CD20 binding molecule and its use

Abstract

The present disclosure provides single-domain antibodies that bind to CD20 and chimeric antigen receptors comprising the same. Additionally, engineered immune effector cells (e.g., T cells) comprising the chimeric antigen receptor are provided. Pharmaceutical compositions, kits, and methods for treating diseases or disorders are also provided.

Description

[Technical Field]

[0001] cross reference This application claims priority to International Patent Application No. PCT / CN2020 / 102463, filed on 16 July 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application incorporates the sequence listing submitted with this application by reference as a text file named 14651-026-228_SEQ_LISTING.txt, created on July 9, 2021, and has a size of 236,191 bytes.

[0003] 1. Field This disclosure relates to anti-CD20 single-domain antibodies, chimeric antigen receptors, engineered immune effector cells, and methods of using them. This disclosure further relates to cell activation and augmentation for therapeutic use, in particular to chimeric antigen receptor-based T-cell immunotherapy. [Background technology]

[0004] 2.Background CD20 is a surface antigen expressed at specific stages of B cell differentiation. Targeting CD20-positive B cells with therapeutic monoclonal antibodies (mAbs) has been an effective strategy in treating hematological malignancies, such as non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). The initial success of rituximab (RTX) has accelerated the creation and development of more effective CD20-based therapies. However, conventional mAb treatment has not been sufficient to overcome obstacles such as refractory / relapsed disease (Shanehbandi et al., Current Cancer Drug Targets, 17(5):423-444 (2007) (Non-patent Literature 1)).

[0005] Chimeric antigen receptor T (CAR-T) cell therapy is an emerging and effective cancer immunotherapy. In particular, CAR-T cells have achieved interesting results in hematological malignancies. Early clinical studies of CD20 CAR-T cells have been conducted, showing that CD20 CAR-T cells have promising therapeutic potential in B-cell NHL (Brudno and Kochenderfer, Nature Reviews Clinical Oncology, 15:31-46 (2018) (Non-patent Literature 2)). However, the application of CAR-T cells is naturally hindered by side effects such as cytokine release syndrome and on-target off-tumor toxicity (Yu et al., Molecular Cancer 18(1):125 (2019) (Non-patent Literature 3)).

[0006] Therefore, improved CD20-binding molecules and engineered CD20-targeting cells are needed. For example, there is a need to develop stable, therapeutically effective, and small-sized CD20-binding molecules for use in more effective or efficient CAR-T therapies. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Shanehbandi et al.,Current Cancer Drug Targets,17(5):423-444(2007) [Non-Patent Document 2] Brudno and Kochenderfer,Nature Reviews Clinical Oncology,15:31-46(2018) [Non-Patent Document 3] Yu et al.,Molecular Cancer 18(1):125(2019) [Overview of the Initiative]

[0008] 3. Overview In one embodiment, an anti-CD20 single-domain antibody (sdAb) comprising the following is provided herein: (i) CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and SEQ ID NO: 8 (iv) CDR3 containing the sequence, CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 9, and CDR3 containing the amino acid sequence of SEQ ID NO: 10, (v) CDR1 containing the amino acid sequence of SEQ ID NO: 11 or 196, CDR2 containing the amino acid sequence of SEQ ID NO: 12, and CDR3 containing the amino acid sequence of SEQ ID NO: 8, (vi) CDR1 containing the amino acid sequence of SEQ ID NO: 13, CDR2 containing the amino acid sequence of SEQ ID NO: 9, and CDR3 containing the amino acid sequence of SEQ ID NO: 10, (vii) the amino acid sequence of SEQ ID NO: 14 or 197 (viii) CDR1 containing the amino acid sequence of SEQ ID NO: 15, CDR2 containing the amino acid sequence of SEQ ID NO: 16, (ix) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 21, and CDR3 containing the amino acid sequence of SEQ ID NO: 16, (x) CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 23, and CDR3 containing the amino acid sequence of SEQ ID NO: 19, (xi) CDR1 containing the amino acid sequence of SEQ ID NO: 24 or 198, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 26, (xii) CDR1 containing the amino acid sequence of SEQ ID NO: 27, CDR2 containing the amino acid sequence of SEQ ID NO: 28, and CDR3 containing the amino acid sequence of SEQ ID NO: 29, (xiii) CDR1 containing the amino acid sequence of SEQ ID NO: 14 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 30, and CDR3 containing the amino acid sequence of SEQ ID NO: 16,(xiv) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 31, and CDR3 containing the amino acid sequence of SEQ ID NO: 19, (xv) CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 containing the amino acid sequence of SEQ ID NO: 32, and CDR3 containing the amino acid sequence of SEQ ID NO: 3, (xvi) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 6, (xvii) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, sequence number CDR2 containing the amino acid sequence of sequence number 34, and CDR3 containing the amino acid sequence of sequence number 16, (xviii) CDR1 containing the amino acid sequence of sequence number 22, CDR2 containing the amino acid sequence of sequence number 35, and CDR3 containing the amino acid sequence of sequence number 19, (xix) CDR1 containing the amino acid sequence of sequence number 20 or 197, CDR2 containing the amino acid sequence of sequence number 36, and CDR3 containing the amino acid sequence of sequence number 16, (xx) CDR1 containing the amino acid sequence of sequence number 20 or 197, CDR2 containing the amino acid sequence of sequence number 37, and sequence number CDR3 containing 16 amino acid sequences, (xxi) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 16, (xxii) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 39, (xxiii) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 40, and (xxiv) SEQ ID NO: 165 Alternatively, CDR1 containing the amino acid sequence of 199, CDR2 containing the amino acid sequence of SEQ ID NO: 166, and CDR3 containing the amino acid sequence of SEQ ID NO: 167, (xxv) CDR1 containing the amino acid sequence of SEQ ID NO: 168, CDR2 containing the amino acid sequence of SEQ ID NO: 169, and CDR3 containing the amino acid sequence of SEQ ID NO: 170, (xxvi) CDR1 containing the amino acid sequence of SEQ ID NO: 171 or 200, CDR2 containing the amino acid sequence of SEQ ID NO: 172, and CDR3 containing the amino acid sequence of SEQ ID NO: 173, (xxvii) CDR1 containing the amino acid sequence of SEQ ID NO: 174,CDR2 containing the amino acid sequence of SEQ ID NO: 175, and CDR3 containing the amino acid sequence of SEQ ID NO: 176; (xxviii) CDR1 containing the amino acid sequence of SEQ ID NO: 177 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 178, and CDR3 containing the amino acid sequence of SEQ ID NO: 179; or (xxix) CDR1 containing the amino acid sequence of SEQ ID NO: 180, CDR2 containing the amino acid sequence of SEQ ID NO: 181, and CDR3 containing the amino acid sequence of SEQ ID NO: 182.

[0009] In another embodiment, anti-CD20 single-domain antibodies (sdAbs) are provided herein, including: (i) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as described in SEQ ID NO: 41, (ii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as described in SEQ ID NO: 42, (iii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as described in SEQ ID NO: 43, (iv) CDR1 as described in SEQ ID NO: 44 (v) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR2 and CDR3 respectively, (v) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 45, (vi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 46, (vii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 47, (viii) CDR1 described in SEQ ID NO: 48, (ix) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR2 and CDR3 respectively, (x) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 49, (x) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 50, (xi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 51, (xii) CDR1, CD described in SEQ ID NO: 52 CDR1, CDR2, and CDR3 having the amino acid sequences of R2 and CDR3 respectively, (xiii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 53, (xiv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 54, (xv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 55, (xvi) CDR1 described in SEQ ID NO: 56,CDR1, CDR2, and CDR3 having the amino acid sequences of CDR2 and CDR3 respectively, (xvii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 57, (xviii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 58, (xix) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 59, (xx) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 60, (xxi) CD having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 61 R1, CDR2, and CDR3, CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in (xxii) SEQ ID NO. 62, respectively, or CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in (xxiii) SEQ ID NO. 63, CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in (xxiv) SEQ ID NO. 183, CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in (xxv) SEQ ID NO. 184, respectively, or CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in (xxvi) SEQ ID NO. 185, respectively.

[0010] In some embodiments, CDR1, CDR2, or CDR3 are determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or a combination thereof.

[0011] In some embodiments, the anti-CD20 sdAb provided herein further comprises one or more FR regions described in SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 183, SEQ ID NO: 184, and / or SEQ ID NO: 185.

[0012] In some embodiments, the anti-CD20 sdAb provided herein comprises the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 183, SEQ ID NO: 184, or SEQ ID NO: 185.

[0013] In other embodiments, the anti-CD20 sdAb provided herein comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 183, SEQ ID NO: 184, or SEQ ID NO: 185.

[0014] In some embodiments, the anti-CD20 sdAb is a camelid sdAb. In some embodiments, the anti-CD20 sdAb is a humanized sdAb. In some embodiments, the anti-CD20 sdAb is a VHH domain. In some embodiments, the anti-CD20 sdAb is a humanized VHH domain.

[0015] In some embodiments, the anti-CD20 sdAb is genetically fused to or chemically conjugated to the drug.

[0016] In another embodiment, a chimeric antigen receptor is provided herein, comprising (a) an extracellular antigen-binding domain containing an anti-CD20 sdAb provided herein, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains. In some embodiments, the extracellular antigen-binding domain further comprises one additional antigen-binding domain. In other embodiments, the extracellular antigen-binding domain further comprises two additional antigen-binding domains. In some embodiments, one or more additional antigen-binding domains bind to one or more antigens selected from the group consisting of CD20, CD19, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the CD20 CAR is monospecific. In some embodiments, the CD20 CAR is monovalent. In some embodiments, the CD20 CAR is polyvalent. In some embodiments, the extracellular antigen-binding domain comprises at least two anti-CD20 sdAbs provided herein.

[0017] In some embodiments, polyvalent (e.g., bivalent and trivalent) CARs comprising a polypeptide including (a) an extracellular antigen-binding domain containing two or more single-domain antibodies (sdAbs) that specifically bind to CD20, (b) a transmembrane domain, and (c) an intracellular signaling domain are provided herein. In some embodiments, the extracellular antigen-binding domain comprises two single-domain antibodies (sdAbs) that specifically bind to CD20 as provided herein. In other embodiments, the extracellular antigen-binding domain comprises three single-domain antibodies (sdAbs) that specifically bind to CD20 as provided herein.

[0018] In some embodiments using any one of the above CARs, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α.

[0019] In some embodiments using any one of the above CARs, the intracellular signaling domain includes the primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0020] In other embodiments using any one of the above CARs, the intracellular signaling domain further comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, the co-stimulatory signaling domain is derived from CD137.

[0021] In some embodiments, the CAR provided herein further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α.

[0022] In some embodiments, the CAR provided herein further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD8α.

[0023] In a particular embodiment, (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 189, 190, and 191, or (ii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84, and 191. Chimeric antigen receptors (CARs) are provided herein, comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the sequence of number 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 191.

[0024] In another embodiment, an isolated nucleic acid comprising a nucleic acid sequence encoding an anti-CD20 sdAb provided herein is provided herein. In another embodiment, a vector comprising a nucleic acid encoding an anti-CD20 sdAb provided herein is provided herein.

[0025] In yet another embodiment, an isolated nucleic acid comprising a nucleic acid sequence encoding a CAR provided herein is provided herein. In yet another embodiment, a vector comprising a nucleic acid encoding a CAR provided herein is provided herein.

[0026] In yet another embodiment, engineered immune effector cells comprising CARs, isolated nucleic acids, or vectors provided herein are provided herein. In some embodiments, the engineered immune effector cells are T cells or B cells.

[0027] In another embodiment, a pharmaceutical composition comprising an anti-CD20 sdAb, engineered immunoeffector cells, or a vector provided herein, and a pharmaceutically acceptable excipient is provided herein.

[0028] In yet another embodiment, a method for treating a disease or disorder of interest is provided herein, the method comprising administering an effective amount of anti-CD20 sdAb, engineered immune effector cells, or pharmaceutical composition provided herein to the subject. In some embodiments, the disease or disorder is a B-cell related disease or disorder and / or a CD20-related disease or disorder. In some embodiments, the disease or disorder is cancer. In other embodiments, the disease or disorder is a B-cell malignancy. In some embodiments, the B-cell malignancy is a B-cell leukemia or a B-cell lymphoma. In some embodiments, the disease or disorder is selected from the group consisting of marginal zone lymphoma (e.g., splenic marginal zone lymphoma), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, primary mediastinal B-cell lymphoma (PMBL), small lymphocytic lymphoma (SLL), B-cell prelymphoblastic leukemia (B-PLL), follicular lymphoma (FL), Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia (HCL), precursor B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), high-grade B-cell lymphoma (HGBL), and multiple myeloma (MM). In other embodiments, the disease or disorder is an autoimmune and / or inflammatory disease. In some embodiments, autoimmune and / or inflammatory diseases are associated with inadequate or increased B cell counts and / or activation. [Invention 1001] Anti-CD20 single-domain antibody (sdAb), (i) CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO: 3 (ii) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 5, and CDR3 containing the amino acid sequence of SEQ ID NO: 6 (iii) CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 containing the amino acid sequence of SEQ ID NO: 7, and CDR3 containing the amino acid sequence of SEQ ID NO: 8 (iv) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 9, and CDR3 containing the amino acid sequence of SEQ ID NO: 10. (v) CDR1 containing the amino acid sequence of SEQ ID NO: 11 or 196, CDR2 containing the amino acid sequence of SEQ ID NO: 12, and CDR3 containing the amino acid sequence of SEQ ID NO: 8 (vi) CDR1 containing the amino acid sequence of SEQ ID NO: 13, CDR2 containing the amino acid sequence of SEQ ID NO: 9, and CDR3 containing the amino acid sequence of SEQ ID NO: 10. (vii) CDR1 containing the amino acid sequence of SEQ ID NO: 14 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 15, and CDR3 containing the amino acid sequence of SEQ ID NO: 16. (viii) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 18, and CDR3 containing the amino acid sequence of SEQ ID NO: 19. (ix) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 21, and CDR3 containing the amino acid sequence of SEQ ID NO: 16. (x) CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 23, and CDR3 containing the amino acid sequence of SEQ ID NO: 19. (xi) CDR1 containing the amino acid sequence of SEQ ID NO: 24 or 198, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 26. (xii) CDR1 containing the amino acid sequence of SEQ ID NO: 27, CDR2 containing the amino acid sequence of SEQ ID NO: 28, and CDR3 containing the amino acid sequence of SEQ ID NO: 29. (xiii) CDR1 containing the amino acid sequence of SEQ ID NO: 14 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 30, and CDR3 containing the amino acid sequence of SEQ ID NO: 16. (xiv) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 31, and CDR3 containing the amino acid sequence of SEQ ID NO: 19. (xv) CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 containing the amino acid sequence of SEQ ID NO: 32, and CDR3 containing the amino acid sequence of SEQ ID NO: 3. (xvi) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 6 (xvii) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 34, and CDR3 containing the amino acid sequence of SEQ ID NO: 16. (xviii) CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 35, and CDR3 containing the amino acid sequence of SEQ ID NO: 19. (xix) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 36, and CDR3 containing the amino acid sequence of SEQ ID NO: 16. (xx) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 37, and CDR3 containing the amino acid sequence of SEQ ID NO: 16. (xxi) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 16. (xxii) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 39. (xxiii) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 40, (xxiv) CDR1 containing the amino acid sequence of SEQ ID NO: 165 or 199, CDR2 containing the amino acid sequence of SEQ ID NO: 166, and CDR3 containing the amino acid sequence of SEQ ID NO: 167. (xxv) CDR1 containing the amino acid sequence of SEQ ID NO: 168, CDR2 containing the amino acid sequence of SEQ ID NO: 169, and CDR3 containing the amino acid sequence of SEQ ID NO: 170. (xxvi) CDR1 containing the amino acid sequence of SEQ ID NO: 171 or 200, CDR2 containing the amino acid sequence of SEQ ID NO: 172, and CDR3 containing the amino acid sequence of SEQ ID NO: 173. (xxvii) CDR1 containing the amino acid sequence of SEQ ID NO: 174, CDR2 containing the amino acid sequence of SEQ ID NO: 175, and CDR3 containing the amino acid sequence of SEQ ID NO: 176. (xxviii) CDR1 containing the amino acid sequence of SEQ ID NO: 177 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 178, and CDR3 containing the amino acid sequence of SEQ ID NO: 179, or (xxix) CDR1 containing the amino acid sequence of SEQ ID NO: 180, CDR2 containing the amino acid sequence of SEQ ID NO: 181, and CDR3 containing the amino acid sequence of SEQ ID NO: 182. The anti-CD20 sdAb, including the aforementioned anti-CD20 sdAb. [Invention 1002] Anti-CD20 single-domain antibody (sdAb), (i) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 41, (ii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 42, respectively (iii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 43, (iv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 44, (v) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 45, respectively (vi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 46, respectively (vii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 47, respectively. (viii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 48, respectively (ix) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 49, (x) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 50, (xi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 51, (xii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 52, respectively. (xiii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 53, respectively. (xiv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 54, (xv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 55, respectively. (xvi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 56, respectively. (xvii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 57, respectively. (xviii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 58, respectively. (xix) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 59, respectively. (xx) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 60, respectively. (xxi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 61, respectively. (xxii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 62, respectively. (xxiii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 63, respectively. (xxiv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 183, respectively. (xxv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 184, respectively, or (xxvi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 185, respectively. The anti-CD20 sdAb, including the aforementioned anti-CD20 sdAb. [Invention 1003] The anti-CD20 sdAb of the present invention 1002, wherein the CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. [Invention 1004] Any anti-CD20 sdAb of the present invention 1001 to 1003 further comprising one or more FR regions described in SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 183, SEQ ID NO: 184, and / or SEQ ID NO: 185. [Invention 1005] An anti-CD20 sdAb according to any of the present invention 1001 to 1004, comprising the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 183, SEQ ID NO: 184, or SEQ ID NO: 185. [Invention 1006] An anti-CD20 sdAb according to any of the present invention 1001 to 1004, comprising or consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 183, SEQ ID NO: 184, or SEQ ID NO: 185. [Invention 1007] Anti-CD20 sdAb of the present invention 1001 or 1002, which is an sdAb for camelid animals. [Invention 1008] A humanized sdAb, the anti-CD20 sdAb of the present invention 1001 or the present invention 1002. [Invention 1009] An anti-CD20 sdAb according to any of the inventions 1001 to 1008, which is genetically fused to or chemically conjugated to a drug. [Invention 1010] (a) An extracellular antigen-binding domain comprising any of the anti-CD20 sdAb of the present invention 1001 to 1009; (b) Transmembrane domain; and (c) Intracellular signaling domain Chimeric antigen receptors (CARs), including those mentioned above. [Invention 1011] The CAR of the present invention 1010, wherein the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains. [Invention 1012] The CAR of the present invention 1011, wherein the extracellular antigen-binding domain further comprises one additional antigen-binding domain, or the extracellular antigen-binding domain further comprises two additional antigen-binding domains. [Invention 1013] A CAR according to any of the invention 1011 to 1012, wherein the one or more additional antigen-binding domains(s) mentioned above bind to one or more antigens(s) selected from the group consisting of CD20, CD19, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. [Invention 1014] The extracellular antigen-binding domain comprises two or three anti-CD20 sdAbs from any of the inventions 1001 to 1009, wherein the CAR is one of the inventions 1011 to 1013. [Invention 1015] The transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, and is a CAR according to any of the present invention 1010 to 1014. [Invention 1016] The transmembrane domain is derived from CD8α, according to the CAR of the present invention 1015. [Invention 1017] The CAR according to any one of the present invention 1010 to 1016, wherein the intracellular signaling domain includes the primary intracellular signaling domain of an immune effector cell. [Invention 1018] The CAR of the present invention 1017, wherein the primary intracellular signaling domain is derived from CD3ζ. [Invention 1019] The CAR of the present invention 1017 or 1018, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain. [Invention 1020] The CAR of the present invention 1019, wherein the aforementioned co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. [Invention 1021] The CAR of the present invention 1020, wherein the aforementioned co-stimulus signaling domain is derived from CD137. [Invention 1022] A CAR according to any one of the present invention 1010 to 1021, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. [Invention 1023] The CAR of the present invention 1022, wherein the hinge domain is derived from CD8α. [Invention 1024] A CAR according to any one of the present invention 1010 to 1023, further comprising a signal peptide located at the N-terminus of the polypeptide. [Invention 1025] The signal peptide is the CAR of the present invention 1024, derived from CD8α. [Invention 1026] (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 189, 190, and 191, or (ii) Amino acid sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the sequences of SEQ ID NOs. 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 189, 190, or 191. Chimeric antigen receptors (CARs), including those mentioned above. [Invention 1027] An isolated nucleic acid comprising a nucleic acid sequence encoding any of the anti-CD20 sdAb of the present invention 1001 to 1009. [Invention 1028] A vector comprising the isolated nucleic acid of the present invention 1027. [Invention 1029] An isolated nucleic acid comprising a nucleic acid sequence encoding any of the CARs described in invention 1010 to 1026. [Invention 1030] A vector comprising the isolated nucleic acid of the present invention 1029. [Invention 1031] Engineered immunoeffector cells comprising any CAR of Invention 1010-1026, isolated nucleic acid of Invention 1029, or vector of Invention 1030. [Invention 1032] The manipulated immune effector cells of the present invention 1031, which are T cells or B cells. [Invention 1033] A pharmaceutical composition comprising an anti-CD20 sdAb of any of Invention 1001 to 1009, engineered immunoeffector cells of Invention 1031 or Invention 1032, or a vector of Invention 1028 or Invention 1030, and a pharmaceutically acceptable excipient. [Invention 1034] A method for treating a target disease or disorder, comprising administering to the target an effective amount of any anti-CD20 sdAb of Invention 1001 to 1009, engineered immune effector cells of Invention 1031 or Invention 1032, or a pharmaceutical composition of Invention 1033. [Invention 1035] The method of the present invention 1034, wherein the disease or disorder is a B cell-related disease or disorder and / or a CD20-related disease or disorder. [Invention 1036] The method of the present invention 1035, wherein the disease or disorder is cancer. [Invention 1037] The method of the present invention 1036, wherein the disease or disorder is a B-cell malignant tumor. [Invention 1038] The method of the present invention 1037, wherein the B-cell malignant tumor is B-cell leukemia or B-cell lymphoma. [Invention 1039] The method of the present invention 1034, wherein the disease or disorder is selected from the group consisting of marginal zone lymphoma (e.g., splenic marginal zone lymphoma), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, primary mediastinal B-cell lymphoma (PMBL), small lymphocytic lymphoma (SLL), B-cell prelymphoblastic leukemia (B-PLL), follicular lymphoma (FL), Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia (HCL), precursor B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), high-grade B-cell lymphoma (HGBL), and multiple myeloma (MM). [Invention 1040] The method of the present invention 1034, wherein the disease or disorder is an autoimmune and / or inflammatory disease. [Invention 1041] The method of the present invention 1040, wherein the autoimmune and / or inflammatory disease is associated with an inappropriate or increased number and / or activation of B cells. [Brief explanation of the drawing]

[0029] 4. Brief explanation of the drawing [Figure 1A] This shows the exemplary transduction efficiency of VHH-based CAR-T cells. UnT refers to T cells that have not been transduced by CAR. [Figure 1B] This shows the exemplary transduction efficiency of scFv-based CAR-T cells. UnT refers to T cells that have not been transduced by CAR. [Figure 2A] This shows the in vitro cytotoxicity of VHH-based CAR-T cells compared to scFv-based CAR-T cells against CD20-positive cell lines (Figures 2A-2B and 2E-2F) or CD20-negative cell lines (Figures 2C-2D). [Figure 2B] This shows the in vitro cytotoxicity of VHH-based CAR-T cells compared to scFv-based CAR-T cells against CD20-positive cell lines (Figures 2A-2B and 2E-2F) or CD20-negative cell lines (Figures 2C-2D). [Figure 2C]This shows the in vitro cytotoxicity of VHH-based CAR-T cells compared to scFv-based CAR-T cells against CD20-positive cell lines (Figures 2A-2B and 2E-2F) or CD20-negative cell lines (Figures 2C-2D). [Figure 2D] This shows the in vitro cytotoxicity of VHH-based CAR-T cells compared to scFv-based CAR-T cells against CD20-positive cell lines (Figures 2A-2B and 2E-2F) or CD20-negative cell lines (Figures 2C-2D). [Figure 2E] This shows the in vitro cytotoxicity of VHH-based CAR-T cells compared to scFv-based CAR-T cells against CD20-positive cell lines (Figures 2A-2B and 2E-2F) or CD20-negative cell lines (Figures 2C-2D). [Figure 2F] This shows the in vitro cytotoxicity of VHH-based CAR-T cells compared to scFv-based CAR-T cells against CD20-positive cell lines (Figures 2A-2B and 2E-2F) or CD20-negative cell lines (Figures 2C-2D). [Figure 3A] The in vitro cytotoxicity of mono-, bi-, and tri-VHH CAR-T cells compared to CD20 scFv CAR-T cells against CD20-positive cell lines (Figures 3A-3B) or CD20-negative cell lines (Figures 3C-3D) is shown. [Figure 3B] The in vitro cytotoxicity of mono-, bi-, and tri-VHH CAR-T cells compared to CD20 scFv CAR-T cells against CD20-positive cell lines (Figures 3A-3B) or CD20-negative cell lines (Figures 3C-3D) is shown. [Figure 3C] The in vitro cytotoxicity of mono-, bi-, and tri-VHH CAR-T cells compared to CD20 scFv CAR-T cells against CD20-positive cell lines (Figures 3A-3B) or CD20-negative cell lines (Figures 3C-3D) is shown. [Figure 3D]The in vitro cytotoxicity of mono-, bi-, and tri-VHH CAR-T cells compared to CD20 scFv CAR-T cells against CD20-positive cell lines (Figures 3A-3B) or CD20-negative cell lines (Figures 3C-3D) is shown. [Figure 4A] This study demonstrates in vitro cytotoxicity of bi-VHH CAR-T cells with peptide linkers of different lengths against Raji.Luc cells. [Figure 4B] This study demonstrates in vitro cytotoxicity of bi-VHH CAR-T cells with peptide linkers of different lengths against Raji.Luc cells. [Figure 5A] This shows the IFN-γ release levels of VHH-based CAR-T cells compared to scFv-based CAR-T cells after 24 hours of co-culture with Raji.Luc or K562-CD20.Luc cells at different E:T ratios. [Figure 5B] This shows the IFN-γ release levels of VHH-based CAR-T cells compared to scFv-based CAR-T cells after 24 hours of co-culture with Raji.Luc or K562-CD20.Luc cells at different E:T ratios. [Figure 6] The IFN-γ release levels of mono-, bi-, and tri-VHH CAR-T cells are shown compared to CD20 scFv CAR-T cells after 24 hours of co-culture with Raji.Luc or K562.Luc cells. [Figure 7A] This shows the proliferative capacity and CAR positivity rate of exemplary CD20 VHH CAR-T cells compared with and without antigen-specific stimulation. [Figure 7B] This shows the proliferative capacity and CAR positivity rate of exemplary CD20 VHH CAR-T cells compared with and without antigen-specific stimulation. [Figure 7C] This shows the proliferative capacity and CAR positivity rate of exemplary CD20 VHH CAR-T cells compared with and without antigen-specific stimulation. [Figure 7D]This shows the proliferative capacity and CAR positivity rate of exemplary CD20 VHH CAR-T cells compared with and without antigen-specific stimulation. [Figure 8] The results of the evaluation of fatigue markers in exemplary CD20 VHH CAR-T cells and CD20 scFv CAR-T cells are shown. [Figure 9A] The results of memory marker evaluation of exemplary CD20 VHH CAR-T cells and CD20 scFv CAR-T cells are shown. [Figure 9B] The results of memory marker evaluation of exemplary CD20 VHH CAR-T cells and CD20 scFv CAR-T cells are shown. [Figure 9C] The results of memory marker evaluation of exemplary CD20 VHH CAR-T cells and CD20 scFv CAR-T cells are shown. [Figure 9D] The results of memory marker evaluation of exemplary CD20 VHH CAR-T cells and CD20 scFv CAR-T cells are shown. [Figure 10A] This study demonstrates the in vivo antitumor and reimmunization effects of VHH-273 and VHH-496 CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 10A-10B), body weight (Figure 10C), and CAR copies in mouse peripheral blood (Figure 10D). Next, for the reimmunization assay, mice were selected that had been tumor-free for at least 5 weeks and for which no CAR copy number (copies of genomic DNA / ng) was detected in PBMCs. Raji tumors were monitored weekly using bioluminescence imaging (Figure 10E), and mouse body weight was measured (Figure 10F). [Figure 10B]This study demonstrates the in vivo antitumor and reimmunization effects of VHH-273 and VHH-496 CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 10A-10B), body weight (Figure 10C), and CAR copies in mouse peripheral blood (Figure 10D). Next, for the reimmunization assay, mice were selected that had been tumor-free for at least 5 weeks and for which no CAR copy number (copies of genomic DNA / ng) was detected in PBMCs. Raji tumors were monitored weekly using bioluminescence imaging (Figure 10E), and mouse body weight was measured (Figure 10F). [Figure 10C] This study demonstrates the in vivo antitumor and reimmunization effects of VHH-273 and VHH-496 CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 10A-10B), body weight (Figure 10C), and CAR copies in mouse peripheral blood (Figure 10D). Next, for the reimmunization assay, mice were selected that had been tumor-free for at least 5 weeks and for which no CAR copy number (copies of genomic DNA / ng) was detected in PBMCs. Raji tumors were monitored weekly using bioluminescence imaging (Figure 10E), and mouse body weight was measured (Figure 10F). [Figure 10D] This study demonstrates the in vivo antitumor and reimmunization effects of VHH-273 and VHH-496 CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 10A-10B), body weight (Figure 10C), and CAR copies in mouse peripheral blood (Figure 10D). Next, for the reimmunization assay, mice were selected that had been tumor-free for at least 5 weeks and for which no CAR copy number (copies of genomic DNA / ng) was detected in PBMCs. Raji tumors were monitored weekly using bioluminescence imaging (Figure 10E), and mouse body weight was measured (Figure 10F). [Figure 10E]This study demonstrates the in vivo antitumor and reimmunization effects of VHH-273 and VHH-496 CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 10A-10B), body weight (Figure 10C), and CAR copies in mouse peripheral blood (Figure 10D). Next, for the reimmunization assay, mice were selected that had been tumor-free for at least 5 weeks and for which no CAR copy number (copies of genomic DNA / ng) was detected in PBMCs. Raji tumors were monitored weekly using bioluminescence imaging (Figure 10E), and mouse body weight was measured (Figure 10F). [Figure 10F] This study demonstrates the in vivo antitumor and reimmunization effects of VHH-273 and VHH-496 CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 10A-10B), body weight (Figure 10C), and CAR copies in mouse peripheral blood (Figure 10D). Next, for the reimmunization assay, mice were selected that had been tumor-free for at least 5 weeks and for which no CAR copy number (copies of genomic DNA / ng) was detected in PBMCs. Raji tumors were monitored weekly using bioluminescence imaging (Figure 10E), and mouse body weight was measured (Figure 10F). [Figure 11A] This study demonstrates the in vivo antitumor and reimmunization effects of mono-, bi-, and tri-VHH CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 11A–11B), body weight (Figure 11C), and CAR copies in mouse peripheral blood (Figures 11D–11E; Figure 11D is taken from Figure 11E, and the Y-axis scale is adjusted to improve the resolution of genomic DNA copies / ng). Next, tumor-free mice were selected for reimmunization assays for at least 6 weeks. Raji tumors were monitored weekly with bioluminescence imaging (Figure 11F). [Figure 11B]This study demonstrates the in vivo antitumor and reimmunization effects of mono-, bi-, and tri-VHH CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 11A–11B), body weight (Figure 11C), and CAR copies in mouse peripheral blood (Figures 11D–11E; Figure 11D is taken from Figure 11E, and the Y-axis scale is adjusted to improve the resolution of genomic DNA copies / ng). Next, tumor-free mice were selected for reimmunization assays for at least 6 weeks. Raji tumors were monitored weekly with bioluminescence imaging (Figure 11F). [Figure 11C] This study demonstrates the in vivo antitumor and reimmunization effects of mono-, bi-, and tri-VHH CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 11A–11B), body weight (Figure 11C), and CAR copies in mouse peripheral blood (Figures 11D–11E; Figure 11D is taken from Figure 11E, and the Y-axis scale is adjusted to improve the resolution of genomic DNA copies / ng). Next, tumor-free mice were selected for reimmunization assays for at least 6 weeks. Raji tumors were monitored weekly with bioluminescence imaging (Figure 11F). [Figure 11D] This study demonstrates the in vivo antitumor and reimmunization effects of mono-, bi-, and tri-VHH CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 11A–11B), body weight (Figure 11C), and CAR copies in mouse peripheral blood (Figures 11D–11E; Figure 11D is taken from Figure 11E, and the Y-axis scale is adjusted to improve the resolution of genomic DNA copies / ng). Next, tumor-free mice were selected for reimmunization assays for at least 6 weeks. Raji tumors were monitored weekly with bioluminescence imaging (Figure 11F). [Figure 11E]This study demonstrates the in vivo antitumor and reimmunization effects of mono-, bi-, and tri-VHH CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 11A–11B), body weight (Figure 11C), and CAR copies in mouse peripheral blood (Figures 11D–11E; Figure 11D is taken from Figure 11E, and the Y-axis scale is adjusted to improve the resolution of genomic DNA copies / ng). Next, tumor-free mice were selected for reimmunization assays for at least 6 weeks. Raji tumors were monitored weekly with bioluminescence imaging (Figure 11F). [Figure 11F] This study demonstrates the in vivo antitumor and reimmunization effects of mono-, bi-, and tri-VHH CAR-T cells in a Raji xenograft NCG mouse model. Mice were regularly evaluated to monitor tumor growth using bioluminescence imaging (Figures 11A–11B), body weight (Figure 11C), and CAR copies in mouse peripheral blood (Figures 11D–11E; Figure 11D is taken from Figure 11E, and the Y-axis scale is adjusted to improve the resolution of genomic DNA copies / ng). Next, tumor-free mice were selected for reimmunization assays for at least 6 weeks. Raji tumors were monitored weekly with bioluminescence imaging (Figure 11F). [Figure 12A] The results from a study evaluating the binding affinity of anti-CD20 VHH-huIgG1Fc mAbs are shown. MFI = Mean Fluorescence Intensity. [Figure 12B] The results from a study evaluating the binding affinity of anti-CD20 VHH-huIgG1Fc mAbs are shown. MFI = Mean Fluorescence Intensity. [Figure 12C] The results from a study evaluating the binding affinity of anti-CD20 VHH-huIgG1Fc mAbs are shown. MFI = Mean Fluorescence Intensity. [Figure 12D] The results from a study evaluating the binding affinity of anti-CD20 VHH-huIgG1Fc mAbs are shown. MFI = Mean Fluorescence Intensity. [Figure 12E] The results from a study evaluating the binding affinity of anti-CD20 VHH-huIgG1Fc mAbs are shown. MFI = Mean Fluorescence Intensity. [Figure 13A] Exemplary humanized CD20 VHH CAR-T cells exhibit cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. Cytotoxicity of huVHH-746 CAR-T cells, huVHH-750 CAR-T cells, and huVHH-753 CAR-T cells is also shown. [Figure 13B] Exemplary humanized CD20 VHH CAR-T cells exhibit cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. Cytotoxicity of huVHH-746 CAR-T cells, huVHH-750 CAR-T cells, and huVHH-753 CAR-T cells is also shown. [Figure 13C] Exemplary humanized CD20 VHH CAR-T cells exhibit cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. Cytotoxicity of huVHH-746 CAR-T cells, huVHH-750 CAR-T cells, and huVHH-753 CAR-T cells is also shown. [Figure 13D] Exemplary humanized CD20 VHH CAR-T cells exhibit cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. Cytotoxicity of huVHH-253 CAR-T cells and huVHH-256 CAR-T cells is also demonstrated. [Figure 13E]Exemplary humanized CD20 VHH CAR-T cells exhibit cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. Cytotoxicity of huVHH-253 CAR-T cells and huVHH-256 CAR-T cells is also demonstrated. [Figure 13F] Exemplary humanized CD20 VHH CAR-T cells exhibit cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. Cytotoxicity of huVHH-253 CAR-T cells and huVHH-256 CAR-T cells is also demonstrated. [Figure 13G] Exemplary humanized CD20 VHH CAR-T cells exhibit cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. Cytotoxicity of huVHH-253 CAR-T cells and huVHH-256 CAR-T cells is also demonstrated. [Figure 13H] This shows the cytotoxicity of exemplary humanized CD20 VHH CAR-T cells against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. It also shows the cytotoxicity of huVHH-260 CAR-T cells. [Figure 13I] This shows the cytotoxicity of exemplary humanized CD20 VHH CAR-T cells against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc cells at different effector cell-to-target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1. It also shows the cytotoxicity of huVHH-260 CAR-T cells. [Figure 13J]Exemplary humanized CD20 VHH CAR-T cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc at different effector cell to target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1 is shown. Cytotoxicity of huVHH-260 CAR-T cells is shown. [Figure 13K] Exemplary humanized CD20 VHH CAR-T cytotoxicity against Raji.Luc cells, K652-CD20.Luc cells, K562.Luc cells, and Nalm.6.Luc at different effector cell to target cell ratios (E:T) of 15:1, 10:1, 5:1, or 2.5:1 is shown. Cytotoxicity of huVHH-260 CAR-T cells is shown.

Mode for Carrying Out the Invention

[0030] 5. Detailed Description The present disclosure is based, in part, on novel single domain antibodies (e.g., VHH domains) that bind to CD20, chimeric antigen receptors, or engineered cells containing the same, and their improved properties.

[0031] 5.1. Definitions The methods and procedures described or referenced herein include those generally well understood and / or commonly used by those skilled in the art, such as the commonly used methodologies by those skilled in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010). Unless otherwise defined herein, the technical and scientific terms used herein have meanings generally understood by those skilled in the art. For the purpose of interpreting this specification, the following definitions of terms apply, and wherever appropriate, a term used in the singular also includes the plural, and vice versa. If any of the definitions of the terms provided herein conflict with any document incorporated herein by reference, the definitions of the terms provided below shall prevail.

[0032] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and in their broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having polyepitope or monoepitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, and multispecific antibodies (e.g., bispecific antibodies insofar as they exhibit the desired biological activity), and are formed from at least two intact antibodies, single-chain antibodies, and their fragments (e.g., domain antibodies), as described below. Antibodies may be derived from humans, humanized, chimeric, and / or affinity-matured antibodies, as well as from other species, such as mice, rabbits, llamas, etc. The term "antibody" is intended to include B cell polypeptide products within the immunoglobulin class of polypeptides that can bind to specific molecular antigens and consist of two identical pairs of polypeptide chains, each pair comprising one heavy chain (approximately 50–70 kDa) and one light chain (approximately 25 kDa), with each amino-terminus of each chain containing a variable region of approximately 100–130 or more amino acids, and each carboxy-terminus of each chain containing a constant region. See, for example, Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). Antibodies include, but are not limited to, synthetic antibodies, recombinant antibodies, single-domain antibodies containing camelid species (e.g., llamas or alpacas) or their humanized variants, intrabodies, anti-idiotype (anti-Id) antibodies, and any of the functional fragments of the above (e.g., antigen-binding fragments), and refer to a portion of the heavy or light chain polypeptide of an antibody that retains some or all of the antibody-binding activity from which the fragment originates.Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv(scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv(dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, the antibodies provided herein contain immunoglobulin molecules and molecules containing immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or antigen-binding sites that bind to antigens (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in: Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd ed. 1990). The antibodies provided herein may be of any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibodies may be agonist antibodies or antagonist antibodies. Antibodies can be neither agonists nor antagonists.

[0033] An "antigen" is a structure to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen associates with cells and is present, for example, on or inside the cell.

[0034] An "intact" antibody includes an antigen-binding site and CL and at least a heavy chain constant region, CH1, CH2, and CH3. The constant region may include a human constant region or an amino acid sequence variant thereof. In certain embodiments, the intact antibody has one or more effector functions.

[0035] A "sFv" or "scFv," also abbreviated as "sFv," is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0036] The term "heavy-chain-only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in four-chain antibodies. Camelids (e.g., camels, llamas, or alpacas) are known to produce HCAbs.

[0037] As used herein, “single-domain antibody” or “sdAb” refers to a single monomeric variable antibody domain that is capable of antigen binding (e.g., a single-domain antibody that binds to CD20). A single-domain antibody contains a VHH domain as described herein. Examples of single-domain antibodies include, but are not limited to, antibodies that naturally lack a light chain, such as those from camelid species (e.g., llamas), single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain skeletons other than those derived from antibodies. Single-domain antibodies may be derived from any species, including, but are not limited to, mice, humans, camels, llamas, goats, rabbits, and cattle. For example, a single-domain antibody may be derived from an antibody produced in camelid species such as camels, llamas, dromedaries, alpacas, and guanacos, as described herein. Other non-camelid species may produce heavy-chain antibodies that naturally lack a light chain, and VHHs derived from such other species are within the scope of this disclosure. In some embodiments, the single-domain antibodies (e.g., VHH) provided herein have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The single-domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., a drug) described herein. The single-domain antibodies may be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0038] The term "binding" or "bonding" refers to intermolecular interactions, including, for example, the formation of a complex. Interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. Complexes can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of all non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule such as an antigen is the affinity of the antibody or functional fragment to that epitope. The dissociation rate (k) of the binding molecule (e.g., antibody) versus monovalent antigen is... off ) Pair bond rate (k on ) ratio (k off / k on) is the dissociation constant K D and is inversely proportional to the affinity. The lower the K D value, the higher the affinity of the antibody. The value of K D varies depending on the complex of the antibody and the antigen and depends on both k on and k off . The dissociation constant K D of the antibodies provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants such as a multivalent antigen contacts an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site will increase the likelihood of a reaction at the second site. The strength of such multiple interactions between multivalent antibodies and antigens is called avidity.

[0039] In relation to the binding molecules described herein, terms such as "binding," "specifically binding to," and similar terms are used interchangeably herein and refer to binding molecules of antigen-binding domains that specifically bind to an antigen, e.g., a polypeptide. Binding molecules or antigen-binding domains that bind to or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other methods known to those skilled in the art. In some embodiments, a binding molecule or antigen-binding domain binds to or specifically binds to an antigen if it binds to the antigen with a higher affinity (measured using experimental techniques such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA)) than any cross-reactive antigen. Typically, a specific or selective reaction is at least twice the background signal or noise, and may exceed 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2 ded. 1989). In certain embodiments, the degree of binding of the binding molecule or antigen-binding domain to a “non-target” protein is less than approximately 10% of the binding of the binding molecule or antigen-binding domain to its particular target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. Antigen-binding molecules or antigen-binding domains include those capable of binding to the antigen with sufficient affinity so that the binding molecule is useful, for example, as an antigen-targeting therapeutic and / or diagnostic agent. In certain embodiments, the binding molecule or antigen-binding domain that binds to the antigen has a dissociation constant (K) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. D ) has. In certain embodiments, the binding molecule or antigen-binding domain binds to an antigen epitope that is conserved among antigens of different species.

[0040] In certain embodiments, the binding molecule or antigen-binding domain may contain a “chimeric” sequence in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody originating from a particular species or belonging to a particular antibody class or subclass, but the remainder of the chain is identical or homologous to a corresponding sequence of an antibody originating from another species or belonging to another antibody class or subclass, and of a fragment of such an antibody, as long as it exhibits the desired biological activity (see below: U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). The chimeric sequence may also contain a humanized sequence.

[0041] In certain embodiments, the binding molecule or antigen-binding domain may include a portion of a “humanized” form of a non-human (e.g., camelid, mouse, non-human primate) antibody containing a sequence derived from a human immunoglobulin (e.g., recipient antibody), and the native CDR residues are replaced with residues derived from the corresponding CDRs of a non-human species (e.g., donor antibody), e.g., camelid, mouse, rat, rabbit, or a non-human mammal having the desired specificity, affinity, and capability. In some cases, one or more FR region residues of the human immunoglobulin sequence are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications are further made to improve the performance of the antibody. The heavy or light chain of the humanized antibody may contain substantially all of at least one variable region, with all or substantially all of the CDRs corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being from the human immunoglobulin sequence. In certain embodiments, the humanized antibody will contain at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0042] In certain embodiments, the binding molecule or antigen-binding domain may include a “fully human antibody” or a “human antibody,” the terms used interchangeably herein, and refer to an antibody containing a human variable region, e.g., a human constant region. The binding molecule may also include a single-domain antibody sequence. In certain embodiments, the terms refer to an antibody containing a variable and constant region of human origin. In certain embodiments, “fully human” antibodies may also include antibodies that bind to a polypeptide and encode a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulins as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). “Human antibody” has an amino acid sequence corresponding to an antibody produced by a human and / or is produced using any of the methods for producing human antibodies. This definition of human antibodies explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of methods known in the art, including phage display libraries (Hoogenboom and Winter, J.Mol.Biol.227:381(1991); Marks et al., J.Mol.Biol.222:581(1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68(2006)). Furthermore, methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77(1985); Boerner et al., J.Immunol.147(1):86-95(1991); and van Dijk and van de Winkel, Curr.Opin.Pharmacol.5:368-74(2001) are available for the preparation of human monoclonal antibodies.Human antibodies can be prepared by administering an antigen to transgenic animals, such as mice, that have been modified to produce such antibodies in response to antigen administration, but whose endogenous gene locus has been deactivated (e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and, with respect to XENOMOUSE® technology, U.S. Patents 6,075,181 and 6,150,584). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006), with respect to human antibodies produced via human B-cell hybridoma technology.

[0043] In certain embodiments, the binding molecule or antigen-binding domain may include part of a “recombinant human antibody,” which includes human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice or cattle) that are transgenic and / or transchromosomes of human immunoglobulin genes (see, e.g., Taylor, L D et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, created, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies may have undergone in vitro mutagenesis (or in vivo somatic mutagenesis, if transgenic animals for human Ig sequences are used), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody may be derived from and related to human germline VH and VL sequences, but may not be naturally present in the human antibody germline repertoire in vivo.

[0044] In certain embodiments, the binding molecule or antigen-binding domain may constitute part of the “monoclonal antibody,” and the term as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, for example, the individual antibodies constituting the population being identical except for possible naturally occurring mutations or well-known post-translational modifications that may be present in small amounts, such as amino acid iomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation, and each monoclonal antibody will typically recognize a single epitope on an antigen. In certain embodiments, the “monoclonal antibody” as used herein is an antibody produced by a single hybridoma or other cell. The term “monoclonal” is not limited to a particular method for producing the antibody. For example, monoclonal antibodies useful in this disclosure may be prepared by the hybridoma methodology first described in Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods in bacterial or eukaryotic or plant cells (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using, for example, the methods described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J.Mol.Biol.222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0045] A typical quadruple antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, a quadruple unit generally has a weight of approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has intrachain disulfide bridges arranged at regular intervals. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) in the case of α and γ chains, respectively, and four CH domains in the case of μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at the other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains. The pairing of VH and VL leads to the formation of a single antigen-binding site. For the structures and properties of various classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5 th (ed. 2001).

[0046] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. Typically, conventional IgG contains two Fab regions, each located on one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region in both the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in a Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in a Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions within a Fab region can be arranged in various ways to confer the antigen-binding ability according to this disclosure. For example, as with the Fab regions of conventional IgG, the VH and CH1 regions may be on one polypeptide, and the VL and CL regions may be on another polypeptide. Alternatively, the VH, CH1, VL, and CL regions may all be on the same polypeptide and oriented in different orders, as will be described in more detail in the following sections.

[0047] The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody's light or heavy chain, typically located at the amino terminus of the light or heavy chain, approximately 120–130 amino acids long in the heavy chain and approximately 100–110 amino acids long in the light chain, used for the binding and specificity of each particular antibody to a specific antigen. The variable region of the heavy chain is sometimes called "VH." The variable region of the light chain is sometimes called "VL." The term "variable" refers to the fact that specific segments of the variable region have significantly different sequences between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody to a specific antigen. However, variability is not evenly distributed across the entire 110-amino acid span of the variable region. Instead, the V region consists of a less variable (e.g., relatively invariant) stretch of approximately 15–30 amino acids called the framework region (FR), which is separated by shorter regions of greater variability (e.g., extreme variability), called the "hypervariable region," each approximately 9–12 amino acids long. The variable regions of the heavy and light chains each contain four FRs, which primarily employ a β-sheet structure and are linked by three hypervariable regions, forming loops that link the β-sheet structures in some cases, and forming parts of the β-sheet structures in others. The hypervariable regions of each chain are held together in close proximity by FRs and, together with hypervariable regions from other chains, contribute to the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, such as the antibody's involvement in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC). The sequence of the variable regions differs significantly between different antibodies. In certain embodiments, the variable region is a human variable region.

[0048] The terms “Kabat variable region residue numbering” or “Kabat amino acid position numbering,” and their variations, refer to the numbering system used for the heavy-chain or light-chain variable region of antibody edits in Kabat et al., cited above. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or CDR in the variable domain. For example, the heavy-chain variable domain may contain a single amino acid insertion after residue 52 (Kabat residue 52a) and three inserted residues after residue 82 (e.g., Kabat residues 82a, 82b, and 82c). The Kabat numbering of residues may be determined for a given antibody by alignment in homologous regions of the sequence of an antibody having a “standard” Kabat numbering sequence. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately light-chain residues 1-107 and heavy-chain residues 1-113) (e.g., Kabat et al., cited above). The "EU numbering system" or "EU index" is commonly used to refer to residues in the constant region of immunoglobulin heavy chains (e.g., the EU index reported by Kabat et al. above). "Kabat's EU index" refers to the residue numbering of human IgG1 EU antibodies. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0049] When used in reference to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50–70 kDa, with a variable region of approximately 120–130 or more amino acids at the amino terminus and a constant region at the carboxy terminus. The constant region can be one of five different types (e.g., isotypes) called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. Different heavy chains differ in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies, namely IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4, respectively.

[0050] When used in reference to antibodies, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, with a variable region of approximately 100 to 110 or more amino acids at the amino terminus and a constant region at the carboxy terminus. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two distinct types called kappa (κ) or lambda (λ).

[0051] As used herein, the terms “hypervariable region,” “HVR,” “complementarity-determining region,” and “CDR” are used interchangeably. “CDR” refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VHβ sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VLβ sheet framework. Therefore, a CDR is a variable region sequence scattered within a framework region sequence.

[0052] The CDR region is well known to those skilled in the art and is defined by well known numbering systems. For example, the Kabat complementarity determination region (CDR) is based on sequence variability and is the most commonly used (see, e.g., Kabat et al. cited above). Chothia, on the other hand, refers rather to the location of structural loops (see, e.g., Chothia and Lesk, J.Mol.Biol.196:901-17(1987)). When numbered using the Kabat numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places inserts at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; and if both 35A and 35B exist, the loop ends at 34). The AbM hypervariable region represents an intermediate between the Kabat CDR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel eds., 2d ed. 2010)). The “contact” hypervariable region is based on the analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) information system (registered trademark) (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. In this specification, CDRs are referred to in terms of both amino acid sequence and position within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides in structures called loops, CDRs and framework residues can be easily identified by using a numbering system that aligns variable domain sequences according to their structural characteristics.This information can be used when transplanting and substituting CDR residues from one species of immunoglobulin into an acceptor framework, typically derived from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, J.Mol.Biol.309:657-70 (2001). Correspondence between numbering systems, including Kabat numbering and IMGT-specific numbering systems, is well known to those skilled in the art (see, for example, Kaba above; Chothia and Lesk above; Martin above; and Lefranc et al. above). Residues derived from each of these hypervariable regions or CDRs are exemplified in Table 1 below.

[0053] (Table 1) Exemplary CDRs using various numbering systems TIFF0007846667000001.tif132165

[0054] The boundaries of a particular CDR may vary depending on the scheme used for identification. Therefore, unless otherwise specified, the terms “CDR” and “complementarity-determining region” of a given antibody or region, e.g., the variable region, as well as the individual CDRs of the antibody or region (e.g., CDR-H1, CDR-H2), should be understood to encompass the complementarity-determining region as defined in any of the known schemes described herein. In some cases, a scheme is specified for identifying a particular CDR or multiple CDRs, such as CDRs defined by IMGT, Kabat, Chothia, or Contact methods. In some cases, one or more positions by Kabat numbering may not be occupied by the actual sequence, or the actual sequence may contain more amino acid residues than permitted by Kabat numbering. See, for example, Deschacht et al., 2010. J Immunol 184:5696-704 (on exemplary numbering of the VHH domain by Kabat). In other cases, a specific amino acid sequence of the CDR is given. It should be noted that CDR areas can also be defined by various numbering system combinations, such as a combination of the Kabat and Chothia numbering systems, or a combination of the Kabat and IMGT numbering systems. Therefore, terms such as "a CDR specified in a particular VH or VHH" include, but are not limited to, any CDR1 defined in the exemplary CDR numbering systems described above. Those skilled in the art will understand that given a variable area (e.g., VHH, VH, or VL), CDRs within that area can be defined by different numbering systems or combinations thereof.

[0055] The hypervariable region may include the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0056] The term "constant region" or "constant domain" refers to the carboxyl-terminal portions of the light and heavy chains that do not directly participate in antibody binding to an antigen but exhibit various effector functions, such as interaction with the Fc receptor. This term refers to a portion of the immunoglobulin molecule that has a more conserved amino acid sequence compared to the variable region, which is the other part of the immunoglobulin that contains the antigen-binding site. The constant region may include the CH1, CH2, and CH3 regions of the heavy chain, as well as the CL region of the light chain.

[0057] The term "framework" or "FR" refers to variable region residues adjacent to the CDR. FR residues are found, for example, in chimeric, humanized, human, domain antibodies (e.g., single-domain antibodies), diabolic, linear, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region residues or CDR residues.

[0058] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, and includes, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain may differ, the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinant operations on the nucleic acid encoding the antibody heavy chain. Thus, compositions of intact antibodies may include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having mixtures of antibodies with and without the K447 residue. A “functional Fc region” has the “effector function” of the native sequence Fc region. Examples of “effector functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors). Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be evaluated using various assays known to those skilled in the art. A “variant Fc region” includes a variant Fc region that differs in amino acid sequence from the native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions, compared to the native sequence Fc region or the parent polypeptide Fc region. The variant Fc region described herein may have at least about 80% homology to the natural sequence Fc region and / or the Fc region of the parent polypeptide, or at least about 90% homology, for example, at least about 95% homology.

[0059] As used herein, “epitope” is a term of the art and refers to a localization region of an antigen to which a binding molecule (e.g., an antibody comprising a single-domain antibody sequence) can specifically bind. Epitopes can be linear, conformal, nonlinear, or discontinuous epitopes. In the case of polypeptide antigens, for example, an epitope may be a sequence of amino acids in the polypeptide (a “linear” epitope), or an epitope may consist of amino acids from two or more discontinuous regions of the polypeptide (a “conformal,” “nonlinear,” or “discontinuous” epitope). Generally, those skilled in the art will understand that linear epitopes may or may not depend on the secondary, tertiary, or quaternary structure. For example, in some embodiments, the binding molecule binds to an amino acid group regardless of whether the amino acid group folds into a native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues constituting the epitope exhibit a specific conformation (e.g., bend, twist, rotate, or fold) in order to recognize and bind to the epitope.

[0060] A "blocking" antibody or "antagonist" antibody inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen.

[0061] An "agonist" or activating antibody enhances or initiates signaling by the antigen to which it binds. In some embodiments, the agonist antibody induces or activates signaling in the absence of a native ligand.

[0062] "Percent (%) amino acid sequence identity" and "homology" for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0063] The term "specificity" refers to the selective recognition of an antigen-binding protein (e.g., CAR or sdAb) for a specific epitope of an antigen. For example, natural antibodies are monospecific. As used herein, the term "multispecificity" means that an antigen-binding protein (e.g., CAR or sdAb) has two or more antigen-binding sites that bind to at least two different antigens. As used herein, "bispecificity" means that an antigen-binding protein (e.g., CAR or sdAb) has two different antigen-binding specificities. As used herein, the term "monospecific" CAR means an antigen-binding protein (e.g., CAR or sdAb) that has one or more binding sites, each of which binds to the same antigen.

[0064] As used herein, the term “valence” refers to the presence of a specific number of binding sites in an antigen-binding protein (e.g., CAR or sdAb). For example, a natural antibody or a full-length antibody has two binding sites and is divalent. Thus, the terms “trivalent,” “tetravalent,” “pentavalent,” and “hexavalent” refer to the presence of two, three, four, five, and six binding sites, respectively, in an antigen-binding protein (e.g., CAR or sdAb).

[0065] As used herein, “chimeric antigen receptor” or “CAR” refers to a genetically engineered receptor that can be used to transfer one or more antigen specificities to immune effector cells such as T cells. Some CARs are also known as “artificial T cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” In some embodiments, a CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain for T cells and / or other receptors that are specific to one or more antigens (e.g., tumor antigens). “CAR-T cell” refers to a T cell that expresses a CAR.

[0066] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymers may be linear or branched, and may contain modified amino acids or be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification. For example, polypeptides containing one or more analogs of amino acids, including non-natural amino acids, as well as other modifications known in the art, are also included in the definition. Polypeptides of this disclosure may be based on antibodies or other members of the immunoglobulin superfamily, and in certain embodiments, it is understood that a “polypeptide” may arise as a single chain or as two or more associated chains.

[0067] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. A nucleotide may be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or a synthetic reaction. Polynucleotides may also include modified nucleotides, e.g., methylated nucleotides and their analogs. As used herein, “oligonucleotide” refers to a short, generally single-stranded synthetic polynucleotide, generally less than approximately 200 nucleotides in length, although this is not strictly necessary. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells producing the binding molecules of this disclosure may include parental hybridoma cells, as well as bacterial and eukaryotic host cells into which antibody-encoding nucleic acids have been introduced. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition from 5' to 3' of a newly generated RNA transcript is called the transcription direction; the sequence region on the DNA strand at the 5' to 5' end of the RNA transcript, which has the same sequence as the RNA transcript, is called the "upstream sequence"; and the sequence region on the DNA strand at the 3' to 3' end of the RNA transcript, which has the same sequence as the RNA transcript, is called the "downstream sequence."

[0068] "Isolated nucleic acids" are nucleic acids, e.g., RNA, DNA, or mixed nucleic acids, substantially isolated from other genomic DNA sequences, as well as proteins or complexes, e.g., ribosomes and polymerases, which naturally accompany the native sequence. "Isolated" nucleic acid molecules are those isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, "isolated" nucleic acid molecules, such as cDNA molecules, may substantially contain other cellular material or culture media if produced by recombinant methods, or substantially contain chemical precursors or other chemicals if chemically synthesized. In certain embodiments, one or more nucleic acid molecules encoding a single-domain antibody or an antibody described herein are isolated or purified. This term encompasses nucleic acid sequences removed from their naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or biosynthesized analogs by heterologous systems. Substantially pure molecules may include isolated forms of molecules. Specifically, "isolated" nucleic acid molecules encoding CARs or sdAbs as described herein are nucleic acid molecules identified and isolated from at least one contaminating nucleic acid molecule that normally associates in the environment in which they are produced.

[0069] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a coding sequence that is operablely linked in a particular host organism. Regulatory sequences suitable for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome-binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0070] As used herein, the term “operatably linked” and similar phrases (e.g., gene-fused) refer, when used in relation to nucleic acids or amino acids, to operatably linked nucleic acid sequences or amino acid sequences arranged in a functional relationship with each other. For example, operatably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the precise production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatably linked nucleic acid elements result in the transcription of an open reading frame, ultimately leading to the production of a polypeptide (i.e., expression of the open reading frame). As another example, operatably linked peptides are arranged with functional domains spaced appropriately apart from each other to confer the intended function of each domain.

[0071] The term "vector" refers to a substance used to contain or encapsulate a nucleic acid sequence, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce the nucleic acid sequence into a host cell. Applicable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selectable sequences or markers that can be implemented for stable integration into the chromosome of a host cell. Furthermore, a vector may contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes that may be included may, for example, provide resistance to antibiotics or toxins, complement nutritional deficiencies, or supply essential nutrients that are not present in the culture medium. Expression regulatory sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, etc., which are well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., both antibody heavy and light chains, or antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. In the case of single-vector expression, the coding nucleic acid can be operably ligated to one common expression regulatory sequence, or to different expression regulatory sequences such as one inductive promoter and one constitutive promoter. The introduction of the nucleic acid molecule into host cells can be confirmed using methods well known in the art. Such methods include nucleic acid analysis, e.g., Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for the expression of a gene product, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product, and it is further understood that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

[0072] As used herein, the term “host” refers to an animal, such as a mammal (e.g., a human).

[0073] As used herein, the term “host cell” refers to a specific target cell that can be transfected with a nucleic acid molecule, and its offspring or potential offspring. Such offspring may not be identical to the parent cell transfected with the nucleic acid molecule due to the influence of the environment that may occur in mutation or subsequent generation or integration of the nucleic acid molecule into the host genome.

[0074] As used herein, the term “self-derived” means any substance that originates from the same organism and is later reintroduced into the organism.

[0075] "Homogenous, dissimilar" refers to grafts derived from different individuals of the same species.

[0076] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is one that has been transfected, transformed, or transduced with an exogenous nucleic acid. Cells include primary target cells and their offspring.

[0077] As used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory agency, or that its use in animals, particularly in humans, is listed in the United States Pharmacopeia, the European Pharmacopeia, or any other generally accepted pharmacopoeia.

[0078] "Excipient" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coatings, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, wetting agents, lubricants, fragrances, preservatives, propellants, release agents, bactericides, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)) or vehicle.

[0079] In some embodiments, the excipients are pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugar alcohols, e.g., mannitol or sorbitol; salt-forming counterions, e.g., sodium; and / or nonionic surfactants, e.g., TWEEN®, polyethylene glycol (PEG), and PLURONICS®. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0080] In one embodiment, each component is “pharmaceutically acceptable” in the sense that it is compatible with other components of the pharmaceutical formulation, and is suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. See, for example: Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is non-toxic to cells or mammals to which it is exposed at the dosage and concentration used. In some embodiments, the pharmaceutically acceptable excipient is a pH-buffered aqueous solution.

[0081] In some embodiments, excipients are sterile liquids, water, and oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is an exemplary excipient when the composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solution and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly as injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, and ethanol. The composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, as needed. The composition may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral compositions containing the formulation may include standard excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.

[0082] A composition containing a pharmaceutical compound may, for example, contain a binding molecule (e.g., an antibody) in an isolated or purified form, along with an appropriate amount of excipients.

[0083] As used herein, the terms “effective dose” or “therapeutic dose” refer to a quantity of the drug and single-domain antibody or therapeutic molecule, including the single-domain antibody or pharmaceutical composition provided herein, that is sufficient to produce the desired result.

[0084] The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, the subject is a mammal, e.g., a non-primate or primate (e.g., a human). In certain embodiments, the subject is a human. In one embodiment, the subject is a mammal, e.g., a human, that has been diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, e.g., a human, that is at risk of developing a disease or disorder.

[0085] "Administer" or "Deliver" means the act of injecting a substance that is present outside the body into a patient or otherwise physically delivering it, for example, by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art.

[0086] As used herein, the terms “to treat,” “treatment,” and “to treat” refer to the reduction or improvement of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there has been a reduction, relief, and / or reduction of one or more symptoms associated with the underlying disease, to the extent that improvement is observed in the patient, even though the patient may still have the underlying disease. The term “to treat” includes both management and improvement of the disease. The terms “to manage,” “to manage,” and “manage” refer to the beneficial effect that a subject receives from a therapy that does not necessarily result in a cure for the disease.

[0087] The terms “prevent,” “prevention,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated condition (e.g., diabetes or cancer).

[0088] As used herein, “delaying” the onset of cancer means delaying, interfering with, slowing, preventing, stabilizing, and / or postponing the onset of the disease. This delay can be of varying lengths depending on the disease history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may effectively encompass prevention in that the individual does not develop the disease. A method for “delaying” the onset of cancer is a method that reduces the likelihood of the disease developing within a given time frame and / or the severity of the disease within a given time frame compared to not using the method. Such comparisons are usually based on clinical studies using statistically significant populations. Cancer onset can be detected using standard methods, including, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasonography, coagulation tests, angiography, or biopsy. Onset may also refer to the progression of cancer, which may initially be undetectable, and includes development, recurrence, and onset.

[0089] As used herein, “B-cell related disease or disorder” refers to a disease or disorder mediated by B cells or conferred by abnormal B-cell function (e.g., dysregulation of B-cell function). As used herein, “B-cell related disease or disorder” includes, but is not limited to, B-cell malignancies, such as B-cell leukemia or B-cell lymphoma. This also includes marginal zone lymphoma (e.g., splenic marginal zone lymphoma), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, primary mediastinal B-cell lymphoma (PMBL), small lymphocytic lymphoma (SLL), B-cell prelymphoblastic leukemia (B-PLL), follicular lymphoma (FL), Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia (HCL), precursor B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), high-grade B-cell lymphoma (HGBL), and multiple myeloma (MM). "B-cell-related disorders or conditions" also include certain autoimmune and / or inflammatory diseases associated with inappropriate or increased B cell counts and / or activation.

[0090] As used herein, “CD20-related disease or disorder” refers to a disease or disorder that includes cells or tissues expressing CD20. In some embodiments, the CD20-related disease or disorder includes cells that abnormally express CD20. In other embodiments, the CD20-related disease or disorder includes cells that lack CD20 internally or externally.

[0091] The terms "approximately" and "about" refer to a specific value or range of 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.

[0092] As used in this disclosure and in the claims, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly stated in the context.

[0093] Where an embodiment is described herein using the term “comprising,” it will be understood that other similar embodiments are also provided, described with respect to “consisting of” and / or “consisting essentially of.” Where an embodiment is described herein using the expression “consisting essentially of,” it will be understood that other similar embodiments are also provided, described with respect to “consisting of.”

[0094] The term "between" or "between A and B" refers to a range that includes both A and B.

[0095] The term "and / or" as used in expressions such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in expressions such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0096] 5.2. Single-domain antibodies 5.2.1. Single-domain antibodies that bind to CD20 In one embodiment, a single-domain antibody (e.g., a VHH domain) capable of binding to CD20 is provided herein.

[0097] In some embodiments, the single-domain antibody (e.g., VHH domain) provided herein binds to human CD20. In some embodiments, the anti-CD20 single-domain antibody provided herein modulates one or more CD20 activities. In some embodiments, the anti-CD20 single-domain antibody provided herein is an antagonist antibody.

[0098] In some embodiments, the anti-CD20 single-domain antibodies provided herein are ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 The dissociation constant (K) of M D) binds to CD20 (e.g., human CD20). Various methods for measuring binding affinity are known in the art and any of them may be used for the purposes of this disclosure, including, for example, by RIA performed using the Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biolayer interferometry (BLI) or by surface plasmon resonance (SPR) assay by Octet® using, for example, the Octet® Red96 system; or by Biacore® using, for example, Biacore® TM-2000 or Biacore® TM-3000. The "on rate," "association rate," "association velocity," or "kon" may also be determined by the above-mentioned biolayer interferometry (BLI) or surface plasmon resonance (SPR) using, for example, the Octet® Red96, Biacore® TM-2000, or Biacore® TM-3000 systems.

[0099] In some embodiments, the anti-CD20 single-domain antibody provided herein is a VHH domain. As described in Section 6 below, exemplary VHH domains provided herein are generated, and these VHH domains are referred to as VHH-273, VHH-283, VHH-313, VHH-440, VHH-466, VHH-496, VHH-653, huVHH-253, huVHH-256, huVHH-260, huVHH-746, huVHH-750, huVHH-753, huVHH-836, huVHH-840, huVHH-843, huVHH-846, 2082H1, 2082H2, 2082H3, 2082H4, 2082H5, 2082H6, VHH-623, VHH-640, and / or VHH-657.

[0100] Accordingly, in some embodiments, the single-domain antibodies provided herein include one or more CDR sequences, any one of VHH-273, VHH-283, VHH-313, VHH-440, VHH-466, VHH-496, VHH-653, huVHH-253, huVHH-256, huVHH-260, huVHH-746, huVHH-750, huVHH-753, huVHH-836, huVHH-840, huVHH-843, huVHH-846, 2082H1, 2082H2, 2082H3, 2082H4, 2082H5, 2082H6, VHH-623, VHH-640, and / or VHH-657. In some embodiments, single-domain antibodies that bind to CD20, comprising the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, are provided herein, and the CDR sequences are VHH-273, VHH-283, VHH-313, VHH-440, VHH-466, VHH-496, VHH-653, huVHH-253, huVHH-256, h Select from uVHH-260, huVHH-746, huVHH-750, huVHH-753, huVHH-836, huVHH-840, huVHH-843, huVHH-846, 2082H1, 2082H2, 2082H3, 2082H4, 2082H5, 2082H6, VHH-623, VHH-640, and / or VHH-657.

[0101] (Table 2) Exemplary single-domain antibodies TIFF0007846667000002.tif217165TIFF0007846667000003.tif200165

[0102] In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 41. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 43. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 44. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 45. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 46. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 47. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 48. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 49. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 50. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 51. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 52. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 53. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 54. In some embodiments, an anti-CD20 single-domain antibody is provided that contains one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 55.In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 56. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 57. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 58. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 59. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 60. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 61. In some embodiments, an anti-CD20 single-domain antibody is provided containing one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 62. In some embodiments, an anti-CD20 single-domain antibody is provided that contains one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 63. In some embodiments, an anti-CD20 single-domain antibody is provided that contains one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 183. In some embodiments, an anti-CD20 single-domain antibody is provided that contains one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 184. In some embodiments, an anti-CD20 single-domain antibody is provided that contains one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 185. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0103] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 41. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 41. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 6) having the amino acid sequence of CDR3 described in SEQ ID NO: 41. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 41. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 41. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 41. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 41. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0104] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 42. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 42. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 8 or 10) having the amino acid sequence of CDR3 described in SEQ ID NO: 42. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 42. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 42. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 42. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 42. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0105] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 43. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 43. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 8 or 10) having the amino acid sequence of CDR3 described in SEQ ID NO: 43. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 43. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 43. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 43. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 43. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0106] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 44. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 44. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 16 or 19) having the amino acid sequence of CDR3 described in SEQ ID NO: 44. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 44. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 44. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 44. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 44. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0107] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 45. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 45. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 16 or 19) having the amino acid sequence of CDR3 described in SEQ ID NO: 45. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 45. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 45. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 45. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 45. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0108] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 46. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 46. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 26 or 29) having the amino acid sequence of CDR3 described in SEQ ID NO: 46. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 46. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 46. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 46. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 46. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0109] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 47. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 47. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 16 or 19) having the amino acid sequence of CDR3 described in SEQ ID NO: 47. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 47. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 47. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 47. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 47. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0110] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 48. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 48. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 6) having the amino acid sequence of CDR3 described in SEQ ID NO: 48. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 48. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 48. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 48. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 48. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0111] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 49. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 49. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 6) having the amino acid sequence of CDR3 described in SEQ ID NO: 49. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 49. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 49. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 49. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 49. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0112] In some embodiments, the single-domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 50. In some embodiments, the single-domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 50. In other embodiments, the single-domain antibody has a CDR3 (e.g., SEQ ID NO: 8 or 10) having the amino acid sequence of CDR3 set forth in SEQ ID NO: 50. In some embodiments, the single-domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 50. In some embodiments, the single-domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 50. In some embodiments, the single-domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 50. In some embodiments, the single-domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 50. The CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs follow the IMGT numbering. In some embodiments, the CDRs follow the Kabat numbering. In some embodiments, the CDRs follow the AbM numbering. In other embodiments, the CDRs follow the Chothia numbering. In other embodiments, the CDRs follow the Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0113] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 51. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 51. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 26 or 29) having the amino acid sequence of CDR3 described in SEQ ID NO: 51. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 51. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 51. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 51. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 51. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0114] In some embodiments, the single-domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 52. In some embodiments, the single-domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 52. In other embodiments, the single-domain antibody has a CDR3 (e.g., SEQ ID NO: 26 or 29) having the amino acid sequence of CDR3 set forth in SEQ ID NO: 52. In some embodiments, the single-domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 52. In some embodiments, the single-domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 52. In some embodiments, the single-domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 52. In some embodiments, the single-domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 52. The CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs follow IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In some embodiments, the CDRs follow AbM numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0115] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 53. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 53. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 26 or 29) having the amino acid sequence of CDR3 described in SEQ ID NO: 53. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 53. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 53. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 53. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 53. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0116] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 54. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 54. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 16 or 19) having the amino acid sequence of CDR3 described in SEQ ID NO: 54. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 54. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 54. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 54. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 54. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0117] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 55. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 55. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 16 or 19) having the amino acid sequence of CDR3 described in SEQ ID NO: 55. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 55. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 55. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 55. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 55. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0118] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 56. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 56. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 16 or 19) having the amino acid sequence of CDR3 described in SEQ ID NO: 56. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 56. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 56. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 56. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 56. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0119] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 57. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 57. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 16 or 19) having the amino acid sequence of CDR3 described in SEQ ID NO: 57. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 57. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 57. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 57. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 57. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0120] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 58. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 58. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 6) having the amino acid sequence of CDR3 described in SEQ ID NO: 58. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 58. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 58. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 58. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 58. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0121] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 59. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 59. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 39) having the amino acid sequence of CDR3 described in SEQ ID NO: 59. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 59. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 59. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 59. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 59. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0122] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 60. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 60. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 6) having the amino acid sequence of CDR3 described in SEQ ID NO: 60. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 60. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 60. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 60. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 60. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0123] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 61. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 61. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 40) having the amino acid sequence of CDR3 described in SEQ ID NO: 61. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 61. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 61. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 61. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 61. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0124] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 62. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 62. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 39) having the amino acid sequence of CDR3 described in SEQ ID NO: 62. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 62. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 62. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 62. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 62. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0125] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 63. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 63. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 3 or 6) having the amino acid sequence of CDR3 described in SEQ ID NO: 63. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 63. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 63. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 63. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 63. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0126] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 183. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 183. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 167 or 170) having the amino acid sequence of CDR3 described in SEQ ID NO: 183. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 183. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 183. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 183. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 183. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0127] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 184. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 184. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 173 or 176) having the amino acid sequence of CDR3 described in SEQ ID NO: 184. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 184. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 184. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 184. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 184. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0128] In some embodiments, the single-domain antibody has CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 185. In some embodiments, the single-domain antibody has CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 185. In other embodiments, the single-domain antibody has CDR3 (e.g., SEQ ID NO: 179 or 182) having the amino acid sequence of CDR3 described in SEQ ID NO: 185. In some embodiments, the single-domain antibody has CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 185. In some embodiments, the single-domain antibody has CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 185. In some embodiments, the single-domain antibody has CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 185. In some embodiments, the single-domain antibody has CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NO: 185. The CDR sequences can be determined according to a well-known numbering system. In some embodiments, the CDR follows IMGT numbering. In some embodiments, the CDR follows Kabat numbering. In some embodiments, the CDR follows AbM numbering. In other embodiments, the CDR follows Chothia numbering. In other embodiments, the CDR follows Contact numbering. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0129] In some embodiments, single-domain antibodies that bind to CD20, comprising the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, are provided herein, (i) CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 195, SEQ ID NO: 4, SEQ ID NO: 11 or 196, SEQ ID NO: 13, SEQ ID NO: 14 or 197, SEQ ID NO: 17, SEQ ID NO: 20 or 197, SEQ ID NO: 22, SEQ ID NO: 24 or 198, SEQ ID NO: 27, SEQ ID NO: 165 or 199, SEQ ID NO: 168, SEQ ID NO: 171 or 200, SEQ ID NO: 174, SEQ ID NO: 177 or 197, SEQ ID NO: 180, (ii) CDR2 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: (iii) CDR3 comprises the amino acid sequence of SEQ ID NO. 15, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 166, SEQ ID NO. 169, SEQ ID NO. 172, SEQ ID NO. 175, SEQ ID NO. 178, or SEQ ID NO. 181, and / or (iii) CDR3 comprises the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 26, SEQ ID NO. 29, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 167, SEQ ID NO. 170, SEQ ID NO. 173, SEQ ID NO. 176, SEQ ID NO. 179, or SEQ ID NO. 182. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0130] In other embodiments, the anti-CD20 sdAb in the CAR comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where (i) CDR1 is sequence number 1 or 195, sequence number 4, sequence number 11 or 196, sequence number 13, sequence number 14 or 197, sequence number 17, sequence number 20 or 197, sequence number 22, sequence number 24 or 198, sequence number 27, sequence number 165 or 199, sequence number 168, sequence number 171 or 200, sequence number 174, sequence number 177 or (ii) CDR2 contains an amino acid sequence having sequence identity of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with sequence number 197, and (ii) CDR2 contains an amino acid sequence having sequence identity of at least 75%, 80%, 85%, 86%, 87%, 88%, 94%, 95%, 96%, 97%, 98%, or 99% with sequence number 180, and (ii) CDR2 contains an amino acid sequence having sequence identity of at least 75%, 80%, 85%, 86%, 87%, 88%, 94%, 95%, 96%, 97%, 98%, or 99% with sequence number 2, sequence number 5, sequence number 7, sequence number 9, sequence number 12, sequence number 15, sequence number 18, sequence number 21, sequence number 23, sequence number 25, sequence number 28, sequence number 30, sequence number 31, sequence number 32 , comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 166, SEQ ID NO: 169, SEQ ID NO: 172, SEQ ID NO: 175, SEQ ID NO: 178, or SEQ ID NO: 181, and / or (iii) CDR3 is sequence number The amino acid sequence contains at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 3, sequence number 6, sequence number 8, sequence number 10, sequence number 16, sequence number 19, sequence number 26, sequence number 29, sequence number 39, sequence number 40, sequence number 167, sequence number 170, sequence number 173, sequence number 176, sequence number 179, or sequence number 182. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized.In some embodiments, the anti-CD20 single-domain antibody includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0131] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 195, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0132] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0133] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and CDR3 comprising the amino acid sequence of SEQ ID NO: 8 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and CDR3 comprising the amino acid sequence of SEQ ID NO: 8 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 195, CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and CDR3 comprising the amino acid sequence of SEQ ID NO: 8 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0134] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0135] In some embodiments, provided herein is an anti-CD20 single-domain antibody (sdAb) comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 11 or 196, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, provided herein is an anti-CD20 single-domain antibody (sdAb) comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, provided herein is an anti-CD20 single-domain antibody (sdAb) comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 196, a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0136] In some embodiments, provided herein is an anti-CD20 single-domain antibody (sdAb) comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0137] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 14 or 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 14, CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0138] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 17, CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and CDR3 comprising the amino acid sequence of SEQ ID NO: 19 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0139] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0140] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 22, CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR3 comprising the amino acid sequence of SEQ ID NO: 19 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0141] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 24 or 198, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 24, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 198, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0142] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR3 comprising the amino acid sequence of SEQ ID NO: 29 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0143] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 14 or 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 14, CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0144] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 17, CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR3 comprising the amino acid sequence of SEQ ID NO: 19 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0145] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 195, CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0146] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0147] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0148] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 22, CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR3 comprising the amino acid sequence of SEQ ID NO: 19 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0149] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0150] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0151] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0152] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0153] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR3 comprising the amino acid sequence of SEQ ID NO: 40 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0154] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 165 or 199, CDR2 comprising the amino acid sequence of SEQ ID NO: 166, and CDR3 comprising the amino acid sequence of SEQ ID NO: 167 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 165, CDR2 comprising the amino acid sequence of SEQ ID NO: 166, and CDR3 comprising the amino acid sequence of SEQ ID NO: 167 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 199, CDR2 comprising the amino acid sequence of SEQ ID NO: 166, and CDR3 comprising the amino acid sequence of SEQ ID NO: 167 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0155] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 168, CDR2 comprising the amino acid sequence of SEQ ID NO: 169, and CDR3 comprising the amino acid sequence of SEQ ID NO: 170 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0156] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 171 or 200, CDR2 comprising the amino acid sequence of SEQ ID NO: 172, and CDR3 comprising the amino acid sequence of SEQ ID NO: 173 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 171, CDR2 comprising the amino acid sequence of SEQ ID NO: 172, and CDR3 comprising the amino acid sequence of SEQ ID NO: 173 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 200, CDR2 comprising the amino acid sequence of SEQ ID NO: 172, and CDR3 comprising the amino acid sequence of SEQ ID NO: 173 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0157] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 174, CDR2 comprising the amino acid sequence of SEQ ID NO: 175, and CDR3 comprising the amino acid sequence of SEQ ID NO: 176 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0158] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 177 or 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 178, and CDR3 comprising the amino acid sequence of SEQ ID NO: 179 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 177, CDR2 comprising the amino acid sequence of SEQ ID NO: 178, and CDR3 comprising the amino acid sequence of SEQ ID NO: 179 are provided herein. In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 197, CDR2 comprising the amino acid sequence of SEQ ID NO: 178, and CDR3 comprising the amino acid sequence of SEQ ID NO: 179 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0159] In some embodiments, anti-CD20 single-domain antibodies (sdAbs) comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 180, CDR2 comprising the amino acid sequence of SEQ ID NO: 181, and CDR3 comprising the amino acid sequence of SEQ ID NO: 182 are provided herein. In some embodiments, the anti-CD20 single-domain antibody is a camelid. In some embodiments, the anti-CD20 single-domain antibody is humanized. In some embodiments, the anti-CD20 single-domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0160] In some embodiments, the single-domain antibody further comprises one or more framework regions from among VHH-273, VHH-283, VHH-313, VHH-440, VHH-466, VHH-496, VHH-653, huVHH-253, huVHH-256, huVHH-260, huVHH-746, huVHH-750, huVHH-753, huVHH-836, huVHH-840, huVHH-843, huVHH-846, 2082H1, 2082H2, 2082H3, 2082H4, 2082H5, 2082H6, VHH-623, VHH-640, and / or VHH-657. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 41. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 42. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 43. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 44. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 45. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 46. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 47. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 48. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 49. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 50.In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 51. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 52. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 53. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 54. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 55. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 56. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 57. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 58. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 59. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 60. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 61. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 62. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 63. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 183.In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 184. In some embodiments, the single-domain antibody comprises one or more frameworks derived from a VHH domain containing the sequence of SEQ ID NO: 185.

[0161] In some embodiments, the single-domain antibodies provided herein are humanized single-domain antibodies. In some embodiments, humanized single-domain antibodies can be produced using the methods illustrated in Section 6 below or the methods described in the following sections.

[0162] The framework region described herein is determined based on the boundaries of the CDR numbering system. In other words, if the CDR is determined by, for example, Kabat, IMGT, or Chothia, the framework region is the amino acid residues surrounding the CDR within a variable region of the form N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the N-terminal amino acid residue of the CDR1 amino acid residue as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residue between the CDR1 and CDR2 amino acid residues as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residue between the CDR2 and CDR3 amino acid residues as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the C-terminal amino acid residue of the CDR3 amino acid residue as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0163] In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 41. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 42. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 42 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 43. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 43 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 44. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 44 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 45. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 45 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 46. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 46 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO: 47 is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 47 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO: 48 is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 48 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO: 49 is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 49 is provided.In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 50. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 50 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 51. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 51 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 52. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 52 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 53. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 53 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 54. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 54 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 55. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 55 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO: 56 is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 56 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO: 57 is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 57 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO: 58 is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 58 is provided.In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 59. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 59 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 60. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 60 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 61. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 61 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 62. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 62 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 63. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 63 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 183. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 183 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO: 184 is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 184 is provided. In some embodiments, an isolated anti-CD20 single-domain antibody comprising a VHH domain having the amino acid sequence of SEQ ID NO: 185 is provided. In some embodiments, a polypeptide comprising the amino acid sequence of SEQ ID NO: 185 is provided.

[0164] In certain embodiments, the antibodies or antigen-binding fragments described herein contain amino acid sequences having a specific percentage identity with any one of the antibodies VHH-273, VHH-283, VHH-313, VHH-440, VHH-466, VHH-496, VHH-653, huVHH-253, huVHH-256, huVHH-260, huVHH-746, huVHH-750, huVHH-753, huVHH-836, huVHH-840, huVHH-843, huVHH-846, 2082H1, 2082H2, 2082H3, 2082H4, 2082H5, 2082H6, VHH-623, VHH-640, and VHH-657.

[0165] The determination of percentage identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using mathematical algorithms. A non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), which is modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, a BLAST nucleotide search can be performed with NBLAST nucleotide program parameters set, for example, score = 100 and word length = 12. To obtain amino acid sequences homologous to the protein molecules described herein, a BLAST protein search can be performed using XBLAST program parameters, for example, set to a score of 50 and a word length of 3. To obtain gap alignments for comparison purposes, the gap BLAST described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997) can be used. Alternatively, PSI BLAST can be used to perform iterative searches to detect distant relationships between molecules (ibid.). When using the BLAST, gap BLAST, and PSI Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Percentage identity between two sequences can be determined using similar methods as described above, with or without gaps. In percentage identity calculations, only exact matches are typically counted.

[0166] In some embodiments, an anti-CD20 single-domain antibody is provided comprising a VHH domain having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NOs. In some embodiments, the VHH sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to a reference sequence, but the anti-CD20 single-domain antibody comprising that sequence retains the ability to bind to CD20. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in an amino acid sequence selected from SEQ ID NOs. 41-63 and 183-185. In some embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., the FR). Optionally, an anti-CD20 single-domain antibody contains an amino acid sequence (including post-translational modifications of that sequence) selected from SEQ ID NOs. 41-63 and 183-185.

[0167] In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 41, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 42, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 43, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 44, and the single-domain antibody binds to CD20.In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 45, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 46, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 47, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 48, and the single-domain antibody binds to CD20.In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 49, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 50, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 51, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 52, and the single-domain antibody binds to CD20.In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 53, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 54, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 55, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 56, and the single-domain antibody binds to CD20.In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 57, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 58, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 59, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 60, and the single-domain antibody binds to CD20.In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 61, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 62, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 63, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 183, and the single-domain antibody binds to CD20.In certain embodiments, the single-domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 184, and the single-domain antibody binds to CD20. In certain embodiments, the single-domain antibody described herein comprises at least 75%, at least 80%, and at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 185. A single-domain antibody contains a VHH domain having sequence identity of %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and binds to CD20.

[0168] In some embodiments, functional epitopes can be mapped, for example, by combinatorial alanine scanning, to identify amino acids in the CD20 protein necessary for interaction with anti-CD20 single-domain antibodies provided herein. In some embodiments, the three-dimensional and crystalline structures of anti-CD20 single-domain antibodies bound to CD20 may be used to identify epitopes. In some embodiments, the disclosure provides antibodies that specifically bind to the same epitopes as any of the anti-CD20 single-domain antibodies provided herein. For example, in some embodiments, antibodies are provided that bind to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 41. In some embodiments, antibodies are provided that bind to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, antibodies are provided that bind to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 43. In some embodiments, antibodies are provided that bind to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 44. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 45. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 46. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 47. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 49. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 50. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 51.In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 52. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 53. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 54. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 55. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 56. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 57. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 58. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 59. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 60. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 61. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 62. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 63. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 183. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 184. In some embodiments, an antibody is provided that binds to the same epitope as an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 185.

[0169] In some embodiments, anti-CD20 antibodies or antigen-binding fragments thereof that specifically bind to CD20 in competition with any of the anti-CD20 single-domain antibodies described herein are provided. In some embodiments, competitive binding may be determined using an ELISA assay. For example, in some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 47. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 49. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 50. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 51. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 52.In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 53. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 54. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 55. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 56. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 57. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 58. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 59. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 60. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 61. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 62. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 63. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 183. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 184. In some embodiments, an antibody is provided that specifically binds to CD20 in competition with an anti-CD20 single-domain antibody containing the amino acid sequence of SEQ ID NO: 185.

[0170] In some embodiments, CD20-binding proteins comprising any one of the anti-CD20 single-domain antibodies described herein are provided. In some embodiments, the CD20-binding protein is a monoclonal antibody comprising a camelid, chimeric, humanized, or human antibody. In some embodiments, the anti-CD20 antibody is an antibody fragment, e.g., a VHH fragment. In some embodiments, the anti-CD20 antibody is a full-length heavy-chain-only antibody containing an Fc region of any antibody class or isotype, such as IgG1 or IgG4. In some embodiments, the Fc region has reduced or minimized effector function. In some embodiments, the CD20-binding protein is a fusion protein comprising an anti-CD20 single-domain antibody provided herein. In other embodiments, the CD20-binding protein is a multispecific antibody comprising an anti-CD20 single-domain antibody provided herein. Other exemplary CD20-binding molecules are described in more detail in the following sections.

[0171] In some embodiments, the anti-CD20 antibody (e.g., anti-CD20 single-domain antibody) or antigen-binding protein according to any of the embodiments described above may incorporate any of the features, individually or in combination, as described in sections 5.2.2 to 5.2.7 below.

[0172] 5.2.2. Humanized single-domain antibodies The single-domain antibodies described herein include humanized single-domain antibodies. General strategies for humanizing single-domain antibodies derived from camelid species are described (see, for example, Vinckeetal, J. Biol. Chem., 284(5):3273-3284 (2009)) and may be useful for producing the humanized VHH domains disclosed herein. The design of humanized single-domain antibodies derived from camelid species may include characteristic VHH residues such as residues 11, 37, 44, 45, and 47 (residue numbering by Kabat) (Muyldermans, Reviews Mol Biotech 74:277-302 (2001)).

[0173] Humanized antibodies, such as the humanized single-domain antibodies disclosed herein, can also be prepared using various techniques known in the art, including, but not limited to, the following: CDR transplantation (European Patent No. EP239,400; International Publication No. WO91 / 09967; and U.S. Patents Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (European Patent Nos. EP592,106 and EP519,596; Padlan, Molecular Immunology 28(4 / 5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814(1994); and Roguska et al., PNAS 91:969-973 (1994), Chain shuffling (US Patent No. 5,565,332), and, for example, US Patent No. 6,407,213, US Patent No. 5,766,886, WO9317105, Tan et al., J.Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J.Biol.Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer The methods disclosed in Res.55(23 Supp):5973s-5977s(1995), Couto et al., Cancer Res.55(8):1717-22(1995), Sandhu JS, Gene 150(2):409-10(1994), and Pedersen et al., J.Mol.Biol.235(3):959-73(1994). See also U.S. Patent Publication No. 2005 / 0042664A1 (February 24, 2005) (each of these is incorporated herein by reference in whole).

[0174] In some embodiments, the single-domain antibodies provided herein may be humanized single-domain antibodies that bind to CD20, including human CD20. For example, the humanized single-chain antibodies of this disclosure may include one or more CDRs described in SEQ ID NOs. 41-63 and 183-185. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called “import” residues, which are usually obtained from an “import” variable domain. Humanization may be carried out, for example, by substituting a hypervariable region sequence with the corresponding sequence of a human antibody, according to the methods of Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); and Verhoeyen et al., Science 239:1534-36 (1988). In certain embodiments, the humanization of the single-domain antibody provided herein is carried out as described in Section 6 below.

[0175] In some cases, humanized antibodies are constructed using CDR transplantation, where the amino acid sequence of the CDR of the parent non-human antibody is transplanted into the human antibody framework. For example, Padlan et al. determined that only about one-third of the residues in the CDR actually contact the antigen, and these are called "specificity-determining residues" or SDRs (Padlan et al., FASEB J.9:133-39 (1995)). In the SDR transplantation method, only the SDR residues are transplanted into the human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34 (2005)).

[0176] The selection of human variable domains used in the production of humanized antibodies can be crucial for reducing antigenicity. For example, following a so-called "best-fit" method, the sequences of variable domains from non-human antibodies are screened against an entire library of known human variable domain sequences. The sequence that most closely resembles the human sequence of the non-human antibody may be selected as the human framework for the humanized antibody (Sims et al., J.Immunol.151:2296-308 (1993); and Chothia et al., J.Mol.Biol.196:901-17 (1987)). Another method involves using a specific framework derived from the consensus sequences of all human antibodies in a particular subgroup of the light or heavy chain. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); and Presta et al., J. Immunol. 151:2623-32 (1993)). In some cases, the framework is the most abundant human subclass V L 6 Subgroup I(V L 6I), and V H Subgroup III (V H It originates from the consensus sequence in (III). Alternatively, human germline genes are used as a source of framework regions.

[0177] In another paradigm based on CDR comparison, called hyperhumanization, FR homology is irrelevant. The method involves comparing non-human sequences with a functional human germline gene repertoire. Next, genes encoding canonical structures identical to or closely related to the mouse sequence are selected. Then, among the genes sharing a canonical structure with a non-human antibody, the gene with the highest homology within the CDR is selected as the FR donor. Finally, non-human CDRs are transplanted into these FRs (see, e.g., Tan et al., J.Immunol. 169:1119-25 (2002)).

[0178] Furthermore, it is generally desirable that antibodies be humanized while retaining affinity for the antigen and other desirable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by an analytical process of the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the expected three-dimensional conformational structure of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-24 (2002)), Modeller (Sali and Blundell, J. Mol. Biol. 234:779-815 (1993)), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-23 (1997)). By investigating these indications, it becomes possible to analyze the potential roles of residues in the function of candidate immunoglobulin sequences, for example, the residues that influence the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from recipient and transfer sequences to achieve desired antibody properties, such as increased affinity for the target antigen(s). Generally, hypervariable region residues are directly and most substantially involved in the influence of antigen binding.

[0179] Another method for humanizing antibodies is based on a metric called Human String Content (HSC), which measures the humanity of an antibody. This method compares a mouse sequence to a repertoire of human germline genes, and the differences are scored as HSC. The target sequence is then humanized by maximizing the HSC, instead of using a global identity scale to generate multiple diverse humanized variants (Lazar et al., Mol.Immunol. 44:1986-98 (2007)).

[0180] In addition to the methods described above, experimental methods may be used to generate and select humanized antibodies. These methods include those based on the generation and enrichment of large libraries of humanized variants or the selection of the best clones using high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as by bacterial colony screening (see, for example: Hoogenboom, Nat. Biotechnol. 23:1105-16 (2005); Dufner et al., Trends Biotechnol. 24:523-29 (2006); Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003); and Schlapschy et al., Protein Eng. Des. Sel. 17:847-60 (2004)).

[0181] In the FR library approach, a collection of residue variants is introduced at specific positions in the FR, and then the library is screened to select the FR that best supports the transplanted CDR. The residues to be substituted may include some or all of the "Vernier" residues that have been identified as potentially contributing to the CDR structure (see, for example, Foote and Winter, J.Mol.Biol.224:487-99 (1992)), or those derived from a more restricted set of target residues identified by Baca et al. J.Biol.Chem.272:10678-84 (1997).

[0182] In FR shuffling, instead of creating a combinatorial library of selected residue variants, the entire FR is combined with a non-human CDR (see, e.g., Dall'Acqua et al., Methods 36:43-60 (2005)). A one-step FR shuffling process may also be used. Such a process has been shown to be efficient, as the resulting antibodies exhibited improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol.Immunol. 44:3049-60 (2007)).

[0183] The "human ringing" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and involves sequentially substituting non-human fragments into a library of human FRs and evaluating their binding. This methodology typically results in the epitope retention and identification of antibodies from multiple subclasses with different human V-segment CDRs.

[0184] The “human engineering” method involves modifying non-human antibodies or antibody fragments by making specific changes to the amino acid sequence of an antibody in order to produce a modified antibody with reduced human immunogenicity (while nevertheless retaining its desirable binding properties as the original non-human antibody). Generally, this method involves classifying amino acid residues of non-human antibodies as “low-risk,” “medium-risk,” or “high-risk” residues. The classification is made using an overall risk / reward calculation that assesses the expected benefit of making a specific substitution (e.g., human immunogenicity) against the risk that the substitution will affect the resulting antibody folding. By aligning the amino acid sequence derived from the variable region of the non-human antibody with the corresponding region of a specific or consensus human antibody sequence, specific human amino acid residues to be substituted at a given position in the non-human antibody sequence (e.g., low-risk or medium-risk) can be selected. According to the alignment, amino acid residues at low-risk or medium-risk positions in the non-human sequence can be substituted with corresponding residues in the human antibody sequence. The methods for producing human-generated proteins are described in detail in Studnicka et al. Protein Engineering 7:805-14 (1994); U.S. Patents No. 5,766,886; No. 5,770,196; No. 5,821,123; and No. 5,869,619; and PCT Publication WO93 / 11794.

[0185] For example, composite human antibodies can be generated using Composite Human Antibody (trademark) technology (Antitope Ltd., Cambridge, United Kingdom). To generate composite human antibodies, the variable region sequence is designed from fragments of multiple human antibody variable region sequences in a way that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.

[0186] Deimmunized antibodies are antibodies from which T cell epitopes have been removed. Methods for producing deimmunized antibodies are described. See, for example, Jones et al., Methods Mol Biol. 525:405-23 (2009), xiv, and De Groot et al., Cell. Immunol. 244:148-153 (2006). Deimmunized antibodies contain a T cell epitope depletion variable region and a human constant region. In summary, the variable region of an antibody is cloned, and then the T cell epitopes are identified by testing for duplicate peptides derived from the antibody's variable region in a T cell proliferation assay. The T cell epitopes are identified by an in silico method that identifies peptides that bind to human MHC class II. Mutations are introduced into the variable region to inactivate binding to human MHC class II. The mutated variable region is then used to produce deimmunized antibodies.

[0187] 5.2.3. Single-domain antibody variants In some embodiments, amino acid sequence modifications (may be multiple) of the CD20-binding single-domain antibodies described herein are intended. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including, but not limited to, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Accordingly, it is intended that, in addition to the CD20-binding single-domain antibodies described herein, variants of the CD20-binding single-domain antibodies described herein can be prepared. For example, single-domain antibody variants can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art will recognize that amino acid changes can alter the post-translational processes of single-domain antibodies.

[0188] chemical modification In some embodiments, the single-domain antibodies provided herein are chemically modified, for example, by covalent bonding of any type of molecule to the single-domain antibody. Antibody derivatives may include antibodies chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., Fc or a portion of Fc). Any of the numerous chemical modifications can be carried out by known methods, including, but not limited to, specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. Furthermore, the antibody may contain one or more non-classical amino acids.

[0189] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0190] When a single-domain antibody provided herein is fused to an Fc region, the carbohydrate bound to it may be modified. Natural antibodies produced by mammalian cells typically contain branched or bibranched oligosaccharides commonly bound by an N-link to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of a bibranched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the binding molecule provided herein may be made to create variants having specific improved properties.

[0191] In other embodiments, if the single-domain antibody provided herein is fused to an Fc region, the antibody variant provided herein may have a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, the amount of fucose was determined by calculating the average amount of fucose in the glycans of Asn297 relative to the total amount of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297 (measured by MALDI-TOF mass spectrometry), as described in WO2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 (EU numbering of Fc region residues) within the Fc region. However, due to slight sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may improve ADCC function. See, for example, U.S. Patent Publications US2003 / 0157108 and US2004 / 0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108;WO2000 / 61739;WO2001 / 29246;US2003 / 0115614;US2002 / 0164328;US2004 / 0093621;US2004 / 01321 40;US2004 / 0110704;US2004 / 0110282;US2004 / 0109865;WO2003 / 085119;WO2003 / 084570;WO2005 / 035586;WO2005 / 035778;WO2005 / 053742;WO2002 / 031140;Okazaki et al.J.Mol.Biol.336:1239-1249(2004);Yamane-Ohnuki et al.Biotech.Bioeng.87:614(2004) are cited.Examples of cell lines capable of producing defucosylated antibodies include: Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108; and WO2004 / 056312, in particular, Example 11), as well as knockout cell lines, e.g., α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, for example: Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0192] The conjugated molecules containing single-domain antibodies provided herein are also provided together with bisected oligosaccharides, for example, a bisected oligosaccharide bound to the Fc region being bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and U.S.2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such variants may have improved CDC function. Such variants are described, for example, in WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0193] In this single-domain antibody and molecule containing an Fc region, one or more amino acid modifications can be introduced into the Fc region, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0194] In some embodiments, this application examines variants that possess some, but not all, effector functions, thereby making them desirable candidates for applications where the half-life of the binding molecule in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the binding molecule lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are listed below: U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, for example, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, for example: ACTI® Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc., Madison, Wisconsin) and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wisconsin)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, for example, as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity.For example, see: C1q and C3c binding ELISAs WO2006 / 029879 and WO2005 / 100402. CDC assays can be performed to assess complement activation (e.g., see: Gazzano-Santoro et al., J.Immunol.Methods 202:163(1996); Cragg, MS et al., Blood 101:1045-1052(2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance / half-life can also be determined using methods known in the art (e.g., see: Petkova, S B et al., Int'l.Immunol.18(12):1759-1769(2006)).

[0195] Binding molecules with reduced effector function include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants having two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant having alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581).

[0196] Specific variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0197] In some embodiments, the variant includes an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region. In some embodiments, modifications are made in the Fc region that result in alterations (i.e., improvements or reductions) to C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642 and Idusogie et al. J.Immunol. 164:4178-4184 (2000).

[0198] Binding molecules with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which are involved in the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These molecules contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Examples of such Fc variants include substitutions of one or more Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, those having a substitution of Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see Duncan & Winter, Nature 322:738-40 (1988); U.S. Patents No. 5,648,260, U.S. Patent No. 5,624,821, and WO94 / 29351.

[0199] In some embodiments, it may be desirable to produce a cysteine-modified antibody, in which one or more residues of the antibody are replaced with cysteine ​​residues. In some embodiments, the substituted residues are located in an accessible site of the antibody. Furthermore, as described herein, these residues may be replaced with cysteine ​​to create an immunoconjugate, thereby placing a reactive thiol group in an accessible site of the antibody, which may be used to conjugate the antibody to other parts, such as a drug part or a linker-drug part.

[0200] Replacement, deletion, or insertion The change may be a substitution, deletion, or insertion of one or more codons encoding a single-domain antibody or polypeptide, resulting in a change in the amino acid sequence compared to the original antibody or polypeptide. The target sites for substitutional mutagenesis include CDR and FR.

[0201] Amino acid substitutions may result from replacing one amino acid with another amino acid having similar structural and / or chemical properties, for example, replacing leucine with serine, or, for example, a conservative amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertions or deletions may optionally range from about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions include, compared to the original molecule, substitutions of less than 25, less than 20, less than 15, less than 10, less than 5, less than 4, less than 3, or less than 2 amino acids. In certain embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Acceptable changes may be determined by systematically performing insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity indicated by a parent antibody.

[0202] Amino acid sequence insertions include the fusion of amino and / or carboxyl termini ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequences of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue.

[0203] Single-domain antibodies produced by conservative amino acid substitutions are included in this disclosure. In conservative amino acid substitutions, an amino acid residue is substituted with an amino acid residue having a side chain having a similar charge. As described above, families of amino acid residues having side chains having a similar charge are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example, by saturated mutagenesis, and the biological activity can be screened to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and its activity can be determined. Conservative substitutions (e.g., within amino acid groups with similar properties and / or side chains) may be performed to maintain or significantly alter the properties. Exemplary substitutions are shown in Table 3 below.

[0204] (Table 3) Amino Acid Substitutions TIFF0007846667000004.tif91165

[0205] Amino acids may be grouped according to the similarity of their side chain properties (see, for example, Lehninger, Biochemistry 73-75 (2nd ed. 1975)): (1) Nonpolar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M); (2) Non-charged: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q); (3) Acidic: Asp(D), Glu(E); and (4) Basic: Lys(K), Arg(R), His(H). Alternatively, naturally occurring residues may be divided into the following groups based on common side-chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. For example, any cysteine ​​residue not involved in maintaining the proper conformation of a single-domain antibody may also be substituted with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Non-conservative substitutions require replacing a member of one of these classes with another.

[0206] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) and / or substantially retain specific biological properties of the parent antibody compared to the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which may be readily generated using, for example, a phage display-based affinity-mature technique as described herein. In summary, one or more CDR residues are mutated, the variant antibody is presented on a phage, and screened for specific biological activity (e.g., binding affinity).

[0207] For example, modifications (e.g., substitutions) may be made in the CDR to improve antibody affinity. Such modifications may be made in the CDR "hotspots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDR (α-CDR), and the resulting variant antibody or fragment is tested for binding affinity. Affinity maturation by secondary library construction and re-selection is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). A secondary library is then constructed. Next, the library is screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves a CDR-specific approach in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling. A more detailed explanation of affinity maturation is provided in the following sections.

[0208] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made in the CDRs. In some embodiments of the variant VHH sequences provided herein, each CDR is either unchanged or contains one, two, or three or fewer amino acid substitutions.

[0209] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described in Cunningham and Wells, Science, 244:1081-1085 (1989). In this method, target residues or groups (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) are identified and substituted with charged amino acids (e.g., alanine or polyalanine) to determine whether they are neutral or whether the interaction of the antigen with the antibody is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be used to identify contact sites between the antibody and antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0210] Amino acid sequence insertions include the fusion of amino and / or carboxyl terminals ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of the antibody to the N-terminus or C-terminus of the antibody.

[0211] Changes can be introduced using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. To generate single-domain antibody variant DNA, site-directed mutagenesis (see, e.g., Carter, Biochem J.237:1-7 (1986); and Zoller et al., Nucl. Acids Res.10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques can be applied to cloned DNA.

[0212] 5.2.4. In vitro affinity maturation In some embodiments, antibody variants with improved properties such as affinity, stability, or expression level compared to the parent antibody may be prepared by in vitro affinity maturation. Similar to natural prototypes, in vitro affinity maturation is based on the principles of mutation and selection. A library of antibodies is presented on the surface of an organism (e.g., a phage, bacterium, yeast, or mammalian cell) or associated (e.g., covalently or noncovalently) with their encoding mRNA or DNA. Affinity selection of the displayed antibodies allows for the separation of the organism or complex carrying the genetic information encoding the antibody. Two or three mutations and selections using display methods such as phage display typically result in antibody fragments with affinities in the low nanomolar range. Affinity-matured antibodies may have nanomolar or even picomolar affinities to a target antigen.

[0213] Phage display is a broad method for displaying and selecting antibodies. Antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions to bacteriophage coat proteins. Selection involves exposure to an antigen to allow the antibody presented on the phage to bind to its target; this process is called "panning." For further selection rounds, phages bound to the antigen are recovered and used to infect bacteria to obtain phages. For an overview, see, for example, Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002); and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0214] In yeast display systems (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997); and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies may be fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond to Aga1p. Aga2p-mediated protein display causes the protein to protrude from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen libraries and select antibodies with improved affinity or stability. Binding to the soluble antigen of interest is determined by labeling the yeast with a secondary reagent conjugated with a biotinylated antigen and a fluorescent, e.g., streptavidin. Changes in antibody surface expression can be measured by immunofluorescence labeling of either a hemagglutinin or c-Myc epitope tag adjacent to a single-chain antibody (e.g., scFv). Expression has been shown to correlate with the stability of the presented protein, thereby allowing for antibody selection to improve stability and affinity (see, e.g., Shusta et al., J.Mol.Biol.292:949-56(1999)). An additional advantage of yeast display is that the presented protein folds in the endoplasmic reticulum of eukaryotic yeast cells, utilizing endoplasmic reticulum chaperones and quality control mechanisms. Once maturation is complete, antibody affinity can be easily "titrated" while the protein is presented on the yeast surface, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface presentation is that the functional library size may be smaller than that of other presentation methods, although recent approaches have used the yeast cell mating system to achieve a size of 10 14 Combinatorial diversity is presumed to arise (see, for example, U.S. Patent Publication 2003 / 0186374; and Blaise et al., Gene 342:211-18 (2004)).

[0215] In ribosome display, an antibody-ribosome-mRNA (ARM) complex is generated for selection in a cell-free system. A DNA library encoding a specific antibody library is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, still binds to peptidyl-tRNA, occupying a ribosomal tunnel, thereby allowing the target protein to protrude from the ribosome and fold. The resulting mRNA-ribosome-protein complex can bind to a surface-bound ligand, allowing for simultaneous isolation of the antibody and its encoding mRNA by ligand-assisted affinity capture. The ribosome-bound mRNA is then reverse-transcribed back to cDNA, subsequently undergoing mutational induction and usable in the next selection round (see, e.g., Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, puromycin is used as an adapter molecule to establish covalent bonding between the antibody and mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0216] These methods, being entirely in vitro, offer two main advantages over other selection techniques. First, library diversity is limited only by the number of ribosomes and different mRNA molecules present in the test tube, rather than by the transformation efficiency of bacterial cells. Second, because there is no need to transform the library after any diversification step, random mutations can be easily introduced after each selection round, for example, by using an uncorrected polymerase.

[0217] In some embodiments, a mammalian display system may be used.

[0218] Diversity can also be introduced into the CDRs of an antibody library, either in a targeted manner or through random introduction. The former approach includes sequentially targeting all CDRs of an antibody through high or low levels of mutagenesis, or targeting isolated hotspots of somatic hypermutation (e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)) or residues suspected to affect affinity for experimental or structural reasons. Diversity can also be introduced by substitution of naturally diverse regions via DNA shuffling or similar techniques (e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003); see U.S. Patents 5,565,332 and 6,989,250). Alternative approaches target hypervariable loops extending into framework region residues (see, e.g., Bond et al., J.Mol.Biol.348:699-709 (2005)), utilize loop deletions and insertions within the CDR, or employ hybridization-based diversity (see, e.g., U.S. Patent Publication 2004 / 0005709). Additional methods for generating diversity in the CDR are disclosed, for example, in U.S. Patent No. 7,985,840. Further methods that can be used to generate antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Patents 8,685,897 and 8,603,930, and U.S. Publications 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0219] Library screening can be achieved by various techniques known in the art. For example, single-domain antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed on host cells immobilized on adsorption plates, used for cell sorting, conjugated to biotin for capture using streptavidin-coated beads, or used in any other way for panning display libraries.

[0220] For an overview of in vitro affinity maturation methods, see, for example, Hoogenboom, Nature Biotechnology 23:1105-16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010); and the references within them.

[0221] 5.2.5. Modification of single-domain antibodies Covalent modifications of single-domain antibodies are included within the scope of this disclosure. Covalent modifications include reacting target amino acid residues of a single-domain antibody with an organic derivatizer. This organic derivatizer can react with selected side chains or N- or C-terminal residues of a single-domain antibody. Other modifications include deamidation of glutamyl and asparaginyl residues to their corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0222] Other types of covalent modifications of single-domain antibodies included within the scope of this disclosure include: alteration of the natural glycosylation pattern of the antibodies or polypeptides described above (see, for example, Beck et al., Curr. Pharm. Biotechnol. 9:482-501 (2008); and Walsh, Drug Discov. Today 15:773-80 (2010)); and conjugating antibodies to various non-proteinoid polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene, by methods described in, for example, U.S. Patents No. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. The CD20-binding single-domain antibodies of this disclosure may also be genetically fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) to extend their half-lives and / or confer known Fc-mediated effector functions.

[0223] The CD20-binding single-chain antibodies of this disclosure may also be modified to form chimeric molecules containing the CD20-binding single-chain antibody fused to: another heterologous polypeptide or amino acid sequence, e.g., an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)). The CD20-binding single-chain antibodies may also be used to obtain CD20-binding chimeric antigen receptors (CARs), as described in more detail below.

[0224] Furthermore, fusion proteins comprising single-chain antibodies and heterologous polypeptides that bind to CD20 are also provided herein. In some embodiments, heterologous polypeptides to which antibodies are genetically fused or chemically conjugated are useful for targeting antibodies to cells having cell surface-expressed CD20.

[0225] A panel of antibodies that bind to the CD20 antigen is also provided herein. In certain embodiments, the antibody panel has different association rates, different dissociation rates, different affinities to the CD20 antigen, and / or different specificities to the CD20 antigen. In some embodiments, the panel contains or consists of about 10 to about 1000 or more antibodies. The antibody panel can be used in assays such as ELISA, for example, in 96-well or 384-well plates.

[0226] 5.2.6. Preparation of single-domain antibodies Methods for preparing single-domain antibodies are described. See, for example, Els Pardon et al, Nature Protocol, 9(3):674 (2014). Single-domain antibodies (e.g., VHHs) can be obtained by using methods known in the art, for example, by immunizing camelid species (e.g., camels or llamas) and obtaining hybridomas therefrom, or by cloning a library of single-domain antibodies using molecular biological techniques known in the art, followed by selection using ELISA or phage display with individual clones of the unselected library.

[0227] The single-domain antibodies provided herein may be produced by culturing cells transformed or transfected with a vector containing the nucleic acid encoding the single-domain antibody. The polynucleotide sequence encoding the polypeptide component of the antibodies disclosed herein can be obtained using standard recombinant techniques. The desired polynucleotide sequence may be isolated and sequenced from antibody-producing cells such as hybridoma cells or B cells. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR technique. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells. Many vectors available and known in the art can be used for the purposes of this disclosure. The selection of a suitable vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Host cells suitable for expressing the antibodies disclosed herein include prokaryotes such as archaea and bacteria, including Gram-negative or Gram-positive organisms; eukaryotic microorganisms such as filamentous fungi or yeasts; invertebrate cells such as insect or plant cells; and vertebrate cells such as mammalian host cell lines. Host cells are transformed with the expression vector described above and cultured in a conventional nutrient medium modified to be suitable for promoter induction, transformant selection, or amplification of the gene encoding the desired sequence. Antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0228] Methods for antibody production, including vector construction, expression, and purification, are further described in Pluckthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (McCafferty et al. eds., 1996); Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, in Current Protocols in Protein Science (2009); Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, in Antibody Expression and Production (Al-Rubeai ed., 2011); and Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed., 2009).

[0229] It is naturally assumed that other methods well known in the art may be used to prepare anti-CD20 single-domain antibodies. For example, a suitable amino acid sequence or part thereof may be produced by direct peptide synthesis using solid-phase techniques (see, for example, Stewart et al., Solid-Phase Peptide Synthesis (1969); and Merrifield, J.Am.Chem.Soc.85:2149-54 (1963)). In vitro protein synthesis may be carried out using manual or automated methods. To obtain the desired anti-CD20 antibody, various parts of the anti-CD20 antibody may be chemically synthesized separately and combined using chemical or enzymatic methods. Alternatively, as described, for example, in U.S. Patents 5,545,807 and 5,827,690, the antibody may be purified from cells or bodily fluids such as milk of a transgenic animal engineered to express the antibody.

[0230] Specifically, single-domain antibodies or other CD20 conjugates provided herein can be generated by immunizing llamas, performing single B-cell sorting, extracting the V gene, cloning CD20 conjugates such as VHH-Fc fusions, and then performing small-scale expression and purification. ELISA-positive, BLI-positive, and K < 100 nM D Additional screening of single-domain antibodies and other molecules that bind to CD20 can be performed, including one or more of the selection criteria. These selection criteria can be combined, as described in Section 6 below. Furthermore, individual VHH conjugates (and other molecules that bind to CD20) can be assayed for their ability to bind to cells expressing CD20. Such assays can be performed by measuring the mean fluorescence intensity (MFI) of fluorescently labeled VHH molecules using FACS analysis with cells expressing CD20. The various aspects described above are described in more detail below.

[0231] Polyclonal antibodies Polyclonal antibodies are generally produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the associated antigen and adjuvant. Difunctional or derivatizing agents, such as maleimide-benzoylsulfosuccinimide (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR(where R and R 1 It may be useful to conjugate an antigen associated with an immunogenic protein in the species to be immunized, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine cytoglobulin, or soy trypsin inhibitors, using an independent lower alkyl group. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvants (monophosphoryl lipid A, synthetic trehalose dicolinomicolate). Immunization protocols can be selected by those skilled in the art without excessive experimentation.

[0232] For example, animals are immunized with an antigen, immunogenic conjugate, or derivative by intradermal injection of a solution containing 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) in three volumes of Freund's complete adjuvant at multiple sites. After one month, the animals are boost-immunized with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, blood is collected from the animals and the serum is assayed for antibody titer. The animals are boost-immunized until the titer stabilizes. Conjugates can also be prepared as protein fusions in recombinant cell culture. Agglutinants such as alum are also suitable for enhancing the immune response.

[0233] Monoclonal antibodies Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or trace amounts of post-translational modifications (e.g., isomerization, amidation). Thus, the modifier "monoclonal" indicates an antibody characteristic that it is not a mixture of distinct antibodies.

[0234] For example, monoclonal antibodies may be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by the recombinant DNA method (U.S. Patent No. 4,816,567).

[0235] In the hybridoma method, a suitable host animal is immunized to produce antibodies that specifically bind to proteins used in immunity, or to induce lymphocytes capable of producing such antibodies. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0236] Immunotherapeutic agents will typically contain antigen proteins or fusion variants thereof. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103. Immortalized cell lines are typically transformed mammalian cells. Therefore, prepared hybridoma cells are seeded and grown in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. Preferred immortalized myeloma cells are those that fuse efficiently, support stable high levels of antibody production by selected antibody-producing cells, and are sensitive to media such as HAT medium.

[0237] The culture medium in which hybridoma cells are grown can be assayed for the production of monoclonal antibodies against an antigen. The culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against a desired antigen. Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980).

[0238] After hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, the clones may be subcloned using limiting dilution procedures and grown by standard methods (Goding, cited above). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can grow in vivo as tumors in mammals.

[0239] Monoclonal antibodies secreted by subclones can be appropriately isolated from culture media, ascites fluid, or serum using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0240] Monoclonal antibodies may also be synthesized by recombinant DNA methods, e.g., those described in U.S. Patent No. 4,816,567, and as described above. The DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells serve as a preferred source of such DNA. The isolated DNA may be placed in an expression vector and then transfected into host cells, e.g., Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, in order to synthesize monoclonal antibodies in such recombinant host cells. Reviews on the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Revs. 130:151-188 (1992).

[0241] In further embodiments, antibodies can be isolated from antibody phage libraries generated using the method described in McCafferty et al., Nature, 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J.Mol.Biol., 222:581-597 (1991). Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl.Acids Res., 21:2265-2266 (1993)). Therefore, these techniques are a practical alternative to conventional monoclonal antibody hybridoma methods for separating monoclonal antibodies.

[0242] Furthermore, DNA can be modified, for example, by substituting the coding sequence (U.S. Patent No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to the coding sequence. Such non-immunoglobulin polypeptides can be substituted to produce a chimeric bivalent antibody containing one antigen-binding site with specificity for a particular antigen and another antigen-binding site with specificity for a different antigen.

[0243] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutylimidates.

[0244] Recombination production in prokaryotic cells The polynucleotide sequences encoding the antibodies of this disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequences may be isolated and sequenced from antibody-producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizers or PCR techniques. Once obtained, the polypeptide-encoding sequences are inserted into recombinant vectors capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of this disclosure. The selection of a suitable vector will primarily depend on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains a variety of components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it exists. Vector components generally include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome-binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0245] Generally, plasmid vectors containing replicons and regulatory sequences derived from a species compatible with the host cell are used in association with these hosts. The vectors typically have replication sites and marking sequences that can provide phenotypic selection in transformed cells. For example, Escherichia coli is usually transformed using pBR322, a plasmid derived from the Escherichia coli species. Examples of pBR322 derivatives used for the expression of specific antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

[0246] In addition, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors for these hosts. For example, bacteriophages such as GEM(trademark)-11 may be used to create recombinant vectors that can be used to transform susceptible host cells such as Escherichia coli LE392.

[0247] The expression vector of this application may include two or more promoter-cistron pairs encoding each of the polypeptide components. The promoter is a non-translating regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. An inducible promoter is a promoter that, under its control, initiates an increased transcriptional level of cistron in response to changes in culture conditions, such as the presence or absence of nutrients or temperature changes.

[0248] Numerous promoters recognized by various potential host cells are well known. A selected promoter can be operably ligated to the cistron DNA encoding the antibody by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of this application. Both native promoter sequences and many heterologous promoters may be used to induce amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow for more transcription and higher yields of expressed target genes compared to native target polypeptide promoters.

[0249] Promoters suitable for use in prokaryotic hosts include the PhoA promoter, β-galactamase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as tac or trc promoters. However, other promoters that function in bacteria (e.g., other known bacterial promoters or phage promoters) are equally suitable. Their nucleic acid sequences are publicly available, so that those skilled in the art can ligate them into activatable cistrons encoding target peptides using linkers or adapters to provide any necessary restriction sites (Siebenlist et al. Cell 20:269 (1980)).

[0250] In one embodiment, each cistron in the recombinant vector contains a secretory signal sequence component that induces the transmembrane movement of the expressed polypeptide. Generally, the signal sequence may be a component of the vector or part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention must be recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and process the signal sequence specific to the heterologous polypeptide, the signal sequence may be replaced with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or a heat-stable enterotoxin II (STII) reader, LamB, PhoE, PelB, OmpA, and MBP.

[0251] In some embodiments, antibody production according to this disclosure may occur in the cytoplasm of the host cell and therefore does not require the presence of secretory signaling sequences within each cistron. - The company provides cytoplasmic conditions favorable for disulfide bond formation, thereby enabling proper folding and assembly of expressed protein subunits.

[0252] Suitable prokaryotic host cells for expressing the antibodies of this disclosure include archaea and bacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Enterobacteriaceae, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreosilla, or Paracoccus. Gram-negative cells are used in some embodiments. In one embodiment, Escherichia coli cells are used as the host. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol.2 (Washington, DC: American Society for Microbiology, 1987), pp.1190-1219; ATCC Deposit No.27, 325) and its derivatives (genotype W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompT A(nmpc-fepE)degP41 kan) R Examples include strain 33D3 (containing the specified genotype) (U.S. Patent No. 5,639,635). Other strains and their derivatives, such as Escherichia coli 294 (ATCC 31,446), Escherichia coli B, Escherichia coli 1776 (ATCC 31,537), and Escherichia coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having the specified genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, it is necessary to select an appropriate bacterium considering the replication potential of the replicon within the bacterial cell. When well-known plasmids, such as pBR322, pBR325, pACYC177, or pKN410, are used to supply the replicon, Escherichia coli, Serratia, or Salmonella species can be appropriately used as hosts.

[0253] Normally, host cells should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may, if desired, be incorporated into the cell culture.

[0254] Host cells are transformed with the expression vector described above and cultured in a conventional nutrient medium modified to suit promoter induction, transformant selection, or amplification of the gene encoding the desired sequence. Transformation means introducing DNA into a prokaryotic host so that the DNA can replicate, either as an extrachromosomal element or by chromosomal integrants. Depending on the host cell used, transformation is carried out using standard techniques suitable for such cells. Calcium treatment using calcium chloride is commonly used for bacterial cells that contain a substantial cell wall barrier. Another method for transformation uses polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0255] Prokaryotic cells used to produce the antibodies of this application were grown in a culture medium suitable for the art and for the culture of selected host cells. Examples of suitable media include Luria broth (LB) and necessary nutritional supplements. In some embodiments, the medium also contains a selectant selected based on the construction of the expression vector in order to selectively enable the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin resistance gene.

[0256] Other necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included in appropriate concentrations, either alone or in mixtures with other adjuvants or media, such as a complex nitrogen source. Optionally, the media may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at a suitable temperature and pH.

[0257] When an inducible promoter is used in the expression vector of this application, protein expression is induced under conditions suitable for promoter activation. In one aspect of this application, the PhoA promoter is used to control polypeptide transcription. Thus, transformed host cells are cultured in phosphate-restricted medium for induction. Preferably, the phosphate-restricted medium is CRAP medium (see, for example, Simmons et al., J.Immunol.Methods 263:133-147 (2002)). Various other inducing factors known in the art may be used, depending on the vector construct used.

[0258] The expressed antibodies in this disclosure are secreted into the periplasm of host cells and recovered therefrom. Protein recovery typically involves disrupting the microorganisms by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, the proteins can be transferred to a culture medium and isolated therein. The cells may be removed from the culture, the culture supernatant filtered, and concentrated for further purification of the produced proteins. The expressed polypeptides can be further separated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0259] Alternatively, protein production can be carried out on a large scale through fermentation processes. Various large-scale batch fermentation procedures are available for the production of recombinant proteins. Various fermentation conditions can be modified to improve the production yield and quality of the antibodies of this disclosure. For example, chaperone proteins have been demonstrated to promote the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. J Bio Chem 274:19601-19605 (1999); U.S. Patent No. 6,083,715; U.S. Patent No. 6,027,888; Bothmann and Pluckthun, J. Biol. Chem. 275:17100-17105 (2000); Ramm and Pluckthun, J. Biol. Chem. 275:17106-17113 (2000); Arie et al., Mol. Microbiol. 39:199-210 (2001).

[0260] For example, as described in U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996), specific host strains lacking proteolytic enzymes can be used in the present invention to minimize the proteolysis of expressed heterologous proteins (particularly those that are proteolytically sensitive). Escherichia coli strains lacking proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins may be used as host cells in the expression systems encoding the antibodies of this application.

[0261] To obtain substantially homogeneous preparations for further assays and use, the antibodies produced herein can be further purified. Standard protein purification methods known in the art can be used. The following procedure is an exa...

Claims

1. Anti-CD20 single-domain antibody (sdAb), (i) CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO: 3 (ii) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 5, and CDR3 containing the amino acid sequence of SEQ ID NO: 6 (iii) CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 containing the amino acid sequence of SEQ ID NO: 7, and CDR3 containing the amino acid sequence of SEQ ID NO:

8. (iv) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 9, and CDR3 containing the amino acid sequence of SEQ ID NO:

10. (v) CDR1 containing the amino acid sequence of SEQ ID NO: 11 or 196, CDR2 containing the amino acid sequence of SEQ ID NO: 12, and CDR3 containing the amino acid sequence of SEQ ID NO: 8 (vi) CDR1 containing the amino acid sequence of SEQ ID NO: 13, CDR2 containing the amino acid sequence of SEQ ID NO: 9, and CDR3 containing the amino acid sequence of SEQ ID NO:

10. (vii) CDR1 containing the amino acid sequence of SEQ ID NO: 14 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 15, and CDR3 containing the amino acid sequence of SEQ ID NO:

16. (viiii) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 18, and CDR3 containing the amino acid sequence of SEQ ID NO:

19. (ix) CDR1 containing the amino acid sequence of SEQ ID NO: 20 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 21, and CDR3 containing the amino acid sequence of SEQ ID NO:

16. (x) CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 23, and CDR3 containing the amino acid sequence of SEQ ID NO: 19 (xi) CDR1 containing the amino acid sequence of SEQ ID NO: 24 or 198, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO:

26. (xi) CDR1 containing the amino acid sequence of SEQ ID NO: 27, CDR2 containing the amino acid sequence of SEQ ID NO: 28, and CDR3 containing the amino acid sequence of SEQ ID NO:

29. (xiiii) CDR1 containing the amino acid sequence of SEQ ID NO: 14 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 30, and CDR3 containing the amino acid sequence of SEQ ID NO:

16. (xiv) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 31, and CDR3 containing the amino acid sequence of SEQ ID NO:

19. (xv) CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 containing the amino acid sequence of SEQ ID NO: 32, and CDR3 containing the amino acid sequence of SEQ ID NO:

3. (xvi) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 6 (xxiv) CDR1 containing the amino acid sequence of SEQ ID NO: 165 or 199, CDR2 containing the amino acid sequence of SEQ ID NO: 166, and CDR3 containing the amino acid sequence of SEQ ID NO:

167. (xxv) CDR1 containing the amino acid sequence of SEQ ID NO: 168, CDR2 containing the amino acid sequence of SEQ ID NO: 169, and CDR3 containing the amino acid sequence of SEQ ID NO: 170, (xxvi) CDR1 containing the amino acid sequence of SEQ ID NO: 171 or 200, CDR2 containing the amino acid sequence of SEQ ID NO: 172, and CDR3 containing the amino acid sequence of SEQ ID NO:

173. (xxvii) CDR1 containing the amino acid sequence of SEQ ID NO: 174, CDR2 containing the amino acid sequence of SEQ ID NO: 175, and CDR3 containing the amino acid sequence of SEQ ID NO:

176. (xxviiii) CDR1 containing the amino acid sequence of SEQ ID NO: 177 or 197, CDR2 containing the amino acid sequence of SEQ ID NO: 178, and CDR3 containing the amino acid sequence of SEQ ID NO: 179, or (xxix) CDR1 containing the amino acid sequence of SEQ ID NO: 180, CDR2 containing the amino acid sequence of SEQ ID NO: 181, and CDR3 containing the amino acid sequence of SEQ ID NO:

182. The anti-CD20 sdAb includes the aforementioned anti-CD20 sdAb.

2. CDR1 containing the amino acid sequence of SEQ ID NO: 1 or 195, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO: 3 The anti-CD20 sdAb according to claim 1, including the following:

3. Anti-CD20 single-domain antibody (sdAb), (i) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 41, (ii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 42, (iii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 43, (iv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 44, (v) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 45, (vi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 46, (vii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 47, (viiii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 48, (ix) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 49, (x) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 50, (xi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 51, (xi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 52, (xiiii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 53, (xxiv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 183, respectively, (xxv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 184, respectively, or (xxvi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 185, respectively. Includes, Here, the anti-CD20 sdAb is determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, or Contact numbering scheme, where CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, or Contact numbering scheme.

4. The anti-CD20 sdAb according to claim 3, comprising CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in Sequence ID No. 41, respectively.

5. The amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 183, SEQ ID NO: 184, or SEQ ID NO: 185 is included, or A sequence containing, or consisting of, an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 183, SEQ ID NO: 184, or SEQ ID NO: 185 Anti-CD20 sdAb according to claim 1 or 3.

6. comprising the amino acid sequence of Sequence ID No. 41, or It contains, or consists of, an amino acid sequence having at least 90% sequence identity with the sequence of Sequence ID No.

41. Anti-CD20 sdAb according to claim 5.

7. The anti-CD20 sdAb according to claim 1 or claim 3, which is a camelid sdAb or a humanized sdAb.

8. The anti-CD20 sdAb according to any one of claims 1 to 7, wherein the drug is genetically fused to or chemically conjugated with the anti-CD20 sdAb.

9. (a) an extracellular antigen-binding domain comprising anti-CD20 sdAb as described in any one of claims 1 to 8; (b) Transmembrane domains; and (c) Intracellular signal transduction domain Chimeric antigen receptors (CARs), including those mentioned above.

10. The CAR according to claim 9, wherein the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains.

11. The CAR according to claim 10, wherein the one or more additional antigen-binding domains(s) bind to one or more antigens(s) selected from the group consisting of CD20, CD19, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77.

12. The CAR according to claim 10, wherein the extracellular antigen-binding domain further comprises an additional antigen-binding domain for CD19.

13. The CAR according to any one of claims 9 to 12, wherein the extracellular antigen-binding domain comprises two or three of the anti-CD20 sdAbs described in any one of claims 1 to 8.

14. The CAR according to any one of claims 9 to 13, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

15. The CAR according to any one of claims 9 to 14, wherein the intracellular signaling domain includes the primary intracellular signaling domain of an immune effector cell.

16. The CAR according to claim 15, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain.

17. The CAR according to any one of claims 9 to 16, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

18. The CAR according to any one of claims 9 to 17, further comprising a signal peptide located at the N-terminus of the polypeptide.

19. A chimeric antigen receptor (CAR) comprising anti-CD20 sdAb as described in claim 1, wherein the CAR is (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 98, 99, 100, 101, 102, 103, 104, 105, 106, 189, 190, and 191, or (ii) Amino acid sequences having at least 90% sequence identity with the sequences of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 98, 99, 100, 101, 102, 103, 104, 105, 106, 189, 190, or 191. Chimeric antigen receptors (CARs), including those mentioned above.

20. The CAR according to claim 19, comprising the amino acid sequence of SEQ ID NO: 81, or comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:

81.

21. An isolated nucleic acid comprising a nucleic acid sequence encoding a CAR as described in any one of claims 9 to 20.

22. A vector comprising the isolated nucleic acid described in claim 21.

23. The vector according to claim 22, further comprising an additional isolated nucleic acid containing a nucleic acid sequence encoding an additional CAR.

24. Engineered immunoeffector cells comprising a CAR according to any one of claims 9 to 20, an isolated nucleic acid according to claim 21, or a vector according to claim 22 or 23.

25. The manipulated immune effector cell according to claim 24, comprising the CAR according to any one of claims 9 to 20, wherein the manipulated immune effector cell further comprises an additional CAR.

26. The manipulated immune effector cell according to claim 24 or 25, which is a T cell or a B cell.

27. A pharmaceutical composition comprising an anti-CD20 sdAb according to any one of claims 1 to 8, an engineered immunoeffector cell according to any one of claims 24 to 26, an isolated nucleic acid according to claim 21, or a vector according to claim 22 or 23, and a pharmaceutically acceptable excipient.

28. Use of anti-CD20 sdAb according to any one of claims 1 to 8, engineered immune effector cells according to any one of claims 24 to 26, or the pharmaceutical composition according to claim 27 in the manufacture of a drug for treating a target disease or disorder.

29. The use according to claim 28, wherein the disease or disorder is a B-cell malignant tumor.

30. The CAR according to claim 14, wherein the transmembrane domain is derived from CD8α.

31. The CAR according to claim 15, wherein the primary intracellular signaling domain is derived from CD3ζ.

32. The CAR according to claim 16, wherein the aforementioned co-stimulus signaling domain is derived from CD137.

33. The CAR according to claim 17, wherein the hinge domain is derived from CD8α.

34. The CAR according to claim 18, wherein the signal peptide is derived from CD8α.

35. The use according to claim 28, wherein the disease or disorder is a B cell-related disease or disorder and / or a CD20-related disease or disorder.

36. The use according to claim 29, wherein the B-cell malignant tumor is B-cell leukemia or B-cell lymphoma.

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