Anti-CD22 single-domain antibody and therapeutic construct

Abstract

Provided herein are anti-CD22 single domain antibodies (sdAbs) prepared by immunizing llamas with the extracellular domain of the predominant human CD22 isoform. By constructing a library of generated heavy chain repertoires, VHH antibody domains specific for the immunogen were isolated. The 27 exemplary antibodies initially produced include CDR1, CDR2, and CDR3 sequences corresponding to SEQ ID NOs: 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, 22-24, 25-27, 28-30, 31-33, 34-36, 37-39, 40-42, 43-45, 46-48, 49-51, 52-54, 55-57, 58-60, 61-63, 64-66, 67-69, 70-72, 73-75, 75-78, and 79-81, respectively; and related sequences. Also provided are multivalent antibodies comprising any one of the sdAbs, such as bispecific T cell engagers, bispecific killer cell engagers (BiKE), trispecific killer cell engagers (TriKE), etc. Furthermore, chimeric antigen receptors (CARs) for CAR-T therapy comprising any one of the aforementioned sdAbs are described. The use of these molecules in the treatment of cancer is also described.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 058,203, filed on 29 July 2020, entitled “ANTI-CD22 SINGLE DOMAIN ANTIBODIES AND THERAPEUTIC CONSTRUCTS,” the contents of which are incorporated herein by reference.

[0002] field This disclosure generally relates to anti-CD22 antibodies. More specifically, this disclosure relates to anti-CD22 single-domain antibodies. [Background technology]

[0003] background Cancer is a major public health problem and the second leading cause of death worldwide. Traditional cancer treatments include surgery, radiation, and chemotherapy. These have achieved some success in treating some cancers, especially those diagnosed early. However, there are no effective treatments for many highly invasive cancers. Recent technological advancements suggest that immunotherapy (stimulating or restoring the patient's own immune system to fight cancer) may provide powerful and long-lasting responses to many cancers, including invasive cancers that are difficult to treat.

[0004] Immunotherapy has achieved remarkable success in treating hematological cancers, leading to regulatory approval for many forms of these therapies; therapeutic antibodies (i.e., multiple approved monoclonal antibodies targeting CD20, CD30, CD33, and CD52), antibody-drug conjugates targeting CD22 and CD33, and multivalent antibodies such as bispecific T-cell engagers (blinatumomab targeting CD19 on B cells).

[0005] Such antibodies have also been used in therapeutic constructs.

[0006] For example, bispecific T cell engagers, as well as bispecific and trispecific killer cell engagers (BiKE and TriKE) incorporating single-chain variable fragments (scFv), have been developed to induce the host immune system to target cancer cells.

[0007] Chimeric antigen receptor (CAR) constructs have been created to integrate multiple facets of T cell activation into a single protein. These molecules link an extracellular antigen-recognition domain to an intracellular signaling domain, activating T cells when an antigen binds to them.

[0008] Chimeric antigen receptor (CAR) modified immunotherapy is a newly emerging form of cancer immunotherapy that combines one or more antigen-binding domains of an antibody that specifically targets cell surface proteins of cancer cells with an immune cell activation domain to create "armored" immune cells that seek out and kill specific cells possessing the target antigen. CAR-T therapy has yielded unprecedented responses in patients suffering from incurable and highly invasive forms of B-cell leukemia and lymphoma, leading to FDA and Health Canada approvals for CD19-targeted CAR-T cell products, such as tisagenlecleucel (Kymriah®) and axicabtagene ciloleucel (Yescarta®); these products are used to treat pediatric and young adult patients with relapsed or refractory B-cell ALL, and adult patients with relapsed or refractory large B-cell lymphoma, including diffuse large B-cell lymphoma unspecified (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma. While these CD19-targeted CAR therapies have significant clinical value, responses are not always long-lasting, and relapse after CAR-T therapy remains a problem, with approximately one-third of CAR-T relapse cases showing a clear disappearance of the CD19 antigen on leukemia cells. The development of new CAR receptors targeting leukemia antigens other than CD19 is an actively researched area, and similar B-cell-specific antigens such as CD22 and CD20 are highly anticipated as alternative targets for CAR-T therapy. Furthermore, molecular optimization of the signaling characteristics of CD22-targeted CAR-T receptors is likely to lead to improved treatment response rates, and the use of multiple CAR therapies targeting different B-cell-specific antigens has been shown to be an effective strategy for improving treatment outcomes.

[0009] For example, patients with relapsed and chemotherapy-resistant B-cell malignancies (a group of diseases including various forms of leukemia and lymphoma) are currently candidates for CD19-targeted CAR-T therapy. While response statistics vary depending on the specific clinical application of CAR-T therapy, on average, more than half of adult patients experience relapse after CD19-targeted treatment.

[0010] Therefore, there is a need to provide immune response-inducing molecules that have affinity for cancer-related therapeutic targets. [Overview of the project]

[0011] overview The purpose of this disclosure is to avoid or mitigate at least one drawback of previous approaches.

[0012] In one embodiment, an isolated single-domain antibody (sdAb) that specifically binds to human CD22 is provided, the sdAb comprising the following sequence: a) CDR1 amino acid sequence GX1X2X3DX4YX5 (SEQ ID NO: 126), where, X1 is either F or V. X2 is either T or S, X3 is L, F, or S. X4 is Y, D, or S. X5 is either V or A; CDR2 amino acid sequence X6X7X8X9X 10 GX 11 T (SEQ ID NO: 127), here, X6 is either I or M. X7 is T, R, G, or S. X8 is either S or N. X9 is either D or S. X 10 It is either D or does not exist. X 11 is V or A; and CDR3 amino acid sequence AVDKPFYDGGX 12 X13 YTCPVDFX 14 S (SEQ ID NO: 128), where X 12 is I, N, Y, or S, X 13 is Q, Y, R, or L, X 14 is D or G; b) The CDR1 amino acid sequence GSX1FX2X3X4X5V (SEQ ID NO: 129), where X1 is I or T, X2 is R or absent, X3 is R, S, or I, X4 is I, S, or T, X5 is A, S, or T; The CDR2 amino acid sequence ITSX6GX7X8 (SEQ ID NO: 130), where X6 is G, S, or A, X7 is E, D, or S, X8 is T or S; and The CDR3 amino acid sequence NAX9X 10 GX 11 X 12 X 13 X 14 (SEQ ID NO: 131), where X9 is K or Q, X 10 is W or Y, X 11 is Q, R, or G, X 12 is Y, D, or R, X 13 is E or S, X 14 is Y, D, or H; c) The CDR1 amino acid sequence GRIX1RSYV (SEQ ID NO: 132), where X1 is F or S; The CDR2 amino acid sequence is IGX2SDT (SEQ ID NO: 133), where, X2 is W or C; and The CDR3 amino acid sequence is AX3X4SPPYGPQRDEFX5Y (SEQ ID NO: 134), where, X3 is either A or E. X4 is either N or Y. X5 is either G or D; d) CDR1 amino acid sequence GX1TX2SVYX3 (SEQ ID NO: 135), where, X1 is either R or G. X2 is either S or F. X3 is either G or T; The CDR2 amino acid sequence is X4X5X6SX7GX8T (SEQ ID NO: 136), where, X4 is M or I, X5 is either S or R. X6 is either W or G. X7 is either G or does not exist. X8 is P or G; and The CDR3 amino acid sequence is AVRIRRTLX9EPLTKETLYDY (SEQ ID NO: 137), where, X9 is L or V; e) CDR1 amino acid sequence as described in SEQ ID NO: 49, The CDR2 amino acid sequence described in SEQ ID NO: 50, and CDR3 amino acid sequence as described in SEQ ID NO: 51; f) CDR1 amino acid sequence as described in SEQ ID NO: 4, The CDR2 amino acid sequence described in SEQ ID NO: 5, and CDR3 amino acid sequence as described in SEQ ID NO: 6; g) CDR1 amino acid sequence as described in SEQ ID NO: 7, The CDR2 amino acid sequence described in SEQ ID NO: 8, and CDR3 amino acid sequence as described in SEQ ID NO: 9; h) CDR1 amino acid sequence as described in SEQ ID NO: 13, The CDR2 amino acid sequence described in SEQ ID NO: 14, and CDR3 amino acid sequence as described in SEQ ID NO: 15; i) CDR1 amino acid sequence described in SEQ ID NO: 16, The CDR2 amino acid sequence described in SEQ ID NO: 17, and CDR3 amino acid sequence as described in SEQ ID NO: 18; j) CDR1 amino acid sequence described in SEQ ID NO: 40, The CDR2 amino acid sequence described in SEQ ID NO: 41, and CDR3 amino acid sequence as described in SEQ ID NO: 42; k) CDR1 amino acid sequence described in SEQ ID NO: 55, The CDR2 amino acid sequence described in SEQ ID NO: 56, and CDR3 amino acid sequence as described in SEQ ID NO: 57; l) CDR1 amino acid sequence as described in SEQ ID NO: 64, The CDR2 amino acid sequence described in SEQ ID NO: 65, and CDR3 amino acid sequence as described in SEQ ID NO: 66; or m) CDR1 amino acid sequence described in SEQ ID NO: 70, The CDR2 amino acid sequence described in SEQ ID NO: 71, and CDR3 amino acid sequence as described in SEQ ID NO: 72.

[0013] In the above: Group a) provides consensus sequences defined herein by antibodies referred to as hCD221ug-80, hCD22100ng-2, hCD221ug-74, hCD22100ng-66, hCD221ug-6, hCD22pas-10, and hCD22pass-33; Group b) provides consensus sequences defined by antibodies referred to herein as hCD221ug-77, hCD221ug-87, hCD221ug-75, hCD221ug-93, hCD22pas-82, and hCD22pas-23; Group c) provides consensus sequences defined by antibodies referred to herein as hCD22pas-32, hCD221ug-14, and hCD22pas-55; Group d) provides consensus sequences defined by antibodies referred to herein as hCD22pas-79 and hCD22pas-72; Group f) is defined by the CDR from the antibody called hCD22pas-16; Group f) is defined by the CDR from the antibody called hCD221ug-10; Group g) is defined by the CDR from the antibody called hCD221ug-13; Group h) is defined by the CDR from the antibody called hCD221ug-36; Group i) is defined by the CDR from the antibody called hCD221ug-61; Group j) is defined by the CDR from the antibody called hCD22100ug-62; Group k) is defined by the CDR from an antibody called hCD22pas-24; Group l) is defined by CDR from an antibody called hCD22pas-48; and Group m) is defined by the CDR from the antibody called hCD22pas-64.

[0014] In another embodiment, an isolated VHH single-domain antibody (sdAb) that specifically binds to human CD22 is provided, the sdAb comprising the following sequence: A) The CDR1 amino acid sequence described in SEQ ID NO: 1, the CDR2 amino acid sequence described in SEQ ID NO: 2, and the CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6); The CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10); The CDR1 amino acid sequence described in SEQ ID NO: 7, the CDR2 amino acid sequence described in SEQ ID NO: 8, and the CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13); The CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14); The CDR1 amino acid sequence described in SEQ ID NO: 13, the CDR2 amino acid sequence described in SEQ ID NO: 14, and the CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36); The CDR1 amino acid sequence described in SEQ ID NO: 16, the CDR2 amino acid sequence described in SEQ ID NO: 17, and the CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61); The CDR1 amino acid sequence described in SEQ ID NO: 19, the CDR2 amino acid sequence described in SEQ ID NO: 20, and the CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74); The CDR1 amino acid sequence described in SEQ ID NO: 22, the CDR2 amino acid sequence described in SEQ ID NO: 23, and the CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75); The CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77); The CDR1 amino acid sequence described in SEQ ID NO: 28, the CDR2 amino acid sequence described in SEQ ID NO: 29, and the CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80); The CDR1 amino acid sequence described in SEQ ID NO: 31, the CDR2 amino acid sequence described in SEQ ID NO: 32, and the CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87); The CDR1 amino acid sequence described in SEQ ID NO: 34, the CDR2 amino acid sequence described in SEQ ID NO: 35, and the CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93); The CDR1 amino acid sequence described in SEQ ID NO: 37, the CDR2 amino acid sequence described in SEQ ID NO: 38, and the CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2); The CDR1 amino acid sequence described in SEQ ID NO: 40, the CDR2 amino acid sequence described in SEQ ID NO: 41, and the CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62); The CDR1 amino acid sequence described in SEQ ID NO: 43, the CDR2 amino acid sequence described in SEQ ID NO: 44, and the CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66); The CDR1 amino acid sequence described in SEQ ID NO: 46, the CDR2 amino acid sequence described in SEQ ID NO: 47, and the CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10); The CDR1 amino acid sequence described in SEQ ID NO: 49, the CDR2 amino acid sequence described in SEQ ID NO: 50, and the CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16); The CDR1 amino acid sequence described in SEQ ID NO: 52, the CDR2 amino acid sequence described in SEQ ID NO: 53, and the CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23); The CDR1 amino acid sequence described in SEQ ID NO: 55, the CDR2 amino acid sequence described in SEQ ID NO: 56, and the CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24); The CDR1 amino acid sequence described in SEQ ID NO: 58, the CDR2 amino acid sequence described in SEQ ID NO: 59, and the CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32); The CDR1 amino acid sequence described in SEQ ID NO: 61, the CDR2 amino acid sequence described in SEQ ID NO: 62, and the CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33); The CDR1 amino acid sequence described in SEQ ID NO: 64, the CDR2 amino acid sequence described in SEQ ID NO: 65, and the CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48); The CDR1 amino acid sequence described in SEQ ID NO: 67, the CDR2 amino acid sequence described in SEQ ID NO: 68, and the CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55); The CDR1 amino acid sequence described in SEQ ID NO: 70, the CDR2 amino acid sequence described in SEQ ID NO: 71, and the CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64); The CDR1 amino acid sequence described in SEQ ID NO: 73, the CDR2 amino acid sequence described in SEQ ID NO: 74, and the CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72); The CDR1 amino acid sequence described in SEQ ID NO: 76, the CDR2 amino acid sequence described in SEQ ID NO: 77, and the CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79); or The CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82).

[0015] In another embodiment, an isolated VHH single-domain antibody (sdAb) that specifically binds to human CD22 is provided, the sdAb comprising the following sequence: A) The CDR1 amino acid sequence described in SEQ ID NO: 1, the CDR2 amino acid sequence described in SEQ ID NO: 2, and the CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6); The CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10); The CDR1 amino acid sequence described in SEQ ID NO: 7, the CDR2 amino acid sequence described in SEQ ID NO: 8, and the CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13); The CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14); The CDR1 amino acid sequence described in SEQ ID NO: 13, the CDR2 amino acid sequence described in SEQ ID NO: 14, and the CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36); The CDR1 amino acid sequence described in SEQ ID NO: 16, the CDR2 amino acid sequence described in SEQ ID NO: 17, and the CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61); The CDR1 amino acid sequence described in SEQ ID NO: 19, the CDR2 amino acid sequence described in SEQ ID NO: 20, and the CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74); The CDR1 amino acid sequence described in SEQ ID NO: 22, the CDR2 amino acid sequence described in SEQ ID NO: 23, and the CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75); The CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77); The CDR1 amino acid sequence described in SEQ ID NO: 28, the CDR2 amino acid sequence described in SEQ ID NO: 29, and the CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80); The CDR1 amino acid sequence described in SEQ ID NO: 31, the CDR2 amino acid sequence described in SEQ ID NO: 32, and the CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87); The CDR1 amino acid sequence described in SEQ ID NO: 34, the CDR2 amino acid sequence described in SEQ ID NO: 35, and the CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93); The CDR1 amino acid sequence described in SEQ ID NO: 37, the CDR2 amino acid sequence described in SEQ ID NO: 38, and the CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2); The CDR1 amino acid sequence described in SEQ ID NO: 40, the CDR2 amino acid sequence described in SEQ ID NO: 41, and the CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62); The CDR1 amino acid sequence described in SEQ ID NO: 43, the CDR2 amino acid sequence described in SEQ ID NO: 44, and the CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66); The CDR1 amino acid sequence described in SEQ ID NO: 46, the CDR2 amino acid sequence described in SEQ ID NO: 47, and the CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10); The CDR1 amino acid sequence described in SEQ ID NO: 49, the CDR2 amino acid sequence described in SEQ ID NO: 50, and the CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16); The CDR1 amino acid sequence described in SEQ ID NO: 52, the CDR2 amino acid sequence described in SEQ ID NO: 53, and the CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23); The CDR1 amino acid sequence described in SEQ ID NO: 55, the CDR2 amino acid sequence described in SEQ ID NO: 56, and the CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24); The CDR1 amino acid sequence described in SEQ ID NO: 58, the CDR2 amino acid sequence described in SEQ ID NO: 59, and the CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32); The CDR1 amino acid sequence described in SEQ ID NO: 61, the CDR2 amino acid sequence described in SEQ ID NO: 62, and the CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33); The CDR1 amino acid sequence described in SEQ ID NO: 64, the CDR2 amino acid sequence described in SEQ ID NO: 65, and the CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48); The CDR1 amino acid sequence described in SEQ ID NO: 67, the CDR2 amino acid sequence described in SEQ ID NO: 68, and the CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55); The CDR1 amino acid sequence described in SEQ ID NO: 70, the CDR2 amino acid sequence described in SEQ ID NO: 71, and the CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64); The CDR1 amino acid sequence described in SEQ ID NO: 73, the CDR2 amino acid sequence described in SEQ ID NO: 74, and the CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72); The CDR1 amino acid sequence described in SEQ ID NO: 76, the CDR2 amino acid sequence described in SEQ ID NO: 77, and the CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79); or The CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82); or B) CDR1, CDR2, and CDR3 amino acid sequences that are at least 80% identical to the CDR1, CDR2, and CDR3 sequences defined in any one of Part A) i) to xxviii).

[0016] In another embodiment, an isolated VHH single-domain antibody (sdAb) that specifically binds to human CD22 is provided, the sdAb comprising the following sequence: A) CDR3 amino acid sequence as described in SEQ ID NO: 3; CDR3 amino acid sequence as described in SEQ ID NO: 6; CDR3 amino acid sequence as described in SEQ ID NO: 9; CDR3 amino acid sequence as described in SEQ ID NO: 12; CDR3 amino acid sequence as described in SEQ ID NO: 15; CDR3 amino acid sequence as described in SEQ ID NO: 18; CDR3 amino acid sequence as described in SEQ ID NO: 21; CDR3 amino acid sequence as described in SEQ ID NO: 24; CDR3 amino acid sequence as described in SEQ ID NO: 27; CDR3 amino acid sequence as described in SEQ ID NO: 30; CDR3 amino acid sequence as described in SEQ ID NO: 33; CDR3 amino acid sequence as described in SEQ ID NO: 36; CDR3 amino acid sequence as described in SEQ ID NO: 39; CDR3 amino acid sequence as described in SEQ ID NO: 42; CDR3 amino acid sequence as described in SEQ ID NO: 45; CDR3 amino acid sequence as described in SEQ ID NO: 48; CDR3 amino acid sequence as described in SEQ ID NO: 51; CDR3 amino acid sequence as described in SEQ ID NO: 54; CDR3 amino acid sequence as described in SEQ ID NO: 57; CDR3 amino acid sequence as described in SEQ ID NO: 60; CDR3 amino acid sequence as described in SEQ ID NO: 63; CDR3 amino acid sequence as described in SEQ ID NO: 66; CDR3 amino acid sequence as described in SEQ ID NO: 69; CDR3 amino acid sequence as described in SEQ ID NO: 72; CDR3 amino acid sequence as described in SEQ ID NO: 75; CDR3 amino acid sequence as described in SEQ ID NO: 79; or CDR3 amino acid sequence as described in SEQ ID NO: 81.

[0017] In one embodiment, the isolated sdAb contains the following sequence: The CDR1 amino acid sequence described in SEQ ID NO: 1, and the CDR3 amino acid sequence described in SEQ ID NO: 3; The CDR1 amino acid sequence described in SEQ ID NO: 4, and the CDR3 amino acid sequence described in SEQ ID NO: 6; The CDR1 amino acid sequence described in SEQ ID NO: 7, and the CDR3 amino acid sequence described in SEQ ID NO: 9; The CDR1 amino acid sequence described in SEQ ID NO: 10, and the CDR3 amino acid sequence described in SEQ ID NO: 12; The CDR1 amino acid sequence described in SEQ ID NO: 13, and the CDR3 amino acid sequence described in SEQ ID NO: 15; The CDR1 amino acid sequence described in SEQ ID NO: 16, and the CDR3 amino acid sequence described in SEQ ID NO: 18; The CDR1 amino acid sequence described in SEQ ID NO: 19, and the CDR3 amino acid sequence described in SEQ ID NO: 21; The CDR1 amino acid sequence described in SEQ ID NO: 22, and the CDR3 amino acid sequence described in SEQ ID NO: 24; The CDR1 amino acid sequence described in SEQ ID NO: 25, and the CDR3 amino acid sequence described in SEQ ID NO: 27; The CDR1 amino acid sequence described in SEQ ID NO: 28, and the CDR3 amino acid sequence described in SEQ ID NO: 30; The CDR1 amino acid sequence described in SEQ ID NO: 31, and the CDR3 amino acid sequence described in SEQ ID NO: 33; The CDR1 amino acid sequence described in SEQ ID NO: 34, and the CDR3 amino acid sequence described in SEQ ID NO: 36; The CDR1 amino acid sequence described in SEQ ID NO: 37, and the CDR3 amino acid sequence described in SEQ ID NO: 39; The CDR1 amino acid sequence described in SEQ ID NO: 40, and the CDR3 amino acid sequence described in SEQ ID NO: 42; The CDR1 amino acid sequence described in SEQ ID NO: 43, and the CDR3 amino acid sequence described in SEQ ID NO: 45; The CDR1 amino acid sequence described in SEQ ID NO: 46, and the CDR3 amino acid sequence described in SEQ ID NO: 48; The CDR1 amino acid sequence described in SEQ ID NO: 49, and the CDR3 amino acid sequence described in SEQ ID NO: 51; The CDR1 amino acid sequence described in SEQ ID NO: 52, and the CDR3 amino acid sequence described in SEQ ID NO: 54; The CDR1 amino acid sequence described in SEQ ID NO: 55, and the CDR3 amino acid sequence described in SEQ ID NO: 57; The CDR1 amino acid sequence described in SEQ ID NO: 58, and the CDR3 amino acid sequence described in SEQ ID NO: 60; The CDR1 amino acid sequence described in SEQ ID NO: 61, and the CDR3 amino acid sequence described in SEQ ID NO: 63; The CDR1 amino acid sequence described in SEQ ID NO: 64, and the CDR3 amino acid sequence described in SEQ ID NO: 66; The CDR1 amino acid sequence described in SEQ ID NO: 67, and the CDR3 amino acid sequence described in SEQ ID NO: 69; The CDR1 amino acid sequence described in SEQ ID NO: 70, and the CDR3 amino acid sequence described in SEQ ID NO: 72; The CDR1 amino acid sequence described in SEQ ID NO: 73, and the CDR3 amino acid sequence described in SEQ ID NO: 75; The CDR1 amino acid sequence described in SEQ ID NO: 76, and the CDR3 amino acid sequence described in SEQ ID NO: 78; or The CDR1 amino acid sequence described in SEQ ID NO: 79, and the CDR3 amino acid sequence described in SEQ ID NO: 81.

[0018] In one embodiment, a VHH single-domain antibody (sdAb) is provided that competes with the isolated sdAbs described herein for specific binding to CD22.

[0019] In one embodiment, a recombinant polypeptide comprising sdAb as defined herein is provided.

[0020] In a further embodiment, the disclosure provides an anti-CD22 sdAb as defined herein, linked to a cargo molecule.

[0021] In one embodiment, sdAb, recombinant polypeptide, or V as defined herein H H:F c A recombinant nucleic acid molecule encoding the fusion is provided.

[0022] In one embodiment, a composition is provided comprising an sdAb as defined herein, or a polypeptide containing such an sdAb, together with an acceptable excipient, diluent, or carrier.

[0023] In one embodiment, a treatment for cancer or autoimmune disease using sdAb as defined herein, or one or more V as defined herein. H H:F c The use of antibodies containing fusion compounds is provided.

[0024] In one embodiment, a preparation of a pharmaceutical product for the treatment of cancer or autoimmune disease, comprising sdAb as defined herein, or one or more V as defined herein. H H:F c The use of antibodies containing fusion compounds is provided.

[0025] In one embodiment, for use in the treatment of cancer or autoimmune diseases, sdAb as defined herein, or one or more V as defined herein.H H:F c Antibodies containing fusion compounds are provided.

[0026] In one embodiment, sdAb as defined herein, or one or more V as defined herein. H H:F c A method for treating cancer or autoimmune disease in a subject is provided, which includes administering an antibody containing a fusion to the subject.

[0027] In one embodiment, a polyvalent antibody comprising an sdAb as defined herein is provided.

[0028] In one embodiment, a polyvalent antibody is provided comprising a first antigen-binding moiety, an amino acid linker containing a polypeptide hinge derived from human CD8, and a second antigen-binding moiety.

[0029] In one embodiment, a recombinant nucleic acid molecule encoding a polyvalent antibody as defined herein is provided.

[0030] In one embodiment, a composition is provided comprising a polyvalent antibody as defined herein, together with an acceptable excipient, diluent, or carrier.

[0031] In one embodiment, the use of a polyvalent antibody as defined herein for the treatment of cancer or an autoimmune disease is provided. In one embodiment, cancer is a hematological malignancy.

[0032] In one embodiment, the use of a polyvalent antibody as defined herein is provided for preparing a pharmaceutical product for the treatment of cancer or an autoimmune disease.

[0033] In one embodiment, a polyvalent antibody as defined herein is provided for use in the treatment of cancer or an autoimmune disease. In one embodiment, cancer is a hematological malignancy.

[0034] In one embodiment, a method is provided for treating cancer or an autoimmune disease in a subject, comprising administering a polyvalent antibody as defined herein to the subject.

[0035] In one embodiment, a chimeric antibody receptor (CAR) that binds to human CD22 is provided, comprising a VHH sdAb as defined herein.

[0036] In one embodiment, a recombinant nucleic acid molecule encoding a CAR as defined herein is provided.

[0037] In one embodiment, a vector comprising a recombinant nucleic acid molecule as defined herein is provided.

[0038] In one embodiment, recombinant virus particles comprising recombinant nucleic acids as defined herein are provided.

[0039] In one embodiment, cells containing recombinant nucleic acid molecules as defined herein are provided.

[0040] In one embodiment, the use of nucleic acids, vectors, or viral particles described herein for producing cells for CAR-T is provided.

[0041] In one embodiment, a method for producing cells for CAR-T is provided, which includes contacting T cells with the viral particles described herein.

[0042] In one embodiment, a method for producing cells for CAR-T is provided, comprising introducing a nucleic acid or vector described herein into T cells.

[0043] In one embodiment, the use of CARs or engineered cells described herein for the treatment of cancer or autoimmune diseases is provided.

[0044] In one embodiment, the use of CARs or genetically modified cells as described herein is provided for preparing a pharmacopoeia for the treatment of cancer or autoimmune diseases.

[0045] In one embodiment, CARs or genetically modified cells described herein are provided for use in the treatment of cancer or autoimmune diseases.

[0046] In one embodiment, a method for treating cancer or an autoimmune disease in a subject is provided, comprising administering the genetically modified cells described herein to the subject.

[0047] Other aspects and features of this disclosure will become apparent to those skilled in the art by examining the following descriptions of specific embodiments in conjunction with the accompanying drawings. Examples of embodiments of the present invention include the following. [Embodiment 1] An isolated single-domain antibody (sdAb) that specifically binds to human CD22, with the following sequence: a) CDR1 amino acid sequence GX 1 X 2 X 3 DX 4 YX 5 (SEQ ID NO: 126), here, X 1 is F or V, X 2 is T or S, X 3 It is L, F, or S, X 4 is Y, D, or S, X 5 is either V or A; CDR2 amino acid sequence X 6 X 7 X 8 X 9 X 10 GX 11 T (SEQ ID NO: 127), here, X 6 is I or M, X 7 is T, R, G, or S, X 8 is S or N, X 9 is either D or S, X 10 It is either D or does not exist. X 11 is V or A; and CDR3 amino acid sequence AVDKPFYDGGX 12 X 13 YTCPVDFX 14 S (SEQ ID NO: 128), here, X 12 is I, N, Y, or S, X 13 is Q, Y, R, or L, X 14 is either D or G; b) CDR1 amino acid sequence GSX 1 FX 2 X 3 X 4 X 5 V (SEQ ID NO: 129), here, X 1 is I or T, X 2 is either R or does not exist. X 3 is R, S, or I, X 4 is I, S, or T, X 5 is A, S, or T; CDR2 amino acid sequence ITSX 6 GX 7 X 8 (SEQ ID NO: 130), here, X 6 is G, S, or A, X 7 is E, D, or S, X 8 is T or S; and CDR3 amino acid sequence NAX 9 X 10 GX 11 X 12 X 13 X 14 (SEQ ID NO: 131), here, X 9 is either K or Q, X 10 is either W or Y, X 11 is Q, R, or G, X 12 is Y, D, or R, X 13 is E or S, X 14 is Y, D, or H; c) CDR1 amino acid sequence GRIX 1 RSYV (SEQ ID NO: 132), here, X 1 is either F or S; CDR2 amino acid sequence IGX 2 SDT (SEQ ID NO: 133), here, X 2 is W or C; and CDR3 amino acid sequence AX 3 X 4 SPPYGPQRDEFX 5 Y (SEQ ID NO: 134), here, X 3 is A or E, X 4 is either N or Y, X 5 is G or D; d) CDR1 amino acid sequence GX 1 TX 2 SVYX 3 (SEQ ID NO: 135), here, X 1 is R or G, X 2 is S or F, X 3 is either G or T; CDR2 amino acid sequence X 4 X 5 X 6 SX 7 GX 8 T (SEQ ID NO: 136), here, X 4 is M or I, X 5 is either S or R, X 6 is W or G, X 7 is either G or does not exist. X 8 is P or G; and CDR3 amino acid sequence AVRIRRTLX 9 EPLTKETLYDY (SEQ ID NO: 137), here, X 9 is L or V; e) CDR1 amino acid sequence as described in SEQ ID NO: 49, The CDR2 amino acid sequence described in SEQ ID NO: 50, and CDR3 amino acid sequence (hCD22pas-16) as described in SEQ ID NO: 51; f) CDR1 amino acid sequence as described in SEQ ID NO: 4, The CDR2 amino acid sequence described in SEQ ID NO: 5, and CDR3 amino acid sequence (hCD221ug-10) as described in SEQ ID NO: 6; g) CDR1 amino acid sequence as described in SEQ ID NO: 7, The CDR2 amino acid sequence described in SEQ ID NO: 8, and CDR3 amino acid sequence (hCD221ug-13) as described in SEQ ID NO: 9; h) CDR1 amino acid sequence as described in SEQ ID NO: 13, The CDR2 amino acid sequence described in SEQ ID NO: 14, and CDR3 amino acid sequence (hCD221ug-36) as described in SEQ ID NO: 15; i) CDR1 amino acid sequence described in SEQ ID NO: 16, The CDR2 amino acid sequence described in SEQ ID NO: 17, and CDR3 amino acid sequence (hCD221ug-61) as described in SEQ ID NO: 18; j) CDR1 amino acid sequence described in SEQ ID NO: 40, The CDR2 amino acid sequence described in SEQ ID NO: 41, and CDR3 amino acid sequence (hCD22100ug-62) as described in SEQ ID NO: 42; k) CDR1 amino acid sequence described in SEQ ID NO: 55, The CDR2 amino acid sequence described in SEQ ID NO: 56, and CDR3 amino acid sequence (hCD22pas-24) described in SEQ ID NO: 57; l) CDR1 amino acid sequence as described in SEQ ID NO: 64, The CDR2 amino acid sequence described in SEQ ID NO: 65, and CDR3 amino acid sequence (hCD22pas-48) as described in SEQ ID NO: 66; or m) CDR1 amino acid sequence described in SEQ ID NO: 70, The CDR2 amino acid sequence described in SEQ ID NO: 71, and CDR3 amino acid sequence (hCD22pas-64) as described in SEQ ID NO: 72; Isolated sdAb, including. [Embodiment 2] An isolated single-domain antibody (sdAb) that specifically binds to human CD22, with the following sequence: A) i) CDR1 amino acid sequence described in SEQ ID NO: 1, CDR2 amino acid sequence described in SEQ ID NO: 2, and CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6); ii) The CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10); iii) CDR1 amino acid sequence described in SEQ ID NO: 7, CDR2 amino acid sequence described in SEQ ID NO: 8, and CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13); iv) The CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14); v) CDR1 amino acid sequence described in SEQ ID NO: 13, CDR2 amino acid sequence described in SEQ ID NO: 14, and CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36); vi) CDR1 amino acid sequence described in SEQ ID NO: 16, CDR2 amino acid sequence described in SEQ ID NO: 17, and CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61); vii) CDR1 amino acid sequence described in SEQ ID NO: 19, CDR2 amino acid sequence described in SEQ ID NO: 20, and CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74); viii) CDR1 amino acid sequence described in SEQ ID NO: 22, CDR2 amino acid sequence described in SEQ ID NO: 23, and CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75); ix) The CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77); x) CDR1 amino acid sequence described in SEQ ID NO: 28, CDR2 amino acid sequence described in SEQ ID NO: 29, and CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80); xi) CDR1 amino acid sequence described in SEQ ID NO: 31, CDR2 amino acid sequence described in SEQ ID NO: 32, and CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87); xii) CDR1 amino acid sequence described in SEQ ID NO: 34, CDR2 amino acid sequence described in SEQ ID NO: 35, and CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93); xiii) CDR1 amino acid sequence described in SEQ ID NO: 37, CDR2 amino acid sequence described in SEQ ID NO: 38, and CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2); xiv) CDR1 amino acid sequence described in SEQ ID NO: 40, CDR2 amino acid sequence described in SEQ ID NO: 41, and CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62); xv) CDR1 amino acid sequence described in SEQ ID NO: 43, CDR2 amino acid sequence described in SEQ ID NO: 44, and CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66); xvi) CDR1 amino acid sequence described in SEQ ID NO: 46, CDR2 amino acid sequence described in SEQ ID NO: 47, and CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10); xvii) CDR1 amino acid sequence described in SEQ ID NO: 49, CDR2 amino acid sequence described in SEQ ID NO: 50, and CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16); xviii) CDR1 amino acid sequence described in SEQ ID NO: 52, CDR2 amino acid sequence described in SEQ ID NO: 53, and CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23); xix) CDR1 amino acid sequence described in SEQ ID NO: 55, CDR2 amino acid sequence described in SEQ ID NO: 56, and CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24); xx) CDR1 amino acid sequence described in SEQ ID NO: 58, CDR2 amino acid sequence described in SEQ ID NO: 59, and CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32); xxi) CDR1 amino acid sequence described in SEQ ID NO: 61, CDR2 amino acid sequence described in SEQ ID NO: 62, and CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33); xxii) CDR1 amino acid sequence described in SEQ ID NO: 64, CDR2 amino acid sequence described in SEQ ID NO: 65, and CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48); xxiii) CDR1 amino acid sequence described in SEQ ID NO: 67, CDR2 amino acid sequence described in SEQ ID NO: 68, and CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55); xxiv) CDR1 amino acid sequence described in SEQ ID NO: 70, CDR2 amino acid sequence described in SEQ ID NO: 71, and CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64); xxv) CDR1 amino acid sequence described in SEQ ID NO: 73, CDR2 amino acid sequence described in SEQ ID NO: 74, and CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72); xxvi) CDR1 amino acid sequence described in SEQ ID NO: 76, CDR2 amino acid sequence described in SEQ ID NO: 77, and CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79); or xxvii) CDR1 amino acid sequence described in SEQ ID NO: 79, CDR2 amino acid sequence described in SEQ ID NO: 80, and CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82); Isolated sdAb, including. [Embodiment 3] An isolated single-domain antibody (sdAb) that specifically binds to human CD22, with the following sequence: A) i) CDR1 amino acid sequence described in SEQ ID NO: 1, CDR2 amino acid sequence described in SEQ ID NO: 2, and CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6); ii) The CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10); iii) CDR1 amino acid sequence described in SEQ ID NO: 7, CDR2 amino acid sequence described in SEQ ID NO: 8, and CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13); iv) The CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14); v) CDR1 amino acid sequence described in SEQ ID NO: 13, CDR2 amino acid sequence described in SEQ ID NO: 14, and CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36); vi) CDR1 amino acid sequence described in SEQ ID NO: 16, CDR2 amino acid sequence described in SEQ ID NO: 17, and CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61); vii) CDR1 amino acid sequence described in SEQ ID NO: 19, CDR2 amino acid sequence described in SEQ ID NO: 20, and CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74); viii) CDR1 amino acid sequence described in SEQ ID NO: 22, CDR2 amino acid sequence described in SEQ ID NO: 23, and CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75); ix) The CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77); x) CDR1 amino acid sequence described in SEQ ID NO: 28, CDR2 amino acid sequence described in SEQ ID NO: 29, and CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80); xi) CDR1 amino acid sequence described in SEQ ID NO: 31, CDR2 amino acid sequence described in SEQ ID NO: 32, and CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87); xii) CDR1 amino acid sequence described in SEQ ID NO: 34, CDR2 amino acid sequence described in SEQ ID NO: 35, and CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93); xiii) CDR1 amino acid sequence described in SEQ ID NO: 37, CDR2 amino acid sequence described in SEQ ID NO: 38, and CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2); xiv) CDR1 amino acid sequence described in SEQ ID NO: 40, CDR2 amino acid sequence described in SEQ ID NO: 41, and CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62); xv) CDR1 amino acid sequence described in SEQ ID NO: 43, CDR2 amino acid sequence described in SEQ ID NO: 44, and CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66); xvi) CDR1 amino acid sequence described in SEQ ID NO: 46, CDR2 amino acid sequence described in SEQ ID NO: 47, and CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10); xvii) CDR1 amino acid sequence described in SEQ ID NO: 49, CDR2 amino acid sequence described in SEQ ID NO: 50, and CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16); xviii) CDR1 amino acid sequence described in SEQ ID NO: 52, CDR2 amino acid sequence described in SEQ ID NO: 53, and CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23); xix) CDR1 amino acid sequence described in SEQ ID NO: 55, CDR2 amino acid sequence described in SEQ ID NO: 56, and CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24); xx) CDR1 amino acid sequence described in SEQ ID NO: 58, CDR2 amino acid sequence described in SEQ ID NO: 59, and CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32); xxi) CDR1 amino acid sequence described in SEQ ID NO: 61, CDR2 amino acid sequence described in SEQ ID NO: 62, and CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33); xxii) CDR1 amino acid sequence described in SEQ ID NO: 64, CDR2 amino acid sequence described in SEQ ID NO: 65, and CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48); xxiii) CDR1 amino acid sequence described in SEQ ID NO: 67, CDR2 amino acid sequence described in SEQ ID NO: 68, and CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55); xxiv) CDR1 amino acid sequence described in SEQ ID NO: 70, CDR2 amino acid sequence described in SEQ ID NO: 71, and CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64); xxv) CDR1 amino acid sequence described in SEQ ID NO: 73, CDR2 amino acid sequence described in SEQ ID NO: 74, and CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72); xxvi) CDR1 amino acid sequence described in SEQ ID NO: 76, CDR2 amino acid sequence described in SEQ ID NO: 77, and CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79); or xxvii) The CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82); or B) CDR1, CDR2, and CDR3 amino acid sequences that are at least 80% identical to the CDR1, CDR2, and CDR3 sequences defined in any one of Part A) i) to xxviii); Isolated sdAb, including. [Embodiment 4] An isolated single-domain antibody (sdAb) that specifically binds to human CD22, with the following sequence: A) i) CDR3 amino acid sequence as described in SEQ ID NO: 3; ii) CDR3 amino acid sequence as described in SEQ ID NO: 6; iii) CDR3 amino acid sequence as described in SEQ ID NO: 9; iv) CDR3 amino acid sequence as described in SEQ ID NO: 12; v) CDR3 amino acid sequence as described in SEQ ID NO: 15; vi) CDR3 amino acid sequence as described in SEQ ID NO: 18; vii) CDR3 amino acid sequence as described in SEQ ID NO: 21; viii) CDR3 amino acid sequence as described in SEQ ID NO: 24; ix) CDR3 amino acid sequence as described in SEQ ID NO: 27; x) CDR3 amino acid sequence as described in SEQ ID NO: 30; xi) CDR3 amino acid sequence as described in SEQ ID NO: 33; xii) CDR3 amino acid sequence as described in SEQ ID NO: 36; xiii) CDR3 amino acid sequence as described in SEQ ID NO: 39; xiv) CDR3 amino acid sequence described in SEQ ID NO: 42; xv) CDR3 amino acid sequence as described in SEQ ID NO: 45; xvi) CDR3 amino acid sequence described in SEQ ID NO: 48; xvii) CDR3 amino acid sequence as described in SEQ ID NO: 51; xviii) CDR3 amino acid sequence as described in SEQ ID NO: 54; xix) CDR3 amino acid sequence described in SEQ ID NO: 57; xx) CDR3 amino acid sequence as described in SEQ ID NO: 60; xxi) CDR3 amino acid sequence as described in SEQ ID NO: 63; xxii) CDR3 amino acid sequence described in SEQ ID NO: 66; xxiii) CDR3 amino acid sequence as described in SEQ ID NO: 69; xxiv) CDR3 amino acid sequence described in SEQ ID NO: 72; xxv) CDR3 amino acid sequence as described in SEQ ID NO: 75; xxvi) CDR3 amino acid sequence as described in SEQ ID NO: 79; or xxvii) CDR3 amino acid sequence as described in SEQ ID NO: 81; Isolated sdAb, including. [Embodiment 5] The following array: A) i) The CDR1 amino acid sequence described in SEQ ID NO: 1, and the CDR3 amino acid sequence described in SEQ ID NO: 3; ii) The CDR1 amino acid sequence described in SEQ ID NO: 4, and the CDR3 amino acid sequence described in SEQ ID NO: 6; iii) The CDR1 amino acid sequence described in SEQ ID NO: 7, and the CDR3 amino acid sequence described in SEQ ID NO: 9; iv) The CDR1 amino acid sequence described in SEQ ID NO: 10, and the CDR3 amino acid sequence described in SEQ ID NO: 12; v) The CDR1 amino acid sequence described in SEQ ID NO: 13, and the CDR3 amino acid sequence described in SEQ ID NO: 15; vi) The CDR1 amino acid sequence described in SEQ ID NO: 16, and the CDR3 amino acid sequence described in SEQ ID NO: 18; vii) The CDR1 amino acid sequence described in SEQ ID NO: 19, and the CDR3 amino acid sequence described in SEQ ID NO: 21; viii) The CDR1 amino acid sequence described in SEQ ID NO: 22, and the CDR3 amino acid sequence described in SEQ ID NO: 24; ix) The CDR1 amino acid sequence described in SEQ ID NO: 25, and the CDR3 amino acid sequence described in SEQ ID NO: 27; x) The CDR1 amino acid sequence described in SEQ ID NO: 28, and the CDR3 amino acid sequence described in SEQ ID NO: 30; xi) The CDR1 amino acid sequence described in SEQ ID NO: 31, and the CDR3 amino acid sequence described in SEQ ID NO: 33; xii) The CDR1 amino acid sequence described in SEQ ID NO: 34, and the CDR3 amino acid sequence described in SEQ ID NO: 36; xiii) The CDR1 amino acid sequence described in SEQ ID NO: 37, and the CDR3 amino acid sequence described in SEQ ID NO: 39; xiv) The CDR1 amino acid sequence described in SEQ ID NO: 40, and the CDR3 amino acid sequence described in SEQ ID NO: 42; xv) The CDR1 amino acid sequence described in SEQ ID NO: 43, and the CDR3 amino acid sequence described in SEQ ID NO: 45; xvi) The CDR1 amino acid sequence described in SEQ ID NO: 46, and the CDR3 amino acid sequence described in SEQ ID NO: 48; xvii) The CDR1 amino acid sequence described in SEQ ID NO: 49, and the CDR3 amino acid sequence described in SEQ ID NO: 51; xviii) The CDR1 amino acid sequence described in SEQ ID NO: 52, and the CDR3 amino acid sequence described in SEQ ID NO: 54; xix) The CDR1 amino acid sequence described in SEQ ID NO: 55, and the CDR3 amino acid sequence described in SEQ ID NO: 57; xx) The CDR1 amino acid sequence described in SEQ ID NO: 58, and the CDR3 amino acid sequence described in SEQ ID NO: 60; xxi) CDR1 amino acid sequence described in SEQ ID NO: 61, and CDR3 amino acid sequence described in SEQ ID NO: 63; xxii) The CDR1 amino acid sequence described in SEQ ID NO: 64, and the CDR3 amino acid sequence described in SEQ ID NO: 66; xxiii) The CDR1 amino acid sequence described in SEQ ID NO: 67, and the CDR3 amino acid sequence described in SEQ ID NO: 69; xxiv) The CDR1 amino acid sequence described in SEQ ID NO: 70, and the CDR3 amino acid sequence described in SEQ ID NO: 72; xxv) CDR1 amino acid sequence described in SEQ ID NO: 73, and CDR3 amino acid sequence described in SEQ ID NO: 75; xxvi) The CDR1 amino acid sequence described in SEQ ID NO: 76, and the CDR3 amino acid sequence described in SEQ ID NO: 78; or xxvii) CDR1 amino acid sequence described in SEQ ID NO: 79, and CDR3 amino acid sequence described in SEQ ID NO: 81; An isolated single-domain antibody (sdAb) as described in Embodiment 4, including the above. [Embodiment 6] A) an isolated sdAb according to Embodiment 2 or 3, comprising one amino acid sequence from SEQ ID NO: 85-112 and 120-125, or B) an amino acid sequence that is at least 80% identical over its entire length to one from SEQ ID NO: 82-108 and 120-125. [Embodiment 7] A) An isolated sdAb according to Embodiment 1 or 2, comprising one amino acid sequence from SEQ ID NO: 82-108 and 120-125. [Embodiment 8] An isolated sdAb according to any one of Embodiments 1 to 5, which is a camelid sdAb. [Embodiment 9] The isolated sdAb described in Embodiment 8 is a llama sdAb. [Embodiment 10] A humanized isolated sdAb according to any one of Embodiments 1 to 5. [Embodiment 11] 2.5×10 -7 An isolated sdAb according to any one of Embodiments 1 to 10, having affinity for human CD22 of a magnitude of 1 nm or less. [Embodiment 12] 3×10 -8 An isolated sdAb according to any one of Embodiments 1 to 10, having affinity for human CD22 of a magnitude of 1 nm or less. [Embodiment 13] 9.6×10 -9 An isolated sdAb according to any one of Embodiments 1 to 10, having affinity for human CD22 of a magnitude of 1 nm or less. [Embodiment 14] 9.3×10 -10 An isolated sdAb according to any one of Embodiments 1 to 10, having affinity for human CD22 of a magnitude of 1 nm or less. [Embodiment 15] 7×10 -12 An isolated sdAb according to any one of Embodiments 1 to 10, having affinity for human CD22 of a magnitude of 1 nm or less. [Embodiment 16] An isolated sdAb according to any one of Embodiments 1 to 15, which binds to the first Ig-like domain of human CD22. [Embodiment 17] The isolated sdAb according to Embodiment 16, wherein the antibody contains SEQ ID NO: 91 (hCD221ug-80). [Embodiment 18] An isolated sdAb according to any one of Embodiments 1 to 15, which binds to the fourth Ig-like domain of human CD22. [Embodiment 19] The isolated sdAb according to Embodiment 18, wherein the antibody contains SEQ ID NO: 84 (hCD221ug-13). [Embodiment 20] An isolated sdAb according to any one of Embodiments 1 to 15, which binds to the sixth Ig-like domain of human CD22. [Embodiment 21] The isolated sdAb according to Embodiment 20, wherein the antibody comprises SEQ ID NO: 84 (hCD221ug-13), SEQ ID NO: 91 (hCD221ug-80), SEQ ID NO: 86 (hCD221ug-36), or SEQ ID NO: 105 (hCD221pas-64). [Embodiment 22] An isolated sdAb according to any one of Embodiments 1 to 15, which binds to the seventh Ig-like domain of human CD22. [Embodiment 23] The isolated sdAb according to Embodiment 22, wherein the antibody comprises SEQ ID NO: 87 (hCD221ug-61), SEQ ID NO: 83 (hCD221ug-10), or SEQ ID NO: 100 (hCD22pas-24). [Embodiment 24] A single-domain antibody (sdAb) that competes with the isolated sdAb described in Embodiment 3 or 7 for specific binding to CD22. [Embodiment 25] The sdAb according to Embodiment 24, which is a camelid sdAb. [Embodiment 26] The sdAb described in Embodiment 25 is a ram sdAb. [Embodiment 27] A humanized sdAb according to any one of embodiments 24 to 26. [Embodiment 28] A recombinant polypeptide comprising one or more sdAbs as described in any one of Embodiments 1 to 27. [Embodiment 29] A first antigen-binding moiety comprising sdAb as described in any one of Embodiments 1 to 27; and The second antigen-binding site; A multivalent antibody containing [specific antibody type]. [Embodiment 30] The polyvalent antibody according to Embodiment 29, wherein the second antigen-binding moiety specifically binds to a cell surface marker of an immune cell. [Embodiment 31] A polyvalent antibody according to Embodiment 30, wherein the cell surface marker of immune cells includes a T cell marker. [Embodiment 32] A polyvalent antibody according to Embodiment 31, comprising the T cell marker human CD3. [Embodiment 33] From the N-terminus towards the C-terminus, - The first antigen-binding site; - Amino acid linkers; and - Second antigen-binding site; A polyvalent antibody according to any one of embodiments 29 to 32, including the above. [Embodiment 34] A polyvalent antibody according to Embodiment 33, further comprising an N-terminal signal peptide. [Embodiment 35] The polyvalent antibody according to Embodiment 34, wherein the signal peptide is a signal peptide derived from human CD28, preferably containing SEQ ID NO: 110. [Embodiment 36] A polyvalent antibody according to any one of embodiments 33 to 35, wherein the amino acid linker comprises a polypeptide hinge derived from human CD8. [Embodiment 37] A polyvalent antibody according to Embodiment 36, wherein the polypeptide hinge derived from human CD8 contains SEQ ID NO: 112. [Embodiment 38] A polyvalent antibody according to Embodiment 36 or 37, wherein the amino acid linker includes SEQ ID NO: 111, 112, and 118 in the direction from the N-terminus to the C-terminus. [Embodiment 39] A polyvalent antibody according to any one of embodiments 33 to 38, coded by SEQ ID NO: 119. [Embodiment 40] A polyvalent antibody according to Embodiment 30, wherein the cell surface marker of the immune cell marker includes a natural killer (NK) cell marker. [Embodiment 41] A polyvalent antibody according to Embodiment 40, wherein the NK cell marker contains human CD16. [Embodiment 42] A polyvalent antibody according to embodiment 40 or 41, further comprising cytokines for stimulating the activation, proliferation, and / or survival of NK cells. [Embodiment 43] The polyvalent antibody according to Embodiment 42, wherein the cytokine for stimulating the proliferation of NK cells is interleukin 15 (IL15). [Embodiment 44] A polyvalent antibody according to any one of embodiments 40 to 43, further comprising a third antigen-binding moiety that binds to a second NK cell marker. [Embodiment 45] The polyvalent antibody according to Embodiment 44, wherein the second NK cell marker is human NKp46. [Embodiment 46] A polyvalent antibody according to any one of Embodiments 40 to 43, further comprising at least a third antigen-binding moiety that binds to a tumor-associated antigen, preferably the tumor-associated antigen being different from human CD22. [Embodiment 47] A polyvalent antibody according to any one of Embodiments 29 to 39, further comprising at least a third antigen-binding moiety that binds to a tumor-associated antigen, preferably the tumor-associated antigen being different from human CD22. [Embodiment 48] The third antigen-binding portion is V H H, V NAR A polyvalent antibody according to either embodiment 46 or 47, comprising scVF. [Embodiment 49] The second antigen-binding region is V H H, V NAR A polyvalent antibody according to any one of Embodiments 29 to 47, comprising scVF. [Embodiment 50] A polyvalent antibody according to any one of Embodiments 29 to 49, comprising an antigen-binding moiety that binds to human serum albumin. [Embodiment 51] A polyvalent antibody according to any one of embodiments 31 to 39, which is a bispecific T cell engager. [Embodiment 52] A polyvalent antibody according to embodiment 40 or 41, which is a bispecific killer cell engager (BiKE). [Embodiment 53] A polyvalent antibody according to any one of embodiments 42 to 48, which is a triple-specific killer cell engager (TriKE). [Embodiment 54] sdAb is, CDR1 amino acid sequence as described in SEQ ID NO: 13, The CDR2 amino acid sequence described in SEQ ID NO: 14, and CDR3 amino acid sequence (hCD221ug36) as described in SEQ ID NO: 15; or CDR1 amino acid sequence as described in SEQ ID NO: 7, The CDR2 amino acid sequence described in SEQ ID NO: 8, and CDR3 amino acid sequence (hCD221ug13) as described in SEQ ID NO: 9; A polyvalent antibody according to any one of embodiments 29 to 53, including the above. [Embodiment 55] A polyvalent antibody according to any one of Embodiments 29 to 53, wherein sdAb comprises SEQ ID NO: 86 (hCD221ug36) or SEQ ID NO: 84 (hCD221ug13). [Embodiment 56] A recombinant nucleic acid molecule encoding a recombinant polypeptide as described in Embodiment 28, or a polyvalent antibody as described in any one of Embodiments 29 to 55. [Embodiment 57] Use of a polyvalent antibody according to any one of embodiments 29 to 55 for the treatment of cancer or autoimmune disease. [Embodiment 58] A method for treating cancer or an autoimmune disease, comprising administering a polyvalent antibody described in any one of embodiments 29 to 55 to a target. [Embodiment 59] A chimeric antibody receptor (CAR) that binds to human CD22, comprising the VHH sdAb described in any one of Embodiments 1 to 27. [Embodiment 60] From the N-terminus towards the C-terminus, - A CD22-binding domain containing sdAb as described in any one of Embodiments 1 to 27; - Polypeptide hinge; - Transmembrane domain; and - A cytoplasmic domain comprising a signaling domain, preferably further comprising a co-stimulatory domain; The CAR according to Embodiment 59, including the CAR described in Embodiment 59. [Embodiment 61] The CAR according to Embodiment 60, wherein the polypeptide hinge is a CD8 hinge domain. [Embodiment 62] The CAR according to Embodiment 61, wherein the CD8 hinge domain includes SEQ ID NO: 112. [Embodiment 63] The CAR according to any one of embodiments 60 to 62, wherein the transmembrane domain is a CD28 transmembrane domain. [Embodiment 64] The CAR according to Embodiment 63, wherein the CD28 transmembrane domain includes SEQ ID NO: 113. [Embodiment 65] A CAR according to any one of embodiments 60 to 64, wherein the signal transduction domain is a CD3ζ signal transduction domain. [Embodiment 66] The CAR according to Embodiment 65, wherein the CD3ζ signaling domain includes SEQ ID NO: 115. [Embodiment 67] A CAR according to any one of embodiments 60 to 66, wherein the co-stimulatory domain is the 4-1BB co-stimulatory domain. [Embodiment 68] The CAR according to Embodiment 67, wherein the 4-1BB signaling domain includes SEQ ID NO: 114. [Embodiment 69] A CAR according to any one of embodiments 60 to 68, further comprising a flexible amino acid linker between the sdAb and the polypeptide hinge. [Embodiment 70] The CAR according to Embodiment 69, wherein a flexible amino acid linker comprises SEQ ID NO: 111. [Embodiment 71] A CAR according to any one of embodiments 59 to 70, further comprising a signal peptide. [Embodiment 72] The CAR according to Embodiment 71, wherein the signal peptide is a signal peptide derived from human CD28, and preferably the signal peptide derived from human CD28 contains SEQ ID NO: 110. [Embodiment 73] A CAR according to any one of embodiments 59 to 72, coded by SEQ ID NO: 119. [Embodiment 74] A CAR according to any one of Embodiments 58 to 72, wherein sdAb includes SEQ ID NO: 86 (hCD221ug-36), SEQ ID NO: 83 (hCD221ug-10), SEQ ID NO: 87 (hCD221ug-61), SEQ ID NO: 82 (hCD221ug-6), SEQ ID NO: 88 (hCD221ug-74), SEQ ID NO: 85 (hCD221ug-14), SEQ ID NO: 102 (hCD22pos-33), or SEQ ID NO: 84 (hCD221ug-13). [Embodiment 75] VHH sdAb is, CDR1 amino acid sequence as described in SEQ ID NO: 4, The CDR2 amino acid sequence described in SEQ ID NO: 5, and CDR3 amino acid sequence (hCD221ug-10) as described in SEQ ID NO: 6 A CAR according to any one of embodiments 59 to 72, including the CAR described therein. [Embodiment 76] VHH sdAb is, CDR1 amino acid sequence as described in SEQ ID NO: 13, The CDR2 amino acid sequence described in SEQ ID NO: 14, and CDR3 amino acid sequence (hCD221ug-36) as described in SEQ ID NO: 15 A CAR according to any one of embodiments 59 to 72, including the CAR described therein. [Embodiment 77] VHH sdAb is, CDR1 amino acid sequence as described in SEQ ID NO: 19, The CDR2 amino acid sequence described in SEQ ID NO: 20, and CDR3 amino acid sequence (hCD221ug-74) as described in SEQ ID NO: 21 A CAR according to any one of embodiments 59 to 72, including the CAR described therein. [Embodiment 78] VHH sdAb is, CDR1 amino acid sequence as described in SEQ ID NO: 61, The CDR2 amino acid sequence described in SEQ ID NO: 62, and CDR3 amino acid sequence (hCD221pas-33) as described in SEQ ID NO: 63 A CAR according to any one of embodiments 59 to 72, including the CAR described therein. [Embodiment 79] VHH sdAb is, CDR1 amino acid sequence as described in SEQ ID NO: 1, The CDR2 amino acid sequence described in SEQ ID NO: 2, and CDR3 amino acid sequence (hCD221ug-6) as described in SEQ ID NO: 3 A CAR according to any one of embodiments 59 to 72, including the CAR described therein. [Embodiment 80] The CAR according to any one of embodiments 59 to 79, further comprising an additional binding domain located at the N-terminus or C-terminus of the CD22 binding domain and linked to the CD22 binding domain by an amino acid linker. [Embodiment 81] The CAR according to embodiment 80, wherein an additional binding domain binds to CD22. [Embodiment 82] The CAR according to Embodiment 81, wherein the additional binding domain includes a second sdAb that is different from the sdAb of the CD22 binding domain. [Embodiment 83] The CAR according to Embodiment 82, wherein the second sdAb binds to a different epitope of CD22 compared to the sdAb of the CD22-binding domain. [Embodiment 84] The CAR according to Embodiment 81, wherein the additional binding domain includes a second sdAb containing the same CDR1, CDR2, and CDR3 sdAbs as the CD22 binding domain, preferably the second sdAb being identical to the sdAb of the CD22 binding domain. [Embodiment 85] The CAR according to embodiment 80, wherein an additional binding domain binds to a molecule different from CD22. [Embodiment 86] The CAR according to Embodiment 80, wherein the additional binding domain includes a second sdAb. [Embodiment 87] The CAR according to embodiment 85 or 86, wherein CD22 and a different molecule are expressed by the same target cells as CD22. [Embodiment 88] A recombinant nucleic acid molecule encoding a CAR as described in any one of embodiments 59 to 87. [Embodiment 89] A vector comprising the recombinant nucleic acid molecule described in Embodiment 88. [Embodiment 90] A viral vector, as described in Embodiment 89. [Embodiment 91] The vector according to embodiment 90, wherein the viral vector is a lentiviral vector. [Embodiment 92] Recombinant virus particles comprising the recombinant nucleic acid molecule described in Embodiment 88. [Embodiment 93] Recombinant virus particles according to Embodiment 92, which are recombinant lentivirus particles. [Embodiment 94] A cell containing the recombinant nucleic acid molecule described in Embodiment 88. [Embodiment 95] A genetically modified cell expressing the CAR described in any one of embodiments 59 to 87 on its cell surface membrane. [Embodiment 96] A genetically modified cell, which is an immune cell, as described in Embodiment 95. [Embodiment 97] Genetically modified cells according to embodiment 96, wherein the immune cells are derived from T lymphocytes. [Embodiment 98] Use of genetically modified cells according to any one of embodiments 95 to 97 for the treatment of cancer or autoimmune disease. [Embodiment 99] The use according to Embodiment 57 or 98, wherein the cancer is a hematological malignancy. [Embodiment 100] The use according to Embodiment 99, wherein the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. [Embodiment 101] The use according to Embodiment 100, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). [Embodiment 102] The use according to Embodiment 100, wherein the lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. [Embodiment 103] A method for treating cancer in a subject, comprising administering genetically modified cells described in any one of embodiments 92 to 97 to the subject. [Embodiment 104] The method according to embodiment 58 or 103, wherein the cancer is a hematological malignancy. [Embodiment 105] The method according to Embodiment 104, wherein the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. [Embodiment 106] The method according to Embodiment 105, wherein the leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). [Embodiment 107] The method according to Embodiment 105, wherein the lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. [Embodiment 108] - The first antigen-binding site; - Amino acid linkers containing a polypeptide hinge derived from human CD8; and - Second antigen-binding site; A multivalent antibody containing [specific antibody type]. [Embodiment 109] A polyvalent antibody according to Embodiment 108, wherein the polypeptide hinge derived from human CD8 contains SEQ ID NO: 112. [Embodiment 110] A polyvalent antibody according to Embodiment 108 or 109, wherein the amino acid linker further comprises at least one G4S at the N-terminus of a human CD8-derived polypeptide hinge and at least one G4S at the C-terminus of a human CD8-derived polypeptide hinge. [Embodiment 111] A polyvalent antibody according to any one of embodiments 108 to 110, wherein the amino acid linker is at least 47 residues long, preferably at least 52 residues long, preferably at least 57 residues long, more preferably at least 62 residues long, and even more preferably at least 67 residues long. [Embodiment 112] A polyvalent antibody according to any one of embodiments 108 to 111, wherein the amino acid linker includes SEQ ID NO: 111, 112, and 118 in the direction from the N-terminus to the C-terminus. [Embodiment 113] A polyvalent antibody according to any one of embodiments 108 to 112, wherein the first antigen-binding portion specifically binds to human CD22. [Embodiment 114] The first antigen-binding site is V H H, V NAR A polyvalent antibody according to Embodiment 113, which is either scVF or scVF. [Embodiment 115] A polyvalent antibody according to any one of embodiments 108 to 111, wherein the first antigen-binding portion is an sdAb according to any one of embodiments 1 to 27. [Embodiment 116] A polyvalent antibody according to any one of embodiments 108 to 115, wherein the second antigen-binding moiety specifically binds to a cell surface marker of an immune cell. [Embodiment 117] A polyvalent antibody according to Embodiment 116, wherein the cell surface marker of immune cells includes a T cell marker. [Embodiment 118] A polyvalent antibody according to Embodiment 117, comprising the T cell marker human CD3. [Embodiment 119] The second antigen-binding site is V H H, V NAR A polyvalent antibody according to any one of embodiments 108 to 118, which is either scVF or scVF.

[0048] Next, with reference to the attached drawings, embodiments of the present disclosure will be described as mere examples. [Brief explanation of the drawing]

[0049] [Figure 1] Figure 1 shows the structure of the human CD22 molecule encoded by the CD22 gene located on chromosome 19q13.12. [Figure 2] Figure 2 shows SDS-PAGE of IMAC-purified CD22 extracellular domains (CD22-ECD) from two different expression batches under non-reducible and reduced conditions. [Figure 3] Figure 3 shows the rama heavy chain immune response to CD22-ECD from the final blood sample, along with preimmune serum as a negative control. [Figure 4A] Figure 4A shows the first portion of the amino acid sequence alignment of 27 VHH sequences. CDR sequences VHH that have distinguishable sequence similarities between CDRs are enclosed in boxes. [Figure 4B] Figure 4B shows the second part of the sequence alignment of the 27 VHHs, continuing from Figure 4A. [Figure 5] Figure 5 shows the SDS-PAGE of 14 anti-CD22 VHH antibodies expressed in TG1 Escherichia coli (E. coli) and purified by IMAC. [Figure 6] Figure 6 shows the binding of biotin-conjugated anti-CD22 VHH to CD22-expressing tumor cells (Raji, left panel), Ramos cells (center panel), or Ramos cells manipulated to lack CD22 expression by CRISPR gene knockout (right panel). [Figure 7] Figure 7 is a schematic diagram showing the perceptive binding of the sdAb described in Example 1 to the subdomains of the CD22 ectodomain at cell surface locations, based on the epitope mapping / binning results from Tables 5 and 6. [Figure 8] Figure 8 shows the results of the CAR-Jurkat assay when Jurkat cells were transiently electroporated with various CD22 single-domain antibody CAR plasmids and cultured alone or co-cultured with CD22-positive (Ramos) or CD22-negative (Ramos-CD22ko) cell lines. [Figure 9] Figure 9 shows the results of a CAR-T tonic activation assay, in which various CAR constructs were introduced into primary T cells derived from donor blood, and target-independent proliferation was examined. [Figure 10] Figure 10 shows the results of CAR-T target-specific activation assays performed using donor blood-derived T cells into which various CD22 single-domain antibodies or control (FMC63) CAR constructs were introduced. [Figure 11]Figure 11 shows the results of CAR-T antigen-specific target cell growth inhibition assays performed using donor blood-derived T cells transfected with various CD22 single-domain antibodies or control (FMC63) CAR constructs described herein. Mocks refer to unmodified donor-derived T cells without CAR expression, subjected to similar treatment conditions. CAR-T cells were co-cultured with CD22+ target cells (left graph - Raji target, center graph - Ramos target) or CD22-negative target cells (right graph - Ramos-CD22ko target) and observed using live fluorescence microscopy. [Figure 12] Figure 12 shows the results of CAR-T target-specific serial killing assays performed using donor blood-derived T cells into which various CD22 single-domain antibody CAR constructs prepared as described herein were introduced. Mock cells refer to unmodified donor-derived T cells without CAR expression, subjected to similar treatment conditions. [Figure 13] Figure 13 shows the results of a consistency analysis and comparison with a benchmark CD22-targeted scFv CAR for single-domain antibody-targeted CAR-T cells generated from two separate donors as described herein. [Figure 14] Figure 14 shows the results of direct tumor lysis measurements using a radioactive chromium (51Cr) release assay. [Figure 15] Figure 15 shows the results of CAR-T cell-induced direct tumor lysis using a chromium release assay at different effector:target ratios. [Figure 16] Figure 16 shows the results of direct tumor lysis measurements using a chromium release assay from CAR-T cells after restimulation with CD22-carrying tumor cells. [Figure 17] Figure 17 shows the schema of the experimental protocol for the in vivo model. [Figure 18] Figure 18 shows the results of survival analysis of NSG mice inoculated with Ramos-Luc and subsequently treated with various CAR-T cells. [Figure 19]Figure 19 shows the tumor burden results of mice inoculated with Ramos-FLUC and treated with various CAR-T cells. [Figure 20] Figure 20 shows the results of different phenotypic tumor loading, total CAR-T cell population, and circulating CAR-T cell population in peripheral blood of mice inoculated with Ramos-FLUC and treated with various CAR-T cells. [Figure 21] Figure 21 shows the molecular structure of a CD22-specific single-domain antibody bispecific T cell engager protein; the 5' end of the DNA construct contains a CD22-sdAb sequence, followed by a linker sequence that can have various compositions, and then a CD3-specific single-chain variable fragment. [Figure 22] Figure 22 shows the results of a Jurkat cell bispecific T cell engager activation activity assay when HEK293T supernatant containing various bispecific T cell engager molecules was placed on a co-culture containing Jurkat cells and CD22-positive (Ramos) or CD22-negative (U87vIII) target cells. [Figure 23] Figure 23 shows the results of a bispecific T cell engager activity assay using primary human T cells co-cultured with CD22-positive target cells (Ramos). [Figure 24] Figure 24 shows the timing of treatment and testing in in vivo studies of CAR-T constructs. [Figure 25] Figure 25 shows the survival rates of mice in the treatments and tests shown in Figure 24. [Figure 26] Figure 26 shows the CAR-T cells counted in the peripheral blood of mice during the treatment and testing described in Figure 24. [Figure 27] Figure 27 shows the survival rate of mice after initial tumor challenge and CAR-T treatment. [Figure 28] Figure 28 shows the survival rate of mice after the second attempt. [Figure 29] Figure 29 shows a graph of tumor growth after the challenge. [Figure 30]Figure 30 shows the molecular structure of the multi-binder domain containing the CD22-specific CAR molecule. [Figure 31] Figure 31 shows the results of the CAR-Jurkat assay when Jurkat cells were transiently electroporated with various CAR plasmids, including single and multi-binder types. [Figure 32] Figure 32 shows the results of a similar CAR activation test using primary T cells derived from human blood into which a lentiviral vector encoding a multi-sdAb-containing CAR construct had been introduced. [Figure 33] Figure 33 shows the molecular structures of a CD22-specific CAR (left), a BCMA-specific CAR (right), or a tandem CD22-BCMA-CAR molecule (center). [Figure 34] Figure 34 shows the results of a CAR-Jurkat assay in which Jurkat cells were transiently electroporated with various CD22, BCMA, or CD22 / BCMA-specific CAR plasmids and cultured either alone or co-cultured with BCMA+ / CD22+(Ramos), BCMAlow / CD22+(NALM6), or BCMA+ / CD22-(Ramos-CD22ko) target cell lines to examine their activation state (CD69 expression). [Modes for carrying out the invention]

[0050] Detailed explanation Generally, this disclosure provides anti-CD22 single-domain antibodies (sdAbs) produced by immunizing llamas with the extracellular domain of a dominant human CD22 isoform. By constructing a library of heavy chain repertoire, the VHH antibody domain specific to this immunogen was isolated by phage panning. The first 27 exemplary antibodies produced consist of CDR1, CDR2, and CDR3 sequences corresponding to SEQ ID NO: 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, 22-24, 25-27, 28-30, 31-33, 34-36, 37-39, 40-42, 43-45, 46-48, 49-51, 52-54, 55-57, 58-60, 61-63, 64-66, 67-69, 70-72, 73-75, 75-78, and 79-81, respectively, and antibodies having related sequences are also described and included. Also provided are multivalent antibodies containing any one of the sdAbs, such as bispecific T cell engagers, bispecific killer cell engagers (BiKEs), and triplicate killer cell engagers (TriKEs). Furthermore, chimeric antigen receptors (CARs) for CAR-T therapy containing any one of the aforementioned sdAbs are also described.

[0051] Single-domain antibodies and polypeptides containing them Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. sdAbs are derived from heavy-chain antibodies found in camelid species (camels, llamas, dromedary camels, alpacas, guanacos, etc.) using molecular biological techniques. H H fragment (referred to as "V" in this specification) H Also known as "H" or "VHH". Another example is V, which originates from heavy chain antibodies found in cartilaginous fish such as sharks. NAR Fragments are included. sdAbs are also synthesized using engineering techniques from the heavy / light chains of conventional immunoglobulin G (IgG).

[0052] V HH molecules are about one-tenth the size of IgG molecules. These single polypeptides are generally very stable and can often withstand extreme pH and temperature conditions that can be problematic for conventional antibodies and antibody fragments. Furthermore, V H H tends to be more resistant to the action of proteases. In addition, V H Expressing H in vitro tends to yield well-folded / functional VHH in high yield. Furthermore, heavy chain antibodies produced in camelid species and their modified fragments (i.e., VHH) may recognize cryptic or hidden epitopes that are inaccessible to larger conventional antibodies and antibody fragments produced in vitro via antibody libraries or obtained by immunization of other mammals.

[0053] In one embodiment, an isolated single-domain antibody (sdAb) that specifically binds to human CD22 is provided, the sdAb comprising the following sequence: a) CDR1 amino acid sequence GX1X2X3DX4YX5 (SEQ ID NO: 126), where, X1 is either F or V. X2 is either T or S, X3 is L, F, or S. X4 is Y, D, or S. X5 is either V or A; CDR2 amino acid sequence X6X7X8X9X 10 GX 11 T (SEQ ID NO: 127), here, X6 is either I or M. X7 is T, R, G, or S. X8 is either S or N. X9 is either D or S. X 10 It is either D or does not exist. X 11 is V or A; and CDR3 amino acid sequence AVDKPFYDGGX 12 X13 YTCPVDFX 14 S (SEQ ID NO: 128), here, X 12 is I, N, Y, or S, X 13 is Q, Y, R, or L, X 14 is either D or G; b) CDR1 amino acid sequence GSX1FX2X3X4X5V (SEQ ID NO: 129), where, X1 is either I or T. X2 is either R or does not exist. X3 is R, S, or I. X4 is I, S, or T. X5 is A, S, or T; The CDR2 amino acid sequence is ITSX6GX7X8 (SEQ ID NO: 130), where, X6 is G, S, or A. X7 is E, D, or S. X8 is T or S; and CDR3 amino acid sequence NAX9X 10 GX 11 X 12 X 13 X 14 (SEQ ID NO: 131), here, X9 is either K or Q. X 10 is either W or Y, X 11 is Q, R, or G, X 12 is Y, D, or R, X 13 is E or S, X 14 is Y, D, or H; c) CDR1 amino acid sequence GRIX1RSYV (SEQ ID NO: 132), where, X1 is either F or S; The CDR2 amino acid sequence is IGX2SDT (SEQ ID NO: 133), where, X2 is W or C; and The CDR3 amino acid sequence is AX3X4SPPYGPQRDEFX5Y (SEQ ID NO: 134), where, X3 is either A or E. X4 is either N or Y. X5 is either G or D; d) CDR1 amino acid sequence GX1TX2SVYX3 (SEQ ID NO: 135), where, X1 is either R or G. X2 is either S or F. X3 is either G or T; The CDR2 amino acid sequence is X4X5X6SX7GX8T (SEQ ID NO: 136), where, X4 is M or I, X5 is either S or R. X6 is either W or G. X7 is either G or does not exist. X8 is P or G; and The CDR3 amino acid sequence is AVRIRRTLX9EPLTKETLYDY (SEQ ID NO: 137), where, X9 is L or V; e) CDR1 amino acid sequence as described in SEQ ID NO: 49, The CDR2 amino acid sequence described in SEQ ID NO: 50, and CDR3 amino acid sequence as described in SEQ ID NO: 51; f) CDR1 amino acid sequence as described in SEQ ID NO: 4, The CDR2 amino acid sequence described in SEQ ID NO: 5, and CDR3 amino acid sequence as described in SEQ ID NO: 6; g) CDR1 amino acid sequence as described in SEQ ID NO: 7, The CDR2 amino acid sequence described in SEQ ID NO: 8, and CDR3 amino acid sequence as described in SEQ ID NO: 9; h) CDR1 amino acid sequence as described in SEQ ID NO: 13, The CDR2 amino acid sequence described in SEQ ID NO: 14, and CDR3 amino acid sequence as described in SEQ ID NO: 15; i) CDR1 amino acid sequence described in SEQ ID NO: 16, The CDR2 amino acid sequence described in SEQ ID NO: 17, and CDR3 amino acid sequence as described in SEQ ID NO: 18; j) CDR1 amino acid sequence described in SEQ ID NO: 40, The CDR2 amino acid sequence described in SEQ ID NO: 41, and CDR3 amino acid sequence as described in SEQ ID NO: 42; k) CDR1 amino acid sequence described in SEQ ID NO: 55, The CDR2 amino acid sequence described in SEQ ID NO: 56, and CDR3 amino acid sequence as described in SEQ ID NO: 57; l) CDR1 amino acid sequence as described in SEQ ID NO: 64, The CDR2 amino acid sequence described in SEQ ID NO: 65, and CDR3 amino acid sequence as described in SEQ ID NO: 66; or m) CDR1 amino acid sequence described in SEQ ID NO: 70, The CDR2 amino acid sequence described in SEQ ID NO: 71, and CDR3 amino acid sequence as described in SEQ ID NO: 72.

[0054] In the above: Group a) provides consensus sequences defined herein by antibodies referred to as hCD221ug-80, hCD22100ng-2, hCD221ug-74, hCD22100ng-66, hCD221ug-6, hCD22pas-10, and hCD22pass-33; Group b) provides consensus sequences defined by antibodies referred to herein as hCD221ug-77, hCD221ug-87, hCD221ug-75, hCD221ug-93, hCD22pas-82, and hCD22pas-23; Group c) provides consensus sequences defined by antibodies referred to herein as hCD22pas-32, hCD221ug-14, and hCD22pas-55; Group d) provides consensus sequences defined by antibodies referred to herein as hCD22pas-79 and hCD22pas-72; Group f) is defined by the CDR from the antibody called hCD22pas-16; Group f) is defined by the CDR from the antibody called hCD221ug-10; Group g) is defined by the CDR from the antibody called hCD221ug-13; Group h) is defined by the CDR from the antibody called hCD221ug-36; Group i) is defined by the CDR from the antibody called hCD221ug-61; Group j) is defined by the CDR from the antibody called hCD22100ug-62; Group k) is defined by the CDR from an antibody called hCD22pas-24; Group l) is defined by CDR from an antibody called hCD22pas-48; and Group m) is defined by the CDR from the antibody called hCD22pas-64.

[0055] A "CDR" or "complementarity-determining region" is a portion of the variable chain of an immunoglobulin that aggregates to form a paratope, thereby conferring binding specificity and affinity to an antibody. As used herein, this term refers to a CDR mapped to an sdAb according to the standards or rules established by IMGT® (International ImMunoGeneTics Information System).

[0056] The antibodies described herein were produced against the recombinant extracellular domain (ECD) of the dominant human CD22β isoform. An example of the mRNA sequence of this isoform can be found in GenBank entry NM_001771.4, where amino acids 1–19 of the encoded protein correspond to the leader sequence and amino acids 20–687 correspond to the ECD (see also UniProt entry P20273).

[0057] In another embodiment, an isolated VHH single-domain antibody (sdAb) that specifically binds to human CD22 is provided, the sdAb comprising the following sequence: A) The CDR1 amino acid sequence described in SEQ ID NO: 1, the CDR2 amino acid sequence described in SEQ ID NO: 2, and the CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6); The CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10); The CDR1 amino acid sequence described in SEQ ID NO: 7, the CDR2 amino acid sequence described in SEQ ID NO: 8, and the CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13); The CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14); The CDR1 amino acid sequence described in SEQ ID NO: 13, the CDR2 amino acid sequence described in SEQ ID NO: 14, and the CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36); The CDR1 amino acid sequence described in SEQ ID NO: 16, the CDR2 amino acid sequence described in SEQ ID NO: 17, and the CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61); The CDR1 amino acid sequence described in SEQ ID NO: 19, the CDR2 amino acid sequence described in SEQ ID NO: 20, and the CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74); The CDR1 amino acid sequence described in SEQ ID NO: 22, the CDR2 amino acid sequence described in SEQ ID NO: 23, and the CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75); The CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77); The CDR1 amino acid sequence described in SEQ ID NO: 28, the CDR2 amino acid sequence described in SEQ ID NO: 29, and the CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80); The CDR1 amino acid sequence described in SEQ ID NO: 31, the CDR2 amino acid sequence described in SEQ ID NO: 32, and the CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87); The CDR1 amino acid sequence described in SEQ ID NO: 34, the CDR2 amino acid sequence described in SEQ ID NO: 35, and the CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93); The CDR1 amino acid sequence described in SEQ ID NO: 37, the CDR2 amino acid sequence described in SEQ ID NO: 38, and the CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2); The CDR1 amino acid sequence described in SEQ ID NO: 40, the CDR2 amino acid sequence described in SEQ ID NO: 41, and the CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62); The CDR1 amino acid sequence described in SEQ ID NO: 43, the CDR2 amino acid sequence described in SEQ ID NO: 44, and the CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66); The CDR1 amino acid sequence described in SEQ ID NO: 46, the CDR2 amino acid sequence described in SEQ ID NO: 47, and the CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10); The CDR1 amino acid sequence described in SEQ ID NO: 49, the CDR2 amino acid sequence described in SEQ ID NO: 50, and the CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16); The CDR1 amino acid sequence described in SEQ ID NO: 52, the CDR2 amino acid sequence described in SEQ ID NO: 53, and the CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23); The CDR1 amino acid sequence described in SEQ ID NO: 55, the CDR2 amino acid sequence described in SEQ ID NO: 56, and the CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24); The CDR1 amino acid sequence described in SEQ ID NO: 58, the CDR2 amino acid sequence described in SEQ ID NO: 59, and the CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32); The CDR1 amino acid sequence described in SEQ ID NO: 61, the CDR2 amino acid sequence described in SEQ ID NO: 62, and the CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33); The CDR1 amino acid sequence described in SEQ ID NO: 64, the CDR2 amino acid sequence described in SEQ ID NO: 65, and the CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48); The CDR1 amino acid sequence described in SEQ ID NO: 67, the CDR2 amino acid sequence described in SEQ ID NO: 68, and the CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55); The CDR1 amino acid sequence described in SEQ ID NO: 70, the CDR2 amino acid sequence described in SEQ ID NO: 71, and the CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64); The CDR1 amino acid sequence described in SEQ ID NO: 73, the CDR2 amino acid sequence described in SEQ ID NO: 74, and the CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72); The CDR1 amino acid sequence described in SEQ ID NO: 76, the CDR2 amino acid sequence described in SEQ ID NO: 77, and the CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79); or The CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82).

[0058] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 1, the CDR2 amino acid sequence described in SEQ ID NO: 2, and the CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6).

[0059] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10).

[0060] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 7, the CDR2 amino acid sequence described in SEQ ID NO: 8, and the CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13).

[0061] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14).

[0062] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 13, the CDR2 amino acid sequence described in SEQ ID NO: 14, and the CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36).

[0063] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 16, the CDR2 amino acid sequence described in SEQ ID NO: 17, and the CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61).

[0064] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 19, the CDR2 amino acid sequence described in SEQ ID NO: 20, and the CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74).

[0065] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 22, the CDR2 amino acid sequence described in SEQ ID NO: 23, and the CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75).

[0066] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77).

[0067] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 28, the CDR2 amino acid sequence described in SEQ ID NO: 29, and the CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80).

[0068] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 31, the CDR2 amino acid sequence described in SEQ ID NO: 32, and the CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87).

[0069] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 34, the CDR2 amino acid sequence described in SEQ ID NO: 35, and the CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93).

[0070] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 37, the CDR2 amino acid sequence described in SEQ ID NO: 38, and the CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2).

[0071] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 40, the CDR2 amino acid sequence described in SEQ ID NO: 41, and the CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62).

[0072] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 43, the CDR2 amino acid sequence described in SEQ ID NO: 44, and the CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66).

[0073] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 46, the CDR2 amino acid sequence described in SEQ ID NO: 47, and the CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10).

[0074] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 49, the CDR2 amino acid sequence described in SEQ ID NO: 50, and the CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16).

[0075] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 52, the CDR2 amino acid sequence described in SEQ ID NO: 53, and the CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23).

[0076] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 55, the CDR2 amino acid sequence described in SEQ ID NO: 56, and the CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24).

[0077] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 58, the CDR2 amino acid sequence described in SEQ ID NO: 59, and the CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32).

[0078] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 61, the CDR2 amino acid sequence described in SEQ ID NO: 62, and the CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33).

[0079] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 64, the CDR2 amino acid sequence described in SEQ ID NO: 65, and the CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48).

[0080] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 67, the CDR2 amino acid sequence described in SEQ ID NO: 68, and the CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55).

[0081] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 70, the CDR2 amino acid sequence described in SEQ ID NO: 71, and the CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64).

[0082] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 73, the CDR2 amino acid sequence described in SEQ ID NO: 74, and the CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72).

[0083] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 76, the CDR2 amino acid sequence described in SEQ ID NO: 77, and the CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79).

[0084] In one embodiment, the antibody comprises the CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82).

[0085] In another embodiment, an isolated VHH single-domain antibody (sdAb) that specifically binds to human CD22 is provided, the sdAb comprising the following sequence: A) The CDR1 amino acid sequence described in SEQ ID NO: 1, the CDR2 amino acid sequence described in SEQ ID NO: 2, and the CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6); The CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10); The CDR1 amino acid sequence described in SEQ ID NO: 7, the CDR2 amino acid sequence described in SEQ ID NO: 8, and the CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13); The CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14); The CDR1 amino acid sequence described in SEQ ID NO: 13, the CDR2 amino acid sequence described in SEQ ID NO: 14, and the CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36); The CDR1 amino acid sequence described in SEQ ID NO: 16, the CDR2 amino acid sequence described in SEQ ID NO: 17, and the CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61); The CDR1 amino acid sequence described in SEQ ID NO: 19, the CDR2 amino acid sequence described in SEQ ID NO: 20, and the CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74); The CDR1 amino acid sequence described in SEQ ID NO: 22, the CDR2 amino acid sequence described in SEQ ID NO: 23, and the CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75); The CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77); The CDR1 amino acid sequence described in SEQ ID NO: 28, the CDR2 amino acid sequence described in SEQ ID NO: 29, and the CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80); The CDR1 amino acid sequence described in SEQ ID NO: 31, the CDR2 amino acid sequence described in SEQ ID NO: 32, and the CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87); The CDR1 amino acid sequence described in SEQ ID NO: 34, the CDR2 amino acid sequence described in SEQ ID NO: 35, and the CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93); The CDR1 amino acid sequence described in SEQ ID NO: 37, the CDR2 amino acid sequence described in SEQ ID NO: 38, and the CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2); The CDR1 amino acid sequence described in SEQ ID NO: 40, the CDR2 amino acid sequence described in SEQ ID NO: 41, and the CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62); The CDR1 amino acid sequence described in SEQ ID NO: 43, the CDR2 amino acid sequence described in SEQ ID NO: 44, and the CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66); The CDR1 amino acid sequence described in SEQ ID NO: 46, the CDR2 amino acid sequence described in SEQ ID NO: 47, and the CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10); The CDR1 amino acid sequence described in SEQ ID NO: 49, the CDR2 amino acid sequence described in SEQ ID NO: 50, and the CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16); The CDR1 amino acid sequence described in SEQ ID NO: 52, the CDR2 amino acid sequence described in SEQ ID NO: 53, and the CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23); The CDR1 amino acid sequence described in SEQ ID NO: 55, the CDR2 amino acid sequence described in SEQ ID NO: 56, and the CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24); The CDR1 amino acid sequence described in SEQ ID NO: 58, the CDR2 amino acid sequence described in SEQ ID NO: 59, and the CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32); The CDR1 amino acid sequence described in SEQ ID NO: 61, the CDR2 amino acid sequence described in SEQ ID NO: 62, and the CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33); The CDR1 amino acid sequence described in SEQ ID NO: 64, the CDR2 amino acid sequence described in SEQ ID NO: 65, and the CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48); The CDR1 amino acid sequence described in SEQ ID NO: 67, the CDR2 amino acid sequence described in SEQ ID NO: 68, and the CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55); The CDR1 amino acid sequence described in SEQ ID NO: 70, the CDR2 amino acid sequence described in SEQ ID NO: 71, and the CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64); The CDR1 amino acid sequence described in SEQ ID NO: 73, the CDR2 amino acid sequence described in SEQ ID NO: 74, and the CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72); The CDR1 amino acid sequence described in SEQ ID NO: 76, the CDR2 amino acid sequence described in SEQ ID NO: 77, and the CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79); or The CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82); or B) CDR1, CDR2, and CDR3 amino acid sequences that are at least 80% identical to the CDR1, CDR2, and CDR3 sequences defined in any one of Part A) i) to xxviii).

[0086] In one embodiment, in B), the CDR1, CDR2, and CDR3 amino acid sequences are at least 90% identical to the CDR1, CDR2, and CDR3 sequences defined in any one of i) to xxviii) of Part A). In one embodiment, in B), the CDR1, CDR2, and CDR3 amino acid sequences are at least 95% identical to the CDR1, CDR2, and CDR3 sequences defined in any one of i) to xxviii) of Part A). In one embodiment, in B), the CDR1, CDR2, and CDR3 amino acid sequences have up to three substitutions compared to the CDR1, CDR2, and CDR3 sequences defined in any one of i) to xxviii) of Part A). In one embodiment, in B), the CDR1, CDR2, and CDR3 amino acid sequences have up to two substitutions compared to the CDR1, CDR2, and CDR3 sequences defined in any one of i) to xxviii) of Part A). In one embodiment, in B), the CDR1, CDR2, and CDR3 amino acid sequences have at most one substitution compared to the CDR1, CDR2, and CDR3 sequences defined in any one of i) to xxviii) of Part A). In one embodiment, the sequence differences from the sequences described in A) are conservative sequence substitutions.

[0087] The term “conservative amino acid substitution” is known in the art and is defined herein as follows (candidate amino acids that can be conservedly substituted are shown in parentheses): Ala (Gly, Ser); Arg (Gly, Gln); Asn (Gln; His); Asp (Glu); Cys (Ser); Gln (Asn, Lys); Glu (Asp); Gly (Ala, Pro); His (Asn; Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg; Gln); Met (Leu, Ile); Phe (Met, Leu, Tyr); Ser (Thr; Gly); Thr (Ser; Val); Trp (Tyr); Tyr (Trp; Phe); Val (Ile; Leu).

[0088] Sequence variants according to specific embodiments are intended to encompass molecules whose binding affinity and / or specificity are substantially altered compared to the parent molecule from which they are derived. Such parameters can be readily tested, for example, using the techniques described herein and techniques known in the art. Such embodiments may encompass sequence substitutions, insertions, or deletions.

[0089] In another embodiment, an isolated VHH single-domain antibody (sdAb) that specifically binds to human CD22 is provided, the sdAb comprising the following sequence: A) CDR3 amino acid sequence as described in SEQ ID NO: 3; CDR3 amino acid sequence as described in SEQ ID NO: 6; CDR3 amino acid sequence as described in SEQ ID NO: 9; CDR3 amino acid sequence as described in SEQ ID NO: 12; CDR3 amino acid sequence as described in SEQ ID NO: 15; CDR3 amino acid sequence as described in SEQ ID NO: 18; CDR3 amino acid sequence as described in SEQ ID NO: 21; CDR3 amino acid sequence as described in SEQ ID NO: 24; CDR3 amino acid sequence as described in SEQ ID NO: 27; CDR3 amino acid sequence as described in SEQ ID NO: 30; CDR3 amino acid sequence as described in SEQ ID NO: 33; CDR3 amino acid sequence as described in SEQ ID NO: 36; CDR3 amino acid sequence as described in SEQ ID NO: 39; CDR3 amino acid sequence as described in SEQ ID NO: 42; CDR3 amino acid sequence as described in SEQ ID NO: 45; CDR3 amino acid sequence as described in SEQ ID NO: 48; CDR3 amino acid sequence as described in SEQ ID NO: 51; The CDR3 amino acid sequence set forth in SEQ ID NO: 54; The CDR3 amino acid sequence set forth in SEQ ID NO: 57; The CDR3 amino acid sequence set forth in SEQ ID NO: 60; The CDR3 amino acid sequence set forth in SEQ ID NO: 63; The CDR3 amino acid sequence set forth in SEQ ID NO: 66; The CDR3 amino acid sequence set forth in SEQ ID NO: 69; The CDR3 amino acid sequence set forth in SEQ ID NO: 72; The CDR3 amino acid sequence set forth in SEQ ID NO: 75; The CDR3 amino acid sequence set forth in SEQ ID NO: 79; or The CDR3 amino acid sequence set forth in SEQ ID NO: 81.

[0090] Recognizing that CDR3 is often the major determinant of V H H sdAb binding, it will be appreciated that other CDRs can be mutagenized or diversified in other ways and the resulting library (or candidate molecules) screened to find antibodies that bind to CD22 and / or cross-compete with the parental molecule for binding to CD22. These embodiments are intended to cover, inter alia, the molecules identified by this method.

[0091] In one embodiment, the isolated sdAb comprises the following sequences: The CDR1 amino acid sequence set forth in SEQ ID NO: 1, and the CDR3 amino acid sequence set forth in SEQ ID NO: 3; The CDR1 amino acid sequence set forth in SEQ ID NO: 4, and the CDR3 amino acid sequence set forth in SEQ ID NO: 6; The CDR1 amino acid sequence set forth in SEQ ID NO: 7, and the CDR3 amino acid sequence set forth in SEQ ID NO: 9; The CDR1 amino acid sequence set forth in SEQ ID NO: 10, and the CDR3 amino acid sequence set forth in SEQ ID NO: 12; The CDR1 amino acid sequence described in SEQ ID NO: 13, and the CDR3 amino acid sequence described in SEQ ID NO: 15; The CDR1 amino acid sequence described in SEQ ID NO: 16, and the CDR3 amino acid sequence described in SEQ ID NO: 18; The CDR1 amino acid sequence described in SEQ ID NO: 19, and the CDR3 amino acid sequence described in SEQ ID NO: 21; The CDR1 amino acid sequence described in SEQ ID NO: 22, and the CDR3 amino acid sequence described in SEQ ID NO: 24; The CDR1 amino acid sequence described in SEQ ID NO: 25, and the CDR3 amino acid sequence described in SEQ ID NO: 27; The CDR1 amino acid sequence described in SEQ ID NO: 28, and the CDR3 amino acid sequence described in SEQ ID NO: 30; The CDR1 amino acid sequence described in SEQ ID NO: 31, and the CDR3 amino acid sequence described in SEQ ID NO: 33; The CDR1 amino acid sequence described in SEQ ID NO: 34, and the CDR3 amino acid sequence described in SEQ ID NO: 36; The CDR1 amino acid sequence described in SEQ ID NO: 37, and the CDR3 amino acid sequence described in SEQ ID NO: 39; The CDR1 amino acid sequence described in SEQ ID NO: 40, and the CDR3 amino acid sequence described in SEQ ID NO: 42; The CDR1 amino acid sequence described in SEQ ID NO: 43, and the CDR3 amino acid sequence described in SEQ ID NO: 45; The CDR1 amino acid sequence described in SEQ ID NO: 46, and the CDR3 amino acid sequence described in SEQ ID NO: 48; The CDR1 amino acid sequence described in SEQ ID NO: 49, and the CDR3 amino acid sequence described in SEQ ID NO: 51; The CDR1 amino acid sequence described in SEQ ID NO: 52, and the CDR3 amino acid sequence described in SEQ ID NO: 54; The CDR1 amino acid sequence described in SEQ ID NO: 55, and the CDR3 amino acid sequence described in SEQ ID NO: 57; The CDR1 amino acid sequence described in SEQ ID NO: 58, and the CDR3 amino acid sequence described in SEQ ID NO: 60; The CDR1 amino acid sequence described in SEQ ID NO: 61, and the CDR3 amino acid sequence described in SEQ ID NO: 63; The CDR1 amino acid sequence described in SEQ ID NO: 64, and the CDR3 amino acid sequence described in SEQ ID NO: 66; The CDR1 amino acid sequence described in SEQ ID NO: 67, and the CDR3 amino acid sequence described in SEQ ID NO: 69; The CDR1 amino acid sequence described in SEQ ID NO: 70, and the CDR3 amino acid sequence described in SEQ ID NO: 72; The CDR1 amino acid sequence described in SEQ ID NO: 73, and the CDR3 amino acid sequence described in SEQ ID NO: 75; The CDR1 amino acid sequence described in SEQ ID NO: 76, and the CDR3 amino acid sequence described in SEQ ID NO: 78; or The CDR1 amino acid sequence described in SEQ ID NO: 79, and the CDR3 amino acid sequence described in SEQ ID NO: 81.

[0092] These embodiments are intended to encompass, among other things, embodiments in which molecules are recovered after mutagenesis / diversification of CDR2 and screening for variant molecules (defined from the parent molecule) that bind to and / or cross-compete with the parent molecule for binding to CD22. As described above, it is also possible to screen libraries or test individual candidate molecules.

[0093] In one embodiment, sdAb includes either A) one amino acid sequence with SEQ ID NO: 85-112 and 120-125, or B) an amino acid sequence that is at least 80% identical to one with SEQ ID NO: 82-108 and 120-125 over its entire length. In one embodiment, the amino acid sequence of B) is at least 85% identical to one of the amino acid sequences of A) over its entire length. In one embodiment, the amino acid sequence of B) is at least 90% identical to one of the amino acid sequences of A) over its entire length. In one embodiment, the amino acid sequence of B) is at least 95% identical to one of the amino acid sequences of A) over its entire length. In one embodiment, the amino acid sequence of B) is at least 98% identical to one of the amino acid sequences of A) over its entire length. In one embodiment, the amino acid sequence of B) is at least 98% identical to one of the amino acid sequences of A) over its entire length. In some of these embodiments, the sequence differences from sequence A) are outside the CDR sequence.

[0094] In one embodiment, sdAb contains one amino acid sequence from A) SEQ ID NO: 82-108 and 120-125. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 82. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 83. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 84. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 85. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 86. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 87. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 88. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 89. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 90. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 91. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 92. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 93. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 94. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 95. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 96. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 97. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 98. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 99. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 100. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 101. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 102. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 103. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 104. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 105. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 106. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 107. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 108. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 120. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 121. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 122. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 123. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 124. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 125.

[0095] In one embodiment, sdAb includes SEQ ID NO: 82. In one embodiment, sdAb includes SEQ ID NO: 83. In one embodiment, sdAb includes SEQ ID NO: 84. In one embodiment, sdAb includes SEQ ID NO: 85. In one embodiment, sdAb includes SEQ ID NO: 86. In one embodiment, sdAb includes SEQ ID NO: 87. In one embodiment, sdAb includes SEQ ID NO: 88. In one embodiment, sdAb includes SEQ ID NO: 89. In one embodiment, sdAb includes SEQ ID NO: 90. In one embodiment, sdAb includes SEQ ID NO: 91. In one embodiment, sdAb includes SEQ ID NO: 92. In one embodiment, sdAb includes SEQ ID NO: 93. In one embodiment, sdAb includes SEQ ID NO: 94. In one embodiment, sdAb includes SEQ ID NO: 95. In one embodiment, sdAb includes SEQ ID NO: 96. In one embodiment, sdAb includes SEQ ID NO: 97. In one embodiment, sdAb includes SEQ ID NO: 98. In one embodiment, sdAb includes SEQ ID NO: 99. In one embodiment, sdAb includes SEQ ID NO: 100. In one embodiment, sdAb includes SEQ ID NO: 101. In one embodiment, sdAb includes SEQ ID NO: 102. In one embodiment, sdAb includes SEQ ID NO: 103. In one embodiment, sdAb includes SEQ ID NO: 104. In one embodiment, sdAb includes SEQ ID NO: 105. In one embodiment, sdAb includes SEQ ID NO: 106. In one embodiment, sdAb includes SEQ ID NO: 107. In one embodiment, sdAb includes SEQ ID NO: 108. In one embodiment, sdAb includes SEQ ID NO: 120. In one embodiment, sdAb includes SEQ ID NO: 121. In one embodiment, sdAb includes SEQ ID NO: 122. In one embodiment, sdAb includes SEQ ID NO: 123. In one embodiment, sdAb includes SEQ ID NO: 124. In one embodiment, sdAb includes SEQ ID NO: 125. In one embodiment, sdAb is a Camelidae V H H sdAb. In one embodiment, sdAb is Rama V H H sdAb. In one embodiment, sdAb is a humanized camelid V H It is H.

[0096] As used herein, the term "humanized" means mutated such that it has little or no immunogenicity when administered to a human patient. Humanizing a polypeptide according to the present invention involves replacing one or more camelid amino acids with their human counterparts found in the human consensus sequence without the polypeptide losing its typical properties, i.e., without significantly affecting the antigen-binding ability of the polypeptide for which humanization is achieved. Humanized antibodies can be made using various techniques known in the art, including but not limited to CDR grafting, veneering or resurfacing, chain shuffling, etc.

[0097] In one embodiment, the sdAb has an affinity for human CD22 of 2.5×10 -7 nm or less. In one embodiment, the sdAb has an affinity for human CD22 of 3×10 -8 nm or less. In one embodiment, the sdAb has an affinity for human CD22 of 9.6×10 -9 nm or less. In one embodiment, the sdAb has an affinity for human CD22 of 9.3×10 -10 nm or less. In one embodiment, the sdAb has an affinity for human CD22 of 7×10 -12 nm or less. The binding affinity can be determined, for example, according to the assays described herein.

[0098] CD22 will be understood to contain seven Ig-like domains, numbered 1–7 from distal to proximal to the membrane (see Figure 7 for reference). In some embodiments, sdAb exhibits selective or preferential binding to one or more of these Ig-like domains. The mature human CD22 ECD extends from amino acids 20 to 687 of UniProt entry P20273. Within this subsequence, the seven Ig-like domains generally correspond to the regions of amino acids 1–119(1), 124–216(2), 223–302(3), 312–397(4), 400–481(5), 486–563(6), and 574–657(7). Elsewhere, the domains named 1–7 herein are commonly referred to as IgL-V and IgL-1 through IgL-6, respectively. For simplicity, in this specification, the domains are numbered sequentially from 1 ("first") to 7 ("seventh"). In epitope mapping, the seven fragments each contain an Ig-like domain as bins 1-7, corresponding to the regions of amino acids 1-135 (bin 1), 111-230 (bin 2), (211-317) (bin 3), 303-405 (bin 4), 391-490 (bin 5), 476-575 (bin 6), and 561-668 (bin 7).

[0099] In one embodiment, the sdAb binds to a first Ig-like domain of human CD22. In one such particular embodiment, the sdAb that binds to the first Ig-like domain includes SEQ ID NO: 91(hCD221ug-80). In one embodiment, the sdAb binds to a fourth Ig-like domain of human CD22. In one such particular embodiment, the sdAb that binds to the fourth Ig-like domain includes SEQ ID NO: 84(hCD221ug-13). In one embodiment, the sdAb binds to a sixth Ig-like domain of human CD22. In one such particular embodiment, the sdAb binding to the sixth Ig-like domain includes SEQ ID NO: 84 (hCD221ug-13), SEQ ID NO: 91 (hCD221ug-80), SEQ ID NO: 86 (hCD221ug-36), or SEQ ID NO: 105 (hCD221pas-64). In one embodiment, the sdAb binds to the seventh Ig-like domain of human CD22. In one such particular embodiment, the sdAb binding to the seventh Ig-like domain includes SEQ ID NO: 87 (hCD221ug-61), SEQ ID NO: 83 (hCD221ug-10), or SEQ ID NO: 100 (hCD22pas-24).

[0100] In one embodiment, a VHH single-domain antibody (sdAb) is provided that competes with the isolated sdAb described above for specific binding to CD22. The sdAb of the present invention can be identified by a method comprising a binding assay to evaluate whether a test antibody can cross-compete with a known antibody of the present invention for a binding site on a target molecule. For example, the antibody described above herein can be used as a reference antibody. Methods for carrying out competitive binding assays are well known in the art. For example, they include contacting a known antibody of the present invention and a target molecule under conditions in which the antibody can bind to the target molecule. The antibody / target complex is then contacted with a test antibody to evaluate the extent to which the test antibody can displace the antibody of the present invention from the antibody / target complex. Another method may include contacting a test antibody and a target molecule under conditions in which antibody binding is possible, then adding an antibody of the present invention that can bind to the target molecule, and evaluating the extent to which the antibody of the present invention can displace the test antibody from the antibody / target complex. Such antibodies can be identified by constructing new sdAbs for CD22 and screening the resulting library for cross-competition. Alternatively, one of the antibodies described herein may be used as a starting point for diversification, library construction, and screening. Further alternatives may include testing individual variants of the antibodies described herein.

[0101] In one embodiment, the sdAb as defined herein is a camelid sdAb.

[0102] In one embodiment, sdAb as defined herein is llama sdAb.

[0103] In one embodiment, the sdAb as defined herein is a humanized form of a camelid sdAb.

[0104] Table 1 shows the full-length sequences of the various sdAb disclosed herein. CDR1, CDR2, and CDR3 sequences are underlined. The identification and numbering of CDRs used herein are in accordance with IMGT (Trademark) rules.

[0105] [Table 1] TIFF0007869193000002.tif210168TIFF0007869193000003.tif106168

[0106] Table 2 provides the names of the antibodies used herein, their VHH# numbers, and the corresponding CDR1, CDR2, CDR3, and full-length sequence SEQ ID NOs for each sdAb.

[0107] [Table 2] TIFF0007869193000005.tif96159

[0108] Recombinant polypeptides In one embodiment, a recombinant polypeptide comprising an sdAb as defined herein is provided. In one embodiment, a recombinant polypeptide comprising one or more sdAbs as defined herein is provided. In one embodiment, a recombinant polypeptide comprising two or more sdAbs as defined herein is provided. In one embodiment, a recombinant polypeptide comprising two or more sdAbs as defined herein is provided.

[0109] V H H:F c fusion In one embodiment, sdAb("V") as defined herein is fused to human Fc. H H:F c A "fusion" is provided. For example, V H H:F c The fusion may contain at least CH2 and CH3 of an IgG, IgA, or IgD isotype. H H:F cThe fusion may contain at least CH2, CH3, and CH4 of an IgM or IgE isotype. Such embodiments may be useful for activating the immune system with higher-order recombinant molecules. For example, according to one embodiment, two such F c Contains V H H:F c Fusions can be assembled to form monomeric recombinant antibodies. In some embodiments, such monomeric antibodies have the ability to activate the immune system. Such monomeric antibodies may be IgG, IgA, IgD, IgE, or IgM isotypes. In one embodiment, IgA F c Contains V H H:F c The fusion can also be assembled into a recombinant dimer (secreted type). In some embodiments, polymers are also conceivable. For example, assembling five IgM monomers can form a pentamer recombinant antibody. In some embodiments, the polyvalent antibodies described herein may be assemblies of the same VHH:Fc fusion.

[0110] In some embodiments, the polyvalent antibodies described herein may be assemblies of different VHH:Fc fusions having the same binding target. For example, these may be capable of binding to different epitopes on the same target molecule. Examples include assemblies of different VHH:Fc fusions, each containing a different anti-CD22 sdAb as defined herein.

[0111] In some embodiments, the polyvalent antibodies described herein may be assemblies of a VHH:Fc fusion as defined herein (containing an anti-CD22 sdAb as defined herein) and another VHH:Fc fusion containing a paratope directed to a different target. Fusion into cargo molecules

[0112] In further embodiments, the Disclosure provides anti-CD22 sdAb as defined herein, conjugated to a cargo molecule. The cargo molecule may include a therapeutic moiety, such as a cytotoxic agent, a cell proliferation inhibitor, an anticancer agent, or a radiotherapeutic agent. In certain embodiments of the Disclosure, the antibody-drug conjugate may include a cytotoxic agent. Another particular embodiment of the Disclosure relates to an antibody-drug conjugate including a radiotherapeutic agent.

[0113] Recombinant nucleic acid molecules In one embodiment, sdAb, recombinant polypeptide, or V as defined herein H H:F c A recombinant nucleic acid molecule encoding the fusion is provided.

[0114] composition In one embodiment, a composition is provided comprising an sdAb as defined herein, or a polypeptide containing such an sdAb, together with an acceptable excipient, diluent, or carrier. In one embodiment, the composition is a pharmaceutical composition, and the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0115] Usage and Method In one embodiment, a treatment for cancer or autoimmune disease using sdAb as defined herein, or one or more V as defined herein. H H:F cThe use of antibodies containing fusions is provided. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes.

[0116] In one embodiment, a preparation of a pharmaceutical product for the treatment of cancer or autoimmune disease, comprising sdAb as defined herein, or one or more V as defined herein. H H:F c The use of antibodies containing fusions is provided. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes.

[0117] In one embodiment, for use in the treatment of cancer or autoimmune diseases, sdAb as defined herein, or one or more V as defined herein. H H:F cAntibodies containing fusions are provided. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes.

[0118] In one embodiment, sdAb as defined herein, or one or more V as defined herein. H H:F c A method is provided for treating cancer or an autoimmune disease in a subject, comprising administering an antibody containing a fusion to the subject. In one embodiment, the cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, the leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, the lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes.

[0119] Polyvalent antibodies and related embodiments In one embodiment, a polyvalent antibody containing the sdAb defined above is provided.

[0120] In this specification, "polyvalent antibody" is used to mean a molecule that contains two or more variable regions or paratopes for binding to one or more antigens within the same or different target molecules.

[0121] In some embodiments, the paratope can bind to different epitopes on the same target molecule. In some embodiments, the paratope can bind to different target molecules. In these embodiments, the polyvalent antibody is referred to as bispecific, tripspecific, or multispecific, depending on the number of paratopes with different specificities present. Since the polyvalent antibody contains one of the anti-CD22 sdAbs as defined herein, the polyvalent antibody has CD22 binding affinity.

[0122] For example, as described above, in some embodiments, the polyvalent antibody is V as defined herein. H H:F c A fusion (including sdAb as defined herein) and another V containing different paratopes that confer different specificities. H H:F c It could be an assembly with a fused entity.

[0123] In one embodiment, a bispecific antibody is provided comprising the sdAb defined above and a second antigen-binding moiety. In some embodiments, the second antigen-binding moiety is a monoclonal antibody, Fab, F(ab')2, Fab', scFv, or sdAb, for example, V H H or V NAR It can include...

[0124] The term "antigen-binding moiety" refers to a polypeptide comprising an antibody or its antigen-binding fragment having antigen-binding activity, such as an engineered antibody or its fragment.

[0125] In some embodiments, the second antigen-binding moiety can bind to human serum albumin, for example, for the purpose of stabilization / half-life extension.

[0126] In one embodiment, a trispecific antibody is provided comprising the sdAb defined above, a second antigen-binding moiety, and a third antigen-binding moiety. In some embodiments, the second antigen-binding moiety is a monoclonal antibody, Fab, F(ab')2, Fab', scFv, or sdAb, for example, V H H or V NAR , including. In some embodiments, the third antigen-binding moiety is independently a monoclonal antibody, Fab, F(ab')2, Fab', scFv, or sdAb, for example V H H or V NAR , including.

[0127] The second and / or third antigen-binding moieties can bind to human serum albumin, for example, for stabilization / half-life extension.

[0128] In some embodiments, a triplicate antibody can be multispecific and may contain one or more additional antigen-binding moieties. In such embodiments, the additional antigen-binding moieties may independently be Fab, F(ab')2, Fab', scFv, or sdAb, for example, V H H or V NAR That is possible.

[0129] In one embodiment, the multispecific antibody comprises a first antigen-binding moiety containing an sdAb as defined herein and a second antigen-binding moiety. In one embodiment, the second antigen-binding moiety specifically binds to a cell surface marker of an immune cell.

[0130] A "cell surface marker" is a molecule expressed on the surface of a cell that is specific to (or abundant in) a particular cell type and can be bound to or recognized by an antigen-binding moiety. Bispecific T cell engagers

[0131] In one embodiment, the polyvalent antibody is a bispecific T cell engager comprising an sdAb as defined herein and a second antigen-binding moiety that specifically binds to a T cell surface marker. In one embodiment, the T cell marker includes human CD3.

[0132] Human CD3 is a multi-subunit antigen, as recognized, and various subunits of it can be involved in CD3 activation. One such subunit is CD3ε (see, e.g., GenBank NP_000724.1). Other non-restrictive examples include CD3γ (see, e.g., GenBank NP_000064.1) and CD3δ (see, e.g., GenBank NP_000723.1 for δ isoform A, and, e.g., GenBank NP_001035741.1 for δ isoform B).

[0133] In some embodiments, the T cell marker includes CD3ε, CD3γ, or CD3δ. In a particular embodiment, the T cell marker includes CD3ε.

[0134] As used herein, the term “bispecific T cell engager” refers to a recombinant bispecific protein having two linked variable regions from two different antibodies, one targeting a cell surface molecule on a T cell (e.g., CD3ε) and the other targeting an antigen on the surface of a diseased cell (typically a malignant cell). For example, a bispecific T cell engager may include sdAb as defined herein and scFv. A bispecific T cell engager may also include sdAb as defined herein and a second VHH / sdAb. The two variable regions are typically linked together by a short, flexible linker, such as a GlySer linker. By simultaneously binding to tumor antigens and T cells, bispecific T cell engagers mediate the T cell response and tumor cell killing. The adhesion to T cells / target cells facilitated by bispecific T cell engagers is independent of MHC haplotype.

[0135] In one embodiment, the bispecific T cell engager is directed from the N-terminus to the C-terminus. - The first antigen-binding site; - Amino acid linkers; and - Second antigen-binding site; Includes. In one embodiment, the signal peptide is further included at the N-terminus of the first antigen-binding moiety.

[0136] The “signal peptides” referred to herein can direct nascent proteins toward the endoplasmic reticulum and then toward the cell surface, where they are expressed. The core of a signal peptide may contain a long stretch of hydrophobic amino acids that tend to form a single α-helix. A signal peptide can begin with a short stretch of positively charged amino acids, which helps to enhance the proper topology of the polypeptide during translocation. The terminus of a signal peptide typically contains a stretch of amino acids that are recognized and cleaved by a signal peptidase. The signal peptidase can cleave the signal peptide during or after transport to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides can be located at the amino terminus of a molecule.

[0137] In one embodiment, the signal peptide is a signal peptide derived from human CD28. In one embodiment, the signal peptide derived from human CD28 contains SEQ ID NO: 110. In one embodiment, the signal peptide is at least 80% identical to SEQ ID NO: 110. In one embodiment, the signal peptide is at least 90% identical to SEQ ID NO: 110. In one embodiment, the signal peptide is at least 95% identical to SEQ ID NO: 110. In one embodiment, the signal peptide is at least 98% identical to SEQ ID NO: 110.

[0138] In this context, an "amino acid linker" is understood to be a sequence of sufficient length, flexibility, and composition that allows a bispecific T cell engager to function properly and engage with both of its targets. The amino acid linker may contain a hinge. The hinge may be derived from human CD8, for example, as shown in SEQ ID NO: 12.

[0139] In one embodiment, the amino acid linker contains a sequence that is at least 80% identical to SEQ ID NO: 111 - SEQ ID NO: 112 - SEQ ID NO: 118 (from the N-terminus to the C-terminus). In one embodiment, the amino acid linker contains a sequence that is at least 80% identical to SEQ ID NO: 111. In one embodiment, the amino acid linker contains a sequence that is at least 90% identical to SEQ ID NO: 111. In one embodiment, the amino acid linker contains a sequence that is at least 95% identical to SEQ ID NO: 111. In one embodiment, the amino acid linker contains a sequence that is at least 98% identical to SEQ ID NO: 111. In one embodiment, the amino acid linker contains a sequence that is at least 80% identical to SEQ ID NO: 112. In one embodiment, the amino acid linker includes a sequence that is at least 90% identical to SEQ ID NO: 112. In one embodiment, the amino acid linker includes a sequence that is at least 95% identical to SEQ ID NO: 112. In one embodiment, the amino acid linker includes a sequence that is at least 98% identical to SEQ ID NO: 112. In one embodiment, the amino acid linker includes a sequence that is at least 80% identical to SEQ ID NO: 118. In one embodiment, the amino acid linker includes a sequence that is at least 90% identical to SEQ ID NO: 118. In one embodiment, the amino acid linker includes a sequence that is at least 95% identical to SEQ ID NO: 118. In one embodiment, the amino acid linker includes a sequence that is at least 98% identical to SEQ ID NO: 118.

[0140] In one embodiment, the polyvalent antibody is encoded by SEQ ID NO: 119.

[0141] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 1, the CDR2 amino acid sequence described in SEQ ID NO: 2, and the CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6).

[0142] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10).

[0143] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 7, the CDR2 amino acid sequence described in SEQ ID NO: 8, and the CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13).

[0144] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14).

[0145] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 13, the CDR2 amino acid sequence described in SEQ ID NO: 14, and the CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36).

[0146] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 16, the CDR2 amino acid sequence described in SEQ ID NO: 17, and the CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61).

[0147] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 19, the CDR2 amino acid sequence described in SEQ ID NO: 20, and the CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74).

[0148] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 22, the CDR2 amino acid sequence described in SEQ ID NO: 23, and the CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75).

[0149] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77).

[0150] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 28, the CDR2 amino acid sequence described in SEQ ID NO: 29, and the CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80).

[0151] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 31, the CDR2 amino acid sequence described in SEQ ID NO: 32, and the CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87).

[0152] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 34, the CDR2 amino acid sequence described in SEQ ID NO: 35, and the CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93).

[0153] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 37, the CDR2 amino acid sequence described in SEQ ID NO: 38, and the CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2).

[0154] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 40, the CDR2 amino acid sequence described in SEQ ID NO: 41, and the CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62).

[0155] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 43, the CDR2 amino acid sequence described in SEQ ID NO: 44, and the CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66).

[0156] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 46, the CDR2 amino acid sequence described in SEQ ID NO: 47, and the CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10).

[0157] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 49, the CDR2 amino acid sequence described in SEQ ID NO: 50, and the CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16).

[0158] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 52, the CDR2 amino acid sequence described in SEQ ID NO: 53, and the CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23).

[0159] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 55, the CDR2 amino acid sequence described in SEQ ID NO: 56, and the CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24).

[0160] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 58, the CDR2 amino acid sequence described in SEQ ID NO: 59, and the CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32).

[0161] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 61, the CDR2 amino acid sequence described in SEQ ID NO: 62, and the CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33).

[0162] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 64, the CDR2 amino acid sequence described in SEQ ID NO: 65, and the CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48).

[0163] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 67, the CDR2 amino acid sequence described in SEQ ID NO: 68, and the CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55).

[0164] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 70, the CDR2 amino acid sequence described in SEQ ID NO: 71, and the CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64).

[0165] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 73, the CDR2 amino acid sequence described in SEQ ID NO: 74, and the CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72).

[0166] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 76, the CDR2 amino acid sequence described in SEQ ID NO: 77, and the CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79).

[0167] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82).

[0168] In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 82. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 83. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 84. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 85. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 86. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 87. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 88. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 89. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 90. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 91. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 92. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 93. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 94. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 95. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 96. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 97. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 98. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 99. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 100. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 101. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 102. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 103. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 104. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 105. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 106. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 107. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 108. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 120. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 121. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 122. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 123. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 124. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 125.

[0169] In one embodiment, sdAb includes SEQ ID NO: 82. In one embodiment, sdAb includes SEQ ID NO: 83. In one embodiment, sdAb includes SEQ ID NO: 84. In one embodiment, sdAb includes SEQ ID NO: 85. In one embodiment, sdAb includes SEQ ID NO: 86. In one embodiment, sdAb includes SEQ ID NO: 87. In one embodiment, sdAb includes SEQ ID NO: 88. In one embodiment, sdAb includes SEQ ID NO: 89. In one embodiment, sdAb includes SEQ ID NO: 90. In one embodiment, sdAb includes SEQ ID NO: 91. In one embodiment, sdAb includes SEQ ID NO: 92. In one embodiment, sdAb includes SEQ ID NO: 93. In one embodiment, sdAb includes SEQ ID NO: 94. In one embodiment, sdAb includes SEQ ID NO: 95. In one embodiment, sdAb includes SEQ ID NO: 96. In one embodiment, sdAb includes SEQ ID NO: 97. In one embodiment, sdAb includes SEQ ID NO: 98. In one embodiment, sdAb includes SEQ ID NO: 99. In one embodiment, sdAb includes SEQ ID NO: 100. In one embodiment, sdAb includes SEQ ID NO: 101. In one embodiment, sdAb includes SEQ ID NO: 102. In one embodiment, sdAb includes SEQ ID NO: 103. In one embodiment, sdAb includes SEQ ID NO: 104. In one embodiment, sdAb includes SEQ ID NO: 105. In one embodiment, sdAb includes SEQ ID NO: 106. In one embodiment, sdAb includes SEQ ID NO: 107. In one embodiment, sdAb includes SEQ ID NO: 108. In one embodiment, sdAb includes SEQ ID NO: 120. In one embodiment, sdAb includes SEQ ID NO: 121. In one embodiment, sdAb includes SEQ ID NO: 122. In one embodiment, sdAb includes SEQ ID NO: 123. In one embodiment, sdAb includes SEQ ID NO: 124. In one embodiment, sdAb includes SEQ ID NO: 125.

[0170] In some embodiments, BiKE is a sequence variant of BiKE having 80%, 90%, 95%, 98%, or 99% identity to one of the BiKEs. In some embodiments, the variant retains substantially the same binding specificity as the parent molecule from which it originates. In some embodiments, the variant retains substantially the same binding affinity as the parent molecule from which it originates.

[0171] BiKE and TriKE In one embodiment, the polyvalent antibody is a bispecific killer cell engager.

[0172] The term "BiKE" refers to a recombinant bispecific protein in which two variable regions from two different antibodies are linked, one targeting cell surface molecules (e.g., CD16) on natural killer (NK) cells and the other targeting antigens on the surface of diseased cells (typically malignant cells). For example, BiKE may consist of two scFv, two VHH, or a combination thereof. These two are usually linked together by a short, flexible linker. By simultaneously binding to tumor antigens and NK cells, BiKE mediates the NK cell response and the killing of tumor cells.

[0173] In one embodiment, the cell surface markers of immune cells include natural killer (NK) cell markers. In one embodiment, the NK cell markers include human CD16.

[0174] In one embodiment, the polyvalent antibody is a triple-specific killer cell engager (BiKE).

[0175] The term "TriKE" refers to BiKE, which has been further modified to possess another functionality. This term is used to encompass various approaches. One approach involves inserting intervening immunomodulatory molecules (modified human IL-15 crosslinkers) to promote the activation, proliferation, and / or survival of NK cells (Vallera et al. IL-15 Trispecific Killer Engagers (TriKEs) Make Natural Killer Cells Specific to CD33+ Targets While Also Inducing In Vivo Expansion, and Enhanced Function. Clinical Cancer Research. 2012;22(14):3440-50). Other TriKE approaches are trispecific molecules containing three antibody variable regions: one targeting NK cell receptors and two targeting tumor-associated antigens (Gleason et al. Bispecific and Trispecific Killer Cell Engagers Directly Activate Human NK Cells Through CD16 Signaling and Induce Cytotoxicity and Cytokine Production. Mol Cancer Ther. 2012; 11(12): 2674-84). Yet another TriKE approach targets two NK cell receptors (e.g., CD16 and NKp46) and one tumor-associated antigen (Gauthier et al. Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity. Cell. 2019; 177(7): 1701-13).

[0176] In one embodiment, the polyvalent antibody further comprises cytokines for stimulating the activation, proliferation, and / or survival of NK cells. In one embodiment, the cytokine for stimulating NK cell proliferation is interleukin-15 (IL-15), a variant thereof, or a functional fragment thereof.

[0177] In one embodiment, the polyvalent antibody further comprises at least a third antigen-binding moiety that binds to a second NK cell marker. In one embodiment, the second NK cell marker is human NKp46.

[0178] In one embodiment, the polyvalent antibody further comprises at least a third antigen-binding moiety that binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is different from human CD22.

[0179] In one embodiment, the third antigen-binding portion is V H H, V NAR , or including scVF.

[0180] In one embodiment, the second antigen-binding portion is V H Includes H.

[0181] In one embodiment, the third antigen-binding moiety binds to human serum albumin. In such an embodiment, affinity for human serum albumin may contribute to increased stabilization / half-life.

[0182] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 1, the CDR2 amino acid sequence described in SEQ ID NO: 2, and the CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6).

[0183] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10).

[0184] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 7, the CDR2 amino acid sequence described in SEQ ID NO: 8, and the CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13).

[0185] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14).

[0186] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 13, the CDR2 amino acid sequence described in SEQ ID NO: 14, and the CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36).

[0187] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 16, the CDR2 amino acid sequence described in SEQ ID NO: 17, and the CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61).

[0188] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 19, the CDR2 amino acid sequence described in SEQ ID NO: 20, and the CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74).

[0189] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 22, the CDR2 amino acid sequence described in SEQ ID NO: 23, and the CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75).

[0190] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77).

[0191] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 28, the CDR2 amino acid sequence described in SEQ ID NO: 29, and the CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80).

[0192] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 31, the CDR2 amino acid sequence described in SEQ ID NO: 32, and the CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87).

[0193] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 34, the CDR2 amino acid sequence described in SEQ ID NO: 35, and the CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93).

[0194] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 37, the CDR2 amino acid sequence described in SEQ ID NO: 38, and the CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2).

[0195] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 40, the CDR2 amino acid sequence described in SEQ ID NO: 41, and the CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62).

[0196] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 43, the CDR2 amino acid sequence described in SEQ ID NO: 44, and the CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66).

[0197] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 46, the CDR2 amino acid sequence described in SEQ ID NO: 47, and the CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10).

[0198] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 49, the CDR2 amino acid sequence described in SEQ ID NO: 50, and the CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16).

[0199] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 52, the CDR2 amino acid sequence described in SEQ ID NO: 53, and the CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23).

[0200] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 55, the CDR2 amino acid sequence described in SEQ ID NO: 56, and the CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24).

[0201] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 58, the CDR2 amino acid sequence described in SEQ ID NO: 59, and the CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32).

[0202] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 61, the CDR2 amino acid sequence described in SEQ ID NO: 62, and the CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33).

[0203] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 64, the CDR2 amino acid sequence described in SEQ ID NO: 65, and the CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48).

[0204] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 67, the CDR2 amino acid sequence described in SEQ ID NO: 68, and the CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55).

[0205] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 70, the CDR2 amino acid sequence described in SEQ ID NO: 71, and the CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64).

[0206] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 73, the CDR2 amino acid sequence described in SEQ ID NO: 74, and the CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72).

[0207] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 76, the CDR2 amino acid sequence described in SEQ ID NO: 77, and the CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79).

[0208] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82).

[0209] In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 82. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 83. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 84. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 85. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 86. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 87. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 88. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 89. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 90. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 91. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 92. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 93. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 94. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 95. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 96. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 97. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 98. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 99. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 100. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 101. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 102. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 103. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 104. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 105. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 106. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 107. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 108. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 120. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 121. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 122. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 123. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 124. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 125.

[0210] In one embodiment, sdAb includes SEQ ID NO: 82. In one embodiment, sdAb includes SEQ ID NO: 83. In one embodiment, sdAb includes SEQ ID NO: 84. In one embodiment, sdAb includes SEQ ID NO: 85. In one embodiment, sdAb includes SEQ ID NO: 86. In one embodiment, sdAb includes SEQ ID NO: 87. In one embodiment, sdAb includes SEQ ID NO: 88. In one embodiment, sdAb includes SEQ ID NO: 89. In one embodiment, sdAb includes SEQ ID NO: 90. In one embodiment, sdAb includes SEQ ID NO: 91. In one embodiment, sdAb includes SEQ ID NO: 92. In one embodiment, sdAb includes SEQ ID NO: 93. In one embodiment, sdAb includes SEQ ID NO: 94. In one embodiment, sdAb includes SEQ ID NO: 95. In one embodiment, sdAb includes SEQ ID NO: 96. In one embodiment, sdAb includes SEQ ID NO: 97. In one embodiment, sdAb includes SEQ ID NO: 98. In one embodiment, sdAb includes SEQ ID NO: 99. In one embodiment, sdAb includes SEQ ID NO: 100. In one embodiment, sdAb includes SEQ ID NO: 101. In one embodiment, sdAb includes SEQ ID NO: 102. In one embodiment, sdAb includes SEQ ID NO: 103. In one embodiment, sdAb includes SEQ ID NO: 104. In one embodiment, sdAb includes SEQ ID NO: 105. In one embodiment, sdAb includes SEQ ID NO: 106. In one embodiment, sdAb includes SEQ ID NO: 107. In one embodiment, sdAb includes SEQ ID NO: 108. In one embodiment, sdAb includes SEQ ID NO: 120. In one embodiment, sdAb includes SEQ ID NO: 121. In one embodiment, sdAb includes SEQ ID NO: 122. In one embodiment, sdAb includes SEQ ID NO: 123. In one embodiment, sdAb includes SEQ ID NO: 124. In one embodiment, sdAb includes SEQ ID NO: 125.

[0211] In some embodiments, BiKE or TriKE is a sequence variant of the above BiKE and TriKE having 80%, 90%, 95%, 98%, or 99% identity. In some embodiments, the variant retains substantially the same binding specificity as the parent molecule from which it is derived. In some embodiments, the variant retains substantially the same binding affinity as the parent molecule from which it is derived.

[0212] In one embodiment, a polyvalent antibody is provided comprising a first antigen-binding moiety, an amino acid linker containing a polypeptide hinge derived from human CD8, and a second antigen-binding moiety. In one embodiment, the polypeptide hinge derived from human CD8 includes SEQ ID NO: 112. In one embodiment, the amino acid linker further comprises at least one G4S at the N-terminus of the polypeptide hinge derived from human CD8 and at least one G4S at the C-terminus of the polypeptide hinge derived from human CD8. In one embodiment, the amino acid linker is at least 47 amino acids (aa) long, preferably at least 52 residues long, preferably at least 57 residues long, more preferably at least 62 residues long, and even more preferably at least 67 residues long. In one embodiment, the amino acid linker comprises SEQ ID NO: 111, 112, and 118 in the direction from the N-terminus to the C-terminus. In one embodiment, the first antigen-binding moiety specifically binds to human CD22. In one embodiment, the first antigen-binding moiety is V H H, V NAR, or scVF. In one embodiment, the first antigen-binding moiety is one of the anti-CD22 sdAbs described herein. In one embodiment, the second antigen-binding moiety specifically binds to a cell surface marker of an immune cell. In one embodiment, the cell surface marker of an immune cell includes a T cell marker. In one embodiment, the T cell marker includes human CD3. In one embodiment, the second antigen-binding moiety is V H H, V NAR , or scVF. Recombinant nucleic acid molecules In one embodiment, a recombinant nucleic acid molecule encoding a polyvalent antibody as defined herein is provided. In one embodiment, the nucleic acid is a vector.

[0213] composition In one embodiment, a composition is provided comprising a polyvalent antibody as defined herein, together with an acceptable excipient, diluent, or carrier. In one embodiment, the composition comprises a bispecific T cell engager as defined herein. In one embodiment, the composition comprises BiKE as defined herein. In one embodiment, the composition comprises TriKE as defined herein. In one embodiment, the composition is a pharmaceutical composition, and the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0214] Usage and Method In one embodiment, the use of a multivalent antibody as defined herein for the treatment of cancer or an autoimmune disease is provided. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes mellitus.

[0215] In one embodiment, the use of a polyvalent antibody as defined herein is provided for preparing a medicament for the treatment of cancer or an autoimmune disease. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes mellitus.

[0216] In one embodiment, a multivalent antibody as defined herein is provided for use in the treatment of cancer or an autoimmune disease. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes mellitus.

[0217] In one embodiment, a method is provided for treating cancer or an autoimmune disease in a subject, comprising administering a multivalent antibody as defined herein to the subject. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes.

[0218] Table 3 shows exemplary sequences and modules of the polyvalent antibodies and CARs described herein, according to specific aspects and embodiments.

[0219] [Table 3] TIFF0007869193000007.tif207169TIFF0007869193000008.tif192169TIFF0007869193000009.tif212170TIFF0007869193000010.tif143169

[0220] Regarding the exemplary CAR construct (SEQ ID NO: 138) containing the antibody hCD221ug36: - Positions 1-54 correspond to signal peptides; - Positions 55-396 correspond to antibody 1ug36 ABD (however, alternatively, any other anti-CD22 sdAb described herein may be present); - Positions 397-606 correspond to the (G4S)3-human CD8a hinge and restriction scar; - Positions 607-702 correspond to the human CD28 transmembrane domain; - Positions 703-828 correspond to the human 41BB co-stimulatory domain; - Positions 829-1170 correspond to the human CD3ζ signaling domain.

[0221] For experimental and testing purposes, the construct subsequently includes an optional in-frame P2A-GFP marker.

[0222] Regarding an exemplary bispecific T cell engager construct (SEQ ID NO: 139) containing the antibody hCD221ug36: - Positions 1-72 correspond to the signal peptide and restriction scar; - Positions 64-405 correspond to the antibody hCD221ug36 (however, alternatively, any other anti-CD22 sdAb described herein); - Positions 406-633 correspond to the (G4S)3-hCD8a-(G4S) linker domain (including the limiting portion of the modular hinge); - Positions 633 and 634 are separated by the heavy chain of CD3-specific mouse scFv; - Positions 634-678 correspond to the (G4S)3 linker; - Positions 678-679 are separated by the light chain of CD3-specific mouse scFv.

[0223] For experimental and testing purposes, the construct subsequently includes positions 679–699 corresponding to the 6xHis tag and the stop codon.

[0224] Note that the exemplary antibody sequences corresponding to SEQ ID NO: 120-125 shown in Table 3 have sequence differences, particularly towards the amino terminus. Specifically, positions 1, 3, and 5 are Q, Q, and V, respectively (compare SEQ ID NO: 120 and 121 in Table 3 with VHH#22 (SEQ ID NO: 86) and VHH#11 (SEQ ID NO: 93) in Tables 1 and 3). While not theoretically bound, these sequence differences may contribute to stability and / or protease resistance (see Hussack G et al. Protein Engineering Design & Selection. 2014; 27(6); 191-198). Therefore, the polyvalent antibodies and CARs of the embodiments disclosed herein include variants containing any one of the sdAbs disclosed herein modified to contain Q, Q, and V at positions 1, 3, and 5, respectively.

[0225] Chimeric antibody receptors and related embodiments In one embodiment, a chimeric antibody receptor (CAR) that binds to human CD22 is provided, comprising a VHH sdAb as defined herein.

[0226] A "chimeric antigen receptor" is a receptor protein that has been engineered to confer a new ability to target specific proteins to T cells. This receptor is chimeric because it combines both antigen-binding and T-cell activation functions into a single receptor (see Stoiber et al. Limitations in the Design of Chimeric Antigen Receptors for Cancer Therapy. Cells. 2012; 8(5): 472 and van der Stegen et al. The pharmacology of second-generation chimeric antigen receptors. Nat Rev Drug Discov. 2019; 14(7): 499-509).

[0227] In one embodiment, the CAR is directed from the N-terminus to the C-terminus. - A CD22-binding domain containing an sdAb as defined in any one of claims 1 to 27; - Polypeptide hinge; - Transmembrane domain; and - Cytoplasmic domain containing a co-stimulatory domain and a signal transduction domain; Includes.

[0228] As used herein, the term “polypeptide hinge” generally refers to any oligopeptide or polypeptide that functions to link an extracellular ligand-binding domain to a transmembrane domain. In particular, hinge regions are used to give extracellular ligand-binding domains greater flexibility and accessibility. Hinge regions may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. Hinge regions may be derived from all or part of naturally occurring molecules, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of the constant region of an antibody. Alternatively, hinge regions may be synthetic sequences equivalent to naturally occurring hinge sequences, or entirely synthetic hinge sequences.

[0229] In one embodiment, the polypeptide hinge is a CD8 hinge domain. In one embodiment, the CD8 hinge domain contains SEQ ID NO: 112. In one embodiment, the CD8 hinge domain is at least 80% identical to SEQ ID NO: 112. In one embodiment, the hinge domain is at least 90% identical to SEQ ID NO: 112. In one embodiment, the hinge domain is at least 95% identical to SEQ ID NO: 112. In one embodiment, the hinge domain is at least 98% identical to SEQ ID NO: 112.

[0230] The term "transmembrane domain" refers to a polypeptide that spans the cell membrane and thereby connects the extracellular portion of a CAR (including the CD22-binding portion) with the intracellular portion involved in signal transduction. Commonly used transmembrane domains in CARs are derived from CD4, CD8α, CD28, and CD3ζ.

[0231] In one embodiment, the transmembrane domain is the CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain contains SEQ ID NO: 113. In one embodiment, the transmembrane domain is at least 80% identical to SEQ ID NO: 113. In one embodiment, the transmembrane domain is at least 90% identical to SEQ ID NO: 113. In one embodiment, the transmembrane domain is at least 95% identical to SEQ ID NO: 113. In one embodiment, the transmembrane domain is at least 98% identical to SEQ ID NO: 113.

[0232] The term "cytoplasmic domain" (also called "signaling domain") refers to the intracellular portion of a CAR that carries out intracellular signaling after the extracellular ligand-binding domain binds to a target, leading to the activation of immune cells and the immune response. In other words, the cytoplasmic domain is involved in the activation of at least one normal effector function of the immune cell on which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "cytoplasmic domain" refers to the portion of a protein that transmits effector signals, causing cells to perform specialized functions. Such cytoplasmic domains typically include a co-stimulatory domain in addition to the signaling domain.

[0233] The term "signaling domain" refers to a portion of a protein that transmits effector signals, causing cells to perform specialized functions. Examples of signaling domains for use in CARs may be cytoplasmic sequences of T cell receptors and co-receptors that act cooperatively to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences having the same function. Signaling domains include two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to result in secondary or co-stimulatory signals. Primary cytoplasmic signaling sequences may include signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for syk / zap70 class tyrosine kinases. Non-limiting examples of signal transduction domains used in the present invention include those derived from TCRζ, common FcRγ (FCERIG), Fcγ Rlla, FcRβ (FcεRib), FcRε, CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, DAP10, or DAP12. In preferred embodiments, the signal transduction domain of CAR may include the CD3ζ signal transduction domain.

[0234] In one embodiment, the signaling domain is a CD3ζ signaling domain. In one embodiment, the CD3ζ signaling domain includes SEQ ID NO: 115. In one embodiment, the signaling domain is at least 80% identical to SEQ ID NO: 115. In one embodiment, the signaling domain is at least 90% identical to SEQ ID NO: 115. In one embodiment, the signaling domain is at least 95% identical to SEQ ID NO: 115. In one embodiment, the signaling domain is at least 98% identical to SEQ ID NO: 115.

[0235] The term "costimulatory domain" refers to a cognitive-binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by T cells (such as proliferation, but not limited to this). Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include: ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, C D8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD lla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or combinations thereof.

[0236] In one embodiment, the co-stimulatory domain is the 4-1BB co-stimulatory domain. In one embodiment, the 4-1BB signaling domain includes SEQ ID NO: 114. In one embodiment, the co-stimulatory domain is at least 80% identical to SEQ ID NO: 114. In one embodiment, the co-stimulatory domain is at least 90% identical to SEQ ID NO: 114. In one embodiment, the co-stimulatory domain is at least 95% identical to SEQ ID NO: 114. In one embodiment, the co-stimulatory domain is at least 98% identical to SEQ ID NO: 114.

[0237] In one embodiment, the CAR further comprises a flexible amino acid linker between the sdAb and the polypeptide hinge. In one embodiment, the amino acid linker comprises SEQ ID NO: 111. In one embodiment, the amino acid linker is at least 80% identical to SEQ ID NO: 111. In one embodiment, the amino acid linker is at least 90% identical to SEQ ID NO: 111. In one embodiment, the amino acid linker is at least 95% identical to SEQ ID NO: 111. In one embodiment, the amino acid linker is at least 98% identical to SEQ ID NO: 111.

[0238] In one embodiment, the CAR further comprises a signal peptide.

[0239] In one embodiment, the signal peptide is a signal peptide derived from human CD28. In one embodiment, the signal peptide derived from human CD28 contains SEQ ID NO: 110. In one embodiment, the signal peptide is at least 80% identical to SEQ ID NO: 110. In one embodiment, the signal peptide is at least 90% identical to SEQ ID NO: 110. In one embodiment, the signal peptide is at least 95% identical to SEQ ID NO: 110. In one embodiment, the signal peptide is at least 98% identical to SEQ ID NO: 110.

[0240] In one embodiment, the CAR is coded by SEQ ID NO: 119.

[0241] In one embodiment, sdAb includes SEQ ID NO: 86 (hCD221ug-36), SEQ ID NO: 83 (hCD221ug-10), SEQ ID NO: 87 (hCD221ug-61), SEQ ID NO: 82 (hCD221ug-6), SEQ ID NO: 88 (hCD221ug-74), SEQ ID NO: 85 (hCD221ug-14), SEQ ID NO: 102 (hCD22pos-33), or SEQ ID NO: 84 (hCD221ug-13).

[0242] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 1, the CDR2 amino acid sequence described in SEQ ID NO: 2, and the CDR3 amino acid sequence described in SEQ ID NO: 3 (hCD221ug-6).

[0243] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 4, the CDR2 amino acid sequence described in SEQ ID NO: 5, and the CDR3 amino acid sequence described in SEQ ID NO: 6 (hCD221ug-10).

[0244] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 7, the CDR2 amino acid sequence described in SEQ ID NO: 8, and the CDR3 amino acid sequence described in SEQ ID NO: 9 (hCD221ug-13).

[0245] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 10, the CDR2 amino acid sequence described in SEQ ID NO: 11, and the CDR3 amino acid sequence described in SEQ ID NO: 12 (hCD221ug-14).

[0246] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 13, the CDR2 amino acid sequence described in SEQ ID NO: 14, and the CDR3 amino acid sequence described in SEQ ID NO: 15 (hCD221ug-36).

[0247] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 16, the CDR2 amino acid sequence described in SEQ ID NO: 17, and the CDR3 amino acid sequence described in SEQ ID NO: 18 (hCD221ug-61).

[0248] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 19, the CDR2 amino acid sequence described in SEQ ID NO: 20, and the CDR3 amino acid sequence described in SEQ ID NO: 21 (hCD221ug-74).

[0249] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 22, the CDR2 amino acid sequence described in SEQ ID NO: 23, and the CDR3 amino acid sequence described in SEQ ID NO: 24 (hCD221ug-75).

[0250] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 25, the CDR2 amino acid sequence described in SEQ ID NO: 26, and the CDR3 amino acid sequence described in SEQ ID NO: 27 (hCD221ug-77).

[0251] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 28, the CDR2 amino acid sequence described in SEQ ID NO: 29, and the CDR3 amino acid sequence described in SEQ ID NO: 30 (hCD221ug-80).

[0252] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 31, the CDR2 amino acid sequence described in SEQ ID NO: 32, and the CDR3 amino acid sequence described in SEQ ID NO: 33 (hCD221ug-87).

[0253] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 34, the CDR2 amino acid sequence described in SEQ ID NO: 35, and the CDR3 amino acid sequence described in SEQ ID NO: 36 (hCD221ug-93).

[0254] In one embodiment, sdAb comprises the CDR1 amino acid sequence described in SEQ ID NO: 37, the CDR2 amino acid sequence described in SEQ ID NO: 38, and the CDR3 amino acid sequence described in SEQ ID NO: 39 (hCD22100ug-2).

[0255] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 40, the CDR2 amino acid sequence described in SEQ ID NO: 41, and the CDR3 amino acid sequence described in SEQ ID NO: 42 (hCD22100ug-62).

[0256] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 43, the CDR2 amino acid sequence described in SEQ ID NO: 44, and the CDR3 amino acid sequence described in SEQ ID NO: 45 (hCD22100ug-66).

[0257] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 46, the CDR2 amino acid sequence described in SEQ ID NO: 47, and the CDR3 amino acid sequence described in SEQ ID NO: 48 (hCD22pas-10).

[0258] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 49, the CDR2 amino acid sequence described in SEQ ID NO: 50, and the CDR3 amino acid sequence described in SEQ ID NO: 51 (hCD22pas-16).

[0259] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 52, the CDR2 amino acid sequence described in SEQ ID NO: 53, and the CDR3 amino acid sequence described in SEQ ID NO: 54 (hCD22pas-23).

[0260] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 55, the CDR2 amino acid sequence described in SEQ ID NO: 56, and the CDR3 amino acid sequence described in SEQ ID NO: 57 (hCD22pas-24).

[0261] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 58, the CDR2 amino acid sequence described in SEQ ID NO: 59, and the CDR3 amino acid sequence described in SEQ ID NO: 60 (hCD22pas-32).

[0262] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 61, the CDR2 amino acid sequence described in SEQ ID NO: 62, and the CDR3 amino acid sequence described in SEQ ID NO: 63 (hCD22pas-33).

[0263] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 64, the CDR2 amino acid sequence described in SEQ ID NO: 65, and the CDR3 amino acid sequence described in SEQ ID NO: 66 (hCD22pas-48).

[0264] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 67, the CDR2 amino acid sequence described in SEQ ID NO: 68, and the CDR3 amino acid sequence described in SEQ ID NO: 69 (hCD22pas-55).

[0265] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 70, the CDR2 amino acid sequence described in SEQ ID NO: 71, and the CDR3 amino acid sequence described in SEQ ID NO: 72 (hCD22pas-64).

[0266] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 73, the CDR2 amino acid sequence described in SEQ ID NO: 74, and the CDR3 amino acid sequence described in SEQ ID NO: 75 (hCD22pas-72).

[0267] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 76, the CDR2 amino acid sequence described in SEQ ID NO: 77, and the CDR3 amino acid sequence described in SEQ ID NO: 78 (hCD22pas-79).

[0268] In one embodiment, sdAb includes the CDR1 amino acid sequence described in SEQ ID NO: 79, the CDR2 amino acid sequence described in SEQ ID NO: 80, and the CDR3 amino acid sequence described in SEQ ID NO: 81 (hCD22pas-82).

[0269] In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 82. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 83. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 84. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 85. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 86. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 87. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 88. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 89. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 90. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 91. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 92. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 93. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 94. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 95. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 96. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 97. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 98. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 99. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 100. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 101. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 102. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 103. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 104. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 105. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 106. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 107. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 108. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 120. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 121. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 122. In one embodiment, the sdAb comprises CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 123. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 124. In one embodiment, the sdAb includes CDR1, CDR2, and CDR2 of the sdAb sequence described in SEQ ID NO: 125.

[0270] In one embodiment, sdAb includes SEQ ID NO: 82. In one embodiment, sdAb includes SEQ ID NO: 83. In one embodiment, sdAb includes SEQ ID NO: 84. In one embodiment, sdAb includes SEQ ID NO: 85. In one embodiment, sdAb includes SEQ ID NO: 86. In one embodiment, sdAb includes SEQ ID NO: 87. In one embodiment, sdAb includes SEQ ID NO: 88. In one embodiment, sdAb includes SEQ ID NO: 89. In one embodiment, sdAb includes SEQ ID NO: 90. In one embodiment, sdAb includes SEQ ID NO: 91. In one embodiment, sdAb includes SEQ ID NO: 92. In one embodiment, sdAb includes SEQ ID NO: 93. In one embodiment, sdAb includes SEQ ID NO: 94. In one embodiment, sdAb includes SEQ ID NO: 95. In one embodiment, sdAb includes SEQ ID NO: 96. In one embodiment, sdAb includes SEQ ID NO: 97. In one embodiment, sdAb includes SEQ ID NO: 98. In one embodiment, sdAb includes SEQ ID NO: 99. In one embodiment, sdAb includes SEQ ID NO: 100. In one embodiment, sdAb includes SEQ ID NO: 101. In one embodiment, sdAb includes SEQ ID NO: 102. In one embodiment, sdAb includes SEQ ID NO: 103. In one embodiment, sdAb includes SEQ ID NO: 104. In one embodiment, sdAb includes SEQ ID NO: 105. In one embodiment, sdAb includes SEQ ID NO: 106. In one embodiment, sdAb includes SEQ ID NO: 107. In one embodiment, sdAb includes SEQ ID NO: 108. In one embodiment, sdAb includes SEQ ID NO: 120. In one embodiment, sdAb includes SEQ ID NO: 121. In one embodiment, sdAb includes SEQ ID NO: 122. In one embodiment, sdAb includes SEQ ID NO: 123. In one embodiment, sdAb includes SEQ ID NO: 124. In one embodiment, sdAb includes SEQ ID NO: 125.

[0271] In one embodiment, the CAR further comprises a second CD22-binding domain located at the N-terminus or C-terminus relative to the first CD22-binding domain, the second CD22-binding domain being positioned away from the first CD22-binding domain by an amino acid linker.

[0272] In one embodiment, the second CD22-binding domain contains the same sdAb as the first CD22-binding domain. These embodiments are referred to herein as “double binders.” For example, both the first and second CD22-binding domains may contain antibody 1ug36.

[0273] In another embodiment, the second CD22-binding domain contains an sdAb different from the sdAb of the first CD22-binding domain. These embodiments are referred to herein as “bi-paratopic.” In this embodiment, the sdAb of the second CD22-binding domain can bind to a different CD22 epitope than the one bound by the sdAb of the first CD22-binding domain. For example, a CAR may contain a first CD22-binding domain containing sdAb 1ug36 and a second CD22-binding domain containing sdAb 1ug74. For example, 1ug36 may be located at the N-terminus of 1ug74 (see, for example, the schematic diagram in Figure 30). The “different epitope” may alternatively be an epitope that overlaps with the epitope bound by the sdAb of the first CD22-binding domain. Alternatively, sdAb may bind to the same epitope that is bound by sdAb in the first CD22-binding domain.

[0274] In one embodiment, the CAR further includes an additional binding domain that binds to a target molecule other than CD22. These embodiments are referred to herein as “tandem constructs.” The additional binding domain may include an additional sdAb. The additional binding domain may be located at the N-terminus or C-terminus relative to the CD22 binding domain. The additional binding domain may be separated from the CD22 binding domain by an amino acid linker. In one embodiment, the target molecule bound by the additional binding domain is expressed by a target cell that also expresses CD22, thereby providing a CAR with dual affinity to the same target cell. For example, a target molecule other than CD22 may be a B cell maturation antigen (BCMA). The CD22 binding domain may include, for example, sdAb 1ug36, and the additional binding domain may include an anti-BCMA sdAb (see, for example, the schematic diagram shown in Figure 33).

[0275] In some embodiments, the tandem construct may include a third binding domain that targets a different target molecule than CD22 and is not bound by the additional binding domain. Such constructs are referred to herein as “multi-binders.”

[0276] In some embodiments, CAR is a sequence variant of one of the CARs having 80%, 90%, 95%, 98%, or 99% identity with the CAR. In some embodiments, the variant retains substantially the same binding specificity as the parent molecule from which it originates. In some embodiments, the variant retains substantially the same binding affinity as the parent molecule from which it originates.

[0277] nucleic acids and vectors In one embodiment, a recombinant nucleic acid molecule encoding a CAR as defined herein is provided.

[0278] In one embodiment, a vector comprising a recombinant nucleic acid molecule as defined herein is provided. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is a lentiviral vector.

[0279] Virus particles In one embodiment, recombinant virus particles comprising recombinant nucleic acids as defined herein are provided. In one embodiment, the recombinant virus particles are recombinant lentivirus particles.

[0280] cell In one embodiment, cells containing recombinant nucleic acid molecules as defined herein are provided.

[0281] In one embodiment, genetically modified cells are provided that express a CAR as defined herein on their cell surface membrane. In one embodiment, the genetically modified cells are immune cells. In one embodiment, the immune cells are T lymphocytes or derived from T lymphocytes.

[0282] Usage and Method CAR-T cell therapy uses T cells modified with CARs to treat cancer. The premise of CAR-T immunotherapy is to modify T cells to recognize diseased cells (usually cancer cells) in order to more effectively target and destroy them. Generally, T cells are genetically modified to express CARs, and these cells are injected into the patient to attack the tumor. CAR-T cells can be derived from T cells in the patient's own blood (autologous cells) or from T cells of another healthy donor (allogeneic cells).

[0283] In one embodiment, the use of nucleic acids, vectors, or viral particles described herein for producing cells for CAR-T is provided.

[0284] In one embodiment, a method for producing cells for CAR-T therapy is provided, comprising contacting T cells with the viral particles described herein. In one embodiment, the T cells are donor-derived. In one embodiment, the T cells are patient-derived.

[0285] In one embodiment, a method for producing cells for CAR-T therapy is provided, comprising introducing nucleic acids or vectors described herein into T cells. In one embodiment, the T cells are donor-derived. In one embodiment, the T cells are patient-derived.

[0286] In one embodiment, the use of CARs or genetically modified cells as described herein for treating cancer or autoimmune diseases is provided. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes mellitus.

[0287] In one embodiment, the method further includes an initial step of obtaining cells from a patient or donor and introducing a recombinant nucleic acid molecule or vector encoding a CAR as described herein. In one embodiment, the method further includes an initial step of obtaining cells from a patient or donor and contacting the cells with the viral particles described herein.

[0288] In one embodiment, the use of CARs or genetically modified cells as described herein is provided for preparing a medicament for the treatment of cancer or an autoimmune disease. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes mellitus.

[0289] In one embodiment, CARs or genetically modified cells described herein are provided for use in the treatment of cancer or autoimmune diseases. In one embodiment, cancer is a hematological malignancy. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes mellitus.

[0290] In one embodiment, a method is provided for treating cancer or an autoimmune disease in a subject, comprising administering genetically modified cells as defined herein to the subject. In one embodiment, the hematological malignancy is leukemia, lymphoma, or myelodysplastic syndrome. In one embodiment, the leukemia is acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL). In one embodiment, the lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell follicular lymphoma, and large cell follicular lymphoma. In one embodiment, the autoimmune disease is an inflammatory disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the autoimmune disease is autoimmune diabetes. [Examples]

[0291] The following examples illustrate embodiments of the present invention and / or provide an overview of research conducted in relation to the present invention. These examples are illustrative, and the present invention is not limited to the following exemplary embodiments.

[0292] Overview of Implementation Examples Previous clinical trials using CD22-targeted CAR-T receptors have all utilized single-chain variable fragments (scFv) based on mouse monoclonal antibodies, which have introduced numerous complex issues related to domain pairing (VH:VL pairing), including expression levels and stability. This can ultimately limit the productivity and efficacy of the final CAR-T construct. The approach described herein utilizes a camelid single-domain antibody (nanobody) for CAR targeting as an alternative to the scFv antibody domain. The potential for many therapeutic applications of camelid single-domain antibodies as soluble, stable modular domains has been well established by the bivalent nanobody, first FDA-approved in 2018. Thus, nanobodies provide excellent building blocks for CAR-T molecules, enabling the construction of a pool of functional CAR-T constructs; thus increasing opportunities to screen for more effective CAR-T cells for the treatment of non-solid tumor cells.

[0293] Furthermore, these nanobodies may also be used to develop additional safe and effective immunotherapy regimens, including but not limited to naked antibodies or drug-conjugated antibody therapies and bispecific immune cell engagers.

[0294] The approach described herein utilizes single-domain antibodies (sdAbs) derived from immunized llamas. These sdAb sequences specifically bind to the CD22 antigen, which is specifically expressed on human B cells and B-cell leukemia. Using these sdAb sequences, novel chimeric receptor sequences were constructed (in the form of 41BB, CD28 or other costimulatory domains and CD3ζ signaling domains) by combining CD22-specific sdAbs with T cell signaling molecules. In addition to applications as chimeric antigen receptors, these CD22-targeting antibodies may be useful for developing other forms of immunotherapy, including but not limited to applications on bispecific / trispecific T or NK cell engagers, antibody-drug conjugates, or naked antibodies.

[0295] Example 1: Preparation of sdAb antibody Introduction Single-domain antibodies (sdAbs) (also known as VHHs or nanobodies) are derived from the variable domain of the camelid heavy chain and are characterized by their stability, accompanied by a functional N-terminal domain with sufficient antigen-binding ability. In addition to their small size, sdAbs possess high affinity, solubility, low immunogenicity in humans due to high homology with the human VH3 family, high expression levels in microorganisms such as E. coli, and excellent stability at high temperatures, extreme pH, and high salt concentrations. Due to their excellent antibody engineering potential, sdAbs are considered ideal building blocks for bivalent and multispecific therapeutic reagents. Notable examples include the first FDA-approved bivalent anti-vWF nanobody (Caplacizumab, 2019) and 10 other therapeutic nanobodies in bivalent / multivalent or bivalent / multispecific formats; these have been advanced to preclinical and clinical development by Ablynx / Sanofi and other biopharmaceutical companies.

[0296] sdAbs are also ideal building blocks for creating chimeric antigen receptors (CARs); in this case, the cancer-specific antigen-binding domain (scFv, Fab) of conventional IgG is genetically fused with an immune cell activation domain to create "armed" immune T lymphocytes (CAR-Ts) that seek out and kill specific cells with a target antigen. Applying sdAbs to CAR-T constructs reduces the domain complexity of the scFv / Fab fragment and greatly improves the productivity and efficacy of the final CAR-T construct. It also allows for adding further specificity to the CAR-T construct (specificity to a second cancer biomarker) (i.e., creating bispecific CAR-T cells), thus increasing the opportunity to screen for more effective CAR-T cells for treating hematological malignancies. Similarly, these sdAbs are also ideal candidates for the development of other forms of immunotherapy, such as bi, tri, and multispecific immune cell engagers.

[0297] This study aimed to develop immunotherapies, including but not limited to CAR-T therapies, bi-, tri-, and multi-specific immune engager therapies, and naked therapeutic antibodies with appropriate human IgG fusions, by creating functional camelid sdAbs against the ectodomain of CD22, a B-cell-specific leukemia antigen. These therapies are intended for use in the treatment of cancer, autoimmune diseases, and inflammatory diseases. Examples of developing CAR-T and bi-specific immune engagers with effective antitumor activity using these sdAb sequences are presented.

[0298] material and method Cloning and expression of CD22-ECD The gene encoding the extracellular domain of the dominant human CD22β isoform was cloned into a proprietary pTT5:NRC mammalian expression vector. After transfection into NRC CHO-3E7 cells, the cells were cultured in 1L flasks, and the expressed protein containing a 6xHis tag at the C-terminus was purified by immunoaffinity chromatography (IMAC) followed by size exclusion chromatography and analyzed by SDS-PAGE.

[0299] Llama immunization Lama were immunized with the recombinant ECD domain of the CD22 antigen. In each injection, 100 μg of recombinant human CD22 protein in a total volume of 0.5 mL was mixed with equal volumes of complete Freund's adjuvant (first injection) and incomplete Freund's adjuvant (subsequent injection) and injected subcutaneously. Five injections were administered at approximately two-week intervals, and blood samples were collected after the third injection and 7 days after the last injection.

[0300] RNA isolation and PCR amplification The QIAamp RNA blood mini-kit (QIAGEN Sciences, Mississauga, ON) was used according to the kit instructions to collect approximately 1 x 10⁶ blood samples on day 49 of the immunization protocol. 7Total RNA was isolated from individual lymphocytes. Approximately 5 μg of total RNA was used as a template for first-chain cDNA synthesis using oligo-dT primers with a first-chain cDNA synthesis kit (Amersham Biosciences, USA). Three variable domain sense primers (MJ1-3) and two CH2 domain antisense primers (CH2 and CH2b3) were designed based on camelid and llama immunoglobulin databases (Baral TN et al 2013). Initial PCR was performed using cDNA as a template, and the variable regions of both conventional antibodies (IgG1) and heavy-chain antibodies (IgG2 and IgG3) were amplified in two separate reactions using MJ1-3 / CH2 and MJ1-3 / CH2b primer combinations. The PCR reaction mixture contained the following components: 2 μL of cDNA, 5 pmol of MJ1-3 primer mixture, 5 pmol of CH2 or CH2b primer, 5 μL of 10X reaction buffer, 3 μL of 2.5 mM dNTPs, 2.5 units of Taq DNA polymerase (Roche Applied Science, Indianapolis, IN), and water up to a final volume of 50 μL. The PCR protocol consisted of an initial stage of 3 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 55°C, and 1 minute at 72°C, and a final extension stage of 7 minutes at 72°C. The amplified PCR product, when run on a 2% agarose gel, consisted of two main bands: approximately 850 bp corresponding to conventional IgG1 and approximately 600 bp (550-650 bp) corresponding to heavy chain antibodies. The smaller band was excised from the gel, purified using the QIAquick gel extraction kit (QIAGEN), and re-amplified in a second PCR reaction; this PCR reaction involved 1 μL of purified DNA template and 5 pmol each of MJ7 (VH sense primers with underlined SfiI restriction sites; 5'-CAT GTG TAG ACT CGC) GGC CCA GCC GGC C AT GGC C-3') and MJ8 (antisense primers with underlined SfiI restriction enzyme sites; 5'-CAT GTG TAG ATT CCT GGC CGG CCT GGC CThe reagents included TG AGG AGA CGG TGA CCT GG, 5 μL of 10X reaction buffer, 3 μL of 2.5 mM dNTPs, 2.5 units of Taq DNA polymerase (Roche Applied Science, Indianapolis, IN), and water up to a final volume of 50 μL. The PCR protocol consisted of an initial stage of 3 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 55°C, and 1 minute at 72°C, and a final extension stage of 7 minutes at 72°C. The amplified PCR products (approximately 400-450 bp) corresponding to the VHH fragments of the heavy chain antibody were purified using the QIAquick PCR purification kit (QIAGEN), digested using SfiI (New England BioLabs), and purified again using the same kit.

[0301] Building a Library 30 μg of pMED1 (Arbabi-Ghahroudi et al. 2009) DNA was digested overnight at 50°C using SfiI. To minimize the opportunity for self-ligation, 20 units each of XhoI and PstI restriction enzymes were added, and digestion was continued at 37°C for a further 2 hours. For library construction, 10 μg of phagemid DNA was ligated with 1.75 μg of VHH fragment, and incubated at room temperature for 2 hours using the LigaFast DNA ligation system (Promega, Madison, WI) according to the recommended protocol. The ligation product was electroporated into competent E. coli TG1 cells (Stratagene, Cedar Creek, TX). The transformed bacterial cells were diluted in SOC medium and incubated at 37°C for 1 hour with gentle shaking. The library size was calculated by plating aliquots onto LB-Amp. VHH fragments from 96 colonies were amplified by PCR and sequenced for diversity analysis. This library was aliquoted and stored at -80°C.

[0302] Library panning and screening Two strategies were used for panning the CD22 immunotherapy library: biotinylation and passive adsorption.

[0303] a) Panning of biotinylated CD22 ECD : The constructed size is 3.3 x 10 7 The LPAR1 library 2 was phage rescued, and 1.4 × 10⁶ 9 Using phage titers of cfu / μL, panning was performed against CD22 ECD antigen pre-biotinized with EZ-link sulfo-NHS-LC-LC-Biotin (Thermoscientific catalog #21338). Four rounds of panning were performed using alternating blocking buffers [for example, Starter Block (Thermo Fisher catalog #37559) was used for rounds 1 and 3, and biotin-free casein was used for rounds 2 and 4]. Panning was performed alternately between wells coated with Pierce® streptavidin (rounds 1 and 3) (Thermoscientific catalog #15501; lot #TF252884) and wells coated with Pierce® neutraavidin (rounds 2 and 4) (Thermoscientific catalog #15508; lot #SK253835). First, two wells and one or two microfuge tubes were incubated overnight at 4°C in a suitable blocking buffer.

[0304] The next day, library input phage (approximately 3 x 10) 11 The phages were added to one of the streptavidin wells blocked with appropriate blocking buffer at room temperature for 1 hour, then transferred to a blocked microtube and mixed with biotinylated CD22 antigen. In subsequent rounds, approximately 3 × 10⁶ of amplified phages were obtained from each round. 11The input phage was used. In the first round, 1 μg of biotinylated CD22 antigen was used. In subsequent rounds, both 1 μg and 100 ng of antigen were used. After incubation at room temperature for 1 hour, the input phage / biotinylated CD22 mixture was transferred to other blocked streptavidin wells and incubated at room temperature for 30 minutes. This was followed by a washing step {3 × 300 μL of PBS-T (PBS + 0.05% Tween 20) (quick); 2 × 300 μL of PBS-T + (PBS + 0.05% Tween 20) (incubate 5 minutes after each wash); 3 × 300 μL of PBS (quick); 2 × 300 μL of PBS (incubate 5 minutes after each wash)} and elution with 100 μL of 100 mM TEA. The phages were then removed from the wells and neutralized in a new tube with 50 μL of 1 M Tris-HCl pH 7.4. OD in 15 mL Falcon tube with 2YT + 2% glucose 600 3 mL of exponentially growing TG1 E. coli culture, pre-cultured to =0.5 at 37°C and 250 rpm, was infected with the eluted phages. A 100 μL aliquot of uninfected TG1 E. coli cells was set aside as a control. The eluted phages were incubated at 37°C for 30 minutes without shaking, and the aliquots were titrated (10 2 ~10 8 The culture was used at the following dilution ratio and plated overnight on a 2YT plate at 32°C. The remaining 3 mL of the infected TG1 culture was treated with M13KO7 helper phage (approximately 1 × 10⁻¹⁶). 10 Phage amplification was carried out overnight using cfu.

[0305] The following day, the eluted titers were calculated to determine the amount of input phage for subsequent rounds. The cell cultures containing the amplified phages were centrifuged at 5000 rpm for 30 minutes, the supernatant was filtered through a 0.22 μM filter unit (Millipore), precipitated with 20% PEG / 2.5 M sodium chloride (NaCl), centrifuged, and resolubilized in PBS (pH 7.5). The titers of the amplified phages were measured in pre-cultured TG1 E. coli cells (10 4 ~10 12(Dilution ratio). After 4 rounds of panning, the sequences of positive colonies from phage ELISA were analyzed.

[0306] b) Passive panning of CD22-ECD The same rescued phages were used for panning against the CD22 ECD antigen. Four rounds of panning were performed using alternating blocking buffers [e.g., Starter Block (Thermo Fisher catalog #37559) was used for rounds 1 and 3, and 4% milk PBS was used for rounds 2 and 4]. The amount of CD22 ECD passively adsorbed into the Nunc well was reduced from 40 μg to 10 μg over the four rounds of panning (round 1: 40 μg, round 2: 30 μg, round 3: 20 μg, round 4: 10 μg). First, the wells were blocked with the appropriate buffer at room temperature for 2 hours, followed by input phages (approximately 3 × 10⁶). 11 ) was added. In subsequent rounds, approximately 3 × 10 from the amplified phage of each round. 11 The input phage was used. After incubation, washing steps were performed {R1: 5×PBS-T; 2×PBS; R2: 5×PBS-T; 5×PBS; R3: 7×PBS-T; 5×PBS; R4: 10×PBS-T; 10×PBS} and elution with 100 μL of 100 mM TEA. Then, the phage was removed from the well and neutralized in a new tube with 50 μL of 1 M Tris-HCl pH 7.4. Oxidation was performed in a 15 mL Falcon tube with 2YT + 2% glucose. 600 2 mL of exponentially growing TG1 E. coli culture, pre-cultured to 0.5 at 37°C and 250 rpm, was infected with the eluted phages. A 100 μL aliquot of uninfected TG1 E. coli cells was set aside as a control. The eluted phages were incubated at 37°C for 30 minutes without shaking, and the aliquots were titrated (10 2 ~10 8 The sample was used (dilution ratio) and plated on a 2YT plate overnight at 32°C. M13KO7 helper phage (approximately 1 × 10⁻¹⁶) 10Phage amplification was carried out overnight using cfu. The following day, the eluted titer was calculated to determine the amount of input phage for subsequent rounds. Cell cultures containing the amplified phages were centrifuged at 5000 rpm for 30 minutes, the supernatant was filtered through a 0.22 μM filter unit (Millipore), precipitated with 20% PEG / 2.5 M NaCl, centrifuged, and resolubilized with PBS (pH 7.5). The titer of the amplified phages was measured in pre-cultured TG1 E. coli cells (10 4 ~10 12 (Dilution ratio). After 4 rounds of panning, the sequences of positive colonies from phage ELISA were analyzed.

[0307] result Llamas were immunized with recombinant CD22-ECD, and serum titers were monitored and analyzed by ELISA. As shown in Figure 3, CD22 induced a strong heavy-chain immune response in llamas. The serum heavy-chain immune response was measured using two monoclonal antibodies that specifically bind to the hinge-CH2 boundary of llama IgG2b and to the Fc of llama IgG2c, respectively (Henry KA et al. Llama Peripheral B-cell Populations Producing Conventional and Heavy Chain-Only IgG Subtypes Are Phenotypically Indistinguishable but Immunogenetically Distinct. Immunogenetics 2019 Apr;71(4):307-320).

[0308] The heavy chain repertoire of llama immunoglobulin was amplified using gene-specific primers and cloned into a phagemid vector (pMED1). A medium-sized library (3.3 × 10⁶) was created. 7The complexity of the library was analyzed by constructing a single-domain antibody and sending 96 colonies for sequencing. Sequencing data showed that the library was highly complex, as all VHH sequences were full-length and free of repeats. This library was phage-rescue using the M13 helper phage, as described elsewhere (Baral TN, MacKenzie R, Arbabi Ghahroudi M. Single-domain antibodies and their utility. Curr Protoc Immunol. 2013 Nov 18;103:2.17.1-2.17.57), and the phage antibodies were used in two separate panning experiments. After four rounds of panning, 96 colonies from each panning strategy were cultured and co-infected with the M13 helper phage, as described elsewhere (Baral TN et al 2013), and these phages were used in ELISA. Positive colonies were sent for sequencing, and the sequencing data was analyzed. Sequence alignment was performed using OPIG software. http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / ) and IMGT numbering were used (see Figures 4A and 4B). Twenty-seven unique VHH sequences were selected for gene synthesis and cloning into expression vectors (pMROs).

[0309] Figure 1 shows the structure of the human CD22 molecule encoded by the CD22 gene located on chromosome 19q13.12. CD22β is the dominant isoform (having seven domains; 847aa). The cytoplasmic tail (140aa), which contains four immunoreceptor motifs, is connected to the extracellular domain (669aa) by a transmembrane domain (18aa).

[0310] Figure 2 shows SDS-PAGE of IMAC-purified CD22 extracellular domains (CD22-ECD) from two different expression batches under non-reducible and reduced conditions. The purified proteins have a predicted molecular weight of approximately 75 kDa.

[0311] Figure 3 shows the llama heavy chain immune response to CD22-ECD from the final blood collection (August 3). Pre-immunized llama serum was used as a control. Heavy chain antibody binding was detected by anti-llama mAb (our company's NRC), followed by donkey anti-mouse HRP. As shown, a potent and specific anti-CD22-ECD heavy chain immune response exists.

[0312] Consideration The extracellular domain of the dominant human CD22 isoform was successfully expressed in the mammalian CHO system, and recombinant CD22-ECD showed favorable results in all downstream assays (data not shown). Immunizing llamas with recombinant CD22-ECD resulted in a potent heavy chain immune response, as confirmed by ELISA using heavy chain-specific mAbs. By constructing a library of heavy chain repertoire, a VHH domain antibody specific to the immunogen (CD22-ECD) was isolated.

[0313] Example 2: Characterization of sdAb antibodies Introduction After obtaining a positive immune response to human CD22-ECD, a library of heavy chain repertoire proteins from immunized llamas was constructed. Subsequently, two different panning strategies were performed using biotinylated and unbiotinylated human CD22-ECD proteins, followed by screening of over 200 individual colonies by phage ELISA. Each VHH clone was sequenced and grouped based on their CDR1-3 sequences, resulting in 27 unique VHH sequences. The genes encoding these VHHs were cloned into NRC bacterial expression vectors, and the purified proteins were characterized.

[0314] material and method Soluble V H H expression OD by phage ELISA 450The DNA sequence of the most repeated clone showing >0.8 was sent to TWIST Bioscience for gene synthesis and subsequently cloned into a pMRO (pET28a derivative, Novagen) expression vector. E. coli TG1 cells were transformed with the VHH construct, and each clone was cultured in 0.25 L of 2×YT medium containing 0.1% glucose + ampicillin (100 mg·mL-1) until the OD600 reached 0.8. The cultures were induced with 1 mM IPTG and cultured overnight at 37°C on a rotary shaker. After confirming expression by SDS-PAGE and Western blotting, recombinant VHH protein was extracted from bacterial cells using a standard lysis method, purified by immobilized metal affinity chromatography (IMAC), and quantified as described elsewhere (Baral TN, Arbabi-Ghahroudi M. Expression of single-domain antibodies in bacterial systems. Methods Mol Biol. 2012;911:257-75). The VHH protein was subjected to Supdex 75 size exclusion chromatography to recover the monomer fraction.

[0315] SPR analysis For surface plasmon resonance (SPR), 21 selected VHHs were passed through a Superdex 75 size exclusion column (GE Healthcare) in 10 mM HEPES containing 150 mM NaCl and 3 mM EDTA, pH 7.4. The monomer sdAb fraction was collected, and the protein concentration was determined by measuring the absorbance at 280 nm (A280). The analysis was performed using a Biacore T200 instrument (GE Healthcare). All measurements were performed at 25°C in 10 mM HEPES containing 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.4 (GE Healthcare). Approximately 1919 RU of recombinant monomer CD22-ECD (obtained after SEC purification of CD22-ECD) was captured on an SA sensor tip (GE Healthcare) at a flow rate of 5 μL / min. Various concentrations of monomer VHH were injected into the CD22-ECD surface at a flow rate of 40 μL / min, using the SA surface as a reference. The surface was prepared by washing with running buffer. The data were analyzed using BIAevaluation 4.1 software.

[0316] Epitope binning by SPR In addition to obtaining binding reaction rate data, Biacore co-injection experiments were also performed on 11 selected VHHs to investigate whether these anti-CD22 VHHs could bind to unique or overlapping epitopes on the CD22-ECD protein surface. Briefly, 80 μL of the first VHH was diluted in HBS-EP buffer to a concentration five times its KD value and injected at 40 μL / min onto 1919 RU immobilized CD22-ECD. After the injection of the first VHH, buffer or a second VHH (total volume 80 μL, 5 × KD) was injected at 40 μL / min onto the CD22-ECD surface already saturated with the first VHH. For all possible pair combinations of the 11 VHHs, data was collected and evaluated as described above in both orientations (i.e., using each VHH as both the first and second VHH).

[0317] Epitope mapping using yeast surface displays The hCD22 ectodomain (ECD) and its derived fragments were expressed and covalently displayed on the surface of yeast cells using a yeast surface display (Feldhaus et al., 2003, Nat. Biotechnol. Vol. 21, 163-170). The YSD vector (pPNL6) was obtained from the Pacific Northwest National Laboratory in the United States. Seven hCD22 fragments, named bins 1-7, with overlapping ends (15-25aa), covering the entire hCD22-ECD (668aa), were cloned together with the full-length hCD22-ECD and expressed as a fusion protein (Aga2-HA-(hAXL)-MYC) on the surface of yeast cells. The displayed hCD22 fragments were used to map the domain of hCD22 to which the anti-hCD22 sdAb of Example 1 binds. Binding of (biotinylated) sdAb to the CD22 fragment on yeast cells was performed using whole yeast cell ELISA probed with HRP-labeled streptavidin. The relative amount of the expressed fusion protein was measured by probing with anti-MYC antibody, then with HRP-labeled secondary antibody, and used to normalize the sdAb binding signal. HRP activity was assayed using the substrate TMB (tetramethylbenzidine) according to the manufacturer's conditions, and OD 450 The data was read using [method / tool]. To investigate the properties of the epitope, whether it was linear or three-dimensional, yeast cells displaying the CD22 fragment were heated at 80°C for 30 minutes, then cooled on ice for 20 minutes, and finally labeled with antibody.

[0318] Evaluation of target specificity of sdCD22 VHH The target specificity of sdCD22 Ab was evaluated by flow cytometry using biotinylated VHH. Human Burkitt lymphoma cell lines Raji and Ramos, which express CD22, and Ramos cells in which CD22 expression was deleted using CRISPR gene knockout, were incubated with biotin-labeled sdCD22 VHH at a 5-fold dilution of 7.5–0.06 μg / mL. Binding of CD22-targeted VHH to cell surface CD22 was detected by flow cytometry using fluorescently labeled streptavidin.

[0319] result Gene synthesis and subcloning were performed at TWIST Bioscience (USA), and TG1 E. coli were transformed with plasmid DNA for protein expression. Because the pMRO vector contains a histidine tag and a biotinylation signal sequence, not only is purification by IMAC column easy, but specific addition of the biotin moiety at the VHH C-terminus is also possible. Single biotin addition facilitates the detection of VHH in future epitope mapping assays. IMAC-purified VHH proteins were subjected to SDS-PAGE (Figure 5; showing only 14 types of VHH). As shown, the VHH antibody exhibits the expected molecular weight of approximately 15–17 kDa.

[0320] The aggregation state of the purified proteins was confirmed by size exclusion chromatography, and as expected, all were non-aggregating monomers (data not shown). The reactivity of individual VHH proteins was also confirmed by ELISA, in which case a rabbit anti-His6 antibody conjugated to HRP was used to detect VHH binding to immobilized CD22-ECD (data not shown).

[0321] A total of 21 monomer fractions of VHH were used in the SPR experiment. Here, human CD22-ECD was immobilized on a CM5 dextran chip, and VHH at various concentrations (0.6 to 400 nM) was passed through the sensor chip. SPR analysis revealed that all 21 VHHs specifically bound to CD22-ECD with equilibrium constants ranging from 250 nM for hCD22pas-64 to 6 pM for hCD221ug-14. All collected data fit a 1:1 binding model.

[0322] In epitope binning of anti-CD22 VHHs, co-injection SPR experiments were performed using pairs of VHHs in both orderings to investigate whether the antibodies could simultaneously bind to CD22-ECD. An increase in response when any two VHHs are simultaneously injected would indicate that these antibodies recognize independent epitopes, as the binding (saturation concentration) of the first VHH does not hinder the binding of the second VHH. However, if the response does not change significantly when two VHHs are simultaneously injected, it would indicate that they recognize overlapping / identical epitopes, as the two VHHs cannot simultaneously bind to the same region. Co-injection SPR experiments were performed on 11 anti-CD22 VHHs, and seven bindings were identified as shown in Table 5. The remaining 10 VHHs showed high sequence similarity in the CDR region to the aforementioned 11 VHHs binned by SPR. Therefore, these 10 VHHs were placed in bins with the closest amino acid similarity, as shown in Table 5.

[0323] Figures 4A and 4B show the amino acid sequence alignments of 27 VHH molecules.

[0324] Table 1 (above) shows the amino acid sequences of 27 types of VHH. The CDR region (underlined) and framework region are in the IMGT numbering system. www.IMGT.com They are numbered according to the following criteria.

[0325] Table 2 (above) shows the correspondence between the VHH name, number, and SEQ ID NO.

[0326] Figure 5 shows the SDS-PAGE of 14 anti-CD22 VHH antibodies expressed in TG1 E. coli and purified by IMAC. The purified proteins showed the expected molecular weight of 15-17 kDa, and no signs of degradation were observed in any of the protein samples.

[0327] Table 4 shows the measured affinity of all 27 types of VHH described in the text. The affinity data range from pM (6 pM for hCD221ug-14) to high nM (250 nM for hCD22pass-64).

[0328] [Table 4] TIFF0007869193000012.tif111170

[0329] Table 5 shows epitope binning by co-injection SPR experiments. Pairs of VHHs were co-injected in both orderings, and the increase in response units was measured for 11 anti-CD22 VHHs. As shown, seven bins were confirmed by 1-4 members. The remaining 10 VHHs were placed in their respective bins because they showed high amino acid sequence similarity to the 11 VHHs binned by SPR.

[0330] [Table 5] TIFF0007869193000014.tif85153

[0331] Figure 6 shows the binding of biotin-labeled anti-CD22 VHH to CD22-expressing tumor cells (Raji, left panel), Ramos cells (center panel), or Ramos cells manipulated to lack CD22 expression by CRISPR gene knockout (right panel). The cell binding analysis included nine VHH sequences from representative panels; this analysis shows varying degrees of dose-dependent binding to CD22-positive Raji and Ramos cells. In all cases, binding strength was significantly reduced or absent when tested with CD22-negative Ramos cells.

[0332] Table 6 shows the physical binning of CD22 sdAbs shown in Example 1 against hCD22 ectodomain fragments displayed on the yeast surface using cell ELISA. The displayed CD22 fragments are defined as physical bins indicating the coverage of mature CD22 (in amino acid residues according to the numbering). Binding of each sdAb (biotinylated) at 250 nM to a specific CD22 fragment(s) on the yeast cell surface was elucidated by the enzymatic activity of streptavidin-bound HRP. OD > 0.25 450 Wells that produced a reaction are highlighted, indicating significant antibody-antigen interactions. All tested sdAbs except hCD22_1ug-14 were assigned to specific bins. Both hCD221ug-13 and hCD221ug-80 were assigned to two bins, suggesting that these sdAbs recognize epitopes shared at multiple locations on CD22.

[0333] [Table 6]

[0334] Table 7 shows the evaluation of the properties of CD22 sdAb epitopes to determine whether the CD22 sdAb epitopes are continuous (linear) or discontinuous (conventional). Various fragments of the CD22 domain were expressed on yeast cells, and their binding to sdAbs was assayed using the cell ELISA described in Table 6, using either native (ND) or heat-denatured (D) YSD cells as described. The binding of each sdAb to native and denatured CD22 fragments was assayed using OD. 450 The binding ratio between denatured and undenatured CD22 was calculated by measuring the ratio; a ratio ≥ 0.5 indicates a linear (L) epitope, and a ratio < 0.5 indicates a structural (C) epitope.

[0335] [Table 7]

[0336] Figure 7 is a schematic diagram showing the sensory binding of sdAb of Example 1 to subdomains of the CD22 ectodomain relative to cell surface locations, based on the epitope mapping / binning results in Tables 5 and 6. Each subdomain / bin is defined and numbered as in Table 6, and represented as an ellipse with bin 1 distal to the cell membrane and bin 7 proximal. Binding of sdAb (indicated by specific shape and name) to a subdomain / bin is indicated by its proximity to the subdomain / bin it binns.

[0337] Consideration Anti-CD22-ECD VHH was expressed in E. coli, and the resulting protein was purified and biotinylated. The antibody exhibited non-aggregated monomeric behavior when confirmed by size exclusion chromatography. Binding rate reactions of 21 VHHs were measured by SPR, showing that the antibody specifically bound to human CD22-ECD with affinities ranging from low to high nanomolar, with the exception of one VHH with an affinity of 6 picomoles. This diverse set of affinities allows for investigation of the effect of affinity on the productivity of CAR-T constructs. Epitope binning of 11 VHHs by SPR showed that some VHHs bound to overlapping epitopes, while others bound to unique epitopes. This was confirmed through antibody epitope binning using a yeast surface display library of human CD22 ectodomain fragments and by using cell ELISA. All tested sdCD22 Abs, except for 1ug-14, were assigned to specific bins. Both 1ug-13 and 1ug-80 could be assigned to two bins, suggesting that these sdAbs recognize epitopes shared at multiple locations on CD22. This epitope binning information can be used to design biparatopic therapeutics containing multiple sdCD22 VHH sequences.

[0338] Example 3: In vitro testing of CAR-T Introduction Following the identification of the novel CD22-binding single-domain antibody (sdAb) sequence described above, it was desirable to validate its activity in the context of a chimeric antigen receptor (CAR) molecule; this CAR molecule can be used to direct the human T cell response towards cells possessing specific surface antigens. Therefore, using the previously described high-throughput technique (Bloemberg, et. al. 2020), novel CD22-sdAb-targeted CAR constructs were constructed, and their relative T cell activation activity was tested using various assays described below.

[0339] material and method The antigen-binding sequence of a single-domain antibody was transferred to a modular CAR plasmid backbone [SEQ ID No: 119] containing a restriction site to enable efficient recombination; in this case, the antigen-binding domain was removed and replaced with a novel CD22 antigen-binding domain (ABD) sequence. The specific CAR designs used were as follows: one of the following: human CD28 signal peptide [SEQ ID NO: 110], VHH antibody (ABD) [SEQ ID NO: 82-108], flexible linker domain [SEQ ID NO: 111], human CD8 hinge domain [SEQ ID NO: 112], human CD28 transmembrane domain [SEQ ID NO: 113], human 4-1BB costimulatory domain [SEQ ID NO: 114], and human CD3ζ signaling domain [SEQ ID NO: 115]. The control construct was also constructed using a sequence derived from either a previously shown CD19-specific CAR sequence (in this case, the ABD being the FMC63-scFv sequence [SEQ ID NO: 117]) or a CD22-specific CAR sequence (in this case, the ABD being the M971-scFv sequence [SEQ ID NO: 116]).

[0340] Subsequently, the novel CD22-targeted CAR construct was tested for activity in an immortalized human T cell line (Jurkat), similar to the method described in Bloemberg et al. 2020. Briefly, the plasmid was electroporated into Jurkat T cells and allowed to recover for several hours. Then, Jurkat-CAR cells were mixed with target cell lines exhibiting different levels of human CD22 expression at various doses. This study utilized a target cell line (Ramos) that highly expressed CD22 to confirm CAR activation activity in Jurkat cells. Cells with CD22 expression removed using CRISPR genome editing were also used to confirm the specificity of the novel CAR construct to the human CD22 antigen. Subsequently, co-cultures of CAR-expressing Jurkat cells and target cells were incubated overnight under standard mammalian cell culture conditions to allow CAR activation to occur. To quantify CAR-mediated Jurkat cell activation, CD69 expression was measured using specific antibody staining and flow cytometry. CAR-expressing cells were gated using GFP marker expression, and the level of T cell activation was measured using the CD69 surface marker. In various Jurkat cells expressing CD22-sdAb-targeted CAR constructs, CD69 marker levels were significantly elevated when these cells were co-cultured with CD22-expressing Ramos cells, but not when co-cultured with CD22 knockout cells (Figure 8).

[0341] Following the CAR-J trial, several CD22-CAR constructs were selected for testing in primary human T cells. To achieve this, lentiviruses were generated by simultaneous transfection of CAR plasmids with lentiviral packaging cell lines. Lentiviral particles were collected from the cell supernatant and concentrated using ultracentrifugation. Next, primary human T cells were isolated from donor blood samples using magnetic bead separation and polyclonally activated with anti-CD3 and anti-CD28 beads. Subsequently, the activated human T cells were introduced with concentrated lentiviruses containing various CD22-targeted CAR constructs at infection multiplicity of more than 10. After viral introduction, cell expression of CARs was confirmed using flow cytometry analysis of the GFP marker. Subsequently, the transmuted T cells (CAR-T cells) were grown for 9 days before testing CAR activity.

[0342] Several assays were used to investigate CAR activity in virus-transfected CAR-T cells. First, cells were placed under controlled cell culture conditions without additional stimulation, and nonspecific cell proliferation over 6 days was observed using a live microscope with an IncuCyte® S3 instrument (Sartorius, USA). Total cell count was measured using an automated cell counter. Primary human T cells stably transfected with various CD22-sdAb-targeted CAR constructs did not show significant cell proliferation when left unstimulated from day 9 to day 15 after polyclonal activation (Figure 9). These results indicate that the tested CD22-sdAb-targeted CAR constructs do not confer target-independent tonic T cell activation to primary human T cells.

[0343] Subsequently, primary CAR-T cells were tested for antigen-specific activation and target cell killing in response to CD22-expressing and non-CD22-expressing cells. CAR-T cells were co-cultured with various target cells expressing the red fluorescent protein tag Nuclight-Lenti (Sartorius, USA) and monitored for 6 days using an IncuCyte S3 live microscope. When examining the number of GFP-labeled CAR-T cells, two CAR constructs (1ug36 and 1ug10) showed clear proliferation of GFP+ cells in response to CD22+ cell lines (Raji, Ramos), but did not respond to CD22 knockout cells (Ramos-CD22ko), demonstrating antigen-specific activation and proliferation (Figure 10). When examining the number of Nuclight-labeled target cells co-cultured with the most active CD22-CAR construct (1ug36), clear suppression of CD22+ target cell growth was observed, but there was little effect on CD22 knockout cells (Figure 11). Based on these results, the lead molecule (1ug36) was selected for further testing.

[0344] Next, we conducted experiments to demonstrate the serial killing ability of novel CD22-sdAb-targeted primary CAR-T cells. As described above, CAR-T cells were generated from donor blood-derived T cells using lentiviral transduction and grown in cell culture for 9 days. Next, the CAR-T cells were co-cultured with fluorescently labeled Ramos cells to examine the proliferation of target cells. As described above, the lead construct showed the most effective target cell suppression among the constructs tested (Figure 12, top). After 1 week of co-culture, the CAR-T cells were rechallenged with fresh Ramos target cells to examine their ability to continue killing target cells (Figure 12, bottom). In this case as well, the 1ug36-CAR construct showed the most clear suppression of target cell proliferation among the constructs tested, and was therefore selected for further analysis.

[0345] To investigate the intradonor and interdonor variability of the novel CD22-sdAb-targeted CAR construct, additional CAR-T cells were generated from two different donor blood samples as described above. In this experiment, the novel CAR-T construct was also compared to a benchmark CD22-targeted CAR (a previously demonstrated CAR construct consisting of a human CD22-specific single-chain variable fragment [SEQ ID NO: 116] within a CAR construct similar to the one used to test the novel sdAb construct described herein). Subsequently, the CAR-T cells were co-cultured with CD22-expressing target cells (Raji) and the inhibition of tumor cell growth (Figure 13 top) and CAR-T cell proliferation (Figure 13 bottom) were examined. The results demonstrate that the lead CAR construct exhibits a consistent response across various donors, similar to the benchmark CAR response.

[0346] Next, we investigated the specific lysis of CD22-sdAb-targeted CAR-T cells from two donors, targeting CD22-expressing cells (Raji) and CD22-negative cells (MCF7). Various CAR-T cells prepared as described above were co-cultured with radioactive chromium-loaded target cells for 4.5 hours under standard cell culture conditions. Following co-culture, the cell supernatant was removed, and the chromium content was counted using a scintillation counter. The experiments were performed using various CAR constructs from CAR-T cells derived from the two donors, either with a single effector:target ratio (Figure 14), or with various effector:target ratios using the lead CD22-sdAb CAR construct identified above (Figure 15). The results demonstrate that specific target cell lysis is induced by CAR-T cells introduced with the novel CD22-sdAb CAR construct.

[0347] Finally, the ability of CD22-sdAb-targeted CAR-T cells to be restimulated by CD22-expressing tumor cells (Ramos) was evaluated. Various CAR-T cells containing the lead 1ug36-CAR construct were generated as described above and cultured from the proliferative expansion phase on day 9 to the quiescent phase on day 17. Subsequently, the CAR-T cells were stimulated with irradiated CD22-expressing Ramos cells for an additional 5 days, at which point their potential cytotoxicity against chromium-loaded CD22-expressing target cells (Raji) and CD22-negative target cells (MCF7) was evaluated. After co-culturing for 4.5 hours in various effector:target ratios, the released chromium was quantified using a scintillation counter (Figure 16). The results indicate that restimulation of quiescent CD22-targeted CAR-T cells containing the lead 1ug36-CAR construct with CD22-carrying Ramos cells resulted in the retention of specific lytic activity against CD22-expressing Raji target cells at a level similar to that of the scFv CAR benchmark.

[0348] result Figure 8 shows the results of the CAR-Jurkat assay when Jurkat cells were transiently electroporated with various CAR plasmids and cultured alone or co-cultured with CD22-positive (Ramos) or CD22-negative (Ramos-CD22ko) cell lines. T cell activation levels were measured by human CD69-specific antibody staining and flow cytometry. The graph shows the average fluorescence intensity of CD69 staining for 28 single-domain antibody-targeted CAR constructs, cultured with either no target cells (white bars), Ramos-targeted cells (black bars), or Ramos-CD22ko cells (gray bars), with each experiment performed in pairs. Error bars indicate the standard error of the mean values ​​in the pairs of wells. The results demonstrate antigen-specific responses from approximately half (15 / 28) of the novel CAR constructs tested.

[0349] Figure 9 shows the results of a CAR-T tonic activation assay when various CAR constructs were introduced into primary T cells derived from donor blood to investigate target-independent proliferation. Mock cells refer to donor-derived T cells subjected to similar treatment conditions in the absence of lentiviruses expressing the CAR. As described in the methods, CAR-T cells were observed to proliferate in cell culture under live microscopy from day 9 to day 15 after polyclonal activation. The graph shows the multiplicative change in cell number relative to the initial cell number of this assay, measured using automated cell counting. The results demonstrate that the tested CAR constructs did not result in antigen-independent T cell proliferation.

[0350] Figure 10 shows the results of CAR-T target-specific activation assays performed using donor blood-derived T cells transfected with various CD22 single-domain antibodies or a control (FMC63) CAR constructs. Mock cells refer to unmodified donor-derived T cells without CAR expression, subjected to similar treatment conditions. As described above, CAR-T cells were co-cultured with CD22+ target cells (left graph - Raji, middle graph - Ramos target) or CD22-negative target cells (right graph - Ramos-CD22ko target) and observed for proliferation by live fluorescence microscopy from days 9 to 15 after polyclonal activation. Graphs show the proliferation rate of CAR-expressing cells marked with green fluorescent protein, measured using automated cell counting. The results demonstrate specific proliferation of CAR-T cells in response to CD22-expressing target cells, with the 1ug36-BBz CAR construct showing the greatest activity compared to the other constructs tested.

[0351] Figure 11 shows the results of a CAR-T antigen-specific target cell growth suppression assay performed using donor blood-derived T cells transfected with various CD22 single-domain antibodies or a control (FMC63) CAR construct. Mock cells refer to unmodified donor-derived T cells without CAR expression, subjected to similar treatment conditions. CAR-T cells were co-cultured with CD22+ target cells (left graph - Raji, center graph - Ramos target) or CD22-negative target cells (right graph - Ramos-CD22ko target) and observed using live fluorescence microscopy. Graphs show the proliferation rate of target cells marked with red fluorescent protein (Nuclight), measured using automated cell counting. The results demonstrate CAR-T specific suppression of CD22-expressing target cell growth, with the 1ug36-BBz CAR construct showing the greatest activity compared to the other constructs tested.

[0352] Figure 12 shows the results of a CAR-T target-specific serial killing assay performed using donor blood-derived T cells into which various CD22 single-domain antibody CAR constructs were introduced, as prepared as described above. "Mock" refers to unmodified donor-derived T cells without CAR expression, subjected to similar treatment conditions. CAR-T cells or mock T cells were co-cultured with CD22+ target cells (upper graph - Ramos target). Six days after the initial challenge, the cells were divided into fifths in fresh medium and challenged with additional CD22+ target cells (lower graph - Ramos target). The graphs show the proliferation rate of target cells marked with red fluorescent protein (Nuclight), measured using automated cell counting. The results demonstrate CAR-T specific serial suppression of CD22-expressing target cell growth, with the 1ug36-BBz CAR construct showing the greatest activity compared to the other constructs tested.

[0353] Figure 13 shows the results of consistency analysis and comparison with benchmark CD22-targeted scFv CARs for single-domain antibody-targeted CAR-T cells generated from two separate donors, as described above. Mock cells refer to donor-derived T cells exposed to similar treatment conditions in the absence of lentivirus expressing the CAR. CAR-T cells were co-cultured with CD22+ target cells (Raji) in two-well pairs and observed using a live fluorescence microscope. Graphs show the proliferation rate of target cells marked with red fluorescent protein (Nuclight) (upper graph) or CAR cells marked with green fluorescent protein (lower graph), measured using automated cell counting. The results demonstrate consistency within and between donors for CAR-T specific suppression of CD22+ target cell growth and target-induced proliferation of CAR-T cells similar to that of benchmark CD22-scFv CARs.

[0354] Figure 14 shows radioactive chromium ( 51 The results of direct tumor lysis measurements using a Cr) release assay are shown. As described above, various CD22 single-domain antibodies and control CAR-T cells were prepared. Mocks refer to donor-derived T cells exposed to similar treatment conditions in the absence of lentivirus expressing the CAR. Nine days after lentivirus induction, CAR-T cells were co-cultured with radioactive chromium-loaded CD22-positive (Raji) or CD22-negative (MCF7) target cells. The graph shows the relative lysis of target cells at a single effector:target ratio, based on the quantification of chromium release into the supernatant. Error bars indicate the standard deviation of measurements from a pair of wells. The results demonstrate comparable direct lysis of the lead CD22-sdAb CAR molecule compared to the benchmark CD22-scFv CAR.

[0355] Figure 15 shows the results of direct tumor lysis induced by CAR-T cells at different effector:target ratios using a chromium release assay. CD22-CAR construct lug36 CAR-T cells, scFv benchmark M971 CAR-T cells, and CD19 CAR construct FMC-63 CAR-T cells were prepared as described above. Mock cells refer to donor-derived T cells exposed to similar treatment conditions in the absence of CAR-expressing lentiviruses. After growing the CAR-T cells for 9 days, these cells were co-cultured with chromium-loaded CD22-positive Raji or CD22-negative MCF7 target cells for 4.5 hours, gradually decreasing the effector:target ratio. Specific lysis was calculated based on released chromium measured with a scintillation counter. The results demonstrate that lug36-CAR-T cells specifically lyse CD22-expressing Raji targets in a dose-dependent manner, similar to the scFV benchmark. When these CAR constructs were co-cultured with CD22 or CD19-negative MCF-7 tumor cells, no target cell killing beyond the background (mock control) was observed.

[0356] Figure 16 shows the results of direct tumor lysis measurements using a chromium release assay from CAR-T cells after restimulation with CD22-carrying tumor cells. As described above, various CD22 single-domain antibodies and control CAR-T cells were prepared. Mock cells refer to donor-derived T cells exposed to similar treatment conditions in the absence of CAR-expressing lentiviruses. After culturing CAR-T cells until growth arrest (day 17), they were stimulated with irradiated CD22-expressing Ramos cells to induce target-specific activation. After 5 days of co-culture, activated CAR-T cells were mixed with radioactive chromium-loaded CD22-positive (Raji) or CD22-negative (MCF7) target cells, gradually decreasing the effector:target ratio. The graph shows the relative lysis of target cells based on the quantification of chromium release into the supernatant. The results demonstrate efficient reactivation of quiescent CAR-T cells by specific CD22 antigens and dose-dependent Raji oncolysis (CD22-positive) of the lead CD22-sdAb CAR molecule, comparable to that of the benchmark CD22-scFv CAR.

[0357] Consideration Overall, these results illustrate that CD22-specific single-domain binders can elicit potent antigen-driven T cell activation signaling, promoting target cell killing and serial killing, prolonged tumor cell growth inhibition, CAR-T proliferation, and direct target cell lysis. While one lead molecule was identified in the exemplary data provided here, further optimization of the molecule using additional CD22-specific single-domain antibody sequences is possible to construct highly functional CAR molecules. Furthermore, combining multiple CD22-specific single-domain antibody sequences into a single molecule may be an effective strategy for enhancing target-specific CAR activation activity.

[0358] Example 4: In vivo testing of CAR-T Introduction To further confirm the in vitro antitumor effect of CD22-binding single-domain CAR-T cells, NOD / SCID / IL2rγ chains were introduced prior to the injection of CD22-binding single-domain CAR-T cells.null A xenograft model was established by intravenously inoculating (NSG) mice with Ramos tumor cells expressing firefly luciferase as a reporter. material and method

[0359] In in vivo studies, luciferase-expressing cell lines were created by stably introducing a lentiviral vector encoding firefly luciferase (FLUC) into wild-type tumor cells, followed by selecting luciferase-positive cells using puromycin resistance as a selection marker. Ramos-FLUC cells were maintained in RPMI 1640 supplemented with 10% thermoactivated fetal bovine serum, 2 mM L-glutamine, and 1 mM sodium pyruvate. All cell culture reagents were purchased from Gibco. Cell lines were confirmed to be free from mycoplasma contamination by PCR.

[0360] 6-8 week old female NOD / SCID / IL2Ry - / - (NSG) mice were obtained from Jackson Laboratories and maintained at the Animal Resource Group of the National Research Council of Canada. The mice were housed in pathogen-free, individually ventilated cages under controlled barrier systems. The animals were given free access to certified rodent feed and sterile water from water bottles. NSG mice lack mature T cells, B cells, and natural killer cells; therefore, they are superior to nu / nu mice for research. At 8 weeks of age, NSG mice were given 5 × 10⁶ cells in 100 μL of HBSS. 4Ramos-FLUC cells were intravenously injected into the tail vein. Four days after tumor cell injection, mice were intravenously injected into the retro-orbital plexus with CD22-targeted single-domain CAR-T cells, CD19-targeted CAR-T cells (whose target-directing domain is similar to the clinically validated CD19 CAR constructs Kymriah and Yescarta), or mock CAR-T cells without CAR expression. Tumor growth in the mice was monitored using bioluminescence (IVIS imager; PerkinElmer), and blood was collected weekly to monitor circulating CAR-T cells and tumor cells. Mice were monitored daily for signs of disease, and were immediately sacrificed if they met pre-specified humane endpoints, including but not limited to hind limb paralysis, respiratory distress, or 20% weight loss, as approved by the research center's animal care committee.

[0361] Mouse blood samples were washed with PBS / 0.1% BSA and resuspended in 50 μl of Brilliant buffer (BD Biosciences, USA). Cells were stained with antibody fluorescent dye conjugates against the following (all from BD Biosciences, USA unless otherwise noted): hCD45-APC-H7, hCD45RA-BV650, hCD45RO-PE-CF594, hCD27-BUV737, hCCR7-PE, hCD4-BUV395, and hCD8-PerCP-Cy5.5.

[0362] CD45 is a protein tyrosine phosphatase that regulates the src family of kinases and is expressed in all hematopoietic cells. Thus, an anti-human CD45 antibody was used to detect human hematopoietic cells in mice. CD45 can be expressed as one of several isoforms by alternative splicing of exons including the extracellular domain; the expression of various isoforms indicates the differentiation state of T cells. CD45RA is expressed in naive T cells and in effector cells of both CD4 and CD8. After antigen experience, central memory T cells and effector memory T cells acquire CD45RO expression and lose CD45RA expression. Therefore, antibodies against human CD45RA and CD45RO were used to distinguish between naive T cell populations and memory T cell populations. Distinction between central memory populations and effector memory populations, and between naive populations and effector populations, can be achieved by adding a second marker. Several markers have been used for this purpose, and these markers tend to mark these populations at slightly different stages of the differentiation pathway that are thought to occur in T cells when they change from central memory cells to effector memory cells. The chemokine receptor CCR7 distinguishes these two populations; therefore, for this purpose, an antibody against human CCR7 was used in this study. CAR expression was detected by GFP incorporated into the plasmid. To assess exhaustion, an antibody against hPD-1-BV421 (BioLegend, USA) was applied. T cell activation was detected by fluorescently labeled antibodies against hCD25-PE-Cy7 and hCD69-BV786. In in vivo studies, mouse cells were gated with mouse CD45-BV711, and leukemia cells were identified using human CD19 expression with anti-human CD19-BUV496 antibody. Samples were incubated in the dark at 4°C for 30 minutes. Subsequently, RBC lysis buffer (Sigma-Aldrich, USA) was added to dissolve the blood. The sample was briefly mixed to resuspend the cells and incubated at room temperature for 10 minutes.Subsequently, the cells were washed with PBS / 0.1% BSA and resuspended in PBS / 0.1% BSA. Data were acquired using a BD Fortessa cytometer (BD Biosciences). UltraComp® eBeads (eBiosciences, USA) were used for compensation. Analysis was performed using FlowJo software (FloJo, USA).

[0363] In vivo bioluminescence imaging to monitor tumor growth in mice was performed using the IVIS Lumina III imaging system (Perkin Elmer, Waltham, MA, USA). Mice were repeatedly anesthetized with isoflurane (3.0% during induction, reduced to 2.0% during maintenance) and their hair was removed. At time 0, 150 mg / kg of Redi-Ject D-luciferin (Perkin Elmer, Waltham, MA, USA) was subcutaneously injected into the mice. Subsequently, the animals were transferred to the imaging system under maintenance anesthesia, and imaging was performed using an open filter 20 minutes after D-luciferin administration to ensure uniform distribution of D-luciferin and a plateau in the signal. To calculate the relative expression level of the luciferase gene from the images, we used Living Image software (Perkin Elmer, Waltham, MA, USA) to determine the total radiance in photons / second / square centimeter / steradian (p / s / cm2 / sr) in the region of interest (ROI) throughout the body. result

[0364] Figure 17 shows the schema of the experimental protocol for this in vivo model. To evaluate the activity of CD22-binding single-domain CAR-T in the xenograft model, 8-week-old NOD / SCID mice were intravenously inoculated with 50,000 Ramos-FLUC cells on day 0, followed by 1 × 10⁶ cells on day 4. 7Mice were treated with posterior orbital injection of CD22-targeted single-domain CAR-T cells (1ug13 or 1ug36), FMC63-CAR-T cells (CD19-targeted CAR-T cells using the same antigen-binding domain as clinically validated CAR constructs), or mock T cells lacking CAR expression. Mice were imaged by bioluminescence in vivo imaging, and blood was collected weekly from the mice.

[0365] Figure 18 shows the results of survival analysis of NSG mice inoculated with Ramos-Luc and subsequently treated with various CAR-T cells. Mock refers to donor-derived T cells exposed to similar treatment conditions in the absence of lentivirus expressing CAR. The animal experiment setup is as described above. The graph shows the survival time of mice in this study in days. The results demonstrate the long-term survival observed in mice treated with CD22-targeting single-domain antibodies 1ug13 and 1ug36 CAR-T cells compared to those treated with FMC63 and mock T cells.

[0366] Figure 19 shows the tumor burden results for mice inoculated with Ramos-FLUC and treated with various CAR-T cells. Tumor burden was monitored in mice by quantifying bioluminescence using IVIS Lumina III. The graph shows the total flux (photons / second) (black circles) for individual animals within each treatment group and the average total flux per group (horizontal bars) at 18 days after tumor cell injection, the final experimental point in time when mice from all groups were still alive. Mice treated with 1ug36 CAR-T cells or FMC63 CAR-T cells showed a significant reduction in tumor burden compared to mice administered with mock T cells (p<0.001 and p=0.0004, respectively).

[0367] Figure 20 shows the results of different phenotypic tumor loading, total CAR-T cell population, and circulating CAR-T cell population in peripheral blood of mice inoculated with Ramos-FLUC and treated with various CAR-T cells. Blood was collected 23 days after tumor cell challenge, erythrocytes were lysed, and the remaining cells were stained with various fluorescently labeled antibodies as described above. The left graph shows the number of human CD19+ leukemia cells per million total blood cell events analyzed by flow cytometry. The center graph shows the number of CD45+ / GFP+ CAR-T cells detected per million blood cells analyzed by flow cytometry. The right graph shows the differentiation state of gated CAR-T cells, or the differentiation state of ungated CD45+ cells in mock T cell populations, based on the expression of T cell surface markers CD45RA and CCR7. In summary, the results demonstrate an overall reduction in leukemia burden in mice treated with 1ug36-sdAb-targeted CAR-T cells, proliferation of CAR-T cells in all CAR-T-treated groups (showing clear proliferation in treated mice), and a balanced differentiation profile favoring central memory T cells in the group treated with sdCD22 CAR-T cells.

[0368] Consideration NSG mice are widely used to study the interaction between the human immune system and cancer, and have become a practical platform for evaluating immunotherapies in the context of human immune cells and human tumors. Overall, these results clearly demonstrate the anti-leukemic activity of CD22-targeted single-domain CAR-modified T cells in vivo as well as in vitro, and demonstrate that the efficacy of CD22-targeted single-domain CAR-T cells is equivalent to or greater than the efficacy of CD19 (FMC63)-CAR-T cells, which have already shown clinical responses in clinical settings.

[0369] Example 5: Bispecific T cell engager construct Introduction Similar to chimeric antigen receptor technology, it is also possible to create soluble molecules that can simultaneously bind to T cells and cellular target molecules, resulting in antigen-specific T cell activation signals, by linking a novel antigen-binding element to an antibody element that engages with CD3. This type of molecule is called a bispecific T cell engager, exemplified by blinatumomab; a single molecule that simultaneously engages with human CD19 and human CD3 is used as a therapeutic agent for malignancies of the CD19-expressing B cell family. To evaluate whether the human CD22-specific single-domain antibody created herein could be used as such a bispecific T cell engager molecule, molecules were created with one end consisting of a CD22-specific single-domain antibody sequence and the other end consisting of a CD3 engager molecule. These novel bispecific T cell engagers were then screened for nonspecific and antigen-specific induction of T cell activation and T cell killing of target cells.

[0370] material and method The antigen-binding sequence of a single-domain antibody was transferred to a modular bispecific T cell engager DNA sequence [SEQ ID NO: 119] within the plasmid backbone; the DNA sequence used contained a restriction site that allowed for efficient recombination, where the antigen-binding domain could be replaced with a novel CD22 antigen-binding domain (ABD) sequence. The specific bispecific T cell engager design used was as follows: human CD28 signal peptide [SEQ ID NO: 110], VHH antibody (ABD) (one of SEQ ID NO: 82-108), flexible linker domain [SEQ ID NO: 111], human CD8 hinge domain [SEQ ID NO: 112], short flexible linker domain [SEQ ID NO: 118], and CD3-specific single-chain variable fragment sequence. A model of the CD22-CD3 bispecific T cell engager molecule is provided (Figure 21). The construct was fabricated using a golden gate assembly and verified using Sanger sequencing before proceeding to downstream testing.

[0371] To obtain purified protein forms of bispecific T cell engager molecules, plasmid DNA containing various constructs was transfected into HEK293 T cells using polyethyleneimine via a standard process. The transfected cells were cultured, and the supernatant was collected over several days. Next, to test the bispecific T cell engager activity of the supernatant, it was placed directly onto Jurkat cells alone or onto co-cultures of Jurkat cells with CD22-positive (Ramos) or CD22-negative (U87vIII) target cells and incubated overnight under standard conditions. Subsequently, Jurkat cells were examined for T cell activation using antibody staining against human CD69 markers and flow cytometry (Figure 24). The results demonstrate that CD22-sdAb-targeted bispecific T cell engagers, when delivered in solution, can induce target-dependent T cell activation, although there are differences in activity between different constructs.

[0372] To verify whether these results extended to the induction of a specific antitumor response in primary human T cells, supernatants containing the novel bispecific T cell engagers prepared above were used in assays with primary T cells. Specifically, T cells were isolated from human donor blood and polyclonally grown for 10 days. After polyclonal growth, the T cells were co-cultured with CD22-expressing target cells (Ramos) expressing a stable fluorescent protein (Nuclight; Sartorius, USA) in the presence of supernatants containing various bispecific T cell engagers or control supernatants (mocks). Subsequently, the co-cultures were monitored for target cell growth using an IncuCyte (Sartorius, USA) live microscope. Relative growth of target cells was quantified over 3 days using automated cell counting of fluorescently labeled target cells (Figure 25). The results demonstrate that bispecific T cell engager molecules, including CD22-sdAb, can retarget the cytolytic human T cell response to target cells expressing CD22.

[0373] result Figure 21 shows the molecular structure of a CD22-specific single-domain antibody bispecific T cell engager protein; the 5' end of the DNA construct contains a CD22-sdAb sequence, followed by a linker sequence which may have various compositions, and then a CD3-specific single-chain variable fragment.

[0374] Figure 22 shows the results of a Jurkat cell bispecific T cell engager activation activity assay; in this assay, HEK293T supernatant containing various bispecific T cell engager molecules was placed on a co-culture containing Jurkat cells and CD22-positive (Ramos) or CD22-negative (U87vIII) target cells. The graph shows the mean values ​​of CD69-specific antibody staining of Jurkat cells measured by flow cytometry. Error bars indicate the standard error of the mean values ​​for the pair of co-culture wells. The results demonstrate CD22 antigen-specific activation of T cells in the presence of novel CD22-sdAb bispecific T cell engager molecules.

[0375] Figure 23 shows the results of a bispecific T cell engager activity assay using primary human T cells co-cultured with CD22-positive target cells (Ramos). As described above, donor blood-derived T cells were co-cultured with fluorescently labeled target cells in the presence of a control supernatant (mock) or a supernatant containing CD22-specific bispecific T cell engagers, and observed hourly for 3 days using a live fluorescence microscope. The graph shows the growth rate of the fluorescently labeled target cells, measured using automated cell counting. Error bars indicate the standard error of the mean values ​​for the pair of co-culture wells. The results demonstrate T cell-mediated inhibition of tumor growth in the presence of the CD22-sdAb-targeted bispecific T cell engager molecule.

[0376] Notably, this construct, which contains extended linkers including SEQ ID NO: 111, SEQ ID NO: 112 (human CD8 hinge domain), and SEQ ID NO: 118, showed higher activity than a similar construct containing only the shorter G4S linker (data not shown).

[0377] Consideration Overall, these results illustrate that CD22-specific single-domain binders, when incorporated into bispecific T cell engager molecules, can generate potent antigen-driven T cell activation signaling. The CD22-sdAb-targeted bispecific T cell engager molecule is demonstrated to promote target-specific T cell activation and direct target cell killing by primary human T cells. Exemplary data for two CD22-specific single-domain antibodies are provided, suggesting that further high-affinity CD22 binders may exhibit similar activity. Furthermore, molecular optimization can be performed to further enhance the functionality of the bispecific T cell engager molecule. Additionally, combining multiple CD22-specific single-domain antibody sequences into a single molecule may be an effective strategy for increasing target-specific activation activity.

[0378] Example 6: In vivo study of CAR-T constructs 1 x 10 5 After inoculating with 2.5 × 10⁶ Ramos-Luc tumor cells, 6 Survival analysis and circulating CAR-T cell counts were performed on NSG mice treated with individual sdCD22 (1ug13 or 1ug36) CAR-T cells or benchmark scFvCD22 CAR-T cells. A mock cell refers to donor-derived T cells exposed to similar treatment conditions in the absence of a lentivirus expressing the CAR. Tx-less mice were administered a vehicle (Hanks equilibrium salt solution).

[0379] Figure 24 is a schematic diagram of the procedure and test.

[0380] Figure 25 shows the survival rate of mice.

[0381] Figure 26 shows CAR-T cells counted in mouse peripheral blood.

[0382] The results demonstrate that mice treated with CD22-CAR-T cells showed extended CAR-T cell survival and persistence compared to mice treated with mock T cells or untreated mice. Mice treated with sdCD22-1ug36 CAR-T cells showed improved survival and proliferation / persistence of circulating CAR-T cells compared to benchmark scFv CAR-T or sdCD22-1ug13 CAR-T cells.

[0383] Also, 0.5 × 10 5 After inoculating NSG mice with Ramos-Luc tumor cells, survival analysis and tumor growth dynamics were investigated in NSG mice treated with various doses of sdCD22-1ug36 CAR-T or benchmark scFvCD22-m971 CAR-T cells. A mock cell refers to donor-derived T cells exposed to similar treatment conditions in the absence of a lentivirus expressing the CAR. Tx-less mice were administered a vehicle (Hanks equilibrium salt solution).

[0384] Figure 27 shows the survival rates of mice after initial tumor challenge and CAR-T treatment. Compared to mice treated with mock T cells or vehicle controls, CAR-T treated mice showed a dose-dependent increase in survival time. More than 50% of mice treated with the highest dose of CAR-T cells survived for an extended period; they survived 88 days after the initial tumor challenge (0.5 × 10⁶). 5 We re-challenged individual Ramos-Luc tumor cells and monitored their survival rate and tumor growth.

[0385] Figure 28 shows the survival rate of mice after the second challenge. All untreated (vehicle control) animals died from the disease, but all (sdCD22-1ug36) or most (benchmark scFvCD22-m971) of the CAR-T treated mice survived the second tumor challenge.

[0386] Figure 29 shows a graph of tumor growth after challenge. All untreated control mice developed tumors, but CAR-T treated mice were able to resist rechallenge, demonstrating the long-term persistence of CAR-T cells and therefore the persistence of the antitumor response.

[0387] Example 7: Biparatopic and Multiparatopic CAR Construction Figure 30 shows the molecular structure of a multibinder domain containing a CD22-specific CAR molecule, which (left) contains an N-terminal CD22-sdAb sequence followed by a linker sequence, which can be followed by (center) another copy of the same sdAb binder sequence, or (right) a different sdAb binder. This may result in a CAR molecule exhibiting higher affinity and / or higher activity.

[0388] Figure 31 shows the results of the CAR-Jurkat assay when Jurkat cells were transiently electroporated with various CAR plasmids (encoding single-binder and multi-binder types) and cultured alone or co-cultured with CD22-positive (Ramos) target cell lines.

[0389] Figure 32 shows the results of a similar CAR activation test using primary T cells derived from human blood into which a lentiviral vector encoding a multi-sdAb-containing CAR construct had been introduced. These data demonstrate that multi-CD22 sdAb-containing CAR molecules can show a higher molecular responsiveness to CD22-expressing target cells.

[0390] Figure 33 shows the molecular structures of a CD22-specific CAR molecule (left), a BCMA-specific CAR molecule (right), or a tandem CD22-BCMA-CAR molecule (center). The tandem construct includes an N-terminal CD22-sdAb sequence followed by a linker sequence, which can be followed by another antigen-specific sdAb, such as a BCMA-targeting sdAb as shown here.

[0391] Figure 34 shows the results of a CAR-Jurkat assay in which Jurkat cells were transiently electroporated with various CD22, BCMA, or CD22 / BCMA-specific CAR plasmids and cultured alone or co-cultured with BCMA+ / CD22+(Ramos), BCMAlow / CD22+(NALM6), or BCMA+ / CD22-(Ramos-CD22ko) target cell lines to examine their activation state (CD69 expression). These data demonstrate that multi-CD22 sdAb-containing CAR molecules can maintain responsiveness to both target antigens and enhance responsiveness to low-antigenic target cells.

[0392] Consideration of the Examples This is the first demonstration experiment of a single-domain antibody for application in CAR-T therapy targeting CD22. Compared to single-chain variable fragment antibodies typically used as antigen-recognition domains in CAR constructs, single-domain antibodies offer significant advantages, including extremely small size, high homology to human antibody sequences, high modularity, and the ability to target epitopes that scFv may not be able to access. The present invention can later be combined with single-domain antibodies targeting CD20, CD19, or BCMA to create a single CAR construct targeting multiple B cell-specific antigens.

[0393] The above description includes numerous details for illustrative purposes to provide a complete understanding of the embodiments. However, it will be apparent to those skilled in the art that these specific details are unnecessary.

[0394] The embodiments described above are intended to be illustrative only. Those skilled in the art can modify, alter, and change specific embodiments. The claims should not be limited by any specific embodiment described herein, but should be construed to be consistent with the Specified Terms as a whole.

[0395] References TIFF0007869193000017.tif137164 All references mentioned herein are expressly incorporated herein by reference in their entirety.

[0396] [Table 8] TIFF0007869193000019.tif210166TIFF0007869193000020.tif205165TIFF0007869193 000021.tif198165TIFF0007869193000022.tif205165TIFF0007869193000023.tif20916 2TIFF0007869193000024.tif213164TIFF0007869193000025.tif211163TIFF0007869193 000026.tif212164TIFF0007869193000027.tif213164TIFF0007869193000028.tif64166

Claims

1. An isolated single-domain antibody (sdAb) that specifically binds to human CD22, with the following sequence: CDR1 amino acid sequence as described in Sequence ID No. 13, The CDR2 amino acid sequence described in Sequence ID No. 14, and CDR3 amino acid sequence as described in Sequence ID No. 15; Isolated sdAb, including.

2. The isolated sdAb according to claim 1, comprising an amino acid sequence that is at least 90% identical to sequence number 86 over its entire length.

3. An isolated sdAb according to claim 1 or 2, comprising the amino acid sequence of SEQ ID NO:

86.

4. An isolated sdAb according to claim 1 or 2, which is a camelid sdAb or has been humanized.

5. A recombinant polypeptide comprising one or more sdAbs according to any one of claims 1 to 4.

6. A first antigen-binding moiety comprising sdAb according to any one of claims 1 to 4; and The second antigen-binding site; A multivalent antibody containing [specific antibody type].

7. From the N-terminus towards the C-terminus, - The first antigen-binding site; - Amino acid linkers; and - Second antigen-binding site; A polyvalent antibody according to claim 6, comprising:

8. The polyvalent antibody according to claim 7, wherein the amino acid linker comprises a polypeptide hinge derived from human CD8.

9. The polyvalent antibody according to claim 7, wherein the amino acid linker comprises SEQ ID NO:

112.

10. A polyvalent antibody according to any one of claims 6 to 9, further comprising an N-terminal signal peptide.

11. The polyvalent antibody according to claim 10, wherein the signal peptide is a signal peptide derived from human CD28.

12. The polyvalent antibody according to claim 10, wherein the signal peptide comprises SEQ ID NO:

110.

13. The polyvalent antibody according to any one of claims 6 to 12, wherein the second antigen-binding portion specifically binds to a cell surface marker of an immune cell.

14. The polyvalent antibody according to claim 13, wherein the cell surface marker of an immune cell includes a T cell marker or a natural killer (NK) cell marker.

15. The polyvalent antibody according to claim 14, wherein the T cell marker contains human CD3, or the NK cell marker contains human CD16.

16. A polyvalent antibody according to any one of claims 6 to 15, for use in the treatment of cancer or autoimmune disease.

17. A chimeric antibody receptor (CAR) that binds to human CD22, comprising the sdAb described in any one of claims 1 to 4.

18. From the N-terminus towards the C-terminus, - A CD22-binding domain containing sdAb according to any one of claims 1 to 4; - Polypeptide hinge; - Transmembrane domain; and - A cytoplasmic domain comprising a signaling domain, preferably further comprising a co-stimulatory domain; The CAR according to claim 17, including the following:

19. The CAR according to claim 18, wherein the polypeptide hinge is a CD8 hinge domain, the transmembrane domain is a CD28 domain or a CD8 domain, the signaling domain is a CD3ζ signaling domain, and / or the co-stimulatory domain is a 4-1BB co-stimulatory domain.

20. The CAR according to claim 19, wherein the CD8 hinge domain comprises SEQ ID NO: 112, the CD28 transmembrane domain comprises SEQ ID NO: 113, the CD3ζ signaling domain comprises SEQ ID NO: 115, and / or the 4-1BB signaling domain comprises SEQ ID NO:

114.

21. The CAR according to any one of claims 17 to 20, further comprising a flexible amino acid linker between the sdAb and the polypeptide hinge.

22. The CAR according to claim 21, wherein the flexible amino acid linker comprises SEQ ID NO:

111.

23. The CAR according to any one of claims 17 to 22, further comprising a signal peptide.

24. The CAR according to claim 23, wherein the signal peptide is a signal peptide derived from human CD28.

25. The CAR according to claim 23, wherein the signal peptide comprises SEQ ID NO:

110.

26. The CAR according to any one of claims 17 to 25, further comprising an additional binding domain located at the N-terminus or C-terminus of the CD22 binding domain and linked to the CD22 binding domain by an amino acid linker.

27. A recombinant polypeptide according to claim 5, a polyvalent antibody according to any one of claims 6 to 16, or a recombinant nucleic acid molecule encoding a CAR according to any one of claims 17 to 26.

28. A genetically modified cell expressing the CAR described in any one of claims 17 to 26 on its cell surface membrane.

29. Genetically modified cells according to claim 28, for use in the treatment of cancer or autoimmune disease.

30. The genetically modified cell according to claim 29, wherein the cancer is a hematological malignancy.

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