ANTI-CD22 NANO ANTIBODY AND USE THEREOF
20230416360 · 2023-12-28
Inventors
- Qiong Wang (Shanghai, CN)
- Cuiqing Yang (Shanghai, CN)
- Zhuoxiao Cao (Shanghai, CN)
- Renhong Tang (Shanghai, CN)
- Jinsheng Ren (Nanjing, Jiangsu, CN)
Cpc classification
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61K39/464413
HUMAN NECESSITIES
C07K2317/33
CHEMISTRY; METALLURGY
International classification
Abstract
Provided are an CD22 nano antibody, and a preparation method therefor and an application thereof. The CD22 nano antibody has high affinity with a CD22 protein, and can be used for preparing drugs for treating tumors, autoimmune diseases, etc.
Claims
1. A nanobody or an antigen-binding fragment that specifically binds to CD22, wherein the nanobody or the antigen-binding fragment comprises a combination of CDRs, the combination of CDRs comprises: CDR1, CDR2, and CDR3; the CDR1, CDR2 and CDR3 have any sequence combination selected from the following, or a sequence combination with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination: TABLE-US-00021 SEQ ID NO. No. CDR1 CDR2 CDR3 VH1 SEQ ID NO. 57 SEQ ID NO. 58 SEQ ID NO. 59 VH2 SEQ ID NO. 60 SEQ ID NO. 61 SEQ ID NO. 62 VH3 SEQ ID NO. 63 SEQ ID NO. 64 SEQ ID NO. 65 VH4 SEQ ID NO. 66 SEQ ID NO. 67 SEQ ID NO. 68 VH5 SEQ ID NO. 69 SEQ ID NO. 70 SEQ ID NO. 71 VH6 SEQ ID NO. 72 SEQ ID NO. 73 SEQ ID NO. 74 VH7 SEQ ID NO. 75 SEQ ID NO. 76 SEQ ID NO. 77 VH8 SEQ ID NO. 78 SEQ ID NO. 79 SEQ ID NO. 80 VH9 SEQ ID NO. 81 SEQ ID NO. 82 SEQ ID NO. 83 VH10 SEQ ID NO. 84 SEQ ID NO. 85 SEQ ID NO. 86 VH11 SEQ ID NO. 87 SEQ ID NO. 88 SEQ ID NO. 89 VH12 SEQ ID NO. 90 SEQ ID NO. 91 SEQ ID NO. 92 VH13 SEQ ID NO. 93 SEQ ID NO. 94 SEQ ID NO. 95 VH14 SEQ ID NO. 96 SEQ ID NO. 97 SEQ ID NO. 98 VH15 SEQ ID NO. 99 SEQ ID NO. 100 SEQ ID NO. 101 VH16 SEQ ID NO. 102 SEQ ID NO. 103 SEQ ID NO. 104 VH17 SEQ ID NO. 105 SEQ ID NO. 106 SEQ ID NO. 107 VH18 SEQ ID NO. 108 SEQ ID NO. 109 SEQ ID NO. 110 VH19 SEQ ID NO. 111 SEQ ID NO. 112 SEQ ID NO. 113 VH20 SEQ ID NO. 114 SEQ ID NO. 115 SEQ ID NO. 116 VH21 SEQ ID NO. 117 SEQ ID NO. 118 SEQ ID NO. 119 VH22 SEQ ID NO. 120 SEQ ID NO. 121 SEQ ID NO. 122 VH23 SEQ ID NO. 123 SEQ ID NO. 124 SEQ ID NO. 125 VH24 SEQ ID NO. 126 SEQ ID NO. 127 SEQ ID NO. 128 VH25 SEQ ID NO. 129 SEQ ID NO. 130 SEQ ID NO. 131 VH26 SEQ ID NO. 132 SEQ ID NO. 133 SEQ ID NO. 134 VH27 SEQ ID NO. 135 SEQ ID NO. 136 SEQ ID NO. 137 VH28 SEQ ID NO. 138 SEQ ID NO. 139 SEQ ID NO. 140 VH29 SEQ ID NO. 141 SEQ ID NO. 142 SEQ ID NO. 143 VH30 SEQ ID NO. 144 SEQ ID NO. 145 SEQ ID NO. 146 VH31 SEQ ID NO. 147 SEQ ID NO. 148 SEQ ID NO. 149 VH32 SEQ ID NO. 150 SEQ ID NO. 151 SEQ ID NO. 152 VH33 SEQ ID NO. 153 SEQ ID NO. 154 SEQ ID NO. 155 VH34 SEQ ID NO. 156 SEQ ID NO. 157 SEQ ID NO. 158 VH35 SEQ ID NO. 159 SEQ ID NO. 160 SEQ ID NO. 161 VH36 SEQ ID NO. 162 SEQ ID NO. 163 SEQ ID NO. 164 VH37 SEQ ID NO. 165 SEQ ID NO. 166 SEQ ID NO. 167 VH38 SEQ ID NO. 168 SEQ ID NO. 169 SEQ ID NO. 170 VH39 SEQ ID NO. 171 SEQ ID NO. 172 SEQ ID NO. 173 VH40 SEQ ID NO. 174 SEQ ID NO. 175 SEQ ID NO. 176 VH41 SEQ ID NO. 177 SEQ ID NO. 178 SEQ ID NO. 179 VH42 SEQ ID NO. 180 SEQ ID NO. 181 SEQ ID NO. 182 VH43 SEQ ID NO. 183 SEQ ID NO. 184 SEQ ID NO. 185 VH44 SEQ ID NO. 186 SEQ ID NO. 187 SEQ ID NO. 188 VH45 SEQ ID NO. 189 SEQ ID NO. 190 SEQ ID NO. 191 VH46 SEQ ID NO. 192 SEQ ID NO. 193 SEQ ID NO. 194 VH47 SEQ ID NO. 195 SEQ ID NO. 196 SEQ ID NO. 197 VH48 SEQ ID NO. 198 SEQ ID NO. 199 SEQ ID NO. 200 VH49 SEQ ID NO. 201 SEQ ID NO. 202 SEQ ID NO. 203 VH50 SEQ ID NO. 204 SEQ ID NO. 205 SEQ ID NO. 206 VH51 SEQ ID NO. 207 SEQ ID NO. 208 SEQ ID NO. 209 VH52 SEQ ID NO. 210 SEQ ID NO. 211 SEQ ID NO. 212 VH53 SEQ ID NO. 213 SEQ ID NO. 214 SEQ ID NO. 215 VH54 SEQ ID NO. 216 SEQ ID NO. 217 SEQ ID NO. 218 each CDR1, CDR2 and CDR3 is coded according to the prevailing analysis methods of KABAT, Chothia or IMGT; preferably, the substitution is a conservative amino acid substitution.
2. The nanobody or the antigen-binding fragment of claim 1, wherein (1) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 57, 58 and 59, respectively; (2) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 60, 61 and 62, respectively; (3) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 63, 64 and respectively; (4) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 66, 67 and 68, respectively; (5) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 69, 70 and 71, respectively; (6) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 72, 73 and 74, respectively; (7) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 75, 76 and 77, respectively; (8) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 78, 79 and respectively; (9) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 81, 82 and 83, respectively; (10) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 84, 85 and 86, respectively; (11) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 87, 88 and 89, respectively; (12) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 90, 91 and 92, respectively; (13) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 93, 94 and respectively; (14) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 96, 97 and 98, respectively; (15) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 99, 100 and 101, respectively; (16) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 102, 103 and 104, respectively; (17) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 105, 106 and 107, respectively; (18) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 108, 109 and 110, respectively; (19) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 111, 112 and 113, respectively; (20) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 114, 115 and 116, respectively; (21) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 117, 118 and 119, respectively; (22) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 120, 121 and 122, respectively; (23) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 123, 124 and 125, respectively; (24) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 126, 127 and 128, respectively; (25) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 129, 130 and 131, respectively; (26) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 132, 133 and 134, respectively; (27) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 135, 136 and 137, respectively; (28) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 138, 139 and 140, respectively; (29) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 141, 142 and 143, respectively; (30) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 144, 145 and 146, respectively; (31) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 147, 148 and 149, respectively; (32) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 150, 151 and 152, respectively; (33) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 153, 154 and 155, respectively; (34) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 156, 157 and 158, respectively; (35) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 159, 160 and 161, respectively; (36) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 162, 163 and 164, respectively; (37) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 165, 166 and 167, respectively; (38) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 168, 169 and 170, respectively; (39) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 171, 172 and 173, respectively; (40) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 174, 175 and 176, respectively; (41) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 177, 178 and 179, respectively; (42) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 180, 181 and 182, respectively; (43) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 183, 184 and 185, respectively; (44) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 186, 187 and 188, respectively; (45) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 189, 190 and 191, respectively; (46) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 192, 193 and 194, respectively; (47) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 195, 196 and 197, respectively; (48) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 198, 199 and 200, respectively; (49) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 201, 202 and 203, respectively; (50) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 204, 205 and 206, respectively; (51) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 207, 208 and 209, respectively; (52) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 210, 211 and 212, respectively; (53) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 213, 214 and 215, respectively; (54) the CDR1, CDR2 and CDR3 have sequences as shown in SEQ ID NO. 216, 217 and 218, respectively; or, the CDR1, CDR2 and CDR3 have a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared with the above sequence combinations (1)-(54).
3. The nanobody or the antigen-binding fragment of claim 1, wherein the nanobody or the antigen-binding fragment comprises: (1) a variable region having a sequence as shown in SEQ ID NO: 21, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (2) a variable region having a sequence as shown in SEQ ID NO: 23, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (3) a variable region having a sequence as shown in SEQ ID NO: 25, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (4) a variable region having a sequence as shown in SEQ ID NO: 27, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (5) a variable region having a sequence as shown in SEQ ID NO: 29, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (6) a variable region having a sequence as shown in SEQ ID NO: 31, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (7) a variable region having a sequence as shown in SEQ ID NO: 33, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (8) a variable region having a sequence as shown in SEQ ID NO: 35, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (9) a variable region having a sequence as shown in SEQ ID NO: 37, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (10) a variable region having a sequence as shown in SEQ ID NO: 39, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (11) a variable region having a sequence as shown in SEQ ID NO: 41, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (12) a variable region having a sequence as shown in SEQ ID NO: 43, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (13) a variable region having a sequence as shown in SEQ ID NO: 45, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (14) a variable region having a sequence as shown in SEQ ID NO: 47, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (15) a variable region having a sequence as shown in SEQ ID NO: 49, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (16) a variable region having a sequence as shown in SEQ ID NO: 51, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; (17) a variable region having a sequence as shown in SEQ ID NO: 53, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above; or, (18) a variable region having a sequence as shown in SEQ ID NO: 55, or a sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity with the sequence shown above.
4. The nanobody or the antigen-binding fragment of claim 1, wherein the nanobody or the antigen-binding fragment binds to human CD22 with a dissociation constant (KD) of no more than 50 nM.
5. The nanobody or the antigen-binding fragment of claim 1, wherein the antibody or the antigen-binding fragment comprises a sequence of the constant region of any one of human or murine antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, comprises a sequence of the constant region of human or murine antibody IgG1, IgG2, IgG3 or IgG4.
6. The nanobody or the antigen-binding fragment of claim 1, wherein the nanobody or the antigen-binding fragment further comprises a heavy chain constant region sequence without a CH1 fragment.
7. The nanobody or the antigen-binding fragment of claim 1, wherein the nanobody or the antigen-binding fragment further comprises a heavy chain constant region sequence with CH2 and CH3 fragments.
8. The nanobody or the antigen-binding fragment of claim 1, wherein the antibody or the antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or, (3) a fully human antibody or a fragment thereof; preferably, the antibody or the antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (for example, a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab, F(ab)2, Fd, Fv, scFv, a diabody or a single domain antibody.
9. The nanobody or the antigen-binding fragment of claim 1, wherein the nanobody or the antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescence label, a ultrasound contrast agent or a photosensitizer.
10. A multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module, the first antigen-binding module comprises the nanobody or the antigen-binding fragment of claim 1, the second antigen-binding module specifically binds to other antigens than CD22 or binds to a CD22 epitope different from the first antigen-binding module; preferably, the other antigens are selected from CD3, CD16, CD16A, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66(a-d), CD74, CD80, CD126, CD138, B7, MUC, Ia, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, oncogene products, IL-2, IL-6, TRAIL-R1 or TRAIL-R2; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.
11. A chimeric antigen receptor (CAR), wherein the chimeric antigen receptor at least comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen-binding domain comprises the nanobody or the antigen-binding fragment of claim 1.
12. An immune effector cell, wherein the immune effector cell comprises the chimeric antigen receptor of claim 11 or comprises a nucleic acid fragment encoding the chimeric antigen receptor of claim 11; preferably, the immune effector cell is selected from a T cell, a NK cell (a natural killer cell), a NKT cell (a natural killer T cell), a monocyte, a macrophage, a dendritic cell or a mast cell; the T cell can be selected from: an inflammatory T cell, a cytotoxic T cell, a regulatory T cell (Treg) or a helper T cell; preferably, the immune effector cell is an allogeneic immune effector cell or an autologous immune cell.
13. An isolated nucleic acid molecule, wherein the nucleic acid molecule encodes the nanobody or the antigen-binding fragment of claim 1.
14. An expression vector, comprising the isolated nucleic acid molecule of claim 13.
15. An isolated host cell, comprising the isolated nucleic acid molecule of claim 13; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and/or Bacillus subtilis; more preferably, the host cell is selected from HEK293E cell or CHO cell.
16. A method for preparing the antibody or the antigen-binding fragment of claim 1, comprising: culturing a host cell under appropriate conditions, wherein the host cell is an isolated host cell comprising an isolated nucleic acid molecule, and the nucleic acid molecule encodes the nanobody or the antigen-binding fragment, and isolating the antibody or the antigen-binding fragment from the host cell.
17. A method for preparing an immune effector cell, comprising: introducing a nucleic acid fragment encoding the chimeric antigen receptor of claim 11 into the immune effector cell, and optionally, enabling the immune effector cell to express the chimeric antigen receptor of claim 11.
18. A pharmaceutical composition, comprising the antibody or the antigen-binding fragment of claim 1; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional antineoplastic agent.
19. (canceled)
20. A method for preventing and/or treating a B-cell disease, wherein the method comprises administering an effective amount of the antibody or the antigen-binding fragment of claim 1 to a patient in need thereof; the B cell disease is preferably a tumor or an autoimmune disease; preferably, the tumor is selected from lymphoma or leukemia, more preferably, the lymphoma or leukemia is selected from B-cell lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, primary mediastinal B-cell lymphoma, diffuse large B-cell lymphoma, precursor B-cell acute lymphocytic leukemia (pre-B ALL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, multiple myeloma; preferably, the autoimmune disease is selected from systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, Sjogren's syndrome, Guillain-Barre syndrome, myasthenia gravis, large vessel vasculitis, medium vessel vasculitis, polyarteritis nodosa, pemphigus, scleroderma, pulmonary hemorrhage-nephritic syndrome, glomerular nephritis, primary biliary cirrhosis, Graves' disease, membranous nephropathy, autoimmune hepatitis, sprue, Addison's disease, polymyositis/dermatomyositis, monoclonal gamma globulin disease, factor VIII deficiency, cryoglobulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, and chronic lymphocytic thyroiditis.
21. (canceled)
22. A kit, comprising the antibody or the antigen-binding fragment of claim 1, and instructions for use.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0169] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.
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DETAILED DESCRIPTION OF EMBODIMENTS
[0185] The present invention will be further described below in conjunction with specific examples, and the advantages and characteristics of the present invention will become clearer along with the description. If specific conditions are not specified in the examples, conventional conditions or conditions recommended by a manufacturer are followed. The reagents or instruments used therein for which manufacturers are not specified are all conventional products that are commercially available.
[0186] The examples of the present invention are merely exemplary, and do not limit the scope of the present invention in any way. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.
Example 1 Screening of Single Domain Antibody Against Human CD22
[0187] 1.1 Immunization and Serum Titer Detection of Alpaca
[0188] The human CD22 (Asp20-Arg687)-His protein used for immunization was purchased from ACRO Biosystems (catalog number: CD2-H52H8). Two alpacas (Llama) were selected for immunization, and each alpaca was immunized four times with an interval of 3 weeks. After the third immunization and after the fourth immunization, peripheral blood was collected and serum was separated, and enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) were used to detect the antibody titer and specificity against human CD22 in serum, and the results are shown in
TABLE-US-00002 TABLE 1 The serum antibody titer of alpaca immunized with human CD22 protein detected by ELISA OD450 nm Batch Serum dilution NB150 (TB2) NB150 (TB3) NB151 (TB2) NB151 (TB3) 1:100 2.36 1.98 1.96 2.09 1:300 2.36 1.98 1.89 2.02 1:900 2.36 1.93 1.82 2.00 1:2700 2.18 1.86 1.80 1.89 1:8100 1.96 1.71 1.67 1.72 1:24300 1.42 1.41 1.52 1.49 1:72900 0.92 0.93 1.22 1.12 1:218700 0.48 0.50 0.79 0.68 1:656100 0.24 0.26 0.47 0.37 1:1968300 0.12 0.14 0.23 0.18 1:5904900 0.09 0.09 0.14 0.11 Blank control 0.050 0.05 0.06 0.05
[0189] 1.2 Library Construction
[0190] A total of 100 mL of alpaca peripheral blood was collected after three immunizations and after four immunizations; Lymphocyte separation medium was used to isolate PBMC, RNAiso Plus reagent (Takara, catalog number: #9108/9109) was used to extract total RNA, and PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, catalog number: 6210A) was used to reverse transcribe the extracted RNA into cDNA. Nested PCR was used to amplify the variable region nucleic acid fragment encoding the nanobody: [0191] the first round of PCR:
TABLE-US-00003 upstreamprimer: (SEQIDNO.16) CTTGGTGGTCCTGGCTGC downstreamprimer: (SEQIDNO.17) GGTACGTGCTGTTGAACTGTTCC [0192] the second round of PCR: [0193] the first round PCR product was used as a template,
TABLE-US-00004 upstreamprimer: (SEQIDNO.18) CATGCCATGACTGTGGCCCAGGCGGCCCAGKT GCAGCTCGTGGAGTC downstreamprimer-1: (SEQIDNO.19) CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTG GTGCG downstreamprimer-2: (SEQIDNO.20) CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGT CTTGGG
[0194] The nucleic acid fragment of the nanobody of interest was recovered and cloned into the phage display vector pcomb3XSS (from Sichuan NB Biolab Co., Ltd) using the restriction endonuclease SfiI (NEB, catalog number: R0123S). The product was then electrotransformed into Escherichia coli electroporation competent cell TG1, and a nanobody phage display library target against CD22 was constructed and tested. By serial dilution plating, the calculated capacity is 2.010.sup.9. To test the insertion rate of the library, 48 clones were randomly selected for colony PCR. The results showed that the insertion rate reached 100%.
[0195] 1.3 Panning of Nanobody VHH Against CD22
[0196] The plate was coated with human CD22-llama Fc fusion protein (ACRO Biosystems, catalog number: 512-H525a) at 0.5 g/well, and placed at 4 C. overnight; The next day, after blocking with 3% BSA-PBS at 37 C. for 1 hour, 100 l of phage display library was added and incubated at 37 C. for 1 hour; After that, the plate was washed 6 times with PBST and 2 times with PBS to wash away unbound phages. Finally, 100 L of Gly-HCl eluent was added to elute the phages that specifically bind to CD22 to enrich positive clones.
[0197] 1.4 Screening of Specific Single Positive Clones by Phage Enzyme-Linked Immunoassay
[0198] After panning, blank Escherichia coli were infected with the obtained CD22-binding positive phages and plated. Then 96 single colonies were selected for proliferation and culture. The plates were coated with human CD22-llama Fc and human CD22-His proteins respectively at 4 C. overnight, the phage culture supernatant was added, and incubated at 37 C. for 1 hour. After washing, TMB chromogenic solution was added to develop color, and the optical density was measured at a wavelength of 450 nm. Human CD22-llama Fc and human CD22-His double-positive clones were selected for sequencing. The sequencing results were analyzed using MOE software, and the evolutionary tree was constructed according to the amino acid sequence coding the VHH protein. According to the sequence similarity, 18 clones were obtained by eliminating the sequences that were close to each other on the evolutionary tree, and the CDR sequences of the 18 clones were analyzed by KABAT, Chothia or IMGT software respectively, and the corresponding sequence information was shown in the following table 2-4. Wherein Table 2 showed the antibody sequences represented by amino acids of 18 nanobody molecules, and Table 3 showed the antibody sequences represented by nucleotides of 18 nanobody molecules, and Table 4 showed the results of IMGT, Kabat and Chothia analysis of the CDRs of 18 nanobody molecules. Subsequently, the production and identification of VHH nanobody Fc fusion protein were carried out.
TABLE-US-00005 TABLE2 Specificaminoacidsequenceinformationoftheheavychainvariable regionoftheanti-CD22antibody Antibody Sequence number number Heavychainvariableregionsequence(VH) S002-NB SEQIDNO.21 QVQLVESGGGLVQAGDSLKLSCLASGITFSSYYMSWFRQAPGKEREF 150-42 VAGVGRSGAPSHHADSVKGRFTISRDNAKSVYLQMNSLQPEDTAVY FCAAKWMAASTTVADYWGQGTQVTVSS S002-NB SEQIDNO.23 QLQLVESGGGLVQAGGSLRLSCVGSGDRVSIALMGWFRQAPGKERE 150-5 FVAAISPSGGRITYSQSVKGRFTISRDNAKNTVYLQMNSLKRDDTAVY VCAANFRGYLSSSRGSDYAYWGQGTQVTVSS S002-NB SEQIDNO.25 QVQLVESGGGLVQDGGSLRLSCAASGLTFSFYNMGWFRQAPGKERE 150-40 FVAAISQSGLSTHYADSMKGRFTISRQNAKNTVYLQMNSLNPVDTAAY YCAAGRRGYLASTTNDYEYWGQGTQVTVSS S002-NB SEQIDNO.27 QVQLVESGGGLVQAGDSLRLSCAVTGRAYSRSDMGWFRQAPGKER 150-57 EIVAVITFGGGTDYADSVKGRFTISSEDTKNTLYLQMNSLQPEDTAVYY CAAGFRGSRWSTAARRKEDDYAFWGQGTQVTVSS S002-NB SEQIDNO.29 QVQLVESGGGLVQPGGSLRLSCAASGSWFNIAMGWFRQAPGKEREF 150-2 VAGIRWSDSKTYYADSVKGRATISGDNAKNTTYLQMNSLKPEDTAVYY CAAQRYYRGSYNRQTNYDHWGQGTQVTVSS S002-NB SEQIDNO.31 QVQLVESGGGLVQAGGSLRLSCTTSGGTLSRITMGWFRQAPGKERE 151-14 FVAAIKWGGSSTPYSDSVKGRFTISRQNAKNTVYLQMDRLVPEDTAVY YCAGGLRGPYSDSVTSSVQYNYWGQGTQVTVSS S002-NB SEQIDNO.33 QVQLVESGGGLVQAGGSLRLSCAASGITSDDYAIGWFRQAPGKEREG 151-51 VSCMYSSDGSTYYADSVKGRFTISRQNAKNTVYLQMNSLRPEDTAVY HCAAGPWCFVTGVGREEWGQGTQVTVSS S002-NB SEQIDNO.35 QVQLVESGGGLVQAGDSLRLSCAASGRTFSPSTMAWFRQAPGKERE 151-2 LVARIGWTYGDTYYVDSAKGISMQNVKNTVYLQMNSLKPEDTAV YYCAALFRGGLSYNFSNRGTQVTVSS S002-NB SEQIDNO.37 EVQVVESGGGLVQAGDSLRLSCTT5GRTFYGDYIAWFRQAPGKEREF 151-82 VAAIAVRGRGTFYGQSVKGRFTISRDNAENTLSLQMNSLKPEDTAVYY CAADAGSYASRGFPVRETGRYDYWGQGTQVTVSS S002-NB SEQIDNO.39 QVQLVESGGGLVQAGGSLTVSCTNSGRTLSTSTIAWFRQKPGKDSEF 151-23 VAAIRWSGGMGWYADSVKGRFTISRDNAENTVSLQMNTLQPEDTAV YFCAVSTVPYYGSIYTRVGDYPYWGQGTQVTVSS S002-NB SEQIDNO.41 QVQLVESGGGLVQAGGSLRLACASGDTVSIYYMGWFRQAPGKERE 151-66 FVGALSASGQFTDHADSVKGRFTISRDDAQNTVFLHMNDLKPEDTAV YYCAVQRYGWVYRTDKTMYNYWGQGTQVTVSS S002-NB SEQIDNO.43 QVQLVESGGGLVQSGGSLRLSCAASGFTFSDYTMNWVRQAPGKGLE 151-92 WWSEISSGGHFKSNADSVKDRFTISRQNAKNTLYLQMNSLKPEDTAVY FCAKCPRPSVYQRDYESTSLYRGQGTQVTVSS S002-NB SEQIDNO.45 QVQLVESGGGLVQPGGSLTLSCAASGFQFSFYTMTWVRQAQGKGLE 151-64 WWVSTISTGGGGSNYADSVKGRFTISRDNAKKTLYLQMTSLKPEDTAVY YCASHRRGYMFVSSRKNEYDYWGRGTQVTVSS S002-NB SEQIDNO.47 QVQLVESGGGLVQAGGSLRLSCAASGRASGSGAMGWFRQAPGQER 151-30 RFVAGIEWSSGGPVYADSVKGRFTLSRQNAKNTVHLQMNSLKPEDTA VYYCAAAPWWFSSPDDYMYWGQGTQVTVSS S002-NB SEQIDNO.49 QVQLVESGGGLVQAGGSLRLSCAASGLTFSYYGMGWFRQAPGKERE 151-36 FVAAINWSGTSTYYADSVKDRFTVSRDNAKNTAYLHMNNLKPEDTAV YYCAARKYSSSQAMGLPNDIDYWGQGTQVTVSS S002-NB SEQIDNO.51 QVQLVESGGGLVQAGDSLRLSCAASTRIFRSYVMGWFRQAPGKERE 151-13 AVASIRWSNDTGTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY SCAADSPPWGRTWYTGYDYWGQGTQVTVSS S002-NB SEQIDNO.53 EVQVVESGGGLVQTGGSLRLSCAASGRPFDWLTVGWFRQAPGKER 150-152n EFVAAISQSGGRIFYSDSVKGRFTISRDNAKNTAFLQMNSLKPEDTAV YSCAAQMRGYSRAAYPDEYDYWGQGTQVTVSS S002-NB SEQIDNO.55 QVQLVESGGGLVQAGDSLRLACEASGRTATISTMGWFRQAPGKERE 150-159n FVGRIDWSAESTYIVDSVKGRFIISRDNAKNAGYLQMNSLKPVDTAVY YCAAIVVRERSYKYWGQGTQVTVSS
TABLE-US-00006 TABLE3 Specificnucleotidesequenceinformationoftheheavychainvariable regionoftheanti-CD22antibody Antibody Sequence number number Heavychainvariableregionsequence(VH) S002-NB SEQIDNO.22 CAGGTGCAGCTGGTGGAGAGCGGGGGGGGCCTGGTGCAGGCCG 150-42 GCGACAGCCTGAAGCTGAGCTGCCTGGCCAGCGGCATCACCTTC AGCAGCTACTACATGAGCTGGTTCAGGCAGGCCCCCGGCAAGGA GAGGGAGTTCGTGGGGGGCGTGGGCAGGAGGGGCGCCCCCAGC CACCACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGAGCGCCGTGTACCTGCAGATGAACAGCCTGCAGC CCGAGGACACCGCCGTGTACTTCTGCGCCGCCAAGTGGATGGCC GCCAGCACCACCGTGGCCGACTACTGGGGCCAGGGCACCCAGG TGACCGTGAGCAGC S002-N8 SEQIDNO.24 CAGCTGCAGCTGGTGGAGAGCGGGGGGGGCCTGGTGGAGGCCG 150-5 GCGGCAGCCTGAGGCTGAGCTGCGTGGGCAGCGGCGACAGGGT GAGCATCGCCCTGATGGGCTGGTTCAGGCAGGCCCCCGGCAAG GAGAGGGAGTTCGTGGCCGCCATCAGCCCCAGCGGCGGCAGGA TCACCTAGAGCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GAGAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAA GAGGGACGACACCGCCGTGTACGTGTGCGCCGCCAACTTCAGGG GCTACCTGAGCAGCAGCAGGGGCAGCGACTACGCCTACTGGGG CCAGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.26 CAGGTGCAGGTGGTGGAGAGGGGCGGCGGCCTGGTGCAGGAOG 150-40 GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCCTGACCTT CAGCTTCTACAACATGGGCTGGTTCAGGCAGGCCCCCGGCAAGG AGAGGGAGTTCGTGGCCGCCATCAGCCAGAGCGGCCTGAGCAC CCACTACGCCGACAGCATGAAGGGCAGGTTCACCATCAGCAGGG ACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAACC CCGTGGACACCGCCGCCTACTACTGCGCCGCCGGCAGGAGGGG CTACCTGGCCAGCACCACCAACGACTACGAGTACTGGGGCCAGG GCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.28 CAGGTGCAGCTGGTGGAGAGCGGGGGGGGCCTGGTGCAGGCCG 150-57 GCGACAGCCTGAGGCTGAGCTGCGCCGTGACCGGCAGGGCCTA CAGCAGGAGCGACATGGGCTGGTTCAGGCAGGCCCCCGGCAAG GAGAGGGAGATCGTGGCCGTGATCACCTTCGGCGGCGGCACCG ACTACGCCGACAGCGTGAAGGGGAGGTTCACCATCAGCAGCGAG GACACCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCAGCC CGAGGACACCGCCGTGTACTACTGCGCCGCCGGCTTCAGGGGCA GCAGGTGGAGCACCGCCGCCAGGAGGAAGGAGGACGACTACGC CTTCTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.30 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCG 150-2 GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCAGCTGGTT CAACATCGCCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGA GGGAGTTCGTGGCCGGCATCAGGTGGAGCGACAGCAAGACCTAC TACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCGGCGACAA CGCCAAGAACACCACCTACCTGCAGATGAACAGCCTGAAGCCCG AGGACACCGCCGTGTACTACTGCGCCGCCCAGAGGTACTACAGG GGCAGCTACAACAGGCAGACCAACTACGACCACTGGGGCCAGGG CACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.32 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-14 GCGGCAGCCTGAGGCTGAGCTGCACCACCAGCGGCGGCACCCT GAGCAGGATCACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAG GAGAGGGAGTTCGTGGCCGCCATCAAGTGGGGCGGCAGCAGCA CCCCCTACAGCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAACGCCAAGAACACCGTGTACCTGCAGATGGACAGGCTGGT GCCCGAGGACACCGCCGTGTACTACTGCGCCGGCGGCCTGAGG GGCCCCTACAGCGACAGCGTGACCAGCAGCGTGCAGTACAACTA CTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.34 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-51 GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCATCACCAG CGACGACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAG GAGAGGGAGGGCGTGAGCTGCATGTACAGCAGCGACGGCAGCA CCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAG GCCCGAGGACACCGCCGTGTACCACTGCGCCGCCGGCCCCTGG TGCTTCGTGACCGGCGTGGGCAGGGAGGAGTGGGGCCAGGGCA CCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.36 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-2 GCGACAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCAGGACCTT CAGCCCCAGCACCATGGCCTGGTTCAGGCAGGCCCCCGGCAAG GAGAGGGAGCTGGTGGCCAGGATCGGCTGGACCTACGGCGACA CCTACTACGTGGACAGCGCCAAGGGCAGGTTCACCATCAGCATG GACAACGTGAAGAACACCGTGTACCTGCAGATGAACAGCCTGAA GCCCGAGGACACCGCCGTGTACTACTGCGCCGCCCTGTTCAGGG GCGGCCTGAGCTACAACTTCAGCAACAGGGGCACCCAGGTGACC GTGAGCAGC S002-NB SEQIDNO.38 GAGGTGCAGGTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-82 GCGACAGCCTGAGGCTGAGCTGCACCACCAGCGGCAGGACCTTC TACGGCGACTACATCGCCTGGTTCAGGCAGGCCCCCGGCAAGGA GAGGGAGTTCGTGGCCGCCATCGCCGTGAGGGGCAGGGGCACC TTCTACGGCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCGAGAACACCCTGAGCCTGCAGATGAACAGCCTGAAGC CCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGCCGGCAG CTACGCCAGCAGGGGCTTCCCCGTGAGGGAGACCGGCAGGTAC GACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.40 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-23 GCGGCAGCCTGACCGTGAGCTGCACCAACAGCGGCAGGACCCT GAGCACCAGCACCATCGCCTGGTTCAGGCAGAAGCCCGGCAAGG ACAGCGAGTTCGTGGCCGCCATCAGGTGGAGCGGCGGCATGGG CTGGTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGG ACAACGCCGAGAACACCGTGAGCCTGCAGATGAACACCCTGCAG CCCGAGGACACCGCCGTGTACTTCTGCGCCGTGAGCACCGTGCC CTACTACGGCAGCATCTACACCAGGGTGGGCGACTACCCCTACT GGGGCCAGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.42 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-66 GCGGCAGCCTGAGGCTGGCCTGCGCCGCCAGCGGCGACACCGT GAGCATCTACTACATGGGCTGGTTCAGGCAGGCCCCCGGCAAGG AGAGGGAGTTCGTGGGCGCCCTGAGCGCCAGCGGCCAGTTCAC CGACCACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGG ACGACGCCCAGAACACCGTGTTCCTGCACATGAACGACCTGAAG CCCGAGGACACCGCCGTGTACTACTGCGCCGTGCAGAGGTACGG CTGGGTGTACAGGACCGACAAGACCATGTACAACTACTGGGGCC AGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.44 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGAGCG 151-92 GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTC AGCGACTACACCATGAACTGGGTGAGGCAGGCCCCCGGCAAGG GCCTGGAGTGGGTGAGCGAGATCAGCAGCGGCGGCCACTTCAA GAGCAACGCCGACAGCGTGAAGGACAGGTTCACCATCAGCAGGG ACAACGCCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAGC CCGAGGACACCGCCGTGTACTTCTGCGCCAAGTGCCCCAGGCCC AGCGTGTACTGCAGGGACTACGAGAGCACCAGCCTGTACAGGGG CCAGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.46 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCG 151-64 GCGGCAGCCTGACCCTGAGCTGCGCCGCCAGCGGCTTCGACTTC AGCTTCTACACCATGACCTGGGTGAGGCAGGCCCAGGGCAAGGG CCTGGAGTGGGTGAGCACCATCAGCACCGGCGGCGGCGGCAGC AACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGAAGACCCTGTACCTGCAGATGACCAGCCTGAAGC CCGAGGACACCGCCGTGTACTACTGCGCCAGCCACAGGAGGGG CTACATGTTCGTGAGCAGCAGGAAGAACGAGTACGACTACTGGG GCAGGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.48 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-30 GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCAGGGCCAG CGGCAGCGGCGCCATGGGCTGGTTCAGGCAGGCCCCCGGCCAG GAGAGGAGGTTCGTGGCCGGCATCGAGTGGAGCAGCGGCGGCC CCGTGTACGCCGACAGCGTGAAGGGCAGGTTCACCCTGAGCAGG GACAACGCCAAGAACACCGTGCACCTGCAGATGAACAGCCTGAA GCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGCCCCCTGG GTGTTCAGCAGCCCCGACGACTACATGTACTGGGGCCAGGGCAC CCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.50 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-36 GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCCTGACCTT CAGCTACTACGGCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGG AGAGGGAGTTCGTGGCCGCCATCAACTGGAGCGGCACCAGCACC TACTACGCCGACAGCGTGAAGGACAGGTTCACCGTGAGCAGGGA CAACGCCAAGAACACCGCCTACCTGCACATGAACAACCTGAAGCC CGAGGACACCGCCGTGTACTACTGCGCCGCCAGGAAGTACAGCA GCAGCCAGGCCATGGGCCTGCCCAACGACATCGACTACTGGGGC CAGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.52 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 151-13 GCGACAGCCTGAGGCTGAGCTGCGCCGCCAGCACCAGGATCTTC AGGAGCTACGTGATGGGCTGGTTCAGGCAGGCCCCCGGCAAGG AGAGGGAGGCCGTGGCCAGCATCAGGTGGAGCAACGACACCGG CACCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGG ACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAG CCCGAGGACACCGCCGTGTACAGCTGCGCCGCCGACAGCCCCC CCTGGGGCAGGACCTGGTACACCGGCTACGACTACTGGGGCCAG GGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.54 GAGGTGCAGGTGGTGGAGAGCGGCGGCGGCCTGGTGCAGACCG 150-152n GCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCAGGCCCTT CGACTGGCTGACCGTGGGCTGGTTCAGGCAGGCCCCCGGCAAG GAGAGGGAGTTCGTGGCCGCCATCAGCCAGAGCGGCGGCAGGA TCTTCTACAGCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAACGCCAAGAACACCGCCTTCCTGCAGATGAACAGCCTGAA GCCCGAGGACACCGCCGTGTACAGCTGCGCCGCCCAGATGAGG GGCTACAGCAGGGCCGCCTACCCCGACGAGTACGACTACTGGGG CCAGGGCACCCAGGTGACCGTGAGCAGC S002-NB SEQIDNO.56 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCG 150-159n GCGACAGCCTGAGGCTGGCCTGCGAGGCCAGCGGCAGGACCGC CACCATCAGCACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGG AGAGGGAGTTCGTGGGCAGGATCGACTGGAGCGCCGAGAGCAC CTACATCGTGGACAGCGTGAAGGGCAGGTTCATCATCAGCAGGG ACAACGCCAAGAACGCCGGCTACCTGCAGATGAACAGCCTGAAG CCCGTGGACACCGCCGTGTACTACTGCGCCGCCATCGTGGTGAG GGAGAGGAGCTACAAGTACTGGGGCCAGGGCACCCAGGTGACC GTGAGCAGC
TABLE-US-00007 TABLE4 SpecificsequenceinformationofCDRsofCD22nanobodyanalyzed byIMGT,KABATandChothiasoftware IMGTanalysis Antibody Sequence Sequence Sequence number number CDR1 mimber CDR2 number CDR3 S002-NB SEQID GITFSSYY SEQID VGRSGAPS SEQID AAKWMAAST 150-42 NO.37 NO.58 NO.59 TVADY S002-NB SEQID GDRVSIAL SEQID ISPSGGRI SEQID AANFRGYLSS 150-5 NO.66 NO.67 NO.68 SRGSDYAY S002-NB SEQID GLIFSFYN SEQID ISQSGLST SEQID AAGRRGYLAS 150-40 NO.75 NO.76 NO.77 TTNDYEY S002-NB SEQID GRAYSRSD SEQID ITFGGGT SEQID AAGFRGSRWS 150-57 NO.84 NO.85 NO.86 TAARRKEDDY AF S002-NB SEQID GSWENIA SEQID IRWSDSKT SEQID AAQRYYRGSY 150-2 NO.93 NO.94 NO.95 NRQTNYDH S002-NB SEQID GGILSRIT SEQID IKWGGSST SEQID AGGLRGPYSD 151-14 NO.102 NO.103 NO.104 SVTSSVWYNY S002-NB SEQID GITSDDYA SEQID MYSSDGST SEQID AAGPWCEVYG 151-51 NO.111 NO.112 NO.113 VGREE S002-NB SEQID GRTFSPST SEQID IGWTYGDT SEQID AALFRGGLSY 151-2 NO.120 NO.121 NO.122 NP S002-NB SEQID GRTFYGDY SEQID IAVRGRGT SEQID AADAGSYASR 151-82 NO.29 NO.130 NO.131 GFPVRETGRY DY S002-NB SEQID GRTLSTST SEQID IRWSGGMG SEQID AVSTVPYYGSI 151-23 NO.138 NO.139 NO.140 YTRVGDYPY S002-NB SEQID GDTVSIYY SEQID LSASGQFT SEQID AVQRYGWVY 151-66 NO.147 NO.148 NO.149 RTDKTMYNY S002-NB SEQID GFTESDYT SEQID ISSGGHFK SEQID AKCPRPSVYC 151-92 NO.156 NO.157 NO.158 RDYESTSLY 8002-NB SEQID GFDFSFYT SEQID ISTGGGGS SEQID ASHRRGYMFV 151-64 NO.165 NO.166 NO.167 SSRKNEYDY S002-NB SEQID GRASGSGA SEQID IEWSSGGP SEQID AAAPWVFSSP 151-30 NO.174 NO.175 NO.176 DDYMY S002-NB SEQID GLTFSYYG SEQID INWSGTST SEQID AARKYSSSQA 151-36 NO.183 NO.184 NO.185 MGLPNDIDY SO02-NB SEQID TRIFRSYV SEQID IRWSNDTG SEQID AADSPPWGRT 151-13 NO.192 NO.193 NO.194 WYTGYDY S002-NB SEQID GRPFDWLT SEQID ISQSGGRI SEQID AAQMRGYSR 150-152n NO.201 NO.202 NO.203 AAYPDEYDY S002-NB SEQID GRTATIST SEQID IDWSAEST SEQID AAIVVRERSY 150-1590 NO.210 NO.211 NO.212 KY Kabatanalysis Antibody Sequence Sequence Sequence povober number CDR1 number CDR2 number CDR3 S002-NB SEQID SYYMS SEQID GVGRSGAPSHHA SEQID KWMAASTTV 150-42 NO.60 NO.61 DSVKG NO.62 ADY S002-NB SEQID IALMG SEQID AISPSGGRITYSDS SEQID NFRGYLSSSRG 150-5 NO.69 NO.70 VKG NO.71 SDYAY S002-NB SEQID FYNMG SEQID AISQSGLSTHYAD SEQID GRRGYLASTI 150-40 NO.78 NO.79 SMKG NO.80 NDYEY S002-NB. SEQID RSDMG SEQID VITFGGGTDYADS SEQID GFRGSRWSTA 150-57 NO.87 NO.88 VKG NO.89 ARRKEDDYAF S002-NB SEQID IAMG SEQID GIRWSDSKTYYA SEQID QRYYRGSYNR 150-2 NO.96 NO.97 DSVKG NO.98 QTNYDH S002-NB SEQID RITMG SEQID AIKWGGSSTPYS SEQID GLRGPYSDSV 151-14 NO.105 NO.106 DSVKG NO.107 TSSVQYNY S002-NB SEQID DYAIG SEQID CMYSSDGSTYYA SEQID GPWCFVTGVG 151-51 NO.114 NO.115 DSVKG NO.116 REE S002-NB SEQID PSTMA SEQID RIGWTYGDTYYV SEQID LFRGGLSYNF 151-2 NO.123 NO.124 DSAKG NO.125 S002-NB SEQID GDYIA SEQID AIAVRGRGTFYG SEQID DAGSYASRGF 151-82 NO.132 NO.133 DSVKG NO.134 PVRETGRYDY S002-NB SEQID TSTIA SEQID AIRWSGGMGWY SEQID STVPYYGSIYT 151-23 NO.141 NO.142 ADSVKG NO.143 RVGDYPY S002-NB SEQID IYYMG SEQID ALSASGQFTDHA SEQID QRYGWVYRT 151-66 NO.156 NO.151 DSVKG NO.152 DKTMYNY S002-NB SEQID DYTMN SEQID EISSGGHFKSNAD SEQID CPRPSVYCRD 151-92 NO.159 NO.160 SVKD NO.161 YESTSLY S002-NB SEQID FYTMT SEQID TISTGGGGSNYAD SEQID HRRGYMFVSS 151.64 NO.168 NO.169 SVKG NO.170 RKNEYDY S002-NB SEQID SGAMG SEQID GIEWSSGGPVYA SEQID APWVFSSPDD 151-30 NO.177 NO.178 DSVKG NO.179 YMY S002-NB SEQID YYGMG SEQID AINWSGTSTYYA SEQID RKYSSSQAMG 151-36 NO.186 NO.187 DSVKD NO.188 LPNDIDY S002-NB SEQID SYVMG SEQID SIRWSNDTGTYA SEQID DSPPWGRTWY 151-13 NO:195 NO.196 DSVKG NO.197 TGYDY S002-NB SEQID WLTVG SEQID AISQSGGRIFYSD SEQID QMRGYSRAAY 150-152n NO.204 NO.205 SVKG NO.206 PDEYDY S002-NB SEQID ISTMG SEQID RIDWSAESTYTVD SEQID IVVRERSYKY 150-159n NO.213 NO.214 SVKG NO.215 Chothiaanalysis Antibody Sequence Sequence Sequence number number CDR1 number CDR2 number CDR3 S002-NB SEQID GITFSSY SEQID GRSGAP SEQID KWMAASTTV 150-42 NO.63 NO.64 NO.63 ADY S002-NB SEQID GDRVSIA SEQID SPSGGR SEQID NFRGYLSSSRG 150.5 NO.72 NO.73 NO.74 SDYAY $002-NB SEQID GLTFSFY SEQID SQSGLS SEQID GRRGYLASTT 150-40 NO.81 NO.82 NO.83 NDYEY S002-NB SEQID GRAYSRS SEQID TFGGG SEQID GFRGSRWSTA 150-57 NO.90 NO.91 NO.92 ARRKEDDYAF 6002-NB SEQID GSWFNI SEQID RWSDSK SEQID QRYYRGSYNR 150-2 NO.99 NO.100 NO.101 QTNYDH S002-NB SEQID GGTLSRI SEQID KWGGSS SEQID GLRGPYSDSV 151-14 NO.108 NO.109 NO.110 TSSVQYNY S002-NB SEQID GITSDDY SEQID YSSDGS SEQID GPWCFVTGVG 151-51 NO.117 NO.118 NO.119 REE 6002-NB SEQID GRTFSPS SEQID GWTYGD SEQID LFRGGLSYNF 151-2 NO.126 NO.127 NO.128 S002-NB SEQID GRTFYGD SEQID AVRGRG SEQID DAGSYASRGP 151-82 NO.135 NO.136 NO.137 PVRETGRYDY $002-NB SEQID GRTLSTS SEQID RWSGGM SEQID STVPYYGSIYT 151-23 NO.144 NO.145 NO.146 RVGDYPY S002-NB SEQID GDTVSIY SEQID SASGQP SEQID QRYGWVYRT 151-66 NO.153 NO.154 NO.155 DKTMYNY S002-NB SEQID GFTFSDY SEQID SSGGHF SEQID CPRPSVYCRD 161-92 NO.162 NO.163 NO.164 YESTSLY S002-NB SEQID GFDFSFY SEQID STGGGG SEQID HRRGYMFVSS 151-64 NO.171 NO.172 NO.173 RKNEYDY S002-NB SEQID GRASGSG SEQID EWSSGG SEQID APWVFSSPDD 151-30 NO.180 NO.181 NO.182 YMY 6002-NB SEQID GLTFSVY SEQID NWSGTS SEQID RKYSSSQAMG 161-36 NO.189 NO.190 NO.191 LPNDIDY S002-NB SEQID TRIFRSY SEQID RWSNDT SEQID DSPPWGRTWY 151-13 NO.198 NO.199 NO.200 TGYDY S002-NB SEQID GRPFDWL SEQID SQSGGR SEQID QMRGYSRAAY 150-152n NO.207 NO.208 NO.209 PDEYDY S002-NB SEQID GRTATIS SEQID DWSAES SEQID IVVRERSYKY 150-159n NO.216 NO.217 NO.218
Example 2 Preparation of VHH Antibody, Control Antibody, Polyclonal Antibody Serum and CD22 Protein 2.1 Expression and Purification of VHH Antibody
[0199] The VHH variable region sequence was recombined into the expression vector BI3.4-huIgG1 containing a signal peptide and human IgG1 Fc (the human IgG1 Fc sequence was shown in SEQ ID NO: 14, the hinge region sequence was shown in SEQ ID NO: 15) by Taizhou Biointron Biotechnology Co., Ltd, and plasmids were prepared according to the established standard molecular biology methods. For the specific method, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). The expression vector was transiently transfected into HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) according to the instructions of PEI (purchased from Polysciences, catalog number: 24765-1), and the transfected cells were continuously cultured at 37 C. for 5 days using FreeStyle 293 (Thermo Fisher Scientific, catalog number: 12338018), and the cell components were removed by centrifugation to obtain the culture supernatant containing the VHH antibody. The culture supernatant was loaded onto the protein A chromatography column (the protein A filler AT Protein A Diamond and the chromatography column BXK 16/26 were both purchased from Bestchrom (Shanghai) Biosciences Ltd., and the catalog numbers were: AA0273 and B-1620, respectively), the column was washed with PBS phosphate buffer (pH 7.4), then washed with 20 mM PB, 1M NaCl (pH 7.2), and finally eluted with citric acid buffer (pH 3.4). The antibody with Fc label eluted from the protein A chromatography column was collected, neutralized with 1/10 volume of 1M Tris (pH 8.0), and dialyzed with PBS at 4 C. overnight, and the dialyzed protein was aseptically filtered by 0.22 micron filter membrane and then subpackaged for storage at 80 C.
TABLE-US-00008 SEQIDNO:14SequenceofhumanIgG1Fc: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQIDNO:15Hingeregionsequence: EPKSADKTHTCPPCP
[0200] 2.2 Preparation of Control Antibody
[0201] The CD22 protein has 7 IgG-like domains outside the cell, in which domain 1 is located at the farthest end from the membrane and domain 7 is located at the nearest end from the membrane. HA22, m971 and hL22 are antibodies that recognize human CD22, wherein the antigen-binding epitopes of HA22 and hL22 are located in domain 2-3, and the antigen-binding epitope of m971 is located in domain 5-7. The heavy chain variable region and light chain variable region sequences of HA22 were obtained according to U.S. Pat. No. 9,580,461 B (which is incorporated herein by reference), the heavy chain variable region and light chain variable region sequences of m971 were obtained according to U.S. Pat. No. 8,591,889 B (which is incorporated herein by reference), and the heavy chain variable region and light chain variable region amino acid sequences of hL22 were obtained according to U.S. Pat. No. 5,789,554 B (which is incorporated herein by reference). The VH and VL of the antibody HA22, m971 and hL22 that recognize human CD22 and the human IgG1 Fc were linked in order from the N-terminal to the C-terminal, wherein the VH and VL were linked by three GGGGS linkers to form scFv-hFc, and the corresponding amino acid sequence information was shown in Table 5 below. The corresponding nucleotide sequences were respectively cloned into the pTT5 vector by GENERAL Biosystems (Anhui) Corporation Limited and expressed in HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) and purified according to the method of Example 2.1.
TABLE-US-00009 TABLE5 SpecificsequenceinformationofVHandVLofanti-humanCD22 antibodyHA22,m971andhL22andhumanIgG1FcandscFv-hFcformed thereby Designationof Sequence sequence number Aminoacidsequence HA22VH SEQID EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCL NO:5 EWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDT AMYYCARHSGYGTHWGVLFAYWGQGTLVTVSA HA22VL SBOID DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGVKL NO:6 LIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQGNTL PWTFGCGTKLEIK HA22 SEQID EVQLVESGGGLVKPGGSLKLSCAASGFAPSIYDMSWVRQTPEKCL scFv-hFc NO:7 EWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDT AMYYCARHSGYGTHWGVLFAYWGQGTLVTVSAGGGGSGGGGSG GGGSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDG TVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQ GNTLPWTFGCGTKLEIKEPKSADKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK m971VH SEQID QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG NO:8 LEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPE DTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS In971VL SEQID DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNL NO:9 LIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSI PQTPGQGTKLEIK m971 SEQID QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG scFv-hFc NO:10 LEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPE DTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGG GGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQORPGK APNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQ QSYSIPQTFGQGTKLEIKEPKSADKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDQVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDESRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK hL22VH SEQID QVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVR NO:11 QAPGQGLEWIGYINPRNDYTEY NQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRDI TTFYWGQGTTVTVSS hL22VL SEQID DIQLTQSPSSLSASYGDRVTMSCKSSQSVLYSANHKNYLA NO:12 WYQQKPGKAPKLLIYWASTR ESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSW TFGGGTKLEIK HL22scFv-Fc SEQID QVQLQOSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVR NO:13 QAPGQGLEWIGYINPRNDYTEY NQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRDI TTFYWGQGTTVTVSSGGGGGGGGSGGGGSDIQLTQSPSS LSASVGDRVTMSCKSSQSVLYSANHKNYLAWYQQKPGK APKLLIYWASTR ESGVPSRESGSGSGTDFTFTISSLQPEDIATYYCHQYLSSW TFGGGTKLEIKEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISKTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK
[0202] 2.3 Preparation of Human CD22-His Tag Protein
[0203] The CD22 protein has 7 IgG-like domains outside the cell, in which domain 1 is located at the farthest end from the membrane and domain 7 is located at the nearest end from the membrane, the antigen-binding epitopes of HA22 and hL22 are located in domain 2-3, and the antigen-binding epitope of m971 is located in domain 5-7. The nucleotide sequences encoding the amino acid sequence of human CD22 protein (NCBI: NP_001762.2, SEQ ID NO: 1), the extracellular region (ECD, extra-cellular domain) amino acid sequence Asp 20-Arg 687 (SEQ ID NO: 2), the domain 1-4 Asp 20-Val 425 amino acid sequence (SEQ ID NO: 3) and the domain 5-7 Asp 414-Arg 687 amino acid sequence (SEQ ID NO: 4) were cloned into the pTT5 vector by GENERAL Biosystems (Anhui) Corporation Limited, respectively, and plasmids were prepared according to the established standard molecular biology methods. The corresponding amino acid sequence information was shown in Table 6 below. For the specific method, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) were transiently transfected (PEI, Polysciences, catalog number: 24765-1) and FreeStyle293 (Thermofisher scientific, catalog number: 12338018) was used for scale-up culture at 37 C. After 6 days, the cell culture fluid was collected, centrifuged to remove cell components, and the culture supernatant containing the extracellular region of human CD22 protein was obtained. The culture supernatant was loaded onto a nickel ion affinity chromatography column HisTrap Excel (GE Healthcare, catalog number: GE17-3712-06), and the change in the ultraviolet absorbance (A280 nm) was monitored with an ultraviolet (UV) detector. After sample loading, the nickel ion affinity chromatography column was washed with 20 mM PB, 0.5M NaCl (pH 7.4) until the ultraviolet absorbance returned to the baseline, and then gradient elutions (2%, 4%, 8%, 16%, 50%, 100%) were performed with Buffer A: mM PB, 0.5M NaCl (pH 7.4) and Buffer B: 20 mM PB, 0.5M NaCl, and 500 mM imidazole. His-tagged human CD22 protein eluted from the nickel ion affinity chromatography column was collected and dialyzed against PBS phosphate buffer (pH 7.4) overnight in a refrigerator at 4 C. The dialyzed protein was aseptically filtered by 0.22 micron filter membrane and then subpackaged for storage at 80 C. to obtain purified human CD22 protein. The bands of interest of samples detected by SDS-PAGE reducing gel and non-reducing gel were shown in
TABLE-US-00010 TABLE6 AminoacidsequencesofhumanCD22proteinandextracellular region Designation Sequence ofsequence number Aminoacidsequence Human SEQID MHLLGPWLLLLVLEYLAFSDSSKWVFHPETLYAWEGACVWIPCTYRALD CD22 NO:1 GDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPDEQRRVQFLGDKNKN protein CTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPHIQLPPEIQES QEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTLTIKSVFTRSELKFS PQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTPSDAIVREG DSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNREVTKDQSGKYC CQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPL PTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPG AELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGA WEEPSLGVLKIQNVGWDNTTIACAACNSWCSWASPVALNVQYAPRDVRVR KIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLLGKESQLNFDSISPE DAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSPGDQVMEGKSATL TCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVKVQHSGAYWCQGTN SVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILILAICGLKLQRRWKRTQ SQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRF PEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGDYENVIPDFPEDE GIHYSELIQFGVGERPQAQENVDYVILKH Human SEQID DSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNPEYNKNTSK CD22 NO:2 FDGTRLYESTEDGKYPSEQKRVQFLGDKNKNCTLSIHPVHENDSGQLGLRM protein ESKTEKWMERIHLNVSERPFPPHIQLFPEIQESQEVTLTCLLNFSCYGYPIQL extracellular QWLLEGVPMRQAAVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDAD region GKFLSNDTVQLNVKHTPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTV SWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDNGPGRSEEVFLQV QYAPEPSTVQILHSPAVEGSQVEFLCMSLANPLPTNYTWYHNGKEMQGRTE EKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPKKVTTVIQNP MPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDN TTIAVAACNSWCSWASPVALNVQYAPRDVRVRKIKPLSEHSGNSVSLQCDF SSSHPKEVQFFWEKNGRLLGKESQLNFDSISPEDAGDYCWVNNSIGQTASK AWTLEVLYAPRRLRVSMSPGDQVMEGKSATLTCESDANPPVSHYTWFDWN NQSLPYHSQKLRLEPVKVQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETI GRR Human SEQID DSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNPEYNKNTSK CD22 NO:3 FDGTRLYESTKDGKVPSEQKRVQFLGDKNKKNCTLSIHPVHLNDSGQLGLRM proteins ESKTEKWMERIHLNVSERPFFPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQL domain1- QWLLEGVPMRQAAVTSTSLTIKSVFTRSELFFSPQWSHHGKIVTCQLQDAD 4 GKFLSNDTVQLNVKHTPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTV SWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDVGPGRSEEVFLQV QYAPEPSTVQILHSPAVEGSQVEPLCMSLANPLPTNYTWYHNGKEMQGRTE EKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPKKVTTV Human SEQID DVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEE CD22 NO:4 PSLGLKIQNVGWDNTTIACAACNSWCSWASPVALNVQYAPRDVRVRKIK protein PLSEIHGNSVSLQCDFSSSHPKEVQFFWEKNGRLLGKESQLNFDSISPEDA domain5- GSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSPGDQVMEGKSATLTCE 7 SDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVKVQHSGAYWCQGTNSVG KGRSPLSTLTVYYSPETIGRR
Example 3 Identification of Endogenous Expression Cell Line and Preparation of Overexpression Cell Line
[0204] 3.1 Identification of Cell Line Expressing CD22 Endogenously
[0205] Raji cells (purchased from China Center for Type Culture Collection, Wuhan University) were scale-up cultured in T-25 cell culture flasks to the logarithmic growth phase, the supernatant of the medium was discarded by centrifugation, and the cell pellet was washed 2 times with PBS. The HA22 and m971 antibodies were used as primary antibodies, APC-labeled secondary antibody (purchased from Biolegend, catalog number: 409306) was used and FACS (FACS Canto, purchased from BD company) was used for detection and result analysis. The analysis results were shown in Table 7 and
TABLE-US-00011 TABLE 7 FACS detection results of the endogenous cell line Raji cells Cell mean fluorescence density Designation IgG subtype CD22 No. of antibody control antibody 1 HA22 158 6308 2 m971 158 3955
[0206] 3.2 Preparation of CHO-K1 Monoclonal Cell Line Stably Transfected with Human CD22
[0207] The nucleotide sequence encoding the full-length amino acid sequence of human CD22 (NCBI: NP_001762.2, SEQ ID NO: 1) was cloned into the pcDNA3.1 vector and a plasmid was prepared by GENERAL Biosystems (Anhui) Corporation Limited. Plasmid transfection (Lipofectamine 3000Transfection Kit, purchased from Invitrogen, catalog number: L3000-015) was performed on CHO-K1 cell line (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and then the transfected cells were selectively incubated for 2 weeks in DMEM/F12 medium containing 10 g/ml of puromycin and 10% (w/w) of fetal bovine serum. The FITC-labeled anti-CD22 antibody (Thermofisher scientific, catalog number: 11-0229-42) was used to sort the positive monoclonal cells into a 96-well plate on flow cytometer FACS Ariall (BD Biosciences) and the plate was placed in a cell incubator at 37 C. and 5% (v/v) CO.sub.2 for cell culture. Some wells containing monoclonal cells were selected for amplification after approximately 2 weeks. The amplified clones were screened by flow cytometry. The monoclonal cell line with better growth and higher fluorescence intensity was selected for further scale-up culture and then freezed in liquid nitrogen.
[0208] The specific selection results were shown in Table 8 and
TABLE-US-00012 TABLE 8 FACS detection results of CHO-K1 cell line stably transfected with human CD22 protein Cell mean fluorescence intensity Clone number of stably IgG subtype CD22 No. transfected cell line control antibody 1 CHO-K1-human CD22 2C4 66 4621 2 CHO-K1-human CD22 1G5 66 3154 3 CHO-K1-human CD22 1D9 66 2488
Example 4 Identification of VHH Antibody in Alpaca
[0209] 4.1 Binding of VHH-Fc Antibodies to Human CD22 Protein Detected by Enzyme-Linked Immunosorbent Assay (ELISA)
[0210] In order to detect the binding activity of VHH-Fc to human CD22 protein, the purified human CD22-ECD-His protein obtained in Example 2 was diluted with PBS to a final concentration of 2 g/mL, and then added to 96-well ELISA plate at 100 l/well. The plate was sealed with plastic film and incubated overnight at 4 C., the plate was washed 2 times with PBS the next day, and then a blocking solution [PBS+2% (w/w) BSA] was added for blocking at room temperature for 2 hours. The blocking solution was poured off, and 100 nM of serially diluted VHH-Fc antibody or negative control antibody was added at 50 l/well. After incubation at 37 C. for 2 hours, the plate was washed 3 times with PBS. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma, catalog number: A0170) was added, and incubated at 37 C. for 2 hours, and the plate was washed 5 times with PBS. TMB substrate was added at 50 l/well, and incubated at room temperature for 30 minutes, then a stop solution (1.0 N HCl) was added at 50 l/well. An ELISA plate reader (Multimode Plate Reader, EnSight, purchased from PerkinElmer) was used to read the OD450nm value, and the ELISA results of VHH-Fc and human CD22-ECD are shown in
TABLE-US-00013 TABLE 9 Binding reaction of VHH-Fc antibodies with human CD22 protein detected by ELISA OD450 Designation of Antibody concentration (nM) antibody 100 10 0.10 0.01 0.001 0.0001 0.00001 Blank S002-NB151-14 2.01 1.71 1.28 0.58 0.33 0.22 0.25 0.05 S002-NB151-23 1.33 1.24 1.24 0.69 0.35 0.25 0.26 0.05 S002-NB151-30 1.80 1.72 1.35 0.53 0.22 0.11 0.11 0.05 S002-NB151-51 1.68 1.69 1.54 0.60 0.18 0.10 0.07 0.05 S002-NB151-66 1.38 1.43 1.29 0.64 0.26 0.15 0.14 0.05 S002-NB151-82 1.54 1.68 1.30 0.59 0.23 0.13 0.13 0.05 S002-NB150-2 2.29 1.89 1.83 1.15 0.44 0.26 0.17 0.06 S002-NB150-5 2.56 2.04 1.91 1.10 0.46 0.24 0.15 0.06 S002-NB150-40 2.26 1.95 1.65 0.68 0.25 0.15 0.11 0.06 S002-NB150-42 2.02 1.82 1.87 0.93 0.28 0.12 0.09 0.05 S002-NB150-57 2.08 1.90 1.90 1.00 0.30 0.13 0.08 0.05 S002-NB151-2 1.93 1.86 1.82 1.17 0.54 0.30 0.17 0.05 S002-NB151-13 2.12 1.87 1.65 0.86 0.39 0.25 0.20 0.06 S002-NB151-36 2.58 2.21 1.78 0.69 0.24 0.11 0.85 0.05 S002-NB151-64 2.18 2.03 1.95 1.08 0.35 0.17 0.11 0.05 S002-NB151-92 1.61 2.15 1.86 0.71 0.17 0.08 0.06 0.05 S002-NB150-152n 3.33 2.82 2.66 1.21 0.29 0.13 0.10 0.07 S002-NB150-159n 3.20 2.58 2.22 0.90 0.20 0.09 0.07 0.06 HA22 2.01 1.92 1.50 0.45 0.13 0.07 0.06 0.05 m971 1.77 1.87 1.55 0.56 0.15 0.08 0.06 0.05 hIgG1 0.15 0.08 0.06 0.06 0.06 0.06 0.05 0.06
[0211] 4.2 The Binding of Antibody to Different CD22 Expressing Cells Detected by Flow Cytometry (FACS)
[0212] The required cells were scale-up cultured in a T-75 cell culture flask to the logarithmic growth phase. For the adherent cell CHO-K1, the medium was aspirated, the cells were washed 2 times with PBS buffer, and then digested with trypsin. After the digestion was terminated, the cells were washed 2 times with PBS buffer. For suspension cell Raji, the medium supernatant was directly centrifuged and discarded, and the cell pellet was washed 2 times with PBS. After counting the cells in the previous step, the cell pellet was resuspended with [PBS+2% (w/w) BSA] blocking solution to 210 6 cells/ml, and added to a 96-well FACS reaction plate at 50 l/well, and then the VHH-Fc antibody test sample was added at 50 l/well, and incubated on ice for 2 hours. The mixture was centrifuged and washed 3 times with PBS buffer, Alexa Flour 488-labeled secondary antibody (purchased from Invitrogen, catalog number: A-11013) was added at 50 l/well, and incubated on ice for 1 hour. The obtained mixture was centrifuged and washed 5 times with PBS, and FACS (FACS Canto, purchased from BD Company) was used for detection and result analysis. Data analysis was performed by software (CellQuest) to obtain the mean fluorescence density (MFI) of the cells. And then software (GraphPad Prism8) was used for analysis, data fitting, and EC50 value calculation. The analysis results are shown in Table 10 and
TABLE-US-00014 TABLE 10 Binding reaction of VHH-Fc antibodies with Raji and CHO-K1-human CD22 2C4 cells detected by FACS Raji CHO-K1-human CD22 2C4 Maximum mean Maximum mean fluorescence fluorescence Designation intensity intensity of antibody Max MFI Ec50 (nM) Max MFI Ec50 (nM) S002-NB151-2 4197 0.32 17895 1.17 S002-NB151-13 3958 0.45 17556 1.36 S002-NB151-14 2370 1.03 11135 3.7 S002-NB151-23 2203 0.49 11799 2.02 S002-NB151-30 2327 0.80 12810 2.46 S002-NB151-36 4761 0.41 22344 1.37 S002-NB151-51 2377 0.40 12810 1.45 S002-NB151-64 4690 0.26 24458 0.66 S002-NB151-66 2313 0.48 12540 2.14 S002-NB151-82 2169 0.31 12967 1.67 S002-NB151-92 3922 0.68 13705 1.30 S002-NB150-2 4106 0.25 22178 0.87 S002-NB150-5 4615 0.17 25743 0.71 S002-NB150-40 4066 0.36 22482 1.44 S002-NB150-42 4465 0.20 24363 0.73 S002-NB150-57 4655 0.17 26675 0.76 S002-NB150-152n 2113 0.12 10587 1.04 S002-NB150-159n 1871 0.37 9655 2.14 HA22 2521 0.83 9968 1.89 m971 2039 4.54 8839 3.07 hL22 3080 3.80 15153 2.50 hIgG1 374 Negative 340 Negative
Example 5 Detection of Species Cross-Binding Activity of VHH-Fc Antibodies
[0213] 5.1 Binding of VHH-Fc Antibodies to Murine CD22 Protein Detected by ELISA
[0214] In order to detect the species cross-binding activity of the VHH-Fc antibodies, an ELISA plate was coated with commercial mouse CD22 (ACROBiosystems, catalog number: SI2-M52Ha), and the ELISA detection was performed according to the method in Example 4.1. The ELISA results of VHH-Fc and murine CD22-ECD are shown in
TABLE-US-00015 TABLE 11 Binding reaction of VHH-Fc antibodies with murine CD22 protein detected by ELISA OD450 Designation of Antibody concentration (nM) antibody 100 10 0.10 0.01 0.001 0.0001 0.00001 Blank S002-NB151-14 0.15 0.08 0.06 0.06 0.07 0.07 0.07 0.09 S002-NB151-23 0.14 0.07 0.06 0.06 0.06 0.06 0.07 0.08 S002-NB151-30 0.12 0.07 0.06 0.06 0.06 0.07 0.07 0.09 S002-NB151-51 2.31 2.77 2.33 0.69 0.13 0.08 0.07 0.09 S002-NB151-66 0.14 0.08 0.06 0.06 0.06 0.07 0.08 0.10 S002-NB151-82 0.12 0.08 0.06 0.07 0.06 0.08 0.07 0.09 S002-NB150-2 0.09 0.06 0.05 0.05 0.05 0.05 0.05 0.05 S002-NB150-5 0.13 0.06 0.06 0.05 0.05 0.05 0.05 0.05 S002-NB150-40 0.06 0.05 0.05 0.05 0.05 0.05 0.05 0.06 S002-NB150-42 0.07 0.06 0.05 0.05 0.05 0.05 0.05 0.06 S002-NB150-57 0.07 0.06 0.05 0.05 0.05 0.05 0.05 0.05 S002-NB151-2 0.08 0.06 0.05 0.06 0.05 0.06 0.05 0.05 S002-NB151-13 0.38 0.11 0.06 0.06 0.06 0.06 0.06 0.05 S002-NB151-36 0.07 0.05 0.05 0.05 0.05 0.05 0.06 0.06 S002-NB151-64 0.10 0.06 0.11 0.08 0.05 0.05 0.06 0.05 S002-NB151-92 0.07 0.05 0.07 0.05 0.05 0.05 0.06 0.05 S002-NB150-152n 0.10 0.06 0.06 0.07 0.06 0.06 0.06 0.07 S002-NB150-159n 0.07 0.05 0.05 0.05 0.05 0.05 0.05 0.06 983* 2.81 2.87 2.34 1.04 0.27 0.12 0.10 0.11 hIgG1 0.10 0.09 0.09 0.08 0.09 0.08 0.09 0.09 *983 serum was 5-fold serially diluted from 1:100.
[0215] 5.2 Binding of VHH-Fc Antibodies to Peripheral Blood B Cells of Cynomolgus Monkey (Latin Name: Macaca fascicularis) Detected by FACS
[0216] The monkey peripheral blood mononuclear cells were extracted from fresh cynomolgus monkey peripheral blood (purchased from Shanghai Medicilon Inc.)
[0217] according to the instructions of Ficoll-Paque Plus (purchased from GE Healthcare, catalog number: 171440-02). After the cell suspension was centrifuged, the cells were resuspended in PBS containing 1% BSA, and then the cells were counted. At the same time, the murine antibody Brilliant Violet 605 anti-human CD20 (catalog number: 302334, purchased from Biolegend) with monkey CD20 cross-binding activity and the VHH-Fc antibodies to be tested (1 nM, 10 nM and 100 nM) were added. The mixture was incubated for 1 hour at room temperature. After washing the cells three times, APC-labeled secondary antibody anti-human IgG Fc (catalog number: 409306, purchased from Biolegend) was added. After incubation at room temperature in the dark for 30 minutes, the cells were washed 5 times, gently resuspended with PBS containing 1% BSA, and detected and analyzed by FACS (FACS Canto, purchased from BD Company). Wherein CD20 was used as a marker of B cells, and the CD20-positive B cell population was gated, the proportion of VHH-Fc positive cells was analyzed, and the proportion of VHH-Fc positive cell population to B cell population was calculated after treatments with VHH-Fc antibodies at the concentrations of 100 nM, 10 nM and 1 nM, respectively. The results are shown in Table 12. The scatter plot of double-stained cells by Brilliant Violet 605-labeled CD20 and APC secondary antibody indirectly labeled VHH-Fc is shown in
TABLE-US-00016 TABLE 12 Binding reaction of VHH-Fc antibodies with cynomolgus monkey B cells detected by FACS Proportion of VHH-Fc antibody positive cells to B cells (%) Designation Antibody concentration of antibody 100 nM 10 nM 1 nM S002-NB151-2 84 86 67 S002-NB151-13 61 23 3 S002-NB151-14 94 90 84 S002-NB151-23 94 88 85 S002-NB151-30 90 70 32 S002-NB151-36 40 10 5 S002-NB151-51 96 93 90 S002-NB151-64 19 4 3 S002-NB151-66 22 5 20 S002-NB151-82 2 2 3 S002-NB151-92 75 69 54 S002-NB150-2 89 80 73 S002-NB150-5 36 3 3 S002-NB150-40 3 0 3 S002-NB150-42 80 36 3 S002-NB150-57 58 16 3 S002-NB150-152n 14 6 0 S002-NB150-159n 30 18 4 HA22 96 84 59 hIgG1 3 0 0
Example 6 CD22 Antibody Affinity Assay
[0218] 6.1 Assay of Affinity of VHH-Fc to Human CD22-ECD-his Protein
[0219] Anti-human CD22 VHH-hFc antibody was captured using a Protein A chip (GE Healthcare; 29-127-558). Sample buffer and running buffer were HBS-EP+(10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69). The flow-through cell was set to 25 C. The sample block was set to 16 C. Both were pretreated with the running buffer. In each cycle, the antibody to be tested was first captured with a Protein A chip, then a single concentration of CD22 antigen protein was injected. The binding and dissociation process of the antibody and the antigen protein was recorded, and finally Glycine pH 1.5 (GE Healthcare; BR-1003-54) was used to complete chip regeneration. Binding was measured by injecting different concentrations of recombinant human CD22-ECD His in solution for 240 s with a flow rate of 30 L/min. The concentration started from 200 nM (see the detailed results for the actual concentration in the test) and was diluted at 1:1, making a total of 5 concentrations. The dissociation phase was monitored for up to 600 s and was triggered by switching from sample solution to running buffer. The surface was regenerated by washing with a 10 mM of glycine solution (pH 1.5) for 30 s at a flow rate of 30 L/min. Bulk refractive index difference was corrected by subtracting the response obtained from the goat anti-human Fc surface. Blank injection was also subtracted (=double reference). For calculation of apparent KD and other kinetic parameters, Langmuir 1:1 model was used. The binding rate (Ka), dissociation rate (Kd) and binding affinity (KD) of the VHH-Fc antibodies to human CD22-His protein are shown in Table 13, and the antibody HA22 was used as a control. As shown in
TABLE-US-00017 TABLE 13 Affinity of VHH-Fc antibodies to human CD22 detected by SPR (biacore) Designation of antibody ka (1/Ms) kd (1/s) KD (M) S002-NB151-2 1.69E+04 8.27E04 4.89E08 S002-NB151-13 5.98E+04 3.58E05 5.98E10 S002-NB151-14 1.60E+04 2.54E04 1.59E08 S002-NB151-23 2.04E+05 4.76E04 2.33E09 S002-NB151-30 8.43E+04 5.14E04 6.09E09 S002-NB151-36 8.73E+04 5.20E04 5.97E09 S002-NB151-51 1.42E+05 7.39E05 5.20E10 S002-NB151-64 2.39E+05 2.07E03 8.68E09 S002-NB151-66 2.49E+05 5.62E04 2.25E09 S002-NB151-82 6.00E+04 1.74E03 2.90E08 S002-NB151-92 4.40E+04 1.11E03 2.52E08 S002-NB150-2 3.17E+04 7.61E05 2.40E09 S002-NB150-5 8.32E+04 3.09E04 3.71E09 S002-NB150-40 8.63E+04 5.41E04 6.27E09 S002-NB150-42 1.96E+05 4.23E04 2.16E09 S002-NB150-57 1.10E+05 4.07E04 3.70E09 S002-NB150-152n 2.12E+05 4.42E04 2.08E09 S002-NB150-159n 2.89E+04 6.95E05 2.40E09 HA22 8.15E+04 1.12E04 1.37E09 m971 2.51E+05 7.36E03 2.93E08
[0220] 6.2 Assay of Affinity of VHH-Fc to Cynomolgus Monkey CD22-ECD-his Protein
[0221] According to the method of Example 6.1, the affinity of VHH-Fc antibodies to cynomolgus monkey CD22-ECD-His (purchased from R&D, catalog number: 9864-SL-050) protein was determined, and the results are shown in
TABLE-US-00018 TABLE 14 Affinity of VHH-Fc antibodies to cynoCD22 detected by SPR (biacore) Designation of antibody ka (1/Ms) kd (1/s) KD (M) S002-NB151-2 There is binding/the fit is poor S002-NB151-23 There is binding/the fit is poor S002-NB151-14 There is binding/the fit is poor S002-NB151-51 1.64E+05 8.31E05 5.07E10 S002-NB151-92 There is binding/the fit is poor S002-NB150-2 There is binding/the fit is poor HA22 1.27E+04 3.76E04 2.97E07
Example 7 Antibody Antigen-Binding Epitope Analysis
[0222] 7.1 Identification of Antigen-Binding Region of Antibody
[0223] The CD22 protein has 7 IgG-like domains outside the cell, in which domain 1 is located at the farthest end from the membrane and domain 7 is located at the nearest end from the membrane, the antigen-binding epitopes of HA22 and hL22 are located in domain 2-3, and the antigen-binding epitope of m971 is located in domain5-7. In order to identify the antigen-binding epitope distribution of the VHH antibodies, according to the ELISA method in Example 4.1, human CD22-domain1-4-His (distal end of membrane) and human CD22 domain5-7-His (proximal end of membrane) were used for coating, respectively. The VHH antibodies were classified into the type of distal end of membrane and the type of proximal end of membrane, as shown in
TABLE-US-00019 TABLE 15 Epitopes at the distal end of membrane and epitopes at the proximal end of membrane of the VHH antibodies classified by ELISA method Binding region Designation of antibody domain 1-4 domain 5-7 S002-NB151-13 + S002-NB151-14 + S002-NB151-23 + S002-NB151-30 + S002-NB151-36 + S002-NB151-51 + S002-NB151-64 + S002-NB151-66 + S002-NB151-82 + S002-NB150-2 + S002-NB150-5 + S002-NB150-40 + S002-NB150-42 + S002-NB150-57 + S002-NB150-152n + S002-NB151-2 + S002-NB151-92 + S002-NB150-159n +
[0224] 7.2 Antibody Antigen-Binding Epitope Competition Experiment (Epitope Binning)
[0225] Competitive ELISA was used to classify the epitopes of VHH antibodies and control antibody with known epitopes. According to the method in Example 4.2, the ELISA plate was coated with 2 g/mL of the antibody, and the human CD22-ECD-his protein was serially diluted starting from 30 g/mL, and the EC80 value was calculated (Table 16). The ELISA plate was coated with 2 g/mL of the antibody, 25 g/mL of the antibody to be detected was added, and then human CD22-ECD-his protein corresponding to each antibody to be detected was added at the EC80 concentration, incubated for 2 hours, and washed 5 times with PBS, and then HRP-labeled anti-His antibody was added for detection. If there is no competitive relationship between the antibody coated on the plate and the antibody to be detected in the solution, the antibody coated on the plate can bind to the complex of the antibody to be detected and the human CD22-ECD-his antigen in solution, and the absorption at OD450nm was detected, and the inhibition rate between each pair of antibodies was calculated according to the absorbance at OD450nm (
TABLE-US-00020 TABLE 16 EC80 value of human CD22-ECD-his protein corresponding to VHH antibody Designation of antibody EC80 (nM) domain 1-4 S002-NB151-23 3.20 HA22 0.80 S002-NB151-64 17.60 S002-NB151-66 2.80 S002-NB150-2 1.73 S002-NB150-5 3.73 S002-NB150-40 2.40 S002-NB150-42 2.27 S002-NB150-57 1.60 S002-NB150-152n 4.00 hL22 0.93 S002-NB151-82 4.00 S002-NB151-36 11.07 S002-NB151-14 32.27 S002-NB151-30 8.13 S002-NB151-51 1.60 S002-NB151-13 80.13 domain 5-7 S002-NB151-2 20.80 S002-NB151-92 15.60 S002-NB150-15911 5.73 111971 2.13