Patent Application
20230295343
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy is named GBYZL022-PKG Sequence Listing.txt, created on 02/14/2023, and is 54,394 bytes in size.
The present application relates to a nanobody against a factor XII (FXII) heavy chain and a use of FXII as a target for vasculitis.
FXII is a plasma protein with a molecular weight of 80 kDa and is present in the form of an inactive proenzyme at a quiesced state. FXII is a starting molecule of the contact system. FXII present in the form of a proenzyme has the function of limiting the activity of a proteolytic enzyme in plasma. When contacting with some negatively charged molecules such as collagen and polyphosphate, FXII will be activated. Activated FXII (FXIIa) is a protein consisting of two peptide chains linked through disulfide bonds and still has a molecular weight of 80 kDa. Studies have shown that the knockout of an FXII gene or the pharmacological blockade of FXII in mice can significantly inhibit arterial thrombosis, and the deletion or blockade of FXII does not influence bleeding in vivo. At present, FXII is a hot target for the treatment of thrombosis, and some anti-FXII antibodies or FXII inhibitors have entered clinical trials.
At present, the inhibition of thrombosis by antagonizing FXII is mainly divided into two directions: (1) inhibition of FXII activation (an antagonist binds to a functional domain of an FXII heavy chain and occupies a position to inhibit the FXII activation by an FXII activator) and (2) inhibition of FXIIa activity (an antagonist binds to a light chain of activated FXII to inhibit its thrombin activity). It is now clear that thrombosis can be significantly inhibited in both directions. The present application is intended to prepare an FXII-specific antibody for inhibiting FXII activation. There are two reasons for choosing this direction: (1) Inflammation plays a vital role in the occurrence and development of thrombosis and even other cardiovascular diseases (CVDs). A large number of studies have shown that FXII is closely related to the occurrence and development of inflammation, and in recent years, studies have shown that an FXII heavy chain is involved in an FXII-associated inflammatory response. Therefore, an FXII heavy chain-specific antibody can be screened out to inhibit the FXII activation, thereby reducing the production of FXIIa and inhibiting an inflammatory response regulated by FXII. (2) Studies have shown that the inhibitor rHA-infestin-4 for FXIIa can significantly inhibit thrombosis but has an obvious side effect, that is, a plasmin content in the body increases after injection of the inhibitor. Therefore, an FXIIa-specific antibody may not avoid this side effect.
Vasculitis refers to the infiltration of inflammatory cells in vessel walls and around blood vessels and is accompanied by vascular damage, including cellulose deposition, collagen fibrosis, and endothelial cell and muscle cell necrosis. Vasculitis is also known as angiitis. The vasculitis occurrence caused by anaphylaxis-induced immune complex deposition is a common form of vasculitis. The immune complex deposition will destroy small blood vessels in skin tissue, resulting in tissue redness and local bleeding. A traditional treatment method is primarily a combination of a glucocorticoid and an immunosuppressant (such as cyclophosphamide and methotrexate), but this treatment method has a risk of causing immunosuppression or hormonal dysregulation in the body. The present application discovers for the first time that the knockout of an FXII gene or the pharmacological blockade of FXII by a nanobody can significantly improve immune complex-induced vasculitis.
FXII is a protein that includes a multifunctional domain and has a complicated spatial conformation, and a heavy chain of the protein includes six different functional domains, which contribute to the complicated biological functions of FXII. Multiple domains play important roles in the activation of FXII. In a traditional antibody, a complementarity-determining region (CDR) is short, an antigen-antibody binding position is relatively small, and an antigen-binding epitope is a linear epitope. Compared with the traditional antibody, the nanobody has a longer CDR and can bind to a conformational epitope of an antigen. Therefore, the screening of an FXII-specific nanobody has promising prospects.
Currently, the literatures about an antibody for FXII or FXIIa are introduced as follows: an inhibitor rHA-infestin-4 for FXIIa can significantly inhibit arterial thrombosis in mice as reported in Larsson, Magnus, et al. “A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk.” Science translational medicine 6.222 (2014): 222ra17-222ra17. A genetically engineered antibody for FXIIa can significantly inhibit arterial thrombosis in mice as reported in Hagedorn I, Schmidbauer S, Pleines I, et al. Factor XIIa Inhibitor Recombinant Human Albumin Infestin-4 Abolishes Occlusive Arterial Thrombus Formation Without Affecting Bleeding [J]. Circulation, 2010, 121 (13): 1510-1517. A monoclonal antibody (mAb) for an FXII heavy chain can significantly inhibit arterial thrombosis in baboons as reported in Matafonov, Anton, et al. “Factor XII inhibition reduces thrombus formation in a primate thrombosis model.” Blood, The Journal of the American Society of Hematology 123.11 (2014): 1739-1746. There are currently no published clinical trial data.
According to a first aspect of the present application, an anti-FXII nanobody or an antigen-binding fragment thereof is provided to solve the problems in the background. In the present application, a neutralizing antibody for FXII is screened out by phage display, and it has been found that the neutralizing antibody exhibits prominent anti-thrombosis and anti-vasculitis effects in mouse arterial thrombosis and rat extracorporeal membrane oxygenation (ECMO) models.
The anti-FXII nanobody or the antigen-binding fragment thereof binds to FXII through a binding epitope for FXII to block the activation of FXII, and the binding epitope for FXII includes a conformational epitope.
Optionally, the binding epitope of the anti-FXII nanobody or the antigen-binding fragment thereof to FXII is a conformational epitope and can bind to both a fibronectin type II domain and a kringle domain of FXII.
Optionally, the anti-FXII nanobody or the antigen-binding fragment thereof has almost no ability to block an activity of FXIIa.
Optionally, when a molar ratio of the anti-FXII nanobody or the antigen-binding fragment thereof to FXII is 1:(0.1-0.3), a blocking efficiency of the anti-FXII nanobody or the antigen-binding fragment thereof for FXII is no less than 50%.
Optionally, the anti-FXII nanobody or the antigen-binding fragment thereof is derived from an alpaca.
Optionally, the anti-FXII nanobody or the antigen-binding fragment thereof is obtained based on an immune library obtained after immunization of an FXII protein.
Optionally, the anti-FXII nanobody or the antigen-binding fragment thereof is a recombinant antibody.
Optionally, the nanobody is an alpaca nanobody.
Optionally, the recombinant antibody includes a nanobody fused with an immunoglobulin Fc fragment. The fusion with an immunoglobulin Fc fragment significantly increases a half-life of the antibody.
Optionally, based on Kabat Database analysis,
n-1. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 1, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 2, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 3; or
n-2. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 5, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 6, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 7; or
n-3. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 9, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 10, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 11; or
n-4. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 13, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 14, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 15; or
n-5. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 17, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 18, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 19; or
n-6. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 21, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 22, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 23; or
n-7. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 25, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 26, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 27; or
n-8. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 29, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 30, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 31; or
n-9. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 33, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 34, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 35; or
n-10. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 37, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 38, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 39; or
n-11. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 41, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 42, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 43; or
n-12. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 45, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 46, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 47; or
n-13. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 49, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 50, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 51; or
n-14. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 53, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 54, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 55; or
n-15. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 57, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 58, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 59; or
n-16. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 61, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 62, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 63; or
n-17. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 65, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 66, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 67; and based on IMGT Database analysis,
n-1. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 69, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 70, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 71; or
n-2. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 73, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 74, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 75; or
n-3. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 77, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 78, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 79; or
n-4. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 81, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 82, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 83; or
n-5. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 85, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 86, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 87; or
n-6. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 89, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 90, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 91; or
n-7. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 93, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 94, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 95; or
n-8. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 97, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 98, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 99; or
n-9. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 101, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 102, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 103; or
n-10. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 105, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 106, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 107; or
n-11. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 109, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 110, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 111; or
n-12. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 113, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 114, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 115; or
n-13. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 117, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 118, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 119; or
n-14. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 121, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 122, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 123; or
n-15. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 125, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 126, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 127; or
n-16. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 129, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 130, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 131; or
n-17. the anti-FXII nanobody includes a heavy chain CDR-H1 with a sequence shown in SEQ ID NO: 133, a heavy chain CDR-H2 with a sequence shown in SEQ ID NO: 134, and a heavy chain CDR-H3 with a sequence shown in SEQ ID NO: 135.
Optionally, based on Kabat Database analysis,
n-101. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 4; or
n-102. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 8; or
n-103. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 12; or
n-104. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 16; or
n-105. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 20; or
n-106. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 24; or
n-107. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 28; or
n-108. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 32; or
n-109. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 36; or
n-110. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 40; or
n-111. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 44; or
n-112. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 48; or
n-113. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 52; or
n-114. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 56; or
n-115. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 60; or
n-116. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 64; or
n-117. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 68; and based on IMGT Database analysis,
n-101. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 72; or
n-102. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 76; or
n-103. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 80; or
n-104. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 84; or
n-105. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 88; or
n-106. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 92; or
n-107. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 96; or
n-108. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 100; or
n-109. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 104; or
n-110. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 108; or
n-111. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 112; or
n-112. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 116; or
n-113. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 120; or
n-114. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 124; or
n-115. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 128; or
n-116. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 132; or
n-117. the anti-FXII nanobody includes a heavy chain variable region with a sequence shown in SEQ ID NO: 136.
The CDR sequence and heavy chain variable region sequence of the anti-FXII nanobody are shown in Tables 1 and 2 below (The sequences in Table 1 are obtained according to the Kabat Database analysis, and the sequences in Table 2 are obtained according to the IMGT Database analysis).
According to a second aspect of the present application, a nucleic acid encoding the anti-FXII nanobody or the antigen-binding fragment thereof described above is provided.
According to a third aspect of the present application, a vector including the nucleic acid that is effectively linked to an appropriate promoter sequence is provided.
According to a fourth aspect of the present application, a prokaryotic cell, cell line, yeast cell, or viral system including the vector is provided.
According to a fifth aspect of the present application, a method for preparing the antibody or the antigen-binding fragment thereof described above is provided, including:
cultivating the prokaryotic cell, cell line, yeast cell, or viral system under conditions suitable for expression of the antibody, and isolating and purifying the antibody from a culture supernatant.
According to a sixth aspect of the present application, a use of the antibody or the antigen-binding fragment thereof in medicine is provided.
According to a seventh aspect of the present application, a use of the antibody or the antigen-binding fragment thereof in the preparation of an antithrombotic drug is provided.
According to an eighth aspect of the present application, an antithrombotic use of the antibody or the antigen-binding fragment thereof in an artificial medical device in contact with blood is provided.
According to a ninth aspect of the present application, an antithrombotic use of the antibody or the antigen-binding fragment thereof in ECMO is provided.
According to a tenth aspect of the present application, a use of the antibody or the antigen-binding fragment thereof in the preparation of an antivasculitis drug is provided.
According to an eleventh aspect of the present application, a use of the antibody or the antigen-binding fragment thereof in the preparation of an anti-cardiac ischemia-reperfusion injury (IRI) drug is provided.
According to a twelfth aspect of the present application, a pharmaceutical composition including the antibody or the antigen-binding fragment thereof is provided.
According to a thirteenth aspect of the present application, a target for inhibiting vasculitis is provided, and the target is FXII.
According to a fourteenth aspect of the present application, a drug for inhibiting vasculitis is provided, and the drug is an antibody for inhibiting FXII.
According to a fifteenth aspect of the present application, a pharmaceutical composition for inhibiting vasculitis is provided, and the pharmaceutical composition is an antibody for inhibiting FXII.
In the present application, the “recombinant antibody” refers to an antibody obtained through artificial modification or recombinant expression based on a natural mAb, a nanobody, or another single epitope-recognizing antibody.
In the present application, the “conformational epitope” refers to the fibronectin type II domain and kringle domain of FXII.
Possible beneficial effects of the present application:
1) The present application provides an anti-FXII nanobody or an antigen-binding fragment thereof. Compared with the traditional antibodies, the nanobody has a longer CDR, and a binding epitope of the nanobody to FXII is a conformational epitope and can bind to both of the two epitopes with important functions of FXII.
2) The present application provides an anti-FXII nanobody or an antigen-binding fragment thereof, which can block FXII activity. The treatment with the anti-FXII nanobody or the antigen-binding fragment thereof can significantly prolong the time of FeCl3-induced carotid arterial thrombosis in mice and the time of laser-induced cremaster arteriolar thrombosis in mice, and can significantly reduce the thrombus deposition on the oxygenator membrane during ECMO in rats.
3) The present application provides an anti-FXII nanobody or an antigen-binding fragment thereof, where the anti-FXII nanobody is an immunoglobulin-modified nanobody, Fc is linked in series to a C-terminus of the nanobody, and a half-life of the anti-FXII nanobody is extended to 6 h.
4) The present application provides an anti-FXII nanobody or an antigen-binding fragment thereof, and the knockout of an FXII gene or the treatment with the nanobody, of which C-terminus is tandem to Fc, can significantly improve the immune complex-induced cutaneous vasculitis in mice.
The present application will be described in detail below with reference to examples, but the present application is not limited to these examples.
Unless otherwise specified, the raw materials in the examples of the present application are all purchased from commercial sources.
Analysis methods in the examples of the present application are as follows: DNA amplification is conducted with an ABI Veriti PCR instrument.
An OD value is determined with a Tecan Infinite M200 Pro microplate reader.
Human FXII (Haematologic Technologies Inc, 800 μg/animal) was mixed with a complete Freund's adjuvant (CFA) (a concentration of FXII in the CFA was 0.50 mg/mL), and a resulting mixture was emulsified and then used to immunize an alpaca for the first time; and 21 d, 35 d, and 49 d later, the alpaca was immunized with a mixture of human FXII (400 μg/animal) and incomplete freund's adjuvant (IFA) (sigma) (a concentration of FXII in the IFA was 0.50 mg/mL) for the second time, the third time, and the fourth time, respectively. 7 d after the last immunization, peripheral blood was collected from the alpaca, and a titer of an anti-FXII antibody in the peripheral blood was detected.
When the titer met criteria, lymphocytes in the peripheral blood were isolated, RNA was extracted and reverse-transcribed to obtain a cDNA fragment, and then a nanobody fragment was obtained through nested PCR. The nanobody fragment was inserted into a phage display vector pHEN1 to construct an anti-FXII phage display library, which was directly used for affinity screening of specific phages. The library was screened using human FXII protein for five rounds through liquid phase screening, then the clones were randomly picked from plates for screening and eluting phages, and positive clones were identified by PHAGE-ELISA and sent for sequencing to obtain different nanobody sequences. A nanobody sequence was linked to a prokaryotic expression vector PET26b, a constructed plasmid was transformed into Escherichia coli (E. coli), and isopropylthiogalactoside (IPTG) was added at 0.25 mmol/L to induce the expression of a nanobody. Bacteria were collected and subjected to ultrasonic disruption, a resulting mixture was centrifuged, and a resulting supernatant was subjected to AKTA purifier Ni affinity chromatography for purification to obtain a nanobody protein with a purity of higher than 85%.
The titer detection method in Example 1 was as follows: An ELISA plate was coated with human FXII at a protein concentration of 1 μg/mL overnight at 4° C.; then the ELISA plate was washed with PBST buffer and blocked with 5% skimmed milk powder at room temperature for 2 h; the ELISA plate was washed with PBST buffer (which was prepared with 1 L of PBS and 500 μL of Tween 20), then nanobodies at different dilutions were added, and the ELISA plate was incubated for 2 h at room temperature; the ELISA plate was washed with PBST buffer, then an anti-horseradish peroxidase (HRP)-labeled 6×His tag antibody (Abcam) was added, and the ELISA plate was incubated for 1 h at room temperature in the dark; the ELISA plate was washed with PBST buffer, a tetramethylbenzidine (TMB) (R&D Systems) substrate solution was added, and a chromogenic reaction was conducted for 5 min to 10 min; and a stop solution (sulfuric acid with a concentration of 2 M) was added, and an OD value of each well was determined at 450 nm by a microplate reader.
Titer detection results are shown in
1 μg of human FXII was thoroughly mixed with 140 μl of the nanobody at different concentrations, and a resulting mixture was incubated at 37° C. for 30 min; then 20 μl of ellagic acid (final concentration: 4 μg/mL) was added, and a resulting mixture was thoroughly mixed and incubated at 37° C. for 10 min; then 40 μl of S-2302 (kallikrein chromogenic substrate, 4 mmol/mL) was added, and a resulting mixture was thoroughly mixed and incubated at 37° C. for 15 min; and finally 40 μl of 20% acetic acid was added, and an OD value was detected at 405 nm.
1 μg of human FXIIa was thoroughly mixed with 140 μl of the nanobody at different concentrations, and a resulting mixture was incubated at 37° C. for 30 min; then 40 μl of S-2302 (4 mmol/mL) was added, and a resulting mixture was thoroughly mixed and incubated at 37° C. for 15 min; and finally 40 μl of 20% acetic acid was added, and an OD value was detected at 405 nm.
(1) A Fortebio Octet special solution M (a PBST solution including BSA with a mass fraction of 0.5%) was prepared, filtered through a 0.22 μm filter membrane, and stored at 4° C. for later use; (2) a biotinylated FXII protein was diluted to 10 μg/mL, a nanobody N4-38 was diluted to 50 μg/mL, 20 μg/mL, and 10 μg/mL, and resulting samples each were added; (3) a biosensor was placed in a corresponding clean well of a spare box, and 200 mL of the Fortebio Octet special solution M was added to each well corresponding to the biosensor; (4) a program was set as follows: first stage Baseline: 60 s, second stage Loading: 200 s, third stage Baseline 2: 100 s, fourth stage Association: 300 s, fourth stage Dissociation: 300 s, and reaction temperature during the whole process: 37° C.; and (5) after a reaction was completed, data analysis was conducted by software.
The three curves from top to bottom in
An NC membrane was taken, and 5 mg of natural human FXII, denatured human FXII (which was treated with 100 Mm DTT for 10 min and then boiled at a high temperature for 10 min), and BSA were added dropwise on the NC membrane; the NC membrane was dried and then blocked with 5% skimmed milk powder at room temperature for 2 h; then the N4-38 antibody (1:10,000) was added, and the NC membrane was incubated at room temperature for 2 h; the NC membrane was washed with PBST, an HRP-labeled anti-6HIS tag antibody (1:1,000) was added, and the NC membrane was incubated at room temperature for 1 h; and the NC membrane was washed with PBST, and an ECL luminescent solution was added for exposure.
A 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was prepared, then the treated fibronectin domain type II (FBII), epidermal-growth-factor-like domain (EGFI), fibronectin domain type I (FBI), second epidermal-growth-factor-like domain (EFGII), kringle (KNG), and proline rich region (PRO) protein samples were spotted into the gel, and electrophoresis was conducted. When the samples reached a bottom of the gel, the SDS-PAGE gel was removed and placed in a membrane transfer instrument for membrane transfer. After the membrane transfer was completed, an NC membrane was taken out and blocked with 5% skimmed milk powder at room temperature for 2 h; then the N4-38 antibody (a volume ratio of the antibody to an antibody dilution was 1:10,000) was added to the NC membrane, and the NC membrane was incubated at room temperature for 2 h; the NC membrane was washed with PBST, an HRP-labeled anti-6×HIS tag antibody (a volume ratio of the antibody to an antibody dilution was 1:1,000) was added, and the NC membrane was incubated at room temperature for 1 h; and the NC membrane was washed with PBST, and an ECL solution was added for exposure.
A gene fragment for an antibody fragment obtained by linking a Fc fragment of human IgG through 7 repeated GS linkers in series to a C terminus of the nanobody N38 was prepared through gene synthesis, and the gene fragment was inserted into a prokaryotic expression vector PET26b; and a constructed plasmid was transformed into E. coli, and IPTG was added to induce the expression. Bacteria were collected and subjected to ultrasonic disruption, a resulting mixture was centrifuged, and a resulting supernatant was subjected to AKTA purifier Ni affinity chromatography for purification to obtain a N38-Fc protein.
(A) The N38 and N38-Fc proteins each were injected intravenously into C57BL/6 mice (1 mg/kg), and 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, and 16 h after the injection, peripheral blood was collected from the mice, and plasma was isolated through centrifugation; 50 μl of the plasma was taken and mixed with 50 μl of a coating solution, and a resulting mixture was added to an ELISA plate and incubated overnight at 4° C.; the ELISA plate was washed with PBST and then blocked with 5% skimmed milk powder at room temperature for 2 h; the ELISA plate was washed with PBST, then an anti-HRP-labeled 6×His tag antibody (Abeam) was added, and the ELISA plate was incubated for 1 h at room temperature in the dark; the ELISA plate was washed with PBST, a TMB substrate solution was added, and a chromogenic reaction was conducted for 5 min to 10 min; and a stop solution was added, and an OD value of each well was determined at 450 nm by a microplate reader.
(B) 1 μg of human FXII was thoroughly mixed with 140 μl of N38 or N38-Fc at different concentrations, and a resulting mixture was incubated at 37° C. for 30 min; then 20 μl of ellagic acid (final concentration: 4 μg/mL) was added, and a resulting mixture was thoroughly mixed and incubated at 37° C. for 10 min; then 40 μl of S-2302 (4 mmol/mL) was added, and a resulting mixture was thoroughly mixed and incubated at 37° C. for 15 min; and finally 40 μl of 20% acetic acid was added, and an OD value was detected at 405 nm.
(A) 8-week-old male C57BL/6 mice were selected, grouped, and intraperitoneally injected with the N38-Fc protein at different doses. 30 min later, the mice were anesthetized with pentobarbital (80 mg/kg), and the left carotid artery was collected; a round filter paper (r=1.0 mm) was placed above the blood vessel, then 0.5 μL of 7.5% FeCl3 (sigma) was added dropwise on the filter paper, and the filter paper was removed 3 min later; and a Doppler sonography blood flow monitor (Transonic Systems Inc.) was used to measure a blood flow of the carotid artery. (B) 8-week-old male C57BL/6 mice were selected, grouped, and intraperitoneally injected with the N38-Fc protein at different doses. 30 min later, the mice are anesthetized with pentobarbital (80 mg/kg) and simultaneously injected with 5% dextran-FITC (500 mg/kg); a cremaster muscle was collected, and a blood vessel was subjected to thermal damage with 488 nm laser (power: 5 mw) of a confocal microscope; and a time of cremasteric arterial thrombosis was observed.
An ECMO system composed of a peristaltic pump connected to a silicone tube, a 10 mL syringe, and a customized small-volume oxygenator (500 cm2 gas exchange membrane, Dongguan Kewei Medical Equipment Co., Ltd.) was adopted. The entire circuit was pre-filled with 8 mL of a 6% hydroxyethyl starch (HES) injection. The entire circuit was free of heparin coating. A 400 g male SD rat was anesthetized with an isoflurane gas, subjected to endotracheal intubation, connected to a ventilator, and then injected with heparin (500 U/kg) alone or heparin (500 U/kg) in combination with N38-Fc (2 mg/kg). The femoral artery and jugular vein of the rat were collected, and the ECMO system was connected to each of the femoral artery and jugular vein of the rat through a catheter to form a complete rat ECMO circuit. An ACT value was detected once and circulation was conducted for 2 h every 30 min. 200 μL of peripheral blood was collected from the rat at 5 min and 2 h of the circulation, 30 μL of whole blood was collected, and a small animal whole blood cell counter (HEMAVET 950FS) was used to measure a change of blood cells during an ECMO process; and the remaining peripheral blood was centrifuged to obtain plasma, and a change of TNF-αlevel in rat peripheral blood before and after the circulation was detected by a rat TNF-αELISA detection kit (RayBiotech). After the 2 h of circulation was completed, the oxygenator membrane was taken out, rinsed with PBS, and observed by scanning electron microscopy (SEM) (as shown in
8-week-old C57BL/6 mice or FXII-knockout mice were selected; and the C57BL/6 mice each were treated with N38-Fc (2 mg/kg) and an isotype control thereof and then anesthetized with pentobarbital, hair on the back was removed with depilatory paste, and then the mice each were intravenously injected with BSA (7511 g/g, sigma) and then immediately injected with 20 μL of an anti-BSA polyclonal antibody (pAb) (60 sigma) intracutaneously through the dorsal skin. 4 h later, the mice were euthanized, the dorsal skin was collected, and a diameter of an inflammatory spot at an inner side of the skin was measured (results were shown in
It was reported by Gao et al. (Circ Res. 2010; 107: 1445-1453) that myocardial IRI was induced using an artificial ventilation-free method. 8-10 week-old C57BL/6 mice were selected and divided into an experimental group and a control group; and the experimental group (N38-Fc) was injected with N38-Fc (8 mg/kg) and the control group (Vehicle) was injected with a same volume of PBS. The first injection was conducted 5 min before surgery, and then injection was conducted every 6 h. The mice were allowed to inhale 3% isoflurane for anaesthetization and then to inhale 1.5% to 2% isoflurane for anaesthetization maintenance; the mice were placed at a supine position, the skin on the left chest was cut, a chest muscle was simply separated, and then the chest cavity was rapidly exposed by left fourth intercostal thoracotomy; the pericardium was opened to expose the mice, and the left anterior descending (LAD) coronary artery 2 mm to 3 mm from the start was ligated with a 7-0 silk suture through a slipknot; the success ligation was confirmed by the simultaneous occurrence of anterior wall whitening of the left ventricle and ST segment elevation of electrocardiogram (ECG); then the heart was quickly returned to the chest cavity, the air was manually evacuated, and the chest cavity was closed with a 4-0 suture; one inner end of a slipknot suture was cut as short as possible, and the other end was about 0.8 cm long and remained outside the chest cavity; and the anaesthetization was then stopped, and the animal was allowed to recover. After 30 min of ischaemia, the mice were anesthetized once again, and the slipknot was loosened by smoothly pulling a long end of the suture until a complete release was achieved, at which point myocardial reperfusion was started; and 24 h after ischemia-reperfusion, the cardiac function and ventricular structure were determined through echocardiography (VisualSonics VeVo 2100 imaging system) by assessing EF, left ventricular FS, left ventricular anterolateral wall (LVAW) thickness, left ventricular posterior wall (LVPW), left ventricular volume, and left ventricular mass. The groups had a similar mortality of about 20%. After 24 h of cardiac reperfusion, the LAD was occluded once again in the previous position, and a 2% Evans blue dye (Sigma, Darmstadt, Germany) was injected into the cardiac chamber through the ascending aorta; the mice were then euthanized, the heart was collected, rinsed with PBS, frozen at −80° C. for 30 min, and cut crosswise below the ligation line into 5 sections; the sections were incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Amresco, America) for 10 min in a dark room at 37° C. and then fixed with formalin for 2 h; images were acquired with a stereomicroscope (Zeiss, Germany) (results were shown in
The above examples are merely few examples of the present application, and do not limit the present application in any form. Although the present application is disclosed as above with preferred examples, the present application is not limited thereto. Some changes or modifications made by any technical personnel familiar with the profession using the technical content disclosed above without departing from the scope of the technical solutions of the present application are equivalent to equivalent implementation cases and fall within the scope of the technical solutions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010547554.9 | Jun 2020 | CN | national |
This application is the national phase entry of International Application No. PCT/CN2020/116566, filed on Sep. 21, 2020, which is based upon and claims priority to Chinese Patent Application No. 202010547554.9, filed on Jun. 16, 2020, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/116566 | 9/21/2020 | WO |
