差异。同样的，由于E64AK-G6特异性结合E64表位(图5A)，鉴于E64CK-B9与E64AK-G6序列的同源性，我们预计E64CK-B9应该也结合E64表位。与预期的一致，E64CK-B9-mFc能竞争抑制E64AK-G6和E64AK-A2与hIL-1β的结合(图7E)，提示这些克隆(E64CK-B9，E64AK-G6，E64AK-A2)均结合在hIL-1β上相似的区域。
接着，我们表达纯化了E64CK-G9-mFc和E64CK-A5-mFc，在蛋白水平上验证其结合表位。与噬菌体ELISA结果一致，E64CK-A5-mFc和E64CK-G9-mFc结合hIL-1β63- 66A的亲和力(图8A,B)显著降低。另外，canakinumab能与E64CK-A5-mFc和E64CK-G9-mFc竞争结合hIL-1β(图8C，D)，与噬菌体竞争ELISA结果一致(图8E，F)。
为了确定hIL-1βE64CK诱导的表位特异性的抗体的富集是否是由于CK的化学交联活性所致，我们将E64CK-G9-mFc或E64CK-A5-mFc分别与hIL-1βE64CK在前面所述的交联条件下孵育48h，WB未检测到交联复合物(图8H)。但是，将孵育时间延长至5天后，检测到一条弱的交联条带(图8H)。相反，将这两个抗体与hIL-1βE64AK孵育48h后均检测到交联复合物条带(图8G,H)；这两个抗体与hIL-1βQ15AK的孵育未能检测到交联复合物条带(图8G,H)。在同样的交联条件下，canakinumab和hIL-1βE64CK也未发生交联反应(图2D)。综合上述结果说明，hIL-1βE64CK或hIL-1βE64AK免疫的小鼠在克隆选择和B细胞超频突变过程中，通过具有位点特异性交联活性的抗原特性能够直接靶向目的表位产生抗体反应和富集。
4.表位特异性的抗体反应是由于IL-1β掺入的AK或CK的化学交联活性所致
为了排除针对hIL-1βE64CK和hIL-1βE64AK表位特异性的抗体反应的诱发是由于hIL-1βE64表位的序列特异性所致，我们将WT IL-1β免疫小鼠后，构建免疫噬菌体抗体文库，按照前面所述的步骤进行了常规淘选。经过2轮的淘选，随机挑选96个克隆测序，根据序列的同源性将其分为12个抗体簇。87个克隆为鼠全长scfv序列，在这些噬菌体中，只有一个克隆(WT E21)可结合在E64表位(图9)。此外，我们还将无交联活性的非天然氨基酸Nε-Boc-L-Lysine(BK)在hIL-1β的E64位掺入，将hIL-1βE64BK免疫小鼠后构建噬菌体文库，通过2轮的淘选，随机挑选72个克隆测序，结果显示只有一簇抗体(E64BK-A11,频次为2)看起来结合hIL-1βE64表位(图10)。综合上述结果，表位定向的抗体反应可能是由于目标表位内掺入的AK和CK的化学交联活性所致。
5.CK诱导的表位特异性抗体的反应不依赖于表位序列
我们的数据显示CK或AK掺入的抗原能有效诱导针对E64附近表位的抗体反应。为了研究该机制是否不依赖于表位序列，我们选择hIL-1β的另一个表位进行CK的插入。Q15是IL-1β与IL-1RI结合的重要位点(Evans et al.,1995)。我们按照前面所述的步骤构建、表达、纯化了hIL-1βQ15CK，将其免疫小鼠后构建噬菌体抗体文库。经过2轮的淘选，随机挑选96个克隆测序，根据氨基酸序列的同源性将其分为12个抗体簇，其中有89个output克隆含有完整的鼠scfv序列。从每个抗体簇中选择1-2个代表性的克隆以及3个不在抗体簇内的克隆包装成单克隆噬菌体。为便于表位分析，我们构建并表达纯化 了不与IL1RI结合的hIL-1βQ15G突变体，并对其性质进行了鉴定(图11A,B,C)。噬菌体ELISA结果显示16个噬菌体克隆均能与WT hIL-1β交叉结合，其中8个与hIL-1βQ15CK的亲和力显著降低(图12)。这些结果提示这8个克隆所在的抗体簇结合hIL-1βQ15表位。值得注意的是，这些结合hIL-1βQ15CK或hIL-1βQ15G亲和力无明显差异的噬菌体克隆可能也会结合hIL-1βQ15表位，就像前面所述的E64表位抗体筛选情况一样。然而，通过单一的表位鉴定方法发现这些克隆均能结合在hIL-1βQ15表位，如果加权每簇抗体出现的频次，那么结合hIL-1βQ15表位的抗体在96个分析的克抗体中占比60％。
接着，我们构建和纯化了Q15CK-G8scfv-Fc融合蛋白(该克隆所在的抗体簇抗体序列频次最高)，其与hIL-1β和hIL-1βQ15CK的亲和力分别为3.8±0.9nM和2.4±0.6nM(Figure 6C)，而与hIL-1βQ15G的亲和力下降了约10倍(图13)，这些结果与噬菌体ELISA结果一致(图12)。综合上述结果，我们推断CK诱导的表位定向的抗体反应不依赖于抗原表位序列。
6.CK掺入的IL-1β有望用于开发亚单位疫苗
IL-1β是一种结合IL-1RI和IL1RII的促炎症细胞因子(Afonina et al.,2015)。阻断IL-1β与IL-1RI信号通路可用于治疗一系列自体免疫性疾病，如II型糖尿病、类风湿性关节炎、痛风等(Dinarello et al.,2012)。基于IL-1β的一些疫苗构架已被用于评估其作为疫苗的可能性，但是具体效果有待临床验证(Cavelti-Weder et al.,2016；Spohn et al.,2008；Spohn et al.,2014)。虽然可以通过工程化亚单位疫苗或者将亚单位疫苗与佐剂联用增强传统亚单位疫苗的抗体反应，但是产生的中和性抗体的滴度在总抗体滴度中所占的比例非常低，并且通过目前现有的手段难以提高该比例。
我们推测如果在IL-1β与IL-1RI结合的关键位点掺入CK，该突变体抗原引发的抗体反应将靶向该突变表位富集抗体，从而可能中和IL-1β的IL1RI受体激活活性。根据hIL-1β-IL1RI(ECD)复合物的结构(Vigers et al.,1997)，Q15,G33,N53和I106均位于与受体结合界面，我们在这些位点分别掺入CK，构建相应的突变体(分别命名为hIL-1βG33CK,hIL-1βN53CK和hIL-1βI106CK)，将这些突变体分别免疫小鼠，第3次免疫后10天检测血清中IgG抗体滴度。结果显示WT hIL-1Β和CK掺入的IL-1β突变体免疫的小鼠其IgG抗体滴度相当(～1:10 6,图14A)。接着，我们收集了小鼠血清，protein A纯化后，评估每组IgG的中和效果。WT IL-1β和DBPS免疫小鼠的IgG均未观察到任何抑制效果，而来自hIL-1βQ15CK,hIL-1βG33CK,hIL-1βN53CK或hIL-1βI106CK免疫小鼠的IgG 均能显著抑制IL-1β诱导的HEK-Blue IL-1R的激活(图14B)，其中，来自hIL-1βQ15CK免疫小鼠的IgG的抑制效果最强，IC 50约为137.5±0.1nM，相同实验条件下，与IL-1β具有高亲和力的canikizumab，其IC50为4.7±0.1nM(图14C)。
将CK掺入非IL1RI结合的K138表位形成的hIL-1βK138CK(图14A)免疫小鼠后，从血清中纯化得到的总IgG并不能抑制IL-1β激活HEK-Blue IL-1R。将pNO2F掺入到Q15位点，构建hIL-1βQ15pNO2F免疫小鼠后，其血清中总IgG并未表现出中和活性(图14B)，与之前文献报道中所述的pNO2F掺入的TNFa仅增加了总抗体滴度，但是并没有提高对掺入表位的抗体的滴度一致(Grünewald et al.,2008；Kessel et al.,2014)。结果表明，在IL-1β/IL1RI界面掺入CK可诱导高滴度的中和抗体，有效阻断IL-1β与IL1RI的结合，具有IL-1β疫苗开发的具大潜力。
非天然氨基酸AK/CK标记新冠RBD设计产生针对特定表位的中和抗体和疫苗应用实验方法与步骤
1，新冠病毒S蛋白RBD区域及其突变体的表达和纯化
我们根据文献中新冠病毒RBD和ACE2相互作用界面，在突变株β和γ选择K417位点，δ突变株和Lambda突变株L452选择位点Y453插入AK，在RBD远离与ACE2作用界面的位置，选择K386位点插入AK作为阴性对照，参照前面实施例“WT IL-1β及其突变体的表达和纯化”的方法，在E.coli中表达了AK或CK插入的新冠病毒S蛋白RBD区域，将RBD蛋白免动物后，通过化学共价交联刺激B细胞抗体体内进化，产生针对Acrk/Kcr插入位点附近表位附近的抗体，使产生的抗体大多数针对与ACE2结合的表位，从而阻断RBD与ACE2的结合，提高疫苗的阻断效果。
另外，我们将前述插入AK或CK的新冠病毒S蛋白RBD区域包被成为完整的假病毒，结果显示，假病毒能够侵染293T-ACE2细胞；此外利用假病毒纳米颗粒表面展示的多个S蛋白和S蛋白上AK/CK能够诱导产生针对AK或CK插入位点附近表位附近的抗体，使产生的抗体大多数针对与ACE2结合的表位，从而阻断RBD与ACE2的结合，提高疫苗的阻断效果。
掺入具有化学交联基团的多肽提高针对多肽的免疫应答
选取三角褐指藻蛋白一段20个氨基酸的多肽(序列：AKPAADNEQSIKPKKKKPKM)(命名为PTN-WT)、以及第12位氨基酸侧链带有Nε-巴豆酰基的突变体多肽(序列：APKAADNEQSIK(cr)PKKKKPKM)(命名为：PTN- CK)，在多肽N端添加一个cys，以便于下一步的偶联。将PTN-WT和PTN-CK使用SMCC(一种N-羟基琥珀酰亚胺(NHS)活性酯和马来酰亚胺的双功能偶联剂)通过N端的Cys分别偶联到血蓝蛋白KLH，制备出KLH-PTN-WT和KLH-PTN-CK。将合成的多肽PTN-WT、PTN-CK，KLH-PTN-WT,KLH-PTN-CK分为四组，作为免疫原免疫小鼠BAlb/C，初次免疫所用佐剂为完全弗氏佐剂，免疫量为30ug。再次免疫所用佐剂为不完全弗氏佐剂。在免疫后35天取血清，根据实验的需求，采用不同的蛋白(如PTN-WT、PTN-CK、KLH-PTN-WT或KLH-PTN-CK)作为包被蛋白测效价。
结果如图15所示：PTN-WT免疫组血清为检测到与PTN-WT结合的抗体，而PTN-CK免疫组血清，其效价与PTN-WT免疫组相比具有显著的提升(图15A)，提示进行巴豆酰修饰后的PTN(PTN-CK)能够增强PTN多肽本身的免疫原性；KLH-PTN-WT或KLH-PTN-CK免疫组血清对其各自免疫原的效价均能达到较高水平，且两组的效价类似(图15B)；对KLH蛋白的效价，KLH-PTN-WT、KLH-PTN-CK免疫组差别很大，且KLH-PTN-WT组针对KLH的效价明显高于KLH-PTN-CK组针对KLH的效价(图15C)；KLH-PTN-WT免疫血清中针对PTN-WT的抗体显著低于KLH-PTN-CK免血清中针对PTN-CK的抗体(图15D)，但是KLH-PTN-WT免疫血清中能够结合PTN-CK的抗体含量，与KLH-PTN-CK免疫血清中能够结合PTN-WT的抗体含量，基本接近，提示两组血清能够产生交叉反应(图15E)。
进一步将两组(KLH-PTN-WT、KLH-PTN-CK)免疫后的血清针对KLH和PTN多肽部分的效价进行比较(图15F)，KLH-PTN-WT组产生针对KLH和PTN多肽部分的抗体，大部分针对KLH。KLH-PTN-Kcr组产生针对KLH和PTN多肽部分的抗体，大部分针对PTN多肽部分。
此外，我们进一步发现两组免疫血清中的不同亚型抗体含量也表现出明显差异(图16)，KLH-PTN-WT组产生针对KLH蛋白的抗体主要抗体类型为IgG1，而KLH-PTN-Kcr组产生针对KLH蛋白的抗体类型几乎检测不到(图16A)；KLH-PTN-WT组产生针对PTN-WT的抗体主要抗体类型为IgG1，而KLH-PTN-Kcr组产生针对PTN-CK的抗体类型主要包括IgG1，IgG2a，IgG2b(图16B)

Claims (30)

  1. 一种用于免疫动物的试剂，其包含：
    -免疫有效量的突变体抗原，其是在野生型抗原中的一个或多个目标表位上掺有具有化学交联活性的基团或其衍生物的抗原，和
    -生理上可接受的载体，
    其中该试剂施用于动物后可刺激或增强该动物中针对所述一个或多个目标表位的抗体的生成。
  2. 权利要求1所述的试剂，其中所述具有化学交联活性的基团为天然氨基酸或非天然氨基酸。
  3. 权利要求1所示的试剂，其中所述具有化学交联活性的基团为Nε-巴豆酰基或Nε-丙烯酰基。
  4. 权利要求2所述的试剂，其中所述具有化学交联活性基团的非天然氨基酸为Nε-巴豆酰基-L-赖氨酸(CK)或Nε-丙烯酰基-L-赖氨酸(AK)。
  5. 权利要求1-4中任一项所述的试剂，其中所述具有化学交联活性的基团或其衍生物是通过扩展基因密码子或者化学合成掺入的。
  6. 权利要求1-5中任一项所述的试剂，其中所述动物为啮齿类动物、非人哺乳动物或哺乳动物。
  7. 权利要求1-6中任一项所述的试剂，其中所述野生型抗原为可溶性蛋白、可溶性多肽、表达在磷脂膜结构上的跨膜蛋白、或表达在磷脂膜结构上的多肽。
  8. 权利要求1-7中任一项所述的试剂，其中所述试剂是抗体制备用试剂。
  9. 权利要求1-7中任一项所述的试剂，其中所述试剂是预防或治疗用试剂。
  10. 权利要求9所述的试剂，其中所述试剂是疫苗组合物。
  11. 一种调节抗原的免疫原性的方法，包括在抗原的一个或多个目标表位上掺入具有化学交联活性的基团或其衍生物。
  12. 权利要求11的方法，其中所述调节导致抗原激发抗体的分布(profile)改变。
  13. 一种提高动物针对抗原的目标表位的免疫应答的方法，所述方法包括：对所述动物施用一定量的突变体抗原，所述突变体抗原为野生型抗原的目标表位上掺有具有化学交联活性的基团或其衍生物的抗原。
  14. 一种筛选针对野生型抗原的目标表位的抗体的方法，所述方法包括如下步骤：
    (a)提供突变体抗原，所述突变体抗原为在野生型抗原的目标表位上掺有具有化学交联活性的基团或其衍生物的抗原；
    (b)对动物施用步骤(a)所述的突变体抗原。
  15. 权利要求14所述的方法，其进一步包括：
    (i)从所述动物中分离血清；
    (ii)利用所述野生型抗原从所述血清中筛选出特异性结合目标表位的抗体；
    或者
    (i)从所述动物中分离B细胞，将所述B细胞与骨髓瘤细胞融合以产生杂交瘤细胞；
    (ii)利用所述野生型抗原从所述杂交瘤细胞的培养上清筛选出特异性结合目标表位的抗体；
    或者
    (i)从所述动物中分离B细胞，利用所述B细胞构建抗体文库；
    (ii)利用所述野生型抗原从所述抗体文库中筛选出特异性结合目标表位的抗体。
  16. 权利要求14所述的方法，其进一步包括：
    (i)从所述动物中分离血清；
    (ii)将所述突变体抗原与所述血清在一定的条件下孵育，以便所述突变体抗原与抗体共价交联形成突变体抗原-抗体复合物；
    或者
    (i)从所述动物中分离B细胞，将所述B细胞与骨髓瘤细胞融合以产生杂交瘤细胞；
    (ii)将所述突变体抗原与所述杂交瘤细胞的培养上清在一定的条件下孵育，以便所述突变体抗原与抗体共价交联形成突变体抗原-抗体复合物；
    或者
    (i)从所述动物中分离B细胞，利用所述B细胞构建抗体文库；
    (ii)将所述突变体抗原与所述抗体文库在一定的条件下孵育，以便所述突变体抗原与抗体共价交联形成突变体抗原-抗体复合物；
    以及
    (iii)以一定的条件洗脱除去未与所述突变体抗原共价交联的抗体，并释放与所述突变体抗原共价交联的抗体；
    (iv)利用野生型抗原，从所述与突变体抗原共价交联的抗体中进一步筛选特异性结合目标表位的抗体。
  17. 权利要求16所述的方法，其中所述孵育的条件为：pH8.8的溶液，37℃，时间24-48h。
  18. 权利要求16所述的方法，其中所述的洗脱条件为：
    (a)用高pH洗脱缓冲液进行碱性洗脱；
    (b)用低pH洗脱缓冲液进行酸性洗脱。
  19. 权利要求16所述的方法，其中所述释放为通过酶消化释放与所述抗原共建交联的抗体。
  20. 权利要求11-19中任一项所述的方法，其中所述具有化学交联活性的基团为天然氨基酸或非天然氨基酸。
  21. 权利要求11-19中任一项所述的方法，其中所述具有化学交联活性的基团为Nε-巴豆酰基或Nε-丙烯酰基。
  22. 权利要求20所述的方法，其中所述非天然氨基酸为Nε-巴豆酰基-L-赖氨酸(CK)或Nε-丙烯酰基-L-赖氨酸(AK)。
  23. 权利要求11-19中任一项所述的方法，其中所述具有化学交联活性的基团或其衍生物是通过扩展基因密码子或者化学合成掺入的。
  24. 权利要求11-19中任一项所述的方法，其中所述动物为啮齿类动物、非人哺乳动物或哺乳动物。
  25. 权利要求11-19中任一项所述的方法，其中所述抗原为可溶性蛋白、可溶性多肽、表达在磷脂膜结构上的跨膜蛋白、或表达在磷脂膜结构上的多肽。
  26. 权利要求14-19中任一项所述的突变体抗原在用于制备预防和治疗疾病的疫苗中的用途。
  27. 根据权利要求11-25任一项所述的方法获得的抗体。
  28. 根据权利要求11-25中任一项的方法获得的抗血清，其包含针对所述目标表位的抗体。
  29. 权利要求28所述的抗体，其中所述抗体可特异性结合IL-1b。
  30. 权利要求29所述的抗体，其中所述抗体包括如下VH和VL：
    a)SEQ ID NO.6所示的VH和SEQ ID NO.4所示的VL；
    b)SEQ ID NO.10所示的VH和SEQ ID NO.8所示的VL；
    c)SEQ ID NO.14所示的VH和SEQ ID NO.12所示的VL；
    d)SEQ ID NO.18所示的VH和SEQ ID NO.16所示的VL；
    e)SEQ ID NO.22所示的VH和SEQ ID NO.20所示的VL；
    f)SEQ ID NO.26所示的VH和SEQ ID NO.24所示的VL；
    g)SEQ ID NO.30所示的VH和SEQ ID NO.28所示的VL。
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