ANTI-HUMAN CD22 MONOCLONAL ANTIBODIES AND USE THEREOF

Abstract

CD22 antibodies, a preparation method therefor, and an application thereof. The CD22 antibodies have a high affinity to CD22 protein Therefore, the CD22 antibodies can be used in the preparation of drugs for the treatment of diseases such as tumors and autoimmune diseases.

Description

The present application claims the right of priority of Chinese patent application no. 202110060035.4 titled “ANTI-HUMAN CD22 MONOCLONAL ANTIBODIES AND USE THEREOF” and filed with the China Patent Office on Monday, Jan. 18, 2021, which is incorporated in the present disclosure by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the fields of bioengineering and biomedicine, and mainly relates to a monoclonal antibody targeting human CD22 or an antigen-binding fragment thereof, and an encoding nucleic acid, an expression vector and an expression cell, a preparation method, a pharmaceutical composition therefor, and their use for treating diseases, such as use for treating tumors and autoimmune diseases.

BACKGROUND ART

CD22 is a type I transmembrane glycoprotein, which belongs to the sialic acid-binding immunoglobulinlike lectins (Siglec) family. As a B lineage differentiation antigen, it is specifically expressed in B cells. The expression of CD22 begins from the pre-B cell (pre-B cell) stage, and stops after B cells differentiate into plasma cells. The broad-spectrum expression of CD22 in B cell development makes it an attractive molecule for targeting B cells.

The extracellular region of CD22 consists of 7 Ig-like domains and 12 predicted N-linked glycosylation sites, and its N-terminal (i.e., distal end of membrane) domain 1 is V Type Ig-like domain, which, as a ligand binding site, can recognize α2,6-coupled sialic acid. The intracellular region of CD22 has immunoreceptor tyrosine-based inhibitory motifs (ITIMs). When the tyrosine on the ITIMs is phosphorylated by the Src family protein kinase, binding sites for molecules containing SH2 (Src homology2) domain would be generated, then SHP-1 (Src homology region 2 domain-containing phosphatase-1) was recruited to inhibit the BCR (B-cell receptor) signaling pathway of normal B cells.

α2,6-coupled sialoglycoprotein exists in hematopoietic cells, some endothelial cells, T cells and B cells, and CD22 protein itself also produces α2,6-coupled sialic acid, so CD22 can form cis-interaction with itself and other sialoglycoproteins on the surface of B cells, and trans-interaction with sialoglycoproteins on the surface of other types of cells. In resting B cells, the cis-interaction between CD22 molecules makes the ligand-binding site of CD22 masked, but once the ligand is presented by an adjacent cell, the masked ligand-binding site of CD22 is exposed and interacts with the ligand of the adjacent cells to form trans-interaction. The cis-interaction between CD22 molecules forms homo-oligomers on the B cell surface and the homo-oligomers can form a dynamic nanocluster and generate an antigen binding signal threshold that must be reached before B cell activation, thereby regulating B cell signaling pathway.

CD22 is expressed in 60% to 90% of B cell malignancies and is not expressed in hematopoietic stem cells. In an early clinical study on acute lymphoblastic leukemia (ALL), CD22 is expressed in 60% to 85% of ALL. In another study, the positive rate of CD22 in B-lineage ALL patients reaches 93%. CD22 is expressed in more than 85% of patients with diffuse large B-cell lymphomas (DLBCLs). There are many clinical trials investigating the effectiveness of drugs that target CD22. Epratuzumab is a CD22 monoclonal antibody that has certain effects in adults and children with B-ALL. CD22 antibody-conjugated drugs have a certain therapeutic effect on B-ALL.

Monoclonal antibodies, due to their advantages such as targeting ability, specificity, selectivity, high affinity, etc., are becoming a new type of therapeutic drug. However, early clinical trials revealed that the use of non-human monoclonal antibodies in humans often resulted in severe immune responses due to human anti-mouse antibody (HAMA) and human anti-rat antibody (HARA) responses, and the antibodies were rapidly cleared. Less immunogenic antibodies were subsequently developed, including chimeric, humanized, and fully human antibodies. According to the degree of humanization, therapeutic monoclonal antibody drugs can be divided into 4 types: a murine antibody (without human amino acid sequence), a chimeric antibody (with 60%-70% humanized amino acid sequence), a CDR-grafted antibody (with 90%-95% humanized amino acid sequence) and a fully human antibody (with 100% human amino acid sequence). As the degree of humanization increases, a non-murine monoclonal antibody can reduce human anti-mouse antibody reactions (HAMA and HARA reactions) during human treatment, gradually eliminate the immunogenicity of a heterologous antibody, and maintain high affinity to an antigen and improve pharmacokinetics of the antibody at the same time, and these antibody drugs have been widely used in clinical targeted therapy.

SUMMARY OF THE INVENTION

The present invention provides an antibody or an antigen-binding fragment that specifically binds to human CD22, a nucleic acid encoding the antibody and the antigen-binding fragment, a pharmaceutical composition and a kit comprising the antibody and the antigen-binding fragment, and their use in the preparation of drugs for treating tumors, etc.

In some embodiments, the antibody or the antigen-binding fragment that specifically binds to human CD22 comprises a combination of CDRs comprising: 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:

	SEQ ID NO.
No.	CDR1-VH	CDR2-VH	CDR3-VH
VH1	SEQ ID NO. 83	SEQ ID NO. 84	SEQ ID NO. 85
VH2	SEQ ID NO. 86	SEQ ID NO. 87	SEQ ID NO. 88
VH3	SEQ ID NO. 89	SEQ ID NO. 90	SEQ ID NO. 91
VH4	SEQ ID NO. 101	SEQ ID NO. 102	SEQ ID NO. 103
VH5	SEQ ID NO. 104	SBQ ID NO. 105	SEQ ID NO. 106
VH6	SEQ ID NO. 107	SEQ ID NO. 108	SEQ ID NO. 109
VH7	SEQ ID NO. 119	SEQ ID NO. 120	SEQ ID NO. 121
VH8	SEQ ID NO. 122	SEQ ID NO. 123	SEQ ID NO. 124
VH9	SEQ ID NO. 125	SEQ ID NO. 126	SEQ ID NO. 127
VH10	SEQ ID NO. 137	SEQ ID NO. 138	SEQ ID NO. 139
VH11	SEQ ID NO. 140	SEQ ID NO. 141	SEQ ID NO. 142
VH12	SEQ ID NO. 143	SEQ ID NO. 144	SEQ ID NO. 145
VH13	SEQ ID NO. 155	SEQ ID NO. 156	SEQ ID NO. 157
VH14	SEQ ID NO. 158	SBQ ID NO. 159	SEQ ID NO. 160
VH15	SEQ ID NO. 161	SEQ ID NO. 162	SEQ ID NO. 163
VH16	SEQ ID NO. 173	SEQ ID NO. 174	SEQ ID NO. 175
VH17	SEQ ID NO. 176	SEQ ID NO. 177	SEQ ID NO. 178
VH18	SEQ ID NO. 179	SEQ ID NO. 180	SEQ ID NO. 181
VH19	SEQ ID NO. 191	SEQ ID NO. 192	SEQ ID NO. 193
VH20	SEQ ID NO. 194	SEQ ID NO. 195	SEQ ID NO. 196
VH21	SEQ ID NO. 197	SEQ ID NO. 198	SEQ ID NO. 199
VH22	SEQ ID NO. 209	SEQ ID NO. 210	SEQ ID NO. 211
VH23	SEQ ID NO. 212	SEQ ID NO. 213	SEQ ID NO. 214
VH24	SEQ ID NO. 215	SEQ ID NO. 216	SEQ ID NO. 217
VH25	SEQ ID NO. 227	SEQ ID NO. 228	SEQ ID NO. 229
VH26	SEQ ID NO. 230	SEQ ID NO. 231	SEQ ID NO. 232
VH27	SEQ ID NO. 233	SEQ ID NO. 234	SEQ ID NO. 235
VH28	SEQ ID NO. 245	SEQ ID NO. 246	SEQ ID NO. 247
VH29	SEQ ID NO. 248	SEQ ID NO. 249	SEQ ID NO. 250
VH30	SEQ ID NO. 251	SEQ ID NO. 252	SEQ ID NO. 253
VH31	SEQ ID NO. 263	SEQ ID NO. 264	SEQ ID NO. 265
VH32	SEQ ID NO. 266	SEQ ID NO. 267	SEQ ID NO. 268
VH33	SEQ ID NO. 269	SEQ ID NO. 270	SEQ ID NO. 271
VH34	SEQ ID NO. 281	SEQ ID NO. 282	SEQ ID NO. 283
VH35	SEQ ID NO. 284	SEQ ID NO. 285	SEQ ID NO. 286
VH36	SEQ ID NO. 287	SEQ ID NO. 288	SEQ ID NO. 289
VH37	SEQ ID NO. 299	SEQ ID NO. 300	SEQ ID NO. 301
VH38	SEQ ID NO. 302	SEQ ID NO. 303	SEQ ID NO. 304
VH39	SEQ ID NO. 305	SEQ ID NO. 306	SEQ ID NO. 307
VH40	SEQ ID NO. 317	SEQ ID NO. 318	SEQ ID NO. 319
VH41	SEQ ID NO. 320	SEQ ID NO. 321	SEQ ID NO. 322
VH42	SEQ ID NO. 323	SEQ ID NO. 324	SEQ ID NO. 325
VH43	SEQ ID NO. 335	SEQ ID NO. 336	SEQ ID NO. 337
VH44	SEQ ID NO. 338	SEQ ID NO. 339	SEQ ID NO. 340
VH45	SEQ ID NO. 341	SEQ ID NO. 342	SEQ ID NO. 343
VH46	SEQ ID NO. 353	SEQ ID NO. 354	SEQ ID NO. 355
VH47	SEQ ID NO. 356	SEQ ID NO. 357	SEQ ID NO. 358
VH48	SEQ ID NO. 359	SEQ ID NO. 360	SEQ ID NO. 361
VH49	SEQ ID NO. 371	SEQ ID NO. 372	SEQ ID NO. 373
VH50	SEQ ID NO. 374	SEQ ID NO. 375	SEQ ID NO. 376
VH51	SEQ ID NO. 377	SEQ ID NO. 378	SEQ ID NO. 379
VH52	SEQ ID NO. 389	SEQ ID NO. 390	SEQ ID NO. 391
VH53	SEQ ID NO. 392	SEQ ID NO. 393	SEQ ID NO. 394
VH54	SEQ ID NO. 395	SEQ ID NO. 396	SEQ ID NO. 397
VH55	SEQ ID NO. 407	SEQ ID NO. 408	SEQ ID NO. 409
VH56	SEQ ID NO. 410	SEQ ID NO. 411	SEQ ID NO. 412
VH57	SEQ ID NO. 413	SEQ ID NO. 414	SEQ ID NO. 415
VH58	SEQ ID NO. 425	SEQ ID NO. 426	SEQ ID NO. 427
VH59	SEQ ID NO. 428	SEQ ID NO. 429	SEQ ID NO. 430
VH60	SEQ ID NO. 431	SEQ ID NO. 432	SEQ ID NO. 433
VH61	SEQ ID NO. 443	SEQ ID NO. 444	SEQ ID NO. 445
VH62	SEQ ID NO. 446	SEQ ID NO. 447	SEQ ID NO. 448
VH63	SEQ ID NO. 449	SEQ ID NO. 450	SEQ ID NO. 451
VH64	SEQ ID NO. 461	SEQ ID NO. 462	SEQ ID NO. 463
VH65	SEQ ID NO. 464	SEQ ID NO. 465	SEQ ID NO. 466
VH66	SEQ ID NO. 467	SEQ ID NO. 468	SEQ ID NO. 469
VH67	SEQ ID NO. 479	SEQ ID NO. 480	SEQ ID NO. 481
VH68	SEQ ID NO. 482	SEQ ID NO. 483	SEQ ID NO. 484
VH69	SEQ ID NO. 485	SEQ ID NO. 486	SEQ ID NO. 487
VH70	SEQ ID NO. 497	SEQ ID NO. 498	SEQ ID NO. 499
VH71	SEQ ID NO. 500	SEQ ID NO. 501	SEQ ID NO. 502
VH72	SEQ ID NO. 503	SEQ ID NO. 504	SEQ ID NO. 505
VH73	SEQ ID NO. 515	SEQ ID NO. 516	SEQ ID NO. 517
VH74	SEQ ID NO. 518	SEQ ID NO. 519	SEQ ID NO. 520
VH75	SEQ ID NO. 521	SEQ ID NO. 522	SEQ ID NO. 523
VH76	SEQ ID NO. 533	SEQ ID NO. 534	SEQ ID NO. 535
VH77	SEQ ID NO. 536	SEQ ID NO. 537	SEQ ID NO. 538
VH78	SEQ ID NO. 539	SEQ ID NO. 540	SEQ ID NO. 541
VH79	SEQ ID NO. 551	SEQ ID NO. 552	SEQ ID NO. 553
VH80	SEQ ID NO. 554	SEQ ID NO. 555	SEQ ID NO. 556
VH81	SEQ ID NO. 557	SEQ ID NO. 558	SEQ ID NO. 559
VH82	SEQ ID NO. 569	SEQ ID NO. 570	SEQ ID NO. 571
VH83	SEQ ID NO. 572	SEQ ID NO. 573	SEQ ID NO. 574
VH84	SEQ ID NO. 575	SEQ ID NO. 576	SEQ ID NO. 577
VH85	SEQ ID NO. 587	SEQ ID NO. 588	SEQ ID NO. 589
VH86	SEQ ID NO. 590	SEQ ID NO. 591	SEQ ID NO. 592
VH87	SEQ ID NO. 593	SEQ ID NO. 594	SEQ ID NO. 595
VH88	SEQ ID NO. 605	SEQ ID NO. 606	SEQ ID NO. 607
VH89	SEQ ID NO. 608	SEQ ID NO. 609	SEQ ID NO. 610
VH90	SEQ ID NO. 611	SEQ ID NO. 612	SEQ ID NO. 613
VH91	SEQ ID NO. 623	SEQ ID NO. 624	SEQ ID NO. 625
VH92	SEQ ID NO. 626	SEQ ID NO. 627	SEQ ID NO. 628
VH93	SEQ ID NO. 629	SEQ ID NO. 630	SEQ ID NO. 631
VH94	SEQ ID NO. 641	SEQ ID NO. 642	SEQ ID NO. 643
VH95	SEQ ID NO. 644	SEQ ID NO. 645	SEQ ID NO. 646
VH96	SEQ ID NO. 647	SEQ ID NO. 648	SEQ ID NO. 649
VH97	SEQ ID NO. 659	SEQ ID NO. 660	SEQ ID NO. 661
VH98	SEQ ID NO. 662	SEQ ID NO. 663	SEQ ID NO. 664
VH99	SEQ ID NO. 665	SEQ ID NO. 666	SEQ ID NO. 667
VH100	SEQ ID NO. 677	SEQ ID NO. 678	SEQ ID NO. 679
VH101	SEQ ID NO. 680	SEQ ID NO. 681	SEQ ID NO. 682
VH102	SEQ ID NO. 683	SEQ ID NO. 684	SEQ ID NO. 685
VH103	SEQ ID NO. 695	SEQ ID NO. 696	SEQ ID NO. 697
VH104	SEQ ID NO. 698	SEQ ID NO. 699	SEQ ID NO. 700
VH105	SEQ ID NO. 701	SEQ ID NO. 702	SEQ ID NO. 703

• • and,
  • (2) the combination of light chain CDRs comprises: CDR1-VL, CDR2-VL, and CDR3-VL, the CDR1-VL, CDR2-VL and CDR3-VL 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:

	SEQ ID NO.
No.	CDR1-VL	CDR2-VL	CDR3-VL
VL1	SEQ ID NO. 92	SEQ ID NO. 93	SEQ ID NO. 94
VL2	SEQ ID NO. 95	SEQ ID NO. 96	SEQ ID NO. 97
VL3	SEQ ID NO. 98	SEO ID NO. 99	SEQ ID NO. 100
VL4	SEQ ID NO. 110	SEQ ID NO. 111	SEQ ID NO. 112
VL5	SEQ ID NO. 113	SEQ ID NO. 114	SEQ ID NO. 115
VL6	SEQ ID NO. 116	SEQ ID NO. 117	SEQ ID NO. 118
VL7	SEQ ID NO. 128	SEQ ID NO. 129	SEQ ID NO. 130
VL8	SEQ ID NO. 131	SEQ ID NO. 132	SEQ ID NO. 133
VL9	SEQ ID NO. 134	SEQ ID NO. 135	SEQ ID NO. 136
VL10	SEQ ID NO. 146	SEQ ID NO. 147	SEQ ID NO. 148
VL11	SEQ ID NO. 149	SEQ ID NO. 150	SEQ ID NO. 151
VL12	SEQ ID NO. 152	SEQ ID NO. 153	SEQ ID NO. 154
VL13	SEQ ID NO. 164	SEQ ID NO. 165	SEQ ID NO. 166
VL14	SEQ ID NO. 167	SEQ ID NO. 168	SEQ ID NO. 169
VL15	SEQ ID NO. 170	SEQ ID NO. 171	SEQ ID NO. 172
VL16	SEQ ID NO. 182	SEQ ID NO. 183	SEQ ID NO. 184
VL17	SEQ ID NO. 185	SEQ ID NO. 186	SEQ ID NO. 187
VL18	SEQ ID NO. 188	SEQ ID NO. 189	SEQ ID NO. 190
VL19	SEQ ID NO. 200	SEQ ID NO. 201	SEQ ID NO. 202
VL20	SEQ ID NO. 203	SEQ ID NO. 204	SEQ ID NO. 205
VL21	SEQ ID NO. 206	SEQ ID NO. 207	SEQ ID NO. 208
VL22	SEQ ID NO. 218	SEQ ID NO. 219	SEQ ID NO. 220
VL23	SEQ ID NO. 221	SEQ ID NO. 222	SEQ ID NO. 223
VL24	SEQ ID NO. 224	SEQ ID NO. 225	SEQ ID NO. 226
VL25	SEQ ID NO. 236	SEQ ID NO. 237	SEQ ID NO. 238
VL26	SEQ ID NO. 239	SEQ ID NO. 240	SEQ ID NO. 241
VL27	SEQ ID NO. 242	SEQ ID NO. 243	SEQ ID NO. 244
VL28	SEQ ID NO. 254	SEQ ID NO. 255	SEQ ID NO. 256
VL29	SEQ ID NO. 257	SEQ ID NO. 258	SEQ ID NO. 259
VL30	SEQ ID NO. 260	SEQ ID NO. 261	SEQ ID NO. 262
VL31	SEQ ID NO. 272	SEQ ID NO. 273	SEQ ID NO. 274
VL32	SEQ ID NO. 275	SEQ ID NO. 276	SEQ ID NO. 277
VL33	SEQ ID NO. 278	SEQ ID NO. 279	SEQ ID NO. 280
VL34	SEQ ID NO. 290	SEQ ID NO. 291	SEQ ID NO. 292
VL35	SEQ ID NO. 293	SEQ ID NO. 294	SEQ ID NO. 295
VL36	SEQ ID NO. 296	SEQ ID NO. 297	SEQ ID NO. 298
VL37	SEQ ID NO. 308	SEQ ID NO. 309	SEQ ID NO. 310
VL38	SEQ ID NO. 311	SEQ ID NO. 312	SEQ ID NO. 313
VL39	SEQ ID NO. 314	SEQ ID NO. 315	SEQ ID NO. 316
VL40	SEQ ID NO. 326	SEQ ID NO. 327	SEQ ID NO. 328
VL41	SEQ ID NO. 329	SEQ ID NO. 330	SEQ ID NO. 331
VL42	SEQ ID NO. 332	SEQ ID NO. 333	SEQ ID NO. 334
VL43	SEQ ID NO. 344	SEQ ID NO. 345	SEQ ID NO. 346
VL44	SEQ ID NO. 347	SEQ ID NO. 348	SEQ ID NO. 349
VL45	SEQ ID NO. 350	SEQ ID NO. 351	SEQ ID NO. 352
VL46	SEQ ID NO. 362	SEQ ID NO. 363	SEQ ID NO. 364
VL47	SEQ ID NO. 365	SEQ ID NO. 366	SEQ ID NO. 367
VL48	SEQ ID NO. 368	SEQ ID NO. 369	SEQ ID NO. 370
VL49	SEQ ID NO. 380	SEQ ID NO. 381	SEQ ID NO. 382
VL50	SEQ ID NO. 383	SEQ ID NO. 384	SEQ ID NO. 385
VL51	SEQ ID NO. 386	SEQ ID NO. 387	SEQ ID NO. 388
VL52	SEQ ID NO. 398	SEQ ID NO. 399	SEQ ID NO. 400
VL53	SEQ ID NO. 401	SEQ ID NO. 402	SEQ ID NO. 403
VL54	SEQ ID NO. 404	SEQ ID NO. 405	SEQ ID NO. 406
VL55	SEQ ID NO. 416	SEQ ID NO. 417	SEQ ID NO. 418
VL56	SEQ ID NO. 419	SEQ ID NO. 420	SEQ ID NO. 421
VL57	SEQ ID NO. 422	SEQ ID NO. 423	SEQ ID NO. 424
VL58	SEQ ID NO. 434	SEQ ID NO. 435	SEQ ID NO. 436
VL59	SEQ ID NO. 437	SEQ ID NO. 438	SEQ ID NO. 439
VL60	SEQ ID NO. 440	SEQ ID NO. 441	SEQ ID NO. 442
VL61	SEQ ID NO. 452	SEQ ID NO. 453	SEQ ID NO. 454
VL62	SEQ ID NO. 455	SEQ ID NO. 456	SEQ ID NO. 457
VL63	SEQ ID NO. 458	SEQ ID NO. 459	SEQ ID NO. 460
VL64	SEQ ID NO. 470	SEQ ID NO. 471	SEQ ID NO. 472
VL65	SEQ ID NO. 473	SEQ ID NO. 474	SEQ ID NO. 475
VL66	SEQ ID NO. 476	SEQ ID NO. 477	SEQ ID NO. 478
VL67	SEQ ID NO. 488	SEQ ID NO. 489	SEQ ID NO. 490
VL68	SEQ ID NO. 491	SEQ ID NO. 492	SEQ ID NO. 493
VL69	SEQ ID NO. 494	SEQ ID NO. 495	SEQ ID NO. 496
VL70	SEQ ID NO. 506	SEQ ID NO. 507	SEQ ID NO. 508
VL71	SEQ ID NO. 509	SEQ ID NO. 510	SEQ ID NO. 511
VL72	SEQ ID NO. 512	SEQ ID NO. 513	SEQ ID NO. 514
VL73	SEQ ID NO. 524	SEQ ID NO. 525	SEQ ID NO. 526
VL74	SEQ ID NO. 527	SEQ ID NO. 528	SEQ ID NO. 529
VL75	SEQ ID NO. 530	SEQ ID NO. 531	SEQ ID NO. 532
VL76	SEQ ID NO. 542	SEQ ID NO. 543	SEQ ID NO. 544
VL77	SEQ ID NO. 545	SEQ ID NO. 546	SEQ ID NO. 547
VL78	SEQ ID NO. 548	SEQ ID NO. 549	SEQ ID NO. 550
VL79	SEQ ID NO. 560	SEQ ID NO. 561	SEQ ID NO. 562
VL80	SEQ ID NO. 563	SEQ ID NO. 564	SEQ ID NO. 565
VL81	SEQ ID NO. 566	SEQ ID NO. 567	SEQ ID NO. 568
VL82	SEQ ID NO. 578	SEQ ID NO. 579	SEQ ID NO. 580
VL83	SEQ ID NO. 581	SEQ ID NO. 582	SEQ ID NO. 583
VL84	SEQ ID NO. 584	SEQ ID NO. 585	SEQ ID NO. 586
VL85	SEQ ID NO. 596	SEQ ID NO. 597	SEQ ID NO. 598
VL86	SEQ ID NO. 599	SEQ ID NO. 600	SEQ ID NO. 601
VL87	SEQ ID NO. 602	SEQ ID NO. 603	SEQ ID NO. 604
VL88	SEQ ID NO. 614	SEQ ID NO. 615	SEQ ID NO. 616
VL89	SEQ ID NO. 617	SEQ ID NO. 618	SEQ ID NO. 619
VL90	SEQ ID NO. 620	SEQ ID NO. 621	SEQ ID NO. 622
VL91	SEQ ID NO. 632	SEQ ID NO. 633	SEQ ID NO. 634
VL92	SEQ ID NO. 635	SEQ ID NO. 636	SEQ ID NO. 637
VL93	SEQ ID NO. 638	SEQ ID NO. 639	SEQ ID NO. 640
VL94	SEQ ID NO. 650	SEQ ID NO. 651	SEQ ID NO. 652
VL95	SEQ ID NO. 653	SEQ ID NO. 654	SEQ ID NO. 655
VL96	SEQ ID NO. 656	SEQ ID NO. 657	SEQ ID NO. 658
VL97	SEQ ID NO. 668	SEQ ID NO. 669	SEQ ID NO. 670
VL98	SEQ ID NO. 671	SEQ ID NO. 672	SEQ ID NO. 673
VL99	SEQ ID NO. 674	SEQ ID NO. 675	SEQ ID NO. 676
VL100	SEQ ID NO. 686	SEQ ID NO. 687	SEQ ID NO. 688
VL101	SEQ ID NO. 689	SEQ ID NO. 690	SEQ ID NO. 691
VL102	SEQ ID NO. 692	SEQ ID NO. 693	SEQ ID NO. 694
VL103	SEQ ID NO. 704	SEQ ID NO. 705	SEQ ID NO. 706
VL104	SEQ ID NO. 707	SEQ ID NO. 708	SEQ ID NO. 709
VL105	SEQ ID NO. 710	SEQ ID NO. 711	SEQ ID NO. 712

• • each CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is coded according to the prevailing analysis methods of KABAT, Chothia or IMGT.

In particular, for example, the antibody or the antigen-binding fragment thereof of the invention comprises a combination of a heavy chain CDR and a light chain CDR selected from: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6, VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17+VL17, VH18+VL18, VH19+VL19, VH20+VL20, VH21+VL21, VH22+VL22, VH23+VL23, VH24+VL24, VH25+VL25, VH26+VL26, VH27+VL27, VH28+VL28, VH29+VL29,

VH30+VL30, VH31+VL31, VH32+VL32, VH33+VL33, VH34+VL34, VH35+VL35, VH36+VL36, VH37+VL37, VH38+VL38, VH39+VL39, VH40+VL40, VH41+VL41, VH42+VL42, VH43+VL43, VH44+VL44, VH45+VL45, VH46+VL46, VH47+VL47, VH48+VL48, VH49+VL49, VH50+VL50, VH51+VL51, VH52+VL52, VH53+VL53, VH54+VL54, VH55+VL55, VH56+VL56, VH57+VL57, VH58+VL58, VH59+VL59, VH60+VL60, VH61+VL61, VH62+VL62, VH63+VL63, VH64+VL64, VH65+VL65, VH66+VL66, VH67+VL67, VH68+VL68, VH69+VL69, VH70+VL70, VH71+VL71, VH72+VL72, VH73+VL73, VH74+VL74, VH75+VL75, VH76+VL76, VH77+VL77, VH78+VL78, VH79+VL79, VH80+VL80, VH81+VL81, VH82+VL82, VH83+VL83, VH84+VL84, VH85+VL85, VH86+VL86, VH87+VL87, VH88+VL88, VH89+VL89, VH90+VL90, VH91+VL91, VH92+VL92, VH93+VL93, VH94+VU4, VH95+VL95, VH96+VL96, VH97+VL97, VH98+VL98, VH99+VL99, VH100+VL100, VH101+VL101, VH102+VL102, VH103+VL103, VH104+VL104, or VH105+VL105, and a combination of CDRs with insertions, deletions and/or substitutions of 1, 2, 3 or more amino acids compared to the sequence of the heavy chain CDR and the light chain CDR in these combinations.

In a specific embodiment, the invention provides an antibody or an antigen-binding fragment thereof comprising:

• • (1) a heavy chain variable region having a sequence as shown in SEQ ID NO: 13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81; a light chain variable region having a sequence as shown in SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 82;
  • (2) an amino acid sequence having at least 90% identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in (1) above; or,
  • (3) a framework region of the antibody or the antigen-binding fragment having at least 90% identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the framework region of the amino acid sequence as shown in (1) above.

In a preferred embodiment, the dissociation constant (KD) of the antibody or the antigen-binding fragment of the present invention binding to human CD22 is no more than 10⁻⁶ M, and the dissociation constant (KD) of the antibody or the antigen-binding fragment of the present invention binding to rhesus monkey CD22 (KD) is no more than 10⁻⁸ M.

Or, optionally, the antibody or the antigen-binding fragment binds or does not bind to monkey CD22;

Optionally, the antibody or the antigen-binding fragment binds to or does not bind to murine CD22.

In a preferred embodiment, the antibody or the antigen-binding fragment of the invention is chimeric or humanized or fully human; 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.

In a preferred embodiment, the antibody or the antigen-binding fragment thereof of the present invention 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; or a sequence of the constant region of human or murine antibody IgG1, IgG2, IgG3 or IgG4 carrying a mutation.

In a preferred embodiment, the antigen-binding fragment of the present invention is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv bispecific antibody, a nanobody and an antibody minimum recognition unit.

In a preferred embodiment, the antibody or the antigen-binding fragment thereof of the invention 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.

In a preferred embodiment, the present invention also provides a multispecific antigen-binding molecule; preferably, the multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module, the first antigen-binding module comprises the antibody or the antigen-binding fragment described in any one of the above, the second antigen-binding module specifically binds to other antigens than CD22 or binds to a different CD22 epitope than the first antigen-binding module;

• • preferably, the other antigens are selected from CD3, CD16, CD16A, CD4, CDS, 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.

In a preferred embodiment, the present invention provides a chimeric antigen receptor (CAR); preferably, 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 any of the CD22 antibody or the antigen-binding fragment described above.

In a preferred embodiment, the present invention provides an immune effector cell; preferably, the immune effector cell comprises the chimeric antigen receptor described above or a nucleic acid fragment encoding the chimeric antigen receptor described above;

• • 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 may 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.

In a preferred embodiment, the present invention provides an isolated nucleic acid molecule encoding the nanobody, the antigen-binding fragment, or any combination thereof according to any one of the above aspects of the present invention, the multispecific antigen-binding molecule described above or the chimeric antigen receptor described above.

In some embodiments, the present invention provides an expression vector comprising the isolated nucleic acid molecule of the present invention described above.

In some embodiments, the present invention provides a host cell comprising the isolated nucleic acid molecule or the expression vector of the present invention described above.

In a preferred embodiment, 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 or Chinese hamster ovary (CHO) cell.

In some embodiments, the present invention provides a method for preparing an antibody or an antigen-binding fragment or a multispecific antigen-binding molecule, the method comprises culturing or culturing, under appropriate conditions, the host cell of the present invention described above, and isolating the antibody or the antigen-binding fragment or the multispecific antigen-binding molecule.

In some embodiments, the present invention provides a method for preparing an immune effector cell, wherein the CAR nucleic acid fragment described above is introduced into the immune effector cell, preferably, the method further comprises enabling the immune effector cell to express the CAR described above.

In some embodiments, the present invention provides a pharmaceutical composition comprising the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, or a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, and a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; more preferably, the pharmaceutical composition further comprises an additional antineoplastic agent.

In some embodiments, the present invention provides a method for preventing and/or treating a B cell disease, the method comprises administering the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, or the pharmaceutical composition of the invention described above 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, 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.

In some embodiments, the present invention provides the use of the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, or the pharmaceutical composition of the invention described above in the preparation of a drug for preventing and/or treating a B cell disease, and the B cell disease is preferably a tumor or an autoimmune disease;

• • preferably, the tumor is selected from lymphoma or leukemia, 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.

In some embodiments, the present invention provides the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, or the pharmaceutical composition of the invention described above for preventing and/or treating a B cell disease; The B cell disease is preferably a tumor or an autoimmune disease;

• • preferably, the tumor is selected from lymphoma or leukemia, 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.

In some embodiments, the present invention provides a kit comprising the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, or a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, or the pharmaceutical composition of the invention described above, and instructions for use.

Definition and Description of Terminology

As used herein, the term “antibody” (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with an antigen of interest and includes polyclonal, monoclonal, genetically engineered, and other modified forms of an antibody (including but not limited to a chimeric antibody, a humanized antibody, a fully human antibody, a heteroconjugate antibody (e.g. a bispecific, trispecific and tetraspecific antibody, a diabody, a triabody and a tetrabody, an antibody conjugate)) and an antigen-binding fragment of an antibody (including, for example, Fab′, F(ab′)2, Fab, Fv, rIgG and scFv fragment). Furthermore, unless otherwise indicated, the term “monoclonal antibody” (mAb) is intended to include both an intact antibody molecule and an incomplete antibody fragment (such as Fab and F(ab′)2 fragment, which lacks the Fc fragment of the intact antibody (cleared more quickly from circulation in animals), thus lacking Fc-mediated effector function) capable of specifically binding to a target protein (see Wahl et al., J. Nucl. Med. 24: 316, 1983; the content of which is incorporated herein by reference).

An “antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanaco, alpaca, sheep, rabbits, mice, rats or cartilaginous fishes (such as sharks).

The term “natural antibody” herein refers to an antibody produced and paired by the immune system of a multicellular organism. The term “engineered antibody” herein refers to a non-natural antibody obtained through genetic engineering, antibody engineering, etc. For example, “engineered antibody” includes a humanized antibody, a small molecule antibody (such as scFv, etc.), a bispecific antibody, etc.

The term “monospecific” herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.

The term “multispecific” herein refers to having at least two antigen binding sites, each of which binds a different epitope of the same antigen or a different epitope of a different antigen. Thus, terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/an antigen-binding molecule can bind.

The term “valence” herein refers to the presence of a defined number of binding sites in an antibody/an antigen-binding molecule. Thus, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to the presence of one binding site, two binding sites, four binding site and six binding sites in an antibody/an antigen-binding molecule, respectively.

“Full-length antibody”, “complete antibody” and “intact antibody” are used interchangeably herein to mean that they have a structure substantially similar to that of a natural antibody.

As used herein, the term “antigen-binding fragment” refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. The antibody fragment can be Fab, F(ab′)2, scFv, SMIP, a diabody, a triabody, an affibody, a nanobody, an aptamer or a domain antibody. Examples of a binding fragment encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) an Fab fragment, which is a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) an F(ab)2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (V) dAb comprising VH and VL domains; (vi) dAb fragment consisting of a VH domain (Ward et al., Nature 341: 544-546, 1989); (vii) dAb consisting of VH or VL domains; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, the CDRs may optionally be linked by a synthetic linker.

Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be joined using a recombinant method through a linker that enables forming a single protein chain (referred to as a single chain Fv (scFv); see e.g., Bird et al., Science 242: 423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988) in which the VL and VH regions pair to form a monovalent molecule. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as an intact antibody. The antigen-binding fragment can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of an intact immunoglobulin, or in some embodiments by chemical peptide synthesis procedures known in the art.

As used herein, the term “CD22” refers to Siglec-2, a molecule belonging to the SIGLEC lectin family, which is present on the surface of mature B cells and to a lesser extent on certain immature B cells. The term “CD22” includes CD22 proteins of any human and non-human animal species, and specifically includes human CD22 as well as CD22 of non-human mammals.

As used herein, the term “bispecific antibody” refers to an antibody, typically a human or humanized antibody, that has monoclonal binding specificities for at least two different antigens. In the present invention, one of the binding specificities can be detected against an antigen epitope of CD22, and the other can be detected against another antigen epitope of CD22 or any other antigen except CD22, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterium-derived protein, or a bacterial surface protein.

As used herein, the term “chimeric” antibody refers to an antibody that has a variable sequence derived from an immunoglobulin of one organism (such as a rat or mouse) and a constant region derived from an immunoglobulin of a different organism, such as human. Methods for producing the chimeric antibody are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, Bio Techniques 4: 214-221; Gillies et al., 1985 J Immunol Methods 125: 191-202; The above documents are incorporated herein by reference.

As used herein, the term “heavy chain antibody” refers to an antibody that lacks the light chains of an conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

As used herein, the term “nanobody” refers to a natural heavy chain antibody without light chains in camel and cloning its variable region can obtain a single domain antibody, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which only consists of a heavy chain variable region, and is the smallest functional antigen-binding fragment. For a further description of VHH and nanobody, reference is made to the review article by Muyldermans (2001, Reviews in Molecular Biotechnology 74:277-302), and to the following patent applications mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Free University of Brussels; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of

Algonomics N.V. and Ablynx N.V.; WO 01/90190 of the National Research Council of Canada; WO 03/025020 (=EP 1433793) of the Institute of Antibodies; and WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825 of Ablynx N.V., and further published patent applications of Ablynx N.V.

Reference is also made to the additional prior art mentioned in these applications, in particular the list of references mentioned on pages 41-43 of International Application WO 06/040153, which list and references are incorporated herein by reference. As described in these references, nanobody (in particular VHH sequences and partially humanized nanobody) may inter alia be characterized by the presence of one or more “signature residues” in one or more framework sequences. Further descriptions of nanobody can be found in, for example, WO 08/101985 and WO 08/142164, including humanization and/or camelization of nanobody, as well as other modifications, parts or fragments, derivatives or “nanobody fusion”, multivalent constructs (including some non-limiting examples of linker sequences) and various modifications that increase the half-life of a nanobody and a formulation thereof. For a further general description of nanobody , reference is made to the prior art cited herein, for example WO 08/020079 (page 16).

As used herein, the term “complementarity determining region” (CDR) refers to hypervariable regions found in both light and heavy chain variable domains. The more conserved portions in variable domains are called the framework regions (FR). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary according to the context and various definitions known in the art. Some positions within variable domains can be considered heterozygous hypervariable positions because these positions can be considered to be within the hypervariable regions under one set of criteria (such as IMGT or KABAT) but outside the hypervariable regions under a different set of criteria (such as KABAT or IMGT).

One or more of these positions may also be found in extended hypervariable regions. The invention includes an antibody comprising modifications in these hybrid hypervariable positions. The variable domains of the native heavy and light chains respectively comprise four framework regions that largely adopt a sheet configuration and connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets, and in some cases form part of the sheet structure. The CDRs in each chain are held tightly together by the FR regions in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference). For example, herein, CDR1-VH, CDR2-VH, and CDR3-VH refer to the first CDR, the second CDR, and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region); CDR1-VL, CDR2-VL, and CDR3-VL refer to the first CDR, the second CDR, and the third CDR of the light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).

As used herein, the term “monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation by the method by which the antibody is produced.

As used herein, the term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv or Fab. The term “VL” refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.

The term “heavy chain constant region” herein refers to the carboxy-terminal portion of the heavy chain of an antibody, which is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, and has a more conserved amino acid sequence relative to the antibody's variable domain. A “heavy chain constant region” comprises at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof “Heavy chain constant region” includes “full-length heavy chain constant region” and “heavy chain constant region fragment”, the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “a part of the full-length heavy chain constant region”. For example, a typical “full-length antibody heavy chain constant region” consists of CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. For example, a typical “heavy chain constant region fragment” can be selected from CH1, Fc or CH3 domains.

The term “light chain constant region” herein refers to the carboxy-terminal portion of the light chain of an antibody, which is not directly involved in the binding of the antibody to an antigen. The light chain constant region may be selected from a constant η domain or a constant λ domain.

The term “Fc” herein refers to the carboxy-terminal portion of an antibody obtained by papain hydrolysis of the intact antibody, which typically includes the CH3 and CH2 domains of the antibody. Fc region includes, for example, a native sequence Fc region, a recombinant Fc region and a variant Fc region. Although the boundary of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, thus the Fc region may comprise or may not comprise Lys447.

The term “humanized antibody” herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody. Generally speaking, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (for example, variable region FR and/or constant region) are derived from human Immunoglobulin (recipient antibody). Humanized antibody usually retains or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.

The term “fully human antibody” herein refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibody herein may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, a “fully human antibody” herein is not intended to include an antibody in which CDR sequences derived from the germline of another mammalian species (for example, mouse) have been grafted onto human framework sequences.

The term “naked antibody” herein refers to an antibody that is not linked, fused or conjugated to another agent or molecule (for example, label or drug), peptide or polypeptide. In specific embodiments, the naked antibody expressed by a mammalian host cell can be glycosylated by the host cell's glycosylation machinery (for example, glycosylase). In certain embodiments, the naked antibody is not glycosylated when expressed by a host cell that does not have its own glycosylation machinery (for example, glycosylase). In certain embodiments, the naked antibody is an intact antibody, while in other embodiments, the naked antibody is the antigen-binding fragment of an intact antibody, such as Fab antibody.

The term “conjugated antibody” herein refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent and can be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.

The term “diabody” herein refers to bivalent bispecific antibody that can bind to different epitopes on the same or different antigens.

As used herein, the term “percent (%) sequence identity” refers to the percentage of amino acid (or nucleotide) residues of the candidate sequence that are identical to those of the reference sequence after aligning the sequences and introducing gaps (if necessary) in order to achieve maximum percentage sequence identity (for example, for optimal alignment, gaps can be introduced in one or both of the candidate sequence and the reference sequence, and non-homologous sequences can be ignored for comparison purpose). For purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence shows sequence identity from 50% to 100% in the full length of the candidate sequence or in the selected part of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purpose is at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

The term “conservative amino acid” herein generally refers to amino acids that belong to the same class or have similar characteristics (for example, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). For example, the amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:

• • (1) acidic amino acids: Asp (D) and Glu (E);
  • (2) basic amino acids: Lys (K), Arg (R) and His (H):
  • (3) hydrophilic uncharged amino acids: Ser (S), Thr (T), Asn (N) and Gin (Q);
  • (4) Aliphatic uncharged amino acids: Gly (G), Ala (A), Val (V), Leu (L) and Ile (I);
  • (5) nonpolar uncharged amino acids: Cys (C), Met (M) and Pro (P);
  • (6) aromatic amino acids: Phe (F), Tyr (Y) and Trp (W).

The term “Kabat numbering system” herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

The term “Chothia numbering system” herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol . Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883).

The term “IMGT numbering system” herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol . Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883).

As used herein, the term “specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, the proteins and other biomolecules are for example specifically recognized by an antibody or an antigen-binding fragment thereof. An antibody or an antigen-binding fragment thereof that specifically binds to an antigen would bind to the antigen with a KD of less than 100 nM. For example, an antibody or an antigen-binding fragment thereof that specifically binds to an antigen would bind to the antigen with a KD of up to 100 nM (for example, between 1 pM and 100 nM). An antibody or an antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or an epitope thereof would exhibit a KD for the particular antigen or the epitope thereof of greater than 100 nM (for example, greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM). Various immunoassays are available to select for an antibody that reacts specifically with a specific protein or carbohydrate. For example, solid-phase ELISA immunoassay is routinely used to select for an antibody that reacts specifically with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describes immunoassay methods and conditions that can be used to determine specific immunoreactivity.

As used herein, the term “antibody conjugate” refers to a coupled entity/conjugate formed by chemically bonding an antibody molecule to another molecule either directly or through a linker. Examples include antibody-drug conjugate (ADC), in which the drug molecule is said another molecule.

The term “chimeric antigen receptor (CAR)” herein refers to a recombinant protein comprising at least (1) an extracellular antigen-binding domain, such as a variable heavy or light chain of an antibody, and (2) a transmembrane domain used to make the anchored CAR enter immune effector cells, and (3) an intracellular signaling domain. In certain embodiments, the extracellular antigen binding domain of the CAR comprises a scFv. The scFv can be derived from the variable heavy and light regions of a fusion antibody. Alternatively or additionally, the scFv may be derived from Fab's (rather than an antibody, for example obtained from a Fab library). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain.

Herein the term “nucleic acid” includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, the nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually expressed as 5′ to 3′. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and a polymer containing a mixture of two or more of these molecules. The nucleic acid molecule can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense strand and antisense strand, as well as single-stranded form and double-stranded form. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. The nucleic acid molecule also encompasses DNA and RNA molecules which are suitable as vectors for direct expression of the antibody of the invention in vitro and/or in vivo, for example in a host or patient. Such DNA (for example cDNA) or RNA (for example mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate an antibody in vivo (see, for example, Stadler et al., Nature Medicine 2017, Published online Jun. 12, 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).

As used herein, the term “vector” includes a nucleic acid vector, such as a DNA vector (such as a plasmid), a RNA vector, a virus or other suitable replicons (such as viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vector of the invention contains polynucleotide sequences together with additional sequence elements, for example, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibody and the antibody fragment of the invention include plasmids that contain regulatory sequences (such as promoter and enhancer regions) that direct transcription of the gene. Other useful vectors for expressing the antibody and the antibody fragment contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA transcripted from the genes. These sequence elements include, for example, 5′ and 3′ untranslated regions, internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct the efficient transcription of the genes carried on the expression vector. The expression vector of the present invention may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothyrcin.

The term “host cell” herein refers to a cell into which a foreign nucleic acid is introduced, including the progenys of such a cell. The host cell includes “transformant” and “transformed cell” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical to the parental cell in nucleic acid content, and may contain a mutation.

The mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

As used herein, the term “pharmaceutical composition” refers to a preparation that is present in a form which allows the active ingredients contained therein to be biologically effective and does not contain additional ingredients that would be unacceptably toxic to the subject to which the pharmaceutical composition is administered.

As used herein, the terms “subject”, “object” and “patient” refer to an organism receiving treatment for a particular disease or condition, such as a cancer or an infectious disease, as described herein. Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovid family (cattle, bison, buffalo, elk, yak, etc.), sheep, and horses receiving treatment for a disease or a condition (for example, a cell proliferative disorder, such as a cancer or an infectious disease).

As used herein, the term “treatment” refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology in the subject being treated, such as the progress of a cell proliferative disorder (such as a cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial response or complete response), whether detectable or undetectable. Subjects in need of treatment include subjects who already have a condition or a disease, subjects who are prone to a condition or a disease or subjects who intend to prevent a condition or a disease. When referring to the terms slow down, alleviation, diminishment, palliation, remission, etc., the meaning of eliminate, disappear, not occur, etc. is also included.

The term “effective amount” herein refers to an amount of a therapeutic agent effective to prevent or relieve a disease or a condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. “Effective amount” also refers to an amount of a compound sufficient to relieve symptoms, for example, treat, cure, prevent or relieve the associated medical conditions, or to increase the rate of treatment, cure, prevent or relieve such conditions. When the active ingredient is administered to an individual alone, a therapeutically effective dose refers to the ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.

The term “appropriate condition” herein refers to a condition suitable for culturing various host cells, including eukaryotic cells and prokaryotic cells.

The term “cancer” herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.

The term “tumor” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “tumor” are not mutually exclusive when referred to herein.

The term “anti-tumor agent” herein refers to an anti-tumor drug, which is a class of drugs for the treatment of tumor diseases, for example, a chemotherapy drug, a biological agent and the like.

The term “EC50” herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and which can be measured by methods known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG. 1 shows the detection results of SDS-PAGE reducing and non-reducing gels of CD22-ECD-His, CD22 domain1-4-His and CD22 domain5-7-His protein samples. Lane 1 is the protein band of hCD22-ECD-His under non-reducing conditions, Lane 2 is the protein band of hCD22 domain5-7-His under non-reducing conditions, Lane 3 is the protein band of hCD22 domain5-7-His under reducing conditions, Lane 4 is the protein band of hCD22-ECD-His under reducing conditions, Lane 5 is the protein band of hCD22 domain1-4-His under non-reducing conditions, Lane 6 is the protein band of hCD22 domain1-4-His under reducing conditions, and Lane M is the protein marker band.

FIG. 2A is the FACS result of detecting CD22 expression quantity in Raji cells by HA22 antibody; FIG. 2B shows the FACS result of detecting CD22 expression quantity in Raji cells by m971 antibody.

FIG. 3A shows the FACS screen and detection results of CHO-K1-human CD22 2C4 cells transfected with human CD22 protein; FIG. 3B shows the FACS screen and detection results of CHO-K1-human CD22 1G5 cells transfected with human CD22 protein; FIG. 3C shows the FACS screen and detection results of CHO-K1-human CD22 1D9 cells transfected with human CD22 protein.

FIG. 4 shows the FACS result of detecting HEK293T cells transfected with monkey CD22 protein by hL22 antibody.

FIG. 5 shows the binding reaction of chimeric antibody with human CD22-ECD-His protein detected by ELISA. The anti-CD22 positive control antibody is: HA22 and m971, the negative control is hIgG1.

FIG. 6A shows the binding reaction of the chimeric antibody of the present invention with Raji detected by FACS; FIG. 6B shows the binding reaction of the chimeric antibody of the present invention with CHO-K1-human CD22 detected by FACS; The anti-CD22 positive control antibody is: HA22 and m971, the negative control is hIgG1; FIG. 6C shows the binding reaction of the chimeric antibody of the present invention at 1 nM and 10 nM with Raji cells and MOLT4 cells detected by FACS; FIG. 6D shows the binding reaction of the chimeric antibody of the present invention at 1 nM and 10 nM with CHO-K1 cells and CHO-K1-human CD22 cells detected by FACS.

FIG. 7 shows the binding reaction of the chimeric antibody of the present invention with murine CD22-ECD-HiS protein detected by ELISA; The positive control is 983; the negative control is hIgG1.

FIG. 8 shows the binding reaction of the chimeric antibody of the present invention with monkey CD22-ECD-His protein detected by ELISA; The positive control is HA22; the negative control is hIgG1.

FIG. 9A shows the binding reaction of the chimeric antibody of the present invention with HEK293T-monkey CD22 detected by FACS; The anti-CD22 positive control antibody is: HA22, the negative control is hIgG1; FIG. 9B shows the binding reaction of the chimeric antibody of the present invention at 1 nM and 10 nM with HEK293T cells and HEK293T-monkey-CD22 detected by FACS.

FIG. 10A-10B show a scatter plot of peripheral blood mononuclear cells of cynomolgus monkeys stained with both CD20 antibody and 1 nM of the chimeric antibody of the present invention detected by FACS, CD20 is a B cell marker, and the ratio shown in the figure is the ratio of chimeric antibody positive cells to CD20 positive cells, and the anti-CD22 positive control antibody is: HA22 and hL22, the negative control is hIgG1.

FIG. 11A shows the binding reaction of the chimeric antibody of the present invention with human CD22 domain1-4-His protein detected by ELISA; FIG. 11B shows the binding reaction of the chimeric antibody of the present invention with human CD22 domain5-7-His protein detected by ELISA. The anti-CD22 domain1-4 positive control antibody is HA22, the anti-CD22 domain5-7 positive control antibody is m971, and the negative control is hIgG1.

DETAILED DESCRIPTION OF EMBODIMENTS

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.

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

1.1 Preparation of Human CD22-His Tag Protein

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 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 1 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 FreeStyle™ 293 (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: 20 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 FIG. 1.

TABLE 1
Amino acid sequences of human CD22 protein and extracellular region
of sequence
Designation
of sequence	Sequence	Amino acid sequence
Human CD22	SEQ ID NO: 1
protein
Haman CD22	SEQ ID NO: 2
extracellular
region
Human CD22	SEQ ID NO: 3
protein
domain1-4
Human CD22	SEQ ID NO: 4
protein
indicates data missing or illegible when filed

1.2 Preparation of Human CD22 Control Antibody

HA22 and m971 clones were antibodies that recognize human CD22, wherein the antigen-binding epitope of the HA22 clone was located in domain 2-3, and the antigen-binding epitope of the m971 clone was located in domain5-7. The heavy chain variable region sequence and the light chain variable region sequence of the HA22 clone were obtained according to patent U.S. Pat. No. 9,580,461 B (which was incorporated herein by reference), and the heavy chain variable region sequence and the light chain variable region sequence of the m971 clone were obtained according to patent U.S. Pat. No. 8,591,889 B (which was incorporated herein by reference). Taizhou Biointron Biotechnology Co., Ltd. cloned the light chain variable region sequences of the HA22 and m971 into the expression vector pcDNA3.4-B1HH1 containing a signal peptide and the light chain constant region of human antibody IgG1 (private vector of Taizhou Biointron Biotechnology Co., Ltd.), respectively, and cloned the heavy chain variable region sequences into the expression vector pcDNA3.4-B1HLK containing a signal peptide and the heavy chain constant region of human antibody IgG1 (private vector of Taizhou Biointron Biotechnology Co., Ltd.), respectively, and thus sequences of m971-hIgG1 and HA22-hIgG1 were obtained. Unless otherwise specified, HA22 and m971 referred to m971-hIgG1 and HA22-hIgG1 respectively hereinafter. 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 TM 293 (Thermo Fisher Scientific, catalog number: 12338018), and the cell components were removed by centrifugation to obtain the culture supernatant containing the 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 2
Sequence information of heavy and light chains of anti-human CD22
antibodies HA22-hIgG1 and m971-hIgG1
Designation	Sequence
of sequence	number	Amino acid sequence
HA22-hIgH1	SEQ ID
heavy chain	NO: 5
HA22-hIgG1	SEQ ID
light chain	NO: 6
m971-hIgG1	SEQ ID
heavy chain	NO: 7
m971-hIgG1	SEQ ID
light chain	NO: 8
indicates data missing or illegible when filed

EXAMPLE 2

Construction and Identification of Cell Lines Overexpressing Human CD22 and Monkey CD22

2.1 Identification of Cell Line Expressing CD22 Endogenously

Raji cells (purchased from China Center for Type Culture Collection, Wuhan University, catalog number: TCHu 44) 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 antibody 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 3 and FIGS. 2A-2B, the Raji cells can bind to HA22 and m971.

TABLE 3
FACS detection results of the endogenous cell line Raji cells
	Cell mean fluorescence
	density
	Designation of	IgG subtype	CD22
No.	antibody	control	antibody
1	HA22	44	22952
2	m971	44	12686

2.2 Preparation of CHO-K1 Monoclonal Cell Line Stably Transfected With Human CD22

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® 3000 Transfection 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, catalog number: SCSP-507), and then the transfected cells were selectively incubated for 2 weeks in DMEM/F12 medium containing 10 μg/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 AriaII (BD Biosciences) and the plate was placed in a cell incubator at 37° C. and 5% (v/v) CO₂ 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.

The specific selection results were shown in Table 4 and FIG. 3A-FIG. 3C, and the IgG subtype control was human IgG1 control. Table 4 showed that a series of CHO-K1 monoclonal cell lines positive for CD22 expression was prepared. In FIG. 3A-FIG. 3C, the abscissa was the fluorescence intensity of cells, and the ordinate was the number of cells. The results in FIG. 3A-FIG. 3C indicated that CHO-K1-human CD22 2C4, CHO-K1-human CD22 1G5 and CHO-K1-human CD22 1D9 were cell lines with high expression of CD22. CHO-K1-human 2C4 cell line was finally selected for the following examples.

TABLE 4
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-A CD22 2C4	66	4621
2	CHO-K1-A CD22 1G5	66	3154
3	CHO-K1-A CD22 1D9	66	2488

2.3 Preparation of HEK293T Cell Line Stably Transfected With Monkey CD22

The nucleotide sequence encoding the full-length amino acid sequence of the monkey CD22 (NCBI: XP_014979161.2, SEQ ID NO: 9) was cloned into the pcDNA3.1 vector (purchased from Thermofisher scientific, catalog number: V79020) and a plasmid was prepared. After plasmid transfection of the HEK293T cell line with FuGENE® HD (Promega, catalog number: #E2311), the transfected cells were selectively cultured in DMEM medium containing 10 μg/ml puromycin and 10%(w/w) fetal bovine serum for 2 weeks, subcloned in 96-well culture plates by limited dilution method, and cultured in a 37° C., 5% (v/v) CO₂ incubator. After about 2 weeks, some wells contained polyclones were selected and amplification was performed into 6-well plates. The amplified clones were screened by CD22 antibody hL22 with monkey cross activity (purchased from Enzo Life Sciences; catalog number: ENZ-ABS619-0200) by flow cytometry analysis, the cell line with better growth and higher fluorescence intensity was selected for further scale-up culture and then freezed and stored in liquid nitrogen. FIG. 4 is the result of flow cytometry analysis of the HEK293T cell line detected by hL22 antibody. The result shows a single positive cell peak overexpressing monkey CD22 after puromycin screening, which could be used to detect the cross-activity of antibodies.

Monkey CD22 full-length amino acid sequence
(SEQ ID NO: 9)
MHLLGPWLLLLVLEYLAFSDSSKWNIEHPGTIYAWEGACVWVPCTYRVLDGALETFILFHNPEYNQNMSKFEGTRL
YESTKDGKLPSGQKRVQRLGNKINNNCTLSHIPVHVNDSGQLGLRMVSKTEKWMERHILNVSERPFPPRIQLPPKLQESQ
EVTLTCLLNFSCYGYQIQLQWLLEGVPMRQAAVTLTSLSTKSVFTRSELKFSPQWSHHGKIVTCELHDVDGKVLSEDMVQ
LNVKHTPKLTIEVTPNETTVRKGDSVTMTCKVNSSNPEYTTVSWLKDGIPLKEQNTLMLTLHKVTKSQSGRYCCRVSNDV
GPATSEKVFLQVQYAPESSRVQISQSPAVEGSEVNFLCISPANPLPTNYTWYHNGKEVQGRTEKQFQIQKILPWHAGTYS
CEAENILGIGERGPGTELDVQYPPKKVTMVIENPTPIREGDTVTLSCNYSSSNPIVNHYEWRPRGAWEEPSLGVLKIQNI
GWNNTAVACAACNNWCSWASPVTLNVLYAPRGVRVRKTKPLSETHSGNSVSLQCDESSSHPKEVQFFWEKNGSLLGKESQ
LNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSQGNQVMEGKTATLICESDANPPVYSYAWFDWNVQ
SLPYSGRMLRLEPVKVQHSGAYWCQGTNRVGKGHSPLITLTVYYSPQTIGRRVAVGLGSCLAILILAMCGFKVQRRWKRT
QSQQGLQENSSGQSFFVRNKKVRRTPLSEGPHSLGCYNPMMEDGISYATLRFPETNTPRTGDAETSELQRPPPDCDDTVT
YSVLQKRQVGDYENVTPDFPEDEGTHYSELTQFGFGERPQAQENVDYVTVKH

EXAMPLE 3

Preparation of Anti-Human CD22 Hybridoma Monoclonal Antibody

3.1 Animal Immunity

An anti-human CD22 monoclonal antibody was produced by immunizing mice. BALB/c AnNCrl mice and SJL/JorllcoCrl mice (purchased from Shanghai SLAC Co., Ltd.), female, aged 6-8 weeks, were used in the experiment. Breeding environment: SPF rating. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12/12-hour light/dark cycle adjustment, and a temperature of 20-25° C.; humidity 40%-60%. The acclimatized mice were immunized according to the following scheme. The immune antigen was human CD22 (Asp20-Arg687)-His protein (purchased from ACRO Biosystems, catalog number: CD2-H52H8). For the first immunization, the immunogen was emulsified with TiterMax (purchased from Sigma, catalog number: T2684) and injected subcutaneously and intraperitoneally at 0.1 ml respectively, that is, each mouse was injected with 50 micrograms of immunogen protein A. For booster immunization, the immunogen was injected subcutaneously and intraperitoneally at 0.1 ml with Imject Alum Adjuvant (purchased from Thermofisher scientific, catalog number: 77161), that is, each mouse was injected with 25 micrograms of the immunogen. The frequency of immunization was once a week, blood was collected on the 4th, 18th, 46th, and 70th day, and the antibody titer in the mouse serum was detected by ELISA and FACS methods. The results are shown in Table 5-8. After immunization, the sera of mice immunized with human CD22-his protein binded to the immunogen to varying degrees, showing antigen-antibody reactions, and the highest dilution was about 6 million. The blank control is 1% (w/w) BSA, the batch refers to the mouse serum on the seventh day after the seventh booster immunization, and the data in the table are OD450 nm and MFI values.

TABLE 5
Serum antibody titer of Balb/c mice after immunization detected by ELISA
	OD450 nm
	Serum dilution
												Blank
Batch	1:100	1:300	1:900	1:2700	1:8100	1:24300	1:72900	1:218700	1:656100	1:1968300	1:5904900	control
261 (TB3)	2.55	2.60	2.57	2.54	2.25	2.10	1.69	1.23	0.65	0.33	0.20	0.09
262 (TB3)	2.33	2.26	2.22	2.27	2.07	1.94	1.51	0.95	0.45	0.23	0.13	0.08
263 (TB3)	2.40	2.24	2.15	2.13	2.01	1.82	1.41	0.85	0.41	0.20	0.12	0.08
264 (TB3)	2.40	2.31	2.30	2.27	2.07	1.82	1.30	0.71	0.31	0.16	0.10	0.07
265 (TB3)	2.35	2.31	2.32	2.16	2.08	1.84	1.41	0.85	0.41	0.19	0.11	0.08

TABLE 6
Serum antibody titer of SJL mice after immunization detected by ELISA
	OD450 nm
	Serum dilution
												Blank
Batch	1:100	1:300	1:900	1:2700	1:8100	1:24300	1:72900	1:218700	1:656100	1:1968300	1:5904900	control
266 (TE3)	2.40	2.36	2.35	2.24	2.05	1.79	1.34	0.75	0.37	0.19	0.12	0.08
267 (TB3)	2.31	2.16	2.02	1.82	1.51	1.06	0.53	0.26	0.13	0.09	0.07	0.09
268 (TB3)	2.29	2.10	1.96	1.64	1.29	0.78	0.35	0.19	0.12	0.10	0.10	0.11
269 (TB3)	2.61	2.49	2.36	2.29	2.07	1.88	1.45	0.88	0.43	0.21	0.13	0.09
270 (TB3)	1.83	1.23	0.63	0.28	0.14	0.08	0.08	0.06	0.09	0.09	0.07	0.08

TABLE 7
Serum antibody titer of Balb/c mice after immunization detected by FACS
	MFI
	Serum dilution
												Blank
Batch	1:100	1:300	1:900	1:2700	1:8100	1:24300	1:72900	1:218700	1:656100	1:1968300	1:5904900	control
261 (TB3)	64458	52926	45069	39113	32293	20440	9577	4202	1860	821	459	131
262 (TB3)	63113	54421	48096	41627	30655	17728	7976	3348	1476	679	399	112
263 (TB3)	68470	54517	46623	39177	29120	16910	7792	3301	1374	641	340	89
264 (TB3)	62190	48925	40670	32314	19921	9761	3979	1703	698	347	292	150
265 (TB3)	63768	52435	45919	37727	26351	14061	6023	2590	1122	504	284	110

TABLE 8
Serum antibody titer of SJL mice after immunization detected by FACS
	MFI
	Serum dilution
												Blank
Batch	1:100	1:300	1:900	1:2700	1:8100	1:24300	1:72900	1:218700	1:656100	1:1968300	1:5904900	control
266 (TB3)	67436	53379	45330	37604	26313	13406	5887	2427	1095	514	310	195
267 (TB3)	37379	27314	14870	6493	2661	1102	496	245	157	209	98	116
268 (TB3)	34130	26195	14039	5895	2365	971	449	270	140	136	134	156
269 (TB3)	9657	4490	1894	797	398	258	127	133	160	97	165	139
270 (TB3)	12544	5962	2715	1054	494	261	182	102	129	113	142	117

After the 7th to 8th immunization, the mice with high antibody titer in serum which tended to plateau were selected for splenocyte fusion. Immunization was boosted 3 days before splenocyte fusion, an antigen solution prepared with normal saline was injected subcutaneously and intraperitoneally (IP) at 50 μg/mouse.

3.2 Splenocyte Fusion and Hybridoma Screening

ACK Lysing Buffer (purchased from Gibco, catalog number: A1049201) was added to lyse the red blood cells doped in the splenocytes to obtain a splenocyte suspension. The cells were washed with DMEM (purchased from Gibco, catalog number: 11995081) basal medium 3 times by centrifugation at 1000 rpm, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC, catalog number: CRL-1581) at a ratio of 2:1 in terms of living cells. BTX ECM2001+ high-efficiency electrofusion method (see METHODS IN ENZYMOLOGY, VOL. 220) was used for cell fusion. The fused cells were diluted into DMEM medium containing 20% fetal bovine serum (ExCell Bio, catalog number: FSD500), 1× HAT (purchased from Sigma, catalog number: H0262) (the percentages were mass percentages), then added into a 96-well cell culture plate at 2×10⁴/200 microliters per well, and put in 5% CO₂, 37° C. incubator (the percentage is a volume percentage). After 14 days, ELISA was used to screen the supernatant in the cell fusion plate, ELISA positive clones were amplified into a 24-well plate, and DMEM (purchased from Gibco, catalog number: 11995081) containing 10% (w/w) HT (purchased from Sigma, catalog number: H0137) and fetal bovine serum was used for scale-up culture at 37° C., 5% (v/v) CO₂. After culturing for 3 days, the culture medium of the scale-up culture in the 24-well plate was taken for centrifugation, and the supernatant was collected. the antibody subtype was analyzed for the supernatant, and ELISA and FACS were used to determine the binding activity to human CD22 protein and human CD22 positive cells (for the detection methods of binding activity, please refer to Example 5.1 and Example 5.2 respectively).

According to the screening results of the 24-well plate, the positive hybridoma cells in the ELISA and FACS experiments were selected as eligible positive clones, and were subcloned in DMEM medium containing 10% (w/w) FBS (purchased from Gibco, catalog number: 11995081) in a 96-well plate by limited dilution method, and cultured at 37° C. and 5% (v/v) CO₂. 10 days after subcloning, ELISA and FACS were used for preliminary screening, and a single positive clone was selected and amplified into a 24-well plate for further culture. According to the detection results of the samples in the 24-well plate, the optimal clone was selected and placed in DMEM medium containing 10% (w/w) FBS (purchased from Gibco, catalog number: 11995081) at 37° C. and 5% (v/v) CO₂ condition for scale-up culture. The obtained cells were frozen in liquid nitrogen to obtain the hybridoma cells of the present invention.

EXAMPLE 4

Determination of Amino Acid Sequence of Variable Regions of Light and Heavy Chains of Positive Hybridoma Clones

The hybridoma cells in the logarithmic growth phase were collected, and the cells were fully lysed with Trizol (Invitrogen, catalog number: 15596-018) and stored at −80° C. for testing. Suzhou GENEWIZ Biotechnology Co., Ltd. was entrusted to complete the determination of the amino acid sequences of the light and heavy chain variable regions of hybridoma positive clones. The sequencing results were analyzed by MOE software, and the phylogenetic tree was constructed according to the amino acid sequence of the protein encoded by the variable region. After eliminating the sequences that were close to each other on the phylogenetic tree according to the sequence similarity, 35 clones were obtained. Among them, there are 23 (F1.236.15, F1.214.8, F1.273.12, F1.231.15, F1.11.7, F1.77.9, F1.105.11, F1.267.9, F1.7.6, F1.224.1, F1.250.16, F1.120.15, F1.216.2, F1.280.1, F1.200.11, F1.192.1, F1.245.2, F1.60.9, F1.172.13, F1.17.1, F1.161.7, F1.257.3 and F1.62.10) for F1 series, and 12 (F2.70.2, F2.104.10, F2.180.16, F2.121.9, F2.173.9, F2.343.16, F2.205.9, F2.99.1, F2.127.11, F2.55.1, F2.42.9 and F2.151.13) for F2 series.

Taizhou Biointron Biotechnology Co., Ltd. cloned the heavy chain variable region sequences of the 35 clones into the expression vector pcDNA3.4-B1HH1 containing a signal peptide and human antibody IgG1 heavy chain constant region (SEQ ID NO: 10) (private vector of Taizhou Biointron Biotechnology Co., Ltd.), cloned the light chain variable region sequences of the clones of the F1 series into the expression vector pcDNA3.4-B1HLK containing a signal peptide and human antibody IgG1 Kappa light chain constant region (SEQ ID NO: 11) (private vector of Taizhou Biointron Biotechnology Co., Ltd.), cloned the light chain variable region sequences of the clones of the F2 series into the expression vector pcDNA3.4-BIHLS containing a signal peptide and human antibody IgG1 Lambda light chain constant region (SEQ ID NO: 12) (private vector of Taizhou Biointron Biotechnology Co., Ltd.), and the expression vector of human-murine chimeric antibody was obtained and the antibody was prepared according to the method of Example 1.2. The CDRs of the antibody sequences were analyzed by KABAT, Chothia or IMGT software respectively, and the corresponding sequence information is shown in the following Tables 9-10. Among them, Table 9 shows the antibody sequences represented by amino acids of the heavy and light chain variable regions of the 35 chimeric antibody molecules, and Table 10 shows the analysis results of IMGT, Kabat and Chothia of the CDRs of the 35 chimeric antibody molecules.

TABLE 9
Specific amino acid sequence information of the heavy chain variable
region and light chain variable region of the anti-CD22 antibody
Antibody	Sequence
number	number	Heavy chain variable region sequence (VH)
	SEQ ID NO. 13
	SEQ ID NO. 15
	SEQ ID NO. 17
	SEQ ID NO. 19
	SEQ ID NO. 21
	SEQ ID NO. 23
	SEQ ID NO. 25
	SEQ ID NO. 27
	SEQ ID NO. 29
	SEQ ID NO. 31
	SEQ ID NO. 33
	SEQ ID NO. 35
	SEQ ID NO. 37
		YCVRGDYYNSRYWYFDVWGTGTTVTVSS
F1.120.15	SEQ ID NO. 39	QVQLQQSGPELVKPGASVKISCKASGNAFSNSWMNWVKQRPGKGLE
		WIGRVYSEDGDTQYNGKFRDKATLTADRSTSTAYMQLSSLTSEDSAV
		YFCARWLIYYGTYGAMDYWGQGTSVTVSS
F1.62.10	SEQ ID NO. 41	QVQLQQSGPELVKPGASVKISCKASGYEFSSIWMNWVKQRPGEGLE
		WIGRIFPGDGEINYNEKFKGKATLTADRSSTTAYMQLSSLTSEDSAVY
		FCAGWRIYYGTYGAMDYWGQGTSVTVSS
F1.216.2	SEQ ID NO. 43	QVQLQQSGPEVVRPGASVKISCKASGYAFSSSWMNWMKQRPGKALE
		WIGRIYPGDGDTNYSGEFKVKATLTADKSSSTAYMQLSSLTSEDSAV
		YFCAREGSYYSNPWYFDYWGQGTTLTVSS
F1.280.1	SEQ ID NO. 45	QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQTPGKGLTW
		MGWINTDSGVPTYADDFKRRFAFSLEASATTAYLQINNLKNEDTATY
		FCARGGPDYWGQGTTLTVSS
F1.200.11	SEQ ID NO. 47	QNQLVQSGPELKKPGETVKISCKASGYTFTTAGMQWVQKMPGKGFK
		WIGWINTLSGEPKYAEDFKGRFAFSLETSATTAYLQISNLKNEDTATY
		FCARAYYSNLYWYFDVWGTGTTVTVSS
F1.192.1	SEQ ID NO. 49	AVQLQQSGVELVRPGASVKLSCTGSDFNIKEDYIHWVKQRPEQGLEW
		IGWIDPENGDTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYY
		CATYGSLDYWGQGTTLTVSS
F1.245.2	SEQ ID NO. 51	EVQLQQSGAELVKPGASVKLSCTGSGFNIKDYYIHWVKQRTEQGLEW
		MGRIDPEDGETEYAPKFQDEATITSDTSSNTAYLQLSSLTYEDTAVYY
		CAREAAGYFYRGSSYGYFDVWGTGTTVTVSS
F1.60.9	SEQ ID NO. 53	QVQLKESGPGLVAPSQSLSITCTVSGLSLNNYGVSWVRQPPGKGLEW
		LGVIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNSLHTDDTATYYC
		AINWGDYWGQGTTLIVSS
F1.172.13	SEQ ID NO. 55	QVTLKESGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEW
		LAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYC
		ARAPPPNWDEYYFDYWGQGTTLTVSS
F1.17.1	SEQ ID NO. 57	QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYTVHWVRQPPGKGLEWL
		GMIWGGGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYY
		CARPHDFDAGGFAYWGQGTLVTVSA
F2.151.13	SEQ ID NO. 59	QVHLQQPGTELMKPGASVKLSCKATGYTFRDYWLEWVKQRPGHGL
		EWIGEILPGSGNAYYNEKFKGKATFTADTSSNTAYMQLSSLTTEDSAI
		YYCARVYSNWYFDAWGKGTTVTVSS
F2.70.2	SEQ ID NO. 61	EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMNWFRQAPEKGLE
		WVAYISRGSHTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSNDTAM
		YYCVRMAGYYAMDYWGQGTSVTVSS
F2.104.10	SEQ ID NO. 63	EVQLVESGGGFVKPGGSLKLSCAASGFTFSDYGMNWFRQAPEKGLE
		WVAYISRGSHTIYYADTVKGRFTISRDNAENTLFLQMTSLRSNDTAM
		YYCVRMAGYYAMDYWGQGTSLTVSS
F2.180.16	SEQ ID NO. 65	EVKLVESGGGLVLPGGSLKISCAASGFTFSDYYMYWIRQTPDKRLEW
		VAYISNTGYSTFYPDSVKGRFTVSRDNAKNTLYLQMSRLQSEDTAMY
		YCARHGILYAMDYWGQGTSVTVSS
F2.121.9	SEQ ID NO. 67	EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYVMSWIRQTPEKRLEW
		VATISDAGSYTFYPDNLKGRFTISRDNAKNNLYLQMNHLTSEDTAFY
		YCAITYFGNYGGYWGQGTLVTVSV
F2.173.9	SEQ ID NO. 69	EVQLVESGGDLVKPGESLKLSCEASGFTFSNYAMSWVRQTPDRRLEW
		VATISSIGSFTYYSDSVKGRFTISRDNVKSTLHLQMNNLKSGDTAIYFC
	SEQ ID NO. 71
	SEQ ID NO. 73
	SEQ ID NO. 75
	SEQ ID NO. 77
	SEQ ID NO  79
	SEQ ID NO. 81
Antibody	Sequence
number	number	Light chain variable region sequence (VL)
	SEQ ID NO. 14
	SEQ ID NO. 16
	SEQ ID NO. 18
	SEQ ID NO. 20
	SEQ ID NO. 22
	SEQ ID NO. 24
	SEQ ID NO. 26
F1 257.3	SEQ ID NO. 28
F1.105.11	SEQ ID NO. 30
F1.267.9	SEQ ID NO. 32	DIVLTQSPASLAVSLGQRATISCRASESVDNYGFDFIHWYQQKPGQPP
		KLLIYRASNLESGIPARFSGSGSRPDFTLTINPVETDDVATYYCQQSIKD
		PWTFGGGTKLEIK
F1.7.6	SEQ ID NO. 34	DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNNQKNYLAWYQQKPG
		QSPKKLIYWASTRESGVPDRLTGSGSGTDFTLTISSVTAEDLAVYYCQ
		QFYSYPWTFGGGTKLEIK
F1.224.1	SEQ ID NO. 36	DIVLTQSPASLAVSLGQRATISCRASQSVSTPRYSFMHWYQQKPGQPP
		KLLIKYASNLESGFPARFTGSGSGTDFTLNIHPVEEEDTAAYYCQQSW
		AIPWTFGGGTKLEIK
F1.250.16	SEQ ID NO. 38	DIVMTQSQKFMSTTVGDRVSITCKAGQNVRTAVAWYQQKPGQSPKL
		LIYSASTRYIGVPERFTGSGSGTDFTLTITNMQSEDLADYFCQQYSSFP
		LTFGTGTKLELK
F1.120.15	SEQ ID NO. 40	DIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYHQKPDGTVKLLI
		YFTSRPYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWT
		FGGGTKLEIK
F1.62.10	SEQ ID NO. 42	DIQMTQITSSLSASLGDRVTISCRASQDIGNYLNWYQQKPDGTVKLLI
		YYTSRLHSGVPSRFSGSGSRTDYSLTISNLQQEDIATYFCQQGNTLPWT
		FGGGTKLEIK
F1.216.2	SEQ ID NO. 44	DVLVTQTPLSLPVSLGDQASISCRSSQSIVQSNGDTYLEWYLQKPGQS
		PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS
		HVPFTFGSGTKLEIK
F1.280.1	SEQ ID NO. 46	DILLTQSPATLSVTPGETVSLSCRASQSIYKNLHWYHQKSHRSPRLLIK
		YASDSISGIPSRFTGSGSGTNFTLSINSVKPEDEGIYYCLQGYIVPLTFG
		AGTKLELK
F1.200.11	SEQ ID NO. 48	DIVMTQSQKFMSTTVGDRVTITCKASQNVVTAVAWYQQKPGQSPKV
		LIYSASNRYSGVPDRFTGSGSGTDFTLTISNMHSEDLANYFCHQYSSYP
		FTFGSGTKLEIK
F1.192.1	SEQ ID NO. 50	DIKMTQSPSSMYAFLGERVTITCKASQGINSFLTWFQQKPGKSPKTLIY
		RANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDVGIYYCLQYDEFPRTF
		GGGTKLEIK
F1.245.2	SEQ ID NO. 52	DFQMTQTTSFLSASLGDRVTISCSASQDISNYLNWYQQKPDGTVKLLI
		YYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSMLPYT
		FGGGTKLEME
F1.60.9	SEQ ID NO. 54	DIKMTQSPSSMNASLGERVTITCKASQDINSYLSWFQQKPGKSPKILIY
		RANRLIDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYGVFPLTF
		GAGTKLELK
F1.172.13	SEQ ID NO. 56	DIQMTQSPASLSASVGETVTITCRASGNIHNSLAWYQQKQGKSPQLLV
		HNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPP
		FGGGTKLEIK
F1.17.1	SEQ ID NO. 58	QIVLTQSPAILSTSPGEKVTMTCSASSRVNYMYWYQQKPGSSPRLLIY
		DTSNLASGVPVRESGSGSGTSYSLTISRMEAEDAATYYCQQWTSYPLT
		FGAGTKLELK
F2.151.13	SEQ ID NO. 60	QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFT
		GLIGGTNNQAPGVPARFSGSLIGDKAALTISGAQTEDEAIYFCALWFS
		NHWVFGGGTKLTVL
F2.70.2	SEQ ID NO. 62	QLVLTQSSSASFSLGASAKLTCTLSSQHSSYIIEWYQQQPLKPPKYVME
		LKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIK
		EQFVYVFGGGTKVTVL
F2.104.10	SEQ ID NO. 64	QLVLTQSSSASFSLGASAKLTCTLSSQHSSYIIEWYQQQPLKPPKYVME
		VKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQSEDEAIYICGVGDTIK
		EQFVYVFGGGTKVTVL
F2.180.16	SEQ ID NO. 66	QPVLTQSSSASFSLGASAKLTCTLSSQYSTYTIGWYQQQPLKPPKYVM
		ELKHDGSRHTGDGIPDRFSGSSSGADRYLSISNIQPEDEATYICGVGDTI
		KEQFMYVFGGGTKVTVL
F2.121.9	SEQ ID NO. 68	QAVVTQESALTTSPGGTVVLTCRSNTGAVTTSNYANWVQEKPDHLFT
		GLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYS
		THWVFGGGTKLTVL
F2.173.9	SEQ ID NO. 70	QLVLTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPHKPPKFVM
		EIKKDGSHYTGDGIPDRFSGSSSGADRYLSISNIQPEDETIYICGVSDMI
		KDQFVYVFGGGTKVTVL
F2.343.16	SEQ ID NO. 72	QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFT
		GLIGGTYNRVPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYS
		NHFWVFGGGTKLTVL
F2.205.9	SEQ ID NO. 74	QAVVTQESALTTSPGETVTLTCRSSTGAVTTRNYANWVQEKPDHLFT
		GLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYS
		NHFIFGSGTKVTVL
F2.99.1	SEQ ID NO. 76	QLVLTQSSSASFSLGASAKLTCTLSRQHSAYTIEWYQQQPLKPPKYVM
		EVKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTI
		KEHFVFGGGTKVTVL
F2.127.11	SEQ ID NO. 78	QPVITQSSSASFSLGASAELTCTLSSQHSTYTIEWYQQQPLKPPKYVMG
		LKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVSDTIK
		EQFVYVFGGGTKVTVL
F2.55.1	SEQ ID NO. 80	QLVLTQSSSASFSLGASAKLTCTLSSQHSAYTVEWYQQQPLKPPKYV
		MELKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGD
		TVKEQFVFGGGTKVTVL
F2.42.9	SEQ ID NO. 82	QLVLTQSSSASFSLGASAKLTCTLSSQHSAYTIEWYQQQPLKPPKFVM
		DLKKDGSHSTGDGIPARFSGSSSGADRYLTISNIQPEDEAIYICGVGDTI
		KEQYVFGGGTKVTVL
indicates data missing or illegible when filed

TABLE 10
Specific sequence information of CDRs of CD22 antibody analyzed by
IMGT, KABAT and Chothia software
IMGT analysis
Antibody	Sequence		Sequence		Sequence
number	number	CDR1-HC	number	CDR2-HC	number	CDR3-HC
	SEQ ID		SEQ ID		SEQ ID
	NO: 83		NO. 84		NO. 85
	SEQ ID		SEQ ID		SEQ ID
	NO. 101		NO. 102		NO. 103
	SEQ ID		SEQ ID		SEQ ID
	NO. 119		NO. 120		NO. 121
	SEQ ID		SEQ ID		SEQ ID
	NO. 137		NO. 138		NO. 139
	SEQ ID		SEQ ID		SEQ ID
	NO. 155		NO. 156		NO. 157
	SEQ ID		SEQ ID		SEQ ID
	NO. 173		NO. 174		NO. 175
	SEQ ID		SEQ ID		SEQ ID
	NO. 191		NO. 192		NO. 193
F1.257.3	SEQ ID	GYTFTSYG	SEQ ID	IYPKLGTT	SEQ ID	ACPHYYATR
	NO. 209		NO. 210		NO. 211	GGDY
F1.105.11	SEQ ID	GYTFSDYF	SEQ ID	INSYSGGT	SEQ ID	ARWMDY
	NO. 227		NO. 228		NO. 229
F1.267.9	SEQ ID	GYTFTDFY	SEQ ID	INPYNGGI	SEQ ID	ARRMEYHA
	NO. 245		NO. 246		NO. 247	MDY
F1.7.6	SEQ ID	GYDFTRYN	SEQ ID	IDPYNGDT	SEQ ID	EAIYYDMEG
	NO. 263		NO. 264		NO. 265	YALDY
F1.224.1	SEQ ID	GYTFTDFN	SEQ ID	VNPNNGGT	SEQ ID	VRLGTSDYG
	NO. 281		NO. 282		NO. 283	EAWFIS
F1.250.16	SEQ ID	GYTFTTYP	SEQ ID	IHPYNDDT	SEQ ID	VRGDYYNSR
	NO. 299		NO. 300		NO. 301	YWYFDV
F1.120.15	SEQ ID	GNAFSNSW	SEQ ID	VYSEDGDT	SEQ ID	ARWLIYYGT
	NO. 317		NO. 318		NO. 319	YGAMDY
F1.62.10	SEQ ID	GYEFSSIW	SEQ ID	IFPGDGEI	SEQ ID	AGWRIYYGT
	NO. 335		NO. 336		NO. 337	YGAMDY
F1.216.2	SEQ ID	GYAFSSSW	SEQ ID	IYPGDGDT	SEQ ID	AREGSYYSNP
	NO. 353		NO. 354		NO. 355	WYFDY
F1.280.1	SEQ ID	GYTFTTYG	SEQ ID	INTDSGVP	SEQ ID	ARGGPDY
	NO. 371		NO. 372		NO. 373
F1.200.11	SEQ ID	GYTFTTAG	SEQ ID	INTLSGEP	SEQ ID	ARAYYSNLY
	NO. 389		NO. 390		NO. 391	WYFDV
F1.192.1	SEQ ID	DFNIKEDY	SEQ ID	IDPENGDT	SEQ ID	ATYGSLDY
	NO. 407		NO. 408		NO. 409
F1.245.2	SEQ ID	GFNIKDYY	SEQ ID	IDPEDGET	SEQ ID	AREAAGYFY
	NO. 425		NO. 426		NO. 427	RGSSYGYFD
						V
F1.60.9	SEQ ID	GLSLNNYG	SEQ ID	IWGDGST	SEQ ID	AINWGDY
	NO. 443		NO. 444		NO. 445
F1.172.13	SEQ ID	GFSLSTSGM	SEQ ID	IYWDDDK	SEQ ID	ARAPPPNWD
	NO. 461	G	NO. 462		NO. 463	EYYFDY
F1.17.1	SEQ ID	GFSLSRYT	SEQ ID	IWGGGST	SEQ ID	ARPHDFDAG
	NO. 479		NO. 480		NO. 481	GFAY
F2.151.13	SEQ ID	GYTFRDYW	SEQ ID	ILPGSGNA	SEQ ID	ARVYSNWYF
	NO. 497		NO. 498		NO. 499	DA
F2.70.2	SEQ ID	GFTFSDYG	SEQ ID	ISRGSHTI	SEQ ID	VRMAGYYA
	NO. 515		NO. 516		NO. 517	MDY
F2.104.10	SEQ ID	GFTFSDYG	SEQ ID	ISRGSHTI	SEQ ID	VRMAGYYA
	NO. 533		NO. 534		NO. 535	MDY
F2.180.16	SEQ ID	GFTFSDYY	SEQ ID	ISNTGYST	SEQ ID	ARHGILYAM
	NO. 551		NO. 552		NO. 553	DY
F2.121.9	SEQ ID	GFTFSDYV	SEQ ID	ISDAGSYT	SEQ ID	AIIYFGNYGG
	NO. 569		NO. 570		NO. 571	Y
F2.173.9	SEQ ID	GFTFSNYA	SEQ ID	ISSIGSFT	SEQ ID	ARLGVYFDS
	NO. 587		NO. 588		NO. 589
F2.343.16	SEQ ID	GFSLTGYG	SEQ ID	IWGGGGT	SEQ ID	ARYSKYGHF
	NO. 605		NO. 606		NO. 607	DV
F2.205.9	SEQ ID	GFSLSTSGM	SEQ ID	IWWDDDE	SEQ ID	ARISIPYGYY
	NO. 623		NO. 624		NO. 623	DWFFDV
	SEQ ID		SEQ ID		SEQID
	NO. 641		NO. 642		NO. 643
	SEQ ID		SEQ ID		SEQ ID
	NO. 659		NO. 660		NO. 661
	SEQ ID		SEQ ID		SEQ ID
	NO. 677		NO. 678		NO. 679
	SEQ ID		SEQ ID		SEQ ID
	NO. 695		NO. 696		NO. 697
Antibody	Sequence		Sequence		Sequence
number	number	CDR1-LC	number	CDR2-LC	number	CDR3-LC
	SEQ ID		SEQ ID	RA	SEQ ID
	NO. 92		NO. 93		NO. 94
	SEQ ID		SEQ ID	RA	SEQ ID
	NO. 110		NO. 111		NO. 112
	SEQ ID		SEQ ID	RA	SEQ ID
	NO. 128		NO. 129		NO. 130
	SEQ ID		SEQ ID	TV	SEQ ID
	NO. 146		NO. 147		NO. 148
	SEQ ID		SEQ ID	RT	SEQ ID
	NO. 164		NO. 165		NO. 166
	SEQ ID		SEQ ID	RA	SEQ ID
	NO. 182		NO. 183		NO. 184
	SEQ ID		SEQ ID		SEQ ID
	NO. 200		NO. 201		NO. 202
	SEQ ID		SEQ ID	YA	SEQ ID
	NO. 218		NO. 219		NO. 220
	SEQ ID		SEQ ID	SG	SEQ ID
	NO. 236		NO. 237		NO. 238
	SEQ ID		SEQ ID	RA	SEQ ID
	NO. 254		NO. 255		NO. 256
	SEQ ID		SEQ ID	WA	SEQ ID
	NO. 272		NO. 273		NO. 274
	SEQ ID		SEQ ID	YA	SEQ ID
	NO. 290		NO. 291		NO. 292
	SEQ ID		SEQ ID	SA	SEQ ID
	NO. 308		NO. 309		NO. 310
	SEQ ID		SEQ ID	FT	SEQ ID
	NO. 326		NO. 327		NO. 328
	SEQ ID		SEQ ID	YT	SEQ ID
	NO. 344		NO. 345		NO. 346
	SEQ ID		SEQ ID	KV	SEQ ID
	NO. 362		NO. 363		NO. 364
	SEQ ID		SEQ ID	YA	SEQ ID
	NO. 380		NO. 381		NO. 382
	SEQ ID		SEQ ID	SA	SEQ ID
	NO. 398		NO. 399		NO. 400
	SEQ ID		SEQ ID	RA	SEQ ID
	NO. 416		NO. 417		NO. 418
	SEQ ID		SEQ ID	YT	SEQ ID
	NO. 434		NO. 435		NO. 436
	SEQ ID		SEQ ID	RA	SEQ ID
	NO. 452		NO. 453		NO. 454
	SEQ ID		SEQ ID	NA	SEQ ID
	NO. 470		NO. 471		NO. 472
	SEQ ID		SEQ ID	DT	SEQ ID
	NO. 488		NO. 489		NO. 490
	SEQ ID		SEQ ID	GT	SEQ ID
	NO. 506		NO. 507		NO. 508
	SEQ ID		SEQ ID		SEQ ID
	NO. 524		NO. 525		NO. 526
	SEQ ID		SEQ ID		SEQ ID
	NO. 542		NO. 543		NO. 544
	SEQ ID		SEQ ID		SEQ ID
	NO. 560		NO. 561		NO. 562
	SEQ ID		SEQ ID	GT	SEQ ID
	NO. 578		NO. 579		NO. 580
	SEQ ID		SEQ ID		SEO ID
	NO. 596		NO. 597		NO. 598
	SEQ ID		SEQ ID	GT	SEQ ID
	NO. 614		NO. 615		NO. 616
	SEQ ID		SEQ ID	GT	SEQ ID
	NO. 632		NO. 633		NO. 634
	SEQ ID		SEQ ID		SEQ ID
	NO. 650		NO. 651		NO. 652
	SEQ ID		SEQ ID		SEQ ID
	NO. 668		NO. 669		NO. 670
	SEQ ID		SEQ ID		SEQ ID
	NO. 686		NO. 687		NO. 688
	SEO ID		SEQ ID		SEQ ID
	NO. 704		NO. 705		NO. 706
KABAT analysis
Antibody	Sequence		Sequence		Sequence
number	number	CDR1-HC	number	CDR2-HC	number	CDR3-HC
	SEQ ID		SEQ ID		SEQ ID
	NO. 86		NO. 87		NO. 88
	SEQ ID		SEQ ID		SEQ ID
	NO. 104		NO. 105		NO. 106
	SEQ ID		SEQ ID		SEQ ID
	NO. 122		NO. 123		NO. 124
	SEQ ID		SEQ ID		SEQ ID
	NO. 146		NO. 147		NO. 148
	SEQ ID		SEQ ID		SEQ ID
	NO. 158		NO. 159		NO. 160
	SEQ ID		SEQ ID		SEQ ID
	NO. 176		NO. 177		NO. 178
F1.161.7	SEQ ID	SYWIS	SEQ ID	DIYPGTGSSNYN	SEQ ID	LKFEGIGY
	NO. 194		NO. 195	EKFKS	NO. 196
F1.257.3	SEQ ID	SYGIS	SEQ ID	EIYPKLGTTYYN	SEQ ID	PHYYATRGG
	NO. 212		NO. 213	EKFKD	NO. 214	DY
F1.105.11	SEQ ID	DYFMN	SEQ ID	IINSYSGGTSYNQ	SEQ ID	WMDY
	NO. 230		NO. 231	KFKG	NO. 232
F1.267.9	SEQ ID	DFYMN	SEQ ID	VINPYNGGINYN	SEQ ID	RMEYHAMD
	NO. 248		NO. 249	QKFKG	NO. 250	Y
F1.7.6	SEQ ID	RYNMY	SEQ ID	YIDPYNGDTRYN	SEQ ID	IYYDMEGYA
	NO. 266		NO. 267	QKFKG	NO. 268	LDY
F1.224.1	SEQ ID	DFNMD	SEQ ID	DVNPNNGGTIYN	SEQ ID	LGTSDYGEA
	NO. 284		NO. 285	QKFKG	NO. 286	WFIS
F1.250.16	SEQ ID	TYPIE	SEQ ID	NIHPYNDDTEYN	SEQ ID	GDYYNSRYW
	NO. 302		NO. 303	EKFKG	NO. 304	YFDV
F1.120.15	SEQ ID	NSWMN	SEQ ID	RVYSEDGDTQY	SEQ ID	WLIYYGTYG
	NO. 320		NO. 321	NGKFRD	NO. 322	AMDY
F1.62.10	SEQ ID	SIWMN	SEQ ID	RIFPGDGEINYNE	SEQ ID	WRIYYGTYG
	NO. 338		NO. 339	KFKG	NO. 340	AMDY
F1.216.2	SEQ ID	SSWMN	SEQ ID	RIYPGDGDTNYS	SEQ ID	EGSYYSNPW
	NO. 356		NO. 357	GEFKV	NO. 358	YFDY
F1.280.1	SEQ ID	TYGMS	SEQ ID	WINTDSGVPTYA	SEQ ID	GGPDY
	NO. 374		NO. 375	DDFKR	NO. 376
F1.200.11	SEQ ID	TAGMQ	SEQ ID	WINTLSGEPKYA	SEQ ID	AYYSNLYWY
	NO. 392		NO. 393	EDFKG	NO. 394	FDV
F1.192.1	SEQ ID	EDYIH	SEQ ID	WIDPENGDTEYA	SEQ ID	YGSLDY
	NO. 410		NO. 411	SKFQG	NO. 412
F1.245.2	SEQ ID	DYYIH	SEQ ID	RIDPEDGETEYAP	SEQ ID	EAAGYFYRG
	NO. 428		NO. 429	KFQD	NO. 430	SSYGYFDV
F1.60.9	SEQ ID	NYGVS	SEQ ID	VIWGDGSTNYHS	SEQ ID	NWGDY
	NO. 446		NO. 447	ALIS	NO. 448
F1.172.13	SEQ ID	TSGMGVS	SEQ ID	HIYWDDDKRYN	SEQ ID	APPPNWDEY
	NO. 464		NO. 465	PSLKS	NO. 466	YFDY
F1.17.1	SEQ ID	RYTVH	SEQ ID	MIWGGGSTDYN	SEQ ID	PHDFDAGGF
	NO. 482		NO. 483	SALKS	NO. 484	AY
F2.151.13	SEQ ID	DYWLE	SEQ ID	EILPGSGNAYYN	SEQ ID	VYSNWYFDA
	NO. 500		NO. 501	EKFKG	NO. 502
F2.70.2	SEQ ID	DYGMN	SEQ ID	YISRGSHTIYYAD	SEQ ID	MAGYYAMD
	NO. 518		NO. 519	TVKG	NO. 520	Y
F2.104.10	SEQ ID	DYGMN	SEQ ID	YISRGSHTIYYAD	SEQ ID	MAGYYAMD
	NO. 536		NO. 537	TVKG	NO. 538	Y
F2.180.16	SEQ ID	DYYMY	SEQ ID	YISNTGYSTFYPD	SEQ ID	HGILYAMDY
	NO. 554		NO. 555	SVKG	NO. 556
F2.121.9	SEQ ID	DYVMS	SEQ ID	TISDAGSYTFYPD	SEQ ID	IYFGNYGGY
	NO. 572		NO. 573	NLKG	NO. 574
F2.173.9	SEQ ID	NYAMS	SEQ ID	TISSIGSFTYYSDS	SEQ ID	LGVYFDS
	NO. 590		NO. 591	VKG	NO. 592
F2.343.16	SEQ ID	GYGVQ	SEQ ID	VIWGGGGTDYN	SEQ ID	YSKYGHFDV
	NO. 608		NO. 609	AAFIS	NO. 610
	SEQ ID		SEQ ID		SEQ ID
	NO. 626		NO. 627		NO. 628
	SEQ ID		SEQ ID		SEQ ID
	NO. 644		NO. 645		NO. 646
	SEQ ID		SEQ ID		SEQ ID
	NO. 662		NO. 663		NO. 664
	SEQ ID		SEQ ID		SEQ ID
	NO. 680		NO. 681		NO. 682
	SEQ ID		SEQ ID		SEQ ID
	NO. 698		NO. 699		NO. 700
Antibody	Sequence		Sequence		Sequence
number	number	CDR1-LC	number	CDR2-LC	nuraber	CDR3-LC
	SEQ ID		SEQ ID		SEQ ID
	NO. 95		NO. 96		NO. 97
	SEQ ID		SEQ ID		SEQ ID
	NO. 113		NO. 114		NO. 115
	SEQ ID		SEQ ID		SEQ ID
	NO. 131		NO. 132		NO. 133
	SEQ ID		SEQ ID		SEQ ID
	NO. 149		NO. 150		NO. 151
	SEQ ID		SEQ ID		SEQ ID
	NO. 167		NO. 168		NO. 169
	SEQ ID		SEQ ID		SEQ ID
	NO. 185		NO. 186		NO. 187
	SEQ ID		SEQ ID		SEQ ID
	NO. 203		NO. 204		NO. 205
	SEQ ID		SEQ ID		SEQ ID
	NO. 221		NO. 222		NO. 223
	SEQ ID		SEQ ID		SEQ ID
	NO. 239		NO. 240		NO: 241
	SEQ ID		SEQ ID		SEQ ID
	NO. 257		NO. 258		NO. 259
	SEQ ID		SEQ ID		SEQ ID
	NO. 275		NO. 276		NO. 277
	SEQ ID		SEQ ID		SEQ ID
	NO. 293		NO. 294		NO. 295
	SEQ ID		SEQ ID		SEQ ID
	NO. 311		NO. 312		NO. 313
	SEQ ID		SEQ ID		SEQ ID
	NO. 329		NO. 330		NO. 331
	SEQ ID		SEQ ID		SEQ ID
	NO. 347		NO. 348		NO. 349
	SEQ ID		SEQ ID		SEQ ID
	NO. 365		NO. 366		NO. 367
	SEQ ID		SEQ ID		SEQ ID
	NO. 383		NO. 384		NO. 385
	SEQ ID		SEQ ID		SEQ ID
	NO. 401		NO. 402		NO. 403
	SEQ ID		SEQ ID		SEQ ID
	NO. 419		NO. 420		NO. 421
	SEQ ID		SEQ ID		SEQ ID
	NO. 437		NO. 438		NO. 439
	SEQ ID		SEQ ID		SEQ ID
	NO. 453		NO. 456		NO. 457
	SEQ ID		SEQ ID		SEQ ID
	NO. 473		NO. 474		NO. 475
	SEQ ID		SEQ ID		SEQ ID
	NO. 491		NO. 492		NO. 493
	SEQ ID:		SEQ ID		SEQ ID
	NO. 509		NO. 510		NO. 511
	SEQ ID		SEQ ID		SEQ ID
	NO. 527		NO. 528		NO. 529
	SEQ ID		SEQ ID		SEQ ID
	NO. 545		NO. 546		NO. 547
	SEQ ID		SEQ ID		SEQ ID
	NO. 563		NO. 564		NO. 565
	SEQ ID		SEQ ID		SEQ ID
	NO. 581		NO. 582		NO. 583
	SEQ ID		SEQ ID		SEQ ID
	NO. 599		NO. 600		NO. 601
	SEQ ID		SEQ ID		SEQ ID
	NO. 617		NO. 618		NO. 619
	SEQ ID		SEQ ID		SEQ ID
	NO. 635		NO. 636		NO. 637
	SEQ ID		SEQ ID		SEQ ID
	NO. 653		NO. 654		NO. 655
	SEQ ID		SEQ ID		SEQ ID
	NO. 671		NO. 672		NO. 673
	SEQ ID		SEQ ID		SEQ ID
	NO. 689		NO. 690		NO. 691
	SEQ ID		SEQ ID		SEQ ID
	NO. 707		NO.  708		NO.709
Chothia analysis
Antibody	Sequence		Sequence		Sequence
number	number	CDR1-HC	number	CDR2-HC	number	CDR3-HC
	SEQ ID		SEQ ID		SEQ ID
	NO. 89		NO. 90		NO. 91
	SEQ ID		SEQ ID		SEQ ID
	NO. 107		NO. 108		NO. 109
	SEQ ID		SEQ ID		SEQ ID
	NO. 125		NO. 126		NO. 127
	SEQ ID		SEQ ID		SEQ ID
	NO. 143		NO. 144		NO. 145
	SEQ ID		SEQ ID		SEQ ID
	NO. 161		NO. 162		NO. 163
F1.77.9	SEQ ID	GYTFTTY	SEQ ID	NPSSGY	SEQ ID	QLDY
	NO. 179		NO. 180		NO. 181
F1.161.7	SEQ ID	GYTFTSY	SEQ ID	YPGTGS	SEQ ID	LKFEGIGY
	NO. 197		NO. 198		NO. 199
F1.257.3	SEQ ID	GYTFTSY	SEQ ID	YPKLGT	SEQ ID	PHYYATRGG
	NO. 215		NO. 216		NO. 217	DY
F1.105.11	SEQ ID	GYTFSDY	SEQ ID	NSYSGG	SEQ ID	WMDY
	NO. 233		NO. 234		NO. 235
F1.267.9	SEQ ID	GYTFTDF	SEQ ID	NPYNGG	SEQ ID	RMEYHAMD
	NO. 251		NO. 252		NO. 253	Y
F1.7.6	SEQ ID	GYDFTRY	SEQ ID	DPYNGD	SEQ ID	IYYDMEGYA
	NO. 269		NO. 270		NO. 271	LDY
F1.224.1	SEQ ID	GYTFTDF	SEQ ID	NPNNGG	SEQ ID	LGTSDYGEA
	NO. 287		NO. 288		NO. 289	WFIS
F1.250.16	SEQ ID	GYTFTTY	SEQ ID	HPYNDD	SEQ ID	GDYYNSRYW
	NO. 305		NO. 306		NO. 307	YFDV
F1.120.15	SEQ ID	GNAFSNS	SEQ ID	YSEDGD	SEQ ID	WLIYYGTYG
	NO. 323		NO. 324		NO. 325	AMDY
F1.62.10	SEQ ID	GYEFSSI	SEQ ID	FPGDGE	SEQ ID	WRIYYGTYG
	NO. 341		NO. 342		NO. 343	AMDY
F1.216.2	SEQ ID	GYAFSSS	SEQ ID	YPGDGD	SEQ ID	EGSYYSNPW
	NO. 359		NO. 360		NO. 361	YFDY
F1.280.1	SEQ ID	GYTFTTY	SEQ ID	NTDSGV	SEQ ID	GGPDY
	NO. 377		NO. 378		NO. 379
F1.200.11	SEQ ID	GYTFTTA	SEQ ID	NTLSGE	SEQ ID	AYYSNLYWY
	NO. 395		NO. 396		NO. 397	FDV
F1.192.1	SEQ ID	DFNIKED	SEQ ID	DPENGD	SEQ ID	YGSLDY
	NO. 413		NO. 414		NO. 415
F1.245.2	SEQ ID	GFNIKDY	SEQ ID	DPEDGE	SEQ ID	EAAGYFYRG
	NO. 431		NO. 432		NO. 433	SSYGYFDV
F1.60.9	SEQ ID	GLSLNNY	SEQ ID	WGDGS	SEQ ID	NWGDY
	NO. 449		NO. 450		NO. 451
F1.172.13	SEQ ID	GFSLSTSGM	SEQ ID	YWDDD	SEQ ID	APPPNWDEY
	NO. 467		NO. 468		NO. 469	YFDY
F1.17.1	SEQ ID	GFSLSRY	SEQ ID	WGGGS	SEQ ID	PHDFDAGGF
	NO. 485		NO. 486		NO. 487	AY
F2.151.13	SEQ ID	GYTFRDY	SEQ ID	LPGSGN	SEQ ID	VYSNWYFDA
	NO. 503		NO. 504		NO. 505
F2.70.2	SEQ ID	GFTFSDY	SEQ ID	SRGSHT	SEQ ID	MAGYYAMD
	NO. 521		NO. 522		NO. 523	Y
F2.104.10	SEQ ID	GFTFSDY	SEQ ID	SRGSHT	SEQ ID	MAGYYAMD
	NO. 539		NO. 540		NO. 541	Y
F2.180.16	SEQ ID	GFTFSDY	SEQ ID	SNTGYS	SEQ ID	HGILYAMDY
	NO. 557		NO. 558		NO. 559
F2.121.9	SEQ ID	GFTESDY	SEQ ID	SDAGSY	SEQ ID	TYFGNYGGY
	NO. 575		NO. 576		NO. 577
F2.173.9	SEQ ID	GFTFSNY	SEQ ID	SSIGSF	SEQ ID	LGVYFDS
	NO. 593		NO. 594		NO. 595
F2.343.16	SEQ ID	GFSLTGY	SEQ ID	WGGGG	SEQ ID	YSKYGHFDV
	NO. 611		NO. 612		NO. 613
	SEQ ID		SEQ ID		SEQ ID
	NO. 629		NO. 630		NO. 631
	SEQ ID		SEQ ID		SEQ ID
	NO. 647		NO. 648		NO. 649
	SEQ ID		SEQ ID		SEQ ID
	NO. 665		NO. 666		NO. 667
	SEQ ID		SEQ ID		SEQ ID
	NO. 683		NO. 684		NO. 685
	SEQ ID		SEQ ID		SEQ ID
	NO: 701		NO. 702		NO. 703
Antibody	Sequence		Sequence		Sequence
number	number	CDR1-LC	number	CDR2-LC	number	CDR3-LC
	SEQ ID		SEQ ID		SEQ ID
	NO. 98		NO. 99		NO 100
	SEQ ID		SEQ ID		SEQ ID
	NO. 116		NO. 117		NO. 118
	SEQ ID		SEQ ID		SEQ ID
	NO. 134		NO. 135		NO. 135
	SEQ ID		SEQ ID		SEQ ID
	NO. 152		NO. 153		NO. 154
	SEQ ID		SEQ ID		SEQ ID
	NO. 170		NO. 171		NO. 172
	SEQ ID		SEQ ID		SEQ ID
	NO. 188		NO. 189		NO. 190
	SEQ ID		SEQ ID		SEQ ID
	NO. 206		NO. 207		NO. 208
	SEQ ID		SEQ ID		SEQ ID
	NO. 224		NO. 225		NO. 226
	SEQ ID		SEQ ID		SEQ ID
	NO. 242		NO. 243		NO. 244
	SEQ ID		SEQ ID		SEQ ID
	NO. 260		NO. 261		NO. 262
	SEQ ID		SEQ ID		SEQ ID
	NO. 278		NO. 279		NO. 280
	SEQ ID		SEQ ID		SEQ ID
	NO. 296		NO. 297		NO. 298
	SEQ ID		SEQ ID		SEQ ID
	NO. 314		NO. 315		NO. 316
	SEQ ID		SEQ ID		SEQ ID
	NO. 332		NO. 333		NO. 334
	SEQ ID		SEQ ID		SEQ ID
	NO. 350		NO. 351		NO. 352
	SEQ ID		SEQ ID		SEQ ID
	NO. 368		NO. 369		NO. 370
	SEQ ID		SEQ ID		SEQ ID
	NO. 386		NO. 387		NO. 388
	SEQ ID		SEQ ID		SEQ ID
	NO. 404	AVA	NO. 405		NO. 406
F1.192.1	SEQ ID	KASQGINSF	SEQ ID	RANRLVD	SEQ ID	LQYDEFPRT
	NO. 422	LT	NO. 423		NO. 424
F1.245.2	SEQ ID	SASQDISNY	SEQ ID	YTSSLHS	SEQ ID	QQYSMLPYT
	NO. 440	LN	NO. 441		NO. 442
F1.60.9	SEQ ID	KASQDINSY	SEQ ID	RANRLID	SEQ ID	LQYGVFPLT
	NO. 458	LS	NO. 459		NO. 460
F1.172.13	SEQ ID	RASGNIHNS	SEQ ID	NAKTLAD	SEQ ID	QHFWSTPP
	NO. 476	LA	NO. 477		NO. 478
F1.17.1	SEQ ID	SASSRVNY	SEQ ID	DTSNLAS	SEQ ID	QQWTSYPLT
	NO. 494	MY	NO. 495		NO. 496
F2.151.13	SEQ ID	RSSTGAVTT	SEQ ID	GTNNQAP	SEQ ID	ALWFSNHWV
	NO. 512	SNYAN	NO. 513		NO. 514
F2.70.2	SEQ ID	TLSSQHSSY	SEQ ID	LKKDGSHSTGD	SEQ ID	GVGDTIKEQF
	NO. 530	IIE	NO. 531		NO. 532	VYV
F2.104.10	SEQ ID	TLSSQHSSY	SEQ ID	VKKDGSHSTGD	SEQ ID	GVGDTIKEQF
	NO. 548	IIE	NO. 549		NO. 550	VYV
F2.180.16	SEQ ID	TLSSQYSTY	SEQ ID	LKHDGSRHTGD	SEQ ID	GVGDTIKEQF
	NO. 566	TIG	NO. 567		NO. 568	MYV
F2.121.9	SEQ ID	RSNTGAVT	SEQ ID	GTSNRAP	SEQ ID	ALWYSTHWV
	NO. 584	TSNYAN	NO. 585		NO. 586
F2.173.9	SEQ ID	TLSSQHSTY	SEQ ID	IKKDGSHYTGD	SEQ ID	GVSDMIKDQ
	NO. 602	TIE	NO. 603		NO. 604	FVYV
F2.343.16	SEQ ID	RSSTGAVTT	SEQ ID	GTYNRVP	SEQ ID	ALWYSNHFW
	NO. 620	SNYAN	NO. 621		NO. 622	V
F2.205.9	SEQ ID	RSSTGAVTT	SEQ ID	GTNNRAP	SEQ ID	ALWYSNHFI
	NO. 638	RNYAN	NO. 639		NO. 640
F2.99.1	SEQ ID	TLSRQHSA	SEQ ID	VKKDGSHSTGD	SEQ ID	GVGDTIKEHF
	NO. 656	YTIE	NO. 657		NO. 658	V
F2.127.11	SEQ ID	TLSSQHSTY	SEQ ID	LKKDGSHSTGD	SEQ ID	GVSDTIKEQF
	NO. 674	TIE	NO. 675		NO. 676	VYV
F2.55.1	SEQ ID	TLSSQHSAY	SEQ ID	LKKDGSHSTGD	SEQ ID	GVGDTVKEQ
	NO. 692	TVE	NO. 693		NO. 694	FV
F2.42.9	SEQ ID	TLSSQHSAY	SEQ ID	LKKDGSHSTGD	SEQ ID	GVGDTIKEQ
	NO. 710	TIE	NO. 711		NO. 712	YV
indicates data missing or illegible when filed

The heavy chain constant region containing a signal peptide and murine antibody IgG1 (SEQ ID NO: 10)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWYSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDTAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK

The Kappa light chain constant region containing a signal peptide and human antibody IgG1 (SEQ ID NO: 11)

indicates data missing or illegible when filed

The Lambda light chain constant region containing a signal peptide and human antibody IgG1 (SEQ ID NO: 12)

indicates data missing or illegible when filed

EXAMPLE 5

Identification of CD22 Human-Murine Chimeric Antibodies

5.1 Binding of Chimeric Antibodies to CD22 Protein Detected By Enzyme-Linked Immunosorbent Assay (ELISA)

In order to detect the binding activity of CD22 human-murine chimeric antibodies to CD22 protein, the purified human CD22-ECD-His protein obtained in Example 2 was diluted with PBS to a final concentration of 2 μg/mL, and then added to 96-well ELISA plate at 100 μl/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 chimeric antibodies or negative control antibody was added at 50 μl/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 μl/well, and incubated at room temperature for 30 minutes, then a stop solution (1.0 N HCl) was added at 50 μl/well. An ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer) was used to read the OD450 nm value, and the ELISA results of the chimeric antibodies and human CD22-ECD protein are shown in FIG. 5 and Table 11. Table 11 shows that the purified antibodies are all can bind to human CD22-ECD at ELISA level. The negative control antibody hIgG1 was the antibody anti-hel-hIgG1 (purchased from Biointron, catalog number: B117901) against chicken egg lysozyme. The data in the table are OD450 nm values.

TABLE 11
Binding reaction of chimeric antibodies
with human CD22 protein detected by ELISA
	OD450
	Antibody concentration (nM)
Antibody	100.00	10.00	0.10	0.01	0.001	0.0001	0.00001	Blank control
F1.236.15	2.98	2.62	2.51	2.05	0.57	0.14	0.09	0.08
F1.214.8	2.95	2.50	2.34	2.14	0.81	0.18	0.08	0.06
F1.273.12	3.07	2.72	2.58	2.20	0.77	0.17	0.07	0.06
F1.231.15	2.64	2.44	2.42	2.14	1.08	0.27	0.13	0.06
F1.11.7	2.89	2.63	2.42	1.64	0.37	0.11	0.06	0.06
F1.77.9	2.84	2.64	2.58	2.12	0.69	0.19	0.11	0.06
F1.105.11	2.79	2.68	2.52	1.93	0.53	0.14	0.08	0.07
F1.267.9	2.56	2.55	2.39	1.47	0.34	0.10	0.06	0.07
F1.7.6	2.88	2.46	2.51	2.20	1.19	0.34	0.15	0.08
F1.224.1	3.12	2.43	2.37	2.21	1.08	0.34	0.17	0.07
F1.250.16	3.07	2.56	2.58	2.24	0.86	0.21	0.12	0.06
F1.120.15	2.89	2.61	2.63	2.28	1.21	0.42	0.12	0.06
F1.216.2	2.69	2.53	2.55	2.48	1.71	0.45	0.20	0.06
F1.280.1	2.46	2.30	2.38	1.92	0.81	0.24	0.13	0.06
F1.200.11	2.34	2.33	2.40	2.00	0.85	0.51	0.33	0.07
F1.192.1	2.71	2.57	2.52	1.79	0.46	0.10	0.06	0.07
F1.245.2	2.70	2.54	2.57	2.30	1.24	0.44	0.22	0.07
F1.60.9	2.82	2.55	2.59	1.75	0.47	0.13	0.09	0.08
F1.172.13	2.87	2.51	2.53	2.11	0.74	0.17	0.10	0.10
F1.17.1	2.86	2.21	2.41	1.85	0.75	0.20	0.11	0.10
F1.161.7	2.97	2.63	2.61	2.34	1.22	0.43	0.25	0.06
F1.257.3	2.83	2.64	2.49	1.66	0.60	0.28	0.20	0.07
F1.62.10	2.93	2.67	2.79	2.16	1.07	0.37	0.24	0.06
F2.70.2	2.24	2.03	1.86	1.56	0.57	0.18	0.09	0.07
F2.104.10	2.44	2.00	1.94	1.70	0.66	0.32	0.22	0.06
F2.180.16	2.61	2.08	1.80	1.55	0.61	0.31	0.21	0.06
F2.121.9	2.11	1.92	1.84	1.60	0.69	0.33	0.23	0.06
F2.173.9	2.32	1.97	1.93	1.52	0.65	0.32	0.18	0.06
F2.343.16	1.94	1.84	1.93	1.29	0.43	0.25	0.18	0.06
F2.205.9	2.22	2.11	2.10	1.48	0.40	0.24	0.19	0.06
F2.99.1	2.10	2.06	2.10	1.45	0.51	0.27	0.08	0.06
F2.127.11	1.92	1.98	1.91	1.34	0.50	0.26	0.21	0.06
F2.55.1	2.08	2.16	2.03	1.30	0.44	0.23	0.16	0.07
F2.42.9	3.14	2.68	2.58	1.95	0.68	0.19	0.09	0.08
F2.151.13	3.28	2.60	2.55	2.10	0.64	0.20	0.10	0.07
HA22	2.65	2.61	2.57	2.12	0.89	0.33	0.19	0.07
m971	2.63	2.48	2.58	2.12	0.83	0.17	0.08	0.06
hIgG1	0.15	0.08	0.06	0.05	0.06	0.06	0.05	0.06

5.2 The Binding of Chimeric Antibodies to Different CD22 Expressing Cells Detected by Flow Cytometry (FACS)

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 2×10⁶ cells/ml, and added to a 96-well FACS reaction plate at 50 μl/well, and then the chimeric antibody test sample was added at 50 μl/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 μl/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 Tables 12-13 and FIGS. 6A-6B. The chimeric antibodies all can bind to human CD22 protein on the surface of Raji cells and CHO-K1-human CD22 2C4 cells (FIGS. 6A-6B). The same method was used to detect the binding of the chimeric antibodies to endogenous CD22-negative cell MoLT4 cells (purchased from ATCC, CRL-1582) and CHO-K1 cells. The results are shown in FIGS. 6C-6D: FIGS. 6C-6D do not show the histograms of binding to MoLT4 cells and CHO-K1 cells. It can be seen that all the chimeric antibodies do not bind to MoLT4 cells and CHO-K1 cells, and have good specificity.

TABLE 12
Binding reaction of chimeric antibodies with Raji
and CHO-K1-human CD22 2C4 cells detected by FACS
	Raji	CHO-K1-human CD22 2C4
	Maximum		Maximum
	mean		mean
Designation	fluorescence	Ec50	fluorescence	Ec50
of antibody	intensity	(nM)	intensity	(nM)
F1.236.15	26025	0.34	30207	0.81
F1.214.8	24067	0.33	24120	0.81
F1.273.12	25310	0.38	28225	0.99
F1.231.15	28757	0.45	35404	1.18
F1.11.7	25492	0.42	29596	0.89
F1.77.9	24148	0.29	20158	0.71
F1.105.11	24407	1.39	28288	2.53
F1.267.9	26741	1.66	31189	2.08
F1.7.6	25861	0.23	29720	0.63
F1.224.1	23595	0.65	30372	0.99
F1.250.16	24999	0.16	37373	0.49
F1.120.15	23746	0.89	27851	1.07
F1.216.2	25288	0.16	31494	0.32
F1.280.1	25039	0.5	43819	0.92
F1.200.11	20844	2.41	23041	11.2
F1.192.1	24398	0.37	27170	0.81
F1.245.2	29927	0.07	43303	0.33
F1.60.9	25323	0.29	30139	0.61
F1.172.13	23765	0.17	33783	0.59
F1.17.1	28521	0.17	36915	0.44
F1.161.7	27853	0.22	37135	0.59
F1.257.3	29336	0.27	46611	0.68
F1.62.10	25601	1.1	29180	1.31
HA22	25015	0.29	32749	0.81
m971	13043	3.32	23659	2.45
hIgG1	2772	Negative	5221	Negative

TABLE 13
Binding reaction of chimeric antibodies with Raji
and CHO-K1-human CD22 2C4 cells detected by FACS
	Raji	CHO-K1-human CD22 2C4
	Maximum		Maximum
	mean		mean
Designation	fluorescence	Ec50	fluorescence	Ec50
of antibody	intensity	(nM)	intensity	(nM)
F2.70.2	16564	0.35	39021	0.76
F2.104.10	16258	0.4	40596	0.88
F2.180.16	15686	0.88	34374	1.63
F2.121.9	18917	0.86	39749	1.09
F2.173.9	18947	0.84	40264	1.23
F2.343.16	19332	0.8	50169	1.15
F2.205.9	15548	0.38	40245	0.72
F2.99.1	18104	0.59	44796	0.83
F2.127.11	24364	0.59	58514	1.22
F2.55.1	18574	0.41	42004	0.82
F2.42.9	16701	0.33	40953	0.87
F2.151.13	18763	1.26	36975	3.28
HA22	19668	0.71	43414	1.1
m971	8917	4.61	30114	2.86
hIgG1	564	Negative	150	Negative

EXAMPLE 6

Detection of Species Cross-Binding Activity of Chimeric Antibodies

6.1 Binding of Chimeric Antibodies to CD22 Proteins of Different Species Detected by ELISA

In order to detect the species cross-binding activity of the chimeric antibodies, an ELISA plate was coated with commercial murine CD22 protein (ACROBiosystems, catalog number: SI2-M52Ha) and monkey CD22 protein (ACROBiosystems, catalog number: SI2-R52Ha), respectively, and the ELISA detection was performed according to the method in Example 5.1. The ELISA results of the chimeric antibodies and the murine CD22-ECD are shown in FIG. 7 and Table 14. Table 14 shows that all the purified chimeric antibodies do not bind to murine CD22-ECD at the ELISA level. The negative control is hIgG1, and 983 is the serum of human CD22-ECD-His immunized mice as a positive control, and the data in the table are OD450 nm values.

TABLE 14
Binding reaction of chimeric antibodies with
murine CD22 protein detected by ELISA
	OD450
Designation of	Antibody concentration (nM)
antibody	100	10	0.1	0.01	0.001	0.0001	0.00001	Blank control
F1.236.15	0.07	0.08	0.07	0.07	0.07	0.07	0.08	0.08
F1.214.8	0.06	0.05	0.05	0.05	0.05	0.05	0.06	0.07
F1.273.12	0.06	0.05	0.05	0.05	0.05	0.05	0.05	0.07
F1.231.15	0.06	0.06	0.05	0.05	0.05	0.05	0.06	0.06
F1.11.7	0.07	0.05	0.05	0.06	0.05	0.05	0.06	0.07
F1.77.9	0.08	0.06	0.05	0.05	0.05	0.06	0.06	0.06
F1.105.11	0.08	0.05	0.05	0.05	0.05	0.05	0.05	0.06
F1.267.9	0.07	0.08	0.06	0.06	0.07	0.07	0.07	0.07
F1.7.6	0.07	0.06	0.06	0.06	0.06	0.06	0.06	0.07
F1.224.1	0.07	0.10	0.05	0.06	0.05	0.05	0.06	0.06
F1.250.16	0.06	0.06	0.05	0.05	0.05	0.05	0.06	0.06
F1.120.15	0.06	0.06	0.05	0.05	0.05	0.05	0.06	0.07
F1.216.2	0.08	0.06	0.05	0.05	0.05	0.06	0.06	0.07
F1.280.1	0.16	0.07	0.05	0.05	0.05	0.05	0.06	0.07
F1.200.11	0.07	0.06	0.05	0.05	0.05	0.05	0.06	0.07
F1.192.1	0.08	0.08	0.07	0.07	0.06	0.07	0.07	0.08
F1.245.2	0.09	0.07	0.06	0.06	0.06	0.07	0.06	0.07
F1.60.9	0.08	0.06	0.05	0.06	0.05	0.06	0.06	0.07
F1.172.13	0.08	0.06	0.05	0.05	0.05	0.06	0.06	0.07
F1.17.1	0.07	0.06	0.05	0.05	0.05	0.05	0.06	0.07
F1.161.7	0.08	0.06	0.05	0.05	0.05	0.05	0.06	0.07
F1.257.3	0.20	0.07	0.05	0.05	0.05	0.05	0.06	0.07
F1.62.10	0.08	0.06	0.05	0.05	0.05	0.06	0.07	0.07
F2.70.2	0.10	0.07	0.06	0.06	0.06	0.06	0.06	0.07
F2.104.10	0.07	0.07	0.06	0.07	0.06	0.06	0.05	0.07
F2.180.16	0.05	0.06	0.05	0.05	0.05	0.07	0.06	0.06
F2.121.9	0.06	0.06	0.05	0.05	0.06	0.06	0.06	0.07
F2.173.9	0.09	0.06	0.05	0.05	0.05	0.05	0.06	0.09
F2.343.16	0.07	0.06	0.05	0.05	0.05	0.05	0.06	0.07
F2.205.9	0.07	0.06	0.05	0.05	0.05	0.05	0.06	0.06
F2.99.1	0.07	0.06	0.05	0.06	0.05	0.05	0.06	0.07
F2.127.11	0.10	0.07	0.06	0.05	0.05	0.06	0.06	0.06
F2.55.1	0.09	0.06	0.06	0.05	0.05	0.06	0.06	0.06
F2.42.9	0.09	0.08	0.08	0.07	0.07	0.08	0.08	0.09
F2.151.13	0.09	0.08	0.07	0.07	0.07	0.07	0.07	0.07
983*	2.58	2.13	1.12	0.25	0.08	0.06	0.10	0.09
HA22	0.10	0.07	0.06	0.06	0.05	0.06	0.06	0.07
m971	0.11	0.08	0.07	0.07	0.06	0.07	0.07	0.08
hIgG1	0.16	0.09	0.08	0.08	0.08	0.07	0.08	0.08
*983 serum was 5-fold serially diluted from 1:100.

The ELISA results of the chimeric antibodies and the monkey CD22-ECD are shown in FIG. 8 and Table 15. Table 15 shows that F1.236.15, F1.11.7, F1.105.11, F1.267.9, F1.224.1, F1.250.16, F1.120.15, F1.216.2, F1.200.11, F1.192.1, F1.172.13, F1.17.1, F2.121.9, F2.173.9, F1.205.9, F2.99.1, F2.55.1, F2.42.9 and F2.151.13 have binding activity to the monkey CD22-ECD protein.

TABLE 15
Binding reaction of chimeric antibodies with
monkey CD22 protein detected by ELISA
	OD450
Designation of	Antibody concentration (nM)
antibody	100	10	0.1	0.01	0.001	0.0001	0.00001	Blank control
F1.236.15	1.03	0.81	0.49	0.23	0.08	0.06	0.06	0.07
F1.214.8	0.23	0.15	0.12	0.08	0.06	0.07	0.06	0.06
F1.273.12	0.23	0.22	0.09	0.07	0.06	0.06	0.06	0.06
F1.231.15	0.45	0.31	0.17	0.09	0.06	0.06	0.06	0.06
F1.11.7	1.82	1.72	1.72	0.86	0.21	0.08	0.06	0.06
F1.77.9	0.44	0.67	0.14	0.08	0.06	0.06	0.06	0.06
F1.105.11	1.29	1.40	1.31	0.68	0.17	0.07	0.06	0.06
F1.267.9	2.27	1.89	1.07	0.27	0.10	0.07	0.06	0.07
F1.7.6	0.44	0.17	0.07	0.06	0.06	0.06	0.06	0.06
F1.224.1	2.87	2.48	2.30	1.86	0.60	0.15	0.08	0.06
F1.250.16	3.08	2.97	2.81	2.62	1.02	0.23	0.10	0.07
F1.120.15	1.64	1.90	1.56	0.65	0.16	0.07	0.06	0.06
F1.216.2	1.94	2.15	2.24	2.04	0.91	0.21	0.09	0.06
F1.280.1	0.20	0.08	0.06	0.05	0.06	0.06	0.06	0.07
F1.200.11	1.25	1.23	0.62	0.19	0.08	0.06	0.06	0.06
F1.192.1	1.21	0.84	0.53	0.19	0.07	0.07	0.07	0.07
F1.245.2	0.08	0.06	0.06	0.06	0.06	0.06	0.06	0.07
F1.60.9	0.09	0.07	0.06	0.05	0.05	0.06	0.06	0.06
F1.172.13	3.21	2.93	2.78	2.47	0.82	0.18	0.09	0.07
F1.17.1	1.91	2.16	1.99	1.75	0.51	0.13	0.07	0.06
F1.161.7	0.34	0.13	0.06	0.05	0.05	0.05	0.06	0.06
F1.257.3	0.18	0.07	0.06	0.05	0.05	0.06	0.06	0.07
F1.62.10	0.36	0.18	0.07	0.05	0.05	0.06	0.06	0.06
F2.70.2	0.11	0.08	0.06	0.06	0.07	0.06	0.06	0.06
F2.104.10	0.07	0.07	0.05	0.06	0.05	0.05	0.05	0.06
F2.180.16	0.06	0.06	0.05	0.05	0.05	0.05	0.05	0.06
F2.121.9	0.72	0.56	0.34	0.13	0.06	0.05	0.05	0.06
F2.173.9	1.18	1.12	1.25	0.38	0.12	0.06	0.06	0.06
F2.343.16	0.08	0.06	0.05	0.05	0.05	0.05	0.05	0.06
F2.205.9	2.33	2.28	2.21	1.78	0.63	0.16	0.08	0.06
F2.99.1	1.37	1.03	0.27	0.07	0.05	0.05	0.05	0.06
F2.127.11	0.39	0.09	0.05	0.05	0.05	0.05	0.05	0.06
F2.55.1	1.91	2.00	1.73	0.85	0.18	0.07	0.06	0.08
F2.42.9	2.42	1.65	0.95	0.25	0.09	0.07	0.07	0.08
F2.151.13	2.77	1.89	1.60	0.94	0.27	0.10	0.07	0.07
HA22	2.20	2.41	2.24	1.54	0.47	0.14	0.08	0.06
m971	0.12	0.09	0.07	0.07	0.06	0.07	0.07	0.08
hIgG1	0.13	0.10	0.07	0.07	0.07	0.07	0.07	0.07

6.2 The Binding of Chimeric Antibodies to Monkey CD22 Expressing Cells Detected by FACS

HEK293T-monkey CD22 cells were subjected to FACS detection and data analysis according to the method in Example 5.2. The analysis results are shown in Tables 16-17 and FIG. 9A, except for F1.280.1, F1.245.2, F1.60.9, F1.257.3, F2.70.2, F2.104.10, F2.180.16, F2.343.16, F2.99.1, F2.127.11 and F2.42.9, other chimeric antibodies have binding activity to HEK293T cells overexpressing monkey CD22, and the EC50 shows that the highest binding activity is up to 0.26 nM. The same method was used to detect the binding of 1 nm and 10 nm of the chimeric antibodies to HEK293T cells at the same time, and the results are shown in FIG. 9B: FIG. 9B does not show the histograms of binding to HEK293T cells. It can be seen that all the chimeric antibodies do not bind to HEK293T cells, and have good specificity.

TABLE 16
Binding reaction of chimeric antibodies with
HEK293T-monkey CD22 cells detected by FACS
	HEK293T-monkey CD22
		Maximum mean
	Designation of	fluorescence	Ec50
	antibody	intensity	(nM)
	F1.236.15	6754	2.47
	F1.214.8	12120	0.55
	F1.273.12	9998	0.82
	F1.231.15	9254	12.34
	F1.11.7	16077	0.63
	F1.77.9	10879	0.71
	F1.105.11	7528	1.3
	F1.267.9	15314	1.15
	F1.7.6	9223	2.22
	F1.224.1	16267	0.79
	F1.250.16	19655	0.31
	F1.120.15	13565	0.95
	F1.216.2	13880	0.26
	F1.280.1	1488	Negative
	F1.200.11	16187	4.32
	F1.192.1	16286	0.62
	F1.245.2	124	Negative
	F1.60.9	531	Negative
	F1.172.13	19039	0.47
	F1.17.1	17192	0.38
	F1.161.7	6027	31.2
	F1.257.3	147	Negative
	F1.62.10	4326	41.08
	HA22	17054	0.91
	m971	49	Negative
	hIgG1	129	Negative

TABLE 17
Binding reaction of chimeric antibodies with
HEK293T-monkey CD22 cells detected by FACS
	HEK293T-monkey
		Maximum mean
	Designation of	fluorescence	Ec50
	antibody	intensity	(nM)
	F2.70.2	409	Negative
	F2.104.10	251	Negative
	F2.180.16	69	Negative
	F2.121.9	19947	0.97
	F2.173.9	22679	2.1
	F2.343.16	83	Negative
	F2.205.9	25370	0.4
	F2.99.1	144	Negative
	F2.127.11	109	Negative
	F2.55.1	5765	34.7
	F2.42.9	580	Negative
	F2.151.13	30502	1.26
	HA22	31169	1.59
	m971	104	Negative
	hIgG1	167	Negative

6.3 Binding of Chimeric Antibodies to Peripheral Blood B Cells of Cynomolgus Monkey (Latin Name: Macaca fascicularis) Detected by FACS

The monkey peripheral blood mononuclear cells were extracted from fresh cynomolgus monkey peripheral blood (purchased from Shanghai Medicilon Inc.) 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 chimeric antibodies to be tested (1 nM, 10 nM and 100 nM) were added and incubated at room temperature for 1 hour. 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 chimeric antibody positive cells was analyzed, and the proportion of chimeric antibody positive cell population to B cell population was calculated after treatments with the chimeric antibodies at the concentrations of 100 nM, 10 nM and 1 nM, respectively. The results are shown in Table 18. The scatter plot of double-stained cells by Brilliant Violet 605-labeled CD20 and APC secondary antibody indirectly labeled chimeric antibody is shown in FIG. 10A-FIG. 10B (under the condition of 1 nM of chimeric antibody concentration). It can be seen from the results that F1.11.7, F1.77.9, F1.105.11, F1.267.9, F1.224.1, F1.250.16, F1.120.15, F1.17.1, F2.121.9 and F2.151.13 still bind to B cells of cynomolgus monkeys in a high proportion even under the condition of low concentration of 1 nM, and has equivalent or better binding activity than positive antibodies HA22 and hL22.

TABLE 18
Binding reaction of chimeric antibodies with
cynomolgus monkey B cells detected by FACS
Proportion of chimeric antibody positive cells to B cells (%)
	Designation of	Antibody concentration
	antibody	100 nM	10 nM	1 nM
	F1.236.15	22	17	12
	F1.214.8	52	35	24
	F1.273.12	45	33	26
	F1.231.15	29	16	11
	F1.11.7	91	84	74
	F1.77.9	61	39	27
	F1.105.11	69	59	46
	F1.267.9	95	91	39
	F1.7.6	43	28	17
	F1.224.1	97	96	83
	F1.250.16	97	95	92
	F1.120.15	93	86	27
	F1.216.2	34	25	22
	F1.280.1	35	18	19
	F1.200.11	56	37	21
	F1.192.1	38	30	22
	F1.245.2	17	17	16
	F1.60.9	18	16	16
	F1.172.13	34	31	30
	F1.17.1	68	62	60
	F1.161.7	29	21	16
	F1.257.3	34	20	17
	F1.62.10	24	19	17
	F2.70.2	34	20	20
	F2.104.10	26	21	19
	F2.180.16	23	20	16
	F2.121.9	64	56	27
	F2.173.9	71	43	19
	F2.343.16	22	21	21
	F2.205.9	46	41	40
	F2.99.1	22	16	19
	F2.127.11	22	21	20
	F2.55.1	66	28	22
	F2.42.9	23	20	20
	F2.151.13	60	48	38
	HA22	93	89	33
	hL22	66	42	30
	hIgG1	10	8	10

EXAMPLE 7

CD22 Antibody Affinity Assay

7.1 Affinity Assay of Chimeric Antibodies to Human CD22-ECD-His Protein

Anti-human CD22 chimeric antibodies were captured using Protein A chips (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 protein in solution for 240 s with a flow rate of 30 μL/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 μL/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 chimeric antibodies to human CD22-His protein are shown in Table 19, and the antibodies HA22 and m971 were used as positive l controls. As shown in Table 19, the highest affinity of the chimeric antibodies to human CD22 can reach 2.54E-10M.

TABLE 19
Affinity of chimeric antibodies to human
CD22 detected by SPR (biacore)
	Designation of	ka	kd	KD
	antibody	(1/Ms)	(1/s)	(M)
	F1.11.7	2.27E+05	7.80E−05	3.43E−10
	F1.105.11	2.99E+05	1.54E−04	5.14E−10
	F1.236.15	2.62E+05	1.56E−04	5.96E−10
	F1.257.3	2.70E+05	1.13E−04	4.17E−10
	F1.60.9	3.01E+05	7.64E−05	2.54E−10
	F1.245.2	2.60E+06	1.19E−03	4.58E−10
	F1.17.1	2.41E+05	4.98E−04	2.07E−09
	F1.77.9	1.90E+05	3.59E−04	1.89E−09
	F1.216.2	3.73E+04	9.44E−04	2.53E−08
	F1.161.7	2.01E+05	1.18E−03	5.88E−09
	F1.280.1	6.29E+04	4.23E−04	6.73E−09
	F1.267.9	5.44E+03	2.23E−04	4.09E−08
	F1.7.6	1.20E+05	2.03E−04	1.70E−09
	F1.62.10	1.36E+04	3.99E−04	2.93E−08
	F1.192.1	2.25E+05	2.44E−04	1.08E−09
	F1.214.8	1.51E+05	2.42E−03	1.60E−08
	F1.224.1	1.94E+04	2.67E−04	1.38E−08
	F1.273.12	1.21E+05	3.75E−04	3.10E−09
	F1.231.15	5.45E+04	2.44E−03	4.48E−08
	F1.250.16	4.22E+05	6.07E−04	1.44E−09
	F1.120.15	1.19E+04	2.32E−04	1.95E−08
	F1.172.13	3.06E+05	5.39E−03	1.76E−08
	F1.200.11	9.98E+03	1.02E−03	1.02E−07
	F2.205.9	2.46E+05	3.86E−04	1.57E−09
	F2.99.1	1.05E+05	1.62E−04	1.55E−09
	F2.70.2	6.95E+04	7.44E−04	1.07E−08
	F2.127.11	2.26E+05	6.60E−04	2.93E−09
	F2.55.1	1.27E+05	7.42E−05	5.86E−10
	F2.104.10	7.14E+04	6.10E−04	8.54E−09
	F2.42.9	6.61E+04	6.63E−04	1.00E−08
	F2.180.16	8.45E+03	1.55E−04	1.84E−08
	F2.173.9	4.90E+04	2.32E−04	4.74E−09
	F2.121.9	4.91E+03	3.93E−04	8.00E−08
	F2.343.16	2.04E+04	1.01E−01	4.97E−06
	F2.151.13	2.35E+04	5.33E−04	2.27E−08
	HA22	6.74E+04	8.96E−05	1.33E−09
	m971	1.87E+05	1.31E−02	7.02E−08

7.2 Affinity Assay of Chimeric Antibodies to Rhesus Monkey CD22-ECD-His Protein

According to the method in Example 7.1, the affinity of the chimeric antibodies to rhesus monkey CD22 (ACROBiosystems, catalog number: S12-R52Ha) protein was determined, and the results are shown in Table 20. The highest affinity of the chimeric antibodies to rhesus monkey CD22 can reach 2.04E-09M.

TABLE 20
Affinity of chimeric antibodies to rhesus
monkey CD22 detected by SPR (biacore)
	Designation	ka	kd	KD
	of antibody	(1/Ms)	(1/s)	(M)
	F1.11.7	5.94E+04	1.11E−03	1.87E−08
	F1.105.11	2.59E+04	4.48E−04	1.73E−08
	F1.17.1	1.44E+05	5.31E−04	3.67E−09
	F1.216.2	7.49E+04	5.38E−04	7.18E−09
	F1.267.9	2.67E+04	6.00E−05	2.25E−09
	F1.192.1	6.92E+04	1.60E−03	2.31E−08
	F1.224.1	5.23E+04	1.07E−04	2.04E−09
	F1.273.12	There is binding/the fit is poor
	F1.231.15	There is binding/the fit is poor
	F1.250.16	3.66E+05	1.27E−03	3.48E−09
	F1.120.15	4.21E+04	6.37E−04	1.51E−08
	F1.172.13	2.00E+05	1.38E−03	6.90E−09
	F1.200.11	1.35E+04	5.96E−04	4.42E−08
	F2.205.9	1.35E+05	1.40E−03	1.03E−08
	F2.99.1	3.76E+04	5.45E−04	1.45E−08
	F2.55.1	There is binding/the fit is poor
	F2.42.9	There is binding/the fit is poor
	F2.173.9	1.89E+04	8.83E−04	4.67E−08
	F2.121.9	There is binding/the fit is poor
	F2.151.13	1.92E+04	5.28E−04	2.75E−08
	HA22	6.86E+04	2.34E−04	3.41E−09
	Remark: “Poor fit” indicates that EC50 value could not be calculated.

EXAMPLE 8

Identification Of Antigen-Binding Region of Antibody

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 domain2-3, and the antigen-binding epitope of m971 is located in domain5-7. In order to identify the antigen-binding epitope distribution of the chimeric antibodies, according to the ELISA method in Example 5.1, 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 chimeric antibodies were classified into the type of distal end of membrane and the type of proximal end of membrane, as shown in FIGS. 11A-11B and Table 21, the antibodies can be divided into three categories: the first category does not bind to CD22 domain5-7, has equivalent binding activity to CD22 domain1-4 and CD22 full-length ECD and has a binding epitope located in domain1-4, such as F1.231.15; the second category does not bind to CD22 domain1-4, has equivalent binding activity to CD22 domain5-7 and CD22 full-length ECD and has a binding epitope located in domain5-7, such as F1.236.15; the third category does not bind to CD22 domain5-7 and has lower binding activity to CD22 domain1-4 than CD22 full-length ECD, such as F1.250.16, F1.172.13 and F1.62.10.

TABLE 21
Classification of the chimeric antibodies by ELISA
method according to epitopes at the distal end of
membrane and epitopes at the proximal end of membrane
	Designation	Binding region
	of antibody	Domain 1-4	Domain5-7
	F1.236.15	−	+
	F1.214.8	−	+
	F1.273.12	−	+
	F1.231.15	+	−
	F1.11.7	−	+
	F1.77.9	−	+
	F1.105.11	−	+
	F1.267.9	+	−
	F1.7.6	+	−
	F1.224.1	+	−
	F1.250.16	Weak binding	−
	F1.120.15	+	−
	F1.216.2	+	−
	F1.280.1	+	−
	F1.200.11	−	+
	F1.192.1	−	+
	F1.245.2	+	−
	F1.60.9	−	+
	F1.172.13	Weak binding	−
	F1.17.1	+	−
	F1.161.7	+	−
	F1.257.3	+	−
	F1.62.10	Weak binding	−
	F2.70.2	+	−
	F2.104.10	+	−
	F2.180.16	−	+
	F2.121.9	+	−
	F2.173.9	+	−
	F2.343.16	+	−
	F2.205.9	+	−
	F2.99.1	+	−
	F2.127.11	+	−
	F2.55.1	+	−
	F2.42.9	+	−
	F2.151.13	−	+
	HA22	+	−
	m971	−	+
	“+” indicates that there is binding in this region.
	“−” indicates that there is no binding in this region.

Claims

1. An isolated antibody or an antigen-binding fragment that specifically binds to human CD22, characterized in that, the antibody or the antigen-binding fragment comprises a combination of heavy chain CDRs and a combination of light chain CDRs: (1) the combination of the heavy chain CDRs comprises: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH 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:

2. The antibody or the antigen-binding fragment of claim 1, characterized in that, the antibody or the antigen-binding fragment comprises a combination of a heavy chain CDR and a light chain CDR selected from: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6, VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17+VL17, VH18+VL18, VH19+VL19, VH20+VL20, VH21+VL21, VH22+VL22, VH23+VL23, VH24+VL24, VH25+VL25, VH26+VL26, VH27+VL27, VH28+VL28, VH29+VL29, VH30+VL30, VH31+VL31, VH32+VL32, VH33+VL33, VH34+VL34, VH35+VL35, VH36+VL36, VH37+VL37, VH38+VL38, VH39+VL39, VH40+VL40, VH41+VL41, VH42+VL42, VH43+VL43, VH44+VL44, VH45+VL45, VH46+VL46, VH47+VL47, VH48+VL48, VH49+VL49, VH50+VL50, VH51+VL5 1, VH52+VL52, VH53+VL53, VH54+VL54, VH55+VL55, VH56+VL56, VH57+VL57, VH58+VL58, VH59+VL59, VH60+VL60, VH61+VL61, VH62+VL62, VH63+VL63, VH64+VL64, VH65+VL65, VH66+VL66, VH67+VL67, VH68+VL68, VH69+VL69, VH70+VL70, VH71+VL71, VH72+VL72, VH73+VL73, VH74+VL74, VH75+VL75, VH76+VL76, VH77+VL77, VH78+VL78, VH79+VL79, VH80+VL80, VH81+VL81, VH82+VL82, VH83+VL83, VH84+VL84, VH85+VL85, VH86+VL86, VH87+VL87, VH88+VL88, VH89+VL89, VH90+VL90, VH91+VL91, VH92+VL92, VH93+VL93, VH94+VU4, VH95+VL95, VH96+VL96, VH97+VL97, VH98+VL98, VH99+VL99, VH100+VL100, VH101+VL101, VH102+VL102, VH103+VL103, VH104+VL104, or VH105+VL105, and a combination of CDRs with insertions, deletions and/or substitutions of 1, 2, 3 or more amino acids compared to the sequence of the heavy chain CDR and the light chain CDR in these combinations.

3. The antibody or the antigen-binding fragment of claim 1, characterized in that, the antibody or the antigen-binding fragment comprises: (1) a heavy chain variable region having a sequence as shown in SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81; a light chain variable region having a sequence as shown in SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 82;(2) an amino acid sequence having at least 90% identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in (1) above; or, (3) a framework region of the antibody or the antigen-binding fragment having at least 90% identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the framework region of the amino acid sequence as shown in (1) above.

4. The antibody or the antigen-binding fragment of claim 1, characterized in that, the dissociation constant (1(D) of the antibody or the antigen-binding fragment binding to human CD22 is no more than 10−6 M, and the dissociation constant (KD) of the antibody or the antigen-binding fragment binding to rhesus monkey CD22 (KD) is no more than 10−8 M; or, optionally, the antibody or the antigen-binding fragment binds or does not bind to monkey CD22;optionally, the antibody or the antigen-binding fragment binds to or does not bind to murine CD22.

5. The antibody or the antigen-binding fragment of claim 1, characterized in that, the antibody or the antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof;(2) a humanized antibody or a fragment thereof;(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.

6. The antibody or the antigen-binding fragment of claim 1, characterized in that, the antibody 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.

7. The antibody or the antigen-binding fragment of claim 1, characterized in that, the antigen-binding fragment is selected from one or more of F(ab)2, Fab′, Fab, Fv, scFv, a bispecific antibody, a nanobody and an antibody minimum recognition unit.

8. The antibody or the antigen-binding fragment of claim 1, characterized in that, the antibody 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.

9. A multispecific antigen-binding molecule, characterized in that, the multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module, the first antigen-binding module comprises the antibody 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.

10. A chimeric antigen receptor (CAR), characterized in that, 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 CD22 antibody or the antigen-binding fragment of claim 1.

11. An immune effector cell, characterized in that, the immune effector cell comprises the chimeric antigen receptor of claim 10 or comprises a nucleic acid fragment encoding the chimeric antigen receptor of claim 10; 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 may 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.

12. An isolated nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the antibody or the antigen-binding fragment of claim 1.

13. An expression vector, characterized in that the expression vector comprises the isolated nucleic acid molecule of claim 12.

14. An isolated host cell, characterized in that the isolated host cell comprises the isolated nucleic acid molecule of claim 12; 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 an HEK293E cell or a CHO cell.

15. A method for preparing the antibody or the antigen-binding fragment of claim 1, characterized in that, a host cell is cultured or cultured under appropriate conditions, and the antibody or the antigen-binding fragment is isolated, and wherein the host cell is an isolated host cell comprising an isolated nucleic acid molecule, and the nucleic acid molecule encodes the antibody or the antigen-binding fragment.

16. A method for preparing an immune effector cell, characterized in that, the method comprises introducing a nucleic acid fragment encoding the CAR of claim 10 into the immune effector cell, optionally, the method further comprises enabling the immune effector cell to express the CAR.

17. A pharmaceutical composition, wherein the composition comprises 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.

18. (canceled)

19. A method for preventing and/or treating a B-cell disease, characterized in that, 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.

20. (canceled)

21. A kit, characterized in that the kit comprises the antibody or the antigen-binding fragment thereof of claim 1; optionally, the kit further comprises an instruction for use.